Mitochondrial Ca2+ Uniporter Is a Mitochondrial Luminal Redox Sensor that Augments MCU Channel Activity.

Ca2+ dynamics and oxidative signaling are fundamental mechanisms for mitochondrial bioenergetics and cell function. The MCU complex is the major pathway by which these signals are integrated in mitochondria. Whether and how these coactive elements interact with MCU have not been established. As an approach toward understanding the regulation of MCU channel by oxidative milieu, we adapted inflammatory and hypoxia models. We identified the conserved cysteine 97 (Cys-97) to be the only reactive thiol in human MCU that undergoes S-glutathionylation. Furthermore, biochemical, structural, and superresolution imaging analysis revealed that MCU oxidation promotes MCU higher order oligomer formation. Both oxidation and mutation of MCU Cys-97 exhibited persistent MCU channel activity with higher [Ca2+]m uptake rate, elevated mROS, and enhanced [Ca2+]m overload-induced cell death. In contrast, these effects were largely independent of MCU interaction with its regulators. These findings reveal a distinct functional role for Cys-97 in ROS sensing and regulation of MCU activity.

Iron alters the cell wall composition and intracellular lactate to affect Candida albicans susceptibility to antifungals and host immune response.

Fungal pathogen Candida albicans has a complex cell wall consisting of an outer layer of mannans and an inner layer of ß-glucans and chitin. The fungal cell wall is the primary target for antifungals and is recognized by host immune cells. Environmental conditions such as carbon sources, pH, temperature, and oxygen tension can modulate the fungal cell wall architecture. Cellular signaling pathways, including the mitogen-activated protein kinase (MAPK) pathways, are responsible for sensing environmental cues and mediating cell wall alterations. Although iron has recently been shown to affect ß-1,3-glucan exposure on the cell wall, we report here that iron changes the composition of all major C. albicans cell wall components. Specifically, high iron decreased the levels of mannans (including phosphomannans) and chitin; and increased ß-1,3-glucan levels. These changes increased the resistance of C. albicans to cell wall-perturbing antifungals. Moreover, high iron cells exhibited adequate mitochondrial functioning; leading to a reduction in accumulation of lactate that signals through the transcription factor Crz1 to induce ß-1,3-glucan masking in C. albicans We show here that iron-induced changes in ß-1,3-glucan exposure are lactate-dependent; and high iron causes ß-1,3-glucan exposure by preventing lactate-induced, Crz1-mediated inhibition of activation of the fungal MAPK Cek1. Furthermore, despite exhibiting enhanced antifungal resistance, high iron C. albicans cells had reduced survival upon phagocytosis by macrophages. Our results underscore the role of iron as an environmental signal in multiple signaling pathways that alter cell wall architecture in C. albicans, thereby affecting its survival upon exposure to antifungals and host immune response.

Helicobacter pylori cagL amino acid polymorphisms and its association with gastroduodenal diseases.

CagL is a pilus protein of Helicobacter pylori that interacts with host cellular α5ß1 integrins through its arginine-glycine-aspartate (RGD) motif, guiding proper positioning of the T4SS and translocation of CagA. Deletion or sequence variations of cagL significantly diminished the ability of H. pylori to induce secretion of IL-8 by the host cell. Therefore, this study was undertaken to investigate the association of cagL and its amino acid sequence polymorphisms with gastric cancer (GC), peptic ulcer disease (PUD), and non-ulcer dyspepsia (NUD) as there are no such studies from India. In total, 200 adult patients (NUD 120, PUD 30, GC 50) who underwent an upper gastrointestinal endoscopy were enrolled. H. pylori infection was diagnosed by rapid urease test, culture, histopathology, and PCR. The collected isolates were screened for cagL genotype by PCR and assessed for amino acid sequence polymorphisms using sequence translation. The prevalence of H. pylori infection in study population was 52.5%. Most of the isolates were cagL genopositive (86.6%), and all had RGD motif in their amino acid sequences. D58 and K59 polymorphisms in cagL-genopositive strains were significantly higher in GC patients (P < 0.05). Combined D58K59 polymorphism was associated with higher risk of GC (3.8-fold) when compared to NUD. In conclusion, H. pylori cagL amino acid polymorphisms such as D58K59 are correlated with a higher risk of GC in the Indian population. Further studies are required to know the exact role of particular cagL amino acid polymorphisms in the pathogenicity of H. pylori infection.

