Missed phenotypic drug resistance in pediatric tuberculosis: A cause of concern in a resource-limited setting.

BACKGROUND: Multi-drug resistance (MDR) in pediatric tuberculosis (TB) is a growing global threat. Unavailability of conventional or molecular drug susceptibility test (DST) in resource-limited settings often impede the determination of the extent of first line anti-tubercular drugs deployed in national programs. MATERIALS AND METHOD: Pulmonary and extra pulmonary specimens were collected from clinically suspected pediatric TB cases, who were microbiologically confirmed. Resistance to first-line anti-TB was detected by 1% proportion method. KatG315 and inhA-15 genes were amplified by PCR and detection of mutations were done by sequencing. Genotypic resistance for rifampicin was detected by Xpert MTB/RIF assay (Cepheid Inc., Sunnyvale, California). RESULTS: Fifty-one cases of pediatric tuberculosis were confirmed microbiologically. Resistance to isoniazid, streptomycin, rifampicin and ethambutol were 5 (14%), 4 (11%), 2 (5.5%) and 2 (5.5%) respectively by 1% proportion method. Genotypic Rifampicin and isoniazid resistance was found in 2 (5.5%) and 7 (14%) samples respectively. CONCLUSION: Existing genotypic methods, detect targeted mutations conferring rifampicin resistance, however isoniazid (INH) resistance often go undetected. Since the resistance to pivotal anti-TB drugs are often encoded by multiple genes which may not be targeted by widely available molecular tests, discrepancies in molecular and culture-based DST reports should be interpreted with caution.

Deciphering the conformational stability of MazE7 antitoxin in Mycobacterium tuberculosis from molecular dynamics simulation study.

MazEF Toxin-antitoxin (TA) systems are associated with the persistent phenotype of the pathogen, Mycobacterium tuberculosis (Mtb), aiding their survival. Though extensively studied, the mode of action between the antitoxin-toxin and DNA of this family remains largely unclear. Here, the important interactions between MazF7 toxin and MazE7 antitoxin, and how MazE7 binds its promoter/operator region have been studied. To elucidate this, molecular dynamics (MD) simulation has been performed on MazE7, MazF7, MazEF7, MazEF7-DNA, and MazE7-DNA complexes to investigate how MazF7 and DNA affect the conformational change and dynamics of MazE7 antitoxin. This study demonstrated that the MazE7 dimer is disordered and one monomer (Chain C) attains stability after binding to the MazF7 toxin. Both the monomers (Chain C and Chain D) however are stabilized when MazE7 binds to DNA. MazE7 is also observed to sterically inhibit tRNA from binding to MazF7, thus suppressing its toxic activity. Comparative structural analysis performed on all the available antitoxins/antitoxin-toxin-DNA structures revealed MazEF7-DNA mechanism was similar to another TA system, AtaRT_E.coli. Simulation performed on the crystal structures of AtaR, AtaT, AtaRT, AtaRT-DNA, and AtaR-DNA showed that the disordered AtaR antitoxin attains stability by AtaT and DNA binding similar to MazE7. Based on these analyses it can thus be hypothesized that the disordered antitoxins enable tighter toxin and DNA binding thus preventing accidental toxin activation. Overall, this study provides crucial structural and dynamic insights into the MazEF7 toxin-antitoxin system and should provide a basis for targeting this TA system in combating Mycobacterium tuberculosis.Communicated by Ramaswamy H. Sarma.

Studies of Antioxidant and Cytotoxic Activity in Ready-to-Drink Wild Ganoderma Teas: An In Vitro Approach.

