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1
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Huang Y, Li GM. Role of HSP40 proteins in genome maintenance, insulin signaling and cancer therapy. DNA Repair (Amst) 2025; 149:103839. [PMID: 40267605 DOI: 10.1016/j.dnarep.2025.103839] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2024] [Revised: 02/05/2025] [Accepted: 04/11/2025] [Indexed: 04/25/2025]
Abstract
The DnaJ heat shock protein family (HSP40) is the biggest chaperone family in mammalian cells, mainly functioning as cochaperone of HSP70 to maintain proteostasis and cellular homeostasis under both normal and stressful conditions. Although the functions of HSP70s have been extensively studied in diverse biological pathways and senesces including genome maintenance, HSP40s' biological functions at basal state or in response to exogenous insults remain largely under-investigated. Emerging evidence shows that HSP40 proteins participate in genome maintenance pathways and modulate cancer therapy efficacy. This review aims to summarize recent progresses regarding HSP40's functions in genome maintenance and cancer therapy, and provides hints for future studies in the field.
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Affiliation(s)
- Yaping Huang
- Institute for Molecular and Cellular Therapeutics, Chinese Institutes for Medical Research, Beijing, China; School of Basic Medical Sciences, Capital Medical University, Beijing, China.
| | - Guo-Min Li
- School of Basic Medical Sciences, Capital Medical University, Beijing, China; Institute for Cancer Research, Chinese Institutes for Medical Research, Beijing, China
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2
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Wang T, Wang Z. Targeting the "Undruggable": Small-Molecule Inhibitors of Proliferating Cell Nuclear Antigen (PCNA) in the Spotlight in Cancer Therapy. J Med Chem 2025; 68:2058-2088. [PMID: 39904718 DOI: 10.1021/acs.jmedchem.4c00526] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2025]
Abstract
PCNA plays multiple roles in cancer development, including cell proliferation regulation, DNA repair, replication, and serving as a widely used biomarker and therapeutic target. Despite its significant role in oncology, PCNA has historically been considered "undruggable" due to the absence of known endogenous small molecule modulators and identifiable ligand binding sites. Unlike other protein-protein interfaces, PCNA lacks explicit binding grooves, featuring a relatively small and shallow surface pocket, which hinders the discovery of traditional small molecule targets. Recent breakthroughs have introduced promising PCNA-targeting candidates, with ATX-101 and AOH1996 entering phase I clinical trials for cancer therapy, garnering academic and industry interest. These achievements provide new evidence for PCNA as a drug target. This article provides insight and perspective on the application of small-molecule PCNA inhibitors in cancer treatment, covering PCNA function, its relationship with cancer, structural modification of small molecule inhibitors, and discovery strategies.
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Affiliation(s)
- Tiantian Wang
- College of Pharmacy, Jiangxi University of Chinese Medicine, Nanchang 330004, P. R. China
- National Pharmaceutical Engineering Center for Solid Preparation in Chinese Herbal Medicine, Jiangxi University of Chinese Medicine, Nanchang 330006, P. R. China
| | - Zengtao Wang
- College of Pharmacy, Jiangxi University of Chinese Medicine, Nanchang 330004, P. R. China
- Jiangxi Provincial Key Laboratory of TCM Female Reproductive Health and Related Diseases Research and Transformation, Jiangxi University of Chinese Medicine, Nanchang 330004, P. R. China
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3
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Gad F, Abdelghaffar Emam M, Eldeeb AA, Abdelhameed AA, Soliman MM, Alotaibi KS, Albattal SB, Abughrien B. Mitigative Effects of l-Arginine and N-Acetyl Cysteine against Cisplatin-Induced Testicular Dysfunction and Toxicity through the Regulation of Antioxidant, Anti-inflammatory, and Antiapoptotic Markers: Role of miR-155 and miR-34c Expression. ACS OMEGA 2024; 9:27680-27691. [PMID: 38947789 PMCID: PMC11209920 DOI: 10.1021/acsomega.4c03742] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/18/2024] [Revised: 05/28/2024] [Accepted: 06/05/2024] [Indexed: 07/02/2024]
Abstract
Testicular dysfunction is a common adverse effect of cisplatin (CIS) administration as a chemotherapeutic drug. The current study has outlined the role of micro-RNAs (miR-155 and 34c) in CIS-induced testicular dysfunction and evaluated the protective effect of N-acetyl cysteine (NAC) and/or l-arginine (LA). Seven groups of Albino rats were used for this study. The control (C) group received physiological saline; the CIS group was injected CIS (7 mg/kg IP, once) on day 21 of the experiment; the NAC group was administered NAC (150 mg/kg intragastric, for 28 days); and the LA group was injected LA (50 mg/kg IP, for 28 days). NAC+CIS, LA+CIS, and NAC+LA+CIS groups received the above regime. CIS significantly reduced serum testosterone, LH, and FSH concentrations with decline of testicular enzyme activities. CIS caused significant elevation in testicular oxidative-stress biomarkers, inflammation-associated cytokines, and apoptosis markers, along with overexpression of miR-155 and low miR-34c expression. Additionally, marked testicular degenerative changes were observed in the examined histological section; a significant decrease in the expression of PCNA with significant increase in expressions of F4/80 and BAX was confirmed. The administration of NAC or LA upregulated testicular functions and improved histopathological and immunohistochemical changes as well as miRNA expression compared with the CIS-administered group. Rats receiving both NAC and LA showed a more significant ameliorative effect compared with groups receiving NAC or LA alone. In conclusion, NAC or LA showed an ameliorative effect against CIS-induced testicular toxicity and dysfunction through the regulation of antioxidant, anti-inflammatory, and antiapoptotic markers and via modulating miR-155 and miR-34c expression.
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Affiliation(s)
- Fatma
A. Gad
- Clinical
Pathology Department, Faculty of Veterinary Medicine, Benha University, P.O. Box13736 Benha, Egypt
| | - Mahmoud Abdelghaffar Emam
- Histology
Department., Faculty of Veterinary Medicine, Benha University, P.O. Box 13736 Benha, Egypt
| | - Abeer A. Eldeeb
- Clinical
Pharmacology Department, Faculty of Medicine, Benha University, 13511 Benha, Egypt
| | - Abeer A. Abdelhameed
- Clinical
Pharmacology Department, Faculty of Medicine, Benha University, 13511 Benha, Egypt
| | - Mohamed Mohamed Soliman
- Department
of Clinical Laboratory Sciences, Turabah University College, Taif University, P.O.
Box 11099, Taif 21944, Saudi Arabia
| | - Khalid S. Alotaibi
- General
Science and English Language Department, College of Applied Sciences, AlMaarefa University, Riyadh 71666, Saudi Arabia
| | - Shatha B. Albattal
- General
Science and English Language Department, College of Applied Sciences, AlMaarefa University, Riyadh 71666, Saudi Arabia
| | - Badia Abughrien
- Anatomy and
Histology Department, Faculty of Veterinary Medicine, Tripoli University, 15673 Tripoli, Libya
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4
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Huber HF, Li C, Xie D, Gerow KG, Register TC, Shively CA, Cox LA, Nathanielsz PW. Female baboon adrenal zona fasciculata and zona reticularis regulatory and functional proteins decrease across the life course. GeroScience 2024; 46:3405-3417. [PMID: 38311700 PMCID: PMC11009170 DOI: 10.1007/s11357-024-01080-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2023] [Accepted: 01/09/2024] [Indexed: 02/06/2024] Open
Abstract
Debate exists on life-course adrenocortical zonal function trajectories. Rapid, phasic blood steroid concentration changes, such as circadian rhythms and acute stress responses, complicate quantification. To avoid pitfalls and account for life-stage changes in adrenocortical activity indices, we quantified zonae fasciculata (ZF) and reticularis (ZR) across the life-course, by immunohistochemistry of key regulatory and functional proteins. In 28 female baboon adrenals (7.5-22.1 years), we quantified 12 key proteins involved in cell metabolism, division, proliferation, steroidogenesis (including steroid acute regulatory protein, StAR), oxidative stress, and glucocorticoid and mitochondrial function. Life-course abundance of ten ZF proteins decreased with age. Cell cycle inhibitor and oxidative stress markers increased. Seven of the 12 proteins changed in the same direction for ZR and ZF. Importantly, ZF StAR decreased, while ZR StAR was unchanged. Findings indicate ZF function decreased, and less markedly ZR function, with age. Causes and aging consequences of these changes remain to be determined.
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Affiliation(s)
- Hillary Fries Huber
- Southwest National Primate Research Center, Texas Biomedical Research Institute, 8715 W. Military, San Antonio, TX, 78227, USA.
| | - Cun Li
- Texas Pregnancy & Life-Course Health Research Center, Animal Science, University of Wyoming, Laramie, WY, USA
| | - Dongbin Xie
- Texas Pregnancy & Life-Course Health Research Center, Animal Science, University of Wyoming, Laramie, WY, USA
| | | | - Thomas C Register
- Pathology-Comparative Medicine, Wake Forest School of Medicine, Winston-Salem, NC, USA
| | - Carol A Shively
- Pathology-Comparative Medicine, Wake Forest School of Medicine, Winston-Salem, NC, USA
| | - Laura A Cox
- Southwest National Primate Research Center, Texas Biomedical Research Institute, 8715 W. Military, San Antonio, TX, 78227, USA
- Center for Precision Medicine, Wake Forest School of Medicine, Winston-Salem, NC, USA
| | - Peter W Nathanielsz
- Southwest National Primate Research Center, Texas Biomedical Research Institute, 8715 W. Military, San Antonio, TX, 78227, USA
- Texas Pregnancy & Life-Course Health Research Center, Animal Science, University of Wyoming, Laramie, WY, USA
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5
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Ershova ES, Savinova EA, Kameneva LV, Porokhovnik LN, Veiko RV, Salimova TA, Izhevskaya VL, Kutsev SI, Veiko NN, Kostyuk SV. Antipsychotics Affect Satellite III (1q12) Copy Number Variations in the Cultured Human Skin Fibroblasts. Int J Mol Sci 2023; 24:11283. [PMID: 37511043 PMCID: PMC10380077 DOI: 10.3390/ijms241411283] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2023] [Revised: 07/01/2023] [Accepted: 07/05/2023] [Indexed: 07/30/2023] Open
Abstract
The fragment of satellite III (f-SatIII) is located in pericentromeric heterochromatin of chromosome 1. Cell with an enlarged f-SatIII block does not respond to various stimuli and are highly stress-susceptible. The fraction of f-SatIII in the cells of schizophrenia patients changed during antipsychotic therapy. Therefore, antipsychotics might reduce the f-SatIII content in the cells. We studied the action of haloperidol, risperidone and olanzapine (3 h, 24 h, 96 h) on human skin fibroblast lines (n = 10). The f-SatIII contents in DNA were measured using nonradioactive quantitative hybridization. RNASATIII were quantified using RT-qPCR. The levels of DNA damage markers (8-oxodG, γ-H2AX) and proteins that regulate apoptosis and autophagy were determined by flow cytometry. The antipsychotics reduced the f-SatIII content in DNA and RNASATIII content in RNA from HSFs. After an exposure to the antipsychotics, the autophagy marker LC3 significantly increased, while the apoptosis markers decreased. The f-SatIII content in DNA positively correlated with RNASATIII content in RNA and with DNA oxidation marker 8-oxodG, while negatively correlated with LC3 content. The antipsychotics arrest the process of f-SatIII repeat augmentation in cultured skin fibroblasts via the transcription suppression and/or through upregulated elimination of cells with enlarged f-SatIII blocks with the help of autophagy.
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Affiliation(s)
- Elizaveta S Ershova
- Research Centre for Medical Genetics, 1 Moskvorechye St., 115522 Moscow, Russia
| | | | - Larisa V Kameneva
- Research Centre for Medical Genetics, 1 Moskvorechye St., 115522 Moscow, Russia
| | - Lev N Porokhovnik
- Research Centre for Medical Genetics, 1 Moskvorechye St., 115522 Moscow, Russia
| | - Roman V Veiko
- Research Centre for Medical Genetics, 1 Moskvorechye St., 115522 Moscow, Russia
| | - Tatiana A Salimova
- Research Centre for Medical Genetics, 1 Moskvorechye St., 115522 Moscow, Russia
| | - Vera L Izhevskaya
- Research Centre for Medical Genetics, 1 Moskvorechye St., 115522 Moscow, Russia
| | - Sergey I Kutsev
- Research Centre for Medical Genetics, 1 Moskvorechye St., 115522 Moscow, Russia
| | - Natalia N Veiko
- Research Centre for Medical Genetics, 1 Moskvorechye St., 115522 Moscow, Russia
| | - Svetlana V Kostyuk
- Research Centre for Medical Genetics, 1 Moskvorechye St., 115522 Moscow, Russia
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6
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Quan J, Li X, Li Z, Wu M, Zhu B, Hong SB, Shi J, Zhu Z, Xu L, Zang Y. Transcriptomic Analysis of Heat Stress Response in Brassica rapa L. ssp. pekinensis with Improved Thermotolerance through Exogenous Glycine Betaine. Int J Mol Sci 2023; 24:ijms24076429. [PMID: 37047402 PMCID: PMC10094913 DOI: 10.3390/ijms24076429] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2023] [Revised: 03/23/2023] [Accepted: 03/23/2023] [Indexed: 04/03/2023] Open
Abstract
Chinese cabbage (Brassica rapa L. ssp. pekinensis) is sensitive to high temperature, which will cause the B. rapa to remain in a semi-dormancy state. Foliar spray of GB prior to heat stress was proven to enhance B. rapa thermotolerance. In order to understand the molecular mechanisms of GB-primed resistance or adaptation towards heat stress, we investigated the transcriptomes of GB-primed and non-primed heat-sensitive B. rapa ‘Beijing No. 3’ variety by RNA-Seq analysis. A total of 582 differentially expressed genes (DEGs) were identified from GB-primed plants exposed to heat stress relative to non-primed plants under heat stress and were assigned to 350 gene ontology (GO) pathways and 69 KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways. The analysis of the KEGG enrichment pathways revealed that the most abundantly up-regulated pathways were protein processing in endoplasmic reticulum (14 genes), followed by plant hormone signal transduction (12 genes), ribosome (8 genes), MAPK signaling pathway (8 genes), homologous recombination (7 genes), nucleotide excision repair metabolism (5 genes), glutathione metabolism (4 genes), and ascorbate and aldarate metabolism (4 genes). The most abundantly down-regulated pathways were plant-pathogen interaction (14 genes), followed by phenylpropanoid biosynthesis (7 genes); arginine and proline metabolism (6 genes); cutin, suberine, and wax biosynthesis (4 genes); and tryptophan metabolism (4 genes). Several calcium sensing/transducing proteins, as well as transcription factors associated with abscisic acid (ABA), salicylic acid (SA), auxin, and cytokinin hormones were either up- or down-regulated in GB-primed B. rapa plants under heat stress. In particular, expression of the genes for antioxidant defense, heat shock response, and DNA damage repair systems were highly increased by GB priming. On the other hand, many of the genes involved in the calcium sensors and cell surface receptors involved in plant innate immunity and the biosynthesis of secondary metabolites were down-regulated in the absence of pathogen elicitors in GB-primed B. rapa seedlings. Overall GB priming activated ABA and SA signaling pathways but deactivated auxin and cytokinin signaling pathways while suppressing the innate immunity in B. rapa seedlings exposed to heat stress. The present study provides a preliminary understanding of the thermotolerance mechanisms in GB-primed plants and is of great importance in developing thermotolerant B. rapa cultivars by using the identified DEGs through genetic modification.