Rapid visual detection of Mycobacterium tuberculosis DNA using gold nanoparticles.

Tuberculosis (TB) is one of the world's deadliest infections caused by Mycobacterium tuberculosis (MTB). Though curable, the disease goes undetected in early stages owing to the lack of rapid, simple, cost-effective, and sensitive detection methods. In this investigation, we describe a procedure which is superior, more sensitive, and easier to handle, as compared to the previously reported, nanoparticle-based visual colorimetric assays for rapid detection of TB DNA, after its PCR amplification. This assay employs plasmonic gold nanoparticles (GNP) as a colorimetric agent and ethanol to promote aggregation of GNPs, thereby specifically detecting the amplified MTB DNA. An unambiguous response was achieved within 3 min after adding the DNA amplicon to the reaction tube. This conclusion was supported by spectroscopic data. The assay is sensitive up to ∼340 femtomole levels of MTB DNA, which was amplified using 0.125 ng µL-1 of the MTB DNA template. Thus, the technique developed here may be employed as a sensitive screening tool for early diagnosis of TB infection and is valuable for low-resource settings in remote areas, because of its simplicity. This ethanol-based visual TB DNA detection method is more sensitive, and fool-proof as compared to the commonly used salt-based colorimetric TB DNA assays, to the best of our knowledge.

Analytical methods for 25-hydroxyvitamin D: advantages and limitations of the existing assays.

Vitamin D plays an important role in the absorption of calcium and phosphorus, bone metabolism. Various analytical methods are being used to measure the circulating vitamin D levels in the blood. Among all vitamin D metabolites, 25 hydroxyvitamin (25(OH) D) is the most well-known member of the vitamin D family. It is available in two forms, viz. 25 hydroxyvitamin D3 (25(OH)D3) and 25 hydroxyvitamin D2(25(OH)D2). These are stable and available in ample amounts in serum and hence are the most commonly measured during estimation of vitamin D. Although, immunoassay was the most common analytical technique, it could not differentiate between 25(OH) D2 and 25(OH) D3. HPLC is another low-cost technique but lacks sufficient sensitivity for determining trace amounts of 25(OH) D2 and D3. Further, more advanced techniques like liquid chromatography-mass spectrometry have been developed to provide better specificity, higher degree of separation and identification of different forms of vitamin D. However, this technique is not very user friendly. Thus, standardization of these analytical methods in clinical and research laboratories is essential for developing and implementing evidence based clinical guidelines. This will also enable achievement of comparable results via different methods, based on diverse principles. In this manuscript, we have presented a comprehensive overview of the efforts for standardization in vitamin D assessment, along with latest advances in methods used for 25(OH) D2 and 25(OH) D3 analysis. Finally, a summary of clinical trials associated with vitamin D has been presented to discuss all the analytical methods employed for measuring 25(OH) D2 and D3.

High iron-mediated increased oral fungal burden, oral-to-gut transmission, and changes to pathogenicity of Candida albicans in oropharyngeal candidiasis.

BACKGROUND: Iron affects the diversity of the oral microbial landscape. Laboratory-strain CAI4 of Candida albicans that causes oropharyngeal candidiasis (OPC) exhibits iron-induced changes to the cell wall, impacting phagocytosis (by macrophages) and susceptibility of fungal cells to cell wall-perturbing antifungals, in vitro. AIM: To understand the effect of iron on the CAI4-strain, wild type (WT) SC5314-strain, and oral isolates of C. albicans. METHODS: An immunosuppressed murine model of OPC was used to assess the effect of iron on oral-to-gut infection and antifungal susceptibility of the CAI4-strain. In vitro antifungal susceptibility, cell wall analysis, and phagocytic assays were performed under low and high iron, for the SC5314-strain and oral isolates. RESULTS: High iron enhanced oral and gut fungal levels for the CAI4-strain in mice; CAI4 cells from low iron mice were more susceptible to antifungals. The SC5314-strain and oral isolates showed enhanced antifungal-resistance towards most antifungals tested, under high iron. Iron-mediated cell wall changes and phagocytic response in the SC5315-strain were similar to CAI4; oral isolates showed a variable response. CONCLUSION: Host iron can potentially alter infection severity and dissemination, efficacy of antifungal treatment, and host immune response during OPC. Clinical isolates showed most of these effects of iron, despite exhibiting a varied cell wall composition-change response to iron.

