DIA-Based Proteomic Analysis Reveals MYOZ2 as a Key Protein Affecting Muscle Growth and Development in Hybrid Sheep.

Hybridization of livestock can be used to improve varieties, and different hybrid combinations produce unique breeding effects. In this study, male Southdown and Suffolk sheep were selected to hybridize with female Hu sheep to explore the effects of male parentage on muscle growth and the development of offspring. Using data-independent acquisition technology, we identified 119, 187, and 26 differentially abundant proteins (DAPs) between Hu × Hu (HH) versus Southdown × Hu (NH), HH versus Suffolk × Hu (SH), and NH versus SH crosses. Two DAPs, MYOZ2 and MYOM3, were common to the three hybrid groups and were mainly enriched in muscle growth and development-related pathways. At the myoblast proliferation stage, MYOZ2 expression decreased cell viability and inhibited proliferation. At the myoblast differentiation stage, MYOZ2 expression promoted myoblast fusion and enhanced the level of cell fusion. These findings provide new insights into the key proteins and metabolic pathways involved in the effect of male parentage on muscle growth and the development of hybrid offspring in sheep.

Revealing the degrading-possibility of methyl red by two azoreductases of Anoxybacillus sp. PDR2 based on molecular docking.

Azo dyes, as the most widely used synthetic dyes, are considered to be one of the culprits of water resources and environmental pollution. Anoxybacillus sp. PDR2 is a thermophilic bacterium with the ability to degrade azo dyes, whose genome contains two genes encoding azoreductases (named AzoPDR2-1 and AzoPDR2-2). In this study, through response surface methodology (RSM), when the initial pH, inoculation volume and Mg2+ addition amount were 7.18, 10.72% and 0.1 g/L respectively, the decolorization rate of methyl red (MR) (200 mg/L) could reach its maximum (98.8%). The metabolites after biodegradation were detected by UV-Vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), and liquid chromatography mass spectrometry (LC-MS/MS), indicating that MR was successfully decomposed into 4-aminobenzoic acid and other small substrates. In homologous modeling, it was found that both azoreductases were flavin-dependent azoreductases, and belonged to the α/ß structure, using the Rossmann fold. In their docking results with the cofactor flavin mononucleotide (FMN), FMN bound to the surface of the protein dimer. Nicotinamide adenine dinucleotide (NADH) was superimposed on the plane of the pyrazine ring between FMN and the activity pocket of protein. Besides, both azoreductase complexes (azoreductase-FMN-NADH) exhibited a substrate preference for MR. Asn104 and Tyr74 played an important role in the combination of the azoreductase AzoPDR2-1 complex and the azoreductase AzoPDR2-2 complex with MR, respectively. This provided assistance for studying the mechanism of azoreductase biodegradation of azo dyes in thermophilic bacteria.

Single-Cell Transcriptome Sequence Profiling on the Morphogenesis of Secondary Hair Follicles in Ordos Fine-Wool Sheep.

The Ordos fine-wool sheep is a high-quality breed in China that produces superior natural textiles and raw materials such as wool and lamb meat. However, compared to the Australian Merino sheep, there is still a gap in terms of the wool fiber fineness and wool yield. The hair follicle is the main organ that controls the type of wool fiber, and the morphological changes in the secondary hair follicle are crucial in determining wool quality. However, the process and molecular mechanisms of hair follicle morphogenesis in Ordos fine-wool sheep are not yet clear. Therefore, analyzing the molecular mechanisms underlying the process of follicle formation is of great significance for improving the fiber diameter and wool production of Ordos fine-wool sheep. The differential expressed genes, APOD, POSTN, KRT5, and KRT15, which related to primary hair follicles and secondary hair follicles, were extracted from the dermal papillae. Based on pseudo-time analysis, the differentiation trajectories of dermal lineage cells and epidermal lineage cells in the Ordos fine-wool sheep were successfully constructed, providing a theoretical basis for breeding research in Ordos fine-wool sheep.

Lawsonia intracellularis flagellin protein LfliC stimulates NF-κB and MAPK signaling pathways independently of TLR5 interaction.

