Vitellogenin receptor (VgR) plays a crucial role in oogenesis by mediating endocytosis of vitellogenin and a portion of the yolk proteins in many insect species. However, the function of VgR in minute parasitoid wasps is largely unknown. Here, we applied Trichogramma dendrolimi, a minute egg parasitoid, as a study model to investigate the function of VgR in parasitoids. We developed RNA interference (RNAi) methods based on microinjection of prepupae in T. dendrolimi. RNAi employs nanomaterial branched amphipathic peptide capsules (BAPC) as a carrier for double-stranded RNA (dsRNA), significantly enhancing delivery efficiency. Also, artificial hosts without medium were used to culture the injected prepupae in vitro. Utilizing these methods, we found that ovarian growth was disrupted after knockdown of TdVgR, as manifested by the suppressed development of the ovariole and the inhibition of nurse cell internalization by oocytes. Also, the initial mature egg load in the ovary was significantly reduced. Notably, the parasitic capacity of the female adult with ovarian dysplasia was significantly decreased, possibly resulting from the low availability of mature eggs. Moreover, ovarian dysplasia in T. dendrolimi caused by VgR deficiency are conserved despite feeding on different hosts. The results confirmed a critical role of TdVgR in the reproductive ability of T. dendrolimi and provided a reference for gene functional studies in minute insects.

The transforming growth factor-beta (TGF-β) signaling pathway is a key conserved developmental pathway, demonstrating remarkable conservation in its components, structures, and functions across diverse animal species. This study presents a comprehensive characterization and comparative analysis of receptor-regulated Smads (R-Smads) in Fasciola gigantica, a major parasite affecting cattle, buffalo, and ruminants in tropical regions. Four R-Smads were identified: FgSmad1/5, FgSmad2, FgSmad3, and FgSmad8. Full-length cDNAs for these R-Smads, isolated using the rapid amplification of cDNA ends (RACE) approach, were 3348, 3010, 5172, and 1991 bp in length, encoding proteins of 464, 506, 678, and 404 amino acids, respectively. FgSmad1/5 and FgSmad8 were classified as bone morphogenetic protein (BMP)-specific R-Smads (BR-Smads) due to their conserved signature motifs and similarity to those found in parasitic platyhelminths and mammalian hosts. Conversely, FgSmad2 and FgSmad3 were identified as TGF-β/activin-specific R-Smads (AR-Smads), displaying a distinctive pattern of conserved motifs commonly observed among trematode parasites. Notably, FgSmad2 exhibited an atypical feature for R-Smads, as it lacked the MH1 domain. The expressions of F. gigantica R-Smad genes were observed across all developmental stages, with the highest levels of most R-Smads occurring in the unembryonated egg stage (primarily BR-Smads) and the metacercaria stage (predominantly AR-Smads), underscoring a significant shift in TGF-β and BMP signaling dynamics during development. These findings greatly advance our understanding of R-Smad proteins within the TGF-β signaling pathway of F. gigantica and provide a solid foundation for future research into their roles and regulatory mechanisms.

G protein-coupled receptors (GPCRs) are cell-surface proteins that are targeted therapeutically for a range of disorders, including diabetes. Adhesion GPCRs (aGPCRs) are the second largest class of the GPCR superfamily and some members of this family have been implicated in appropriate organ development. However, the role of aGPCRs in endocrine pancreas specification is not yet known.

Here, we systematically characterised expression of mRNAs encoding aGPCRs and their ligands in developing mouse and human pancreas using our own and publicly available single-cell RNA sequencing and spatial transcriptomics data, and we conducted qPCR analysis of aGPCR expression in human pancreas at different gestational stages. We then investigated the role of GPR56 (ADGRG1), the most abundant aGPCR in pancreatic endocrine progenitors, in islet development using Gpr56 null mice and their wildtype littermates.

We demonstrated that aGPCRs are dynamically expressed during mouse and human pancreas development, with specific aGPCR mRNAs expressed in distinct endocrine, endothelial, mesenchymal, acinar, ductal, and immune cell clusters. aGPCR ligand mRNAs were mostly expressed by non-endocrine cells, and the most highly expressed receptor-ligand interacting mRNA pairs were those encoding GPR56 and COL3A1. Deletion of Gpr56 in neonatal mice was associated with an altered α-/β-/δ-cell ratio and reduced β-cell proliferation.

Our data show that aGPCRs are expressed at key stages of human and mouse pancreas endocrine lineage decisions, and analysis of pancreases from Gpr56 knockout mice implicate this aGPCR in the development of a full complement of β-cells.

Molecular biology techniques, including qPCR, are reliable, powerful, and commonly used tools for diagnostic purposes. qPCR might be useful for determining the etiology of yellow semen syndrome (YSS), an endemic condition in the turkey population, leading to decreased reproductive potential of this species. The current study aimed to evaluate the most accurate qPCR internal controls that might be used as first-choice-reference genes in studies on the reproductive tract, liver, and immune tissues and cells of adult male turkey breeders. RNA was isolated from testis, epididymis, ductus deferens, liver, thymus, bursa of Fabricius, spleen, whole blood, and peripheral blood mononuclear cells (PBMC) from healthy adult individuals producing white semen (WS, n = 6) and with yellow semen syndrome (YSS, n = 6). The expression of seven commonly used housekeeping genes, i.e., glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin beta (ACTB), phosphoglycerate kinase (PGK1), 60S ribosomal protein L13 (RPL13),ribosomal protein L19 (RPL19), transferrin receptor protein (TFRC), and vimentin (VIM) was tested with qPCR followed by computational calculation of these genes' expression stability in selected tissues/cells. For YSS studies we recommend using ACTB/GAPDH gene pair as a reference for male reproductive organs i.e. testis, epididymis, and ductus deferens as well as in the liver, RPL13/RPL19 for central immune organs, i.e. bursa of Fabricius and thymus, and RPL13/VIM, ACTB/RPL19, and PGK1/RPL19 in the spleen, whole blood, and PBMC research, respectively. A careful investigation of the stability of these genes in each following experiment is required to maintain high-accuracy results. Our results may help optimize protocols for better investigation of molecular mechanisms in physiological and pathological conditions in male turkey breeders for further improving commercial flock welfare and livestock production, and are a prerequisite for future studies determining YSS etiology.

The Anopheles Hyrcanus Group comprises 25 recognized mosquito species that exhibit significant variations in critical vector characteristics despite having similar morphology and ecological niches. Cross-species transcriptional profiling with appropriate reference genes is essential to evaluate the physiological differences among these closely related species. This study aimed to identify reliable pan-species reference genes among six Anopheles Hyrcanus Group species found in South Korea. We assessed the expression stability of eight candidate genes across five developmental stages in each of the six species. Utilizing qPCR and analysis programs, our analysis revealed that the transcription of ribosomal protein L8 (RPL8) and ribosomal protein L13a was the most stable at the larval stage. In contrast, ribosomal protein L32 and ribosomal protein S17 (RPS17) exhibited stability across all tested adult stages. In comparisons across different developmental stages within individual species, RPS17 emerged as a reliable reference gene for Anopheles belenrae, Anopheles pullus, Anopheles sinensis, and Anopheles sineroides, whereas ribosomal protein S7 and RPL8 were suitable for Anopheles kleini and Anopheles lesteri. These selected reference genes may facilitate comparative gene expression profiling within the Anopheles Hyrcanus Group, thereby providing insights into the molecular mechanisms underlying their differences, such as distinct vector competence and baseline insecticide susceptibility.

