Dynamical hallmarks of cancer: Phenotypic switching in melanoma and epithelial-mesenchymal plasticity.

Phenotypic plasticity was recently incorporated as a hallmark of cancer. This plasticity can manifest along many interconnected axes, such as stemness and differentiation, drug-sensitive and drug-resistant states, and between epithelial and mesenchymal cell-states. Despite growing acceptance for phenotypic plasticity as a hallmark of cancer, the dynamics of this process remains poorly understood. In particular, the knowledge necessary for a predictive understanding of how individual cancer cells and populations of cells dynamically switch their phenotypes in response to the intensity and/or duration of their current and past environmental stimuli remains far from complete. Here, we present recent investigations of phenotypic plasticity from a systems-level perspective using two exemplars: epithelial-mesenchymal plasticity in carcinomas and phenotypic switching in melanoma. We highlight how an integrated computational-experimental approach has helped unravel insights into specific dynamical hallmarks of phenotypic plasticity in different cancers to address the following questions: a) how many distinct cell-states or phenotypes exist?; b) how reversible are transitions among these cell-states, and what factors control the extent of reversibility?; and c) how might cell-cell communication be able to alter rates of cell-state switching and enable diverse patterns of phenotypic heterogeneity? Understanding these dynamic features of phenotypic plasticity may be a key component in shifting the paradigm of cancer treatment from reactionary to a more predictive, proactive approach.

Multiplexed and Extraction-Free Amplification for Simplified SARS-CoV-2 RT-PCR Tests.

The rapid onset of the global COVID-19 pandemic has led to challenges for accurately diagnosing the disease, including supply shortages for sample collection, preservation, and purification. Currently, most diagnostic tests require RNA extraction and detection by RT-PCR; however, extraction is expensive and time-consuming and requires technical expertise. With these challenges in mind, we report extraction-free, multiplexed amplification of SARS-CoV-2 RNA from 246 clinical samples, resulting in 86% sensitivity and 100% specificity. The multiplex RT-PCR uses the CDC singleplex targets and has an LoD of 2 c/µL. We also report on amplification using a range of master mixes in different transport media. This work can help guide which combinations of reagents will enable accurate results when availability of supplies changes throughout the pandemic. Implementing these methods can reduce complexity and cost, minimize reagent usage, expedite time to results, and increase testing capacity.

Nanoluciferase Reporter Mycobacteriophage for Sensitive and Rapid Detection of Mycobacterium tuberculosis Drug Susceptibility.

Phenotypic testing for drug susceptibility of Mycobacterium tuberculosis is critical to basic research and managing the evolving problem of antimicrobial resistance in tuberculosis management, but it remains a specialized technique to which access is severely limited. Here, we report on the development and validation of an improved phage-mediated detection system for M. tuberculosis We incorporated a nanoluciferase (Nluc) reporter gene cassette into the TM4 mycobacteriophage genome to create phage TM4-nluc. We assessed the performance of this reporter phage in the context of cellular limit of detection and drug susceptibility testing using multiple biosafety level 2 drug-sensitive and -resistant auxotrophs as well as virulent M. tuberculosis strains. For both limit of detection and drug susceptibility testing, we developed a standardized method consisting of a 96-hour cell preculture followed by a 72-hour experimental window for M. tuberculosis detection with or without antibiotic exposure. The cellular limit of detection of M. tuberculosis in a 96-well plate batch culture was ≤102 CFU. Consistent with other phenotypic methods for drug susceptibility testing, we found TM4-nluc to be compatible with antibiotics representing multiple classes and mechanisms of action, including inhibition of core central dogma functions, cell wall homeostasis, metabolic inhibitors, compounds currently in clinical trials (SQ109 and Q203), and susceptibility testing for bedaquiline, pretomanid, and linezolid (components of the BPaL regimen for the treatment of multi- and extensively drug-resistant tuberculosis). Using the same method, we accurately identified rifampin-resistant and multidrug-resistant M. tuberculosis strains.IMPORTANCEMycobacterium tuberculosis, the causative agent of tuberculosis disease, remains a public health crisis on a global scale, and development of new interventions and identification of drug resistance are pillars in the World Health Organization End TB Strategy. Leveraging the tractability of the TM4 mycobacteriophage and the sensitivity of the nanoluciferase reporter enzyme, the present work describes an evolution of phage-mediated detection and drug susceptibility testing of M. tuberculosis, adding a valuable tool in drug discovery and basic biology research. With additional validation, this system may play a role as a quantitative phenotypic reference method and complement to genotypic methods for diagnosis and antibiotic susceptibility testing.

