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Polydatin (PD), a monocrystalline compound isolated from the root and rhizome of Polygonum cuspidatum, is widely used in inhibiting the inflammatory response and oxidative stress. PD has an anti‐inflammatory effect on colitis mice; however, information regulating the mechanism by which maintains the intestinal epithelium barrier is currently scarce. Here, we assessed the anti‐inflammatory and antioxidant of PD in lipopolysaccharide (LPS)‐induced macrophages in vitro, and explored its effects on inhibiting intestinal inflammation and maintaining the intestinal epithelium barrier in dextran sodium sulfate (DSS)‐induced colitis mice. Results showed that PD reduced the level of proinflammatory cytokines and enzymes, including tumor necrosis factor‐α, interleukin‐4 (IL‐4), IL‐6, cyclooxygenase‐2, and inducible nitric oxide synthase, in LPS‐induced macrophages, and improved the expression level of IL‐10. PD maintained the expression of tight junction proteins in medium (LPS‐induced macrophages medium)‐induced MCEC cells. Additionally, PD inhibited the phosphorylation of nuclear factor‐κB (NF‐κB), p65, extracellular signal‐regulated kinase‐1/2, c‐Jun N‐terminal kinase, and p38 signaling pathways in LPS‐induced macrophages and facilitated the phosphorylation of AKT and the nuclear translocation of Nrf2, improving the expression of HO‐1 and NQO1. Furthermore, PD ameliorated the intestinal inflammatory response and improved the dysfunction of the colon epithelium barrier in DSS‐induced colitis mice. Taken together, our results indicated that PD inhibited inflammation and oxidative stress, maintained the intestinal epithelium barrier, and the protective role of PD was associated with the NF‐κB p65, itogen‐activated protein kinases, and AKT/Nrf2/HO‐1/NQO1 signaling pathway.

Background Warts, hypogammaglobulinemia, recurrent bacterial infections and myelokathexis (WHIM) syndrome is a primary immunodeficiency disease (PID) usually caused by autosomal dominant mutations in the chemokine receptor CXCR4 gene. To date, a total of nine different mutations including eight truncation mutations and one missense mutation (E343K, CXCR4E343K) distributed in the C-terminus of CXCR4 have been identified in humans. Studies have clarified that the loss of phosphorylation sites in the C-terminus of truncated CXCR4 impairs the desensitization process, enhances the activation of G-protein, prolongs downstream signaling pathways and introduces over immune responses, thereby causing WHIM syndrome. So far, there is only one reported case of WHIM syndrome with a missense mutation, CXCR4E343K, which has a full length of C-terminus with entire phosphorylation sites, no change in all potential phosphorylation sites. The mechanism of the missense mutation (CXCR4E343K) causing WHIM syndrome is unknown. This study aimed to characterize the effect of mutation at the 343 site of CXCR4 causing the replacement of arginine/E with glutamic acid/K on the receptor signal transduction, and elucidate the mechanism underling CXCR4E343K causing WHIM in the reported family. Results We completed a series of mutagenesis to generate different mutations at the 343 site of CXCR4 tail, and established a series of HeLa cell lines stably expressing CXCR4WT or CXCR4E343D (glutamic acid/E replaced with aspartic acid/D) or CXCR4E343K (glutamic acid/E replaced with lysine/K) or CXCR4E343R (glutamic acid/E replaced with arginine/R) or CXCR4E343A (glutamic acid/E replaced with alanine/A) and then systematically analyzed functions of the CXCR4 mutants above. Results showed that the cells overexpressing of CXCR4E343D had no functional changes with comparison that of wild type CXCR4. However, the cells overexpressing of CXCR4E343K or CXCR4E343R or CXCR4E343A had enhanced cell migration, prolonged the phosphorylation of ERK1/2, p38, JNK1/2/3, aggravated activation of PI3K/AKT/NF-κB signal pathway, introduced higher expression of TNFa and IL6, suggesting over immune response occurred in CXCR4 mutants with charge change at the 343 site of receptor tail, as a result, causing WHIM syndrome. Biochemical analysis of those mutations at the 343 site of CXCR4 above shows that CXCR4 mutants with no matter positive or neutral charge have aberrant signal pathways downstream of activated mutated CXCR4, only CXVR4 with negative charge residues at the site shows normal signal pathway post activation with stromal-derived factor (SDF1, also known as CXCL12). Conclusion Taken together, our results demonstrated that the negative charge at the 343 site of CXCR4 plays an essential role in regulating the down-stream signal transduction of CXCR4 for physiological events, and residue charge changes, no matter positive or neutral introduce aberrant activities and functions of CXCR4, thus consequently lead to WHIM syndrome.

BACKGROUND: This study aimed to evaluate the clinical performance of low serum calcium and phosphorus in discriminative diagnosis of the severity of patients with coronavirus disease 2019 (COVID-19). We conducted a single-center hospital-based study and consecutively recruited 122 suspected and 104 confirmed patients with COVID-19 during January 24 to April 25, 2020. Clinical risk factors of COVID-19 were identified. The discriminative power of low calcium and phosphorus regarding the disease severity was evaluated. Low calcium and low phosphorus are more prevalent in severe or critical COVID-19 patients than moderate COVID-19 patients (odds ratio [OR], 15.07; 95% confidence interval [CI], 1.59-143.18 for calcium; OR, 6.90; 95% CI, 2.43-19.64 for phosphorus). The specificity in detecting the severe or critical patients among COVID-19 patients reached 98.5% (95% CI, 92.0%-99.7%) and 84.8% (95% CI, 74.3%-91.6%) by low calcium and low phosphorus, respectively, albeit with suboptimal sensitivity. Calcium and phosphorus combined with lymphocyte count could obtain the best discriminative performance for the severe COVID-19 patients (area under the curve [AUC] = 0.80), and combined with oxygenation index was promising (AUC = 0.71). Similar discriminative performances of low calcium and low phosphorus were found between suspected and confirmed COVID-19 patient. Low calcium and low phosphorus could indicate the severity of COVID-19 patients, and may be utilized as promising clinical biomarkers for discriminative diagnosis.

