Novel phage display-derived mycolic acid-specific antibodies with potential for tuberculosis diagnosis

Tuberculosis is a major cause of mortality and morbidity due to infectious disease. However, current clinical diagnostic methodologies such as PCR, sputum culture, or smear microscopy are not ideal. Antibody-based assays are a suitable alternative but require specific antibodies against a suitable biomarker. Mycolic acid, which has been found in patient sputum samples and comprises a large portion of the mycobacterial cell wall, is an ideal target. However, generating anti-lipid antibodies using traditional hybridoma methodologies is challenging and has limited the exploitation of this lipid as a diagnostic marker. We describe here the isolation and characterization of four anti-mycolic acid antibodies from a nonimmune antibody phage display library that can detect mycolic acids down to a limit of 4.5ng. All antibodies were specific for the methoxy subclass of mycolic acid with weak binding for α mycolic acid and did not show any binding to closely related lipids or other Mycobacterium tuberculosis (Mtb) derived lipids. We also determined the clinical utility of these antibodies based on their limit of detection for mycobacteria colony forming units (CFU). In combination with an optimized alkaline hydrolysis method for rapid lipid extraction, these antibodies can detect 10⁵ CFU of Mycobacterium bovis BCG, a close relative of Mtb and therefore represent a novel approach for the development of diagnostic assays for lipid biomarkers.     

3,5-Diiodo-l-thyronine ameliorates diabetic nephropathy in streptozotocin-induced diabetic rats

3, 5-Diiodothyronine (T2), a natural metabolite of triiodothyronine (T3) from deiodination pathway, can mimic biologic effects of T3 without inducing thyrotoxic effects. Recent studies revealed T3 acted as a protective factor against diabetic nephropathy (DN). Nevertheless, little is known about the effect of T2 on DN. This study was designed to investigate whether and how T2 affects experimental models of DN in vivo and in vitro. Administration of T2 was found to prevent significant decrease in SIRT1 protein expression and activity as well as increases in blood glucose, urine albumin excretion, matrix expansion, transforming growth factor-β1 expression, fibronectin and type IV collagen deposition in the diabetic kidney. Concordantly, similar effects of T2 were exhibited in the cultured rat mesangial cells (RMC) exposed to high glucose and that could be abolished by a known SIRT1 inhibitor, sirtinol. Moreover, enhanced NF-κB acetylation and JNK phosphorylation present in both diabetic rats and high glucose-treated RMC were distinctly dampened by T2. Collectively, these results suggested that T2 was a protective agent against renal damage in diabetic nephropathy, whose action involved regulation of SIRT1.     

Characterization of one novel cry8 gene from Bacillus thuringiensis strain Q52-7

Bacillus thuringiensis (Bt) is the most widely used insecticidal microbe due to its specific toxicity and safe use with respect to animals and the environment. In this study, we isolated Bt strain Q52-7 from a soil sample collected in the Qian Shan District, Liao Ning Province, China. We observed that the Q52-7 strain produced spherical crystals. The Bt Q52-7 strain had high toxicity against Asian Cockchafer (Holotrichia parallela), exhibiting an LC₅₀ of 3.80 × 10⁹ cfu/g, but is not toxic for Anomala corpulenta Motschulsky and Holotrichia oblita. Using general cry8 primers, we amplified a 1.3 kb fragment with the polymerase chain reaction. Specific primers were designed for the amplified fragment to clone the full-length coding region. A novel gene, cry8Na1, had 69 % sequence similarity with cry8Ca1. cry8Na1 gene was successfully expressed in the HD-73^– acrystalliferous mutant of Bt subsp. Kurstaki HD-73. Bioassays demonstrated that the Cry8Na1 protein is highly toxic for the H. parallela, with a 50 % lethal concentration of 8.18 × 10¹⁰ colony forming units per gram.     

Epidemiology and Outcome of Severe Sepsis and Septic Shock in Intensive Care Units in Mainland China

Introduction

Information about sepsis in mainland China remains scarce and incomplete. The purpose of this study was to describe the epidemiology and outcome of severe sepsis and septic shock in mixed ICU in mainland China, as well as the independent predictors of mortality.

Methods

We performed a 2-month prospective, observational cohort study in 22 closed multi-disciplinary intensive care units (ICUs). All admissions into those ICUs during the study period were screened and patients with severe sepsis or septic shock were included.

Results

A total of 484 patients, 37.3 per 100 ICU admissions were diagnosed with severe sepsis (n = 365) or septic shock (n = 119) according to clinical criteria and included into this study. The most frequent sites of infection were the lung and abdomen. The overall ICU and hospital mortality rates were 28.7% (n = 139) and 33.5% (n = 162), respectively. In multivariate analyses, APACHE II score (odds ratio[OR], 1.068; 95% confidential interval[CI], 1.027–1.109), presence of ARDS (OR, 2.676; 95%CI, 1.691–4.235), bloodstream infection (OR, 2.520; 95%CI, 1.142–5.564) and comorbidity of cancer (OR, 2.246; 95%CI, 1.141–4.420) were significantly associated with mortality.

Conclusions

Our results indicated that severe sepsis and septic shock were common complications in ICU patients and with high mortality in China, and can be of help to know more about severe sepsis and septic shock in China and to improve characterization and risk stratification in these patients.     

