Genome-directed discovery of antiproliferative bafilomycins from a deepsea-derived Streptomyces samsunensis.

Genomic bioinformatics analysis identified a bafilomycin biosynthetic gene cluster (named bfl) in the deepsea-derived S. samsunensis OUCT16-12, from which two new (1 and 2, named bafilomycins R and S) along with four known (3-6) bafilomycins were targetly obtained. The structure of 3 was clearly identified for the first time, thus named bafilomycin T herein. Differ from the fumarate substitution at C-21 of known bafilomycins, its location on C-23 is a unique feature of 1 and 2. The stereochemistry of the compounds was established based on NOE correlations, ketoreductase (KR)-types in PKS modules of bfl, and ECD calculations. Moreover, a detailed biosynthetic model of 1-6 in S. samsunensis OUCT16-12 was provided based on the gene function prediction and sequence identity. Compared with the positive control doxorubicin, 1-6 showed more potent antiproliferative activities against drug-resistant lung cancer cell line A549-Taxol, with IC50 values ranging from 0.07 µM to 1.79 µM, which arrested cell cycle in G0/G1 phase to hinder proliferation.

Formation of actin-cofilin rods by depletion forces.

Various stress conditions induce the formation of actin-cofilin rods in either the nucleus or the cytoplasm, although the mechanism of rod formation is unclear. In this study, we constituted actin-cofilin rods using purified actin, cofilin and actin interacting protein 1 (AIP1) in the presence of a physiological buffer containing a crowding agent, 0.8% methylcellulose (MC), which led to bundled actin filaments formed by depletion forces. Most of the F-actin bundles formed with methylcellulose were linear, whereas cofilin-bound F-actin bundles often had bent, looped, and often ring-like shapes. Increasing the amount of AIP1 shortened actin-cofilin bundles into rod-like bundles with tapering at both ends. As much shorter actin-cofilin filaments were formed in the presence of AIP1 before MC was added to the mixture, the rod-like bundle might be a mass of those short filaments. Furthermore, the small rods fused with each other to become larger rods, indicating that these rods were anisotropic liquid droplets. Several minutes after the addition of MC to the F-actin-cofilin-AIP1 mixture, we observed some long bundles in which the thick and thin parts appear alternately, reminiscent of a Plateau-Rayleigh instability observed in fluid columns. Simultaneously, we found images in which thin parts were interrupted, but the thick parts were arranged in a row in the longitudinal direction. These structures were also observed in cytoplasmic actin-cofilin rods in cells overexpressing cofilin-GFP, suggesting that cytoplasmic actin-cofilin rods have the same structure formation process as the rods reconstituted in vitro.

Sphingomyelin Synthase 2 Participate in the Regulation of Sperm Motility and Apoptosis.

Sphingomylin participates in sperm function in animals, and also regulates the Akt and ERK signaling pathways, both of which are associated with the asthenospermia. Sphingomyelin synthase 2 (SMS2) is involved in the biosynthesis of sphingomylin. To determine the relationship between SMS2 and human sperm function, we analyzed the distribution of SMS2 in human sperm and testes, and SMS2 expression in patients with asthenospermia and normozoospermia; human sperm were treated with anti-SMS2, and the sperm motility, penetration ability into methylcellulose, capacitation and acrosome reaction, and sperm [Ca2+]i imaging were evaluated, while the Akt and ERK pathway and cleaved caspase 3 were also analyzed. Results showed that SMS2 was localized in the testis and human sperm, and the protein levels of normozoospermia were higher than asthenospermia. Inhibition of SMS2 activity significantly decreased sperm motility and penetration ability into methylcellulose, but had no influence on capacitation and acrosome reaction, or on intracellular [Ca2+]i compared to IgG-treated control groups. Moreover, the phosphorylation level of Akt was decreased, whereas the phosphorylation of ERK and cleaved-caspase 3 levels were significantly increased. Taken together, SMS2 can affect sperm motility and penetration ability into methylcellulose, and participate in apoptosis associated with the Akt and ERK signaling pathways.

