In vitro leishmanicidal effects of the anti-fungal drug natamycin are mediated through disruption of calcium homeostasis and mitochondrial dysfunction

Natamycin, a Food and Drug Administration approved anti-fungal drug, and also used as a food additive was evaluated for anti-leishmanial activity since it is known to specifically bind to ergosterol, which is essential to these parasites but absent in mammals. Promising anti-proliferative activity was observed in both promastigote and amastigote forms of the parasite with IC₅₀ values of 15 and 8 µM respectively and a selective index of 12.5. The ultrastructural effects of natamycin on both forms of the parasite and physiological effects on promastigotes were studied in detail for the first time. Electron microscopic observations in treated cells revealed sub-cellular changes like plasma membrane alterations, accumulation of vesicles in the flagellar pocket and extensive mitochondrial damage. Natamycin treatment in promastigotes resulted in elevation of cytosolic calcium (Ca²⁺) levels which caused irreversible loss of mitochondrial membrane potential. This resulted in depletion of cellular ATP levels along with ROS generation finally leading to apoptosis-like and necrotic cell death. In view of our observations along with the safety profile of an existing anti-fungal drug, natamycin may be further investigated for repurposing it as a promising drug candidate against Leishmaniasis.     

Bioremediation of cadmium induced renal toxicity in Rattus norvegicus by medicinal plant Catharanthus roseus

Cadmium is the second most hazardous metals with bio-concentration factor (BCF)?>?100 Although WHO permitted cadmium concentration in drinking water is 0.005?mg/L, yet the reality is far above to this limit because of industrial utility of this metal. Oral exposure of cadmium to human results in dreadful symptoms of metabolic disorders especially in liver and kidneys. Endogenous protection could be supported by some exogenous herbal supplement (viz., Catharanthus roseus in this case) to overcome the toxic effects. Present Study has been designed to find out the functional renal changes under the effect of cadmium and Catharanthus roseus in the model organism albino rats. Cadmium significantly (p?>?0.01) increases the level of nitrogenous waste (Urea, BUN, Uric Acid and Creatinin), while decreases the serum protein profile in acute and sub-acute sets. Urea concentration of control ranged from 16.56 to 17.72?mg/dl while that of Group-B and D were 19.84 to 20.87?mg/dl and 17.56 to 17.59?mg/dl respectively. Similarly uric acid concentration ranged in control form 6.98 to 8.01?mg/dl in group-B from 7.58 to 10.25?mg/dl, in Group-D 8.02 to 8.59?mg/dl respectively. Creatinin concentration ranged in control 0.57 to 0.65?mg/dl, in group-B 0.97 to 1.02?mg/dl, in group-D – 0.95 to 0.98?mg/dl respectively.These results might be due to altered filtration rate of kidney because of protein disruption. The studies conclude the efficient nephro-protection offered by Catharanthus roseus extract against Cadmium toxicity.     

Amaranthus caudatus lectin with polyproline II fold: conformational and functional transitions and molecular dynamics

Polyproline II (PPII) fold, a peculiar structural element was detected in the Amaranthus caudatus seed lectin (ACL) based on far UV circular dichroism spectrum, conformational transitions of the lectin, and a distinct isodichroic point in thermal denaturation. It was confirmed using PolyprOnline database to estimate the percentage of amino acids contributing to PPII fold and showed the values as 13.5 and 13.9% for PROSS and XTLSSTR, respectively. Investigations of the functional and conformational transitions of ACL during thermal-, pH-, and guanidine hydrochloride (GdnHCl)-induced denaturation were carried out using biochemical and biophysical techniques and molecular dynamics (MD) simulations approach. The lectin got aggregated at 60°C with instantaneous structural alterations. The aggregation-prone regions in ACL were predicted using online servers viz. AGGRESCAN, AmylPred, FoldAmyloid, and Waltz that were represented by Visual Molecular Dynamics tools. Nine conserved regions were identified by these softwares as being ‘hot-spots’ for aggregation. MD simulation studies of the lectin at 60°C revealed increase in radius of gyration. The loss of PPII fold in 2.0 M GdnHCl was reversible. The partially unfolded intermediate of ACL with diminished PPII fold formed at pH 1.0 was stable up to 90°C. The polyproline II fold has been rarely detected in lectins, ACL being the second after the potato lectin.     

Differentially Expressed Peroxidases from <Emphasis Type="Italic">Artemisia annua</Emphasis> and Their Responses to Various Abiotic Stresses

Artemisia annua is well-known for producing the antimalarial phytomolecule, artemisinin. The role of peroxidases has been hypothesized in artemisinin metabolism owing to the presence of an –O–O– linkage in this sesquiterpene lactone. Earlier, using a microarray, we identified differentially expressed genes, including peroxidases, in plant growth stages having contrasting artemisinin content. Here, three peroxidases—Aa547, having higher expression in low-artemisinin stage, and Aa540 and Aa528, having higher expression in high artemisinin stage, which could be associated with trichomes on the basis of their approximate gene expression pattern inferred from EST counts in UniGene—were selected for full-length cloning, tissue-specific expression profiling, and in silico analyses. The upstream genomic region of Aa547 was cloned and various cis-regulatory elements were identified. All the three candidates were predicted to be class III plant peroxidases. Further, this study aimed to check the responsiveness of the logically selected peroxidase genes to various abiotic stress factors. Taking cues from previous reports and the regulatory elements observed in the Aa547 promoter, hydration, salinity, temperature, salicylic acid, hydrogen peroxide, and methyl jasmonate, were selected to study their effect on the expression of the peroxidase genes through qRT-PCR. The peroxidases were found to be highly sensitive to the various factors but differed in their responses. Broadly, except for responses to high temperature and salicylic acid, the response of Aa547 to various factors was distinct from that of Aa540 and Aa528, which was in line with its distinctness from the other two peroxidases, considering the in planta artemisinin content and predicted structural features.     

