Cardiac sodium channelopathies

Cardiac sodium channel are protein complexes that are expressed in the sarcolemma of cardiomyocytes to carry a large inward depolarizing current (I_Na) during phase 0 of the cardiac action potential. The importance of I_Na for normal cardiac electrical activity is reflected by the high incidence of arrhythmias in cardiac sodium channelopathies, i.e., arrhythmogenic diseases in patients with mutations in SCN5A, the gene responsible for the pore-forming ion-conducting α-subunit, or in genes that encode the ancillary β-subunits or regulatory proteins of the cardiac sodium channel. While clinical and genetic studies have laid the foundation for our understanding of cardiac sodium channelopathies by establishing links between arrhythmogenic diseases and mutations in genes that encode various subunits of the cardiac sodium channel, biophysical studies (particularly in heterologous expression systems and transgenic mouse models) have provided insights into the mechanisms by which I_Na dysfunction causes disease in such channelopathies. It is now recognized that mutations that increase I_Na delay cardiac repolarization, prolong action potential duration, and cause long QT syndrome, while mutations that reduce I_Na decrease cardiac excitability, reduce electrical conduction velocity, and induce Brugada syndrome, progressive cardiac conduction disease, sick sinus syndrome, or combinations thereof. Recently, mutation-induced I_Na dysfunction was also linked to dilated cardiomyopathy, atrial fibrillation, and sudden infant death syndrome. This review describes the structure and function of the cardiac sodium channel and its various subunits, summarizes major cardiac sodium channelopathies and the current knowledge concerning their genetic background and underlying molecular mechanisms, and discusses recent advances in the discovery of mutation-specific therapies in the management of these channelopathies.     

The use of quaternised chitosan-loaded PMMA to inhibit biofilm formation and downregulate the virulence-associated gene expression of antibiotic-resistant staphylococcus

Biomaterial-associated infections remain a serious complication in orthopaedic surgery. Treatments, including the local use of antibiotic-loaded polymethylmethacrylate (PMMA) bone cement, are not always successful because of multiantibiotic-resistant organisms. In this study, we synthesised a new quaternised chitosan derivative (hydroxypropyltrimethyl ammonium chloride chitosan, HACC) that contains a series of substitutions of quaternary ammonium and demonstrated that HACC with a 26% degree of substitution (DS; referred to as 26%HACC) had a strong antibacterial activity and simultaneously good biocompatibility with osteogenic cells. We loaded 26%HACC at 20% by weight into PMMA bone cement to investigate whether HACC in PMMA prevents bacterial biofilm formation on the surface of bone cements. Chitosan-loaded PMMA (at the same weight ratio), gentamicin-loaded PMMA and PMMA with no antibiotic were also investigated and compared. Two clinical isolates, Staphylococcus epidermidis 389 and methicillin-resistant S. epidermidis (MRSE287), and two standard strains, S. epidermidis (ATCC35984) and methicillin-resistant Staphylococcus aureus (ATCC43300), were selected to evaluate the bacterial biofilm formation at 6, 12 and 24 h using the spread plate method, confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). The results showed that 26%HACC-loaded PMMA inhibited biofilm formation on its surface, while the PMMA control and chitosan-loaded PMMA were unable to inhibit biofilm formation. The gentamicin-loaded PMMA decreased the number of viable methicillin-resistant Staphylococcus strains, but its ability to inhibit biofilm formation was lower than 26%HACC-loaded PMMA. Real-time PCR demonstrated that 26%HACC-loaded PMMA markedly downregulated the expression of icaAD, which encodes essential enzymes for polysaccharide intercellular adhesion (PIA) biosynthesis, upregulated the expression level of icaR, which negatively mediates icaAD expression, and also downregulated the expression of MecA, which encodes membrane-bound enzymes known to be penicillin-binding proteins. Our study indicates that 26%HACC-loaded PMMA prevents biofilm formation of Staphylococcus, including antibiotic-resistant strains, on the surface of bone cement, and downregulates the virulence-associated gene expression of antibiotic-resistant staphylococcus, thus providing a promising new strategy for combating implant infections and osteomyelitis.     

Natural mineralized scaffolds promote the dentinogenic potential of dental pulp stem cells via the mitogen-activated protein kinase signaling pathway

The selection of a suitable scaffold material is important for dentin tissue regeneration, as the characteristics of biomaterials can potentially influence cell proliferation and differentiation. We compared the effects of different scaffolds on dentin regeneration based on dental pulp stem cells (DPSCs) and investigated the regulatory mechanisms of odontogenic differentiation of DPSCs by these scaffolds. Five different scaffolds were tested: demineralized dentin matrix (DDM), ceramic bovine bone (CBB), small intestinal submucosa (SIS), poly-L-lactate-co-glycolate, and collagen-chondroitin sulfate-hyaluronic acid. DPSCs cultured on DDM and CBB exhibited higher levels of alkaline phosphatase (ALP) activity and mRNA expression of bone sialoprotein, osteocalcin, dentin sialophosphoprotein (DSPP), and dentin matrix protein-1 (DMP-1) than those cultured on the other three scaffolds. Further, the phosphorylation levels of mitogen-activated protein kinase (MAPK) ERK1/2 and p38 in DPSCs cultured on DDM and CBB were also significantly enhanced compared with the other three scaffolds, and their inhibitors significantly inhibited odontogenic differentiation as assessed by ALP activity and mRNA expression of DSPP and DMP-1. The implantation experiment confirmed these results and showed a large amount of regular-shaped dentin-pulp complex tissues, including dentin, predentin, and odontoblasts only in the DDM and CBB groups. The results indicated that natural mineralized scaffolds (DDM and CBB) have potential as attractive scaffolds for dentin tissue-engineering-promoted odontogenic differentiation of DPSCs through the MAPK signaling pathway.     

