Application of Synergistic Phenomena for Enhanced Production of Alpha 1,4 D- Glucan Glucanohydrolase Using Submerged Fermentation.

The study revealed enhanced production of α 1, 4 D glucan glucanohydrolase utilizing the synergistic phenomena of bacterial hetero-culture. For this purpose, 101 hetero-cultures were screened qualitatively and quantitatively. The bacterial hetero-culture showing the highest amylolytic potential was identified as Bacillus subtilis and Bacillus amyloliquefeciens by 16S rDNA sequencing technique. Different fermentation media were evaluated and M 5 gave maximum GGH production. Different physicochemical parameters like incubation time, temperature, initial pH and inoculum size was optimized. The optimal enzyme production was obtained at 24 h, 37oC, pH 7.0 and 3% inoculum size. Glucose (3%), ammonium sulphate (1.5%) and yeast extract (2.0%) was selected as best carbon and nitrogen source, respectively. The novelty of the present piece of research was the application of the hetero-culture technique for enhanced GGH production using submerged fermentation which was not experienced before with these strains.

Extracellular biosynthesis, characterization, optimization of silver nanoparticles (AgNPs) using Bacillus mojavensis BTCB15 and its antimicrobial activity against multidrug resistant pathogens.

Biosynthesis of metal nanoparticles is an area of interest among researchers because of its eco-friendly approach. Current study focuses at biosynthesis of silver nanoparticles (AgNPs) and optimization of physico-chemical conditions to obtain mono-dispersed and stable AgNPs having antimicrobial activity. Initially Bacillus mojavensis BTCB15 produced silver nanoparticles (AgNPs) of 105 nm. Silver nanoparticles (AgNPs) were characterized by particle size analyzer, UV-Vis Spectroscopy, Fourier transforms infrared spectroscopy (FTIR), Atomic force microscopy (AFM), and X-ray diffraction (XRD). Whereas, under optimal conditions of temperature 55 °C, pH 8, addition of surfactant Tween 20, and metal ion K2SO4, about 104% size reduction was achieved with average size of 2.3nm. Molecular characterization revealed 98% sequence homology with Bacillus mojavensis. AgNPs exhibited antibacterial activity at concentrations ranging from 0.5 to 2.5 µg/µl against Escherichia coli BTCB03, Klebsiella pneumonia BTCB04, Acinetobacter sp. BTCB05, and Pseudomonas aeruginosa BTCB01 but none against Staphylococcus aureus BTCB02. Highest antibacterial activity was observed at 0.27 µg/µl and lowest at 0.05 µg/µl of AgNPs indicated by zone of inhibition. Conclusively, under optimum conditions, Bacillus mojavensis BTCB15 was able to produce AgNPs of 2.3 nm size and had antibacterial activity against multi drug resistant pathogens.

Influence of medium composition and physical factors on enhanced production of endoglucanase by locally isolated fungal strain in solid state fermentation.

Endoglucanase is one of the most important enzymes of the cellulase group.  Endoglucanase are involved in the catalytic hydrolysis of cellulose and plays a pivotal role in different sectors like pharmaceutical, textile, detergent, and food processing as well as paper and pulp industry. With consumers getting more and more aware of environmental issues, industries find enzymes as a better option over other chemical catalysts. In the current research different thermophilic fungal strains were isolated from the different sources. Qualitative screening was carried out on the basis of cellulose hydrolysis zone. The quantitative screening was carried out employing solid state fermentation.  The fungal culture, showing highest EG potential was selected identified and assigned the code Aspergillus fumigatus BBT2. Different fermentation media were evaluated and M 2 containing wheat bran gave maximum EG production. The maximal enzyme productivity was recorded in 72 hours, 40°C, pH 5, inoculum size 1.5ml, and moisture content (1:1). Glucose (1%) and peptone (1%) were optimized as best carbon and nitrogen sources, respectively.

Mesenchymal stem cells (MSCs) as skeletal therapeutics - an update.

