Characterization of CD200 Ectodomain Shedding

We have previously reported the existence of a soluble form of CD200 (sCD200) in human plasma, and found sCD200 to be elevated in the plasma of Chronic Lymphocytic Leukemia (CLL) patients. CLL cells release CD200 at a constitutive level, which could be attenuated partially by ADAM28 silencing. In this study, we further explored mechanisms of CD200 shedding beyond that of ADAM28, and performed biochemical analysis of sCD200 using materials derived from purified CLL cells and Hek293 cells stably transfected with CD200, and antibodies generated specifically against either the extracellular or cytoplasmic regions of CD200. CD200 shedding was enhanced by PMA stimulation, and the loss of cell surface CD200 could be monitored as a reduction in CD200 cell surface expression by flow cytometry, in parallel with an increase in the detection of sCD200 in the supernatant. Western blot analyses and functional studies using CD200R1 expressing Hek293 cells showed that the shed CD200 detected in CLL and Hek293-hCD200 supernatants lacked the cytoplasmic domain of CD200 but retained the functional extracellular domain required for binding to, and phosphorylation of, CD200R. These data confirms that a functionally active CD200 extracellular moiety can be cleaved from the surface of CD200 expressing cells following ectodomain shedding.     

Regional Abnormality of Grey Matter in Schizophrenia: Effect from the Illness or Treatment?

Both schizophrenia and antipsychotic treatment are known to modulate brain morphology. However, it is difficult to establish whether observed structural brain abnormalities are due to disease or the effects of treatment. The aim of this study was to investigate the effects of illness and antipsychotic treatment on brain structures in antipsychotic-naïve first-episode schizophrenia based on a longitudinal short-term design. Twenty antipsychotic-naïve subjects with first-episode schizophrenia and twenty-four age- and sex-matched healthy controls underwent 3T MRI scans. Voxel-based morphometry (VBM) was used to examine the brain structural abnormality in patients compared to healthy controls. Nine patients were included in the follow-up examination after 8 weeks of treatment. Tensor-based morphometry (TBM) was used to identify longitudinal brain structural changes. We observed significantly reduced grey matter volume in the right superior temporal gyrus in antipsychotic-naïve patients with schizophrenia compared with healthy controls. After 8 weeks of treatment, patients showed significantly increased grey matter volume primarily in the bilateral prefrontal cortex, insula, right thalamus, left superior occipital cortex and the bilateral cerebellum. In addition, a greater enlargement of the prefrontal cortex is associated with the improvement in negative symptoms, and a more enlarged thalamus is associated with greater improvement in positive symptoms. Our results suggest the following: (1) the abnormality in the right superior temporal gyrus is present in the early stages of schizophrenia, possibly representing the core region related to schizophrenia; and (2) atypical antipsychotics could modulate brain morphology involving the thalamus, cortical grey matter and cerebellum. In addition, examination of the prefrontal cortex and thalamus might facilitate an efficient response to atypical antipsychotics in terms of symptom improvement.     

Whole-Genome Sequencing for the Investigation of a Hospital Outbreak of MRSA in China

Staphylococcus aureus is a globally disseminated drug-resistant bacterial species. It remains a leading cause of hospital-acquired infection, primarily among immunocompromised patients. In 2012, the Affiliated People’s Hospital of Jiangsu University experienced a putative outbreak of methicillin-resistant S. aureus (MRSA) that affected 12 patients in the Neurosurgery Department. In this study, whole-genome sequencing (WGS) was used to gain insight into the epidemiology of the outbreak caused by MRSA, and traditional bacterial genotyping approaches were also applied to provide supportive evidence for WGS. We sequenced the DNA from 6 isolates associated with the outbreak. Phylogenetic analysis was constructed by comparing single-nucleotide polymorphisms (SNPs) in the core genome of 6 isolates in the present study and another 3 referenced isolates from GenBank. Of the 6 MRSA sequences in the current study, 5 belonged to the same group, clustering with T0131, while the other one clustered closely with TW20. All of the isolates were identified as ST239-SCCmecIII clones. Whole-genome analysis revealed that four of the outbreak isolates were more tightly clustered into a group and SA13002 together with SA13009 were distinct from the outbreak strains, which were considered non-outbreak strains. Based on the sequencing results, the antibiotic-resistance gene status (present or absent) was almost perfectly concordant with the results of phenotypic susceptibility testing. Various toxin genes were also analyzed successfully. Our analysis demonstrates that using traditional molecular methods and WGS can facilitate the identification of outbreaks and help to control nosocomial transmission.     