Enhanced respiration prevents drug tolerance and drug resistance in Mycobacterium tuberculosis.

Persistence, manifested as drug tolerance, represents a significant obstacle to global tuberculosis control. The bactericidal drugs isoniazid and rifampicin kill greater than 99% of exponentially growing Mycobacterium tuberculosis (Mtb) cells, but the remaining cells are persisters, cells with decreased metabolic rate, refractory to killing by these drugs, and able to generate drug-resistant mutants. We discovered that the combination of cysteine or other small thiols with either isoniazid or rifampicin prevents the formation of drug-tolerant and drug-resistant cells in Mtb cultures. This effect was concentration- and time-dependent, relying on increased oxygen consumption that triggered enhanced production of reactive oxygen species. In infected murine macrophages, the addition of N-acetylcysteine to isoniazid treatment potentiated the killing of Mtb Furthermore, we demonstrate that the addition of small thiols to Mtb drug treatment shifted the menaquinol/menaquinone balance toward a reduced state that stimulates Mtb respiration and converts persister cells to metabolically active cells. This prevention of both persister cell formation and drug resistance leads ultimately to mycobacterial cell death. Strategies to enhance respiration and initiate oxidative damage should improve tuberculosis chemotherapies.

A Syringe-Based Biosensor to Rapidly Detect Low Levels of Escherichia Coli (ECOR13) in Drinking Water Using Engineered Bacteriophages.

A sanitized drinking water supply is an unconditional requirement for public health and the overall prosperity of humanity. Potential microbial and chemical contaminants of drinking water have been identified by a joint effort between the World Health Organization (WHO) and the United Nations Children's Fund (UNICEF), who together establish guidelines that define, in part, that the presence of Escherichia coli (E. coli) in drinking water is an indication of inadequate sanitation and a significant health risk. As E. coli is a nearly ubiquitous resident of mammalian gastrointestinal tracts, no detectable counts in 100 mL of drinking water is the standard used worldwide as an indicator of sanitation. The currently accepted EPA method relies on filtration, followed by growth on selective media, and requires 24-48 h from sample to results. In response, we developed a rapid bacteriophage-based detection assay with detection limit capabilities comparable to traditional methods in less than a quarter of the time. We coupled membrane filtration with selective enrichment using genetically engineered bacteriophages to identify less than 20 colony forming units (CFU) E. coli in 100 mL drinking water within 5 h. The combination of membrane filtration with phage infection produced a novel assay that demonstrated a rapid, selective, and sensitive detection of an indicator organism in large volumes of drinking water as recommended by the leading world regulatory authorities.

Early Detection of Emergent Extensively Drug-Resistant Tuberculosis by Flow Cytometry-Based Phenotyping and Whole-Genome Sequencing.

