ERMA (TMEM94) is a P-type ATPase transporter for Mg2+ uptake in the endoplasmic reticulum.

Intracellular Mg2+ (iMg2+) is bound with phosphometabolites, nucleic acids, and proteins in eukaryotes. Little is known about the intracellular compartmentalization and molecular details of Mg2+ transport into/from cellular organelles such as the endoplasmic reticulum (ER). We found that the ER is a major iMg2+ compartment refilled by a largely uncharacterized ER-localized protein, TMEM94. Conventional and AlphaFold2 predictions suggest that ERMA (TMEM94) is a multi-pass transmembrane protein with large cytosolic headpiece actuator, nucleotide, and phosphorylation domains, analogous to P-type ATPases. However, ERMA uniquely combines a P-type ATPase domain and a GMN motif for ERMg2+ uptake. Experiments reveal that a tyrosine residue is crucial for Mg2+ binding and activity in a mechanism conserved in both prokaryotic (mgtB and mgtA) and eukaryotic Mg2+ ATPases. Cardiac dysfunction by haploinsufficiency, abnormal Ca2+ cycling in mouse Erma+/- cardiomyocytes, and ERMA mRNA silencing in human iPSC-cardiomyocytes collectively define ERMA as an essential component of ERMg2+ uptake in eukaryotes.

MCU-complex-mediated mitochondrial calcium signaling is impaired in Barth syndrome.

Calcium signaling via mitochondrial calcium uniporter (MCU) complex coordinates mitochondrial bioenergetics with cellular energy demands. Emerging studies show that the stability and activity of the pore-forming subunit of the complex, MCU, is dependent on the mitochondrial phospholipid, cardiolipin (CL), but how this impacts calcium-dependent mitochondrial bioenergetics in CL-deficiency disorder like Barth syndrome (BTHS) is not known. Here we utilized multiple models of BTHS including yeast, mouse muscle cell line, as well as BTHS patient cells and cardiac tissue to show that CL is required for the abundance and stability of the MCU-complex regulatory subunit MICU1. Interestingly, the reduction in MICU1 abundance in BTHS mitochondria is independent of MCU. Unlike MCU and MICU1/MICU2, other subunit and associated factor of the uniporter complex, EMRE and MCUR1, respectively, are not affected in BTHS models. Consistent with the decrease in MICU1 levels, we show that the kinetics of MICU1-dependent mitochondrial calcium uptake is perturbed and acute stimulation of mitochondrial calcium signaling in BTHS myoblasts fails to activate pyruvate dehydrogenase, which in turn impairs the generation of reducing equivalents and blunts mitochondrial bioenergetics. Taken together, our findings suggest that defects in mitochondrial calcium signaling could contribute to cardiac and skeletal muscle pathologies observed in BTHS patients.

Eco-friendly synthesis of Ag-NPs using Endostemon viscosus (Lamiaceae): Antibacterial, antioxidant, larvicidal, photocatalytic dye degradation activity and toxicity in zebrafish embryos.

Nanotechnology is a multidisciplinary area of study that has grown significantly in serving many functions and impacting human society. New fields of science have been facilitated by the clean, non-toxic, and biocompatible nature of plant-derived nanoparticles. The present study deals with the first green synthesis of silver nanoparticles (Ag-NPs) using Endostemon viscosus, and their synthesized Ag NPs were characterized by different spectral methods (UV-vis Spectroscopy, Fourier Transform Infrared Spectroscopy (FTIR), X-ray diffraction Spectroscopy (XRD), Transmission Electron Microscopy (TEM) and Energy dispersive X-ray Spectroscopy (EDAX). The change initially observed the production of Ag-NPs in color from green to ash and then confirmed by SPR band at 435 nm in UV-vis spectral analysis. The FTIR findings indicate that many functional groups belong to the pharmaceutically useful phytochemicals, which interact as reducing, capping, and stabilizing agents in synthesizing silver nanoparticles. The predominant peaks in the XRD pattern belong to the planes 210°, 111°, 200°, 241°, and 311° and thus demonstrated the Ag-NPs FCC crystal structure. TEM analysis exhibited spherical-shaped particles with an average size of 13 nm, and the EDAX band showed a distinctive metallic silver peak at 3.0 keV. The antibacterial activity of Ag-NPs tested to show a maximum zone of inhibition of 19 mm for Staphylococcus aureus and 15 mm for Escherichia coli at 100 µg/mL, respectively. Bio-fabricated Ag-NPs were assessed for antioxidant activity (DPPH with % inhibition 57.54% and FRAP with % inhibition 70.89%). The biosynthesized Ag-NPs demonstrated potential larvicidal efficacy against Aedes aegypti with more than 90% at 250 µg/mL. Histological profiles were altered while treating with Ag-NPs at 250 µg/mL. The photocatalytic activity of synthesized E. viscosus Ag-NPs was tested against methylene blue (MB) and crystal violet (CV), and the maximum degradation efficiency was found as 90 and 94%, respectively. Furthermore, the toxicity test on zebrafish embryos demonstrated that aberrations have only been induced at concentrations higher than 500 µg/mL. We conclude that the greenly produced Ag-NPs may find use in biomedical applications based on bacteria and cost-effective industrial wastewater treatment.

