Combinatorial Delivery of Gallium (III) Nitrate and Curcumin Complex-Loaded Hollow Mesoporous Silica Nanoparticles for Breast Cancer Treatment.

The main aims in the development of a novel drug delivery vehicle is to efficiently carry therapeutic drugs in the body's circulatory system and successfully deliver them to the targeted site as needed to safely achieve the desired therapeutic effect. In the present study, a passive targeted functionalised nanocarrier was fabricated or wrapped the hollow mesoporous silica nanoparticles with 3-aminopropyl triethoxysilane (APTES) to prepare APTES-coated hollow mesoporous silica nanoparticles (HMSNAP). A nitrogen sorption analysis confirmed that the shape of hysteresis loops is altered, and subsequently the pore volume and pore diameters of GaC-HMSNAP was reduced by around 56 and 37%, respectively, when compared with HMSNAP. The physico-chemical characterisation studies of fabricated HMSNAP, Ga-HMSNAP and GaC-HMSNAP have confirmed their stability. The drug release capacity of the fabricated Ga-HMSNAP and GaC-HMSNAP for delivery of gallium and curcumin was evaluated in the phosphate buffered saline (pH 3.0, 6.0 and 7.4). In an in silico molecular docking study of the gallium-curcumin complex in PDI, calnexin, HSP60, PDK, caspase 9, Akt1 and PTEN were found to be strong binding. In vitro antitumor activity of both Ga-HMSNAP and GaC-HMSNAP treated MCF-7 cells was investigated in a dose and time-dependent manner. The IC50 values of GaC-HMSNAP (25 µM) were significantly reduced when compared with free gallium concentration (40 µM). The mechanism of gallium-mediated apoptosis was analyzed through western blotting and GaC-HMSNAP has increased caspases 9, 6, cleaved caspase 6, PARP, and GSK 3ß(S9) in MCF-7 cells. Similarly, GaC-HMSNAP is reduced mitochondrial proteins such as prohibitin1, HSP60, and SOD1. The phosphorylation of oncogenic proteins such as Akt (S473), c-Raf (S249) PDK1 (S241) and induced cell death in MCF-7 cells. Furthermore, the findings revealed that Ga-HMSNAP and GaC-HMSNAP provide a controlled release of loaded gallium, curcumin and their complex. Altogether, our results depicted that GaC-HMNSAP induced cell death through the mitochondrial intrinsic cell death pathway, which could lead to novel therapeutic strategies for breast adenocarcinoma therapy.

CTX-M-type ESBL-mediated resistance to third-generation cephalosporins and conjugative transfer of resistance in Gram-negative bacteria isolated from hospitals in Tamil Nadu, India.

Clinical pathogens, especially Gram-negative bacteria developing resistance to third-generation cephalosporins, are making clinical outcomes more complicated and serious. This study was undertaken to evaluate the distribution of CTX-M-type extended-spectrum ß-lactamases (ESBLs) in Tamil Nadu, India. For this study, clinical samples were collected from five different hospitals located in Tamil Nadu and the ESBL-producing Gram-negative isolates were characterized. MIC was performed using cefotaxime and ceftazidime. The bla ESBL-producing genes were screened using multiplex PCR for the genes, CTX-M group-1, -2, -8, -9, -26. The conjugation studies were performed using Escherichia coli AB1157 as a recipient for the isolates harbouring plasmid-borne resistance following broth-mating experiment. In total, 1500 samples were collected and 599 Gram-negative bacteria were isolated that included E. coli (n=233), Klebsiella pneumoniae (n=182), Pseudomonas aeruginosa (n=79), Citrobacter spp. (n=30), Proteus mirabilis (n=28), Salmonella spp. (n=21), Acinetobacter baumannii (n=12), Serratia spp. (n=6), Shigella spp. (n=4), Morganella morganii (n=3) and Providencia spp. (n=1). MIC results showed that 358 isolates were resistant to cefotaxime and ceftazidime. Further, ESBL gene-amplification results showed that 19 isolates had CTX-M group-1 gene including E. coli (n=16), K. pneumoniae (n=2) and P. aeruginosa (n=1) whereas one M. morganii isolate had CTX-M group-9, which was plasmid-borne. Through conjugation studies, 12/20 isolates were found to be involved in the transformation of its plasmid-borne resistance gene. Our study highlighted the importance of horizontal gene transfer in the dissemination of plasmid-borne bla CTX-M-type resistance genes among the clinical isolates.

An ingenious non-spherical mesoporous silica nanoparticle cargo with curcumin induces mitochondria-mediated apoptosis in breast cancer (MCF-7) cells.

Curcumin delivery to cancer cells is challenging due to its hydrophobic nature, low bio distribution and low availability. Many nano vehicles suffer from low stability and toxicity, and hence the prerequisite of a non-toxic nano vehicle with effective drug delivery is still being delved. The present study investigates the delivery efficiency of curcumin with non-spherical mesoporous silica nanoparticles (MSNAs). Their mechanism of drug delivery and signalling proteins activated to induce apoptosis was further explored in MCF-7 cells. A non-spherical MSN was synthesised, functionalised with PEI (MSNAP) and analysed its intracellular behaviour. Our result indicates that MSNAP was non-toxic until 20 µg/mL and likely localizes in cytoplasmic vesicles. On contrast, well-known MCM-41P induced autophagosome formation, indicating cellular toxicity. Curcumin was loaded on MSNAP and its effectiveness in inducing cell death was studied in MCF-7 and in MCF-7R cells. Curcumin loading on MSNAP induces better cell death with 30 µM curcumin, better than unbounded curcumin. Western blot analysis suggest, curcumin induce apoptosis through the activation of caspase 9, 6, 12, PARP, CHOP and PTEN. The cell survival protein Akt1 was downregulated by curcumin with and without the nanostructure. Interestingly, cleaved caspase 9 was activated in higher amount in nano-conjugated curcumin compared to the free curcumin. But other ER resident protein like IRE1α, PERK and GRP78 were downregulated indicating curcumin disturbs ER homeostasis. Further, electron microscopic analysis reveled that nanocurcumin induced apoptosis by disrupting mitochondria and nucleus. Our results with doxorubicin resistant MCF-7 cell lines confirm nanodelivery of doxorubicin and curcumin sensitised cells effectively at lesser concentration. Further docking studies of curcumin indicate it interacts with the apoptotic proteins through hydrogen bonding formation and with higher binding energy.