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7
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Caffeic acid abrogates amyloidosis, hypospermatogenesis and cell membrane alterations in the testes and epididymis of fructose-diabetic rats by upregulating steroidogenesis, PCNA and Nrf2 expression. Tissue Cell 2022; 79:101912. [DOI: 10.1016/j.tice.2022.101912] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2022] [Revised: 06/11/2022] [Accepted: 08/28/2022] [Indexed: 11/22/2022]
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8
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Liu X, Liu H, Ye G, Xue M, Yu H, Feng C, Zhou Q, Liu X, Zhang L, Jiao S, Weng C, Huang L. African swine fever virus pE301R negatively regulates cGAS-STING signaling pathway by inhibiting the nuclear translocation of IRF3. Vet Microbiol 2022; 274:109556. [PMID: 36099692 DOI: 10.1016/j.vetmic.2022.109556] [Citation(s) in RCA: 19] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2022] [Revised: 08/16/2022] [Accepted: 09/01/2022] [Indexed: 12/11/2022]
Abstract
African swine fever (ASF) is a highly contagious and lethal infectious disease of domestic pigs and wild boars by the African swine fever virus (ASFV). ASFV infects domestic pigs with the mortality rate approaching 100 % at acute stage of infection. The cGAS-STING-mediated antiviral responses are wildly accepted that cGAS acts as DNA sensor for sensing of viral DNA during DNA virus infection. However, the molecular mechanisms underlying negatively regulation of cGAS-STING signaling and type I IFN (IFN-I) production by ASFV proteins are not fully understood. In this study, we demonstrated that ASFV pE301R antagonize the activities of IFN-β-, NF-κB-, ISRE-luciferase (Luc) reporters-induced by cGAS-STING in a dose dependent manner. Consistent with these results, the mRNA levels of Ifnb1, Isg15, Isg56 are attenuated by ASFV pE301R. Furthermore, ASFV pE301R executes its inhibitory function at the downstream of IFN-regulatory factor 3 (IRF3) phosphorylation. Mechanistically, pE301R interacts with IRF3 via its amino acid (aa) 1-200 region, resulting in inhibition of the nuclear translocation of IRF3 induced by cGAMP and poly(dA:dT). Overall, our findings reveal that pE301R acts as a negatively regulator to inhibit IFN-I production and to subvert host antiviral innate immunity during ASFV infection.
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Affiliation(s)
- Xiaohong Liu
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin 150069, China
| | - Hongyang Liu
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin 150069, China
| | - Guangqiang Ye
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin 150069, China
| | - Mengdi Xue
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin 150069, China
| | - Huibin Yu
- Department of Immunobiology, Yale University School of Medicine, New Haven 06511, CT, USA
| | - Chunying Feng
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin 150069, China
| | - Qiongqiong Zhou
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin 150069, China
| | - Xuemin Liu
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin 150069, China
| | - Longfeng Zhang
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin 150069, China
| | - Shuang Jiao
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin 150069, China
| | - Changjiang Weng
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin 150069, China; Heilongjiang Provincial Key Laboratory of Veterinary Immunology, Harbin 150069, China.
| | - Li Huang
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin 150069, China; Heilongjiang Provincial Key Laboratory of Veterinary Immunology, Harbin 150069, China.
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9
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Blando S, Raffaele I, Chiricosta L, Valeri A, Gugliandolo A, Silvestro S, Pollastro F, Mazzon E. Cannabidiol Promotes Neuronal Differentiation Using Akt and Erk Pathways Triggered by Cb1 Signaling. Molecules 2022; 27:molecules27175644. [PMID: 36080415 PMCID: PMC9457834 DOI: 10.3390/molecules27175644] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2022] [Revised: 08/25/2022] [Accepted: 08/30/2022] [Indexed: 11/17/2022] Open
Abstract
Recently, the scientific community has started to focus on the neurogenic potential of cannabinoids. The phytocompound cannabidiol (CBD) shows different mechanism of signaling on cannabinoid receptor 1 (CB1), depending on its concentration. In this study, we investigated if CBD may induce in vitro neuronal differentiation after treatment at 5 µM and 10 µM. For this purpose, we decided to use the spinal cord × neuroblastoma hybrid cell line (NSC-34) because of its proliferative and undifferentiated state. The messenger RNAs (mRNAs) expression profiles were tested using high-throughput sequencing technology and Western blot assay was used to determine the number of main proteins in different pathways. Interestingly, the treatment shows different genes associated with neurodifferentiation statistically significant, such as Rbfox3, Tubb3, Pax6 and Eno2. The CB1 signaling pathway is responsible for neuronal differentiation at 10 µM, as suggested by the presence of p-ERK and p-AKT, but not at 5 µM. A new correlation between CBD, neurodifferentiation and retinoic acid receptor-related orphan receptors (RORs) has been observed.
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Affiliation(s)
- Santino Blando
- IRCCS Centro Neurolesi “Bonino-Pulejo”, Via Provinciale Palermo, Contrada Casazza, 98124 Messina, Italy,
| | - Ivana Raffaele
- IRCCS Centro Neurolesi “Bonino-Pulejo”, Via Provinciale Palermo, Contrada Casazza, 98124 Messina, Italy,
| | - Luigi Chiricosta
- IRCCS Centro Neurolesi “Bonino-Pulejo”, Via Provinciale Palermo, Contrada Casazza, 98124 Messina, Italy,
| | - Andrea Valeri
- IRCCS Centro Neurolesi “Bonino-Pulejo”, Via Provinciale Palermo, Contrada Casazza, 98124 Messina, Italy,
| | - Agnese Gugliandolo
- IRCCS Centro Neurolesi “Bonino-Pulejo”, Via Provinciale Palermo, Contrada Casazza, 98124 Messina, Italy,
| | - Serena Silvestro
- IRCCS Centro Neurolesi “Bonino-Pulejo”, Via Provinciale Palermo, Contrada Casazza, 98124 Messina, Italy,
| | - Federica Pollastro
- Department of Pharmaceutical Sciences, University of Eastern Piedmont, Largo Donegani 2, 28100 Novara, Italy
| | - Emanuela Mazzon
- IRCCS Centro Neurolesi “Bonino-Pulejo”, Via Provinciale Palermo, Contrada Casazza, 98124 Messina, Italy,
- Correspondence:
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10
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Zheng F, Georgescu RE, Yao NY, Li H, O'Donnell ME. Cryo-EM structures reveal that RFC recognizes both the 3'- and 5'-DNA ends to load PCNA onto gaps for DNA repair. eLife 2022; 11:77469. [PMID: 35829698 PMCID: PMC9293004 DOI: 10.7554/elife.77469] [Citation(s) in RCA: 18] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2022] [Accepted: 06/06/2022] [Indexed: 12/27/2022] Open
Abstract
RFC uses ATP to assemble PCNA onto primed sites for replicative DNA polymerases d and e. The RFC pentamer forms a central chamber that binds 3' ss/ds DNA junctions to load PCNA onto DNA during replication. We show here five structures that identify a 2nd DNA binding site in RFC that binds a 5' duplex. This 5' DNA site is located between the N-terminal BRCT domain and AAA+ module of the large Rfc1 subunit. Our structures reveal ideal binding to a 7-nt gap, which includes 2 bp unwound by the clamp loader. Biochemical studies show enhanced binding to 5 and 10 nt gaps, consistent with the structural results. Because both 3' and 5' ends are present at a ssDNA gap, we propose that the 5' site facilitates RFC's PCNA loading activity at a DNA damage-induced gap to recruit gap-filling polymerases. These findings are consistent with genetic studies showing that base excision repair of gaps greater than 1 base requires PCNA and involves the 5' DNA binding domain of Rfc1. We further observe that a 5' end facilitates PCNA loading at an RPA coated 30-nt gap, suggesting a potential role of the RFC 5'-DNA site in lagging strand DNA synthesis.
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Affiliation(s)
- Fengwei Zheng
- Department of Structural Biology, Van Andel Institute, Grand Rapids, United States
| | - Roxana E Georgescu
- DNA Replication Laboratory, Rockefeller University, New York, United States
| | - Nina Y Yao
- DNA Replication Laboratory, Rockefeller University, New York, United States
| | - Huilin Li
- Department of Structural Biology, Van Andel Institute, Grand Rapids, United States
| | - Michael E O'Donnell
- Howard Hughes Medical Institute, Rockefeller University, New York, United States
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11
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Post-Translational Modifications of PCNA: Guiding for the Best DNA Damage Tolerance Choice. J Fungi (Basel) 2022; 8:jof8060621. [PMID: 35736104 PMCID: PMC9225081 DOI: 10.3390/jof8060621] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2022] [Revised: 06/01/2022] [Accepted: 06/07/2022] [Indexed: 02/01/2023] Open
Abstract
The sliding clamp PCNA is a multifunctional homotrimer mainly linked to DNA replication. During this process, cells must ensure an accurate and complete genome replication when constantly challenged by the presence of DNA lesions. Post-translational modifications of PCNA play a crucial role in channeling DNA damage tolerance (DDT) and repair mechanisms to bypass unrepaired lesions and promote optimal fork replication restart. PCNA ubiquitination processes trigger the following two main DDT sub-pathways: Rad6/Rad18-dependent PCNA monoubiquitination and Ubc13-Mms2/Rad5-mediated PCNA polyubiquitination, promoting error-prone translation synthesis (TLS) or error-free template switch (TS) pathways, respectively. However, the fork protection mechanism leading to TS during fork reversal is still poorly understood. In contrast, PCNA sumoylation impedes the homologous recombination (HR)-mediated salvage recombination (SR) repair pathway. Focusing on Saccharomyces cerevisiae budding yeast, we summarized PCNA related-DDT and repair mechanisms that coordinately sustain genome stability and cell survival. In addition, we compared PCNA sequences from various fungal pathogens, considering recent advances in structural features. Importantly, the identification of PCNA epitopes may lead to potential fungal targets for antifungal drug development.
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12
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Giannos P, Kechagias KS, Bowden S, Tabassum N, Paraskevaidi M, Kyrgiou M. PCNA in Cervical Intraepithelial Neoplasia and Cervical Cancer: An Interaction Network Analysis of Differentially Expressed Genes. Front Oncol 2021; 11:779042. [PMID: 34900731 PMCID: PMC8661029 DOI: 10.3389/fonc.2021.779042] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2021] [Accepted: 10/28/2021] [Indexed: 11/30/2022] Open
Abstract
The investigation of differentially expressed genes (DEGs) and their interactome could provide valuable insights for the development of markers to optimize cervical intraepithelial neoplasia (CIN) screening and treatment. This study investigated patients with cervical disease to identify gene markers whose dysregulated expression and protein interaction interface were linked with CIN and cervical cancer (CC). Literature search of microarray datasets containing cervical epithelial samples was conducted in Gene Expression Omnibus and Pubmed/Medline from inception until March 2021. Retrieved DEGs were used to construct two protein-protein interaction (PPI) networks. Module DEGs that overlapped between CIN and CC samples, were ranked based on 11 topological algorithms. The highest-ranked hub gene was retrieved and its correlation with prognosis, tissue expression and tumor purity in patients with CC, was evaluated. Screening of the literature yielded 9 microarray datasets (GSE7803, GSE27678, GSE63514, GSE6791, GSE9750, GSE29570, GSE39001, GSE63678, GSE67522). Two PPI networks from CIN and CC samples were constructed and consisted of 1704 and 3748 DEGs along 21393 and 79828 interactions, respectively. Two gene clusters were retrieved in the CIN network and three in the CC network. Multi-algorithmic topological analysis revealed PCNA as the highest ranked hub gene between the two networks, both in terms of expression and interactions. Further analysis revealed that while PCNA was overexpressed in CC tissues, it was correlated with favorable prognosis (log-rank P=0.022, HR=0.58) and tumor purity (P=9.86 × 10-4, partial rho=0.197) in CC patients. This study identified that cervical PCNA exhibited multi-algorithmic topological significance among DEGs from CIN and CC samples. Overall, PCNA may serve as a potential gene marker of CIN progression. Experimental validation is necessary to examine its value in patients with cervical disease.