Clinical, Microbiological Profile and Treatment Outcomes of Infants with BCG Adenitis: A Retrospective Study.

BACKGROUND: Bacille Calmette-Guérin (BCG) adenitis is an uncommon complication following BCG vaccination. In rare cases, infants can develop other complications. Controversy exists regarding the diagnosis and management of these cases. Not much information is available in literature regarding their microbiological and immunological characteristics. METHODS: Electronic medical records of children presenting to the Pediatric Infectious Diseases clinic in a tertiary care hospital from January 2011-December 2020 with a diagnosis of BCG adenitis were retrospectively reviewed. Their clinical, microbiological, treatment and follow-up data were noted and analyzed. FINDINGS: During the study period, 40 infants presented with a probable diagnosis of BCG adenitis with or without disseminated BCG. Median age at symptom onset was 4(2.5-5.9) months. Nine infants had disseminated disease at presentation. Fifteen infants were suspected to have underlying immune deficiency of whom 12 had proven defects in immune function. On multivariable logistic regression analysis, presence of disseminated disease was the only factor predictive of underlying immunodeficiency. Isoniazid monoresistance was seen in seven cases (32%) of the 22 samples sent for TB cultures. CONCLUSIONS: Though BCG adenitis runs a benign course, it could rarely be the first manifestation of an underlying immune defect. There is sizable isoniazid monoresistance, hence sending tissue samples for microbiologic evaluation is necessary to guide anti-tubercular therapy.

Identification of plasmids by PCR based replicon typing in bacteremic Klebsiella pneumoniae.

BACKGROUND: Klebsiella pneumoniae is a notorious pathogen with plasmid mediated resistance to all classes of antibiotics. It is important to determine the plasmid profile coding for resistance genes. Plasmid profile varies among geographical regions and tracking the types helps in determining the MDR and XDR K. pneumoniae spread especially in hospital setting. Aim of the present study was to determine the plasmid profile and types among bacteraemic K. pneumoniae. MATERIALS AND METHODS: Ninety consecutive K. pneumoniae collected over a period of three months from blood cultures were characterised by PCR for plasmid profile. Inc plasmid types were determined by PCR based replicon typing (PBRT) and carbapenemases were determined by multiplex PCR. For a subset of isolates hybrid assemblies were developed by sequencing with Ion Torrent and MinIon. RESULTS: Overall, PBRT showed 29% of isolates carried four plasmids including IncHI1B, IncFIA, IncFII(K) and IncR. The most common type of plasmid was IncHI1B (93%) followed by IncFIIK (89%) and IncR (82%). IncFIA was predominant among carbapenem resistant isolates. Almost all plasmids identified in K. pneumoniae were AMR plasmids, except two isolates which had virulence plasmids. IncX3 plasmid observed in this study was previously reported to be self-disseminating. Furthermore, the hybrid genome sequencing revealed complete structural arrangements of plasmids, which would be missed in short-read sequencing. NDM and OXA48-like were co-produced in 59% of the carbapenem resistant isolates. BlaOXA-232 was present on ColKP3; aac(6')-lb3 and rmtF on IncFIB. CONCLUSION: Diverse plasmid profile among the successive K. pneumoniae isolates indicates the transfer of resistance genes through different types of plasmids. IncHI1B, IncFIA, IncFIIK and IncR were the prevalent plasmid types. Hybrid assembly revealed blaOXA-232 was present on ColKP3 unlike global reports of IncL/M. Hybrid assemblies provide better plasmid structure that long and short read assemblies. There was no significant association of ß-lactamases with specific Inc groups in this study.

