[Retrospective analysis for 424 330 first-line screening results of non-invasive prenatal testing in Hebei province].

Objective: To evaluate the effect of noninvasive prenatal testing (NIPT) as first-line screening in fetal chromosome aneuploidy screening practice, and to provide evidence for the prevention and control strategy of birth defects. Methods: Since July 2019, Hebei province had carried out the NIPT project providing first-line screening for eligible pregnant women in the area (except for those who were not applicable). Pregnant women with high risk received genetic counseling, prenatal diagnosis and intervention guidance. Low risk and false-positive ones received continuous detection and moved to prenatal diagnosis center for counseling and diagnosis if abnormities were discovered. All pregnant women were followed up to learn about pregnancy outcomes and newborn health status. Detection results and clinical data of pregnant women participating the NIPT project from July 2019 to July 2020 were collected. The detection results and effect of NIPT were analyzed. Results: (1) Basic information of the screened population: A total of 424 330 pregnant women were screened, and 423 596 were successfully detected, with a success rate of 99.83% (423 596/424 330). The age of pregnant women was (28.8±4.5) years old; the gestational age of screening was (16.6±2.3) weeks; the proportion of advanced-age pregnant women (≥35 years old) was 10.18% (43 132/423 596); in vitro fertilization-embryo transfer (IVF-ET) rate was 1.58% (6 713/423 596); the twin rate was 1.38% (5 849/423 596); the proportion of primipara was 34.23% (144 977/423 596). (2) Screening results and detection performance: totally, 325, 73 and 20 pregnant women were diagnosed with trisomy 21, 18 and 13; the sensitivity were 99.39%, 100.00% and 100.00%; the specificity were 99.98%, 99.99% and 99.98%; the positive predictive value were 75.76%, 68.87% and 21.51%, respectively. Besides, 249 190 pregnant women were received supplementary reports as well, and 255, 10 and 9 were confirmed for sex chromosome aneuploidy, other autosomal aneuploidy and deletion/duplication syndrome; the positive predictive value were 37.78%, 6.06% and 32.14%, respectively. The sensitivity of NIPT for target trisomy (trisomy 21, 18 and 13) screening in advanced-age, IVF-ET and twin pregnant women were 99.29%, 100.00% and 90.00%, respectively; the specificity were 99.93% for all; the positive predictive value were 82.25%, 61.54% and 69.23%, respectively. Conclusions: NIPT has a significant effect and good performance in the first-line screening of fetal chromosome aneuploidy in the whole population, which might provide reference for the improvement of birth defect prevention and control strategy.

Founder effects facilitate the use of a genotyping-based approach to molecular diagnosis in Swedish patients with familial hypercholesterolaemia.

AIM: To investigate whether genotyping could be used as a cost-effective screening step, preceding next-generation sequencing (NGS), in molecular diagnosis of familial hypercholesterolaemia (FH) in Swedish patients. METHODS AND RESULTS: Three hundred patients of Swedish origin with clinical suspicion of heterozygous FH were analysed using a specific array genotyping panel embedding 112 FH-causing mutations in the LDLR, APOB and PCSK9 genes. The mutations had been selected from previous reports on FH patients in Scandinavia and Finland. Mutation-negative cases were further analysed by NGS. In 181 patients with probable or definite FH using the Dutch lipid clinics network (DLCN) criteria (score ≥ 6), a causative mutation was identified in 116 (64%). Of these, 94 (81%) were detected by genotyping. Ten mutations accounted for more than 50% of the positive cases, with APOB c.10580G>A being the most common. Mutations in LDLR predominated, with (c.2311+1_2312-1)(2514)del (FH Helsinki) and c.259T>G having the highest frequency. Two novel LDLR mutations were identified. In patients with DLCN score < 6, mutation detection rate was significantly higher at younger age. CONCLUSION: A limited number of mutations explain a major fraction of FH cases in Sweden. Combination of selective genotyping and NGS facilitates the clinical challenge of cost-effective genetic screening in suspected FH. The frequency of APOB c.10580G>A was higher than previously reported in Sweden. The lack of demonstrable mutations in the LDLR, APOB and PCSK9 genes in ~1/3 of patients with probable FH strongly suggests that additional genetic mechanisms are to be found in phenotypic FH.

Enhancement of immune responses by co-administration of bacterial ghosts-mediated Neisseria gonorrhoeae DNA vaccines.