R-spondin1 (Rspo1) is a member of the secreted furin-like domain-containing protein family, and it is recognized for its significance in mammalian ovarian development. However, its role in teleost ovarian development remains largely uninvestigated. The Qi River crucian carp (Carassius auratus) is a species capable of gynogenesis, and it encounters challenges of premature ovarian maturation in aquaculture settings. Previous research established the essential involvement of Rspo1 in oocyte growth in Qi River crucian carp, but the precise molecular mechanisms underlying its role remain poorly understood. In this study, we categorized the pre-spawning ovarian development process of premature Qi River crucian carp into five stages through meticulous examination of morphology and histology. Immunofluorescence analysis revealed colocalization of Rspo1 with Vasa protein in oogonia, primary growth stage, and cortical vacuolar stage oocytes, and it was also detected in somatic cells. After a 60-day period of RNA interference via injection of Rspo1 double-stranded RNA into late-previtellogenesis stage ovaries, a substantial proportion of oocytes were arrested in the primary growth stage and exhibited a marked reduction in the expression of germ cell marker genes and an increase in apoptosis signaling. RNA-sequencing and real-time PCR analyses indicated a potential association between genes involved in hormone synthesis, lipid storage, and cell proliferation with ovary development in Qi River crucian carp. Furthermore, a significant decrease in levels of serum estrogens and vitellogenin was observed after Rspo1 knockdown. Dual-fluorescence in situ hybridization analysis demonstrated co-expression of Rspo1 with cyp19a1a in ovarian germ and surrounding somatic cells. Furthermore, results of a promoter assay indicated that Rspo1 can dose-dependently activate cyp19a1a expression. Collectively, these findings suggest that Rspo1 plays a role in ovarian development and oocyte growth by modulating cyp19a1a expression and influencing estrogen synthesis. These results provide valuable insights into the molecular mechanisms underlying the involvement of Rspo1 in ovarian development in Qi River crucian carp.

Lathyrus sativus (grass pea) is a valuable crop for sustainable agriculture, offering dietary benefits and desirable agronomic traits. However, its yield stability is limited by diseases such as powdery mildew caused by Erysiphe pisi. Increasing fungal resistance to pesticides and environmental concerns demand the development of resistant crop varieties. To identify key defense mechanisms and effector genes involved in the Lathyrus sativus-Erysiphe pisi interaction we analyzed four L. sativus accessions exhibiting varying resistance to E. pisi (resistant, partially resistant, partially susceptible, and susceptible) using a dual RNA-Seq experiment across different time points. We observed a host biphasic response, characterized by an initial burst of gene expression, followed by a quiescent phase, and a subsequent wave of intense gene expression. Common L. sativus defense mechanisms included antifungal protein expression, cell wall reinforcement, and reactive oxygen species-mediated defense. These defenses involved respectively Bowman-Birk type proteinase inhibitors, peptidyl-prolyl cis-trans isomerases and mannitol dehydrogenases. The resistant accession specifically activated early reinforcement of structural barriers associated with lignin biosynthesis and the phenylpropanoid pathway, along with sustained chemical defenses (e.g. eugenol synthase 1), epigenetic regulation, and oxidative stress responses thorough peroxidases and heat shock proteins. The partial resistant accession exhibited a front-loaded defense response at early infection stages. Contrastingly, the partially susceptible accession exhibited a weaker baseline defense, with a slower and less robust response targeting pathogen infection. We identified potential E. pisi effectors, including genes involved in cell wall hydrolysis (e.g. mannosidase DCW1), nutrient acquisition (e.g. secreted alpha-glucosidase), and virulence (e.g. SnodProt1), with a higher diversity of effectors identified in the susceptible accession. In conclusion, this study identifies novel targets such as NLRs and effectors, antifungal proteins and genes related to cell wall reinforcement, within the complex Lathyrus sativus-Erysiphe pisi interaction to support future breeding programs aimed at enhancing resistance to E. pisi in L. sativus and related species.

Duchenne muscular dystrophy (DMD) is an X-linked disorder that is caused by mutations in the DMD gene, leading to progressive muscle wasting and weakness. There is currently no cure for DMD. The BL10-mdx mouse is the most commonly used model in preclinical DMD studies, but it exhibits a mild disease phenotype compared to DMD patients, limiting research translatability. The newer D2-mdx mouse has a more severe phenotype at an early age and may better recapitulate human disease. To compare these mouse models on a transcriptional level with quantitative RT-PCR, stable and reliable reference genes are indispensable. We aimed to evaluate the stability and reliability of a panel of nine candidate reference genes (Actb, Ap3d1, Gapdh, Hmbs, Htatsf1, Pak1ip1, Rpl13a, Sdha and Zfp91) in the gastrocnemius, diaphragm and heart of mice from both strains and their corresponding wild types aged 4 to 52 weeks. Data was analyzed using geNorm, BestKeeper, deltaCt and NormFinder. We found that Htatsf1, Pak1ip1 and Zfp91 are suitable reference genes for normalization of gene expression in dystrophic and healthy mice, regardless of the tissue type or age. In our hands, Actb, Gapdh and Rpl13a were not suitable as reference genes, exhibiting tissue-, age-, or disease specific changes in expression. This study highlights the importance of the selection of suitable reference genes, as their stability can differ between specific experimental setups.

Reverse transcription-quantitative PCR (RT-qPCR) is a powerful tool for quantifying gene expression. However, reference genes (RGs) for gene expression analysis in peach (Prunus persica) during interactions with Monilinia laxa, a major fungal pathogen that causes brown rot, have not been established. In this study, we analysed 12 candidate RGs in this pathosystem by analysing samples from 12 to 144 HAI. The stability of the RGs was evaluated using the ΔCq method and BestKeeper, NormFinder, and geNorm algorithms. Our results identified AKT3, RNA pol II (RPII) and SNARE (using geNorm), RPII, AKT3 and TEF2 (using NormFinder), AKT3, SNARE and RPII (using BestKeeper) and RPII, MUB6 and AKT3 (using the ΔCq method) as the most stable RGs for mRNA normalisation in this pathosystem across all tested samples. The geNorm algorithm was used to determine the optimal number of suitable RGs required for proper normalisation under these experimental conditions, indicating that the three RGs were sufficient for normalisation. Analysis of the results obtained using different algorithms showed that AKT3, RPII, and SNARE were the three most stable RGs. Furthermore, to confirm the validity of the reference genes, the expression levels of six genes of interest, involved in different metabolic pathways, were normalized in inoculated and uninoculated peach fruit. These findings provide a set of RGs for accurate RT-qPCR analysis in studies involving peach and M. laxa interactions, facilitating deeper insights into the molecular mechanisms underlying this important plant-pathogen relationship.