Utility of Films to Anticipate Effect of Drug Load and Polymer on Dissolution Performance from Tablets of Amorphous Itraconazole Spray-Dried Dispersions.

Because spray-dried dispersion (SDD) performance depends on polymer selection and drug load, time- and resource-sparing methods to screen drug/polymer combinations before spray drying are desirable. The primary objective was to assess the utility of films to anticipate the effects of drug load and polymer grade on dissolution performance of tablets containing SDDs of itraconazole (ITZ). A secondary objective was to characterize the solid-state attributes of films and SDDs to explain drug load and polymer effects on dissolution performance. SDDs employed three different grades of hypromellose acetate succinate (i.e., either HPMCAS-L, HPMCAS-M, or HPMCAS-H). Solid-state characterization employed differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD), and solid-state nuclear magnetic resonance (ssNMR) spectroscopy. Results indicate that films correctly anticipated the effects of drug load and polymer on dissolution performance. The best dissolution profiles were observed under the following conditions: 20% drug loading performed better than 30% for both films and SDDs, and the polymer grade rank order was HPMCAS-L > HPMCAS-M > HPMCAS-H for both films and SDDs. No dissolution was detected from films or SDDs containing HPMCAS-H. Solid-state characterization revealed percent crystallinity and phase miscibility as contributing factors to dissolution, but were not the sole factors. Amorphous content in films varied with drug load (10% > 20% > 30%) and polymer grades (HPMCAS-L > HPMCAS-M > HPMCAS-H), in agreement with dissolution. In conclusion, films anticipated the rank-order effects of drug load and polymer grade on dissolution performance from SDDs of ITZ, in part through percent crystallinity and phase miscibility influences.

A Combined Utilization of Plasdone-S630 and HPMCAS-HF in Ziprasidone Hydrochloride Solid Dispersion by Hot-Melt Extrusion to Enhance the Oral Bioavailability and No Food Effect.

The purpose of this study was to research a novel combination of Plasdone-S630 and HPMCAS-HF as hot-melt carrier used in ziprasidone hydrochloride for enhanced oral bioavailability and dismissed food effect. Ziprasidone hydrochloride solid dispersion (ZH-SD) was prepared by hot-melt extrusion technique, and its optimized formulation was selected by the central composite design (CCD), which was characterized for powder X-ray diffraction (PXRD), fourier transform infrared spectroscopy (FTIR), in vitro dissolution study, and stability study. Finally, the in vivo study in fasted/fed state was carried out in beagle dogs. Based on PXRD analysis, HME technique successfully dispersed ziprasidone with a low crystallinity hydrochloride form in the polymers. According to the analysis of FTIR, hydrogen bonds were formed between drug and polymers during the process of HME. Without any noticeable bulk, crystalline could be found from the micrograph of ZH-SD when analyzed the result of scanning electron microscope (SEM). Pharmacokinetics studies indicated that the bioavailability of ZH-SD formulation had no significant difference in fasted and fed state, and the Cmax and AUC of ZH-SD were two fold higher than Zeldox® in fasted state. This result indicated that ziprasidone has achieved a desired oral bioavailability in fasted state and no food effect.

Impact of Physicochemical Properties of Cellulosic Polymers on Supersaturation Maintenance in Aqueous Drug Solutions.