AIM: Gonorrhoea remains a leading public health burden and the development of vaccine against gonorrhoea becomes more urgent. Here, a novel Neisseria gonorrhoeae DNA vaccine delivered by Salmonella enteritidis ghosts was developed and the immune responses of the vaccine candidate were evaluated. METHODS AND RESULTS: Neisseria gonorrhoeae nspA gene was cloned into the pVAX1 vector. The constructed recombinant plasmid pVAX1-nspA was loaded into the lyophilized SE ghosts to produce SE ghosts (pVAX1-nspA). Then, the immune responses induced by SE ghosts (pVAX1-nspA) alone and co-administrated with SE ghosts (pVAX1-porB) were evaluated in mouse model. Co-administered SE ghosts (pVAX1-nspA) and SE ghosts (pVAX1-porB) could elicited significantly higher levels of specific IgG antibody responses and lymphocyte proliferative responses than the control groups. Furthermore, the group co-administered SE ghosts (pVAX1-nspA) and SE ghosts (pVAX1-porB) had the highest bactericidal antibody titres. CONCLUSIONS: Co-administration of SE ghosts (pVAX1-nspA) and SE ghosts (pVAX1-porB) elicited significant specific humoral and cellular immune responses. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the potential of co-administration of SE ghosts (pVAX1-nspA) and SE ghosts (pVAX1-porB) as an attractive vaccination regimen for gonorrhoea.

Different effects of probiotics and antibiotics on the composition of microbiota, SCFAs concentrations and FFAR2/3 mRNA expression in broiler chickens.

AIMS: The aims of this study were to investigate the effects of probiotics and antibiotics on microbial composition, short chain fatty acids (SCFAs) concentration and free fatty acid receptor 2/3 (FFAR2/3) expression in boiler chickens. METHODS AND RESULTS: A total of 150 1-day-old male broilers were randomly allocated into three groups, control (CON) group, probiotics (PB) group and antibiotics (ATB) group. Results indicated that PB improved the average body weight from 1 to 21 days and feed intake from 21 to 42 days (P < 0·05), while ATB improved the feed efficiency from 1 to 42 days (P < 0·05). Based on 16s rRNA sequencing, PB treatment increased the amount of kingdom bacteria, and the relative abundance of the main bacteria including acetate and butyrate producing bacteria of phylum Firmicutes, family Ruminococcaceae and genus Faecalibacterium. ATB treatment also increased the relative abundance of phylum Firmicutes, family Ruminococcaceae and Lachnospiraceae, however, it introduced some pathogenic bacteria, such as bacteria of family Rikenellaceae and Enterobacteriaceae. Gas chromatography and mass spectrometry (GC-MS) assay revealed that PB increased acetate and butyrate concentrations at both 21 and 42 days, and propionate at 42 days in the colorectum. Moreover qRT-PCR analysis showed PB treatment significantly activated the FFAR2/3 mRNA expressions. On the contrast, ATB treatment lowered the colorectal propionate at 21 days, and decreased acetate, propionate and butyrate concentrations at 42 days, accompanied with decreased FFAR2/3 mRNA expressions. CONCLUSIONS: Compared to the CON birds, an enriched SCFAs producing bacteria with higher SCFAs contents and activated FFAR2/3 expressions are prominent features of PB birds. However, antibiotics treatment plays the reverse effect compared to PB treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: This study brings a significant idea that less SCFAs concentration may be another reason why the antibiotics inhibit the immune system development and immunity of the body.

Lipopolysaccharide inhibits hypothalamic Agouti-related protein gene expression via activating mechanistic target of rapamycin signaling in chicks.

Lipopolysaccharide (LPS) induces profound anorexia in birds. However, the neuronal regulatory network underlying LPS-provoked anorexia is unclear. To determine whether any cross talk occurs among hypothalamic mechanistic target of rapamycin (mTOR) and LPS in the regulation of appetite, we performed an intracerebroventricular injection of rapamycin (an mTOR inhibitor) on LPS-treated chicks. The results indicate that peripheral administrations of LPS decreased the agouti-related protein (AgRP) mRNA level, but increased the phosphorylated mTOR and nuclear factor-кB (NF-кB) protein level. Blocking mTOR significantly attenuated LPS-induced anorexia, AgRP suppression, and p-NF-кB increase. Thus, the results suggest that LPS causes anorexia via the mTOR-AgRP signaling pathway, and mTOR signaling is also associated with the regulation of LPS in p-NF-кB.

[Clinical diagnosis of alcoholic hepatitis in Tongliao City, Inner Mongolia].

