ROLE OF AN ABC TRANSPORTER COMPLEX IN VIOLOGEN TOLERANCE IN STREPTOCOCCUS MUTANS

The gram-positive bacterium Streptococcus mutans is the primary causative agent in the formation of dental caries in humans. To persist in the oral cavity, S. mutans must be able to tolerate rapid and substantial environmental fluctuations and exposure to various toxic chemicals. However, the mechanisms underlying the ability of this cariogenic pathogen to survive and proliferate under harsh environmental conditions remain largely unknown. In the current study we wanted to understand the mechanisms by which S. mutans withstand exposure to various quaternary ammonium compounds (QAC) such as methyl viologen (MV) that also generates superoxide radicals in the cell. To elucidate the genes that are essential for MV tolerance, sensitive mutants of S. mutans were generated via ISS1 mutagenesis. Screening of approximately 3,500 mutants revealed fifteen MV sensitive mutants. Among them, five and four independent insertions had occurred in SMU.905 and SMU.906, respectively. These two genes are organized in an operon and encode a putative ABC-transporter complex. Linkage PCR analysis supports the operon organization of these two genes and also indicates that the transcription start site maps further upstream of SMU.905. To confirm our results, SMU.905 was deleted using an antibiotic resistance marker; the SMU.905 deletion mutant was just as sensitive to MV as the ISS1 insertion mutants. Furthermore, SMU.905 and SMU.906 mutants were sensitive to other viologen compounds such as benzyl- and ethyl- viologen. Sensitivity to various drugs including a wide range of QACs was tested. It appears that a functional SMU.905 is also required for full resistance towards acriflavin, ethidium bromide, and safranin; all are well known QAC. These results indicate that SMU.905/SMU.906 probably constitute a heterodimeric multidrug efflux pump of the ABC family. BLAST-P analysis suggests that this ABC-type efflux pump is widely present in streptococci, enterococci, and clostridia including some important human pathogens

Lantibiotic Smb: Characterization of the immunity protein, identification of a novel receptor-like protein, and a new perspective on regulation

Dental caries, commonly known as tooth decay, is a chronic disease that develops slowly and requires formation of biofilm on tooth surfaces, commonly known as dental plaque. Dental plaque is a highly complex multispecies biofilm containing over ~700 different microorganisms. In this microbiota, Streptococcus mutans is considered to be the primary etiological agent for cariogenesis. To colonize and maintain its dominance over competing non-cariogenic species, S. mutans secretes various antimicrobial peptides called bacteriocins. S. mutans produces two types of bacteriocins: linear unmodified peptides known as non-lantibiotics and extensively modified nonlinear peptides called lantibiotics. S. mutans GS-5 strain is a highly virulent isolate that has been extensively used for genetic and biochemical studies. This strain produces a broad-spectrum lantibiotic called Smb. This lantibiotic is one of the arsenals that S. mutans GS-5 uses to shift the established bacterial flora associated with dental health towards the flora associated with dental caries. A lantibiotic producer strain must contain a self-protection mechanism to protect itself from the lantibiotic-mediated damage. Immunity mechanisms against Smb have not been identified. A previous report by Kuramitsu's group described SmbG, a putative ABC transporter with a peptidase domain, as the immunity protein for Smb. This proposed function of SmbG in providing immunity is not supported by their experimental data. In this study we show that an ABC-transporter encoded by SmbFT functions as an immunity complex. We show that GS-5 becomes sensitized to Smb upon deletion of smbT, which makes the ABC transporter non-functional. We demonstrate that SmbFT can confer protection against Smb when expressed heterologously in four different sensitive streptococci. We also demonstrate that SmbFT can confer protection against structurally similar two-petide lantibiotics such as haloduracin. We conclude that SmbFT truly displays immunity function and confers protection against Smb and structurally similar lantibiotics. Lantibiotics are potent bactericidal agents and usually functional at nanomolar range, whereas other antimicrobial peptides are effective at micromolar concentrations. This fact indicates that the interplay between the lantibiotics and the target organisms must be specific and perhaps it occurs through receptor-mediated interaction. However, to date, no such receptor molecules have been identified for any lantibiotics. In this study we identify in S. pyogenes (a human pathogen) a membrane-bound protein that exhibits a receptor-like function for Smb. This protein, which we named LsrS, belongs to CAAX-protease family. LsrS is widely present in streptococci including S. mutans and is highly conserved. Deletion of the LsrS homolog in sensitive S. mutans strains makes them refractory to Smb inhibition. However, neither LsrS nor its homolog can recognize other structurally similar two-peptide lantibitics. Nevertheless, this is the first protein that displays a receptor-like function for any lantibiotics. It is of great importance to understand how the producer strain regulates the expression of its immunity protein to counteract the cognate lantibiotic produced by the cell as well as by the neighbours. An auto-sensing mechanism may exist to maintain a constant ratio of the immunity protein and the lantibiotic. Little is known about the transcriptional regulation of the smb operon. Unlike most of the other lantibiotic loci that encode their own regulatory factors, smb locus does not encode any factor that can function as an auto-regulator. We provide experimental evidences that Smb peptides function as signaling molecules and auto regulate the smb operon through some yet to be discovered regulators. We attempted to identify the unknown regulators by transposon mutagenesis and identified an operon that seems to be involved in activation of smb operon. Further analysis indicated that a transcriptional regulator encoded within the operon indeed regulates smb production. Our results show that a new regulator and perhaps a new regulatory pathway might control smb expression. Lantibiotics, such as Smb, are highly potent, stable, and active at nanomolar concentrations. Because of the stability and potency lantibiotics are widely used in food industry as preservative. Few other lantibiotics are in clinical trial with the prospective to be used as antimicrobial agents in the healthcare industry. Since Smb can inhibit many pathogenic streptococci, it has the potential to be used as an antimicrobial agent in food and/or healthcare industry