Larvicidal Enzyme Inhibition and Repellent Activity of Red Mangrove Rhizophora mucronata (Lam.) Leaf Extracts and Their Biomolecules Against Three Medically Challenging Arthropod Vectors.

The larvicidal potential of crude leaf extracts of Rhizophora mucronata, the red mangrove, using diverse solvent extracts of the plant against the early fourth instar larvae of Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti mosquito vectors was analyzed. The acetone extract of R. mucronata showed the greatest efficacy: for Cx. quinquefasciatus (LC50 = 0.13 mg/mL; LC90 = 2.84 mg/mL), An. stephensi (LC50 = 0.34 mg/mL; LC90 = 6.03 mg/mL), and Ae. aegypti (LC50 = 0.11 mg/mL; LC90 = 1.35 mg/mL). The acetone extract was further fractionated into four fractions and tested for its larvicidal activity. Fraction 3 showed stronger larvicidal activity against all the three mosquito larvae. Chemical characterization of the acetone extract displayed the existence of several identifiable compounds like phytol, 3,7,11,15-tetramethyl-2-hexadecen-1-ol, 1-hexyl-2-nitrocyclohexane, eicosanoic acid etc. Enzyme assay displayed that R. mucronata active F3-fractions exert divergent effects on all three mosquitos' biochemical defensive mechanisms. The plant fractions displayed significant repellent activity against all the three mosquito vectors up to the maximum repellent time of 210 min. Thus, the bioactive molecules in the acetone extract of R. murconata leaves showed significant larvicidal and enzyme inhibitory activity and displayed novel eco-friendly tool for mosquito control.

Rapidly disseminating blaOXA-232 carrying Klebsiella pneumoniae belonging to ST231 in India: multiple and varied mobile genetic elements.

BACKGROUND: Recently, in India, there has been a shift from NDM to OXA48-like carbapenemases. OXA-181 and OXA-232 are the frequently produced variants of OXA48-like carbapenemases. OXA48-like carbapenemases are also known to be carried on transposons such as Tn1999, Tn1999.2 and it is also associated with IS1R carried on Tn1999. In India, there are no previous reports studying the association of mobile genetic elements (MGEs) with OXA48-like carbapenemases. The present study was aimed at determining the genetic backbone of OXA48-like carbapenemases to determine the role of MGEs in its transfer and to investigate the Inc plasmid type carrying blaOXA48-like. RESULTS: A total of 49 carbapenem resistant K. pneumoniae which included 25 isolates from South India and 24 isolates from North India, were included in the study. Whole genome sequencing using Ion Torrent PGM was performed to study the isolates. OXA-232 was present in 35 isolates (71%). In 19 isolates (39%), blaOXA48-like was associated with MGEs. Insertion sequences such as ISX4, IS1, IS3, ISKpn1, ISKpn26, ISKpn25, ISSpu2, ISKox1, IS4321R, ISEc36, and ISPa38; and transposons such as TnAs3 and Tn2, were present. Isolates from northern and southern India belonging to same sequence type (ST) had diverse genetic backbone for blaOXA48-like. ST14 isolates from north had IS5 and Tn3 families while from south they had IS1, IS5 and IS630 families. ST231 from north had IS5, IS6 and Tn3 families with blaOXA-232 while from south, IS1, IS3 and IS5 families were observed; with ISKpn26 being present among isolates from both the regions. blaOXA48-like was predominantly found on ColKP3 plasmid. ST231 was the predominant ST in 22 isolates (45%). CONCLUSION: OXA-232 is the predominant variant of OXA48-like carbapenemase with ST231 being the commonest ST of OXA48-like carbapenemase producing K. pneumoniae in India. Diverse MGEs have been associated with both blaOXA-232 and blaOXA-181 which contribute to their spread. The MGEs in the present study are different from those reported earlier. There is no clonal expansion of blaOXA48-like producing K. pneumoniae since diverse STs were observed. Monitoring the genetic backbone of OXA48-like carbapenemase is essential to better understand the transmission dynamics of XDR K. pneumoniae.