Objective: To explore the current status of alcoholic hepatitis diagnosis by clinicians' in China. Methods: Clinical data of inpatients confirmed with alcohol-associated liver disease diagnosed at Tongliao Infectious Disease Hospital of Inner Mongolia from June 1, 2018 to May 31, 2019 were retrospectively analyzed. The consistency of clinical diagnosis of alcoholic hepatitis was judged according to the diagnostic criteria recommended by the National Institute of Alcohol Abuse and Alcoholism (USA), and then the alcoholic hepatitis severity assessment model recommended by international guidelines, including Maddrey discriminant function, Model for end-stage liver disease, and Glasgow alcoholic hepatitis score and ABIC scores (age, total bilirubin, international normalized ratio and creatinine) were applied to evaluate this group of cases. Results: Among 79 cases with alcohol-associated liver disease, 75 were males and 4 were females, age ranged between 27~75 (51.1±8.8) years. Alcohol consumption varied from 60 g/d to 600g/d, with an average consumption of 148.8 ± 76.6 g/d. The alcohol consumption duration ranged from 4 to 50 [average (23.9 ± 9.6)] years. According to the initial discharge diagnosis, there were 47 and 32 cases in alcoholic hepatitis and alcoholic liver cirrhosis group, respectively. The mean erythrocyte volume, serum alanine aminotransferase, aspartate aminotransferase and total bilirubin were increased in alcoholic liver cirrhosis than alcoholic hepatitis group, while albumin and total cholesterol were lowered in alcoholic liver cirrhosis than alcoholic hepatitis group, and coagulation indexes were significantly extended. Alpha-fetoprotein of both groups were in the normal range; however, it was significantly higher in the alcoholic hepatitis group than the alcoholic cirrhosis group. The 10 cases in the alcoholic cirrhosis group met the definition and diagnosis of alcoholic hepatitis defined by the National Institute of Alcohol Abuse and Alcoholism (USA), but there was no case in the alcoholic hepatitis group. Among the 10 diagnosed cases of alcoholic hepatitis, 5, 6, 1 and 3 cases met the diagnostic criteria of Maddrey discriminant function, Model for end-stage liver disease, Glasgow alcoholic hepatitis score, and ABIC score for severe alcoholic hepatitis, respectively. The Maddrey discriminant function, ABIC score, and Glasgow alcoholic hepatitis score within the Model for end-stage liver disease scores> 20 points had 5, 1, and 3 cases, respectively. Conclusion: Alcoholic hepatitis is over-diagnosed by clinicians. Alcoholic hepatitis patients have the base of liver cirrhosis who meet the diagnostic criteria of National Institute of Alcohol Abuse and Alcoholism (USA). Patients with Model for end-stage liver disease score > 20 points have good consistency with Maddrey discriminant function score ≥ 32 points, and both can be used to evaluate the alcoholic hepatitis patient clinical severity.

Altered resting-state functional connectivity and effective connectivity of the habenula in irritable bowel syndrome: A cross-sectional and machine learning study.

Irritable bowel syndrome (IBS) is a disorder involving dysfunctional brain-gut interactions characterized by chronic recurrent abdominal pain, altered bowel habits, and negative emotion. Previous studies have linked the habenula to the pathophysiology of negative emotion and pain. However, no studies to date have investigated habenular function in IBS patients. In this study, we investigated the resting-state functional connectivity (rsFC) and effective connectivity of the habenula in 34 subjects with IBS and 34 healthy controls and assessed the feasibility of differentiating IBS patients from healthy controls using a machine learning method. Our results showed significantly enhanced rsFC of the habenula-left dorsolateral prefrontal cortex (dlPFC) and habenula-periaqueductal grey (PAG, dorsomedial part), as well as decreased rsFC of the habenula-right thalamus (dorsolateral part), in the IBS patients compared with the healthy controls. Habenula-thalamus rsFC was positively correlated with pain intensity (r = .467, p = .005). Dynamic causal modeling (DCM) revealed significantly decreased effective connectivity from the right habenula to the right thalamus in the IBS patients compared to the healthy controls that was negatively correlated with disease duration (r = -.407, p = .017). In addition, IBS was classified with an accuracy of 71.5% based on the rsFC of the habenula-dlPFC, habenula-thalamus, and habenula-PAG in a support vector machine (SVM), which was further validated in an independent cohort of subjects (N = 44, accuracy = 65.2%, p = .026). Taken together, these findings establish altered habenular rsFC and effective connectivity in IBS, which extends our mechanistic understanding of the habenula's role in IBS.