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Affiliation(s)
- Panagiotis Giannos
- Society of Meta-Research and Biomedical Innovation, Cancer Research Working Group, London, United Kingdom.,Department of Life Sciences, Faculty of Natural Sciences, Imperial College London, London, United Kingdom
| | - Konstantinos S Kechagias
- Society of Meta-Research and Biomedical Innovation, Cancer Research Working Group, London, United Kingdom.,Department of Metabolism, Digestion and Reproduction, Faculty of Medicine, Imperial College London, London, United Kingdom.,Department of Obstetrics and Gynaecology, Chelsea and Westminster Hospital National Health Service (NHS) Foundation Trust, London, United Kingdom
| | - Sarah Bowden
- Department of Metabolism, Digestion and Reproduction, Faculty of Medicine, Imperial College London, London, United Kingdom.,Institute of Reproductive and Developmental Biology, Imperial College London, London, United Kingdom
| | - Neha Tabassum
- Department of Life Sciences, Faculty of Natural Sciences, Imperial College London, London, United Kingdom
| | - Maria Paraskevaidi
- Department of Metabolism, Digestion and Reproduction, Faculty of Medicine, Imperial College London, London, United Kingdom.,Institute of Reproductive and Developmental Biology, Imperial College London, London, United Kingdom
| | - Maria Kyrgiou
- Department of Metabolism, Digestion and Reproduction, Faculty of Medicine, Imperial College London, London, United Kingdom.,Institute of Reproductive and Developmental Biology, Imperial College London, London, United Kingdom.,Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, London, United Kingdom.,West London Gynaecological Cancer Centre, Imperial College Healthcare National Health Service (NHS) Trust, London, United Kingdom
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13
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Diez A, An HY, Carfagnini N, Bottini C, MacDougall-Shackleton SA. Neurogenesis and the development of neural sex differences in vocal control regions of songbirds. J Comp Neurol 2021; 529:2970-2986. [PMID: 33719029 DOI: 10.1002/cne.25138] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2020] [Revised: 03/08/2021] [Accepted: 03/09/2021] [Indexed: 11/10/2022]
Abstract
The brain regions that control the learning and production of song and other learned vocalizations in songbirds exhibit some of the largest sex differences in the brain known in vertebrates and are associated with sex differences in singing behavior. Song learning takes place through multiple stages: an early sensory phase when song models are memorized, followed by a sensorimotor phase in which auditory feedback is used to modify song output through subsong, plastic song, to adult crystalized song. However, how patterns of neurogenesis in these brain regions change through these learning stages, and differ between the sexes, is little explored. We collected brains from 63 young male and female zebra finches (Taeniopygia guttata) over four stages of song learning. Using neurogenesis markers for cell division (proliferating cell nuclear antigen), neuron migration (doublecortin), and mature neurons (neuron-specific nuclear protein), we demonstrate that there are sex-specific changes in neurogenesis over song development that differ between the caudal motor pathway and anterior forebrain pathway of the vocal control circuit. In many of these regions, sex differences emerged very early in development, by 25 days post hatch, at the beginning of song learning. The emergence of sex differences in other components of the system was more gradual and had specific trajectories depending on the brain region and its function. In conclusion, we found that sex differences occurred early and continued during song learning. Moreover, transitions from the different phases of song development do not seem to depend on large changes in neurogenesis in the vocal control areas measured.
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Affiliation(s)
- Adriana Diez
- Graduate Program in Neuroscience, University of Western Ontario, London, Ontario, Canada.,Advanced Facility for Avian Research, University of Western Ontario, London, Ontario, Canada
| | - Ha Yun An
- Advanced Facility for Avian Research, University of Western Ontario, London, Ontario, Canada
| | - Nicole Carfagnini
- Department of Biology, University of Western Ontario, London, Ontario, Canada
| | - Claire Bottini
- Department of Biology, University of Western Ontario, London, Ontario, Canada.,Department of Psychology, University of Western Ontario, London, Ontario, Canada
| | - Scott A MacDougall-Shackleton
- Advanced Facility for Avian Research, University of Western Ontario, London, Ontario, Canada.,Department of Biology, University of Western Ontario, London, Ontario, Canada.,Department of Psychology, University of Western Ontario, London, Ontario, Canada
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14
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Kirdajova D, Valihrach L, Valny M, Kriska J, Krocianova D, Benesova S, Abaffy P, Zucha D, Klassen R, Kolenicova D, Honsa P, Kubista M, Anderova M. Transient astrocyte-like NG2 glia subpopulation emerges solely following permanent brain ischemia. Glia 2021; 69:2658-2681. [PMID: 34314531 PMCID: PMC9292252 DOI: 10.1002/glia.24064] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2021] [Revised: 07/12/2021] [Accepted: 07/13/2021] [Indexed: 12/13/2022]
Abstract
NG2 glia display wide proliferation and differentiation potential under physiological and pathological conditions. Here, we examined these two features following different types of brain disorders such as focal cerebral ischemia (FCI), cortical stab wound (SW), and demyelination (DEMY) in 3‐month‐old mice, in which NG2 glia are labeled by tdTomato under the Cspg4 promoter. To compare NG2 glia expression profiles following different CNS injuries, we employed single‐cell RT‐qPCR and self‐organizing Kohonen map analysis of tdTomato‐positive cells isolated from the uninjured cortex/corpus callosum and those after specific injury. Such approach enabled us to distinguish two main cell populations (NG2 glia, oligodendrocytes), each of them comprising four distinct subpopulations. The gene expression profiling revealed that a subpopulation of NG2 glia expressing GFAP, a marker of reactive astrocytes, is only present transiently after FCI. However, following less severe injuries, namely the SW and DEMY, subpopulations mirroring different stages of oligodendrocyte maturation markedly prevail. Such injury‐dependent incidence of distinct subpopulations was also confirmed by immunohistochemistry. To characterize this unique subpopulation of transient astrocyte‐like NG2 glia, we used single‐cell RNA‐sequencing analysis and to disclose their basic membrane properties, the patch‐clamp technique was employed. Overall, we have proved that astrocyte‐like NG2 glia are a specific subpopulation of NG2 glia emerging transiently only following FCI. These cells, located in the postischemic glial scar, are active in the cell cycle and display a current pattern similar to that identified in cortical astrocytes. Astrocyte‐like NG2 glia may represent important players in glial scar formation and repair processes, following ischemia.
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Affiliation(s)
- Denisa Kirdajova
- Department of Cellular Neurophysiology, Institute of Experimental Medicine, Czech Academy of Sciences, Prague, Czech Republic.,Second Faculty of Medicine, Charles University, Prague, Czech Republic
| | - Lukas Valihrach
- Laboratory of Gene Expression, Institute of Biotechnology CAS, BIOCEV, Vestec, Czech Republic
| | - Martin Valny
- Department of Cellular Neurophysiology, Institute of Experimental Medicine, Czech Academy of Sciences, Prague, Czech Republic
| | - Jan Kriska
- Department of Cellular Neurophysiology, Institute of Experimental Medicine, Czech Academy of Sciences, Prague, Czech Republic
| | - Daniela Krocianova
- Department of Cellular Neurophysiology, Institute of Experimental Medicine, Czech Academy of Sciences, Prague, Czech Republic
| | - Sarka Benesova
- Laboratory of Gene Expression, Institute of Biotechnology CAS, BIOCEV, Vestec, Czech Republic.,Faculty of Chemical Technology, Laboratory of Informatics and Chemistry, University of Chemistry and Technology, Prague, Czech Republic
| | - Pavel Abaffy
- Laboratory of Gene Expression, Institute of Biotechnology CAS, BIOCEV, Vestec, Czech Republic
| | - Daniel Zucha
- Laboratory of Gene Expression, Institute of Biotechnology CAS, BIOCEV, Vestec, Czech Republic.,Faculty of Science, Charles University, Prague, Czech Republic
| | - Ruslan Klassen
- Laboratory of Gene Expression, Institute of Biotechnology CAS, BIOCEV, Vestec, Czech Republic
| | - Denisa Kolenicova
- Department of Cellular Neurophysiology, Institute of Experimental Medicine, Czech Academy of Sciences, Prague, Czech Republic.,Second Faculty of Medicine, Charles University, Prague, Czech Republic
| | - Pavel Honsa
- Department of Cellular Neurophysiology, Institute of Experimental Medicine, Czech Academy of Sciences, Prague, Czech Republic
| | - Mikael Kubista
- Laboratory of Gene Expression, Institute of Biotechnology CAS, BIOCEV, Vestec, Czech Republic
| | - Miroslava Anderova
- Department of Cellular Neurophysiology, Institute of Experimental Medicine, Czech Academy of Sciences, Prague, Czech Republic.,Second Faculty of Medicine, Charles University, Prague, Czech Republic
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15
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Gaowa N, Li W, Gelsinger S, Murphy B, Li S. Analysis of Host Jejunum Transcriptome and Associated Microbial Community Structure Variation in Young Calves with Feed-Induced Acidosis. Metabolites 2021; 11:414. [PMID: 34201826 PMCID: PMC8303401 DOI: 10.3390/metabo11070414] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2021] [Revised: 06/15/2021] [Accepted: 06/15/2021] [Indexed: 12/05/2022] Open
Abstract
Diet-induced acidosis imposes a health risk to young calves. In this study, we aimed to investigate the host jejunum transcriptome changes, along with its microbial community variations, using our established model of feed-induced ruminal acidosis in young calves. Eight bull calves were randomly assigned to two diet treatments beginning at birth (a starch-rich diet, Aci; a control diet, Con). Whole-transcriptome RNA sequencing was performed on the jejunum tissues collected at 17 weeks of age. Ribosomal RNA reads were used for studying microbial community structure variations in the jejunum. A total of 853 differentially expressed genes were identified (402 upregulated and 451 downregulated) between the two groups. The cell cycle and the digestion and absorption of protein in jejunal tissue were affected by acidosis. Compared to the control, genera of Campylobacter, Burkholderia, Acidaminococcus, Corynebacterium, and Olsenella significantly increased in abundance in the Aci group, while Lachnoclostridium and Ruminococcus were significantly lower in the Aci group. Expression changes in the AXL gene were associated with the abundance variations of a high number of genera in jejunum. Our study provided a snapshot of the transcriptome changes in the jejunum and its associated meta-transcriptome changes in microbial communities in young calves with feed-induced acidosis.
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Affiliation(s)
- Naren Gaowa
- College of Animal Science and Technology, China Agricultural University, No.2 Yuanmingyuan West Road, Haidian, Beijing 100193, China;
| | - Wenli Li
- Cell Wall Biology and Utilization Research Unit, US Dairy Forage Research Center, Agricultural Research Service, US Department of Agriculture, 1925 Linden Drive, Madison, WI 53706, USA;
| | - Sonia Gelsinger
- Department of Dairy Science, University of Wisconsin-Madison, Madison, WI 53706, USA;
| | - Brianna Murphy
- Cell Wall Biology and Utilization Research Unit, US Dairy Forage Research Center, Agricultural Research Service, US Department of Agriculture, 1925 Linden Drive, Madison, WI 53706, USA;
| | - Shengli Li
- College of Animal Science and Technology, China Agricultural University, No.2 Yuanmingyuan West Road, Haidian, Beijing 100193, China;
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16
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Wang C, Guo X, Wang Y, Wang H. Silencing of miR-324-5p alleviates rat spinal cord injury by Sirt1. Neurosci Res 2021; 173:34-43. [PMID: 34051279 DOI: 10.1016/j.neures.2021.05.010] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2021] [Revised: 04/24/2021] [Accepted: 05/23/2021] [Indexed: 12/11/2022]
Abstract
MicroRNAs (miRNAs) are implicated in the pathogenesis of spinal cord injury (SCI) as primary regulators. Previous studies have reported that miR-324-5p is involved in the modulation of neural injury, while the underlying mechanisms of miR-324-5p in SCI remain unclear. In a SCI rat model, miR-324-5p was significantly upregulated in the spinal cord tissues after SCI. Downregulation of miR-324-5p via injection of adeno-associated viruses (AAV) expressing miR-324-5p inhibitor relieved animal motor deficits and pathological changes in the tissues. Furthermore, downregulation of miR-324-5p significantly altered the expression of genes regulating neural growth, apoptosis, and the inflammatory and antioxidant response, which are implicated in SCI pathogenesis. In a H2O2-induced cell injury model, miR-324-5p silencing rescued the elevated apoptosis of PC12 cells. Finally, miR-324-5p directly targeted the 3'-untranslated region of NAD-dependent protein deacetylase sirtuin-1 (Sirt1) and negatively regulated the levels of Sirt1, an anti-inflammatory protein involved in SCI. Silencing of Sirt1 aggravated SCI and rescued the effects of miR-324-5p downregulation in rats. Overall, our findings indicated that silencing of miR-324-5p alleviates the loss of animal locomotion and concurrently mediates several degenerative processes relevant to the pathogenesis of SCI by Sirt1, which may provide clues for SCI treatment.
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Affiliation(s)
- Chuanbao Wang
- Department of Orthopedics, Yantai Mountain Hospital, Yangtai, 264001, Shandong, China.
| | - Xiuli Guo
- Department of Gerontology, Yantai Mountain Hospital, Yangtai, 264001, Shandong, China
| | - Ying Wang
- Department of Neurology, Yantai Mountain Hospital, Yangtai, 264001, Shandong, China
| | - Hai Wang
- Department of Orthopedics, Yantai Mountain Hospital, Yangtai, 264001, Shandong, China
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17
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Öznurlu Y, Özaydın T, Sur E, Özparlak H. The effects of in ovo administered bisphenol A on tibial growth plate histology in chicken. Birth Defects Res 2021; 113:1130-1139. [PMID: 33991407 DOI: 10.1002/bdr2.1925] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2020] [Revised: 04/28/2021] [Accepted: 05/03/2021] [Indexed: 01/13/2023]
Abstract
OBJECTIVES The aim of this study was to determine of the effects of in ovo administered BPA on embryonic development of the tibial growth plate using histological methods in chickens. METHODS Three hundred and ten fertile eggs of Isa Brown laying parent stock were divided into five groups as untreated control, vehicle-injected control, 50, 100, and 250 μg/egg BPA. At the 13th, 18th, and 21st days of incubation, eggs were randomly opened from each group until 10 live embryos were obtained. Embryos were weighed and crown-rump length was measured. Tibial tissue samples were taken from embryos. Tibia weight, relative tibia weight and tibia length were determined. Tissue samples were fixed in 10% buffered formalin solution. Sections were stained with Safranin O staining methods and zones in the growth plate were measured. Also, proliferating cell nuclear antigen (PCNA) was stained immunohistochemically. RESULTS The mortality in the BPA treated groups was higher than untreated control group. The results have revealed that mean relative embryo weights, crown-rump length, mean tibia weight, relative tibia weight, and tibia length of BPA treated groups were significantly lower when compared to the untreated control and vehicle-injected control groups. Also, proliferative zone get significantly narrowed, whereas the transitional and hypertrophic zone thickened and PCNA positive chondrocytes increased in growth plate of BPA treated groups. CONCLUSION These results have suggested that developmental exposure to BPA adversely affected development of the tibial growth plate.
