Identification of copy number variation in Tibetan sheep using whole genome resequencing reveals evidence of genomic selection.

BACKGROUND: Copy number variation (CNV) is an important source of structural variation in the mammalian genome. CNV assays present a new method to explore the genomic diversity of environmental adaptations in animals and plants and genes associated with complex traits. In this study, the genome-wide CNV distribution characteristics of 20 Tibetan sheep from two breeds (10 Oula sheep and 10 Panou sheep) were analysed using whole-genome resequencing to investigate the variation in the genomic structure of Tibetan sheep during breeding. RESULTS: CNVs were detected using CNVnator, and the overlapping regions of CNVs between individual sheep were combined. Among them, a total of 60,429 CNV events were detected between the indigenous sheep breed (Oula) and the synthetic sheep breed (Panou). After merging the overlapping CNVs, 4927 CNV regions (CNVRs) were finally obtained. Of these, 4559 CNVRs were shared by two breeds, and there were 368 differential CNVRs. Deletion events have a higher percentage of occurrences than duplication events. Functional enrichment analysis showed that the shared CNVRs were significantly enriched in 163 GO terms and 62 KEGG pathways, which were mainly associated with organ development, neural regulation, immune regulation, digestion and metabolism. In addition, 140 QTLs overlapped with some of the CNVRs at more than 1 kb, such as average daily gain QTL, body weight QTL, and total lambs born QTL. Many of the CNV-overlapping genes such as PPP3CA, SSTR1 and FASN, overlap with the average daily weight gain and carcass weight QTL regions. Moreover, VST analysis showed that XIRP2, ABCB1, CA1, ASPA and EEF2 differed significantly between the synthetic breed and local sheep breed. The duplication of the ABCB1 gene may be closely related to adaptation to the plateau environment in Panou sheep, which deserves further study. Additionally, cluster analysis, based on all individuals, showed that the CNV clustering could be divided into two origins, indicating that some Tibetan sheep CNVs are likely to arise independently in different populations and contribute to population differences. CONCLUSIONS: Collectively, we demonstrated the genome-wide distribution characteristics of CNVs in Panou sheep by whole genome resequencing. The results provides a valuable genetic variation resource and help to understand the genetic characteristics of Tibetan sheep. This study also provides useful information for the improvement and breeding of Tibetan sheep in the future.

Expression of MSTN/Smad signaling pathway genes and its association with meat quality in Tibetan sheep (Ovis aries).

Tibetan sheep is a unique breed living in Qinghai-Tibet Plateau. Since MSTN/Smad signaling pathway plays a critical role in the regulation of muscle development, we aimed to study the mutton quality, mRNA expression of main transduction genes in the MSTN/Smad signaling pathway, and the effects of those genes on the mutton quality of Tibetan sheep in this study. Six-month-old Qinghai-Tibetan sheep were selected, slaughtered, and their Longissimus lumborum, semitendinosus muscle, arm triceps, and quadriceps femoris muscle were collected. The mutton quality was evaluated, and gene expression and their association with the mutton quality were analyzed using RT-qPCR. The results showed that the indexes of mutton quality were not significantly different between ewes and rams (p > .05) except for Warner-Bratzler shear force (WBSF) (p < .05). A total of 21 different fatty acids were detected in the muscles of Tibetan sheep, including nine types of SFA, four types of MUFA, and eight types of PUFA. The main transduction genes of the MSTN/Smad signaling pathway were found to be widely expressed in muscle tissues, but no significant differences were observed (p > .05). The correlation analysis of the main genes and mutton quality showed that MSTN was significantly correlated with redness and cooking time; Smad2, Smad3, Smad4, and TGFßRI had significant positive correlations with marbling in arm triceps; Smad3 and TGFßRII had strong negative correlations with pH24 h in Longissimus lumborum; Smad2 was negatively correlated with drip loss in Longissimus lumborum. In short, the expression level of MSTN in muscles was positively correlated with Smad2, Smad3, and Smad4 genes and negatively correlated with TGFßRII genes. Thus, the results of this study provide a theoretical basis for the regulation mechanism of the MSTN/Smad pathway on mutton quality.

Whole genome sequencing revealed genetic diversity, population structure, and selective signature of Panou Tibetan sheep.