Clear differentiation of high-grade and clinically insignificant prostate cancer (PCa) is critical for clinical decision-making. Here, we developed a proprietary urinary exosome isolation approach (EVLatch) and established a facile diagnostic workflow. We discovered that EEF1A1 levels, abundantly expressed on urinary exosomes, positively correlate to urinary exosome counts irrespective of source and collection time and demonstrated that EEF1A1 enables in-assay quantification of urinary exosomes. Importantly, a prostate cancer urinary EVLatch-based artificial intelligence diagnostics (PURE-AID) classification system utilizing PCA3, HOXC6, and DLX1 as targets with SPDEF for reference and EEF1A1 for quality checking, trained on 271 patients, achieved an area under the receiver operating characteristic curve (AUROC) of 0.76 in the test set of 351 patients. Combination of PURE-AID with prostate-specific antigen (PSA) and age increases AUROC to 0.80 and reduces 54.3% of unnecessary biopsies with 86.8% sensitivity. Our study provides a new classification system for differentiating high-grade PCa in a workflow- and patient-friendly manner.

Compared with the traditional gene expression techniques of quantitative analysis, RT-qPCR is most widely used because of its low cost, time-saving, and accuracy. It is essential to introduce suitable internal reference genes as reference corrections in RT-qPCR experiments to reduce the RNA quality and reverse transcription efficiency of different samples. In our study, we chose the candidate internal reference genes of alfalfa from transcriptome sequence datasets (162 RNA-seq sequencing data) through comparative analysis. Finally, 10 candidate reference genes were selected. These candidate reference gene expressions were determined by RT-qPCR under five abiotic stresses of drought, alkali, high temperature, low temperature, and acid treatments. The stability index of these candidate genes was calculated and evaluated correspondently using specific software and different lgorithms, such as GeNorm, Normfinder, Bestkeeper, ΔCt method, and an online analysis tool RefFinder. Then the appropriate candidate genes were screened strictly; and validated by the phyA gene. GAPDH and Actin are taken as traditional reference genes on gene expression of quantitative analysis commonly used in alfalfa, Our results showed GAPDH and Actin aren't the most appropriate reference genes of alfalfa under different abiotic stresses, under alkaline stress, the optimal reference gene is UBL-2a, and the optimal combination of reference genes is MS.65,463 (some candidate reference genes haven't been annotated yet, using gene ID abbreviation number of Medicago sativa L. instead )and UBL-2a; Under drought stress, the optimal reference gene is Ms.33,066, and the optimal combination of reference genes is MS.65,463 and UBL-2a; Under high-temperature stress, the optimal reference gene is Actin, and the optimal combination of reference genes is Rer1, Actin, MS.00617, MS.74,923, UBL-2a, MS.33,066, MS.99,505, and MS.65,463; Under low-temperature stress, the optimal reference gene is Actin, and the optimal combination of reference genes is Rer1, Actin, MS.99,505, MS.073307, and UBL-2a. The optimal reference genes and their combinations need further validation under acid stress. This paper provides scientific evidence for quantitative analysis of the genes of alfalfa.

The selection of a stably expressed reference gene is a critical step for the quantitation of gene expression by qRT-PCR. We tested the stability of expression of nine putative reference genes in normoxia and hypoxia in four different human cell types: coronary (HCAECs) and pulmonary endothelial cells (HPAECs), EA.hy926 endothelial cells, and A549 alveolar epithelial cells. Cells were cultured in normoxic and hypoxic conditions for up to 72 h. Total RNA was isolated and used for qRT-PCR. Stability of expression was assessed by calculating the coefficient of variation of the cycle threshold (Ct CV) by pairwise comparison of ΔCt values, and by the NormFinder algorithm. A final rank was calculated for each gene. Finally, we analyzed VEGFA expression by using GAPDH or the optimal candidate reference gene found in this study. Gene expression was variable between cell lines and experimental conditions. The most stable reference gene across all cell lines was TBP, followed by RPLP1 and RPL13A. VEGFA expression was significantly upregulated by 4-fold in hypoxia when using TBP as reference, whilst this result was insignificant when GAPDH was used. The selection of a stably expressed reference gene is a critical step for the generation of reliable and reproducible data in gene expression studies. The most appropriate reference gene may vary in different cell lines and experimental conditions; it should be chosen individually for each experimental set-up.

Real-time quantitative PCR (RT-qPCR) is a widely used method to investigate gene expression in neuroscience studies. Accurate relative quantification of RT-qPCR requires the selection of reference genes that are stably expressed across the experimental conditions and tissues of interest. While RT-qPCR is often performed to investigate gene expression changes during neurodevelopment, few studies have examined the expression stability of commonly used reference genes in the developing mouse cortex.

Here, we evaluated the stability of five housekeeping genes, Actb, Gapdh, B2m, Rpl13a, and Hprt, in cortical tissue from mice at embryonic day 15 to postnatal day 0 to identify optimal reference genes with stable expression during late corticogenesis. The expression stability was assessed using five computational algorithms: BestKeeper, geNorm, NormFinder, DeltaCt, and RefFinder. Our results showed that B2m, Gapdh, and Hprt, or a combination of B2m/Gapdh and B2m/Hprt, were the most stably expressed genes or gene pairs. In contrast, Actb and Rpl13a were the least stably expressed.

This study identifies B2m, Gapdh, and Hprt as suitable reference genes for relative quantification in RT-qPCR-based cortical development studies spanning the period of embryonic day 15 to postnatal day 0.

To identify stable reference genes for qRT-PCR analysis across different developmental stages and color variations of tender leaves in Litsea coreana, seven candidate reference genes were selected based on existing transcriptome data. qRT-PCR was performed on tender leaves of L. coreana at various stages and under different color conditions. The stability of these genes was evaluated using GeNorm (version 2003), NormFinder (version 0953), BestKeeper (version 2003), and ReFinder software (version 2004). The most stable genes were selected, and the stability of the chosen reference genes was validated. RPL and ACT were the most stable genes across different leaf developmental stages, while ACT and EF1-α showed the highest stability across different leaf colors. Overall, ACT and EF1-α were the most stable reference genes for both developmental stages and color variations. ACT and EF1-α can be used as reliable reference genes for gene expression studies in the color change process of L. coreana tender leaves. This will provide a foundation for further research into the molecular mechanisms of leaf color changes and the development of color regulation genes in L. coreana.

Sweet osmanthus (Osmanthus fragrans) is a prominent woody ornamental plant extensively utilized in horticulture, the food industry, cosmetics, and traditional Chinese medicine. MicroRNAs (miRNAs) are crucial regulators of gene regulation, playing a vital role in enabling plants to adapt to environmental fluctuations. Despite their significance, research on miRNA expression in O. fragrans under adverse stress conditions remains limited. Therefore, the selection of appropriate reference miRNAs is essential to ensure accurate miRNA expression analysis.

In this study, qRT-PCR technology was combined with four algorithms (i.e., delta-Ct, geNorm, NormFinder, and BestKeeper) to systematically evaluate the expression stability of 14 candidate miRNAs across eleven environmental conditions, including under abiotic stress, under hormone and metal ion treatments, during flower opening and senescence, and across various tissues.

