Covalent Attachment of Proteins to Solid Supports and Surfaces via Sortase-Mediated Ligation

BACKGROUND: There is growing interest in the attachment of proteins to solid supports for the development of supported catalysts, affinity matrices, and micro devices as well as for the development of planar and bead based protein arrays for multiplexed assays of protein concentration, interactions, and activity. A critical requirement for these applications is the generation of a stable linkage between the solid support and the immobilized, but still functional, protein. METHODOLOGY: Solid supports including crosslinked polymer beads, beaded agarose, and planar glass surfaces, were modified to present an oligoglycine motif to solution. A range of proteins were ligated to the various surfaces using the Sortase A enzyme of S. aureus. Reactions were carried out in aqueous buffer conditions at room temperature for times between one and twelve hours. CONCLUSIONS: The Sortase A transpeptidase of S. aureus provides a general, robust, and gentle approach to the selective covalent immobilization of proteins on three very different solid supports. The proteins remain functional and accessible to solution. Sortase mediated ligation is therefore a straightforward methodology for the preparation of solid supported enzymes and bead based assays, as well as the modification of planar surfaces for microanalytical devices and protein arrays

Increased de novo copy number variants in the offspring of older males

The offspring of older fathers have an increased risk of neurodevelopmental disorders, such as schizophrenia and autism. In light of the evidence implicating copy number variants (CNVs) with schizophrenia and autism, we used a mouse model to explore the hypothesis that the offspring of older males have an increased risk of de novo CNVs. C57BL/6J sires that were 3- and 12–16-months old were mated with 3-month-old dams to create control offspring and offspring of old sires, respectively. Applying genome-wide microarray screening technology, 7 distinct CNVs were identified in a set of 12 offspring and their parents. Competitive quantitative PCR confirmed these CNVs in the original set and also established their frequency in an independent set of 77 offspring and their parents. On the basis of the combined samples, six de novo CNVs were detected in the offspring of older sires, whereas none were detected in the control group. Two of the CNVs were associated with behavioral and/or neuroanatomical phenotypic features. One of the de novo CNVs involved Auts2 (autism susceptibility candidate 2), and other CNVs included genes linked to schizophrenia, autism and brain development. This is the first experimental demonstration that the offspring of older males have an increased risk of de novo CNVs. Our results support the hypothesis that the offspring of older fathers have an increased risk of neurodevelopmental disorders such as schizophrenia and autism by generation of de novo CNVs in the male germline

Is occupation a good predictor of self-rated health in China?

China's rapidly changing economic landscape has led to widening social inequalities. Occupational status in terms of occupational type and prestige may reflect these socio-structural shifts of social position and be more predictive of self-rated health status than income and education, which may only reflect more gradual acquisitions of social status over time. The goals of this study were to understand the role of occupational status in predicting self-rated health, which is well known to be associated with long-term mortality, as well as compare the occupational status to the other major socioeconomic indicators of income and education.Data from the 2010 baseline surveys of the China Family Panel Studies, which utilized multi-stage probability sampling with implicit stratification was used. Logistic regression was used to examine the relationship of various socioeconomic indicators (i.e. occupational status, income, and education) with self-rated health as the primary outcome of interest. A series of models considered the associations of occupational category or occupational prestige with self-rated health.The final sample consisted of 14,367 employed adults aged 18-60, which was nationally representative of working adults in China. We found that occupation was not a major predictor of self-rated health in China when age, ethnicity, location, marital status, physical and mental health status were controlled for, with the exception of women working in lower grade management and professional jobs (OR = 1.82, 95% CI: 1.03-3.22). In comparison, income followed by education exhibited greater association with self-rated health. The highest income group had the least probability to report poor health (In men: OR = 0.30, 95% CI: 0.21-0.43. In women: OR = 0.44, 95% CI: 0.26-0.73). People educated with junior high school had better self-rated health than those with primary and below education level (In men: OR = 0.62, 95% CI: 0.50-0.75. In women: OR = 0.53, 95% CI: 0.42-0.68). Income, education and occupation were correlated with each other.Within the context of rapid societal changes in China, income and its implications for greater healthcare access and benefits had the greatest association with self-rated health followed by education. Occupational status was not associated. Occupational categories and prestige should be better adapted to reflect China's unique sociopolitical and historical context

