Mutations in PB2 and HA enhanced pathogenicity of H4N6 avian influenza virus in mice.

The H4 subtype avian influenza virus (AIV) continues to circulate in both wild birds and poultry, and occasionally infects mammals (e.g. pigs). H4-specific antibodies have also been detected in poultry farm workers, which suggests that H4 AIV poses a potential threat to public health. However, the molecular mechanism by which H4 AIVs could gain adaptation to mammals and whether this has occurred remain largely unknown. To better understand this mechanism, an avirulent H4N6 strain (A/mallard/Beijing/21/2011, BJ21) was serially passaged in mice and mutations were characterized after passaging. A virulent mouse-adapted strain was generated after 12 passages, which was tentatively designated BJ21-MA. The BJ21-MA strain replicated more efficiently than the parental BJ21, both in vivo and in vitro. Molecular analysis of BJ21-MA identified four mutations, located in proteins PB2 (E158K and E627K) and HA (L331I and G453R, H3 numbering). Further studies showed that the introduction of E158K and/or E627K substitutions into PB2 significantly increased polymerase activity, which led to the enhanced replication and virulence of BJ21-MA. Although individual L331I or G453R substitutions in HA did not change the pathogenicity of BJ21 in mice, both mutations significantly enhanced virulence. In conclusion, our data presented in this study demonstrate that avian H4 virus can adapt to mammals by point mutations in PB2 or HA, which consequently poses a potential threat to public health.

Deletion of the LuxR-type regulator VjbR in Brucella canis affects expression of type IV secretion system and bacterial virulence, and the mutant strain confers protection against Brucella canis challenge in mice.

Brucella spp. are facultative intracellular pathogens and zoonotic agents which pose a huge threat to human health and animal husbandry. The B. melitensis, B. abortus, and B. suis cause undulant fever and influenza-like symptoms in humans. However, the effects of B. canis have not been extensively studied. The quorum sensing-dependent transcriptional regulator VjbR influences the Brucella virulence in smooth type Brucella strains, such as B. melitensis, B. abortus and rough type Brucella ovis. However, the function of VjbR in the rough-type B. canis is unknown. In the present study, we discovered that deletion of this regulator significantly affected Brucella virulence in macrophage and mice infection models. The expression levels of virB operon and the ftcR gene were significantly altered in the vjbR mutant strain. We further investigated the protective effect of different doses of the vjbR mutant in mice and the results indicated that VjbR conferred protection against the virulent B. canis strain. This study presents the first evidence that the transcriptional regulator VjbR has important function in B. canis. In addition, according to its reduced virulence and the protective immunity it induces in mice, it can be a potential live attenuated vaccine against B. canis.

RNA-seq reveals the critical role of Lon protease in stress response and Brucella virulence.

The Brucella spp encounter stressful environment inside their host cells. The Lon protein is an important protease related to cellular protein degradation and resistance to stress in Brucella. However, the molecular mechanism between Lon protein and stress response was still unknown. In this study, it was found that the lon mutant exhibited obvious growth defect in TSB medium, compared with its parent strain. In addition, our results indicated that Lon protein was involved in resistance to various stress conditions and all the ß-lactam antibiotics tested. Although deletion of this protease did not affect Brucella virulence in macrophage, the mutant strain was significantly attenuated in mice infection model at 1 week post infection, and the expression level of several cytokine genes was significantly changed in vivo. To gain insight into the genetic basis for the distinctive phenotypic properties exhibited by the lon mutant strain, RNA-seq was performed, and the result showed that various genes involved in stress response, quorum sensing and transcriptional regulation were significantly altered in Δlon strain. Overall, these studies have preliminary uncovered the molecular mechanism between Lon protease, stress response and bacterial virulence.

Search of Somatic Mutations of NKX2-5 and GATA4 Genes in Chinese Patients with Sporadic Congenital Heart Disease.