The results revealed that under hormone treatments, ofr-miR159b-3p, novel8, and novel3 exhibited high expression stability; under abiotic stress, ofr-miR159b-3p, novel8, ofr-miR403-3p, and novel2 demonstrated considerable stability; during metal ion treatments, novel3, ofr-miR159b-3p, novel33, novel2, and ofr-miR395e were identified as stable miRNAs; in different tissues, novel2 and ofr-miR395e were relatively stable; and during flower opening and senescence, novel33 and ofr-miR395e maintained stable expression.

This study represents the first comprehensive assessment of reference miRNA stability in O. fragrans, providing a reliable framework for miRNA expression analysis under diverse conditions, including flower development and senescence, abiotic stress, hormone treatments, and metal ion treatments. These findings carry significant implications for future research into the function of miRNAs.

Given the obesity epidemic and the prevalence of comorbidities, there is an ongoing need to understand the health consequences of this disease state better. Understanding gene expression signals will facilitate the identification of mechanisms of kidney and liver dysfunction/disease often present in individuals with obesity. qPCR is the standard method for studying changes in relative gene expression. Reference genes (RGs) are obligatory for accurately normalizing mRNA transcript levels across samples. Despite the prevalence of qPCR, the reliability of the data is often compromised because RGs are still used without validation or have proven to be unstable in different tissues and various diseases. In this study, we validated seven reference genes (ACTB, B2M, RPLP0, HPRT1, GAPDH, 18S rRNA, and PPIA) using human liver tissue from 15 lean individuals and 17 individuals with a BMI ≥ 25 and human kidney tissue from 13 lean individuals and 15 individuals with a BMI ≥ 25. Cross-validation of expression stability was performed using the RefFinder platform with four algorithms: NormFinder, BestKeeper, geNorm, and the comparative ΔCt method. In obesity-related studies, the most suitable reference genes in gene expression studies are RPLP0 and HPRT1 in human kidney tissue and RPLP0 and GAPDH in the liver.

Vespula vulgaris is an invasive wasp that causes considerable detriment to native birds and invertebrates in New Zealand. Reducing at least 80% of invasive wasp densities is necessary to manage the problems this species presents to its invaded range. To explore the function of target genes for the genetic management of V. vulgaris, screening of appropriate reference genes is crucial for conducting the reverse transcriptase-quantitative real-time PCR (RT-qPCR). The selection of appropriate reference genes is an important but often overlooked consideration when delving into RNA research. Many studies rely on one of two tried and trusted reference genes widely used in the literature, which may not be suitable for the normalization of data under particular variables.

Here, I selected six reference genes of V. vulgaris and evaluated their stability across two conditions: developmental stage and sex by using five different tools for analysis: the ΔCt method, geNorm, NormFinder, BestKeeper, and RefFinder.

Differing appropriate reference genes for different research foci: TBP, EF1A, RPL18X3, and CAPZB for developmental stage treatment, and KTB, EF1A, and CAPZB amongst the sexes.

My study further emphasizes that there is no "one size fits all" reference gene, and advocates for analysis of reference gene suitability when conducting gene quantification experiments.

A major resistance gene of the melon accession PI414723 to zucchini yellow mosaic virus (ZYMV) is located at the Zym-1 locus on chromosome 2, but the underlying defense mechanisms are poorly understood. The physiological responses and expression of selected genes at Zym-1 were assessed in PI414723 and in the susceptible genotype Védrantais. Viral titers and the expression of genes related to systemic acquired resistance were evaluated in inoculated (Inoc) and noninoculated (Non-Inoc) portions of the cotyledons at 3, 7, and 10 days after inoculation and in apical leaves at 10 days after inoculation. ZYMV was detected in both portions of the cotyledons but not in the apical leaves of PI414723 plants. Also, ZYMV was recovered in a susceptible zucchini only from Inoc portions at 3 days after inoculation. By contrast, in Védrantais, ZYMV was detected and recovered from all tissues at high concentrations. Starchy local lesions and accumulation of transcripts of the systemic acquired resistance marker genes PR1 and PR4 were also detected in the resistant genotype. Plus, transcripts of one candidate resistance gene analog previously located at Zym-1 and of two melon homologs of restricted tobacco etch virus movement 2 genes located close to Zym-1 accumulated only in PI414723. It is proposed that resistance results from the combined action of the R gene involved in restricting ZYMV replication after a supposed recognition event and of the restricted tobacco etch virus movement genes that impact viral systemic movement to distal apical tissues.

Reverse transcription quantitative real-time PCR (RT-qPCR) is esteemed for its precision and reliability, positioning it as the standard for evaluating gene expression. Selecting suitable reference genes is crucial for acquiring accurate data on target gene expression. However, identifying appropriate reference genes for specific rice tissues or growth conditions has been a challenge. To overcome this, we introduce the Rice Reference Genes (RRG) tool ( https://www.rrgenes.com/ ), which assists researchers in selecting reference genes for diverse experimental conditions in rice. This tool utilizes 4404 rice-derived RNA-seq datasets, categorized by five tissue types-leaf, root, seedling, panicle, and seed-and seven stress conditions (cold, disease, drought, heat, hormone, metal, and salt), along with corresponding control groups (mock). In this research, we employed the RRG web-based tool to identify candidate reference genes in rice leaves, roots, and seedlings exposed to salt and drought stress. These candidates were rigorously tested against conventionally established reference genes, confirming their accuracy and reliability. The RRG tool is designed to be user-friendly, allowing even those with limited experience to efficiently select optimal reference genes in rice with ease.

Bacillus cereus is a Gram-positive pathogen associated with foodborne illnesses and severe non-gastrointestinal infections. Robust tools for accurate gene transcription analysis are essential for studying toxin gene expression dynamics and deciphering the complex regulatory networks orchestrating the expression of toxin and virulence factors. This study aimed to identify reliable reference genes for normalizing reverse transcription quantitative PCR (RT-qPCR) data in toxigenic B. cereus. An emetic and an enteropathogenic strain were used as model organisms to establish a suitable reference gene set to monitor the dynamics of toxin gene transcription. Ten candidate reference genes were evaluated for their expression stability using geNorm, NormFinder, BestKeeper and the ΔCq method, with the final rankings integrated via RefFinder. Among the tested genes, rho, rpoD and recA were identified as the most stable expressed reference genes across all tested conditions. As shown in this proof-of-principle study, the established reference gene set provides a suitable tool to investigate the influence of extrinsic and intrinsic factors on toxin gene transcription. In conclusion, our newly established reference gene set provides a robust basis for studying toxin gene expression in B. cereus and contributes to a better understanding of its pathogenicity and potential strategies to mitigate its harmful effects.

