(-)-Epigallocatechin-3-gallate in Camellia sinensis leaves from Himalayan region of Sikkim: inhibitory effects against biochemical events and tumor initiation in Sencar mouse skin.

Recently, we and others showed that the components of green tea may be useful cancer chemopreventive agents. It has been suggested that (-)-epigallocatechin-3-gallate (EGCG), the major constituent in green tea, may possess antitumor-promoting and/or anticarcinogenic effects in rodent tumor bioassay systems. During the chemical analysis of various green tea products, we found a traditionally preserved preparation of green tea used by tribes in the Himalayan region of Sikkim, India that was rich in EGCG. EGCG was isolated from this tea product, and its inhibitory effects were evaluated against the binding of topically applied 3H-labeled polycyclic aromatic hydrocarbons (PAHs) to epidermal DNA and 12-O-tetradecanoylphorbol-13-acetate (TPA) caused induction of epidermal ornithine decarboxylase (ODC) activity in Sencar mice, the short-term markers of tumor initiation and tumor promotion, respectively. Preapplication of EGCG resulted in significant inhibition (p less than 0.05) in the binding of [3H]PAH to epidermal DNA. Similarly, the topical application of EGCG resulted in significant inhibition (p less than 0.005) in TPA-caused induction of epidermal ODC activity. In further studies, we assessed the anti-skin tumor-initiating effect of EGCG in Sencar mice in an initiation-promotion protocol. The application of EGCG before challenge with 7,12-dimethylbenz[a]anthracene as tumor initiator resulted in significant reduction both in percentage of mice with tumors and number of tumors per mouse compared with a non-EGCG-pretreated group of animals. The results of the present study suggest that the green tea preparation from Sikkim may be a good source for the isolation of EGCG and that this compound may have significant potential as a cancer chemopreventive agent.     

Hepatic microsomal cytochrome P450 system during experimental hookworm infection

Experimental infection of golden hamsters with the hookworm, Ancylostoma ceylanicum, caused a profound decline in the hepatic microsomal cytochrome P450 content. Concomitant decrease was also noticed in aminopyrine N-demethylase and benzo[a]pyrene hydroxylase activities. However, aniline hydroxylase activity was only marginally elevated during the infection. Microsomal markers, viz., cytochrome b5, NADH-cytochrome-c reductase, and glucose-6-phosphatase, were not significantly altered. Hepatic tissue exhibited an accumulation of lipids, especially phospholipids, triglycerides, and cholesterol, resulting in fatty necrosis around the central vein region. Isolated hepatic microsomes showed a decrease in phosphatidylcholine content. Impairment in hepatic mixed function oxidase (MFO) activities was further confirmed by prolongation in hexobarbital sleeping time and zoxazolamine-induced paralysis. The hepatic MFO system of A. ceylanicum-infected hamsters responded qualitatively and quantitatively in a manner similar to that of control hamsters, upon stimulation with selective chemical inducers like phenobarbitone and 3-methylcholanthrene. Kinetic and in vitro substrate binding studies revealed that for aminopyrine the substrate affinity and the maximum enzyme activity (Vmax) were decreased, while for aniline the binding affinity was decreased and the binding capacity was enhanced. Results indicate specific/selective impairment of the hepatic microsomal cytochrome P450 system during hookworm infection and may have many practical implications in toxicology and pharmacology.     

Studies on the cellular response in avian inflammation using a simple subcutaneous pouch model

A subcutaneous pouch model was developed and used for examination of cellular events in the local acute inflammatory response in the chicken. The model involved insertion of a pair of coverslips-dipped in the irritant-into the prepared subcutaneous pouch, and their replacement with plain coverslips at frequent intervals. The model was simple to operate, caused minimal distress to the experimental bird, and permitted multiple continual sampling of emigrating cells. The cytological features and migratory sequence of inflammatory cells produced by non-immunological and immunological stimuli were studied over a period of 48 h. The initial migration of leukocytes comprised heterophils and monocytes. This was soon followed by emigration of basophils. The basophilic reaction was pronounced in response to Escherichia coli endotoxin. Lymphocytes were prominent in the later stages. Also, in the later stages the monocytes apparently coalesced, formed syncytia and even what appeared to be distinct multinucleated giant cells. The results obtained by the present model corroborated the findings of earlier investigators recorded through the use of tissue sections and impression smears. It is suggested that the subcutaneous pouch model may offer an excellent system in avian inflammation research.     