A critical gap in tuberculosis (TB) treatment is detection of emergent drug resistance. We hypothesized that advanced phenotyping with whole-genome sequencing (WGS) will detect low-frequency Mycobacterium tuberculosis drug resistance. We assessed a reporter mycobacteriophage (Φ2GFP10) in vitro to detect drug-resistant subpopulations and predict M. tuberculosis bactericidal activity in this pilot study. Subsequently, we prospectively studied 20 TB patients with serial Φ2GFP10, Xpert MTB/RIF, and M. tuberculosis culture through end of treatment. WGS was performed, and single nucleotide polymorphisms (SNPs) were examined to detect mixed infection in selected M. tuberculosis isolates. Resistant M. tuberculosis isolates were detected at 1:100,000, and changes in cytometry-gated events were predictive of in vitroM. tuberculosis bactericidal activity using the Φ2GFP10 assay. Emergent drug resistance was detected in one patient by Φ2GFP10 at 3 weeks but not by conventional testing (M. tuberculosis culture and GeneXpert). WGS revealed a phylogeographically distinct extensively drug-resistant tuberculosis (XDR-TB) genome, identical to an XDR-TB isolate from the patient's spouse. Variant lineage-specific SNPs were present early, suggesting mixed infection as the etiology of emergent resistance with temporal trends providing evidence for selection during treatment. Φ2GFP10 can detect low-frequency drug-resistant M. tuberculosis and with WGS characterize emergent M. tuberculosis resistance. In areas of high TB transmission and drug resistance, rapid screening for heteroresistance should be considered.

Noncanonical SMC protein in Mycobacterium smegmatis restricts maintenance of Mycobacterium fortuitum plasmids.

Research on tuberculosis and leprosy was revolutionized by the development of a plasmid transformation system in the fast-growing surrogate, Mycobacterium smegmatis. This transformation system was made possible by the successful isolation of a M. smegmatis mutant strain mc(2)155, whose efficient plasmid transformation (ept) phenotype supported the replication of Mycobacterium fortuitum pAL5000 plasmids. In this report, we identified the EptC gene, the loss of which confers the ept phenotype. EptC shares significant amino acid sequence homology and domain structure with the MukB protein of Escherichia coli, a structural maintenance of chromosomes (SMC) protein. Surprisingly, M. smegmatis has three paralogs of SMC proteins: EptC and MSMEG_0370 both share homology with Gram-negative bacterial MukB; and MSMEG_2423 shares homology with Gram-positive bacterial SMCs, including the single SMC protein predicted for Mycobacterium tuberculosis and Mycobacterium leprae. Purified EptC was shown to bind ssDNA and stabilize negative supercoils in plasmid DNA. Moreover, an EptC-mCherry fusion protein was constructed and shown to bind to DNA in live mycobacteria, and to prevent segregation of plasmid DNA to daughter cells. To our knowledge, this is the first report of impaired plasmid maintenance caused by a SMC homolog, which has been canonically known to assist the segregation of genetic materials.

Noninvasive ventilation for acute exacerbations of asthma: A systematic review of the literature.

BACKGROUND: Asthma is a chronic disease characterised by reversible airway obstruction caused by bronchospasm, mucous and oedema. People with asthma commonly experience acute exacerbations of their disease requiring hospitalisation and subsequent utilisation of economic and healthcare resources. Noninvasive ventilation has been suggested as a treatment for acute exacerbations of asthma due to its ability to provide airway stenting, optimal oxygen delivery and decreased work of breathing. OBJECTIVES: This paper is a systematic review of the available published research focused on the use of noninvasive ventilation for the treatment of acute exacerbations of asthma to determine if this treatment provides better outcomes for patients compared to standard medical therapy. METHOD: Database searches were conducted using EBSCOhost, MEDLINE and PubMed. Search terms used were combinations of 'noninvasive ventilation', 'BiPAP', 'CPAP', 'wheez*' and 'asthma'. Articles were included if they were research papers focused on adult patients with asthma and a treatment of noninvasive ventilation, and were published in full text in English. Included articles were reviewed using the National Health and Medical Research Council (Australia) evidence hierarchy and quality appraisal tools. RESULTS: There were 492 articles identified from the database searches. After application of inclusion/exclusion criteria 13 articles were included in the systematic review. Studies varied significantly in design, endpoints and outcomes. There was a trend in better outcomes for patients with acute asthma who were treated with noninvasive ventilation compared to standard medical therapy, however, the variability of the studies meant that no conclusive recommendations could be made. CONCLUSION: More research is required before noninvasive ventilation can be conclusively recommended for the treatment of acute exacerbations of asthma.

