Deletion of YLR358C Contributes to Reduce Cell Wall Integrity in Saccharomyces Cerevisiae

The mechanism of fungal cell wall synthesis and assembly is still unclear. Saccharomyces cerevisiae (S. cerevisiae) and pathogenic fungi are conserved in cell wall construction and response to stress signals, and often respond to cell wall stress through activated cell wall integrity (CWI) pathways. Whether the YLR358C open reading frame regulates CWI remains unclear. This study found that the growth of S. cerevisiae with YLR358C knockout was significantly inhibited on the medium containing different concentrations of cell wall interfering agents Calcofluor White (CFW), Congo Red (CR) and sodium dodecyl sulfate (SDS). CFW staining showed that the cell wall chitin was down-regulated, and transmission electron microscopy also observed a decrease in cell wall thickness. Transcriptome sequencing and analysis showed that YLR358C gene may be involved in the regulation of CWI signaling pathway. It was found by qRT-PCR that WSC3, SWI4 and HSP12 were differentially expressed after YLR358C was knocked out. The above results suggest that YLR358C may regulate the integrity of the yeast cell walls and has some potential for application in fermentation.

Cytochrome P450 Sterol 14 Alpha-Demethylase Gene SsCI72380 Is Required for Mating/Filamentation and Pathogenicity in Sporisorium scitamineum

Sugarcane smut is a significant sugarcane disease caused by Sporisorium scitamineum and is a large threat to the sugar industry in China and the world. Accordingly, it is important to study the pathogenic mechanism by which this disease occurs to identify effective prevention and control strategies. Gene SsCI72380, which encodes cytochrome P450 sterol 14 alpha-demethylase (CYP51), was screened out from the transcriptome of S. scitamineum. In this study, the functions of gene SsCI72380 were identified via the knockout mutants ΔSs72380+ and ΔSs72380−, which were obtained by polyethylene glycol (PEG)-mediated protoplast transformation technology, as well as the complementary mutants COM72380+ and COM72380−. The results showed that the CYP51 gene SsCI72380 played an important role in sporidial growth, sexual mating/filamentation, hyphae growth, and pathogenicity in S. scitamineum. Gene SsCI72380 may regulate the biosynthesis process of ergosterol by encoding CYP51 enzymes and then affecting the structure and function of the cell membrane. Gene SsCI72380 also played an important role in the response toward different abiotic stresses, including hyperosmotic stress, oxidative stress, and cell wall stress, by regulating the permeability of the cell membrane. In addition, gene SsCI72380 is a new type of pathogenic gene from S. scitamineum that enhances the pathogenicity of S. scitamineum.

The Antibiotic Fosfomycin Mimics the Effects of the Intermediate Metabolites Phosphoenolpyruvate and Glyceraldehyde-3-Phosphate on the Stenotrophomonas maltophilia Transcriptome

Stenotrophomonas maltophilia is an opportunistic pathogen with an environmental origin, which presents a characteristically low susceptibility to antibiotics and is capable of acquiring increased levels of resistance to antimicrobials. Among these, fosfomycin resistance seems particularly intriguing; resistance to this antibiotic is generally due to the activity of fosfomycin-inactivating enzymes, or to defects in the expression or the activity of fosfomycin transporters. In contrast, we previously described that the cause of fosfomycin resistance in S. maltophilia was the inactivation of enzymes belonging to its central carbon metabolism. To go one step further, here we studied the effects of fosfomycin on the transcriptome of S. maltophilia compared to those of phosphoenolpyruvate—its structural homolog—and glyceraldehyde-3-phosphate—an intermediate metabolite of the mutated route in fosfomycin-resistant mutants—. Our results show that transcriptomic changes present a large degree of overlap, including the activation of the cell-wall-stress stimulon. These results indicate that fosfomycin activity and resistance are interlinked with bacterial metabolism. Furthermore, we found that the studied compounds inhibit the expression of the smeYZ efflux pump, which confers intrinsic resistance to aminoglycosides. This is the first description of efflux pump inhibitors that can be used as antibiotic adjuvants to counteract antibiotic resistance in S. maltophilia.

Vacuolar Protein-Sorting Receptor MoVps13 Regulates Conidiation and Pathogenicity in Rice Blast Fungus Magnaporthe oryzae

