Evidence for intraflagellar transport in butterfly spermatocyte cilia.

In the model organism insect Drosophila melanogaster short cilia assemble on spermatocytes that elaborate into 1.8 mm long flagella during spermatid differentiation. A unique feature of these cilia/flagella is their lack of dependence on intraflagellar transport (IFT) for their assembly. Here, we show that in the common butterfly Pieris brassicae, the spermatocyte cilia are exceptionally long: about 40 µm compared to less than 1 µm in Drosophila. By transmission electron microscopy, we show that P. brassicae spermatocytes display several features not found in melanogaster, including compelling evidence of IFT structures and features of motile cilia.

Coordination of Zika Virus Infection and Viroplasm Organization by Microtubules and Microtubule-Organizing Centers.

Zika virus (ZIKV) became a global health concern in 2016 due to its links to congenital microcephaly and other birth defects. Flaviviruses, including ZIKV, reorganize the endoplasmic reticulum (ER) to form a viroplasm, a compartment where virus particles are assembled. Microtubules (MTs) and microtubule-organizing centers (MTOCs) coordinate structural and trafficking functions in the cell, and MTs also support replication of flaviviruses. Here we investigated the roles of MTs and the cell's MTOCs on ZIKV viroplasm organization and virus production. We show that a toroidal-shaped viroplasm forms upon ZIKV infection, and MTs are organized at the viroplasm core and surrounding the viroplasm. We show that MTs are necessary for viroplasm organization and impact infectious virus production. In addition, the centrosome and the Golgi MTOC are closely associated with the viroplasm, and the centrosome coordinates the organization of the ZIKV viroplasm toroidal structure. Surprisingly, viroplasm formation and virus production are not significantly impaired when infected cells have no centrosomes and impaired Golgi MTOC, and we show that MTs are anchored to the viroplasm surface in these cells. We propose that the viroplasm is a site of MT organization, and the MTs organized at the viroplasm are sufficient for efficient virus production.

Design of advanced siRNA therapeutics for the treatment of COVID-19.

COVID-19 is a newly emerged viral disease that is currently affecting the whole globe. A variety of therapeutic approaches are underway to block the SARS-CoV-2 virus. Among these methods, siRNAs could be a safe and specific option, as they have been tested against other viruses. siRNAs are a class of inhibitor RNAs that act promisingly as mRNA expression blockers and they can be designed to interfere with viral mRNA to block virus replication. In order to do this, we designed and evaluated the efficacy of six highly specific siRNAs, which target essential viral mRNAs with no predicted human genome off-targets. We observed a significant reduction in the copy number viral mRNAs after treatment with the siRNAs, and are expected to inhibit virus replication. We propose siRNAs as a potential co-therapy for acute SARS-CoV-2 infection.

MiR-193b deregulation is associated with Parkinson's disease.

PGC-1α/FNDC5/BDNF has found to be a critical pathway in neurodegeneration. MicroRNAs (miR(NA)s) are non-coding regulatory RNAs whose dysregulation has been observed in multiple neurological disorders, and miRNA-mediated gene deregulation plays a decisive role in PD. Here, candidate miRNA was chosen based on the literature survey and in silico studies. Chronic and acute models of PD were created using MPP+-treated SH-SY5Y cells. Twenty PD patients and 20 healthy volunteers were recruited. RT-qPCR was performed to assess the expression of miRNA and genes. Severe mitochondrial dysfunction induced by acute MPP+ treatment instigated compensatory mechanisms through enhancing expression of PGC-1α/FNDC5/BDNF pathway genes, while chronic MPP+ toxicity led to down-regulated levels of the genes in SH-SY5Y cells. PD peripheral blood mononuclear cells (PBMCs) also showed decreased expression of target genes. There were significant changes in the level of miR-193b in both models, as well as PD PBMCs. Moreover, miR-193b overexpression significantly affected PGC-1α, FNDC5 and TFAM levels. Interestingly, down-regulations of PGC-1α, FNDC5, BDNF and TFAM were inversely correlated with miR-193b up-regulation in PD PBMCs. This study showed the deregulation of PGC-1α/FNDC5/BDNF pathway in PD models and PBMCs, verifying its importance in neurodegeneration. Our findings also revealed that miR-193b functions in PD development, possibly through regulating PGC-1α/FNDC5/BDNF pathway, suggesting miR-193b as a potential biomarker for PD diagnosis.

