Development and characterization of polymorphic microsatellite loci in the endangered Yangtze finless porpoise (Neophocaena phocaenoides asiaeorientalis)

The finless porpoise (Neophocaena phocaenoides) is one of the smallest cetacean species widely distributed in the shallow coastal waters of the Indo-Pacific Oceans. The population size of the Yangtze subspecies (N. p. asiaeorientalis) has sharply decreased in the last two decades and access to objective data on its population structure and genetic diversity would be of great assistance for their proper management. Here we report on the isolation of nine polymorphic microsatellite using the “Fast Isolation by AFLP of Sequences Containing repeats” (FIASCO) protocol. Polymorphism was assessed using 30 porpoise individuals randomly sampled in the Yangtze River. The number of alleles per locus varies from 2 to 9, with an average value of 5.56, whereas the ranges of observed and expected heterozygosities were 0.300–0.633 (mean 0.496) and 0.473–0.804 (mean 0.659), respectively.     

人类免疫缺陷病毒1型Tat蛋白N末端缺失突变体融合蛋白的表达及其免疫原性分析

本研究采用PCR方法从人类免疫缺陷病毒1型（Human immunodeficiency virus 1,HIV-1）HXB2株tat基因中扩增编码Tat蛋白N末端1-21位氨基酸缺失的突变体Tat22-101基因片段,构建其原核表达质粒pET32a-Tat22-101,经双酶切及测序验证后,转化大肠埃希菌BL21（DE3）,进行IPTG诱导表达及Ni²⁺-NTA柱亲和层析纯化。纯化后的突变体融合蛋白PET32a-Tat22-101经SDS-PAGE及Western blotting鉴定,其相对分子质量约为26.9kD。该融合蛋白免疫BALB/c小鼠,经ELISA检测结果表明,pET32a-Tat22-101融合蛋白不仅较好地保留其免疫原性,而且能诱导产生高滴度的针对Tat N末端区之外的Tat其他功能区表位的抗体,为进一步研究Tat生物学功能和研制新型HIV Tat疫苗奠定试验基础。     

Progesterone differentially regulates the membrane-type matrix metalloproteinase-1 (MT1 -MMP) compartment of proMMP-2 activation in MG-63 cells.

Osteoblast-derived matrix metalloproteinases (MMPs) are considered to play a crucial role in bone formation and initiation of bone resorption by degrading the bone matrix. MMP-2 is constitutively secreted in a latent zymogen by osteoblasts, and requires the process of activation mediated by membrane-type matrix metalloproteinase-1 (MT1-MMP)/tissue inhibitor of metalloproteinase (TIMP-2) complex in the cell surface. Bone is one target tissue for progestins. In the present study, we observed the effects of progesterone on proMMP-2 activation and MT1-MMP expression, and also TIMP-2 levels in osteoblastic MG-63 cells. Gelatin zymograms and ELISA showed that progesterone have no effects on proMMP-2 activation. Using Western immunoblot analysis, we unexpectedly found that treatment with increasing doses of progesterone in MG-63 cells caused a dose-dependent increase in expression of MT1-MMP protein, and after 48h treatment, progesterone at 10(-8)M increased MT1-MMP protein level. Confocal immunohistochemistry analysis also confirmed that progesterone induced MT1-MMP expression in MG-63 cells. The results of Northern blot analysis showed that progesterone at 10(-8)M increased MT1-MMP protein levels after 48 h treatment. We also found that TIMP-2 levels were undetectable in MG-63 cells. In conclusion, progesterone increases MT1-MMP protein and mRNA levels in MG-63 cells, but has no effects on proMMP-2 activation, which is partly attributable to the undetectable levels of tissue inhibitor of metalloproteinase-2 (TIMP-2). Our studies suggest that TIMP-2 is involved in proMMP-2 activation, and regulation of MT1-MMP by progesterone may contribute to its actions on bone formation.     

