IGF2BP2 regulates the proliferation and migration of endometrial stromal cells through the PI3K/AKT/mTOR signaling pathway in Hu sheep.

Insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2), a significant member of the conserved RNA-binding protein family, plays various roles in numerous physiological and pathological processes. However, the specific function of IGF2BP2 in regulating endometrial function in sheep remains largely unknown. In this study, we observed a significant upregulation in IGF2BP2 mRNA abundance in the endometrium during the luteal phase (LP) compared to the follicular phase (FP) in Hu sheep. The knockdown of IGF2BP2 resulted in accelerated cell proliferation and migration of Hu sheep endometrial stromal cells (ESCs). Moreover, RNA sequencing analysis revealed that genes with significantly altered expression in IGF2BP2 knockdown cells were predominantly enriched in endometrial receptivity-related signaling pathways, such as cytokine-cytokine receptor interaction, NOD-like receptor, PI3K-AKT, and JAK-STAT signaling pathway. Additionally, the knockdown of IGF2BP2 significantly increased the expression of matrix metalloprotein 9 (MMP9), vascular endothelial growth factor (VEGF), and prolactin (PRL) in ESCs. The knockdown of IGF2BP2 was also observed to stimulate the PI3K/AKT/mTOR pathway by upregulating integrin ß4 (ITGB4) expression. Notably, the downregulation of ITGB4 attenuates IGF2BP2 knockdown-induced facilitation of proliferation and migration of Hu sheep ESCs by inhibiting the PI3K/AKT/mTOR pathway. Collectively, these findings highlight the important role of IGF2BP2 in regulating endometrial function, particularly through the modulation of ESC proliferation and migration via the PI3K/AKT/mTOR pathway.

Mechanistic of AMPK/ACC2 regulating myoblast differentiation by fatty acid oxidation of goat.

Myoblast differentiation depends on fatty acid oxidation (FAO)，and its rate-limiting enzyme acetyl-CoA carboxylase 2 (ACC2) participate in the regulation skeletal muscle development. However, the precise regulatory mechanism is still unknown. Using previous RNA-sequencing data from our laboratory, we explored the effect of ACC2 on myoblast differentiation, as a candidate gene, since its expression is higher in myoblasts of lamb (first day of age) than that of the fetus (75th day of pregnancy). Our findings show that siACC2 inhibited myoblast proliferation, promoted differentiation, and boosted mitochondrial and fatty acid oxidation activities. The effect of ACC2 on goat muscle cell differentiation was modulated by Etomoxir, a CPT1A inhibitor. Notably, the AMPK/ACC2 pathway was found to regulate fatty acid oxidation and goat muscle cell differentiation. Inhibiting the AMPK/ACC2 pathway significantly reduced CPT1A expression. These findings indicate that AMPK/ACC2 regulate goat myoblast differentiation via fatty acid oxidation, contributing to understanding the mechanism of goat skeletal muscle development.

circUSP13 facilitates the fast-to-slow myofiber shift via the MAPK/ERK signaling pathway in goat skeletal muscles.

Understanding how skeletal muscle fiber proportions are regulated is essential for understanding muscle function and improving the quality of mutton. While circular RNA (circRNA) has a critical function in myofiber type transformation, the specific mechanisms are not yet fully understood. Prior evidence indicates that circular ubiquitin-specific peptidase 13 (circUSP13) can promote myoblast differentiation by acting as a ceRNA, but its potential role in myofiber switching is still unknown. Herein, we found that circUSP13 enhanced slow myosin heavy chain (MyHC-slow) and suppressed MyHC-fast expression in goat primary myoblasts (GPMs). Meanwhile, circUSP13 evidently enhanced the remodeling of the mitochondrial network while inhibiting the autophagy of GPMs. We obtained fast-dominated myofibers, via treatment with rotenone, and further demonstrated the positive role of circUSP13 in the fast-to-slow transition. Mechanistically, activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway significantly impaired the slow-to-fast shift in fully differentiated myotubes, which was restored by circUSP13 or IGF1 overexpression. In conclusion, circUSP13 promoted the fast-to-slow myofiber type transition through MAPK/ERK signaling in goat skeletal muscle. These findings provide novel insights into the role of circUSP13 in myofiber type transition and contribute to a better understanding of the genetic mechanisms underlying meat quality.