Studies of the aging transcriptome focus on genes that change with age. But what can we learn from age-invariant genes-those that remain unchanged throughout the aging process? These genes also have a practical application: they can serve as reference genes in expression studies. Reference genes have mostly been identified and validated in young organisms, and no systematic investigation has been done across the lifespan. Here, we build upon a common pipeline for identifying reference genes in RNA-seq datasets to identify age-invariant genes across seventeen C57BL/6 mouse tissues (brain, lung, bone marrow, muscle, white blood cells, heart, small intestine, kidney, liver, pancreas, skin, brown, gonadal, marrow, and subcutaneous adipose tissue) spanning 1 to 21+ months of age. We identify 9 pan-tissue age-invariant genes, and many tissue-specific age-invariant genes. These genes are stable across the lifespan and are validated in independent bulk RNA-seq datasets and RT-qPCR. Age-invariant genes have shorter transcripts and are enriched for CpG islands. Interestingly, pathway enrichment analysis for age-invariant genes identifies an overrepresentation of molecular functions associated with some, but not all, hallmarks of aging. Thus, even though hallmarks of aging typically involve change, select genes associated with these hallmarks resist age-related change. Finally, our analysis provides a list of murine tissues where classical reference genes are appropriate for application in aging studies. However, no classical reference gene is appropriate across all aging tissues. Instead, we provide novel tissue-specific and pan-tissue reference genes for assays utilizing gene normalization (RT-qPCR) that can be applied to mice across the lifespan.

Hypoxia is common in breast tumours and is linked to therapy resistance and advanced disease. To understand hypoxia-driven breast cancer progression, RT-qPCR is a widely used technique to quantify transcriptional changes that occur during malignant transformation. Reference genes (RGs) are endogenous RT-qPCR controls used to normalise mRNA levels, allowing accurate assessment of transcriptional changes. However, hypoxia reprograms transcription and post-transcriptional processing of RNA such that favoured RGs including GAPDH or PGK1 are unsuitable for this purpose. To address the need for robust RGs to study hypoxic breast cancer cell lines, we identified 10 RG candidates by analysing public RNA-seq data of MCF-7 and T-47D (Luminal A), and, MDA-MB-231 and MDA-MB-468 (triple negative breast cancer (TNBC)) cells cultured in normoxia or hypoxia. We used RT-qPCR to determine RG candidate levels in normoxic breast cancer cells, removing TBP and EPAS1 from downstream analysis due to insufficient transcript abundance. Assessing primer efficiency further removed ACTB, CCSER2 and GUSB from consideration. Following culture in normoxia, acute, or chronic hypoxia, we ascertained robust non-variable RGs using RefFinder. Here we present RPLP1 and RPL27 as optimal RGs for our panel of two Luminal A and two TNBC cell lines cultured in normoxia or hypoxia. Our result enables accurate evaluation of gene expression in selected hypoxic breast cancer cell lines and provides an essential resource for assessing the impact of hypoxia on breast cancer progression.

Osmanthus fragrans, a well-regarded traditional flower in China, holds extensive applications in gardening, food, cosmetics, and traditional Chinese medicine. Despite its importance, research on long non-coding RNAs (lncRNAs) in O. fragrans has been constrained by the absence of reliable reference genes (RGs).

We employed five distinct algorithms, i.e., delta-Ct, NormFinder, geNorm, BestKeeper, and RefFinder, to evaluate the expression stability of 17 candidate RGs across various experimental conditions.

The results indicated the most stable RG combinations under different conditions as follows: cold stress: lnc00249739 and lnc00042194; drought stress: lnc00042194 and lnc00174850; salt stress: lnc00239991 and lnc00042194; abiotic stress: lnc00239991, lnc00042194, lnc00067193, and lnc00265419; ABA treatment: lnc00239991 and 18S; MeJA treatment: lnc00265419 and lnc00249739; ethephon treatment: lnc00229717 and lnc00044331; hormone treatments: lnc00265419 and lnc00239991; Al3+ treatment: lnc00087780 and lnc00265419; Cu2+ treatment: lnc00067193 and 18S; Fe2+ treatment: lnc00229717 and ACT7; metal ion treatment: lnc00239991 and lnc00067193; flowering stage: lnc00229717 and RAN1; different tissues: lnc00239991, lnc00042194, lnc00067193, TUA5, UBQ4, and RAN1; and across all samples: lnc00239991, lnc00042194, lnc00265419 and UBQ4. The reliability of these selected RGs was further validated by analyzing the expression patterns of lnc00003036, lnc00126603, and lnc00250780. This study represents the first comprehensive evaluation of lncRNA RG stability in O. fragrans, significantly enhancing the accuracy and depth of lncRNA expression analyses in this species, contributing to advancements in plant stress resistance breeding and improving environmental adaptability.

Metschnikowia bicuspidata is the specific pathogen for "milky disease" in the Chinese mitten crab (Eriocheir sinensis), accounting for huge losses to the industry. And yet, there is no precise study describing the pathogenesis of M. bicuspidata, largely hindering the development of novel control methods against its causing diseases. Here, we compared the transcriptomes of M. bicuspidata cells collected from a control group (cultured without E. sinensis hemocytes) and a treatment group (cultured with E. sinensis hemocytes), using RNA sequencing. Through comprehensively analyzing the differentially expressed genes (DEGs), both the most regulated ones and the ones involved in crucial enriched KEGG pathways, we found that certain processes might be required for M. bicuspidata's survival under hemocyte stress. Key genes involved in oxidative phosphorylation, fatty acid metabolism, upper glycolysis, and gluconeogenesis were upregulated, and those for β-glucan unmasking, autophagy, and cell polarity were downregulated, in the treatment group. Our results suggest that M. bicuspidata colonizes and therefore establishes an infection in E. sinensis via enhancing aerobic respiration, glucose-6-phosphate accumulation, and cell-wall masking. In addition, we applied multiple means to evaluate a series of candidate reference genes and found that PMA1 in combination with ACT1 is the most suitable choice for accurate normalization in quantitative real-time PCR (qRT-PCR) assays. Thus, we used this combination as the reference and performed qRT-PCR verification of several DEGs. It is shown that the expression trends of these tested DEGs in qRT-PCR assays are the same as those in RNA-Seq assays. This study not only provides insights into strategies facilitating M. bicuspidata's survival within E. sinensis, initially elucidating the pathogenesis of this yeast, but also recommends a useful molecular tool regarding qRT-PCR assays in this pathogen.

Cell cycle-dependent gene expression analysis is particularly important as numerous genes show tightly regulated expression patterns at different phases of the cell cycle. For cancer cells, analysis of cell cycle-related events is of paramount significance since tumorigenesis is characteristically coupled to cell cycle perturbations. RT-qPCR is a highly sensitive technique to investigate cell cycle-dependent transcriptional regulation. However, for reliable evaluation of qPCR data reference genes with stable mRNA expression are required. Although several studies investigating cell cycle-dependent gene expression employ frequently used reference genes, the suitability of these reference genes has not been thoroughly investigated so far. Moreover, such potential reference genes for cell cycle analysis have not been described in the literature. Therefore, we aimed to identify reference genes characterized with stable expression throughout the cell cycle in MOLT4 and U937 human leukemia cell lines synchronized with RO-3306 CDK1 inhibitor using RT-qPCR. Here we show that for cell cycle-dependent gene expression analysis the commonly used TBP is suitable, while the recently recognized reference genes SNW1 and CNOT4 are applicable in a cell line-dependent manner. We also suggest that proper selection of reference genes for each experimental condition is crucial for reliable normalization as these aspects can severely compromise conclusions.

