Degradation of Components of the Lpt Transenvelope Machinery Reveals LPS-Dependent Lpt Complex Stability in Escherichia coli

Lipopolysaccharide (LPS) is a peculiar component of the outer membrane (OM) of many Gram-negative bacteria that renders these bacteria highly impermeable to many toxic molecules, including antibiotics. LPS is assembled at the OM by a dedicated intermembrane transport system, the Lpt (LPS transport) machinery, composed of seven essential proteins located in the inner membrane (IM) (LptB2CFG), periplasm (LptA), and OM (LptDE). Defects in LPS transport compromise LPS insertion and assembly at the OM and result in an overall modification of the cell envelope and its permeability barrier properties. LptA is a key component of the Lpt machine. It connects the IM and OM sub-complexes by interacting with the IM protein LptC and the OM protein LptD, thus enabling the LPS transport across the periplasm. Defects in Lpt system assembly result in LptA degradation whose stability can be considered a marker of an improperly assembled Lpt system. Indeed, LptA recruitment by its IM and OM docking sites requires correct maturation of the LptB2CFG and LptDE sub-complexes, respectively. These quality control checkpoints are crucial to avoid LPS mistargeting. To further dissect the requirements for the complete Lpt transenvelope bridge assembly, we explored the importance of LPS presence by blocking its synthesis using an inhibitor compound. Here, we found that the interruption of LPS synthesis results in the degradation of both LptA and LptD, suggesting that, in the absence of the LPS substrate, the stability of the Lpt complex is compromised. Under these conditions, DegP, a major chaperone–protease in Escherichia coli, is responsible for LptD but not LptA degradation. Importantly, LptD and LptA stability is not affected by stressors disturbing the integrity of LPS or peptidoglycan layers, further supporting the notion that the LPS substrate is fundamental to keeping the Lpt transenvelope complex assembled and that LptA and LptD play a major role in the stability of the Lpt system.

De Novo Transcriptome Assembly for Venom Gland in Two Spe-Cies of Spiders (Sinopoda Pengi and Trichonephila Clavata)

Natural molecules from spider venom are considered potential drugs for diseases including cancer and pain, as well as the development of new biological insecticides for agricultural use. During coevolution in the long-term predator-prey game, spiders have formed a huge molecular diversity of toxins. As of March 1 of 2021, a total of 49,243 spider species had been described, but studies of venom have been performed in only a few hundred of these species due to the difficulty of collecting venom. Two technologies have helped partially dealing with this limitation in the recent past: the screening of cDNA libraries constructed from venom gland mRNAs and the heterologous expression of the coded peptides for functional characterization. In this study, transcriptomic analysis was performed to describe the predicted toxins of Sinopoda pengi (hereafter S. pengi) and Trichonephila clavata (hereafter T. clavata). The Trinity assembly result in 163,418 transcripts, 114,127 unigene of S. pengi and 125,099 transcripts, 87,084 unigene of T. clavata. A total of 22 and 24 unigenes were identified which were predicted to inhibitor cysteine knot (ICK) toxins from S. pengi and T. clavata, respectively. In summary, molecular templates with potential application value in medical and biological fields were obtained by classifying and characterizing presumed venom components, which lays a foundation for the further study of venom.

gcaPDA: A Haplotype-resolved Diploid Assembler

Generating chromosome-scale haplotype resolved assembly is important for functional studies. However, current de novo assemblers are either haploid assemblers that discard allelic information, or diploid assemblers that can only tackle genomes of low complexity. Here, we report a diploid assembler, gcaPDA (gamete cells assisted Phased Diploid Assembler), which exploits haploid gamete cells to assist in resolving haplotypes. We generate chromosome-scale phased diploid assemblies for the highly heterozygous and repetitive genome of a maize F1 hybrid using gcaPDA and evaluate the assembly result thoroughly. With applicability of coping with complex genomes and fewer restrictions on application than other diploid assemblers, gcaPDA is likely to find broad applications in studies of eukaryotic genomes.

P0407THE INVESTIGATION OF SYNAPTOPODIN EXPRESSION OF PODOCYTES IN GLOMERONEPHRITIS

Background and Aims Synaptopodin, a proline-rich actin-associated protein, plays an important role in the regulation of podocytes processes structures and dynamics. The mutation or lack of synaptopodin may lead to the changes of podocytes structures and functions and cause the occurrence of proteinuria. But the underlying molecular mechanisms remain primarily elusive. Method we used cellular and pathological experiments to observe the expression changes synaptopodin in vivo and vitrio. Results The results showed that the reduction expression of synaptopodin and RhoA were found in the podocytes in different nephriris of human renal biopsy as well as in rat adriamycin nephropathy. The cultured cells treated with inflammatory cytokins such as TNF, IL-1 also showed decreased synaptopodin level in podocyte, which led to low RhoA level and disarrange the actin cytoskeleton assembly, result in the abnormal changes of podocyte morphology. Conclusion These data preliminarily proved that synaptopodin loss in podocyte injury plays an important role in the regulation of podocyte morphology and function through RhoA signaling pathway, and further researches are required to clarify the more mechanism, which may provide new strategies and methods for the prevention and treatment of glomerular diseases.

