Nuclear Segregation Research Articles

Overexpression of SKA Complex Is Associated With Poor Prognosis in Gliomas

The spindle and kinetochore-associated complex is composed of three members: SKA1, SKA2, and SKA3. It is necessary for stabilizing spindle microtubules attaching to kinetochore (KT) in the middle stage of mitosis. The SKA complex is associated with poor prognosis in several human cancers. However, the role of SKA complex in rare malignant diseases, such as gliomas, has not been fully investigated. We investigated several databases, including Oncomine, UALCAN, and cBioPortal to explore the expression profile and prognostic significance of SKA complex in patients with gliomas. Gene ontology and Kyoto Encyclopedia of Genes and Genome pathways were used to analyze the potential enriched pathways. The genes co-expressed with SKA complex were identified and used for developing a protein-protein interaction (PPI) network using the STRING database. We found a significant overexpression of the mRNA levels of SKA1, SKA2, and SKA3 in patients with glioma patients. Higher expression of SKA1 and SKA3, but not SKA2, was significantly correlated with shorter overall survival of patients with glioma. In glioma, SKA complex was found to be involved in nuclear division, chromosome segregation, and DNA replication. The results of PPI network identified 10 hub genes (CCNB2, UBE2C, BUB1B, TPX2, CCNA2, CCNB1, MELK, TOP2A, PBK, and KIF11), all of which were overexpressed and negatively associated with prognosis of patients with glioma. In conclusion, our study sheds new insights into the biological role and prognostic significance of SKA complex in glioma.

Abf1 Is an Essential Protein That Participates in Cell Cycle Progression and Subtelomeric Silencing in Candida glabrata.

Accurate DNA replication and segregation is key to reproduction and cell viability in all organisms. Autonomously replicating sequence-binding factor 1 (Abf1) is a multifunctional protein that has essential roles in replication, transcription, and regional silencing in the model yeast Saccharomyces cerevisiae. In the opportunistic pathogenic fungus Candida glabrata, which is closely related to S. cerevisiae, these processes are important for survival within the host, for example, the regulation of transcription of virulence-related genes like those involved in adherence. Here, we describe that CgABF1 is an essential gene required for cell viability and silencing near the telomeres, where many adhesin-encoding genes reside. CgAbf1 mediated subtelomeric silencing depends on the 43 C-terminal amino acids. We also found that abnormal expression, depletion, or overexpression of Abf1, results in defects in nuclear morphology, nuclear segregation, and transit through the cell cycle. In the absence of ABF1, cells are arrested in G2 but start cycling again after 9 h, coinciding with the loss of cell viability and the appearance of cells with higher DNA content. Overexpression of CgABF1 causes defects in nuclear segregation and cell cycle progression. We suggest that these effects could be due to the deregulation of DNA replication.

A non-reactive natural product precursor of the duocarmycin family has potent and selective antimalarial activity

We identify a selective nanomolar inhibitor of blood-stage malarial proliferation from a screen of microbial natural product extracts. The responsible compound, PDE-I2, is a precursor of the anticancer duocarmycin family that preserves the class's sequence-specific DNA binding but lacks its signature DNA alkylating cyclopropyl warhead. While less active than duocarmycin, PDE-I2 retains comparable antimalarial potency to chloroquine. Importantly, PDE-I2 is >1,000-fold less toxic to human cell lines than duocarmycin, with mitigated impacts on eukaryotic chromosome stability. PDE-I2 treatment induces severe defects in parasite nuclear segregation leading to impaired daughter cell formation during schizogony. Time-of-addition studies implicate parasite DNA metabolism as the target of PDE-I2, with defects observed in DNA replication and chromosome integrity. We find the effect of duocarmycin and PDE-I2 on parasites is phenotypically indistinguishable, indicating that the DNA binding specificity of duocarmycins is sufficient and the genotoxic cyclopropyl warhead is dispensable for the parasite-specific selectivity of this compound class.

Malaria Parasite Schizont Egress Antigen-1 Plays an Essential Role in Nuclear Segregation during Schizogony.