The precipitation inhibitory effect of cellulosic polymers in relation to their physicochemical properties was studied. Using a poorly water-soluble model drug, griseofulvin, the precipitation inhibitory effect of a series of hydroxypropyl methylcellulose (HPMC) and methylcellulose polymers was studied using solvent-shift method. The extent of supersaturation maintenance of each polymer was then quantified by the parameter, supersaturation factor (SF). Partial least square (PLS) regression analysis was employed to understand the relative contribution from viscosity, hydroxypropyl content (HC), methoxyl content, methoxyl/hydroxypropyl ratio, and drug-polymer interaction parameter (χ) on SF. All grades of cellulosic polymers effectively prolonged supersaturation of griseofulvin. PLS regression analysis revealed that HC and χ appeared to have the strongest influence on SF response. A regression model of SF = 1.65-0.16 χ + 0.05 HC with a high correlation coefficient, r of 0.921, was obtained. Since the value of χ is inversely related to the strength of drug-polymer interaction, the result shows that SF increases with increasing drug-polymer interaction and increasing HC. As such, it can be implied that strong drug-polymer interaction and presence of hydroxypropyl groups in cellulosic polymers for hydrogen bonding are two key parameters for effective supersaturation maintenance. This knowledge on the relative contribution of polymer physicochemical properties on precipitation inhibition will allow the selection of suitable cellulosic polymers for systematic development of supersaturating drug delivery systems.

Alignment of actin filament streams driven by myosin motors in crowded environments.

BACKGROUND: Cellular dynamics depend on cytoskeletal filaments and motor proteins. Collective movements of filaments driven by motor proteins are observed in the presence of dense filaments in in vitro systems. As multiple macromolecules exist within cells and the physiological ionic conditions affect their interactions, crowding might contribute to ordered cytoskeletal architecture because of collective behavior. METHODS: Using an in vitro reconstituted system, we observed the emergence of stripe patterns resulting from collective actin filament streaming driven by myosin motors in the presence of the crowding agent, methylcellulose (MC). RESULTS: Although at high KCl concentrations (150mM), actin filaments tended to dissociate from a myosin-coated surface, 1% MC prevented this dissociation and enabled filament movement on myosin molecules. At concentrations of actin filaments above 0.2mg/mL, the moving filaments accumulated and progressively formed long, dense bands. The bands were spaced at about 10-µm intervals. Increasing the KCl concentration up to 300mM resulted in narrowing of the spacing between the aligned bands. On the other hand, low KCl concentrations (≤25mM) induced broad streams, where actin filaments exhibited bidirectional movement. CONCLUSIONS: These results suggest that crowded environments can promote spatial patterning of the actin cytoskeleton, depending on the intensity of the myosin driving force and filament velocity, both modulated by the ionic strength. GENERAL SIGNIFICANCE: The mutual contribution of packing and driving forces provides insight into cytoskeleton organization in living cells, in which various macromolecules mingle.

Angiogenic sprouting is regulated by endothelial cell expression of Slug.

The Snail family of zinc-finger transcription factors are evolutionarily conserved proteins that control processes requiring cell movement. Specifically, they regulate epithelial-to-mesenchymal transitions (EMT) where an epithelial cell severs intercellular junctions, degrades basement membrane and becomes a migratory, mesenchymal-like cell. Interestingly, Slug expression has been observed in angiogenic endothelial cells (EC) in vivo, suggesting that angiogenic sprouting may share common attributes with EMT. Here, we demonstrate that sprouting EC in vitro express both Slug and Snail, and that siRNA-mediated knockdown of either inhibits sprouting and migration in multiple in vitro angiogenesis assays. We find that expression of MT1-MMP, but not of VE-Cadherin, is regulated by Slug and that loss of sprouting as a consequence of reduced Slug expression can be reversed by lentiviral-mediated re-expression of MT1-MMP. Activity of MMP2 and MMP9 are also affected by Slug expression, likely through MT1-MMP. Importantly, we find enhanced expression of Slug in EC in human colorectal cancer samples compared with normal colon tissue, suggesting a role for Slug in pathological angiogenesis. In summary, these data implicate Slug as an important regulator of sprouting angiogenesis, particularly in pathological settings.

Kinetics of myosin light chain kinase activation of smooth muscle myosin in an in vitro model system.

