Wafer scale growth of single crystal two-dimensional van der Waals materials.

Two-dimensional (2D) van der Waals (vdW) materials, including graphene, hexagonal boron nitride (hBN), and metal dichalcogenides (MCs), form the basis of modern electronics and optoelectronics due to their unique electronic structure, chemical activity, and mechanical strength. Despite many proof-of-concept demonstrations so far, to fully realize their large-scale practical applications, especially in devices, wafer-scale single crystal atomically thin highly uniform films are indispensable. In this minireview, we present an overview on the strategies and highlight recent significant advances toward the synthesis of wafer-scale single crystal graphene, hBN, and MC 2D thin films. Currently, there are five distinct routes to synthesize wafer-scale single crystal 2D vdW thin films: (i) nucleation-controlled growth by suppressing the nucleation density, (ii) unidirectional alignment of multiple epitaxial nuclei and their seamless coalescence, (iii) self-collimation of randomly oriented grains on a molten metal, (iv) surface diffusion and epitaxial self-planarization and (v) seed-mediated 2D vertical epitaxy. Finally, the challenges that need to be addressed in future studies have also been described.

Metformin-Loaded Hyaluronic Acid-Derived Carbon Dots for Targeted Therapy against Hepatocellular Carcinoma by Glutamine Metabolic Reprogramming.

Metabolic reprogramming is a significant hallmark of cancer that promotes chemoresistance by allowing tumor tissues to adapt to changes in the tumor microenvironment caused by anticancer therapies. Hepatocellular carcinoma (HCC), one of the most common types of primary tumors, is associated with recurrent metabolic reprogramming that maximizes cancer cell growth and proliferation. Herein, we developed metformin (MET)-loaded hyaluronic acid (HA)-derived carbon dots (HA-CD-MET) by a simple and green method with no involvement of any additives. HA-CD-MET was utilized for specifically binding the CD44 receptor overexpressed in HCC and induced glutamine metabolic rewiring to inhibit HCC cell proliferation. Exposure to HA-CD-MET resulted in ∼6.5-fold better anticancer efficacy against CD44+ Hep3B cells in comparison to CD44-, HepG2, and noncancerous HEK293 cells at a very low dose of 80 µg/mL. Moreover, treatment of three-dimensional (3D) tumor spheroid model of HCC (Hep3B) with HA-CD-MET resulted in ∼4.9-fold reduction in tumor size. This improved anticancer efficacy of HA-CD-MET was attributed to the inhibition of glutaminase-1 (GLS-1), a mitochondrial enzyme that hydrolyzes glutamine into glutamate as confirmed from immunofluorescence and immunoblotting experiments. Furthermore, treatment with HA-CD-MET resulted in downregulation of glucose transporter-1 (GLUT-1) in Hep3B cells. Consequently, cancer cells were starved from essential nutrients, glutamine, and glucose, leading to the enhancement in intracellular ROS generation. This increase in intracellular ROS accumulation activated AMP-activated protein kinase (AMPK) and inhibited AKT phosphorylation, leading to cancer cell apoptosis. Thus, this study offers the targeting of metabolic reprogramming by HA-CD-MET that opens up a promising strategy for therapeutic intervention in hepatocarcinoma.

Naphthalene Diimide-Based Orange Emitting Luminogen: A Fluorometric Probe for Thiol Sensing through the Click Reaction.