Ovaries produce sex hormones, and ovariectomized animals are often used as models for ovarian dysfunction. The liver is a vital organ involved in metabolism and immunity. In the present study, we conducted experiments to investigate the effects of ovariectomy on transcription and metabolic processes in the liver in chicken. Eight Single Comb White Leghorn (SCWL) female chickens were ovariectomized at 17 wk of age, and 8 intact SCWL females served as controls. At 100 wk of age, all chickens were euthanized. High-throughput transcriptome sequencing was performed on liver RNA obtained from ovariectomized and intact females. A total of 267 differentially expressed genes (DEG) were identified in our study. After analysis using DAVID functional annotation tool, one significant Kyoto Encyclopedia of Genes and Genomes pathway, the phosphatidylinositol signaling pathway, was clustered. Gene Ontology enrichment analysis yielded 46 significant Gene Ontology terms. Among terms describing biological processes, the glycerolipid metabolic and lipid localization processes were dominant. The anabolic genes, PEPCK and GK5, and the catabolic genes, VTG1; VTG2; PLD5; DGKQ; DGKE; and FABP3, were detected in ovariectomized chickens. Differentially expressed genes such as ENSGALG00000000162, IL-1Β, SVOPL, and CA12 implied that livers in ovariectomized chickens were subjected to strong inflammatory reactions, whereas defenses against endogenous materials were compromised. A comprehensive view of gene expression in the liver of ovariectomized chickens would advance our understanding of lipid metabolism, glycometabolism, and their relationships to pathologies induced by absence of the ovary. The identified DEG indicated that ovariectomy disturbed lipid metabolism in the liver and was accompanied by an increase in hepatic gluconeogenesis and reductions in phosphatidic acid synthesis and lipid carrier capacity.

Currently, assays for rapid therapeutic drug monitoring (TDM) of β-lactam antibiotics in blood, which might be of benefit in optimizing doses for treatment of critically ill patients, remain challenging. Previously, we developed an assay for determining the penicillin-class antibiotics in blood using a thermometric penicillinase biosensor. The assay eliminates sample pretreatment, which makes it possible to perform semicontinuous penicillin determinations in blood. However, penicillinase has a narrow substrate specificity, which makes it unsuitable for detecting other classes of β-lactam antibiotics, such as cephalosporins and carbapenems. In order to assay these classes of clinically useful antibiotics, a novel biosensor was developed using New Delhi metallo-β-lactamase-1 (NDM-1) as the biological recognition layer. NDM-1 has a broad specificity range and is capable of hydrolyzing all classes of β-lactam antibiotics in high efficacy with the exception of monobactams. In this study, we demonstrated that the NDM-1 biosensor was able to quantify multiple classes of β-lactam antibiotics in blood plasma at concentrations ranging from 6.25 mg/L or 12.5 mg/L to 200 mg/L, which covered the therapeutic concentration windows of the tested antibiotics used to treat critically ill patients. The detection of ceftazidime and meropenem was not affected by the presence of the β-lactamase inhibitors avibactam and vaborbactam, respectively. Furthermore, both free and protein-bound β-lactams present in the antibiotic-spiked plasma samples were detected by the NDM-1 biosensor. These results indicated that the NDM-1 biosensor is a promising technique for rapid TDM of total β-lactam antibiotics present in the blood of critically ill patients.

Background Peutz–Jeghers syndrome (PJS) is a rare autosomal dominantly inherited disease resulting in multiple gastrointestinal hamartomatous polyps, mucocutaneous pigmentation, and an increased risk of various types of cancer, and is caused by variations in the serine/threonine protein kinase STK11 (LKB1). Methods STK11 gene variations were identified by analyzing STK11 cDNA and genomic DNA. Minigenes carrying the wild‐type and mutant sequences were subjected to in vitro splicing assay to dissect the features of these mutations. The different distribution of wild‐type and mutant protein in cells were tested by Immunofluorescence assays and the functional analysis of the variation were performed using Western blot. Results A novel heterozygous splice‐acceptor site variation (c.921‐2 A>C) in intron 7 of the STK11 gene which is co‐segregates with the PJS phenotypes in the proband and all the affected family members and three previously reported variations (c.180C>G, c.580G>A, c.787_790del) were identified in the four families. The c.921‐2 A>C substitution resulted in the inactivation of a splice site and the utilization of a cryptic splice acceptor site surrounding exon 8, generating three different aberrant RNA transcripts, leading to frameshift translation and protein truncation. The results of minigenes indicated that the spliceosome can use a variety of 3’ acceptor site sequences to pair with a given 5’ donor site. The immunofluorescent visualization showed that the distribution of mutant STK11 was different from that of wild‐type STK11, suggesting the mutation may be the causative effect on the dysfunction of the mutant protein. The rescue experiments indicated that the failure of suppressing mTOR phosphorylation by shRNA STK11 could be eliminated by supply of wild‐type STK11 rather than mutant STK11. Conclusion We identified a novel heterozygous mutation (c.921‐2 A>C) in the STK11 in a Chinese PJS family. Haploinsufficiency of STK11 might contribute to the pathogenesis of the disease., We identified a novel heterozygous mutation (c.921‐2 A>C) in the STK11 in a Chinese PJS family. The c.921‐2 A>C substitution resulted in three different aberrant RNA transcripts, leading to frameshift translation and protein truncation. The rescue experiments indicated that the failure of suppressing mTOR phosphorylation by shRNA STK11 could be eliminated by supply of wild type STK11 rather than mutant STK11.

Antimicrobial resistance (AMR) threatens global public health and modern surgical medicine. Expression of β-lactamase genes is the major mechanism by which pathogens become antibiotic resistant. Pathogens expressing extended spectrum β-lactamases (ESBL) and carbapenemases (CP) are especially difficult to treat and are associated with increased hospitalization and mortality rates. Despite considerable effort, identification of ESBLs and CPs in a clinically relevant timeframe remains challenging. In this study, a two-dimensional AMR profiling assay strategy was developed employing panels of antibiotics (penicillins, cephamycins, oximino-cephalosporins and carbapenems) and β-lactamases inhibitors (avibactam and EDTA). The assay required the development of a novel biosensor that employed New Delhi metallo-β-lactamase-1 (NDM-1) as the sensing element. Functionally probing β-lactamase activity using substrates and inhibitors combinatorically increased the informational content that enabled the development of assays capable of simultaneous, differential identification of multiple β-lactamases expressed in a single bacterial isolate. More specifically, the assay enabled the simultaneous identification of ESBL and CP in mock samples, as well as in an engineered construct which co-expressed these β-lactamases. The NDM-1 biosensor assay was 16 times and 8 times more sensitive than the ESBL Nordmann/Dortet/Poirel (NDP) and Carba Nordmann/Poirel (NP) assays, respectively. In a retrospective study, NDM-1 biosensor assays were able to differentially identify ESBLs, metallo-CPs and serine-CPs β-lactamases in 23 clinical isolates with 100% accuracy. An assay algorithm was developed which accelerated data analytics reducing turnaround to

Tuberculosis (TB), caused by Mycobacterium tuberculosis attacking the lungs and other organs, is one of the most common infectious disease worldwide. According to the WHO's 2020 report, a quarter of the world's population were infected with M. tuberculosis, and ~1.4 million people died of TB. Therefore, TB is a significant public health concern, which requires cost-effective strategies for prevention and treatment. The microbiota has been considered as a “forgotten organ” and a complex dynamic ecosystem, which plays a significant role in many physiological processes, and its dysbiosis is closely associated with infectious disease. Recently, a few studies have indicated associations between TB and microbiota. This review summarizes studies concerning the alterations of the gut and respiratory microbiota in TB, and their relationship with host susceptibility to M. tuberculosis infection, indicating that microbiota signatures in different stages in TB progression could be considered as biomarkers for TB diagnosis and control. In addition, the potential role of probiotics and postbiotics in TB treatment was discussed.

