Envelope Defects Research Articles

Evaluation of aerial thermography for measuring the thermal transmittance (U-value) of a building façade

In efforts to mitigate greenhouse gas emissions in the construction sector, policies promoting energy efficiency improvements in existing buildings are being implemented. Accurately assessing the impact of energy retrofit strategies requires precise determination of thermal envelope performance through energy audits. Aerial thermography, employing unmanned aerial vehicles (UAVs) equipped with infrared thermal cameras (IRT), is an underused method overcoming limitations in energy audits of large buildings or complexes. This approach offers an elevated perspective, providing an overview of the building’s thermal patterns, aiding in the identification of thermal envelope defects. By measuring temperature distribution, even in inaccessible envelope areas, the severity of identified defects can be estimated. Beyond detecting thermal bridges, air leaks, and moisture, aerial thermography is valid for estimating thermal transmittance (U-value) of enclosures. The aim of this research is to evaluate this method to calculate the U-value of the façade of an university building located in a Subtropical Mediterranean climate, during a typical full day in different seasons of the year. Concurrently with aerial thermography, the thermometric method (THM) was applied to compare results and refine the operational conditions during U-value measurement using different statistical indices and uncertainty analysis. The results demonstrated the validation of airborne thermography as a method for U-value calculation under specific meteorological and operational conditions, overcoming the common physical limitations of traditional building audit methods.

Abstract Tu086: Repairing nuclear envelope ruptures to ameliorate Lamin-related cardiomyopathy

Heterozygous loss-of-function mutations in LMNA , encoding nuclear lamina protein Lamin A/C, cause severe adult-onset dilated cardiomyopathy. A prevailing hypothesis posits that LMNA insufficiency causes nuclear envelope structural defects that ultimately cause the disease. However, the mechanisms linking defective nuclear envelopes to cardiomyopathy remain undefined. To determine specific nuclear envelope defects and their consequences, we deleted Lmna in cardiomyocytes in adult mice ( Lmna CKO ). Strikingly, a modest (50%) reduction of Lamin A/C caused widespread localized ruptures of the nuclear envelope in cardiomyocytes (En et al. bioRxiv 2023). The nuclear envelope ruptures did not cause immediate cell death, but accompanied a strong inflammatory response in the heart, prior to fatal cardiomyopathy. We hypothesized that DNA leaked from ruptured nuclei might elicit the cGAS-STING cytosolic DNA sensing pathway of innate immunity. Contrary to this hypothesis, we did not observe cGAS-STING activation in Lmna CKO cardiomyocytes. This lack of cGAS-STING activation was likely due to the near absence of cGAS protein in adult cardiomyocytes. To investigate the mechanism underlying cardiac inflammation in Lmna CKO mice, we conducted time-course single-nucleus RNA-seq. This analysis nominated cardiac fibroblasts as the central mediator of the inflammatory response, receiving ECM-mediated signaling from Lmna CKO cardiomyocytes and recruiting immune cells to the mutant hearts. Finally, we found evidence suggesting that nuclear envelope repair activity counteracts nuclear envelope ruptures in Lmna CKO mice. We found that the envelope ruptured sites co-localized with the ESCRT-III membrane remodeling complex, previously implicated in nuclear envelope repair. We further found that overexpression of DNA-binding protein BANF1 promoted ESCRT-III recruitment to the ruptured sites. We are currently investigating whether facilitated nuclear envelope repair ameliorates cardiomyopathy in Lmna CKO mice. If it does, nuclear envelope repair may be a potential therapeutic strategy for LMNA -related dilated cardiomyopathy.

Component-based hurricane vulnerability model for mid/high-rise commercial residential buildings

This paper focuses on the vulnerability of residential mid/high-rise buildings (MHRB) to hurricane-induced wind and wind-driven rain ingress, with a special emphasis on interior and contents damage. A component-based methodology utilizes the physics of rainwater ingress, distribution and propagation to provide the basis for the proposed MHRB risk assessment model. The model combines estimates of wind speed, impinging rain and surface run-off wind driven rain, envelope defects and breaches, interior water distribution and propagation, and component cost analyses to project envelope, interior and contents damage. The paper describes the model, verifies that it satisfies logical relationships to risk, and compares it against existing MHRB models. The new physics-based interior and contents vulnerability model should facilitate the evaluation of the cost effectiveness of mitigation measures.

