Metaphase Arrest Research Articles

Eg5 UFMylation promotes spindle organization during mitosis

UFMylation is a highly conserved ubiquitin-like post-translational modification that catalyzes the covalent linkage of UFM1 to its target proteins. This modification plays a critical role in the maintenance of endoplasmic reticulum proteostasis, DNA damage response, autophagy, and transcriptional regulation. Mutations in UFM1, as well as in its specific E1 enzyme UBA5 and E2 enzyme UFC1, have been genetically linked to microcephaly. Our previous research unveiled the important role of UFMylation in regulating mitosis. However, the underlying mechanisms have remained unclear due to the limited identification of substrates. In this study, we identified Eg5, a motor protein crucial for mitotic spindle assembly and maintenance, as a novel substrate for UFMylation and identified Lys564 as the crucial UFMylation site. UFMylation did not alter its transcriptional level, phosphorylation level, or protein stability, but affected the mono-ubiquitination of Eg5. During mitosis, Eg5 and UFM1 co-localize at the centrosome and spindle apparatus, and defective UFMylation leads to diminished spindle localization of Eg5. Notably, the UFMylation-defective Eg5 mutant (K564R) exhibited shorter spindles, metaphase arrest, spindle checkpoint activation, and a failure of cell division in HeLa cells. Overall, Eg5 UFMylation is essential for proper spindle organization, mitotic progression, and cell proliferation.

MAPK-dependent control of mitotic progression in S. pombe

BackgroundMitogen-activated protein kinases (MAPKs) preserve cell homeostasis by transducing physicochemical fluctuations of the environment into multiple adaptive responses. These responses involve transcriptional rewiring and the regulation of cell cycle transitions, among others. However, how stress conditions impinge mitotic progression is largely unknown. The mitotic checkpoint is a surveillance mechanism that inhibits mitotic exit in situations of defective chromosome capture, thus preventing the generation of aneuploidies. In this study, we investigate the role of MAPK Pmk1 in the regulation of mitotic exit upon stress.ResultsWe show that Schizosaccharomyces pombe cells lacking Pmk1, the MAP kinase effector of the cell integrity pathway (CIP), are hypersensitive to microtubule damage and defective in maintaining a metaphase arrest. Epistasis analysis suggests that Pmk1 is involved in maintaining spindle assembly checkpoint (SAC) signaling, and its deletion is additive to the lack of core SAC components such as Mad2 and Mad3. Strikingly, pmk1Δ cells show up to twofold increased levels of the anaphase-promoting complex (APC/C) activator Cdc20Slp1 during unperturbed growth. We demonstrate that Pmk1 physically interacts with Cdc20Slp1 N-terminus through a canonical MAPK docking site. Most important, the Cdc20Slp1 pool is rapidly degraded in stressed cells undergoing mitosis through a mechanism that requires MAPK activity, Mad3, and the proteasome, thus resulting in a delayed mitotic exit.ConclusionsOur data reveal a novel function of MAPK in preventing mitotic exit and activation of cytokinesis in response to stress. The regulation of Cdc20Slp1 turnover by MAPK Pmk1 provides a key mechanism by which the timing of mitotic exit can be adjusted relative to environmental conditions.

Functional analysis of Cdc20 reveals a critical role of CRY box in mitotic checkpoint signaling

Accurate mitosis is coordinated by the spindle assembly checkpoint (SAC) through the mitotic checkpoint complex (MCC), which inhibits the anaphase-promoting complex or cyclosome (APC/C). As an essential regulator, Cdc20 promotes mitotic exit through activating APC/C and monitors kinetochore-microtubule attachment through activating SAC. Cdc20 requires multiple interactions with APC/C and MCC subunits to elicit these functions. Functionally assessing these interactions within cells requires efficient depletion of endogenous Cdc20, which is highly difficult to achieve by RNA interference (RNAi). Here we generated Cdc20 RNAi-sensitive cell lines which display a penetrant metaphase arrest by a single RNAi treatment. In this null background, we accurately measured the contribution of each known motif of Cdc20 on APC/C and SAC activation. The CRY box, a previously identified degron, was found critical for SAC by promoting MCC formation and its interaction with APC/C. These data reveal additional regulation within the SAC and establish a novel method to interrogate Cdc20.