Mesenchymal stem cells hold the promise to treat not only several congenital and acquired bone degenerative diseases but also to repair and regenerate morbid bone tissues. Utilizing MSCs, several lines of evidences advocate promising clinical outcomes in skeletal diseases and skeletal tissue repair/regeneration. In this context, both, autologous and allogeneic cell transfer options have been utilized. Studies suggest that MSCs are transplanted either alone by mixing with autogenous plasma/serum or by loading onto repair/induction supportive resorb-able scaffolds. Thus, this review is aimed at highlighting a wide range of pertinent clinical therapeutic options of MSCs in the treatment of skeletal diseases and skeletal tissue regeneration. Additionally, in skeletal disease and regenerative sections, only the early and more recent preclinical evidences are discussed followed by all the pertinent clinical studies. Moreover, germane post transplant therapeutic mechanisms afforded by MSCs have also been conversed. Nonetheless, assertive use of MSCs in the clinic for skeletal disorders and repair is far from a mature therapeutic option, therefore, posed challenges and future directions are also discussed. Importantly, for uniformity at all instances, term MSCs is used throughout the review.

Incidence of environmental and genetic factors causing congenital cataract in Children of Lahore.

OBJECTIVE: To check the incidence of environmental and genetic factors causing congenital cataract in infants. METHODS: The descriptive study was conducted at Layton Rahmatullah Benevolent Trust, Lahore, Pakistan, from October 2013 to April 2014, and comprised children under 15 years of age who had rubella syndrome, herpes simplex, birth trauma, trisomy 21, Nance-Horan syndrome or Lowe's syndrome. RESULTS: Of the 38,000 cases examined, 120(0.3%) patients were diagnosed with congenital cataract. Of them, 52(43.33%)were aged between 2 and 5 years,22(18.33%) <11 years and 10(8.33%) ?15 years. Bilateral congenital cataract was observed in 91(75.83%) patients and unilateral congenital cataract in 29(24.17%). Environmental factors caused 72(62.07%) cases and genetic factors caused 44(37.93%).. CONCLUSIONS: Congenital cataract predominated in boys compared to girls. Early diagnosis and adequate therapy requires specific technology, as well as long-term and permanent care..

Aberrant gene expression profiles, during in vitro osteoblast differentiation, of telomerase deficient mouse bone marrow stromal stem cells (mBMSCs).

BACKGROUND: Telomerase deficiency has been associated with inadequate differentiation of mesenchymal stem cells. However, the effect of telomerase deficiency on differential regulation of osteoblast specific genes, based on functional gene grouping, during in vitro osteoblast differentiation has not been reported before. RESULTS: To examine these effects, Terc (-/-) BMSCs (bone marrow stromal stem cells) were employed which exhibited reduced proliferation during in vitro osteogenesis along with increased population doubling time and level compared to wild type (WT) BMSCs during the normal culture. Osteogenic super array at day 10 of osteoblast differentiation revealed that telomerase deficiency strongly affected the osteoblast commitment by down-regulating Runx2, Twist and Vdr - known transcription regulators of osteogenesis. Similarly, in Terc (-/-) BMSCs a marked reduction in other genes engaged in various phases of osteoblast differentiation were observed, such as Fgfr2 involved in bone mineralization, Phex and Dmp1 engaged in ossification, and Col11a1 and Col2a1 involved in cartilage condensation. A similar trend was observed for genes involved in osteoblast proliferation (Tgfb1, Fgfr2 and Pdgfa) and bone mineral metabolism (Col1a1, Col2a1, Col1a2 and Col11a1). More profound changes were observed in genes engaged in extracellular matrix production: Col1a1, Col1a2, Mmp10, Serpinh1 and Col4a1. CONCLUSION: Taken together, these data suggest that telomerase deficiency causes impairment of BMSCs differentiation into osteoblasts affecting commitment, proliferation, matrix mineralization and maturation. Thus, modulating telomerase in BMSCs with advanced aging could improve BMSCs responsiveness towards osteoblast differentiation signals, optimal for osteoblast commitment, proliferation and maturation processes.

Bone Marrow Stromal Cell (BMSC) and skeletal aging: role of telomerase enzyme.