Fluorometric sensors have gained considerable attention in various fields, including environmental monitoring, biomedical research, and clinical diagnostics. This article delineates the fabrication of an orange emitting naphthalene diimide (NDI) derivative consisting of maleimide moiety (NDI-mal) for fluorometric sensing of thiols. Spherical shaped organic nanoparticles (∼100-150 nm) were constructed by NDI-mal in dimethyl sulfoxide (DMSO)/dimethylformamide (DMF)-water through J-type aggregation. NDI-mal displayed self-assembly driven aggregation-induced emission (AIE) through excimer formation at λem= 588 nm at fw = 99 vol % DMSO/DMF-water. Naphthyl residue at both terminals of NDI-mal facilitates intramolecular charge transfer (ICT) from the donor naphthyl residue to the acceptor NDI core. The fluorescence intensity of NDI-mal fluorescent organic nanoparticles (FONPs) got quenched in the presence of thiols due to thiol-maleimide adduct formation (Michael addition). NDI-mal FONPs selectively probed thiol functionalized small molecules (4-aminothiophenol), biomolecules (glutathione (GSH)), and proteins (reduced BSA) with high sensitivity having a limit of detection of 15.3 nM, 6.0 nM, and 9.2 ng/mL, respectively. Importantly, thiol sensing was selective against analogous small molecules, biomolecules, and proteins devoid of thiol moieties. Cellular imaging demonstrated selective diagnosis of cancer cells by NDI-mal FONPs through quenching of its emission upon interaction with thiols in B16F10 cells due to the high abundance of GSH in cancer cells compared to NIH3T3 cells. NDI-mal FONPs emitted their native fluorescence inside cells subjected to reactive oxygen species mediated thiol oxidation via Fenton's reaction. Notably, GSH-maleimide adduct formation by NDI-mal FONPs displayed notable therapeutic efficacy against cancer cells having ∼2.4-fold higher killing of B16F10 in comparison to NIH3T3 cells possibly through oxidative stress induced apoptosis owing to the depletion in the GSH level. Thus, NDI-mal AIE-gen successfully emerged as a selective and sensitive probe toward thiols through thiol-maleimide click chemistry with therapeutic ability against cancer cells in the absence of systematic intervention.

Naphthalimide-Based AIEgens for Sensing Protein Disulfide Isomerase through Thiol-Disulfide Redox Exchange.

Aggregation-induced emission (AIE)-based fluorescent organic nanoparticles (FONPs) with distinctive characteristics are emerging as superior sensors due to their facile fabrication, high signal-to-noise ratio, and good biocompatibility. The present article delineates the detection and analysis of the redox behavior of the protein disulfide isomerase (PDI) enzyme by exploitation of the AIE of novel naphthalimide (NI) derivatives having thiol (-SH) and disulfide (-S-S-) moieties. Self-aggregated spherical-shaped organic nanoparticles were prepared by synthesized NI-based amphiphiles (NISH, NISS, NINSS, and TNINSH) through J-type aggregation in DMSO-water (fw = 99 vol %). Naphthyl residue containing NI-derived amphiphiles (NINSS and TNINSH) exhibited AIE (blue and yellow) at 470 and 550 nm, respectively, in DMSO-water (fw = 99 vol %). NINSS and TNINSH FONPs were suitably utilized in sensing PDI through their redox nature of thiol-disulfide exchange. Fluorescence quenching of NINSS FONPs was observed due to reduction of disulfide to thiol by PDI, whereas emission intensity was progressively red-shifted and enhanced ("Dual-AIE") for TNINSH (containing ER-targeting N-tosylethylenediamine), owing to oxidation of thiol to disulfide by PDI. NINSS and TNINSH FONPs were found to be highly efficient in sensing PDI through the AIE-based "fluorescence off/on" mechanism having limits of detection of ∼12.6-17.7 and ∼11.7-16.5 ng/mL, respectively. In vitro cell imaging for NIH3T3 (noncancer) and B16F10 (melanoma) cells with NINSS and TNINSH FONPs displayed excellent diagnosis of eukaryotic cells upon interaction with indigenous PDI. Notably, detection of cancer cells was more sensitive over the noncancerous cells by these FONPs due to overexpression of PDI within cancer cells.

Faster and accurate identification of clinically important Trichosporon using MALDI TOF MS.

PURPOSE: Trichosporon species are emerging human pathogens, accounting for the second most common cause of non-candidal mycosis. Rapid and reliable identification of these agents allows a better understanding of their epidemiology and therapeutic management. The Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) technique has the potential to be precise, fast and cost-effective. However, the precision of identification totally depends upon the type of protein extraction method used and embedded database in the system. Our objectives were to standardize the protein extraction technique and expand the present Bruker database by creating an in-house database and validating it with diverse clinical Trichosporon species of Indian origin. METHODS: Two different protein extraction protocols (on-plate and off-plate) were evaluated. The off-plate protocol was finalized for the identification. MALDI TOF MS with the existing Bruker database was evaluated for its ability to identify a total of 79 intergenic spacer 1 (IGS1) gene sequence confirmed clinical isolates of 5 different Trichosporon species. RESULTS: As outcome, off plate protocol yielded higher accuracy (73% on the species level and 95% on the genus level) than on-plate (25% on the genus level) in terms of log scores. The existing database for Trichosporon species was enriched with 28 sequence confirmed isolates, which improved accuracy from 73% to 100% and were identified up to species level with a log score >2.3. CONCLUSIONS: Used with standardized protein-extraction protocol along with an expanded database, MALDI-TOF MS could be a rapid and reliable approach to identify clinical Trichosporon species routinely in the laboratory.