Ganoderma is a medicinally important mushroom and has been used since ancient times. However, mostly G. lucidum has been used for therapeutic purposes, in form of tea, dietary and drug supplements but other species of Ganoderma are still remaining underexploited. This study is the first approach to valorize Ganoderma teas prepared from different wild species of Ganoderma other than G. lucidum with respect to both phytochemically and therapeutically through investigation of their phytochemical, carbohydrate contents and exploring their antioxidant activity. Phytochemical contents such as phenol and flavonoids were quantified using spectrophotometry methods. The carbohydrate content of the teas was estimated by phenol sulphuric acid method. The biochemical analysis revealed the teas contained a notable amount of phenolic compounds ranging from 19.15 to 40.2 µg GAE/mg of extract and also showed significant content of flavonoids. Further, antioxidant potential in terms of DPPH and ABTS radical scavenging ability and total antioxidant capacity was also evaluated. According to the results, G. resinaceum tea showed better potential in scavenging DPPH (EC50 36 ug/mL) and ABTS radicals (EC50 3 9 ug/mL) whereas the least effect was shown for the tea of G. ahmedi. Therefore, tea showing the best results, i.e. G. resinaceum tea, was also analyzed for cytotoxicity on breast cancer cells. It was found that the tea made from G. resinaceum inhibited cellular growth and proliferation in a dose-dependent manner with maximum growth inhibition (61%) observed at the highest concentration of 2.3 mg/mL. The presence of a greater quantity of carbohydrates in G. resinaceum tea also justified the remarkable anticancer potential of the tea. Overall, our findings indicated that a few wild species of Ganoderma other than G. lucidum have great potential to be valued as a healthy beverage with immense therapeutic benefits.

Crystal structure of a mycobacterial secretory protein Rv0398c and in silico prediction of its export pathway.

Secretory proteins are used by pathogenic bacteria to manipulate the host systems and compete with other microorganisms, thereby enabling their survival in their host. Similar to other bacteria, secretory proteins of Mycobacterium tuberculosis also play a pivotal role in evading immune response within hosts, thereby leading to acute and latent tuberculosis infection. Prokaryotes have several classes of bacterial secretory systems out of which the Sec and Tat pathways are the most conserved in Mtb to transport proteins across the cytoplasmic membrane. Here, we report the crystal structure of a secretory protein, Rv0398c determined to 1.9 Å resolution. The protein comprises a core of antiparallel ß sheets surrounded by α helices adopting a unique ß sandwich fold. Structural comparison with other secretory proteins in Mtb and other pathogenic bacteria reveals that Rv0398c may be secreted via the Sec pathway. Our structural and in silico analyses thus provide mechanistic insights into the pathway adopted by Mtb to transport out secretory protein, Rv0398c which will facilitate the invasion to the host immune system.

Bilayered skin substitute incorporating rutin nanoparticles for antioxidant, anti-inflammatory, and anti-fibrotic effect.

Hypertrophic scarring in large burns and delayed healing in chronic wounds are consequences of prolonged and aggravated inflammation, sustained infiltration of immune cells, free radical generation, and abundance of inflammatory mediators. Therefore, it is imperative to curb hyperinflammation to expedite wound healing. In this study, rutin nanoparticles (RNPs) were synthesized without an encapsulant and incorporated into eggshell membrane powder-crosslinked gelatin-chitosan cryogels to impart antioxidant and anti-inflammatory properties for treating hyperinflammation. The resultant nanoparticles were found to be 17.53 ± 4.03 nm in size and were stable at room temperature for a month with no visible sedimentation. RNPs were found to be non-cytotoxic and exhibited anti-inflammatory (by increasing IL-10 levels) and antioxidant properties (by controlling the generation of reactive oxygen species and enhancing catalase production in human macrophages). Additionally, RNPs were found to reduce α-SMA expression in fibroblasts, thereby demonstrating their anti-scarring effect. In vivo studies with a bilayered skin substitute constituting an RNP-incorporated cryogel proved that it is biocompatible, does not induce renal toxicity, aids wound healing, and induces better re-epithelialization than the control groups at the initial stages. Thus, RNP-incorporated cryogels containing bilayered skin substitutes are an advanced and novel alternative to commercial dermo-epidermal substitutes that lack anti-inflammatory or anti-scarring properties.

Bioactivity Profiling of Infusion, Decoction, and Hydroalcoholic Fraction Obtained from Wild Golden Chanterelle Mushroom, Cantharellus cibarius (Agaricomycetes).