Fluorescent Reporter DS6A Mycobacteriophages Reveal Unique Variations in Infectibility and Phage Production in Mycobacteria.

Mycobacteriophage DS6A is unique among the more than 8,000 isolated mycobacteriophages due to its ability to form plaques exclusively on mycobacteria belonging to the Mycobacterium tuberculosis complex (MTBC). Speculation surrounding this specificity has led to unsupported assertions in published studies and patents that nontuberculous mycobacteria (NTM) are wholly resistant to DS6A infection. In this study, we identified two independent nonessential regions in the DS6A genome and replaced them with an mVenus-expressing plasmid to generate fluorescent reporter phages Φ2GFP12 and Φ2GFP13. We show that even though DS6A is able to form plaques only on MTBC bacteria, infection of various NTM results in mVenus expression in transduced cells. The efficiency of DS6A in delivering DNA varied between NTM species. Additionally, we saw a striking difference in the efficiency of DNA delivery between the closely related members of the Mycobacterium abscessus complex, M. abscessus and Mycobacterium massiliense We also demonstrated that TM4 and DS6A, two phages that do not form plaques on M. massiliense, differ in their ability to deliver DNA, suggesting that there is a phage-specific restriction between mycobacterial species. Phylogenetic analysis reveals that the DS6A genome has a characteristically mosaic structure but provided few insights into the basis for the specificity for MTBC hosts. This study demonstrates that the inability of the MTBC-specific phage DS6A to form plaques on NTM is more complex than previously thought. Moreover, the DS6A-derived fluorophages provide important new tools for the study of mycobacterial biology. IMPORTANCE: The coevolution of bacteria and their infecting phages involves a constant arms race for bacteria to prevent phage infection and phage to overcome these preventions. Although a diverse array of phage defense systems is well characterized in bacteria, very few phage restriction systems are known in mycobacteria. The DS6A mycobacteriophage is unique in the mycobacterial world in that it forms plaques only on members of the Mycobacterium tuberculosis complex. However, the novel DS6A reporter phages developed in this work demonstrate that DS6A can infect nontuberculous mycobacteria at various efficiencies. By comparing the abilities of DS6A and another phage, TM4, to infect and form plaques on various mycobacterial species, we can begin to discern new phage restriction systems employed within the genus.

A Novel Reporter Phage To Detect Tuberculosis and Rifampin Resistance in a High-HIV-Burden Population.

Improved diagnostics and drug susceptibility testing for Mycobacterium tuberculosis are urgently needed. We developed a more powerful mycobacteriophage (Φ(2)GFP10) with a fluorescent reporter. Fluorescence-activated cell sorting (FACS) allows for rapid enumeration of metabolically active bacilli after phage infection. We compared the reporter phage assay to GeneXpert MTB/RIF for detection of M. tuberculosis and rifampin (RIF) resistance in sputum. Patients suspected to have tuberculosis were prospectively enrolled in Durban, South Africa. Sputum was incubated with Φ(2)GFP10, in the presence and absence of RIF, and bacilli were enumerated using FACS. Sensitivity and specificity were compared to those of GeneXpert MTB/RIF with an M. tuberculosis culture as the reference standard. A total of 158 patients were prospectively enrolled. Overall sensitivity for M. tuberculosis was 95.90% (95% confidence interval (CI), 90.69% to 98.64%), and specificity was 83.33% (95% CI, 67.18% to 93.59%). In acid-fast bacillus (AFB)-negative sputum, sensitivity was 88.89% (95% CI, 73.92% to 96.82%), and specificity was 83.33% (95% CI, 67.18% to 93.59%). Sensitivity for RIF-resistant M. tuberculosis in AFB-negative sputum was 90.00% (95% CI, 55.46% to 98.34%), and specificity was 91.94% (95% CI, 82.16% to 97.30%). Compared to GeneXpert, the reporter phage was more sensitive in AFB smear-negative sputum, but specificity was lower. The Φ(2)GFP10 reporter phage showed high sensitivity for detection of M. tuberculosis and RIF resistance, including in AFB-negative sputum, and has the potential to improve phenotypic testing for complex drug resistance, paucibacillary sputum, response to treatment, and detection of mixed infection in clinical specimens.