Folic Acid-Functionalized Carbon Dot-Enabled Starvation Therapy in Synergism with Paclitaxel against Breast Cancer.

Glucose oxidase (GOx)-induced cancer starvation has recently emerged for halting the abnormal proliferation of triple-negative breast cancer (TNBC). However, monotherapy with GOx or a conventional chemotherapeutic displays suboptimal efficacy in eliminating tumors and poses impending risks to healthy tissues. To augment therapeutic efficacy and tumor selectivity, folic acid (FA)-functionalized carbon dots (CDs) embedded with GOx and paclitaxel (PTX) [FA-CD-(PTX-GOx)] was developed that showed the efficient killing of TNBC, MDA-MB-468 cells over noncancerous HEK 293 cells through synergistic effects of cancer starvation-induced oxidative stress and chemotherapy. The cargo-laden FA-CD complex resulted in a 4-8 fold increase in cancer cell death at 60 µg/mL when compared to standalone therapy with the native compounds and individually loaded cargo on FA-CD. This improved cancer cell killing efficacy of the FA-CD-(PTX-GOx) complex could be endorsed by folate receptor (FR)-mediated target-specific cellular internalization of the FA-CD complex. The antitumorigenic efficacy of the FA-CD-(PTX-GOx) complex was further validated in a three-dimensional (3D) breast tumor spheroid model. A significant 4.5-fold reduction in spheroid dimension along with antiproliferation was observed with time up to 72 h following exposure to the FA-CD-(PTX-GOx) complex. This antitumorigenic potential of FA-CD-(PTX-GOx) could be attributed to the enhanced intratumoral reactive oxygen species generation following glucose depletion by GOx that has been facilitated by the chemotherapeutic efficacy of PTX resulting in the efficient killing of cancer cells. The present study provides a novel strategy of FR-mediated fluorescent CD-enabled combined formulation of GOx and PTX for the target-specific superior killing of TNBC cells in the synergism of glucose starvation with chemotherapy.

Lipase sensing by naphthalene diimide based fluorescent organic nanoparticles: a solvent induced manifestation of self-assembly.

The precise control of supramolecular self-assembly is gaining utmost interest for the demanding applications of manifested nano-architecture across the scientific domain. This study delineates the morphological transformation of naphthalene diimide (NDI) derived amphiphiles with varying water content in dimethyl sulfoxide (DMSO) and the selective sensing of lipase using its aggregation-induced emission (AIE) properties. To this end, NDI-based, benzyl alcohol protected alkyl chain (C1, C5, and C10) linked amphiphilic molecules (NDI-1,2,3) were synthesized. Among the synthesized amphiphiles, benzyl ester linked C5 tailored naphthalene diimide (NDI-2) exhibited AIE with an emission maximum at 490 nm in a DMSO-water binary solvent system from fw = 30% and above water content. The fibrous morphology of NDI-2 at fw = 30% got gradually transformed to spherical aggregated particles along with steady increment in the emission intensity upon increasing the amount of water in DMSO. At fw = 99% water in DMSO, complete transformation to fluorescent organic nanoparticles (FONPs) was observed. Microscopic and spectroscopic techniques demonstrated the solvent driven morphological transformation and the AIE property of NDI-2. Moreover, this AIE of NDI-2 FONPs was employed in the selective turn-off sensing of lipase against many other enzymes including esterase, through hydrolysis of a benzyl ester linkage with a limit of detection 10.0 ± 0.8 µg L-1. The NDI-2 FONP also exhibited its lipase sensing efficiency in vitro using a human serum sample.

AgNPs Ornamented Modified Bacterial Cellulose Based Self-Healable L-B-L Assembly via a Schiff Base Reaction: A Potential Wound Healing Patch.

