Modulation Of Entry Research Articles

Seasonal human coronaviruses OC43, 229E, and NL63 induce cell surface modulation of entry receptors and display host cell-specific viral replication kinetics.

The emergence of the COVID-19 pandemic prompted an increased interest in seasonal human coronaviruses. OC43, 229E, NL63, and HKU1 are endemic seasonal coronaviruses that cause the common cold and are associated with generally mild respiratory symptoms. In this study, we identified cell lines that exhibited cytopathic effects (CPE) upon infection by three of these coronaviruses and characterized their viral replication kinetics and the effect of infection on host surface receptor expression. We found that NL63 produced CPE in LLC-MK2 cells, while OC43 produced CPE in MRC-5, HCT-8, and WI-38 cell lines, while 229E produced CPE in MRC-5 and WI-38 by day 3 post-infection. We observed a sharp increase in nucleocapsid and spike viral RNA (vRNA) from day 3 to day 5 post-infection for all viruses; however, the abundance and the proportion of vRNA copies measured in the supernatants and cell lysates of infected cells varied considerably depending on the virus-host cell pair. Importantly, we observed modulation of coronavirus entry and attachment receptors upon infection. Infection with 229E and OC43 led to a downregulation of CD13 and GD3, respectively. In contrast, infection with NL63 and OC43 leads to an increase in ACE2 expression. Attempts to block entry of NL63 using either soluble ACE2 or anti-ACE2 monoclonal antibodies demonstrated the potential of these strategies to greatly reduce infection. Overall, our results enable a better understanding of seasonal coronaviruses infection kinetics in permissive cell lines and reveal entry receptor modulation that may have implications in facilitating co-infections with multiple coronaviruses in humans.IMPORTANCESeasonal human coronavirus is an important cause of the common cold associated with generally mild upper respiratory tract infections that can result in respiratory complications for some individuals. There are no vaccines available for these viruses, with only limited antiviral therapeutic options to treat the most severe cases. A better understanding of how these viruses interact with host cells is essential to identify new strategies to prevent infection-related complications. By analyzing viral replication kinetics in different permissive cell lines, we find that cell-dependent host factors influence how viral genes are expressed and virus particles released. We also analyzed entry receptor expression on infected cells and found that these can be up- or down-modulated depending on the infecting coronavirus. Our findings raise concerns over the possibility of infection enhancement upon co-infection by some coronaviruses, which may facilitate genetic recombination and the emergence of new variants and strains.

Evaluating Medical Waste Sorting Methods and Ultimate Disposal Across Various Health Units at Kampala International University Teaching Hospital

Globally, the surge in medical and healthcare waste is evident, primarily driven by population growth, expanding healthcare facilities, and increased usage of disposable medical products. This study employed qualitative data collection methods, utilizing direct observation and daily review of completed questionnaires to ensure accuracy. Data sets were meticulously organized using an Excel spreadsheet and the Statistical Products and Service Solution (SPSS) data entry module. The assessment focused on waste segregation practices within various health units at KIU-TH. It was observed that healthcare officers in specific units predominantly managed waste segregation, with 95% of units employing color-coded waste bins. Black bins were the most prevalent, followed by red and yellow bins. Notably, a significant percentage indicated that containers for healthcare waste transportation consistently had securely covered lids (96%). The transportation of waste primarily relied on manual methods, either through hand-pushing containers or via vehicles. The study highlighted the implementation of Standard Operating Procedures (SOPs) and guiding documents across all health units and disposal sites. Overall, the findings underscored the prevalence of healthcare waste segregation using color-coded collection bins. However, instances of non-hazardous waste mixing with hazardous waste in general containers were observed, alongside the widespread presence of instructive posters in nearly all visited health units. Keywords: medical waste, segregation practices, disposal