Congenital heart disease (CHD) usually occurs sporadically, with only a minority of cases associated with a known genetic mechanism. Cardiac-specific transcription factors NKX2-5 and GATA4 play key roles in the mammalian heart development, and the affected cardiac tissues of CHD patients are prone to somatic mutations which thus participate in the pathogenesis of CHD. We collected 98 patients with sporadic CHD, extracted genomic DNA from cardiac tissues and blood, and then screened NKX2-5 and GATA4 genes using PCR-direct sequence analysis. A novel heterozygous missense mutation (c.907G > A, p.V303I) of NKX2-5 gene was identified in a patient with tetralogy of Fallots. Functional assay revealed that this mutant was associated with significantly reduced transcriptional activity. In addition, we found two known single-nucleotide polymorphisms (SNPs) (rs2277923, rs3729753) in NKX2-5 and two known SNPs (rs56166237, rs3729856) in GATA4. All variations identified in cardiac tissues were consistent with those of peripheral blood, and no somatic mutations were found in cardiac tissues. Our study shows no evidence of NKX2-5 and GATA4 somatic mutations playing a role in the pathogenesis of sporadic CHD.

Whole-Exome Sequencing Reveals Novel Genetic Variation for Dilated Cardiomyopathy in Pediatric Chinese Patients.

Dilated cardiomyopathy (DCM) is characterized by left or bilateral ventricular dilation and systolic dysfunction without rational conditions, which can lead to progressive heart failure and sudden cardiac death. Most of the pathogenic genes have been reported in adult population by locus mapping in familial cases and animal model studies. However, it still remains challenging to decipher the role of genetics in the etiology of pediatric DCM. We applied whole-exome sequencing (WES) for 30 sporadic pediatric DCM subjects and 100 non-DCM local controls. We identified the pathogenic mutations using bioinformatics tools based on genomic strategies synergistically and confirmed mutations by Sanger sequencing. We identified compound heterozygous nonsense mutations in DSP (c.3799C > T, p.R1267X; c.4444G > T, p.E1482X). In sporadic cases, the two heterozygous mutations in XIRP2 were identified. Then we performed an exome-wide association study with 30 case and 100 control subjects. Interestingly, we could not identify TTN truncating variants in all cases. Collectively, we observed a significant risk signal between carriers of TTN deleterious missense variants and DCM risk (odds ratio 4.0, 95% confidence interval 1.1-22.2, p = 3.12 × 10-2). Our observations expanded the spectrum of mutations and were valuable in the pre- and postnatal screening and genetic diagnosis for DCM.

[Clinical characteristics and genetic analysis of three pediatric patients with idiopathic restrictive cardiomyopathy].

OBJECTIVE: Restrictive cardiomyopathy (RCM) is rare in children, and little is known about the molecular basis of RCM. The aim of this study was to investigate the clinical and myopathological characteristics and to detect mutations on cardiac sarcomere protein genes in three idiopathic pediatric RCMs. METHODS: Detailed clinical characteristics and familiar history were obtained in three idiopathic pediatric RCMs. One hundred healthy pediatric individuals were recruited as controls. Histological evaluation was performed with heart tissue retrieved at catheterization in case-1 and case-2. The entire coding sequences of four cardiac sarcomere protein genes, including cardiac troponin T (TNNT2), cardiac troponin I(TNNI3), ß-myosin heavy chain (MYH7), and α-actin (ACTC)were screened for mutations. Sequence variants were then tested in the family as well as in 100 healthy control DNAs. RESULTS: All three index cases were diagnosed as primary RCMs without family history, and their clinical conditions deteriorated rapidly. Case-1 was in combination with ventricular septal defect. Case-2 was in combination with mid- and inferoseptal hypertrophy. In case-1, myocardial biopsies displayed extensive an isomorphism and disarray of cardiomyocytes; electron microscopy showed large stacks of severely dysmorphic megamitochondria and focal Z-disc streaming. In case-2, endomyocardial biopsy revealed moderate myocyte hypertrophy with mild interstitial fibrosis; transmission electron microscopy showed misalignment of Z-bands and unequal Z-Z band distances. Genetic analysis identified two heterozygous missense mutations in TNNI3, with R204H in case-1 and R192H in case-3 respectively. A de novo heterozygous deletion in TNNT2 (p. Asn100_Glu101del) was identified in case-2. Sequence analysis shows that all three mutations are located in a position highly conserved across many species. The three mutations were negative for their parents and controls. CONCLUSION: The clinical conditions in all three index cases are deteriorated rapidly after diagnosed as primary RCM. Three heterozygous mutations including two in TNNI3 and one in TNNT2 gene are identified in the three RCMs respectively, which are considered as causative mutations. These findings provide new insights into the molecular etiology responsible for pediatric RCM.