Lawsonia intracellularis, a Gram-negative obligate intracellular bacterium and etiologic agent of porcine proliferative enteropathy, was observed to have a long, single, and unipolar flagellum. Bacterial flagellar filament comprises thousands of copies of the protein flagellin (FliC), and has been reported to be recognized by Toll-like receptor (TLR5) to activate the NF-κB and MAPK signaling pathways, thereby inducing the expression of proinflammatory genes. Recently, two L. intracellularis flagellin proteins, LfliC and LFliC, were reported to be involved in bacterial-host interaction and immune response. Here, to further explore the role of LfliC in proinflammatory response, we purified LfliC, and found that its exposure could activate NF-κB signaling pathway in both HEK293T and IPI-FX cells, as well as activate MAPK p38 and ERK1/2 in HEK293T cells but not in IPI-FX cells. However, our yeast two-hybrid and co-immunoprecipitation assay results revealed that LfliC has no interaction with the porcine TLR5 ECD domain though it harbors the conserved D1-like motif required for the interaction. Moreover, LfliC was identified as a substrate of the virulence-associated type III secretion system (T3SS) by using the heterologous Y. enterocolitica system. Transient expression of LfliC also activated the NF-κB and MAPK signaling pathway in HEK293T cells. Collectively, our results suggest that both the exposure and expression of L. intracellularis LfliC can induce the NF-κB and MAPK signaling pathway in mammalian cells. Our findings may provide important implications and resources for the development of diagnostic tools or vaccines and dissection of the pathogenesis of L. intracellularis.

New insights into the typical nitrogen-containing heterocyclic compound-quinoline degradation and detoxification by microbial consortium: Integrated pathways, meta-transcriptomic analysis and toxicological evaluation.

As the primary source of COD in industrial wastewater, quinoline has aroused increasing attention because of its potential teratogenic, carcinogenic, and mutagenic effects in the environment. The activated sludge isolate quinoline-degrading microbial consortium (QDMC) efficiently metabolizes quinoline. However, the molecular underpinnings of the degradation mechanism of quinoline by QDMC have not been elucidated. High-throughput sequencing revealed that the dominant genera included Diaphorobacter, Bacteroidia, Moheibacter and Comamonas. Furthermore, a positive strong correlation was observed between the key bacterial communities (Diaphorobact and Bacteroidia) and quinoline degradation. According to metatranscriptomics, genes associated with quorum sensing, ABC transporters, component systems, carbohydrate, aromatic compound degradation, energy metabolism and amino metabolism showed high expression, thus improving adaptability of microbial community to quinoline stress. In addition, the mechanism of QDMC in adapting and resisting to extreme environmental conditions in line with the corresponding internal functional properties and promoting biogegradation efficiency was illustrated. Based on the identified products, QDMC effectively mineralized quinoline into low-toxicity metabolites through three major metabolic pathways, including hydroxyquinoline, 1,2,3,4-H-quinoline, 5,6,7,8-tetrahydroquinoline and 1-oxoquinoline pathways. Finally, toxicological, genotoxicity and phytotoxicity studies supported the detoxification of quinoline by the QDMC. This study provided a promising approach for the stable, environmental-friendly and efficient bioremediation applications for quinoline-containing wastewater.

Characterization of translocon proteins in the type III secretion system of Lawsonia intracellularis.

Lawsonia intracellularis, the etiologic agent of proliferative enteropathy (PE), is an obligate intracellular Gram-negative bacterium possessing a type III secretion system (T3SS), which enables the pathogen to translocate effector proteins into targeted host cells to modulate their functions. T3SS is a syringe-like apparatus consisting of a base, an extracellular needle, a tip, and a translocon. The translocon proteins assembled by two hydrophobic membrane proteins can form pores in the host-cell membrane, and therefore play an essential role in the function of T3SS. To date, little is known about the T3SS and translocon proteins of L. intracellularis. In this study, we first analyzed the conservation of the T3S apparatus between L. intracellularis and Yersinia, and characterized the putative T3S hydrophobic major translocon protein LI1158 and minor translocon protein LI1159 in the L. intracellularis genome. Then, by using Yersinia pseudotuberculosis as a surrogate system, we found that the full-length LI1158 and LI1159 proteins, but not the putative class II chaperone LI1157, were secreted in a - Ca2+ and T3SS-dependent manner and the secretion signal was located at the N terminus (aa 1-40). Furthermore, yeast-two hybrid experiments revealed that LI1158 and LI1159 could self-interact, and LI1159 could interact with LI1157. However, unlike CPn0809 and YopB, which are the major hydrophobic translocon proteins of the T3SS of C. pneumoniae and Yersinia, respectively, full-length LI1158 was non-toxic to both yeast and Escherichia coli cells, but full-length LI1159 showed certain toxicity to E. coli cells. Taken together, despite some differences from the findings in other bacteria, our results demonstrate that LI1158 and LI1159 may be the translocon proteins of L. intracellularis T3SS, and probably play important roles in the translocation of effector proteins at the early pathogen infection stage.