Antiviral Lectins of the Plant Family Amaryllidaceae

AbstractPlants have long served as a first line of defence against viral-borne diseases. Their chemical constituents have also afforded a sound basis for antiviral drug discovery. The plant family Amaryllidaceae is distinguished for its isoquinoline alkaloids, some of which have proved to be interesting antiviral drug leads. Its lectin (or agglutinin) principles have likewise attracted considerable attention as potential antiviral drugs. This review focuses on the antiviral activities that have been described for the lectins of the Amaryllidaceae. Of the thirty lectins known in the family, fourteen have been screened against nearly seventy pathogens belonging to thirteen viral families. Whilst good activities were reported in most cases, the lectins from Galanthus nivalis, Narcissus pseudonarcissus and Hippeastrum hybrid were identified with the best overall activities. They displayed potent inhibitory effects against the human immunodeficiency virus HIV-1(IIIB) proliferation in CEM lymphoblastic cells (EC50s 0.005, 0.009 and 0.004 μM, respectively). Although significant effort was dedicated to the Retroviridae, noteworthy effects were also observed against members of other viral families (such as hepatitis C virus of the Flaviviridae). Furthermore, the lectins were shown to be highly selective antiviral agents, devoid of significant toxicities towards the nearly forty cells employed as hosts. Almost all of the details of their modes of operation have emerged from studies carried out on HIV. They were shown to inhibit viral attachment, fusion and adsorption to a variety of host cells. Modulation of viral entry was shown to occur via interference with the virus envelope glycoprotein. These observations fit into the key biological characteristic of lectins, that of sugar-binding proteins. Graphical Abstract

Modulation of calcium signaling “on demand” to decipher the molecular mechanisms responsible for primary aldosteronism

Primary aldosteronism (PA) is the most frequent form of secondary hypertension. Over the past two decades, major advances have been made in our understanding of the disease with the identification of germline or somatic mutations in ion channels and pumps. These mutations enhance calcium signaling, the main trigger of aldosterone biosynthesis. The objective of our work was to elucidate, using chemogenetic tools, the molecular mechanisms underlying the development of PA by modulating sodium entry into the cells, thus mimicking some of the known mutations identified in PA. We have developed an adrenocortical H295R-S2 cell line stably expressing a chimeric ion channel receptor formed by the extracellular ligand-binding domain of the a7 nicotinic acetylcholine receptor fused to the ion pore domain of the serotonin receptor 5HT3a named a7-5HT3. Its activation by a selective agonist named PSEM-817 leads to sodium entry into the cells. This cell line was characterized in terms of intracellular sodium and calcium concentrations, cell proliferation, steroid production electrophysiological properties, and gene expression. Treatment of a7-5HT3 expressing cells with increasing concentration of PSEM-817 (from 10–9 to 10–5 M) induced a significant increase in intracellular calcium concentrations. Interestingly, whereas cells were hyperpolarized in absence of stimulation (around –60 mV), PSEM-817 induced a strong depolarization, with cells rising to a membrane potential of around –10 mV. The stimulation of calcium signaling by PSEM-817 leads to an increase of CYP11B2 (encoding for aldosterone synthase) mRNA expression and aldosterone biosynthesis but did not affect cell proliferation measured at different time points (8 h, 24 h, 48 h, 72 h). Interestingly, calcium kinetic analyses revealed significant differences in response to the modulation of potassium or sodium entry into cells. Gene expression analyses of gene expression by RNAseq highlighted the activation of specific signaling pathways in response to treatment with Angiotensin II, potassium, and PSEM-817. This cell line, in which we can modulate the intracellular calcium concentration “on demand”, is a useful tool for a better understanding of the alterations of intracellular ion balance and calcium signaling in the pathophysiology of PA.