BACKGROUND: The detection of selective traits in different populations can not only reveal current mechanisms of artificial selection for breeding, but also provide new insights into phenotypic variation in new varieties and the search for genes associated with important traits. Panou sheep is a cultivated breed of Tibetan sheep in China with stable genetic performance, consistent appearance and fast growth and development after decades of artificial selection and cultivation. Due to long-term adaptation to the high altitude, cold and hypoxic environment in the plateau area, they may have formed a unique gene pool that is different from other Tibetan sheep breeds. To explore the genetic resources of Panou sheep, we used next-generation sequencing technology for the first time to investigate the genome-wide population structure, genetic diversity, and candidate signatures of positive selection in Panou sheep. RESULTS: Comparative genomic analysis with the closely related species Oula sheep (a native breed of Tibetan sheep in China) was used to screen the population selection signal of Panou sheep. Principal component analysis and neighbor joining tree showed that Panou sheep and Oula sheep had differences in population differentiation. Furthermore, analyses of population structure, they came from the same ancestor, and when K = 2, the two populations could be distinguished. Panou sheep exhibit genetic diversity comparable to Oula sheep, as shown by observed heterozygosity, expected heterozygosity and runs of homozygosity. Genome-wide scanning using the Fst and π ratio methods revealed a list of potentially selected related genes in Panou sheep compared to Oula sheep, including histone deacetylase 9 (HDAC9), protein tyrosine kinase 2 (PTK2), microphthalmia-related transcription factor (MITF), vesicular amine transporter 1 (VAT1), trichohyalin-like 1 (TCHHL1), amine oxidase, copper containing 3 (AOC3), interferon-inducible protein 35 (IFI35). CONCLUSIONS: The results suggest that traits related to growth and development and plateau adaptation may be selection targets for the domestication and breeding improvement of Tibetan sheep. This study provides the fundamental footprints for Panou sheep breeding and management.

Effect of Zanthoxylum bungeanum essential oil on rumen enzyme activity, microbiome, and metabolites in lambs.

Antibiotics were once used in animal production to improve productivity and resistance to pathogenic microbiota. However, due to its negative effects, the search for a new class of substances that can replace its efficacy has become one of the urgent problems to be solved. Plant essential oils (EOs) as a natural feed additive can maintain microbiota homeostasis and improve animal performance. However, its specific mechanism of action needs to be further investigated. Therefore, we added different doses of essential oil of Zanthoxylum bungeanum (EOZB) to the diets of Small Tail Han Sheep hybrid male lambs (STH lambs) to evaluate the effect of EOZB on rumen enzyme activity, rumen microbiology, and its metabolites in STH lambs. Twenty STH lambs were randomly divided into four groups (n = 5/group) and provided with the same diet. The dietary treatments were as follows: basal diet (BD) group; BD+EOZB 5 ml/kg group; BD+EOZB 10 ml/kg group; BD+EOZB 15 ml/kg group. We found that EOZB 10 ml/kg helped to increase rumen pectinase (P<0.05) and lipase (P<0.05) activities. Microbial 16S rRNA gene analysis showed that EOZB significantly altered the abundance of rumen microbiota (P<0.05). LC/GC-MS metabolomic analysis showed that the addition of EOZB produced a total of 1073 differential metabolites, with 58 differential metabolites remaining after raising the screening criteria. These differential metabolites were mainly enriched in glycerophospholipid metabolism, choline metabolism in cancer, retrograde endocannabinoid signaling, benzoxazinoid biosynthesis, and protein digestion and absorption. Correlation analysis showed that some rumen microbiota were significantly correlated with differential metabolite and enzyme activities.

RNA-Seq reveals the functional specificity of epididymal caput, corpus, and cauda genes of cattleyak.

The first filial generation of the cattleyaks demonstrates hybrid vigor; however, the male cattleyaks are infertile and restrict productivity and breeding. The discovery of genes in a segment-specific approach offers valuable information and understanding concerning fertility status, yet the biology of cattleyak epididymis is still progressing. Comparative transcriptome analysis was performed on segment pairs of cattleyak epididymis. The caput versus corpus epididymis provided the highest (57.8%) differentially expressed genes (DEGs), corpus versus cauda (25.1%) followed, whereas caput versus cauda pair (17.1%) had the least DEGs. The expression levels of genes coding EPHB6, TLR1, MUC20, MT3, INHBB, TRPV5, EI24, PAOX, KIF12, DEPDC5, and KRT25, which might have the potentials to regulate the homeostasis, innate immunity, differentiation, motility, transport, and sperm maturation-related function in epididymal cells, were downregulated in the distal segment of epididymis. Top enriched KEGG pathways included mTOR, axon guidance, and taste transduction signaling pathways. EIF4B, EPHB6, and TAS2R42 were enriched in the pathways, respectively. Identifying key, new, and unexplored DEGs among the epididymal segments and further analyzing them could boost cattleyak fertility by maximizing sperm quality from genetically better sires and also facilitate better understanding of the epididymal biology.

Distribution of bacteria in different regions of the small intestine with Zanthoxylum bungeanum essential oil supplement in small-tailed Han sheep.

