Envelope Integrity Research Articles

Severe ACTA1-related nemaline myopathy: intranuclear rods, cytoplasmic bodies, and enlarged perinuclear space as characteristic pathological features on muscle biopsies

Nemaline myopathy (NM) is a muscle disorder with broad clinical and genetic heterogeneity. The clinical presentation of affected individuals ranges from severe perinatal muscle weakness to milder childhood-onset forms, and the disease course and prognosis depends on the gene and mutation type. To date, 14 causative genes have been identified, and ACTA1 accounts for more than half of the severe NM cases. ACTA1 encodes α-actin, one of the principal components of the contractile units in skeletal muscle. We established a homogenous cohort of ten unreported families with severe NM, and we provide clinical, genetic, histological, and ultrastructural data. The patients manifested antenatal or neonatal muscle weakness requiring permanent respiratory assistance, and most deceased within the first months of life. DNA sequencing identified known or novel ACTA1 mutations in all. Morphological analyses of the muscle biopsy specimens showed characteristic features of NM histopathology including cytoplasmic and intranuclear rods, cytoplasmic bodies, and major myofibrillar disorganization. We also detected structural anomalies of the perinuclear space, emphasizing a physiological contribution of skeletal muscle α-actin to nuclear shape. In-depth investigations of the nuclei confirmed an abnormal localization of lamin A/C, Nesprin-1, and Nesprin-2, forming the main constituents of the nuclear lamina and the LINC complex and ensuring nuclear envelope integrity. To validate the relevance of our findings, we examined muscle samples from three previously reported ACTA1 cases, and we identified the same set of structural aberrations. Moreover, we measured an increased expression of cardiac α-actin in the muscle samples from the patients with longer lifespan, indicating a potential compensatory effect. Overall, this study expands the genetic and morphological spectrum of severe ACTA1-related nemaline myopathy, improves molecular diagnosis, highlights the enlargement of the perinuclear space as an ultrastructural hallmark, and indicates a potential genotype/phenotype correlation.

The essential Rhodobacter sphaeroides CenKR two-component system regulates cell division and envelope biosynthesis.

Bacterial two-component systems (TCSs) often function through the detection of an extracytoplasmic stimulus and the transduction of a signal by a transmembrane sensory histidine kinase. This kinase then initiates a series of reversible phosphorylation modifications to regulate the activity of a cognate, cytoplasmic response regulator as a transcription factor. Several TCSs have been implicated in the regulation of cell cycle dynamics, cell envelope integrity, or cell wall development in Escherichia coli and other well-studied Gram-negative model organisms. However, many α-proteobacteria lack homologs to these regulators, so an understanding of how α-proteobacteria orchestrate extracytoplasmic events is lacking. In this work we identify an essential TCS, CenKR (Cell envelope Kinase and Regulator), in the α-proteobacterium Rhodobacter sphaeroides and show that modulation of its activity results in major morphological changes. Using genetic and biochemical approaches, we dissect the requirements for the phosphotransfer event between CenK and CenR, use this information to manipulate the activity of this TCS in vivo, and identify genes that are directly and indirectly controlled by CenKR in Rb. sphaeroides. Combining ChIP-seq and RNA-seq, we show that the CenKR TCS plays a direct role in maintenance of the cell envelope, regulates the expression of subunits of the Tol-Pal outer membrane division complex, and indirectly modulates the expression of peptidoglycan biosynthetic genes. CenKR represents the first TCS reported to directly control the expression of Tol-Pal machinery genes in Gram-negative bacteria, and we predict that homologs of this TCS serve a similar function in other closely related organisms. We propose that Rb. sphaeroides genes of unknown function that are directly regulated by CenKR play unknown roles in cell envelope biosynthesis, assembly, and/or remodeling in this and other α-proteobacteria.

YAP/TAZ activity in stromal cells prevents ageing by controlling cGAS-STING.