Overview of Liquid-Metal PFC R&D at the University of Illinois Urbana-Champaign

The design and implementation of future flowing liquid-lithium plasma-facing components (LLPFCs) will be dependent on several factors. Of course, one of the most important is the need to be able to deal with high heat fluxes incident on the surface of the LLPFCs, but there are also several other important liquid-metal behaviors that have been identified for their critical impact on the feasibility of a LLPFC. One of these is the ability to constantly wet 100% of the plasma-facing component area and the best way to achieve that. Another key point is knowing and understanding the erosion and corrosion of the surfaces subject to a flowing liquid-lithium system and the ability for hydrogen and helium uptake by the system. The Center for Plasma Material Interactions (CPMI) has been tasked with looking at these various issues. The Mock-up Entry module for EAST device was used to investigate wetting and erosion effects and to design a suitable distribution and collection system with a liquid-lithium loop. The vapor shielding effects of lithium on the surface were also modeled and studied. A model coupling CRANE, an open-source global reaction network solver, and Zapdos, a plasma transport solver, is being developed to better understand the dynamics of the vapor cloud. Experiments on the Magnum-PSI at the Dutch Institute for Fundamental Energy Research have been carried out to study the vapor shielding effect and obtain experimental benchmarks to verify the model. Also, initial experiments using the Hybrid Illinois Device for Research and Applications have been performed to understand the pumping effects of lithium on helium. Experiments with a drop of liquid lithium (~100 mg) into a helium plasma have shown the ability of lithium to take out the cold recycling helium gas as well as hydrogen and oxygen impurity gases. The improvement in plasma performance was significant, and further understanding of this effect will have impacts on how future LLPFCs will be designed. Further investigation into the exact mechanism for helium pumping by lithium needs to be performed in the future. This paper presents a summary of the results obtained at the CPMI.

A Review of Protein- and Peptide-Based Chemical Conjugates: Past, Present, and Future.

Over the past few decades, the complexity of molecular entities being advanced for therapeutic purposes has continued to evolve. A main propellent fueling innovation is the perpetual mandate within the pharmaceutical industry to meet the needs of novel disease areas and/or delivery challenges. As new mechanisms of action are uncovered, and as our understanding of existing mechanisms grows, the properties that are required and/or leveraged to enable therapeutic development continue to expand. One rapidly evolving area of interest is that of chemically enhanced peptide and protein therapeutics. While a variety of conjugate molecules such as antibody-drug conjugates, peptide/protein-PEG conjugates, and protein conjugate vaccines are already well established, others, such as antibody-oligonucleotide conjugates and peptide/protein conjugates using non-PEG polymers, are newer to clinical development. This review will evaluate the current development landscape of protein-based chemical conjugates with special attention to considerations such as modulation of pharmacokinetics, safety/tolerability, and entry into difficult to access targets, as well as bioavailability. Furthermore, for the purpose of this review, the types of molecules discussed are divided into two categories: (1) therapeutics that are enhanced by protein or peptide bioconjugation, and (2) protein and peptide therapeutics that require chemical modifications. Overall, the breadth of novel peptide- or protein-based therapeutics moving through the pipeline each year supports a path forward for the pursuit of even more complex therapeutic strategies.

Building a Database System for Electronic Medical Records to Ensure Efficient Data Management and Interpretation

This research presents the design and implementation of an e-database system of medical records. The system comprises various modules, including data entry, user login, data storage, data management, and query analysis. The data entry module enables the input of electronic medical record data, while the user login module provides secure access and identity authentication. The data storage module stores the medical records and user information, and the data management module handles data manipulation and user authorization. The query analysis module allows users to search and analyze medical records based on specified criteria. This system aims to enhance the management and analysis of electronic medical records, providing medical professionals with valuable insights for diagnosis and treatment.

STIM1 and ORAI1 mutations leading to tubular aggregate myopathies are sensitive to the Store-operated Ca2+-entry modulators CIC-37 and CIC-39