Case report: Rare restrictive cardiomyopathy with ventricular fibrillation as initial symptom rescued by automatic external defibrillator in a pediatric patient.

Restrictive cardiomyopathy (RCM) is a rare form of heart muscle disease with poor prognosis. Its primary manifestations were caused by systemic or pulmonary circulation congestion. Here, we reported a case of RCM with ventricular fibrillation as initial symptom in a 7-year-old boy. The child suffered cardiac and respiratory arrest suddenly while exercising at school and immediately was given external chest compression and defibrillation by the school's equipped automatic external defibrillator (AED). The rescue was successful. At the time of the AED discharge, his electrocardiogram (ECG) indicated ventricular fibrillation. Upon further examination, the echocardiogram revealed enlarged bilateral atria, decreased diastolic function and normal ventricular thickness. Genetic analysis identified a heterozygous missense mutation [c.611(exon8)G>A,p.R204H] of TNNI3 in the proband boy. This case contributes to our understanding of RCM in children and emphasizes the importance of having AEDs available in public places.

Case report: Rare abernethy malformation with hepatopulmonary syndrome in a pediatric patient.

Abernethy malformation is a rare abnormality of the hepatic portal vein system with non-specific and diverse clinical manifestations. Here, we described a case of abernethy malformation with hepatopulmonary syndrome in a 10-year-old girl. On physical examination, cyanosed lips and acropachy could be found. Her oxygen saturation fluctuated at 89-94%, and the fasting blood ammonia was 98 umol/L. Furthermore, there were abnormalities in the imaging. The microbubble test with contrast echocardiography was positive. Computer tomography angiography (CTA) showed the splenic vein, and the superior mesenteric drained directly into the inferior vena cave after confluence. The same result was also observed in delayed splenic arteriography. Then, we discovered a tiny branch of the intrahepatic portal vein by the inferior vena cava balloon occlusion test, which could also show the confluence of the splenic vein and superior mesenteric vein with the inferior venacave. According to the evidence above, we concluded that the girl was a patient of type II abernethy malformation. For the severe dysplasia of the portal vein, the girl accepted partial ligation of portosystemic shunt and Rex shunt, which improved her oxygen saturation and exercise tolerance.

ToxMVA: An end-to-end multi-view deep autoencoder method for protein toxicity prediction.

Effectively predicting protein toxicity plays an essential step in the early stage of protein-based drug discovery, which is of great help to speed up novel drug screening and reduce costs. Recently, several relevant datasets have been designed, and then machine learning-based methods have been proposed to predict the toxicity of the protein and have shown satisfactory performance. However, previous studies generally directly concatenate different protein features, which may introduce irrelevant information and decrease model performance. In this study, we present a novel end-to-end deep learning-based method called ToxMVA, to predict protein toxicity. To be specific, we first build comprehensive feature profiles of proteins based on primary sequences, including sequential, physicochemical, and contextual semantic information. Next, an autoencoder network is introduced to integrate the multi-view information for obtaining a more concise and accurate feature representation. Extensive experimental results on three datasets demonstrate that ToxMVA has superior performance for protein toxicity prediction and shows better robustness among three different datasets.

Deletion of the Transcriptional Regulator MucR in Brucella canis Affects Stress Responses and Bacterial Virulence.