Telomere shortening and telomerase deficiency have been linked with several age related degenerative diseases. Moreover, degenerative changes in various tissues/organs have been attributed to derangement of stem cell functions causing regenerative tragedy. Bone marrow stromal cells (BMSCs) are considered the ideal candidates for regenerative approaches owing to their beneficial effects in numerous clinical applications. Thus, the effect of telomerase deficiency in perpetrating age related changes in BMSC functions during in vitro culture; their morphology, proliferation and differentiation, that can be extrapolated and reasoned for skeletal aging is conversed in this review. Besides, information regarding pertinent molecular and biochemical markers that can be employed to examine the earliest events, during the course of BMSC aging, is also provided. Additionally, impact of telomerase deficiency in enacting skeletal aging phenotype and its associated microenvironment is also discussed. In the end, further studies, using tissue specific models of telomerase deficiency, are recommended as a future research strategy to advance our understanding of tissue specific telomerase regulation.

Phytochemical and antimicrobial properties of different plants and in silico investigation of their bioactive compounds in wound healing and rheumatism.

In the current study the assessment of the antimicrobial and phytochemical properties of Cassia fistula, Musa paradisiaca, Ficus religiosa and Murraya koenigii plants extracts was carried out. The antibacterial potential of these plants extracts was tested against S. aureus and E. coli. The Cassia fistula and Ficus religiosa leaves showed the larger zone of inhibition in aqueous and butanolic extract respectively against Escherichia coli. Musa paradisiaca and Murraya koenigii leaves showed larger zone of inhibition in ethanolic extract against S. aureus. Qualitative phytochemical analysis showed the presence of alkaloids, flavonoids, phenols, terpenoids, steroids, glycosides, saponins, carbohydrates, proteins and tannins in all extracts while phylobatannins, emodins, anthocyanins and leucoanthocyanins were not present in these extracts. Quantitative phytochemical analysis showed the highest alkaloid content in the Murraya koenigii leaves. Highest tannin content and flavonoid content was found in Ficus religiosa leaves, while highest phenolic content was found in case of Cassia fistula. In addition to this antioxidant potential of all the extracts was determined. Musa paradisiaca leaves showed highest antioxidant potential as compared to other plant extracts. In silico analysis of bioactive components present in plant extracts was performed by molecular docking. The rutin and Glu from Musa paradisiaca and Murraya koenigii respectively, were docked with Glycogen Synthase Kinase 3 beta (1GSK-3beta) protein. Quercetin and rutin from Cassia fistula and Ficus religiosa respectively, were docked with C- reactive protein (CRP). The tested bioactive compounds showed good binding affinity with significant number of hydrogen bonds and can be used as a good alternative of synthetic drugs to treat rheumatism and wounds.

Prospective Roles of Extremophilic Fungi in Climate Change Mitigation Strategies.

Climate change and the resultant environmental deterioration signify one of the most challenging problems facing humankind in the 21st century. The origins of climate change are multifaceted and rooted in anthropogenic activities, resulting in increasing greenhouse gases in the environment and leading to global warming and weather drifts. Extremophilic fungi, characterized by their exceptional properties to survive extreme habitats, harbor great potential in mitigating climate change effects. This review provides insight into the potential applications of extremophilic fungi in climate change mitigation strategies. They are able to metabolize organic biomass and degrade carbon compounds, thereby safely sequestering carbon and extenuating its release into the environment as noxious greenhouse gases. Furthermore, they possess extremozymes, which break down recalcitrant organic species, including lignocellulosic biomass and hydrocarbons. Enzymatic machinery equips these extremophilic fungi to perform the bioremediation of polluted environments. Extremophilic fungi can also be exploited for various biological interventions, such as biofuels, bioplastics, and other bioprocessing applications. However, these fungi characterize a valued but underexplored resource in the arsenal of climate change mitigation strategies.

In silico approaches to study the human asparagine synthetase: An insight of the interaction between the enzyme active sites and its substrates.

