Cellular Remodeling Research Articles

Virus-Induced Aggregates in Infected Cells

Amid disease, numerous infections actuate cellular remodelling, coming about within the arrangement of insoluble aggregates/inclusions, ordinarily containing viral basic proteins. Recognizable proof of totals has ended up a valuable demonstrative instrument for certain viral contaminations. There's wide assortment of viral totals, which vary by their area, estimate, substance and putative work. The part of accumulation within the context of a particular infection is frequently ineffectively caught on, particularly within the case of plant infections. The totals are utilized by infections to house a expansive complex of proteins of both viral and have root to advance infection replication, interpretation, intra- and intercellular transportation. Amassed structures may secure viral utilitarian complexes from the cellular corruption apparatus. On the other hand, the actuation of have defence components may include sequestration of infection components in totals, taken after by their neutralization as poisonous for the have cell. The diversity of virus-induced aggregates in mammalian and plant cells is the subject of this review.

Cellular Proliferation, Self-Assembly, and Modulation of Signaling Pathways in Silk Fibroin Gelatin-Based 3D Bioprinted Constructs.

Three-dimensional (3D) bioprinting is a highly innovative and promising technology to render precise positioning of biologics together with living cells and extracellular matrix (ECM) constituents. In spite of such enthralling potential, the fabrication of a clinically relevant engineered tissue is quite challenging. A constellation of factors simulating the complex architecture of the native tissue, selection of the "ideal bioink", optimization of the biochemical, mechanical, and topographical functions of the cell-laden printed construct, cellular differentiation, their self-assembly, and remodeling into the desired lineage postprinting present major complications. Keeping this in view, we have attempted to highlight the use of silk fibroin (SF) protein from Bombyx mori silkworm as a promising biomaterial of choice for the formulation of bioink owing to its distinct characteristics involving rheology behavior, self-supporting filamentous extrusion, and a suitable biomaterial to achieve resolution printing. Further, we have elaborated on how SF gelatin bioink can in specific regulate the cellular differentiation pathway of progenitor cells, the mechanism of cellular self-assembly, cell migration, matrix remodeling, and self-orientation, leading to the desired tissue-specific construct. How features of bioink and fabrication design aspects can induce in vitro tissue patterning and anatomically relevant tissue organization have also been explored in this review. Importantly, we have tried to shift the understanding of bioprinted tissue regeneration from a cell-proliferation-centric and gene-expression-centric point of view to the complex role of the microenvironment present within the bioprinted constructs. We believe that shedding light on these factors would help in achieving the so-called "ideal 3D bioprinted construct" to meet the shortages of high-quality donor tissues for the regeneration of the damaged and diseased ones.

Abstract 17259: Identification of Biological Pathways of Candidate Atrial Fibrillation Risk Genes Through the Use of Weighted Gene Coexpression Network Analysis

Introduction: Over 135 genetic loci have been linked to atrial fibrillation (AF), yet the biological pathways of AF pathophysiology remain elusive. Weighted gene coexpression network analysis (WGCNA) constructs gene modules within a network based on correlations in gene expression, and identifies mechanisms related to AF risk. Objective: To identify biological pathways of candidate AF risk genes that will advance our understanding of AF mechanisms. Methods: RNA-sequencing was performed on left atrial appendage tissue from 265 patients. RNA-seq data were adjusted for differences in AF rhythm state and other known AF risk factors. Correlations from adjusted data were further adjusted for latent factors then spatial quantile normalized to correct for mean-variance bias. WGCNA was applied to the resulting adjusted and normalized gene-gene correlations to identify gene modules. Ingenuity Pathway Analysis and gene set over representation analysis (GSOR) were applied to each module. Results: WGCNA identified 63 modules from 17,434 genes; 47 of these contained at least one candidate AF risk gene. AF risk genes were overrepresented in 7 modules (Table 1). Notable top pathways of AF overrepresented modules include apelin signaling, heme metabolism, intracellular ion homeostasis, and the unfolded protein response. These are known to be involved in calcium signaling, iron homeostasis, glucose regulation, heat shock response, and protein ubiquitination during states of high energy demand and stress. These pathways coincide with larger cellular processes of myocyte remodeling, apoptosis, and cell survival, which were also prominent. Conclusions: Biological pathways identified through WGCNA and GSOR suggest that sustained increases in energy demand during AF promotes stress-induced cellular remodeling. Changes in calcium signaling, iron homeostasis, the unfolded protein response and glucose regulation are likely primary mechanisms of AF pathophysiology.