A layer-by-layer (L-B-L) bacterial cellulose (BC)-based transdermal patch has been prepared via a Schiff base reaction. The L-B-L assembly consisting of covalently cross-linked ethylene diamine-modified carboxymethylated BC isolated from the Glucanoacetobacter xylinus (MTCC7795) bacterial strain and aldehyde-modified pectin formed via a Schiff base reaction. The presence of the imine bond assists the self-healing process after being scratched in the presence of a pH 7.4 buffer solution monitored via optical microscopy, atomic force microscopy, and tensile strength analyses. The formation of the L-B-L assembly was confirmed using field-emission scanning electron microscopy (FESEM) analysis. Simultaneously, water swelling and deswelling studies were carried out to test its water retention efficiency. The presence of silver nanoparticles (AgNPs) has been confirmed by ultraviolet-visible spectroscopy and FESEM analyses. The antimicrobial activity of the AgNPs-incorporated transdermal patch has been examined over Staphylococcus aureus and Escherichia coli using the zone of inhibition method. Additionally, the cell viability assay was performed using the fluorescent dyes 4',6-diamidino-2-phenylindole and propidium iodide. The AgNPs in the L-B-L assembly showed antimicrobial property against both types of bacteria. The cytotoxicity and wound healing property of the patch system have been studied over NIH 3T3 fibroblast and A549 epithelial cell lines. The L-B-L film also influenced the wound healing process of these two cell lines.

Nipecotic-Acid-Tethered, Naphthalene-Diimide-Based, Orange-Emitting Organic Nanoparticles as Targeted Delivery Vehicle and Diagnostic Probe toward GABAA-Receptor-Enriched Cancer Cells.

This article demonstrates target-specific cellular imaging of GABA (γ-aminobutyric acid) receptor (GABAAR)-enriched cells (SH-SY5Y and A549) with therapeutic efficacy by naphthalene diimide (NDI)-derived fluorescent organic nanoparticles (FONPs). Self-assembly-driven formation of spherical organic particles by nipecotic-acid-tethered l-aspartic acid appended NDI derivative (NDI-nip) took place in DMSO-water through J-type aggregation. NDI-nip having a naphthyl residue and a nipecotic acid unit at both terminals exhibited aggregation-induced emission (AIE) at and above 60% water content in DMSO because of excimer formation at λem = 579 nm. The orange-emitting NDI-nip FONPs (1:99 v/v DMSO-water) having excellent cell viability and high photostability were used for selective bioimaging and killing of GABAAR-overexpressed cancer cells through target-specific delivery of the anticancer drug curcumin. The fluorescence intensity of NDI-nip FONPs were quenched in GABAAR-enriched neuroblastoma cells (SH-SY5Y) and cancerous cells (A549). Notably, in the presence of GABA, the NDI-nip FONPs exhibited their native fluorescence within the same cell lines. Importantly, no such quenching and regaining of NDI-nip FONP emission in the presence of GABA was noted in the case of the noncancerous cell NIH3T3. The killing efficiency of curcumin-loaded NDI-nip FONPs ([curcumin] = 100 µM and [NDI-nip FONPs] = 50 µM) was significantly higher in the cases of SH-SY5Y (88 ± 3%) and A549 (72 ± 2%) than in NIH3T3 (37 ± 2). The presence of a nipecotic acid moiety facilitated the selective cellular internalization of NDI-nip FONPs into GABAAR-overexpressing cells. Hence, these orange-emitting NDI-nip FONPs may be exploited as a targeted diagnostic probe as well as a drug delivery vehicle for GABAAR-enriched cancer cells.

Candidaemia in a tertiary care centre of developing country: Monitoring possible change in spectrum of agents and antifungal susceptibility.

Purpose: Candidaemia is a major cause of morbidity and mortality of hospitalised patients, especially in developing countries. This study was conducted to monitor any change in species distribution and antifungal susceptibility pattern of Candida species causing candidaemia over the last 20 years. Materials and Methods: The candidaemia cases reported during January 1999 and December 2018 at our centre were reviewed. The yeasts were identified by phenotypic characters (during 1999-2014) and matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) (during 2014-2018). Antifungal susceptibility testing (AFST) was performed in accordance with the Clinical and Laboratory Standards Institute guidelines. Results: A total of 602,963 blood samples from patients with suspected sepsis were processed. Candidaemia was diagnosed in 7927 (1.31%) cases. The frequency of cases rose significantly (P = 0.000) in the last quarter of the study. Candida tropicalis (40.1%) was the most common species, followed by Candida albicans (15.2%), Wickerhamomyces anomalus (13.1%), Candida krusei (6.6%), Candida parapsilosis (4.7%) and others. Rare species such as Candida auris, Candida lambica, Candida orthopsilosis, Candida vishwanathii were identified after the introduction of MALDI-TOF. The minimum inhibitory concentrations of amphotericin B rose significantly from the first to last quarter (0.5%-4.9%). Fluconazole resistance was fairly constant at 7.4%-8.8%. Conclusion: Local epidemiology of candidaemia at our centre was distinct regarding prevalence and change of spectrum of species. The identification of rare species was possible after the introduction of MALDI-TOF. With the emergence of multidrug-resistant C. auris and resistance in other species, routine AFST has become imperative.