A genome-wide association and gene-environment interaction study for serum triglycerides levels in a healthy Chinese male population

Triglyceride (TG) is a complex phenotype influenced by both genetic and environmental factors. Recent genome-wide association studies (GWAS) have identified genes or loci affecting lipid levels; however, such studies in Chinese populations are limited. A two-stage GWAS were conducted to identify genetic variants that were associated with TG in a Chinese population of 3495 men. Gene-environment interactions on serum TG levels were further investigated for the seven single nucleotide polymorphisms (SNPs) that were studied in both stages. Two previously reported SNPs (rs651821 in APOA5, rs328 in LPL) were replicated in the second stage, and the combined P-values were 9.19 × 10(-26) and 1.41 × 10(-9) for rs651821 and rs328, respectively. More importantly, a significant interaction between aldehyde dehydrogenase 2 (ALDH2) rs671 and alcohol consumption on serum TG levels were observed (P = 3.34 × 10(-5)). Rs671 was significantly associated with serum TG levels in drinkers (P = 1.90 × 10(-10)), while no association was observed in non-drinkers (P > 0.05). For drinkers, men carrying the AA/AG genotype have significantly lower serum TG levels, compared with men carrying the GG genotype. For men with the GG genotype, the serum TG levels increased with the quantity of alcohol intake (P = 1.28 × 10(-8) for trend test). We identified a novel, significant interaction effect between alcohol consumption and the ALDH2 rs671 polymorphism on TG levels, which suggests that the effect of alcohol intake on TG occurs in a two-faceted manner. Just one drink can increase TG level in susceptible individuals who carry the GG genotype, while individuals carrying AA/AG genotypes may actually benefit from moderate drinking.     

Ectopic bone regeneration by human bone marrow mononucleated cells, undifferentiated and osteogenically differentiated bone marrow mesenchymal stem cells in beta-tricalcium phosphate scaffolds

Tissue engineering approaches using the combination of porous ceramics and bone marrow mesenchymal stem cells (BMSCs) represent a promising bone substitute for repairing large bone defects. Nevertheless, optimal conditions for constructing tissue-engineered bone have yet to be determined. It remains unclear if transplantation of predifferentiated BMSCs is superior to undifferentiated BMSCs or freshly isolated bone marrow mononucleated cells (BMNCs) in terms of new bone formation in vivo. The aim of this study was to investigate the effect of in vitro osteogenic differentiation (β-glycerophosphate, dexamethasone, and l-ascorbic acid) of human BMSCs on the capability to form tissue-engineered bone in unloaded conditions after subcutaneous implantation in nude mice. After isolation from human bone marrow aspirates, BMNCs were divided into three parts: one part was seeded onto porous beta-tricalcium phosphate ceramics immediately and transplanted in a heterotopic nude mice model; two parts were expanded in vitro to passage 2 before cell seeding and in vivo transplantation, either under osteogenic conditions or not. Animals were sacrificed for micro-CT and histological evaluation at 4, 8, 12, 16, and 20 weeks postimplantation. The results showed that BMSCs differentiated into osteo-progenitor cells after induction, as evidenced by the altered cell morphology and elevated alkaline phosphatase activity and calcium deposition, but their clonogenicity, proliferating rate, and seeding efficacy were not significantly affected by osteogenic differentiation, compared with undifferentiated cells. Extensive new bone formed in the pores of all the scaffolds seeded with predifferentiated BMSCs at 4 weeks after implantation, and maintained for 20 weeks. On the contrary, scaffolds containing undifferentiated BMSCs revealed limited bone formation only in 1 out of 6 cases at 8 weeks, and maintained for 4 weeks. For scaffolds with BMNCs, woven bone was observed sporadically only in one case at 8 weeks. Overall, this study suggests that ectopic osteogenesis of cell/scaffold composites is more dependent on the in vitro expansion condition, and osteo-differentiated BMSCs hold the highest potential concerning in vivo bone regeneration.     

