• EV
• ExoPAS
• isolation
• miRNAs
• miRQuick

• Korea Evaluation Institute of Industrial Technology
• National Research Foundation of Korea

• Bothrops
• RNA purification
• cDNA synthesis
• transcripts
• venom
• venom gland

• N° 079 - 2021 - FONDECYT and N° PE501080181-2022-PROCIENCIA Programa Nacional de Investigación Científica y Estudios Avanzados, PROCIENCIA
• B2110007i Vicerrectorado de Investigación y Posgrado - UNMSM

• AGPs and homogalacturonan immunodetection
• Arvanitolia
• Gaidourelia
• Koroneiki
• Lefkolia
• Olea europeae
• arabinogalactan proteins (AGPs)
• cell wall
• long-term salinity stress
• prolyl 4-hydroxylases (P4Hs)

• Olea
• Prolyl Hydroxylases
• Sodium Chloride/pharmacology
• Salt Stress
• Procollagen-Proline Dioxygenase

• 2018ΣΕ01300000 General Secretariat for Research & Technology (GSRT), Greece under the Emblematic 844 Αction "The Οlive Road"

• clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated proteins biosensing mechanisms
• infectious diseases diagnosis
• integrated detection
• microfluidic platforms

• Humans
• Microfluidics
• Communicable Diseases/diagnosis
• Communicable Diseases/genetics

• 22104090 National Natural Science Foundation of China
• 22ZR1436200 Natural Science Foundation of Shanghai
• National Natural Science Foundation of China
• Natural Science Foundation of Shanghai

• Human metapneumovirus
• Immunochromatography assay
• Nasopharyngeal swab
• Rt-pcr
• Sputum

• Candida albicans
• Cell lysis
• RNA extraction

Accurate and reliable analysis of gene expression depends on the extraction of pure and high-quality RNA. However, while the conventional phenol-chloroform RNA extraction is preferable over silica-based columns, particularly when cost is a concern or higher RNA yield is desired, it can result in significant RNA contamination. Contaminants including excess phenol, chloroform, or salts, can have significant impacts on downstream applications, including RNA quantification and reverse transcription, that can skew data collection and interpretation. To overcome the issue of RNA contamination in the conventional phenol-chloroform based RNA extraction method, we have optimized the protocol by adding one chloroform extraction step, and several RNA washing steps. Importantly, RNA quality and purity and accuracy in the quantification of RNA concentration were significantly improved with the modified protocol, resulting in reliable data collection and interpretation in downstream gene expression analysis.

•

Our protocol is customized by the addition of a second chloroform extraction step. Chloroform is carefully pipetted so as to not disturb the interphase layer. Any contaminants accidentally removed from interphase will be present in subsequent steps and can result in RNA contaminated with protein or phenol. The additional chloroform step increases RNA purity.

• NRVM
• NRVMs
• Phenol-chloroform RNA extraction
• Phenol-chloroform extraction
• RNA contamination
• RNA extraction
• RNA purification
• RNA purity
• RNA quantification
• RT-qPCR

• RNA
• cell culture
• extraction
• silica
• tissue

• Animals
• Classical Swine Fever/blood
• Classical Swine Fever/diagnosis
• Classical Swine Fever/virology
• Classical Swine Fever Virus/genetics
• Classical Swine Fever Virus/isolation & purification
• Heart/virology
• Kidney/virology
• Liver/virology
• Lymph Nodes/virology
• Palatine Tonsil/virology
• RNA, Viral/blood
• RNA, Viral/isolation & purification
• Reverse Transcriptase Polymerase Chain Reaction
• Spleen/virology
• Swine/blood
• Swine/virology
• Urinary Bladder/virology

