Biochemical characterization of spleen tyrosine kinase (SYK) isoforms in platelets.

Alternate splicing is among the regulatory mechanisms imparting functional diversity in proteins. Studying protein isoforms generated through alternative splicing is therefore critical for understanding protein functions in many biological systems. Spleen tyrosine kinase (Syk) plays an essential role in ITAM/hemITAM signaling in many cell types, including platelets. However, the spectrum of Syk isoforms expressed in platelets has not been characterized. Syk has been shown to have a full-length long isoform SykL and a shorter SykS lacking 23 amino acid residues within its interdomain B. Furthermore, putative isoforms lacking another 23 amino acid-long sequence or a combination of the two deletions have been postulated to exist. In this report, we demonstrate that mouse platelets express full-length SykL and the previously described shorter isoform SykS, but lack other shorter isoforms, whereas human platelets express predominantly SykL. These results both indicate a possible role of alternative Syk splicing in the regulation of receptor signaling in mouse platelets and a difference between signaling regulation in mouse and human platelets.

Platelets express two sizes of the Syk molecule with possible alternate functions in the cell. We need to understand how these two differ in their structure so that further studies can be developed by selectively deleting one of them to evaluate their function in platelets. This study shows that platelet Syk molecules differ in their structure with and without a linker region in the molecule.

Hydantoin based dual inhibitors of ALR2 and PARP-1: Design, synthesis, in-vitro and in-vivo evaluation.

Diabetic nephropathy is one of the most dreadful diabetic complications (DCs). The polyol pathway and unified mechanism are two important pathways implicated in the progression of DCs. In this regard, targeting the key enzymes i.e., aldose reductase (ALR2) and poly (ADP-ribose) polymerase-1 (PARP-1), of these pathways can be a relevant strategy. Thus, in this study, the pharmacophoric requirements necessary for the dual inhibition of these two enzymes i.e., ALR2 and PARP-1 were identified and consequently, some hydantoin based molecules were designed. The designed molecules were subjected to structure-based molecular modelling analysis including molecular docking analysis and molecular dynamic simulations. The promising molecules were duly synthesized and examined for their ALR2 and PARP-1 dual inhibitory activities and selectivity over aldehyde reductase (ALR1) using in vitro enzymatic assays. Based on the results of in silico analysis and in vitro assays, the best three molecules were evaluated in vivo for their nephroprotective effect and antioxidant potential in the high-fat diet-streptozotocin induced diabetic rat model. The results showed that the compounds FM6B, FM7B and FM9B were having low micromolar inhibitory potential against ALR2 (IC50; 1.02, 1.14 and 1.08 µM, respectively) and PARP-1 (IC50; 0.95, 0.81 and 1.42 µM, respectively) with selectivity over ALR1 (selectivity index; 43.63, 37.03 and 45.14, respectively).

Drug metabolizing enzymes-associated chemo resistance and strategies to overcome it.

Regardless of continuous research to develop effective chemotherapies and improve patient's prognosis, cancer still remains one of the most deadly diseases worldwide. The reduction in the pace of successfully developing an effective anti-cancer drug is due to the rapid emergence of drug resistance exhibited by tumor cells. One of the resistance mechanisms which is least considered and somewhat overlooked is chemoresistance via drug metabolizing enzymes (DMEs). Therefore, this review emphasizes on pharmacokinetic resistance specifically the DMEs associated chemoresistance, in which drug molecule is rapidly metabolized by DMEs resulting in diminished potential of anti-cancer drugs. The current review will be covering DMEs that are associated with chemoresistance such as ALDH1A1, GST-π, DPD, CYP1B1 and so forth. Although several strategies have been developed to solve this problem such as prodrug designing, analog designing, DMEs inhibitors designing and development of specific pharmaceutical formulations but the inhibition of DMEs is still not considered significantly. Considering the significance of DMEs in chemoresistance, this review shed light on the mechanism of DMEs associated resistance at molecular level, their reported inhibitors that can be used as an adjuvant therapy and strategies (like prodrug designing, analog designing etc.) used so far to combat this problem.