Ythdf2-mediated STK11 mRNA decay supports myogenesis by inhibiting the AMPK/mTOR pathway.

An emerging research focus is the role of m6A modifications in mediating the post-transcriptional regulation of mRNA during mammalian development. Recent evidence suggests that m6A methyltransferases and demethylases play critical roles in skeletal muscle development. Ythdf2 is a m6A "reader" protein that mediates mRNA degradation in an m6A-dependent manner. However, the specific function of Ythdf2 in skeletal muscle development and the underlying mechanisms remain unclear. Here, we observed that Ythdf2 expression was significantly upregulated during myogenic differentiation, whereas Ythdf2 knockdown markedly inhibited myoblast proliferation and differentiation. Combined analysis of high-throughput sequencing, Co-IP, and RIP assay revealed that Ythdf2 could bind to m6A sites in STK11 mRNA and form an Ago2 silencing complex to promote its degradation, thereby regulating its expression and consequently, the AMPK/mTOR pathway. Furthermore, STK11 downregulation partially rescued Ythdf2 knockdown-induced impairment of proliferation and myogenic differentiation by inhibiting the AMPK/mTOR pathway. Collectively, our results indicate that Ythdf2 mediates the decay of STK11 mRNA, an AMPK activator, in an Ago2 system-dependent manner, thereby driving skeletal myogenesis by suppressing the AMPK/mTOR pathway. These findings further enhance our understanding of the molecular mechanisms underlying RNA methylation in the regulation of myogenesis and provide valuable insights for conducting in-depth studies on myogenesis.

LncRNA12097.1 contributes to endometrial cell growth by enhancing YES1 activating ß-catenin via sponging miR-145-5p.

Despite previous investigations elucidating the regulatory mechanisms of long non-coding RNAs (lncRNAs) in endometrial function and reproductive disorders, the precise pathways through which lncRNAs impact endometrial functions and fertility remain unclear. In this study, we performed an expression profile analysis of lncRNAs in the endometrial tissue of Hu sheep with different prolificacy, identifying 13,707 lncRNAs. We discovered a bidirectional lncRNA, designated lncRNA12097.1, exhibiting significant up-regulation exclusively in the endometrium of Hu sheep with high fecundity. Functional analyses revealed lncRNA12097.1 significantly enhanced proliferation and cell cycle progression in both endometrial epithelial cell (EEC) and stromal cells (ESC), while inhibiting apoptosis in these cell types. Mechanistically, we demonstrated a directly interaction between lncRNA12097.1 and miR-145-5p, with YES proto-oncogene 1 (YES1) being identified as a validated target of miR-145-5p. Interference with lncRNA12097.1 resulted in suppressed cell growth through down-regulation of YES1 expression, which could be rescued by miR-145-5p. Furthermore, lncRNA12097.1 functions as a competitive endogenous RNA (ceRNA) for miR-145-5p in ESCs, sequestering miR-145-5p and preventing its binding to the 3'UTR of YES1 mRNA. This interaction led to increased expression of YES1 and subsequent activation of downstream ß-catenin signaling, thereby promoting ESC growth in Hu sheep. These findings provide novel molecular insights into the mechanisms underlying prolificacy in sheep.

CREB1 Is Involved in miR-134-5p-Mediated Endometrial Stromal Cell Proliferation, Apoptosis, and Autophagy.