Construction and Analysis of High-Ethanol-Producing Fusants with Co-Fermentation Ability through Protoplast Fusion and Double Labeling Technology

Double labeling of resistance markers and report genes can be used to breed engineered Saccharomyces cerevisiae strains that can assimilate xylose and glucose as a mixed carbon source for ethanol fermentation and increased ethanol production. In this study Saccharomyces cerevisiae W5 and Candida shehatae 20335 were used as parent strains to conduct protoplast fusion and the resulting fusants were screened by double labeling. High performance liquid chromatography (HPLC) was used to assess the ethanol yield following the fermentation of xylose and glucose, as both single and mixed carbon sources, by the fusants. Interestingly, one fusant (ZLYRHZ7) was demonstrated to have an excellent fermentation performance, with an ethanol yield using the mixed carbon source of 0.424 g g⁻¹, which compares with 0.240 g g⁻¹ (W5) and 0.353 g g⁻¹ (20335) for the parent strains. This indicates an improvement in the ethanol yield of 43.4% and 16.7%, respectively.     

Early-phase clinical trials of anti-HIV Drugs——understanding and discussion

Innovative anti-HIV drugs developed by local sponsors in China have come into the stage of early-phase clinical trials. How to systemically design the clinical trials of innovative anti-HIV drugs still remains a challenge for them. This article references the literature and the experience of reviewers, to introduce general considerations concerning early-phase clinical trials of innovative anti-HIV drugs.     

Tryptase activates PKB in inflammatory reaction in ECV304 cells

Tryptase is involved in proteinase-activated receptor-2 (PAR-2) mediated up-regulation of IL-8 expression. The present report showed the effects of tryptase on gene expression and activation, including up-regulation IL-8 expression. The expression of mRNA for NF-kappaB first increased at 1 h after tryptase-treatment (1 ng/ml) and reached the plateau after 4 h. The NF-kappaB mRNA increased by 3-fold (n = 3, P < 0.05), AP-1 by 2-fold (n = 3, P < 0.05), and PKB by 10-fold (n = 3, P < 0.05). However, tryptase-treatment did not affect the expression of JNK and p38 MAPK when compared with control cells at mRNA level. Furthermore, in addition to increasing phosphorylation of p38 MAPK, tryptase-treatment also increased phosphorylation of PKB by 2-fold at 15 min following the treatment. The up-regulation and phosphorylation of PKB by tryptase could be abolished by either phosphoinositol-3-kinase (PI3K) inhibitor (LY294002) at 10 microM or antisense PKB cDNA transfection. The up-regulation of NF-kappaB expression could be inhibited by LY294002 and antisense PKB cDNA. These results indicate that tryptase can activate PI3K-PKB pathway and enhance IL-8 expression.     

NAAG peptidase inhibitor increases dialysate NAAG and reduces glutamate, aspartate and GABA levels in the dorsal hippocampus following fluid percussion injury in the rat

Traumatic brain injury (TBI) produces a rapid and excessive elevation in extracellular glutamate that induces excitotoxic brain cell death. The peptide neurotransmitter N-acetylaspartylglutamate (NAAG) is reported to suppress neurotransmitter release through selective activation of presynaptic group II metabotropic glutamate receptors. Therefore, strategies to elevate levels of NAAG following brain injury could reduce excessive glutamate release associated with TBI. We hypothesized that the NAAG peptidase inhibitor, ZJ-43 would elevate extracellular NAAG levels and reduce extracellular levels of amino acid neurotransmitters following TBI by a group II metabotropic glutamate receptor (mGluR)-mediated mechanism. Dialysate levels of NAAG, glutamate, aspartate and GABA from the dorsal hippocampus were elevated after TBI as measured by in vivo microdialysis. Dialysate levels of NAAG were higher and remained elevated in the ZJ-43 treated group (50 mg/kg, i.p.) compared with control. ZJ-43 treatment also reduced the rise of dialysate glutamate, aspartate, and GABA levels. Co-administration of the group II mGluR antagonist, LY341495 (1 mg/kg, i.p.) partially blocked the effects of ZJ-43 on dialysate glutamate and GABA, suggesting that NAAG effects are mediated through mGluR activation. The results are consistent with the hypothesis that inhibition of NAAG peptidase may reduce excitotoxic events associated with TBI.     

Synthesis, characterization and biological activity of lanthanum(III) complexes containing 2-methylene-1,10-phenanthroline units bridged by aliphatic diamines

Two novel lanthanum(III) complexes containing 2-methylene-1,10-phenanthroline units bridged by aliphatic diamines were synthesized and characterized by elemental analysis, IR, NMR, thermal analysis and conductance measurements. They have been assayed for anticancer activity in vitro against HL-60 (human leukocytoma) cells, PC-3MIE8 (human prostate carcinoma) cells, BGC-823 (human stomach carcinoma) cells, MDA-MB-435 (human galactophore carcinoma) cells, Bel-7402 (human liver carcinoma) cells, and Hela (human cervix carcinoma) cells. The results show that the two complexes exhibit good cytotoxic activities against different cell lines in general, especially more effective than cisplatin against Bel-7402, BGC-823 and MDA-MB-435 cell lines. DNA-binding studies indicate that, besides the intercalation, the complexes bind to DNA by the other interaction(s), which might be responsible for the production of more compact DNA, coinciding with more A-like feature of DNA as suggested by CD spectra.