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Pyridoxal kinase (PdxK) is a vitamin B 6 salvage pathway enzyme which produces pyridoxal phosphate. We have investigated the impact of PdxK deletion in Leishmania donovani on parasite survivability, infectivity and cellular metabolism. LdPdxK mutants were generated by gene replacement strategy. All mutants showed significant reduction in growth in comparison to wild type. For PdxK mediated biochemical perturbations, only heterozygous mutants and complementation mutants were used as the growth of null mutants were compromised. Heterozygous mutant showed reduction invitro infectivity and higher cytosolic and mitochondrial ROS levels. Glutathione levels decreased significantly in heterozygous mutant indicating its involvement in cellular oxidative metabolism. Pyridoxal kinase gene deletion resulted in reduced ATP levels in parasites and arrest at G 0 /G 1 phase of cell cycle. All these perturbations were rescued by PdxK gene complementation. This is the first report to confirm that LdPdxK plays an indispensable role in cell survival, pathogenicity, redox metabolism and cell cycle progression of L. donovani parasites. These results provide substantial evidence supporting PdxK as a therapeutic target for the development of specific antileishmanial drug candidates., Competing Interests: Declaration of competing interest The authors of the current manuscript ‘Pyridoxal kinase gene deletion leads to impaired growth, deranged redox metabolism and cell cycle arrest in Leishmania donovani’ have no conflict of interest., (Copyright © 2024 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Clotrimazole is an FDA approved drug and is widely used as an antifungal agent. An extensive body of research is available about its mechanism of action on various cell types but its mode of killing of Leishmania donovani parasites is unknown. L. donovani causes Visceral Leishmaniasis which is a public health problem with limited treatment options. Its present chemotherapy is expensive, has adverse effects and is plagued with drug resistance issues. In this study we have explored the possibility of repurposing clotrimazole as an antileishmanial drug. We have assessed its efficacy on the parasites and attempted to understand its mode of action. We found that it has a half-maximal inhibitory concentration (IC 50 ) of 35.75 ± 1.06 μM, 12.75 ± 0.35 μM and 73 ± 1.41 μM in promastigotes, intracellular amastigotes and macrophages, respectively. Clotrimazole is 5.73 times more selective for the intracellular amastigotes as compared to the mammalian cell. Effect of clotrimazole was reduced by ergosterol supplementation. It leads to impaired parasite morphology. It alters plasma membrane permeability and disrupts plasma membrane potential. Mitochondrial function is compromised as is evident from increased ROS generation, depolarized mitochondrial membrane and decreased ATP levels. Cell cycle analysis of clotrimazole treated parasites shows arrest at sub-G 0 phase suggesting apoptotic mode of cell death., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The hexose monophosphate shunt is a crucial pathway in a variety of microorganisms owing to its vital metabolic products and intermediates such as NADPH, ribose 5-phosphate etc. The enzyme 6-phosphogluconolactonase catalyses the second step of this pathway, converting 6-phosphogluconolactone to 6-phosphogluconic acid. This enzyme has been known to have a significant involvement in growth, pathogenesis and sensitivity to oxidative stress in bacterial and protozoal pathogens. However, the functional role of kinetoplastid Leishmania donovani 6-phospohogluconolactonase (Ld6PGL) remains unexplored. L. donovani is the second largest parasitic killer and causative organism of life threatening visceral leishmaniasis. To understand its possible functional role in the parasite, the alleles of Ld6PGL were sequentially knocked-out followed by gene complementation. The Ld6PGL mutant cell lines showed decrease in transcriptional and translational expression as well as in the enzyme activity. In case of Ld6PGL null mutants, approximately 2-fold reduction was observed in growth. The null mutants also showed ∼38% decrease in infectivity, which recovered to ∼15% on complementation. Scanning electron microscopy showed a marked decrease in flagellar length in the knockout parasites. When treated with the standard drug miltefosine, the mutant strains had no significant change in the drug sensitivity. However, the Ld6PGL mutants were more susceptible to oxidative stress. Our findings suggest that 6PGL is required for parasite