The Role of Ki-67 in HR+/HER2- Breast Cancer: A Real-World Study of 956 Patients

Qin Ma,1 Yao-Bang Liu,2 Tong She,3 Xin-Lan Liu4 1Department of Radiation Oncology, General Hospital of Ningxia Medical University, Yinchuan, Ningxia, 750004, People’s Republic of China; 2Department of Surgical Oncology, General Hospital of Ningxia Medical University, Yinchuan, Ningxia, 750004, People’s Republic of China; 3Hospital of Zhongwei, Zhongwei, People’s Republic of China; 4Department of Medical Oncology, General Hospital of Ningxia Medical University, Yinchuan, Ningxia, 750004, People’s Republic of ChinaCorrespondence: Xin-Lan Liu, Tel +8613709577339, Email [email protected]: This study determined the cut-off value of Ki-67 expression and discussed the interaction between Ki-67 and histological grade, further explored the prognostic role of Ki-67 in hormone receptor-positive and human epidermal growth factor receptor 2 negative (HR+/HER2-) breast cancer;.Materials and Methods: We assessed the Ki-67 expression of 956 patients with HR+/HER2 breast cancer diagnosed in the General Hospital of Ningxia Medical University from 2015 to 2019 by immunohistochemistry (IHC), The disease-free survival (DFS) was defined as the time from postoperative to the first local recurrence, distant metastasis or death of the disease. The follow-up by means of inpatient or outpatient medical records and telephone.Results: 22.5% was used as the cut-off for low/high Ki-67 expression in HR+/HER2- breast cancer. Compared with the value of 14%, which is commonly used in clinic at present, the consistency of the two values is moderate (Kappa = 0.484, P 22.5% have a worse prognosis and are more likely to have early recurrence and metastasis.Keywords: Ki-67, breast cancer, cut-off value, histological grade, recurrence and metastasi

Synthesis of network reduced graphene oxide in polystyrene matrix by a two-step reduction method for superior conductivity of the composite

Polymer/graphene composites have attracted much attention due to their unique organic-inorganic hybrid structure and exceptional properties. In this paper, we report the synthesis of polystyrene/reduced graphene oxide (PS/r-GO) composites by a two-step in situ reduction technique, which consists of a hydrazine hydrate reduction and a subsequent thermal reduction at 200 degrees C for 12 h. The structure and micromorphology of PS/r-GO composites were characterized by scanning electron microscopy, transmission electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, Raman spectroscopy, X-ray photoelectron spectroscopy and thermogravimetric analysis. The results show that the GO can be efficiently reduced by the two-step in situ reduction method, and the r-GO sheets are well dispersed and ultimately form a continuous network structure in the polymer matrix. PS/r-GO composite films (5 wt% GO) are prepared by the hot press molding method, possessing a conductivity as high as 22.68 S m(-1). The superior conductivity arises from the high reduction degree of GO and its high dispersion and the formation of a network structure in the polymer matrix. These polymer/r-GO composites are expected to be applied in multiple electric devices. The techniques for preparing polymer/r-GO composite films could be further extended to other similar systems

A facile soft-template synthesis of mesoporous polymeric and carbonaceous nanospheres

Owing to the weak self-assembly ability of precursor components and the serious crosslinking of neighbouring nanospheres during a hydrothermal process, the synthesis of monodisperse mesoporous polymer nanospheres with diameters below 500 nm remains a great challenge. Here we extend the synthesis method of mesoporous silica nanospheres to enable the preparation of ordered mesoporous resorcinol formaldehyde nanospheres with particle size from 80 to 400 nm and mesopores of ~3.5 nm in diameter. By finely tuning the synthesis parameters, multi-layered mesoporous resorcinol formaldehyde hollow nanospheres can be successfully synthesized. Mesoporous carbon nanospheres and hollow nanospheres with high surface area are further obtained through carbonization of the polymer spheres. The resulting mesoporous carbon nanospheres are demonstrated as the host cathode material for lithium–sulphur batteries. The synthesis strategy provides a benchmark for fabricating well-defined porous carbonaceous nanospheres with potential for energy storage and conversion applications.Jian Liu, Tianyu Yang, Da-Wei Wang, Gao Qing (Max) Lu, Dongyuan Zhao & Shi Zhang Qia

Activation of Hippo signaling pathway mediates mitochondria dysfunction and dilated cardiomyopathy in mice

Rationale: Mitochondrial dysfunction facilitates heart failure development forming a therapeutic target, but the mechanism involved remains unclear. We studied whether the Hippo signaling pathway mediates mitochondrial abnormalities that results in onset of dilated cardiomyopathy (DCM). Methods: Mice with DCM due to overexpression of Hippo pathway kinase Mst1 were studied. DCM phenotype was evident in adult animals but contractile dysfunction was identified as an early sign of DCM at 3 weeks postnatal. Electron microscopy, multi-omics and biochemical assays were employed. Results: In 3-week and adult DCM mouse hearts, cardiomyocyte mitochondria exhibited overt structural abnormalities, smaller size and greater number. RNA sequencing revealed comprehensive suppression of nuclear-DNA (nDNA) encoded gene-sets involved in mitochondria turnover and all aspects of metabolism. Changes in cardiotranscriptome were confirmed by lower protein levels of multiple mitochondrial proteins in DCM heart of both ages. Mitochondrial DNA-encoded genes were also downregulated; due apparently to repression of nDNA-encoded transcriptional factors. Lipidomics identified remodeling in cardiolipin acyl-chains, increased acylcarnitine content but lower coenzyme Q10 level. Mitochondrial dysfunction was featured by lower ATP content and elevated levels of lactate, branched-chain amino acids and reactive oxidative species. Mechanistically, inhibitory YAP-phosphorylation was enhanced, which was associated with attenuated binding of transcription factor TEAD1. Numerous suppressed mitochondrial genes were identified as YAP-targets. Conclusion: Hippo signaling activation mediates mitochondrial damage by repressing mitochondrial genes, which causally promotes the development of DCM. The Hippo pathway therefore represents a therapeutic target against mitochondrial dysfunction in cardiomyopathy