Ageing is intimately connected tothe induction of cell senescence1,2, but why this is so remains poorly understood. A key challenge is the identification of pathways thatnormally suppress senescence, arelost during ageing and arefunctionally relevant to oppose ageing3. Here we connected the structural and functional decline of ageing tissues to attenuated function of themaster effectors of cellular mechanosignallingYAP and TAZ. YAP/TAZ activity declines during physiological ageing in stromal cells, and mimicking such decline through genetic inactivation ofYAP/TAZin these cellsleads to accelerated ageing. Conversely, sustaining YAP function rejuvenates old cells and opposes the emergence of ageing-related traits associated with either physiological ageing or accelerated ageing triggered by a mechano-defective extracellular matrix. Ageing traits induced by inactivation ofYAP/TAZ are preceded by induction of tissue senescence. This occurs because YAP/TAZ mechanotransduction suppresses cGAS-STING signalling, to the extent thatinhibition of STING prevents tissue senescence and premature ageing-related tissue degeneration after YAP/TAZ inactivation. Mechanistically, YAP/TAZ-mediated control of cGAS-STING signallingrelies on theunexpected roleof YAP/TAZ in preserving nuclear envelope integrity, at least in part through direct transcriptional regulation of lamin B1 and ACTR2, the latter of which isinvolved in building the peri-nuclear actin cap. The findings demonstrate that decliningYAP/TAZ mechanotransduction drives ageing by unleashing cGAS-STINGsignalling, a pillar of innate immunity. Thus, sustaining YAP/TAZ mechanosignalling or inhibiting STING may represent promising approaches for limitingsenescence-associated inflammation and improving healthy ageing.

The Tol–Pal system of Escherichia coli plays an unexpected role in the import of the oxyanions chromate and phosphate

Chromate is a toxic metal that enters bacteria by using oxyanion importers. Here, we show that each mutant of the Tol–Pal system of Escherichia coli exhibited increased chromate resistance. This system, which spans the cell envelope, plays a major role in envelope integrity and septation. The ΔtolQR mutant accumulated three-fold less chromate than the wild-type. Addition of phosphate but not sulfate to rich medium drastically reduced chromate toxicity and import in the wild-type strain. Furthermore, the intracellular concentration of free inorganic phosphate was significantly reduced for the ΔtolR mutant in comparison to the wild-type strain. Moreover, extracellular labeled phosphate was significantly less incorporated into the ΔtolR mutant. Finally, two distinct TolQR mutant complexes, specifically affected in Tol–Pal energization without affecting the TolQRA complex structure, did not complement the ΔtolQR mutant for inorganic phosphate accumulation. We thus propose that, while the Pst system is well known to import inorganic phosphate, the Tol–Pal system participates to phosphate uptake in particular at medium to high extracellular phosphate concentrations. Since mutations disabling the Tol–Pal system lead to pleiotropic effects, chromate resistance and reduced inorganic phosphate import could occur from an indirect effect of mutations in components of the Tol–Pal system.

Loss of Zfp335 triggers cGAS/STING-dependent apoptosis of post-β selection pre-T cells

Abstract Production of a diverse peripheral T cell compartment requires massive expansion of the bone marrow progenitors that seed the thymus. There are two main phases of expansion during T cell development, following T lineage commitment at the DN2 stage and following successful rearrangement and selection for functional TCRβ chains in DN3 thymocytes, which promotes development of DN4 cells to the DP stage. Signals driving expansion of DN2 thymocytes are well studied, however, factors regulating the proliferation and survival of DN4 cells remain poorly understood. Here, we uncover an unexpected link between the transcription factor Zfp335 and control of cGAS/STING-dependent cell death in post-β-selection DN4 thymocytes. Zfp335 controls survival by sustaining expression of Ankle2 and regulating phosphorylation of Baf which functions to promote nuclear envelope integrity and suppress cGAS/STING-dependent cell death. Together, this study identifies Zfp335 as a key transcription factor controlling the survival of proliferating post-β-selection thymocytes and demonstrates a key role for the cGAS/STING pathway driving apoptosis of developing T cells. Supported by grants from NIH R01-GM059638, P01-AI102853, RO1-AI099100, RO1-NS120417, and RO1-AI160737