The transcriptional regulator MucR is related to normal growth, stress responses and Brucella virulence, and affects the expression of various virulence-related genes in smooth-type Brucella strains. However, the function of MucR in the rough-type Brucella canis remains unknown. In this study, we discovered that MucR protein was involved in resistance to heat stress, iron-limitation, and various antibiotics in B. canis. In addition, the expression level of various bacterial flagellum-related genes was altered in mucR mutant strain. Deletion of this transcriptional regulator in B. canis significantly affected Brucella virulence in RAW264.7 macrophage and mice infection model. To gain insight into the genetic basis for distinctive phenotypic properties exhibited by mucR mutant strain, RNA-seq was performed and the result showed that various genes involved in translation, ribosomal structure and biogenesis, signal transduction mechanisms, energy production, and conversion were significantly differently expressed in ΔmucR strain. Overall, these studies have not only discovered the phenotype of mucR mutant strain but also preliminarily uncovered the molecular mechanism between the transcriptional regulator MucR, stress response and bacterial virulence in B. canis.

Case Report: Rare Iliac Vein Compression (May-Thurner) Syndrome in a Pediatric Patient.

Iliac vein compression syndrome (IVCS) or May-Thurner syndrome occurs predominantly in young to middle-aged women. Here we reported a case of IVCS in a 5-year-old boy. The child was admitted to our vasculocardiology department with left lower extremity that had been swollen for 1 month. Blood tests revealed coagulation routine and platelets in the normal ranges. Computer tomography angiography (CTA) and magnetic resonance imaging (MRI) showed the left common iliac vein had become narrow before it entered the right common iliac vein. To further clarify, we performed angiography, which clearly showed the stenosis and the blood return of the left common iliac vein. So IVCS was diagnosed. What is more, we found the aorta descended to the right of the spine, and this may be the reason for the apparent compression of the left common iliac vein. Given the young age and mild symptoms of the child, the treatment was conservative mainly including elevation of the affected limb, wearing medical elastic socks, and short-term oral aspirin for anticoagulation. Meanwhile, the boy is being followed up closely. If the swelling of the left lower extremity significantly increases, stent placement may need to be considered in the future.

Bacteriological and molecular typing of Clostridium perfringens strains isolated in retail beef in Beijing, China.

Clostridium perfringens is an important zoonotic pathogen. This study was designed to explore the prevalence and toxin types of C. perfringens in retail beef collected from Beijing, China. Among 221 beef samples collected, 53 samples were positive for C. perfringens, resulting in the average prevalence as 23.98%. By toxin gene-based typing, the most C. perfringens strains belong to type A (96.23%, 51/53), only 2 strains were identified as type D. By a multi-locus sequence typing (MLST)-based analysis, a total of 36 sequence types (STs) were detected, and the most STs (n=30) represented just a single strain. These finding suggested that the prevalence of C. perfringens in retail beef in Beijing was considerably high and these bacteria displayed extreme diversity in genetics.

Case Report: A Novel Variant c.2262+3A>T of the SCN5A Gene Results in Intron Retention Associated With Incessant Ventricular Tachycardias.

Objective: Voltage-gated sodium channel Nav1.5 encoded by the SCN5A gene plays crucial roles in cardiac electrophysiology. Previous genetic studies have shown that mutations in SCN5A are associated with multiple inherited cardiac arrhythmias. Here, we investigated the molecular defect in a Chinese boy with clinical manifestations of arrhythmias. Methods: Gene variations were screened using whole-exome sequencing and validated by direct Sanger sequencing. A minigene assay and reverse transcription PCR (RT-PCR) were performed to confirm the effects of splice variants in vitro. Western blot analysis was carried out to determine whether the c.2262+3A>T variant produced a truncated protein. Results: By genetic analysis, we identified a novel splice variant c.2262+3A>T in SCN5A gene in a Chinese boy with incessant ventricular tachycardias (VT). This variant was predicted to activate a new cryptic splice donor site and was identified by in silico analysis. The variant retained 79 bp at the 5' end of intron 14 in the mature mRNA. Furthermore, the mutant transcript that created a premature stop codon at 818 amino acids [p.(R818*)] could be produced as a truncated protein. Conclusion: We verified the pathogenic effect of splicing variant c.2262+3A>T, which disturbed the normal mRNA splicing and caused a truncated protein, suggesting that splice variants play an important role in the molecular basis of early onset incessant ventricular tachycardias, and careful molecular profiling of these patients will be essential for future effective personalized treatment options.