Disrupting Central Carbon Metabolism Increases β-Lactam Antibiotic Susceptibility in Vibrio cholerae.

Antibiotic tolerance, the ability of bacteria to sustain viability in the presence of typically bactericidal antibiotics for extended time periods, is an understudied contributor to treatment failure. The Gram-negative pathogen Vibrio cholerae, the causative agent of cholera, becomes highly tolerant to β-lactam antibiotics (penicillin and related compounds) in a process requiring the two-component system VxrAB. VxrAB is induced by exposure to cell wall damaging conditions, which results in the differential regulation of >100 genes. While the effectors of VxrAB are relatively well known, VxrAB environment-sensing and activation mechanisms remain a mystery. Here, we used transposon mutagenesis to screen for mutants that spontaneously upregulate VxrAB signaling. This screen was answered by genes known to be required for proper cell envelope homeostasis, validating the approach. Unexpectedly, we also uncovered a new connection between central carbon metabolism and antibiotic tolerance in Vibrio cholerae. Inactivation of pgi (vc0374, coding for glucose-6-phosphate isomerase) resulted in an intracellular accumulation of glucose-6-phosphate and fructose-6-phosphate, concomitant with a marked cell envelope defect, resulting in VxrAB induction. Deletion of pgi also increased sensitivity to β-lactams and conferred a growth defect on salt-free LB, phenotypes that could be suppressed by deleting sugar uptake systems and by supplementing cell wall precursors in the growth medium. Our data suggest an important connection between central metabolism and cell envelope integrity and highlight a potential new target for developing novel antimicrobial agents. IMPORTANCE Antibiotic tolerance (the ability to survive exposure to antibiotics) is a stepping stone toward antibiotic resistance (the ability to grow in the presence of antibiotics), an increasingly common cause of antibiotic treatment failure. The mechanisms promoting tolerance are poorly understood. Here, we identified central carbon metabolism as a key contributor to antibiotic tolerance and resistance. A strain with a mutation in a sugar utilization pathway accumulates metabolites that likely shut down the synthesis of cell wall precursors, which weakens the cell wall and thus increases susceptibility to cell wall-active drugs. Our results illuminate the connection between central carbon metabolism and cell wall homeostasis in V. cholerae and suggest that interfering with metabolism may be a fruitful future strategy for the development of antibiotic adjuvants.

LdtC Is a Key l,d-Transpeptidase for Peptidoglycan Assembly in Mycobacterium smegmatis.

The peptidoglycan of mycobacteria has two types of direct cross-links, classical 4-3 cross-links that occur between diaminopimelate (DAP) and alanine residues, and nonclassical 3-3 cross-links that occur between DAP residues on adjacent peptides. The 3-3 cross-links are synthesized by the concerted action of d,d-carboxypeptidases and l,d-transpeptidases (Ldts). Mycobacterial genomes encode several Ldt proteins that can be classified into six classes based upon sequence identity. As a group, the Ldt enzymes are resistant to most β-lactam antibiotics but are susceptible to carbapenem antibiotics, with the exception of LdtC, a class 5 enzyme. In previous work, we showed that loss of LdtC has the greatest effect on the carbapenem susceptibility phenotype of Mycobacterium smegmatis (also known as Mycolicibacterium smegmatis) compared to other ldt deletion mutants. In this work, we show that a M. smegmatis mutant lacking the five ldt genes other than ldtC has a wild-type phenotype with the exception of increased susceptibility to rifampin. In contrast, a mutant lacking all six ldt genes has pleiotropic cell envelope defects, is temperature sensitive, and has increased susceptibility to a variety of antibiotics. These results indicate that LdtC is capable of functioning as the sole l,d-transpeptidase in M. smegmatis and suggest that it may represent a carbapenem-resistant pathway for peptidoglycan biosynthesis. IMPORTANCE Mycobacteria have several enzymes to catalyze nonclassical 3-3 linkages in the cell wall peptidoglycan. Understanding the biology of these cross-links is important for the development of antibiotic therapies to target peptidoglycan biosynthesis. Our work provides evidence that LdtC can function as the sole enzyme for 3-3 cross-link formation in M. smegmatis and suggests that LdtC may be part of a carbapenem-resistant l,d-transpeptidase pathway.