Mucormycosis due to Apophysomyces species complex- 25 years' experience at a tertiary care hospital in southern India.

Apophysomyces elegans species complex is an important cause of cutaneous mucormycosis in India. However, majority of those cases are reported as case reports only. We desired to analyze our patients with Apophysomyces infection reported over 25 years (1992-2017) to understand the epidemiology, management, and outcome of the disease. During the study period 24 cases were reported, and the majority (95.8%) of them presented with necrotizing fasciitis following accidental/surgical/iatrogenic trauma. One patient presented with continuous ambulatory peritoneal dialysis (CAPD) related peritonitis. Healthcare related Apophysomyces infection was noted in 29.2% patients. In addition to trauma, comorbidities were noted in 37.5% patients (type 2diabetes mellitus-6, chronic alcoholism-2, and chronic kidney disease-1). Of the 24 isolates, 11 isolates starting from year 2014 were identified as Apophysomyces variabilis by molecular methods. Majority (95.8%) of the patients were managed surgically with or without amphotericin B deoxycholate therapy, while one patient was treated with amphotericin B deoxycholate alone. Among 24 patients, seven (29.1%) recovered, six (25%) patients could not afford antifungal management and left the hospital against medical advice, and 11 (45.9%) patients died.The present case series highlights that necrotizing fasciitis caused by A. variabilis is prevalent in India, and the disease may be healthcare related. Although diagnosis is not difficult, awareness among surgeons is still limited about the infection, leading to a delay in sending samples to the mycology laboratory. Apophysomyces infection must be considered in the differential diagnosis in apatient with progressive necrosis of a wound who is not responding to antibacterial therapy.

An aero mycological analysis of Mucormycetes in indoor and outdoor environments of northern India.

Mucormycosis is an angio-invasive infection, predominantly acquired by inhalation of sporangiospores from the environment. However, the burden of Mucormycetes sporangiospores in the air is not well studied. We aimed to estimate the burden of Mucormycetes spores in the outdoor and indoor (hospital) environment across different seasons in north India. A total of 380 air samples from outdoor (n = 180) and indoor (n = 200) environment were included in the study. Air samples were suctioned using air sampler (100 l/min) and cultured on Dichloran Rose Bengal Chloramphenicol (DRBC) with benomyl for selective isolation of Mucormycetes. The isolates were identified by phenotypic and genotypic methods. The mean spore count (±SD) of Mucormycetes (cfu/m3) in outdoor samples varied from 0.73 (±0.96) to 8.60 (±5.70) across different seasons. In hospital, the mean spore count varied from 0.68 (±1.07) to 1.12 (±1.07) and 0.88 (±1.01) to 1.72 (±2.17) for air-conditioned wards and non-air-conditioned wards, respectively. Rhizopus arrhizus was the predominant agent isolated from both indoor and outdoor environment followed by Cunninghamella species. We also report a single isolate of the rare mucormycete agent, Apophysomyces variabilis from outdoor environment. The present study highlights the presence of low spore burden of Mucormycetes in outdoor and hospital settings in north India. This study also reports the first isolation of A. variabilis from air samples in the Indian subcontinent.

Fabrication of Orange-Emitting Organic Nanoparticle-Protamine Conjugate: Fluorimetric Sensor of Heparin.