Malaria parasites cause disease through repeated cycles of intraerythrocytic proliferation. Within each cycle, several rounds of DNA replication produce multinucleated forms, called schizonts, that undergo segmentation to form daughter merozoites. Upon rupture of the infected cell, the merozoites egress to invade new erythrocytes and repeat the cycle. In human malarial infections, an antibody response specific for the Plasmodium falciparum protein PF3D7_1021800 was previously associated with protection against malaria, leading to an interest in PF3D7_1021800 as a candidate vaccine antigen. Antibodies to the protein were reported to inhibit egress, resulting in it being named schizont egress antigen-1 (SEA1). A separate study found that SEA1 undergoes phosphorylation in a manner dependent upon the parasite cGMP-dependent protein kinase PKG, which triggers egress. While these findings imply a role for SEA1 in merozoite egress, this protein has also been implicated in kinetochore function during schizont development. Therefore, the function of SEA1 remains unclear. Here, we show that P. falciparum SEA1 localizes in proximity to centromeres within dividing nuclei and that conditional disruption of SEA1 expression severely impacts the distribution of DNA and formation of merozoites during schizont development, with a proportion of SEA1-null merozoites completely lacking nuclei. SEA1-null schizonts rupture, albeit with low efficiency, suggesting that neither SEA1 function nor normal segmentation is a prerequisite for egress. We conclude that SEA1 does not play a direct mechanistic role in egress but instead acts upstream of egress as an essential regulator required to ensure the correct packaging of nuclei within merozoites.IMPORTANCE Malaria is a deadly infectious disease. Rationally designed novel therapeutics will be essential for its control and eradication. The Plasmodium falciparum protein PF3D7_1021800, annotated as SEA1, is under investigation as a prospective component of a malaria vaccine, based on previous indications that antibodies to SEA1 interfere with parasite egress from infected erythrocytes. However, a consensus on the function of SEA1 is lacking. Here, we demonstrate that SEA1 localizes to dividing parasite nuclei and is necessary for the correct segregation of replicated DNA into individual daughter merozoites. In the absence of SEA1, merozoites develop defectively, often completely lacking a nucleus, and, consequently, egress is impaired and/or aberrant. Our findings provide insights into the divergent mechanisms by which intraerythrocytic malaria parasites develop and divide. Our conclusions regarding the localization and function of SEA1 are not consistent with the hypothesis that antibodies against it confer protective immunity to malaria by blocking merozoite egress.

Homotypic clustering of L1 and B1/Alu repeats compartmentalizes the 3D genome

Organization of the genome into euchromatin and heterochromatin appears to be evolutionarily conserved and relatively stable during lineage differentiation. In an effort to unravel the basic principle underlying genome folding, here we focus on the genome itself and report a fundamental role for L1 (LINE1 or LINE-1) and B1/Alu retrotransposons, the most abundant subclasses of repetitive sequences, in chromatin compartmentalization. We find that homotypic clustering of L1 and B1/Alu demarcates the genome into grossly exclusive domains, and characterizes and predicts Hi-C compartments. Spatial segregation of L1-rich sequences in the nuclear and nucleolar peripheries and B1/Alu-rich sequences in the nuclear interior is conserved in mouse and human cells and occurs dynamically during the cell cycle. In addition, de novo establishment of L1 and B1 nuclear segregation is coincident with the formation of higher-order chromatin structures during early embryogenesis and appears to be critically regulated by L1 and B1 transcripts. Importantly, depletion of L1 transcripts in embryonic stem cells drastically weakens homotypic repeat contacts and compartmental strength, and disrupts the nuclear segregation of L1- or B1-rich chromosomal sequences at genome-wide and individual sites. Mechanistically, nuclear co-localization and liquid droplet formation of L1 repeat DNA and RNA with heterochromatin protein HP1α suggest a phase-separation mechanism by which L1 promotes heterochromatin compartmentalization. Taken together, we propose a genetically encoded model in which L1 and B1/Alu repeats blueprint chromatin macrostructure. Our model explains the robustness of genome folding into a common conserved core, on which dynamic gene regulation is overlaid across cells.

Transcriptome Profiling of Human Follicle Dermal Papilla Cells in response to Porphyra-334 Treatment by RNA-Seq.

