[Identification of Cervi Cornu (Cervus elaphus) and its formula granules based on PCR-RFLP].

Cervi Cornu is the ossified antler, or the base antler that falls off in the spring of the following year after the pilose antler is sawn off from Cervus elaphus or C. nippon, as a precious traditional Chinese medicine, has been recognized for its medicinal value and widely used in clinical practice. However, the origins of Cervi Cornu are miscellaneous, and Cervi Cornu is even mixed with adulterants in the market. Currently, there is a shortage of ways to identify Cervi Cornu and no standard to control the quality of Cervi Cornu. So it is valuable to develop a way to effectively identify Cervi Cornu from the adulterants. In this study, the differences in the mitochondrial barcode cytochrome b(Cytb) gene sequences of C. elaphus, C. nippon and their related species were compared and the specific single nucleotide polymorphism(SNP) sites on the Cytb sequences of Cervi Cornu were screened out. According to the screened SNPs, Cervi Cornu-specific primers dishmy-F and dishmy-R were designed. The PCR system was established and optimized, and the tolerance and feasibility of Taq polymerases and PCR systems affecting the repeatability of the PCR method were investigated. The amplification products of C. elaphus and C. nippon were digested using the restriction enzyme MseⅠ. The results showed that after electrophoresis of the product from PCR with the annealing temperature of 56 ℃ and 35 cycles, a single specific band at about 100 bp was observed for C. elaphus samples, and the product of C. elaphus samples was 60 bp shorter than that of C. nippon samples. There was no band for adulterants from other similar species such as Alces alces, Rangifer tarandus, Odocoileus virginianus, O. hemionus, Cap-reolus pygargus, Przewalskium albirostis and negative controls. The polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) method established in this study can quickly and accurately identify Cervi Cornu originated from C. elaphus in crude drugs, standard decoctions, and formula granules, and distinguish the origins of Cervi Cornu products, i.e., C. nippon and similar species. This study can be a reference for other studies on the quality standard of other formula granules of traditional Chinese medicines.

Closing the gap: Solving complex medically relevant genes at scale.

Comprehending the mechanism behind human diseases with an established heritable component represents the forefront of personalized medicine. Nevertheless, numerous medically important genes are inaccurately represented in short-read sequencing data analysis due to their complexity and repetitiveness or the so-called 'dark regions' of the human genome. The advent of PacBio as a long-read platform has provided new insights, yet HiFi whole-genome sequencing (WGS) cost remains frequently prohibitive. We introduce a targeted sequencing and analysis framework, Twist Alliance Dark Genes Panel (TADGP), designed to offer phased variants across 389 medically important yet complex autosomal genes. We highlight TADGP accuracy across eleven control samples and compare it to WGS. This demonstrates that TADGP achieves variant calling accuracy comparable to HiFi-WGS data, but at a fraction of the cost. Thus, enabling scalability and broad applicability for studying rare diseases or complementing previously sequenced samples to gain insights into these complex genes. TADGP revealed several candidate variants across all cases and provided insight into LPA diversity when tested on samples from rare disease and cardiovascular disease cohorts. In both cohorts, we identified novel variants affecting individual disease-associated genes (e.g., IKZF1, KCNE1). Nevertheless, the annotation of the variants across these 389 medically important genes remains challenging due to their underrepresentation in ClinVar and gnomAD. Consequently, we also offer an annotation resource to enhance the evaluation and prioritization of these variants. Overall, we can demonstrate that TADGP offers a cost-efficient and scalable approach to routinely assess the dark regions of the human genome with clinical relevance.

Somatic mutations of esophageal adenocarcinoma: a comparison between Black and White patients.