Gain-of-function mutations on STIM1 and ORAI1 genes are responsible for an increased store-operated calcium entry, and underlie the characteristic symptoms of three overlapping ultra-rare genetic disorders (i.e tubular aggregate myopathy, Stormorken syndrome, York platelet syndrome) that can be grouped as tubular aggregate myopathies. These mutations lead to a wide spectrum of defects, which usually include muscle weakness and cramps.Negative modulators of store-operated Ca2+-entry targeting wild-type STIM1 and ORAI1 have entered clinical trials for a different array of disorders, including pancreatitis, COVID-19, cancer, and autoimmune disorders and, while efficacy data is awaited, safety data indicates tolerability of thisSTIM1/ORAI1 mutations are amenable to pharmacological intervention. If this were so, given that there are no approved treatments or clinical trials ongoing for these rare disorders, it could be envisaged that these agents could also rehabilitate tubular aggregate myopathy patients. In the present contribution we characterized the Ca2+-entry patterns induced by eleven STIM1 and three ORAI1 mutations in heterologous systems or in patient-derived cells, i.e. fibroblasts and myotubes, and evaluated the effect of CIC-37 and CIC-39, two novel store-operated calcium entry modulators. Our data show that all STIM1 and ORAI1 gain-of-function mutations tested, with the possible exception of the R304Q STIM1 mutation, are amenable to inhibition, albeit with slightly different sensitivities, paving the way to the development of SOCE modulators in tubular aggregate myopathies.

H-2Kb and H-2Db class I molecules on cerebral endothelium differentially modulate CD8 T cells dynamics and pathological outcomes in experimental cerebral malaria

Abstract Immune mechanisms of neuropathology in human cerebral malaria are not fully understood. Using the Plasmodium berghei ANKA model of experimental cerebral malaria (ECM), we sought to define the contribution of cerebral endothelial cell (CEC) MHC class I antigen presentation, to the pathogenesis and lethality of ECM. CD8 T cell interactions with class I molecules on CECs has been put forward as a putative mechanism for ECM. However, the importance of this interaction has not been confirmed molecularly in vivo. We therefore hypothesized that conditional ablation of antigen presentation by H-2Kb and H-2Db class I molecules specifically on brain endothelium would result in controlled modulation of activation and entry of parasite-specific CD8 T cells, attenuating pathology. To test this, we crossed endothelium-specific CDH5-Cre mice with our novel H-2Kb and H-2Db LoxP mice to create endothelium-specific cKO of either or both class I molecules. 2-photon imaging revealed that each discreet class I molecule influenced differential T cell dynamics at the neurovasculature during lethal stage of disease. Furthermore, CD8 T cell activation and infiltration of the brain was distinctly altered, per class I interaction, and vascular permeability at the blood-brain barrier was attenuated, resulting in improved survival of H-2Kb and H-2Db endothelial cKO mice. Together these data demonstrate that parasite-specific CD8 T cells undergo Ag-dependent engagement with H-2Kb or H-2Db class I molecules on CECs in order to coordinate activation, entry, and induction of neuropathology. These studies indicate that modulation of CEC class I restricted antigen presentation may hold therapeutic benefit in cases of severe malaria. Supported by Ruth L. Kirschstein National Research Service Award Individual Predoctoral Fellowship F31 1F31NS116924-01. NIH R01 5R01NS103212-05.

SARS-CoV-2 Spike Glycoprotein and ACE2 Interaction Reveals Modulation of Viral Entry in Wild and Domestic Animals.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a viral pathogen causing life-threatening diseases in humans. Interaction between the spike protein of SARS-CoV-2 and angiotensin-converting enzyme 2 (ACE2) is a potential factor in the infectivity of a host. In this study, the interaction of SARS-CoV-2 spike protein with its receptor, ACE2, in different hosts was evaluated to predict the probability of viral entry. Phylogeny and alignment comparison of the ACE2 sequences did not lead to any meaningful conclusion on viral entry in different hosts. The binding ability between ACE2 and the spike protein was assessed to delineate several spike binding parameters of ACE2. A significant difference between the known infected and uninfected species was observed for six parameters. However, these parameters did not specifically categorize the Orders into infected or uninfected. Finally, a logistic regression model constructed using spike binding parameters of ACE2, revealed that in the mammalian class, most of the species of Carnivores, Artiodactyls, Perissodactyls, Pholidota, and Primates had a high probability of viral entry. However, among the Proboscidea, African elephants had a low probability of viral entry. Among rodents, hamsters were highly probable for viral entry with rats and mice having a medium to low probability. Rabbits have a high probability of viral entry. In Birds, ducks have a very low probability, while chickens seemed to have medium probability and turkey showed the highest probability of viral entry. The findings prompt us to closely follow certain species of animals for determining pathogenic insult by SARS-CoV-2 and for determining their ability to act as a carrier and/or disseminator.