Minimal-access retroperitoneal pancreatic necrosectomy for infected necrotizing pancreatitis: a multicentre study of a step-up approach.

BACKGROUND: Various minimally invasive approaches have been described for infected necrotizing pancreatitis. This article describes a modified minimal-access retroperitoneal pancreatic necrosectomy (MARPN) procedure assisted by gas insufflation. METHODS: This retrospective, observational study documented patients who had undergone a step-up MARPN between 1 January 2010 and 31 December 2016. A minimum follow-up of 1 year was required for inclusion. The step-up approach involved percutaneous catheter drainage followed by the modified MARPN and necrosectomy. If more than one access site was needed it was categorized as complex MARPN. RESULTS: Of 212 patients with infected necrotizing pancreatitis, 164 (77·4 per cent) underwent a step-up approach. The median number of percutaneous catheter drains and MARPN procedures was 3 (range 1-7) and 1 (1-6) respectively. Ninety patients (54·9 per cent) underwent complex MARPN. For residual necrosis after MARPN, three patients (1·8 per cent) underwent sinus tract gastroscopy, and 11 (6·7 per cent) had sinography combined with a tube change. However, operations in 13 patients (7·9 per cent) required conversion to open surgery. Postoperative complications developed in 103 patients (62·8 per cent). The mortality rate was 6·1 per cent (10 deaths). CONCLUSION: A step-up approach using a modified MARPN for infected necrotizing pancreatitis is a reasonable option.

ANTECEDENTES: Los procedimientos mínimamente invasivos se han convertido en los más frecuentes para el tratamiento de necrosis pancreáticas infectadas. El objetivo de este estudio fue presentar un procedimiento de necrosectomía pancreática retroperitoneal de acceso mínimo (minimal-access retroperitoneal pancreatic necrosectomy, MARPN) modificado y asistido mediante insuflación de gases, así como evaluar su seguridad y eficacia. MÉTODOS: Se realizó un análisis retrospectivo y observacional de los datos de un hospital desde el 1 de enero de 2010 hasta el 31 de diciembre de 2016. Se incluyeron en el análisis todos los pacientes en los que realizó un abordaje por etapas, que consistía en el drenaje percutáneo mediante la colocación de un catéter seguido de un procedimiento MARPN modificado, en los que se dispusiese de un seguimiento postoperatorio mínimo de 1 año. El MARPN en el lado derecho y la necrosectomía realizada a través de más de un acceso se clasificaron como MARPN complejo. Se evaluaron los resultados radiológicos y quirúrgicos. RESULTADOS: De 212 pacientes con necrosis pancreática infectada, en 164 (77,4%) se realizó un abordaje por etapas. La mediana del número de drenajes percutáneos y procedimientos MARPN fue 3 (rango, 1-7) y 1 (rango, 1-6), respectivamente. En 90 pacientes (54,9%) se realizó un MARPN complejo. Para la exéresis de necrosis residual después de un MARPN, en 3 pacientes (1,8%) se realizó mediante gastroscopia y en 11 pacientes (6,7%) con un recambio de drenaje bajo control radiológico. En 13 pacientes (7,9%) fue necesaria la reconversión a cirugía abierta. Hubo complicaciones postoperatorias en 103 pacientes (62,8%). La tasa de mortalidad fue del 6,1% (n = 10). CONCLUSIÓN: El abordaje por etapas con un MARPN modificado es seguro y efectivo en el tratamiento de la necrosis pancreática infectada.

Mercury oxidation coupled to autotrophic denitrifying branched sulfur oxidation and sulfur disproportionation for simultaneous removal of Hg0 and NO.