Effect of new organic supplement (Panchgavya) on seed germination and soil quality.

We studied the suitability of Panchgavya (five products of cow), new organic amendment, application on seed germination, plant growth, and soil health. After characterization, Panchgavya was mixed with water to form different concentration and was tested for seed germination, germination index, and root and shoot growth of different seedlings. Four percent solution of Panchgavya was applied to different plants to test its efficacy. Panchgavya and other two organic amendments were incorporated in soil to test the change of soil chemical and microbiological parameters. Panchgavya contained higher nutrients as compared to farm yard manure (FYM) and vermicompost. Its application on different seeds has positively influenced germination percentage, germination index, root and shoot length, and fresh and dry weight of the seedling. Water-soluble macronutrients including pH and metal were positively and negatively correlated with the growth parameters, respectively. Four percent solution of Panchgavya application on some plants showed superiority in terms of plant height and chlorophyll content. Panchgavya-applied soil had higher values of macro and micronutrients (zinc, copper, and manganese), microbial activity as compared to FYM, and vermicompost applied soils. Application of Panchgavya can be gainfully used as an alternative organic supplement in agriculture.

Drug tolerance and persistence in bacteria, fungi and cancer cells: Role of non-genetic heterogeneity.

A common feature of bacterial, fungal and cancer cell populations upon treatment is the presence of tolerant and persistent cells able to survive, and sometimes grow, even in the presence of usually inhibitory or lethal drug concentrations, driven by non-genetic differences among individual cells in a population. Here we review and compare data obtained on drug survival in bacteria, fungi and cancer cells to unravel common characteristics and cellular pathways, and to point their singularities. This comparative work also allows to cross-fertilize ideas across fields. We particularly focus on the role of gene expression variability in the emergence of cell-cell non-genetic heterogeneity because it represents a possible common basic molecular process at the origin of most persistence phenomena and could be monitored and tuned to help improve therapeutic interventions.

Cell-state transitions and density-dependent interactions together explain the dynamics of spontaneous epithelial-mesenchymal heterogeneity.

Cancer cell populations comprise phenotypes distributed among the epithelial-mesenchymal (E-M) spectrum. However, it remains unclear which population-level processes give rise to the observed experimental distribution and dynamical changes in E-M heterogeneity, including (1) differential growth, (2) cell-state switching, and (3) population density-dependent growth or state-transition rates. Here, we analyze the necessity of these three processes in explaining the dynamics of E-M population distributions as observed in PMC42-LA and HCC38 breast cancer cells. We find that, while cell-state transition is necessary to reproduce experimental observations of dynamical changes in E-M fractions, including density-dependent growth interactions (cooperation or suppression) better explains the data. Further, our models predict that treatment of HCC38 cells with transforming growth factor ß (TGF-ß) signaling and Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/3) inhibitors enhances the rate of mesenchymal-epithelial transition (MET) instead of lowering that of E-M transition (EMT). Overall, our study identifies the population-level processes shaping the dynamics of spontaneous E-M heterogeneity in breast cancer cells.

Prevalence of Epstein Barr Virus and Herpes Simplex Virus Among Human Papillomavirus Negative Oral Cancer Patients: A Cross-Sectional Study from South India.