Molecular characterization of colistin-resistant Klebsiella pneumoniae & its clonal relationship among Indian isolates.

Background & objectives: Klebsiella pneumoniae (KP), a common cause of invasive infections, is often extensively drug resistant in India. At present, studies on resistance mechanism and clonal relationship of KP from India are limited. The present study was undertaken to determine the resistance mechanism and clonal relationship of colistin-resistant isolates obtained from various specimens. Carbapenemases were also determined since the isolates were carbapenem resistant. Methods: Sixty five isolates from blood, exudates and respiratory specimens collected between 2016 and 2017 were studied. Colistin minimum inhibitory concentration (MIC) was performed by broth-micro dilution method. Multiplex PCR was carried out to determine carbapenemases. Targeted sequencing was performed to determine mutations in mgrB, phoP, phoQ and multilocus sequence typing was performed to determine the prevalent clones. Results: Colistin MIC ranged from 4 to 256 µg/ml. SHV, TEM and CTX-M were co-produced in 60 per cent and OXA48-like in 71 per cent. Thirteen isolates had mutations in mgrB. Mutations included a premature stop codon at 21st amino acid, the presence of insertion sequences such as IS903, IS Kpn 14 and ISK pn 26; and elongation of mgrB. Novel mutations were also observed among phoP and phoQ genes. Colistin resistance due to mcr genes was absent. Fifteen clonal types were seen with ST231, ST14 and ST2096 being predominant. Interpretation & conclusions: This study revealed the changing trend of carbapenem resistance mechanism predominantly to OXA48-like from NDM. Known mgrB mutations and novel mutations in phoP and phoQ were detected. There was no plasmid-mediated colistin resistance. ST14 and ST231 were international clones associated with carbapenem resistance. Colistin-resistant KP was of diverse clones with predominantly ST231, ST14 and ST2096.

Rapid Detection of M. tuberculosis and Its Resistance to Rifampicin and Isoniazid with the mfloDx™ MDR-TB test.

BACKGROUND: Rapid detection of tuberculosis (TB) and its resistance are essential for the prompt initiation of correct drug therapy and for stopping the spread of drug-resistant TB. There is an urgent need for increased use of rapid diagnostic tests to control the threat of increased TB and multidrug-resistant TB (MDR-TB). METHODS: EMPE Diagnostics has developed a multiplex molecular diagnostic platform called mfloDx™ by combining nucleotide-specific padlock probe-dependent rolling circle amplification with sensitive lateral flow biosensors, providing visual signals, similar to a COVID-19 test. The first test kit of this platform, mfloDx™ MDR-TB can identify Mycobacterium tuberculosis (MTB) complex and its clinically significant mutations in the rpoB and katG genes and in the inhA promotor contributing resistance to rifampicin (RIF) and isoniazid (INH), causing MDR-TB. RESULTS: We have evaluated the performance of the mfloDx™ MDR-TB test on 210 sputum samples (110 from suspected TB cases and 100 from TB-negative controls) received from a tertiary care center in India. The clinical sensitivity for detecting MTB compared to acid-fast microscopy and mycobacteria growth indicator tube (MGIT) cultures was 86.4% and 84.9%, respectively. All the 100 control samples were negative indicating excellent specificity. In smear-positive sputum samples, the mfloDx™ MDR-TB test showed a sensitivity of 92.5% and 86.4% against MGIT culture and Xpert MTB/RIF, respectively. The clinical sensitivity for the detection of RIF and INH resistance in comparison with MGIT drug susceptibility testing was 100% and 84.6%, respectively, while the clinical specificity was 100%. CONCLUSION: From the above evaluation, we find mfloDx™ MDR-TB to be a rapid and efficient test to detect TB and its multidrug resistance in 3 h at a low cost making it suitable for resource-limited laboratories.

