qRT-PCR Assay Research Articles

Improved therapeutic effects on vascular intimal hyperplasia by mesenchymal stem cells expressing MIR155HG that function as a ceRNA for microRNA-205.

Coronary artery bypass grafting (CABG) is an effective treatment for coronary heart disease, with vascular transplantation as the key procedure. Intimal hyperplasia (IH) gradually leads to vascular stenosis, seriously affecting the curative effect of CABG. Mesenchymal stem cells (MSCs) were used to alleviate IH, but the effect was not satisfactory. This work aimed to investigate whether lncRNA MIR155HG could improve the efficacy of MSCs in the treatment of IH and to elucidate the role of the competing endogenous RNA (ceRNA). The effect of MIR155HG on MSCs function was investigated, while the proteins involved were assessed. IH was detected by HE and Van Gieson staining. miRNAs as the target of lncRNA were selected by bioinformatics analysis. qRT-PCR and dual-luciferase reporter assay were performed to verify the binding sites of lncRNA-miRNA. The apoptosis, Elisa and tube formation assay revealed the effect of ceRNA on the endothelial protection of MIR155HG-MSCs. We observed that MIR155HG improved the effect of MSCs on IH by promoting viability and migration. MIR155HG worked as a sponge for miR-205. MIR155HG/miR-205 significantly improved the function of MSCs, avoiding apoptosis and inducing angiogenesis. The improved therapeutic effects of MSCs on IH might be due to the ceRNA role of MIR155HG/miR-205.

Hyperandrogenic environment regulates the function of ovarian granulosa cells by modulating macrophage polarization in PCOS.

Polycystic ovary syndrome (PCOS) is a common endocrine-metabolic disorder characterized by oligo-anovulation, hyperandrogenism, and polycystic ovaries, with hyperandrogenism being the most prominent feature of PCOS patients. However, whether excessive androgens also exist in the ovarian microenvironment of patients with PCOS, and their modulatory role on ovarian immune homeostasis and ovarian function, is not clear. Follicular fluid samples from patients participating in their first in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) treatment were collected. Androgen concentration of follicular fluid was assayed by chemiluminescence, and the macrophage M1:M2 ratio was detected by flow cytometry. In an in vitro model, we examined the regulatory effects of different concentrations of androgen on macrophage differentiation and glucose metabolism levels using qRT-PCR, Simple Western and multi-factor flow cytometry assay. In a co-culture model, we assessed the effect of a hyperandrogenic environment in the presence or absence of macrophages on the function of granulosacells using qRT-PCR, Simple Western, EdU assay, cell cycle assay, and multi-factor flow cytometry assay. The results showed that a significantly higher androgen level and M1:M2 ratio in the follicular fluid of PCOS patients with hyperandrogenism. The hyperandrogenic environment promoted the expression of pro-inflammatory and glycolysis-related molecules and inhibited the expression of anti-inflammatory and oxidative phosphorylation-related molecules in macrophages. In the presence of macrophages, a hyperandrogenic environment significantly downregulated the function of granulosa cells. There is a hyperandrogenic microenvironment in the ovary of PCOS patients with hyperandrogenism. Hyperandrogenic microenvironment can promote the activation of ovarian macrophages to M1, which may be associated with the reprogramming of macrophage glucose metabolism. The increased secretion of pro-inflammatory cytokines by macrophages in the hyperandrogenic microenvironment would impair the normal function of granulosa cells and interfere with normal ovarian follicle growth and development.

Glutathione peroxidase 4 restrains temporomandibular joint osteoarthritis progression by inhibiting ferroptosis.