Cantharellus cibarius Fr. is one of the most desirable, popularly known wild edible mushrooms of the genus Cantharellus. Besides having nutritional benefits, this mushroom is reported to harbor significant medicinal potential. In present study, we describe for the first-time bioactive metabolites content and medicinal activities of three different types of extracts including infusion, decoction, and hydroalcoholic fraction from wild Cantharellus cibarius of West Bengal. Bioactive metabolites quantification revealed the phenol and flavonoids present in the range between 0.56-1.46 mg of GAE/g of dried fruit bodies and 0.12-0.29 mg of QE/g of dried fruit bodies, respectively, whereas ascorbic acid contents were also found. Antioxidant activity was assessed using six in vitro systems; decoction displayed better results in all investigated assays with EC50 values ranging between 0.05 and 0.34 mg/ml whereas hydroalcoholic extract showed highest total antioxidant capacity (18 ± 1.61 µg AAE per mg of extract) than other extracts. Moreover, infusion and decoction were found to scavenge highly reactive free radicals like hydroxyl and nitric oxide with their EC50 values ranging between 0.26 and 0.91 mg/ml. Additionally, anti-inflammatory potential of the extracts was evaluated using protein denaturation assay. Hydroalcoholic extract at a concentration range of 0.005-0.1 mg/ml showed comparatively better activity than decoction and infusion, although all the extracts presented dose dependent inhibition of heat induced protein denaturation. Altogether, the present investigation is expected to extend the existing knowledge of this species and recommended its use in pharmaceutical industries as an effective mycomedicine.

Advances in modified antimicrobial peptides as marine antifouling material.

Biofouling is the undesirable attachment of organisms and their by-products on surfaces. It has become a severe problem in the industries that utilize devices and facilities in the marine environment. Several antifouling strategies have been developed, but many have adverse effects on numerous species, the surrounding environment, and marine devices. However, antimicrobial peptides (AMPs) have emerged as a promising non-toxic biomaterial that can modify the submerged surfaces to inhibit biofouling. AMPs are getting recognized as a highly potent material as they exhibit strong antimicrobial activity against fouling organisms and resistance towards biofilm formation. This review discusses the latest developments made in recent years regarding applying AMPs as prominent marine antifouling material. The various properties of AMPs, including structural, functional characteristics, and mechanism of action, are presented. Different types of modification of AMPs to improve their stability, efficacy, and activity against fouling organisms are discussed in detail. Furthermore, future perspectives and significant improvements required to make AMPs an integrative part of the marine antifouling process are reviewed.

Time-dependent electrochemical characteristics of a phenolic and non-phenolic compound in the presence of laccase/ABTS system.

The laccase/ABTS system has found several industrial applications ranging from biodeterioration to biodegradation and bioremediation. However, the capability of the laccase/ABTS system varies depending upon the type of substrate used. Voltammetric studies involving two widely used substrates, i.e., veratryl alcohol (VA) and alkali lignin (AL), were performed to gain new insight into the electrochemical behavior of the reactions. The individual electrochemical reactions established the differential nature of the two compounds over a concentration range, along with the mediator ABTS producing a distinguishing effect on their oxidative reactions, which was further studied over a 12hour period. It was followed by the reaction of both the compounds against the laccase/ABTS system that helped verify the role of the enzyme and the mediator in the electron transfer process and elucidate the mediated oxidations carried out by laccase against the phenolic and non-phenolic substrate through the process of cyclic voltammetry.

Clove Oil-Incorporated Antibacterial Gelatin-Chitosan Cryogels for Tissue Engineering: An In Vitro Study.

Infections are a leading cause of mortality and amputations among patients with burns and chronic wounds, respectively. Moreover, the extensive use of antibiotics has led to the rapid spreading of drug resistance among microorganisms. Alternatively, plant-derived natural products, which have been used as traditional therapies for several centuries, are recently gaining popularity as they are relatively affordable and easily available in many developing countries where modern medications are expensive or unavailable. In this study, clove essential oil is used for its antimicrobial property and is further incorporated into cryogels to increase its bioavailability and prolong its bioactivity. The oil-incorporated cryogels are macroporous, biodegradable, possess mechanical properties similar to commercial skin substitutes, are cytocompatible, antibacterial, and allow long-term sustained release of oil for up to at least 14 days. Additionally, clove oil aids the faster closure of in vitro scratch wounds by improving the migration of fibroblasts. This work presents a novel, bioactive scaffold that has the potential to be used as a dermal substitute and serves as an alternative to commercial skin substitutes.