Aerodynamic design, analysis, and validation techniques for the Tianwen-1 entry module

The clear differences between the atmosphere of Mars and the Earth coupled with the lack of a domestic research basis were significant challenges for the aerodynamic prediction and verification of Tianwen-1. In addition, the Mars entry, descent, and landing (EDL) mission led to specific requirements for the accuracy of the aerodynamic deceleration performance, stability, aerothermal heating, and various complex aerodynamic coupling problems of the entry module. This study analyzes the key and difficult aerodynamic and aerothermodynamic problems related to the Mars EDL process. Then, the study process and results of the design and optimization of the entry module configuration are presented along with the calculations and experiments used to obtain the aerodynamic and aerothermodynamic characteristics in the Martian atmosphere. In addition, the simulation and verification of the low-frequency free oscillation characteristics under a large separation flow are described, and some special aerodynamic coupling problems such as the aeroelastic buffeting response of the trim tab are discussed. Finally, the atmospheric parameters and aerodynamic characteristics obtained from the flight data of the Tianwen-1 entry module are compared with the design data. The data obtained from the aerodynamic design, analysis, and verification of the Tianwen-1 entry module all meet the engineering requirements. In particular, the flight data results for the atmospheric parameters, trim angles of attack, and trim axial forces are within the envelopes of the prediction deviation zones.

Study on Dynamic Characteristics of Mars Entry Module in Transonic and Supersonic Speeds

The aerodynamic configuration of the Tianwen-1 Mars entry module that adopts a blunt-nosed and short body shape has obvious dynamic instability from transonic to supersonic speeds, which may bring risk to parachute deployment. The unsteady detached eddy of the entry module cannot be accurately simulated by the Reynolds-Averaged Navier-Stokes (RANS) model, while the computational cost for direct numerical simulation (DNS) and large eddy simulation (LES) is huge. It is difficult to implement these methods in the coupled engineering calculation of unsteady flow and motion. This paper proposes the integrated numerical simulation method of computational fluid dynamics and rigid body dynamics (CFD/RBD) based on detached eddy simulation (DES) and calculates and studies the dynamic characteristics of attitude oscillation of the Mars entry module in free flight from transonic to supersonic speeds with one degree of freedom (1-DOF) at small releasing angle of attack. In addition, the unstable range of Mach number and angle of attack are determined, and the effect of different afterbody shapes on dynamic stability is analyzed.