• PCR
• RNA/DNA extraction
• bacteria
• coinfection
• mass spectrometry
• virus

The coast of the Bulgarian Black Sea is a popular summer holiday destination. The Dam of Iskar is the largest artificial dam in Bulgaria, with a capacity of 675 million m³. It is the main source of tap water for the capital Sofia and for irrigating the surrounding valley. There is a close relationship between the quality of aquatic ecosystems and human health as many infections are waterborne. Rapid molecular methods for the analysis of highly pathogenic bacteria have been developed for monitoring quality. Mycobacterial species can be isolated from waste, surface, recreational, ground and tap waters and human pathogenicity of nontuberculose mycobacteria (NTM) is well recognized. The objective of our study was to perform molecular analysis for key-pathogens, with a focus on mycobacteria, in water samples collected from the Black Sea and the Dam of Iskar. In a two year period, 38 water samples were collected—24 from the Dam of Iskar and 14 from the Black Sea coastal zone. Fifty liter water samples were concentrated by ultrafiltration. Molecular analysis for 15 pathogens, including all species of genus Mycobacterium was performed. Our results showed presence of Vibrio spp. in the Black Sea. Rotavirus A was also identified in four samples from the Dam of Iskar. Toxigenic Escherichia coli was present in both locations, based on markers for stx1 and stx2 genes. No detectable amounts of Cryptosporidium were detected in either location using immunomagnetic separation and fluorescence microscopy. Furthermore, mass spectrometry analyses did not detect key cyanobacterial toxins. On the basis of the results obtained we can conclude that for the period 2012–2014 no Mycobacterium species were present in the water samples. During the study period no cases of waterborne infections were reported.

• Bulgaria
• PCR
• molecular detection
• monitoring pathogens

• Black Sea
• Bulgaria
• Cryptosporidium/isolation & purification
• Ecosystem
• Environmental Medicine
• Fresh Water
• Humans
• Mycobacterium/isolation & purification
• Recreation
• Seasons
• Water Microbiology
• Water Pollution

• DNA
• NanoDrop
• Oligonucleotides
• Phenol
• RNA
• TRIzol

Diagnostic tests for respiratory viral infections use traditionally either nasopharyngeal washes or swabs. Sputum is representative of the lower respiratory tract but is used rarely for viral testing. The aim of this study was to compare the detection rates of respiratory viruses from nasopharyngeal swabs and sputum using a multiplex real‐time reverse transcription‐polymerase chain reaction (RT‐PCR). Adults who were admitted or presented to the clinics of Gil Medical Center with acute respiratory symptoms were recruited from 1 November 2012 to 31 March 2013. Paired specimens of nasopharyngeal swabs and sputum were obtained from 154 subjects, and RNA was extracted and tested for 16 different respiratory viruses using the Anyplex II RV16 Detection kit (Seegene, Seoul, Korea). The positive rate was 53% (81/154) for nasopharyngeal swabs and 68% (105/154) for sputum (P < 0.001). One hundred thirty‐four viruses were identified for 107 illnesses. Influenza A virus, RSV A, HRV, coronavirus OC43, and adenovirus were detected more frequently in sputum samples than in nasopharyngeal swabs (P < 0.001). Importantly, 12 of 44 (27%) influenza A infections and 11 of 27 (41%) RSV infections were positive in only sputum samples. The detection rates of respiratory viruses from sputum samples were significantly higher than those from nasopharyngeal swabs in adults using real‐time multiplex RT‐PCR. These findings suggest that sputum would benefit for the detection of respiratory viruses by nucleic acid amplification tests (NAATs) in patients who produce sputum. Further studies are needed to establish standardized RNA extraction methods from sputum samples. J. Med. Virol. 86:2122–2127, 2014. © 2014 Wiley Periodicals, Inc.

• nasopharyngeal swab
• reverse transcription-polymerase chain reaction
• sputum

• Animals
• Chickens/virology
• Influenza A virus/genetics
• Influenza A virus/isolation & purification
• Limit of Detection
• Meat/virology
• RNA, Viral/analysis
• Real-Time Polymerase Chain Reaction
• Reverse Transcriptase Polymerase Chain Reaction
• Virology/methods

This chapter discusses the molecular detection of multiple respiratory viruses. Respiratory infections are very common. They are responsible for significant morbidity and mortality and are costly in terms of lost time from work, inappropriate use of antibiotics, and lengthy hospital stays. Laboratory detection of causative agents can influence treatment and allow appropriate infection control measures, potentially saving time and money, promoting quicker recovery for the patient, and lowering the risk of nosocomial transmission of the organism. Molecular testing may be the most sensitive method of detecting respiratory viruses. These methods have been shown to detect multiple viruses in a single sample more often than any of the other methods described and, in addition, can detect viruses that are difficult or impossible to grow. The detection of nonviable organisms may provide additional insight into disease but provides its own challenges in terms of determining clinical significance of positive results. Like many areas of clinical medicine and microbiology, the increasing use of molecular diagnostics for detection of respiratory viruses raises many new questions for the laboratory and the clinical diagnostician, but also promises many benefits as these methods become increasingly available for routine use.