Ligands Binding Peptidoglycan‐Associated Lipoprotein as Biomarkers for Sepsis

Peptidoglycan‐Associated Lipoprotein (Pal) is an outer membrane protein conserved among Gram‐negative bacteria. Pal protein binds to peptidoglycan and several outer membrane and periplasmic proteins as part of the Pal‐Tol complex, a system of interacting proteins that link together the outer membrane, the peptidoglycan layer, and the inner membrane. The Pal‐Tol complex is important for maintaining the integrity of the cell envelope and plays a role in the invagination process during cell division. We proposed to identify small molecules that could disrupt the Pal‐TolB interaction and/or Pal‐peptidoglycan binding, as the two binding sites are thought to overlap. As part of a summer research program, funded by the NSF, our team of researchers used the computational program Dock6 to dock a variety of small molecules from the ZINC database in different rigid and flexible conformations. The small molecules that docked the most favorably were linear polycyclic structures that had alcohol and amine groups, such as FMN, capastat, leucal, and ceftriaxone. We also describe our ongoing efforts to develop a binding assay to test the effect of these small molecules on Pal‐TolB and Pal‐peptidoglycan binding. Due to Pal’s role in cell division and cell envelope integrity, we propose that small molecules that disrupt interactions to Pal’s binding partners could serve as potential antibiotics.

The Wsp system of Pseudomonas aeruginosa links surface sensing and cell envelope stress

Surface sensing is a critical process that promotes the transition to a biofilm lifestyle. Several surface-sensing mechanisms have been described for a range of species, most involving surface appendages, such as flagella and pili. Pseudomonas aeruginosa uses the Wsp chemosensory-like signal transduction pathway to sense surfaces and promote biofilm formation. The methyl-accepting chemotaxis protein WspA recognizes an unknown surface-associated signal and initiates a phosphorylation cascade that activates the diguanylate cyclase WspR. We conducted a screen for Wsp-activating compounds and found that chemicals that impact the cell envelope induce Wsp signaling, increase intracellular c-di-GMP levels, and can promote surface attachment. To isolate the Wsp system from other P. aeruginosa surface-sensing systems, we heterologously expressed it in Escherichia coli and found it sufficient for sensing surfaces and the chemicals identified in our screen. Using well-characterized reporters for different E. coli cell envelope stress responses, we then determined that Wsp sensitivity overlapped with multiple E. coli cell envelope stress-response systems. Using mutational and CRISPRi analysis, we found that misfolded proteins in the periplasm appear to be a major stimulus of the Wsp system. Finally, we show that surface attachment appears to have an immediate, observable effect on cell envelope integrity. Collectively, our results provide experimental evidence that cell envelope stress represents an important feature of surface sensing in P. aeruginosa.

Weight-bearing activity impairs nuclear membrane and genome integrity via YAP activation in plantar melanoma

Acral melanoma commonly occurs in areas that are not exposed to much sunlight, such as the sole of the foot. Little is known about risk factors and mutational processes of plantar acral melanoma. Nuclear envelope rupture during interphase contributes to genome instability in cancer. Here, we show that the nuclear and micronuclear membranes of melanoma cells are frequently ruptured by macroscopic mechanical stress on the plantar surface due to weight-bearing activities. The marginal region of plantar melanoma nodules exhibits increased nuclear morphological abnormalities and collagen accumulations, and is more susceptible to mechanical stress than the tumor center. An increase in DNA damage coincides with nuclear membrane rupture in the tumor margin. Nuclear envelope integrity is compromised by the mechanosensitive transcriptional cofactor YAP activated in the tumor margin. Our results suggest a mutagenesis mechanism in melanoma and explain why plantar acral melanoma is frequent at higher mechanical stress points.

Inhibition of Human Cytomegalovirus Particle Maturation by Activation of Liver X Receptor.

Human cytomegalovirus (HCMV), a herpesvirus family member, is a large, complex enveloped virus. The activation of liver X receptor (LXR) can significantly inhibit the replication of HCMV and weaken the virulence of progeny virus (unpublished data). Our results showed activated LXR affected some important viral protein expression and reduced cholesterol content in HCMV infected cells and virus particles. To further clarify the influence of activated LXR on HCMV replication, HCMV assembly and maturation processes were studied by transmission electron microscopy (TEM) in HCMV infected foreskin fibroblasts treated with LXR agonist GW3965. Results showed that activated LXR could reduce the envelope integrity of maturating virions. The functional stage of activated LXR on viral envelope integrity was mainly at virus assembly compartment (VAC) mediated envelopment but not structurally complete virus nucleocapsid formation and the egress of nucleocapsid from the nucleus to the cytoplasm mediated by nuclear egress complex. Reduced cholesterol synthesis and viral protein expression might interfere with the VAC-mediated envelopment. The nucleocapsid and tegument proteins enter the VAC area for the secondary envelope, which was interfered with and resulted in the defective particle, thereby affecting the amount and infectivity of the mature virus. The results indicate that inhibition of HCMV maturation is one mechanism of activated LXR inhibiting virus replication in infected cells.