Esophageal adenocarcinoma is the most common histological subtype of esophageal cancer in Western countries and shows poor prognosis with rapid growth. EAC is characterized by a strong male predominance and racial disparity. EAC is up to fivefold more common among Whites than Blacks, yet Black patients with EAC have poorer survival rates. The racial disparity remains largely unknown, and there is limited knowledge of mutations in EAC regarding racial disparities. We used whole-exome sequencing to show somatic mutation profiles derived from tumor samples from 18 EAC male patients. We identified three molecular subgroups based on the pre-defined esophageal cancer-specific mutational signatures. Group 1 is associated with age and NTHL1 deficiency-related signatures. Group 2 occurs primarily in Black patients and is associated with signatures related to DNA damage from oxidative stress and NTHL1 deficiency-related signatures. Group 3 is associated with defective homologous recombination-based DNA often caused by BRCA mutation in White patients. We observed significantly mutated race related genes (LCE2B in Black, SDR39U1 in White) were (q-value < 0.1). Our findings underscore the possibility of distinct molecular mutation patterns in EAC among different races. Further studies are needed to validate our findings, which could contribute to precision medicine in EAC.

Genetic sex validation for sample tracking in next-generation sequencing clinical testing.

OBJECTIVE: Data from DNA genotyping via a 96-SNP panel in a study of 25,015 clinical samples were utilized for quality control and tracking of sample identity in a clinical sequencing network. The study aimed to demonstrate the value of both the precise SNP tracking and the utility of the panel for predicting the sex-by-genotype of the participants, to identify possible sample mix-ups. RESULTS: Precise SNP tracking showed no sample swap errors within the clinical testing laboratories. In contrast, when comparing predicted sex-by-genotype to the provided sex on the test requisition, we identified 110 inconsistencies from 25,015 clinical samples (0.44%), that had occurred during sample collection or accessioning. The genetic sex predictions were confirmed using additional SNP sites in the sequencing data or high-density genotyping arrays. It was determined that discrepancies resulted from clerical errors (49.09%), samples from transgender participants (3.64%) and stem cell or bone marrow transplant patients (7.27%) along with undetermined sample mix-ups (40%) for which sample swaps occurred prior to arrival at genome centers, however the exact cause of the events at the sampling sites resulting in the mix-ups were not able to be determined.

Whole-genome resequencing reveals candidate genes associated with milk production trait in Guanzhong dairy goats.

Milk production is one of the most important economic utility of goats. Guanzhong dairy goat is a local dairy goat in Shaanxi Province of China and has high milk yield and quality. However, there are relatively few studies on molecular markers of milk production traits in Guanzhong dairy goats. In this study, we sequenced the whole genomes of 20 Guanzhong dairy goats, 10 of which had high milk yield (HM) and 10 of which had low milk yield (LM). We detected candidate signatures of selection in HM goats using Fst and π-ratio statistics and identified several candidate genes including ANPEP, ADRA1A and PRKG1 associated with milk production. Our results provide the basis for molecular breeding of milk production traits in Guanzhong dairy goats.

Proteomic Analysis of Frozen-Thawed Spermatozoa with Different Levels of Freezability in Dairy Goats.

The results of artificial insemination (AI) are adversely affected by changes in sperm motility and function throughout the cryopreservation procedure. The proteome alterations of frozen-thawed spermatozoa with various levels of freezability in dairy goats, however, remain largely unknown. To discover differentially expressed proteins (DEPs) and their roles in dairy goat sperm with high or low freezability (HF or LF), we conducted 4D-DIA quantitative proteomics analysis, the results of which are presented in this work. Additionally, we explored the underlying processes that may lead to the variations in sperm freezing resistance. A total of 263 DEPs (Fold Change > 2.0, p-value < 0.05) were identified between the HF group and LF group in frozen-thawed dairy goat spermatozoa. In our Gene Ontology (GO) enrichment analysis, the DEPs were mostly associated with the regulation of biological processes, metabolic processes, and responses to stress and cellular component biogenesis. Our Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis also revealed that the DEPs were predominantly engaged in oxidative phosphorylation, N-Glycan biosythesis, and cysteine and methionien metabolism. A protein-protein interaction (PPI) network analysis revealed 14 potential proteins (NUDFB8, SDHC, PDIA4, HSPB1, etc.) that might influence the freezability of dairy goat sperm. These findings shed light on the processes underlying alterations in the proteome and sperm freezability, aiding further research on sperm cryopreservation.

Indocyanine Green Fluorescence Imaging in the Surgical Management of Skin Squamous Cell Carcinoma.