Sequential Water and Headgroup Merger: Membrane Poration Paths and Energetics from MD Simulations

Membrane topology changes such as poration, stalk formation, and hemifusion rupture are essential to cellular function, but their molecular details, energetics, and kinetics are still not fully understood. Here, we present a unified energetic and mechanistic picture of metastable pore defects in tensionless lipid membranes. We used an exhaustive committor analysis to test and select optimal reaction coordinates and also to determine the nucleation mechanism. These reaction coordinates were used to calculate free-energy landscapes that capture the full process and end states. The identified barriers agree with the committor analysis. To enable sufficient sampling of the complete transition path for our molecular dynamics simulations, we developed a “gizmo” potential biasing scheme. The simulations suggest that the essential step in the nucleation is the initial merger of lipid headgroups at the nascent pore center. To facilitate this event, an indentation pathway is energetically preferred to a hydrophobic defect. Continuous water columns that span the indentation were determined to be on-path transients that precede the nucleation barrier. This study gives a quantitative description of the nucleation mechanism and energetics of small metastable pores and illustrates a systematic approach to uncover the mechanisms of diverse cellular membrane remodeling processes.

The Functional Impact of Mitochondrial Structure Across Subcellular Scales.

Mitochondria are key determinants of cellular health. However, the functional role of mitochondria varies from cell to cell depending on the relative demands for energy distribution, metabolite biosynthesis, and/or signaling. In order to support the specific needs of different cell types, mitochondrial functional capacity can be optimized in part by modulating mitochondrial structure across several different spatial scales. Here we discuss the functional implications of altering mitochondrial structure with an emphasis on the physiological trade-offs associated with different mitochondrial configurations. Within a mitochondrion, increasing the amount of cristae in the inner membrane improves capacity for energy conversion and free radical-mediated signaling but may come at the expense of matrix space where enzymes critical for metabolite biosynthesis and signaling reside. Electrically isolating individual cristae could provide a protective mechanism to limit the spread of dysfunction within a mitochondrion but may also slow the response time to an increase in cellular energy demand. For individual mitochondria, those with relatively greater surface areas can facilitate interactions with the cytosol or other organelles but may be more costly to remove through mitophagy due to the need for larger phagophore membranes. At the network scale, a large, stable mitochondrial reticulum can provide a structural pathway for energy distribution and communication across long distances yet also enable rapid spreading of localized dysfunction. Highly dynamic mitochondrial networks allow for frequent content mixing and communication but require constant cellular remodeling to accommodate the movement of mitochondria. The formation of contact sites between mitochondria and several other organelles provides a mechanism for specialized communication and direct content transfer between organelles. However, increasing the number of contact sites between mitochondria and any given organelle reduces the mitochondrial surface area available for contact sites with other organelles as well as for metabolite exchange with cytosol. Though the precise mechanisms guiding the coordinated multi-scale mitochondrial configurations observed in different cell types have yet to be elucidated, it is clear that mitochondrial structure is tailored at every level to optimize mitochondrial function to meet specific cellular demands.

Contractile function of detrusor smooth muscle from children with posterior urethral valves – The role of fibrosis