Magnaporthe oryzae (synonym Pyricularia oryzae) is a filamentous fungal pathogen that causes major yield losses in cultivated rice worldwide. However, the mechanisms of infection of M. oryzae are not well characterized. The VPS13 proteins play vital roles in various biological processes in many eukaryotic organisms, including in the organization of actin cytoskeleton, vesicle trafficking, mitochondrial fusion, and phagocytosis. Nevertheless, the function of the Vps13 protein in plant pathogenic fungi has not been explored. Here, we analysed the biological functions of the Vps13 protein in the development and pathogenicity of M. oryzae. Deletion mutants of MoVps13 significantly reduced the conidiation and decreased the rate of fungal infection on hosts. Moreover, the loss of MoVps13 resulted in defective cell wall integrity (CWI) and plasma membrane (PM) homeostasis when treated with chemicals for inducing cell wall stress (200 mg/mL Congo Red or 0.005% SDS) and sphingolipid synthesis inhibitors (2 μM myriocin or 2 μM amphotericin B). This indicated that MoVps13 is also involved in cell wall synthesis and sphingolipid synthesis. Through immunoblotting, autophagic flux detection, co-localization, and chemical drug sensitivity assays, we confirmed the involvement of Movps13 in ER-phagy and the response to ER stress. Additionally, we generated the C-terminal structure of MoVps13 with high accuracy using the alphaflod2 database. Our experimental evidence indicates that MoVps13 is an important virulence factor that regulates the pathogenicity of M. oryzae by controlling CWI, lipid metabolism and the ER-phagy pathway. These results have expanded our knowledge about pathogenic fungi and will help exploration for novel therapeutic strategies against the rice blast fungus.

Differential Requirement for the Cell Wall Integrity Sensor Wsc1p in Diploids Versus Haploids

The primary role of the Cell Wall Integrity Pathway (CWI) in Saccharomyces cerevisiae is monitoring the state of the cell wall in response to general life cycle stresses (growth and mating) and imposed stresses (temperature changes and chemicals). Of the five mechanosensor proteins monitoring cell wall stress, Wsc1p and Mid2p are the most important. We find that WSC1 has a stringent requirement in zygotes and diploids, unlike haploids, and differing from MID2’s role in shmoos. Diploids lacking WSC1 die frequently, independent of mating type. Death is due to loss of cell wall and plasma membrane integrity, which is suppressed by osmotic support. Overexpression of several CWI pathway components suppress wsc1∆ zygotic death, including WSC2, WSC3, and BEM2, as well as the Rho-GAPS, BEM3 and RGD2. Microscopic observations and suppression by BEM2 and BEM3 suggest that wsc1∆ zygotes die during bud emergence. Downstream in the CWI pathway, overexpression of a hyperactive protein kinase C (Pkc1p-R398P) causes growth arrest, and blocks the pheromone response. With moderate levels of Pkc1p-R398P, cells form zygotes and the wsc1∆ defect is suppressed. This work highlights functional differences in the requirement for Wsc1p in diploids Versus haploids and between Mid2p and Wsc1p during mating.

A Cytochrome B5-Like Heme/Steroid Binding Domain Protein, PlCB5L1, Regulates Mycelial Growth, Pathogenicity and Oxidative Stress Tolerance in Peronophythora litchii

As an electron transport component, cytochrome b5 is an essential component of the Class II cytochrome P450 monooxygenation system and widely present in animals, plants, and fungi. However, the roles of Cyt-b5 domain proteins in pathogenic oomycetes remain unknown. Peronophythora litchii is an oomycete pathogen that causes litchi downy blight, the most destructive disease of litchi. In this study, we identified a gene, designated PlCB5L1, that encodes a Cyt-b5 domain protein in P. litchii, and characterized its function. PlCB5L1 is highly expressed in the zoospores, cysts, germinated cysts, and during early stages of infection. PlCB5L1 knockout mutants showed reduced growth rate and β-sitosterol utilization. Importantly, we also found that PlCB5L1 is required for the full pathogenicity of P. litchii. Compared with the wild-type strain, the PlCB5L1 mutants exhibited significantly higher tolerance to SDS and sorbitol, but impaired tolerance to cell wall stress, osmotic stress, and oxidative stress. Further, the expression of genes involved in oxidative stress tolerance, including peroxidase, cytochrome P450, and laccase genes, were down-regulated in PlCB5L1 mutants under oxidative stress. This is the first report that a Cyt-b5 domain protein contributes to the development, stress response, and pathogenicity in plant pathogenic oomycetes.

Is It Good to Have a Stiff Aorta with Aging? Causes and Consequences

Aortic stiffness increases with advancing age more than doubling during the human lifespan and is a robust predictor of cardiovascular disease (CVD) clinical events independent of traditional risk factors. The aorta increases in diameter and length to accommodate growing body size and cardiac output in youth, but in middle- and older age the aorta continues to remodel to a larger diameter thinning the pool of permanent elastin fibers increasing intramural wall stress resulting in the transfer of load bearing onto stiffer collagen fibers. While aortic stiffening in early middle-age may be a compensatory mechanism to normalize intramural wall stress and therefore theoretically 'good' early in the lifespan, the negative clinical consequences of accelerated aortic stiffening beyond middle-age far outweigh any earlier physiological benefit. Indeed, aortic stiffness and the loss of the "Windkessel effect" with advancing age results in elevated pulsatile pressure and flow in downstream microvasculature that is associated with subclinical damage to high flow, low resistance organs such as brain, kidney, retina and heart. The mechanisms of aortic stiffness include alterations in extracellular matrix proteins (collagen deposition, elastin fragmentation), increased vasodilator tone (oxidative stress and inflammation-related reduced vasodilators and augmented vasoconstrictors; enhanced sympathetic activity), arterial calcification, vascular smooth muscle cell stiffness and extracellular matrix glycosaminoglycans. Given the rapidly aging population of the U.S., aortic stiffening will likely contribute to substantial CVD burden over the next 2-3 decades unless new therapeutic targets and interventions are identified to prevent the potential avalanche of clinical sequelae related to age-related aortic stiffness.

