The mRNAs for the pro-alpha 1(I) and pro-alpha 2(I) chains of type I procollagen are translated at the same rate in normal human fibroblasts and in fibroblasts from two variants of osteogenesis imperfecta with altered steady state ratios of the two mRNAs

Homologous DNA fragments were prepared from cloned cDNAs for the pro-alpha 1(I) and pro-alpha 2(I) chains of human type I procollagen. The DNA fragments were then used to develop a dot blot hybridization assay for mRNAs for pro-alpha 1(I) and pro-alpha 2(I) chains in skin fibroblasts. In normal fibroblasts, the ratio of the steady state levels of the two mRNAs was 1.94 +/- 0.34 S.D. The ratio for the rates of synthesis of the two pro-alpha chains in the same cells was 1.84 +/- 0.13 S.D. Since the two ratios were essentially the same, the results indicated that the mRNAs for the two chains are translated at about the same rates. Therefore, there is no need to invoke translational control or more complex mechanisms to explain synthesis of pro-alpha 1(I) and pro-alpha 2(I) chains in a stoichiometry of 2:1. The dot blot hybridization assay was also used to examine the levels of the mRNAs in fibroblasts from several variants of osteogenesis imperfecta. In two of the variants, the ratios of the steady state levels of mRNAs for pro-alpha 1(I) and pro-alpha 2(I) chains were 3.05 and 2.52, respectively. In the same fibroblasts, the ratios for the rates of synthesis of the two chains were 2.99 +/- 0.43 and 2.45 +/- 0.16, respectively. Therefore, even though the ratios of the levels of the two mRNAs in the fibroblasts were abnormal, the two mRNAs were still translated at the same rates, and there was no evidence of differential regulation at the translational level.     

Improving the limit of detection in portable luminescent assay readers through smart optical design

Critical biomarkers of disease are increasingly being detected by point‐of‐care assays. Chemiluminescence (CL) and electrochemiluminescence (ECL) are often used in such assays due to their convenience and that they do not require light sources or other components that could complicate or add cost to the system. Reports of these assays often include readers built on a cellphone platform or constructed from low‐cost components. However, the impact the optical design has on the limit of detection (LOD) in these systems remains unexamined. Here, we report a theoretical rubric to evaluate different optical designs in terms of maximizing the use of photons emitted from a CL or ECL assay to improve the LOD. We demonstrate that the majority of cellphone designs reported in the literature are not optimized, in part due to misunderstandings of the optical tradeoffs in collection systems, and in part due to limitations imposed on the designs arising from the use of a mobile phone with a very small lens aperture. Based on the theoretical rubric, we design a new portable reader built using off‐the‐shelf condenser optics, and demonstrate a nearly 10× performance enhancement compared to prior reports on an ECL assays running on a portable chip.     

LINC01342 promotes the progression of ovarian cancer by absorbing microRNA-30c-2-3p to upregulate HIF3A

Ovarian cancer (OC) is a highly prevalent gynecologic malignancy and its mortality is extremely high. Therefore, the development of novel therapeutic approaches for OC is of great significance. In this study, LINC01342 was upregulated in OC tissue in the GSE38666 microarray and in tumor tissue samples collected in our center. The silencing of LINC01342 suppressed the proliferative and metastatic capacities of A2780 and HO8910 cells. Subcellular distribution assays showed that LINC01342 was mainly enriched in the cytoplasm. Subsequently, the downregulation of microRNA-30c-2-3p was proven to be the target of LINC01342. The silencing of microRNA-30c-2-3p enhanced the clonality and migratory capacity of OC cells. Moreover, the silencing of microRNA-30c-2-3p could reverse the inhibited migration and clonality in OC cells caused by LINC01342 knockdown. In addition, hypoxia-inducible factor 3 subunit α (HIF3A) was proven to be the target gene of microRNA-30c-2-3p, which was upregulated. HIF3A was negatively regulated by microRNA-30c-2-3p but positively regulated by LINC01342 in OC cells. An RNA binding protein immunoprecipitation assay showed that microRNA-30c-2-3p, LINC01342, and HIF3A could bind to argonaute RISC catalytic component 2. The overexpression of HIF3A reversed the inhibited migration and clonality in OC cells with LINC01342 knockdown. By analyzing the follow-up data from the enrolled OC patients, the LINC01342 and HIF3A levels were negatively correlated with prognosis, while the microRNA-30c-2-3p level was positively correlated with the same. In short, the upregulated LINC01342 in OC absorbs microRNA-30c-2-3p to release HIF3A. Thus, upregulated HIF3A expression accelerates the progression of OC.     

Rapid Immune Colloidal Gold Strip for Cetacean Meat Restraining Illegal Trade and Consumption: Implications for Conservation and Public Health

The consumption of cetacean meat is geographically common and often of undetermined sustainability. Besides, it can expose humans to contaminants and zoonotic pathogens. The illegality of possessing cetacean meat was likely under-reported in some countries due to lack of attention paid by the officials although DNA analysis of market products helped to show such practices. We developed two monoclonal antibodies against synthetic peptides of myoglobin (Mb) for constructing a rapid immune colloidal gold strip. Only cetacean Mb is capable of binding to both antibodies and presents positive signal while the Mb from other animals can bind only 1 of the antibodies and presents negative result. The strip for cetacean meat would be an applicable and cost-effective test for field inspectors and even the general public. It contributes to increase the reporting capacity and coverage of illegal cetacean meat possession, which has implications for global cetacean conservation and public health.     