Among the diverse sensing techniques, fluorimetric detection dominates over the other methods because of its rapid signaling, high selectivity and sensitivity, and operational simplicity. This present article delineates fabrication of a fluorescent organic nanoparticle-protamine (FONP-Pro) conjugate for selective and sensitive detection of heparin simply by exploitation of the aggregation-induced emission (AIE) property of the FONPs. Naphthalene diimide-based bola-type amphiphilic molecules (NDI-1) comprise a naphthyl residue and a 3-aminopyridyl unit at both terminals, forming organic nanoparticles in a dimethyl sulfoxide-water binary solvent mixture, and exhibited AIE through excimer formation. The presence of naphthyl residue in the molecular backbone facilitates the intramolecular charge transfer to generate orange-emitting (λem = 594 nm) AIE-luminogen (AIE-gen). The aminopyridine residues within NDI-1 induced negative surface charge on NDI-1 FONPs, which facilitated interaction with positively charged protamine (Pro) to construct FONP-Pro conjugates. Formation of this NDI-1 FONP-Pro conjugate through the interaction between Pro and FONP drastically reduced the orange emission intensity (fluorescence off) of the AIE-gens. Interestingly, addition of heparin to this FONP-Pro conjugate turned on the fluorescence signal of FONPs through unwinding of the Pro from the FONP surface because of a strong binding affinity between heparin and Pro. Formation of the FONP-Pro conjugate and fluorimetric sensing of heparin was investigated by monitoring the change in emission behavior of NDI-1 FONPs. Also, the heparin-sensing was found to be highly selective against many other biomolecules including proteins, enzymes, and DNA. Hence, a selective and efficient heparin sensor (FONP-Pro) was developed having a limit of detection of 12 nM simply by utilizing the fluorescence "turn-off" and "turn-on" mechanism of NDI-1 FONP.

Evaluation of Liposomal and Conventional Amphotericin B in Experimental Fungal Keratitis Rabbit Model.

PURPOSE: We evaluate the efficacy of liposomal amphotericin (Fungisome) compared to conventional amphotericin (AMB) for the treatment of fungal keratitis (FK) in an experimental rabbit model. METHODS: FK was induced in 48 New Zealand White rabbits using Aspergillus flavus and Candida albicans (24 rabbits each). Rabbits were divided into four groups: 0.1% and 0.05% Fungisome-, and 0.1% AMB-treated groups, and one untreated control group. Clinical scores were recorded throughout the study while fungal burden was estimated by corneal button culture on day 19 (study endpoint). RESULTS: A statistically significant improvement in clinical score was seen on day 11 in the 0.1% and 0.05% Fungisome versus untreated groups (13.91 and 14.4 vs. 19.3; P < 0.001) in the A. flavus model, and on day 9 in the 0.1% Fungisome-treated versus untreated groups (12.96 vs. 14.2; P = 0.006) in the C. albicans model. At endpoint, the mean clinical scores of the untreated controls, and the 0.1% and 0.05% Fungisome-, and 0.1% AMB-treated groups were 20 ± 1.4, 5.33 ± 1.85, 9.66 ± 2.41, and 8.16 ± 1.95, respectively, in the A. flavus model and 15.85 ± 1.87, 3.08 ± 1.31, 4.21 ± 1.370, and 4.13 ± 1.38, respectively, in the C. albicans model. Conjunctival hyperemia score was higher in the 0.1% AMB- versus 0.1% Fungisome-treated groups (1.33 vs. 0.5, P = 0.452). Lowest fungal burden in both models was seen in the 0.1% Fungisome-treated groups. CONCLUSIONS: Clinical improvement was observed with Fungisome relative to untreated controls. However, no statistically significant differences in outcomes were observed between animals treated with Fungisome and AMB. Although the results are encouraging, future studies in humans are warranted. TRANSLATIONAL RELEVANCE: FK is a leading cause of corneal blindness and is on the rise especially in developing countries. Despite the availability of various antifungal agents, heterogeneous treatment outcomes are seen due to lack of a standardized treatment regimen for FK. Although the use of liposomal AMB has been substantiated by clinical evidence in systemic infections, to our knowledge there are no in vivo studies evaluating the role of topical liposomal versus conventional formulation in FK. Our study investigated the efficacy and toxicity profile of liposomal versus conventional formulation of AMB in an experimental rabbit FK model.

A prospective multicenter study on mucormycosis in India: Epidemiology, diagnosis, and treatment.

Mucormycosis due to Mucorales is reported at large numbers in uncontrolled diabetics across India, but systematic multicenter epidemiological study has not been published yet. The present prospective study was conducted at four major tertiary care centers of India (two in north and two in south India) during 2013-2015 to compare the epidemiology, treatment strategies and outcome of mucormycosis between the two regions. Molecular techniques were employed to confirm the identity of the isolates or to identify the agent in biopsy samples. A total of 388 proven/probable mucormycosis cases were reported during the study period with overall mortality at 46.7%. Uncontrolled diabetes (n = 172, 56.8%) and trauma (n = 31, 10.2%) were the common risk factors. Overall, Rhizopus arrhizus (n = 124, 51.9%) was the predominant agent identified, followed by Rhizopus microsporus (n = 30, 12.6%), Apophysomyces variabilis (n = 22, 9.2%) and Rhizopus homothallicus (n = 6, 2.5%). On multivariate analysis, the mortality was significantly associated with gastrointestinal (OR: 18.70, P = .005) and pulmonary infections (OR: 3.03, P = .015). While comparing the two regions, majority (82.7%) cases were recorded from north India; uncontrolled diabetes (n = 157, P = .0001) and post-tubercular mucormycosis (n = 21, P = .006) were significantly associated with north Indian cases. No significant difference was noted among the species of Mucorales identified and treatment strategies between the two regions. The mortality rate was significantly higher in north Indian patients (50.5%) compared to 32.1% in south India (P = .016). The study highlights higher number of mucormycosis cases in uncontrolled diabetics of north India and emergence of R. microsporus and R. homothallicus across India causing the disease.