Manifesting the Dasatinib-gallic acid co-amorphous system to augment anticancer potential: Physicochemical characterization, in silico molecular simulation, ex vivo permeability, and in vitro efficacy.

Dasatinib (DAB) has been explored for repurposing in the treatment of breast cancer (BC) due to its known effectiveness in treating leukemia, in addition to its role as a tyrosine kinase inhibitor. Gallic acid (GA) was chosen as a co-former due to its anticancer potential in BC, as demonstrated in several previous studies. DAB is a low-solubility drug, which is a significant hurdle for its oral bioavailability. To address this limitation, a DAB and GA co-amorphous (DAB-GA-CA) system was developed using liquid-assisted grinding and ball mill technology to enhance solubility, bioavailability, and anti-tumor efficacy. Physical characterization investigation revealed that the emergence of the halo diffractogram in PXRD, single glass transition temperature (Tg) value at 111.7 °C in DSC thermogram, and irregularly shaped blocks with loose, porous surfaces in SEM analysis indicated the formation of the DAB-GA-CA system at 1:1 M ratio. Furthermore, FTIR, Raman spectroscopy, in-silico molecular docking, and molecular dynamic studies confirmed the intermolecular hydrogen connections between DAB and GA. Moreover, the outcomes of the ligands (DAB and GA) and receptors (BCL-2, mTOR, estrogen receptor, and HER-2) docking studies demonstrated that both DAB and GA could interact with those receptors, leading to preventive action on BC cells. Additionally, the solubility and dissolution rate significantly improved at pH 6.8, and the permeability study indicated that DAB-GA-CA showed 1.9 times higher apparent permeability compared to crystalline DAB. Furthermore, in vitro cytotoxicity assessments of the DAB-GA-CA system revealed 3.42 times lower IC50 than free DAB. The mitochondrial membrane depolarization, apoptotic index, and reactive oxygen species formation in MCF-7 cells were also notably higher in the DAB-GA-CA system than in free DAB. Hence, this research suggests that the DAB-GA-CA system could substantially enhance oral delivery, solubility, and therapeutic efficacy.

Dasatinib loaded mucoadhesive lecithin-chitosan hybrid nanoparticles for its augmented oral delivery, in-vitro efficacy and safety.

Dasatinib (DAS) is an oral tyrosine kinase inhibitor; however, its efficacy is significantly subsided by its low oral bioavailability. The present research aimed to improve DAS's oral delivery and efficacy in triple-negative breast cancer by fabricating its mucoadhesive lecithin-chitosan hybrid nanoparticles (DAS-L/CS-NPs). DAS-L/CS-NPs were optimized using Box-Behnken design which showed mean particle size and percent entrapment efficiency of 179.7 ± 5.42 nm and 64.65 ± 0.06 %, respectively. DAS-L/CS-NPs demonstrated sustained release profile in different release media up to 48 h and showed 10 times higher apparent permeability coefficient and flux than free DAS suspension. The binding of DAS-L/CS-NPs to the mucus layer was demonstrated via ex-vivo mucoadhesion study and change in absorbance using turbidimetry. In cell culture studies, DAS-L/CS-NPs revealed a 4.14-fold decrease in IC50, significantly higher cellular uptake and mitochondrial membrane depolarization, 3.82-fold increased reactive oxygen species generation and 2.10-fold enhanced apoptosis in MDA-MB-231 cells than free DAS. In in-vivo pharmacokinetic assessment, DAS-L/CS-NPs showed a 5.08-fold and 3.74-fold rise in AUC (0-t) and Cmax than free DAS suspension, respectively. An acute toxicity study revealed a good safety profile of DAS-L/CS-NPs. In a nutshell, proposed hybrid nanoparticles are promising carriers for improved oral delivery of poorly water-soluble drugs.

Growth of marine diatoms on aquaculture wastewater supplemented with nanosilica.