Porphyra-334 is a kind of mycosporine-like amino acid absorbing ultraviolet-A. Here, we characterized porphyra-334 as a potential antiaging agent. An in vitro assay revealed that porphyra-334 dramatically promoted collagen synthesis in fibroblast cells. The effect of porphyra-334 on cell proliferation was dependent on the cell type, and the increase of cell viability by porphyra-334 was the highest in keratinocyte cells among the three tested cell types. An in vivo clinical test with 22 participants demonstrated the possible role of porphyra-334 in the improvement of periorbital wrinkles. RNA-sequencing using human follicle dermal papilla (HFDP) cells upon porphyra-334 treatment identified the upregulation of metallothionein- (MT-) associated genes, confirming the antioxidant role of porphyra-334 with MT. Moreover, the expression of genes involved in nuclear chromosome segregation and the encoding of components of kinetochores was upregulated by porphyra-334 treatment. Furthermore, we found that several genes associated with the hair follicle cycle, the hair follicle structure, the epidermal structure, and stem cells were upregulated by porphyra-334 treatment, suggesting the potential role of porphyra-334 in hair follicle growth and maintenance. In summary, we provided several new pieces of evidence of porphyra-334 as a potential antiaging cosmetic agent and elucidated the expression network in HFDP cells upon porphyra-334.

Dysregulation of KIF14 regulates the cell cycle and predicts poor prognosis in cervical cancer: a study based on integrated approaches.

Cervical cancer (CC) is the most common malignant tumor in females. Although persistent high-risk human papillomavirus (HPV) infection is a leading factor that causes CC, few women with HPV infection develop CC. Therefore, many mechanisms remain to be explored, such as aberrant expression of oncogenes and tumor suppressor genes. To identify promising prognostic factors and interpret the relevant mechanisms of CC, the RNA sequencing profile of CC was downloaded from the Cancer Genome Atlas and the Gene Expression Omnibus databases. The GSE63514 dataset was analyzed, and differentially expressed genes (DEGs) were obtained by weighted coexpression network analysis and the edgeR package in R. Fifty-three shared genes were mainly enriched in nuclear chromosome segregation and DNA replication signaling pathways. Through a protein-protein interaction network and prognosis analysis, the kinesin family member 14 (KIF14) hub gene was extracted from the set of 53 shared genes, which was overexpressed and associated with poor overall survival (OS) and disease-free survival (DFS) of CC patients. Mechanistically, gene set enrichment analysis showed that KIF14 was mainly enriched in the glycolysis/gluconeogenesis signaling pathway and DNA replication signaling pathway, especially in the cell cycle signaling pathway. RT-PCR and the Human Protein Atlas database confirmed that these genes were significantly increased in CC samples. Therefore, our findings indicated the biological function of KIF14 in cervical cancer and provided new ideas for CC diagnosis and therapies.

Comprehensive Analysis of Key Genes Associated with ceRNA Networks in Nasopharyngeal Carcinoma Based on Bioinformatics Analysis

Nasopharyngeal carcinoma (NPC) is an epithelial malignancy that has high morbidity rates in the east and southeast Asia, especially in South China. However, the molecular mechanisms of NPC remain largely unknown. Therefore, it is urgent to explore the pathogenesis of NPC and search for the potential biomarkers and the prognostic indicators for NPC precision treatment. We comprehensively analyzed the mRNAs, long non-coding RNAs (lncRNAs), and microRNA (miRNAs) from the full transcriptome sequencing data of our hospital (5 normal vs. 5 NPC tissues) and from six microarray datasets (62 normal vs. 334 NPC tissues) downloaded from the Gene Expression Omnibus (GEO) dataset (GSE12452, GSE13597, GSE95166, GSE126683, GSE70970, GSE43039) to see gene expression clustering by using principal components analysis (PCA). Differentially expressed genes (DEGs) were identified using the Limma package of the R software and visualized through volcano plot and Venn diagram. The gene ontology (GO) enrichment, Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis and gene set enrichment analysis (GSEA) were performed for annotation, visualization, and integrated discovery. Furthermore, we constructed mRNA-miRNA-lncRNA (ceRNA) networks using miRanda and TargetScan and obtained the protein-protein interaction (PPI) network of DEGs via STRING database. The hub genes were identified by MCODE, NetworkAnalyzer and CytoHubba plugin. In total, 61 mRNAs, 14miRNAs and 10 lncRNAs were discovered to be DEGs after the intersection of three mRNAs datasets, two miRNAs datasets and three lncRNAs datasets. Based on the GO analysis, which includes annotation of biological processes (BPs), molecular functions (MFs) and cellular components (CCs), we found that changes in NPC were mainly enriched in the chromosomal region, sister chromatid segregation, nuclear chromosome segregation, and DNA replication. The KEGG pathway enrichment analysis indicated that the DEGs were mainly concentrated in DNA replication and cell cycle. Moreover, the GSEA suggested that the MAPK signaling pathway, PI3K-AKT signaling pathway, and apoptotic pathway were largely involved in the initiation and development of NPC. Finally, using the PPI network, a total of 20 hub genes (NUSAP1, RACGAP1, PRC1, KIF4A, TOP2A, PBK, KIF2C, TPX2, CENPU, OIP5, TTK, MAD2L1, NDC80, BIRC5, MELK, CENPF, FOXM1, TYMS, CDK1 and CEP55) were uncovered. Our findings may provide additional insights into the biological characteristics of NPC via integrative analysis of the microarray datasets and the total transcriptome sequencing, offering potential therapeutic targets, biomarkers and prognostic indicators for NPC.