Modulation of the mitochondrial Ca2+ uniporter complex subunit expression by different shear stress patterns in vascular endothelial cells.

Mitochondrial calcium (m Ca2+ ) uptake occurs via the Mitochondrial Ca2+ Uniporter (MCU) complex and plays a critical role in mitochondrial dynamics, mitophagy, and apoptosis. MCU complex activity is in part modulated by the expression of its regulatory subunits. Cardiovascular disease models demonstrated altered gene/protein expression of one or multiple subunits in different cells, including vascular endothelial cells (ECs). MCU complex activity was found necessary for stable flow (s-flow)-induced mitophagy and promotion of an atheroprotective EC phenotype. Disturbed flow (d-flow) is known to lead to an atheroprone phenotype. Despite the role of MCU in flow-regulated EC function, flow-induced alterations in MCU complex subunit expression are currently unknown. We exposed cultured human ECs to atheroprotective (steady shear stress, SS) or atheroprone flow (oscillatory shear stress, OS) and measured mRNA and protein levels of the MCU complex members. SS and OS differentially modulated subunit expression at gene/protein levels. Protein expression changes of the core MCU, m Ca2+ uptake 1 (MICU1) and MCU regulator 1 (MCUR1) subunits in SS- and OS-exposed, compared to static, ECs suggested an enhanced m Ca2+ influx under each flow and a potential contribution to EC dysfunction under OS. In silico analysis of a single-cell RNA-sequencing dataset was employed to extract transcript values of MCU subunits in mouse carotid ECs from regions exposed to s-flow or d-flow. Mcu and Mcur1 genes showed significant differences in expression after prolonged exposure to each flow. The differential expression of MCU complex subunits indicated a tight regulation of the complex activity under physiological and pathological hemodynamic conditions.

Clinical features of human tuberculosis due to Mycobacterium orygis in Southern India.

Mycobacterium orygis is a member of the Mycobacterium tuberculosis complex (MTBC) and causes tuberculosis in a variety of animals, including humans in South Asia. Here, we describe the clinical features associated with 8 human cases of whole genome sequence (WGS) confirmed M. orygis from a tertiary care hospital in South India during 2018-2019. The patient ages ranged from 9 to 51 years, with 5 females and 3 males included. All the patients had extrapulmonary disease with 2 having concomitant pulmonary involvement. Clinical improvement was documented after a full course of anti-tuberculosis therapy in 6 cases for whom follow-up was available. Taken together, the results show that M. orygis causes human tuberculosis in India, with a predominant extrapulmonary disease. Standardized molecular assays of this emerging member of the MTBC are needed to provide further information on the frequency of M. orygis infection in India and other countries where it is found in livestock and domestic wildlife.

Whole genome analysis of Rhizopus species causing rhino-cerebral mucormycosis during the COVID-19 pandemic.

Introduction: Mucormycosis is an acute invasive fungal disease (IFD) seen mainly in immunocompromised hosts and in patients with uncontrolled diabetes. The incidence of mucormycosis increased exponentially in India during the SARS-CoV-2 (henceforth COVID-19) pandemic. Since there was a lack of data on molecular epidemiology of Mucorales causing IFD during and after the COVID-19 pandemic, whole genome analysis of the Rhizopus spp. isolated during this period was studied along with the detection of mutations that are associated with antifungal drug resistance. Materials and methods: A total of 50 isolates of Rhizopus spp. were included in this prospective study, which included 28 from patients with active COVID-19 disease, 9 from patients during the recovery phase, and 13 isolates from COVID-19-negative patients. Whole genome sequencing (WGS) was performed for the isolates, and the de novo assembly was done with the Spades assembler. Species identification was done by extracting the ITS gene sequence from each isolate followed by searching Nucleotide BLAST. The phylogenetic trees were made with extracted ITS gene sequences and 12 eukaryotic core marker gene sequences, respectively, to assess the genetic distance between our isolates. Mutations associated with intrinsic drug resistance to fluconazole and voriconazole were analyzed. Results: All 50 patients presented to the hospital with acute fungal rhinosinusitis. These patients had a mean HbA1c of 11.2%, and a serum ferritin of 546.8 ng/mL. Twenty-five patients had received steroids. By WGS analysis, 62% of the Rhizopus species were identified as R. delemar. Bayesian analysis of population structure (BAPS) clustering categorized these isolates into five different groups, of which 28 belong to group 3, 9 to group 5, and 8 to group 1. Mutational analysis revealed that in the CYP51A gene, 50% of our isolates had frameshift mutations along with 7 synonymous mutations and 46% had only synonymous mutations, whereas in the CYP51B gene, 68% had only synonymous mutations and 26% did not have any mutations. Conclusion: WGS analysis of Mucorales identified during and after the COVID-19 pandemic gives insight into the molecular epidemiology of these isolates in our community and establishes newer mechanisms for intrinsic azole resistance.

