Exploration of chlamydial type III secretion system reconstitution in Escherichia coli

BACKGROUND: Type III secretion system is a virulent factor for many pathogens, and is thought to play multiple roles in the development cycle and pathogenesis of chlamydia, an important human pathogen. However, due to the obligate intracellular parasitical nature of chlamydiae and a lack of convenient genetic methodology for the organisms, very limited approaches are available to study the chlamydial type III secretion system. In this study, we explored the reconstitution of a chlamydial type III secretion in Escherichia coli. RESULTS: We successfully cloned all 6 genomic DNA clusters of the chlamydial type III secretion system into three bacterial plasmids. 5 of the 6 clusters were found to direct mRNA synthesis from their own promoters in Escherichia coli transformed with the three plasmids. Cluster 5 failed to express mRNA using its own promoters. However, fusion of cluster 5 to cluster 6 resulted in the expression of cluster 5 mRNA. Although only two of the type III secretion system proteins were detected transformed E. coli due to limited antibody availability, type III secretion system-like structures were detected in ultrathin sections in a small proportion of transformed E. coli. CONCLUSIONS: We have successfully generated E. coli expressing all genes of the chlamydial type III secretion system. This serves as a foundation for optimal expression and assembly of the recombinant chlamydial type III secretion system, which may be extremely useful for the characterization of the chlamydial type III secretion system and for studying its role in chlamydial pathogenicity

Dynamics simulation research on load vehicle of deep submergence rescue vehicle (LV-DSRV)

Submarine accidents can cause loss of human life and economy, as well as environment damage. Also submarine rescue is difficult for the complexity of rescue process and the uncertainty of the sea state. Success of rescue process is determined by reliability and safety of rescue device. Therefore, research on related device design is rather important. The existing knowledge always supplies some empirical formulas and generates a design scheme by general design rules. The scheme plan obtained may not be a good one due to simplicity of mechanical calculation analysis and particularity of design requirements. To improve design safety and reliability of shipborne device on submarine rescue system, dynamics simulation model of LV-DSRV based on sea state excitation was created in this paper. Wave excitation input at sea state 5 and 8 was considered as the extreme marine working conditions. Kinetic property of LV-DSRV was calculated making use of virtual prototype technology through ADAMS software, and mechanical characteristics of key parts were also analyzed. Optimization strategy was proposed and verified by increasing the number of horizontal wheels and adding gap between horizontal wheels and the track, providing with a case study for similar marine special mechanism design

Estimating Treatment Effects Under Heterogeneous Interference

Treatment effect estimation can assist in effective decision-making in e-commerce, medicine, and education. One popular application of this estimation lies in the prediction of the impact of a treatment (e.g., a promotion) on an outcome (e.g., sales) of a particular unit (e.g., an item), known as the individual treatment effect (ITE). In many online applications, the outcome of a unit can be affected by the treatments of other units, as units are often associated, which is referred to as interference. For example, on an online shopping website, sales of an item will be influenced by an advertisement of its co-purchased item. Prior studies have attempted to model interference to estimate the ITE accurately, but they often assume a homogeneous interference, i.e., relationships between units only have a single view. However, in real-world applications, interference may be heterogeneous, with multi-view relationships. For instance, the sale of an item is usually affected by the treatment of its co-purchased and co-viewed items. We hypothesize that ITE estimation will be inaccurate if this heterogeneous interference is not properly modeled. Therefore, we propose a novel approach to model heterogeneous interference by developing a new architecture to aggregate information from diverse neighbors. Our proposed method contains graph neural networks that aggregate same-view information, a mechanism that aggregates information from different views, and attention mechanisms. In our experiments on multiple datasets with heterogeneous interference, the proposed method significantly outperforms existing methods for ITE estimation, confirming the importance of modeling heterogeneous interference

High temperature strain heterogeneities tracking within hole-specimens of fv566 turbine steel via digital image correlation

This paper presents experimental and numerical investigations to study spatial-temporal distributions of strain heterogeneities within a FV566 turbine rotor steel subjected to high temperature conditions. Strain field were determined during monotonic tensile tests performed on flat specimens containing a circular hole to give rise to heterogeneous strain fields. A home-made digital image correlation system with an image restoration module was developed and employed to record and analyse the deformation process till the macroscopic fracture of the investigated specimens. Noticeable in-plane strain concentration zones and effects of the sample design were highlighted and deeply examined. Experimental results combined with finite element analyses based on a classical Johnson-Cook model indicated that the hole shape was the important factor affecting the strength and the spatio-temporal distribution of the strain fields. Microstructural investigations were also conducted to characterize and assess the microscale damage features of the FV566 steel. The ruptured area for all the investigated temperature tests revealed ductile rupture behaviour. Changes on grain boundaries and misorientation near strain localized regions were also investigated. It was found that the propagation of cracks surrounding the hole in FV566 specimens was mainly affected by the nucleation and propagation of strain localization zones.AVIC (Chine), CSC (Chine

A novel non‑selective atypical PKC agonist could protect neuronal cell line from A β ‑oligomer induced toxicity by suppressing A β generation