The high incidence of oral carcinomas is due to its multifactorial etiology and the presence of various risk factors. Human Papillomavirus (HPV) has a proven role in the pathogenesis of oral carcinomas, but in the recent times there has been an increasing incidence of oral cancers who are negative for HPV infection. Also, these patients are non-smokers and non-drinkers so it could be speculated that these oral cancers are due to some other etiological factor probably of other viral infections. Therefore, this study examined the prevalence of Epstein Barr Virus (EBV) and Herpes Simplex Virus (HSV) among oral cancer patients. This cross-sectional study was conducted from January 2019 to June 2020. Biopsy samples from 47 newly diagnosed untreated patients with oral malignancies were collected along with their demographic and clinicopathological information. DNA extracted from the biopsies was processed for nested PCR for the detection of EBV and HSV. All the samples tested negative for HPV and HSV infection. Nested PCR detected 29 cases (70.7%) to be positive for EBV. The non-cancerous adjacent tissues also were negative for HPV, EBV and HSV. The prevalence of EBV was found to be more in males (62.1%) and the highest number of cases was of the left buccal mucosa compromising 34% of the total cases. From the present study it can be concluded that EBV but not HSV infection is associated with an increased risk of developing oral cancers. Although, 70.7% of the patients were found to be positive for EBV whether the viral infection played any role in the driving the malignancy needs to be further elucidated.

Pili contribute to biofilm formation in vitro in Mycobacterium tuberculosis.

Organized bacterial communities, or biofilms, provide an important reservoir for persistent cells that are inaccessible or tolerant to antibiotics. Curli pili are cell-surface structures produced by certain bacteria and have been implicated in biofilm formation in these species. In order to determine whether these structures, which were suggested to be encoded by the Rv3312A (mtp) gene, have a similar role in Mycobacterium tuberculosis, we generated a Δmtp mutant and a mtp-complemented strain of a clinical isolate of M. tuberculosis and analyzed these strains for their ability to produce pili in comparison to the wild-type strain. Phenotypic analysis by transmission electron microscopy proved the essentiality of mtp for piliation in M. tuberculosis. We then compared biofilm formation of the derived strains in detergent-free Sauton's media. Biofilm mass was quantified spectrophotometrically using crystal violet. Furthermore, we examined mtp gene expression by quantitative real-time PCR in wild-type cells grown under biofilm versus planktonic growth conditions. We found a 68.4 % reduction in biofilm mass in the mutant compared to the wild-type strain (P = 0.002). Complementation of the mutant resulted in a restoration of the wild-type biofilm phenotype (P = 0.022). We, however, found no significant difference between mtp expression in cells of the biofilm to those growing planktonically. Our findings highlight a crucial, but non-specific, role of pili in the biofilm lifestyle of M. tuberculosis and indicate that they may represent an important target for the development of therapeutics to attenuate biofilm formation, thereby potentially reducing persistence.

Epigenetic memory acquired during long-term EMT induction governs the recovery to the epithelial state.

Epithelial-mesenchymal transition (EMT) and its reverse mesenchymal-epithelial transition (MET) are critical during embryonic development, wound healing and cancer metastasis. While phenotypic changes during short-term EMT induction are reversible, long-term EMT induction has been often associated with irreversibility. Here, we show that phenotypic changes seen in MCF10A cells upon long-term EMT induction by TGFß need not be irreversible, but have relatively longer time scales of reversibility than those seen in short-term induction. Next, using a phenomenological mathematical model to account for the chromatin-mediated epigenetic silencing of the miR-200 family by ZEB family, we highlight how the epigenetic memory gained during long-term EMT induction can slow the recovery to the epithelial state post-TGFß withdrawal. Our results suggest that epigenetic modifiers can govern the extent and time scale of EMT reversibility and advise caution against labelling phenotypic changes seen in long-term EMT induction as 'irreversible'.

Balancing Routine and Pandemic: The Synergy of India's Universal Immunization Program and COVID-19 Vaccination Program.