growth and infection, but it is not essential., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The present study aims to evaluate the microstructure, grain size, and mechanical properties of the dissimilar AISI 316L/Inconel 718 (IN 718) rotary friction welded joints under both the as-welded and post-weld heat treatment (PWHT) conditions. Because of reduced flow strength at elevated temperatures, the AISI 316L and IN 718 dissimilar weldments exhibited more flash formation on the AISI 316L side. At higher rotating speeds during friction welding, an intermixing zone was created at the weld joint interface due to the material softening and squeezing. The dissimilar welds exhibited distinctive regions, including the fully deformed zone (FDZ), heat-affected zone (HAZ), thermo-mechanically affected zone (TMAZ), and the base metal (BM), located on either side of the weld interface. The dissimilar friction welds, AISI 316L/IN 718 ST and AISI 316L/IN 718 STA, exhibited yield strength (YS) of 634 ± 9 MPa and 602 ± 3 MPa, ultimate tensile strength (UTS) of 728 ± 7 MPa and 697± 2 MPa, and % elongation (% El) of 14 ± 1.5 and 17 ± 0.9, respectively. Among the welded samples, PWHT samples exhibited high strength (YS = 730 ± 2 MPa, UTS = 828 ± 5 MPa, % El = 9 ± 1.2), and this may be attributed to the formation of precipitates. Dissimilar PWHT friction weld samples resulted in the highest hardness among all the conditions in the FDZ due to the formation of precipitates. On the AISI 316L side, prolonged exposure to high temperatures during PWHT resulted in grain growth and decreased hardness. During the tensile test at ambient temperature, both the as-welded and PWHT friction weld joints failed in the HAZ regions of the AISI 316L side.

The present work aims to investigate the changes in the microstructural and mechanical properties of various pre- and post weld heat treatments (PWHTs) on rotary friction welded dissimilar (AA7075 and AA5083) aluminum alloys. The investigation focused on the evolution of weld macro- and microstructures, as well as the changes in hardness and tensile properties resulting from friction welding. The joint integrity was studied through various characterization techniques, and no cracks or incomplete bonding was observed. The study found that the dissimilar joints of the AA7075 and AA5083 alloys displayed higher flash formation on the AA7075 side, which has a lower melting temperature compared to the AA5083 alloy. Various zones were identified in the weld region, including the dynamic recrystallized zone (DRZ), the thermomechanically affected zone (TMAZ) consisting of TMAZ-1 (elongated grains) and TMAZ-2 (compressed/distorted grains), the heat-affected zone (HAZ), and the base metal (BM) zone. The rotary friction welded sample AA5083/AA7075-PWHT joint exhibited the highest strength (yield strength (YS): 195 ± 3 MPa, ultimate tensile strength (UTS): 387 ± 2 MPa) among all the other welded conditions, and this may be attributed to the major strengthening precipitates MgZn 2 (of AA7075) formed during postweld aging. All dissimilar welds failed in the HAZ region of the AA5083 side due to the formation of coarse grains, indicating the weakest region.

2-Arylquinazolines with a range of alkyl polyamines as side chain/ring functional motifs at the 4th-position were considered for antileishmanial study with the rationale that related heterocyclic scaffolds and polyamine functionalities are present in drugs, clinical trial agents, natural products and anti-parasitic/leishmanial agents. Synthesis involves construction of the 2-arylquinazolin-4-one ring and deoxyamination via deoxychlorination followed by S N Ar-based amination or a methodology of S N Ar-deoxyamination driven by BOP-mediated hydroxyl-activation. Various alkyl-polyamines important for activities were incorporated. A total of 26 compounds were prepared and screened against Leishmania donovani ( Ld ) promastigote cells using the MTT assay. Most of the investigated series of compounds showed characteristic leishmanicidal properties. Several compounds showed pronounced leishmanicidal activities (IC 50 : 5-6.5 μM) with higher efficiency than the antileishmanial drug miltefosine (IC 50 : 10.5 μM), and relatively less cytotoxicity to macrophage host cells (SI: 9.27-13.5) compared to miltefosine (SI: 3.42). Important pharmacophoric skeletons were identified., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Premna integrifolia (Agnimantha brihat) is a traditional medicinal plant with a prominent place in Ayurveda, Siddha and Unani systems of medicine. In this study we have evaluated the anti-inflammatory