Posterior urethral valves (PUV) is the most common cause of congenital bladder outflow obstruction with persistent lower urinary tract and renal morbidities. There is a spectrum of functional bladder disorders ranging from hypertonia to bladder underactivity, but the aetiology of these clinical conditions remains unclear. We tested the hypothesis that replacement of detrusor muscle with non-muscle cells and excessive deposition of connective tissue is an important factor in bladder dysfunction with PUV. We used isolated detrusor samples from children with PUV and undergoing primary or secondary procedures in comparison to age-matched data from children with functionally normal bladders. Invitro contractile properties, as well as passive stiffness, were measured and matched to histological assessment of muscle and connective tissue. We examined if a major pathway for fibrosis was altered in PUV tissue samples. Isometric contractions were measured invitro in response to either stimulation of motor nerves to detrusor or exposure to cholinergic and purinergic receptor agonists. Passive mechanical stiffness was measured by rapid stretching of the tissue and recording changes to muscle tension. Histology measured the relative amounts of detrusor muscle and connective tissue. Multiplex quantitative immunofluorescence labelling using five epitope markers was designed to determine cellular pathways, in particular the Wnt-signalling pathway, responsible for any changes to excessive deposition of connective tissue. PUV tissue showed equally reduced contractile function to efferent nerve stimulation or exposure to contractile agonists. Passive muscle stiffness was increased in PUV tissue samples. The smooth muscle:connective tissue ratio was also diminished and mirrored the reduction of contractile function and the increase of passive stiffness. Immunofluorescence labelling showed in PUV samples increased expression of the matrix metalloproteinase, MMP-7; as well as cyclin-D1 expression suggesting cellular remodelling. However, elements of a fibrosis pathway associated with Wnt-signalling were either reduced (β-catenin) or unchanged (c-Myc). The accumulation of extracellular matrix, containing collagen, will contribute to the reduced contractile performance of the bladder wall. It will also increase tissue stiffness that invivo would lead to reduced filling compliance. Replacement of smooth muscle with fibrosis is a major contributory factor in contractile dysfunction in the hypertonic PUV bladder. This suggests that a potential strategy to restore normal contractile and filling properties is development of the effective use of antifibrotic agents.

Cellular mechanisms of early tachycardia-induced ventricular dysfunction in the human heart

Abstract Background Tachycardia-induced cardiomyopathy (TCM) is a reversible form of ventricular dysfunction caused by persistent tachycardia. Characterization of TCM is mainly based on artificially RV paced animal models. Moreover, the underlying mechanisms and time course from compensation to failure remain unclear. This study aimed to investigate early cellular remodeling of tachycardia-induced myocardial dysfunction in human myocardium. Methods and results To elucidate early cellular electrophysiological targets mediating the transition to TCM, we chronically paced (120bpm vs 60bpm control) human induced pluripotent stem cell cardiomyocytes (hiPS-CM) for up to 7d. As a major substrate of cellular myocardial dysfunction, we investigated the influence of chronic tachycardia on cellular Ca cycling. After 24h of persistent tachycardia we detected a significant decrease in Ca transient (CaT) amplitude and reduced diastolic Ca levels (Fura-2). Meanwhile, Ca elimination time (RT80) was unchanged compared to control (n=44/42 cells / 8 diff.). Caffeine application was performed to evaluate sarcoplasmic reticulum (SR) Ca load. We found a shortening of caffeine-induced CaT relaxation time, whereas SR Ca load was unchanged (n=12/13 /8). Further illustrating the transition to TCM, CaT amplitude was progressively decreased after 7d of chronic tachycardia. In contrast to 24h of tachycardia, 7d persistent stimulation resulted in slowed relaxation (RT80, n=75/65 /7). These findings could be explained by a significant reduction of SERCA activity (Ksys-Kcaff) and SR Ca load (n=14/12 / 7). Diastolic Ca concentration remained reduced (n=75/65 /7), in total suggesting a shift to transsarcolemmal Ca elimination. Sodium measurements (SBFI) revealed a significant increase of intracellular sodium concentration (n=69/69 /5) after 7d of tachycardia. In patch clamp experiments we detected a prolongation of action potential duration as early as 24h after onset of tachycardia (n=26/21 /4), which persisted throughout 7d of pacing (n=8/12 /3). Resting membrane potential and action potential amplitude were not changed. Finally, we investigated tachycardia-mediated effects on pre-existing human heart failure (HF). 8h tachycardic stimulation (120bpm) of human HF ventricular trabeculae compromised systolic force, while diastolic tension and relaxation time were markedly increased compared to control (60bpm) (n=7/6 trabeculae /6 human hearts). The extensive molecular characterization of involved ion channels and pathways mediating transition to TCM is currently under investigation. Conclusion This study demonstrates that a persistent tachycardia adversely alters cardiomyocyte excitation-contraction coupling via early electrophysiological cellular remodeling. In pre-existing HF persistent tachycardia strongly aggravates ventricular dysfunction. Our first translational investigation in human myocardium may help to understand the pathophysiology of an underrated and very prevalent disease. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Else-Kröner-Fresenius-Stiftung