Genomic Selection for Live Weight in the 14th Month in Alpine Merino Sheep Combining GWAS Information.

Alpine Merino Sheep is a novel breed reared from Australian Merino Sheep as the father and Gansu Alpine Fine-Wool Sheep as the mother, living all year in cold and arid alpine areas with exceptional wool quality and meat performance. Body weight is an important economic trait of the Alpine Merino Sheep, but there is limited research on identifying the genes associated with live weight in the 14th month for improving the accuracy of the genomic prediction of this trait. Therefore, this study's sample comprised 1310 Alpine Merino Sheep ewes, and the Fine Wool Sheep 50K Panel was used for genome-wide association study (GWAS) analysis to identify candidate genes. Moreover, the trial population (1310 ewes) in this study was randomly divided into two groups. One group was used as the population for GWAS analysis and screened for the most significant top 5%, top 10%, top 15%, and top 20% SNPs to obtain prior marker information. The other group was used to estimate the genetic parameters based on the weight assigned by heritability combined with different prior marker information. The aim of this study was to compare the accuracy of genomic breeding value estimation when combined with prior marker information from GWAS analysis with the optimal linear unbiased prediction method for genome selection (GBLUP) for the breeding value of target traits. Finally, the accuracy was evaluated using the five-fold cross-validation method. This research provides theoretical and technical support to improve the accuracy of sheep genome selection and better guide breeding. The results demonstrated that eight candidate genes were associated with GWAS analysis, and the gene function query and literature search results suggested that FAM184B, NCAPG, MACF1, ANKRD44, DCAF16, FUK, LCORL, and SYN3 were candidate genes affecting live weight in the 14th month (WT), which regulated the growth of muscle and bone in sheep. In genome selection analysis, the heritability of GBLUP to calculate the WT was 0.335-0.374, the accuracy after five-fold cross-verification was 0.154-0.190, and after assigning different weights to the top 5%, top 10%, top 15%, and top 20% of the GWAS results in accordance with previous information to construct the G matrix, the accuracy of the WT in the GBLUP model was improved by 2.59-7.79%.

Thiocyanate-degrading microflora alleviates thiocyanate stress on tomato seedlings by improving plant and rhizosphere microenvironment.

Thiocyanate in irrigation water can adversely affect plant growth and development. A previously constructed microflora with effective thiocyanate-degrading ability was used to investigate the potential of bacterial degradation for thiocyanate bioremediation. The root and aboveground part dry weight of plants inoculated with the degrading microflora increased by 66.67% and 88.45%, respectively, compared to those plants without the microflora. The supplementation of thiocyanate-degrading microflora (TDM) significantly alleviated the interference of thiocyanate in mineral nutrition metabolism. Moreover, the supplementation of TDM significantly reduced the activities of antioxidant enzymes, lipid peroxidation, and DNA damage and it protected plants from excessive thiocyanate, while the crucial antioxidant enzyme (peroxidase) decreased by 22.59%. Compared with the control without TDM supplementation, the soil sucrase content increased by 29.58%. The abundances of Methylophilus, Acinetobacter, unclassified Saccharimonadales, and Rhodanobacter changed from 19.92%, 6.63%, 0.79%, and 3.90%-13.19%, 0.27%, 3.06%, and 5.14%, respectively, with TDM supplementation. Caprolactam, 5,6-dimethyldecane, and pentadecanoic acid seem to have an effect on the structure of the microbial community in the rhizosphere soil. The above results indicated TDM supplementation can significantly reduce the toxic effects of thiocyanate on the tomato-soil microenvironment.

Integration of proteome and metabolome profiling to reveal heat stress response and tolerance mechanisms of Serratia sp. AXJ-M for the bioremediation of papermaking black liquor.

