Homo-BacPROTAC-induced degradation of ClpC1 as a strategy against drug-resistant mycobacteria.

Antimicrobial resistance is a global health threat that requires the development of new treatment concepts. These should not only overcome existing resistance but be designed to slow down the emergence of new resistance mechanisms. Targeted protein degradation, whereby a drug redirects cellular proteolytic machinery towards degrading a specific target, is an emerging concept in drug discovery. We are extending this concept by developing proteolysis targeting chimeras active in bacteria (BacPROTACs) that bind to ClpC1, a component of the mycobacterial protein degradation machinery. The anti-Mycobacterium tuberculosis (Mtb) BacPROTACs are derived from cyclomarins which, when dimerized, generate compounds that recruit and degrade ClpC1. The resulting Homo-BacPROTACs reduce levels of endogenous ClpC1 in Mycobacterium smegmatis and display minimum inhibitory concentrations in the low micro- to nanomolar range in mycobacterial strains, including multiple drug-resistant Mtb isolates. The compounds also kill Mtb residing in macrophages. Thus, Homo-BacPROTACs that degrade ClpC1 represent a different strategy for targeting Mtb and overcoming drug resistance.

Oligodendroglia Confer Neuroprotection to NSC-34 Motor Neuronal Cells Against the Toxic Insults of Cerebrospinal Fluid from Sporadic Amyotrophic Lateral Sclerosis Patients.

Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disorder with multifactorial pathomechanisms affecting not only motor neurons but also glia. Both astrocytes and microglia get activated and contribute significantly to neurodegeneration. The role of oligodendroglia in such a situation remains obscure, especially in the sporadic form of ALS (SALS), which contributes to 90% of cases. Here, we have investigated the role of oligodendroglia in SALS pathophysiology using a human oligodendroglial cell line, MO3.13, by exposing the cells to cerebrospinal fluid from SALS patients (ALS-CSF; 10% v/v for 48 h). ALS-CSF significantly reduced the viability of MO3.13 cells and down-regulated the expression of oligodendroglia-specific proteins, namely, CNPase and Olig2. Furthermore, to investigate the effect of the observed oligodendroglial changes on motor neurons, NSC-34 motor neuronal cells were co-cultured/supplemented with conditioned/spent medium of MO3.13 cells upon exposure to ALS-CSF. Live cell imaging experiments revealed protection to NSC-34 cells against ALS-CSF toxicity upon co-culture with MO3.13 cells. This was evidenced by the absence of neuronal cytoplasmic vacuolation and beading of neurites, which instead resulted in better neuronal differentiation. Enhanced lactate levels and increased expression of its transporter, MCT-1, with sustained expression of trophic factors, namely, GDNF and BDNF, by MO3.13 cells hint towards metabolic and trophic support provided by the surviving oligodendroglia. Similar metabolic changes were seen in the lumbar spinal cord oligodendroglia of rat neonates intrathecally injected with ALS-CSF. The findings indicate that oligodendroglia are indeed rescuer to the degenerating motor neurons when the astrocytes and microglia turn topsy-turvy.

A cross-sectional study on clinical, demographic, microbiological, and radiological profile of bronchiectasis patients attending a tertiary care teaching center.

BACKGROUND: Bronchiectasis is a chronic pulmonary disease characterized by progressive and irreversible bronchial dilatation. The present study aimed to assess the clinical, demographic, microbiological, and radiological features of patients with bronchiectasis. METHODS: The study population included 60 subjects with bronchiectasis diagnosed by HRCT, who attended the Department of Respiratory Medicine a tertiary care teaching center. A single examiner examined all the 60 participants. Pulmonary function assessment was done on all the subjects on a spirometer, and early-morning sputum samples were taken for culture and sensitivity. Descriptive analysis was carried out by mean and standard deviation for quantitative variables, frequency, and proportion for categorical variables. Categorical outcomes were compared between study groups using the chi-square test. P value <0.05 was considered statistically significant. Co-Guide was used for statistical analysis. RESULT: The majority of the participants were males (62%). The most common sign observed was crepitations (75%). Pseudomonas aeruginosa (36%) was the primary pathogen isolated from sputum, followed by Klebsiella pneumonia (20%). Drug resistance was highest for ampicillin (56%), and imipenem (100%) was the most sensitive drug. CONCLUSION: Bronchiectasis is a heterogeneous entity with varied etiologies and multifarious clinic-radiological patterns. The information on etiology and the causative microorganism and antibiotic sensitivity and resistance aids in providing early treatment and thereby improving the lung function of affected individuals.