Small extracellular vesicles from epicardial fat of patients with atrial fibrillation induce inflammation, fibrosis and re-entrant arrhythmias

Abstract Background and aim Epicardial fat (eFat) has been linked to atrial remodeling and fibrillation (AF). Small extracellular vesicles (sEVs) are heterogeneous membrane vesicles released by all cell types. They can both protect and damage tissues by the delivery of multiple different messengers. Surprisingly, the role of sEVs in the pathogenesis of AF has not been studied. Thus, we aimed to determine whether sEVs derived from eFat play a role in the pathogenesis of AF. Methods and results We collected eFat specimens from patients with and without chronic or paroxysmal AF undergoing open-heart surgery. Isolated eFat specimens were cut into small pieces and incubated as organ cultures. We isolated sEVs from the medium of the explant by differential ultra-centrifugation, high-density gradient or size exclusion chromatography (SEC), and characterized vesicle size distribution, morphology, specific markers, histology and molecular cargo. Immunostaining for macrophage accumulation, fibrosis and apoptosis confirmed the pro-inflammatory and pro-fibrotic properties of eFat sEVs from patients with AF (Fig. 1). eFat sEVs labeled with PKH26 were massively up taken by endothelial cells (Fig. 2). Real-time PCR showed an increased level of oxidative stress genes in endothelial cells. eFat sEVs from patients with AF caused more fibrosis after injection into rat hearts than those without AF. (Fig. 3). Finally, while eFat sEVs from patients with and without AF induced shorter action potential duration, only eFat sEVs from patients with AF induced sustained re-entry (rotor) in human induced pluripotent stem cell (iPS)-derived cardiomyocytes (Fig. 4) Conclusion We show, for the first time, that sEVs from eFat of patients with AF demonstrate unique pro-inflammatory, pro-fibrotic and pro-arrhythmic properties. Our findings suggest that eFat sEVs can induce cellular, molecular and electrophysiological remodeling that can subsequently lead to the development of AF. Funding Acknowledgement Type of funding source: None

In primary airway epithelial cells, the unjamming transition is distinct from the epithelial-to-mesenchymal transition

The epithelial-to-mesenchymal transition (EMT) and the unjamming transition (UJT) each comprises a gateway to cellular migration, plasticity and remodeling, but the extent to which these core programs are distinct, overlapping, or identical has remained undefined. Here, we triggered partial EMT (pEMT) or UJT in differentiated primary human bronchial epithelial cells. After triggering UJT, cell-cell junctions, apico-basal polarity, and barrier function remain intact, cells elongate and align into cooperative migratory packs, and mesenchymal markers of EMT remain unapparent. After triggering pEMT these and other metrics of UJT versus pEMT diverge. A computational model attributes effects of pEMT mainly to diminished junctional tension but attributes those of UJT mainly to augmented cellular propulsion. Through the actions of UJT and pEMT working independently, sequentially, or interactively, those tissues that are subject to development, injury, or disease become endowed with rich mechanisms for cellular migration, plasticity, self-repair, and regeneration.

Potassium starvation induces autophagy in yeast

Autophagy is a conserved process that recycles cellular contents to promote survival. Although nitrogen limitation is the canonical inducer of autophagy, recent studies have revealed several other nutrients important to this process. In this study, we used a quantitative, high-throughput assay to identify potassium starvation as a new and potent inducer of autophagy in the yeast Saccharomyces cerevisiae We found that potassium-dependent autophagy requires the core pathway kinases Atg1, Atg5, and Vps34, and other components of the phosphatidylinositol 3-kinase complex. Transmission EM revealed abundant autophagosome formation in response to both stimuli. RNA-Seq indicated distinct transcriptional responses: nitrogen affects transport of ions such as copper, whereas potassium targets the organization of other cellular components. Thus, nitrogen and potassium share the ability to influence molecular supply and demand but do so in different ways. Both inputs promote catabolism through bulk autophagy, but result in distinct mechanisms of cellular remodeling and synthesis.