The Enigma of Centriole Loss in the 1182-4 Cell Line.

The Drosophilamelanogaster cell line 1182-4, which constitutively lacks centrioles, was established many years ago from haploid embryos laid by females homozygous for the maternal haploid (mh) mutation. This was the first clear example of animal cells regularly dividing in the absence of this organelle. However, the cause of the acentriolar nature of the 1182-4 cell line remained unclear and could not be clearly assigned to a particular genetic event. Here, we detail historically the longstanding mystery of the lack of centrioles in this Drosophila cell line. Recent advances, such as the characterization of the mh gene and the genomic analysis of 1182-4 cells, allow now a better understanding of the physiology of these cells. By combining these new data, we propose three reasonable hypotheses of the genesis of this remarkable phenotype.

Author Correction: Sas-4 provides a scaffold for cytoplasmic complexes and tethers them in a centrosome.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

New insights into the cellular activities of Fndc5/Irisin and its signaling pathways.

Fndc5, a well-defined myokine and also identified as an adipokine, has a critical role in modulation of metabolism and protection against obesity. These important functions are mediated by irisin, a secretory peptide produced from proteolytic processing of Fndc5. The other beneficial physiological effects of irisin are alleviation of oxidative stress, neuroprotective effects, and anti-inflammatory properties and associated anti-metastatic effects. Fndc5/irisin exerts its biological effects through several intracellular signaling pathways. The major signaling pathway is thought to be MAPK signaling pathways which are involved in neural differentiation, browning of white adipocytes, as well as osteoblast proliferation and differentiation. Other essential functions of Fndc5/irisin are mediated through additional pathways including AMPK pathway, PI3K/AKT, and STAT3/Snail. Thorough understanding of the mechanisms of irisin actions are essential in order to develop Fndc5/irisin for therapeutic purposes. In the present review, we focus on the current knowledge of the signaling pathways that elicit irisin actions.

A perinuclear microtubule-organizing centre controls nuclear positioning and basement membrane secretion.

Non-centrosomal microtubule-organizing centres (ncMTOCs) have a variety of roles that are presumed to serve the diverse functions of the range of cell types in which they are found. ncMTOCs are diverse in their composition, subcellular localization and function. Here we report a perinuclear MTOC in Drosophila fat body cells that is anchored by the Nesprin homologue Msp300 at the cytoplasmic surface of the nucleus. Msp300 recruits the microtubule minus-end protein Patronin, a calmodulin-regulated spectrin-associated protein (CAMSAP) homologue, which functions redundantly with Ninein to further recruit the microtubule polymerase Msps-a member of the XMAP215 family-to assemble non-centrosomal microtubules and does so independently of the widespread microtubule nucleation factor γ-Tubulin. Functionally, the fat body ncMTOC and the radial microtubule arrays that it organizes are essential for nuclear positioning and for secretion of basement membrane components via retrograde dynein-dependent endosomal trafficking that restricts plasma membrane growth. Together, this study identifies a perinuclear ncMTOC with unique architecture that regulates microtubules, serving vital functions.

Potential Biomarker and Therapeutic LncRNAs in Multiple Sclerosis Through Targeting Memory B Cells.