In Vitro and In Vivo Activity of Gepotidacin against Drug-Resistant Mycobacterial Infections.

Mycobacterial pathogens, including nontuberculous mycobacteria (NTM) and Mycobacterium tuberculosis, are pathogens of significant worldwide interest owing to their inherent drug resistance to a wide variety of FDA-approved drugs as well as causing a broad range of serious infections. Identifying new antibiotics active against mycobacterial pathogens is an urgent unmet need, especially those antibiotics that can bypass existing resistance mechanisms. In this study, we demonstrate that gepotidacin, a first-in-class triazaacenapthylene topoisomerase inhibitor, demonstrates potent activity against M. tuberculosis and M. fortuitum, as well as against other clinically relevant NTM species, including fluoroquinolone-resistant M. abscessus. Furthermore, gepotidacin exhibits concentration-dependent bactericidal activity against various mycobacterial pathogens, synergizes with several drugs utilized for their treatment, and reduces bacterial load in macrophages in intracellular killing assays comparably to amikacin. Additionally, M. fortuitum ATCC 6841 was unable to generate resistance to gepotidacin in vitro. When tested in a murine neutropenic M. fortuitum infection model, gepotidacin caused a significant reduction in bacterial load in various organs at a 10-fold lower concentration than amikacin. Taken together, these findings show that gepotidacin possesses a potentially new mechanism of action that enables it to escape existing resistance mechanisms. Thus, it can be projected as a potent novel lead for the treatment of mycobacterial infections, particularly for NTM, where present therapeutic interventions are extremely limited.

Phosphorus fractions in the sediment of a tropical reservoir, India: Implications for pollution source identification and eutrophication.

Eutrophication level in lakes and reservoirs depends on both internal and external phosphorus (P) load. Characterization of sediment P fractionation and identifying the P pollution sources are important for assessing the bio-availability of P and the dominant P source, for effectively controlling the water pollution. For determining the availability and sources of sediment P and eutrophication status, spatio-temporal variation in different P fractionation of sediment of hyper-eutrophic Krishnagiri reservoir, Tamil Nadu, India, was investigated. Sediment average total P (TP) content ranged from 4.62 to 5.64 g/kg. Main phosphorus form was the inorganic P (IP), and it makes up to 73.4-87.7% of TP. Among the different P fraction, viz. calcium bound (Ca-P), iron bound (Fe-P), aluminium bound (Al-P), exchangeable (Ex-P) and Organic-P (Org-P), Ca-P was the dominating fraction in both IP and TP. Trend of IP fraction was as follows: Ca-P > Fe-P > Al-P > Ex-P in pre-monsoon season, Fe-P > Ca-P > Al-P > Ex-P in monsoon and Ca-P > Al-P > Fe-P > Ex-P in post-monsoon. Overall the trend was as follows Ca-P > Fe-P > Al-P > Org-P > Ex-P. Bio-available-P (BAP) fractions ranged from 35.2 to 64.0% of TP, indicating its comparative higher value. Pearson's correlation matrix revealed that there was strong correlation among the different P fractions. Factor analysis indicates that different fractions of P were the dominating factor than the other sediment parameters. The observed variation in sediment P fractionation indicate the differences in source and characterization of P which is very helpful for implementation of effective management practices in controlling pollution that arises due to phosphorus in this hyper-eutrophic reservoir.

Molecular docking analysis of piperine with CDK2,CDK4,Cyclin D and Cyclin T proteins.

Piperine is a component of Piper nigrum (Black pepper). It is well known in ayurvedic formulations. Piperine is a bioenhancer as it reduces the activity of drug-metabolizing enzymes in rodents and thereby enhancing the plasma concentrations of several drugs, including the Pglycoprotein substrates. Therefore, it is of interest to understand the molecular docking interactions of piperine with several cell cycle proteins such as Cyclin dependent kinase 2 (CDK2), Cyclin-dependent kinase 4 (CDK4), Cyclin D and Cyclin T for further consideration in drug discovery related to oral cancer.