Antifungal Effect of Piezoelectric Charges on PMMA Dentures.

Candida-associated denture stomatitis is a recurring disease affecting up to 67% of denture wearers. Poly(methyl methacrylate) (PMMA) remains the main material employed in the fabrication of dentures due to its desirable physical, mechanical, and aesthetic properties. However, the improvement of its antimicrobial properties remains a challenge. To address this need, we developed PMMA composite filled with piezoelectric nanoparticles of barium titanate (BaTiO3) for therapeutic effects. Candida albicans biofilms were cultivated on the surface of the composites under continuous cyclic mechanical loading to activate the piezoelectric charges and to resemble mastication patterns. The interactions between biofilms and biomaterials were evaluated by measuring the biofilm biomass, metabolic activity, and the number of viable cells. To explore the antifungal mechanisms, changes in the expression of genes encoding adhesins and superoxide dismutase were assessed using reverse transcription-polymerase chain reaction. With the addition of piezoelectric nanoparticles, we observed a significant reduction in the biofilm formation and interference in the yeast-to-hyphae transition compared to the standard PMMA. Moreover, we observed that the cyclic deformation of biomaterial surfaces without antifungal agents produced increased biomass, metabolic activity, and a number of viable cells compared to the static/no-deformed surfaces. Cyclic deformation appears to be a novel mechanobiological signal that enables pathogenicity and virulence of C. albicans cells with increased expression of the yeast-to-hyphae transition genes. The outcome of this study opens new opportunities for the design of antifungal dentures for improved clinical service and reduced need for cleaning methods.

Blockade of MCU-Mediated Ca2+ Uptake Perturbs Lipid Metabolism via PP4-Dependent AMPK Dephosphorylation.

Mitochondrial Ca2+ uniporter (MCU)-mediated Ca2+ uptake promotes the buildup of reducing equivalents that fuel oxidative phosphorylation for cellular metabolism. Although MCU modulates mitochondrial bioenergetics, its function in energy homeostasis in vivo remains elusive. Here we demonstrate that deletion of the Mcu gene in mouse liver (MCUΔhep) and in Danio rerio by CRISPR/Cas9 inhibits mitochondrial Ca2+ (mCa2+) uptake, delays cytosolic Ca2+ (cCa2+) clearance, reduces oxidative phosphorylation, and leads to increased lipid accumulation. Elevated hepatic lipids in MCUΔhep were a direct result of extramitochondrial Ca2+-dependent protein phosphatase-4 (PP4) activity, which dephosphorylates AMPK. Loss of AMPK recapitulates hepatic lipid accumulation without changes in MCU-mediated Ca2+ uptake. Furthermore, reconstitution of active AMPK, or PP4 knockdown, enhances lipid clearance in MCUΔhep hepatocytes. Conversely, gain-of-function MCU promotes rapid mCa2+ uptake, decreases PP4 levels, and reduces hepatic lipid accumulation. Thus, our work uncovers an MCU/PP4/AMPK molecular cascade that links Ca2+ dynamics to hepatic lipid metabolism.

MIRO-1 Determines Mitochondrial Shape Transition upon GPCR Activation and Ca2+ Stress.

Mitochondria shape cytosolic calcium ([Ca2+]c) transients and utilize the mitochondrial Ca2+ ([Ca2+]m) in exchange for bioenergetics output. Conversely, dysregulated [Ca2+]c causes [Ca2+]m overload and induces permeability transition pore and cell death. Ablation of MCU-mediated Ca2+ uptake exhibited elevated [Ca2+]c and failed to prevent stress-induced cell death. The mechanisms for these effects remain elusive. Here, we report that mitochondria undergo a cytosolic Ca2+-induced shape change that is distinct from mitochondrial fission and swelling. [Ca2+]c elevation, but not MCU-mediated Ca2+ uptake, appears to be essential for the process we term mitochondrial shape transition (MiST). MiST is mediated by the mitochondrial protein Miro1 through its EF-hand domain 1 in multiple cell types. Moreover, Ca2+-dependent disruption of Miro1/KIF5B/tubulin complex is determined by Miro1 EF1 domain. Functionally, Miro1-dependent MiST is essential for autophagy/mitophagy that is attenuated in Miro1 EF1 mutants. Thus, Miro1 is a cytosolic Ca2+ sensor that decodes metazoan Ca2+ signals as MiST.