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Affiliation(s)
- Yasemin Öznurlu
- Department of Histology and Embryology, Faculty of Veterinary Medicine, University of Selcuk, Konya, Turkey
| | - Tuğba Özaydın
- Department of Histology and Embryology, Faculty of Veterinary Medicine, University of Selcuk, Konya, Turkey
| | - Emrah Sur
- Department of Histology and Embryology, Faculty of Veterinary Medicine, University of Selcuk, Konya, Turkey
| | - Haluk Özparlak
- Department of Biology, Faculty of Science, University of Selcuk, Konya, Turkey
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18
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Tohamy HG, El-Kazaz SE, Alotaibi SS, Ibrahiem HS, Shukry M, Dawood MAO. Ameliorative Effects of Boswellic Acid on Fipronil-Induced Toxicity: Antioxidant State, Apoptotic Markers, and Testicular Steroidogenic Expression in Male Rats. Animals (Basel) 2021; 11:1302. [PMID: 33946602 PMCID: PMC8147226 DOI: 10.3390/ani11051302] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2021] [Revised: 04/24/2021] [Accepted: 04/26/2021] [Indexed: 12/28/2022] Open
Abstract
The study investigated the ability of boswellic acid (BA) to alleviate the testicular and oxidative injury FPN insecticide intoxication in the male rat model. Rats were randomly assigned to six equivalent groups (six rats each) as the following: control rats orally administered with 2 mL physiological saline/kg of body weight (bwt); boswellic acid (BA1) rats orally administered 250 mg BA/kg bwt; boswellic acid (BA2) rats orally administered 500 mg BA/kg bwt; fipronil (FPN) rats orally administered 20 mg FPN/kg bwt; (FPN + BA1) rats orally administered 20 mg FPN/kg bwt plus 250 mg BA/kg bwt, and (FPN + BA2) rats orally administered 20 mg FPN/kg bwt plus 500 mg BA/kg bwt. After 60 days, semen viability percentage and live spermatozoa percentage were decreased, and a considerably increased abnormality of the sperm cells in FPN-administered rats improved substantially with the co-administration of BA. BA had refinement of the histological architecture of testes and sexual glands. Quantitative analysis recorded a noticeable decline in the nuclear cell-proliferating antigen (PCNA) percentage area. FPN triggered cell damage, which was suggested by elevated malondialdehyde and interleukin 6, tumor necrosis factors alpha, and decreased glutathione level. Proapoptotic factor overexpression is mediated by FPN administration, while it decreased the antiapoptotic protein expression. Similarly, BA has shown significant upregulation in steroidogenic and fertility-related gene expression concerning the FPN group. Pathophysiological damages induced by FPN could be alleviated by BA's antioxidant ability and antiapoptotic factor alongside the upregulation of steroidogenic and fertility-related genes and regimented the detrimental effects of FPN on antioxidant and pro-inflammatory biomarkers.
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Affiliation(s)
- Hossam G. Tohamy
- Department of Pathology, Faculty of Veterinary Medicine, Alexandria University, Alexandria 22758, Egypt;
| | - Sara E. El-Kazaz
- Animals and Poultry Behavior and Management, Department of Animal Husbandry and Animal Wealth Development, Faculty of Veterinary Medicine, Alexandria University, Alexandria 22758, Egypt;
| | - Saqer S. Alotaibi
- Department of Biotechnology, College of Science, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia;
| | - Hawary S. Ibrahiem
- Department of Veterinary Pharmacology, Faculty of Veterinary Medicine, Alexandria University, Alexandria 22758, Egypt;
| | - Mustafa Shukry
- Department of Physiology, Faculty of Veterinary Medicine, Kafrelsheikh University, Kafrelsheikh 33516, Egypt
| | - Mahmoud A. O. Dawood
- Department of Animal Production, Faculty of Agriculture, Kafrelsheikh University, Kafrelsheikh 33516, Egypt
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19
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Kojima K, Ooka M, Abe T, Hirota K. Pold4, the fourth subunit of replicative polymerase δ, suppresses gene conversion in the immunoglobulin-variable gene in avian DT40 cells. DNA Repair (Amst) 2021; 100:103056. [PMID: 33588156 DOI: 10.1016/j.dnarep.2021.103056] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2020] [Revised: 01/26/2021] [Accepted: 01/28/2021] [Indexed: 10/22/2022]
Abstract
The replicative polymerase δ (Polδ), consisting of four subunits, plays a pivotal role in chromosomal replication. Pold4, the smallest subunit of Polδ, is believed to contribute to the regulation of replication by facilitating repair in response to DNA damage. However, that contribution has not been fully elucidated. We here show that Pold4 contributes to the suppression of gene conversion in immunoglobulin-variable (IgV) gene diversification in the chicken DT40 lymphocyte cell line, where gene conversion diversifies the IgV gene through intragenic homologous recombination (HR) between diverged pseudo-V segments. IgV gene conversion is initiated by activation-induced cytidine deaminase-mediated uracil formation in the IgV gene, which in turn converts into an abasic site, leading to replication arrest. POLD4-/- cells exhibited an increased rate of IgV gene conversion. Moreover, the gene-conversion tract was lengthened and the usage of pseudo-V segments was altered, showing a preference, to use the diverged sequence as a donor in POLD4-/- cells. These data suggest that Pold4 is involved in the regulation of HR-mediated gene conversion in IgV diversification. By contrast, the rate in HR-mediated, sister-chromatid exchange and gene-targeting induced by an I-SceI endonclease-mediated DNA double-strand break exhibited by POLD4-/- cells was indistinguishable from that by wild-type cells. These findings indicate that the functionality of general HR is preserved in POLD4-/- cells. In conclusion, Pold4 is involved in the suppression of IgV-gene conversion without affecting the general functionality of HR.
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Affiliation(s)
- Kota Kojima
- Department of Chemistry, Graduate School of Science, Tokyo Metropolitan University, Minamiosawa 1-1, Hachioji-shi, Tokyo, 192-0397, Japan
| | - Masato Ooka
- Department of Chemistry, Graduate School of Science, Tokyo Metropolitan University, Minamiosawa 1-1, Hachioji-shi, Tokyo, 192-0397, Japan
| | - Takuya Abe
- Department of Chemistry, Graduate School of Science, Tokyo Metropolitan University, Minamiosawa 1-1, Hachioji-shi, Tokyo, 192-0397, Japan
| | - Kouji Hirota
- Department of Chemistry, Graduate School of Science, Tokyo Metropolitan University, Minamiosawa 1-1, Hachioji-shi, Tokyo, 192-0397, Japan.
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20
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Gad FAM, Farouk SM, Emam MA. Antiapoptotic and antioxidant capacity of phytochemicals from Roselle (Hibiscus sabdariffa) and their potential effects on monosodium glutamate-induced testicular damage in rat. ENVIRONMENTAL SCIENCE AND POLLUTION RESEARCH INTERNATIONAL 2021; 28:2379-2390. [PMID: 32888148 DOI: 10.1007/s11356-020-10674-7] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/27/2020] [Accepted: 08/27/2020] [Indexed: 06/11/2023]
Abstract
Monosodium glutamate (MSG), common flavor enhancer and feed additive, causes male reproductive dysfunction. However, Roselle tea, popular Hibiscus sabdariffa (HS) beverage, has a controversial effectiveness on male fertility. Therefore, the current study aimed to investigate either the adverse effect of aqueous HS extract (HSE) on the testicle or its potential ameliorative role including some stress markers, biochemical and immunohistochemical expressions in rats subjected to MSG. Here, the animals were divided into four groups that were given distilled water, HSE, MSG, and HSE + MSG respectively via gavage. After 6 weeks from the beginning of experiment, blood samples were collected for hormonal analysis. Additionally, testicular specimens were excised and processed for oxidative/antioxidant parameters determination, histological examination, and immunohistochemical evaluation of Bax and PCNA positive spermatogenic cells. Preliminary phytochemical analyses as well as antioxidant capacity of the HSE were tested. Our results revealed a strong inhibitory activity of the HSE phytochemical constituents against DPPH radical. MSG group revealed a significant decrease of testosterone, LH, FSH, and antioxidant parameters with elevated MDA compared with control and HSE groups. Additionally, an alteration of the testicular histo-architecture was observed among MSG group along with increased Bax and decreased PCNA positive cells. Meanwhile, the HSE showed a potent protective effect against testicular damage as well as oxidative stress induced by MSG. On the whole, our findings provide evidence that HSE can ameliorate MSG-induced testicular toxicity via induction of cell proliferation along with reduction of oxidative stress and cellular apoptosis in adult rat that attributed to the antioxidant and antiapoptotic effects of its phytochemical constituents.
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Affiliation(s)
- Fatma Abdel-Monem Gad
- Clinical Pathology Department, Faculty of Veterinary Medicine, Benha University, Benha, 13736, Egypt
| | - Sameh Mohamed Farouk
- Department of Cytology and Histology, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, 4152, Egypt.
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21
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Konkova M, Abramova M, Kalianov A, Ershova E, Dolgikh O, Umriukhin P, Izhevskaya V, Kutsev S, Veiko N, Kostyuk S. Mesenchymal Stem Cells Early Response to Low-Dose Ionizing Radiation. Front Cell Dev Biol 2021; 8:584497. [PMID: 33381502 PMCID: PMC7767887 DOI: 10.3389/fcell.2020.584497] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2020] [Accepted: 11/13/2020] [Indexed: 12/13/2022] Open
Abstract
Introduction Mesenchymal stem cells (MSCs) are applied as the therapeutic agents, e.g., in the tumor radiation therapy. Purpose of the Study To evaluate the human adipose MSC early response to low-dose ionizing radiation (LDIR). Materials and Methods We investigated different LDIR (3, 10, and 50 cGy) effects on reactive oxygen species production, DNA oxidation (marker 8-oxodG), and DNA breaks (marker ɣ H2AX) in the two lines of human adipose MSC. Using reverse transcriptase-polymerase chain reaction, fluorescence-activated cell sorting, and fluorescence microscopy, we determined expression of genes involved in the oxidative stress development (NOX4), antioxidative response (NRF2), antiapoptotic and proapoptotic response (BCL2, BCL2A1, BCL2L1, BIRC2, BIRC3, and BAX1), in the development of the nuclear DNA damage response (DDR) (BRCA1, BRCA2, ATM, and P53). Cell cycle changes were investigated by genes transcription changes (CCND1, CDKN2A, and CDKN1A) and using proliferation markers KI-67 and proliferating cell nuclear antigen (PCNA). Results Fifteen to 120 min after exposure to LDIR in MSCs, transient oxidative stress and apoptosis of the most damaged cells against the background of the cell cycle arrest were induced. Simultaneously, DDR and an antiapoptotic response were found in other cells of the population. The 10-cGy dose causes the strongest and fastest DDR following cell nuclei DNA damage. The 3-cGy dose induces a less noticeable and prolonged response. The maximal low range dose, 50 cGy, causes a damaging effect on the MSCs. Conclusion Transient oxidative stress and the death of a small fraction of the damaged cells are essential components of the MSC population response to LDIR along with the development of DDR and antiapoptotic response. A scheme describing the early MSC response to LDIR is proposed.
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Affiliation(s)
- Marina Konkova
- Department of Molecular Biology, Research Centre for Medical Genetics, Moscow, Russia
| | - Margarita Abramova
- Department of Molecular Biology, Research Centre for Medical Genetics, Moscow, Russia
| | - Andrey Kalianov
- Department of Molecular Biology, Research Centre for Medical Genetics, Moscow, Russia
| | - Elizaveta Ershova
- Department of Molecular Biology, Research Centre for Medical Genetics, Moscow, Russia.,I.M. Sechenov First Moscow State Medical University, Department of Normal Physiology, Moscow, Russia
| | - Olga Dolgikh
- Department of Molecular Biology, Research Centre for Medical Genetics, Moscow, Russia
| | - Pavel Umriukhin
- Department of Molecular Biology, Research Centre for Medical Genetics, Moscow, Russia.,I.M. Sechenov First Moscow State Medical University, Department of Normal Physiology, Moscow, Russia.,P.K. Anokhin Institute of Normal Physiology, Moscow, Russia
| | - Vera Izhevskaya
- Department of Molecular Biology, Research Centre for Medical Genetics, Moscow, Russia
| | - Sergey Kutsev
- Department of Molecular Biology, Research Centre for Medical Genetics, Moscow, Russia
| | - Natalia Veiko
- Department of Molecular Biology, Research Centre for Medical Genetics, Moscow, Russia
| | - Svetlana Kostyuk
- Department of Molecular Biology, Research Centre for Medical Genetics, Moscow, Russia.,I.M. Sechenov First Moscow State Medical University, Department of Normal Physiology, Moscow, Russia
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22
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Raslan IR, de Assis Pereira Matos PCA, Boaratti Ciarlariello V, Daghastanli KH, Rosa ABR, Arita JH, Aranda CS, Barsottini OGP, Pedroso JL. Beyond Typical Ataxia Telangiectasia: How to Identify the Ataxia Telangiectasia-Like Disorders. Mov Disord Clin Pract 2021; 8:118-125. [PMID: 33426167 PMCID: PMC7780949 DOI: 10.1002/mdc3.13110] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2020] [Revised: 08/31/2020] [Accepted: 10/18/2020] [Indexed: 12/16/2022] Open
Abstract
BACKGROUND Ataxia telangiectasia is one of the most common causes of autosomal recessive cerebellar ataxias. However, absence of telangiectasia, normal levels of alpha-fetoprotein and negative genetic test may direct to alternative diagnosis with similar phenotypes such as ataxia telangiectasia-like disorders (ATLD). CASES We report two instructive cases of ATLD: the first case with ataxia telangiectasia-like disorder type 1 related to MRE11A gene, and the second case with ataxia telangiectasia-like disorder type 2 related to PCNA gene. LITERATURE REVIEW ATLD is an unusual group of autosomal recessive diseases that share some clinical features and pathophysiological mechanisms with ataxia telangiectasia (AT). ATLD may be associated with mutations in the MRE11A (ATLD type 1) and PCNA (ATLD type 2) genes. ATLD belongs to the group of chromosomal instability syndromes. The reason for the term ATLD is related to the similar pathophysiological mechanisms observed in AT, which is characterized by chromosomal instability and radiosensitivity. CONCLUSIONS In this review, the main clinical features, biomarkers, brain imaging and genetics of ATLD are discussed. Mutations in the MRE11A and PCNA genes should be included in the differential diagnosis for early onset cerebellar ataxia with absence of telangiectasia and normal levels of alpha-fetoprotein.