Plasmodium vivax liver stage assay platforms using Indian clinical isolates.

BACKGROUND: Vivax malaria is associated with significant morbidity and economic loss, and constitutes the bulk of malaria cases in large parts of Asia and South America as well as recent case reports in Africa. The widespread prevalence of vivax is a challenge to global malaria elimination programmes. Vivax malaria control is particularly challenged by existence of dormant liver stage forms that are difficult to treat and are responsible for multiple relapses, growing drug resistance to the asexual blood stages and host-genetic factors that preclude use of specific drugs like primaquine capable of targeting Plasmodium vivax liver stages. Despite an obligatory liver-stage in the Plasmodium life cycle, both the difficulty in obtaining P. vivax sporozoites and the limited availability of robust host cell models permissive to P. vivax infection are responsible for the limited knowledge of hypnozoite formation biology and relapse mechanisms, as well as the limited capability to do drug screening. Although India accounts for about half of vivax malaria cases world-wide, very little is known about the vivax liver stage forms in the context of Indian clinical isolates. METHODS: To address this, methods were established to obtain infective P. vivax sporozoites from an endemic region in India and multiple assay platforms set up to detect and characterize vivax liver stage forms. Different hepatoma cell lines, including the widely used HCO4 cells, primary human hepatocytes as well as hepatocytes obtained from iPSC's generated from vivax patients and healthy donors were tested for infectivity with P. vivax sporozoites. RESULTS: Both large and small forms of vivax liver stage are detected in these assays, although the infectivity obtained in these platforms are low. CONCLUSIONS: This study provides a proof of concept for detecting liver stage P. vivax and provide the first characterization of P. vivax liver stage forms from an endemic region in India.

Comparative study of the efficacy of gentamicin-coated intramedullary interlocking nail versus regular intramedullary interlocking nail in Gustilo type I and II open tibia fractures.

PURPOSE: Open tibia fracture is prone to infection, consequently causing significant morbidity and increasing the hospital stay, occupational loss and onset of chronic osteomyelitis. Intramedullary nailing is one choice for treating tibia shaft fractures. To improve the delivery of antibiotics at the tissue-implant interface, many methods have been proposed as a part of prophylaxis against infection. This study was conducted to study the role of gentamicin-impregnated intramedullary interlocking (IMIL) nail in the prevention of infection in Gustilo type I and II open tibia fractures and to compare the results with regular intramedullary nail. METHODS: The study included 28 patients with open tibia fractures (Gustilo type 1 or type 2); of them 14 underwent regular IMIL nailing and the other 14 were treated with gentamicin-coated nailing. Randomization was done by alternate allocation of the patients. Follow-up was done postoperatively (day 1), 1 week, 6 weeks, and 6 months for bone union, erythrocyte sedimentation rate (ESR), hemoglobin and C-reactive protein (CRP). Statistical significance was tested using unpaired t-test. A p value less than 0.05 was considered significant. RESULTS: There were 4 cases of infection in controls (regular IMIL nail) and no infection among patients treated with gentamicin-coated nail during the follow up (X2 = 4.66, p = 0.031). At 6 months postoperatively, CRP (p = 0.031), ESR (p = 0.046) and hemoglobin level (p = 0.016) showed significant difference between two groups. The bone healing rate was better with gentamicin-coated nail in comparison to regular IMIL nail at 6 months follow-up (p = 0.016). CONCLUSION: Gentamicin-coated IMIL nail has a positive role in preventing infection in Gustilo type I and II open tibia fractures.

Immediate effects of Maitland mobilization versus Mulligan Mobilization with Movement in Osteoarthritis knee- A Randomized Crossover trial.