There are few effective therapeutic strategies for temporomandibular joint osteoarthritis (TMJOA) due to the unclear pathology and mechanisms. We aimed to confirm the roles of GPX4 and ferroptosis in TMJOA progression. ELISA assay was hired to evaluate concentrations of ferroptosis-related markers. The qRT-PCR assay was hired to assess gene mRNA level. Western blot assay and immunohistochemistry were hired to verify the protein level. CCK-8 assay was hired to detect cell viability. Human fibroblast-like synoviocytes (FLSs) were cultured to confirm the effects of GPX4 and indicated inhibitors, and further verified the effects of GPX4 and ferroptosis inhibitors in TMJOA model rats. Markers of ferroptosis including 8-hidroxy-2-deoxyguanosine (8-OHdG) and iron were notably increased in TMJOA tissues and primary OA-FLSs. However, the activity of the antioxidant system including the glutathione peroxidase activity, glutathione (GSH) contents, and glutathione/oxidized glutathione (GSH/GSSG) ratio was notably inhibited in TMJOA tissues, and the primary OA-FLSs. Furthermore, the glutathione peroxidase 4 (GPX4) expression was down-regulated in TMJOA tissues and primary OA-FLSs. Animal and cell experiments have shown that ferroptosis inhibitors notably inhibited ferroptosis and promoted HLS survival as well as up-regulated GPX4 expression. Also, GPX4 knockdown promoted ferroptosis and GPX4 overexpression inhibited ferroptosis. GPX4 also positively regulated cell survival which was the opposite with ferroptosis. In conclusion, GPX4 and ferroptosis regulated the progression of TMJOA. Targeting ferroptosis might be an effective therapeutic strategy for TMJOA patients in the clinic.

The MdVQ37-MdWRKY100 complex regulates salicylic acid content and MdRPM1 expression to modulate resistance to Glomerella leaf spot in apples.

Glomerella leaf spot (GLS), caused by the fungus Colletotrichum fructicola, is considered one of the most destructive diseases affecting apples. The VQ-WRKY complex plays a crucial role in the response of plants to biotic stresses. However, our understanding of the defensive role of the VQ-WRKY complex on woody plants, particularly apples, under biotic stress, remains limited. In this study, we elucidated the molecular mechanisms underlying the defensive role of the apple MdVQ37-MdWRKY100 module in response to GLS infection. The overexpression of MdWRKY100 enhanced resistance to C. fructicola, whereas MdWRKY100 RNA interference in apple plants reduced resistance to C. fructicola by affecting salicylic acid (SA) content and the expression level of the CC-NBS-LRR resistance gene MdRPM1. DAP-seq, Y1H, EMSA, and RT-qPCR assays indicated that MdWRKY100 inhibited the expression of MdWRKY17, a positive regulatory factor gene of SA degradation, upregulated the expression of MdPAL1, a key enzyme gene of SA biosynthesis, and promoted MdRPM1 expression by directly binding to their promotors. Transient overexpression and silencing experiments showed that MdPAL1 and MdRPM1 positively regulated GLS resistance in apples. Furthermore, the overexpression of MdVQ37 increased the susceptibility to C. fructicola by reducing the SA content and expression level of MdRPM1. Additionally, MdVQ37 interacted with MdWRKY100, which repressed the transcriptional activity of MdWRKY100. In summary, these results revealed the molecular mechanism through which the apple MdVQ37-MdWRKY100 module responds to GLS infection by regulating SA content and MdRPM1 expression, providing novel insights into the involvement of the VQ-WRKY complex in plant pathogen defence responses.

Procyanidin B2 alleviates oxidized low-density lipoprotein-induced cell injury, inflammation, monocyte chemotaxis, and oxidative stress by inhibiting the nuclear factor kappa-B pathway in human umbilical vein endothelial cells

BackgroundOxidized low-density lipoprotein (ox-LDL) can initiate and affect almost all atherosclerotic events including endothelial dysfunction. In this text, the role and underlying molecular basis of procyanidin B2 (PCB2) with potential anti-oxidant and anti-inflammatory activities in ox-LDL-induced HUVEC injury were examined.MethodsHUVECs were treated with ox-LDL in the presence or absence of PCB2. Cell viability and apoptotic rate were examined by CCK-8 assay and flow cytometry, respectively. The mRNA and protein levels of genes were tested by RT-qPCR and western blot assays, respectively. Potential downstream targets and pathways of apple procyanidin oligomers were examined by bioinformatics analysis for the GSE9647 dataset. The effect of PCB2 on THP-1 cell migration was examined by recruitment assay. The effect of PCB2 on oxidative stress was assessed by reactive oxygen species (ROS) level, malondialdehyde (MDA) content, and mitochondrial membrane potential (MMP).Resultsox-LDL reduced cell viability, induced cell apoptosis, and facilitated the expression of oxidized low-density lipoprotein receptor 1 (LOX-1), C-C motif chemokine ligand 2 (MCP-1), vascular cell adhesion protein 1 (VCAM-1) in HUVECs. PCB2 alleviated ox-LDL-induced cell injury in HUVECs. Apple procyanidin oligomers triggered the differential expression of 592 genes in HUVECs (|log2fold-change| > 0.58 and adjusted p-value < 0.05). These dysregulated genes might be implicated in apoptosis, endothelial cell proliferation, inflammation, and monocyte chemotaxis. PCB2 inhibited C-X-C motif chemokine ligand 1/8 (CXCL1/8) expression and THP-1 cell recruitment in ox-LDL-stimulated HUVECs. PCB2 inhibited ox-LDL-induced oxidative stress and nuclear factor kappa-B (NF-κB) activation in HUVECs.ConclusionPCB2 weakened ox-LDL-induced cell injury, inflammation, monocyte recruitment, and oxidative stress by inhibiting the NF-κB pathway in HUVECs.