Fasciolosis, caused by the liver flukes Fasciola hepatica and Fasciola gigantica, is a zoonotic parasitic disease associated with substantial economic losses in livestock. The transforming growth factor-beta signalling pathway is implicated in developmental processes and biological functions throughout the animal kingdom, including the Fasciola spp. It may also mediate host-helminth interactions during infection. In this work, we present an exploration of FgSmad4, the sole member of the Co-Smad protein family in F. gigantica. The isolated FgSmad4 cDNA was 4,014 bp in length encoding for a protein comprising 771 amino acids. FgSmad4 exhibited typical Co-Smad protein features, including Mad Homology 1 (MH1) and Mad Homology 2 (MH2) domains, a Nuclear Localisation Signal, a DNA-Binding Motif, and a Nuclear Export Signal. Sequence and phylogenetic analyses of FgSmad4 revealed that its MH1 and MH2 sequences are most similar to those of other trematode species. The MH1 domain, in particular, closely resembles the Co-Smad protein in mammalian hosts more than those in cestodes and nematodes. The expression patterns of FgSmad4 during the liver fluke's developmental stages showed significant variation. Transcript levels were highest at the newly excysted juvenile stage, followed by unembryonated egg, redia, and metacercaria, with the lowest expression in the adult fluke, embryonated egg, and cercaria stages. Our results underscore the conservation and suggest the potential role of FgSmad4, a key transforming growth factor-beta signalling molecule within the liver fluke F. gigantica. As Co-Smad is typically involved in several biological pathways, the precise functions and mechanisms of this identified FgSmad4 necessitate further exploration.

Reverse transcription quantitative real-time polymerase chain reaction Q7 (RT‒qPCR) is a commonly used tool for gene expression quantification. Because the qPCR method depends on several variables that can influence the analysis process, stably expressed genes should be selected for relative gene expression studies. To date, there is insufficient information on the selection of appropriate reference genes for antimicrobial photodynamic inactivation (aPDI) and antimicrobial blue light (aBL) treatment. Therefore, the purpose of the present study was to determine the most stable reference gene under treatment with aBL under sublethal conditions and to evaluate differences in the expression of the selected gene after aBL treatment in comparison to the nontreated control.

Selection of stable reference genes was performed using 4 programs: BestKeeper, geNorm, NormFinder and RefFinder under 409 and 415 nm aBL treatment.

The results revealed that the gene encoding the integration host factor β subunit (ihfB) in Escherichia coli was the most stably expressed gene after both 409 and 415 nm aBL treatment. Three programs, RefFinder, geNorm, and NormFinder, indicated that this gene had the most stable expression in comparison to the other reference gene candidates. The next best candidates were cysG, uidA, and gyrA. NormFinder revealed ihfB as the single gene and cysG - gyrA as the combination of reference genes with the best stability.

Universal reference genes are characterized by stable expression that remains consistent across various stress conditions. Consequently, it is essential to evaluate reference genes for each specific stress factor under investigation. In the case of aBL at different wavelengths, we identified genes that maintain stable expression following irradiation.

Lactation is a complex physiological process regulated by numerous genes and factors. Circular RNA (circRNA), a non-coding RNA, acts as a molecular sponge that sequesters microRNAs (miRNAs) to regulate target gene expression. Although circRNA has been linked to mammary gland lactation, its specific role in yaks remains underexplored. This study employed circular RNA sequencing (circRNA-seq) to examine the differential expression of circRNAs in yak mammary tissues during lactation and the dry period. Additionally, an enrichment analysis of the differentially expressed circRNAs (DECs) was performed. A competing endogenous RNA (ceRNA) network was then constructed to explore the potential of their roles in lactation and mammary gland development. We detected 18,905 circRNAs in yak mammary tissue, among which 302 showed differential expression. The host genes of these DECs were enriched in functions and pathways associated with yak milk synthesis and composition. Through the construction of a ceRNA network and the enrichment analysis of associated mRNAs, this study identified ceRNAs potentially involved in regulating lactation and mammary gland development. In conclusion, circRNAs in yak mammary tissues were identified and analyzed across lactation and dry periods, establishing a ceRNA network related to lactation regulation. These findings provide novel insights into the regulatory mechanisms governing lactation in yaks (Bos grunniens).

A better understanding of secondary metabolites biosynthesis requires comprehensive research at the molecular level. Although the medicinal importance of secondary metabolites extracted from Arnica spp. has been well documented, the very plants themselves have been poorly studied. Characterization of biosynthetic pathways and their complex regulatory mechanisms may be enhanced via transcriptomic approach. To ensure the reliability of data, RT-qPCR based gene expression studies should be preceded by the reference genes selection step. Here, ten candidate reference genes were analyzed in terms of their expression stability in developing flowers of two arnica species, Arnica montana and Arnica chamissonis. After evaluating their expression stability with four distinct algorithms (geNorm, NormFinder, BestKeeper and delta Ct method), we found that SKIP16 and F-box were the most stable reference genes in A. montana, whereas SAND and F-box were best-performing genes in A. chamissonis. On the other hand, ACT1 in combination with F-box could be used as internal control for cross-species investigations. This study will aid in further research on molecular mechanisms underlying the processes of flowering and secondary metabolites production in medicinal plants.

Gene expression profiling is of key importance in all domains of life sciences, as medicine, environment, and plants, for both basic and applied research. Despite the emergence of microarrays and high-throughput sequencing, qPCR remains a standard method for gene expression analyses, with its data normalization step being crucial for ensuring accuracy. Currently, the most widely used normalization method is based on the use of reference genes, assumed to be stably expressed across all experimental conditions. In the present study, we show that finding a stable combination of genes, regardless of their individual stability, outperforms standard reference genes for RT-qPCR data normalization. A stable combination of genes consists of a fixed number of genes whose individual expression balance each other all along experimental conditions of interest. Moreover, the present study shows that such an optimal combination of genes can be found using a comprehensive database of RNA-Seq data. Indeed, assuming that such a comprehensive database contains accurate gene expression profiles, we can extract in silico, by the way of the mathematical variance calculation, a stable combination of genes that reflects in vivo stability. As a case study, this new method was developed using the tomato model plant, with corresponding RNA-Seq data from the TomExpress database. However, the method is potentially applicable to other organisms with available RNA-seq data. Our results demonstrate the superiority of the reported method over commonly used housekeeping genes or other stably expressed genes. We therefore recommend the use of our new method together with classic ones in order to always obtain the best reference genes for a given experimental design.

Real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a powerful tool for analysing target gene expression in biological samples. To achieve reliable results by RT-qPCR, the most stable reference genes must be selected for proper data normalisation, particularly when comparing cells of different types. We aimed to choose the least variable candidate reference genes among eight housekeeping genes tested within a set of human cancer cell lines (HeLa, MCF-7, SK-UT-1B, A549, A431, SK-BR-3), as well as four lines of normal, non-malignant mesenchymal stromal cells (MSCs) of different origins.

The reference gene stability was evaluated using four algorithms (BestKeeper, NormFinder, geNorm and the comparative ΔCt method) and ranked with the RefFinder web-based tool.

We found increased variability in the housekeeping genes' expression in the cancer cell lines compared to that in normal MSCs. POP4 and GAPDH were identified as the most suitable reference genes in cancer cells, while 18S and B2M were the most suitable in MSCs. POP4 and EIF2B1 were shown to be the least variable genes when analysing normal and cancer cell lines together. Epidermal growth factor receptor (EGFR) mRNA relative expression was normalised by the three most stable or three least stable reference genes to demonstrate the reliability of reference genes validation.