Digitization of vibrating wire type load cell for mine support systems

In this study an attempt is made to digitise the output of load cells using electronic circuitry and microcontrollers and its loading profile has been made. The circuit used to excite internal coils of load cell is Wien Bridge Oscillator. The frequency range of each coil was determined for which optimum output is produced from the load cell. The overall frequency range observed during experiment is 60 to 160 kHz. Wien Bridge Oscillator circuit was developed which gives a frequency range of 78 to 143 kHz. Wien Bridge oscillator was interfaced with the instrument and it was loaded from 0 to 25 T using Compression Testing Machine (CTM) and changing the input frequency for a certain load within its range i.e. 78 to 143 kHz. The output rms voltage changed from 1.15 to 0.195 V for red coil, 1.17 to 0.18 V for yellow coil and 1.18 to 0.172 V for green coil. Finally the microcontroller was introduced at the output end for display of digitised load reading from the VW type load cell. The load display in microcontroller display was showing fluctuations. The experimental setup can be further improved by introducing a rectifier along with a suitable filter which would convert the AC output signal to DC. This will minimise the fluctuations of the microcontroller display. It can also be proposed to further extend the work for continuous monitoring using a wireless sensor network

Socio-Demographic and Clinical Profile of Drug Resistant Tuberculosis Patients in a Tertiary Care Centre of Kolkata

Background: Drug resistant tuberculosis is a major public health threat that hinders progress in tuberculosis control worldwide. In 2019, India contributed highest cases of TB (27%) Worldwide. In 2019, 3.3% of new TB cases and 18% of previously treated cases had MDR/RRTB worldwide and India (27%) had the largest share of the global burden. This study was done to find out the sociodemographic and clinical profile of drug resistant TB patients. Methodology: It is an observational descriptive study with cross-sectional design, conducted at the drug resistant tuberculosis centre of R. G. Kar Medical College of Kolkata, West Bengal. Study Population consisted of the patients who were registered for the DR-TB regimen. Study duration was July 2016 –March 2017. Pre-tested, semi-structured schedule was designed to capture their sociodemographic profile, treatment history, clinical findings and available medical records. Data were compiled in MS Excel and analyzed in IBM SPSS 23.0. Results: Out of 159 cases, 27% patients were between 21-30 years. Males were predominant (68.6%). Most patients (56.6%) were underweight (BMI<18.5kg/m2). HIV seropositivity was found in 4 (2.5%) cases. Among all cases, 81.1% had history of taking ATD and 34.4% was cured in previous episode of treatment. The commonest associated comorbidity was DM (15.7%). Rifampicin resistance (93.1%) was most common followed by Isoniazid (8.2%). Pallor was found among 94.3% patients. Most patients had bilateral (62.3%) and moderately extensive (57.2%) lesions in chest x-ray. Conclusion: DR-TB control should focus adequately on younger age group as numbers of resistance is increasing among them. Relapse in previously cured cases was found to be major contributor of DR-TB suspect cases. Though, Rifampicin resistance was so common but prevalence of resistance to 2nd line drugs is still low. Focus should be given on early detection of drug resistance in all TB cases and improvement of nutritional status of the TB patient