Sphingolipids Mediate Differential Echinocandin Susceptibility in Candida albicans and Aspergillus nidulans

The cell wall synthesis-inhibiting echinocandins, including caspofungin and micafungin, play important roles in the treatment of candidiasis and aspergillosis. Previous studies revealed that, in the haploid yeast Candida glabrata, sphingolipid biosynthesis pathway mutations confer caspofungin reduced susceptibility (CRS) but micafungin increased susceptibility (MIS). Here, we describe one Candida albicans strain (of 10 tested) that similarly yields CRS-MIS mutants at relatively high frequency. Mutants demonstrated increased levels of long-chain bases (sphingolipid pathway intermediates) and, unique to this strain, loss of His104/Pro104 heterozygosity in the TSC13-encoded enoyl reductase. CRS-MIS was similarly observed in a C. albicans homozygous fen1Δ fen12Δ laboratory strain and in diverse wild-type strains following exogenous long-chain-base treatment. Analogous to these results, CRS-MIS was demonstrated in an Aspergillus nidulans basA mutant encoding defective sphingolipid C₄-hydroxylase and in its wild-type parent exposed to long-chain bases. Sphingolipids likely modulate echinocandin interaction with their Fks membrane target in all susceptible fungi, with potential implications for optimizing therapy with existing antifungals and the development of novel agents.     

Mitomycin‐C: ‘a ray of hope’ in refractory corrosive esophageal strictures

Increasingly frequent dilation may become a self‐defeating cycle in refractory stricture as recurrent trauma enhance, scar formation, and ultimately recurrence and potential worsening of the stricture. In 12 patients of caustic induced esophageal stricture, who failed to respond despite rigorous dilatation regimen for more than one year, a trial of topical mitomycin‐C application to improve dilatation results was undertaken, considering the recently reported efficacy and safety of this agent. Mitomycin‐C was applied for 2–3 minutes at the strictured esophageal segment after dilation with wire‐guided Savary‐Gilliard dilator. Patient was kept nil by mouth for 2–3 hours. After 4–6 sessions of mitomycin‐C treatment, resolution of symptoms and significant improvement in dysphagia score and periodic dilatation index was seen in all 12 patients. Mitomycin‐C topical application may be a useful strategy in refractory corrosive esophageal strictures and salvage patients from surgery.     

Pre-clinical pharmacokinetic and pharmacodynamic modelling study of 4-hydroxyisoleucine using validated ultra-performance liquid chromatography-tandem mass spectrometry

A reliable and sensitive ultra-performance liquid chromatography-tandem mass spectrometry-based method has been developed for the estimation of 4-hydroxyisoleucine (4-HI), a potent insulinotropic and hypolipidemic agent. The extraction of 4-HI from plasma was accomplished by the protein precipitation technique using l-isoleucine as an internal standard. The separation of analytes was achieved with a mobile phase consisting of acetonitrile and 0.1% formic acid in an isocratic flow system on a BEH Shield RP-18 column (150 mm × 2.1 mm, 1.7 μm). 4-HI and l-isoleucine were detected using an electrospray ionization (ESI) ion source, using multiple reaction monitoring (MRM) in positive ion mode. The precursor to product ion transitions of 4-HI and l-isoleucine were found at m/z values of 148.19 > 74.02 and 132.17 > 69.04, respectively. As per the guidelines for bioanalytical methods, all validation parameter results were within the acceptable range. The method exhibited a robust and reproducible linearity range of 1–5000 ng mL⁻¹ with a coefficient of regression of 0.9999. The method was successfully applied for the estimation of pharmacokinetic parameters after oral administration of 4-HI (10 mg kg⁻¹) in Wistar rats, by using Thoth Pro (version: 4.3) software. Herein, the two-compartment model was statistically fitted based on AIC and SBC values for evaluation of the pharmacokinetic parameters of 4-HI. Pharmacodynamic studies were also performed by measuring the levels of triglyceride and total cholesterol, and showed that the pharmacokinetic and pharmacodynamic data of 4-HI correlated with each other.

A reliable and sensitive UPLC-MS/MS based method has been developed for the estimation of 4-hydroxyisoleucine, a potent insulinotropic and hypolipidemic agent.     