We analysed and selected stable reference genes for RT-qPCR analysis in the wide panel of cancer cell lines and MSCs. The study provides a reliable tool for future research concerning the expression of genes involved in various intracellular signalling pathways and emphasises the need for careful selection of suitable references before analysing target gene expression.

Hypoxia triggers stress, leading to significant alterations in gene expression patterns, which in turn affect fish's growth and development. Real-time quantitative PCR (RT-qPCR) is a pivotal technique for assessing changes in gene expression. However, its accuracy is highly contingent upon the stable expression of reference genes. Ribosomal RNA (18s), β-actin (actb), elongation factor 1-α (ef1a), α tubulin (tuba), and ribosomal protein L17 (rpl17) are the widely used reference genes, but their expression stability in the tissues of black rockfish under hypoxic conditions remains unclear.

The expression of genes was detected by RT-qPCR and the stability was assessed by Delta Ct, geNorm, NormFinder, and BestKeeper algorithms.

Results showed that tuba exhibited stable expression in liver, heart, gill tissues under normoxic conditions, and in the liver and head kidney under hypoxic conditions. Ef1a was identified as the most stably expressed gene in gill tissue under hypoxia. For hypoxic heart studies, rpl17 and tuba were recommended as reference genes. 18s showed high stability in spleen tissue under hypoxic conditions. Actb was the most stably expressed gene in spleen and head kidney tissues under normoxic conditions.

The identified reference genes exhibited tissue-specific stability, and it was necessary to select appropriate reference genes based on the specific tissue type for gene expression studies under hypoxic conditions. These findings help in enhancing the accuracy of gene expression analysis in the mechanism of hypoxia for black rockfish.

Pearl millet (Pennisetum glaucum (L.) R. Br.) is a vital cereal crop, predominantly cultivated in arid and semi-arid regions of Asia and Africa. It serves as a staple food for millions, while also being utilized as forage and an energy crop. The crop's resistance to heat and drought, coupled with its high biomass, positions it as a promising candidate for climate-resilient agriculture. A detailed understanding of its gene expression patterns across various tissues and developmental stages is essential for enhancing its yield and quality. This study aims to fill this knowledge gap by employing RNA-seq to identify housekeeping genes (HKGs) and tissue-specific genes (TSGs) in pearl millet.

Our analysis of RNA-seq data from nine tissues (seed, germ, radicle, leaf, root, tillering tissue, stem, spike, and grain) across eight developmental stages in pearl millet accession Tifleaf3 revealed a comprehensive gene expression profile. We identified 461 HKGs that exhibited stable expression across all tissues and stages, providing robust internal references for RT-qPCR. Additionally, 8091 TSGs were discovered, many of which showed distinctive expression patterns in tissues such as spike, stem, and leaf. Functional enrichment analysis of these genes using GO and KEGG pathways highlighted their roles in key biological processes and pathways, indicating their potential in crop trait enhancement. Protein-protein interaction networks constructed for stem and leaf tissues further illuminated the regulatory mechanisms underlying the transition from vegetative to reproductive growth stages.

This study presents a detailed transcriptomic landscape of pearl millet, identifying a set of HKGs and TSGs that are crucial for understanding the molecular basis of its growth and development. We provided valuable options for transcript normalization and crucial targets for exploring gene function for the plant growth and development in pearl millet. The insights gained from this work are instrumental for breeding programs aimed at enhancing the productivity of pearl millet, thereby contributing to food and energy security.

Friedreich's Ataxia (FRDA) is the most common autosomal recessive ataxia worldwide and is caused by biallelic unstable intronic GAA expansions at FXN. With its limited therapy and the recent approval of the first disease-modifying agent for FRDA, the search for biological markers is urgently needed to assist and ease the development of therapies. MiRNAs have emerged as promising biomarkers in various medical fields such as oncology, cardiology, epilepsy and neurology as well. Cell-free plasmatic miRNAs have potential advantages as biomarkers because of their size, stability against blood RNases, relative ease of obtaining, storage and measurement. In this study, we attempted to characterize the plasma miRNA signature (RNA-Seq followed by qRT-PCR) and its clinical/structural correlates in a cohort of Brazilian patients with FRDA. Our results showed that miR-26a-5p is upregulated and miR-15a-5p is downregulated. The first was correlated with age at onset, cerebellum volume, spinal cord cross-sectional area (C2-CSA) and the left ventricle mass (LV_Mass). For the miR-15a-5p, significant correlations were found with cerebellum volume, spinal cord eccentricity and LV_Mass. It has been previously hypothesized that these miRs target BDNF, modulating its expression and, when this gene is downregulated, it leads to neuronal loss, explaining the ataxic phenotype and our results reinforce this hypothesis. The miR-26a-5p was already associated with cardiomyocyte hypertrophy through the increased NLRP3 inflammasome activity, which is indirectly linked with cardiac hypertrophy. Considering that, we propose these miRNAs as possible prognostic biomarkers for FRDA. However, longitudinal studies are still needed to validate their clinical use.

In recent years, there has been a growing number of adult orthodontic patients with periodontal disease. The progression of periodontal disease is well-linked to oxidative stress (OS). Nevertheless, the impact of OS on orthodontic tooth movement (OTM) is not fully clarified. Therefore, we applied an OS in vitro-model utilizing H2O2 to study its effect on tension-induced mechanotransduction in human osteoblasts (hOBs). Experimental parameters were established based on cell viability and proliferation. Apoptosis detection was based on caspase-3/7 activity. Gene expression related to bone-remodeling (RUNX2, P2RX7, TNFRSF11B/OPG), inflammation (CXCL8/IL8, IL6, PTRGS2/COX2), autophagy (MAP1LC3A/LC3, BECN1), and apoptosis (CASP3, CASP8) was analyzed by RT-qPCR. IL6 and PGE2 secretion were determined by ELISA. Tension increased the expression of PTRGS2/COX2 in all groups, especially after stimulation with higher H2O2 concentration. This corresponds also to the measured PGE2 concentrations. CXCL8/IL8 was upregulated in all groups. Cells subjected to tension alone showed a general upregulation of osteogenic differentiation-related genes; however, pre-stimulation with OS did not induce significant changes especially towards downregulation. MAP1LC3A/LC3, BECN1 and CASP8 were generally upregulated in cells without OS pre-stimulation. Our results suggest that OS might have considerable impacts on cellular behavior during OTM.

Oxidative stress (OS) is a common feature of many inflammatory diseases, oral pathologies, and aging processes. The impact of OS on periodontal ligament cells (PDLCs) in relation to oral pathologies, including periodontal diseases, has been investigated in different studies. However, its impact on orthodontic tooth movement (OTM) remains poorly understood. This study used an in vitro model with human PDLCs previously exposed to H2O2 to investigate the effects of OS under a static compressive force which simulated the conditions of OTM. Human PDLCs were treated with varying concentrations of H2O2 to identify sub-lethal doses that affected viability minimally. To mimic compromised conditions resembling OTM under OS, the cells were pretreated with the selected H2O2 concentrations for 24 h. Using an in vitro loading model, a static compressive force (2 g/cm2) was applied for an additional 24 h. The cell viability, proliferation, and cytotoxicity were evaluated using live/dead and resazurin assays. Apoptosis induction was assessed based on caspase-3/7 activity. The gene expression related to bone remodeling (RUNX2, TNFRSF11B/OPG, BGLAP), inflammation (IL6, CXCL8/IL8, PTGS2/COX2), apoptosis (CASP3, CASP8), and autophagy (MAP1LC3A/LC3, BECN1) was analyzed using RT-qPCR. This study suggests an altering effect of previous OS exposure on static-compression-related mechanosensing. Further research is needed to fully elucidate these mechanisms.