First study on detection of cryptic resistance to linezolid among clinical isolates of methicillin resistant Staphylococcus aureus from India.

PURPOSE: Linezolid is an oral antibiotic which is widely used for serious infections caused by Methicillin Resistant Staphylococcus aureus (MRSA). With emergence of vancomycin MIC creep among clinical strains of MRSA, it is essential to know the possible emergence of subclinical resistance against linezolid as well. With this background, we aimed to detect evident (phenotypic) and cryptic (hidden or genotypic) linezolid resistance among MRSA isolates. METHODS: 250 clinical isolates of MRSA were collected and their susceptibility patterns were determined. Every third MRSA isolate was subjected to PCR for domain V of the 23S rRNA for the mutation hotspot in the 746bp segment which harbors the classical mutation for linezolid resistance. Restriction Fragment Length Polymorphism was done to confirm presence of the G2576U mutation. RESULTS: Six isolates (2.4%) were phenotypically resistant to linezolid. Among these six LRSA isolates, 5 demonstrated the G2576U mutation by PCR - RFLP. Cryptic resistance to Linezolid was identified in two isolates among linezolid susceptible isolates. CONCLUSIONS: In the present study, hidden resistance to linezolid was observed in linezolid susceptible clinical isolates. Emergence of resistance against over-the-counter drugs like linezolid is major challenge. Identification of cryptic resistance among patients implies impending resistance to linezolid. Judicious use of antimicrobials, application of strict infection control practices and prescription audit needs to be made mandatory to preserve such drugs.

Early detection of recurrent lung cancer: Enhancing-nodule in post-radiation fibrosis.

Early detection of lung cancer recurrence on imaging is critical for better clinical prognosis. The 'enhancing nodule in post-radiation fibrosis sign' is an important sign which helps detect recurrent lung cancer early on CT chest.

Electrochemical analysis of Catechol polymerization in presence of Trametes versicolor laccase and the mediator ABTS.

The phenolic compound catechol has found various applications in the industry but is often discharged untreated in industrial effluents. Catechol is highly toxic and adversely affects the environment. This has increased extensive investigation into elucidating the effects of various synthetic elements or different biocatalysts on catechol, thereby leading the way to its bioremediation. Hence, an electrochemical-based study on catechol in the presence of the enzyme laccase could provide a basic understanding of the unique characteristics exhibited by catechol, thus facilitating a distinct perspective to its subsequent treatment and removal. The present study focuses on the electrochemical characterization of catechol based on the oxidation of laccase and the redox mediator 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). Catechol exhibited distinct electrochemical behavior across various concentrations. The unique electroactive nature of ABTS assisted in the polymerization of catechol which was found to be concentration-dependent. Laccase produced a higher oxidation-reduction rate, thereby producing a much more stable condition for the polymerization of catechol. However, with the laccase-mediator system (LMS), the catechol polymerization rate was distinctly higher and more gradual with the enzyme utilizing the electroactive species produced by ABTS to increase the electron transfer and producing a combinatorial impact on the phenolic compound. This study could rightly serve as the building block in developing future technologies like wastewater treatment and biosensors for catechol bioremediation.

A bilayered skin substitute developed using an eggshell membrane crosslinked gelatin-chitosan cryogel.