Cancer is a leading concern and important cause of death worldwide. Cancer is a non-communicable illness defined as uncontrolled division of cells. It can develop into metastatic cancer when tumor cells migrate to other organs. In recent years evidence has emerged that the bioavailability of Asn play a crucial role in cancer metastasis. Asn is a non-essential amino acid formed from an ATP dependent catalyzed reaction by the enzyme asparagine synthetase (ASNS), where Asp and Gln are converted to Asn and Glu, respectively. The human ASNS enzyme consist of 561 amino acids, with a molecular weight of 64 KDa. ASNS governs the activation of transcriptional factors that regulate the process of metastasis. In this work the 3D model of ASNS in E. coli (AS-B) and the human ASNS docked with its different ligands have been used to study the 3D mechanism of the conversion of Asp and Gln to Asn and Glu, in human ASNS. The stability evaluation of the docked complexes was checked by molecular dynamic simulation through the bioinformatic tool Desmond. The binding residues and their interactions can be exploited for the development of inhibitors, as well as for finding new drug molecules against ASNS and prevention of metastatic cancer.

Recent developments in targeting breast cancer stem cells (BCSCs): a descriptive review of therapeutic strategies and emerging therapies.

Despite recent advancements in the diagnosis and treatment of breast cancer (BC), patient outcomes in terms of survival, recurrence, and disease progression remain suboptimal. A significant factor contributing to these challenges is the cellular heterogeneity within BC, particularly the presence of breast cancer stem cells (BCSCs). These cells are thought to serve as the clonogenic nexus for new tumor growth, owing to their hierarchical organization within the tumor. This descriptive review focuses on the evolving strategies to target BCSCs, which have become a pivotal aspect of therapeutic development. We explore a variety of approaches, including targeting specific tumor surface markers (CD133 and CD44), transporters, heat shock proteins, and critical signaling pathways like Notch, Akt, Hedgehog, KLF4, and Wnt/ß-catenin. Additionally, we discuss the modulation of the tumor microenvironment through the CXCR-12/CXCR4 axis, manipulation of pH levels, and targeting hypoxia-inducible factors, vascular endothelial growth factor, and CXCR1/2 receptors. Further, this review focuses on the roles of microRNA expression, strategies to induce apoptosis and differentiation in BCSCs, dietary interventions, dendritic cell vaccination, oncolytic viruses, nanotechnology, immunotherapy, and gene therapy. We particularly focused on studies reporting identification of BCSCs, their unique properties and the efficacy of various therapeutic modalities in targeting these cells. By dissecting these approaches, we aim to provide insights into the complex landscape of BC treatment and the potential pathways for improving patient outcomes through targeted BCSC therapies.

Levofloxacin loaded chitosan and poly-lactic-co-glycolic acid nano-particles against resistant bacteria: Synthesis, characterization and antibacterial activity.