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Affiliation(s)
- Ivana Rocha Raslan
- Department of Neurology, Ataxia UnitUniversidade Federal de São PauloSão PauloBrazil
| | | | | | | | | | | | | | | | - José Luiz Pedroso
- Department of Neurology, Ataxia UnitUniversidade Federal de São PauloSão PauloBrazil
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New Perspectives on Unscheduled DNA Synthesis: Functional Assay for Global Genomic DNA Nucleotide Excision Repair. Methods Mol Biol 2020; 2102:483-507. [PMID: 31989573 DOI: 10.1007/978-1-0716-0223-2_27] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
The unscheduled DNA synthesis (UDS) assay measures the ability of a cell to perform global genomic nucleotide excision repair (NER). This chapter provides instructions for the application of this technique by creating 6-4 photoproducts and pyrimidine dimers using UV-C (254 nm) irradiation. This procedure is designed specifically for quantification of the 6-4 photoproducts. Repair is quantified by the amount of radioactive thymidine incorporated during repair synthesis after this insult, and radioactivity is evaluated by grain counting after autoradiography. The results have been used to clinically diagnose human DNA repair deficiency disorders, and provide a basis for investigation of repair deficiency in human tissues or tumors. Genomic sequencing to establish the presence of specific mutations is also used now for clinical diagnosis of DNA repair deficiency syndromes. Few functional assays are available which directly measure the capacity to perform NER on the entire genome. Since live cells are required for this assay, explant culture techniques must be previously established. Host cell reactivation (HCR). As discussed in Chap. 28 is not an equivalent technique, as it measures only transcription-coupled repair (TCR) at active genes, a small subset of total NER. Our laboratory also explored the fluorescent label-based Click-iT assay that uses EdU as the label, rather than 3H thymidine. Despite emerging studies in the literature finding this assay to be useful for other purposes, we found that the EdU-based UDS assay was not consistent or reproducible compared with the 3H thymidine-based assay.
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24
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Ochsner SA, Pillich RT, McKenna NJ. Consensus transcriptional regulatory networks of coronavirus-infected human cells. Sci Data 2020; 7:314. [PMID: 32963239 PMCID: PMC7509801 DOI: 10.1038/s41597-020-00628-6] [Citation(s) in RCA: 26] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2020] [Accepted: 08/05/2020] [Indexed: 02/08/2023] Open
Abstract
Establishing consensus around the transcriptional interface between coronavirus (CoV) infection and human cellular signaling pathways can catalyze the development of novel anti-CoV therapeutics. Here, we used publicly archived transcriptomic datasets to compute consensus regulatory signatures, or consensomes, that rank human genes based on their rates of differential expression in MERS-CoV (MERS), SARS-CoV-1 (SARS1) and SARS-CoV-2 (SARS2)-infected cells. Validating the CoV consensomes, we show that high confidence transcriptional targets (HCTs) of MERS, SARS1 and SARS2 infection intersect with HCTs of signaling pathway nodes with known roles in CoV infection. Among a series of novel use cases, we gather evidence for hypotheses that SARS2 infection efficiently represses E2F family HCTs encoding key drivers of DNA replication and the cell cycle; that progesterone receptor signaling antagonizes SARS2-induced inflammatory signaling in the airway epithelium; and that SARS2 HCTs are enriched for genes involved in epithelial to mesenchymal transition. The CoV infection consensomes and HCT intersection analyses are freely accessible through the Signaling Pathways Project knowledgebase, and as Cytoscape-style networks in the Network Data Exchange repository.
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Affiliation(s)
- Scott A Ochsner
- The Signaling Pathways Project and Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, 77030, USA
| | - Rudolf T Pillich
- Department of Medicine, University of California San Diego, La Jolla, CA, 92093, USA
| | - Neil J McKenna
- The Signaling Pathways Project and Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, 77030, USA.
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25
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Ochsner SA, Pillich RT, McKenna NJ. Consensus transcriptional regulatory networks of coronavirus-infected human cells. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2020:2020.04.24.059527. [PMID: 32511379 PMCID: PMC7263508 DOI: 10.1101/2020.04.24.059527] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Establishing consensus around the transcriptional interface between coronavirus (CoV) infection and human cellular signaling pathways can catalyze the development of novel anti-CoV therapeutics. Here, we used publicly archived transcriptomic datasets to compute consensus regulatory signatures, or consensomes, that rank human genes based on their rates of differential expression in MERS-CoV (MERS), SARS-CoV-1 (SARS1) and SARS-CoV-2 (SARS2)-infected cells. Validating the CoV consensomes, we show that high confidence transcriptional targets (HCTs) of CoV infection intersect with HCTs of signaling pathway nodes with known roles in CoV infection. Among a series of novel use cases, we gather evidence for hypotheses that SARS2 infection efficiently represses E2F family target genes encoding key drivers of DNA replication and the cell cycle; that progesterone receptor signaling antagonizes SARS2-induced inflammatory signaling in the airway epithelium; and that SARS2 HCTs are enriched for genes involved in epithelial to mesenchymal transition. The CoV infection consensomes and HCT intersection analyses are freely accessible through the Signaling Pathways Project knowledgebase, and as Cytoscape-style networks in the Network Data Exchange repository.
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Affiliation(s)
- Scott A Ochsner
- The Signaling Pathways Project and Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030
| | - Rudolf T Pillich
- Department of Medicine, University of California San Diego, La Jolla, CA 92093
| | - Neil J McKenna
- The Signaling Pathways Project and Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030
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26
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A plant DNA virus replicates in the salivary glands of its insect vector via recruitment of host DNA synthesis machinery. Proc Natl Acad Sci U S A 2020; 117:16928-16937. [PMID: 32636269 PMCID: PMC7382290 DOI: 10.1073/pnas.1820132117] [Citation(s) in RCA: 62] [Impact Index Per Article: 12.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
Viruses pose a great threat to animal and plant health worldwide. Whereas most plant viruses only replicate in plant hosts, some also replicate in their animal (insect) vector. A detailed knowledge of host expansion will give a better understanding of virus evolution, and identification of virus and host components involved in this process can lead to new strategies to combat virus spread. Here, we reveal that a plant DNA virus has evolved to induce and recruit insect DNA synthesis machinery to support its replication in vector salivary glands. Our study sheds light on the understanding of TYLCV–whitefly interactions and provides insights into how a plant virus may evolve to infect and replicate in an insect vector. Whereas most of the arthropod-borne animal viruses replicate in their vectors, this is less common for plant viruses. So far, only some plant RNA viruses have been demonstrated to replicate in insect vectors and plant hosts. How plant viruses evolved to replicate in the animal kingdom remains largely unknown. Geminiviruses comprise a large family of plant-infecting, single-stranded DNA viruses that cause serious crop losses worldwide. Here, we report evidence and insight into the replication of the geminivirus tomato yellow leaf curl virus (TYLCV) in the whitefly (Bemisia tabaci) vector and that replication is mainly in the salivary glands. We found that TYLCV induces DNA synthesis machinery, proliferating cell nuclear antigen (PCNA) and DNA polymerase δ (Polδ), to establish a replication-competent environment in whiteflies. TYLCV replication-associated protein (Rep) interacts with whitefly PCNA, which recruits DNA Polδ for virus replication. In contrast, another geminivirus, papaya leaf curl China virus (PaLCuCNV), does not replicate in the whitefly vector. PaLCuCNV does not induce DNA-synthesis machinery, and the Rep does not interact with whitefly PCNA. Our findings reveal important mechanisms by which a plant DNA virus replicates across the kingdom barrier in an insect and may help to explain the global spread of this devastating pathogen.
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27
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Grant GD, Kedziora KM, Limas JC, Cook JG, Purvis JE. Accurate delineation of cell cycle phase transitions in living cells with PIP-FUCCI. Cell Cycle 2019; 17:2496-2516. [PMID: 30421640 DOI: 10.1080/15384101.2018.1547001] [Citation(s) in RCA: 72] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
Cell cycle phase transitions are tightly orchestrated to ensure efficient cell cycle progression and genome stability. Interrogating these transitions is important for understanding both normal and pathological cell proliferation. By quantifying the dynamics of the popular FUCCI reporters relative to the transitions into and out of S phase, we found that their dynamics are substantially and variably offset from true S phase boundaries. To enhance detection of phase transitions, we generated a new reporter whose oscillations are directly coupled to DNA replication and combined it with the FUCCI APC/C reporter to create "PIP-FUCCI". The PIP degron fusion protein precisely marks the G1/S and S/G2 transitions; shows a rapid decrease in signal in response to large doses of DNA damage only during G1; and distinguishes cell type-specific and DNA damage source-dependent arrest phenotypes. We provide guidance to investigators in selecting appropriate fluorescent cell cycle reporters and new analysis strategies for delineating cell cycle transitions.
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Affiliation(s)
- Gavin D Grant
- a Department of Biochemistry and Biophysics , The University of North Carolina , Chapel Hill , NC , USA.,b Lineberger Comprehensive Cancer Center , The University of North Carolina , Chapel Hill , NC , USA
| | - Katarzyna M Kedziora
- c Department of Genetics , The University of North Carolina , Chapel Hill , NC , USA
| | - Juanita C Limas
- d Department of Pharmacology , The University of North Carolina , Chapel Hill , NC , USA
| | - Jeanette Gowen Cook
- a Department of Biochemistry and Biophysics , The University of North Carolina , Chapel Hill , NC , USA.,b Lineberger Comprehensive Cancer Center , The University of North Carolina , Chapel Hill , NC , USA.,d Department of Pharmacology , The University of North Carolina , Chapel Hill , NC , USA
| | - Jeremy E Purvis
- b Lineberger Comprehensive Cancer Center , The University of North Carolina , Chapel Hill , NC , USA.,c Department of Genetics , The University of North Carolina , Chapel Hill , NC , USA
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28
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Zhang H, Liu J, He Y, Xie Z, Zhang S, Zhang Y, Lin L, Liu S, Wang D. Quantitative proteomics reveals the key molecular events occurring at different cell cycle phases of the in situ blooming dinoflagellate cells. THE SCIENCE OF THE TOTAL ENVIRONMENT 2019; 676:62-71. [PMID: 31029901 DOI: 10.1016/j.scitotenv.2019.04.216] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/18/2019] [Revised: 04/01/2019] [Accepted: 04/13/2019] [Indexed: 05/26/2023]
Abstract
Dinoflagellate blooms are the results of rapid cell proliferation governed by cell cycle, a highly-ordered series of events that culminates in cell division. However, little is known about cell cycle progression of the in situ bloom cells. Here, we compared proteomes of the in situ blooming cells of a dinoflagellate Prorocentrum donghaiense collected at different cell cycle phases. The blooming P. donghaiense cells completed a cell cycle within 24 h with a high synchronization rate of 82.7%. Proteins associated with photosynthesis, porphyrin and chlorophyll synthesis, carbon, nitrogen and amino acid metabolisms exhibited high expressions at the G1 phase; DNA replication and mismatch repair related proteins were more abundant at the S phase; while protein synthesis and oxidative phosphorylation were highly enriched at the G2/M phase. Cell cycle proteins presented similar periodic diel patterns to other eukaryotic cells, and higher expressions of proliferating cell nuclear antigen and cyclin dependent kinase 2 at the S phase ensured the smooth S-G2/M transition. Strikingly, four histones were first identified in P. donghaiense and highly expressed at the G2/M phase, indicating their potential roles in regulating cell cycle. This study presents the first quantitative survey, to our knowledge, of proteome changes at different cell cycle phases of the in situ blooming cells in natural environment and provides insights into cell cycle regulation of the blooming dinoflagellate cells.
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Affiliation(s)
- Hao Zhang
- State Key Laboratory of Marine Environmental Science, College of the Environment and Ecology, Xiamen University, 361005, China
| | - Jiuling Liu
- State Key Laboratory of Marine Environmental Science, College of the Environment and Ecology, Xiamen University, 361005, China; Key Laboratory of Ocean and Marginal Sea Geology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China
| | - Yanbin He
- BGI-Shenzhen, Beishan Industrial Zone 11th building, Yantian District, Shenzhen, Guangdong 518083, China
| | - Zhangxian Xie
- State Key Laboratory of Marine Environmental Science, College of the Environment and Ecology, Xiamen University, 361005, China
| | - Shufei Zhang
- State Key Laboratory of Marine Environmental Science, College of the Environment and Ecology, Xiamen University, 361005, China; South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, China
| | - Yong Zhang
- State Key Laboratory of Marine Environmental Science, College of the Environment and Ecology, Xiamen University, 361005, China
| | - Lin Lin
- State Key Laboratory of Marine Environmental Science, College of the Environment and Ecology, Xiamen University, 361005, China
| | - Siqi Liu
- BGI-Shenzhen, Beishan Industrial Zone 11th building, Yantian District, Shenzhen, Guangdong 518083, China
| | - Dazhi Wang
- State Key Laboratory of Marine Environmental Science, College of the Environment and Ecology, Xiamen University, 361005, China.