During activation of smooth muscle contraction, one myosin light chain kinase (MLCK) molecule rapidly phosphorylates many smooth muscle myosin (SMM) molecules, suggesting that muscle activation rates are influenced by the kinetics of MLCK-SMM interactions. To determine the rate-limiting step underlying activation of SMM by MLCK, we measured the kinetics of calcium-calmodulin (Ca²âºCaM)-MLCK-mediated SMM phosphorylation and the corresponding initiation of SMM-based F-actin motility in an in vitro system with SMM attached to a coverslip surface. Fitting the time course of SMM phosphorylation to a kinetic model gave an initial phosphorylation rate, kp(o), of ~1.17 heads s⁻¹ MLCK⁻¹. Also, we measured the dwell time of single streptavidin-coated quantum dot-labeled MLCK molecules interacting with surface-attached SMM and phosphorylated SMM using total internal reflection fluorescence microscopy. From these data, the dissociation rate constant from phosphorylated SMM was 0.80 s⁻¹, which was similar to the kp(o) mentioned above and with rates measured in solution. This dissociation rate was essentially independent of the phosphorylation state of SMM. From calculations using our measured dissociation rates and Kd values, and estimates of SMM and MLCK concentrations in muscle, we predict that the dissociation of MLCK from phosphorylated SMM is rate-limiting and that the rate of the phosphorylation step is faster than this dissociation rate. Also, association with SMM (11-46 s⁻¹) would be much faster than with pSMM (<0.1-0.2 s⁻¹). This suggests that the probability of MLCK interacting with unphosphorylated versus phosphorylated SMM is 55-460 times greater. This would avoid sequestering MLCK to unproductive interactions with previously phosphorylated SMM, potentially leading to faster rates of phosphorylation in muscle.

The Qdot-labeled actin super-resolution motility assay measures low-duty cycle muscle myosin step size.

Myosin powers contraction in heart and skeletal muscle and is a leading target for mutations implicated in inheritable muscle diseases. During contraction, myosin transduces ATP free energy into the work of muscle shortening against resisting force. Muscle shortening involves relative sliding of myosin and actin filaments. Skeletal actin filaments were fluorescently labeled with a streptavidin conjugate quantum dot (Qdot) binding biotin-phalloidin on actin. Single Qdots were imaged in time with total internal reflection fluorescence microscopy and then spatially localized to 1-3 nm using a super-resolution algorithm as they translated with actin over a surface coated with skeletal heavy meromyosin (sHMM) or full-length ß-cardiac myosin (MYH7). The average Qdot-actin velocity matches measurements with rhodamine-phalloidin-labeled actin. The sHMM Qdot-actin velocity histogram contains low-velocity events corresponding to actin translation in quantized steps of ~5 nm. The MYH7 velocity histogram has quantized steps at 3 and 8 nm in addition to 5 nm and larger compliance compared to that of sHMM depending on the MYH7 surface concentration. Low-duty cycle skeletal and cardiac myosin present challenges for a single-molecule assay because actomyosin dissociates quickly and the freely moving element diffuses away. The in vitro motility assay has modestly more actomyosin interactions, and methylcellulose inhibited diffusion to sustain the complex while preserving a subset of encounters that do not overlap in time on a single actin filament. A single myosin step is isolated in time and space and then characterized using super-resolution. The approach provides a quick, quantitative, and inexpensive step size measurement for low-duty cycle muscle myosin.

NOD-scid IL2R γnull mice engrafted with human peripheral blood mononuclear cells as a model to test therapeutics targeting human signaling pathways.