Thermography evaluation of defect characteristics of building envelopes in urban villages in Guangzhou, China

In many cities, the building envelope is deteriorating and aging. Understanding the defect characteristics of the envelope is essential to increasing the safety of the house and reducing the loss of energy. A 3-day infrared thermography (IRT) experiment was conducted on 5 buildings (11 rooms) in an urban village in Guangzhou, China. The analysis includes qualitative analysis and quantitative analysis. In qualitative analysis, three types of building defects were found, namely, conduction thermal defects, ventilation defects and moisture. In terms of quantitative analysis, (1) an improved temperature factor framework (ITF) is proposed to evaluate conduction thermal defects, (2) the influence of different influencing factors on the thermal performance of the building envelope is analyzed. Qualitative analysis results showed that conduction thermal defects were the most common thermal defects in Nanting Village and occurred 28 times in five residential buildings. It is mainly distributed in the joints of different walls and the joints of walls and other structures. Ventilation defects and moisture were the other two causes of envelope defects. Quantitative analysis showed that orientation has an important effect on thermal defects. The temperature distribution of walls at different heights is different to some extent. Thermal bridges are more likely to occur on extended floors built on the original building.

Maintainability Performance Prediction of Green Building Envelope in Warm-Humid Climate

Climate change and growing energy demands have put maintainability in the limelight for the long-term performance of green building envelopes, especially for warm-humid regions with intense weathering. This study proposed a framework for predicting the maintainability performance of such envelopes in the Indian context. The country is a key player in the global energy sector because green certification is mandatory for large commercial projects. Design-related envelope defects with their precise causes and effects were collated from the literature, field study, and a questionnaire survey to determine their criticalities. The defects of green and conventional buildings portray similar profiles due to common weathering agents. Furthermore, for the envelope components under the broad groupings of wall, fenestration, and roof, a maintainability attribute checklist was developed from the existing standards and guidelines. Common design alternatives for each component were weighted according to their ability to mitigate both critical and noncritical defects. The maintainability performance of a suitably chosen demo building was predicted. Despite its high energy efficiency class and careful maintenance, defects were prevalent, indicating average maintainability. This explains why landmark green projects also deviate from their energy-saving targets. The accuracy of the proposed framework was found to be satisfactory by tallying predicted and actual defects. The work can contribute to the achievement of long-term envelope performance and resource saving.

Taxonomy of Defects in Auxiliary Elements of Facades and Its Relation with Lawsuits Filed by Property Owners

While at first it can be thought that the auxiliary elements of facades are merely ornamental with little practical function, this study shows that these components of the building envelope have a high impact on the envelope’s functioning and performance. This is carried out through the analysis of all relevant lawsuits filed in Spain over a 10 year period, a data set in which a surprisingly high number of 1033 cases of defects was found to affect external windowsills, exterior wainscots or cornices (the three auxiliary elements considered) was found. Considering the total number of lawsuits, this is an objectively unprecedented study. An analysis is carried out regarding the interrelations between elements, defects, causes, and types of buildings, with the aim of obtaining a sorted classification of the data. This constitutes a useful tool to prevent future problems arising from either the design, execution, or maintenance of facades. These include various issues resulting from humidities, one of the most frequent envelope defects, which are found to significantly affect the auxiliary elements of facades.