Molecular Pathways and Animal Models of Hypoplastic Left Heart Syndrome.

Hypoplastic left heart syndrome (HLHS) is a severe congenital heart disease (CHD) with underdevelopment of left-sided heart structures. While previously uniformly fatal, surgical advances now provide highly effective palliation that allows most HLHS patients to survive their critical CHD. Nevertheless, there remains high morbidity and mortality with high risk of heart failure. As hemodynamic compromise from restricted aortic blood flow has been suggested to underlie the poor LV growth, this suggests the possibility of prenatal fetal intervention to recover LV growth. As such interventions have yielded ambiguous results, the optimization of therapy will require more mechanistic insights into the developmental etiology for HLHS. Clinical studies have shown high heritability for HLHS, with an oligogenic etiology indicated in conjunction with genetic heterogeneity. This is corroborated with the recent recovery of mutant mice with HLHS. With availability-induced pluripotent stem cell (iPSC)-derived cardiomyocytes from HLHS mice and patients, new insights have emerged into the cellular and molecular etiology for the LV hypoplasia in HLHS. Cell proliferation defects were observed in conjunction with metaphase arrest and the disturbance of Hippo-YAP signaling. The left-sided restriction of the ventricular hypoplasia may result from epigenetic perturbation of pathways regulating left-right patterning. These findings suggest new avenues for fetal interventions with therapies using existing drugs that target the Hippo-YAP pathway and/or modulate epigenetic regulation.

Ameliorating effect of nanoparticles and seeds’ heat pre-treatment on soybean plants exposed to sea water salinity

Impairing plant growth and reducing crop production, salinity is considered as major problem in modern agriculture. The current study aimed to investigate the role of seeds' heat pretreatment at 45 °C as well as application of two different nanoparticles nanosilica (N1) and nanoselenium (N2) in reducing salinity stress in three genotypes of Egyptian commercial soybeans (Glycine max L.). Two levels of salt stress using diluted sea water (1/12 and 1/6) were tested either alone or in combination with protective treatments. Obtained results revealed that salinity caused a significant reduction in all tested physiological parameters such as germination rate and membrane stability in soybean plants. A significant reduction in mitotic index and arrest in metaphase were recorded under both tested levels of salinity. It was also revealed that chromosomal abnormalities in soybean plants were positively correlated with the applied salinity concentrations. The fragmentation effect of salinity on the nuclear DNA was investigated and confirmed using Comet assay analysis. Seeds heat pre-treatment (45 °C) and both types of nanoparticles' treatments yielded positive effects on both the salt-stressed and unstressed plants. Quantitative real-time reverse transcription PCR (qRT-PCR) analysis for salt stress responsive marker genes revealed that most studied genes (CAT, APX, DHN2, CAB3, GMPIPL6 and GMSALT3) responded favorably to protective treatments. The modulation in gene expression pattern was associated with improving growth vigor and salinity tolerance in soybean plants. Our results suggest that seeds’ heat pretreatment and nanoparticle applications support the recovery against oxidative stresses and represent a promising strategy for alleviating salt stress in soybean genotypes.

Global SUMOylation in mouse oocytes maintains oocyte identity and regulates chromatin remodeling and transcriptional silencing at the end of folliculogenesis.

Meiotically competent oocytes in mammals undergo cyclic development during folliculogenesis. Oocytes within ovarian follicles are transcriptionally active, producing and storing transcripts required for oocyte growth, somatic cell communication and early embryogenesis. Transcription ceases as oocytes transition from growth to maturation and does not resume until zygotic genome activation. Although SUMOylation, a post-translational modification, plays multifaceted roles in transcriptional regulation, its involvement during oocyte development remains poorly understood. In this study, we generated an oocyte-specific knockout of Ube2i, encoding the SUMO E2 enzyme UBE2I, using Zp3-cre+ to determine how loss of oocyte SUMOylation during folliculogenesis affects oocyte development. Ube2i Zp3-cre+ female knockout mice were sterile, with oocyte defects in meiotic competence, spindle architecture and chromosome alignment, and a premature arrest in metaphase I. Additionally, fully grown Ube2i Zp3-cre+ oocytes exhibited sustained transcriptional activity but downregulated maternal effect genes and prematurely activated genes and retrotransposons typically associated with zygotic genome activation. These findings demonstrate that UBE2I is required for the acquisition of key hallmarks of oocyte development during folliculogenesis, and highlight UBE2I as a previously unreported orchestrator of transcriptional regulation in mouse oocytes.