O04 DETERMINING THE EFFECT OF MESH WIDTH AND FIXATION PATTERNS ON THE STRENGTH OF PROPHYLACTICALLY REINFORCED LAPAROTOMY INCISIONS

Aim Research indicates that prophylactic mesh may help prevent incisional hernia after laparotomy, but best practice patterns in these situations are still evolving. Here, we compare the failure loads (FLs) and biomechanical stiffness (BMS) of 35 porcine abdominal wall laparotomy incisions reinforced with meshes of various widths and fixation distances using biomechanical testing. Material and Methods In each specimen, a ten centimeter (cm) incision was made and closed using continuous 1-0 Maxon suture. Specimens were randomized to mesh width (none, 2.5cm, 3cm, 4cm, 6cm, 8cm) and tack separation (1.5cm, 2cm apart), and the meshes secured in an onlay fashion. Cyclic loads oscillating from 15 Newtons (N) to 140N were applied to stimulate abdominal wall stress, and the specimens subsequently loaded to failure. FLs (N) and BMS (N/mm) were comparatively analyzed. Results All specimens failed via suture pull-through. FLs and BMS were lowest in specimens with suture-only (421.43 N; 11.69 N/mm). FLs and BMS were significantly higher in 4cm mesh specimens (567.51N) than those with suture, 2.5cm, and 3.0cm mesh (all p < 0.05). FLs in specimens with a greater number of tacks were consistently higher in meshes of similar sizes, although these did not reach significance. Conclusions Four cm mesh re-enforcement is superior to suture-only and smaller meshes at preserving strength in laparotomy closure in the early stages of healing, but larger meshes (6cm, 8cm) do not provide additional benefit. Meshes with more fixation points may be advantageous, but additional data is needed to make definitive conclusions.

Listeria monocytogenes GlmR is an accessory uridyltransferase essential for cytosolic survival and virulence

The cytosol of eukaryotic host cells is an intrinsically hostile environment for bacteria. Understanding how cytosolic pathogens adapt to and survive in the cytosol is critical to developing novel therapeutic interventions for these pathogens. The cytosolic pathogen Listeria monocytogenes requires glmR (previously known as yvcK), a gene of unknown function, for resistance to cell wall stress, cytosolic survival, inflammasome avoidance and ultimately virulence in vivo. A genetic suppressor screen revealed that blocking utilization of UDP-GlcNAc by a non-essential wall teichoic acid decoration pathway restored resistance to cell wall stress and partially restored virulence of ΔglmR mutants. In parallel, metabolomics revealed that ΔglmR mutants are impaired in the production of UDP-GlcNAc, an essential peptidoglycan and wall teichoic acid (WTA) precursor. We next demonstrated that purified GlmR can directly catalyze the synthesis of UDP-GlcNAc from GlcNAc-1P and UTP, suggesting that it is an accessory uridyltransferase. Biochemical analysis of GlmR orthologues suggest that uridyltransferase activity is conserved. Finally, mutational analysis resulting in a GlmR mutant with impaired catalytic activity demonstrated that uridyltransferase activity was essential to facilitate cell wall stress responses and virulence in vivo. Taken together these studies indicate that GlmR is an evolutionary conserved accessory uridyltransferase required for cytosolic survival and virulence of L. monocytogenes.

Clinical Management of DMD-Associated Cardiomyopathy

Over the past decade, cardiomyopathy has become the leading cause of mortality among patients with Duchenne muscular dystrophy (DMD). The majority of DMD patients over the age of 18 experience some degree of cardiac involvement. The primary cardiac manifestations of DMD include progressive left ventricular (LV) wall stress leading to LV dilatation and wall thinning, and the development of cardiac fibrosis, all of which culminate in decreased LV contractility and reduced cardiac output. Mortality in these patients is predominantly related to pump failure and fatal arrhythmias leading to sudden cardiac death. While basic guidelines for the management of cardiomyopathy in DMD patients exist, these recommendations are by no means comprehensive, and this chapter aims to provide further insight into appropriate clinical diagnosis and management of DMD-associated cardiomyopathy. Notably, earlier and more frequent cardiac assessment and care can allow for better outcomes for these patients. Pharmacological treatments typically include an angiotensin-converting enzyme inhibitor or angiotensin II receptor blocker, beta-adrenergic receptor blockers, mineralocorticoid receptor antagonists, and corticosteroids. Non-pharmacological therapies include automated implantable cardioverter defibrillators and left ventricular assist devices, as well as in rare cases cardiac transplantation. Additionally, many emerging therapies show great promise for improving standards of care. These novel therapies, based primarily on applied gene therapy and genome editing, have great potential to significantly alter the DMD care landscape in the near future.