Mitochondrial calcium signaling mediated transcriptional regulation of keratin filaments is a critical determinant of melanogenesis.

Mitochondria are versatile organelles that regulate several physiological functions. Many mitochondria-controlled processes are driven by mitochondrial Ca2+ signaling. However, role of mitochondrial Ca2+ signaling in melanosome biology remains unknown. Here, we show that pigmentation requires mitochondrial Ca2+ uptake. In vitro gain and loss of function studies demonstrated that Mitochondrial Ca2+ Uniporter (MCU) is crucial for melanogenesis while the MCU rheostats, MCUb and MICU1 negatively control melanogenesis. Zebrafish and mouse models showed that MCU plays a vital role in pigmentation in vivo. Mechanistically, MCU controls activation of transcription factor NFAT2 to induce expression of three keratins (keratin 5, 7 and 8), which we report as positive regulators of melanogenesis. Interestingly, keratin 5 in turn modulates mitochondrial Ca2+ uptake thereby this signaling module acts as a negative feedback loop that fine-tunes both mitochondrial Ca2+ signaling and melanogenesis. Mitoxantrone, an FDA approved drug that inhibits MCU, decreases physiological melanogenesis. Collectively, our data demonstrates a critical role for mitochondrial Ca2+ signaling in vertebrate pigmentation and reveal the therapeutic potential of targeting MCU for clinical management of pigmentary disorders. Given the centrality of mitochondrial Ca2+ signaling and keratin filaments in cellular physiology, this feedback loop may be functional in a variety of other pathophysiological conditions.

Limiting Mrs2-dependent mitochondrial Mg2+ uptake induces metabolic programming in prolonged dietary stress.

The most abundant cellular divalent cations, Mg2+ (mM) and Ca2+ (nM-µM), antagonistically regulate divergent metabolic pathways with several orders of magnitude affinity preference, but the physiological significance of this competition remains elusive. In mice consuming a Western diet, genetic ablation of the mitochondrial Mg2+ channel Mrs2 prevents weight gain, enhances mitochondrial activity, decreases fat accumulation in the liver, and causes prominent browning of white adipose. Mrs2 deficiency restrains citrate efflux from the mitochondria, making it unavailable to support de novo lipogenesis. As citrate is an endogenous Mg2+ chelator, this may represent an adaptive response to a perceived deficit of the cation. Transcriptional profiling of liver and white adipose reveals higher expression of genes involved in glycolysis, ß-oxidation, thermogenesis, and HIF-1α-targets, in Mrs2-/- mice that are further enhanced under Western-diet-associated metabolic stress. Thus, lowering mMg2+ promotes metabolism and dampens diet-induced obesity and metabolic syndrome.

Myrtenal attenuates oxidative stress and inflammation in a rat model of streptozotocin-induced diabetes.

