Highly Efficient Production of Soluble Proteins from Insoluble Inclusion Bodies by a Two-Step-Denaturing and Refolding Method

The production of recombinant proteins in a large scale is important for protein functional and structural studies, particularly by using Escherichia coli over-expression systems; however, approximate 70% of recombinant proteins are over-expressed as insoluble inclusion bodies. Here we presented an efficient method for generating soluble proteins from inclusion bodies by using two steps of denaturation and one step of refolding. We first demonstrated the advantages of this method over a conventional procedure with one denaturation step and one refolding step using three proteins with different folding properties. The refolded proteins were found to be active using in vitro tests and a bioassay. We then tested the general applicability of this method by analyzing 88 proteins from human and other organisms, all of which were expressed as inclusion bodies. We found that about 76% of these proteins were refolded with an average of >75% yield of soluble proteins. This “two-step-denaturing and refolding” (2DR) method is simple, highly efficient and generally applicable; it can be utilized to obtain active recombinant proteins for both basic research and industrial purposes

SalK/SalR, a Two-Component Signal Transduction System, Is Essential for Full Virulence of Highly Invasive Streptococcus suis Serotype 2

BACKGROUND: Streptococcus suis serotype 2 (S. suis 2, SS2) has evolved into a highly infectious entity, which caused the two recent large-scale outbreaks of human SS2 epidemic in China, and is characterized by a toxic shock-like syndrome. However, the molecular pathogenesis of this new emerging pathogen is still poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: 89K is a newly predicted pathogenicity island (PAI) which is specific to Chinese epidemic strains isolated from these two SS2 outbreaks. Further bioinformatics analysis revealed a unique two-component signal transduction system (TCSTS) located in the candidate 89K PAI, which is orthologous to the SalK/SalR regulatory system of Streptococcus salivarius. Knockout of salKR eliminated the lethality of SS2 in experimental infection of piglets. Functional complementation of salKR into the isogenic mutant DeltasalKR restored its soaring pathogenicity. Colonization experiments showed that the DeltasalKR mutant could not colonize any susceptible tissue of piglets when administered alone. Bactericidal assays demonstrated that resistance of the mutant to polymorphonuclear leukocyte (PMN)-mediated killing was greatly decreased. Expression microarray analysis exhibited a transcription profile alteration of 26 various genes down-regulated in the DeltasalKR mutant. CONCLUSIONS/SIGNIFICANCE: These findings suggest that SalK/SalR is requisite for the full virulence of ethnic Chinese isolates of highly pathogenic SS2, thus providing experimental evidence for the validity of this bioinformatically predicted PAI

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Sequential mediation of early temperament and eating behaviors in the pathways from feeding practices to childhood overweight and obesity

IntroductionChildhood eating behaviors and temperament may have important implication for constructing the pathways from maternal feeding practices to childhood overweight and obesity (OW/OB). Examining multiple feeding styles simultaneously to childhood OW/OB is critical through the mediators of early childhood temperament and eating behaviors.MethodsThis cross-sectional study recruited mothers mainly responsible for child care from two hospitals and two healthcare centers in eastern China. Sociodemographic characteristics, and data from the Infant Feeding Style Questionnaire (IFSQ), the short form of Children Behavior Questionnaire [Revised (IBQ-RSF)], and the Child Eating Behavior Questionnaire for toddler (CEBQ-T) were collected. Weight and recumbent length were measured to calculate the age- and sex-specific body mass index (BMI) z-scores (BMIz). The structural equation modeling (SEM) approach was used to examine direct and indirect pathways from five maternal feeding styles to childhood OW/OB through temperament and eating behaviors.ResultsA total of 486 children were recruited, 73 (15.02%) children were OW/OB; the age of the children was 14.55 (SD = 5.14) months, and the age of the mothers was 29.90 (SD = 3.63) years. The responsive feeding exerted significant direct (β = −0.098), indirect (β = −0.136) and total (β = −0.234) effects on childhood OW/OB. Restrictive feeding had significant direct (β = 0.222), indirect (β = 0.102) and total (β = 0.324) effects on childhood OW/OB. Indulgent feeding had significant direct (β = 0.220), indirect (β = 0.063), and total (β = 0.283) effects on childhood OW/OB. Pressuring feeding had significant direct (β = −0.116), indirect (β = −0.096) and total (β = −0.212) effects on childhood OW/OB.DiscussionThere was a direct effect of feeding practices on childhood OW/OB; feeding practices indirectly predicted childhood OW/OB through temperament and eating behaviors in children aged 6–23 months. This study could help governments agencies, policymakers, and healthcare workers to establish optimal intervention programs targeting feeding practices through childhood eating behaviors and temperament to prevent childhood OW/OB

Posttranslational modifications of CXCR4: implications in cancer metastasis

CXCR4, the most widely expressed chemokine receptor in solid malignancies, has been implicated in cancer metastasis. However, how the activity of CXCR4 is regulated during carcinogenesis especially at the metastatic stage remains largely unknown. As with other G protein-coupled receptors, CXCR4 is subjected to posttranslational medications such as phosphorylation, ubiquitination, glycosylation, and sulfation. These posttranslational modifications contribute significantly to the heterogeneity of CXCR4 in terms of intracellular location, signaling, and functionality. We have shown that the difference in the sulfation level of CXCR4 is responsible for, if not all, the difference in the activities of CXCR4 between the highly metastatic and non-metastatic nasopharyngeal carcinoma (NPC) cell lines. Molecular mechanistic studies revealed that the Epstein-Barr virus-encoded oncoprotein LMP1 induces the expression of tyrosylprotein sulfotransferase 1 (TPST-1) through nuclear translocation of the epidermal growth factor receptor. This LMP1-regulated TPST-1 expression accounts for tyrosine sulfation of CXCR4 and is associated with the metastatic phenotype of NPC cell lines. Finally, in NPC patient specimens, there was a positive correlation between the expression of LMP1 and TPST-1 and the metastatic potential of NPC. Our findings provide the first evidence that the posttranslational modification of a chemokine receptor plays a role in cancer metastatic progression. Understanding the role of posttranslational modifications of chemokine receptors in cancer biology may provide new insights for developing attractive therapeutic targets in cancer therapy

Characteration and comparative analysis of the whole chloroplast genomes of five common millet (Panicum miliaceum)

Common millet (Panicum miliaceum) is the most valuable and ancient domesticated important crops in the world. We compared five common millet complete chloroplast genomes. A complete map of the variability across the genomes of the five common millet was produced that included single nucleotide variants, InDels, and structural variants, as well as differences in simple sequence repeats and repeat sequences. Molecular phylogeny strongly supported division of the five walnut species into single monophyly with a 100% bootstrap value. The availability of these genomes will provide genetic information for identifying species and hybrids, taxonomy, phylogeny, and evolution in common millet

Forensic investigation of atypical asphysia

Smothering, choking, confined spaces, traumatic asphyxia, positional asphyxia, and other kinds of atypical mechanical asphyxia are not rare in forensic practice. However, these are not commonly well demonstrated in forensic monographs worldwide. The authors researched related works and literatures and summarized these with a view to contribute to the existing teaching resources and provide help to forensic practitioners who are involved in scene investigation and identification of such deaths