BACKGROUND: Maitland Mobilization or Mulligan Mobilization with Movement (MWM) approaches have been widely used clinically for pain relief and improving mobility in Osteoarthritis knee. However the experimental evidence supporting the usage of these mobilization techniques as sole interventions in management of Osteoarthritis knee is insufficient. OBJECTIVE: To determine from Maitland Mobilization and Mulligan MWM, which mobilization technique will be more effective in reducing pain and improving mobility and function in OA knee immediately after the intervention. STUDY DESIGN: Randomized Crossover trial. MATERIALS AND METHODS: 30 subjects with osteoarthritis knee were recruited and 15 each were randomly allocated to two intervention sequences-one sequence was where Maitland was given first followed by Mulligan and the other was where Mulligan was given first followed by Maitland with a washout period of 48 h in between the two interventions. Numeric Pain Rating Scale (NPRS), Timed Up and Go (TUG) test and Pain free Squat Angle were the outcome measures measured before and immediately after both interventions. RESULTS: Using Repeated Measures ANOVA for analysis of outcomes between and within interventions, no significant differences were seen between Maitland Mobilization and Mulligan MWM, for NPRS, TUG and Pain free Squat Angle (p = 0.18, p = 0.27,p = 0.17) respectively whereas within the interventions both Maitland and Mulligan all outcome measures showed significant changes (p < 0.001). CONCLUSION: Thus it can be seen that Maitland mobilization and Mulligan MWM, both are equally effective in osteoarthritis knee in reducing pain and improving functional mobility and pain free squat angle immediately post treatment.

The promise of image-guided brachytherapy of better clinical outcomes in treatment of cervical cancer: Does it deliver? An Indian scenario.

PURPOSE: The purpose of this series is to study the effectiveness of MRI based image-guided brachytherapy (IGBT) in Indian patients with cervical cancer who mostly present in later stages with bulky diseases. PATIENTS AND METHODS: 151 cervical cancer patients treated at our institution in last four years, with definitive chemoradiation followed by MRI-based brachytherapy were reviewed. With median follow up of 26 months, Kaplan Meier estimates at two years were calculated for local control (LC), pelvic control (PC), disease-free survival (DFS) and overall survival (OS). Also, severe late sequelae were reported. RESULTS: The patients predominantly presented with locally advanced cervical cancer in FIGO stages IIB (53.6%) and IIIB (23.2%). Tumour dimensions at diagnosis were ≥5 cm in 56.3% and pelvic nodal involvement was found in 38.4% of the patients. 94% of the patients received curative chemoradiation. Mean HRCTV volume at the time of brachytherapy was 42.2 ±â€¯19 cm3 and mean cumulative dose to HRCTV was 78.9 ±â€¯5.6 Gy. Overall LC, PC, DFS and OS at 2 years were 88.7%, 88.1%, 82.2% and 94% respectively. The predictors for local failure were FIGO stage (p = 0.002) and tumour size at diagnosis (p = 0.009). Late grade 3-4 bladder and bowel toxicities were observed in 3.8% of the patients. CONCLUSION: Our review demonstrates that IGBT is an effective strategy to improve locoregional control with limited long-term sequelae in patients with locally advanced extensive cervical cancer in the setting of a developing country.

Culture characterization of the skin microbiome in Type 2 diabetes mellitus: A focus on the role of innate immunity.

AIMS: The current study aimed at determining the differences between the cutaneous microbial flora of patients with Type 2 diabetes mellitus (T2DM) and those without, and thus evaluate for cutaneous microbiome dysbiosis in diabetes. METHODS: We employed a case-control study design with 41 participants in each group. The skin over the toe-web space was swabbed and cultured aerobically. Data was analyzed for differences in microbial prevalences and growths between the two groups. Predictors for heavy colonization by microbes were analysed using logistic regression. RESULTS: We found significantly higher prevalences of Staphylococcus epidermidis among patients with T2DM (77.5% vs. 53.7%, p=0.02). Further, when prevalent, these bacteria showed a significantly greater degree of skin colonization i.e. CFUs/cm2 among these patients, p=0.03. Highly pathogenic bacteria such as S. aureus were more prevalent among patients with T2DM. The regression model determined a significant association between T2DM status and heavy colonization by S. epidermidis (OR - 5.40, p=0.02). Also, agricultural workers were significantly more likely to have heavy colonization by S. epidermidis (OR - 3.75, p=0.02). Other predictor variables did not show significant association with heavy colonization by any of the isolated microbes. CONCLUSIONS: Our findings support the existence of cutaneous microbiome dysbiosis among patients with T2DM. Literature suggests that microbiome dybiosis in T2DM could stem from the same activated innate immune response that is thought to be central to the development of T2DM. This dysbiosis could increase the risk of developing skin infections.

Inflammatory Myofibroblastic Tumour of Thyroid with its Prominent Spindle Cell Pattern: A Rare Case Report.