Genome-wide analysis of two different regions of brain reveals the molecular changes of fertility related genes in rln3a-/- mutants in male Nile tilapia (Oreochromis niloticus)

Relaxin3 (rln3) has been associated with various emotional and cognitive processes, including stress, anxiety, learning, memory, motivational behavior, and circadian rhythm. Notably, previous report revealed that Rln3a played an indispensable role in testicular development and male fertility in Nile tilapia (Oreochromis niloticus). However, the underlying molecular mechanisms remain largely unknown. We found that Rln3a is expressed exclusively in the diencephalon* (Di*) of the brain. Deficiency of Rln3a resulted in a significant increase in serum dopamine level and an upregulation of gene expression of gnrh1 and kisspeptin2. To further elucidate the role of Rln3a in fish fertility, we collected two different regions of Di* and hypothalamus (Hyp) tissues for subsequent RNA-seq analysis of both wild-type (rln3a+/+) and rln3a-/- male tilapia. Upon the transcriptomic data, 1136 and 755 differentially expressed genes (DEGs) were identified in the Di* and Hyp tissues, respectively. In Di*, the up-regulated genes were enriched in circadian rhythm, chemical carcinogenesis, while the down-regulated genes were enriched in type II diabetes mellitus, dopaminergic synapse, and other pathways. In Hyp, the up-regulated genes were enriched in circadian rhythm, pyrimidine metabolism, while the down-regulated genes were enriched in type I diabetes mellitus, autoimmune thyroid disease, and other pathways. Subsequently, the results of both qRT-PCR and FISH assays highlighted a pronounced up-regulation of core circadian rhythm genes, cry1b and per3, whereas genes such as clocka, clockb, and arntl exhibited down-regulation. Furthermore, the genes associated with dopamine biosynthesis were significantly increased in the Hyp. In summary, the mutation of rln3a in male tilapia resulted in notable changes in circadian rhythm and disease-linked signaling pathways in the Di* and Hyp. These changes might account for the fertility defects observed in rln3a-/- male mutants in tilapia.

Bacitracin-resistant Staphylococcus aureus induced in chicken gut and in vitro under bacitracin exposure

It is common knowledge that prolonged and excessive use of antibiotics can lead to antimicrobial resistance. However, the characteristics and mechanism of resistant-bacteria induced by clinically recommended and prophylactic dose drugs remain largely unclear. This study aimed to observe the trends of drug resistance of the bacitracin-susceptible Staphylococcus aureus strain FS127 under exposure to bacitracin (BAC), which were induced in vitro and in chicken gut. Antimicrobial susceptibility testing was used to detect the susceptibility of S. aureus induced in vitro and in the chicken gut to gentamicin, chloramphenicol, tetracycline, doxycycline, penicillin and chloramphenicol. The research results showed that bacitracin could induce drug resistance in S. aureus both in vitro and in vivo. The bacitracin-resistance rate of S. aureus isolated from chicken gut was positively correlated with the dose and time of bacitracin administration. The findings revealed that bacitracin-resistant S. aureus induced in vivo had enhanced susceptibility to chloramphenicol but no such change in vitro. Meanwhile, RT-qPCR assay was used to detect the expression levels of vraD, braD, braR and bacA in typical strains with different bacitracin-resistance levels. It was found that BacA may play a key role in the bacitracin resistance of S. aureus. In conclusion, this work reveals the characteristics and mechanism of bacitracin-resistant S. aureus induced by bacitracin in vivo and in vitro respectively.

