Unlocking the Gut's Treasure: Lipase-Producing Bacillus subtilis Probiotic from the Intestine of Microstomus kitt (Lemon sole).

The main objective of this research was to identify potential probiotic candidates belonging to the Bacillus species that could demonstrate tolerance to bile salt and acidic conditions. The study focused on isolating Bacillus strains from the intestine of marine fish-Microstomus kitt. The isolation process involved the use of selective MRS media through the pour plate method. After 24 h, one particular isolate was identified based on its morphological and biochemical traits as Bacillus species. To confirm the identity, molecular characterization of the 16S RNA from the isolated strain was performed, and the sequence analysis verified it as Bacillus subtilis strain ACL_BS 001. With the molecular confirmation, the next step was to assess the probiotic characteristics of this B. subtilis strain. Various tests were conducted to evaluate its acid/pH tolerance, NaCl tolerance, and bile salt tolerance. The results indicated that B. subtilis exhibited high viability percentages even under acidic pH, in the presence of 1.5% bile salt, and at high salt concentrations. Subsequently, we investigated the strain's ability to produce lipase, an important enzyme with potential industrial applications. B. subtilis was grown in MRS agar amended with olive oil as a lipase substrate. After incubation, the presence of lipase activity was confirmed, and the enzymatic assay revealed a significant lipase enzyme activity of 100.23 µmoles/ml of the sample. In conclusion, the study successfully isolated and identified B. subtilis from the intestine of Microstomus kitt, and the strain exhibited promising probiotic characteristics, including resistance to bile salt and acidic conditions. Furthermore, the strain was found to produce lipase, which opens up possibilities for future research focusing on isolating and purifying the lipase from this potential probiotic B. subtilis strain.

The centrosomal recruitment of γ-tubulin and its microtubule nucleation activity is α-fodrin guided.

The regulation and recruitment of γ-TuRCs, the prime nucleator of microtubules, to the centrosome are still thrust areas of research. The interaction of fodrin, a sub-plasmalemmal cytoskeletal protein, with γ-tubulin is a new area of interest. To understand the cellular significance of this interaction, we show that depletion of α-fodrin brings in a significant reduction of γ-tubulin in neural cell centrosomes making it functionally under-efficient. This causes a loss of nucleation ability that cannot efficiently form microtubules in interphase cells and astral microtubules in mitosis. Fluorescence Recovery after Photobleaching (FRAP) experiment implies that α-fodrin is important in the recruitment of γ-tubulin to the centrosome resulting in the aforementioned effects. Further, our experiments indicate that the interaction of α-fodrin with certain pericentriolar matrix proteins such as Pericentrin and CDK5RAP2 are crucial for the recruitment of γ-tubulin to the centrosome. Earlier we reported that α-fodrin limits the nucleation potential of γ-TuRC. In that context, this study suggests that α-fodrin is a γ-tubulin recruiting protein to the centrosome thus preventing cytoplasmic microtubule nucleation and thereby compartmentalizing the process to the centrosome for maximum efficiency. Summary statementα-fodrin is a γ-tubulin interacting protein that controls the process of γ-tubulin recruitment to the centrosome and thereby regulates the microtubule nucleation capacity spatially and temporally.

α-Fodrin in Cytoskeletal Organization and the Activity of Certain Key Microtubule Kinesins.

Cortical cytoskeletal proteins are significant in controlling various cellular mechanisms such as migration, cell adhesion, intercellular attachment, cellular signaling, exo- and endocytosis and plasma membrane integrity, stability and flexibility. Our earlier studies involving in vitro and ex vivo approaches led us to identify certain undiscovered characteristics of α-fodrin, a prominent cortical protein. The conventional functions attributed to this protein mainly support the plasma membrane. In the present study, we utilized a global protein expression analysis approach to detect underexplored functions of this protein. We report that downregulation of α-fodrin in glioblastoma cells, U-251 MG, results in upregulation of genes affecting the regulation of the cytoskeleton, cell cycle and apoptosis. Interestingly, certain key microtubule kinesins such as KIF23, KIF2B and KIF3C are downregulated upon α-fodrin depletion, as validated by real-time PCR studies.

A Fresh Look at the Structure, Regulation, and Functions of Fodrin.

Fodrin and its erythroid cell-specific isoform spectrin are actin-associated fibrous proteins that play crucial roles in the maintenance of structural integrity in mammalian cells, which is necessary for proper cell function. Normal cell morphology is altered in diseases such as various cancers and certain neuronal disorders. Fodrin and spectrin are two-chain (αß) molecules that are encoded by paralogous genes and share many features but also demonstrate certain differences. Fodrin (in humans, typically a heterodimer of the products of the SPTAN1 and SPTBN1 genes) is expressed in nearly all cell types and is especially abundant in neuronal tissues, whereas spectrin (in humans, a heterodimer of the products of the SPTA1 and SPTB1 genes) is expressed almost exclusively in erythrocytes. To fulfill a role in such a variety of different cell types, it was anticipated that fodrin would need to be a more versatile scaffold than spectrin. Indeed, as summarized here, domains unique to fodrin and its regulation by Ca2+, calmodulin, and a variety of posttranslational modifications (PTMs) endow fodrin with additional specific functions. However, how fodrin structural variations and misregulated PTMs may contribute to the etiology of various cancers and neurodegenerative diseases needs to be further investigated.