• Animals
• Cryopreservation
• Electrophoresis, Agar Gel
• Female
• Genetic Techniques
• Guanidines/chemistry
• Isothiocyanates/chemistry
• Nitrogen/chemistry
• Pancreas/metabolism
• Phenol/chemistry
• RNA/chemistry
• RNA/isolation & purification
• Rats
• Reverse Transcriptase Polymerase Chain Reaction

The reliability of gene expression profiling-based technologies to detect transcriptional differences representative of the original samples is affected by the quality of the extracted RNA. It strictly depends upon the technique that has been employed. Hence, the present study aimed at systematically comparing silica-gel column (SGC) and guanidine isothiocyanate (GTC) techniques of RNA isolation to answer the question which technique is preferable when frozen, long-term stored or fresh lung tissues have to be evaluated for the downstream molecular analysis.

Frozen lungs (n = 3) were prepared by long-term storage (2.5 yrs) in -80°C while fresh lungs (n = 3) were harvested and processed immediately. The purity and quantification of RNA was determined with a spectrophotometer whereas the total amounted copy numbers of target sequences were determined with iCycler detection system for assessment of RNA intactness (28S and 18S) and fragment sizes, i.e. short (GAPDH-3' UTR), medium (GAPDH), and long (PBGD) with 200 bp, 700 bp, and 1400 bp distance to the 3'ends of mRNA motif, respectively.

Total yield of RNA was higher with GTC than SGC technique in frozen as well as fresh tissues while the purity of RNA remained comparable. The quantitative reverse transcriptase-polymerase chain reaction data revealed that higher mean copy numbers of 28S and a longer fragment (1400 bp) were obtained from RNA isolated with SGC than GTC technique using fresh as well as frozen tissues. Additionally, a high mean copy number of 18S and medium fragment (700 bp) were obtained in RNA isolated with SGC technique from fresh tissues, only. For the shorter fragment, no significant differences between both techniques were noticed.

Our data demonstrated that although the GTC technique has yielded a higher amount of RNA, the SGC technique was much more superior with respect to the reliable generation of an intact RNA and effectively amplified longer products in fresh as well as in frozen tissues.

• Cell Nucleus/metabolism
• Electrophoresis, Polyacrylamide Gel
• Female
• Guanidines/chemistry
• Humans
• Isothiocyanates/chemistry
• Molecular Weight
• Phenols/chemistry
• Proteins/chemistry
• Proteins/isolation & purification
• RNA/chemistry
• RNA/isolation & purification
• Sensitivity and Specificity
• Solubility
• Thiourea/chemistry
• Urea/chemistry

• Antibiotics, Antineoplastic/pharmacology
• Antineoplastic Agents, Phytogenic/pharmacology
• Benzothiazoles
• Breast Neoplasms/pathology
• Cell Line, Tumor
• DNA, Complementary/genetics
• Diamines
• Doxorubicin/pharmacology
• Drug Resistance, Multiple/genetics
• Female
• Gene Amplification
• Gene Expression
• Humans
• Microarray Analysis
• Organic Chemicals
• Paclitaxel/pharmacology
• Quinolines
• RNA/analysis
• RNA, Messenger/metabolism
• Reverse Transcriptase Polymerase Chain Reaction

• Adult
• Albuterol/therapeutic use
• Anti-Asthmatic Agents/therapeutic use
• Asthma/drug therapy
• Asthma/physiopathology
• Beclomethasone/therapeutic use
• Bronchodilator Agents/therapeutic use
• Cells, Cultured
• Chemokine CCL5/metabolism
• Drug Interactions
• Drug Therapy, Combination
• Ethanolamines/therapeutic use
• Female
• Formoterol Fumarate
• Granulocyte-Macrophage Colony-Stimulating Factor/metabolism
• Humans
• Interleukin-8/metabolism
• Male
• Middle Aged
• Receptors, Adrenergic, beta-2/metabolism
• Receptors, Glucocorticoid/metabolism
• Severity of Illness Index
• Sputum/chemistry
• Sputum/metabolism
• Tissue Distribution