Apophysomyces variabilis: draft genome sequence and comparison of predictive virulence determinants with other medically important Mucorales.

BACKGROUND: Apophysomyces species are prevalent in tropical countries and A. variabilis is the second most frequent agent causing mucormycosis in India. Among Apophysomyces species, A. elegans, A. trapeziformis and A. variabilis are commonly incriminated in human infections. The genome sequences of A. elegans and A. trapeziformis are available in public database, but not A. variabilis. We, therefore, performed the whole genome sequence of A. variabilis to explore its genomic structure and possible genes determining the virulence of the organism. RESULTS: The whole genome of A. variabilis NCCPF 102052 was sequenced and the genomic structure of A. variabilis was compared with already available genome structures of A. elegans, A. trapeziformis and other medically important Mucorales. The total size of genome assembly of A. variabilis was 39.38 Mb with 12,764 protein-coding genes. The transposable elements (TEs) were low in Apophysomyces genome and the retrotransposon Ty3-gypsy was the common TE. Phylogenetically, Apophysomyces species were grouped closely with Phycomyces blakesleeanus. OrthoMCL analysis revealed 3025 orthologues proteins, which were common in those three pathogenic Apophysomyces species. Expansion of multiple gene families/duplication was observed in Apophysomyces genomes. Approximately 6% of Apophysomyces genes were predicted to be associated with virulence on PHIbase analysis. The virulence determinants included the protein families of CotH proteins (invasins), proteases, iron utilisation pathways, siderophores and signal transduction pathways. Serine proteases were the major group of proteases found in all Apophysomyces genomes. The carbohydrate active enzymes (CAZymes) constitute the majority of the secretory proteins. CONCLUSION: The present study is the maiden attempt to sequence and analyze the genomic structure of A. variabilis. Together with available genome sequence of A. elegans and A. trapeziformis, the study helped to indicate the possible virulence determinants of pathogenic Apophysomyces species. The presence of unique CAZymes in cell wall might be exploited in future for antifungal drug development.

A Peptide Based Pro-Drug Ameliorates Amyloid-ß Induced Neuronal Apoptosis in In Vitro SH-SY5Y Cells.

BACKGROUND: Alzheimer's disease (AD), a common protein misfolding progressive neurodegenerative disorder, is one of the most common forms of dementia. Amyloid precursor protein (APP) derived amyloid-ß (Aß) protein accumulate into interneuronal spaces and plays a crucial role in the disease progression and its pathology. The aggregated Aß exerts its neurotoxic effects by inducing apoptosis and oxidative damage in neuronal cells. OBJECTIVES: We have investigated the effects of a synthesized Pro-Drug peptide (PDp) on Aß1-40 induced cytotoxicity in human neuroblastoma SH-SY5Y cells, represents one of the most effective strategies in combating human AD. METHODS: Cells were treated with Aß1-40 to induce cytotoxicity in the experimental model of AD to screen the inhibitory effect of PDp. Assays for cell viability, reactive oxygen species (ROS) generation, levels of intracellular free Ca2+ and expression of key apoptotic proteins were assessed by Western Blotting. RESULTS: Our results showed that Aß1-40 induces for 24h caused reduce cell viability, imbalance in Ca2+ homeostasis and increase in neuronal apoptosis in vitro. Treatment with PDp could effectively ameliorated Aß1-40 induced neurotoxicity and attenuates ROS generation that mediates apoptotic signaling through Bcl-2, Bax, Caspase-3 activity and cytochrome c in the cells. CONCLUSION: These findings suggested that PDp has potential role as a neuroprotective and therapeutic agent for combating human AD.