Zanthoxylum bungeanum essential oil (EOZB) as an extract of Zanthoxylum bungeanum has a range of pharmacological effects such as antibacterial, anti-inflammatory, and antioxidant. However, there were no relevant studies on the regulation of gut microbes by EOZB in ruminants. In this study, the effects of different doses of EOZB on the structure and distribution of microorganisms in the small intestine of small-tailed Han sheep (STH) were investigated by 16s rRNA gene sequencing technique. We found that with the intervention of EOZB. The differential bacteria of duodenal at the phylum level were Firmicutes, Bacteroidetes, Tenericutes and Proteobacteria, and genus level differential bacteria were Prevotella 1, Ruminococcus 2 and Eubacterium coprostanoligenes group. The differential bacteria of jejunal at the phylum level were Firmicutes, Bacteroidetes, Tenericutes and Proteobacteria, and genus level differential bacteria were Prevotella 1, Rikenellaceae RC9 gut group, Christensenellaceae R-7 group, Ruminococcaceae UCG-014, Saccharofermentans, Ruminococcaceae NK4A214 group and Prevotellaceae UCG-001. The differential bacteria of ileal at the phylum level were Firmicutes, Bacteroidetes and Tenericutes, and genus level differential bacteria were Prevotella 1, Christensenellaceae R-7 group, Romboutsia and Ruminococcaceae UCG-014. In addition, at the same dose of EOZB, the five most abundant genera of bacteria varied in different regions of the small intestine. Among them, the abundance of Prevotella 1, Christensenellacea R-7 group and Ruminococcus 2 in ALW group was the highest in jejunum, duodenum and ileum, respectively. The abundance of Prevotella 1, Christensenellacea R-7 group and Rikenellacea RC9 gut group in BLW group was the highest in duodenum, jejunum and ileum, respectively. The abundance of Prevotella 1, Christensenellacea R-7 group and Ruminococcaeae NK4A214 group in CLW group was the highest in jejunum, duodenum and ileum, respectively. The abundance of Prevotella 1, Ruminococcus 2 and Ruminococcus NK4A214 groups in DLW group was the highest in jejunum, duodenum and ileum, respectively. Differential bacteria formed under the regulation of EOZB are associated with the digestion and absorption of nutrients and the state of intestinal health in the host. This study is the first to investigate the effect of EOZB on the distribution and structure of bacteria in the small intestine of STH. The results of the study enriched the structure and distribution of bacteria in the small intestine of ruminants and provided new insights into the future application of herbal medicine in ruminant production. Additionally, it provides a theoretical basis for the selection of probiotic bacteria for ruminants and the development and application of microecological preparations.

High-throughput sequencing reveals differential expression of miRNAs in yak and cattleyak epididymis.

Cattleyaks (CY) are interspecific hybrids between cattle (Bos taurus) and yak (Bos gruniens, YK) exhibiting the same prominent adaptability and higher performances than YK. The main problem of this crossbreeding is that the males are sterile. Different series of events of spermatogenesis coordinate to regulate gene expressing, involving microRNAs (miRNAs). As non-coding ribonucleic acids (ncRNAs), miRNAs predominantly facilitate the regulation of gene expression at post-transcriptional stages and play important roles in the acquisition and maintenance of male fertility in reproduction. The function of miRNA in the male reproductive system extends from the testis into the epididymis, regulating gene expression and contributing to regional gene expression variations. RNA sequencing on biological replicates, we described differentially expressed miRNAs profiles for tissue from epididymis of YK and CY. In the present study, High-throughput sequencing analysis showed that 55 differentially expressed (DE) miRNAs were identified in the epididymis of YK and CY. Among these, 43 DE miRNAs were upregulated while the remaining 12 DE miRNAs were downregulated between epididymis of YK and CY. In addition, we identified that the top most important DE miRNAs, bta-miR-449c, bta-miR-539, bta- miR-136, bta-miR-504, bta-miR-31 and bta-miR-222 were involved in the process of sperm maturation in epididymis CY. It was identified that the bta-miR-449c and bta-miR-222 may play major roles in the process of sperm maturation, sperm quality, sperm count, sperm production and male infertility of CY. Furthermore, GO and KEGG analyses were performed to classify the functions of target genes for DE miRNAs. In addition, RT-qPCR validation of the DE miRNAs and its targeted genes revealed that putative miRNAs are involved in the male CY infertility by altering the gene expression. Present findings may not only increase our understanding of the molecular mechanisms regulated by the miRNAs in epididymis, but also provide a valuable information to understand the male infertility mechanism of CY.

Comparative transcriptome and histological analyses provide insights into the skin pigmentation in Minxian black fur sheep (Ovis aries).