The use of thermophilic bacteria for treating paper black liquor seems to be an efficient bioremediation strategy. In our previous work, the lignin-degrading bacterium Serratia sp. AXJ-M exhibited excellent heat tolerance ability. However, the molecular mechanism of its response to heat stress is unknown. Therefore, the heat stress response of AXJ-M was investigated using morphological and analytical methods. A comparative genomics analysis revealed interesting insights into the adaptability of the genetic basis of AXJ-M to harsh environments. Moreover, TMT quantitative proteomic analysis and parallel reaction monitoring (PRM) assays revealed that proteins related to both component systems, ABC transporters, carbohydrate, and amino metabolism, energy metabolism, etc., were differentially expressed. The non-targeted metabolome analysis revealed that the metabolic pathways associated with the fatty acid and amino acid biosynthesis and metabolism, together with the TCA cycle were most significantly enriched. Furthermore, integrated omics suggested that AXJ-M made metabolic adaptations to compensate for the increased energy demand caused by adverse environmental stimuli. The dominant heat regulator HspQ mediated heat adaptation of AXJ-M at high temperatures and modulated DyP expression. To summarize, the present study sheds light on the effect of high temperature on the lignin-degrading bacterium and its tolerance and underlying regulatory mechanisms.

Biodegradation characteristics of lignin in pulping wastewater by the thermophilic Serratia sp. AXJ-M: Performance, genetic background, metabolic pathway and toxicity assessment.

The key to the efficient removal of pulping wastewater lies in the effective degradation of lignin at high temperature. There is thus an urgent need to seek effective eco-environmental techniques to overcome this environmental limit for lignin degradation. The soil isolate thermophilic Serratia sp. AXJ-M efficiently metabolizes lignin. Nevertheless, the underlying comprehensive molecular mechanism of lignin degradation by thermophilic AXJ-M is poorly understood. Here, strain AXJ-M showed excellent degradation ability toward diverse lignin-related aromatic compounds. Functional genome analysis and RNA-Seq disclosed several traits which in joint consideration suggest a high efficiency of AXJ-M representative to the lignin degradation and environmental adaptation. Multiomics analyses combined with GC-MS revealed seven potential lignin biodegradation pathways. DyP was predicted to be involved in the breakdown of the ß-O-4 ether bond, Cα-Cß bond and Cα oxidation of lignin by prokaryotic expression and gene knockout and complementation. Molecular docking deepens the understanding of the interaction between DyP and lignin. Toxicity assessment experiments clearly indicated that AXJ-M significantly reduced the toxicity of the metabolites. This work expands the knowledge about the degradation mechanism of thermophilic lignin-degrading bacteria, most importantly, offers a new perspective on potential applications in utilizing this strain in pulping wastewater bioremediation.

Comprehensive Analysis of the Longissimus Dorsi Transcriptome and Metabolome Reveals the Regulatory Mechanism of Different Varieties of Meat Quality.

The beef quality significantly varies between breeds. Pingliang Red Cattle resembles Wagyu in fat deposition and flavor. To screen key factors affecting beef quality, we performed meat quality trait testing, RNA-seq, and metabolomics on the longissimus dorsi of Pingliang Red Cattle, Wagyu cross F1 generation, and Simmental cattle. The gene and metabolite expression profiles were similar between Pingliang Red Cattle and Wagyu cross F1 generation. Genes such as FASN, ACACA, PLIN1, and FABP4 were significantly upregulated in the Pingliang Red Cattle and Wagyu cross F1 generation (P < 0.05). Similarly, numerous metabolites, such as 3-iodo-l-tyrosine, arachidonic acid, and cis-aconitate, which may improve the beef quality such as fat deposition and tenderness, were found in higher levels in the Pingliang Red Cattle and Wagyu cross F1 generation. This study revealed differences in the transcriptional and metabolic levels between Pingliang Red Cattle and premium beef breeds, suggesting that Pingliang Red Cattle harbors the genetic potential for breeding high-grade beef cattle.

Differential tissue expression of sex steroid-synthesizing enzyme CYP11A1 in male Tibetan sheep (Ovis aries).