Safety and Transcriptome Analysis of Live Attenuated Brucella Vaccine Strain S2 on Non-pregnant Cynomolgus Monkeys Without Abortive Effect on Pregnant Cynomolgus Monkeys.

Brucellosis, caused by Brucella spp., is an important zoonotic disease leading to enormous economic losses in livestock, posing a great threat to public health worldwide. The live attenuated Brucella suis (B. suis) strain S2, a safe and effective vaccine, is widely used in animals in China. However, S2 vaccination in animals may raise debates and concerns in terms of safety to primates, particularly humans. In this study, we used cynomolgus monkey as an animal model to evaluate the safety of the S2 vaccine strain on primates. In addition, we performed transcriptome analysis to determine gene expression profiling on cynomolgus monkeys immunized with the S2 vaccine. Our results suggested that the S2 vaccine was safe for cynomolgus monkeys. The transcriptome analysis identified 663 differentially expressed genes (DEGs), of which 348 were significantly upregulated and 315 were remarkably downregulated. The Gene Ontology (GO) classification and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that these DEGs were involved in various biological processes (BPs), including the chemokine signaling pathway, actin cytoskeleton regulation, the defense response, immune system processing, and the type-I interferon signaling pathway. The molecular functions of the DEGs were mainly comprised of 2'-5'-oligoadenylate synthetase activity, double-stranded RNA binding, and actin-binding. Moreover, the cellular components of these DEGs included integrin complex, myosin II complex, and blood microparticle. Our findings alleviate the concerns over the safety of the S2 vaccine on primates and provide a genetic basis for the response from a mammalian host following vaccination with the S2 vaccine.

Complete Genome Sequence of Mycoplasma bovis Strain XBY01, Isolated from Henan Province, China.

We report the complete genome sequence of Mycoplasma bovis strain XBY01, which was isolated from a severely diseased young calf in Henan Province, China, in 2019. The genome of XBY01 contains a single circular chromosome of 986,067 bp, with a GC content of 29.30%.

Case Report: Identification of Mutations in LAMP2 in Two Chinese Infants With Danon Disease.

Danon disease (DD) is a monogenic lysosomal storage disorder characterized by cardiomyopathy, skeletal myopathy, and variable degrees of intellectual disability. It is caused by a deficiency of lysosomal-associated membrane protein 2 (LAMP2). Two unrelated boys who presented with severe hypertrophic cardiomyopathy and elevated levels of liver enzymes, and were diagnosed with Danon disease at a very young age, were investigated. One boy was diagnosed at 4 months old and died soon after; his mother also died of hypertrophic cardiomyopathy shortly after his birth. Another developed hypertrophic cardiomyopathy at 3 months old but reported no significant cardiovascular symptoms during more than 5 years follow-up. Genetic screening found compound variants of LAMP2 and MYH7 in both of them. This report highlights the clinical heterogeneity in DD. The timely identification of LAMP2 mutation plays a critical role in their treatment and family counseling.

Left Bundle Pacing for Left Bundle Branch Block and Intermittent Third-Degree Atrioventricular Block in a MYH7 Mutation-Related Hypertrophic Cardiomyopathy With Restrictive Phenotype in a Child.

Hypertrophic cardiomyopathy (HCM) is a group of myocardial diseases defined by cardiac hypertrophy which cannot be explained by secondary causes with a non-dilated left ventricle and preserved or increased ejection fraction. Sometimes it can be combined with restrictive cardiomyopathy. Here we describe a very rare case of a 12-year-old girl with non-obstructive hypertrophic cardiomyopathy accompanied by restrictive phenotype, complete left bundle branch block and intermittent third-degree atrioventricular block, who presented with recurrent syncope. Her father was also found to have hypertrophic cardiomyopathy and treated with implantable cardioverter defibrillator for ventricular tachycardia. Her younger brother is currently asymptomatic but echocardiogram showed hypertrophic cardiomyopathy. Genetic analysis identified a heterozygous missense mutation (c.2155C>T, p.R719W) of MYH7 in the proband girl, her father and her brother. The girl was treated with left bundle pacing and recovered well. The case we present further demonstrates the feasibility of left bundle pacing in children.