BACKGROUND: With the global increase in antibacterial resistance, the challenge faced by developing countries is to utilize the available antibiotics, alone or in combination, against resistant bacterial strains. We aimed to encapsulate the levofloxacin (LVX) into polymeric nanoparticles using biodegradable polymers i.e. Chitosan and PLGA, estimating their physicochemical characteristics followed by functional assessment as nanocarriers of levofloxacin against the different resistant strains of bacteria isolated from biological samples collected from tertiary care hospital in Lahore, Pakistan. METHODS: LVX-NPs were synthesized using ion gelation and double emulsion solvent-evaporation method employing chitosan (CS) and poly-lactic-co-glycolic acid (PLGA), characterized via FTIR, XRD, SEM, and invitro drug release studies, while antibacterial activity was assessed using Kirby-Bauer disc-diffusion method. RESULTS: Data revealed that the levofloxacin-loaded chitosan nanoparticles showed entrapment efficiency of 57.14% ± 0.03 (CS-I), 77.30% ± 0.08(CS-II) and 87.47% ± 0.08 (CS-III). The drug content, particle size, and polydispersity index of CS-I were 52.22% ± 0.2, 559 nm ± 31 nm, and 0.030, respectively, whereas it was 66.86% ± 0.17, 595 nm ± 52.3 nm and 0.057, respectively for CS-II and 82.65% ± 0.36, 758 nm ± 24 nm and 0.1, respectively for CS-III. The PLGA-levofloxacin nanoparticles showed an entrapment efficiency of 42.80% ± 0.4 (PLGA I) and 23.80% ± 0.4 (PLGA II). The drug content, particle size and polydispersity index of PLGA-I were 86% ± 0.21, 92 nm ± 10 nm, and 0.058, respectively, whereas it was 52.41% ± 0.45, 313 nm ± 32 nm and 0.076, respectively for PLGA-II. The XRD patterns of both polymeric nanoparticles showed an amorphous nature. SEM analysis reflects the circular-shaped agglomerated nanoparticles with PLGA polymer and dense spherical nanoparticles with chitosan polymer. The in-vitro release profile of PLGA-I nanoparticles showed a sustained release of 82% in 120 h and it was 58.40% for CS-III. Both types of polymeric nanoparticles were found to be stable for up to 6 months without losing any major drug content. Among the selected formulations, CS-III and PLGA-I, CS-III had better antibacterial potency against gram+ve and gram-ve bacteria, except for K. pneumonia, yet, PLGA-I demonstrated efficacy against K. pneumonia as per CSLI guidelines. All formulations did not exhibit any signs of hemotoxicity, nonetheless, the CS-NPs tend to bind on the surface of RBCs. CONCLUSION: These data suggested that available antibiotics can effectively be utilized as nano-antibiotics against resistant bacterial strains, causing severe infections, for improved antibiotic sensitivity without compromising patient safety.

Assessment of angiotensin converting enzyme inhibitory activity and quality attributes of yoghurt enriched with Cinnamomum verum, Elettaria cardamomum, Beta vulgaris and Brassica oleracea.

The new concept of functional foods has led to the varieties in the production of foods that provide not only basic nutrition, but can also warrant good health and longevity. This study deals with the production and evaluation of fortified yogurts' with Cinnamomum verum, Elettaria cardamomum, Beta vulgaris and Brassica oleracea. The qualitative and quantitative phytochemical analysis of above mentioned plant extracts before using them into the preparation of functional yoghurt was carried out. The sensory evaluation of enriched yogurts with plant extracts carried out using 9 point hedonic scale. Comparative analysis between enriched yogurts and plain yogurt was carried. The results indicated increase in ash contents, water holding capacity, titratable acidity, total soluble solids, total phenolic content, tannin content, and total flavonoid content in fortified yogurt as compared to plain yogurt. In addition to this fortified yogurts showed greater antioxidant and antibacterial activity in contrast to plain yogurt. However, moisture contents, pH and susceptibility to syneresis of yogurt decreases with the addition of plant extracts. Shelf life of plain and fortified yogurt was determined both at room and refrigerated temperature. The results revealed that shelf life of fortified yogurt was greater as compared to plain yogurt. In silico analysis was carried out by using the galaxy web software. The results indicated that bioactive compounds including ascorbic acid, sinapinic acid, cinnamaldehyde and linalool acetate present in the flavored yogurts binds with angiotensin converting enzyme. All enriched yogurts showed higher anti-Angiotensin converting enzyme activity as compared to plain-yogurt.

Correction: Mutational analysis in sodium-borate cotransporter SLC4A11 in consanguineous families from Punjab, Pakistan.

[This corrects the article DOI: 10.1371/journal.pone.0273685.].

Docetaxel-Loaded Methoxy poly(ethylene glycol)-poly (L-lactic Acid) Nanoparticles for Breast Cancer: Synthesis, Characterization, Method Validation, and Cytotoxicity.