Steroid metabolism is a fundament to testicular development and function. The cytochrome P450, family 11, subfamily A, polypeptide 1 (CYP11A1) is a key rate-limiting enzyme for catalyzing the conversion of cholesterol to pregnenolone. However, despite its importance, what expression and roles of CYP11A1 possesses and how it regulates the testicular development and spermatogenesis in Tibetan sheep remains largely unknown. Based on this, we evaluated the expression and localization patterns of CYP11A1 in testes and epididymides of Tibetan sheep at three developmental stages (three-month-old, pre-puberty; one-year-old, sexual maturity and three-year-old, adult) by quantitative real-time PCR (qPCR), western blot and immunofluorescence. The results showed that CYP11A1 mRNA and protein were expressed in testes and epididymides throughout the development stages and obviously more intense in one- and three-year-old groups than three-month-old group (except for the caput epididymidis). Immunofluorescence assay showed that the CYP11A1 protein was mainly located in Leydig cells and epididymal epithelial cells. In addition, positive signals of CYP11A1 protein were observed in germ cells, epididymal connective tissue and sperms stored in the epididymal lumen. Collectively, these results suggested that the CYP11A1 gene might be mainly involved in regulating spermatogenesis and androgen synthesis in developmental Tibetan sheep testis and epididymis.

A newly developed consortium with a highly efficient thiocyanate degradation capacity: A comprehensive investigation of the degradation and detoxification potential.

Thiocyanate-containing wastewater harms ecosystems and can cause serious damage to animals and plants, so it is urgent to treat it effectively. In this study, a new efficient thiocyanate-degrading consortium was developed and its degradation characteristics were studied. It was found that up to 154.64 mM thiocyanate could be completely degraded by this consortium over 6 days of incubation, with a maximum degradation rate of 1.53 mM h-1. High-throughput sequencing analysis showed that Thiobacillus (77.78%) was the predominant thiocyanate-degrading bacterial genus. Plant toxicology tests showed that the germination index of mung bean and rice seeds cultured with media obtained after thiocyanate degradation by the consortium increased by 94% and 84.83%, respectively, compared with the control group without thiocyanate degradation. Cytotoxicity tests showed that thiocyanate without degradation significantly decreased the Neuro-2a cell activity and mitochondrial membrane potential; induced reactive oxygen species generation and apoptosis; increased the cellular Ca2+ concentration; and damaged the cell nucleus and DNA. Furthermore, the thiocyanate degradation products produced the consortium were almost totally non-toxic, revealing the same characteristics as those of the control using distilled water. This study shows that the consortium has a high degradation efficiency and detoxification characteristics, as well as great application potential in bioremediation of industrial thiocyanate-containing wastewater.

Treatment of thiocyanate-containing wastewater: a critical review of thiocyanate destruction in industrial effluents.

Thiocyanate is a common pollutant in gold mine, textile, printing, dyeing, coking and other industries. Therefore, thiocyanate in industrial wastewater is an urgent problem to be solved. This paper reviews the chemical properties, applications, sources and toxicity of thiocyanate, as well as the various treatment methods for thiocyanate in wastewater and their advantages and disadvantages. It is emphasized that biological systems, ranging from laboratory to full-scale, are able to successfully remove thiocyanate from factories. Thiocyanate-degrading microorganisms degrade thiocyanate in autotrophic manner for energy, while other biodegrading microorganisms use thiocyanate as a carbon or nitrogen source, and the biochemical pathways and enzymes involved in thiocyanate metabolism by different bacteria are discussed in detail. In the future, degradation mechanisms should be investigated at the molecular level, with further research aiming to improve the biochemical understanding of thiocyanate metabolism and scaling up thiocyanate degradation technologies from the laboratory to a full-scale.

Effect of moisture content on the evolution of bacterial communities and organic matter degradation during bioaugmented biogas residues composting.

Composting is an excellent way to recycle biogas residues into a stable, non-toxic agricultural end product. In this study, the dynamic changes of physical-chemical parameters and bacterial community in three groups of bioaugmentation composting systems at different moisture contents (MC) of 50% (MC50), 60% (MC60) and 70% (MC70) were monitored. The differences of bacterial communities in composts with different initial MC were compared, and the interaction between biological and non-biological parameters was also explored. The results revealed that after 30 days of composting, the biogas residues compost in MC60 reached highest temperature of 64 °C, total Kjeldahl nitrogen (TKN) of 2%, seed germination index (GI) of 110%, and the longest thermophilic period duration of 5 days (55 °C). Additionally, the result of high-throughput sequencing showed that the diversity of bacterial communities in MC60 was the highest, and the abundance of Actinobacteria (16.93-52.63%), Firmicutes (8.71-56.75%), and Proteobacteria (16.88-46.95%) in all groups were the highest at phylum level. The LEfSe analysis indicated that the abundance of Ochrobactrum and Cellulomonadaceae in MC60 was significantly (p < 0.05) higher than with other treatments. Moreover, canonical correspondence analysis (CCA) indicated thermophilic period duration is significantly (p < 0.05) positively correlated with Paenibacillus. Besides, it was found the relative abundance of Nocardiopsis and Georgenia has a significant (p < 0.01) correlation with the fertilizer efficiency of compost. These results showed that controlling the initial moisture content at 60% can improve the maturity and fertilizer efficiency of compost, and enable the bacteria beneficial to composting to gain the advantage of proliferation.

