Enhancement of proteinase inhibitory activity of recombinant human cystatin C using random-centroid optimization

We had previously written a random-centroid optimization computer program for genetics (RCG) to optimize protein engineering, which was successfully applied to modify single site of the 16 amino acid residues at the active site of B. stearothermophilys neutral protease for improving thermostability [J. Agric. Food Chem., 46 (1998) 1655]. The same program was applied in this study to double-site mutation of the entire sequence of human cystatin C (HCC) with 120 residues for improving its protease inhibitory activity. The RCG program selected two sites simultaneously and amino acid residues to replace the sites selected in the sequence in order to find the best papain-inhibitory activity and stability of the protease inhibitor. Twenty-three double mutants and twenty-two single mutants were expressed by Pichia pastoris. Of the total 45 mutants, G12W/H86V mutant showed a 5-fold increase in the bioactivity over the recombinant wild-type (WT) cystatin. Also, P13F mutant exhibited a half-life temperature (T1/2) 5.2 degrees C higher than 68.2 degrees C of WT in addition to a 56% greater papain inhibitory activity. Mutation for diminishing beta-sheet content reduced polymerization of cystatin C, thus improving papain-inhibitory activity. The approach using RCG was able to improve the functional properties of cystatin by least relying on the prior knowledge of its molecular structure.     

Highly efficient conversion of lactate to pyruvate using whole cells of Acinetobacter sp

On an industrial scale, the production of pyruvate at a high concentration from the cheaper lactate substrate is a valuable process. To produce pyruvate from lactate by whole cells, various lactate-utilizing microorganisms were isolated from soil samples. Among them, strain WLIS, identified as Acinetobacter sp., was screened as a pyruvate producer. For the pyruvate preparation from lactate, the preparative conditions were optimized with whole cells of the strain. The cells cultivated in the medium containing 100 mM of l-lactate showed the highest biotransformation efficiency from lactate to pyruvate. The optimized dry-cell concentration, pH, and temperature of reaction were 6 g/L, pH 7.0-7.5, and 30 degrees C, respectively. The influences of ethylenediaminetetraacetic acid (EDTA) and aeration on a biotransformation reaction were carried out under the test conditions. Under the optimized reaction conditions, l-lactate at concentrations of 200 and 500 mM were almost totally stoichiometrically converted into pyruvate in 8 and 12 h, respectively. About 60% of 800 mM of l-lactate was transformed into pyruvate in 24 h. This reduced conversion rate is probably due to the high substrate inhibition in biotransformation.     

LATE JURASSIC PALAEONTINIDS (HOMOPTERA: AUCHENOR-RHYNCHA) FROM HEBEI AND LIAONING PROVINCES IN CHINA*

Abstract Two new genera and six new species of Palaeontinidae are named and described: Liaocossus hui gen. et sp. nov., L. beipiaoensis gen. et sp. nov., L. exiguus gen. et sp. nov., L. fengningensis gen. et sp. nov., L. pingquanensis gen. et sp. nov., and Miracossus ingentius gen. et sp. nov., The entire body of these new fossils are perfectly preserved and enable us to fix accurately the body characteristics of primitive palaeontinids. All the specimens were collected from Late Jurassic Yixian Formation of Northern Hebei and Western Liaoning Provinces in China.     

Biophysical meanings of orientation and deformation of RBCs in shear flow field of low viscosity with new Ektacytometry

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Influenza Vaccine Induces Intracellular Immune Memory of Human NK Cells

Influenza vaccines elicit antigen-specific antibodies and immune memory to protect humans from infection with drift variants. However, what supports or limits vaccine efficacy and duration is unclear. Here, we vaccinated healthy volunteers with annual vaccine formulations and investigated the dynamics of T cell, natural killer (NK) cell and antibody responses upon restimulation with heterologous or homologous influenza virus strains. Influenza vaccines induced potential memory NK cells with increased antigen-specific recall IFN-γ responses during the first 6 months. In the absence of significant changes in other NK cell markers (CD45RO, NKp44, CXCR6, CD57, NKG2C, CCR7, CD62L and CD27), influenza vaccines induced memory NK cells with the distinct feature of intracellular NKp46 expression. Indeed, surface NKp46 was internalized, and the dynamic increase in NKp46(intracellular)⁺CD56^dim NK cells positively correlated with increased IFN-γ production to influenza virus restimulation after vaccination. In addition, anti-NKp46 antibodies blocked IFN-γ responses. These findings provide insights into a novel mechanism underlying vaccine-induced immunity and NK-related diseases, which may help to design persisting and universal vaccines in the future.     