Multiple sclerosis (MS) is a chronic autoimmune disease that degenerates the central nervous system (CNS). B cells exacerbate the progression of CNS lesions in MS by producing auto-antibodies, pro-inflammatory cytokines, and presenting auto-antigens to activated T cells. Long non-coding RNAs (lncRNAs) play a crucial role in complex biological processes and their stability in body fluids combined with their tissue specificity make these biomolecules promising biomarker candidates for MS diagnosis. In the current study, we investigated memory B cell-specific lncRNAs located, on average, less than 50 kb from differentially expressed protein-coding genes in MS patients compared to healthy individuals. Moreover, we included in our selection criteria lncRNA transcripts predicted to interact with microRNAs with established involvement in MS. To assess the expression levels of lncRNAs and their adjacent protein-coding genes, quantitative reverse transcription PCR was performed on peripheral blood mononuclear cells samples of 50 MS patients compared to 25 controls. Our results showed that in relapsing MS patients, compared to remitting MS patients and healthy controls, lncRNA RP11-530C5.1 was up-regulated while AL928742.12 was down-regulated. Pearson's correlation tests showed positive correlations between the expression levels of RP11-530C5.1 and AL928742.12 with PAWR and IGHA2, respectively. The results of the ROC curve test demonstrated the potential biomarker roles of AL928742.12 and RP11-530C5.1. We conclude that these lncRNAs are potential markers for detection of relapsing MS patients.

Coordination of Embryogenesis by the Centrosome in Drosophila melanogaster.

The first 3 h of Drosophila melanogaster embryo development are exemplified by rapid nuclear divisions within a large syncytium, transforming the zygote to the cellular blastoderm after 13 successive cleavage divisions. As the syncytial embryo develops, it relies on centrosomes and cytoskeletal dynamics to transport nuclei, maintain uniform nuclear distribution throughout cleavage cycles, ensure generation of germ cells, and coordinate cellularization. For the sake of this review, we classify six early embryo stages that rely on processes coordinated by the centrosome and its regulation of the cytoskeleton. The first stage features migration of one of the female pronuclei toward the male pronucleus following maturation of the first embryonic centrosomes. Two subsequent stages distribute the nuclei first axially and then radially in the embryo. The remaining three stages involve centrosome-actin dynamics that control cortical plasma membrane morphogenesis. In this review, we highlight the dynamics of the centrosome and its role in controlling the six stages that culminate in the cellularization of the blastoderm embryo.

LncRNAs associated with multiple sclerosis expressed in the Th1 cell lineage.

Multiple sclerosis (MS) is a type of inflammatory and demyelinating disorder of the central nervous system in which immune-mediated inflammatory processes are elicited by secreted cytokines from T helper (Th)-1 and Th17 cells. While some protein-coding genes expressed in T cell types have established involvement in MS disease progression, little is understood about the roles of long noncoding RNAs (lncRNAs) within the disease landscape. LncRNAs, noncoding RNAs longer than 200 nucleotides, likely control gene expression and function of Th1 cells, and offer the potential to act as therapeutic and biomarker candidates for MS. We identified lncRNAs in Th1 cells linked to MS. Expression levels of candidate lncRNAs and genes were evaluated in 50 MS patients and 25 healthy controls using quantitative real-time polymerase chain reaction, and their correlations were assessed. LncRNAs encoded by AC007278.2 and IFNG-AS1-001 showed significantly higher expression in relapsing Phase MS patients whereas IFNG-AS1-003 was elevated in patients in the remitting phase compared with relapsing patients. Collectively, these misregulated lncRNAs may provide valuable tools to understand the relationships between lncRNAs and MS, and possibly other related disorders.

Microvascular endothelial cells engulf myelin debris and promote macrophage recruitment and fibrosis after neural injury.