Rsr1 Palmitoylation and GTPase Activity Status Differentially Coordinate Nuclear, Septin, and Vacuole Dynamics in Candida albicans.

Directional growth and tissue invasion by hyphae of the pathogenic fungus, Candida albicans, are disrupted by deletion of the small GTPase, Rsr1, which localizes Cdc42 and its kinase, Cla4, to the site of polarized growth. We investigated additional abnormalities observed in rsr1Δ hyphae, including vacuole development, cytoplasm inheritance, mitochondrial morphology, septin ring organization, nuclear division and migration, and branching frequency, which together demonstrate a fundamental role for Rsr1 in cellular organization. Rsr1 contains a C-terminal CCAAX box, which putatively undergoes both reversible palmitoylation and farnesylation for entry into the secretory pathway. We expressed variants of Rsr1 with mutated C244 or C245, or which lacked GTPase activity (Rsr1K16N and Rsr1G12V), in the rsr1Δ background and compared the resulting phenotypes with those of mutants lacking Bud5 (Rsr1 GEF), Bud2 (Rsr1 GAP), or Cla4. Bud5 was required only for cell size and bud site selection in yeast, suggesting there are alternative activators for Rsr1 in hyphae. Septin ring and vacuole dynamics were restored by expression of unpalmitoylated Rsr1C244S, which localized to endomembranes, but not by cytoplasmic Rsr1C245A or GTP/GDP-locked Rsr1, suggesting Rsr1 functions at intracellular membranes in addition to the plasma membrane. Rsr1K16N or cytoplasmic Rsr1C245A restored normal nuclear division but not septin ring or vacuole dynamics. Rsr1-GDP therefore plays a specific role in suppressing START, which can be signaled from the cytosol. Via differential palmitoylation and activity states, Rsr1 operates at diverse cell sites to orchestrate proper nuclear division and inheritance during constitutive polarized growth. As cla4Δ phenocopied rsr1Δ, it is likely these functions involve Cdc42-Cla4 activity.IMPORTANCE Understanding how single eukaryotic cells self-organize to replicate and migrate is relevant to health and disease. In the fungal pathogen, Candida albicans, the small GTPase, Rsr1, guides the directional growth of hyphae that invade human tissue during life-threatening infections. Rsr1 is a Ras-like GTPase and a homolog of the conserved Rap1 subfamily, which directs migration in mammalian cells. Research into how this single GTPase delivers complex intracellular patterning is challenging established views of GTPase regulation, trafficking, and interaction. Here, we show that Rsr1 directly and indirectly coordinates the spatial and temporal development of key intracellular macrostructures, including septum formation and closure, vacuole dynamics, and nuclear division and segregation, as well as whole-cell morphology by determining branching patterns. Furthermore, we categorize these functions by differential Rsr1 localization and activity state and provide evidence to support the emerging view that the cytosolic pool of Ras-like GTPases is functionally active.