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29
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Hicks SD, Miller MW. Ethanol-induced DNA repair in neural stem cells is transforming growth factor β1-dependent. Exp Neurol 2019; 317:214-225. [PMID: 30853389 DOI: 10.1016/j.expneurol.2019.02.003] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2018] [Revised: 12/13/2018] [Accepted: 02/07/2019] [Indexed: 12/18/2022]
Abstract
Following neurotoxic damage, cells repair their DNA, and survive or undergo apoptosis. This study tests the hypothesis that ethanol induces a DNA damage response (DDR) in neural stem cells (NSCs) that promotes excision repair (ER) and this repair is influenced by the growth factor environment. Non-immortalized NSCs treated with fibroblast growth factor 2 or transforming growth factor (TGF) β1 were exposed to ethanol. Ethanol increased total DNA damage, reactive oxygen species, and oxidized DNA bases. TGFβ1 potentiated these toxic effects. Transcriptional analyses of cultured NSCs revealed ethanol-induced increases in transcripts related to the DDR (e.g., Hus1 and p53), base ER (e.g., Mutyh and Nthl1), and nucleotide ER (e.g., Xpc), particularly in the presence of TGFβ1. Expression and activity of ER proteins were affected by ethanol. Similar changes occurred in proliferating cells of ethanol-treated mouse fetuses. Ethanol-induced DNA repair in NSCs depends on the ambient growth factors. Gene products for DNA repair in stem cells are among the first biomarkers identifying fetal alcohol-induced damage.
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Affiliation(s)
- Steven D Hicks
- Department of Neuroscience and Physiology, State University of New York - Upstate Medical University, Syracuse, NY 13210, USA; Developmental Exposure Alcohol Research Center, Binghamton NY 13902, Cortland NY 13045, and Syracuse, NY 13210, USA
| | - Michael W Miller
- Department of Neuroscience and Physiology, State University of New York - Upstate Medical University, Syracuse, NY 13210, USA; Developmental Exposure Alcohol Research Center, Binghamton NY 13902, Cortland NY 13045, and Syracuse, NY 13210, USA; Department of Anatomy, Touro College of Osteopathic Medicine, Middletown, NY 10940, USA; Research Service, Veterans Affairs Medical Center, Syracuse, NY 13210, USA.
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30
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Role of estrogen receptors, P450 aromatase, PCNA and p53 in high-fat-induced impairment of spermatogenesis in rats. C R Biol 2018; 341:371-379. [PMID: 30150094 DOI: 10.1016/j.crvi.2018.07.002] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2018] [Revised: 07/30/2018] [Accepted: 07/31/2018] [Indexed: 12/17/2022]
Abstract
Obesity and overweight are frequently associated with male subfertility. To address new findings on the players involved in the obesity-induced impairment of spermatogenesis, we used a high-fat diet-induced overweight-rat model. Following four weeks of high-fat diet, the organization of seminiferous epithelium was affected, and tubules lumen showed immature/degenerated cells, typical signs of hormonal imbalance and testicular damage. Real-time PCR analysis allowed us to detect increased levels of ERα and decreased levels of aromatase CYP19 transcripts in testis, suggesting an increase in circulating estrogens derived from the accumulating adipose tissue rather than the induction of testicular estrogen synthesis. Moreover, in situ hybridization analysis showed an increased susceptibility towards estrogens in testis from high-fat fed rats, being ERs expressed not only in spermatogonia, as in control testis, but also in spermatids. Western blot and immunohistochemical analyses revealed an increase in the amount of p53 and PCNA, together with a change in their immunodetection, being the proteins localised on germ cells at different stages of maturation. Differences in p53 and PCNA expression may give evidence and be part of a cellular response to stress conditions and damage caused by the excessive intake of saturated fatty acids.
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31
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Li W, Zhou Y, Tang G, Wong NK, Yang M, Tan D, Xiao Y. Chemoproteomics Reveals the Antiproliferative Potential of Parkinson’s Disease Kinase Inhibitor LRRK2-IN-1 by Targeting PCNA Protein. Mol Pharm 2018; 15:3252-3259. [DOI: 10.1021/acs.molpharmaceut.8b00325] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Affiliation(s)
- Weichao Li
- CAS Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China
| | - Yiqing Zhou
- CAS Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China
| | - Guanghui Tang
- School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen 518055, China
| | - Nai-Kei Wong
- State Key Discipline of Infection Diseases, Shenzhen Third People’s Hospital, The Second Affiliated Hospital, Shenzhen University, Shenzhen 518112, China
| | - Mengquan Yang
- CAS Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China
- University of Chinese Academy of Sciences, Beijing 100039, China
| | - Dan Tan
- Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China
| | - Youli Xiao
- CAS Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China
- University of Chinese Academy of Sciences, Beijing 100039, China
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32
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Yang S, Xing L, Gu L, Cheng H, Feng Y, Zhang Y. Combination of RIZ1 Overexpression and Radiotherapy Contributes to Apoptosis and DNA Damage of HeLa and SiHa Cervical Cancer Cells. Basic Clin Pharmacol Toxicol 2018; 123:137-146. [PMID: 29575614 DOI: 10.1111/bcpt.13008] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2017] [Accepted: 02/07/2018] [Indexed: 12/15/2022]
Abstract
Although radiotherapy has been widely applied to treating cervical cancer in the clinic, its therapeutic efficacy is often restricted to the radioresistance of cancer cells. Retinoblastoma protein-interacting zinc finger gene 1 (RIZ1) has been suggested as a tumour suppressor gene, whereas its role in cervical cancer with or without radiotherapy has been unclear. In this study, two cervical cancer cell lines, HeLa and SiHa cells, stably transfected with RIZ1 overexpression plasmid were subjected to ionizing radiation, and their survival fractions were calculated by assessing their clonogenic abilities. Our results showed that the forced overexpression of RIZ1 significantly reduced the clonogenic survival rates of both HeLa and SiHa cells exposed to ionizing radiation. By analysing the cell apoptotic status, we found that the RIZ1-overexpressed cervical cancer cells under ionizing radiation were more vulnerable to damage, and more γ-H2AX foci were found in these cells. Furthermore, the volumes of tumour xenografts formed by the RIZ1-overexpressed cells in nude mice under ionizing radiation were smaller than those generated by the control cells. There were more morphological changes, apoptosis cells and lower expression of PCNA in RIZ1-overexpressed tumour tissues of mice after exposure to ionizing radiation. Taken together, our study demonstrates that the overexpression of RIZ1 combined with radiotherapy facilitates apoptosis and DNA damage of cervical cancer cells.
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Affiliation(s)
- Shanshan Yang
- Department of Gynecological Radiotherapy, Harbin Medical University Cancer Hospital, Harbin, China
| | - Linan Xing
- Department of Gynecological Radiotherapy, Harbin Medical University Cancer Hospital, Harbin, China
| | - Lina Gu
- Department of Gynecological Radiotherapy, Harbin Medical University Cancer Hospital, Harbin, China
| | - Haiyan Cheng
- Department of Gynecological Radiotherapy, Harbin Medical University Cancer Hospital, Harbin, China
| | - Yue Feng
- Department of Gynecological Radiotherapy, Harbin Medical University Cancer Hospital, Harbin, China
| | - Yunyan Zhang
- Department of Gynecological Radiotherapy, Harbin Medical University Cancer Hospital, Harbin, China
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33
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Vigliani MC, Chiò A, Pezzulo T, Soffietti R, Giordana MT, Schiffer D. Proliferating Cell Nuclear Antigen (PCNA) in Low-Grade Astrocytomas: Its Prognostic Significance. TUMORI JOURNAL 2018; 80:295-300. [PMID: 7974802 DOI: 10.1177/030089169408000411] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Aims and background A clear line cannot be drawn between well-differentiated and anaplastic astrocytomas, and a subset of low-grade tumors, histologically indistinguishable from the others, behaves similarly to anaplastic astrocytomas. The proliferative index could aid in the identification of this subgroup, for which a different therapeutic approach would be indicated. Methods We immunohistochemically evaluated the proliferating ceil nuclear antigen (PCNA) expression in 77 well-differentiated astrocytomas, since PCNA has been considered a good proliferation marker. The prognostic significance of PCNA labeling index (LI) was assessed in univariate and multivariate analysis, taking into consideration some clinical and histologic factors known to affect prognosis. Results PCNA immunostaining identified a subgroup of tumors, characterized by a LI > 5%, with a median survival close to that observed in anaplastic astrocytomas. The survival table of such a group was significantly different from that of the group with a lower LI (p = 0.0009). Multivariate analysis confirmed that PCNA-LI is an independent prognostic factor (p = 0.001). Conclusion These data suggest that PCNA immunostaining can be a useful tool to define the prognosis of low-grade astrocytomas on routine biopsy material.
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Affiliation(s)
- M C Vigliani
- II Department of Neurology, University of Turin, Italy
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Mazengenya P, Bhagwandin A, Manger PR, Ihunwo AO. Putative Adult Neurogenesis in Old World Parrots: The Congo African Grey Parrot ( Psittacus erithacus) and Timneh Grey Parrot ( Psittacus timneh). Front Neuroanat 2018; 12:7. [PMID: 29487507 PMCID: PMC5816827 DOI: 10.3389/fnana.2018.00007] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2017] [Accepted: 01/15/2018] [Indexed: 11/21/2022] Open
Abstract
In the current study, we examined for the first time, the potential for adult neurogenesis throughout the brain of the Congo African grey parrot (Psittacus erithacus) and Timneh grey parrot (Psittacus timneh) using immunohistochemistry for the endogenous markers proliferating cell nuclear antigen (PCNA), which labels proliferating cells, and doublecortin (DCX), which stains immature and migrating neurons. A similar distribution of PCNA and DCX immunoreactivity was found throughout the brain of the Congo African grey and Timneh grey parrots, but minor differences were also observed. In both species of parrots, PCNA and DCX immunoreactivity was observed in the olfactory bulbs, subventricular zone of the lateral wall of the lateral ventricle, telencephalic subdivisions of the pallium and subpallium, diencephalon, mesencephalon and the rhombencephalon. The olfactory bulb and telencephalic subdivisions exhibited a higher density of both PCNA and DCX immunoreactive cells than any other brain region. DCX immunoreactive staining was stronger in the telencephalon than in the subtelencephalic structures. There was evidence of proliferative hot spots in the dorsal and ventral poles of the lateral ventricle in the Congo African grey parrots at rostral levels, whereas only the dorsal accumulation of proliferating cells was observed in the Timneh grey parrot. In most pallial regions the density of PCNA and DCX stained cells increased from rostral to caudal levels with the densest staining in the nidopallium caudolaterale (NCL). The widespread distribution of PCNA and DCX in the brains of both parrot species suggest the importance of adult neurogenesis and neuronal plasticity during learning and adaptation to external environmental variations.
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Affiliation(s)
- Pedzisai Mazengenya
- School of Anatomical Sciences, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa
| | - Adhil Bhagwandin
- School of Anatomical Sciences, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa
| | - Paul R Manger
- School of Anatomical Sciences, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa
| | - Amadi O Ihunwo
- School of Anatomical Sciences, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa
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35
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Mizamtsidi M, Nastos C, Mastorakos G, Dina R, Vassiliou I, Gazouli M, Palazzo F. Diagnosis, management, histology and genetics of sporadic primary hyperparathyroidism: old knowledge with new tricks. Endocr Connect 2018; 7:R56-R68. [PMID: 29330338 PMCID: PMC5801557 DOI: 10.1530/ec-17-0283] [Citation(s) in RCA: 29] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/20/2017] [Accepted: 01/12/2018] [Indexed: 12/25/2022]
Abstract
Primary hyperparathyroidism (pHPT) is a common endocrinopathy resulting from inappropriately high PTH secretion. It usually results from the presence of a single gland adenoma, multiple gland hyperplasia or rarely parathyroid carcinoma. All these conditions require different management, and it is important to be able to differentiate the underlined pathology, in order for the clinicians to provide the best therapeutic approach. Elucidation of the genetic background of each of these clinical entities would be of great interest. However, the molecular factors that control parathyroid tumorigenesis are poorly understood. There are data implicating the existence of specific genetic pathways involved in the emergence of parathyroid tumorigenesis. The main focus of the present study is to present the current optimal diagnostic and management protocols for pHPT as well as to review the literature regarding all molecular and genetic pathways that are to be involved in the pathophysiology of sporadic pHPT.
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Affiliation(s)
- Maria Mizamtsidi
- Department of EndocrinologyDiabetes and Metabolism, Hellenic Red Cross Hospital, Athens, Greece
| | - Constantinos Nastos
- Second Department of SurgeryEndocrine Surgery Unit, Aretaieion University Hospital, National and Kapodistrian University of Athens, Athens, Greece
| | - George Mastorakos
- Unit of EndocrinologyDiabetes and Metabolism, Aretaieion University Hospital, National and Kapodistrian University of Athens, Athens, Greece
| | - Roberto Dina
- Department of PathologyHammersmith Hospital, Imperial College Healthcare NHS Trust, London, UK
| | - Ioannis Vassiliou
- Second Department of SurgeryEndocrine Surgery Unit, Aretaieion University Hospital, National and Kapodistrian University of Athens, Athens, Greece
| | - Maria Gazouli
- Department of Basic Medical SciencesLaboratory of Biology, School of Medicine, National and Kapodistrian University of Athens, Athens, Greece
| | - Fausto Palazzo
- Department of Thyroid and Endocrine SurgeryImperial College London, London, UK
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A proteome view of structural, functional, and taxonomic characteristics of major protein domain clusters. Sci Rep 2017; 7:14210. [PMID: 29079755 PMCID: PMC5660162 DOI: 10.1038/s41598-017-13297-0] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2016] [Accepted: 09/21/2017] [Indexed: 12/28/2022] Open
Abstract
Proteome-scale bioinformatics research is increasingly conducted as the number of completely sequenced genomes increases, but analysis of protein domains (PDs) usually relies on similarity in their amino acid sequences and/or three-dimensional structures. Here, we present results from a bi-clustering analysis on presence/absence data for 6,580 unique PDs in 2,134 species with a sequenced genome, thus covering a complete set of proteins, for the three superkingdoms of life, Bacteria, Archaea, and Eukarya. Our analysis revealed eight distinctive PD clusters, which, following an analysis of enrichment of Gene Ontology functions and CATH classification of protein structures, were shown to exhibit structural and functional properties that are taxa-characteristic. For examples, the largest cluster is ubiquitous in all three superkingdoms, constituting a set of 1,472 persistent domains created early in evolution and retained in living organisms and characterized by basic cellular functions and ancient structural architectures, while an Archaea and Eukarya bi-superkingdom cluster suggests its PDs may have existed in the ancestor of the two superkingdoms, and others are single superkingdom- or taxa (e.g. Fungi)-specific. These results contribute to increase our appreciation of PD diversity and our knowledge of how PDs are used in species, yielding implications on species evolution.