The aquaculture industry is a significant producer of highly nutritious food for the increasing global population. However, the wastewater generated from aquaculture ponds is an emerging global issue. The present study demonstrates the culturing of marine diatoms (Chaetoceros gracilis and Thalassiosira weissflogii) in different proportions of aquaculture wastewater (AQW) coupled with inductively coupled plasma nanosilica (ICP-SiO2) and further explores their biorefinery potential concomitant nutrient removal. Thalassiosira weissflogii showed maximum carbohydrate content (79.47 ± 0.21 mg g-1) in 10% and protein content (27.09 ± 0.21 mg g-1) in 30% AQW: ICP-SiO2. Chaetoceros gracilis showed maximum carbohydrate content (91.64 ± 0.11 mg g-1) in 50% and protein content (27.75 ± 0.05 mg g-1) in 10% AQW: ICP-SiO2 respectively. Additionally, Chaetoceros gracilis showed maximum nitrate uptake in 30%, phosphate in 50%, and ammonia in 50% AQW: ICP-SiO2. While Thalassiosira weissflogii showed maximum nitrate, phosphate, and ammonia removal efficiency in 50%, 50%, and 10% AQW: ICP-SiO2 respectively. The study draws attention towards the utilization of diatoms in AQW treatment, aquafeed potential thus imparting a global circular bioeconomy.

Some Scaffolds as Anti-leishmanial Agents: A Review.

Leishmaniasis is a parasitic infectious neglected tropical disease transmitted to humans by the parasites of Leishmania species. Mainly, three types of leishmaniases are usually observed: visceral (VL), cutaneous (CL), and mucocutaneous leishmaniasis. In many western countries, almost 700,000 to 1 million people suffer from leishmaniasis, and it is estimated that around 26000 to 65000 deaths occur from leishmaniasis. Few drugs are available for its treatment; however, none of them are ideal for leishmaniasis due to long treatment, discomfort mode of administration, risk of high-level toxicity, high resistance, etc. Hence, so many patients are unable to take complete treatment due to the high drug resistance. The present review will focus on antileishmanial activity of reported derivatives of betacarboline, chalcone, azole, quinoline, quinazoline, benzimidazole, benzadiazapine, thiaazoles, semicarbazone, and hydontoin analogues. We believe that this present study will be helpful for researchers to design new antileishmanial agents.

Production of lipids and proteins from marine diatoms under changing pH and silica.

Diatom algae are increasingly explored as an alternative sustainable source for functional biomolecules likes fucoxanthin, and eicosapentaenoic acid. But biomolecule quantity and quantity are influenced by growth conditions. So, effect of differential silica concentration (0-120 mg L-1) and medium pH (5.5-9.5) on growth and cellular biochemical composition of commercially important marine diatom species were studied. Growth rate of Thalassiosira sp., Skeletonema sp., and Chaetoceros sp., was higher with 30 mg L-1 Si at a pH of 7.5-8.5. Highest carbohydrate (153.71 mg g-1) and protein (17.34 mg g-1) content was found in Skeletonema sp. Silica concentration positively influenced chlorophyll and carotenoid content in a dose dependent manner. A medium pH of 8.5 and Si concentration between 60 and 120 mg L-1 was ideal for lipid production. The optimum concentration of Si and pH for maximum biomolecule production have been reported with further scope of utilizing these conditions in commercial scale systems.

Faecal microbiome analysis reveals Clostridiales and Bacteroidales as signature gut microbes during estrus of buffalo.

The non-pathogenic intestinal microbes that conquer our intestines are not an accidental jumble of organisms, but rather a disparate community of microbes that coexist, and sustain a mutualistic and symbiotic relationship with the host. The gut microbiome has been shown to be influenced by animal physiology and vice versa. However, information is still scanty. The present study aimed to analyse the variation between faecal bacteria of three different stages (proestrus, estrus and postestrus) of the estrous cycle of Murrah buffalos. A phylogenetic study of buffalo faeces derived from three different stages of estrous cycle was conducted in order to compare the bacterial diversity among these three stages. We performed an exploratory microbiome analysis of buffalo faeces using 16S rRNA sequencing during these stages of the buffalo estrous cycle. A total of three bacterial phyla with six different bacterial orders and twenty-three different genera were identified among all the three comparative phases of the estrous cycle. Among them, the Clostridiales were found to be the most abundant, and Bacteroidales were present exclusive during the estrus phase. As faeces is a source of gut microbes and a non-invasive representative of the metabolic steroids and perceptible pheromones, the profiling of gut microbes during estrous cycle would provide clues towards the major microbes contributing to the perceptible pheromones during estrus stage. To the best of our knowledge, this is the first ever report describing the faecal bacterial diversity during estrous cycle of any ruminant species. Although future studies are required to understand the role of Clostridiales and Bacteroidales in faecal pheromone metabolism.