Quantitative secretome analysis reveals that COL6A1 is a metastasis-associated protein using stacking gel-aided purification combined with iTRAQ labeling

In cancer metastasis, secreted proteins play an important role in promoting cancer cell migration and invasion and thus also in the increase of cancer metastasis in the extracellular microenvironment. In this study, we developed a strategy that combined a simple gel-aided protein purification with iTRAQ labeling to quantify and discover the metastasis-associated proteins in the lung cancer cell secretome. Secreted proteins associated with lung cancer metastasis were produced using CL1-0 and CL1-5 cells with different metastatic abilities. Quantitative secretomics analysis identified a total of 353 proteins, 7 of which were considered to be metastasis-associated proteins. These included TIMP1, COL6A1, uPA, and AAT, all of which were higher in CL1-5, and AL1A1, PRDX1, and NID1, which were higher in CL1-0. Six of these metastasis-associated proteins were validated with Western blot analysis. In addition, pathway analysis was performed in building the interaction network between the identified metastasis-associated proteins. Further functional analysis of COL6A1 on the metastatic abilities of CL1 cells was also carried out. An RNA interference-based knock-down of COL6A1 suppressed the metastatic ability of CL1-5 cells; in contrast, a plasmid-transfected overexpression of COL6A1 increased the metastatic ability of CL1-0 cells. This study describes a simple and high throughput sample purification method that can be used for the quantitative secretomics analysis of metastasis-associated proteins.     

Lack of reversal effect of EDTA treatment on cadmium induced renal dysfunction: a fourteen-year follow-up

The aim of this study was to evaluate the effect of ethylenediaminetetraacetic acid (EDTA) on cadmium (Cd) induced renal dysfunction. Seventeen workers (14 males, 3 females) were diagnosed with occupational Cd poisoning in 1986. These individuals had between 7 to 39 years of Cd exposure. From 1986 to 1999, patients received periodic EDTA therapy as part of their follow-up, all at the same hospital. Levels of urinary cadmium (UCd) and urinary beta2-microglobulin (B2M) were measured before and after each annual EDTA treatment period. Renal dysfunction was defined as urinary B2M > 0.8 mg/g Cr (creatinine). In these workers, patients with UCd level higher than 10 microg/g Cr in 1986 had abnormal B2M excretions (> or = 0.8 mg/g Cr) or trended to have abnormal B2M levels during the treatment period. However, in subjects with UCd concentration lower than 10 microg/g Cr in 1986, their urinary B2M excretions either remained normal (< 0.8 mg/g Cr) or returned to normal during the treatment period. The prevalence of renal dysfunction increased during the follow up period regardless of whether UCd levels increased or not, indicating a progressive renal dysfunction despite removal from Cd exposure. Our results suggest that reversibility of renal dysfunction caused by Cd related to the level of Cd exposure at the time of removal from exposure: renal dysfunction could be reversed if initial UCd < 10 microg/g Cr, but was irreversible when UCd > 10 microg/g Cr. Repeated examinations on these 17 Cd exposed workers from 1986 to 1999 also revealed that periodic administration of EDTA had no beneficial effects on chronic Cd-induced renal dysfunction.     

A comprehensive synthesis unveils the mysteries of phosphate-solubilizing microbes

Phosphate-solubilizing microbes (PSMs) drive the biogeochemical cycling of phosphorus (P) and hold promise for sustainable agriculture. However, their global distribution, overall diversity and application potential remain unknown. Here, we present the first synthesis of their biogeography, diversity and utility, employing data from 399 papers published between 1981 and 2017, the results of a nationwide field survey in China consisting of 367 soil samples, and a genetic analysis of 12986 genome-sequenced prokaryotic strains. We show that at continental to global scales, the population density of PSMs in environmental samples is correlated with total P rather than pH. Remarkably, positive relationships exist between the population density of soil PSMs and available P, nitrate-nitrogen and dissolved organic carbon in soil, reflecting functional couplings between PSMs and microbes driving biogeochemical cycles of nitrogen and carbon. More than 2704 strains affiliated with at least nine archaeal, 88 fungal and 336 bacterial species were reported as PSMs. Only 2.59% of these strains have been tested for their efficiencies in improving crop growth or yield under field conditions, providing evidence that PSMs are more likely to exert positive effects on wheat growing in alkaline P-deficient soils. Our systematic genetic analysis reveals five promising PSM genera deserving much more attention.