Introduction: Indocyanine green (ICG) fluorescence imaging has been used in the resection surgery and sentinel lymph node biopsy of many tumors. The aim of the present study is to verify the feasibility and effectiveness of ICG fluorescence imaging used for guiding the biopsy and resection of skin squamous cell carcinoma (SSCC). Methods: Sixty patients were enrolled, including 18 patients of suspected SSCC and 42 patients of diagnosed SSCC on admission. The ICG fluorescence imaging-guided skin biopsy was performed preoperatively in the 18 cases of suspected SSCC. Fifty-three patients underwent ICG fluorescence imaging-guided radical excision. Results: The results showed that 138 skin tissue samples in 60 patients with preoperative or intraoperative ICG fluorescence imaging-guide biopsy were collected. For a total number of 138 biopsies, 122 specimens were squamous cell carcinoma, and the accuracy rate was 88.4%, which was significantly higher than that of the group without preoperative ICG fluorescence imaging (41/62, 66.1%, P < 0.05). Fifty-three patients underwent surgery guided with ICG fluorescence imaging. Residual fluorescent signals in 24 patients were intraoperatively found and the excision was then expanded until the signals disappeared. Follow-up to November 2022, 12 patients died, of which 5 cases died from the tumor recurrence, and the others died due to advanced ages or other reasons. The recurrence rate was 9.4%, which was not significantly different from that of the group received routine radical resection (4/35, 11.4%, P > 0.05). Moreover, sentinel lymph nodes were successfully detected under ICG fluorescence imaging in the 4 patients with suspected lymph node metastases, and the location of lymph nodes can be precisely identified. Conclusion: ICG fluorescence imaging technique can guide the pathology biopsy to improve the accuracy of pathological examination, and help to identify the boundaries of tumor tissues and sentinel lymph nodes to resect tumor radically during operation.

Genetic Sex Validation for Sample Tracking in Clinical Testing.

Objective: Data from DNA genotyping via a 96-SNP panel in a study of 25,015 clinical samples were utilized for quality control and tracking of sample identity in a clinical sequencing network. The study aimed to demonstrate the value of both the precise SNP tracking and the utility of the panel for predicting the sex-by-genotype of the participants, to identify possible sample mix-ups. Results: Precise SNP tracking showed no sample swap errors within the clinical testing laboratories. In contrast, when comparing predicted sex-by-genotype to the provided sex on the test requisition, we identified 110 inconsistencies from 25,015 clinical samples (0.44%), that had occurred during sample collection or accessioning. The genetic sex predictions were confirmed using additional SNP sites in the sequencing data or high-density genotyping arrays. It was determined that discrepancies resulted from clerical errors, samples from transgender participants and stem cell or bone marrow transplant patients along with undetermined sample mix-ups.

The master growth regulator DELLA binding to histone H2A is essential for DELLA-mediated global transcription regulation.

The DELLA genes, also known as 'Green Revolution' genes, encode conserved master growth regulators that control plant development in response to internal and environmental cues. Functioning as nuclear-localized transcription regulators, DELLAs modulate expression of target genes via direct protein-protein interaction of their carboxy-terminal GRAS domain with hundreds of transcription factors (TFs) and epigenetic regulators. However, the molecular mechanism of DELLA-mediated transcription reprogramming remains unclear. Here by characterizing new missense alleles of an Arabidopsis DELLA, repressor of ga1-3 (RGA), and co-immunoprecipitation assays, we show that RGA binds histone H2A via the PFYRE subdomain within its GRAS domain to form a TF-RGA-H2A complex at the target chromatin. Chromatin immunoprecipitation followed by sequencing analysis further shows that this activity is essential for RGA association with its target chromatin globally. Our results indicate that, although DELLAs are recruited to target promoters by binding to TFs via the LHR1 subdomain, DELLA-H2A interaction via the PFYRE subdomain is necessary to stabilize the TF-DELLA-H2A complex at the target chromatin. This study provides insights into the two distinct key modular functions in DELLA for its genome-wide transcription regulation in plants.

Circulating tumor DNA sequencing of pediatric solid and brain tumor patients: An institutional feasibility study.