Effect of MYC and PARP Inhibitors in Ovarian Cancer Using an In Vitro Model.

The 8q24 chromosomal region, which contains the MYC and PVT1 candidate oncogenes, is amplified in carcinomas. Both genes have been involved in the etiopathogenesis of ovarian cancer (OC). In this study, we used an in vitro OC model with a known 8q24 copy number increase and in silico tools to investigate the expression of MYC/PVT1 loci and copy number variation in OC. We also assessed the effects of rucaparib (a PARP inhibitor) in the presence or absence of 10058F4 (a MYC inhibitor) on the expression of MYC/linear PVT1/circular PVT1. Tissue culture, chromosome preparation, RNA extraction, RT-qPCR, FISH, and wound healing assays were employed. OncoDB, cBioportal, UALKAN, and ROC Plotter in silico tools were also utilized. Although PVT1 and MYC expression levels remained unaltered in OC, putative copy number alterations across all cancers showed a marked difference between the two genes, particularly in gain and amplification for MYC. PVT1 expression demonstrated prognostic value for the treatment of patients with serous and endometrioid OC. Both genes correlated with PARP10, FAM83H, and DEPTOR. The use of rucaparib in the presence or absence of the MYC inhibitor (10058F4) in vitro, led to a significant down-regulation in the expression of MYC, linear, and circular PVT1. Our data provide a novel insight into the potential interactions of MYC and PVT1 with other genes. Moreover, we identified a new PARP inhibition mechanism down-regulating MYC, as well as the linear and circular PVT1 transcripts. Future work should expand on clinical studies to better understand the prognostic role of PVT1 in OC.

Identification and characterization of STAT family in silver pomfret (Pampus argenteus) involved in different exogenous stresses

Members of the Signal Transducer and Activator of Transcription (STAT) family function pivotally as transcriptional activators integral to the modulation of inflammatory responses. The aquaculture of silver pomfret is frequently compromised by the imposition of exogenous stressors, which include thermal fluctuations, notably low-temperatures, diminished oxygen levels, and the onslaught of bacterial pathogens. Notwithstanding the critical impact of these stressors, the scientific literature presents a notable gap in our understanding of the STAT pathway's role in the silver pomfret's adaptive response mechanisms. To address this lacuna, we identified stat genes in the silver pomfret—denominated as Pastat1, Pastat2, Pastat3, Pastat4, and Pastat5—through a thorough and systematic bioinformatics analysis. Further scrutiny of the gene configurations and constituent motifs has elucidated that STAT proteins possess analogous structural frameworks and exhibit significant evolutionary preservation. Subsequently, the expression patterns of five stat genes were verified by RT-qPCR in twelve different tissues and four growth periods in healthy fish, showing that the expression of Pastat genes was temporally and spatially specific, with most of the stat genes expressed at higher levels in the spleen, following muscle, gill, and liver. Transcriptomic analysis of exposure to exogenous stressors, specifically formaldehyde and low-temperature conditions, elucidated that Pastat1 and Pastat2 genes exhibited a heightened sensitivity to these environmental challenges. RT-qPCR assays demonstrated a marked alteration in the expression profiles of jak1 and Pastat gene suites in PaS upon prolonged bacterial infection subsequent to these exogenous insults. Moreover, the gene expression of the downstream effectors involved in innate immunity and apoptosis displayed marked deviations. This study additionally elucidated the Pastat gene family's role in modulating the innate immune response and apoptotic regulation within the silver pomfret during exogenous stressors and subsequent pathogenic incursions.