Inhibiting Mitochondrial Complex II Exposes a Novel Metabolic Vulnerability in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a hematological malignancy, characterized by an increased reliance on mitochondria-related energetic pathways including oxidative phosphorylation (OXPHOS). Consistent with this, the electron transport chain (ETC), a component of OXPHOS has been demonstrated to be a suitable anti-leukemia target, with ETC complex I inhibitors currently in clinical development. Relative to its counterparts, complex II (CII) is unique in that it directly links the ETC to the tricarboxylic acid (TCA) cycle through succinate dehydrogenase (SDH) activity. Moreover, it is the only ETC complex with elevated activity in AML, relative to normal hematopoietic samples, with indirect inhibition selectively targeting AML cells. However, direct CII inhibition in AML has not been previously investigated, nor have the mechanisms underlying the divergent fates of AML and normal cells upon CII inhibition.A genetic approach was first used to assess the effects of CII impairment on AML growth in vitro and in vivo. Using lentiviral mediated shRNA we generated AML cell lines lacking succinate dehydrogenase assembly factor 1 (Sdhaf1). Sdhaf1 knockdown suppressed CII activity, cell proliferation and clonogenic growth across all three cell lines and delayed leukemia growth in vivo. To recapitulate these effects through a pharmacological approach, we aimed to identify a novel CII inhibitor, since currently available inhibitors are only effective at high doses and are neurotoxic. Through an in silico structural screen and molecular docking study, shikonin was identified as a small molecule that selectively binds to CII. Shikonin inhibited CII activity in the AML cells lines and patient-derived samples, and selectively killed AML cells (EC 50: 1.0μM ± 0.04) while sparing normal progenitors. In murine engraftment models, shikonin (2.0-3.0 mg/kg, 3x/week for 5 weeks) significantly reduced engraftment of patient-derived AML cells but had no effect on normal hematopoiesis.To further characterize the mechanisms governing the divergent cell fates of CII inhibition, we performed stable isotope metabolic tracing using 13C 6- glucose and 13C 5, 15N 2-glutamine in patient-derived AML cells and normal mobilized peripheral blood mononuclear cells (MNCs). Both pharmacological and genetic loss of CII resulted in TCA cycle truncation by impairing oxidative metabolism of both glucose and glutamine. In Sdhaf1 knockdown and primary AML cells, this led to a depletion in steady state levels of TCA metabolites proceeding SDH. Inhibition of CII most notably suppressed levels of aspartate, a nucleotide precursor whose levels dictate the proliferative capacity of a cell under ETC dysfunction. Remarkably, MNCs maintained aspartate levels despite inhibition of CII, which was attributed to reductive carboxylation of glutamine, an alternate metabolic pathway that can regenerate TCA intermediates when OXPHOS is impaired. In contrast, while reductive carboxylation was also active in AML cells after CII inhibition, this activity was insufficient to maintain aspartate levels and resulted in metabolite depletion and cell death. Thus, loss of CII activity results in diverse cell fates whereby normal haematopoietic, but not AML cells sufficiently use reductive carboxylation of glutamine to overcome TCA cycle truncation, sustain aspartate levels and avert cell death. This is further evident through modulation of glutamine entry into the TCA cycle, where supplementation of cell-permeable α-ketoglutarate abrogated shikonin-mediated cell death while concomitant treatment with the glutaminase inhibitor CB-839, sensitized cells.Together, these results expose reductive carboxylation to support aspartate biosynthesis, as a novel metabolic vulnerability in AML that can be pharmacologically targeted through CII inhibition for clinical benefit. DisclosuresMinden: Astellas: Consultancy. D'Alessandro: Omix Thecnologies: Other: Co-founder; Rubius Therapeutics: Consultancy; Forma Therapeutics: Membership on an entity's Board of Directors or advisory committees.

The board is set, the pieces are moving: Modulation of New World arenavirus entry by host proteins.

The board is set, the pieces are moving: Modulation of New World arenavirus entry by host proteins.

Zhurong: Features and mission of China's first Mars rover

On May 22, 2021, at 10:40 am (Beijing Time), China’s Zhurong rover successfully reached the Mars surface and began its exploration. China is only the second nation to have conducted a successful soft landing and exploration of the red planet. The Mars probe consists of an orbiter and landing rover. The spacecraft includes an entry module consisting of a back shell, landing platform, and outsole, as well as a rover (Figure 1). Before release, the orbiter, which carries the landing rover, completed the Earth–Mars orbital transfer and an orbit reduction maneuver.

Modulation of Calcium Signaling by Chemogenetic Tools to Elucidate the Pathogenesis of Primary Aldosteronism