Effects of Pgam1-mediated glycolysis pathway in Sertoli cells on Spermatogonial stem cells based on transcriptomics and energy metabolomics.

Spermatogenesis is a complex process involving a variety of intercellular interactions and precise regulation of gene expression. Spermatogenesis is sustained by a foundational Spermatogonial stem cells (SSCs) and in mammalian testis. Sertoli cells (SCs) are the major component of SSC niche. Sertoli cells provide structural support and supply energy substrate for developing germ cells. Phosphoglycerate mutase 1 (Pgam1) is a key enzyme in the glycolytic metabolism and our previous work showed that Pgam1 is expressed in SCs. In the present study, hypothesized that Pgam1-depedent glycolysis in SCs plays a functional role in regulating SSCs fate decisions. A co-culture system of murine SCs and primary spermatogonia was constructed to investigate the effects of Pgam1 knockdown or overexpression on SSCs proliferation and differentiation. Transcriptome results indicated that overexpression and knockdown of Pgam1 in SCs resulted in up-regulation of 458 genes (117 down-regulated, 341 up-regulated) and down-regulation of 409 genes (110 down-regulated, 299 up-regulated), respectively. Further analysis of these DEGs revealed that GDNF, FGF2 and other genes that serve key roles in SSCs niche maintenance were regulated by Pgam1. The metabolome results showed that a total of 11 and 16 differential metabolites were identified in the Pgam1 gene overexpression and knockdown respectively. Further screening of these metabolites indicated that Sertoli cell derived glutamate, glutamine, threonine, leucine, alanine, lysine, serine, succinate, fumarate, phosphoenolpyruvate, ATP, ADP, and AMP have potential roles in regulating SSCs proliferation and differentiation. In summary, this study established a SCs-SSCs co-culture system and identified a list of genes and small metabolic molecules that affect the proliferation and differentiation of SSCs. This study provides additional insights into the regulatory mechanisms underlying interactions between SCs and SSCs during mammalian spermatogenesis.

miR-1285-3p targets TPI1 to regulate the glycolysis metabolism signaling pathway of Tibetan sheep Sertoli cells.

Glycolysis in Sertoli cells (SCs) can provide energy substrates for the development of spermatogenic cells. Triose phosphate isomerase 1 (TPI1) is one of the key catalytic enzymes involved in glycolysis. However, the biological function of TPI1 in SCs and its role in glycolytic metabolic pathways are poorly understood. On the basis of a previous research, we isolated primary SCs from Tibetan sheep, and overexpressed TPI1 gene to determine its effect on the proliferation, glycolysis, and apoptosis of SCs. Secondly, we investigated the relationship between TPI1 and miR-1285-3p, and whether miR-1285-3p regulates the proliferation and apoptosis of SCs, and participates in glycolysis by targeting TPI1. Results showed that overexpression of TPI1 increased the proliferation rate and decreased apoptosis of SCs. In addition, overexpression of TPI1 altered glycolysis and metabolism signaling pathways and significantly increased amount of the final product lactic acid. Further analysis showed that miR-1285-3p inhibited TPI1 by directly targeting its 3'untranslated region. Overexpression of miR-1285-3p suppressed the proliferation of SCs, and this effect was partially reversed by restoration of TPI1 expression. In summary, this study shows that the miR-1285-3p/TPI1 axis regulates glycolysis in SCs. These findings add to our understanding on the regulation of spermatogenesis in sheep and other mammals.

Proteome Informatics in Tibetan Sheep (Ovis aries) Testes Suggest the Crucial Proteins Related to Development and Functionality.