Functionalized poly(lactic acid) based nano-fabric for anti-viral applications

This study endeavoured to explore and fabricate antiviral and antibacterial facemasks using zinc (oligo-lactate) (ZL), developed through a microwave synthesis technique. The prepared nano-fabric layer has excellent antiviral and antibacterial properties against Newcastle Disease Virus (NDV) and E. coli and S. aureus, respectively. Thermogravimetric analysis (TGA) of ZL shows a two-step thermal degradation, which confirms the formation of low molecular weight end group lactyl units with zinc ions. Another investigation using varying ZL concentration and silk nanocrystal (SNC) with poly(lactic acid) (PLA) and electrospinning them into nanofibres led to the fabrication of a facile and sustainable nanofabric that can be utilized as a protective layer for facemasks. Morphological analysis revealed the successful preparation of the nanofabric with proper distribution and uniformity in fibre diameter. Hydrophobicity of the prepared nanofabric confirmed excellent protection from water droplets that may transpire during coughing or sneezing by an infected individual. Breathability and reusability tests confirmed that the prepared facemask could be reused by ethanol washing without compromising its surface properties till 4 cycles. The PLA/ZL nanofabric layer demonstrated 97% antiviral efficacy against NDV in 10 minutes. In conclusion, the electrospun nanofabric layer can be used as a facemask having high hydrophobicity, good breathability, antibacterial, and antiviral properties to control the spread of contagious diseases.

PLA based electrospun nanofabric prepared using ZL and SNC. Incorporation of SNC conferred hydrophobicity. Breathable and reusable nanofabric. PLA/ZL nanofabric demonstrated significant antibacterial & antiviral properties.     

Restoring nervous system structure and function using tissue engineered living scaffolds

Neural tissue engineering is premised on the integration of engineered living tissue with the host nervous system to directly restore lost function or to augment regenerative capacity following nervous system injury or neurodegenerative disease. Disconnection of axon pathways – the long-distance fibers connecting specialized regions of the central nervous system or relaying peripheral signals – is a common feature of many neurological disorders and injury. However, functional axonal regeneration rarely occurs due to extreme distances to targets, absence of directed guidance, and the presence of inhibitory factors in the central nervous system, resulting in devastating effects on cognitive and sensorimotor function. To address this need, we are pursuing multiple strategies using tissue engineered “living scaffolds”, which are preformed three-dimensional constructs consisting of living neural cells in a defined, often anisotropic architecture. Living scaffolds are designed to restore function by serving as a living labeled pathway for targeted axonal regeneration – mimicking key developmental mechanisms– or by restoring lost neural circuitry via direct replacement of neurons and axonal tracts. We are currently utilizing preformed living scaffolds consisting of neuronal clusters spanned by long axonal tracts as regenerative bridges to facilitate long-distance axonal regeneration and for targeted neurosurgical reconstruction of local circuits in the brain. Although there are formidable challenges in preclinical and clinical advancement, these living tissue engineered constructs represent a promising strategy to facilitate nervous system repair and functional recovery.     

Primary vs delayed primary closure in patients undergoing lower limb amputation following trauma: A randomised control study

Lower limb crush injury is a major source of mortality and morbidity in trauma patients. Complications, especially surgical site infections (SSIs) are a major source of financial burden to the institute and to the patient as it delays rehabilitation. As such, every possible attempt should be made to reduce any complications. We, thus, aimed to compare the outcomes in early vs delayed closure of lower extremity stumps in cases of lower limb crush injury requiring amputation, so as to achieve best possible outcome. A randomised controlled study was conducted in the Division of Trauma Surgery & Critical Care at Jai Prakash Narayan Apex Trauma Centre, All India Institute of Medical Sciences, New Delhi from 1 September 2018 to 30 June 2019 and included patients undergoing lower limb amputation below hip joint. Patients were randomised in two groups, in one group amputation stump was closed primarily, while in the second group delayed primary closure of stump was performed. We compared rate of SSI, length of hospital stay, and number of surgeries in both the groups. Fifty‐six patients with 63 amputation stumps were recruited in the study. Mean age of patients in the study was 34 years, of which about 95% patients were males. The most common mechanism of injury was road traffic injury in 66% of patients. Mean injury severity score was 12.28 and four patients had diabetes preoperatively. Total 63 extremities were randomised with 30 cases in group I and 33 cases in group II as per computer‐generated random number. Above knee amputations was commonest (57.14%) followed by below knee amputations (33.3%). Two patients died in the current study. In group I, In‐hospital infection was detected in 7 cases (23.3%) and in group II 9 cases (27.3%) had SSI during hospital admission (P > .05). Mean hospital stay in group I was 10.32 ± 7.68 days and in group II was 11 ± 8.17 days (P > .05). Road traffic injuries and train‐associated injuries are a major cause of lower limb crush injuries, leading to limb loss. Delayed primary closure of such wounds requires extra number of surgical interventions than primary closure. There is no difference in extra number of surgical interventions required in both the groups. Thus, primary closure can be safely performed in patients undergoing lower limb amputations following trauma, provided that a good lavage and wound debridement is performed.