Objective: Wolcott–Rallison syndrome is a rare autosomal recessive inheritance disorder caused by the defectiveness of eukaryotic translation initiation factor 2 alpha kinase 3 (EIF2AK3), which encodes the PKR-like endoplasmic reticulum kinase (PERK). Defect in EIF2AK3 results in a permanent diabetes in early infancy or newborn period, a tendency to develop skeletal fractures and other associated disorders such as severe liver and renal dysfunction, and central hypothyroidism. Two patients with Wolcott–Rallison syndrome-like manifestations in a Chinese family and family members were genetically analyzed to identify if any variations that occurred in EIF2AK3, which may cause Wolcott–Rallison syndrome.Methods: Whole-exome sequencing (WES) was performed to identify genetic variations, and Sanger sequencing was conducted to verify the identified variations in the family members with Wolcott–Rallison syndrome (WRS) clinical manifestations. Several bioinformatics tools were employed to predict the effect of EIF2AK3 variations on the protein function. The impact on PERK protein was analyzed by sequential analysis and evolution conservation study.Results: Two novel EIF2AK3 heterozygous single base variations (c.2818C>T and c.2980G>C) were detected in the proband. PERK has two functional domains: one is regulatory domain (aa 1–576), and the other is catalytic domain (aa 577–1,115). Both variations are missense mutations and locate in catalytic domain of PERK; c.2818C>T resulted in a residue substitution of proline for serine at amino acid site 940 (p.Pro940Ser), and variation c.2980G>C caused an amino acid change at position 994 from glutamic acid to glutamine (p.Glu994Gln). These novel missense variations may affect the physiological functions of PERK protein.Conclusions: Two novel compound heterozygous EIF2AK3 variations (c.2818C>T, p.Pro940Ser and c.2980G>C, p.Glu994Gln) were found in a Chinese family. The identification of the variations and verification of their pathogenicity extended the variation spectrum of EIF2AK3 variations causing Wolcott–Rallison syndrome and enriched valuable information for precise medical intervention for Wolcott–Rallison syndrome in China.

WDR45 is an autophagy-related protein that involves in the formation of autophagosome. Mutations in WDR45 lead to the impairment of autophagy which is associated with the human β-propeller protein-associated neurodegeneration (BPAN). However, the relationship between autophagy and brain iron accumulation in patients with BPAN remains unclear. Here, we demonstrated that transferrin receptor (TfRC) which is critical for the iron import of cells was degraded via autophagy. TfRC was accumulated after the inhibition of autophagy by treatment with autophagic inhibitor chloroquine or knockdown of ATG2A. The intracellular iron content was increased in cells overexpressing TfRC or mutant WDR45, however, ferritin H (FTH) chain was decreased. Increased TfRC and simultaneously decreased FTH consequently resulted in an elevated level of ferrous iron (Fe2+) which further promoted cell ferroptosis, demonstrated by the increased lipid peroxidation and reactive oxygen species (ROS) and the decreased glutathione peroxidase 4 (GPX4) and cell viability. Taken together, these findings provide a piece of important evidence that WDR45 deficiency impairs autophagic degradation of TfRC, therefore leading to iron accumulation, and the elevated iron promotes ferroptosis which may contribute to the progression of BPAN.

Vitamin D was empirically applied for Tuberculosis (TB) treatment in the past, and is currently used as an adjuvant for TB therapy. Although an increasing pile of evidences suggests that vitamin D has no therapeutic effect against TB infection, the prophylactic effect of vitamin D in preventing TB remains largely undetermined. To experimentally valuate the potential prophylactic effect of calcitriol (the active form of vitamin D) against mycobacterium infection, we performed dose-gradient calcitriol soaking in 30-day-old zebrafish before Mycobacterium marinum (M. marinum) challenge through tail vein injection. 1H-NMR metabolomics analysis was further performed for illustration of potential mechanisms underlying the prophylactic effect of calcitriol against M. marinum. The results suggested that calcitriol exerts dose-dependent prophylactic anti-mycobacterium effects, i.e., the bacterial load and the corresponding inflammatory factors (IL-1β, TNF-α, and IFN-γ) expressions in M. marinum challenged zebrafish were reduced by low-dose (25 µg/L) or high-dose (2500 µg/L) calcitriol soaking, rather than by moderate-dose (250 µg/L) calcitriol soaking. Body weight of the M. marinum challenged zebrafish was recovered by high-dose prophylactic calcitriol soaking rather than by low-dose or moderate-dose calcitriol. The 1H-NMR metabolomic profiling identified 29 metabolites with altered abundance among the dose-gradient calcitriol groups, among which 22 metabolites were co-varied with the dose of calcitriol, the rest 7 metabolites were co-varied with the bacterial load and the inflammatory response in term of cytokine expression. Further pathway analysis indicated that the glycine, serine, and threonine metabolism pathway was the activated in both of the two metabolite groups, indicating that the pathway was altered by dose-gradient of calcitriol and was in response to M. marinum infection in zebrafish. The results of the present study suggested that the activation of glycine, serine and threonine metabolism pathway may play a potential role for the dose-dependent anti-mycobacterium effect induced by prophylactic calcitriol soaking.

Mitochondria are known to generate approximately 90% of cellular reactive oxygen species (ROS). The imbalance between mitochondrial reactive oxygen species (mtROS) production and removal due to overproduction of ROS and/or decreased antioxidants defense activity results in oxidative stress (OS), which leads to oxidative damage that affects several cellular components such as lipids, DNA, and proteins. Since the kidney is a highly energetic organ, it is more vulnerable to damage caused by OS and thus its contribution to the development and progression of chronic kidney disease (CKD). This article aims to review the contribution of mtROS and OS to CKD progression and kidney function deterioration.