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Sales CF, Santos KPED, Rizzo E, Ribeiro RIMDA, Santos HBD, Thomé RG. Proliferation, survival and cell death in fish gills remodeling: From injury to recovery. FISH & SHELLFISH IMMUNOLOGY 2017; 68:10-18. [PMID: 28676337 DOI: 10.1016/j.fsi.2017.07.001] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/28/2017] [Revised: 06/30/2017] [Accepted: 07/01/2017] [Indexed: 06/07/2023]
Abstract
Pollutants found dispersed in water can cause irritations on the gills, challenge the immune system and prejudice the welfare of the fish. Here we investigated molecules linked to proliferation, survival, and cell death, as well as inflammatory and vascular control, in a model of fish gill remodeling, from injury to recovery. We assessed the gill histology and immunohistochemistry for PCNA, iNOS, HSP70, and Bax in Hypostomus francisci obtained from a river subjected to chronic anthropic influences and then after they were placed in water of good quality. A total of 30 H. francisci adult individuals were collected and distributed into two groups: euthanized on the day of capture (group 1) and maintained for 30 days in an aquarium (group 2). In all the fish from group 1, the primary and secondary lamellae showed hypertrophy of the respiratory epithelium, lamellar fusion, lifting of the epithelium, aneurysm, hyperemia, and vascular congestion. On the other hand, in all the fish from group 2, restoration of gill integrity was observed, and the primary and secondary lamellae showed a simple epithelium, absence of lamellar fusion, hypertrophy, and aneurysm. Gills of fish from group 1 had higher frequency of cells immunopositive for PCNA, iNOS, HSP70, and Bax than those of fish from group 2 (p < 0.05). The molecular and cellular mechanisms from injury to recovery were proposed, with a balance between survival and cell death signals being essential for determining the gill structure. In addition, the findings indicate that recovery of the structural organization of gills is possible if fishes are maintained in good-quality water, indicating the importance of the conservation of aquatic environments.
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Affiliation(s)
- Camila Ferreira Sales
- Universidade Federal de São João Del Rei, Campus Centro Oeste, Laboratório de Processamento de Tecidos - LAPROTEC, Rua Sebastião Gonçalves Coelho, 400, 35501-296, Divinópolis, Minas Gerais, Brazil; Universidade Federal de Minas Gerais, Instituto de Ciências Biológicas, Departamento de Morfologia, Laboratório de Ictiohistologia, Avenida Presidente Antônio Carlos, 6627, 31270-901, Belo Horizonte, Minas Gerais, Brazil
| | - Keiza Priscila Enes Dos Santos
- Universidade Federal de São João Del Rei, Campus Centro Oeste, Laboratório de Processamento de Tecidos - LAPROTEC, Rua Sebastião Gonçalves Coelho, 400, 35501-296, Divinópolis, Minas Gerais, Brazil
| | - Elizete Rizzo
- Universidade Federal de Minas Gerais, Instituto de Ciências Biológicas, Departamento de Morfologia, Laboratório de Ictiohistologia, Avenida Presidente Antônio Carlos, 6627, 31270-901, Belo Horizonte, Minas Gerais, Brazil
| | - Rosy Iara Maciel de Azambuja Ribeiro
- Universidade Federal de São João Del Rei, Campus Centro Oeste, Laboratório de Patologia Experimental - LAPATEX, Rua Sebastião Gonçalves Coelho, 400, 35501-296, Divinópolis, Minas Gerais, Brazil
| | - Hélio Batista Dos Santos
- Universidade Federal de São João Del Rei, Campus Centro Oeste, Laboratório de Processamento de Tecidos - LAPROTEC, Rua Sebastião Gonçalves Coelho, 400, 35501-296, Divinópolis, Minas Gerais, Brazil
| | - Ralph Gruppi Thomé
- Universidade Federal de São João Del Rei, Campus Centro Oeste, Laboratório de Processamento de Tecidos - LAPROTEC, Rua Sebastião Gonçalves Coelho, 400, 35501-296, Divinópolis, Minas Gerais, Brazil.
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Kim HS, Lee SM, Kim YJ, Jang W, Seo YR. Integrative toxicogenomic analysis for elucidating molecular interference on DNA integrity and repair system with underlying signaling networks in response to low-level lead acetate in rat liver model. Mol Cell Toxicol 2017. [DOI: 10.1007/s13273-017-0019-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
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Elmallah MIY, Elkhadragy MF, Al-Olayan EM, Abdel Moneim AE. Protective Effect of Fragaria ananassa Crude Extract on Cadmium-Induced Lipid Peroxidation, Antioxidant Enzymes Suppression, and Apoptosis in Rat Testes. Int J Mol Sci 2017; 18:957. [PMID: 28475120 PMCID: PMC5454870 DOI: 10.3390/ijms18050957] [Citation(s) in RCA: 68] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2017] [Revised: 04/12/2017] [Accepted: 04/20/2017] [Indexed: 12/25/2022] Open
Abstract
Cadmium is a deleterious environmental pollutant that threats both animals and human health. Oxidative stress and elevated levels of reactive oxygen species (ROS) have recently been reported to be the main cause of cellular damage as a result of cadmium exposure. We investigate, here, the protective effect of strawberry crude extracts on cadmium-induced oxidative damage of testes in rats. Four groups (n = 8) of 32 adult male Wistar rats weighing 160-180 g were used. The control group received 0.9% saline solution all over the experimental period (5 days). Group 2 was intraperitoneally injected with 6.5 mg/kg CdCl₂. Group 3 was provided only with an oral administration of strawberry methanolic extract (SME) at a dose of 250 mg/kg. Group 4 was treated with SME before cadmium injection with the same mentioned doses. It was shown that cadmium exposure results in a significant decrease in both relative testicular weight and serum testosterone level. Analyzing the oxidative damaging effect of cadmium on the testicular tissue revealed the induction of oxidative stress markers represented in the elevated level of lipid peroxidation (LPO), nitric oxide (NO), and a decrease in the reduced glutathione (GSH) content. Considering cadmium toxicity, the level of the antioxidant enzyme activities including catalase (CAT), superoxide dismutase (SOD2), glutathione peroxidase (GPx1), and glutathione reductase (GR) were markedly decreased. Moreover, gene expression analysis indicated significant upregulation of the pro-apoptotic proteins, bcl-2-associated-X-protein (BAX), and tumor necrosis factor-α (TNFA) in response to cadmium intoxication, while significant downregulation of the anti-apoptotic, B-cell lymphoma 2 (BCL2) gene was detected. Immunohistochemistry of the testicular tissue possessed positive immunostaining for the increased level of TNF-α, but decreased number of proliferating cell nuclear antigen (PCNA) stained cells. Administration of SME debilitated the deleterious effect of cadmium via reduction of both LPO and NO levels followed by a significant enhancement in the gene expression level of CAT, SOD2, GPX1, GR, nuclear factor-erythroid 2-related factor 2 (NFE2L2), heme oxygenase-1 (HMOX1), Bcl-2, and PCNA. In addition, the SME treated group revealed a significant increase in the level of testosterone and GSH accompanied by a marked decrease in the gene expression level of Bax and TNF-α. In terms of the summarized results, the SME of Fragaria ananassa has a protective effect against cadmium-induced oxidative damage of testes.
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Affiliation(s)
- Mohammed I Y Elmallah
- Chemistry Department, Faculty of Science, Helwan University, Cairo 11795, Egypt.
- Marine Natural Product Unit (MNPRU), Faculty of Science, Helwan University, Cairo 11795, Egypt.
| | - Manal F Elkhadragy
- Department of Zoology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia.
- Department of Zoology and Entomology, Faculty of Science, Helwan University, Cairo 11795, Egypt.
- Chair Vaccines Research of Infectious Diseases, Faculty of Science, King Saud University, Riyadh 11472, Saudi Arabia.
| | - Ebtesam M Al-Olayan
- Department of Zoology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia.
| | - Ahmed E Abdel Moneim
- Department of Zoology and Entomology, Faculty of Science, Helwan University, Cairo 11795, Egypt.
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Leonel EC, Falleiros LR, Campos SG, Taboga SR. Histological and immunohistochemical characterization of the Mongolian gerbil's mammary gland during gestation, lactation and involution. Acta Histochem 2017; 119:273-283. [PMID: 28238410 DOI: 10.1016/j.acthis.2017.02.003] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2016] [Revised: 02/10/2017] [Accepted: 02/10/2017] [Indexed: 01/22/2023]
Abstract
The morphological description of normal tissues is fundamental for making comparisons and in order to identify injuries and lesions. The aim of this work was to describe the morphological characteristics of the female Mongolian gerbil's (Meriones unguiculatus) normal mammary gland, the average expression of hormone receptors, and the average proliferation rates in the epithelial cells during the periods of lactation, pregnancy and involution. Dams were euthanized on the 14th and 21st gestational days, 7 and 14days after parturition, and 3 and 5days after weaning. The dams' mammary tissues were processed and were submitted to haematoxylin and eosin staining, Periodic Acid Schiff (PAS) staining, and Gomori's Reticulin staining. Additionally, immunohistochemistry was performed for the characterization of myoepithelial cells with α-actin, the proliferation rates with proliferating cell nuclear antigen (PCNA), the estrogen hormonal receptors (ESR1 and ESR2), and progesterone receptor (PR) quantifications. It was observed that the abundant adipose tissues were replaced by glandular epithelia and there was an increase in the epithelial cell's height (from 5.97 to 32.4μm in 14th and 21st gestational days and from 20.64 to 25.4μm in 7th and 14th lactational days, respectively) and the acini diameters (from 24.88 to 69.92μm in 14th and 21st gestational days and from 139.69 to 118.59μm in 7th and 14th lactational days, respectively) with the progression of gestation and lactation. The PAS staining intensity varied throughout the glands and between the stages that were evaluated. The extracellular matrix showed different phenotypes too, with more of a presence of the Type I collagen during the early gestation and involution and with more reticular fibers (Type III collagen) during the late gestation period and lactation. The myoepithelial layers showed alterations in their distribution with thick patterns as verified by the α-actin labeling. The PCNA showed higher rates of the marked cells in 14th and 21st gestational days (40.25 and 60.28%) and in 7th and 14th lactational days (64.08 and 65.08%). The hormone receptor quantifications showed a high variation in the rates: the average PR staining decreased from 14th to 21st gestational days (from 42.3 to 8.54%), from 7th to 14th lactational days (from 59.83 to 23.18%) and from 3rd to 5th days after weaning (from 39.98 to 12.72). There were higher averages of ESR1 staining in gestational days 14 and 21(from 58.06 to 30.02%). ESR2 staining decreased during gestation (25.7 and 12.94% in 14th and 21st gestational days)and involution (from 50.97 to 30.18% in 3rd and 5th days after weaning). The Mongolian gerbils showed similar morphological characteristics when they were compared to mice and rats. However, the higher proliferation rates with a smaller involution period compared to other murine characterized this species as being adequate for mammary pathologies studies.
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Application of Immunohistochemistry in Toxicologic Pathology of the Hematolymphoid System. IMMUNOPATHOLOGY IN TOXICOLOGY AND DRUG DEVELOPMENT 2017. [DOI: 10.1007/978-3-319-47377-2_10] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
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The Pol30-K196 residue plays a critical role in budding yeast DNA postreplication repair through interaction with Rad18. DNA Repair (Amst) 2016; 47:42-48. [PMID: 27707542 DOI: 10.1016/j.dnarep.2016.09.002] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2016] [Revised: 09/20/2016] [Accepted: 09/21/2016] [Indexed: 01/14/2023]
Abstract
PCNA plays critical roles in DNA replication and various DNA repair pathways including DNA damage tolerance (DDT). In budding yeast Saccharomyces cerevisiae, DDT (aka DNA postreplication repair, PRR) is achieved by sequential ubiquitination of PCNA encoded by POL30. Our previous studies revealed that two Arabidopsis PCNA genes were able to complement the essential function of POL30 in budding yeast, but failed to rescue the PRR activity. Here we hypothesize that a certain amino acid variation(s) is responsible for the difference, and identified K196 as a critical residue for the PRR activity. It was found that the pol30-K196V mutation abolishes Rad18 interaction and PRR activity, whereas nearby amino acid substitutions can partially restore Rad18 interaction and PRR activity. Together with the Pol30-Ub fusion data, we believe that we have identified a putative Rad18-binding pocket in Pol30 that is required for PCNA monoubiquitination and PRR.
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Shahrour MA, Nicolae CM, Edvardson S, Ashhab M, Galvan AM, Constantin D, Abu-Libdeh B, Moldovan GL, Elpeleg O. PARP10 deficiency manifests by severe developmental delay and DNA repair defect. Neurogenetics 2016; 17:227-232. [PMID: 27624574 DOI: 10.1007/s10048-016-0493-1] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2016] [Revised: 08/30/2016] [Accepted: 09/01/2016] [Indexed: 11/26/2022]
Abstract
DNA repair mechanisms such as nucleotide excision repair (NER) and translesion synthesis (TLS) are dependent on proliferating cell nuclear antigen (PCNA), a DNA polymerase accessory protein. Recently, homozygosity for p.Ser228Ile mutation in the PCNA gene was reported in patients with neurodegeneration and impaired NER. Using exome sequencing, we identified a homozygous deleterious mutation, c.648delAG, in the PARP10 gene, in a patient suffering from severe developmental delay. In agreement, PARP10 protein was absent from the patient cells. We have previously shown that PARP10 is recruited by PCNA to DNA damage sites and is required for DNA damage resistance. The patient cells were significantly more sensitive to hydroxyurea and UV-induced DNA damage than control cells, resulting in increased apoptosis, indicating DNA repair impairment in the patient cells. PARP10 deficiency joins the long list of DNA repair defects associated with neurodegenerative disorders, including ataxia telangiectasia, xeroderma pigmentosum, Cockayne syndrome, and the recently reported PCNA mutation.