Bioprospecting of marine diatoms Thalassiosira, Skeletonema and Chaetoceros for lipids and other value-added products.

Diatoms are a major storehouse of valuable fucoxanthin and polyunsaturated fatty acids, with enormous nutraceuticals and biofuel potential. Three marine diatom species isolated from the southern coast of India has been screened and their results show that highest biomass concentration and fucoxanthin yield was obtained in Chaetoceros sp. as 0.217 g L-1 and 0.403 mg L-1 respectively. Lipid % as dry cell weight was maximum in Thalassiosira sp. (52%) followed by Skeletonema sp. (44%) and Chaetoceros sp. (22%). However, protein and secondary metabolites content besides the total antioxidant activity was estimated highest in Skeletonema sp. Having strong inhibition zones of 18-20 mm against all the five strains of bacteria also highlights the highest antibacterial prospect in Skeletonema sp. This work manifests the plasticity of diatoms and may provide useful insights for further species-specific selection for large-scale production of eicosapentaenoic acid, docosahexaenoic acid, fucoxanthin and other metabolites with potential health benefits.

In silico guided development of imine-based inhibitors for resistance-deriving kinases.

Two major mechanisms involved in resistant NSCLC (non-small cell lung cancer) include secondary acquired mutation in EGFR (epidermal growth factor receptor), that is, EGFR T790M and amplification of c-MET (hepatocyte growth factor receptor). Thus, already established pharmacophore models of EGFR T790M and c-MET were employed to filter-out an in-house database. Further fitness score led to the selection of imino-pyrimidine scaffold. Followed by sketching of imino-pyrimidine derivatives having varied aryl substitutions, which were then docked and subjected to molecular dynamic simulations, to study the orientations and conformations of the designed molecules in the catalytic domain. Molecules with hydrophobic interaction with mutant residue M790 were selected. Finally, MM-GBSA (Molecular Mechanics-Generalized Born Surface Area) calculations were performed, to study the effect of substitutions on the binding affinity of the double mutant EGFR towards these small molecules. Finally, the designed compounds were synthesized and evaluated for their kinase inhibitory potential using in-vitro experiments. Two compounds were found to possess sub-micromolar range inhibitory potential against EGFR (T790M), while one of the compound showed significant selective inhibitory potential against c-MET. Additionally, one compound was found to possess significant dual inhibitory potential against these target kinases. Communicated by Ramaswamy H. Sarma.

Exploration of the therapeutic aspects of Lck: A kinase target in inflammatory mediated pathological conditions.

Lck, a non-receptor src family kinase, plays a vital role in various cellular processes such as cell cycle control, cell adhesion, motility, proliferation and differentiation. As a 56 KDa protein, Lck phosphorylates tyrosine residues of various proteins such as ZAP-70, ITK and protein kinase C. The structure of Lck is comprised of three domains, one SH3 in tandem with a SH2 domain at the amino terminal and the kinase domain at the carboxy terminal. Physiologically, Lck is involved in the development, function and differentiation of T-cells. Additionally, Lck regulates neurite outgrowth and maintains long-term synaptic plasticity in neurons. Given a major role of Lck in cytokine production and T cell signaling, alteration in expression and activity of Lck may result in various diseased conditions like cancer, asthma, diabetes, rheumatoid arthritis, psoriasis, inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, atherosclerosis etc. This article provides evidence and information establishing Lck as one of the therapeutic targets in various inflammation mediated pathophysiological conditions.