Uip4p modulates nuclear pore complex function in Saccharomyces cerevisiae

ABSTRACT A double membrane bilayer perforated by nuclear pore complexes (NPCs) governs the shape of the nucleus, the prominent distinguishing organelle of a eukaryotic cell. Despite the absence of lamins in yeasts, the nuclear morphology is stably maintained and shape changes occur in a regulated fashion. In a quest to identify factors that contribute to regulation of nuclear shape and function in Saccharomyces cerevisiae, we used a fluorescence imaging based approach. Here we report the identification of a novel protein, Uip4p, that is required for regulation of nuclear morphology. Loss of Uip4 compromises NPC function and loss of nuclear envelope (NE) integrity. Our localization studies show that Uip4 localizes to the NE and endoplasmic reticulum (ER) network. Furthermore, we demonstrate that the localization and expression of Uip4 is regulated during growth, which is crucial for NPC distribution.

The Cpx Stress Response Regulates Turnover of Respiratory Chain Proteins at the Inner Membrane of Escherichia coli.

The Cpx envelope stress response is a major signaling pathway monitoring bacterial envelope integrity, activated both internally by excessive synthesis of membrane proteins and externally by a variety of environmental cues. The Cpx regulon is enriched with genes coding for protein folding and degrading factors, virulence determinants, and large envelope-localized complexes. Transcriptional repression of the two electron transport chain complexes, NADH dehydrogenase I and cytochrome bo3, by the Cpx pathway has been demonstrated, however, there is evidence that additional regulatory mechanisms exist. In this study, we examine the interaction between Cpx-regulated protein folding and degrading factors and the respiratory complexes NADH dehydrogenase I and succinate dehydrogenase in Escherichia coli. Here we show that the cellular need for Cpx-mediated stress adaptation increases when respiratory complexes are more prevalent or active, which is demonstrated by the growth defect of Cpx-deficient strains on media that requires a functional electron transport chain. Interestingly, deletion of several Cpx-regulated proteolytic factors and chaperones results in similar growth-deficient phenotypes. Furthermore, we find that the stability of the NADH dehydrogenase I protein complex is lower in cells with a functional Cpx response, while in its absence, protein turnover is impaired. Finally, we demonstrated that the succinate dehydrogenase complex has reduced activity in E. coli lacking the Cpx pathway. Our results suggest that the Cpx two-component system serves as a sentry of inner membrane protein biogenesis, ensuring the function of large envelope protein complexes and maintaining the cellular energy status of the cell.

The role of TolA, TolB, and TolR in cell morphology, OMVs production, and virulence of Salmonella Choleraesuis

The Tol–Pal system of Gram-negative bacteria is necessary for maintaining outer membrane integrity. It is a multiprotein complex of five envelope proteins, TolQ, TolR, TolA, TolB, and Pal. These proteins were first investigated in E. coli, and subsequently been identified in many other bacterial genera. However, the function of the Tol–Pal system in Salmonella Choleraesuis pathogenesis is still unclear. Here, we reported the role of three of these proteins in the phenotype and biology of S. Choleraesuis. We found that mutations in tolA, tolB, and tolR caused severe damage to the cell wall, which was supported by observing the microstructure of spherical forms, long chains, flagella defects, and membrane blebbing. We confirmed that all the mutants significantly decreased S. Choleraesuis survival when exposed to sodium deoxycholate and exhibited a high sensitivity to vancomycin, which may be explained by the disruption of envelope integrity. In addition, tolA, tolB, and tolR mutants displayed attenuated virulence in a mouse infection model. This could be interpreted as a series of defective phenotypes in the mutants, such as severe defects in envelope integrity, growth, and motility. Further investigation showed that all the genes participate in outer membrane vesicles (OMVs) biogenesis. Interestingly, immunization with OMVs from ΔtolB efficiently enhanced murine viability in contrast to OMVs from the wild-type S. Choleraesuis, suggesting its potential use in vaccination strategies. Collectively, this study provides an insight into the biological role of the S. Choleraesuis Tol–Pal system.