In vitro characterization of xeno-free clinically relevant human collagen and its applicability in cell-laden 3D bioprinting.

Collagen type I, commonly derived from xenogenic sources, is extensively used as a biomaterial for tissue engineering applications. However, the use of xenogenic collagen is typically associated with species specific variation in mechanical, structural, and biological properties that are known to influence cellular response and remodeling. In addition, immunological complications and risks of disease transmission are also major concerns. The goal of this study is to characterize a new xeno-free human skin-derived collagen and assess its applicability as a bioink for cell-laden 3 D bioprinting. Four different concentrations of human collagen (i.e., 0.5 mg/mL, 1 mg/mL, 3 mg/mL and 6 mg/mL) were employed for the synthesis of collagen hydrogels. In addition, bovine collagen was used as a xenogenic control. Results from SDS-PAGE analysis showed the presence of α1, α2, and β chains, confirming that the integrity of type I human collagen is maintained post isolation. Polymerization rate and compressive modulus increased significantly with increase in the concentration of human collagen. When comparing two different sources of collagen, the polymerization rate of xenogenic collagen was significantly faster (p < 0.05) than human collagen while the compressive modulus was comparable. Raman spectroscopy showed a large peak in the Amide I band around 1600 cm-1, indicating a dense and supraorganized fibrillar structure in human collagen hydrogels. Conversely, Amide I band intensity for xenogenic collagen was comparable to that of Amide II and Amide III bands. Further, the use of 6 mg/mL human collagen as a bioink yielded 3 D printed constructs with high shape fidelity and cell viability. On the other hand, xenogenic collagen failed to yield stable 3 D printed constructs. Together, the results from this study provides an impetus for using human-derived collagen as a viable alternative to xenogenic sources for 3 D bioprinting of clinically relevant scaffolds for tissue engineering applications.

A thermodynamic transient cross-bridge model for prediction of contractility and remodelling of the ventricle

Cardiac hypertrophy is an adaption of the heart to a change in cardiovascular loading conditions. The current understanding is that progression may be stress or strain driven, but the multi-scale nature of the cellular remodelling processes have yet to be uncovered. In this study, we develop a model of the contractile left ventricle, with the active cell tension described by a thermodynamically motivated cross-bridge cycling model. Simulation of the transient recruitment of myosin results in correct patterns of ventricular pressure predicted over a cardiac cycle. We investigate how changes in tissue loading and associated deviations in transient force generation can drive restructuring of cellular myofibrils in the heart wall. Our thermodynamic framework predicts in-series sarcomere addition (eccentric remodelling) in response to volume overload, and sarcomere addition in parallel (concentric remodelling) in response to valve and signalling disfunction. This framework provides a significant advance in the current understanding of the fundamental sub-sarcomere level biomechanisms underlying cardiac remodelling. Simulations reveal that pathological tissue loading conditions can significantly alter actin-myosin cross-bridge cycling over the course of the cardiac cycle. The resultant variation in sarcomere stress pushes an imbalance between the internal free energy of the myofibril and that of unbound contractile proteins, initiating remodelling. The link between cross-bridge thermodynamics and myofibril remodelling proposed in this study may significantly advance current understanding of cardiac disease onset.

Non-coding RNAs in Cardiac Intercellular Communication.

Intercellular communication allows for molecular information to be transferred from cell to cell, in order to maintain tissue or organ homeostasis. Alteration in the process due to changes, either on the vehicle or the cargo information, may contribute to pathological events, such as cardiac pathological remodeling. Extracellular vesicles (EVs), namely exosomes, are double-layer vesicles secreted by cells to mediate intercellular communication, both locally and systemically. EVs can carry different types of cargo, including non-coding RNAs (ncRNAs), which, are major regulators of physiological and pathological processes. ncRNAs transported in EVs are functionally active and trigger a cascade of processes in the recipient cells. Upon cardiac injury, exosomal ncRNAs can derive from and target different cardiac cell types to initiate cellular and molecular remodeling events such as hypertrophic growth, cardiac fibrosis, endothelial dysfunction, and inflammation, all contributing to cardiac dysfunction and, eventually, heart failure. Exosomal ncRNAs are currently accepted as crucial players in the process of cardiac pathological remodeling and alterations in their presence profile in EVs may attenuate cardiac dysfunction, suggesting that exosomal ncRNAs are potential new therapeutic targets. Here, we review the current research on the role of ncRNAs in intercellular communication, in the context of cardiac pathological remodeling.