BACKGROUND: Minxian black fur (MBF) sheep are found in the northwestern parts of China. These sheep have developed several special traits. Skin color is a phenotype subject to strong natural selection and diverse skin colors are likely a consequence of differences in gene regulation. METHODS: Skin structure, color differences, and gene expression (determined by RNA sequencing) were evaluated the Minxian black fur and Small-tail Han sheep (n = 3 each group), which are both native Chinese sheep breeds. RESULTS: Small-tail Han sheep have a thicker skin and dermis than the Minxian black fur sheep (P < 0.01); however, the quantity of melanin granules is greater (P < 0.01) in Minxian black fur sheep with a more extensive distribution in skin tissue and hair follicles. One hundred thirty-three differentially expressed genes were significantly associated with 37 ontological terms and two critical KEGG pathways for pigmentation ("tyrosine metabolism" and "melanogenesis" pathways). Important genes from those pathways with known involvement in pigmentation included OCA2 melanosomal transmembrane protein (OCA2), dopachrome tautomerase (DCT), tyrosinase (TYR) and tyrosinase related protein (TYRP1), melanocortin 1 receptor (MC1R), and premelanosome protein (PMEL). The results from our histological and transcriptome analyses will form a foundation for additional investigation into the genetic basis and regulation of pigmentation in these sheep breeds.

Dietary supplementation with oregano essential oil and monensin in combination is antagonistic to growth performance of yearling Holstein bulls.

Our previous work indicated that feeding oregano essential oil (OEO) in combination with monensin (MON) may not be mutually beneficial to dairy calf growth performance. To evaluate this observation further, a 240-d long-term growth experiment was conducted using 12 young growing Holstein bulls using a 2 × 2 factorial treatment arrangement. Main factors were OEO and MON arranged in 4 individual treatments: (1) ration fed without OEO or MON (control), (2) OEO fed at 26 mg/kg of dry matter (DM), (3) MON fed at 25 mg/kg of DM, and (4) OEO and MON fed in combination (OEO+MON). Holstein bulls were 70 d of age and similar in body weight (BW; 93.3 ± 4.54 kg) and individually fed for 240 d. The targeted feeding rates of OEO and MON were blended into 200 g of concentrate and top dressed each morning to a corn stalklage-based ration. Body weights, frame measurements, and blood samples were collected monthly. Interactions of OEO by MON were detected for BW, BW gain, average daily gain, and a trend for feed conversion. Bulls fed OEO or MON demonstrated greater final BW (368, 385, 381, and 358 kg for control, OEO, MON, and OEO+MON, respectively), and BW gains (278, 292, 285, and 265 kg) and average daily gain (1.16, 1.22, 1.19, 1.11 kg/d) were greatest for bulls fed OEO or MON compared with bulls fed OEO+MON; bulls fed the control were intermediate and similar to bulls fed MON. Intake of DM was greater for bulls fed OEO (6.55, 6.99, 6.60, and 6.42 kg/d) compared with bulls fed remaining treatments. Frame growth gain measurements for heart girth, abdominal girth, withers height, body length, and cannon bone circumference were similar for bulls fed all treatments. Serum triglyceride (0.23, 0.25, 0.28, and 0.24 mmol/L) concentrations were greater for bulls fed MON compared with bulls fed the control and OEO+MON, and bulls fed OEO were intermediate and similar. Cholesterol (2.06, 2.29, 2.20, and 2.07 mmol/L) concentrations were greater for bulls fed OEO compared with bulls fed the control and OEO+MON, and bulls fed MON were intermediate and similar. Serum antioxidant measurements were similar for bulls fed all treatments. Serum IgA, IgG, and IgM concentrations were similar for bulls fed all treatments. Feeding OEO or MON separately can improve growth performance of growing Holstein bulls. We do not know why the combination of OEO and MON is antagonistic to growth performance of Holstein bulls. However, these technologies should not be fed in combination to growing dairy cattle.

Study on the correlation between BMPR1B protein in sheep blood and reproductive performance.

Sheep reproductive performance is one of the important economic traits in sheep farming. The bone morphogenetic protein receptor 1B (BMPR1B) gene and protein may play an important role in sheep fertility. This study was to investigate the association of blood BMPR1B protein expression with reproductive performance in sheep. Mongolian sheep with single and twin births and polytocous Small Tail Han sheep were selected due to differences in birth numbers. The BMPR1B mRNA in sheep blood was measured by a reverse transcription-polymerase chain reaction as well as the BMPR1B protein was measured by enzyme-linked immunosorbent assay in blood samples of Mongolian and Small Tail Han sheep. The results demonstrated that blood BMPR1B concentration in Mongolian sheep with twin birth was higher (P < 0.05) than Small Tail Han sheep and Mongolian sheep with single birth. The protein concentration in the anestrus season was higher (P < 0.045) than those in the estrus season for both Mongolian and Small Tail Han sheep. Moreover, BMPR1B concentration in Mongolian sheep increased (P < 0.05) at the age of 6 to 12 mo and that in Small Tail Han sheep increased (P < 0.05) at the age of 3 to 6 mo. The result indicates that the increase in BMPR1B protein concentrations in the blood of Mongolian ewes and Small Tail Han ewes may be beneficial to follicular development, but too high or too low of this blood protein concentration in Mongolian and Small Tail Han sheep is not conducive to ovulation.