Chemoprevention curcumin analog 1.1 promotes metaphase arrest and enhances intracellular reactive oxygen species levels on TNBC MDA-MB-231 and HER2-positive HCC1954 cells.

Previous studies highlighted that chemoprevention curcumin analog-1.1 (CCA-1.1) demonstrated an antitumor effect on breast, leukemia, and colorectal cancer cells. By utilizing immortalized MDA-MB-231 and HCC1954 cells, we evaluated the anticancer properties of CCA-1.1 and its mediated activity to promote cellular death. Cytotoxicity and anti-proliferation were assayed using trypan blue exclusion. The cell cycle profile after CCA-1.1 treatment was established through flow cytometry. May-Grünwald-Giemsa and Hoechst staining were performed to determine the cell cycle arrest upon CCA-1.1 treatment. The involvement of CCA-1.1 in mitotic kinases (aurora A, p-aurora A, p-PLK1, and p-cyclin B1) expression was investigated by immunoblotting. CCA-1.1-treated cells were stained with the X-gal solution to examine the effect on senescence. ROS level and mitochondrial respiration were assessed by DCFDA assay and mitochondrial oxygen consumption rate, respectively. CCA-1.1 exerted cytotoxic activity and inhibited cell proliferation with an irreversible effect, and the flow cytometry analysis demonstrated that CCA-1.1 significantly halted during the G2/M phase, and further assessment revealed that CCA-1.1 caused metaphase arrest. Immunoblot assays confirmed CCA-1.1 suppressed aurora A kinase in MDA-MB-231 cells. The ROS level was elevated after treatment with CCA-1.1, which might promote cellular senescence and suppress basal mitochondrial respiration in MDA-MB-231 cells. Our data suggested the in vitro proof-of-concept that supports the involvement in cell cycle regulation and ROS generation as contributors to the effectiveness of CCA-1.1 in suppressing breast cancer cell growth.

Chronic levamisole exposure in male rats alters sexual behavior and induces apoptosis in the testis

Levamisole is an anti-helminthic drug developed and introduced in veterinary medicine, and it has been used more frequently after the inclusion of its usage in human medicine regarding disorders with immunomodulatory properties. In recent years, it has started to attract attention since it has beneficial effects on the treatment of COVID-19 due to its immunomodulatory properties. To investigate the effects of levamisole on sexual behavior and the reproductive system in male rats, two groups were formed the vehicle (n = 10) and levamisole (n = 10) groups. The vehicle group was given purified water whereas the levamisole group was administered with levamisole (2 mg/kg) by oral gavage daily for 4 weeks. Levamisole treatment significantly increased the mount latency (ML, P < 0.001) as well as the intromission latency (IL, P < 0.01). It also significantly prolonged postejaculatory interval (PEI, P < 0.01), decreased copulatory rate (CR, P < 0.05), and sexual activity index (SAI, P < 0.05). It significantly decreased serum monoamine oxidase A (MAO-A) levels (P < 0.05). Additionally, levamisole induced disorganizations of germinal epithelial cells of seminiferous tubules, congestion, edema in the interstitial area, and metaphase arrest in some spermatocytes (P < 0.001), and it significantly increased the immunohistochemical expressions of apoptotic Bax and cytochrome c, which is crucial proapoptotic protein, in the testis (P < 0.001). Also, levamisole significantly upregulated the mRNA levels of the apoptosis-related key regulatory genes, including Bax (Bcl-2-associated X protein, P = 0.05) and Bax/Bcl-2 ratio (P < 0.01) in testis. The current research is the first to show that levamisole may decrease sexual performance, potency, sexual motivation, and libido and induce apoptosis in the testis.

Notch pathway is required for protection against heat stress in spermatogonial stem cells in medaka.