Coupling elemental mercury (Hg0) oxidation, autotrophic denitrifying sulfur oxidation, and sulfur disproportionation offers technological potential for simultaneous Hg0 and nitric oxide (NO) removal. This study shed light on simultaneous demercuration and denitration of flue gas by a sulfur-oxidizing membrane biofilm reactor (MBfR). Removal efficiency of Hg0 and NO attained 92% and 83%, respectively in long-term operation. Taxonomic and metagenomic study revealed that a tremendous variety of Hg0-oxidizing bacteria (MOB) (Thiobacillus, Truepera, etc.), denitrifying/sulfur-oxidizing bacteria (DSOB) (Thioalkalivibrio, Thauera, etc.), sulfur-disproportionating bacteria (SDB) (Desulfobulbus, Desulfomicrobium, etc.), and multi-functional bacteria (Halothiobacillus, Thiobacillus, etc.) significantly increased in abundance during growth under feeding of Hg0 and NO in simulated flue gas. The comprehensive employment of sequential chemical extraction processes, inductive coupled mass spectrometry, X-ray diffraction, X-ray photoelectron spectroscopy, and scanning electron microscopy coupled to energy disperse spectroscopy confirmed that Hg0 was finally biologically oxidized to crystallized metacinnabar (ß-HgS) extracellular micromolecular particles. Our findings provided mechanistic insights that MOB, DSOB, and multi-functional bacteria synergistically bio-oxidized Hg0 as the initial electron donor to Hg2+ and denitrified NO as the terminal electron acceptor to N2. SDB disproportionated S0 branched from S2O32- into S2- and SO42-, and ß-HgS formation from Hg2+ and disproportionation-derived S2-, thermodynamically favored Hg0 bio-oxidation. This novel biotechnique can be a cost-effective and environmentally friendly alternative to flue gas Hg0 and NO treatment. KEY POINTS: • Combination of Hg0 bio-oxidation and autotrophic denitrifying sulfur oxidation achieved simultaneous Hg0 and NO removal. • Thiosulfate disproportionation reinforced Hg0 bio-oxidation for Hg0 removal. • Mercury-oxidizing bacteria, denitrifying/sulfur-oxidizing bacteria, and sulfur-disproportionating bacteria synergistically accomplished Hg0 and NO removal.

[Efficiency Analysis of EX16+10Y Kit on Detection of the Uygur Population in Xinjiang Province].

OBJECTIVES: To analyse the efficiency of EX16+10Y kit on the forensic detection of the Uygur in Xinjiang province. METHODS: The blood samples were extracted from 4 620 male individuals of Uygur in Xinjiang province, and amplified by EX16+10Y kit. The typing of amplification products was performed by 3130xl genetic analyzer. RESULTS: The genotyping graphs of 15 autosomal STR loci and 10 Y-chromosomal STR loci from 4 620 male individuals of Uygur in Xinjiang province were acquired completely. The genotype distribution of 15 autosomal STR loci was consistent with Hardy-Weinberg equilibrium. The heterozygosity, polymorphism information content and discrimination power of STR loci were 0.637-0.838, 0.580-0.860 and 0.811-0.978, respectively. There were 766 haplotypes in 10 Y -chromosomal STR loci. CONCLUSIONS: The test results of EX16+10Y kit is accurate and trustworthy, which can simultaneously be used for the individual identification and the screening of paternal pedigree in practical work.

[Assessment of Height Prediction Model Based on SNPs Loci].

OBJECTIVES: To establish a height prediction model of Chinese Han male based on the reported 547 height-associated single nucleotide polymorphisms （SNPs） loci in Europeans, and assess its accuracy for height estimation. METHODS: The DNA typing was analyzed in 59 Han male samples of Shandong province by Affymetrix SNP Array 6.0 chip and HiSeq 4000 sequencing platform. Prediction model was established using 547 height-associated SNPs loci as predictors and weight allele sums （WAS） as computing method. The accuracy of height prediction model was analysed using receiver operating characteristic （ROC） curve and area under curve （AUC）. RESULTS: There was no height-associated SNPs locus was found by genome-wide association studies. In present study, height prediction model was established by WAS and obtained an AUC of 0.67 （95% CI: 0.53-0.90）. CONCLUSIONS: It has reference value for predicting the height of Han male in Shandong province by WAS model based on 547 SNPs loci, while it is still necessary to further promote the accuracy of the prediction model by screening more height-associated SNPs loci with population heterogeneity.

PLS3 sequencing in childhood-onset primary osteoporosis identifies two novel disease-causing variants.