The successful establishment of endometrial receptivity is a key factor in ensuring the fertility of ewes and their economic benefits. Hu sheep have attracted attention due to their high fecundity and year-round estrus. In this study, we found that in the luteal phase, the uterine gland density, uterine coefficient, and number of uterine caruncles of high-fertility Hu sheep were higher than those of low-fertility Hu sheep. Thousands of differentially expressed genes were identified in the endometrium of Hu sheep with different fertility potential using RNA sequencing (RNA-Seq). Several genes involved in endometrial receptivity were screened using bioinformatics analysis. The qRT-PCR analysis further revealed the differential expression of cAMP reactive element binding protein-1 (CREB1) in the Hu sheep endometrium during the estrous cycle. Functionally, our results suggested that CREB1 significantly affected the expression level of endometrial receptivity marker genes, promoted cell proliferation by facilitating the transition from the G1 phase to the S phase, and inhibited cell apoptosis and autophagy. Moreover, we observed a negative linear correlation between miR-134-5p and CREB1 in the endometrium. In addition, CREB1 overexpression prevented the negative effect of miR-134-5p on endometrial stromal cell (ESC) growth. Taken together, these data indicated that CREB1 was regulated by miR-134-5p and may promote the establishment of uterine receptivity by regulating the function of ESCs. Moreover, this study provides new theoretical references for identifying candidate genes associated with fertility.

Multi-laboratory validation study of a real-time PCR method for detection of Salmonella in baby spinach.

The FDA Bacteriological Analytical Manual (BAM) Salmonella culture method takes at least 3 days for a presumptive positive result. The FDA developed a quantitative PCR (qPCR) method to detect Salmonella from 24-h preenriched cultures, using ABI 7500 PCR system. The qPCR method has been evaluated as a rapid screening method for a broad range of foods by single laboratory validation (SLV) studies. The present multi-laboratory validation (MLV) study was aimed to measure the reproducibility of this qPCR method and compare its performance with the culture method. Sixteen laboratories participated in two rounds of MLV study to analyze twenty-four blind-coded baby spinach test portions each. The first round yielded ∼84% and ∼82% positive rates across laboratories for the qPCR and culture methods, respectively, which were both outside the fractional range (25%-75%) required for fractionally inoculated test portions by the FDA's Microbiological Method Validation Guidelines. The second round yielded ∼68% and ∼67% positive rates. The relative level of detection (RLOD) for the second-round study was 0.969, suggesting that qPCR and culture methods had similar sensitivity (p > 0.05). The study demonstrated that the qPCR yields reproducible results and is sufficiently sensitive and specific for the detection of Salmonella in food.

RUNX1T1 modulates myogenic differentiation by regulating the calcium signaling pathway and the alternative splicing of ROCK2.

RUNX1T1 (Runt-related transcription factor 1, translocated to 1) plays a wide-ranging and diverse role in cellular development, including hematopoiesis and adipogenesis. However, little is known about the function of RUNX1T1 in the skeletal muscle development. Here, we assessed the impact of RUNX1T1 on the proliferation and myogenic differentiation of goat primary myoblasts (GPMs). It was observed that RUNX1T1 is highly expressed during the early stages of myogenic differentiation and the fetal stage. Moreover, the knockdown of RUNX1T1 promotes the proliferation and inhibits myogenic differentiation and mitochondrial biogenesis of GPMs. RNA sequencing analysis revealed that significantly differentially expressed genes in RUNX1T1 knockdown cells were enriched in the calcium signaling pathway. Additionally, we discovered that RUNX1T1 regulates alternative splicing (AS) events involved in myogenesis. We also show that silencing RUNX1T1 blocked the Ca2+ -CAMK signaling pathway and reduced the expression levels of muscle-specific isoforms of recombinant rho associated coiled coil containing crotein kinase 2 (ROCK2) during myogenic differentiation, partially explaining why RUNX1T1 deficiency leads to the impairment of myotube formation. These findings suggest that RUNX1T1 is a novel regulator of myogenic differentiation that regulates the calcium signaling pathway and AS of ROCK2. Overall, our results highlight the critical role of RUNX1T1 in myogenesis and broaden our understanding of myogenic differentiation.

Successful Detection of Delta and Omicron Variants of SARS-CoV-2 by Veterinary Diagnostic Laboratory Participants in an Interlaboratory Comparison Exercise.