Gamete production is a fundamental process for reproduction; however, exposure to stress, such as increased environmental temperature, can decrease or even interrupt this process, affecting fertility. Thus, the survival of spermatogonial stem cells (SSCs) is crucial for the recovery of spermatogenesis upon stressful situations. Here, we show that the Notch pathway is implicated in such survival, by protecting the SSCs against thermal stress. First, we corroborated the impairment of spermatogenesis under heat stress in medaka, observing an arrest in metaphase I at 10days of heat treatment, an increase in the number of spermatocytes, and downregulation of ndrg1b and sycp3. In addition, at 30days of treatment, an interruption of spermatogenesis was observed with a strong loss of spermatocytes and spermatids. Then, the exposure of adult males to thermal stress condition induced apoptosis mainly in spermatogenic and supporting somatic cells, with the exception of the germinal region, where SSCs are located. Concomitantly, the Notch pathway-related genes were upregulated, including the ligands (dll4, jag1-2) and receptors (notch1a-3). Moreover, during thermal stress presenilin enhancer-2 (pen-2), the catalytic subunit of γ-secretase complex of the Notch pathway was restricted to the germinal region of the medaka testis, observed in somatic cells surrounding type A spermatogonia (SGa). The importance of Notch pathway was further supported by an ex vivo approach, in which the inhibition of this pathway activity induced a loss of SSCs. Overall, this study supports the importance of Notch pathways for the protection of SSCs under chronic thermal stress.

Abstract 1663: Development of a novel oral microtubule targeting agent with pan-cancer efficacy

Abstract Microtubule-targeting agents (MTAs) are among the most effective chemotherapeutics used in the treatment of cancer. However, the clinical utility of current MTAs, such as paclitaxel and vinblastine, are often limited due to adverse side effects or multidrug resistance (MDR) driving the continuous pursuit for the development of novel microtubule interactors. Here, we report the development of a novel non P-gp substrate MTA that is metabolically stable, and that displays broad-spectrum anti-cancer activity in vitro and ex vivo. Anti-cancer activity was assessed by screening a panel of 102 cancer cell lines and in a multi-tumor type panel of patient-derived organoids (PDOs). In vitro, double to triple digit nanomolar (ranging from 17nM to 318 nM) potency was observed in 101 cell lines, while viability was reduced by more than 70% in 76 cell lines out of 102. Similarly, 23 out of 34 (68%) of ex vivo treated PDOs were highly or partially responsive as indicated by a 37-73% decrease in viability. Studies using spindle assembly checkpoint (SAC) inhibitors demonstrated a mechanism at or before microtubule-kinetochore attachment in M phase of the cell cycle. Subsequent microscopic evaluation of kinetochore assembly revealed a lack of proper mitotic spindle formation highlighted with an increase in Mad1 signal intensity emphasizing metaphase arrest. Furthermore, in vitro tubulin polymerization assays demonstrated that the anti-cancer effect is a result of cytoskeleton targeting to inhibit microtubule assembly. Additionally, the antimicrotubule agent inhibited the formation of EBI (N,N’-ethylene-bis(iodoacetamide)):β-tubulin adducts indicating an occupied colchicine-binding site of tubulin. Six multi-tumor preclinical oncology models were evaluated for in vivo anti-tumor efficacy by oral administration of the compound. Further, tumor growth inhibition (%) was demonstrated in preclinical murine models of Acute Myeloid Leukemia (AML) (MV-4-11, 51%), colorectal (COLO205, 35%), prostate (DU 145, 38%), and gastric cancer (HS746T, 74%; SNU-5, 48%). Moreover, tumor tissue analysis for prominent mitotic cell cycle markers, Cyclin B1 and pHH3, revealed a dose-dependent elevation of both markers revealing mitotic arrest phenotype. Together, our data supports further preclinical and clinical development of a novel oral MTA notably in Taxane resistant and/or Taxane sensitive tumors. Citation Format: Maria-Dorothea Nastke, Jacob Matson, Andressa Mota, Archna Ravi, Alex Batrouni, Marcus O'Hara, Shiva Kazerounian, Arcan Guven, Nicole Pellegrino, Kashni Grover, Kayleigh Gray, Anne Diers, Michael Kiebish, Vivek Vishnudas, Stephane Gesta. Development of a novel oral microtubule targeting agent with pan-cancer efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1663.