Eggs sampling as an effective tool for identifying the incidence of viruses in honey bees involved in artificial queen rearing

The Carniolan honey bee (Apis mellifera carnica) plays an essential role in crop pollination, environment diversity, and the production of honey bee products. However, the health of individual honey bees and their colonies is under pressure due to multiple stressors, including viruses as a significant threat to bees. Monitoring various virus infections could be a crucial selection tool during queen rearing. In the present study, samples from all developmental stages (eggs, larvae, pupae, and queens) were screened for the incidence of seven viruses during queen rearing in Slovenia. The screening of a total of 108 samples from five queen breeders was performed by the RT-qPCR assays. The results showed that the highest incidence was observed for black queen cell virus (BQCV), Lake Sinai virus 3 (LSV3), deformed wing virus B (DWV-B), and sacbrood virus (SBV). The highest viral load was detected in queens (6.07 log10 copies/queen) and larvae (5.50 log10 copies/larva) for BQCV, followed by SBV in larvae (5.47 log10 copies/larva). When comparing all the honey bee developmental stages, the eggs exhibited general screening for virus incidence and load in queen mother colonies. The results suggest that analyzing eggs is a good indicator of resilience to virus infection during queen development.

PAX1 represses canonical Wnt signaling pathway and plays dual roles during endoderm differentiation

BackgroundPaired box 1 (PAX1) is a transcription factor and essential for the development of pharyngeal pouches-derived tissues, including thymus. PAX1 mutations are identified in Severe Combined Immunodeficiency (SCID) patients with Otofaciocervical Syndrome Type 2 (OTFCS2). However, despite the critical roles of PAX1 in embryonic development and diseases, detailed insights into its molecular mode of action are critically missing.MethodsThe repressing roles of PAX1 and SCID associated mutants on Wnt signaling pathway were investigated by luciferase reporter assays, qRT-PCR and in situ hybridization in HEK293FT, HCT116 cells and zebrafish embryos, respectively. Co-immunoprecipitation (co-IP) and western blotting assays were carried out to identify the molecular mechanisms underlying PAX1’s role on Wnt signaling pathway. hESC based endoderm differentiation, flow cytometry, high-throughput sequencing data analysis, and qRT-PCR assays were utilized to determine the roles of PAX1 during endoderm differentiation.ResultsHere, we show that PAX1 represses canonical Wnt signaling pathway in vertebrate cells. Mechanically, PAX1 competes with SUMO E3 ligase PIASy to bind to TCF7L2, thus perturbing TCF7L2 SUMOylation level, further reducing its transcriptional activity and protein stability. Moreover, we reveal that PAX1 plays dual roles in hESC-derived definitive and foregut/pharyngeal endoderm cells, which give rise to the thymus epithelium, by inhibiting Wnt signaling. Importantly, our data show PAX1 mutations found in SCID patients significantly compromise the suppressing ability of PAX1 on Wnt signaling.ConclusionsOur study presents a novel molecular mode of action of PAX1 in regulation of canonical Wnt signaling and endoderm differentiation, thus providing insights for the molecular basis of PAX1 associated SCID, offering better understanding of the behavior of PAX1 in embryogenesis.

Hyaluronidase inhibits TGF-β-mediated rat periodontal ligament fibroblast expression of collagen and myofibroblast markers: An in vitro exploration of periodontal tissue remodeling

ObjectiveTo determine the effect of hyaluronic acid (HA) degradation by hyaluronidase (HYAL) in inhibiting collagen fiber production by rat periodontal ligament cells (rPDLCs). DesignPrimary rPDLCs were isolated from the euthanized rats and used for in vitro experiments. The appropriate HYAL concentration was determined through CCK-8 testing for cytotoxicity detection and Alizarin red staining for mineralization detection. RT-qPCR and western blot assays were conducted to assess the effect of HYAL, with or without TGF-β, on generation of collagen fiber constituents and expression of actin alpha 2, smooth muscle (ACTA2) of rPDLCs. ResultsNeither cell proliferation nor mineralization were significantly affected by treatment with 4 U/mL HYAL. HYAL (4 U/mL) alone downregulated type I collagen fiber (Col1a1 and Col1a2) and Acta2 mRNA expression; however, ACTA2 and COL1 protein levels were only downregulated by HYAL treatment after TGF-β induction. ConclusionsTreatment of rPDLCs with HYAL can inhibit TGF-β-induced collagen matrix formation and myofibroblast transformation.

Transition to a mesenchymal state in neuroblastoma may be characterized by a high expression of GD2 and by the acquisition of immune escape from NK cells.