The potential of circulating tumor DNA (ctDNA) analysis to serve as a real-time "liquid biopsy" for children with central nervous system (CNS) and non-CNS solid tumors remains to be fully elucidated. We conducted a study to investigate the feasibility and potential clinical utility of ctDNA sequencing in pediatric patients enrolled on an institutional clinical genomics trial. A total of 240 patients had tumor DNA profiling performed during the study period. Plasma samples were collected at study enrollment from 217 patients and then longitudinally from a subset of patients. Successful cell-free DNA extraction and quantification occurred in 216 of 217 (99.5%) of these initial samples. Twenty-four patients were identified whose tumors harbored 30 unique variants that were potentially detectable on a commercially-available ctDNA panel. Twenty of these 30 mutations (67%) were successfully detected by next-generation sequencing in the ctDNA from at least one plasma sample. The rate of ctDNA mutation detection was higher in patients with non-CNS solid tumors (7/9, 78%) compared to those with CNS tumors (9/15, 60%). A higher ctDNA mutation detection rate was also observed in patients with metastatic disease (9/10, 90%) compared to non-metastatic disease (7/14, 50%), although tumor-specific variants were detected in a few patients in the absence of radiographic evidence of disease. This study illustrates the feasibility of incorporating longitudinal ctDNA analysis into the management of relapsed or refractory patients with childhood CNS or non-CNS solid tumors.

Metformin improves sheep sperm cryopreservation via vitalizing the AMPK pathway.

Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) is a key regulator of sperm function and physiological metabolism. Metformin, an inexpensive and effective antioxidant, is known to play an important role in the activation of AMPK. Therefore metformin has potential to improve sperm cryopreservation. The aim of this study was to investigate the effect of metformin during semen cryopreservation of sheep and to find the most effective concentration in freezing extender. Semen were cryopreserved with extender containing different concentrations of metformin (0, 0.25, 0.5, 1.0, 2.0 and 4.0 mmol/L). Sperm motility, acrosome integrity and plasma membrane integrity were measured after semen freezing and thawing. All results showed that sperm quality was significantly increased in the 1.0 mmol/L metformin-treated group compared with the control group (P < 0.05). In addition, the study showed that metformin effectively reduced the content of malondialdehyde (MDA) and reactive oxygen species (ROS), and increased the activity of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase (CAT) and total antioxidant capacity (T-AOC) of freeze-thawed sperm (P < 0.05). The optimal concentration of metformin was 1.0 mmol/L. Moreover, the results showed that AMPK was localized in the acrosome region, junction and midsection of sperm, and p-AMPK was distributed in the post-acrosomal region, junction and midsection. Western blot analysis indicated that 1.0 mmol/L metformin stimulated the phosphorylation of AMPK in sperm. Further results showed that 1.0 mmol/L metformin significantly increased the mitochondrial membrane potential (ΔΨm), ATP content, glucose uptake and lactate efflux of post-thawed sperm through the AMPK pathway, improved sperm quality, and increased the cleavage rate of in vitro fertilization (P < 0.05).

Glucose Starvation Inhibits Ferroptosis by Activating the LKB1/AMPK Signaling Pathway and Promotes the High Speed Linear Motility of Dairy Goat Sperm.

In mammals, sperm acquire fertilization ability after capacitation in vitro or when in the female reproductive tract. The motility patterns of sperm undergo continuous changes from the moment of ejaculation until fertilization in the female reproductive tract. In vitro, hyperactivated motility can be induced through high glucose mediums, while in vivo, it is induced by oviduct fluids. Conversely, sperm maintain linear motility in seminal plasma or uterine fluids that contain low glucose levels. In dairy goat sperm, energy metabolism associated with capacitation depends on the energy sources in vitro, seminal plasma, or the female reproductive tract, especially the glucose levels. However, there is little experimental knowledge that glucose levels affect sperm energy metabolism in dairy goats. To clarify these hypotheses, we incubated dairy goat spermatozoa with different concentrations of rotenone-glucose (ROT), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and tigecycline (TIG) in vitro. Sperm motility attributes, ATP content, pyruvate and lactate levels, mitochondrial permeability transition pore fluorescence intensity, mitochondrial membrane potential (MMP), and protein synthesis were analyzed. Sperm motility patterns changed from circular to linear under low glucose conditions compared with those in high glucose conditions and showed a significant improvement in progressive motility and straight line speed, whereas lactate and pyruvate levels and MMP decreased remarkably. Incubation of spermatozoa with ROT, FCCP, and TIG inhibited sperm mitochondrial activity, protein synthesis, oxidative phosphorylation, and ATP levels, thereby reducing sperm motility, including the progressive motility, straight line speed, and total motility. Simultaneously, incubation of spermatozoa with Compound C under low glucose conditions significantly decreased the ATP levels and MMP, as well as liver kinase B1 and AMPK protein expression. Under low glucose conditions, sperm mainly produce ATP through mitochondrial OXPHOS to achieve high speed linear movement, inhibit ferroptosis through the LKB1/AMPK signaling pathway, and further maintain energy metabolism homeostasis.