BACKGROUND: Throughout the COVID-19 pandemic, veterinary diagnostic laboratories have tested diagnostic samples for SARS-CoV-2 both in animals and over 6 million human samples. An evaluation of the performance of those laboratories is needed using blinded test samples to ensure that laboratories report reliable data to the public. This interlaboratory comparison exercise (ILC3) builds on 2 prior exercises to assess whether veterinary diagnostic laboratories can detect Delta and Omicron variants spiked in canine nasal matrix or viral transport medium. METHODS: The ILC organizer was an independent laboratory that prepared inactivated Delta variant at levels of 25 to 1000 copies per 50 µL of nasal matrix for blinded analysis. Omicron variant at 1000 copies per 50 µL of transport medium was also included. Feline infectious peritonitis virus (FIPV) RNA was used as a confounder for specificity assessment. Fourteen test samples were prepared for each participant. Participants used their routine diagnostic procedures for RNA extraction and real-time reverse transcriptase-PCR. Results were analyzed according to International Organization for Standardization (ISO) 16140-2:2016. RESULTS: Overall, laboratories demonstrated 93% detection for Delta and 97% for Omicron at 1000 copies per 50 µL. Specificity was 97% for blank samples and 100% for blank samples with FIPV. No differences in Cycle Threshold (Ct) values were significant for samples with the same virus levels between N1 and N2 markers, nor between the 2 variants. CONCLUSIONS: The results indicated that all ILC3 participants were able to detect both Delta and Omicron variants. The canine nasal matrix did not significantly affect SARS-CoV-2 detection.

Targeted Demethylation of the TGFß1 mRNA Promotes Myoblast Proliferation via Activating the SMAD2 Signaling Pathway.

Recent evidence suggested that N6-methyladenosine (m6A) methylation can determine m6A-modified mRNA fate and play an important role in skeletal muscle development. It was well known that transforming growth factor beta 1 (TGFß1) is involved in a variety of cellular processes, such as proliferation, differentiation, and apoptosis. However, little is known about the m6A-mediated TGFß1 regulation in myogenesis. Here, we observed an increase in endogenous TGFß1 expression and activity during myotube differentiation. However, the knockdown of TGFß1 inhibits the proliferation and induces cell apoptosis of myoblast. Moreover, we found that m6A in 5'-untranslated regions (5'UTR) of TGFß1 promote its decay and inhibit its expression, leading to the blockage of the TGFß1/SMAD2 signaling pathway. Furthermore, the targeted specific demethylation of TGFß1 m6A using dCas13b-FTO significantly increased the TGFß1-mediated activity of the SMAD2 signaling pathway, promoting myoblast proliferation. These findings suggest that TGFß1 is an essential regulator of myoblast growth that is negatively regulated by m6A. Overall, these results highlight the critical role of m6A-mediated post-transcriptional regulation in myogenesis.

PPP2R2A promotes testosterone secretion in Hu sheep Leydig cells via activation of the AKT/mTOR signaling pathway.

The serine-threonine protein phosphatase 2A (PP2A) is a heterotrimeric enzyme complex that plays a vital role in regulating male reproductive activities. However, as an essential member of the PP2A family, the physiological functions of PP2A regulatory subunit B55α (PPP2R2A) in testis remain inconclusive. Hu sheep are noted for their reproductive precocity and fertility, and are ideal models for the study of male reproductive physiology. Here, we analyzed the expression patterns of PPP2R2A in the male Hu sheep reproductive tract at different developmental stages and further investigated its role in testosterone secretion and its underlying mechanisms. In this study, we found that there were temporal and spatial differences in PPP2R2A protein expression in the testis and epididymis, especially the expression abundance in the testis at 8 months old (8M) was higher than that at 3 months old (3M). Interestingly, we observed that PPP2R2A interference reduced the testosterone levels in the cell culture medium, which is accompanied by a reduction in Leydig cell proliferation and an elevation in Leydig cell apoptosis. The level of reactive oxygen species in cells increased significantly, while the mitochondrial membrane potential (ΔΨm) decreased significantly after PPP2R2A deletion. Meanwhile, the mitochondrial mitotic protein DNM1L was significantly upregulated, while the mitochondrial fusion proteins MFN1/2 and OPA1 were significantly downregulated after PPP2R2A interference. Furthermore, PPP2R2A interference suppressed the AKT/mTOR signaling pathway. Taken together, our data indicated that PPP2R2A enhanced testosterone secretion, promoted cell proliferation, and inhibited cell apoptosis in vitro, all of which were associated with the AKT/mTOR signaling pathway.