VARIATION IN THE PERCENTAGE OF ENDOMETRIAL p16-POSITIVE SENESCENT CELLS IN CONSECUTIVE MENSTRUAL CYCLES

Recently, it was found that p16ink4a-positive senescent cells are involved in endometrial receptivity and participate in the acute cellular remodelling at the time of embryo implantation. Our previous research showed that the percentage of glandular p16-positive cells and luminal epithelial p16-positive cells is strongly associated with successful implantation and live births. The aim of the present study was to compare the percentage of p16-positive cells in the glandular and luminal epithelial compartments of the human endometrium during the mid-luteal phase in two consecutive menstrual cycles. We measured the percentage of p16ink4a-positive cells by immunohistochemistry in endometrial biopsy samples of 94 women in two consecutive menstrual cycles. This is a prospective observational study of 94 fertile women who had two endometrial biopsies during the mid-lutheal phase (7 days after LH surge) in two consecutive natural cycles. Patients older than 40 years, with BMI<18 kg/m2 or BMI≥30 kg/m2, endometriosis, polycystic ovary syndrome (PCOS), endometrial polyps, abnormal uterine development and hydrosalpinx were excluded from the study. Endometrial biopsies were obtained by pipelle suction and they were immediately fixed in 10% formalin. The endometrial tissue was submitted to paraffin embedding for histological determination and subsequent analysis. Immunohistochemistry (IHC) was performed on the paraffin-embedded sections by Novolink Polymer Detection System (Leica Biosystems, Wetzlar, Hesse, Germany). We used monoclonal antibody against p16ink4a (Master Diagnostica, Granada, Spain) to identify senescent endometrial epithelial cells. The percentage of p16+ cells in each tissue compartment was calculated after enumeration by two independent investigators in multiple endometrial sections. Values were expressed as mean ± SD. Paired t-test was used to compare the percentage of p16-positive cells between the two time points. P<0.05 was considered statistically significant. The percentage of p16-positive cells in the endometrial glands during the mid-luteal phase of the cycle ranged between 0.12% and 53.22%, while it varied between 1.39% and 85.29% in the luminal epithelium. The temporal change in the percentage of p16-positive cells varied at both time points between 0.55% and 15.56% in the endometrial glands, and between 1.58% and 30.74% in the endometrial luminal epithelium. In addition, for both endometrial glands and luminal epithelium, the proportion of p16+ senescent cells was not significantly different between the two time measurements (p=0.56 and p=0.93, respectively). In 80% of the patients the difference in the percentage of p16-positive cells at both time points was lower than 20% in the endometrial luminal epithelium and lower than 9% in the luminal epithelium. The percentage of endometrial epithelial p16-positive senescent cells is relatively constant in human endometrium during the mid-luteal phase in consecutive menstrual cycles.

Glycolaldehyde-Derived High-Molecular-Weight Advanced Glycation End-Products Induce Cardiac Dysfunction through Structural and Functional Remodeling of Cardiomyocytes.