Building envelope anomaly characterization and simulation using drone time-lapse thermography

Defects in building envelopes deteriorate over time without being visible to the human eye, while significantly impacting energy performance due to unaccounted heat transfer. Defects can be characterized in the infrared (IR) spectrum. However, IR readings are typically recorded at singular points in time, when in several cases anomalies can only be revealed at specific times of the day, possibly in different seasons of the year. This paper presents a novel workflow for 3D envelope defect characterization and modeling using aerial time-lapse IR data collection using drones. A comprehensive envelope thermal profile is developed for a case study building employing the photogrammetry software Agisoft Photoscan, which generates temporal IR inspections of building skins using multiple thermography orthomosaics. Point-cloud data is then translated into a CAD model and thermal zones for whole Building Energy Modeling (BEM) using Honeybee as a frontend to EnergyPlus to showcase the potential of inclusion of detailed 4D data. Envelope contributions in this case study’s anomalies showed heat losses of 6447.6 kWh, and Energy Use Intensity (EUI) differences of ∼2 kWh/m2/year from the baseline. Why there is currently little translation of this work in BEM software is discussed, while identifying limitations and future research in the employment of time-lapse thermography using drones for more accurate building envelope inspection and modeling.

Modulation of muscle redox and protein aggregation rescues lethality caused by mutant lamins

Mutations in the human LMNA gene cause a collection of diseases called laminopathies, which includes muscular dystrophy and dilated cardiomyopathy. The LMNA gene encodes lamins, filamentous proteins that form a meshwork on the inner side of the nuclear envelope. How mutant lamins cause muscle disease is not well understood, and treatment options are currently limited. To understand the pathological functions of mutant lamins so that therapies can be developed, we generated new Drosophila models and human iPS cell-derived cardiomyocytes. In the Drosophila models, muscle-specific expression of the mutant lamins caused nuclear envelope defects, cytoplasmic protein aggregation, activation of the Nrf2/Keap1 redox pathway, and reductive stress. These defects reduced larval motility and caused death at the pupal stage. Patient-derived cardiomyocytes expressing mutant lamins showed nuclear envelope deformations. The Drosophila models allowed for genetic and pharmacological manipulations at the organismal level. Genetic interventions to increase autophagy, decrease Nrf2/Keap1 signaling, or lower reducing equivalents partially suppressed the lethality caused by mutant lamins. Moreover, treatment of flies with pamoic acid, a compound that inhibits the NADPH-producing malic enzyme, partially suppressed lethality. Taken together, these studies have identified multiple new factors as potential therapeutic targets for LMNA-associated muscular dystrophy.

The unusual cell wall of the Lyme disease spirochaete Borrelia burgdorferi is shaped by a tick sugar

Peptidoglycan—a mesh sac of glycans that are linked by peptides—is the main component of bacterial cell walls. Peptidoglycan provides structural strength, protects cells from osmotic pressure and contributes to shape. All bacterial glycans are repeating disaccharides of N-acetylglucosamine (GlcNAc) β-(1–4)-linked to N-acetylmuramic acid (MurNAc). Borrelia burgdorferi, the tick-borne Lyme disease pathogen, produces glycan chains in which MurNAc is occasionally replaced with an unknown sugar. Nuclear magnetic resonance, liquid chromatography–mass spectroscopy and genetic analyses show that B. burgdorferi produces glycans that contain GlcNAc–GlcNAc. This unusual disaccharide is chitobiose, a component of its chitinous tick vector. Mutant bacteria that are auxotrophic for chitobiose have altered morphology, reduced motility and cell envelope defects that probably result from producing peptidoglycan that is stiffer than that in wild-type bacteria. We propose that the peptidoglycan of B. burgdorferi probably evolved by adaptation to obligate parasitization of a tick vector, resulting in a biophysical cell-wall alteration to withstand the atypical torque associated with twisting motility.