超顺磁性氧化铁标记对骨髓间充质干细胞多向分化诱导的影响

研究兔骨髓间充质干细胞(BMSCs)经超顺磁性氧化铁(SPIO)标记后,体外成骨、成脂及成软骨诱导能力的变化。体外贴壁培养和扩增兔BMSCs,采用SPIO(25μg/ml)联合硫酸鱼精蛋白转染剂标记兔BMSCs,分别采用适宜的成骨、成脂及成软骨诱导培养液对磁标记BMSCs进行体外定向诱导培养3周,诱导过程中观察细胞形态学变化。3周后对成骨定向诱导组进行钙结节茜素红染色和碱性磷酸酶(ALP)组化染色;对成脂肪定向诱导组行油红-O染色;对成软骨定向诱导组行番红O染色、II型胶原免疫组化染色检测观察胞外基质糖胺多糖、II型胶原的分泌和表达。利用Image-Pro Plus图像分析系统对免疫细胞化学染色进行光密度半定量分析。结果表明:超顺磁性氧化铁粒子标记BMSCs后普鲁士染色和电镜检查显示细胞胞浆内含致密铁颗粒;磁标记BMSCs在成骨、成脂及成软骨潜在多向分化诱导能力上与对照组未标记细胞相比无统计学差异。提示,SPIO联合硫酸鱼精蛋白转染剂能成功标记BMSCs,超顺磁性氧化铁标记对兔BMSCs体外多向分化诱导能力无明显影响,合理应用这种新型细胞标记技术将促进对组织工程种子细胞的研究。     

人类新基因CTRP4的原核表达及多克隆抗体的制备

目的:构建新基因CTRP4的原核表达载体,在大肠杆菌中表达并纯化rhCTRP4-his蛋白,制备和鉴定小鼠抗人CTRP4多克隆抗体,为进一步研究人类新基因CTRP4的生物学功能奠定基础。方法:从pcDNA3.1-myc/his(-)B-hCTRP4重组载体中通过PCR的方法扩增CTRP4基因ORF中不含信号肽的目的基因片段,将得到的片段双酶切定向插入pET-32a中,构建原核表达载体pET-32a-hCTRP4。构建好的质粒在E.coli BL21(DE3)中进行诱导、表达,通过镍亲和层析柱纯化融合蛋白,SDS-PAGE电泳分析蛋白纯度。将pcDNA3.1-myc/his(-)B-hCTRP4重组质粒和纯化好的融合蛋白依次免疫BALB/c小鼠,制备多克隆抗体。并对抗体进行纯化、效价测定以及特异性鉴定。结果:DNA测序证实构建的pET-32a-hCTRP4重组表达载体含有hCTRP4编码序列,其序列比对分析与GenBank中公布序列一致,质粒在E.coli中表达相对分子质量(Mr)为35 000的目的蛋白,SDS-PAGE电泳分析纯度为95%以上。ELISA法检测抗体效价为1∶20 000,Westernblot、免疫荧光细胞化学和免疫组化分析显示抗体能特异性识别重组hCTRP4以及天然状态hCTRP4蛋白。结论:成功构建了hCTRP4蛋白的原核表达载体,并获得较高纯度的融合蛋白,制备了高效价、高特异性的多克隆抗体。     

Characterization of Baylisascaris schroederi from Qinling subspecies of giant panda in China by the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA

In the present study, a total of 20 nematode isolates, (including 10 male and 10 female worms) representing Baylisascaris schroederi from 5 Qinling subspecies of giant pandas (Ailuropoda melanoleuca) in Shaanxi Province of China, were characterized and grouped genetically by the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA (rDNA). The rDNA fragment spanning 3′ end of 18S rDNA, complete ITS-1 rDNA, and 5′ end of 5.8S rDNA were amplified and sequenced. The sequence variability in ITS-1 rDNA was examined within B. schroederi and among parasites in order Ascaridata available in GenBank™, and their phylogenetic relationships were also reconstructed. The sequences of ITS-1 rDNA for all the B. schroederi isolates were 427 bp in length, with no genetic variation detected among these isolates. Phylogenetic analyses based on the ITS-1 rDNA sequences revealed that all the male and female B. schroederi isolates sequenced in the present study were posited into the clade of genus Baylisascaris, sistered to zoonotic nematodes in genus Ascaris, and the ITS-1 rDNA sequence could distinguish different species in order Ascaridata. These results showed that the ITS-1 rDNA provides a suitable molecular marker for the inter-species phylogenetic analysis and differential identification of nematodes in order Ascaridata.     

Increased program cell death-1 expression on T lymphocytes of patients with progressive multifocal leukoencephalopathy

The cellullar immune response is important in the containment of progressive multifocal leukoencephalopathy (PML). We examined program cell death-1 (PD-1) expression, a marker of cellular immune exhaustion, on T lymphocytes in PML. PD-1 expression was elevated on total CD4(+) and CD8(+) T cells (medians 36% and 24%) in PML patients compared with healthy control subjects (medians 14% and 18%; P = 0.0015 and P = 0.033). In PML patients, JC virus (JCV)-specific CD8(+) cytotoxic T lymphocytes expressed PD-1 more frequently than total CD8 T lymphocytes (means 39% and 78%, P = 0.0004). Blocking the PD-1 receptor increased JCV-specific T-cell immune response in a subgroup of PML patients.