This study aimed to synthesize and characterize DTX-mPEG-PLA-NPs along with the development and validation of a simple, accurate, and reproducible method for the determination and quantification of DTX in mPEG-PLA-NPs. The prepared NPs were characterized using AFM, DLS, zetasizer, and drug release kinetic profiling. The RP-HPLC assay was developed for DTX detection. The cytotoxicity and anti-clonogenic effects were estimated using MTT and clonogenic assays, respectively, using both MCF-7 and MDA-MB-231 cell lines in a 2D and 3D culture system. The developed method showed a linear response, high precision, accuracy, RSD values of ≤2%, and a tailing factor ≤2, per ICH guidelines. The DTX-mPEG-PLA-NPs exhibited an average particle size of 264.3 nm with an encapsulation efficiency of 62.22%. The in vitro drug kinetic profile, as per the Krosmeyers-Peppas model, demonstrated Fickian diffusion, with initial biphasic release and a multistep sustained release over 190 h. The MTT assay revealed improved in vitro cytotoxicity against MCF-7 and MDA-MB-231 in the 2D cultures and MCF-7 3D mammosphere cultures. Significant inhibitions of the clonogenic potential of MDA-MB-231 were observed for all concentrations of DTX-mPEG-PLA-NPs. Our results highlight the feasibility of detecting DTX via the robust RP-HPLC method and using DTX-mPEG-PLA-NPs as a perceptible and biocompatible delivery vehicle with greater cytotoxic and anti-clonogenic potential, supporting improved outcomes in BC.

Purification, characterization and thermodynamic analysis of cellulases produced from Thermomyces dupontii and its industrial applications.

Cellulases involved in the hydrolysis of cellulose and plays a vital role in different industries like textile, detergent paper and Feed industry. Cellulases have been a prospective target for research by both the academic and industrial sectors because of the intricacy of the enzyme system and the enormous industrial potential. In the present work Thermomyces dupontii, which had previously been isolated and recorded as a promising cellulase producer were used. Both endoglucanases and betaglucosidases were purified to its homogeneity by ammonium sulfate followed by anion exchange and gel filtration chromatography. The recovery and purification fold for endoglucanases and betaglucosidases were 13.7, 10.7 % and 5.9, 2.7, respectively. The molecular weight of endoglucanases and betaglucosidases were estimated as 37 and 66 kDa on SDS-PAGE. Upon kinetic analysis the purified endoglucanases and betaglucosidases showed Km 0.63; 28.56 mg/ml and Vmax 82; 80 U/ml/min, respectively. Characterization revealed that enzyme was found to be acidophilic cellulase having optimal pH of 5.5 and 70 °C. Furthermore, cellulases were accelerated in the presence of Ca2+ and EDTA. The cellulases had activation energy (Ea) of -44.55; -50.02 kJ/mol for carboxy-methyl-cellulose hydrolysis and Enthalpy (ΔH) 42.20; 47.70 kJ/mol and entropy ΔS -5.1 and -5.7 kJ/mol for EG and BGL, respectively. In addition to this the enzyme had a secondary structure of protein as represented by FTIR spectrum The current study suggested that purified cellulases can be used as a detergent additive to improve washing. Furthermore, it shows the biostoning ability when applied on jean fabric.

Mutational analysis in sodium-borate cotransporter SLC4A11 in consanguineous families from Punjab, Pakistan.

AIM: To identify the molecular basis of Congenital Hereditary Endothelial Dystrophy CHED caused by mutations in SLC4A11, in the consanguineous Pakistani families. METHODS: A total of 7 consanguineous families affected with Congenital Hereditary Endothelial Dystrophy were diagnosed and registered with the help of ophthalmologists. Blood samples were collected from affected and unaffected members of the enrolled families. Mutational analysis was carried out by DNA sequencing using both Sanger and Whole Exome Sequencing (WES). Probands of each pedigree from the 7 families were used for WES. Results were analyzed with the help of different bioinformatics tools. RESULTS: The sequencing results demonstrated three known homozygous mutations in gene SLC4A11 in probands of 7 families. These mutations p.Glu675Ala, p.Val824Met, and p.Arg158fs include 2 missense and 1 frameshift mutation. The mutations result in amino acids that were highly conserved in SLC4A11 across different species. The mutations were segregated with the disease phenotype in the families. CONCLUSION: This study reports 3 mutations in 7 families. One of the pathogenic mutations (p.R158fs) was identified for the first time in the Pakistani population. However, two mutations (p.Glu675Ala, p.Val824Met) were previously reported in two and one Pakistani family respectively. As these mutations segregate with the disease phenotype and bioinformatics tool also liable them as pathogenic, they are deemed as probable cause of underlying disease.