Altogether 95 children with primary bone fragility were screened for variants in PLS3, the gene underlying X-linked osteoporosis. Two children with multiple peripheral and spinal fractures and low BMD had novel disease-causing PLS3 variants. Children with milder phenotypes had no pathogenic variants. PLS3 screening is indicated in childhood-onset primary osteoporosis. INTRODUCTION: The study aimed to determine the role of pathogenic PLS3 variants in children's bone fragility and to elucidate the associated phenotypic features. METHODS: Two cohorts of children with bone fragility were screened for variants in PLS3, the gene underlying X-linked osteoporosis. Cohort I comprised 31 patients with childhood-onset primary osteoporosis of unknown etiology. Cohort II comprised 64 children who had sustained multiple fractures but were otherwise healthy. Clinical and radiological data were reviewed. Peripheral blood DNA was Sanger sequenced for coding exons and flanking intronic regions of PLS3. RESULTS: In two patients of cohort I, where other common genetic causes had been excluded, we identified two novel disease-causing PLS3 variants. Patient 1 was a male with bilateral femoral fractures at 10 years, low BMD (Z-score -4.1; 18 years), and multiple vertebral compression fractures. He had a novel nonsense variant in PLS3. Patient 2 was a girl with multiple long bone and vertebral fractures and low BMD (Z-score -6.6 at 6 years). She had a de novo missense variant in PLS3; whole exome sequencing and array-CGH identified no other genetic causes. Iliac crest bone biopsies confirmed low-turnover osteoporosis in both patients. In cohort II, no pathogenic PLS3 variants were identified in any of the subjects. CONCLUSIONS: Two novel disease-causing variants in PLS3 were identified in a boy and a girl with multiple peripheral and spinal fractures and very low BMD while no pathogenic variants were identified in children with less severe skeletal fragility. PLS3 screening is warranted in male and female patients with childhood-onset primary osteoporosis.

Live performance, carcass characteristic and blood metabolite responses of broilers to two distinct corn types with different extent of grinding.

The major objective of this research was to establish the main and interactive effects of corn type and extent of grinding on broiler performance including carcass characteristics. A completely randomized experimental design with a 2 (corn type) × 2 (fine and coarse) factorial arrangement, each with six replicates of 45 male Ross chicks, was applied. Experimental diets, containing dent or hard corn, were formulated with two extents of grinding (3.00 or 6.00 mm screens) for three growing phases. In comparison with dent corn, the hard corn increased body weight (BW) gain and thigh muscle yield (p < 0.05), while decreasing feed conversion ratio (p < 0.01) and abdominal fat deposition (p < 0.05), some aspects of which were age-dependent and appeared to vary with extent of grinding. Coarser grinding increased the weight of proventriculus (p < 0.01), gizzard (p < 0.05) and small + large intestine (p < 0.10) relative to BW, particularly towards market size. These results suggest that feeding hard corn or large-particle-size corn have some favourable effects on growth performance or gastrointestinal development for finishing broilers.

Calcyon stimulates neuregulin 1 maturation and signaling.

Neuregulin1 (NRG1) is a single transmembrane protein that plays a critical role in neural development and synaptic plasticity. Both NRG1 and its receptor, ErbB4, are well-established risk genes of schizophrenia. The NRG1 ecto-domain (ED) binds and activates ErbB4 following proteolytic cleavage of pro-NRG1 precursor protein. Although several studies have addressed the function of NRG1 in brain, very little is known about the cleavage and shedding mechanism. Here we show that the neuronal vesicular protein calcyon is a potent activator and key determinant of NRG1 ED cleavage and shedding. Calcyon stimulates clathrin-mediated endocytosis and endosomal targeting; and its levels are elevated in postmortem brains of schizophrenics. Overexpression of calcyon stimulates NRG1 cleavage and signaling in vivo, and as a result, GABA transmission is enhanced in calcyon overexpressing mice. Conversely, NRG1 cleavage, ErbB4 activity and GABA transmission are decreased in calcyon null mice. Moreover, stimulation of NRG1 cleavage by calcyon was recapitulated in HEK 293 cells suggesting the mechanism involved is cell-autonomous. Finally, studies with site-specific mutants in calcyon and inhibitors for the major sheddases indicate that the stimulatory effects of calcyon on NRG1 cleavage and shedding depend on clathrin-mediated endocytosis, ß-secretase 1, and interaction with clathrin adaptor proteins. Together these results identify a novel mechanism for NRG1 cleavage and shedding.

Characterization of novel expressed sequence tag-simple sequence repeat markers and analysis of genetic diversity in four geographic populations of Thais luteostoma.