BACKGROUND: Animal models of human inflammatory diseases have limited predictive quality for human clinical trials for various reasons including species specific activation mechanisms and the immunological background of the animals which markedly differs from the genetically heterogeneous and often aged patient population. OBJECTIVE: Development of an animal model allowing for testing therapeutics targeting pathways involved in the development of Atopic Dermatitis (AD) with better translatability to the patient. METHODS: NOD-scid IL2R γnull mice engrafted with human peripheral blood mononuclear cells (hPBMC) derived from patients suffering from AD and healthy volunteers were treated with IL-4 and the antagonistic IL-4 variant R121/Y124D (Pitrakinra). Levels of human (h)IgE, amount of B-, T- and plasma- cells and ratio of CD4 : CD8 positive cells served as read out for induction and inhibition of cell proliferation and hIgE secretion. Results were compared to in vitro analysis. RESULTS: hIgE secretion was induced by IL-4 and inhibited by the IL-4 antagonist Pitrakinra in vivo when formulated with methylcellulose. B-cells proliferated in response to IL-4 in vivo; the effect was abrogated by Pitrakinra. IL-4 shifted CD4 : CD8 ratios in vitro and in vivo when hPBMC derived from healthy volunteers were used. Pitrakinra reversed the effect. Human PBMC derived from patients with AD remained inert and engrafted mice reflected the individual responses observed in vitro. CONCLUSION: NOD-scid IL2R γnull mice engrafted with human PBMC reflect the immunological history of the donors and provide a complementary tool to in vitro studies. Thus, studies in this model might provide data with better translatability from bench to bedside.

Arginine, soy isoflavone and hydroxypropylmethylcellulose have protective effects against obesity in broiler breeder hens fed on high-energy diets.

1. The present study was undertaken to determine the effects of arginine, soy isoflavone (ISF) and hydroxypropylmethylcellulose (HPMC) on obesity in broiler breeder hens. 2. A total of 320 Cobb 500 hens, 45 weeks of age, were assigned to 64 floor pens. The experiment was conducted as a completely randomised design in a factorial arrangement (2 × 2 × 2 × 2) with 4 replicates of 5 hens in each pen. Factors included two concentrations of HPMC (0 and 1%), two concentrations of arginine (8.4 and 12 g/kg), two concentrations of ISF (zero and three times more than that present in basal diets) and two contents of energy (11.7 and 14.6 MJ/kg). Performance criteria and blood characteristics of hens were measured during the experimental period. Expression of genes involved in lipid metabolism was determined in the liver at 55 weeks of age. 3. Hens given high-energy diets showed increased BW (body weight), ovary weight and abdominal fat pad and enhanced plasma glucose, triglyceride (TG), cholesterol, haemoglobin, haematocrit and low lymphocyte percentages. The expression of malic enzyme, peroxisome proliferator-activated receptor-α (PPARα), peroxisome proliferator-activated receptor-γ (PPARγ) and inducible nitric oxide (iNOS) increased and sterol regulatory element binding protein-1c (SREBP1c) decreased with increasing energy content of diets. Arginine addition decreased TG, cholesterol and A1-c haemoglobin concentration and increased PPARα, PPARγ and iNOS expression. Inclusion of ISF and HPMC decreased BW, egg weight, plasma TG, cholesterol and increased egg production and also enhanced PPARγ and iNOS expression. Significant interactions were observed between energy concentration and ISF and HPMC on BW. 4. The results of the current study revealed that ISF, HPMC and arginine have beneficial effects on controlling the metabolism of obese broiler breeder hens and using a mix of these products minimises the harmful effects of obesity.

The pivotal role of Bifida Ferment Lysate on reinforcing the skin barrier function and maintaining homeostasis of skin defenses in vitro.

BACKGROUND: The semiactive or inactive probiotics or their extracts used in dermatology have interesting properties to ameliorate signs of irritated skin and enhance the skin barrier. Bifidobacterium, as the most common probiotics, which has been found to be effective in reducing acne and improving the skin barrier function of atopic dermatitis. Bifida Ferment Lysate (BFL) can be obtained from Bifidobacterium by fermentation and extraction. PURPOSE: In this study, we investigated the effect of a topically used BFL on the skin using in vitro evaluation methods. RESULTS: The results showed that upregulation of skin physical barrier gene (FLG, LOR, IVL, TGM1, and AQP3) and antimicrobial peptide gene (CAMP and hBD-2) in HaCaT cells by BFL might be responsible for skin barrier resistance. In addition, BFL had strong antioxidant properties representing a dose-dependent increasing of the scavenging capacity of DPPH, ABTS, hydroxyl, and superoxide radicals. BFL treatment also fundamentally inhibited the intracellular ROS and MDA production and improved the activities of antioxidant enzymes (CAT and GSH-Px) in H2 O2 -stimulated HaCaT cells. As a good immunomodulatory factor, BFL efficiently decreased the secretion of IL-8 and TNF-α cytokines, and COX-2 mRNA expression in LPS-induced THP-1 macrophages. CONCLUSION: BFL can strengthen the skin barrier function and stimulate skin barrier resistance, to reinforce the skin against oxidative stress and inflammatory stimuli.