The COVID-19 pandemic posed substantial challenges to healthcare systems globally and severely disrupted essential health services, including routine immunization programs. In India, these disruptions were exacerbated due to the sudden emergence of the pandemic and lockdown measures, leading to mass migrations and a shortage of healthcare workers. Caregivers' concerns about routine immunization sessions further compounded the problem, resulting in a sharp increase in zero-dose children. This review paper examines India's strategies for conducting one of the world's largest COVID-19 vaccination programs while effectively restoring and perpetuating its Universal Immunization Program (UIP). The UIP played a pivotal role in sustaining immunization services during the pandemic, ultimately improving immunization coverage compared to pre-pandemic levels. India's accomplishments in this regard are highlighted through key performance indicators, the reach of immunization services, a reduction in zero-dose children, and antigen-wise coverage. The paper also discusses the successful integration of COVID-19 vaccination within the UIP framework, underscoring the significance of existing infrastructure, technology, and capacity building. India's dedication to concurrently managing routine immunization and COVID-19 vaccination showcases the adaptability and resilience of its healthcare system. India's journey serves as a global example of efficient mass immunization during challenging times, emphasizing the importance of political will, healthcare infrastructure investment, skilled healthcare workforces, and comprehensive vaccination programs. In a world grappling with the dual challenge of COVID-19 and routine immunization, India's experience provides a roadmap for strengthening healthcare systems and promoting public health as the critical agenda in challenging times.

φ(2)GFP10, a high-intensity fluorophage, enables detection and rapid drug susceptibility testing of Mycobacterium tuberculosis directly from sputum samples.

The difficulty of diagnosing active tuberculosis (TB) and lack of rapid drug susceptibility testing (DST) at the point of care remain critical obstacles to TB control. This report describes a high-intensity mycobacterium-specific-fluorophage (φ(2)GFP10) that for the first time allows direct visualization of Mycobacterium tuberculosis in clinical sputum samples. Engineered features distinguishing φ(2)GFP10 from previous reporter phages include an improved vector backbone with increased cloning capacity and superior expression of fluorescent reporter genes through use of an efficient phage promoter. φ(2)GFP10 produces a 100-fold increase in fluorescence per cell compared to existing reporter phages. DST for isoniazid and oxofloxacin, carried out in cultured samples, was complete within 36 h. Use of φ(2)GFP10 detected M. tuberculosis in clinical sputum samples collected from TB patients. DST for rifampin and kanamycin from sputum samples yielded results after 12 h of incubation with φ(2)GFP10. Fluorophage φ(2)GFP10 has potential for clinical development as a rapid, sensitive, and inexpensive point-of-care diagnostic tool for M. tuberculosis infection and for rapid DST.

Characterization of DNA topoisomerase I from Mycobacterium tuberculosis: DNA cleavage and religation properties and inhibition of its activity.

Type I DNA topoisomerases from bacteria catalyse relaxation of negatively supercoiled DNA in a Mg(2+) dependent manner. Although topoisomerases of distinct classes have been subjected for anti-cancer and anti-infective drug development, bacterial type I enzymes are way behind in this regard. Our studies with Mycobacterium smegmatis topoisomerase I (MstopoI) revealed several of its distinct properties compared to the well studied Escherichia coli topoisomerase I (EctopoI) suggesting the possibility of targeting the mycobacterial enzyme for inhibitor development. Here, we describe Mycobacterium tuberculosis topoisomerase I (MttopoI) and compare its properties with MstopoI and EctopoI. The enzyme cleaves DNA at preferred sites in a pattern similar to its ortholog from M. smegmatis. Oligonucleotides containing the specific recognition sequence inhibited the activity of the enzyme in a manner similar to that of MstopoI. Substitution of the acidic residues, D111 and E115 which are involved in Mg(2+) co-ordination, to alanines affected the DNA relaxation activity. Unlike the wild type enzyme, D111A was dependent on Mg(2+) for DNA cleavage and both the mutants were compromised in religation. The monoclonal antibody (mAb), 2F3G4, developed against MstopoI inhibited the relaxation activity of MttopoI. These studies affirm the characteristics of MttopoI to be similar to MstopoI and set a stage to target it for the development of specific small molecule inhibitors.