In this study, the genetic diversity in four geographic populations (Yushan Island, Zhoushan, Wenzhou, and Xiamen) of Thais luteostoma was analyzed using 21 microsatellite loci. The results of this study showed that the alleles obtained from different populations ranged from 2 to 8. The average number of alleles and effective alleles were 4.59 and 3.16, respectively. The observed heterozygosity and expected heterozygosity values were in the range 0.338-0.372 and 0.452-0.495, respectively. The polymorphism information content ranged from 0.273 to 0.785. We observed a high level of genetic diversity at 9 of the 21 microsatellite markers in these populations. The genetic differentiation indices of the four geographic populations ranged from 0.0312 to 0.0565, showing a medium level of genetic differentiation. The genetic distances among populations ranged from 0.158 to 0.465. The UPGMA tree indicated that the Yushan Island and Zhoushan populations clustered first, and these subsequently clustered with the Wenzhou and Xiamen populations, indicating that the Xiamen shellfish population was least related to the other populations. The information regarding the shellfish population structure obtained in this study would facilitate the genetic breeding and conservation of T. luteostoma.

Ovalbumin expression in the oviduct magnum of hens is related to the rate of egg laying and shows distinct stress-type-specific responses.

Three trials were performed to evaluate the association of ovalbumin (OVA) abundance in the oviduct magnum with egg production and the underlying regulatory mechanism by glucocorticoids. In trial 1, twenty Hy-Line Brown layers (56-60 weeks of age) with different combinations (n = 5/combination) of laying rate (high or low) and egg weight (high or low) were selected from an initial group of 300. An upregulated expression of magnum OVA was observed (p < 0.05) in hens with higher laying rate, regardless of egg weight. In trial 2, eighty Hy-Line Brown layers (80-90 weeks of age) were subjected to the forced moulting (n = 8). The abundance of OVA transcript and protein in the magnum was significantly decreased during moulting (p < 0.01), and the same was true for laying rate (p < 0.01) and serum oestrogen (p < 0.05). In trial 3, forty-five 56-week-old Hy-Line Brown layers were kept individually (n = 15) in the following conditions for 10 days: constant optimal ambient temperature at 23 °C and ad libitum feeding, high ambient temperature at 32 °C for 6 h/day (10:00-16:00) and ad libitum feeding (32AL), and constant optimal ambient temperature at 23 °C and pair-fed to the 32AL hens. In spite of elevated corticosterone in circulation, OVA synthesis, blood oestrogen and laying rate were not affected by heat exposure (p > 0.05). These results allow concluding that OVA expression in the oviduct magnum of hens is related to the rate of egg laying and shows distinct stress-type-specific responses.

Rapamycin, a specific inhibitor of the target of rapamycin complex 1, disrupts intestinal barrier integrity in broiler chicks.

To uncover the molecular mechanisms underlying the intestinal barrier integrity, this study determined whether the rapamycin (RAPA)-sensitive target of rapamycin complex 1 (TORC1) pathway was involved in this process. Three groups of 4-day-old male chicks were randomly subjected to one of the following treatments for 6 days: high-dose RAPA [a specific inhibitor of TORC1; an intraperitoneal injection of 1.0 mg/kg body weight (BW), once daily at 09:00 hours], low-dose RAPA (0.4 mg/kg BW) and RAPA vehicle (control). Results showed that the RAPA treatment increased mortality, while decreasing villus height (p < 0.01), claudin 1 expression, content of immunoglobulin A (IgA), extent of TORC1 phosphorylation (p < 0.05), ratio of villus height to crypt depth (p < 0.01), and population of IgA-positive B cells in intestinal mucosa, particularly for the jejunum. Some aspects of these responses were dose dependent and appeared to result from weight loss. Together, RAPA exerts the expected inhibition of small intestinal development and IgA production in birds, suggesting the important role of TORC1 in gut barrier integrity.

[Analysis of 8-hydroxy-2'-deoxyguanosine in human urine by ultra-high performance liquid chromatography mass spectrometer method].