Inflammatory myofibroblastic tumour of thyroid is very rare. Only 18 cases reported so far. Here we report a case of Inflammatory myofibroblastic tumour with its prominent spindle cell (fibrohistiocytic) pattern in a 61-year-old male patient. The dominant histological pattern in our case was myofibroblastic in contrast to prominent lymphoplasmocytic pattern in other previously reported cases. The tumour was strongly positive for vimentin, Anaplastic lymphoma kinase and showed focal positivity for Smooth Muscle Actin. The patient was treated with total thyroidectomy and he is comfortable after surgery.

Membrane Sweeping for Vaginal Birth after Caesarean Section and its Outcome -A Comparative Study.

INTRODUCTION: Sweeping of membrane is a method of induction of labour. This is used to avoid prolonged labour. However, there is paucity of data about the use of this method for induction of labour and reducing prolonged labour in pregnancy with previous caesarean section. This study is an effort to find the effect of membrane sweeping in previous caesarean section. OBJECTIVE: To initiate labour in previous LSCS patients by membrane sweeping and maternal outcome. STUDY SETTING: This prospective randomised control study was conducted in Mahatma Gandhi Medical College and Research Institute, Puducherry between January 2011 to June 2012. MATERIALS AND METHODS: Seventy five women were randomly assigned to membrane sweeping and seventy five to control. In study group serial membrane sweeping was done once weekly from 39 weeks of gestation until the onset of labour up to 41weeks of gestation. In control group, no intervention up to 41 weeks of gestation. All the cases were monitored by biophysical profile. OUTCOME MEASURES: The primary outcomes measured were number of patients who had onset of labour. The secondary outcome included the successful vaginal delivery, number of membrane sweeping to initiate labour, sweeping to delivery interval and amount of oxytocin required. RESULTS: The onset of labour in study group was 61.3% similar in control group 64% with p 0.736. The mean interval from sweeping to labour onset was 50.15±8 hours. The rate of VBAC was 17.3% in study group in compared to 18.7% in control group and LSCS was 82.7% in study group in compared to 81.3% in control group respectively. The mean gestation age at delivery 40±0.56 weeks for study group compared with 39.92±0.55 weeks for control group. CONCLUSION: Although membrane sweeping is an easy way of inducing labour, present study failed to demonstrate its beneficial effect on obstetrical outcome.

Crystal structure of 4-{(E)-[2-(pyridin-4-ylcarbon-yl)hydrazin-1-yl-idene]meth-yl}phenyl acetate monohydrate.

The asymmetric unit of the title compound, C15H13N3O3·H2O, comprises a 4-{(E)-[2-(pyridin-4-ylcarbon-yl)hydrazinyl-idene]meth-yl}phenyl acetate mol-ecule and a solvent water mol-ecule linked by O-H⋯O and O-H⋯N hydrogen bonds from the water mol-ecule and a C-H⋯O contact from the organic mol-ecule. The compound adopts an E conformation with respect to the azomethine bond and the dihedral angle between the pyridine and benzene rings is 21.90 (7)°. The azomethine bond [1.275 (2) Å] distance is very close to the formal C=N bond length, which confirms the azomethine bond formation. An extensive set of O-H⋯O, O-H⋯N, N-H⋯O and C-H⋯O hydrogen bonds builds a two-dimensional network progressing along the c axis.

Novel N-linked aminopiperidine-based gyrase inhibitors with improved hERG and in vivo efficacy against Mycobacterium tuberculosis.

DNA gyrase is a clinically validated target for developing drugs against Mycobacterium tuberculosis (Mtb). Despite the promise of fluoroquinolones (FQs) as anti-tuberculosis drugs, the prevalence of pre-existing resistance to FQs is likely to restrict their clinical value. We describe a novel class of N-linked aminopiperidinyl alkyl quinolones and naphthyridones that kills Mtb by inhibiting the DNA gyrase activity. The mechanism of inhibition of DNA gyrase was distinct from the fluoroquinolones, as shown by their ability to inhibit the growth of fluoroquinolone-resistant Mtb. Biochemical studies demonstrated this class to exert its action via single-strand cleavage rather than double-strand cleavage, as seen with fluoroquinolones. The compounds are highly bactericidal against extracellular as well as intracellular Mtb. Lead optimization resulted in the identification of potent compounds with improved oral bioavailability and reduced cardiac ion channel liability. Compounds from this series are efficacious in various murine models of tuberculosis.