[Risk factors for acute fulminant myocarditis in children].

OBJECTIVE: To investigate the risk factors for fulminant myocarditis by analyzing clinical symptoms/signs or laboratory findings in children with viral myocarditis. METHODS: The medical data of 71 children with acute viral myocarditis from March 2005 to September 2008 were retrospectively studied. They were classified into fulminant (n=16) and non-fulminant myocarditis groups (n=55). Chi-square and Student's t-test were used to analyze the clinical presentations, laboratory data, EEG and cardiac ultrasound findings on admission. The multiple regression analysis was used to identify the independent risk factors for fulminant myocarditis. RESULTS: Eight children (50%) died in the fulminant myocarditis group, but none in the non-fulminant group. The following factors were closely related to the fulminant course of myocarditis: lower blood pressure, higher serum CK-MB level, positive cTnI, complete atrioventricular block and left bundle branch block, ST segment alterations, prolonged QRS complex, and decreased left ventricular ejection fraction and short axis fractional shortening. Multiple regression analysis revealed that prolonged QRS complex (OR=1.139; CI=1.014-1.279, P<0.05) and decreased left ventricular ejection fraction (OR=0.711; CI=0.533-0.949, P<0.05) were independent risk factors for fulminant myocarditis. CONCLUSIONS: The mortality of fulminant myocarditis is high in children. Prolonged QRS complex and decreased left ventricular ejection fraction on admission are independent risk factors for fulminant myocarditis in children.

A confined space pyrolysis strategy for controlling the structure of hollow mesoporous carbon spheres with high supercapacitor performance.

Hollow mesoporous carbon spheres (HMCSs) have been widely used in energy storage due to their high chemical stability, high surface area, thermal insulation, low effective density and high compressive strength. The electrochemical properties of HMCSs are related to their inner structure. In this work, we demonstrate a facile and controllable synthesis of HMCSs with a tunable inner structure by a confined space pyrolysis strategy. In this process, a phenolic resin oligomer solid sphere is coated with a layer of compact silica. The solid resin sphere can be transformed into a HMCS with a hollow or yolk-shell structure by direct annealing treatment in the silica shell. The compact silica shell can provide a confined space for the pyrolysis of the solid phenolic resin, producing carbon spheres with a hollow cavity, abundant mesopores and a high specific surface area without the use of a template agent. Moreover, the amount of silica precursor and the polymerization time of the resin sphere have great influence on the inner structure of the HMCSs. As an electrode material for supercapacitors, HMCSs display excellent performance with a specific capacitance of 352 F g-1 at a current density of 0.5 A g-1, which is promising for high performance energy storage.

Comparative Transcriptome Analysis of Artificially Induced Rough-Mutant Brucella Strain RM57 and Its Parent Strain Brucella melitensis M1981.

Brucellosis is one of the most common zoonotic epidemics with a serious threat to public health and livestock development in many countries across the world. Vaccination is a key control strategy toward preventing brucellosis in high-prevalence regions. Recently, a rough-type Brucella melitensis mutant strain (RM57) induced from a B. melitensis strain M1981 showed protective effects in guinea pigs indicating that it is a good vaccine candidate. In this study, stress response assays were performed to reveal the mechanisms underlying virulence attenuation of RM57. In addition, a genome-wide transcriptome profile of RM57 was analyzed relative to the parent strain M1981 in order to reveal genetic factors controlling the phenotypes. Our results indicated a similar sensitivity to various stress conditions in RM57 owing to a lack of significant differences from its parent strain. Transcriptome analysis showed that a total of 1,205 genes were differentially expressed between RM57 and M1981 with gene ontology terms revealing that these genes are involved in energy production and conversion, translation, ribosomal structure, and biogenesis. Pathway enrichment analysis revealed that genes involved in oxidative phosphorylation, ribosome, nitrogen metabolism, tyrosine metabolism, and two-component system were significantly affected. As a result of these differences at the molecular level, the function of type IV secretion system in RM57 was found to be affected leading to reduced virulence of the RM57 mutant strain in both macrophage and mice infection models.