A Randomized,Double Blind,Placebo-Controlled,Multicenter Phase II Trial of Allisartan Isoproxil in Essential Hypertensive Population at Low-Medium Risk

BackgroundAngiotensin II receptor blockers (ARBs) is a well-tolerated class of antihypertensive agents, exhibiting effective antihypertensive and cardiovascular protective function. The objective of the study was to examine the efficacy and safety of Allisartan Isoproxil, a newly developed, selective, nonpeptide blocker of the angiotensin II type 1 receptor (AT1R), in essential hypertensive patients at low-medium risk.Conclusions/SignificanceAllisartan Isoproxil 240mg was effective and safe for essential hypertension patients at low-medium risk.

Trial Registration

http://www.chictr.org/cn/ ChiCTR-TRC-10000886     

Synergistic and Independent Actions of Multiple Terminal Nucleotidyl Transferases in the 3’ Tailing of Small RNAs in Arabidopsis

All types of small RNAs in plants, piwi-interacting RNAs (piRNAs) in animals and a subset of siRNAs in Drosophila and C. elegans are subject to HEN1 mediated 3’ terminal 2’-O-methylation. This modification plays a pivotal role in protecting small RNAs from 3’ uridylation, trimming and degradation. In Arabidopsis, HESO1 is a major enzyme that uridylates small RNAs to trigger their degradation. However, U-tail is still present in null hen1 heso1 mutants, suggesting the existence of (an) enzymatic activities redundant with HESO1. Here, we report that UTP: RNA uridylyltransferase (URT1) is a functional paralog of HESO1. URT1 interacts with AGO1 and plays a predominant role in miRNA uridylation when HESO1 is absent. Uridylation of miRNA is globally abolished in a hen1 heso1 urt1 triple mutant, accompanied by an extensive increase of 3’-to-5’ trimming. In contrast, disruption of URT1 appears not to affect the heterochromatic siRNA uridylation. This indicates the involvement of additional nucleotidyl transferases in the siRNA pathway. Analysis of miRNA tailings in the hen1 heso1 urt1 triple mutant also reveals the existence of previously unknown enzymatic activities that can add non-uridine nucleotides. Importantly, we show HESO1 may also act redundantly with URT1 in miRNA uridylation when HEN1 is fully competent. Taken together, our data not only reveal a synergistic action of HESO1 and URT1 in the 3’ uridylation of miRNAs, but also independent activities of multiple terminal nucleotidyl transferases in the 3’ tailing of small RNAs and an antagonistic relationship between uridylation and trimming. Our results may provide further insight into the mechanisms of small RNA 3’ end modification and stability control.     

Effects of particulate matter exposure on blood 5-hydroxymethylation: results from the Beijing truck driver air pollution study

Previous studies have reported epigenetic changes induced by environmental exposures. However, previous investigations did not distinguish 5-methylcytosine (5mC) from a similar oxidative form with opposite functions, 5-hydroxymethylcytosine (5hmC). Here, we measured blood DNA global 5mC and 5hmC by ELISA and used adjusted mixed-effects regression models to evaluate the effects of ambient PM₁₀ and personal PM_2.5 and its elemental components—black carbon (BC), aluminum (Al), calcium (Ca), potassium (K), iron (Fe), sulfur (S), silicon (Si), titanium (Ti), and zinc (Zn)—on blood global 5mC and 5hmC levels. The study was conducted in 60 truck drivers and 60 office workers in Beijing, China from The Beijing Truck Driver Air Pollution Study at 2 exams separated by one to 2 weeks. Blood 5hmC level (0.08%) was ∼83-fold lower than 5mC (6.61%). An inter-quartile range (IQR) increase in same-day PM₁₀ was associated with increases in 5hmC of 26.1% in office workers (P = 0.004), 20.2% in truck drivers (P = 0.014), and 21.9% in all participants combined (P < 0.001). PM₁₀ effects on 5hmC were increasingly stronger when averaged over 4, 7, and 14 d preceding assessment (up to 132.6% for the 14-d average in all participants, P < 0.001). PM₁₀ effects were also significant after controlling for multiple testing (family-wise error rate; FWER < 0.05). 5hmC was not correlated with personal measures of PM_2.5 and elemental components (FWER > 0.05). 5mC showed no correlations with PM₁₀, PM_2.5, and elemental components measures (FWER > 0.05). Our study suggests that exposure to ambient PM₁₀ affects 5hmC over time, but not 5mC. This finding demonstrates the need to differentiate 5hmC and 5mC in environmental studies of DNA methylation.