• Base Sequence
• Cell Line, Tumor
• Gene Expression/genetics
• Glyceraldehyde-3-Phosphate Dehydrogenases/genetics
• Humans
• Keratins/analysis
• Microarray Analysis/methods
• Nucleic Acid Hybridization
• Oligonucleotide Array Sequence Analysis/methods
• Plasminogen Activators/analysis
• Polymerase Chain Reaction/methods
• RNA, Messenger/genetics
• RNA, Neoplasm/genetics
• Receptors, Cell Surface/analysis
• Receptors, Urokinase Plasminogen Activator
• Reproducibility of Results
• Urokinase-Type Plasminogen Activator/analysis

AIM: To investigate the effects of traditional Chinese medicinal enema (TCME) on inflammatory and immune response of colonic mucosa of rats with ulcerative colitis (UC), and to observe the pathogenic mechanism.

METHODS: Thirty UC rats, induced by intestinal enema together with 2.4-dinitrochlorobenzene (DNCB) and acetic acid, were randomly divided into 3 groups, i.e., G I, G II and G III. Groups G I and G II were administered with TCME and salazosulfapyridine enema (SASPE), respectively. Group G III was clystered with only normal saline (NSE), served as control. Group G IV was taken from normal rats as reference, once daily, from the 7th day after the establishment of UC for total 28 d. Interleukin-6 (IL-6) in the colonic mucosa was assayed by ³H-TdR incorporation assay. Colonic mucosal lymphocyte subpopulation adhesive molecules, CD₄⁺CD_11a⁺, CD₄⁺CD₁₈⁺, CD₈⁺CD_11a⁺, CD₈⁺CD₁₈⁺ (LSAM), tumor necrosis factor (TNF)-α, and interferon-γ (IFN-γ), were detected by enzyme linked immunosorbent assay (ELISA). Moreover, the expression of TNF-α mRNA and IFN-γ mRNA in colonic mucosa were detected by polymerase chain reaction (RT-PCR).

RESULTS: Before therapies, in model groups, G I, G II and G III, levels of IL-6, TNF-α, IFN-γ, CD₈⁺CD_11a⁺ and CD₈⁺CD₁₈⁺ were significantly different (38.29 ± 2.61 U/mL, 16.54 ± 1.23 ng/L, 8.61 ± 0.89 ng/L, 13.51% ± 2.31% and 12.22% ± 1.13%, respectively) compared to those in G IV group (31.56 ± 2.47 U/mL, 12.81 ± 1.38 ng/L, 5.28 ± 0.56 ng/L, 16.68% ± 1.41% and 16.79% ± 1.11%, respectively). After therapeutic enemas, in G I group, the contents of IL-6 (32.48 ± 2.53 U/m), TNF-α (13.42 ± 1.57 ng/L) and IFN-γ (5.87 ± 0.84 ng/L) were reduced; then, the contents of CD₈⁺CD_11a⁺ (16.01% ± 1.05 %) and CD₈⁺CD₁₈⁺ (16.28% ± 0.19%) were raised. There was no significant difference between groups G I and G IV, but the difference between groups G I and G II was quite obvious (P < 0.05). The expressions of TNF-α mRNA and IFN-γ mRNA in group G III were much higher than those of group G IV, but those in group G I were significantly suppressed by TCME therapy.

CONCLUSION: Ulcerative colitis is related to colonic regional mucosal inflammatory factors and immune imbalance. TCME can effectively inhibit regional mucosal inflammatory factors and improve their disorder of immunity.