High-molecular-weight advanced glycation end-products (HMW-AGEs) are abundantly present in our Western diet. There is growing evidence reporting that HMW-AGEs contribute to the development of cardiovascular dysfunction in vivo, next to the well-known low-molecular-weight AGEs. The goal of our study is to assess the ultrastructure and function of cardiomyocytes after chronic exposure to HMW-AGEs. A better understanding of underlying mechanisms is essential to create new opportunities for further research on the specific role of HMW-AGEs in the development and progression of cardiovascular diseases. Adult male rats were randomly assigned to daily intraperitoneal injection for six weeks with either HMW-AGEs (20 mg/kg/day) or a control solution. Hemodynamic measurements were performed at sacrifice. Single cardiomyocytes from the left ventricle were obtained by enzymatic dissociation through retrograde perfusion of the aorta. Unloaded cell shortening, time to peak and time to 50% relaxation were measured during field stimulation and normalized to diastolic length. L-type Ca2+ current density (ICaL) and steady-state inactivation of ICaL were measured during whole-cell ruptured patch clamp. Myofilament functional properties were measured in membrane-permeabilized cardiomyocytes. Ultrastructural examination of cardiac tissue was performed using electron microscopy. Rats injected with HMW-AGEs displayed in vivo cardiac dysfunction, characterized by significant changes in left ventricular peak rate pressure rise and decline accompanied with an increased heart mass. Single cardiomyocytes isolated from the left ventricle revealed concentric hypertrophy, indicated by the increase in cellular width. Unloaded fractional cell shortening was significantly reduced in cells derived from the HMW-AGEs group and was associated with slower kinetics. Peak L-type Ca2+ current density was significantly decreased in the HMW-AGEs group. L-type Ca2+ channel availability was significantly shifted towards more negative potentials after HMW-AGEs injection. The impact of HMW-AGEs on myofilament function was measured in membrane-permeabilized cardiomyocytes showing a reduction in passive force, maximal Ca2+ activated force and rate of force development. Ultrastructural examination of cardiac tissue demonstrated adverse structural remodeling in HMW-AGEs group characterized by a disruption of the cyto-architecture, a decreased mitochondrial density and altered mitochondrial function. Our data indicate that HMW-AGEs induce structural and functional cellular remodeling via a different working mechanism as the well-known LMW-AGEs. Results of our research open the door for new strategies targeting HMW-AGEs to improve cardiac outcome.

Structure and assembly of ESCRT-III helical Vps24 filaments.

ESCRT-III proteins mediate a range of cellular membrane remodeling activities such as multivesicular body biogenesis, cytokinesis, and viral release. Critical to these processes is the assembly of ESCRT-III subunits into polymeric structures. In this study, we determined the cryo-EM structure of a helical assembly of Saccharomyces cerevisiae Vps24 at 3.2-Å resolution and found that Vps24 adopts an elongated open conformation. Vps24 forms a domain-swapped dimer extended into protofilaments that associate into a double-stranded apolar filament. We demonstrate that, upon binding negatively charged lipids, Vps24 homopolymer filaments undergo partial disassembly into shorter filament fragments and oligomers. Upon the addition of Vps24, Vps2, and Snf7, liposomes are deformed into neck and tubular structures by an ESCRT-III heteropolymer coat. The filamentous Vps24 homopolymer assembly structure and interaction studies reveal how Vps24 could introduce unique geometric properties to mixed-type ESCRT-III heteropolymers and contribute to the process of membrane scission events.

Exploring sunflower responses to Sclerotinia head rot at early stages of infection using RNA-seq analysis

Sclerotinia head rot (SHR), caused by the necrotrophic fungus Sclerotinia sclerotiorum, is one of the most devastating sunflower crop diseases. Despite its worldwide occurrence, the genetic determinants of plant resistance are still largely unknown. Here, we investigated the Sclerotinia-sunflower pathosystem by analysing temporal changes in gene expression in one susceptible and two tolerant inbred lines (IL) inoculated with the pathogen under field conditions. Differential expression analysis showed little overlapping among ILs, suggesting genotype-specific control of cell defense responses possibly related to differences in disease resistance strategies. Functional enrichment assessments yielded a similar pattern. However, all three ILs altered the expression of genes involved in the cellular redox state and cell wall remodeling, in agreement with current knowledge about the initiation of plant immune responses. Remarkably, the over-representation of long non-coding RNAs (lncRNA) was another common feature among ILs. Our findings highlight the diversity of transcriptional responses to SHR within sunflower breeding lines and provide evidence of lncRNAs playing a significant role at early stages of defense.

Exposure of Agrobacterium tumefaciens to agroinfiltration medium demonstrates cellular remodelling and may promote enhanced adaptability for molecular pharming.