Cellular adhesion and chondrogenic differentiation inside an alginate-based bioink in response to tailorable artificial matrices and tannic acid treatment.

Many established bioinks fulfill important requirements regarding fabrication standards and cytocompatibility. Current research focuses on development of functionalized bioinks with an improved support of tissue-specific cell differentiation. Many approaches primarily depend on decellularized extracellular matrices or blood components. In this study, we investigated the combination of a highly viscous alginate-methylcellulose (algMC) bioink with collagen-based artificial extracellular matrix (aECM) as a finely controllable and tailorable system composed of collagen type I (col) with and without chondroitin sulfate (CS) or sulfated hyaluronan (sHA). As an additional stabilizer, the polyphenol tannic acid (TA) was integrated into the inks. The assessment of rheological properties and printability as well as hydrogel microstructure revealed no adverse effect of the integrated components on the inks. Viability, adhesion, and proliferation of bioprinted immortalized human mesenchymal stem cells (hTERT-MSC) was improved indicating enhanced interaction with the designed microenvironment. Furthermore, chondrogenic matrix production (collagen type II and sulfated glycosaminoglycans) by primary human chondrocytes (hChon) was enhanced by aECM. Supplementing the inks with TA was required for these positive effects but caused cytotoxicity as soon as TA concentrations exceeded a certain amount. Thus, combining tailorable aECM with algMC and balanced TA addition proved to be a promising approach for promoting adhesion of immortalized stem cells and differentiation of chondrocytes in bioprinted scaffolds.

Interfacial interaction between transmembrane ocular mucins and adhesive polymers and dendrimers analyzed by surface plasmon resonance.

PURPOSE: Development of the first in vitro method based on biosensor chip technology designed for probing the interfacial interaction phenomena between transmembrane ocular mucins and adhesive polymers and dendrimers intended for ophthalmic administration. METHODS: The surface plasmon resonance (SPR) technique was used. A transmembrane ocular mucin surface was prepared on the chip surface and characterized by QCM-D (Quartz Crystal Microbalance with Dissipation) and XPS (X-ray photoelectron spectroscopy). The mucoadhesive molecules tested were: hyaluronic acid (HA), carboxymethyl cellulose (CMC), hydroxypropylmethyl cellulose (HPMC), chitosan (Ch) and polyamidoamine dendrimers (PAMAM). RESULTS: While Ch originated interfacial interaction with ocular transmembrane mucins, for HA, CMC and HPMC, chain interdiffusion seemed to be mandatory for bioadherence at the concentrations used in ophthalmic clinical practise. Interestingly, PAMAM dendrimers developed permanent interfacial interactions with transmembrane ocular mucins whatever their surface chemical groups, showing a relevant importance of co-operative effect of these multivalent systems. Polymers developed interfacial interactions with ocular membrane-associated mucins in the following order: Ch(1 %) > G4PAMAM-NH(2)(2 %) = G4PAMAM-OH(2 %) > G3.5PAMAM-COOH(2 %)>> CMC(0.5 %) = HA(0.2 %) = HPMC(0.3 %). CONCLUSIONS: The method proposed is useful to discern between the mucin-polymer chemical interactions at molecular scale. Results reinforce the usefulness of chitosan and dendrimers as polymers able to increase the retention time of drugs on the ocular surface and hence their bioavailability.