and immunomodulatory properties of the Premna integrifolia root extracts employing cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), and 5-lipoxygenase (5-LOX) enzyme-based assays, lymphocyte proliferation assay, pro-and anti-inflammatory cytokines measurement. Petroleum ether extract (PEE) of Premna integrifolia showed potent inhibition of COX-2 and 5-LOX with IC 50 values of 6.15 μg/mL and 11.33 μg/mL respectively. In in vitro studies on RAW 264.7 cell line, PEE showed inhibition in the formation of nitric oxide (NO), pro-inflammatory cytokines (IL-1β, IL-6), prostaglandin E 2 (PGE 2 ) production, induction of anti-inflammatory cytokine (IL-2) and down-regulation of expression of COX-2, 5-LOX, TNF-α, IL-1β and iNOS. PEE also significantly reduced carrageenan-induced paw edema in mouse model of inflammation. Further, attempts in isolating the active principle(s) involved in these anti-inflammatory effects of PEE by separation on RP-HPLC resulted in the isolation of four active peaks, H1, H2, H3 and H5, inhibiting COX-1, COX-2 and 5-LOX, out of which H3 was identified as 6- hydroxy salvinolone (6-HS). Present findings reveal that PEE of roots of Premna integrifolia exhibits potent anti-inflammatory and immunomodulatory activities, which could form a potential source for development of anti-inflammatory drugs. 6-HS, a COX-2/5-LOX dual inhibitor along with other lead molecules isolated from PEE of Premna integrifolia may form lead molecules for the development of COX-LOX dual inhibitors., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Abstract: In the present investigation, a novel fungus was isolated from leather watch strap and identified as Aspergillus versicolor TANCK-1 by 18 s rRNA sequencing. The isolated fungus was evaluated against three structurally different fungicides such as 2-(thiocyanomethylthio)benzothiazole (TCMTB), potassium dimethyldithiocarbamate (KDDC) and 2,2-dibromo-3-nitrilopropionamide (DBNP) to control the fungal growth. Among these, TCMTB was found to effectively inhibit the growth at a minimum concentration of 31.2 µg/mL as compared to 1250 and 625 µg/mL for KDDC and DBNP respectively. Increased membrane permeability in the fungicide treated samples was evident from cellular release and decrease in cellular ergosterol content. Nevertheless, SEM analysis revealed a considerable change in fungicide treated mycelium with the collapse of hyphae structure and shrunken spores, which was observed to be very pronounced in KDDC and DBNP. Results indicated that all three biocides bring about inhibition through membrane damage with almost negligible effect on the cell wall., Graphical Abstract: Schematic diagram explaining the fungicidal action on Aspergillus versicolor TANCK-1., Competing Interests: Compliance with Ethical StandardsThe authors declare that there is no conflict of interest.

Leishmaniasis is a group of tropical diseases caused by protozoan parasites of the genus Leishmania. Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis, a fatal disease if left untreated. Chemotherapy for leishmaniasis is problematic as the available drugs are toxic, costly and shows drug resistance, hence, there is a necessity to look out for the novel drug targets, chemical entities and vaccine. Glutamine synthetase (GS) catalyzes the synthesis of glutamine from glutamate and ammonia. In the present study, we have identified and characterized GS from L. donovani. The nucleotide sequence encoding putative glutamine synthetase like sequence from L. donovani (LdGS, LDBPK_060370) was cloned. A 43.5 kDa protein with 6X-His tag at the C-terminal end was obtained by overexpression of LdGS in Escherichia coli BL21 (DE3) strain. Expression of native LdGS in promastigotes and recombinant L. donovani glutamine synthetase (rLdGS) was confirmed by western blot analysis. An increase in expression of GS was observed at different phases of growth of the parasite. Expression of LdGS in promastigote and amastigote was confirmed by western blot analysis. Immunofluorescence studies of both the promastigote and amastigote stages of the parasite revealed the presence of LdGS in cytoplasm. GS exists as a single copy gene in parasite genome. Kinetic analysis of GS enzyme revealed K m value of 26.3 ± 0.4 mM for l- glutamate and V max value of 2.15 ± 0.07 U mg -1 . Present study confirms the presence of glutamine synthetase in L. donovani and provides comprehensive overview of LdGS for further validating it as a potential drug target., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Hydroxyapatite (HA) ceramics serve as an alternative to autogenous-free bone grafting by