Overview of Lignocellulolytic Enzyme Systems with Special Reference to Valorization of Lignocellulosic Biomass.

Lignocellulosic biomass, one of the most valuable natural resources, is abundantly present on earth. Being a renewable feedstock, it harbors a great potential to be exploited as a raw material, to produce various value-added products. Lignocellulolytic microorganisms hold a unique position regarding the valorization of lignocellulosic biomass as they contain efficient enzyme systems capable of degrading this biomass. The ubiquitous nature of these microorganisms and their survival under extreme conditions have enabled their use as an effective producer of lignocellulolytic enzymes with improved biochemical features crucial to industrial bioconversion processes. These enzymes can prove to be an exquisite tool when it comes to the eco-friendly manufacturing of value-added products using waste material. This review focuses on highlighting the significance of lignocellulosic biomass, microbial sources of lignocellulolytic enzymes and their use in the formation of useful products.

Investigation of Olea ferruginea Roylebark extracts for potential in vitroantidiabetic and anticancer effects.

This study was conducted to investigate the physicochemical, phytochemical, in vitro antidiabetic and anticancer potential of Olea ferruginea R bark. After extraction using Soxhlet, in vitro antidiabetic and cytotoxic activity on human hepatocellular carcinoma (HepG2) cells was assessed by nonenzymatic glycosylation of hemoglobin assay, alpha-amylase inhibition assay, glucose uptake by yeast cells, and 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay, respectively, and gene expression via real-time polymerase chain reaction. Primary and secondary metabolites were present in the extractants; polyphenols (35.61 ± 0.03) and flavonoids (64.33 ± 0.35 ) in the chloroform; and polysaccharides in the ethanol (268.75 ± 0.34), and glycosaponins (78.01 ± 0.07) in the methanol. The chloroform extract exhibited maximum antidiabetic potential, showing inhibition of nonenzymatic glycosylation of hemoglobin (65%), and alpha-amylase inhibition (32%) with maximum percent glucose uptake by the ethanol extract (78%). Only the ethanol extract had dose-dependent cytotoxic potential against the HepG2 cells. After 24-h exposure to the ethanol-extract, the expression of protein kinase B (Akt) remained unchanged, while the expression of B-cell lymphoma 2 (BCL2) and BCL2 associated X (BAX) changed significantly. After 48-h exposure, the expression of Akt decreased significantly, while that of BCL2 and BAX increased significantly. Olea ferruginea R bark possessed in vitro antidiabetic potential and anticancer/cytotoxic effects, attributable to the decline in the prosurvival signals of the Akt signaling pathway.

Finding inhibitors for PCSK9 using computational methods.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is one of the key targets for atherosclerosis drug development as its binding with low-density lipoprotein receptor leads to atherosclerosis. The protein-ligand interaction helps to understand the actual mechanism for the pharmacological action. This research aims to discover the best inhibitory candidates targeting PCSK9. To start with, reported ACE inhibitors were incorporated into pharmacophore designing using PharmaGist to produce pharmacophore models. Selected models were later screened against the ZINC database using ZINCPHARMER to define potential drug candidates that were docked with the target protein to understand their interactions. Molecular docking revealed the top 10 drug candidates against PCSK9, with binding energies ranging from -9.8 kcal·mol-1 to -8.2 kcal·mol-1, which were analyzed for their pharmacokinetic properties and oral bioavailability. Some compounds were identified as plant-derived compounds like (S)-canadine, hesperetin or labetalol (an antihypertensive drug). Molecular dynamics results showed that these substances formed stable protein-ligand complexes. (S)-canadine-PCSK9 complex was the most stable with the lowest RMSD. It was concluded that (S)-canadine may act as a potential inhibitor against atherosclerosis for the development of new PCSK9 inhibitory drugs in future in vitro research.