Dietary Fibers Differ in Their Effects on Large Bowel Epithelial Proliferation and Fecal Fermentation-Dependent Events in Rats

The effects of different fiber types and processing on putative protective mechanisms for colorectal cancer were evaluated. Rats were fed diets of similar nutrient balance containing either no added fiber or 10% fiber from various sources. The rate of distal colonic epithelial proliferation, measured by the metaphase arrest method, was dependent on fiber type; ranking of fibers from highest to lowest yielded the following order: methylcellulose > coarse wheat bran > fine wheat bran ≈ parboiled and extruded rice brans > no fiber (P = 0.012). Effect on stool output ranked identically. Ranking of effect on fecal pH, from most to least acidic was as follows: coarse wheat bran ≈ the rice brans > fine wheat bran > no fiber ≈ methylcellulose (P = 0.00001). Coarse wheat bran gave significantly higher fecal butyrate concentrations than did the rice brans, which in turn gave higher levels than fine wheat bran, methylcellulose and the no-fiber diet. Proximal colon epithelial proliferation was unaffected by diet although cecal short-chain fatty acid concentrations and pH were affected. Different fibers have different effects on events in the fecal environment and distal colonic epithelium. Putative protective events (increased output, low fecal pH, high butyrate, low proliferation) are not equally affected and are unlikely in themselves to allow prediction of the protective effect of a fiber.

Nitrous Oxide-Induced Metaphase Arrest: A Technique for Somatic Chromosome Analysis.

Procedures to arrest metaphase chromosomes are used for determining chromosome numbers, chromosomal aberrations, and natural chromosome variation, as well as chromosome sorting. Here is described a technique of nitrous oxide gas treatment of freshly harvested root tips that is highly effective at producing an excellent mitotic index together with well-spread chromosomes. The details of the treatment and equipment used are provided. The metaphase spreads can be used directly for determining chromosome numbers or for in situ hybridization to reveal chromosomal features.

Kinesin-5 Eg5 is essential for spindle assembly, chromosome stability and organogenesis in development

Chromosome stability relies on bipolar spindle assembly and faithful chromosome segregation during cell division. Kinesin-5 Eg5 is a plus-end-directed kinesin motor protein, which is essential for spindle pole separation and chromosome alignment in mitosis. Heterozygous Eg5 mutations cause autosomal-dominant microcephaly, primary lymphedema, and chorioretinal dysplasia syndrome in humans. However, the developmental roles and cellular mechanisms of Eg5 in organogenesis remain largely unknown. In this study, we have shown that Eg5 inhibition leads to the formation of the monopolar spindle, chromosome misalignment, polyploidy, and subsequent apoptosis. Strikingly, long-term inhibition of Eg5 stimulates the immune responses and the accumulation of lymphocytes in the mouse spleen through the innate and specific immunity pathways. Eg5 inhibition results in metaphase arrest and cell growth inhibition, and suppresses the formation of somite and retinal development in zebrafish embryos. Our data have revealed the essential roles of kinesin-5 Eg5 involved in cell proliferation, chromosome stability, and organogenesis during development. Our findings shed a light on the cellular basis and pathogenesis in microcephaly, primary lymphedema, and chorioretinal dysplasia syndrome of Eg5-mutation-positive patients.

A phylogenetically-restricted essential cell cycle progression factor in the human pathogen Candida albicans

Chromosomal instability caused by cell division errors is associated with antifungal drug resistance in fungal pathogens. Here, we identify potential mechanisms underlying such instability by conducting an overexpression screen monitoring chromosomal stability in the human fungal pathogen Candida albicans. Analysis of ~1000 genes uncovers six chromosomal stability (CSA) genes, five of which are related to cell division genes of other organisms. The sixth gene, CSA6, appears to be present only in species belonging to the CUG-Ser clade, which includes C. albicans and other human fungal pathogens. The protein encoded by CSA6 localizes to the spindle pole bodies, is required for exit from mitosis, and induces a checkpoint-dependent metaphase arrest upon overexpression. Thus, Csa6 is an essential cell cycle progression factor that is restricted to the CUG-Ser fungal clade, and could therefore be explored as a potential antifungal target.