virtue of their excellent biocompatibility. However, chemically synthesized HA lacks the strong load-bearing capacity as required by bone. The bio-mimetic growth of HA crystals on collagen surface provides a feasible solution for synthesizing bone substitutes with the desired properties. This study deals with the utilization of the collagen hydrolysate recovered from leather waste as a substrate for promoting HA crystal growth. Bio-mimetic growth of HA was induced by subjecting the hydrolysate to various mineralization conditions. Parameters that would have a direct effect on crystal growth were varied to determine the optimal conditions necessary. Maximum mineralization was achieved with a combination of 10mM of CaCl2, 5mM of Na2HPO4, 100mM of NaCl and 0.575% glutaraldehyde at a pH of 7.4. The metal-protein interactions leading to formation of HA were identified through Fourier-transform infrared (FTIR) spectroscopy and x-ray diffraction (XRD) studies. The crystal dimensions were determined to be in the nanoscale range by atomic force microscopy (AFM) and scanning electron microscopy (SEM). The size and crystallinity of bio-mimetically grown HA indicate that hydrolysate from leather waste can be used as an ideal alternative substrate for bone growth., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Leishmaniasis is a group of tropical diseases caused by protozoan parasites of the genus Leishmania. Due to the emergence of resistance to the available antileishmanial drugs there is an immediate need to identify molecular targets on which to base future treatment strategies. Ribose 5-phosphate isomerase (Rpi; EC 5.3.1.6) is a key enzyme of the pentose phosphate pathway (PPP) which catalyses the reversible aldose-ketose isomerization between Ribose 5-phosphate (R5P) and Ribulose 5-phosphate (Ru5P). It exists in two isoforms A and B. These two are completely unrelated enzymes catalyzing the same reaction. Analysis of the Leishmania infantum genome revealed that though the RpiB gene is present, RpiA homologs are completely absent. An absence of RpiBs in the genomes of higher animals makes this enzyme a possible target for the chemotherapy of Leishmaniasis. In this paper, we report for the first time the presence of B isoform of the Rpi enzyme in Leishmania donovani (LdRpiB) by cloning and molecular characterization of the enzyme. An amplified L. donovani RpiB gene is 519 bp and encodes for a putative 172 amino acid protein with a molecular mass of ∼19 kDa. An ∼19 kDa protein with poly-His tag at the C-terminal end was obtained by heterologous expression of LdRpiB in Escherichia coli. The recombinant form of RpiB was obtained in soluble and active form. The LdRpiB exists as a dimer of dimers i.e. the tetramer form. The polyclonal antibody against Trypanosoma cruzi RpiB could detect a band of ∼19 kDa with the purified recombinant RpiB as well as native RpiB from the L. donovani promastigotes. Recombinant RpiB obeys the classical Michaelis-Menten kinetics utilizing R5P as the substrate with a K(m) value of 2.4±0.6 mM and K(cat) value of 30±5.2 s(-1). Our study confirms the presence of Ribose 5-phosphate isomerase B in L. donovani and provides functional characterization of RpiB for further validating it as a potential drug target., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Triphala is an Ayurvedic herbal formulation consisting of equal parts of three myrobalans: Terminalia chebula, Terminalia bellerica and Emblica officinalis. We recently reported that chebulagic acid (CA) isolated from Terminalia chebula is a potent COX-2/5-LOX dual inhibitor. In this study, compounds isolated from Terminalia bellerica were tested for inhibition against COX and 5-LOX. One of the fractionated compounds showed potent inhibition against COX enzymes with no inhibition against 5-LOX. It was identified as gallic acid (GA) by LC-MS, NMR and IR analyses. We report here the inhibitory effects of GA, with an IC(50) value of 74 nM against COX-2 and 1500 nM for COX-1, showing ≈20 fold preference towards COX-2. Further docking studies revealed that GA binds in the active site of COX-2 at the non-steroidal anti-inflammatory drug (NSAID) binding site. The carboxylate moiety of GA interacts with Arg120 and Glu524. Based on substrate dependent kinetics, GA was found to be a competitive inhibitor of both COX-1 and COX-2, with more affinity towards COX-2. Taken together, our studies indicate that GA is a selective inhibitor of COX-2. Being a small natural product with selective and reversible inhibition of COX-2, GA would form a lead molecule for developing potent anti-inflammatory drug candidates.