Primary aldosteronism (PA) is the most frequent form of secondary arterial hypertension. The identification of germline or somatic mutations in different genes coding for ion channels (KCNJ5, CACNA1D, CACNA1H and CLCN2) and ATPases (ATP1A1 and ATP2B3) defines PA as a channelopathy. These mutations promote increased intracellular calcium concentrations and activation of calcium signaling, the main trigger for aldosterone biosynthesis.The aim of our study was to elucidate the mechanisms underlying the development of PA by modulating calcium signaling using chemogenetic tools.We have generated two different adrenocortical H295R_S2 cell lines stably expressing different chimeric ion channels generated by fusing the mutated extracellular ligand binding domain of the α7 nicotinic acetylcholine receptor to the ion pore domain of large Cys-loop receptor ion channel family; these receptors constitute PSAM (Pharmacologically Selective Actuator Modules). The mutations introduced in the ligand-binding domain allow to use synthetic ligands, PSEM (Pharmacologically Selective Effector Molecules) to activate the PSAM. We used two different PSAM: the chimeric receptor α7-5HT3 or a mutated acetylcholine receptor nAchR, allowing respectively modulation of sodium or calcium entry into the cells in response to the specific PSEM: Varenicline for α7-5HT3 or Compound 9S for mutated nAChR. The cells lines were characterized in terms of intracellular calcium concentrations, cell proliferation, aldosterone production and steroidogenic gene expression.Cells expressing α7-5HT3 treated for 24h with increasing concentrations of Varenicline (10–9 to 10-5M) showed increased intracellular calcium concentrations and an increase in expression of steroidogenic genes such as StAR CYP17A1, CYP21A2 and CYP11B2. Cell proliferation was not affected. Calcium entry into cells expressing the mutated nAChR receptor treated for 24h with increasing concentrations of Compound 9S (10–9 to 10-5M) induced an increase in expression of steroidogenic genes such as StAR, CYP21A2 and HSD3B2, but not CYP11B2. Similarly to the results obtained in cells expressing α7-5HT3, cell proliferation was unaffected in response to Compound 9S.These cell lines, in which we can modulate the intracellular calcium concentration « on demand », are a useful tool for a better understanding of the alterations of intracellular ion balance and calcium signaling in the pathophysiology of PA.

BacSJ-Another Bacteriocin with Distinct Spectrum of Activity that Targets Man-PTS.

Lactic acid bacteria produce diverse antimicrobial peptides called bacteriocins. Most bacteriocins target sensitive bacteria by binding to specific receptors. Although a plethora of bacteriocins have been identified, for only a few of them the receptors they recognize are known. Here, we identified permease IIC and surface protein IID, two membrane subunits of the mannose-specific quaternary phosphotransferase system (Man-PTS), as a receptor for BacSJ, a subclass IId bacteriocin produced by Lactobacillus paracasei subsp. paracasei BGSJ2-8. BacSJ shares 45% identity with another Man-PTS binding bacteriocin, garvicin Q (GarQ). Similarly to GarQ, BacSJ has a relatively broad activity spectrum acting against several Gram-positive bacteria, such as Lactococcus lactis and Listeria monocytogenes, harboring fairly similar Man-PTSs, but not against Lactococcus garvieae. To identify specific Man-PTS amino acids responsible for the L. lactis sensitivity to BacSJ, and thus likely involved in the interaction with this bacteriocin, we generated eight independent BacSJ resistant L. lactis mutants harboring five distinct missense mutations in the ptnC or ptnD genes encoding the IIC and IID subunits. Concurrently with the resistance to BacSJ, the mutants efficiently utilized mannose as a carbon source, which indicated functionality of their mutated Man-PTS. The amino acid substitutions in the mutants localized to the intracellular region of the IIC permease or to the extracellular parts of IID. This localization coincides with regions targeted by GarQ and some other Man-PTS-binding garvicins, pointing to similarities between all these bacteriocins in the mechanism of their interaction with Man-PTS. During the attack by these bacteriocins, subunits IID and IIC are assumed to function sequentially as a docking and an entry module allowing the toxic peptide to bind the cell and then open the pore. However, since not all of the BacSJ-resistant mutants exhibited cross-resistance to GarQ, we propose that BacSJ interacts with Man-PTS in a manner slightly different from that of GarQ.

Laboratory information system requirements to manage the COVID-19 pandemic: A report from the Belgian national reference testing center.