Testis has an indispensable function in male reproduction of domestic animals. Tibetan sheep (Ovis aries) is a locally adapted breed of sheep raised in the Qinghai-Tibet Plateau, with outsized roles in providing the livelihood for millions of residents. Nevertheless, less is known on how protein expression and their functional roles in developmental testes of such breed limit their use in breeding efforts. In this study, we obtained comprehensive protein profiles from testes of Tibetan sheep at three developmental stages (including pre-puberty, post-puberty, and adulthood) using data-independent acquisition-based proteomic strategy to quantitatively identify the differentially abundant proteins (DAPs) associated with testicular development and function and to unravel the molecular basis of spermatogenesis. A total of 6,221 proteins were differentially expressed in an age-dependent manner. The reliability of the gene expression abundance was corroborated by quantitative PCR and targeted parallel reaction monitoring. These DAPs were significantly enriched to biological processes concerning spermatid development and sperm deformation, mitosis, glycolytic process, cell-cell/extracellular matrix (ECM) junctions, cell proliferation, apoptosis, and migration and to the pathways including, developmental process and sexual reproduction-related (such as VEGF, estrogen, insulin, GnRH, Hippo, PI3K-Akt, mTOR, MAPK, and AMPK), and testicular cell events-related pathways (such as tight/gap/adherens junctions, ECM-receptor interaction, regulation of actin cytoskeleton, glycolysis, cell cycle, and meiosis). Based on these bioinformatics analysis, we constructed four protein-protein interaction network, among which the proteins are involved in mitosis, meiosis, spermiogenesis, and testicular microenvironment, respectively. Altogether, these bioinformatics-based sequencing results suggest that many protein-coding genes were expressed in a development-dependent manner in Tibetan sheep testes to contribute to the testicular cell development and their surrounding microenvironment remodeling at various stages of spermatogenesis. These findings have important implications for further understanding of the mechanisms underlying spermatogenesis in sheep and even other plateau-adapted animals.

Molecular response of Anoxybacillus sp. PDR2 under azo dye stress: An integrated analysis of proteomics and metabolomics.

Treating azo dye wastewater using thermophilic bacteria is considered a more efficient bioremediation strategy. In this study, a thermophilic bacterial strain, Anoxybacillus sp. PDR2, was regarded as the research target. This strain was characterized at different stages of azo dye degradation by using TMT quantitative proteomic and non-targeted metabolome technology. A total of 165 differentially expressed proteins (DEPs) and 439 differentially metabolites (DMs) were detected in comparisons between bacteria with and without azo dye. It was found that Anoxybacillus sp. PDR2 can degrade azo dye Direct Black G (DBG) through extracellular electron transfer with glucose serving as electron donors. Most proteins related to carbohydrate metabolism, including acetoacetate synthase, and malate synthase G, were overexpressed to provide energy. The bacterium can also self-synthesize riboflavin as a redox mediator of in vitro electron transport. These results lay a theoretical basis for industrial bioremediation of azo dye wastewater.

Lawsonia intracellularis LI0666 is a new EPIYA effector exported by the Yersinia enterocolitica type III secretion system.

Lawsonia intracellularis is the causative agent of proliferative enteropathy. While it harbors genes encoding the entire apparatus required for the type III secretion system (T3SS) and the expression of some of these components has been detected during experimental infection, the identification of L. intracellularis T3SS substrates (effector proteins) has been hampered. The Yersinia T3SS and yeast growth inhibition assays are two important heterologous systems used for the characterization of effector proteins. Bacterial EPIYA effectors are a distinct class of bacterial effectors defined by the presence of EPIYA or the EPIYA-related motif. When delivered into host cells via a T3SS or type IV secretion system, these effectors undergo tyrosine phosphorylation of the EPIYA motif, which enables them to manipulate host cell signaling by promiscuously interacting with multiple SH2 domain-containing proteins. A previous study showed that L. intracellularis LI0666 contains two EPIYA motifs and speculated that this protein could be a T3SS effector. In this study, we show that LI0666 is secreted by Yersinia in a T3SS-dependent manner and inhibits yeast growth. LI0666 is phosphorylated at tyrosine residues in porcine intestinal epithelial cells and in human epithelial cells. Like the archetypal EPIYA effector CagA, the EPIYA-containing region is not required for LI0666 association with yeast and mammalian cell membranes. Our results indicate that LI0666 is an authentic bacterial EPIYA effector. Identification of the tyrosine kinases that are responsible for LI0666 phosphorylation and the SH2 domain-containing host proteins that LI0666 interacts with will help to explore the molecular mechanisms of LI0666 in disease development.