Background: Vitiligo is an acquired disorder characterized by circumscribed depigmented macules devoid of identifiable melanocytes. Complex genetic, immunological, neural and self destructive mechanisms interplay in its pathogenesis. According to autocytotoxic hypothesis, oxidative stress has been suggested to be the initial pathogenic event in melanocyte degeneration., Aims: The aim of our investigation was to evaluate the role of oxidative stress by measuring levels of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) in lesional and normal skin of patients with vitiligo and in the skin of normal controls., Methods: We determined the activity of SOD in lesional and non-lesional skin and CAT in lesional skin only of 25 vitiligo patients and 25 controls by using the spectrophotometric assay and Aebi's method, respectively., Results: There was statistically significant increase in the levels of SOD in vitiliginous and non vitiliginous skin of patient group compared to the control group (P < 0.001). No significant difference was found between the levels of SOD in lesional skin and non-lesional skin of vitiligo patients. The levels of CAT in the skin of patients were found to be significantly lower than those of controls (P < 0.001)., Conclusions: There is increased oxidative stress in vitiligo as is indicated by high levels of SOD and low levels of CAT in the skin of vitiligo patients.

The conventional chemically based method of dehairing and fiber-opening discharges an enormous amount of pollutants in the processing of skins. Hence, bioprocessing of skin through a two-step process, dehairing using protease and fiber opening using alpha-amylase, has been developed. However, because this process involves two steps, we characterized commercial protease and alpha-amylase for their optimum activity and determine the influence of one enzyme on the activity of the other, in order to develop an integrated enzymatic dehairing and fiber-opening process. The influence of various factors, substrate concentration, time, pH, and temperature, on the activity of both protease and alpha-amylase was determined. Furthermore, the activity of protease on mixing with alpha-amylase and vice versa was investigated. It was found that there was no significant change in the activity of one enzyme in the presence of the other. Lineweaver-Burk plots showed K(m) and V(max) values of 31.6 mg/mL and 0.0106 mg/(mL@min) for protease and 8.79 mg/mL and 0.0912 mg/(mL@min) for alpha-amylase. This study provides substantial evidence for integrating the enzyme-based dehairing and fiber-opening processes using both the selected protease and alpha-amylase in one step.

The present paper illustrates our attempt to design and test the reproducibility of low-cost patient positioning devices prepared in-house in our radiotherapy department. Rigid thermocole boards with angulations, scales and support were designed as breast, pelvis and head positioning devices. Reproducibility and accuracy were tested by serial electronic portal imaging detector imaging.The positioning devices (with or without superimposed moulds) showed variations within 2-3 mm on serial treatment days which were within acceptable limits.It is therefore concluded that low-cost patient positioning devices for head, breast and pelvis (the common sites of treatments in radiotherapy) can be fabricated from available materials in-house. These have been shown to be resulting in accurate immobilization, can be customized for particular techniques and are considerably cheaper than commercially available solutions.

The genetic map of Bremia lactucae was expanded utilizing 97 F(1) progeny derived from a cross between Finnish and Californian isolates (SF5xC82P24). Genetic maps were constructed for each parent utilizing 7 avirulence genes, 83 RFLP markers, and 347 AFLP markers, and a consensus map was constructed from the complete data set. The framework map for SF5 contained 24 linkage groups distributed over 835cM; the map for C82P24 contained 21 linkage groups distributed over 606cM. The consensus map contained 12 linkage groups with markers from both parents and 24 parent-specific groups. Six avirulence genes mapped to different linkage groups; four were located at the ends of linkage groups. The closest linkages between molecular markers and avirulence genes were 3cM to Avr4 and 1cM to Avr7. Mating type seemed to be determined by a single locus, where the heterozygote determined the B(2) type and the homozygous recessive genotype determined the B(1) type.