CircUBE3A promotes myoblasts proliferation and differentiation by sponging miR-28-5p to enhance expression.

circRNAs have been found to be involved in the regulatory network of skeletal muscle development in studies. However, their precise functions and regulatory mechanisms remain unknown. The expression patterns and alterations of circRNAs in the longissimus dorsi muscle of two major developmental stages of goats (D75 fetus and D1 kid) were studied using high-throughput sequencing technology and bioinformatics tools in this study. In kid skeletal muscles, 831 differently expressed circRNAs were found, comprising 486 up-regulated circRNAs and 345 down-regulated circRNAs. In skeletal muscle, we focused on the highly expressed and variably expressed circUBE3A. CircUBE3A levels were discovered to be much higher in kid skeletal muscle and differentiated myoblasts. Knocking down circUBE3A resulted in a significant increase in cell proliferation and differentiation in goat myoblasts. CircUBE3A specifically binds to and inhibits miR-28-5p, boosting the expression of Hydroxyacyl Coenzyme A Dehydrogenase Beta (HADHB) and contributing to goat myoblast proliferation and differentiation, according to the mechanistic investigation. The above results indicated that circUBE3A could regulate HADHB expression by targeting miR-28-5p, consequently increasing goat myoblast proliferation and differentiation. Our findings offer fresh perspectives on goat breeding and growth regulation, as well as substantial theoretical basis.

Second round of an interlaboratory comparison of SARS-CoV2 molecular detection assays used by 45 veterinary diagnostic laboratories in the United States.

The COVID-19 pandemic presents a continued public health challenge. Veterinary diagnostic laboratories in the United States use RT-rtPCR for animal testing, and many laboratories are certified for testing human samples; hence, ensuring that laboratories have sensitive and specific SARS-CoV2 testing methods is a critical component of the pandemic response. In 2020, the FDA Veterinary Laboratory Investigation and Response Network (Vet-LIRN) led an interlaboratory comparison (ILC1) to help laboratories evaluate their existing RT-rtPCR methods for detecting SARS-CoV2. All participating laboratories were able to detect the viral RNA spiked in buffer and PrimeStore molecular transport medium (MTM). With ILC2, Vet-LIRN extended ILC1 by evaluating analytical sensitivity and specificity of the methods used by participating laboratories to detect 3 SARS-CoV2 variants (B.1; B.1.1.7 [Alpha]; B.1.351 [Beta]) at various copy levels. We analyzed 57 sets of results from 45 laboratories qualitatively and quantitatively according to the principles of ISO 16140-2:2016. More than 95% of analysts detected the SARS-CoV2 RNA in MTM at ≥500 copies for all 3 variants. In addition, for nucleocapsid markers N1 and N2, 81% and 92% of the analysts detected ≤20 copies in the assays, respectively. The analytical specificity of the evaluated methods was >99%. Participating laboratories were able to assess their current method performance, identify possible limitations, and recognize method strengths as part of a continuous learning environment to support the critical need for the reliable diagnosis of COVID-19 in potentially infected animals and humans.

The regulation of LncRNA GTL2 expression by DNA methylation during sheep skeletal muscle development.

DNA methylation has crucial roles in regulating the expression of genes involved in skeletal muscle development. However, the DNA methylation pattern of lncRNA during sheep skeletal muscle development remains unclear. This study investigated previous WGBS and LncRNA data in skeletal muscle of sheep (fetus and adult). We then focused on LncRNA GTL2, which is differentially expressed in skeletal muscle and has multiple DMRs. We found that the expression level of GTL2 decreased with age. GTL2 DMRs methylation levels were significantly higher in adult muscle than in fetal muscle. After 5AZA treatment, GTL2 expression was significantly increased in a dose-dependent manner.The dCas9-DNMT3A-sgRNA significantly reduced the expression level of GTL2 in cells, but increased GTL2 DMR methylation levels. The above studies indicate that dCas9-DNMT3A can effectively increase the methylation level in the DMR region of GTL2, the expression level of GTL2 is regulated by DNA methylation during muscle development.

MicroRNA profiling reveals miR-145-5p inhibits goat myoblast differentiation by targeting the coding domain sequence of USP13.