Uncoupling of Mitosis and Cytokinesis Upon a Prolonged Arrest in Metaphase Is Influenced by Protein Phosphatases and Mitotic Transcription in Fission Yeast.

Depletion of the Anaphase-Promoting Complex/Cyclosome (APC/C) activator Cdc20 arrests cells in metaphase with high levels of the mitotic cyclin (Cyclin B) and the Separase inhibitor Securin. In mammalian cells this arrest has been exploited for the treatment of cancer with drugs that engage the spindle assembly checkpoint and, recently, with chemical inhibitors of the APC/C. While most cells arrested in mitosis for prolonged periods undergo apoptosis, others skip cytokinesis and enter G1 with unsegregated chromosomes. This process, known as mitotic slippage, generates aneuploidy and increases genomic instability in the cancer cell. Here, we analyze the behavior of fission yeast cells arrested in mitosis through the transcriptional silencing of the Cdc20 homolog slp1. While depletion of slp1 readily halts cells in metaphase, this arrest is only transient and a majority of cells eventually undergo cytokinesis and show steady mitotic dephosphorylation. Notably, this occurs in the absence of Cyclin B (Cdc13) degradation. We investigate the involvement of phosphatase activity in these events and demonstrate that PP2A-B55Pab1 is required to prevent septation and, during the arrest, its CDK-mediated inhibition facilitates the induction of cytokinesis. In contrast, deletion of PP2A-B56Par1 completely abrogates septation. We show that this effect is partly due to this mutant entering mitosis with reduced CDK activity. Interestingly, both PP2A-B55Pab1 and PP2A-B56Par1, as well as Clp1 (the homolog of the budding yeast mitotic phosphatase Cdc14) are required for the dephosphorylation of mitotic substrates during the escape. Finally, we show that the mitotic transcriptional wave controlled by the RFX transcription factor Sak1 facilitates the induction of cytokinesis and also requires the activity of PP2A-B56Par1 in a mechanism independent of CDK.

Female meiosis II and pronuclear fusion require the microtubule transport factor Bicaudal D.

Bicaudal D (BicD) is a dynein adaptor that transports different cargoes along microtubules. Reducing the activity of BicD specifically in freshly laid Drosophila eggs by acute protein degradation revealed that BicD is needed to produce normal female meiosis II products, to prevent female meiotic products from re-entering the cell cycle, and for pronuclear fusion. Given that BicD is required to localize the spindle assembly checkpoint (SAC) components Mad2 and BubR1 to the female meiotic products, it appears that BicD functions to localize these components to control metaphase arrest of polar bodies. BicD interacts with Clathrin heavy chain (Chc), and both proteins localize to centrosomes, mitotic spindles and the tandem spindles during female meiosis II. Furthermore, BicD is required to localize clathrin and the microtubule-stabilizing factors transforming acidic coiled-coil protein (D-TACC/Tacc) and Mini spindles (Msps) correctly to the meiosis II spindles, suggesting that failure to localize these proteins may perturb SAC function. Furthermore, immediately after the establishment of the female pronucleus, D-TACC and Caenorhabditis elegans BicD, tacc and Chc are also needed for pronuclear fusion, suggesting that the underlying mechanism might be more widely used across species.

Kinesin-7 CENP-E is essential for chromosome alignment and spindle assembly of mouse spermatocytes

Genome stability depends on chromosome congression and alignment during cell division. Kinesin-7 CENP-E is critical for kinetochore-microtubule attachment and chromosome alignment, which contribute to genome stability in mitosis. However, the functions and mechanisms of CENP-E in the meiotic division of male spermatocytes remain largely unknown. In this study, by combining the use of chemical inhibitors, siRNA-mediated gene knockdown, immunohistochemistry, and high-resolution microscopy, we have found that CENP-E inhibition results in chromosome misalignment and metaphase arrest in dividing spermatocyte during meiosis. Strikingly, we have revealed that CENP-E regulates spindle organization in metaphase I spermatocytes and cultured GC-2 spd cells. CENP-E depletion leads to spindle elongation, chromosome misalignment, and chromosome instability in spermatocytes. Together, these findings indicate that CENP-E mediates the kinetochore recruitment of BubR1, spindle assembly checkpoint and chromosome alignment in dividing spermatocytes, which finally contribute to faithful chromosome segregation and chromosome stability in the male meiotic division.