OBJECTIVE: To develop an ultra-high performance liquid chromatography mass spectrometer method for the rapid determination of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in human urines. METHODS: 8-OHdG standard solution with the concentration from 0.01 to 0.1 µg/ml was formulated. The solution was implanted into ion source with a rate of 7 µl/min, the mass-to-charge ratio of parent ion and product ions, and ion mass to charge ratio was identified. The mass spectrum parameters of each Ion pairs, such as DP, EP and EXP, were gradually optimized. The urine sample with a concentration of 10.0 µg/L was detected, and the pH of the sample was adjusted using 1 mol/L ammonium formate and formic acid solution with a volume ratio of 5∶1, 4∶1, 3∶1, 2∶1, and 1∶1. It was tested using three different polarity of SPE, i.e.: HLB, MCX, and MAX. The elution effect of methanol and water mixture with the proportion of 90∶10, 80∶20, 50: 50, 20: 80, and 10: 90 were tested, and then acetonitrile and water mixture with the proportion of 90∶10, 80∶20, 50∶50, 20∶80, 10∶90 were also tested. The standard curve was constructed using the ratio of a standard series application fluid concentration to corresponding compounds quantitative ion liquid concentration of the peak area. The detection limit was determined as 3 times of the signal to noise ratio corresponding to the concentration of 8-OHdG, and the quantitative lower limit was determined as 10 times of the signal to noise ratio corresponding to the concentration of 8-OHdG. The blank urine spiked recovery method was used to evaluate the precision and recovery rate. RESULTS: The mass to charge ratio of parent ion was 284.1 and the product ions was 168.1, 140.1, 123.0, and 112.0, respectively. The collision voltage of quantitative ion-pair 284.1/168.1 was 18 V, the 284.1/140.1 collision voltage was 42 V, the 284.1/123.0 collision voltage was 48 V, and the 284.1/112.0 collision voltage was 53 V. The recovery rate was the highest (87.9%-104.3%) when the pH of urine was adjusted by a 10 ml 1 mol/L ammonium formate solution, 2 ml of formic acid, 88 ml of water are mixed with the sample solution volume ratio of 1∶5, and then purified with 3 ml of methanol and 3 ml water activated HLB extraction column. Within 1.0-100.0 µg/L concentration range, 8-OHdG standard application solution test results showed a good linear relationship. The regression equation was y= 1.25x+0.74, and the correlation coefficient was r=0.999 5. The detection limit was 0.2 µg/L, and the limit of quantification was 0.7 µg/L. The method of recovery rate was in the range of 87.9% to 104.3%, the precision was in the range from 1.5% to 3.7% and inter-assay precision was in the range from 1.6% to 5.4%. CONCLUSION: The method developed in this study had high sensitivity, good precision and accuracy, and a wide range of testing concentrations.

[Experience of minimal-access video-assisted retroperitoneal debridement in treatment of infected pancreatic necrosis].

Objective: To explore the experience of minimal-access video-assisted retroperitoneal debridement in treatment of infected pancreatic necrosis(IPN). Methods: A retrospective review was performed on 12 patients with IPN who underwent minimal-access video-assisted retroperitoneal debridement between June 2008 and June 2013 in People's Liberation Army General Hospital and First Affiliated Hospital of People's Liberation Army General Hospital, respectively.There were 10 male patients and 2 female patients aging from 33 to 55 years with mean age of(43±8)years.Pancreas infective necrosis, the serious complications of severe acute pancreatitis occurred in all of the patients among which there were 2 patients with infection after percutaneous catheter drainage(PCD)in early phase of disease, and 12 patients with spontaneous during the late phase. The technical strategies of the minimally invasive treatment mainly included PCD, minimal-access video-assisted retroperitoneal debridement, and irrigation. Results: Ten patients received PCD and the median time from onset of acute necrotizing pancreatitis to PCD was mean of 24 days(range 8-86 days). Minimal-access video-assisted retroperitoneal debridement was performed after 18 days(range 3-29 days) after PCD.Three patients died after surgery.Five patients had hemorrhage complication and 3 had colonic fistula.Pancreatic fistula occurred in 2 patients. Conclusions: The technique of minimal-access video-assisted retroperitoneal debridement has advantage and some minor disadvantage.Delayed minimal-access video-assisted retroperitoneal debridement is recommended.

Genotype/phenotype analysis in Chinese laminin-α2 deficient congenital muscular dystrophy patients.

Laminin-α2 deficient congenital muscular dystrophy (CMD) is an autosomal recessive disorder characterized by severe muscular dystrophy, which is typically associated with abnormal white matter. In this study, we assessed 43 CMD patients with typical white matter abnormality and laminin-α2 deficiency (complete or partial) diagnosed by immunohistochemistry to determine the clinical and molecular genetic characteristics of laminin-α2 deficient CMD. LAMA2 gene mutation analysis was performed by direct sequencing of genomic DNAs. Exonic deletion or duplication was identified by multiplex ligation-dependent probe amplification (MLPA) and verified by high-density oligonucleotide-based CGH microarrays. Gene mutation analysis revealed 86 LAMA2 mutations (100%); 15 known and 37 novel. Among these mutations, 73.9% were nonsense, splice-site or frameshift and 18.8% were deletions of one or more exons. Genetic characterization of affected families will be valuable in prenatal diagnosis of CMD in the Chinese population.