N'-[(E)-Furan-2-ylmethyl-idene]-4-hydroxy-benzohydrazide.

The title compound, C12H10N2O3, exists in the E conformation. The five-membered ring and the phenyl rings form dihedral angles of 36.73 (10) and 12.22 (10)°, respectively, with the central C(=O)N2C unit. The crystal packing is dominated by strong N-H⋯O and O-H⋯N hydrogen bonds. Together with weaker C-H⋯O inter-actions, these establish a three-dimensional supra-molecular network.

Performance evaluation of low cost microfluidic chips made using a digital craft cutter for point of care applications in nucleic acid tests.

A point of care (POC) diagnostic system development for nucleic acid testing (NAT) for developing countries faces several challenges and barriers among which affordability is a very critical one [1,4]. Hence a study was made to evaluate the effectiveness of microfluidic chips made from a digital craft cutter to be used as a disposable cartridge. Low cost materials like double sided tapes, transparent sheets and connectors were used to realize the microfluidic chip [2]. An in-house IVD sample preparation kit for nucleic acid extraction was used as a representative assay. Modifications were made to the assay workflow considering the feature sizes, design and volume of the microfluidic chip made from the paper cutter and other POC system requirements like turnaround time (TAT). The workflow was optimized by reducing overall TAT from 50min to 15min, sample volume from 150 µL to 12.5 µL and reduced reagent volumes. The method was also optimized to work at an isothermal condition. The results showed good correlation and yield in terms of both quality and quantity when compared to results obtained from the established baseline protocol. Thus microfluidic chips made using a digital craft cutter can very well be a low cost alternative to manufacture disposable chips for POC applications in nucleic acid tests.

Development of chitosan conjugated DNA vaccine against nodavirus in Macrobrachium rosenbergii (De Man, 1879).

The protective efficacy of a DNA construct containing extra small virus antisense (XSVAS) gene of nodavirus encapsulated with chitosan nanoparticles (NPs) was investigated in giant freshwater prawn Macrobrachium rosenbergii (De Man, 1879). The delivery was carried out using oral and immersion methods. A plasmid concentration of 100 ng µL(-1) when conjugated with chitosan NPs was found to be more effective in increasing the survivability of the infected prawn. The particle mean size, zeta potential and loading efficiency percentage were 297 nm, 27 mV and 85%, respectively. The ability of the chitosan to form a complex with the plasmid was studied by agarose gel electrophoresis. The NPs were characterized by atomic force microscopy (AFM). Persistence study showed the presence of the DNA construct up to 30th day post-treatment. The oral treatment was found to be better than the immersion treatment for delivery of the chitosan-conjugated DNA construct. This is probably the first report on the delivery of nanoconjugated DNA construct in M. rosenbergii, against nodavirus.

Bactericidal activity and mechanism of action of AZD5847, a novel oxazolidinone for treatment of tuberculosis.

Treatment of tuberculosis (TB) is impaired by the long duration and complexity of therapy and the rising incidence of drug resistance. There is an urgent need for new agents with improved efficacy, safety, and compatibility with combination chemotherapies. Oxazolidinones offer a potential new class of TB drugs, and linezolid-the only currently approved oxazolidinone-has proven highly effective against extensively drug-resistant (XDR) TB in experimental trials. However, widespread use of linezolid is prohibited by its significant toxicities. AZD5847, a novel oxazolidinone, demonstrates improved in vitro bactericidal activity against both extracellular and intracellular M. tuberculosis compared to that of linezolid. Killing kinetics in broth media and in macrophages indicate that the rate and extent of kill obtained with AZD5847 are superior to those obtained with linezolid. Moreover, the efficacy of AZD5847 was additive when tested along with a variety of conventional TB agents, indicating that AZD5847 may function well in combination therapies. AZD5847 appears to function similarly to linezolid through impairment of the mycobacterial 50S ribosomal subunit. Future studies should be undertaken to further characterize the pharmacodynamics and pharmacokinetics of AZD5847 in both in vitro and animal models as well is in human clinical trials.