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Affiliation(s)
| | - Claudia M Nicolae
- Department of Biochemistry and Molecular Biology, The Pennsylvania State University College of Medicine, Hershey, PA, 17033, USA
| | - Simon Edvardson
- Pediatric Neurology Unit, Hadassah, Hebrew University Medical Center, Jerusalem, Israel
- Monique and Jacques Roboh Department of Genetic Research, Hadassah, Hebrew University Medical Center, Jerusalem, Israel
| | - Motee Ashhab
- Department of Pediatrics, Al-Makassed Islamic Hospital, Jerusalem, Israel
| | - Adri M Galvan
- Department of Biochemistry and Molecular Biology, The Pennsylvania State University College of Medicine, Hershey, PA, 17033, USA
| | - Daniel Constantin
- Department of Biochemistry and Molecular Biology, The Pennsylvania State University College of Medicine, Hershey, PA, 17033, USA
| | - Bassam Abu-Libdeh
- Department of Pediatrics, Al-Makassed Islamic Hospital, Jerusalem, Israel
| | - George-Lucian Moldovan
- Department of Biochemistry and Molecular Biology, The Pennsylvania State University College of Medicine, Hershey, PA, 17033, USA.
| | - Orly Elpeleg
- Monique and Jacques Roboh Department of Genetic Research, Hadassah, Hebrew University Medical Center, Jerusalem, Israel.
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Muskhelishvili L, Wingard SK, Latendresse JR. Proliferating Cell Nuclear Antigen—A Marker for Ovarian Follicle Counts. Toxicol Pathol 2016; 33:365-8. [PMID: 15805074 DOI: 10.1080/01926230590930164] [Citation(s) in RCA: 40] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/25/2022]
Abstract
Enumerating ovarian follicles is an effective way to estimate the extent of ovarian toxicity in female rodents exposed to xenobiotics. Differential follicle counts are useful in safety assessment bioassays and in interspecies extrapolation of ovarian toxicity. Counting the follicles in H&E-stained sections is labor intensive, tedious, and costly. In the present study we demonstrated that in rat formalin-fixed, paraffin-embedded ovary sections follicles of all degrees of maturity can be visualized by the use of antibody directed against proliferating cell nuclear antigen (PCNA). Follicles are easily distinguished from ovarian background with the ability to detect and identify primordial follicles being enhanced. This translates into a significant decrease in variability of follicle counts, labor, and cost. Specifically, variability dropped from 11% to 0.2%, the counting time was reduced by 46%, and the cost by 48%.
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Affiliation(s)
- Levan Muskhelishvili
- Toxicologic Pathology Associates at National Center for Toxicological Research, Jefferson, Arkansas 72079, USA.
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Toxic and DNA damaging effects of a functionalized fullerene in human embryonic lung fibroblasts. MUTATION RESEARCH-GENETIC TOXICOLOGY AND ENVIRONMENTAL MUTAGENESIS 2016; 805:46-57. [PMID: 27402482 DOI: 10.1016/j.mrgentox.2016.05.004] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/30/2015] [Revised: 05/04/2016] [Accepted: 05/12/2016] [Indexed: 12/11/2022]
Abstract
Water-soluble fullerenes have been studied as potential nanovectors and therapeutic agents, but their possible toxicity is of concern. We have studied the effects of F-828, a soluble fullerene [C60] derivative, on diploid human embryonic lung fibroblasts (HELFs) in vitro. F-828 causes complex time-dependent changes in ROS levels. Inhibition of Nox4 activity by plumbagin blocks F-828-dependent ROS elevation. F-828 induces DNA breaks, as measured by the comet assay and γH2AX expression, and the activities of the transcription factors NF-kB and p53 increase. F-828 concentrations>25μM are cytotoxic; cell death occurs by necrosis. Expression levels of TGF-β, RHOA, RHOC, ROCK1, and SMAD2 increase following exposure to F-828. Our results raise the possibility that fullerene F-828 may induce pulmonary fibrosis in vivo.
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Model-based contextualization of in vitro toxicity data quantitatively predicts in vivo drug response in patients. Arch Toxicol 2016; 91:865-883. [PMID: 27161439 PMCID: PMC5306109 DOI: 10.1007/s00204-016-1723-x] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2016] [Accepted: 04/20/2016] [Indexed: 12/13/2022]
Abstract
Understanding central mechanisms underlying drug-induced toxicity plays a crucial role in drug development and drug safety. However, a translation of cellular in vitro findings to an actual in vivo context remains challenging. Here, physiologically based pharmacokinetic (PBPK) modeling was used for in vivo contextualization of in vitro toxicity data (PICD) to quantitatively predict in vivo drug response over time by integrating multiple levels of biological organization. Explicitly, in vitro toxicity data at the cellular level were integrated into whole-body PBPK models at the organism level by coupling in vitro drug exposure with in vivo drug concentration–time profiles simulated in the extracellular environment within the organ. PICD was exemplarily applied on the hepatotoxicant azathioprine to quantitatively predict in vivo drug response of perturbed biological pathways and cellular processes in rats and humans. The predictive accuracy of PICD was assessed by comparing in vivo drug response predicted for rats with observed in vivo measurements. To demonstrate clinical applicability of PICD, in vivo drug responses of a critical toxicity-related pathway were predicted for eight patients following acute azathioprine overdoses. Moreover, acute liver failure after multiple dosing of azathioprine was investigated in a patient case study by use of own clinical data. Simulated pharmacokinetic profiles were therefore related to in vivo drug response predicted for genes associated with observed clinical symptoms and to clinical biomarkers measured in vivo. PICD provides a generic platform to investigate drug-induced toxicity at a patient level and thus may facilitate individualized risk assessment during drug development.
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47
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Activation of the DNA Damage Response by RNA Viruses. Biomolecules 2016; 6:2. [PMID: 26751489 PMCID: PMC4808796 DOI: 10.3390/biom6010002] [Citation(s) in RCA: 86] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2015] [Revised: 11/17/2015] [Accepted: 11/24/2015] [Indexed: 12/11/2022] Open
Abstract
RNA viruses are a genetically diverse group of pathogens that are responsible for some of the most prevalent and lethal human diseases. Numerous viruses introduce DNA damage and genetic instability in host cells during their lifecycles and some species also manipulate components of the DNA damage response (DDR), a complex and sophisticated series of cellular pathways that have evolved to detect and repair DNA lesions. Activation and manipulation of the DDR by DNA viruses has been extensively studied. It is apparent, however, that many RNA viruses can also induce significant DNA damage, even in cases where viral replication takes place exclusively in the cytoplasm. DNA damage can contribute to the pathogenesis of RNA viruses through the triggering of apoptosis, stimulation of inflammatory immune responses and the introduction of deleterious mutations that can increase the risk of tumorigenesis. In addition, activation of DDR pathways can contribute positively to replication of viral RNA genomes. Elucidation of the interactions between RNA viruses and the DDR has provided important insights into modulation of host cell functions by these pathogens. This review summarises the current literature regarding activation and manipulation of the DDR by several medically important RNA viruses.
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Roy VK, Chenkual L, Gurusubramanian G. Protection of testis through antioxidant action of Mallotus roxburghianus in alloxan-induced diabetic rat model. JOURNAL OF ETHNOPHARMACOLOGY 2015; 176:268-280. [PMID: 26549273 DOI: 10.1016/j.jep.2015.11.006] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/05/2015] [Revised: 09/28/2015] [Accepted: 11/02/2015] [Indexed: 06/05/2023]
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE Mallotus roxburghianus is used for its antihyperglycaemic properties in Southeast Asia especially in Northeast India (Mizoram) and is also recognized in traditional medicine. About 90% of diabetic patients have been associated with reproductive impairments. The primary aim of this investigation is to examine the effects of diabetes on oxidative stress, steroidogenesis, histopathology, proliferation of germ cells with proliferative cell nuclear antigen (PCNA) and antioxidant status, and alleviative effect of M. roxburghianus on the testis dysfunction. MATERIALS AND METHODS Methanolic leaf extract of M. roxburghianus was given to male albino Wistar rats by oral gavage to study the acute toxicity. Phyto-chemical composition of the methanol extract of M. roxburghianus was analyzed by GC-MS. Male Wistar rats were divided into six groups with seven animals in each group: untreated control; M. roxburghianus methanolic extract control (MRME, 400mg/kg); Alloxan diabetic control group (150 mg/kg); diabetic with 100mg/kg MRME treatment; diabetic with 400mg/kg MRME treatment; and diabetic with glibenclamide (0.1mg/kg) treatment. Diabetes was induced by a single intraperitoneal injection of 150 mg/kg alloxan and was confirmed by testing fasting plasma blood glucose levels 5 days after injection. MRME was administered orally for 28 days. Body and testis weights, serum testosterone, testis malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), glutathione (GSH), glutathione S transferase (GST) and protein levels were measured, and testis tissue was examined histopathologically and immunohistochemically (PCNA). RESULTS No sign of mortality and organ toxicity was observed up to 3000 mg/kg in acute toxicity assay of MRME and inferred to be non-toxic and safe. Bergenin and betulinic acid are the major components of MRME with many biological activities. MRME treatment rendered significant increases in body weight, testis weight, testes-body weight ratio, down regulated the MDA levels, reduced the degeneration and disruption of seminiferous tubule structure, restored the antioxidant enzymes and serum testosterone levels, increased the PCNA activities and attenuated the testes injury. CONCLUSION MRME treatment to diabetic rats improves diabetes induced oxidative damage in testis as well as provides protection to testis. Phenols (Bergenin) and terpenes (Betulinic acid) were the main compounds of MRME that show antioxidant and antidiabetic activities and indeed validated its traditional use in the management of diabetes related testicular impairment.
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Affiliation(s)
- Vikas Kumar Roy
- Department of Zoology, Mizoram Central University, Aizawl 796004, Mizoram, India.
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The kin17 Protein in Murine Melanoma Cells. Int J Mol Sci 2015; 16:27912-20. [PMID: 26610484 PMCID: PMC4661930 DOI: 10.3390/ijms161126072] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2015] [Revised: 10/28/2015] [Accepted: 11/02/2015] [Indexed: 11/16/2022] Open
Abstract
kin17 has been described as a protein involved in the processes of DNA replication initiation, DNA recombination, and DNA repair. kin17 has been studied as a potential molecular marker of breast cancer. This work reports the detection and localization of this protein in the murine melanoma cell line B16F10-Nex2 and in two derived subclones with different metastatic potential, B16-8HR and B16-10CR. Nuclear and chromatin-associated protein fractions were analyzed, and kin17 was detected in all fractions, with an elevated concentration observed in the chromatin-associated fraction of the clone with low metastatic potential, suggesting that the kin17 expression level could be a marker of melanoma.
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50
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Lee CC, Yang YC, Goodman SD, Chen S, Huang TY, Cheng WC, Lin LI, Fang WH. Deoxyinosine repair in nuclear extracts of human cells. Cell Biosci 2015; 5:52. [PMID: 26357532 PMCID: PMC4563847 DOI: 10.1186/s13578-015-0044-8] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2014] [Accepted: 09/02/2015] [Indexed: 01/10/2023] Open
Abstract
BACKGROUND Deamination of adenine can occur spontaneously under physiological conditions generating the highly mutagenic lesion, hypoxanthine. This process is enhanced by ROS from exposure of DNA to ionizing radiation, UV light, nitrous acid, or heat. Hypoxanthine in DNA can pair with cytosine which results in A:T to G:C transition mutations after DNA replication. In Escherichia coli, deoxyinosine (hypoxanthine deoxyribonucleotide, dI) is removed through an alternative excision repair pathway initiated by endonuclease V. However, the correction of dI in mammalian cells appears more complex and was not fully understood. RESULTS All four possible dI-containing heteroduplex DNAs, including A-I, C-I, G-I, and T-I were introduced to repair reactions containing extracts from human cells. The repair reaction requires magnesium, dNTPs, and ATP as cofactors. We found G-I was the best substrate followed by T-I, A-I and C-I, respectively. Moreover, judging from the repair requirements and sensitivity to specific polymerase inhibitors, there were overlapping repair activities in processing of dI in DNA. Indeed, a hereditable non-polyposis colorectal cancer cell line (HCT116) demonstrated lower dI repair activity that was partially attributed to lack of mismatch repair. CONCLUSIONS A plasmid-based convenient and non-radioisotopic method was created to study dI repair in human cells. Mutagenic dI lesions processed in vitro can be scored by restriction enzyme cleavage to evaluate the repair. The repair assay described in this study provides a good platform for further investigation of human repair pathways involved in dI processing and their biological significance in mutation prevention.
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Affiliation(s)
- Chia-Chia Lee
- />Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, #7, Chung-Shan South Road, Taipei, 10002 Taiwan ROC
| | - Ya-Chien Yang
- />Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, #7, Chung-Shan South Road, Taipei, 10002 Taiwan ROC
- />Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, 10002 Taiwan ROC
| | - Steven D. Goodman
- />Center for Microbial Pathogenesis, Nationwide Children’s Hospital, Columbus, OH USA
| | - Shi Chen
- />Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, #7, Chung-Shan South Road, Taipei, 10002 Taiwan ROC
| | - Teng-Yung Huang
- />Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, #7, Chung-Shan South Road, Taipei, 10002 Taiwan ROC
| | - Wern-Cherng Cheng
- />Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, 10002 Taiwan ROC
| | - Liang-In Lin
- />Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, #7, Chung-Shan South Road, Taipei, 10002 Taiwan ROC
- />Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, 10002 Taiwan ROC
| | - Woei-horng Fang
- />Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, #7, Chung-Shan South Road, Taipei, 10002 Taiwan ROC
- />Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, 10002 Taiwan ROC
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