Mucoadhesive tablets for controlled release of acyclovir.

Mucoadhesive chitosan (CS) and/or hydroxypropyl-methylcellulose (HPMC) tablets for gastric drug delivery of acyclovir (ACV) have been developed in order to improve the ACV oral bioavailability. Swelling, bioadhesive and dissolution studies were carried out in two acidic media (pH 1.5 and 4) in order to determine the tablets behaviour in both fed and fasted states. All the designed tablets showed good mucoadhesive properties on gastric mucosa due to the presence of CS and/or HPMC. In vitro dissolution of ACV from tablets was influenced by the swelling behaviour of each polymer. All data release of the studied tablets fitted to Hopfenberg model, which describes drug release from tablets displaying heterogeneous erosion. HPMC and CS/HPMC tablets revealed a sustained release for 24 h, but a complete dissolution of the tablets was not produced at this time. On the contrary, tablets which contained only CS as polymer were able to release the total amount of ACV for 4 h, due to the CS imbibition and erosion processes in pH 1.5 medium. These results allowed us to conclude that CS is the excipient to be chosen to obtain gastroretentive formulations, due to its demonstrated gastric compatibility.

Statistical approach for assessing the influence of calcium silicate and HPMC on the formulation of novel alfuzosin hydrochloride mucoadhesive-floating beads as gastroretentive drug delivery systems.

Multiparticulate floating drug delivery systems have proven potential as controlled-release gastroretentive drug delivery systems that avoid the "all or none" gastric emptying nature of single-unit floating dosage forms. An objective of the presence investigation was to develop calcium silicate (CaSi)/calcium alginate (Ca-Alg)/hydroxypropyl methylcellulose (HPMC) mucoadhesive-floating beads that provide time- and site-specific drug release of alfuzosin hydrochloride (Alf). Beads were prepared by simultaneous internal and external gelation method utilizing 3(2) factorial design as an experimental design; with two main factors evaluated for their influence on the prepared beads; the concentration of CaSi as floating aid (X (1)) and the percentage of HPMC as viscosity enhancer and mucoadhesive polymer (X (2)), each of them was tested in three levels. Developed formulations were evaluated for yield, entrapment efficiency, particle size, surface topography, and buoyancy. Differential scanning calorimetry, Fourier transform infrared spectroscopy, in vitro drug release, as well as in vitro mucoadhesion using rat stomach mucosal membrane were also conducted. Percentage yield and entrapment efficiency ranged from 57.03% to 78.51% and from 49.78% to 83.26%, respectively. Statistical analysis using ANOVA proved that increasing the concentration of either CaSi or HPMC significantly increased the beads yield. Both CaSi and HPMC concentrations were found to significantly affect Alf release from the beads. Additionally, higher CaSi concentration significantly increased the beads diameter while HPMC concentration showed significant positive effect on the beads mucoadhesive properties. CaSi/Ca-Alg/HPMC beads represent simple floating-mucoadhesive gastroretentive system that could be useful in chronopharmacotherapy of benign prostatic hyperplasia.

Discovery of a selective and covalent small-molecule inhibitor of BFL-1 protein that induces robust apoptosis in cancer cells.

Induction of apoptosis by the FDA-approved drug Venetoclax in cancer cells mainly derives from blocking the interactions between BCL-2 and BH3-only proteins. Anti-apoptotic BFL-1, a homolog of BCL-2, also competitively binds to the BH3-only proteins and is responsible for Venetoclax-induced drug resistance. Compared to BCL-2, small-molecule inhibitors of BFL-1 are relatively underexplored. In order to tackle this issue, in-house compound library was screened and a hit compound was identified and optimized to obtain 12 (ZH97) functioning as a covalent and selective inhibitor of BFL-1. 12 modifies BFL-1 at the C55 residue, blocks BFL-1/BID interaction in vitro, promotes cellular cytochrome c release from mitochondria, and induced apoptosis in BFL-1 overexpressing cancer cells. Mechanistic studies show that 12 inhibited BFL-1/PUMA interaction in cell lysate and is effective in cancer cells that harboring high expression level of BFL-1. In summary, blockade of BFL-1/BH3-only proteins interactions with a covalent small-molecule inhibitor induced apoptosis and elicited antitumor activity. Thus, our study demonstrates an appealing strategy for selective modulation of cellular BFL-1 for cancer therapy.