Insights on the involvement of (-)-epigallocatechin gallate in ER stress-mediated apoptosis in age-related macular degeneration.

Endoplasmic reticulum (ER) stress-mediated apoptosis is a well-known factor in the pathogenesis of age-related macular degeneration (AMD). ER stress leads to accumulation of misfolded proteins, which in turn activates unfolded protein response (UPR) of the cell for its survival. The prolonged UPR of ER stress promotes cell death; however, the transition between adaptation and ER stress-induced apoptosis has not been clearly understood. Hence, the present study investigates the regulatory effect of (-)-epigallocatechin gallate (EGCG) on ER stress-induced by hydrogen peroxide (H2O2) and disturbance of calcium homeostasis by thapsigargin (TG) in mouse retinal pigment epithelial (MRPE) cells. The oxidant molecules influenced MRPE cells showed an increased level of intracellular calcium [Ca2+]i in ER and transferred to mitochondria through ER-mitochondrial tether site then increased ROS production. EGCG restores [Ca2+]i homeostasis by decreasing ROS production through inhibition of prohibitin1 which regulate ER-mitochondrial tether site and inhibit apoptosis. Effect of EGCG on ER stress-mediated apoptosis was elucidated by exploring the UPR signalling pathways. EGCG downregulated GRP78, CHOP, PERK, ERO1α, IRE1α, cleaved PARP, cleaved caspase 3, caspase 12 and upregulated expression of calnexinin MRPE cells. In addition to this, inhibition of apoptosis by EGCG was also confirmed with expression of proteins Akt, PTEN and GSK3ß. MRPE cells with EGCG upregulates phosphorylation of Akt at ser473 and phospho ser380 of PTEN, but phosphorylation at ser9 of GSK3ß was inhibited. Further, constitutively active (myristoylated) CA-Akt transfected in MRPE cells had an increased Akt activity in EGCG influenced cells. These findings strongly suggest that antioxidant molecules inhibit cell death through the proper balancing of [Ca2+]i and ROS production in order to maintain UPR of ER in MRPE cells. Thus, modulation of UPR signalling may provide a potential target for the therapeutic approaches of AMD.

A new frog species from the central western Ghats of India, and its phylogenetic position.

Tropical evergreen forests of Indian subcontinent, especially of the Western Ghats, are known hot spots of amphibian diversity, where many new anuran species await to be identified. Here we describe from the Sharavathi River basin of central Western Ghats a new shrub-frog taxon related to the anuran family Rhacophoridae. The new frog possesses the characteristic features of rhacophorids (dilated digit tips with differentiated pads circumscribed by a complete groove, intercalary cartilages on digits, T-shaped terminal phalanges and granular belly, the adaptive characters for arboreal life forms), but also a suite of unique features that distinguish it from all known congeners in the region. Morphogenetic analysis based on morphological characteristics and diversity in the mitochondrial 12S and 16S rRNA genes revealed it to be a new Philautus species that we named Philautus neelanethrus sp. nov. The phylogenetic analysis suggests the new frog to represent a relatively early Philautus species lineage recorded from the region. The distribution pattern of the species suggests its importance as a bioindicator of habitat health. In general, this relatively widespread species was found distributed only in non-overlapping small stretches, which indirectly indicates the fragmentation of the evergreen to moist deciduous forests that characterize the Western Ghats. Thus the discovery of the new rhacophorid species described here not only further reinforces the significance of the Western Ghats as a major hotspot of amphibian biodiversity, but also brings into focus the deterioration of forest habitats in the region and the need for prioritization of their conservation.

Triose phosphate isomerase, a novel enzyme-crystallin, and tau-crystallin in crocodile cornea. High accumulation of both proteins during late embryonic development.

Several enzymes are known to accumulate in the cornea in unusually high concentrations. Based on the analogy with lens crystallins, these enzymes are called corneal crystallins, which are diverse and species-specific. Examining crystallins in lens and cornea in multiple species provides great insight into their evolution. We report data on major proteins present in the crocodile cornea, an evolutionarily distant taxon. We demonstrate that tau-crystallin/alpha-enolase and triose phosphate isomerase (TIM) are among the major proteins expressed in the crocodile cornea as resolved by 2D gel electrophoresis and identified by MALDI-TOF. These proteins might be classified as putative corneal crystallins. tau-Crystallin, known to be present in turtle and crocodile lens, has earlier been identified in chicken and bovine cornea, whereas TIM has not been identified in the cornea of any species. Immunostaining showed that tau-crystallin and TIM are concentrated largely in the corneal epithelium. Using western blot, immunofluorescence and enzymatic activity, we demonstrate that high accumulation of tau-crystallin and TIM starts in the late embryonic development (after the 24th stage of embryonic development) with maximum expression in a two-week posthatched animal. The crocodile corneal extract exhibits significant alpha-enolase and TIM activities, which increases in the corneal extract with development. Our results establishing the presence of tau-crystallin in crocodile, in conjunction with similar reports for other species, suggest that it is a widely prevalent corneal crystallin. Identification of TIM in the crocodile cornea reported here adds to the growing list of corneal crystallins.