Offsetting anthropogenic carbon emissions from biomass waste and mineralised carbon dioxide.

The present work investigates biomass wastes and their ashes for re-use in combination with mineralised CO2 in cement-bound construction products. A range of biomass residues (e.g., wood-derived, nut shells, fibres, and fruit peels) sourced in India, Africa and the UK were ashed and exposed to CO2 gas. These CO2-reactive ashes could mineralise CO2 gas and be used to cement 'raw' biomass in solid carbonated monolithic composites. The CO2 sequestered in ashes (125-414 g CO2/kg) and that emitted after incineration (400-500 g CO2/kg) was within the same range (w/w). The CO2-reactive ashes embodied significant amounts of CO2 (147-424 g equivalent CO2/kg ash). Selected ashes were combined with raw biomass and Portland Cement, CEM 1 and exposed to CO2. The use of CEM 1 in the carbonated products was offset by the CO2 mineralised (i.e. samples were 'carbon negative', even when 10% w/w CEM 1 was used); furthermore, biomass ashes were a suitable substitute for CEM 1 up to 50% w/w. The approach is conceptually simple, scalable, and can be applicable to a wide range of biomass ashes in a closed 'emission-capture' process 'loop'. An extrapolation of potential for CO2 offset in Europe provides an estimate of CO2 sequestration potential to 2030.

α-Fodrin is required for the organization of functional microtubules during mitosis.

The cytoskeleton protein α-fodrin plays a major role in maintaining structural stability of membranes. It was also identified as part of the brain γ-tubulin ring complex, the major microtubule nucleator. Here, we investigated the requirement of α-fodrin for microtubule spindle assembly during mitotic progression. We found that α-fodrin depletion results in abnormal mitosis with uncongressed chromosomes, leading to prolonged activation of the spindle assembly checkpoint and a severe mitotic delay. Further, α-fodrin repression led to the formation of shortened spindles with unstable kinetochore-microtubule attachments. We also found that the mitotic kinesin CENP-E had reduced levels at kinetochores to likely account for the chromosome misalignment defects in α-fodrin-depleted cells. Importantly, we showed these cells to exhibit reduced levels of detyrosinated α-tubulin, which primarily drives CENP-E localization. Since proper microtubule dynamics and chromosome alignment are required for completion of normal mitosis, this study reveals an unforeseen role of α-fodrin in regulating mitotic progression. Future studies on these lines of observations should reveal important mechanistic insight for fodrin's involvement in cancer.

Binding of alpha-fodrin to gamma-tubulin accounts for its role in the inhibition of microtubule nucleation.

Non-erythroid spectrin or fodrin is present as part of the γ-tubulin ring complex (γ-TuRC) in brain tissue and brain derived cells. Here, we show that fodrin, which is otherwise known for providing structural support to the cell membrane, interacts directly with γ-tubulin within the γ-TuRC through a GRIP2-like motif. Turbidometric analysis of microtubule polymerization with nucleation-potent γ-TuRC isolated from HEK-293 cells that lack fodrin and the γ-TuRC from goat brain that contains fodrin shows inefficiency of the latter to promote nucleation. The involvement of fodrin was confirmed by the reduction in the microtubule polymerization efficiency of HEK-293 derived γ-TuRCs upon addition of purified brain fodrin. Thus, the interaction of fodrin with gamma-tubulin is responsible for its inhibitory effect on γ-tubulin mediated microtubule nucleation.

Insights on the persistence of pines (Pinus species) in the Late Cretaceous and their increasing dominance in the Anthropocene.

Although gymnosperms were nearly swept away by the rise of the angiosperms in the Late Cretaceous, conifers, and pines (Pinus species) in particular, survived and regained their dominance in some habitats. Diversification of pines into fire-avoiding (subgenus Haploxylon) and fire-adapted (subgenus Diploxylon) species occurred in response to abiotic and biotic factors in the Late Cretaceous such as competition with emerging angiosperms and changing fire regimes. Adaptations/traits that evolved in response to angiosperm-fuelled fire regimes and stressful environments in the Late Cretaceous were key to pine success and are also contributing to a new "pine rise" in some areas in the Anthropocene. Human-mediated activities exert both positive and negative impacts of range size and expansion and invasions of pines. Large-scale afforestation with pines, human-mediated changes to fire regimes, and other ecosystem processes are other contributing factors. We discuss traits that evolved in response to angiosperm-mediated fires and stressful environments in the Cretaceous and that continue to contribute to pine persistence and dominance and the numerous ways in which human activities favor pines.

Pharmacological credence of the folklore use of Bauhinia malabarica in the management of jaundice.