Streptomycin affinity depends on 13 amino acids forming a loop in homology modelled ribosomal S12 protein (rpsL gene) of Lysinibacillus sphaericus DSLS5 associated with marine sponge (Tedania anhelans)

Streptomycin, an antibiotic used against microbial infections, inhibits the protein synthesis by binding to ribosomal protein S12, encoded by rpsL12 gene, and associated mutations cause streptomycin resistance. A streptomycin resistant, Lysinibacillus sphaericus DSLS5 (MIC >300 µg/mL for streptomycin), was isolated from a marine sponge (Tedania anhelans). The characterisation of rpsL12 gene showed a region having similarity to long terminal repeat sequences of murine lukemia virus which added 13 amino acids for loop formation in RpsL12; in addition, a K56R mutation which corresponds to K43R mutation present in streptomycin-resistant Escherichia coli is also present. The RpsL12 protein was modelled and compared with that of Lysinibacillus boronitolerans, Escherichia coli and Mycobacterium tuberculosis. The modelled proteins docked with streptomycin indicate compound had less affinity. The effect of loop on streptomycin resistance was analysed by constructing three different models of RpsL12 by, (i) removing both loop and mutation, (ii) removing the loop alone while retaining the mutation and (iii) without mutation having loop. The results showed that the presence of loop causes streptomycin resistance (decreases the affinity), and it further enhanced in the presence of mutation at 56th codon. Further study will help in understanding the evolution of streptomycin resistance in organisms.     

The KRAS-regulated kinome identifies WEE1 and ERK coinhibition as a potential therapeutic strategy in KRAS-mutant pancreatic cancer

Oncogenic KRAS drives cancer growth by activating diverse signaling networks, not all of which have been fully delineated. We set out to establish a system-wide profile of the KRAS-regulated kinase signaling network (kinome) in KRAS-mutant pancreatic ductal adenocarcinoma (PDAC). We knocked down KRAS expression in a panel of six cell lines and then applied multiplexed inhibitor bead/MS to monitor changes in kinase activity and/or expression. We hypothesized that depletion of KRAS would result in downregulation of kinases required for KRAS-mediated transformation and in upregulation of other kinases that could potentially compensate for the deleterious consequences of the loss of KRAS. We identified 15 upregulated and 13 downregulated kinases in common across the panel of cell lines. In agreement with our hypothesis, all 15 of the upregulated kinases have established roles as cancer drivers (e.g., SRC, TGF-β1, ILK), and pharmacological inhibition of one of these upregulated kinases, DDR1, suppressed PDAC growth. Interestingly, 11 of the 13 downregulated kinases have established driver roles in cell cycle progression, particularly in mitosis (e.g., WEE1, Aurora A, PLK1). Consistent with a crucial role for the downregulated kinases in promoting KRAS-driven proliferation, we found that pharmacological inhibition of WEE1 also suppressed PDAC growth. The unexpected paradoxical activation of ERK upon WEE1 inhibition led us to inhibit both WEE1 and ERK concurrently, which caused further potent growth suppression and enhanced apoptotic death compared with WEE1 inhibition alone. We conclude that system-wide delineation of the KRAS-regulated kinome can identify potential therapeutic targets for KRAS-mutant pancreatic cancer.     

Antimicrobial potential and taxonomic investigation of piezotolerant <Emphasis Type="Italic">Streptomyces</Emphasis> sp. NIOT-Ch-40 isolated from deep-sea sediment

Microbial-derived natural products from extreme niches such as deepsea are known to possess structural and functional novelty. With this background, the present study was designed to investigate the bioprospecting potential and systematics of a deep-sea derived piezotolerant bacterial strain NIOT-Ch-40, showing affiliation to the genus Streptomyces based on 16S RNA gene similarity. Preliminary screening for the presence of biosynthetic genes like polyketide synthase I, polyketide synthase II, non ribosomal peptide synthase, 3-amino-5-hydroxybenzoic acid synthase and spiroindimicin followed by antibacterial activity testing confirmed the presence of potent bioactivity. The secondary metabolites produced during fermentation in Streptomyces broth at 28?°C for 7 days were extracted with ethyl acetate. The extract exhibited a specific inhibitory activity against Gram-positive bacteria and was significantly effective (p?<?0.0001) against methicillin-resistant Staphylococcus aureus (MRSA). The minimum inhibitory concentration and minimum bactericidal concentration against MRSA was 1.5 µg/mL, which was statistically significant in comparison with erythromycin. A multifaceted analysis of the Streptomyces spp. was carried out to delineate the strain NIOT-Ch-40 at a higher resolution which includes morphological, biochemical and molecular studies. Piezotolerance studies and comparison of fatty acid profiles at high pressures revealed that it could be considered as one of the taxonomic markers, especially for the strains isolated from the deep sea environments. In conclusion, the observation of comparative studies with reference strains indicated towards the strain NIOT-Ch-40 as an indigenous marine piezotolerant Streptomyces sp. with a higher probability of obtaining novel bioactive metabolites.