Increasing Trend of Heterogeneous Vancomycin Intermediate Staphylococcus aureus in a Tertiary Care Center of Northern India.

Heterogeneous vancomycin intermediate Staphylococcus aureus (hVISA) strains are increasingly reported, and their association with vancomycin treatment failure is a well-known problem worldwide. A total of 500 clinical isolates of methicillin-resistant S. aureus were screened for hVISA by four different methods from May 2011 to May 2014. The number of strains screened for hVISA from May to April in 2011-2012, 2012-2013, and 2013-2014 were 198, 123, and 179, respectively. hVISA strains were studied by transmission electron microscopy (TEM) for cell wall thickness and also for their ability to form biofilm on a polystyrene microtiter plate. hVISA strains detected by four different methods-brain heart infusion agar with vancomycin with 4 mg/L/gradient plate/macro E-test/and glycopeptide resistance detection (GRD) E test-were as follows: 11.6%/10%/9%, and 9.5% in 2011-2012, 12.1%/9.7%/8.9%, and 10.5% in 2012-2013, and 13.9%/11.7%/11.1%, and 12.8% in 2013-2014, respectively. Population analysis profile-area under curve analysis confirmed hVISA in 4.5% (9/198), 6.5% (8/123), and 6.7% (12/179) in respective years; 24% (7/29) of hVISA isolates were nonsusceptible to daptomycin. TEM showed a significant increase in cell wall thickness of hVISA isolates (p<0.001) with a distinct reduction in their biofilm formation ability.

Glycopeptide and daptomycin susceptibility trends among clinical isolates of methicillin-resistant Staphylococcus aureus in a tertiary care center in North India.

Increased vancomycin minimum inhibitory concentration (MIC) levels in Staphylococcus aureus and their association with vancomycin treatment failure are well-known problems. Few studies have recognized progressive increases in glycopeptide MIC levels for S. aureus strains in recent years. This study determined glycopeptide and daptomycin susceptibility among methicillin resistant S. aureus (MRSA) strains. A total of 776 clinical isolates of MRSA recovered from 2009 to 2012 were studied for glycopeptide and daptomycin susceptibility using the E-test method. The vancomycin MIC geometric mean (GM) of the MRSA isolates was 0.923, 0.944, 1.134 and 1.294 mg/L in the years 2009, 2010, 2011 and 2012, respectively, and the trend significantly increased over the years (P < 0.0001). Similarly, the teicoplanin MIC GM was 1.47, 1.49, 1.8 and 2.04 mg/L in the years from 2009 to 2012, respectively (P < 0.0001). MIC shifts were not found for daptomycin (P > 0.232). A significant increase in the MIC for glycopeptides was observed among the clinical MRSA isolates at our center over a 4-year period. However, the daptomycin MIC did not increase in the observed MRSA isolates.

Prevalence and molecular characterisation of New Delhi metallo-ß-lactamases NDM-1, NDM-5, NDM-6 and NDM-7 in multidrug-resistant Enterobacteriaceae from India.

The growing prevalence of carbapenem resistance in Enterobacteriaceae worldwide is a major concern. New Delhi metallo-ß-lactamase (NDM)-mediated carbapenem resistance has been identified in Enterobacteriaceae from numerous countries including those of the Indian subcontinent. Currently, seven NDM ß-lactamase variants (NDM-1 to -7) have been identified. This study evaluated the detection and molecular characterisation of NDM variants in Enterobacteriaceae at a tertiary care hospital in India. A total of 464 isolates were tested; 57 (12.3%) were resistant or showed reduced susceptibility to imipenem and meropenem. All carbapenem-resistant isolates were blaNDM-positive by PCR, but 13 isolates bore variants that differed in sequence from blaNDM-1. NDM-5, NDM-6 and NDM-7 were identified in two, eight and three isolates, respectively. blaNDM variants were located on plasmids of >100kb with IncF, IncA/C and untypeable replicon types. Genes encoding the 16S rRNA methyltransferases RmtB, RmtC and ArmA as well as those for AmpC ß-lactamases were also located on the same plasmids as blaNDM in different combinations. The prevalence of NDM-5 to -7 variants was significantly higher in Escherichia coli (P=0.015) and they were more frequently isolated from the urology ward (P=0.037) than NDM-1. The mortality rate was comparable between patients infected with isolates positive for blaNDM-1 and blaNDM variants [25% (11/44) vs. 23% (3/13)]. Expression of blaNDM variants in E. coli using the same promoter showed that NDM-7 conferred higher resistance to imipenem. The diverse genotypic features of blaNDM indicate rapid evolution of NDM resulting from their wide spread in the Indian subcontinent.