Black hole, neutron star, and white dwarf merger rates in AGN discs

ABSTRACT Advanced LIGO and Advanced Virgo are detecting a large number of binary stellar origin black hole (BH) mergers. A promising channel for accelerated BH merger lies in active galactic nucleus (AGN) discs of gas around supermasssive BHs. Here, we investigate the relative number of compact object (CO) mergers in AGN disc models, including BH, neutron stars (NS), and white dwarfs, via Monte Carlo simulations. We find the number of all merger types in the bulk disc grows ∝ t1/3 which is driven by the Hill sphere of the more massive merger component. Median mass ratios of NS–BH mergers in AGN discs are $\tilde{q}=0.07\pm 0.06(0.14\pm 0.07)$ for mass functions (MF) M−1(− 2). If a fraction fAGN of the observed rate of BH–BH mergers (RBH–BH) come from AGN, the rate of NS–BH (NS–NS) mergers in the AGN channel is ${R}_{\mathrm{ BH}\!-\!\mathrm{ NS}} \sim f_{\mathrm{ AGN}}[10,300]\, \rm {Gpc}^{-3}\, \rm {yr}^{-1},({\mathit{ R}}_{NS\!-\!NS} \le \mathit{ f}_{AGN}400\, \rm {Gpc}^{-3}\, \rm {yr}^{-1}$). Given the ratio of NS–NS/BH–BH LIGO search volumes, from preliminary O3 results the AGN channel is not the dominant contribution to observed NS–NS mergers. The number of lower mass gap events expected is a strong function of the nuclear MF and mass segregation efficiency. CO merger ratios derived from LIGO can restrict models of MF, mass segregation, and populations embedded in AGN discs. The expected number of electromagnetic (EM) counterparts to NS–BH mergers in AGN discs at z < 1 is $\sim [30,900]\, {\rm {yr}}^{-1}(f_{\mathrm{ AGN}}/0.1)$. EM searches for flaring events in large AGN surveys will complement LIGO constraints on AGN models and the embedded populations that must live in them.

Comprehensive analysis of key genes associated with ceRNA networks in nasopharyngeal carcinoma based on bioinformatics analysis

BackgroundNasopharyngeal carcinoma (NPC) is an epithelial malignancy with high morbidity rates in the east and southeast Asia. The molecular mechanisms of NPC remain largely unknown. We explored the pathogenesis, potential biomarkers, and prognostic indicators of NPC.MethodsWe analyzed mRNAs, long non-coding RNAs (lncRNAs), and microRNAs (miRNAs) in the whole transcriptome sequencing dataset of our hospital (five normal tissues vs. five NPC tissues) and six microarray datasets (62 normal tissues vs. 334 NPC tissues) downloaded from the Gene Expression Omnibus (GSE12452, GSE13597, GSE95166, GSE126683, and GSE70970, GSE43039). Differential expression analyses, gene ontology (GO) enrichment, kyoto encyclopedia of genes and genomes (KEGG) analysis, and gene set enrichment analysis (GSEA) were conducted. The lncRNA-miRNA-mRNA competing endogenous RNA (ceRNA) networks were constructed using the miRanda and TargetScan database, and a protein–protein interaction (PPI) network of differentially expressed genes (DEGs) was built using Search Tool for the Retrieval of Interacting Genes (STRING) software. Hub genes were identified using Molecular Complex Detection (MCODE), NetworkAnalyzer, and CytoHubba.ResultsWe identified 61 mRNAs, 14miRNAs, and 10 lncRNAs as shared DEGs related to NPC in seven datasets. Changes in NPC were enriched in the chromosomal region, sister chromatid segregation, and nuclear chromosome segregation. GSEA indicated that the mitogen-activated protein kinase (MAPK) pathway, phosphatidylinositol-3 OH kinase/protein kinase B (PI3K-Akt) pathway, apoptotic pathway, and tumor necrosis factor (TNF) were involved in the initiation and development of NPC. Finally, 20 hub genes were screened out via the PPI network.ConclusionsSeveral DEGs and their biological processes, pathways, and interrelations were found in our current study by bioinformatics analyses. Our findings may offer insights into the biological mechanisms underlying NPC and identify potential therapeutic targets for NPC.

Pseudogene DUXAP8 Promotes Cell Proliferation and Migration of Hepatocellular Carcinoma by Sponging MiR-490-5p to Induce BUB1 Expression.

Hepatocellular Carcinoma (HCC) currently remains one of the most lethal malignancies worldwide. Recently, long non-coding RNAs (lncRNAs) had been demonstrated to play a crucial role in the progression of multiple human cancers, including HCC. In this study, we found that lncRNA DUXAP8 was upregulated in tumor samples and served as an oncogene in HCC. Bioinformatics analysis showed that DUXAP8 was significantly associated with the regulation of centrosome organization, homologous recombination, meiotic cell cycle process, sister chromatid segregation, nuclear chromosome segregation, and RNA export from the nucleus. The knockdown of DUXAP8 significantly suppresses cell proliferation and the cell cycle but induces cell apoptosis in HCC. Mechanically, the present study showed that DUXAP8 serves as a sponge of MiR-490-5p to promote the expression of BUB1 in HCC. Although the underlying regulatory mechanisms of DUXAP8 in HCC require further investigation, this study, for the first time, showed that DUXAP8 can serve as a new therapeutic target for HCC.