ERp57 is up‐regulated in free fatty acids‐induced steatotic L‐02 cells and human nonalcoholic fatty livers

Pathogenesis of nonalcoholic fatty liver disease (NAFLD) is not clear. In this study we aimed to identify proteins involved in NAFLD development in free fatty acids (FFA)‐induced hepatosteatotic cells and in human liver biopsies. Steatosis was induced by incubating a normal human hepatocyte‐derived cell line L‐02 with FFA. Differentially expressed proteins in the steatotic cells were analyzed by two‐dimensional gel electrophoresis‐based proteomics. Involvement of one of the up‐regulated proteins in steatosis was characterized using the RNA interference approach with the steatotic cells. Protein expression levels in liver biopsies of patients with NAFLD were assessed by immunohistochemistry. Proteomic analysis of L‐02 steatotic cells revealed the up‐regulation of ERp57, a condition not previously implicated in NAFLD. Knockdown of ERp57 expression with siRNA significantly reduced fat accumulation in the steatotic cells. ERp57 expression was detected in 16 out of 17 patient biopsies and correlated with inflammation grades or fibrosis stages, while in 5 normal biopsies ERp57 expression was not detectable in hepatocytes. In conclusion, ERp57 was up‐regulated in FFA‐induced steatotic hepatic cells and in NAFLD patient livers and demonstrated steatotic properties in cultured cells. Further investigations are warranted to verify the involvement of ERp57 in NAFLD development. J. Cell. Biochem. 110: 1447–1456, 2010. © 2010 Wiley‐Liss, Inc.     

Influence of Plasma Polymerized Dielectric Buffer Layer and Gold Film on the Excitation of Long-Range Surface Plasmon Resonance

Recently, long-range surface plasmon resonance (LRSPR) sensor has attracted a great deal of attention as a potentially non-destructive and label-free technique for cellular studies in real time. Thus, much effort has been placed on the fabrication and optimization of multilayered structure required for the excitation of LRSPR. In this work, a detailed study about the influence of both plasma polymerized dielectric buffer layer (DBL) and thin gold film on the excitation of LRSPR was performed. The DBLs of different thicknesses were deposited directly onto SF11 glass slides by radio frequency plasma polymerization (pp) of perfluorooctyl ethylene (PFOE). Thereafter, Au films of different thicknesses were thermally evaporated onto the ppPFOE layers. Atomic force microscopy (AFM) results suggest that the resulting SF11/ppPFOE/Au structure has a smooth surface regardless of Au film’s thickness. LRSPR measurements indicate that the excitation of LRSPR relies not only on the thickness of the ppPFOE buffer layer, but also on the thickness and optical property of thin Au film. Theoretical simulation based on Fresnel’s equation allows for the determination of both the thickness and optical constant of each layer supporting the LRSPR, and also enables us to predict the optimum combination of ppPFOE and Au film in a LRSPR sensor. The performance of various LRSPR sensors to monitor the bulk refractive index variation has also been investigated.     

Using intraspecific variation of functional traits and environmental factors to understand the formation of nestedness patterns of a local forest community

从功能性状的种内变异和环境因子的角度理解局域森林群落嵌套格局的成因     

Directed evolution reveals the mechanism of HitRS signaling transduction in Bacillus anthracis

Two component systems (TCSs) are a primary mechanism of signal sensing and response in bacteria. Systematic characterization of an entire TCS could provide a mechanistic understanding of these important signal transduction systems. Here, genetic selections were employed to dissect the molecular basis of signal transduction by the HitRS system that detects cell envelope stress in the pathogen Bacillus anthracis. Numerous point mutations were isolated within HitRS, 17 of which were in a 50-residue HAMP domain. Mutational analysis revealed the importance of hydrophobic interactions within the HAMP domain and highlighted its essentiality in TCS signaling. In addition, these data defined residues critical for activities intrinsic to HitRS, uncovered specific interactions among individual domains and between the two signaling proteins, and revealed that phosphotransfer is the rate-limiting step for signal transduction. Furthermore, this study establishes the use of unbiased genetic selections to study TCS signaling and provides a comprehensive mechanistic understanding of an entire TCS.     

Probing Early Tumor Response to Radiation Therapy Using Hyperpolarized [1-13C]pyruvate in MDA-MB-231 Xenografts

Following radiation therapy (RT), tumor morphology may remain unchanged for days and sometimes weeks, rendering anatomical imaging methods inadequate for early detection of therapeutic response. Changes in the hyperpolarized [1-¹³C]lactate signals observed in vivo following injection of pre-polarized [1-¹³C]pyruvate has recently been shown to be a marker for tumor progression or early treatment response. In this study, the feasibility of using ¹³C metabolic imaging with [1-¹³C]pyruvate to detect early radiation treatment response in a breast cancer xenograft model was demonstrated in vivo and in vitro. Significant decreases in hyperpolarized [1-¹³C]lactate relative to [1-¹³C]pyruvate were observed in MDA-MB-231 tumors 96 hrs following a single dose of ionizing radiation. Histopathologic data from the treated tumors showed higher cellular apoptosis and senescence; and changes in the expression of membrane monocarboxylate transporters and lactate dehydrogenase B were also observed. Hyperpolarized ¹³C metabolic imaging may be a promising new tool to develop novel and adaptive therapeutic regimens for patients undergoing RT.     

A General Synthetic Method for 2′,3′-Dideoxynucleosldes: Total Synthetic Approach

Abstract

A general method for the total synthesis of 2′,3′dideoxynucleosides is described.