Agroinfiltration is used to treat plants with modified strains of Agrobacterium tumefaciens for the purpose of transient in planta expression of genes transferred from the bacterium. These genes encode valuable recombinant proteins for therapeutic or industrial applications. Treatment of large quantities of plants for industrial-scale protein production exposes bacteria (harboring genes of interest) to agroinfiltration medium that is devoid of nutrients and carbon sources for prolonged periods of time (possibly upwards of 24 h). Such conditions may negatively influence bacterial viability, infectivity of plant cells, and target protein production. Here, we explored the role of timing in bacterial culture preparation for agroinfiltration using mass spectrometry-based proteomics to define changes in cellular processes. We observed distinct profiles associated with bacterial treatment conditions and exposure timing, including significant changes in proteins involved in pathogenesis, motility, and nutrient acquisition systems as the bacteria adapt to the new environment. These data suggest a progression towards increased cellular remodelling over time. In addition, we described changes in growth- and environment-specific processes over time, underscoring the interconnectivity of pathogenesis and chemotaxis-associated proteins with transport and metabolism. Overall, our results have important implications for the production of transiently expressed target protein products, as prolonged exposure to agroinfiltration medium suggests remodelling of the bacterial proteins towards enhanced infection of plant cells.

Gene expression profile of adhesion and extracellular matrix molecules during early stages of skeletal muscle regeneration.

Skeletal muscle regeneration implies the coordination of myogenesis with the recruitment of myeloid cells and extracellular matrix (ECM) remodelling. Currently, there are no specific biomarkers to diagnose the severity and prognosis of muscle lesions. In order to investigate the gene expression profile of extracellular matrix and adhesion molecules, as premises of homo‐ or heterocellular cooperation and milestones for skeletal muscle regeneration, we performed a gene expression analysis for genes involved in cellular cooperation, migration and ECM remodelling in a mouse model of acute crush injury. The results obtained at two early time‐points post‐injury were compared to a GSE5413 data set from two other trauma models. Third day post‐injury, when inflammatory cells invaded, genes associated with cell‐matrix interactions and migration were up‐regulated. After day 5, as myoblast migration and differentiation started, genes for basement membrane constituents were found down‐regulated, whereas genes for ECM molecules, macrophage, myoblast adhesion, and migration receptors were up‐regulated. However, the profile and the induction time varied according to the experimental model, with only few genes being constantly up‐regulated. Gene up‐regulation was higher, delayed and more diverse following more severe trauma. Moreover, one of the most up‐regulated genes was periostin, suggestive for severe muscle damage and unfavourable architecture restoration.

Cellular Organelles Reorganization During Zika Virus Infection of Human Cells.

Zika virus (ZIKV) is an enveloped positive stranded RNA virus belonging to the genus Flavivirus in the family Flaviviridae that emerged in recent decades causing pandemic outbreaks of human infections occasionally associated with severe neurological disorders in adults and newborns. The intracellular steps of flavivirus multiplication are associated to cellular membranes and their bound organelles leading to an extensive host cell reorganization. Importantly, the association of organelle dysfunction with diseases caused by several human viruses has been widely reported in recent studies. With the aim to increase the knowledge about the impact of ZIKV infection on the host cell functions, the present study was focused on the evaluation of the reorganization of three cell components, promyelocytic leukemia nuclear bodies (PML-NBs), mitochondria, and lipid droplets (LDs). Relevant human cell lines including neural progenitor cells (NPCs), hepatic Huh-7, and retinal pigment epithelial (RPE) cells were infected with the Argentina INEVH116141 ZIKV strain and the organelle alterations were studied by using fluorescent cell imaging analysis. Our results have shown that these three organelles are targeted and structurally modified during ZIKV infection. Considering the nuclear reorganization, the analysis by confocal microscopy of infected cells showed a significantly reduced number of PML-NBs in comparison to uninfected cells. Moreover, a mitochondrial morphodynamic perturbation with an increased fragmentation and the loss of mitochondrial membrane potential was observed in ZIKV infected RPE cells. Regarding lipid structures, a decrease in the number and volume of LDs was observed in ZIKV infected cells. Given the involvement of these organelles in host defense processes, the reported perturbations may be related to enhanced virus replication through protection from innate immunity. The understanding of the cellular remodeling will enable the design of new host-targeted antiviral strategies.