Effects of oregano essential oil on in vitro ruminal fermentation, methane production, and ruminal microbial community.

Different inclusion rates of oregano essential oil (OEO) were investigated for their effects on ruminal in vitro fermentation parameters, total gas, methane production, and bacterial communities. Treatments were (1) control, 0 mg/L of OEO (CON); 13 mg/L (OEO1); 52 mg/L (OEO2); 91 mg/L (OEO3); and 130 mg/L (OEO4), each incubated with 150 mL of buffered rumen fluid and 1,200 mg of substrate for 24 h using the Ankom in vitro gas production system (Ankom Technology Corp., Fairport, NY). Treatment responses were statistically analyzed using polynomial contrasts. Digestibility of DM, NDF, and ADF increased quadratically with increasing OEO inclusion rates. Digestibility of DM and NDF were highest for OEO2, whereas ADF digestibility was highest for OEO3, compared with CON, with the remaining treatments being intermediate and similar. Ammonia nitrogen concentrations decreased from CON at a quadratic rate with increasing OEO inclusion rates, and OEO2 had the lowest concentration compared with the other groups. Total VFA, acetate, propionate, butyrate, valerate, and isovalerate concentrations linearly decreased with increasing OEO inclusion rates. Total gas production levels by CON and OEO4 were greater than those of OEO1, OEO2, and OEO3 in a quadratic response, and methane production linearly decreased from CON, compared with OEO4, at a decreasing rate with OEO inclusion rates. As determined by 16S rRNA sequencing, the α biodiversity of ruminal bacteria was similar among OEO inclusion rates. Increasing OEO inclusion rates linearly increased the relative abundance of Prevotella and Dialister bacteria. Several bacteria demonstrated different polynomial responses, whereas several bacteria were similar among increasing OEO inclusion rates. These results suggested that OEO supplementation can modify ruminal fermentation to alter VFA concentrations and reduce methane emissions by extensively altering the ruminal bacterial community, suggesting an optimal feeding rate for future animal studies of approximately 52 mg/L for mature ruminants.

Effects of oregano essential oil on the ruminal pH and microbial population of sheep.

Oregano essential oil (OEO), which has antimicrobial properties, may be used for altering the ruminal pH and microbial populations of sheep, as observed by the altered volatile fatty acid patterns. To further elucidate the effects of OEO on ruminal pH and microbial populations of sheep, 3 German merino sheep × local sheep crossbred rams with permanent ruminal fistulas were randomly assigned to a 3 × 3 Latin square design with 12-d periods. The treatments were as follows: control (CON); OEO4: OEO supplied at 4 g•d-1; and OEO7: OEO supplied at 7 g•d-1. Starting on day 11, rumen fluid was collected at 0 h, and at 4, 8, 12, 24 and 48 h after supplying OEO, and then pH values of rumen fluid were immediately measured. The abundance of microbial populations was determined by using qPCR. The ruminal pH values were similar among the sheep from all treatments. The abundance of ruminal fungi was higher for the sheep supplied OEO7 compared with the sheep supplied CON and OEO4, especially at 4 and 12 h. The abundance of ruminal protozoa decreased with supplied OEO, indicating that OEO could inhibit the protozoa. The abundance of the total ruminal bacteria was similar for the sheep from all treatments, but R. flavefaciens, R. albus and F. succinogenes increased in the sheep supplied OEO4 compared with those in the sheep supplied CON, however, the sheep supplied OEO7 had higher abundances of R. flavefaciens than the sheep supplied CON. These results demonstrated that supplying OEO to sheep did not affect the ruminal pH but could shift the rumen microbial population to one with less protozoa. Supplying OEO can preferentially enhance the growth of certain rumen microbial populations, but the shifts were influenced by the supply rate. Therefore, supplying low amount (i.e. 4 g•d-1) of OEO could have positive effects on ruminal microbial populations, whereas supplying elevated doses of OEO could be detrimental to those same ruminal microbial populations.

Regulation and related mechanism of GSN mRNA level by hnRNPK in lung adenocarcinoma cells.

Gelsolin (GSN) is an actin filament-capping protein that plays a key role in cell migration. Here we show that heterogeneous nuclear ribonucleoprotein K (hnRNPK) regulates GSN expression level by binding to the 3'-untranslated region (3'UTR) of GSN mRNA in non-small cell lung cancers (NSCLC) H1299 cells which are highly metastatic and express high level of GSN. We found that hnRNPK overexpression increased the mRNA and protein level of GSN, whereas hnRNPK knockdown by siRNA decreased the mRNA and protein level of GSN in both H1299 and A549 cells, indicating a positive role of hnRNPK in the regulation of GSN expression. Furthermore, hnRNPK knockdown affected the migration ability of H1299 and A549 cells which could be rescued by ectopic expression of GSN in those cells. Conversely, GSN knockdown in hnRNPK-overexpressing cells could abort the stimulatory effect of hnRNPK on the cell migration. These results suggest that hnRNPK function in the regulation of cell migration is GSN-dependent. Taken together, these data unveiled a new mechanism of regulation of the GSN expression by hnRNPK and provides new clues for the discovery of new anti-metastatic therapy.