Associations of house characteristics with indoor dampness and measured moisture: Results from three New Zealand House Condition Surveys in 2005, 2010 and 2015

Associations between house characteristics and inspector-assessed subjective indoor dampness (yes/no) and measured floor and ceiling joist timber moisture were measured, using the 2005, 2010 and 2015 New Zealand House Condition Surveys, involving 1572 timber-framed houses. We conducted logistic (dampness) and linear regression (moisture) for each survey separately and mutually adjusted for other house characteristics (ventilation, insulation, subfloor defects, building envelope condition (BEC) defects, tenure, number of occupants), climate zone (latitude), rainfall and outdoor temperature. The odds of subjective damp increased with: more BEC defects (p for trend <0.001), with adjusted odds ratios (aORs) of 3.9–9.6 (p < 0.001) across surveys for houses with 4 or 5 (of 5) defects, compared with houses with ≤1 defect; more subfloor defects (p for trend <0.01 for 2010 and 2015 surveys); less ventilation (p for trend <0.05 for 2010 and 2015 surveys); less insulation (p for trend ≤0.05 for 2010); and increased occupancy (aORs 1.2–2.3, for ≥5 occupants compared to 1–2, not significant). Dampness was more common in rental houses (aORs 1.6 to 2.2, p < 0.05 in 2015). Floor joist moisture content was higher in houses with more subfloor (1.2%–1.9% increase per defect, p for trend ≤0.01) and BEC defects (1.5%–1.8% for houses with 4–5 defects, p < 0.001 for 2005 and 2015 surveys). In conclusion, subfloor and building envelope defects were associated with both inspector-assessed dampness and objectively measured moisture in floor joists. Less insulation, ventilation and higher occupancy were associated with increased subjective dampness but not with measured moisture.

Shewanella oneidensis arcA Mutation Impairs Aerobic Growth Mainly by Compromising Translation.

Arc (anoxic redox control), one of the most intensely investigated two-component regulatory systems in γ-proteobacteria, plays a major role in mediating the metabolic transition from aerobiosis to anaerobiosis. In Shewanella oneidensis, a research model for respiratory versatility, Arc is crucial for aerobic growth. However, how this occurs remains largely unknown. In this study, we demonstrated that the loss of the response regulator ArcA distorts the correlation between transcription and translation by inhibiting the ribosome biosynthesis. This effect largely underlies the growth defect because it concurs with the effect of chloramphenicol, which impairs translation. Reduced transcription of ArcA-dependent ribosomal protein S1 appears to have a significant impact on ribosome assembly. We further show that the lowered translation efficiency is not accountable for the envelope defect, another major defect resulting from the ArcA loss. Overall, our results suggest that although the arcA mutation impairs growth through multi-fold complex impacts in physiology, the reduced translation efficacy appears to be a major cause for the phenotype, demonstrating that Arc is a primary system that coordinates proteomic resources with metabolism in S. oneidensis.

The Rcs System in Enterobacteriaceae: Envelope Stress Responses and Virulence Regulation.

The bacterial cell envelope is a protective barrier at the frontline of bacterial interaction with the environment, and its integrity is regulated by various stress response systems. The Rcs (regulator of capsule synthesis) system, a non-orthodox two-component regulatory system (TCS) found in many members of the Enterobacteriaceae family, is one of the envelope stress response pathways. The Rcs system can sense envelope damage or defects and regulate the transcriptome to counteract stress, which is particularly important for the survival and virulence of pathogenic bacteria. In this review, we summarize the roles of the Rcs system in envelope stress responses (ESRs) and virulence regulation. We discuss the environmental and intrinsic sources of envelope stress that cause activation of the Rcs system with an emphasis on the role of RcsF in detection of envelope stress and signal transduction. Finally, the different regulation mechanisms governing the Rcs system’s control of virulence in several common pathogens are introduced. This review highlights the important role of the Rcs system in the environmental adaptation of bacteria and provides a theoretical basis for the development of new strategies for control, prevention, and treatment of bacterial infections.