Enhanced autophagy reversed aflatoxin B1-induced decrease in lactate secretion of dairy goat Sertoli cells.

The deleterious effects of aflatoxins, especially aflatoxin B1 (AFB1) which are widespread at all stages of food production, on the reproductive system have been widely reported in males. However, it is still far from fully understood about the toxic effect and molecular mechanism after exposure to AFB1 in various testicular cells, especially Sertoli cells (SCs) which provide various energy materials and support to the developing germ cells as nurse cells. In this work, we examined the effects of AFB1 in dairy goat SCs on lactate production and autophagy, and the role of autophagy on AFB1-induced reduction in lactate production. Mechanistically, AFB1 destroyed the energy balance and reduced the secretion of lactate in dairy goat SCs (P < 0.01), resulting in a reduced level of ATP (P < 0.01) and phosphorylation of AMPK (P < 0.01). Subsequently, activated AMPK triggers autophagy by directly phosphorylating ULK1 (P < 0.05). The enhancement of autophagy partially reversed the AFB1-induced decrease in lactate secretion by promoting glucose utilization (P < 0.01) and increasing the expression of proteins related to lactate secretion in dairy goat SCs (P < 0.05) such as GLUT1, GLUT3, LDHA, and MCT4. Collectively, our study suggests that AFB1 inhibits the secretion of lactate which supply for germ cell development by damaging the "Warburg-like" metabolism of dairy goat SCs. Moreover, autophagy contributes to the resistance of glucose metabolism damage induced by AFB1. DATA AVAILABILITY: All data generated or analyzed in this study are available from the corresponding authors upon request.

Four class A AUXIN RESPONSE FACTORs promote tomato fruit growth despite suppressing fruit set.

In flowering plants, auxin produced in seeds after fertilization promotes fruit initiation. The application of auxin to unpollinated ovaries can also induce parthenocarpy (seedless fruit production). Previous studies have shown that auxin signalling components SlIAA9 and SlARF7 (a class A AUXIN RESPONSE FACTOR (ARF)) are key repressors of fruit initiation in tomato (Solanum lycopersicum). A similar repressive role of class A ARFs in fruit set has also been observed in other plant species. However, evidence is lacking for a role of any class A ARF in promoting fruit development as predicted in the current auxin signalling model. Here we generated higher-order tomato mutants of four class A SlARFs (SlARF5, SlARF7, SlARF8A and SlARF8B) and uncovered their precise combinatorial roles that lead to suppressing and promoting fruit development. All four class A SlARFs together with SlIAA9 inhibited fruit initiation but promoted subsequent fruit growth. Transgenic tomato lines expressing truncated SlARF8A/8B lacking the IAA9-interacting PB1 domain displayed strong parthenocarpy, further confirming the promoting role of SlARF8A/8B in fruit growth. Altering the doses of these four SlARFs led to biphasic fruit growth responses, showing their versatile dual roles as both negative and positive regulators. RNA-seq and chromatin immunoprecipitation-quantitative PCR analyses further identified SlARF8A/8B target genes, including those encoding MADS-BOX transcription factors (AG1, MADS2 and AGL6) that are key repressors of fruit set. These results support the idea that SlIAA9/SlARFs directly regulate the transcription of these MADS-BOX genes to inhibit fruit set. Our study reveals the previously unknown dual function of four class A SlARFs in tomato fruit development and illuminates the complex combinatorial effects of multiple ARFs in controlling auxin-mediated fruit set and fruit growth.

Plasmonic nanorod probes' journey inside plant cells for in vivo SERS sensing and multimodal imaging.