The Tibetan chicken ( Gallus gallus; TBC) is an indigenous breed found in the Qinghai-Tibet Plateau that are well adapted to a hypoxic environment. The energy metabolism of embryonic brains in TBCs under hypoxia has been little reported. This study investigated changes in energy metabolism of the TBC brain during embryo development under hypoxia. We found that TBCs exhibited a change of glycolysis and the tricarboxylic acid cycle during embryo development under hypoxia. Hypoxia-inducible factor (HIF)-1 was potentially involved in this by directly inducing overexpression of pyruvate dehydrogenase kinase 1 ( PDK1) and the glycolytic genes hexokinase 1 ( HK1) and lactate dehydrogenase A ( LDHA) to increase glycolysis of TBCs to adapt to hypoxia. Although these may not be unique to TBCs, as we had also found similar results in Dwarf Laying Chickens, a lowland chicken breed, TBCs had a stronger regulating ability. In summary, our study revealed that HIF-1 induced energy metabolism changes in the TBC brain via upregulating expressions of PDK1 and other HIF-1 target genes like HK1 and LDHA to increase glycolysis for TBC hypoxic adaptations during embryo development. It indicates the potential application of TBC energy metabolism research for other animals living on the Qinghai-Tibet Plateau.

A total of 31 differentially expressed genes in the mammary glands were identified in our previous study using RNA sequencing (RNA-Seq), for lactating cows with extremely high and low milk protein and fat percentages. To determine the regulation of milk composition traits, we herein investigated the expression profiles of microRNA (miRNA) using small RNA sequencing based on the same samples as in the previous RNA-Seq experiment. A total of 497 known miRNAs (miRBase, release 22.1) and 49 novel miRNAs among the reads were identified. Among these miRNAs, 71 were found differentially expressed between the high and low groups (p < 0.05, q < 0.05). Furthermore, 21 of the differentially expressed genes reported in our previous RNA-Seq study were predicted as target genes for some of the 71 miRNAs. Gene ontology and KEGG pathway analyses showed that these targets were enriched for functions such as metabolism of protein and fat, and development of mammary gland, which indicating the critical role of these miRNAs in regulating the formation of milk protein and fat. With dual luciferase report assay, we further validated the regulatory role of 7 differentially expressed miRNAs through interaction with the specific sequences in 3′UTR of the targets. In conclusion, the current study investigated the complexity of the mammary gland transcriptome in dairy cattle using small RNA-seq. Comprehensive analysis of differential miRNAs expression and the data from previous study RNA-seq provided the opportunity to identify the key candidate genes for milk composition traits.

Backgrounds Genetic polymorphism of the toll-like receptor 2, 4 (TLR2, TLR4) and natural resistance-associated macrophage protein 1 (NRAMP1) genes may affect host immune response to Mycobacterium tuberculosis (Mtb) and lead to the variation of susceptibility to tuberculosis (TB) in humans. However, the association of single nucleotide polymorphisms (SNP) in these genes and the susceptibility to TB in Mongolian population has not been investigated. Methods We conducted a genetic association study including 197 Mongolian TB patients and 217 Mongolian healthy controls in Inner Mongolia, China. DNA of blood samples was extracted and genotyped for 5 SNPs in TLR4, 4 SNPs in TLR2 and 5 SNPs in NRAMP1 by next-generation sequencing. A logistic regression was performed and odds ratios (OR) with 95% confidence intervals (CI) were calculated to estimate the risk at TB by each SNP. Results The most significant locus associated with the susceptibility to TB was TLR4 rs11536889. The frequency for allele C of TLR4 rs11536889 was 16.0% in TB patients and 23.5% in healthy controls, respectively. Rs11536889 C/C genotype of TLR4 was significantly associated with the low susceptibility against TB compared to G/G genotype in the dominant model (OR 0.62, 95% CI 0.41–0.94). Conclusions The TLR4 rs11536889 polymorphisms might be an indicative of the low susceptibility to TB in Mongolian population, which provides valuable information for the generation of effective strategy or measurement against TB in Mongolian population.

DIP2A mutation is associated with abnormal brain development and diseases including dyslexia, autism and Alzheimer's disease. However, the role and the involved mechanisms remain unknown. To study the biological function of DIP2A during mESCs neural differentiation in early neural development, we generated a Dip2a homozygous knockout 46C ESC cell line using CRISPR/Cas9 genome editing technology. The eighth exon of Dip2a gene was replaced with PGK-Puro-P2A-mCherry. This 46C-Dip2a KO cell line offers a useful resource to investigate the molecular mechanisms of DIP2A in the process of cell fate determination, as well as a potential source of building disease mouse model.

It is known that the male hypogonadism plays an important role in regulating adipose metabolism. In the present study, fifteen pairs of full male sibs were divided into a castrated group and an intact group with a paired experiment design. The pigs were slaughtered at an age of 175days. The carcass characteristics and fat deposit of the studied animal were measured, and the hormone and serum lipid levels of the peripheral blood samples were determined, and the differentially expressed genes of the back fat between the two groups were screened with porcine genome array. Our results showed that the absence of male gonadal steroids attributed to castration significantly raised the serum lipid levels and increased fat accumulation in the pigs. A total of 225 differentially expressed genes were identified between the boars and barrows and 135 of them were upregulated. The analysis of Gene Ontology categories and KEGG pathway indicated that these differentially expressed genes were mainly involved in metabolism of lipid, carbohydrate, amino acid, xenobiotics biodegradation, and immune diseases pathways. Our results indicated that there were higher capacity of fatty acid of synthesis, enhanced uptaking capacity of fatty acids and cholesterol, inhibited lipolysis, and enhanced carbohydrate metabolism in the adipose tissue of barrows compared to boars. The findings of the present study provide new insight into the mechanisms of adipose metabolism induced by hormone deficiency.

Acute intermittent porphyria (AIP) is an autosomal dominant genetic disease caused by a lack or decrease in hydroxymethylbilane synthase (HMBS) activity. It is characterized by acute nerve and visceral attacks caused by factors in the process of heme synthesis. The penetrance rate of this disease is low, and the heterogeneity is strong. Here, we reported two novel HMBS mutations from two unrelated Chinese AIP patients and confirmed the pathogenicity of these two mutations. We found the HMBS c.760–771+2delCTGAGGCACCTGGTinsGCTGCATCGCTGAA and HMBS c.88-1G&gt, C mutations by second-generation sequencing and Sanger sequencing. The in vitro expression analysis showed that these mutations caused abnormal HMBS mRNA splicing and premature termination or partial missing of HMBS protein. Homologous modeling analysis showed that the HMBS mutants lacked the amino acids which are crucial for the enzyme activity or the protein stability. Consistently, enzyme activity analysis confirmed that the HMBS mutants’ overexpression cells exhibited the reduced enzyme activity compared with the HMBS wildtype overexpression cells. Our study identified and confirmed two novel pathogenic HMBS mutations which will expand the molecular heterogeneity of AIP and provide further scientific basis for the clinical diagnosis of AIP.