The information on the hepatoprotective effect of Bauhinia malabarica Roxb. (Family Leguminosae) used in the folkloric medical practice in Malabar coast and Walayar valley of southern India for the treatment of liver related disorders is completely unknown. Hence, the efficacy of the aqueous methanolic extract of stem bark of B. malabarica (AqMeOH-Ba) was evaluated for liver function serum biochemical markers along with the antioxidant markers in liver tissues of Wistar albino rats. The biochemical observations as well as the histopathological examination of liver sections manifested considerable hepatoprotective activity of B. malabarica stem bark, and thus validated the folkloric claim.

+TIP EB1 downregulates paclitaxel­induced proliferation inhibition and apoptosis in breast cancer cells through inhibition of paclitaxel binding on microtubules.

Microtubule plus­end­binding protein (+TIP) EB1 has been shown to be upregulated in breast cancer cells and promote breast tumor growth in vivo. However, its effect on the cellular actions of microtubule­targeted drugs in breast cancer cells has remained poorly understood. By using cellular and biochemical assays, we demonstrate that EB1 plays a critical role in regulating the sensitivity of breast cancer cells to anti­microtubule drug, paclitaxel (PTX). Cell viability assays revealed that EB1 expression in the breast cancer cell lines correlated with the reduction of their sensitivity to PTX. Knockdown of EB1 by enzymatically­prepared siRNA pools (esiRNAs) increased PTX­induced cytotoxicity and sensitized cells to PTX­induced apoptosis in three breast cancer cell lines, MCF­7, MDA MB­231 and T47D. Apoptosis was associated with activation of caspase­9 and an increase in the cleavage of poly(ADP­ribose) polymerase (PARP). p53 and Bax were upregulated and Bcl2 was downregulated in the EB1­depleted PTX­treated MCF­7 cells, indicating that the apoptosis occurs via a p53­dependent pathway. Following its upregulation, the nuclear accumulation of p53 and its association with cellular microtubules were increased. EB1 depletion increased PTX­induced microtubule bundling in the interphase cells and induced formation of multiple spindle foci with abnormally elongated spindles in the mitotic MCF­7 cells, indicating that loss of EB1 promotes PTX­induced stabilization of microtubules. EB1 inhibited PTX­induced microtubule polymerization and diminished PTX binding to microtubules in vitro, suggesting that it modulates the binding sites of PTX at the growing microtubule ends. Results demonstrate that EB1 downregulates inhibition of PTX­induced proliferation and apoptosis in breast cancer cells through a mechanism in which it impairs PTX­mediated stabilization of microtubule polymerization and inhibits PTX binding on microtubules.

Microtubule +TIP protein EB1 binds to GTP and undergoes dissociation from dimer to monomer on binding GTP.

The +TIP protein EB1 autonomously tracks the growing plus end of microtubules and regulates plus-end dynamics. Previous studies have indicated that EB1 can recognize GTP-bound tubulin structures at the plus end, and it localizes on the microtubule surface at a site close to the exchangeable GTP-binding site of tubulin. Although the GTP-dependent structural change in tubulin has been demonstrated to be a critical determinant for recognition of plus ends by EB1, the effect of GTP on the structure of EB1 has remained unclear. Here, we have used spectroscopic, calorimetric, and biochemical methods to analyze the effect of GTP on EB1 in vitro. Isothermal titration calorimetry and tryptophan fluorescence quenching experiments demonstrated that EB1 binds to GTP with a dissociation constant ~30 µM. Circular dichroism measurements showed that EB1 undergoes changes in its secondary structure on binding GTP. Size-exclusion chromatography and urea-induced unfolding analyses revealed that GTP binding induces dissociation of the EB1 dimer to monomers. Size-exclusion chromatography followed by biochemical analysis further determined that EB1-GTP binding involves association of approximately one molecule of GTP per EB1 monomer. The results reveal a hitherto unknown GTP-dependent mechanism of dimer-to-monomer transition in EB1 and further implicate its possible role in regulating the stability of the EB1 dimer vs monomer as well as plus-end regulation in cells.

Complementary and alternative drug therapy in arthritis.

UNLABELLED: Utilization of complementary and alternative medicine (CAM) is universal phenomenon. They are used commonly in chronic diseases like arthritis. To understand the pattern of utilization we undertook this study focussing mainly on the systems, where drug is administered orally. MATERIAL AND METHODS: One hundred and fourteen patients suffering from rheumatoid arthritis (RA), satisfying American College of Rheumatology (ACR) criteria were interviewed for the modalities of therapy and drugs used. We analysed prescriptions of both conventional and CAM practitioners. Direct questionnaires regarding CAM were avoided. RESULTS: Fourty three percent (46/114) had used CAM drugs and 50% of them had used more than two modalities. Ayurveda followed by homeopathy were the two common CAM utilized by the patients. Majority believed conventional medicine has no cure for RA and adverse reactions were rare in CAM. These factors predominantly influenced their decision to use CAM. Family income, urban and rural living did not influence usage of CAM. The use of CAM increased as the duration of disease increased. CONCLUSION: Majority of the patients utilize CAM drugs, for treatment of arthritis. Their knowledge is essential to avoid drug interactions, recognise their reactions and also appreciate their risks and benefits. A scientific scrutiny to these practices and absolving them if beneficial is needed.