Neuroblastoma (NB) is characterized by both adrenergic (ADRN) and undifferentiated mesenchymal (MES) subsets. The ganglioside sialic acid-containing glycosphingolipid (GD2) is widely overexpressed on tumors of neuroectodermal origin promoting malignant phenotypes. MES cells are greatly enriched in post-therapy and relapsing tumors and are characterized by decreased expression of GD2. This event may cause failure of GD2-based immunotherapy. NK cells represent a key innate cell subset able to efficiently kill tumors. However, the tumor microenvironment (TME) that includes tumor cells and tumor-associated (TA) cells could inhibit their effector function. We studied eight NB primary cultures that, in comparison with commercial cell lines, more faithfully reflect the tumor cell characteristics. We studied four primary NB-MES cell cultures and two pairs of MES/ADRN (691 and 717) primary cultures, derived from the same patient. In particular, in the six human NB primary cultures, we assessed their phenotype, the expression of GD2, and the enzymes that control its expression, as well as their interactions with NK cells, using flow cytometry, RT-qPCR, and cytotoxicity assays. We identified mature (CD105+/CD133-) and undifferentiated (CD133+/CD105-) NB subsets that express high levels of the MES transcripts WWTR1 and SIX4. In addition, undifferentiated MES cells display a strong resistance to NK-mediated killing. On the contrary, mature NB-MES cells display an intermediate resistance to NK-mediated killing and exhibit some immunomodulatory capacities on NK cells but do not inhibit their cytolytic activity. Notably, independent from their undifferentiated or mature phenotype, NB-MES cells express GD2 that can be further upregulated in undifferentiated NB-MES cells upon co-culture with NK cells, leading to the generation of mature mesenchymal GD2bright neuroblasts. Concerning 691 and 717, they show high levels of GD2 and resistance to NK cell-mediated killing that can be overcome by the administration of dinutuximab beta, the anti-GD2 monoclonal antibody applied in the clinic. NB is a heterogeneous tumor representing a further hurdle in NB immunotherapy. However, different from what was reported with NB commercial cells and independent of their MES/ADRN phenotype, the expression of GD2 and its displayed sensitivity to anti-GD2 mAb ADCC indicated the possible effectiveness of anti-GD2 immunotherapy.

Identification of a novel prognostic cuproptosis-associated LncRNA signature for predicting prognosis and immunotherapy response in patients with esophageal cancer

Nowadays, effective prognostic models for esophageal cancer (ESCA) are still lacking. Long noncoding RNAs (lncRNAs) are commonly utilized as indicators for diagnosing cancer and forecasting patient outcomes. Cuproptosis is regulated by multiple genes and is crucial to the progression of ESCA. However, it is not yet clear what role the cuproptosis-associated lncRNAs (CuALs) play in ESCA. To tackle this problem, a prognostic signature incorporating three CuALs was created. This signature was constructed by the use of the least absolute shrinkage and selection operator (LASSO) and multivariate Cox regression. Subsequently, the signature effectively stratified ESCA samples into a high-risk group and a low-risk group. Those in the low-risk group demonstrated extended overall survival (OS), as well as increased infiltration of T cells, macrophages, and NK cells, suggesting a potentially enhanced response to immunotherapy. The ROC curve analysis demonstrated that this prognostic signature outperformed conventional clinical factors in predicting patient prognosis (AUC = 0.708). K-M survival analysis and correlation analysis identified UGDH-AS1 (a CuAL) as a protective factor positively associated with patient prognosis. The results of RT-qPCR and wound healing assays indicated that UGDH-AS1 is overexpressed in ESCA and could inhibit cancer cell migration. In general, the prognostic signature of CuALs demonstrated a robust capability in forecasting the immune environment and patient prognosis, highlighting its potential as a tool for enhancing personalized treatment strategies in ESCA.

Pregnancy influences expression of interferon-stimulated genes, progesterone receptor and progesterone-induced blocking factor in ovine thyroid.