Breast cancer is the most frequently diagnosed malignant tumor in women. Foxtail millet is an important cereal food and exhibits a wide range of nutrition. We previously extracted total polyphenols composed of 12 compounds from foxtail millet bran (named BPIS). In this study, we find that BPIS inhibits the proliferation of breast cancer cells by restraining the cellular membrane synthesis via suppressing the conversion of saturated fatty acids (SFA) to monounsaturated fatty acids (MUFA). Mechanically, BPIS decreases the glucose regulated protein 78 (GRP78) level, which further inhibits the expression of SREBP-1 and downstream-target SCD1. Combining molecular docking, MTT screening and in vivo model, we ultimately identify that vitexin and syringic acid are the main active ingredients. Our data showed that the combination of vitexin and syringic acid suppress the proliferation of breast cancer cells, which support further study of BPIS as a new dietary assistant strategy to improve breast cancer.

Tuberculosis is initiated by the entry of Mycobacterium tuberculosis (Mtb) into macrophages in the lungs. A study of the cellular factors responsible for the entry of Mtb into host cells will potentially benefit the development of therapeutic treatments or preventive agents against Mtb infection. Using human THP1-derived macrophages as a model, we found that infection of Mtb H37Ra transiently reduced the level of ASAP1, an ADP ribosylation factor (Arf)-GTPase activating protein. Furthermore, knockdown of ASAP1 increased the efficiency of H37Ra entry into the cell and altered the status of actin remodeling as indicated by the enhanced aggregation of F-actin and the increased numbers of vinculin- and paxillin-rich puncta. Collectively, the results in this report identified ASAP1 as a regulator controlling the entry of Mtb H37Ra into macrophage by remodeling actin cytoskeleton.

G protein coupled Receptor 161 (GPR161) is a ciliary orphan GPCR. It is reported to play critical roles in regulating vertebrate Hedgehog (Hh) signaling pathway, that is conserved in metazoan and functions in earlier embryogenesis and homeostasis of adult metabolism. However, to date, all GPR161 functional studies were performed only in mouse. Knock out gpr161 in NIH3T3 cell lines, the common material for Hh mechanism research, failed to give any obvious Hh pathway defects, raising the question that whether GPR161 functions in Hh pathway is conserved in vertebrate system. Here, we described the characterization and spatiotemporal expression of two zebrafish gpr161 homologs, gpr161a and gpr161b. gpr161a was renamed of the gpr161 previously identified, while gpr161b was novel identified. The whole-mount in situ hybridization and quantitative PCR results showed that gpr161a is initially expressed in maternal manner while gpr161b is not. Although these two gpr161 showed ubiquitously expressed at early embryonic stages, each of them had tissue specific accumulation. gpr161a is abundant in the central nervous system (CNS) and adaxial cells, where are rich of Hh responding cells. Together gpr161a was highly expressed in muscle and intestine in adult fishes. These results strongly suggest the regulating roles of Gpr161 a in zebrafish Hh signal transduction. gpr161b was also accumulated in the CNS but mainly at the midline in the neural tube, similar pattern as wnt5b expression in such area, suggesting its potential function correlated with WNT signaling pathway. Interestingly, we also found the specific accumulation of gpr161 in posterior blood island (PBI) at 24 hours post fertilization (hpf), indicating the gpr161 may play roles in early hematopoiesis in zebrafish. Our work provides a starting point to unveil the divergent functions of gpr161 in vertebrate and will shed light on the studies of mechanism of Hh and WNT pathways, as well as early hematopoiesis.

Background Fabry disease (FD), a rare X‐linked α‐galactosidase A (GLA) deficiency, resulting in progressive lysosomal accumulation of globotriaosylceramide in a variety of cell types. More and more disease‐causing mutations in GLA have been identified in FD due to the advancement of molecular diagnostic tools. We found a novel mutation in a Chinese family with predominant Fabry's disease nephropathy. Methods All coding regions and exon–intron splice junctions of the GLA gene were sequenced to find sequence variations. We evaluated the impact on the GLA protein by analysis of the GLA mRNA, by sequential analysis and homology modeling, and by site‐directed mutagenesis and in vitro expression studies. Results We identified a novel heterozygous missense mutation c.280T>C in our patient with variable phenotypic presentations of renal involvement. The novel GLA variant results in low expression of GLA mRNAs, impaired or loss of the disulfate bridge structure of wild‐type GLA, reduced GLA activity and defected nuclear shape in the GFP‐GLA‐MT transfected HEK293T cells. Conclusion A novel GLA missense mutation, c.280T>C (Cys94Arg), was found in a Chinese family with predominant renal manifestations of FD. Our study reveals the pathogenesis of c.280T>C mutation to FD and provides scientific foundation for accurate diagnosis and precise medical intervention for FD.

Dwarfism is a condition defined by low harvest weight in fish, but also results in strange body figures which may have potential for the selective breeding of new ornamental fish strains. The objectives of this study are to reveal the physiological causes of dwarfism and identify the genetic loci controlling this trait in the white sailfin molly. Skeletons of dwarf and normal sailfin mollies were observed by X-ray radioscopy and skeletal staining. Genome-wide association studies based on genotyping-by-sequencing (n = 184) were used to map candidate genomic regions associated with the dwarfism trait. Quantitative real-time PCR was performed to determine the expression level of candidate genes in normal (n = 8) and dwarf (n = 8) sailfin mollies. We found that the dwarf sailfin molly has a short and dysplastic spine in comparison to the normal fish. Two regions, located at NW_015112742.1 and NW_015113621.1, were significantly associated with the dwarfism trait. The expression level of three candidate genes, ADAMTS like 1, Larp7 and PPP3CA, were significantly different between the dwarf and normal sailfin mollies in the hepatopancreas, with PPP3CA also showing significant differences in the vertebrae and Larp7 showing significant differences in the muscle. This study identified genomic regions and candidate genes associated with the dwarfism trait in the white sailfin molly and would provide a reference to determine dwarf-causing variations.