Atypical protein kinase C (aPKCs) serve key functions in embryonic development by regulating apical-basal polarity. Previous studies have shed light on their roles during adulthood, especially in the development of Alzheimer\u27s disease (AD). Although the crystal structure of PKCι has been resolved, an agonist of aPKCs remains to be discovered. In the present study, by using the Discovery Studio program and LibDock methodology, a small molecule library (K66-X4436 KINA Set) of compounds were screened for potential binding to PKCι. Subsequently, the computational docking results were validated using affinity selection-mass spectrometry, before in vitro kinase activity was used to determine the function of the hit compounds. A cell-based model assay that can mimic the pathology of AD was then established and used to assess the function of these hit compounds. As a result, the aPKC agonist Z640 was identified, which could bind to PKCι in silico, in vitro and in this cell-based model. Z640 was further confirmed as a non-selective aPKC agonist that can activate the kinase activity of both PKCι and PKCζ. In the cell-based assay, Z640 was found to protect neuronal cell lines from amyloid-β (Aβ) oligomer-induced cell death by reducing reactive oxygen species production and restore mitochondrial function. In addition, Z640 could reduce Aβ40 generation in a dose-dependent manner and shift amyloid precursor protein processing towards the non-amyloid pathway. To conclude, the present study is the first, to the best of the authors\u27 knowledge to identify an aPKC agonist by combining computer-assisted drug discovery and cell-based assays. The present study also revealed that aPKC agonists have therapeutic potential for the treatment of AD

Distinct Roles For ROCK1 and ROCK2 in the Regulation of Oxldl-Mediated Endothelial Dysfunction

Background/Aims: This study used Rho-associated protein kinase (ROCK) isoform-selective suppression or a ROCK inhibitor to analyze the roles of ROCK1 and ROCK2 in regulating endothelial dysfunction triggered by oxidized low-density lipoprotein (oxLDL). Methods: ROCK1 or ROCK2 expression in human umbilical vein endothelial cells (HUVECs) was suppressed by small interfering RNA (siRNA). HUVECs were pretreated with 30 μM Y27632 (pan ROCK inhibitor) for 30 min before exposure to 200 μg/mL oxLDL for an additional 24 h. Cell viability was determined by the MTT assay, and cell apoptosis was evaluated by the TUNEL assay. Protein expression and phosphorylation were assessed by Western blot analysis. The morphology of total and phosphorylated vimentin (p-vimentin) and the co-localization of vimentin with vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) were detected by the immunofluorescence assay. The adhesion of promonocytic U937 cells to HUVECs was observed by light microscopy. Results: ROCK2 suppression or Y27632 treatment, rather than ROCK1 deletion, effectively reduced endothelial cell apoptosis and preserved cell survival. ROCK2 suppression exhibited improved vimentin and p-vimentin cytoskeleton stability and decreased vimentin cleavage by attenuating caspase-3 activity. In addition, increased p-vimentin expression induced by oxLDL was significantly inhibited by ROCK2 deletion or Y27632 treatment. In contrast, ROCK1 suppression showed no obvious effects on the vimentin cytoskeleton, but significantly regulated the expression of adhesion molecules. Endothelial ICAM-1 or VCAM-1 expression induced by oxLDL was obviously inhibited by ROCK1 suppression or Y27632 treatment. Moreover, the expression of ICAM-1 induced by oxLDL could also be reduced by ROCK2 suppression. Furthermore, ROCK2 deficiency or Y27632 treatment inhibited the redistribution of adhesion molecules and their co-localization with vimentin caused by oxLDL. These effects resulted in the significant inhibition of monocyte-endothelial adhesion induced by oxLDL. Conclusion: The results of this study support the novel concept that ROCK1 is involved in oxLDL-induced cell adhesion by regulating adhesion molecule expression, whereas ROCK2 is required for both endothelial apoptosis and adhesion by regulating both the vimentin cytoskeleton and adhesion molecules. Consequently, ROCK1 and ROCK2 have distinct roles in the regulation of oxLDL-mediated endothelial dysfunction

Non-coding nucleotides and amino acids near the active site regulate peptide deformylase expression and inhibitor susceptibility in Chlamydia trachomatis

Chlamydia trachomatis, an obligate intracellular bacterium, is a highly prevalent human pathogen. Hydroxamic-acid-based matrix metalloprotease inhibitors can effectively inhibit the pathogen both in vitro and in vivo, and have exhibited therapeutic potential. Here, we provide genome sequencing data indicating that peptide deformylase (PDF) is the sole target of the inhibitors in this organism. We further report molecular mechanisms that control chlamydial PDF (cPDF) expression and inhibition efficiency. In particular, we identify the σ66-dependent promoter that controls cPDF gene expression and demonstrate that point mutations in this promoter lead to resistance by increasing cPDF transcription. Furthermore, we show that substitution of two amino acids near the active site of the enzyme alters enzyme kinetics and protein stability

Long-term decrease in Asian monsoon rainfall and abrupt climate change events over the past 6,700 years.

Asian summer monsoon (ASM) variability and its long-term ecological and societal impacts extending back to Neolithic times are poorly understood due to a lack of high-resolution climate proxy data. Here, we present a precisely dated and well-calibrated tree-ring stable isotope chronology from the Tibetan Plateau with 1- to 5-y resolution that reflects high- to low-frequency ASM variability from 4680 BCE to 2011 CE. Superimposed on a persistent drying trend since the mid-Holocene, a rapid decrease in moisture availability between ∼2000 and ∼1500 BCE caused a dry hydroclimatic regime from ∼1675 to ∼1185 BCE, with mean precipitation estimated at 42 ± 4% and 5 ± 2% lower than during the mid-Holocene and the instrumental period, respectively. This second-millennium-BCE megadrought marks the mid-to late Holocene transition, during which regional forests declined and enhanced aeolian activity affected northern Chinese ecosystems. We argue that this abrupt aridification starting ∼2000 BCE contributed to the shift of Neolithic cultures in northern China and likely triggered human migration and societal transformation