Phytochemical Screening and Bioactivity Studies of Endophytes Cladosporium sp. Isolated from the Endangered Plant Vateria Indica Using In Silico and In Vitro Analysis.

Vateria indica is persistent tree used in Unani sources for the medication and classified as critically endangered. Thus, endophytes for alternative methods to explore these endangered Plants having rich source pharmaceuticals' active molecules for drug development and production. Endophytes comprises unexplored microbes as a potential source of rich pharmaceutically bioactive compounds attributable to their relationship with the host. In the current study, we have isolated endophyte fungi Cladosporium from the plant Vateria indica and performed phytochemical screening of its ethanolic extract to detect the phytochemicals using thin layer chromatography (TLC), gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC), UV-visible spectrophotometry (UV-VIS), and Fourier transform infrared spectroscopy (FTIR). GC-MS analysis revealed the presence of an anticancer compound hydroxymethyl colchicine, antioxidant compound benzoic acid, and antimicrobial 2-(4-chlorophenoxy)-5-nitro in endophyte fungal extract of plant Vateria indica. Moreover, in silico analysis of bioactive compounds identified by GC-MS analysis using the Autodock Vina and SwissADME confirmed excellent anticancer activity methanone, [4-amino-2-[(phenylmethyl) amino]-5-thiazolyl] (4-fluorophenyl)- and hydroxymethyl colchicine against 6VO4 (Bfl-1 protein) as per Lipinski rule. Furthermore, we also demonstrated the excellent antioxidant of endophytic extract compared to plant extract by DPPH and ABTS assay, as well as antimicrobial activity against both Gram (+ ve) and Gram (- ve) bacteria. Moreover, the endophytic extract also showed its antimitotic activity with a mitotic index of 65.32, greater than the plant extract of 32.56 at 10 mg/ml. Thus endophytic fungi Cladosporium species isolated from plant Vateria indica might be used as a potential source for phytochemical anticancer hydroxymethyl colchicine, an antioxidant benzoic acid, and antimicrobial 2-(4-chlorophenoxy)-5-nitro.

Transformation of actoHMM assembly confined in cell-sized liposome.

To construct a simple model of a cellular system equipped with motor proteins, cell-sized giant liposomes encapsulating various amounts of actoHMM, the complexes of actin filaments (F-actin) and heavy meromyosin (HMM, an actin-related molecular motor), with a depletion reagent to mimic the crowding effect of inside of living cell, were prepared. We adapted the methodology of the spontaneous transfer of water-in-oil (W/O) droplets through a phospholipid monolayer into the bulk aqueous phase and successfully prepared stable giant liposomes encapsulating the solution with a physiological salt concentration containing the desired concentrations of actoHMM, which had been almost impossible to obtain using currently adapted methodologies such as natural swelling and electro-formation on an electrode. We then examined the effect of ATP on the cytoskeleton components confined in those cell-sized liposomes, because ATP is known to drive the sliding motion for actoHMM. We added α-hemolysin, a bacterial membrane pore-forming toxin, to the bathing solution and obtained liposomes with the protein pores embedded on the bilayer membrane to allow the transfer of ATP inside the liposomes. We show that, by the ATP supply, the actoHMM bundles inside the liposomes exhibit specific changes in spatial distribution, caused by the active sliding between F-actin and HMM. Interestingly, all F-actins localized around the inner periphery of liposomes smaller than a critical size, whereas in the bulk solution and also in larger liposomes, the actin bundles formed aster-like structures under the same conditions.