Fabry disease (FD) is a rare X-linked α-galactosidase A (GLA) deficiency, resulting in progressive lysosomal accumulation of globotriaosylceramide (Gb3) in a variety of cell types. Here, we report a novel splicing mutation (c.801 + 1G > A) that results in alternative splicing in GLA of a FD patient with variable phenotypic presentations of renal involvement. Sequencing of the RT-PCR products from the patient’s blood sample reveals a 36-nucleotide (nt) insertion exists at the junction between exons 5 and 6 of the GLA cDNA. Splicing assay indicates that the mutated minigene produces an alternatively spliced transcript which causes a frameshift resulting in an early termination of protein expression. Immunofluorescence shows puncta in cytoplasm for mutated GLA whereas uniform staining small dots evenly distributed inside cytoplasm for wild type GLA in transfected HeLa cells. The increased senescence and decreased GLA enzyme activity suggest that the abnormalities might be due to the altered localization which further might result from the lack of the C-terminal end of GLA. Our study reveals the pathogenesis of splicing mutation c.801 + 1G > A to FD and provides scientific foundation for accurate diagnosis and precise medical intervention for FD.

Background Beta‐propeller protein‐associated neurodegeneration (BPAN, OMIM 300894) is an X‐linked neurodegenerative disorder caused by mutations in WDR45. WDR45 is required for autophagy, defect in WDR45 impaired autophagy which contributes for the pathogenesis of BPAN. Previously, we reported a novel de novo mutation (c.1040_1041del, p.Glu347GlyfsTer7) in WDR45 (NM_007075) in a 3‐year‐old Chinese girl with BPAN. Methods The protein structure was constructed using SWISS‐MODEL and the isoelectric point (pI) was predicted by the online pI/Mw tool at ExPASy. The functional effects of this mutation were predicted by two online software programs: PROVEN and MutationTaster. Stable overexpression of Flag‐tagged wild‐type or mutant WDR45 in HeLa cells was constructed. Protein levels of LC3 and p62 were analyzed by western blot upon treatment with/without autophagy inhibitor Bafilomycin A1, the formation of LC3 puncta were analyzed in HeLa cells transfected with mCherry‐LC3 by confocal microscopy. Results The mutation resulted in a shift of pI from 6.74 to 8.84 and was predicted to be pathogenic. The protein levels of LC3‐II and p62 were increased in cells overexpression of wild‐type and mutant WDR45 while the protein levels were not increased in cells overexpression of mutant WDR45 upon treatment with autophagy inhibitor Bafilomycin A1. Results from confocal microscopy revealed that LC3‐positive puncta were increased in cells expressing both wild‐type and mutant WDR45 while the number of LC3‐positive puncta was not increased in cells expressing mutant WDR45 upon treatment with Bafilomycin A1. Conclusion Our study evidenced that this novel mutation in WDR45 impaired autophagy in cells thus this mutation is the cause for BPAN in this patient.

The eggshell blueness is an interesting object for chicken genetic studies and blue-shelled chicken industry, especially after the discovery of the causative mutation of chicken blue eggshell. In the present study, genome wide association study (GWAS) was conducted in Chinese Dongxiang blue-shelled chicken underlying four traits of blue eggshell pigments: quantity of biliverdin (QB), quantity of protoporphyrin (QP), quantity of total pigment (QT), and color density trait (CD). A total of 139 individuals were randomly collected for GWAS. We detected two SNPs in genome-wise significance and 35 in suggestive significance, 24 out of the 37 SNP were located either within intron/exon or near 15 genes in a range of ~1.17 Mb on GGA21. For further confirmation of the identified SNP loci by GWAS, the follow-up replication studies were performed in two populations. A total of 146 individuals of the second generation derived from the former GWAS population, as well as 280 individuals from an alternative independent population were employed for genotyping by MALDI-TOF MS in a genotype-phenotype association study. Eighteen SNPs evenly distributed on the GGA21 significant region were successfully genotyped in the two populations, of which 4 and 6 SNP loci were shown significantly associated with QB, QT and QP in the two repeat populations, respectively. Further, the SNPs were narrowed down to a region of ~ 653.819 Kb on GGA21 that harbors five candidate genes: AJAP1, TNFRSF9, C1ORF174, CAMTA1, and CEP104. Shell gland of chickens laying dark and light blue eggshell was chosen for detection of mRNA expression of the five candidate genes. The results showed differential expression levels of these genes in the two groups. The specific function of these genes has not yet been defined clearly in chickens and further in-depth studies are needed to explore the new functional role in chicken eggshell blueness.

Humans as models of human disease Mice are a convenient model for exploring the functions of cellular signaling pathways. Occasionally, however, an “experiment of nature” highlights the perils of overreliance on mice. RIPK1 is a well studied protein kinase that regulates cell death. Mice deficient in RIPK1 die soon after birth because of the protein's widespread role in multiple tissues and organs. Cuchet-Lourenço et al. studied patients with inherited immunodeficiency of unknown cause (see the Perspective by Pasparakis and Kelliher). They identified inactivating mutations in the RIPK1 gene in four individuals. Unlike what has been seen in mice, the deleterious effects of RIPK1 loss in humans were confined to the immune system, a finding with potential therapeutic implications. Science , this issue p. 810 ; see also p. 756

Multiple epiphyseal dysplasia (MED) is a rare osteochondrodysplasia characterized by moderate short limb dwarfism and earlyonset osteoarthrosis. By whole-exome sequencing (WES), we identified a dominantly inherited mutation (c.1201G>A; p.D401N) in cartilage oligomeric matrix protein (COMP) in a large four-generation Chinese family. Immunofluorescence analysis revealed mutant COMP secretion was severely impaired. Our result expands the mutational spectrum of COMP and provides strong evidence for the genotype-phenotype correlation of COMP pathogenicity in MED.

Spondyloepiphyseal dysplasia congenita (SEDC) is an extremely rare autosomal dominant chondrodysplasia that is usually caused by substitution of glycine with another amino acid in the triple helical region of COL2A1. Herein, we describe a case of SEDC in a Chinese family with a novel de novo mutation in the COL2A1 gene, c.1150G>A (p.Gly384Ser), which may impair protein stability and lead to dysfunction of type II collagen.