Haspin regulates Ras localization to promote Cdc24-driven mitotic depolarization

Cell polarization is of paramount importance for proliferation, differentiation, development, and it is altered during carcinogenesis. Polarization is a reversible process controlled by positive and negative feedback loops. How polarized factors are redistributed is not fully understood and is the focus of this work. In Saccharomyces cerevisiae, mutants defective in haspin kinase exhibit stably polarized landmarks and are sensitive to mitotic delays. Here, we report a new critical role for haspin in polarisome dispersion; failure to redistribute polarity factors, in turn, leads to nuclear segregation defects and cell lethality. We identified a mitotic role for GTP-Ras in regulating the local activation of the Cdc42 GTPase, resulting in its dispersal from the bud tip to a homogeneous distribution over the plasma membrane. GTP-Ras2 physically interacts with Cdc24 regulateing its mitotic distribution. Haspin is shown to promote a mitotic shift from a bud tip-favored to a homogenous PM fusion of Ras-containing vesicles. In absence of haspin, active Ras is not redistributed from the bud tip; Cdc24 remains hyperpolarized promoting the activity of Cdc42 at the bud tip, and the polarisome fails to disperse leading to erroneously positioned mitotic spindle, defective nuclear segregation, and cell death after mitotic delays. These findings describe new functions for key factors that modulate cell polarization and mitotic events, critical processes involved in development and tumorigenesis.

Corticosterone and testosterone treatment influence expression of gene pathways linked to meiotic segregation in preovulatory follicles of the domestic hen.

Decades of work indicate that female birds can control their offspring sex ratios in response to environmental and social cues. In laying hens, hormones administered immediately prior to sex chromosome segregation can exert sex ratio skews, indicating that these hormones may act directly on the germinal disc to influence which sex chromosome is retained in the oocyte and which is discarded into an unfertilizable polar body. We aimed to uncover the gene pathways involved in this process by testing whether treatments with testosterone or corticosterone that were previously shown to influence sex ratios elicit changes in the expression of genes and/or gene pathways involved in the process of meiotic segregation. We injected laying hens with testosterone, corticosterone, or control oil 5h prior to ovulation and collected germinal discs from the F1 preovulatory follicle in each hen 1.5h after injection. We used RNA-sequencing (RNA-seq) followed by DESeq2 and gene set enrichment analyses to identify genes and gene pathways that were differentially expressed between germinal discs of control and hormone-treated hens. Corticosterone treatment triggered downregulation of 13 individual genes, as well as enrichment of gene sets related to meiotic spindle organization and chromosome segregation, and additional gene sets that function in ion transport. Testosterone treatment triggered upregulation of one gene, and enrichment of one gene set that functions in nuclear chromosome segregation. This work indicates that corticosterone can be a potent regulator of meiotic processes and provides potential gene targets on which corticosterone and/or testosterone may act to influence offspring sex ratios in birds.

Deciphering the role of UBA-like domains in intraflagellar distribution and functions of myosin XXI in Leishmania.