MicroRNAs (miRNAs) are evolutionarily conserved endogenous small non-coding RNAs that play critical roles in skeletal muscle development. In this study, we identified putative miRNAs that were differentially expressed in the longissimus dorsi muscle between fetus (75 days of pregnancy) and lamb (1 day of age). We detected 1208 miRNAs, 313 of which were differentially expressed. In particular, we found that miR-145-5p was differentially and highly expressed in lamb skeletal muscle. In addition, our results demonstrated that overexpression of miR-145-5p inhibited the differentiation and apoptosis of goat primary myoblasts (GPMs), whereas knockdown of miR-145-5p had the opposite effect. The coding domain sequence (CDS) of ubiquitin-specific peptidase 13 (USP13) was predicted and validated as a target of miR-145-5p. We also demonstrated that the influence as a key regulator of GPMs differentiation is primarily mediated by targeting and inhibiting USP13. Taken together, these results revealed a novel pathway in skeletal muscle development in which miR-145-5p targets CDS region of USP13 to regulate differentiation and apoptosis of GPMs.

Circular RNA circUSP13 sponges miR-29c to promote differentiation and inhibit apoptosis of goat myoblasts by targeting IGF1.

Circular RNAs (circRNAs) are an indispensable element of post-transcriptional gene regulation, influencing a variety of biological processes including myogenic differentiation; however, little is known about the function of circRNA in goat myogenic differentiation. Using RNA-sequencing data from our laboratory, we explored the influences of circUSP13, as a candidate circRNA, on myoblast differentiation since its expression is higher in myoblasts of lamb (first day of age) than that of the fetus (75th day of pregnancy). In in vitro experiments, circUSP13 significantly promoted differentiation and inhibited apoptosis in goat primary myoblasts. Mechanistically, circUSP13 localized with miR-29c in the cytoplasm of goat myoblasts to regulate IGF1 expression. We further demonstrated that circUSP13 sponges miR-29c, promoting IGF1 expression that upregulated the expression of MyoG and MyHC. Thus, our results identified circUSP13 as a molecular marker for breeding programs of mutton production, as well as the circUSP13-miR-29c-IGF1 axis as a potential therapeutic target for combating muscle wasting.

PPP2R2A affects embryonic implantation by regulating the proliferation and apoptosis of Hu sheep endometrial stromal cells.

Embryonic implantation is a complex reproductive physiological process in mammals. Although several endometrial proteins affecting embryonic implantation have been reported in the past, there are still potential endometrial proteins that have been neglected, and their specific regulatory mechanisms are unclear. This study demonstrated that protein phosphatase 2A regulatory subunit B55α (PPP2R2A) served as a novel regulator in medication of sheep embryonic implantation in vitro. Our results showed that sheep PPP2R2A encoded 447 amino acids and shared 91.74%-92.36% amino acid sequences with its orthologs compared with other species. Meanwhile, PPP2R2A was widely expressed in sheep uterine tissues, and it could regulate the expression levels of key regulators of embryonic implantation in endometrial stromal cells (ESCs). Knockdown of PPP2R2A significantly inhibited cell proliferation by blocking cell cycle transfer G0/G1 into S phase accompanied by downregulation of CDK2, CDK4, CCND1, CCNE1 and upregulation of P21. In contrast to PPP2R2A overexpression, PPP2R2A interference greatly promoted cell apoptosis and the expression of BAX, CASP3, CASP9 and BAX/BCL-2. Taken together, these results suggest that PPP2R2A, as a novel regulatory factor, affects embryonic implantation via regulating the proliferation and apoptosis of Hu sheep ESCs in vitro.

FTO-mediated demethylation of GADD45B promotes myogenesis through the activation of p38 MAPK pathway.