The present study was aimed to investigate the effect of myrtenal on diabetes-associated oxidative stress, lipid peroxidation (LPO), and inflammation using a rat model of streptozotocin (STZ)-induced diabetes. Following the induction of diabetes in male Wistar rats using STZ (40 mg/kg body weight), myrtenal (80 mg/kg body weight) was administered orally to diabetic rats for four weeks and then sacrificed to harvest tissues. We measured the levels of antioxidants, LPO, and proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6), and the p65 subunit of nuclear factor-kappa B (NF-kB p65). Diabetic rats revealed increased levels of LPO, proinflammatory cytokines, and NF-kB p65, and decreased levels of antioxidants in the liver and pancreas. Supplementation with myrtenal significantly attenuated the diabetes-induced changes in the liver and pancreas of diabetic rats. Our findings suggest that myrtenal may serve as an antioxidant and anti-inflammatory agent against diabetes-associated oxidative stress and inflammation.HighlightsOral administration of myrtenal improved the antioxidant status in the liver and pancreas of diabetic rats.Myrtenal treatment diminished inflammation in the liver and pancreas of diabetic rats.Myrtenal supplementation averts oxidative stress and inflammation in diabetic rats.Myrtenal could serve as a potent antioxidant and anti-inflammatory agent in the management of diabetes.

The mitochondrial Ca2+ uniporter channel synergizes with fluid shear stress to induce mitochondrial Ca2+ oscillations.

The mitochondrial calcium (Ca2+) uniporter (MCU) channel is responsible for mitochondrial Ca2+ influx. Its expression was found to be upregulated in endothelial cells (ECs) under cardiovascular disease conditions. Since the role of MCU in regulating cytosolic Ca2+ homeostasis in ECs exposed to shear stress (SS) is unknown, we studied mitochondrial Ca2+ dynamics (that is known to decode cytosolic Ca2+ signaling) in sheared ECs. To understand cause-and-effect, we ectopically expressed MCU in ECs. A higher percentage of MCU-transduced ECs exhibited mitochondrial Ca2+ transients/oscillations, and at higher frequency, under SS compared to sheared control ECs. Transients/oscillations correlated with mitochondrial reactive oxygen species (mROS) flashes and mitochondrial membrane potential (ΔΨm) flickers, and depended on activation of the mechanosensitive Piezo1 channel and the endothelial nitric oxide synthase (eNOS). A positive feedback loop composed of mitochondrial Ca2+ uptake/mROS flashes/ΔΨm flickers and endoplasmic reticulum Ca2+ release, in association with Piezo1 and eNOS, provided insights into the mechanism by which SS, under conditions of high MCU activity, may shape vascular EC energetics and function.

Pulmonary nocardiosis: Risk factors and species distribution from a high burden centre.

Nocardiosis is a clinical and diagnostic challenge. This was a retrospective study carried out on cases of pulmonary nocardiosis presenting over 15 years. Clinical data was retrieved using the electronic patient records. Vitek MS 3.2 (MALDI TOF MS) was carried out on 22 isolates and sequencing on another 9 isolates. Of 71 patients presenting with pulmonary nocardiosis, 58 (81.6%) were on immunosuppressant therapy, 26 (46%) had a previous lung pathology, 11 (8%) were HIV associated. Disseminated disease was seen in 6 (8.4%). There were 8 (11.26%) deaths in this cohort of patients. Of 31/71 identified to species, the most common were Nocardia cyriacigeorgica (n â€‹= â€‹11) followed by Nocardia farcinica (n â€‹= â€‹9).

Oxytocin promotes epicardial cell activation and heart regeneration after cardiac injury.

Cardiovascular disease (CVD) is one of the leading causes of mortality worldwide, and frequently leads to massive heart injury and the loss of billions of cardiac muscle cells and associated vasculature. Critical work in the last 2 decades demonstrated that these lost cells can be partially regenerated by the epicardium, the outermost mesothelial layer of the heart, in a process that highly recapitulates its role in heart development. Upon cardiac injury, mature epicardial cells activate and undergo an epithelial-mesenchymal transition (EMT) to form epicardium-derived progenitor cells (EpiPCs), multipotent progenitors that can differentiate into several important cardiac lineages, including cardiomyocytes and vascular cells. In mammals, this process alone is insufficient for significant regeneration, but it might be possible to prime it by administering specific reprogramming factors, leading to enhanced EpiPC function. Here, we show that oxytocin (OXT), a hypothalamic neuroendocrine peptide, induces epicardial cell proliferation, EMT, and transcriptional activity in a model of human induced pluripotent stem cell (hiPSC)-derived epicardial cells. In addition, we demonstrate that OXT is produced after cardiac cryoinjury in zebrafish, and that it elicits significant epicardial activation promoting heart regeneration. Oxytocin signaling is also critical for proper epicardium development in zebrafish embryos. The above processes are significantly impaired when OXT signaling is inhibited chemically or genetically through RNA interference. RNA sequencing data suggests that the transforming growth factor beta (TGF-ß) pathway is the primary mediator of OXT-induced epicardial activation. Our research reveals for the first time an evolutionary conserved brain-controlled mechanism inducing cellular reprogramming and regeneration of the injured mammalian and zebrafish heart, a finding that could contribute to translational advances for the treatment of cardiac injuries.