A lightweight, mechanically strong, and shapeable copper-benzenedicarboxylate/cellulose aerogel for dye degradation and antibacterial applications

• CuBDC nanosheets were loaded on cellulose aerogels by in-situ growth. • The composite aerogel showed good performance in degradation of methylene blue. • The composite aerogel exhibited excellent killing effect towards various bacteria. • Antibacterial mechanism was related to the damage of bacteria envelope integrity. Metal-organic frameworks (MOFs) suffer from low processability and recyclability due to their fragile powder form. Supporting MOFs on the lightweight and porous cellulose aerogels is an efficient way to dramatically extend their practical applications. Herein, a metal–organic framework (MOF) composite material, named as copper-benzenedicarboxylate/cellulose aerogel (CuBDC/CA), was designed and synthesized by one-pot precursor preparation of cellulose aerogels anchored with copper, followed by in-situ growth of CuBDC in the presence of terephthalic acid. The characterizations (scanning electron microscopy with energy-dispersive X-ray, powder X-ray diffraction, Fourier-transform infrared spectroscopy, thermogravimetric analysis, X-ray photoelectron spectroscopy, stability and mechanical tests) confirmed the successful synthesis of the lightweight (0.0625 ± 0.0010 g/cm 3 for 20% CuBDC/CA), mechanically strong, stable, and porous aerogel composites with well-dispersed CuBDC nanosheets. The aerogel composite showed great potential in wastewater treatment, including dye degradation and antibacterial assays. More than 90% methylene blue could be decomposed by CuBDC/CA in 240 min via the Fenton-like reaction, and the recycling test revealed its good reusability. Moreover, this composite exhibited great antibacterial performance (more than 99.99%) towards commonly used lab strains as well as multidrug-resistant pathogens, including Escherichia coli , Staphylococcus aureus, and Pseudomonas aeruginosa . The screening tests indicated that the mechanism of killing effect was associated with the damage of bacterial envelope integrity. This work has proven that the lightweight and robust MOF/cellulose aerogel is a promising candidate for environmental remediation in practice.

The Involvement of Mycobacterium Type III-A CRISPR-Cas System in Oxidative Stress

Type I and type II CRISPR-Cas systems are employed to evade host immunity by targeting interference of bacteria’s own genes. Although Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis, possesses integrated type III-A CRISPR-Cas system, its role in mycobacteria remains obscure. Here, we observed that seven cas genes (csm2∼5, cas10, cas6) were upregulated in Mycobacterium bovis BCG under oxidative stress treatment, indicating the role of type III-A CRISPR-Cas system in oxidative stress. To explore the functional role of type III-A CRISPR-Cas system, TCC (Type III-A CRISPR-Cas system, including cas6, cas10, and csm2-6) mutant was generated. Deletion of TCC results in increased sensitivity in response to hydrogen peroxide and reduced cell envelope integrity. Analysis of RNA-seq dataset revealed that TCC impacted on the oxidation-reduction process and the composition of cell wall which is essential for mycobacterial envelop integrity. Moreover, disrupting TCC led to poor intracellular survival in vivo and in vitro. Finally, we showed for the first time that TCC contributed to the regulation of regulatory T cell population, supporting a role of TCC in modulating host immunity. Our finding reveals the important role of TCC in cell envelop homeostasis. Our work also highlights type III-A CRISPR-Cas system as an important factor for intracellular survival and host immunoregulation in mycobacteria, thus may be a potential target for therapy.

Integrated transcriptomic and proteomic analysis reveals the response mechanisms of Alicyclobacillus acidoterrestris to heat stress