N6-methyladenosine (m6A) modification plays a critical role in mammalian development. However, the role of m6A in the skeletal muscle development remains largely unknown. Here, we report a global m6A modification pattern of goat skeletal muscle at two key development stages and identified that the m6A modification regulated the expression of the growth arrest and DNA damage-inducible 45B (GADD45B) gene, which is involved in myogenic differentiation. We showed that GADD45B expression increased during myoblast differentiation, whereas the downregulation of GADD45B inhibits myogenic differentiation and mitochondrial biogenesis. Moreover, the expression of GADD45B regulates the expression of myogenic regulatory factors and peroxisome proliferator-activated receptor gamma coactivator 1 alpha by activating the p38 mitogen-activated protein kinase (MAPK) pathway. Conversely, the inactivation of p38 MAPK abolished the GADD45B-mediated myogenic differentiation. Furthermore, we found that the knockdown of fat mass and obesity-associated protein (FTO) increases GADD45B m6A modification and decreases the stability of GADD45B mRNA, which impairs myogenic differentiation. Our results indicate that the FTO-mediated m6A modification in GADD45B mRNA drives skeletal muscle differentiation by activating the p38 MAPK pathway, which provides a molecular mechanism for the regulation of myogenesis via RNA methylation.

Interlaboratory comparison of SARS-CoV2 molecular detection assays in use by U.S. veterinary diagnostic laboratories.

The continued search for intermediate hosts and potential reservoirs for SARS-CoV2 makes it clear that animal surveillance is critical in outbreak response and prevention. Real-time RT-PCR assays for SARS-CoV2 detection can easily be adapted to different host species. U.S. veterinary diagnostic laboratories have used the CDC assays or other national reference laboratory methods to test animal samples. However, these methods have only been evaluated using internal validation protocols. To help the laboratories evaluate their SARS-CoV2 test methods, an interlaboratory comparison (ILC) was performed in collaboration with multiple organizations. Forty-four sets of 19 blind-coded RNA samples in Tris-EDTA (TE) buffer or PrimeStore transport medium were shipped to 42 laboratories. Results were analyzed according to the principles of the International Organization for Standardization (ISO) 16140-2:2016 standard. Qualitative assessment of PrimeStore samples revealed that, in approximately two-thirds of the laboratories, the limit of detection with a probability of 0.95 (LOD95) for detecting the RNA was ≤20 copies per PCR reaction, close to the theoretical LOD of 3 copies per reaction. This level of sensitivity is not expected in clinical samples because of additional factors, such as sample collection, transport, and extraction of RNA from the clinical matrix. Quantitative assessment of Ct values indicated that reproducibility standard deviations for testing the RNA with assays reported as N1 were slightly lower than those for N2, and they were higher for the RNA in PrimeStore medium than those in TE buffer. Analyst experience and the use of either a singleplex or multiplex PCR also affected the quantitative ILC test results.

γ-Linolenic Acid Prevents Lipid Metabolism Disorder in Palmitic Acid-Treated Alpha Mouse Liver-12 Cells by Balancing Autophagy and Apoptosis via the LKB1-AMPK-mTOR Pathway.

Excessive fat deposition is the main character in nonalcoholic fatty liver disease (NAFLD), while γ-linolenic acid (GLA) is a polyunsaturated fatty acid that can reduce lipid deposition. This study investigated the effect and regulatory mechanism of GLA (100 µM) on lipid metabolism in alpha mouse liver 12 (AML-12) cells treated by 400 µM palmitic acid (PA). GLA reduced lipid content and increased fatty acid ß oxidation, as indicated by decreasing triglyceride and cholesterol contents and increasing mRNA and protein expressions of CPT1α and PPARα. GLA relieved oxidative stress caused by PA, upregulated mRNA levels of superoxide dismutase and glutathione peroxidase, and decreased reactive oxygen species content. GLA reduced apoptosis, as indicated by decreases in the BAX/BCL2 expression level and apoptosis percentage. GLA activated autophagy, autophagosome-lysosome fusion, and LKB1-AMPK-mTOR signaling and upregulated mRNA and protein expressions of Beclin-1, autophagy-related 5, and liver kinase B1 (LKB1). These effects of GLA on lipid metabolism disorders of PA-treated hepatocytes were reversed by autophagy inhibitor 3MA and AMPK inhibitor compound C, confirming our conclusions. Overall, GLA can protect AML-12 cells from lipid metabolism disorder caused by PA via balancing autophagy and apoptosis mediated by the LKB1-AMPK-mTOR pathway. Consequently, GLA, as a dietary supplement, can help to prevent and treat NAFLD by regulating lipid metabolism and autophagy.