Protective effects of ß-sheet breaker α/ß-hybrid peptide against amyloid ß-induced neuronal apoptosis in vitro.

Alzheimer's disease is most common neurodegenerative disorder and is characterized by increased production of soluble amyloid-ß oligomers, the main toxic species predominantly formed from aggregation of monomeric amyloid-ß (Aß). Increased production of Aß invokes a cascade of oxidative damages to neurons and eventually leads to neuronal death. This study was aimed to investigate the neuroprotective effects of a ß-sheet breaker α/ß-hybrid peptide (BSBHp) and the underlying mechanisms against Aß40 -induced neurotoxicity in human neuroblastoma SH-SY5Y cells. Cells were pretreated with the peptide Aß40 to induce neurotoxicity. Assays for cell viability, cell membrane damage, cellular apoptosis, generation of reactive oxygen species (ROS), intracellular free Ca2+ , and key apoptotic protein levels were performed in vitro. Our results showed that pretreatment with BSBHp significantly attenuates Aß40 -induced toxicity by retaining cell viability, suppressing generation of ROS, Ca2+ levels, and effectively protects neuronal apoptosis by suppressing pro-apoptotic protein Bax and up-regulating antiapoptotic protein Bcl-2. These results suggest that α/ß-hybrid peptide has neuroprotective effects against Aß40 -induced oxidative stress, which might be a potential therapeutic agent for treating or preventing neurodegenerative diseases.

The environmental source of emerging Apophysomyces variabilis infection in India.

The rare mucoraceous fungus, Apophysomyces species complex ranks second after Rhizopus arrhizus causing mucormycosis in India. The source of this agent in the environment is not clearly known. We conducted an environmental study to find its presence in Indian soil. The soil samples from different geographical locations were analyzed for isolation of Mucorales. Rhizopus arrhizus (24.6%) was most commonly isolated from soil, followed by Lichtheimia spp. (23.2%), Cunninghamella spp. (21.7%), Rhizopus microsporus (14%) and Apophysomyces spp. (4.5%). The isolation of Apophysomyces species complex was significantly associated with low nitrogen content of the soil. Based on sequencing of internal transcribed spacer (ITS) and 28S (D1/D2) regions of ribosomal DNA, the Apophysomyces isolates were identified as Apophysomyces variabilis with 98 to 100% similarity to type strain A. variabilis (CBS658.93). The analysis of amplified fragment length polymorphism (AFLP) fingerprinting data demonstrated genomic diversity of A. variabilis isolates with multiple clades (similarity 40-90%). The minimum inhibitory concentrations (MIC), MIC50 and MIC90 for A. variabilis isolates were 1 and 4 µg/ml for amphotericin B, 0.25 and 0.5 µg/ml for itraconazole, 0.125 and 0.25 µg/ml for posaconazole, 0.06 and 0.12 µg/ml for terbinafine, respectively. The present study revealed abundant presence of A. variabilis in Indian soil with low nitrogen content, its genetic heterogeneity and relatively high MICs for amphotericin B.

Chronic administration of bacopa monniera increases BDNF protein and mRNA expressions: a study in chronic unpredictable stress induced animal model of depression.

OBJECTIVE: The present study aimed to investigate whether graded doses of Bacopa Monniera (BM) extract could produce antidepressant-like effects in chronic unpredictable stress (CUS) induced depression in rats and its possible mechanism(s). METHODS: Rats were subjected to an experimental setting of CUS. The effect of BM extract treatment in CUS-induced depression was examined using behavioral tests including the sucrose consumption, open field test and shuttle box escape test. The mechanism underlying the antidepressant-like action of BM extract was examined by measuring brain-derived neurotrophic factor (BDNF) protein and mRNA expression in brain tissues of CUS-exposed rats. RESULTS: Exposure to CUS for 4 weeks caused depression-like behavior in rats, as indicated by significant decreases in sucrose consumption, locomotor activity and escape latency. In addition, it was found that BDNF protein and mRNA levels in the hippocampus and frontal cortex were lower in CUS-treated rats, as compared to controls. Daily administration of the graded doses of BM extract during the 4-week period of CUS significantly suppressed behavioral changes and attenuated the CUS-induced decrease in BDNF protein and mRNA levels in the hippocampus and frontal cortex. CONCLUSION: The results suggest that BM extract alleviates depression induced by CUS. Present study also confirms that 80-120 mg/kg doses of BM extract have significantly higher antidepressant-like activity.