Human genetic factors predispose to tuberculosis (TB). We studied 7.6 million genetic variants in 5,530 people with pulmonary TB and in 5,607 healthy controls. In the combined analysis of these subjects and the follow-up cohort (15,087 TB patients and controls altogether), we found an association between TB and variants located in introns of the ASAP1 gene on chromosome 8q24 (P = 2.6 × 10(-11) for rs4733781; P = 1.0 × 10(-10) for rs10956514). Dendritic cells (DCs) showed high ASAP1 expression that was reduced after Mycobacterium tuberculosis infection, and rs10956514 was associated with the level of reduction of ASAP1 expression. The ASAP1 protein is involved in actin and membrane remodeling and has been associated with podosomes. The ASAP1-depleted DCs showed impaired matrix degradation and migration. Therefore, genetically determined excessive reduction of ASAP1 expression in M. tuberculosis-infected DCs may lead to their impaired migration, suggesting a potential mechanism of predisposition to TB.

Waardenburg syndrome (WS) is a dominantly inherited, genetically heterogeneous auditory-pigmentary syndrome characterized by non-progressive sensorineural hearing loss and iris discoloration. By whole-exome sequencing (WES), we identified a nonsense mutation (c.598C>T) in PAX3 gene, predicted to be disease causing by in silico analysis. This is the first report of genetically diagnosed case of WS PAX3 c.598C>T nonsense mutation in Chinese ethnic origin by WES and in silico functional prediction methods.

China was one of the first countries to use artificial insemination (AI) in equids, and it achieved international recognition for its widespread application of AI to commercial horse breeding. This article reviews the history of equine AI in China. The technique originated from the high demand for horses to be used in agriculture, transportation, and the military. Artificial insemination was identified as an ideal tool for Chinese horse breeding to improve the productivity of native horses, especially during 1950-1970. Presently, AI is still practiced commonly in China, and it includes the use of fresh semen and transported cooled semen. The use of frozen semen has also been resumed to broaden the range of elite Sporthorse stallions and to preserve threatened or endangered native breeds. Accurate prediction of the optimum time for insemination depended mainly on transrectal palpation of the mare's ovaries. In addition to controlled methods of insemination, factors like volume and number of spermatozoa in the inseminate and timing and frequency of insemination were optimized to accomplish high fertility rates in the field. Production of hinnies and mules for agricultural labor and donkeys for meat and hide gelatin stimulated a nationwide upsurge of research into the reproductive physiology of and AI technology in donkeys. In the future, there will be further increases in the use of traditional and modern AI techniques in the breeding of Chinese equids.

Autophagy and the ubiquitin proteasome system (UPS) are the two major cellular degradation pathways, which are critical for the maintenance of cell homeostasis. The two pathways differ in their mechanisms and clients. The evolutionary conserved ATG16 plays a key role in autophagy and appears to link autophagy with the UPS. Here, we review the role of ATG16 in different species. We summarize the current knowledge of its functions in autophagosome membrane expansion and autophagosome formation, in Crohn’s disease, and in bacterial sequestration. In addition, we provide information on its autophagy-independent functions and its role in the crosstalk between autophagy and the UPS.

Answers from Exomes Exome sequencing, which targets only the protein-coding regions of the genome, has the potential to identify the underlying genetic causes of rare inherited diseases. Angulo et al. (p. 866 , published online 17 October; see Perspective by Conley and Fruman ) performed exome sequencing of individuals from seven unrelated families with severe, recurrent respiratory infections. The patients carried the same mutation in the gene coding for the catalytic subunit of phosphoinositide 3-kinase δ (PI3Kδ). The mutation caused aberrant activation of this kinase, which plays a key role in immune cell signaling. Drugs inhibiting PI3Kδ are already in clinical trials for other disorders.

Hypoxia is a major factor that affects the subsistence and development of multicellular organisms. Tibet chicken, as a unique native chicken breed in altiplano, shows genetic adaptation to hypoxia comparing with the breeds at the low altitude. In the present study, to explore effects of hypoxia on chicken fetal livers, eggs of Tibet chicken and Shouguang chicken were collected and the samples from each breed were divided into two groups, incubated in hypoxia and in normoxia respectively. The blood of embryos on the 16th day of incubation was collected and the serum chemistry parameters indicating liver metabolism were determined, which included glutamic-pyruvic transaminase (GPT), aspartate aminotransferase (GOT), total bilirubin (TB), direct bilirubin (DB), total bile acid (TBA), gamma glutamyltransferase (GGT), alkaline phosphatease (ALP), lactate dehydrogenase (LDH), creatine kinase (CK), glucose and creatinine. The results show that biochemical indices varied significantly between hypoxia and normoxia except for GPT and glucose. Moreover, the concentration of ALP and LDH showed significant differences between the breeds and the incubations. The results suggest that the livers of both Shouguang chicken and Tibet chicken suffered damages in hypoxia, but the former was more serious. The results of this study support the opinion that Tibet chicken had better genetic adaptability on hypoxia, and made a good basis for further study of the genetic mechanism of adaptation to hypoxia. Key words : Hypoxia adaptation, liver metabolism, serum chemistry, Tibet chicken, chicken embryo.

Autosomal dominant polycystic kidney disease (ADPKD) is a common human genetic disease, affecting millions of people worldwide. The progressive growth of cysts in kidneys eventually leads to renal failure in 50 % of patients, and there is currently no effective treatment. Various murine models have been studied to elucidate the disease mechanisms, and much information has been acquired. However, the course of the disease cannot be fully recapitulated using these models. The pig is a suitable model for biomedical research, and pig PKD2 has high similarity to the human ortholog at the molecular level. Here, a mini-pig PKD2 transgenic model was generated, driven by a ubiquitous cytomegalovirus enhancer/promoter. Using somatic cell nuclear transfer, four transgenic pigs with approximately 10 insertion events each were generated. Quantitative real-time PCR and western blotting showed that PKD2 was more highly expressed in transgenic pigs than in wild-type counterparts. Because of the chronic nature of ADPKD, blood urea nitrogen and serum creatinine levels were continuously measured to assess the pig kidney function. The transgenic pigs continue to show no significant alteration in kidney function; it is estimated that 1-2 more years may be required for manifestation of renal cystogenesis in these pigs.