Myosin XXI (Myo21) is a novel class of myosin present in all kinetoplastid parasites, such as Trypanosoma and Leishmania. This protein in Leishmania promastigotes is predominantly localized to the proximal region of the flagellum, and is involved in the flagellum assembly, cell motility and intracellular vesicle transport. As Myo21 contains two ubiquitin associated (UBA)-like domains (UBLD) in its amino acid sequence, we considered it of interest to analyze the role of these domains in the intracellular distribution and functions of this protein in Leishmania cells. In this context, we created green fluorescent protein (GFP)-conjugates of Myo21 constructs lacking one of the two UBLDs at a time or both the UBLDs as well as GFP-conjugates of only the two UBLDs and Myo21 tail lacking the two UBLDs and separately expressed them in the Leishmania cells. Our results show that unlike Myo21-GFP, Myo21-GFP constructs lacking either one or both the UBLDs failed to concentrate and co-distribute with actin in the proximal region of the flagellum. Nevertheless, the GFP conjugate of only the two UBLDs was found to predominantly localize to the flagellum base. Additionally, the cells that expressed only one or both the UBLDs-deleted Myo21-GFP constructs possessed shorter flagellum and displayed slower motility, compared to Myo21-GFP expressing cells. Further, the intracellular vesicle transport and cell growth were severely impaired in the cells that expressed both the UBLDs deleted Myo21-GFP construct, but in contrast, virtually no effect was observed on the intracellular vesicle transport and growth in the cells that expressed single UBLD deleted mutant proteins. Moreover, the observed slower growth of both the UBLDs-deleted Myo21-GFP expressing cells was primarily due to delayed G2/M phase caused by aberrant nuclear and daughter cell segregation during their cell division process. These results taken together clearly reveal that the presence of UBLDs in Myo21 are essentially required for its predominant localization to the flagellum base, and perhaps also in its involvement in the flagellum assembly and cell division. Possible role of UBLDs in involvement of Myo21 during Leishmania flagellum assembly and cell cycle is discussed.

Encapsulation enhances protoplast fusant stability.

A barrier to cost‐efficient biomanufacturing is the instability of engineered genetic elements, such as plasmids. Instability can also manifest at the whole‐genome level, when fungal dikaryons revert to parental species due to nuclear segregation during cell division. Here, we show that by encapsulating Saccharomyces cerevisiae‐Pichia stipitis dikaryons in an alginate matrix, we can limit cell division and preserve their expanded metabolic capabilities. As a proxy to cellulosic ethanol production, we tested the capacity of such cells to carry out ethanologenic fermentation of glucose and xylose, examining substrate use, ploidy, and cell viability in relation to planktonic fusants, as well as in relation to planktonic and encapsulated cell cultures consisting of mixtures of these species. Glucose and xylose consumption and ethanol production by encapsulated dikaryons were greater than planktonic controls. Simultaneous co‐fermentation did not occur; rather the order and kinetics of glucose and xylose catabolism by encapsulated dikaryons were similar to cultures where the two species were encapsulated together. Over repeated cycles of fed‐batch culture, encapsulated S. cerevisiae‐P. stipitis fusants exhibited a dramatic increase in genomic stability, relative to planktonic fusants. Encapsulation also increased the stability of antibiotic‐resistance plasmids used to mark each species and preserved a fixed ratio of S. cerevisiae to P. stipitis cells in mixed cultures. Our data demonstrate how encapsulating cells in an extracellular matrix restricts cell division and, thereby, preserves the stability and biological activity of entities ranging from genomes to plasmids to mixed populations, each of which can be essential to cost‐efficient biomanufacturing.

Fuzzy‐based nuclear analysis segregation model for distinguishing the properties of repository liquids and gas in gas storage

The geophysical well logging analysis is a major task of petroleum research and development, where petroleum oil (gas), transition petroleum layers, water layers, and dry-layer segments are precisely distinguished from each other. The resistivity of the particular oil and gas layers in developing countries is below or close to the water layers. As electrical properties show, the particular oil and gas layer of water layers is not legitimately recognizable. The features of oil and gas strata and their strong components are different in the investigative field from those of different petroleum fields. In this way, various techniques and thoughts ought to be utilized to comprehend the particular issues in the examination zone. A nuclear analysis–based segregation model for distinguishing the properties of repository liquids is worked by utilizing the crude pipeline and distilled unit of oil (gas) layers, transitional oil water layers, water layers, and dry layers using dynamic nonlinear fuzzy clustering model. With directly complex transformation models, reservoir fluids can be separated. The outcomes are analyzed with various model, and the proposed method shows promising outcomes.