Grazing exclosures solely are not the best methods for sustaining alpine grasslands.

BACKGROUND: Grazing is widely regarded as a critical factor affecting the vegetation structure, productivity and nutritional value of natural grasslands. To protect and restore degraded grasslands, non-grazed exclosures are considered as a valuable tool. However, it is not clear whether long term non-grazed exclosures of grazers can improve the condition and nutritional value of vegetation and soil properties. METHODS: We have compared the impact of long-term non-grazed and continuous grazed management strategy on vegetation structure, nutritional values and soil properties of alpine meadow of the Qinghai-Tibet Plateau by field investigation (11-13 years) and indoor analysis during 2015-2017. RESULTS: Our results showed that long-term non-grazed exclosures clearly increased the aboveground biomass and coverage of plant functional types. Long-term non-grazed exclosures improved the development of all vegetation types, except NG (GG, grass species type; SG, sedge species type; LG, leguminous species type; FG, forbs species type and NG, noxious species type). Long-term non-grazed exclosures significantly improved all six measured soil properties (TN, total nitrogen; TP, total phosphorus; TK, total potassium; AN, available nitrogen; AP, available phosphorus and AK, available potassium) in 0-10 cm soil layer, considerable effect on the improvement of all measured soil properties, except TK in 10-20 cm soil layer and all measured soil properties, except TN and TK in 20-30 cm soil layer were observed. However, long-term non-grazed exclosures significantly decreased biodiversity indicators i.e., species richness, Shannon diversity index and Evenness index of vegetation. A substantial decrease in the density, biodiversity and nutritional values (CP (crude protein), IVTD (in vitro ture digestibility) and NDF (neutral detergent fiber)) of all vegetation types, except NG were recorded. While a downward trend in aboveground biomass and all measured soil properties except TP and TK were observed during 2015-2017 in alpine meadows due to long-term grazed treatment. The density, diversity and nutritional value (CP and IVTD) of long-term non-grazed alpine meadows showed a downward trend over time (2015-2017). By considering the biodiversity conservation and grassland livestock production, long-term non-grazed exclosures are not beneficial for the improvement of density, biodiversity and nutritional values of plant functional types. Thus, our study suggests that rotational non-grazed and grazed treatment would be a good management strategy to restore and improve the biodiversity and nutritional values of plant functional types in natural grassland ecosystems.

The effect of periodic energy restriction on growth performance, serum biochemical indices, and meat quality in sheep.

Energy is a key factor regulated by the neuroendocrine system. The objective of this study was to compare growth performance, serum biochemical indices, carcass traits, meat quality, and nutrient composition of lambs fed restricted metabolizable energy (ME) intakes to mimic the seasonal changes of the natural grasslands of northwest China. Nineteen male Dorper × Small Tailed Han lambs were assigned to treatments, control (CON) fed at 1.0 MJ/W0.75 × d-1 throughout the 180 d study, and restricted ME (RES) fed by sequentially restricting ME every 30 d (0.56, 0.84, 1.0, 0.84, 0.56, and 0.28 MJ/W0.75 × d-1). Lambs were harvested at the end of the study. Lambs fed the CON diet demonstrated higher (P < 0.05) average daily gain (ADG) compared with RES-fed lambs (158.79 and 57.01 g/d, for CON and RES, respectively), although ADG were lower in each period with the last period being a negative ADG for RES-fed lambs resulting in a loss of body weight. Both treatments demonstrated lower ADG in the last 3 study periods compared with the first 2 periods, which may be explained by cold stress (<-3 °C) increasing the lamb's energy requirement. Feeding lambs a restricted ME diet resulted in lower (P < 0.05) blood serum glucose, triglycerides, total protein, calcium, phosphorus, immunoglobulin G, immunoglobulin M, and immunoglobulin A concentrations compared with CON-fed lambs, but both treatments were similar (P > 0.05) for cholesterol and high-density lipoprotein-cholesterol. Feeding lambs a restricted ME diet increased (P < 0.05) meat pH, while reducing the cooked meat rate compared with CON-fed lambs. Thus, the carcass yield, rib eye muscle area, and shear force were lower (P < 0.05) for RES-fed lambs compared with CON-fed lambs. The meat water concentration was higher (P < 0.05), whereas the concentrations of dry matter, crude fat, crude protein, and carbohydrate were lower (P < 0.05) for lambs fed a RES ME diet compared to CON-fed lambs. These results demonstrate that sequentially restricting ME intake, as natural grasslands might experience during seasonal forage quality and quantity changes, resulted in a reduction of body weight because lambs were mobilizing body reserves as a source of ME. The reduction in concentrations of key serum nutrients, as well as carcass traits and meat composition, demonstrate the body is responding to the meet the lambs ME requirements, which has implications for both meat quality and the animal's physiological functions.