Commercially available allografts and xenografts pose problems such as high cost, risk of infection transmission and immune rejection of grafts. Thus, bioengineered skin substitutes fabricated from natural biomaterials or synthetic polymers are currently the focus of skin tissue engineering. In this study, eggshell membrane (ESM) powder was used to crosslink a gelatin-chitosan cryogel thereby replacing glutaraldehyde, a known cytotoxic chemical crosslinker. The resultant ESM-crosslinked macroporous cryogel with a pore size ranging between 10 and 350 µm has improved flexibility, biodegradability and biocompatibility compared to a glutaraldehyde-crosslinked cryogel. For healing of large and deep wounds, bilayered scaffolds which exhibit key aspects of skin physiology are being explored. Hence, we fabricated a bilayered substitute by coupling the ESM-crosslinked cryogel (dermal equivalent) to a non-porous, physically-crosslinked gelatin-chitosan film (epidermal equivalent). The epidermal layer provides the requisite barrier properties while the dermal layer facilitates cell attachment and migration for optimal wound healing. Further, chitosan confers antibacterial properties to the cryogel with almost 50% reduction in bacterial viability. Animal studies confirm that the developed bilayered skin substitute is non-allergic, aids wound healing by improving re-epithelialization within 14 days and supports the formation of skin appendages. This system presents a new and alternative treatment option for burn and chronic wounds.

Elucidation of the decolorization of Congo Red by Trametes versicolor laccase in presence of ABTS through cyclic voltammetry.

The azo dye Congo red is heavily used in textile industries and is actively present in the wastewater run-offs. Its structural complexity and physical characteristics make it resistant to the physicochemical treatments employed by the industry. Over time, application of the enzyme laccase has proved to be quite useful due to its ability to oxidize and eventually decolorize the dye. Moreover, the use of ABTS as the electron mediator also helps in enhancing the oxidizing capability of the enzyme with congo red. The present study involves establishing the role of the individual components i.e. laccase, ABTS and the dye, in the LMS electrochemically. Congo red doesn't have any form of electrochemical activity by itself, but the enzyme brings about a substantial change by increasing the rate of reduction. The effect of ABTS, though same, is concentration-dependent. For LMS, laccase helps in bringing about the rate of reduction much faster in the presence of the mediator, initiating the decolorization of the dye.

Predictors for gut colonization of carbapenem-resistant Enterobacteriaceae in neonates in a neonatal intensive care unit.

BACKGROUND: With the emergence of carbapenem-resistant isolates, the therapeutic alternatives have become limited. Various factors are responsible for carbapenem-resistant Enterobacteriaceae (CRE) gut colonization. This study was conducted to determine predictors for CRE gut colonization in neonates who were hospital delivered and admitted in a neonatal intensive care unit (NICU). METHODS: Three rectal swabs were collected from 300 hospital-delivered and NICU-admitted neonates (likely to stay for >3 days). The data collected for the possible risk factors for CRE gut colonization were namely mode of delivery, prolonged rupture of membrane >18 hours, period of gestation, birth weight, meconium-stained liquor, ventilation, intravenous catheter, nasogastric (NG) tube, NG feeding, breastfeeding, katori spoon feeding, top feeding, expressed breastmilk, antibiotics administration, and duration of hospitalization. P < .05 was considered as statistically significant. RESULTS: A total of 26 cases of CRE were isolated from 300 neonates. Statistically significant risk factors were found to be NG tube, breastfeeding, NG feeding, top feeding, expressed breastmilk, ventilation, antibiotic administration, and duration of hospitalization. Top feeding and antibiotics administration were identified as 2 independent risk factors by multiple logistic regression. CONCLUSIONS: Active surveillance of cultures from hospitalized patients and implementation of preventive efforts can reduce the risk of CRE.

Fluorine in medicinal chemistry: a century of progress and a 60-year retrospective of selected highlights.

This perspective explores the origins of both fluorine and medicinal chemistry a century ago and traces the early history of the intersection of these areas and the subsequent roles that fluorine has played in advancing medicinal innovations and diagnoses during the past 60 years. The overview highlights remarkable breakthroughs in many diverse areas of medicinal chemistry, including inter alia, anesthetics, steroidal and nonsteroidal anti-inflammatory drugs, anticancer and antiviral agents, CNS medications, antibacterials and cholesterol biosynthesis inhibitors. The increasing use of fluorine-18-labeled radiotracers in PET for diagnostic imaging of the brain, heart and in oncology is briefly presented. The signature roles of fluorine in medicinal chemistry are now firmly established. The presence of fluorine in pharmaceuticals has had a major impact on a plethora of important medical applications, such as those cited above. Fluorine will very likely continue to contribute significantly by playing multifaceted roles in enhancing future medical advances.