In light of the increasingly adverse environmental conditions and the concomitant challenges to the survival of important crops, there is a pressing need to enhance the resilience of pepper seedlings to extreme weather. Carotenoid plays an important role in plants' resistance to abiotic stress. Nevertheless, the relationship between carotenoid biosynthesis and sweet pepper seedlings' resistance to different abiotic stresses remains uncertain. In this study, the carotenoid content in abiotic-stressed sweet pepper seedling roots was determined, revealing that carotenoid content was extremely significantly elevated by more than 16-fold under salt stress, followed by drought stress (8-fold), and slightly elevated by only about 1-fold under waterlogging stress. After that, serine/threonine-protein phosphatase 2A (PP2A) was found to be the suitable reference gene (RG) in sweet pepper seedling roots under different abiotic stresses by using RT-qPCR and RefFinder analysis. Subsequently, using PP2A as the RG, RT-qPCR analysis showed that the expression level of most genes associated with carotenoid biosynthesis was extremely significantly up-regulated in sweet pepper seedlings under salt and drought stress. Specifically, violoxanthin deepoxidase (VDE) was significantly up-regulated by more than 481- and 36-fold under salt and drought stress, respectively; lycopene epsilon cyclase (LCYE) was significantly up-regulated by more than 840- and 23-fold under salt and drought stress, respectively. This study contributes to a more comprehensive understanding of the carotenoid biosynthesis pathway serving as a major source of retrograde signals in pepper subjected to different abiotic stresses.

Flavin-containing monooxygenases (FMOs) are present in most organisms including plants, fungi, bacteria, invertebrates and vertebrates, where they catalyse the oxidative metabolism of a range of xenobiotics and endogenous metabolites. FMOs have been associated with ageing and longevity in the mouse and in C. elegans. As all five FMOs of C. elegans share an evolutionary root with mouse and human FMO5, it was of interest to discover if effects on ageing and longevity persisted across the whole group. We therefore investigated the impact of fmo gene knockout (KO) in C. elegans. We found that fmo-1, fmo-3 and fmo-4 KO significantly extended C. elegans lifespan relative to wild type and, as previously reported, FMO-2 over-expression did likewise. The transcription levels of C. elegans fmo genes were determined throughout the life cycle (embryo, larva and adult) in wild type and in each mutant to discover if their expression was related to stages in ageing, and expression levels were compared to those in human and mouse. In wild type worms, fmo-1 and fmo-4 were the mostly highly transcribed genes (especially at the larval stage), whereas fmo-2 and fmo-3 were the least transcribed, at all stages. Notably, the knockout of fmo-4 led to a 17- to 30-fold up-regulation of fmo-2, along with significantly increased levels of the other fmos. This parallels recent findings in the long-lived C. elegans tald-1 mutant where fmo-2 was also significantly up-regulated and reinforces its importance in lifespan extension.

A repeatable and reliable reverse transcription quantitative PCR (RT-qPCR) experiment depends upon proper reference genes (RGs) selection. This study aims to examine the expression stability of nine candidate RGs for the Avena sativa - Blumeria graminis experimental setup. B. graminis causes powdery mildew - the most devastating and economically important fungal disease of crops worldwide. RGs were evaluated in Pm3 and Pm4 oat differential lines and the susceptible cultivar Fuchs during compatible and incompatible interactions with different pathotypes of Blumeria graminis f. sp. avenae in six-time points post inoculation. The identification of genes exhibiting high expression stability was done by four algorithms (geNorm, NormFinder, BestKeeper and deltaCt). The results indicated that regardless of the analysed group, two most stable RGs are required for data normalization. The most sufficient RGs combination was HNR (heterogeneous nuclear ribonucleoprotein 27 C) + EIF4A (eukaryotic initiation factor 4 A‑3). ARF (ADP‑ribosylation factor) could also be pondered as demonstrating high expression stability. These genes can be considered universal candidates for RT-qPCR normalization to study interaction with B. graminis as well as Puccinia coronata and Puccinia graminis, as confirmed by our previous research. The worst candidate for data standardisation was TUA (α- tubulin). To our best knowledge, this is the first report regarding RGs' selection in this pathosystem. Identified RGs are proper normalisation candidates for gene expression studies in the A. sativa infected by B. graminis as well as other related pathogens.

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is an essential tool for gene expression analysis; choosing appropriate reference genes for normalization is crucial to ensure data reliability. However, most studies on osteogenic differentiation have had limited success in identifying optimal reference genes. To the best of our knowledge, no optimal reference genes in three-dimensional (3D) osteogenic differentiation culture experiments using human induced pluripotent stem cells (hiPSCs) have been identified. In this study, we aimed to identify stable reference genes that could be used for normalization in gene expression analyses during the 3D osteogenic differentiation of hiPSCs using an atelocollagen sponge as a scaffold. Four algorithms-ΔCt, BestKeeper, NormFinder, and geNorm-were used to evaluate the stability of 14 candidate reference genes. Genes encoding TATA box-binding protein, hypoxanthine phosphoribosyltransferase 1, and 14-3-3 protein zeta polypeptide were identified as the most stable reference genes. In comparison, conventionally used reference genes (beta-2 microglobulin and beta-actin genes) ranked among those with low stability. We also demonstrated the successful 3D osteogenic differentiation of hiPSCs on atelocollagen sponge. Our findings provide valuable insights for reference gene selection and bone tissue regeneration from hiPSCs, which could improve the treatment prospects for bone defects and other similar conditions in regenerative medicine.

Adipogenic differentiation and thermogenesis in brown adipose tissue (BAT) undergo dynamic processes, altering phenotypes and gene expressions. Proper reference genes in gene expression analysis are crucial to mitigate experimental variances and ensure PCR efficacy. Unreliable reference genes can lead to erroneous gene expression quantification, resulting in data misinterpretation. This study focused on identifying suitable reference genes for mouse brown adipocyte research, utilizing brown adipocytes from the Ucp1-luciferase ThermoMouse model. Comparative analysis of gene expression data under adipogenesis and thermogenesis conditions was conducted, validating 13 housekeeping genes through various algorithms, including DeltaCq, BestKeeper, geNorm, Normfinder, and RefFinder. Tbp and Rer1 emerged as optimal references for Ucp1 and Pparg expression in brown adipogenesis, while Tbp and Ubc were ideal for the expression analysis of these target genes in thermogenesis. Conversely, certain conventional references, including Actb, Tubb5, and Gapdh, proved unstable as reference genes under both conditions. These findings stress the critical consideration of reference gene selection in gene expression analysis within specific biological systems to ensure accurate conclusions.