YWHAZ Binds to TRIM21 but Is Not Involved in TRIM21-stimulated Osteosarcoma Cell Proliferation.

OBJECTIVE: Osteosarcoma is the most common type of malignant bone tumor in children and adolescents. The role of E3 ligases in tumorigenesis is currently a focus in tumor research. In the present study, we investigated the role of the E3 ligase tripartite motif 21 (TRIM21) in osteosarcoma cell proliferation. METHODS: 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays were used to assess osteosarcoma cell viability. U2-OS cells stably carrying a recombinant lentivirus expressing tetracycline-regulated TRIM21 were screened. Co-immunoprecipitation was coupled with LCMS/MS analysis to identify novel interacting partners of TRIM21. Co-immunoprecipitation and bimolecular fluorescence complementation (BIFC) were performed to validate the interactions between TRIM21 and its novel partner YWHAZ. A TRIM21-ΔRING construct was generated to test the effects of TRIM21 ligase activity on YWHAZ. RESULTS: TRIM21 positively regulated osteosarcoma cell proliferation. Overexpression of TRIM21 enhanced osteosarcoma cell tolerance toward various stresses. YWHAZ protein was identified as a novel interacting partner of TRIM21 and its expression levels were negatively regulated by TRIM21. The RING domain of TRIM21 was required for TRIM21 negative regulation of YWHAZ expression. However, overexpression of YWHAZ did not affect positive regulation of osteosarcoma cell proliferation by TRIM21. CONCLUSION: Our results further clarify the molecular mechanisms underlying the pathogenesis of osteosarcoma.

Comparison of Three Methods Estimating Baseline Creatinine For Acute Kidney Injury in Hospitalized Patients: a Multicentre Survey in Third-Level Urban Hospitals of China.

BACKGROUND/AIMS: A lack of baseline serum creatinine (SCr) data leads to underestimation of the burden caused by acute kidney injury (AKI) in developing countries. The goal of this study was to investigate the effects of various baseline SCr analysis methods on the current diagnosis of AKI in hospitalized patients. METHODS: Patients with at least one SCr value during their hospital stay between January 1, 2011 and December 31, 2012 were retrospectively included in the study. The baseline SCr was determined either by the minimum SCr (SCrMIN) or the estimated SCr using the MDRD formula (SCrGFR-75). We also used the dynamic baseline SCr (SCrdynamic) in accordance with the 7 day/48 hour time window. AKI was defined based on the KDIGO SCr criteria. RESULTS: Of 562,733 hospitalized patients, 350,458 (62.3%) had at least one SCr determination, and 146,185 (26.0%) had repeat SCr tests. AKI was diagnosed in 13,883 (2.5%) patients using the SCrMIN, 21,281 (3.8%) using the SCrGFR-75 and 9,288 (1.7%) using the SCrdynamic. Compared with the non-AKI patients, AKI patients had a higher in-hospital mortality rate regardless of the baseline SCr analysis method. CONCLUSIONS: Because of the scarcity of SCr data, imputation of the baseline SCr is necessary to remedy the missing data. The detection rate of AKI varies depending on the different imputation methods. SCrGFR-75 can identify more AKI cases than the other two methods.

ERK1/2-mediated Cytoplasmic Accumulation of hnRNPK Antagonizes TRAIL-induced Apoptosis through Upregulation of XIAP in H1299 Cells.

OBJECTIVE: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance greatly limits the clinical therapeutic efficacy of TRAIL. Elucidating the molecular mechanism underlying TRAIL resistance will be fundamental to resolving this problem. METHODS: Nuclear and cytoplasmic protein extraction and immuno？uorescence (IF) assay were used to detect changes in heterogeneous nuclear ribonucleoprotein K (hnRNPK) localization in H1299 cells. The evaluation of cell apoptosis in cells transfected with GFP-hnRNPK, GFP-hnRNPK S284/353A, or GFP-hnRNPK S284/353D mutant was performed using cleaved caspase-3 antibody. The gene expression of XIAP was tested by quantitative RT-PCR. RESULTS: Previously, we reported that hnRNPK antagonized TRAIL-induced apoptosis through inhibition of PKC-mediated GSK3ß phosphorylation. In this study, we further demonstrate that TRAIL treatment induces cytoplasmic accumulation of hnRNPK in H1299 cells. The hnRNPK localized in the cytoplasm has a higher capacity to antagonize TRAIL-induced apoptosis. Both ERK1/2 signaling inhibitor U0126 and ERK-phosphoacceptor-site mutant (GFP-hnRNPK S284/353A) diminish cytoplasmic accumulation of hnRNPK induced by TRAIL. Moreover, we show that XIAP is involved in hnRNPK-mediated TRAIL resistance in H1299 cells. CONCLUSION: Taken together, these results give new insights into the understanding of the molecular mechanism associated with TRAIL resistance in lung adenocarcinoma.