Epistasis Test in Meta-Analysis: A Multi-Parameter Markov Chain Monte Carlo Model for Consistency of Evidence

Conventional genome-wide association studies (GWAS) have been proven to be a successful strategy for identifying genetic variants associated with complex human traits. However, there is still a large heritability gap between GWAS and transitional family studies. The “missing heritability” has been suggested to be due to lack of studies focused on epistasis, also called gene–gene interactions, because individual trials have often had insufficient sample size. Meta-analysis is a common method for increasing statistical power. However, sufficient detailed information is difficult to obtain. A previous study employed a meta-regression-based method to detect epistasis, but it faced the challenge of inconsistent estimates. Here, we describe a Markov chain Monte Carlo-based method, called “Epistasis Test in Meta-Analysis” (ETMA), which uses genotype summary data to obtain consistent estimates of epistasis effects in meta-analysis. We defined a series of conditions to generate simulation data and tested the power and type I error rates in ETMA, individual data analysis and conventional meta-regression-based method. ETMA not only successfully facilitated consistency of evidence but also yielded acceptable type I error and higher power than conventional meta-regression. We applied ETMA to three real meta-analysis data sets. We found significant gene–gene interactions in the renin–angiotensin system and the polycyclic aromatic hydrocarbon metabolism pathway, with strong supporting evidence. In addition, glutathione S-transferase (GST) mu 1 and theta 1 were confirmed to exert independent effects on cancer. We concluded that the application of ETMA to real meta-analysis data was successful. Finally, we developed an R package, etma, for the detection of epistasis in meta-analysis [etma is available via the Comprehensive R Archive Network (CRAN) at https://cran.r-project.org/web/packages/etma/index.html].     

Seamless Insert-Plasmid Assembly at High Efficiency and Low Cost

Seamless cloning methods, such as co-transformation cloning, sequence- and ligation-independent cloning (SLIC) or the Gibson assembly, are essential tools for the precise construction of plasmids. The efficiency of co-transformation cloning is however low and the Gibson assembly reagents are expensive. With the aim to improve the robustness of seamless cloning experiments while keeping costs low, we examined the importance of complementary single-stranded DNA ends for co-transformation cloning and the influence of single-stranded gaps in circular plasmids on SLIC cloning efficiency. Most importantly, our data show that single-stranded gaps in double-stranded plasmids, which occur in typical SLIC protocols, can drastically decrease the efficiency at which the DNA transforms competent E. coli bacteria. Accordingly, filling-in of single-stranded gaps using DNA polymerase resulted in increased transformation efficiency. Ligation of the remaining nicks did not lead to a further increase in transformation efficiency. These findings demonstrate that highly efficient insert-plasmid assembly can be achieved by using only T5 exonuclease and Phusion DNA polymerase, without Taq DNA ligase from the original Gibson protocol, which significantly reduces the cost of the reactions. We successfully used this modified Gibson assembly protocol with two short insert-plasmid overlap regions, each counting only 15 nucleotides.     

Symbiotic Bacteria in Gills and Guts of Chinese Mitten Crab (Eriocheir sinensis) Differ from the Free-Living Bacteria in Water

Aquatic animals have a close relationship with water, but differences in their symbiotic bacteria and the bacterial composition in water remains unclear. Wild or domestic Chinese mitten crabs (Eriocheir sinensis) and the water in which they live were collected from four sampling sites in Jiangsu and Shanghai, China. Bacterial composition in water, gills or guts of E. sinensis, were compared by high-throughput sequencing using 16S rRNA genes. Analysis of >660,000 sequences indicated that bacterial diversity was higher in water than in gills or guts. Tenericutes and Proteobacteria were dominant phyla in guts, while Actinobacteria, Proteobacteria and Bacteroidetes were dominant in gills and water. Non-metric multidimensional scaling analysis indicated that microbiota from gills, guts or water clearly separated into three groups, suggesting that crabs harbor a more specific microbial community than the water in which they live. The dominant OTUs in crab gut were related to Mycoplasmataceae, which were low in abundance in gills, showing that, like mammals, crabs have body-site specific microbiota. OTUs related to Ilumatobacter and Albimonas, which are commonly present in sediment and seawater, were dominant in gills but almost absent from the sampled water. Considering E. sinensis are bottom-dwelling crustacean and they mate in saline water or seawater, behavior and life cycle of crabs may play an important role in shaping the symbiotic bacterial pattern. This study revealed the relationship between the symbiotic bacteria of Chinese mitten crab and their habitat, affording information on the assembly factors of commensal bacteria in aquatic animals.     

Molecular phylogenetic analysis of key <Emphasis Type="Italic">Jatropha</Emphasis> species inferred from nrDNA ITS and chloroplast (<Emphasis Type="Italic">trn</Emphasis>L-F and <Emphasis Type="Italic">rbc</Emphasis>L) sequences

The genus Jatropha (Euphorbiaceae) contains species that are of significant economic and ornamental value. However, Jatropha breeding material is rather limited due to incomplete information regarding phylogenetic relationships among germplasm resources. Phylogenetic analyses were performed based on the internal transcribed spacer of nuclear ribosomal DNA (nrDNA ITS), two chloroplast regions (trnL-F and rbcL), and the combined (ITS+trnL-F+rbcL) dataset among twenty-five specimens representing six key Jatropha species. Phylogenetic relationships of Jatropha were well resolved between subgenus Curcas and subgenus Jatropha, and demonstrated the intermediate position of section Polymorphae among sections of both subgenera. Jatropha curcas and J. integerrima demonstrated a close phylogenetic relationship. The molecular data agreed with the morphological classification that recognized J. multifida and J. podagrica in sec. Peltatae. The distinct intraspecific divergence that occurred in J. curcas could be attributed to restricted gene flow caused by geographical isolation and different ecological conditions. Phylograms produced with trnL-F and rbcL sequence data suggested slow rates of sequence divergence among Jatropha spp., while the ITS gene tree had good resolution suggesting high genetic variation of ITS among Jatropha species.