CovR-Controlled Global Regulation of Gene Expression in Streptococcus mutans

CovR/S is a two-component signal transduction system (TCS) that controls the expression of various virulence related genes in many streptococci. However, in the dental pathogen Streptococcus mutans, the response regulator CovR appears to be an orphan since the cognate sensor kinase CovS is absent. In this study, we explored the global transcriptional regulation by CovR in S. mutans. Comparison of the transcriptome profiles of the wild-type strain UA159 with its isogenic covR deleted strain IBS10 indicated that at least 128 genes (∼6.5% of the genome) were differentially regulated. Among these genes, 69 were down regulated, while 59 were up regulated in the IBS10 strain. The S. mutans CovR regulon included competence genes, virulence related genes, and genes encoded within two genomic islands (GI). Genes encoded by the GI TnSmu2 were found to be dramatically reduced in IBS10, while genes encoded by the GI TnSmu1 were up regulated in the mutant. The microarray data were further confirmed by real-time RT-PCR analyses. Furthermore, direct regulation of some of the differentially expressed genes was demonstrated by electrophoretic mobility shift assays using purified CovR protein. A proteomic study was also carried out that showed a general perturbation of protein expression in the mutant strain. Our results indicate that CovR truly plays a significant role in the regulation of several virulence related traits in this pathogenic streptococcus

Synthesis, X-ray structure and in vitro cytotoxicity studies of Cu(I/II) complexes of thiosemicarbazone: special emphasis on their interactions with DNA

4-(p-X-phenyl)thiosemicarbazone of napthaldehyde {where X = Cl (HL¹) and X = Br (HL²)}, thiosemicarbazone of quinoline-2-carbaldehyde (HL³) and 4-(p-fluorophenyl)thiosemicarbazone of salicylaldehyde (H₂L⁴) and their copper(I) {[Cu(HL¹)(PPh₃)₂Br]·CH₃CN (1) and [Cu(HL²)(PPh₃)₂Cl]·DMSO (2)} and copper(II) {[(Cu₂L³₂Cl)₂(μ-Cl)₂]·2H₂O (3) and [Cu(L⁴)(Py)] (4)} complexes are reported herein. The synthesized ligands and their copper complexes were successfully characterized by elemental analysis, cyclic voltammetry, NMR, ESI-MS, IR and UV-Vis spectroscopy. Molecular structures of all the Cu(I) and Cu(II) complexes have been determined by X-ray crystallography. All the complexes (1–4) were tested for their ability to exhibit DNA-binding and -cleavage activity. The complexes effectively interact with CT-DNA possibly by groove binding mode, with binding constants ranging from 10⁴ to 10⁵ M⁻¹. Among the complexes, 3 shows the highest chemical (60%) as well as photo-induced (80%) DNA cleavage activity against pUC19 DNA. Finally, the in vitro antiproliferative activity of all the complexes was assayed against the HeLa cell line. Some of the complexes have proved to be as active as the clinical referred drugs, and the greater potency of 3 may be correlated with its aqueous solubility and the presence of the quinonoidal group in the thiosemicarbazone ligand coordinated to the metal

Global, regional, and national burden of disorders affecting the nervous system, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021