ObjectiveThe study sought to describe the development, implementation, and requirements of laboratory information system (LIS) functionality to manage test ordering, registration, sample flow, and result reporting during the coronavirus disease 2019 (COVID-19) pandemic.Materials and MethodsOur large (>12 000 000 tests/y) academic hospital laboratory is the Belgian National Reference Center for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing. We have performed a moving total of >25 000 SARS-CoV-2 polymerase chain reaction tests in parallel to standard routine testing since the start of the outbreak. A LIS implementation team dedicated to develop tools to remove the bottlenecks, primarily situated in the pre- and postanalytical phases, was established early in the crisis.ResultsWe outline the design, implementation, and requirements of LIS functionality related to managing increased test demand during the COVID-19 crisis, including tools for test ordering, standardized order sets integrated into a computerized provider order entry module, notifications on shipping requirements, automated triaging based on digital metadata forms, and the establishment of databases with contact details of other laboratories and primary care physicians to enable automated reporting. We also describe our approach to data mining and reporting of actionable daily summary statistics to governing bodies and other policymakers.ConclusionsRapidly developed, agile extendable LIS functionality and its meaningful use alleviates the administrative burden on laboratory personnel and improves turnaround time of SARS-CoV-2 testing. It will be important to maintain an environment that is conducive for the rapid adoption of meaningful LIS tools after the COVID-19 crisis.

Potential Benefits and Harms of Novel Antidiabetic Drugs During COVID-19 Crisis.

Patients with diabetes have been reported to have enhanced susceptibility to severe or fatal COVID-19 infections, including a high risk of being admitted to intensive care units with respiratory failure and septic complications. Given the global prevalence of diabetes, affecting over 450 million people worldwide and still on the rise, the emerging COVID-19 crisis poses a serious threat to an extremely large vulnerable population. However, the broad heterogeneity and complexity of this dysmetabolic condition, with reference to etiologic mechanisms, degree of glycemic derangement and comorbid associations, along with the extensive sexual dimorphism in immune responses, can hamper any patient generalization. Even more relevant, and irrespective of glucose-lowering activities, DPP4 inhibitors and GLP1 receptor agonists may have a favorable impact on the modulation of viral entry and overproduction of inflammatory cytokines during COVID-19 infection, although current evidence is limited and not univocal. Conversely, SGLT2 inhibitors may increase the likelihood of COVID-19-related ketoacidosis decompensation among patients with severe insulin deficiency. Mindful of their widespread popularity in the management of diabetes, addressing potential benefits and harms of novel antidiabetic drugs to clinical prognosis at the time of a COVID-19 pandemic deserves careful consideration.

Mitochondrial TRPC3 promotes cell proliferation by regulating the mitochondrial calcium and metabolism in renal polycystin-2 knockdown cells.

Previous studies indicate that autosomal dominant polycystic kidney disease (ADPKD) cells exhibited dysregulated calcium homeostasis and enhanced cell proliferation. TRPC3 has been shown to function in the modulation of calcium and sodium entry, but whether TRPC3 plays a role in cellular abnormalities of ADPKD cells has not been defined. Human conditionally immortalized proximal tubular epithelial cells and mouse IMCD3 cells were used with polycystin-2 (PC2, TRPP2) knockdown. Cell proliferation assay was used to detect the cell proliferations upon different treatments. QRT-PCR and western blotting were used to measure the expression profiles of TRPP2 and other proteins. High-resolution respirometry, enzymic activities and ROS levels were detected to reflect the mitochondrial functions. Calcium and sodium uptakes were measured using Fura2-AM and SBFI dyes. We showed that PC2 knockdown promoted cell proliferation, ROS productions and ERK phosphorylation, compared with negative control. Meanwhile, we demonstrated that receptor-operated calcium entry (ROCE) exhibited less reductions compared with store-operated calcium entry (SOCE) upon PC2 knockdown. Inhibition of ROCE and SOCE by specific inhibitors partially reversed the enhanced cell proliferation, ROS productions and ERK phosphorylation induced by PC2 knockdown. Moreover, TRPC3 upregulation was observed upon PC2 knockdown, which acted as both SOC and ROC, promoting cation entry, cell proliferation and ERK phosphorylation. Furthermore, we showed that mitochondrial located TRPC3 was upregulated and modulating mitochondrial calcium uptake, thus promoting the ROS productions in the presence of PC2 knockdown. We demonstrated that TRPC3 upregulation upon PC2 knockdown aggravated the mitochondrial abnormalities and cell proliferation by modulating mitochondrial calcium uptake. Targeting TRPC3 might be a promising target for ADPKD treatment.