Nuclear Pore Complexes Cluster in Dysmorphic Nuclei of Normal and Progeria Cells during Replicative Senescence.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare premature aging disease caused by a mutation in LMNA. A G608G mutation in exon 11 of LMNA is responsible for most HGPS cases, generating a truncated protein called “progerin”. Progerin is permanently farnesylated and accumulates in HGPS cells, causing multiple cellular defects such as nuclear dysmorphism, a thickened lamina, loss of heterochromatin, premature senescence, and clustering of Nuclear Pore Complexes (NPC). To identify the mechanism of NPC clustering in HGPS cells, we evaluated post-mitotic NPC assembly in control and HGPS cells and found no defects. Next, we examined the occurrence of NPC clustering in control and HGPS cells during replicative senescence. We reported that NPC clustering occurs solely in the dysmorphic nuclei of control and HGPS cells. Hence, NPC clustering occurred at a higher frequency in HGPS cells compared to control cells at early passages; however, in late cultures with similar senescence index, NPCs clustering occurred at a similar rate in both control and HGPS. Our results show that progerin does not disrupt post-mitotic reassembly of NPCs. However, NPCs frequently cluster in dysmorphic nuclei with a high progerin content. Additionally, nuclear envelope defects that arise during replicative senescence cause NPC clustering in senescent cells with dysmorphic nuclei.

Dysfunction in nonsense-mediated decay, protein homeostasis, mitochondrial function, and brain connectivity in ALS-FUS mice with cognitive deficits

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) represent two ends of the same disease spectrum of adult-onset neurodegenerative diseases that affect the motor and cognitive functions, respectively. Multiple common genetic loci such as fused in sarcoma (FUS) have been identified to play a role in ALS and FTD etiology. Current studies indicate that FUS mutations incur gain-of-toxic functions to drive ALS pathogenesis. However, how the disease-linked mutations of FUS affect cognition remains elusive. Using a mouse model expressing an ALS-linked human FUS mutation (R514G-FUS) that mimics endogenous expression patterns, we found that FUS proteins showed an age-dependent accumulation of FUS proteins despite the downregulation of mouse FUS mRNA by the R514G-FUS protein during aging. Furthermore, these mice developed cognitive deficits accompanied by a reduction in spine density and long-term potentiation (LTP) within the hippocampus. At the physiological expression level, mutant FUS is distributed in the nucleus and cytosol without apparent FUS aggregates or nuclear envelope defects. Unbiased transcriptomic analysis revealed a deregulation of genes that cluster in pathways involved in nonsense-mediated decay, protein homeostasis, and mitochondrial functions. Furthermore, the use of in vivo functional imaging demonstrated widespread reduction in cortical volumes but enhanced functional connectivity between hippocampus, basal ganglia and neocortex in R514G-FUS mice. Hence, our findings suggest that disease-linked mutation in FUS may lead to changes in proteostasis and mitochondrial dysfunction that in turn affect brain structure and connectivity resulting in cognitive deficits.

The Twin-Arginine Translocation System Is Important for Stress Resistance and Virulence of Brucella melitensis.

Brucella, the causative agent of brucellosis, is a stealthy intracellular pathogen that is highly pathogenic to a range of mammals, including humans. The twin-arginine translocation (Tat) pathway transports folded proteins across the cytoplasmic membrane and has been implicated in virulence in many bacterial pathogens. However, the roles of the Tat system and related substrates in Brucella remain unclear. We report here that disruption of Tat increases the sensitivity of Brucella melitensis M28 to the membrane stressor sodium dodecyl sulfate (SDS), indicating cell envelope defects, as well as to EDTA. In addition, mutating Tat renders M28 bacteria more sensitive to oxidative stress caused by H2O2 Further, loss of Tat significantly attenuates B. melitensis infection in murine macrophages ex vivo Using a mouse model for persistent infection, we demonstrate that Tat is required for full virulence of B. melitensis M28. Genome-wide in silico prediction combined with an in vivo amidase reporter assay indicates that at least 23 proteins are authentic Tat substrates, and they are functionally categorized into solute-binding proteins, oxidoreductases, cell envelope biosynthesis enzymes, and others. A comprehensive deletion study revealed that 6 substrates contribute significantly to Brucella virulence, including an l,d-transpeptidase, an ABC transporter solute-binding protein, and a methionine sulfoxide reductase. Collectively, our work establishes that the Tat pathway plays a critical role in Brucella virulence.