BackgroundDisorders affecting the nervous system are diverse and include neurodevelopmental disorders, late-life neurodegeneration, and newly emergent conditions, such as cognitive impairment following COVID-19. Previous publications from the Global Burden of Disease, Injuries, and Risk Factor Study estimated the burden of 15 neurological conditions in 2015 and 2016, but these analyses did not include neurodevelopmental disorders, as defined by the International Classification of Diseases (ICD)-11, or a subset of cases of congenital, neonatal, and infectious conditions that cause neurological damage. Here, we estimate nervous system health loss caused by 37 unique conditions and their associated risk factors globally, regionally, and nationally from 1990 to 2021.MethodsWe estimated mortality, prevalence, years lived with disability (YLDs), years of life lost (YLLs), and disability-adjusted life-years (DALYs), with corresponding 95% uncertainty intervals (UIs), by age and sex in 204 countries and territories, from 1990 to 2021. We included morbidity and deaths due to neurological conditions, for which health loss is directly due to damage to the CNS or peripheral nervous system. We also isolated neurological health loss from conditions for which nervous system morbidity is a consequence, but not the primary feature, including a subset of congenital conditions (ie, chromosomal anomalies and congenital birth defects), neonatal conditions (ie, jaundice, preterm birth, and sepsis), infectious diseases (ie, COVID-19, cystic echinococcosis, malaria, syphilis, and Zika virus disease), and diabetic neuropathy. By conducting a sequela-level analysis of the health outcomes for these conditions, only cases where nervous system damage occurred were included, and YLDs were recalculated to isolate the non-fatal burden directly attributable to nervous system health loss. A comorbidity correction was used to calculate total prevalence of all conditions that affect the nervous system combined.FindingsGlobally, the 37 conditions affecting the nervous system were collectively ranked as the leading group cause of DALYs in 2021 (443 million, 95% UI 378–521), affecting 3·40 billion (3·20–3·62) individuals (43·1%, 40·5–45·9 of the global population); global DALY counts attributed to these conditions increased by 18·2% (8·7–26·7) between 1990 and 2021. Age-standardised rates of deaths per 100 000 people attributed to these conditions decreased from 1990 to 2021 by 33·6% (27·6–38·8), and age-standardised rates of DALYs attributed to these conditions decreased by 27·0% (21·5–32·4). Age-standardised prevalence was almost stable, with a change of 1·5% (0·7–2·4). The ten conditions with the highest age-standardised DALYs in 2021 were stroke, neonatal encephalopathy, migraine, Alzheimer's disease and other dementias, diabetic neuropathy, meningitis, epilepsy, neurological complications due to preterm birth, autism spectrum disorder, and nervous system cancer.InterpretationAs the leading cause of overall disease burden in the world, with increasing global DALY counts, effective prevention, treatment, and rehabilitation strategies for disorders affecting the nervous system are needed

Global burden of 288 causes of death and life expectancy decomposition in 204 countries and territories and 811 subnational locations, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021