Antimycolytic agents: fungistatic and fungicide

Invasive fungal infections are described as a continuous and severe harm to human health and they are associated with at least 1.5 million deaths worldwide each year. Amphotericin B exerts its activity through hydrophobic interactions with cell membrane ergosterol, cause the rupturing or leakage of cell membrane. The antifungal azole medicine group is classified as imidazoles (clotrimazole, ketoconazole, miconazole) and triazoles (fluconazole, itraconazole, voriconazole, posaconazole, isavuconazole) that are named according to the number of nitrogen atoms in the azole ring. Flucytosine is a first-line treatment for the management of cryptococcal meningitis. The most routine adverse effects of fluconazole involve accelerated liver enzymes, gastrointestinal complaints, headache, and skin rash. If antacids, PPIs, H2 blockers administered together with ketoconazole medicines; they will reduce the blood levels of ketoconazole by increasing gastric pH because ketoconazole requires an acidic media for dissolution and systematic absorption. Griseofulvin ruptures mitotic spindle during metaphase by interacting with fungal microtubules-(-), fungal mitosis (metaphase arrest), adequate to block expansion of fungi (drug is static), preventing them from damaging.

Genotoxicity Study of Carboxymethyl Chitosan-based Hydrogel for Clinical Use

A carboxymethyl chitosan-based hydrogel used for hemostatic and anti-adhesive on wounds was assessed by a genotoxicity test. The hydrogel was assessed for its potential to induce bacterial reverse mutation in the histidine auxotroph strains of Salmonella typhimurium and in the tryptophan auxotroph strain of Escherichia coli WP2uvrA. The study was performed with five bacterial test strains at different concentrations of hydrogel, with a negative control, and with five different types of positive controls, both in the presence and absence of a metabolic activation system. In all test strains used, there was no increase in the number of revertant colonies compared to the negative control at any concentration of test item either in the presence of or in the absence of a metabolic activation system. To evaluate additional genotoxicity of carboxymethyl chitosan hydrogel, the chromosome aberration test was performed using a cultured Chinese hamster lung cell line in the absence and presence of a metabolic activation system. The results of the bacterial reverse mutation assay indicated no mutagenic response at the concentration range tested, under the conditions of this study. The chromosome aberration test showed no significant increase of chromosome aberration in cells in metaphase arrest compared to the groups treated with vehicle control. Therefore, the carboxymethyl chitosan-based hydrogel is considered not to induce genotoxicity under the test system utilized.

Functional Assessment of Kinesin-7 CENP-E in Spermatocytes using In Vivo Inhibition, Immunofluorescence and Flow Cytometry.

In eukaryotes, meiosis is essential for genome stability and genetic diversity in sexual reproduction. Experimental analyses of spermatocytes in testes are critical for the investigations of spindle assembly and chromosome segregation in male meiotic division. The mouse spermatocyte is an ideal model for mechanistic studies of meiosis, however, the effective methods for the analyses of spermatocytes are lacking. In this article, a practical and efficient method for the in vivo inhibition of kinesin-7 CENP-E in mouse spermatocytes is reported. A detailed procedure for testicular injection of a specific inhibitor GSK923295 through abdominal surgery in 3-week-old mice is presented. Furthermore, described here is a series of protocols for tissue collection and fixation, hematoxylin-eosin staining, immunofluorescence, flow cytometry and transmission electron microscopy. Here we present an in vivo inhibition model via abdominal surgery and testicular injection, that could be a powerful technique to study male meiosis. We also demonstrate that CENP-E inhibition results in chromosome misalignment and metaphase arrest in primary spermatocytes during meiosis I. Our in vivo inhibition method will facilitate mechanistic studies of meiosis, serve as a useful method for genetic modifications of male germ lines, and shed a light on future clinical applications.