SARS-CoV-2 infection enhances mitochondrial PTP complex activity to perturb cardiac energetics.

SARS-CoV-2 is a newly identified coronavirus that causes the respiratory disease called coronavirus disease 2019 (COVID-19). With an urgent need for therapeutics, we lack a full understanding of the molecular basis of SARS-CoV-2-induced cellular damage and disease progression. Here, we conducted transcriptomic analysis of human PBMCs, identified significant changes in mitochondrial, ion channel, and protein quality-control gene products. SARS-CoV-2 proteins selectively target cellular organelle compartments, including the endoplasmic reticulum and mitochondria. M-protein, NSP6, ORF3A, ORF9C, and ORF10 bind to mitochondrial PTP complex components cyclophilin D, SPG-7, ANT, ATP synthase, and a previously undescribed CCDC58 (coiled-coil domain containing protein 58). Knockdown of CCDC58 or mPTP blocker cyclosporin A pretreatment enhances mitochondrial Ca2+ retention capacity and bioenergetics. SARS-CoV-2 infection exacerbates cardiomyocyte autophagy and promotes cell death that was suppressed by cyclosporin A treatment. Our findings reveal that SARS-CoV-2 viral proteins suppress cardiomyocyte mitochondrial function that disrupts cardiomyocyte Ca2+ cycling and cell viability.

Development of a Multiplex Real-Time PCR Assay for Mycobacterium bovis BCG and Validation in a Clinical Laboratory.

Mycobacterium bovis bacillus Calmette-Guérin (BCG) is a live attenuated vaccine which can result in local or disseminated infection, most commonly in immunocompromised individuals. Differentiation of BCG from other members of the Mycobacterium tuberculosis complex (MTBC) is required to diagnose BCG disease, which requires specific management. Current methods for BCG diagnosis are based on mycobacterial culture and conventional PCR; the former is time-consuming and the latter often unavailable. Further, there are reports that certain BCG strains may be associated with a higher rate of adverse events. This study describes the development of a two-step multiplex real-time PCR assay which uses single nucleotide polymorphisms to detect BCG and identify early or late BCG strains. The assay has a limit of detection of 1 pg BCG boiled lysate DNA and was shown to detect BCG in both pure cultures and experimentally infected tissue. Its performance was assessed on 19 suspected BCG clinical isolates at Christian Medical College in Vellore, India, taken from January 2018 to August 2020. Of these 19 isolates, 10 were identified as BCG (6 early and 4 late strains), and 9 were identified as other MTBC members. Taken together, the results demonstrate the ability of this assay to identify and characterize BCG disease from cultures and infected tissue. The capacity to identify BCG may improve patient management, and the ability to discriminate between BCG strains may enable BCG vaccine pharmacovigilance. IMPORTANCE Vaccination against tuberculosis with bacillus Calmette-Guérin (BCG) can lead to adverse events, including a rare but life-threatening complication of disseminated BCG. This complication often occurs in young children with immunodeficiencies and is associated with an ∼60% mortality rate. A rapid method of reliably identifying BCG infection is important because BCG requires treatment unique to tuberculosis. BCG is resistant to the first-line antituberculosis drug pyrazinamide. Additionally, diagnosis of BCG disease would lead to further investigation of a possible underlying immune condition. We have developed a diagnostic assay to identify BCG which improves upon previously published methods and can reliably identify BCG from bacterial culture or directly from infected tissue. This assay can also differentiate between strains of BCG, which have been suggested to be associated with different rates of adverse events. This assay was validated on 19 clinical isolates collected at Christian Medical College in Vellore, India.