Epstein-Barr virus DNA load and its association with Helicobacter pylori infection in gastroduodenal diseases.

UNLABELLED: Helicobacter pylori and Epstein-Barr virus (EBV) infections are common worldwide. Although H. pylori infection is a major factor in gastroduodenal diseases, its role in association with EBV infection is unknown. OBJECTIVE: To study the association of H. pylori infection and EBV DNA load in patients with gastroduodenal diseases. METHODS: Biopsy samples were collected from 200 adult patients [non-ulcer dyspepsia (NUD) 100, peptic ulcer disease (PUD) 50, gastric carcinoma (GC) 50] undergoing upper gastrointestinal endoscopy. H. pylori infection was diagnosed by rapid urease test, culture, histopathology, PCR and Q-PCR. EBV DNA was detected by non-polymorphic Epstein-Barr nuclear antigen-1 (EBNA-1) gene based Q-PCR. RESULTS: In patients with GC and PUD, EBV DNA was detected more often than NUD (GC versus NUD = 90% versus 37%, p < 0.001; PUD versus NUD = 70% versus 37%, p < 0.001). The dual prevalence of H. pylori infection and EBV DNA was significantly higher in patients with GC and PUD than in those with NUD. Median copy number of EBV DNA was considerably higher in GC and PUD than NUD (p < 0.01). The copy number of EBV DNA was significantly higher in H. pylori infected patients (p = 0.015). The number of ureA gene copies was also found to be significantly higher in PUD and NUD with presence of EBV DNA. However, in GC no significant difference was seen between EBV positive and negative status. CONCLUSION: There was a trend for higher EBV DNA load in H. pylori positive individuals suggesting a probable role of H. pylori in modulating the conversion of EBV to its lytic phase.

Epstein-Barr virus DNA load and its association with Helicobacter pylori infection in gastroduodenal diseases

Helicobacter pylori and Epstein-Barr virus (EBV) infections are common worldwide. Although H. pylori infection is a major factor in gastroduodenal diseases, its role in association with EBV infection is unknown. Objective: To study the association of H. pylori infection and EBV DNA load in patients with gastroduodenal diseases. Methods: Biopsy samples were collected from 200 adult patients [non-ulcer dyspepsia (NUD) 100, peptic ulcer disease (PUD) 50, gastric carcinoma (GC) 50] undergoing upper gastrointestinal endoscopy. H. pylori infection was diagnosed by rapid urease test, culture, histopathology, PCR and Q-PCR. EBV DNA was detected by non-polymorphic Epstein-Barr nuclear antigen-1 (EBNA-1) gene based Q-PCR. Results: In patients with GC and PUD, EBV DNA was detected more often than NUD (GC versus NUD = 90 percent versus 37 percent, p < 0.001; PUD versus NUD = 70 percent versus 37 percent, p < 0.001). The dual prevalence of H. pylori infection and EBV DNA was significantly higher in patients with GC and PUD than in those with NUD. Median copy number of EBV DNA was considerably higher in GC and PUD than NUD (p < 0.01). The copy number of EBV DNA was significantly higher in H. pylori infected patients (p = 0.015). The number of ureA gene copies was also found to be significantly higher in PUD and NUD with presence of EBV DNA. However, in GC no significant difference was seen between EBV positive and negative status. Conclusion: There was a trend for higher EBV DNA load in H. pylori positive individuals suggesting a probable role of H. pylori in modulating the conversion of EBV to its lytic phase.