Embryonic interferon-tau (IFNT) and progesterone affect expression of interferonstimulated genes (ISGs), progesterone receptor (PGR) and progesterone-induced blocking factor (PIBF) in the ovine thyroid. Thyroids of ewes were sampled at day 16 of nonpregnancy, days 13, 16, and 25 of pregnancy, and real-time quantitative polymerase chain reaction assay, western blot and immunohistochemistry were used to detect expression of ISGs, PGR, and PIBF. Free ISG15 protein was undetected, but ISG15 conjugated proteins upregulated at day 16 of pregnancy, and expression levels of ISG15 conjugated proteins, PGR isoform (70 kDa), PIBF, interferon-gamma-inducible protein 10 and myxovirusresistance protein 1 peaked, but expression level of signal transducer and activator of transcription 1 was the lowest at day 16 of pregnancy. In addition, the expression levels of PGR isoform (70 kDa) and signal transducer and activator of transcription 1 (STAT1) decreased, but levels of PGR isoform (43 kDa), 2',5'-oligoadenylate synthetase, IP-10 and MX1 increased at day 25 of pregnancy comparing with day 16 of the estrous cycle. Early pregnancy affects expression of ISGs, PGR, and PIBF in maternal thyroid through IFNT and progesterone, which may regulate thyroid autoimmunity and thyroid hormone secretion in ewes.

PagWOX11/12a from hybrid poplar enhances drought tolerance by modulating reactive oxygen species

WOX11/12 is a homeobox gene of WOX11 and WOX12 in Arabidopsis that plays important roles in crown root development and growth. It has been reported that WOX11/12 participates in adventitious root (AR) formation and different abiotic stress responses, but the downstream regulatory network of WOX11/12 in poplar remains to be further investigated. In this study, we found that PagWOX11/12a is strongly induced by PEG-simulated drought stress. PagWOX11/12a-overexpressing poplar plantlets showed lower oxidative damage levels, greater antioxidant enzyme activities and reactive oxygen species (ROS) scavenging capacity than non-transgenic poplar plants, whereas PagWOX11/12a dominant repression weakened root biomass accumulation and drought tolerance in poplar. RNA-seq analysis revealed that several differentially expressed genes (DEGs) regulated by PagWOX11/12a are involved in redox metabolism and drought stress response. We used RT-qPCR and yeast one-hybrid (Y1H) assays to validate the downstream target genes of PagWOX11/12a. These results provide new insights into the biological function and molecular regulatory mechanism of WOX11/12 in the abiotic resistance processes of poplar.

Evaluation of different genomic regions of rotavirus B and rotavirus C for development of real-time RT‒PCR assays

BackgroundThe causative agents of diarrhea, rotavirus B (RVB) and rotavirus C (RVC) are common in adults and patients of all age groups, respectively. Due to the Rotavirus A (RVA) vaccination program, a significant decrease in the number of gastroenteritis cases has been observed globally. The replacement of RVA infections with RVB, RVC, or other related serogroups is suspected due to the possibility of reducing natural selective constraints due to RVA infections. The data available on RVB and RVC incidence are scant due to the lack of cheap and rapid commercial diagnostic assays and the focus on RVA infections. The present study aimed to develop real-time RT‒PCR assays using the data from all genomic RNA segments of human RVB and RVC strains available in the Gene Bank.ResultsAmong the 11 gene segments, NSP3 and NSP5 of RVB and the VP6 gene of RVC were found to be suitable for real-time RT‒PCR (qRT‒PCR) assays. Fecal specimens collected from diarrheal patients were tested simultaneously for the presence of RVB (n = 192) and RVC (n = 188) using the respective conventional RT‒PCR and newly developed qRT‒PCR assays. All RVB- and RVC-positive specimens were reactive in their respective qRT‒PCR assays and had Ct values ranging between 23.69 and 41.97 and 11.49 and 36.05, respectively. All known positive and negative specimens for other viral agents were nonreactive, and comparative analysis showed 100% concordance with conventional RT‒PCR assays.ConclusionsThe suitability of the NSP5 gene of RVB and the VP6 gene of RVC was verified via qRT‒PCR assays, which showed 100% sensitivity and specificity. The rapid qRT‒PCR assays developed will be useful diagnostic tools, especially during diarrheal outbreaks for testing non-RVA rotaviral agents and reducing the unnecessary use of antibiotics.