The Influence of Temperature Testing on the Technical Data of Cementing Technology

The problematic issues connected with the optical details deformations that arise during the temperature tests of cemented assemblies are considered. Climatic tests of optical devices after assembly result in a change of technical product characteristics. The research problem is to define how N and ∆N of surfaces of cemented optical assemblies are changing after the temperature testing. The research objects are plane convex lenses fabricated of LK7 (LZOS) glass brand and plane concave lenses - of TF7 (LZOS). Before cementing, the bounded surfaces cleaning by the hydromechanical method in the clean room has been purged. The temperature testing of samples is held at 22, 50 and 65 °C. The effect of temperature on a surface deformation of cemented optical assemblies is experimentally established. The comparison of effect of optical cements OK72FT5 and OK72FT15 on the optical surfaces deformation is carried out. In this paper we have compared the results of shape accuracy of the external surfaces of optical assemblies and the flat surfaces. Interferograms of lenses after heat treatment of optical assemblies are presented. It is determined that the cement polymerization temperature equals or higher than the test temperature.

Molecular salt crystals with bis(head-to-tail) interionic complementary assembly for efficient organic THz generators

New ionic organic crystals with bis(head-to-tail) complementary cation–anion assembly result in extremely efficient THz wave generation due to strong interionic binding interactions and parallel alignment of nonlinear optical cationic chromophores.

Local orientational order in self-assembled nanoparticle films: the role of ligand composition and salt

An X-ray cross-correlation study of the local orientational order in self-assembled films made from PEGylated gold nanoparticles is presented. The local structure of this model system is dominated by four- and sixfold order. Coadsorption of shorter ligands in the particle's ligand layer and variation of salt concentration in the suspension prior to self-assembly result in a change of local orientational order. The degree of sixfold order is reduced after salt addition. This decrease of order is less pronounced for the fourfold symmetry. The results presented here suggest complex symmetry-selective order formation upon ligand exchange and salt addition and demonstrate the versatility of X-ray cross-correlation methods for nanoparticle superlattices.

Coarse-grained simulations of actomyosin rings point to a nodeless model involving both unipolar and bipolar myosins

Cytokinesis in many eukaryotic cells is orchestrated by a contractile actomyosin ring. While many of the proteins involved are known, the mechanism of constriction remains unclear. Informed by the existing literature and new three-dimensional (3D) molecular details from electron cryotomography, here we develop 3D coarse-grained models of actin filaments, unipolar and bipolar myosins, actin cross-linkers, and membranes and simulate their interactions. Assuming that local force on the membrane results in inward growth of the cell wall, we explored a matrix of possible actomyosin configurations and found that node-based architectures like those presently described for ring assembly result in membrane puckers not seen in electron microscope images of real cells. Instead, the model that best matches data from fluorescence microscopy, electron cryotomography, and biochemical experiments is one in which actin filaments transmit force to the membrane through evenly distributed, membrane-attached, unipolar myosins, with bipolar myosins in the ring driving contraction. While at this point this model is only favored (not proven), the work highlights the power of coarse-grained biophysical simulations to compare complex mechanistic hypotheses.

Modular assembly of the nucleolar large subunit processome

Early co-transcriptional events of eukaryotic ribosome assembly result in the formation of the small and large subunit processomes. We have determined cryo-EM reconstructions of the nucleolar large subunit processome in different conformational states at resolutions up to 3.4 Ångstroms. These structures reveal how steric hindrance and molecular mimicry are used to prevent premature folding states and binding of later factors. This is accomplished by the concerted activity of 21 ribosome assembly factors that stabilize and remodel pre-ribosomal RNA and ribosomal proteins. Mutually exclusive conformations of these particles suggest that the formation of the polypeptide exit tunnel is achieved through different folding pathways during subsequent stages of ribosome assembly.

Coarse-grained simulations of actomyosin rings point to a nodeless model involving both unipolar and bipolar myosins

Cytokinesis in most eukaryotic cells is orchestrated by a contractile actomyosin ring. While many of the proteins involved are known, the mechanism of constriction remains unclear. Informed by existing literature and new 3D molecular details from electron cryotomography, here we develop 3D coarse-grained models of actin filaments, unipolar and bipolar myosins, actin crosslinkers, and membranes and simulate their nteractions. Exploring a matrix of possible actomyosin configurations suggested that node-based architectures ike those presently described for ring assembly result in membrane puckers not seen in EM images of real cells. Instead, the model that best matches data from fluorescence microscopy, electron cryotomography, and biochemical experiments is one in which actin filaments transmit force to the membrane through evenly-distributed, membrane-attached, unipolar myosins, with bipolar myosins in the ring driving contraction. While at this point this model is only favored (not proven), the work highlights the power of coarse-grained biophysical simulations to compare complex mechanistic hypotheses.Significance StatementIn most eukaryotes, a ring of actin and myosin drives cell division, but how the elements of the ring are arranged and constrict remain unclear. Here we use 3D coarse-grained simulations to explore various possibilities. Our simulations suggest that if actomyosin is arranged in nodes (as suggested by a popular model of ring assembly), the membrane distorts in ways not seen experimentally. Instead, actin and myosin are more ikely uniformly distributed around the ring. In the model that best fits experimental data, ring tension is generated by interactions between bipolar myosins and actin, and transmitted to the membrane via unipolar myosins. Technologically the study highlights how coarse-grained simulations can test specific mechanistic hypotheses by comparing their predicted outcomes to experimental results.