Alicyclobacillus acidoterrestris can survive pasteurization and is implicated in pasteurized fruit juice spoilage. However, the mechanisms underlying heat responses remain largely unknown. Herein, gene transcription changes of A. acidoterrestris under heat stress were detected by transcriptome, and an integrated analysis with proteomic and physiological data was conducted. A total of 911 differentially expressed genes (DEGs) was observed. The majority of DEGs and differentially expressed proteins (DEPs) were exclusively regulated at the mRNA and protein level, respectively, whereas only 59 genes were regulated at both levels and had the same change trends. Comparative analysis of the functions of the specifically or commonly regulated DEGs and DEPs revealed that the heat resistance of A. acidoterrestris was primarily based on modulating peptidoglycan and fatty acid composition to maintain cell envelope integrity. Low energy consumption strategies were established with attenuated glycolysis, decreased ribosome de novo synthesis, and activated ribosome hibernation. Terminal oxidases, cytochrome bd and aa3, in aerobic respiratory chain were upregulated. Meanwhile, the MarR family transcriptional regulator was upregulated, reactive oxygen species (ROS) was discovered, and the concentration of superoxide dismutase (SOD) increased, indicating that the accompanied oxidative stress was induced by high temperature. Additionally, DNA and protein damage repair systems were activated. This study provided a global perspective on the response mechanisms of A. acidoterrestris to heat stress, with implications for better detection and control of its contamination in fruit juice.

The Impact of Rare Human Variants on Barrier-To-Auto-Integration Factor 1 (Banf1) Structure and Function.

Barrier-to-Autointegration Factor 1 (Banf1/BAF) is a critical component of the nuclear envelope and is involved in the maintenance of chromatin structure and genome stability. Banf1 is a small DNA binding protein that is conserved amongst multicellular eukaryotes. Banf1 functions as a dimer, and binds non-specifically to the phosphate backbone of DNA, compacting the DNA in a looping process. The loss of Banf1 results in loss of nuclear envelope integrity and aberrant chromatin organisation. Significantly, mutations in Banf1 are associated with the severe premature ageing syndrome, Néstor–Guillermo Progeria Syndrome. Previously, rare human variants of Banf1 have been identified, however the impact of these variants on Banf1 function has not been explored. Here, using in silico modelling, biophysical and cell-based approaches, we investigate the effect of rare human variants on Banf1 structure and function. We show that these variants do not significantly alter the secondary structure of Banf1, but several single amino acid variants in the N- and C-terminus of Banf1 impact upon the DNA binding ability of Banf1, without altering Banf1 localisation or nuclear integrity. The functional characterisation of these variants provides further insight into Banf1 structure and function and may aid future studies examining the potential impact of Banf1 function on nuclear structure and human health.

Exogenous and Endogenous Phosphoethanolamine Transferases Differently Affect Colistin Resistance and Fitness in Pseudomonas aeruginosa.

Colistin represents a last-line treatment option for infections caused by multidrug resistant Gram-negative pathogens, including Pseudomonas aeruginosa. Colistin resistance generally involves the modification of the lipid A moiety of lipopolysaccharide (LPS) with positively charged molecules, namely phosphoethanolamine (PEtN) or 4-amino-4-deoxy-L-arabinose (Ara4N), that reduce colistin affinity for its target. Several lines of evidence highlighted lipid A aminoarabinosylation as the primary colistin resistance mechanism in P. aeruginosa, while the contribution of phosphoethanolamination remains elusive. PEtN modification can be due to either endogenous (chromosomally encoded) PEtN transferase(s) (e.g., EptA in P. aeruginosa) or plasmid borne MCR enzymes, commonly found in enterobacteria. By individually cloning eptA and mcr-1 into a plasmid for inducible gene expression, we demonstrated that MCR-1 and EptA have comparable PEtN transferase activity in P. aeruginosa and confer colistin resistance levels similar to those provided by lipid A aminoarabinosylation. Notably, EptA, but not MCR-1, negatively affects P. aeruginosa growth and, to a lesser extent, cell envelope integrity when expressed at high levels. Mutagenesis experiments revealed that PEtN transferase activity does not account for the noxious effects of EptA overexpression, that instead requires a C-terminal tail unique to P. aeruginosa EptA, whose function remains unknown. Overall, this study shows that both endogenous and exogenous PEtN transferases can promote colistin resistance in P. aeruginosa, and that PEtN and MCR-1 mediated resistance has no impact on growth and cell envelope homeostasis, suggesting that there may be no fitness barriers to the spread of mcr-1 in P. aeruginosa.