Evaluation of KIAA1549::BRAF fusions and clinicopathological insights of pilocytic astrocytomas

BackgroundPilocytic astrocytoma (PAs) represents a significant portion of childhood primary brain tumors, with distinct histological and radiological features. The prevalence of KIAA1549::BRAF fusion in PAs has been well-established, this study aims to assess the prevalence of KIAA1549::BRAF fusions and explore their associations with tumor characteristics, radiological findings, and patient outcomes in PAs. MethodsHistologically confirmed cases of PAs from a 5-year period were included in the study. Demographic, histopathological, and radiological data were collected, and immunohistochemistry was performed to characterize tumor markers. FISH and qRT-PCR assays were employed to detect KIAA1549::BRAF fusions. Statistical analyses were conducted to examine associations between fusion status and various other parameters. ResultsHistological analysis revealed no significant differences in tumor features based on fusion status. However, younger age groups showed higher fusion prevalence. Radiologically, fusion-positive cases were distributed across different tumor subtypes SE, CWE and NCWE. Survival analysis did not demonstrate a significant impact of fusion status on overall survival, however most cases with recurrence and death harboured KIAA1549::BRAF fusion. Of 200 PAs, KIAA1549::BRAF fusions were detected in 64 % and 74 % of cases via qRT-PCR and FISH, respectively. Concordance between the two platforms was substantial (86 %). ConclusionKIAA1549::BRAF fusions are prevalent in PAs and can be reliably detected using both FISH and qRT-PCR assays. Cost considerations suggest qRT-PCR as a more economical option for fusion detection in routine clinical practice.

Design and Synthesis of Mandelic Acid Derivatives for Suppression of Virulence via T3SS against Citrus Canker.

Citrus canker, a highly contagious bacterial disease caused by Xanthomonas citri subsp. citri (Xcc), poses a substantial threat to citrus crops, leading to serious reductions in fruit yield and economic losses. Most commonly used bactericides against Xcc lead to the rapid development of resistant subpopulations. Therefore, it is imperative to create novel drugs, such as type III secretion system (T3SS) inhibitors, that specifically target bacterial virulence factors rather than bacterial viability. In our study, we designed and synthesized a series of mandelic acid derivatives including 2-mercapto-1,3,4-thiazole. Seven substances were found to reduce the level of transcription of hpa1 without affecting bacterial viability. In vivo bioassays indicated that compound F9 significantly inhibited hypersensitive response and pathogenicity. RT-qPCR assays showed that compound F9 visibly suppressed the expression of Xcc T3SS-related genes as well as citrus canker susceptibility gene CsLOB1. Furthermore, the combination with compound F9 and quorum-quenching bacteria HN-8 can also obviously alleviate canker symptoms.

A Comparative Transcriptome Analysis Unveils the Mechanisms of Response in Feather Degradation by Pseudomonas aeruginosa Gxun-7.

Microbial degradation of feathers offers potential for bioremediation, yet the microbial response mechanisms warrant additional investigation. In prior work, Pseudomonas aeruginosa Gxun-7, which demonstrated robust degradation of feathers at elevated concentrations, was isolated. However, the molecular mechanism of this degradation remains only partially understood. To investigate this, we used RNA sequencing (RNA-seq) to examine the genes that were expressed differentially in P. aeruginosa Gxun-7 when exposed to 25 g/L of feather substrate. The RNA-seq analysis identified 5571 differentially expressed genes; of these, 795 were upregulated and 603 were downregulated. Upregulated genes primarily participated in proteolysis, amino acid, and pyruvate metabolism. Genes encoding proteases, as well as those involved in sulfur metabolism, phenazine synthesis, and type VI secretion systems, were notably elevated, highlighting their crucial function in feather decomposition. Integration of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) taxonomies, combined with a review of the literature, led us to propose that metabolic feather degradation involves environmental activation, reducing agent secretion, protease release, peptide/amino acid uptake, and metabolic processes. Sulfite has emerged as a critical activator of keratinase catalysis, while cysteine serves as a regulatory mediator. qRT-PCR assay results for 11 selected gene subset corroborated the RNA-seq findings. This study enhances our understanding of the transcriptomic responses of P. aeruginosa Gxun-7 to feather degradation and offers insights into potential degradation mechanisms, thereby aiding in the formulation of effective feather waste management strategies in poultry farming.