Summary Myocarditis is an inflammation of the myocardium which often follows virus infections. Coxsackievirus B3 (CVB3), as a marker of the enterovirus group, is one of the most important infectious agents of virus-induced myocarditis. Using a CVB3-induced myocarditis model, we show that injection α-galactosylceramide (α-GalCer), a ligand for invariant natural killer (NK) T (iNK T) cells, can protect the mice from viral myocarditis. After the systemic administration of α-GalCer in CVB3 infected mice, viral transcription and titres in mouse heart, sera and spleen were reduced, and the damage to the heart was ameliorated. This is accompanied by a better disease course with an improved weight loss profile. Compared with untreated mice, α-GalCer-treated mice showed high levels of interferon (IFN)-γ and interleukin (IL)-4, and reduced proinflammatory cytokines and chemokines in their cardiac tissue. Anti-viral immune response was up-regulated by α-GalCer. Three days after CVB3 infection, α-GalCer-administered mice had larger spleens. Besides NK T cells, more macrophages and CD8+ T cells were found in these spleens. Upon stimulation with phorbol myristate acetate plus ionomycin, splenocytes from α-GalCer-treated mice produced significantly more cytokines [including IFN-γ, tumour necrosis factor-α, IL-4 and IL-10] than those from untreated mice. These data suggest that administration of α-GalCer during acute CVB3 infection is able to protect the mice from lethal myocarditis by local changes in inflammatory cytokine patterns and enhancement of anti-viral immune response at the early stage. α-GalCer is a potential candidate for viral myocarditis treatment. Our work supports the use of anti-viral treatment early to reduce the incidence of virus-mediated heart damage.

The aim of this study was to investigate the effect of developmental stage on lipids deposition, composition and oxidative stability of subcutaneous adipose tissue (SAT) and Longissimus dorsi muscle (LDM) in Guizhou mini-pig. Pigs were raised in the same condition, and SAT and LDM were sampled on 90 d, 180 d and 270 d. Lipids content decreased (P < 0.01) from 90 d to 180 d and increased (P < 0.01) from 180 d to 270 d in SAT and LDM. Neutral lipids in both tissues decreased (P < 0.01) from 90 d to 180 d and increased (P < 0.01) from 180 d to 270 d, while phospholipids content changed inversely during the three selected time points. Developmental times had great influence on fatty acids (FAs) composition of neutral lipids, phospholipids and free fatty acids (FFAs) except FAs composition of FFAs in SAT. Lipids oxidative stability in SAT and LDM both decreased between 90 d and 180 d (P < 0.05) and increased by 270 d (P < 0.05). In conclusion, due to the increased contents of unsaturated fatty acids and decreased oxidation stability, Guizhou or other mini-pigs slaughtered at an early age may have a negative effect on meat quality.

Castration of male pig produces significant negative effects on skeletal muscle development. The androgen receptor (AR), two splice variants of insulin-like growth factor-I (IGF-I Ea and MGF) and the myostatin gene may play important roles in this process. In the present study, the expression of AR, IGF-I Ea, MGF and myostatin genes in three skeletal muscles, the brachialis, longissimus and semitendinosus, were studied using real-time quantitative RT-PCR. Our experimental design used 14 pairs of male Landrace sire×Yorkshire dam piglets. The two piglets in each pair were full sibs, one of which was castrated at 21 d of age; the other remained intact. The study group was divided into subgroups of equal size. Animals in the first subgroup were slaughtered at 147 d and those of the second at 210 d of age. Carcass weight and lean meat yield were similar between boars and barrows at 147 d of age (p> 0.05), whereas barrows had lower carcass weight and less lean meat yield at 210 d of age (p 0.05). These results suggest that the down-regulation of AR, IGF-I Ea and MGF gene expression following castration helps to explain the negative effect of castration on skeletal muscle development.

Phenylalanine hydroxylase is assumed to be a key enzyme in drosopterin metabolism, but direct in vivo evidence to support this hypothesis is still absent. In the present study, we found a new natural recessive purple eye mutant of Drosophila melanogaster, Hn ( bp ), which was a 45-nt insertion mutant in the second exon of Henna. The insertion resulted in a predicted protein with 15 additional amino acids as compared to the wild-type protein. Further analysis of protein structure showed that the predicted mutant protein probably had two more beta-sheets, which may cause instability of two alpha-helices near the catalytic centre of the enzyme in the Biopterin-Hydroxyl binding domain. Hn ( bp ) mutant showed eye color defect with decrease of mRNA level, as well as drosopterin content reduction. The drosopterin defect could be fully rescued by expression of wild type Henna in the Hn ( bp ) background by GMR-GAL4 UAS-Henna/UAS-Henna:Hn ( bp )/Hn ( bp ) transgenic line. All taken together, it can be concluded that the mutation in Henna is responsible for drosopterin reduction in mutant Hn ( bp ), which provides key in vivo evidence to support the hypothesis that Henna is involved in drosopterin synthesis.

Tibetan chicken is a precious resource in Qinghai-Tibet Plateau, China. In order to study its growth rhythm and heterosis of its hybrid, three groups comprising Tibetan chicken (T), Dwarf Recessive White (D) and Tibetan × Dwarf Recessive White (TD) were reared under the same management conditions at low and high altitudes. Body weight and shank length were measured, and growth curves were fitted using Richards model. The results showed that the model fitted well with the chickens’ growth courses in weight and shank with R 2 at more than 0.99. The high-altitude might retard the growth of chickens, with decreases in inflection point values, final values and maximal growth rates, and prolongation of inflection point time. The final weight, inflection point weight, and maximal growth rates of Tibetan chicken were 1008.3 g, 477.9 g and 11.69 g respectively at lowaltitude, and 525.3 g, 229.5 g, and 5.12 g at high-altitude. This showed a decline of 47.9%, 52.0%, and 56.8% at the high-altitude; the degree of influence of altitude was less than that of Dwarf Recessive White. The TD had high heterosis in weight gain, and at high-altitude the heterosis of final weight, inflection point weight, and maximal growth rates were 59.5%, 56.8% and 52.3% respectively. Therefore, the Tibetan × Dwarf Recessive White cross was good for improving the Tibetan chicken.

An effective isolation and identification method of primordial follicles would greatly benefit the animal production practice, transgenic animal production and endangered species conservation in the future. This study has not only advanced the isolation method but also developed an identification marker of primordial follicles. After enzymatic digestion, Percoll gradient centrifugation and mesh filtrations, the obtained follicular separations were then subjected to a cell sorter in order to collect primordial follicles. The study greatly improved the yield of primordial follicles (from about 1.85 x 10(5) to 7.79 x 10(5) per prepubertal ovary) by means of increasing cell layer number from 1 ml to 2.5 ml after Percoll gradient centrifugation. Based on traditional morphological criteria, the purity of recovered primordial follicles was averagely about 82.43+/-9.41% (n=5) because of their similar size and appearance with somatic cells. To further exactly appreciate the purity of sorted primordial follicles, a germ cell-specific protein, MSY2, was used to recognize the oocytes of primordial follicles. The results of repeat experiments showed that about 98.31+/-0.73% (n=4) of the primordial follicles cluster was MSY2-positive, which indicated the identification method of primordial follicles was more effective and high-yielded than the previous methods because of its higher purity.