BACKGROUND Regular, detailed reporting on population health by underlying cause of death is fundamental for public health decision making. Cause-specific estimates of mortality and the subsequent effects on life expectancy worldwide are valuable metrics to gauge progress in reducing mortality rates. These estimates are particularly important following large-scale mortality spikes, such as the COVID-19 pandemic. When systematically analysed, mortality rates and life expectancy allow comparisons of the consequences of causes of death globally and over time, providing a nuanced understanding of the effect of these causes on global populations. METHODS The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2021 cause-of-death analysis estimated mortality and years of life lost (YLLs) from 288 causes of death by age-sex-location-year in 204 countries and territories and 811 subnational locations for each year from 1990 until 2021. The analysis used 56 604 data sources, including data from vital registration and verbal autopsy as well as surveys, censuses, surveillance systems, and cancer registries, among others. As with previous GBD rounds, cause-specific death rates for most causes were estimated using the Cause of Death Ensemble model-a modelling tool developed for GBD to assess the out-of-sample predictive validity of different statistical models and covariate permutations and combine those results to produce cause-specific mortality estimates-with alternative strategies adapted to model causes with insufficient data, substantial changes in reporting over the study period, or unusual epidemiology. YLLs were computed as the product of the number of deaths for each cause-age-sex-location-year and the standard life expectancy at each age. As part of the modelling process, uncertainty intervals (UIs) were generated using the 2·5th and 97·5th percentiles from a 1000-draw distribution for each metric. We decomposed life expectancy by cause of death, location, and year to show cause-specific effects on life expectancy from 1990 to 2021. We also used the coefficient of variation and the fraction of population affected by 90% of deaths to highlight concentrations of mortality. Findings are reported in counts and age-standardised rates. Methodological improvements for cause-of-death estimates in GBD 2021 include the expansion of under-5-years age group to include four new age groups, enhanced methods to account for stochastic variation of sparse data, and the inclusion of COVID-19 and other pandemic-related mortality-which includes excess mortality associated with the pandemic, excluding COVID-19, lower respiratory infections, measles, malaria, and pertussis. For this analysis, 199 new country-years of vital registration cause-of-death data, 5 country-years of surveillance data, 21 country-years of verbal autopsy data, and 94 country-years of other data types were added to those used in previous GBD rounds. FINDINGS The leading causes of age-standardised deaths globally were the same in 2019 as they were in 1990; in descending order, these were, ischaemic heart disease, stroke, chronic obstructive pulmonary disease, and lower respiratory infections. In 2021, however, COVID-19 replaced stroke as the second-leading age-standardised cause of death, with 94·0 deaths (95% UI 89·2-100·0) per 100 000 population. The COVID-19 pandemic shifted the rankings of the leading five causes, lowering stroke to the third-leading and chronic obstructive pulmonary disease to the fourth-leading position. In 2021, the highest age-standardised death rates from COVID-19 occurred in sub-Saharan Africa (271·0 deaths [250·1-290·7] per 100 000 population) and Latin America and the Caribbean (195·4 deaths [182·1-211·4] per 100 000 population). The lowest age-standardised death rates from COVID-19 were in the high-income super-region (48·1 deaths [47·4-48·8] per 100 000 population) and southeast Asia, east Asia, and Oceania (23·2 deaths [16·3-37·2] per 100 000 population). Globally, life expectancy steadily improved between 1990 and 2019 for 18 of the 22 investigated causes. Decomposition of global and regional life expectancy showed the positive effect that reductions in deaths from enteric infections, lower respiratory infections, stroke, and neonatal deaths, among others have contributed to improved survival over the study period. However, a net reduction of 1·6 years occurred in global life expectancy between 2019 and 2021, primarily due to increased death rates from COVID-19 and other pandemic-related mortality. Life expectancy was highly variable between super-regions over the study period, with southeast Asia, east Asia, and Oceania gaining 8·3 years (6·7-9·9) overall, while having the smallest reduction in life expectancy due to COVID-19 (0·4 years). The largest reduction in life expectancy due to COVID-19 occurred in Latin America and the Caribbean (3·6 years). Additionally, 53 of the 288 causes of death were highly concentrated in locations with less than 50% of the global population as of 2021, and these causes of death became progressively more concentrated since 1990, when only 44 causes showed this pattern. The concentration phenomenon is discussed heuristically with respect to enteric and lower respiratory infections, malaria, HIV/AIDS, neonatal disorders, tuberculosis, and measles. INTERPRETATION Long-standing gains in life expectancy and reductions in many of the leading causes of death have been disrupted by the COVID-19 pandemic, the adverse effects of which were spread unevenly among populations. Despite the pandemic, there has been continued progress in combatting several notable causes of death, leading to improved global life expectancy over the study period. Each of the seven GBD super-regions showed an overall improvement from 1990 and 2021, obscuring the negative effect in the years of the pandemic. Additionally, our findings regarding regional variation in causes of death driving increases in life expectancy hold clear policy utility. Analyses of shifting mortality trends reveal that several causes, once widespread globally, are now increasingly concentrated geographically. These changes in mortality concentration, alongside further investigation of changing risks, interventions, and relevant policy, present an important opportunity to deepen our understanding of mortality-reduction strategies. Examining patterns in mortality concentration might reveal areas where successful public health interventions have been implemented. Translating these successes to locations where certain causes of death remain entrenched can inform policies that work to improve life expectancy for people everywhere. FUNDING Bill & Melinda Gates Foundation

Regulation of the Glucosyltransferase (gtfBC) Operon by CovR in Streptococcus mutans

Streptococcus mutans is an important etiological agent of dental caries in humans. The extracellular polysaccharides synthesized by cell-associated glucosyltransferases (encoded by gtfBC) from sucrose have been recognized as one of the important virulence factors that promote cell aggregation and adherence to teeth, leading to dental plaque formation. In this study, we have characterized the effect of CovR, a global response regulator, on glucosyltransferase expression. Inactivation of covR in strain UA159 resulted in a marked increase in the GtfB and GtfC proteins, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With the use of a transcriptional reporter system of a single chromosomal copy of the PgtfB-gusA and PgtfC-gusA fusions, we confirmed the transcriptional regulation of these promoters by CovR. By in vitro electrophoretic mobility shift assays with purified CovR protein, we showed that CovR regulates these promoters directly. DNase I footprinting analyses suggest that CovR binds to large regions on these promoters near the transcription start sites. Taken together, our results indicate that CovR negatively regulates the expression of the gtfB and gtfC genes by directly binding to the promoter region