Identification of tumor-infiltrating immune cells and prognostic validation of tumor-infiltrating mast cells in adrenocortical carcinoma: results from bioinformatics and real-world data

ABSTRACT Objective The purpose of this study was to explore the composition of tumor-infiltrating immune cells (TIIC) and prognostic significance of tumor-infiltrating mast cells (TIMC) in adrenocortical carcinoma (ACC). Methods The gene expression profiles of ACC were downloaded from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GSE90713, GSE12368). The abundance of TIICs in ACC samples was calculated by CIBERSORT algorithm and immunohistochemistry was used to identify mast cells of 39 tumor samples from Fudan University Shanghai Cancer Center (FUSCC). Differentially expressed genes (DEGs) were analyzed by LIMMA package using R software. Survival analysis was analyzed by Kaplan-Meier method and Cox regression models. Results The abundance of mast cells (p = .008) was positively correlated with ACC patients’ outcome in TCGA cohort and was also positively correlated with both overall survival (p < .05) and progression-free survival (p < .05) in FUSCC cohort. Different TIMC infiltrations showed significant changes in signaling pathways including DNA replication, nuclear chromosome segregation, and meiotic cell cycle process of ACC. In addition, elevated expression of eight hub genes (KIF18A, CDCA8, SKA1, CEP55, BUB1, CDK1, SGOL1, SGOL2) related to the abundance of TIMC in ACC was significantly correlated with the poor prognosis of the patients. Conclusion In conclusion, higher TIMC infiltration was positively correlated with ACC patients’ outcome in both TCGA and FUSCC cohort. Lower TIMC infiltration and elevated expression of hub genes (KIF18A, CDCA8, SKA1, CEP55, BUB1, CDK1, SGOL1, SGOL2) are markedly correlated with aggressive progression and poor prognosis, which might shed lights on novel targets for treatment strategies.

Three-dimensional electron microscopy analysis reveals endopolygeny-like nuclear architecture segregation in Plasmodium oocyst development

The genus Plasmodium is a unicellular eukaryotic parasite that is the causative agent of malaria, which is transmitted by Anopheline mosquito. There are a total of three developmental stages in the production of haploid parasites in the Plasmodium life cycle: the oocyst stage in mosquitoes and the liver and blood stages in mammalian hosts. The Plasmodium oocyst stage plays an important role in the production of the first generation of haploid parasites. Nuclear division is the most important event that occurs during the proliferation of all eukaryotes. However, obtaining the details of nuclear division at the oocyst stage is challenging owing to difficulties in preparation. In this study, we used focused-ion-beam-milling combined with scanning-electron-microscopy to report the 3D architecture during nuclear segregations in oocyst stage. This advanced technology allowed us to analyse the 3D details of organelle segregation inside the oocyst during sporogony formation. It was revealed that multiple nuclei were involved with several centrosomes in one germ nucleus during sporozoite budding (endopolygeny). Our high-resolution 3D analysis uncovered the endopolygeny-like nuclear architecture of Plasmodium in the definitive host. This nuclear segregation was different from that in the blood stage, and its similarity to other apicomplexan parasite nuclear divisions such as Sarcocystis is discussed.

The Glucan-Remodeling Enzyme Phr1p and the Chitin Synthase Chs1p Cooperate to Maintain Proper Nuclear Segregation and Cell Integrity in Candida albicans.

GH72 family of β-(1,3)-glucanosyltransferases is unique to fungi and is required for cell wall biogenesis, morphogenesis, virulence, and in some species is essential for life. Candida albicans PHR1 and PHR2 are pH-regulated genes that encode GH72 enzymes highly similar to Gas1p of Saccharomyces cerevisiae. PHR1 is expressed at pH ≥ 5.5 while PHR2 is transcribed at pH ≤ 5.5. Both are essential for C. albicans morphogenesis and virulence. During growth at neutral-alkaline pH, Phr1p-GFP preferentially localizes to sites of active cell wall formation as the incipient bud, the mother-daughter neck, the bud periphery, and concentrates in the septum at cytokinesis. We further investigated this latter localization. In chs3Δ cells, lacking the chitin of the chitin ring and lateral cell wall, Phr1p-GFP still concentrated along the thin line of the primary septum formed by chitin deposited by chitin synthase I (whose catalytic subunit is Chs1p) suggesting that it plays a role during formation of the secondary septa. RO-09-3143, a highly specific inhibitor of Chs1p activity, inhibits septum formation and blocks cell division. However, alternative septa are produced and are crucial for cell survival. Phr1p-GFP is excluded from such aberrant septa. Finally, we determined the effects of RO-09-3143 in cells lacking Phr1p. PHR1 null mutant was more susceptible to the drug than the wild type. The phr1Δ cells were larger, devoid of septa, and underwent endomitosis and cell death. Phr1p and Chs1p cooperate in maintaining cell integrity and in coupling morphogenesis with nuclear division in C. albicans.