• Animals
• Cell Adhesion Molecules/metabolism
• Colitis, Ulcerative/drug therapy
• Colitis, Ulcerative/immunology
• Colitis, Ulcerative/metabolism
• Cytokines/metabolism
• Drugs, Chinese Herbal/pharmacology
• Enema
• Female
• Gene Expression/immunology
• Interferon-gamma/genetics
• Intestinal Mucosa/drug effects
• Intestinal Mucosa/immunology
• Intestinal Mucosa/metabolism
• Male
• Medicine, Chinese Traditional
• RNA, Messenger/analysis
• Random Allocation
• Rats
• Rats, Wistar
• Tumor Necrosis Factor-alpha/genetics

One controversial source of infection for hepatitis C virus (HCV) involves the sharing of contaminated implements, such as straws or spoons, used to nasally inhale cocaine and other powdered drugs. An essential precondition for this mode of transmission is the presence of HCV in the nasal secretions of intranasal drug users.

Blood and nasal secretion samples were collected from five plasma-positive chronic intranasal drug users and tested for HCV RNA using RT-PCR.

HCV was detected in all five blood samples and in the nasal secretions of the subject with the highest serum viral load.

This study is the first to demonstrate the presence of HCV in nasal secretions. This finding has implications for potential transmission of HCV through contact with contaminated nasal secretions.

• Apoptosis/genetics
• CD40 Antigens/metabolism
• Carcinoma, Squamous Cell/genetics
• Carcinoma, Squamous Cell/therapy
• Caspase 3
• Caspase 9
• Caspases/metabolism
• Enzyme Activation
• Gene Expression Profiling
• Gene Expression Regulation, Neoplastic
• Genes, p53/genetics
• Humans
• Hyperthermia, Induced
• Interleukin-12/metabolism
• Ki-1 Antigen/metabolism
• Mutation
• Neoplasm Proteins/genetics
• Neoplasm Proteins/metabolism
• Oligonucleotide Array Sequence Analysis
• Tongue Neoplasms/genetics
• Tongue Neoplasms/therapy
• Tumor Cells, Cultured
• Tumor Suppressor Protein p53/metabolism

• Animals
• Arousal/physiology
• Blood Pressure/physiology
• Cerebrospinal Fluid Pressure/physiology
• Dogs
• Heart Rate/physiology
• Hindlimb/injuries
• Hippocampus/pathology
• Hypothalamus/pathology
• Immunoblotting
• Limbic System/pathology
• Microscopy, Electron
• Myelin Basic Protein/cerebrospinal fluid
• Myelin Basic Protein/genetics
• Stress Disorders, Post-Traumatic/cerebrospinal fluid
• Stress Disorders, Post-Traumatic/genetics
• Stress Disorders, Post-Traumatic/pathology
• Wounds, Gunshot/cerebrospinal fluid
• Wounds, Gunshot/genetics
• Wounds, Gunshot/pathology

• Acute Disease
• Adenoviridae
• Administration, Inhalation
• Adult
• Aged
• Aged, 80 and over
• Asthma/epidemiology
• Asthma/therapy
• Asthma/virology
• Comorbidity
• Female
• Follow-Up Studies
• Glucocorticoids/therapeutic use
• Humans
• Male
• Middle Aged
• Patient Admission
• Picornaviridae
• Polymerase Chain Reaction
• Prevalence
• Prospective Studies
• Pulmonary Disease, Chronic Obstructive/epidemiology
• Pulmonary Disease, Chronic Obstructive/therapy
• Pulmonary Disease, Chronic Obstructive/virology
• Respiratory Tract Infections/diagnosis
• Respiratory Tract Infections/therapy
• Respiratory Tract Infections/virology
• Singapore/epidemiology
• Statistics as Topic
• Survival Analysis

• Animals
• Bird Diseases/virology
• Chickens
• Columbidae
• DNA Primers
• Newcastle Disease/virology
• Newcastle disease virus/isolation & purification
• Organ Specificity
• Poultry Diseases/virology
• Reverse Transcriptase Polymerase Chain Reaction/methods

• Acute Disease
• Adult
• Asthma/complications
• Cells, Cultured
• Fluorescent Antibody Technique/methods
• Humans
• Immunoblotting/methods
• Nasopharynx/virology
• RNA Virus Infections/complications
• RNA Virus Infections/diagnosis
• RNA Viruses/genetics
• RNA Viruses/isolation & purification
• RNA, Viral/analysis
• Respiratory Tract Infections/complications
• Respiratory Tract Infections/diagnosis
• Reverse Transcriptase Polymerase Chain Reaction
• Sensitivity and Specificity
• Sputum/virology