Forestalling age-impaired angiogenesis and blood flow by targeting NOX: Interplay of NOX1, IL-6, and SASP in propagating cell senescence

In an aging population, intense interest has shifted toward prolonging health span. Mounting evidence suggests that cellular reactive species are propagators of cell damage, inflammation, and cellular senescence. Thus, such species have emerged as putative provocateurs and targets for senolysis, and a clearer understanding of their molecular origin and regulation is of paramount importance. In an inquiry into signaling triggered by aging and proxy instigator, hyperglycemia, we show that NADPH Oxidase (NOX) drives cell DNA damage and alters nuclear envelope integrity, inflammation, tissue dysfunction, and cellular senescence in mice and humans with similar causality. Most notably, selective NOX1 inhibition rescues age-impaired blood flow and angiogenesis, vasodilation, and the endothelial cell wound response. Indeed, NOX1i delivery in vivo completely reversed age-impaired hind-limb blood flow and angiogenesis while disrupting a NOX1-IL-6 senescence-associated secretory phenotype (SASP) proinflammatory signaling loop. Relevant to its comorbidity with age, clinical samples from diabetic versus nondiabetic subjects reveal as operant this NOX1-mediated vascular senescence and inflammation in humans. On a mechanistic level, our findings support a previously unidentified role for IL-6 in this feedforward inflammatory loop and peroxisome proliferator-activated receptor gamma (PPARγ) down-regulation as inversely modulating p65-mediated NOX1 transcription. Targeting this previously unidentified NOX1-SASP signaling axis in aging is predicted to be an effective strategy for mitigating senescence in the vasculature and other organ systems.

σS-Mediated Stress Response Induced by Outer Membrane Perturbation Dampens Virulence in Salmonella enterica serovar Typhimurium.

Salmonella alters cellular processes as a strategy to improve its intracellular fitness during host infection. Alternative σ factors are known to rewire cellular transcriptional regulation in response to environmental stressors. σs factor encoded by the rpoS gene is a key regulator required for eliciting the general stress response in many proteobacteria. In this study, Salmonella Typhimurium deprived of an outer membrane protein YcfR was attenuated in intracellular survival and exhibited downregulation in Salmonella pathogenicity island-2 (SPI-2) genes. This decreased SPI-2 expression caused by the outer membrane perturbation was abolished in the absence of rpoS. Interestingly, regardless of the defects in the outer membrane integrity, RpoS overproduction decreased transcription from the common promoter of ssrA and ssrB, which encode a two-component regulatory system for SPI-2. RpoS was found to compete with RpoD for binding to the PssrA region, and its binding activity with RNA polymerase (RNAP) to form Eσs holoenzyme was stimulated by the small regulatory protein Crl. This study demonstrates that Salmonella undergoing RpoS-associated stress responses due to impaired envelope integrity may reciprocally downregulate the expression of SPI-2 genes to reduce its virulence.

Result of Ring Fixator in High-energy Schatzker Type VI Fractures of Proximal Tibia

Background: Significant articular depression, separation of both condyles, diaphyseal comminution and dissociation, and loss of soft-tissue envelope integrity are all associated with high-energy proximal tibia fractures (Schatzker VI). Over the past 50 years, there has been a lot of research on plating problems in these complicated fractures. For the care of these complex injuries, Ilizarov devised a new method (ring fixator).
 Aim of the Study: To examine the outcomes of patients who received a ring fixator for the treatment of high-energy proximal tibia fractures (Schatzker VI).
 Materials and Methods: Fourteen patients (mean age 36) were treated with the Ilizarov fixator and transfixion wires for high-energy fractures of the proximal tibia (Schatzker VI). Nine of the patients had open fractures, and five of them had significant soft tissue damage. They were all tracked for an average of 19.4 months. The result was analyzed using the criteria set by Honkonen & Jarvinen (1992).
 Results: Thirteen fractures healed in an average of 14.6 weeks, with one taking six months. Twelve patients recovered complete extension, while eight others regained more than 110 degrees of flexion. All of the patients knees were stable, except one who had a minor varus deformity. Nine patients walked normally, while four had a little limp. Except for one, all of the knees exhibited an articular step-off of less than 4 mm and normal axial alignment. Six knees were found to be outstanding, five to be decent, and three to be fair. There were no instances of postoperative skin infection or septic arthritis, however, three patients did have a pin tract infection that was effectively managed.
 Conclusion: The technique is suitable for the treatment of complex proximal tibia fractures when there is substantial comminution at the fracture site as well as soft tissue damage (Schatzker VI).