Effects of rapamycin against paraquat-induced pulmonary fibrosis in mice.

BACKGROUND AND AIMS: Ingestion of paraquat (PQ), a widely used herbicide, can cause severe toxicity in humans, leading to a poor survival rate and prognosis. One of the main causes of death by PQ is PQ-induced pulmonary fibrosis, for which there are no effective therapies. The aim of this study was to evaluate the effects of rapamycin (RAPA) on inhibiting PQ-induced pulmonary fibrosis in mice and to explore its possible mechanisms. METHODS: Male C57BL/6J mice were exposed to either saline (control group) or PQ (10 mg/kg body weight, intraperitoneally; test group). The test group was divided into four subgroups: a PQ group (PQ-exposed, non-treated), a PQ+RAPA group (PQ-exposed, treated with RAPA at 1 mg/kg intragastrically), a PQ+MP group (PQ-exposed, treated with methylprednisolone (MP) at 30 mg/kg intraperitoneally), and a PQ+MP+RAPA group (PQ-exposed, treated with MP at 30 mg/kg intraperitoneally and with RAPA at 1 mg/kg intragastrically). The survival rate and body weight of all the mice were recorded every day. Three mice in each group were sacrificed at 14 d and the rest at 28 d after intoxication. Lung tissues were excised and stained with hematoxylin-eosin (H&E) and Masson's trichrome stain for histopathological analysis. The hydroxyproline (HYP) content in lung tissues was detected using an enzyme-linked immunosorbent assay (ELISA) kit. The expression of transforming growth factor-ß1 (TGF-ß1) and α-smooth muscle actin (α-SMA) in lung tissues was detected by immunohistochemical staining and Western blotting. RESULTS: A mice model of PQ-induced pulmonary fibrosis was established. Histological examination of lung tissues showed that RAPA treatment moderated the pathological changes of pulmonary fibrosis, including alveolar collapse and interstitial collagen deposition. HYP content in lung tissues increased soon after PQ intoxication but had decreased significantly by the 28th day after RAPA treatment. Immunohistochemical staining and Western blotting showed that RAPA treatment significantly down-regulated the enhanced levels of TGF-ß1 and α-SMA in lung tissues caused by PQ exposure. However, RAPA treatment alone could not significantly ameliorate the lower survival rate and weight loss of treated mice. MP treatment enhanced the survival rate, but had no significant effects on attenuating PQ-induced pulmonary fibrosis or reducing the expression of TGF-ß1 and α-SMA. CONCLUSIONS: This study demonstrates that RAPA treatment effectively suppresses PQ-induced alveolar collapse and collagen deposition in lung tissues through reducing the expression of TGF-ß1 and α-SMA. Thus, RAPA has potential value in the treatment of PQ-induced pulmonary fibrosis.

Protein Phosphatase 2C of Toxoplasma Gondii Interacts with Human SSRP1 and Negatively Regulates Cell Apoptosis.

OBJECTIVE: The protozoan Toxoplasma gondii expresses large amounts of a 37 kDa Type 2C serine-threonine phosphatase, the so-called TgPP2C which has been suggested to contribute to parasite growth regulation. Ectopic expression in mammalian cells also indicated that the enzyme could regulate growth and survival. In this study, we aimed to investigate the interaction of TgPP2C with human SSRP1 (structure-specific recognition protein 1) and the effects of TgPP2C on cell viability. METHODS: The yeast two hybrid system, His-tag pull-down and co-immunoprecipitation assays were used to confirm the interaction of TgPP2C with SSRP1 and determine the binding domain on SSRP1. The evaluation of cell apoptosis was performed using cleaved caspase-3 antibody and Annexin-V/PI kit combined with flow cytometry. RESULTS: We identified human SSRP1 as an interacting partner of TgPP2C. The C-terminal region of SSRP1 including the amino acids 471 to 538 was specifically mapped as the region responsible for interaction with TgPP2C. The overexpression of TgPP2C down-regulated cell apoptosis and negatively regulated apoptosis induced by DRB, casein kinase II (CKII) inhibitor, through enhanced interaction with SSRP1. CONCLUSION: TgPP2C may be a parasitic factor capable of promoting cell survival through interaction with the host protein SSRP1, thereby creating a favorable environment for parasite growth.