Mutual interplay between ArcA and σE orchestrates envelope stress response in Shewanella oneidensis.

To survive and thrive in harsh and ever-changing environments, intricate mechanisms have evolved for bacterial cells to monitor perturbations impacting the integrity of their envelope and to mount an appropriate response to contain or repair the damage. In this study, we report in Shewanella oneidensis a previously undescribed mechanism for the envelope defect resulting from the loss of Arc, a two-component transcriptional regulatory system crucial for respiration. We uncovered σE , a master regulator establishing and maintaining the integrity of the cell envelope in γ-proteobacteria, as the determining factor for the cell envelope defect of the arcA mutant. When ArcA is depleted, σE activity is compromised by enhanced production of anti-σE protein RseA. Surprisingly, S. oneidensis σE is not essential for viability, but becomes so in the absence of ArcA. Furthermore, we demonstrated that there is an interplay between these two regulators as arcA expression is affected by availability of σE . Overall, our results underscore functional interplay of regulatory systems for envelope stress response: although each of the systems may respond to perturbation of particular components of the envelope, they are functionally intertwined, working together to form an interconnected safety net.

Overexpression of lpxT Gene in Escherichia coli Inhibits Cell Division and Causes Envelope Defects without Changing the Overall Phosphorylation Level of Lipid A.

LpxT is an inner membrane protein that transfers a phosphate group from the essential lipid undecaprenyl pyrophosphate (C-55PP) to the lipid A moiety of lipopolysaccharide, generating a lipid A tris-phosphorylated species. The protein is encoded by the non-essential lpxT gene, which is conserved in distantly related Gram-negative bacteria. In this work, we investigated the phenotypic effect of lpxT ectopic expression from a plasmid in Escherichia coli. We found that lpxT induction inhibited cell division and led to the formation of elongated cells, mostly with absent or altered septa. Moreover, the cells became sensitive to detergents and to hypo-osmotic shock, indicating that they had cell envelope defects. These effects were not due to lipid A hyperphosphorylation or C-55PP sequestering, but most likely to defective lipopolysaccharide transport. Indeed, lpxT overexpression in mutants lacking the L,D-transpeptidase LdtD and LdtE, which protect cells with outer membrane defects from osmotic lysis, caused cell envelope defects. Moreover, we found that pyrophosphorylated lipid A was also produced in a lpxT deletion mutant, indicating that LpxT is not the only protein able to perform such lipid A modification in E. coli.

Nuclear Lamins and Emerin Are Differentially Expressed in Osteosarcoma Cells and Scale with Tumor Aggressiveness.

The nuclear lamina is essential for the maintenance of nuclear shape and mechanics. Mutations in lamin genes have been identified in a heterogeneous spectrum of human diseases known as “laminopathies” associated with nuclear envelope defects and deregulation of cellular functions. Interestingly, osteosarcoma is the only neoplasm described in the literature in association with laminopathies. This study aims characterized the expression of A-type and B-type lamins and emerin in osteosarcoma, revealing a higher percentage of dysmorphic nuclei in osteosarcoma cells in comparison to normal osteoblasts and all the hallmarks of laminopathic features. Both lamins and emerin were differentially expressed in osteosarcoma cell lines in comparison to normal osteoblasts and correlated with tumor aggressiveness. We analysed lamin A/C expression in a tissue-microarray including osteosarcoma samples with different prognosis, finding a positive correlation between lamin A/C expression and the overall survival of osteosarcoma patients. An inefficient MKL1 nuclear shuttling and actin depolymerization, as well as a reduced expression of pRb and a decreased YAP nuclear content were observed in A-type lamin deficient 143B cells. In conclusion, we described for the first time laminopathic nuclear phenotypes in osteosarcoma cells, providing evidence for an altered lamins and emerin expression and a deregulated nucleoskeleton architecture of this tumor.