Nanoparticle-based platforms are gaining strong interest in plant biology and bioenergy research to monitor and control biological processes in whole plants. However, in vivo monitoring of biomolecules using nanoparticles inside plant cells remains challenging due to the impenetrability of the plant cell wall to nanoparticles beyond the exclusion limits (5-20 nm). To overcome this physical barrier, we have designed unique bimetallic silver-coated gold nanorods (AuNR@Ag) capable of entering plant cells, while conserving key plasmonic properties in the near-infrared (NIR). To demonstrate cellular internalization and tracking of the nanorods inside plant tissue, we used a comprehensive multimodal imaging approach that included transmission electron microscopy (TEM), confocal fluorescence microscopy, two-photon luminescence (TPL), X-ray fluorescence microscopy (XRF), and photoacoustics imaging (PAI). We successfully acquired SERS signals of nanorods in vivo inside plant cells of tobacco leaves. On the same leaf samples, we applied orthogonal imaging methods, TPL and PAI techniques for in vivo imaging of the nanorods. This study first demonstrates the intracellular internalization of AuNR@Ag inside whole plant systems for in vivo SERS analysis in tobacco cells. This work demonstrates the potential of this nanoplatform as a new nanotool for intracellular in vivo biosensing for plant biology.

CircRNA-104718 promotes glioma malignancy through regulation of miR-218-5p/HMGB1 signalling pathway.

Increasing number of studies have proven that circular RNAs (circRNAs) play a major role in the biological processes of many different cancers, including glioma, especially as competitive molecular sponges of microRNAs (miRNAs). However, the clear molecular mechanism of the circRNA network in glioma is still not well understood. The expression level of circRNA-104718 and microRNA (miR)-218-5p in glioma tissues and cells were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The target protein's expression level was assessed by western blotting. Bioinformatics systems were used to predict the possible microRNAs and target genes of circRNA-104718, after which dual-luciferase reporter assays were used to confirm the predicted interactions. The proliferation, invasion, migration and apoptosis of glioma cells were detected by CCK, EdU, transwell, wound-healing and flow cytometry assays. CircRNA-104718 was upregulated in human glioma tissues, and a higher level of circRNA-104718 indicated poorer outcomes in glioma patients. In contrast, in glioma tissues, miR-218-5p was downregulated. Knockdown of circRNA-104718 suppressed migration and invasion while boosting the apoptosis rate of glioma cells. In addition, the upregulation of miR-218-5p in glioma cells caused the same suppression. Mechanistically, circRNA-104718 inhibited the protein expression level of high mobility group box-1 (HMGB1) by acting as a molecular sponge for miR-218-5p. CircRNA-104718 is a suppressive factor in glioma cells and might represent a new target for the treatment of glioma patients. CircRNA-104718 modulates glioma cell proliferation through the miR-218-5p/HMGB1 signalling axis. CircRNA-104718 provides a possible mechanism for understanding the pathogenesis of glioma.

TLR7/8 agonist (R848) inhibit bovine X sperm motility via PI3K/GSK3α/ß and PI3K/NFκB pathways.

Sex-control technology have great economic value and is one of the hot topics in livestock research. To produce more milk, dairy farmers prefer female offspring. X/Y sperm separation is an effective method for offspring sex control. Currently, the major commercial production method for sperm separation is flow cytometry sorting in cattle. However, flow cytometry requires expensive equipment and long sorting times. So, a simple and inexpensive method for producing a higher number of dairy cows is required. In this study, R848 activates toll-like receptor 7/8 (TLR7/8), thereby separating X from Y sperm. The results showed TLR7/8 is expressed in the tail of X sperm. Immunofluorescence (IF) of testes, epididymis, and ejaculate shows that the number of TLR7+/8+ sperm cells is up to 50 %. Furthermore, TLR7/8 agonist (R848) affects mitochondrial function through the PI3K/GSK3α/ß/hexokinase and PI3K/NFκB/hexokinase signalling pathways, inhibiting X sperm motility, while the motility of Y-sperm remains unchanged. The difference in sperm motility causes Y sperm (with high motility) to move to the upper layer and X-sperm (with low motility) to the lower layer allowing the separation of X and Y sperm. Based on this study, we reveal a simple and effective method for enriched X/Y sperms from cattle.

SPINDLY O-fucosylates nuclear and cytoplasmic proteins involved in diverse cellular processes in plants.

SPINDLY (SPY) is a novel nucleocytoplasmic protein O-fucosyltransferase that regulates target protein activity or stability via O-fucosylation of specific Ser/Thr residues. Previous genetic studies indicate that AtSPY regulates plant development during vegetative and reproductive growth by modulating gibberellin and cytokinin responses. AtSPY also regulates the circadian clock and plant responses to biotic and abiotic stresses. The pleiotropic phenotypes of spy mutants point to the likely role of AtSPY in regulating key proteins functioning in diverse cellular pathways. However, very few AtSPY targets are known. Here, we identified 88 SPY targets from Arabidopsis (Arabidopsis thaliana) and Nicotiana benthamiana via the purification of O-fucosylated peptides using Aleuria aurantia lectin followed by electron transfer dissociation-MS/MS analysis. Most AtSPY targets were nuclear proteins that function in DNA repair, transcription, RNA splicing, and nucleocytoplasmic transport. Cytoplasmic AtSPY targets were involved in microtubule-mediated cell division/growth and protein folding. A comparison with the published O-linked-N-acetylglucosamine (O-GlcNAc) proteome revealed that 30% of AtSPY targets were also O-GlcNAcylated, indicating that these distinct glycosylations could co-regulate many protein functions. This study unveiled the roles of O-fucosylation in modulating many key nuclear and cytoplasmic proteins and provided a valuable resource for elucidating the regulatory mechanisms involved.

Zearalenone induces oxidative stress and autophagy in goat Sertoli cells.

Zearalenone (ZEA), one of the non-steroidal estrogen mycotoxin, can cause male reproductive damage and genotoxicity in mammals. Testicular oxidative injury is an important factor causing male sterility. Testicular Sertoli cells are essential for spermatogenesis and male fertility. At present, the mechanism of oxidative injury in dairy goat Sertoli cells after exposure to ZEA remains unclear. This study explored the effects of ZEA on oxidative stress and autophagy in dairy goat Sertoli cells. It was found that treatment of primary Sertoli cells with 25, 50 and 100 µmol/L ZEA for 24 h can promote ROS production, decrease cell viability, antioxidant enzyme activity and mitochondrial membrane potential, induce caspase-dependent cell apoptosis and autophagy activity. ZEA-induced autophagy was confirmed by LC3-I/LC3-II transformation. More importantly, N-acetylcysteine (NAC) pretreatment can remarkably inhibit ZEA-induced oxidative stress, apoptosis and autophagy in Sertoli cells by eliminating ROS. In conclusion, this study indicates that ZEA induces oxidative stress and autophagy in dairy goat Sertoli cells by promoting ROS production.

FSH-inhibited autophagy protects against oxidative stress in goat Sertoli cells through p62-Nrf2 pathway.

Oxidative stress is a common cause of male infertility. Sertoli cells are one of the target cells of oxidative injury, which leads to impaired testicular function. Follicle-stimulating hormone (FSH) is critical in Sertoli cell function. However, the role of FSH in the response of goat Sertoli cells to H2O2-induced oxidative stress has not been studied yet. To investigate this response, we established an oxidative stress model using goat Sertoli cells. FSH pretreatment significantly enhanced the decreased cell viability (p < 0.05) caused by oxidative injury and inhibited autophagic flux. FSH significantly increased p62 mRNA and protein levels (p < 0.01). Further investigations revealed that FSH also increased the expression level and nuclear translocation of Nrf2 in Sertoli cells (p < 0.01), which resulted in increased antioxidant enzyme activity (p < 0.05). In contrast, treatment with siNrf2 and sip62 abolished this protective effect of FSH. These findings suggest that FSH protects Sertoli cells against oxidative stress via the p62-Nrf2 pathway, and that p62 accumulation maintains persistent activation of Nrf2. Thus, p62 and Nrf2 are required for FSH-mediated protective role in H2O2-induced Sertoli cell injury. The findings reveal new mechanisms by which FSH protects against oxidative injury in goat Sertoli cells.