The clearance of damaged myelin sheaths is critical to ensure functional recovery from neural injury. Here we show a previously unidentified role for microvessels and their lining endothelial cells in engulfing myelin debris in spinal cord injury (SCI) and experimental autoimmune encephalomyelitis (EAE). We demonstrate that IgG opsonization of myelin debris is required for its effective engulfment by endothelial cells and that the autophagy-lysosome pathway is crucial for degradation of engulfed myelin debris. We further show that endothelial cells exert critical functions beyond myelin clearance to promote progression of demyelination disorders by regulating macrophage infiltration, pathologic angiogenesis and fibrosis in both SCI and EAE. Unexpectedly, myelin debris engulfment induces endothelial-to-mesenchymal transition, a process that confers upon endothelial cells the ability to stimulate the endothelial-derived production of fibrotic components. Overall, our study demonstrates that the processing of myelin debris through the autophagy-lysosome pathway promotes inflammation and angiogenesis and may contribute to fibrotic scar formation.

Integrative Analysis of lncRNAs in Th17 Cell Lineage to Discover New Potential Biomarkers and Therapeutic Targets in Autoimmune Diseases.

Th17 cells play a critical role in the pathogenesis of autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, and inflammatory bowel disease. Despite the extensive investigation into this T cell lineage, little is understood regarding the role of Th17 lineage-specific lncRNAs (long non-coding RNAs) > 200 nt. lncRNAs may influence disease through a variety of mechanisms; their expression could be regulated by SNPs. lncRNAs can also affect the expression of neighboring genes or complementary miRNAs, and their expression may have lineage-specific patterns. In the system biology study presented here, the effective lncRNAs from different criteria were predicted for each autoimmune disease, and we then evaluated their expression levels in 50 MS patients compared to 25 controls using qRT-PCR. We identified changes in the expression levels of AL450992.2, AC009948.5, and RP11-98D18.3 as potential peripheral blood mononuclear cell (PBMC) biomarkers for MS among our studied lncRNAs in which co-expression analysis of AL450992.2 had the most AUCs, and the relationship to RORC was also assessed. We propose that the recurrently deregulated lncRNAs identified in this report could provide a valuable resource for studies aimed at delineating the relationship between functional lncRNAs and autoimmune disorders.

Centrosomal and Non-Centrosomal Microtubule-Organizing Centers (MTOCs) in Drosophila melanogaster.

The centrosome is the best-understood microtubule-organizing center (MTOC) and is essential in particular cell types and at specific stages during Drosophila development. The centrosome is not required zygotically for mitosis or to achieve full animal development. Nevertheless, centrosomes are essential maternally during cleavage cycles in the early embryo, for male meiotic divisions, for efficient division of epithelial cells in the imaginal wing disc, and for cilium/flagellum assembly in sensory neurons and spermatozoa. Importantly, asymmetric and polarized division of stem cells is regulated by centrosomes and by the asymmetric regulation of their microtubule (MT) assembly activity. More recently, the components and functions of a variety of non-centrosomal microtubule-organizing centers (ncMTOCs) have begun to be elucidated. Throughout Drosophila development, a wide variety of unique ncMTOCs form in epithelial and non-epithelial cell types at an assortment of subcellular locations. Some of these cell types also utilize the centrosomal MTOC, while others rely exclusively on ncMTOCs. The impressive variety of ncMTOCs being discovered provides novel insight into the diverse functions of MTOCs in cells and tissues. This review highlights our current knowledge of the composition, assembly, and functional roles of centrosomal and non-centrosomal MTOCs in Drosophila.

A Splice Variant of Centrosomin Converts Mitochondria to Microtubule-Organizing Centers.

Non-centrosomal microtubule organizing centers (MTOCs) direct microtubule (MT) organization to exert diverse cell-type-specific functions. In Drosophila spermatids, the giant mitochondria provide structural platforms for MT reorganization to support elongation of the extremely long sperm. However, the molecular basis for this mitochondrial MTOC and other non-centrosomal MTOCs has not been discerned. Here we report that Drosophila centrosomin (cnn) expresses two major protein variants: the centrosomal form (CnnC) and a non-centrosomal form in testes (CnnT). CnnC is established as essential for functional centrosomes, the major MTOCs in animal cells. We show that CnnT is expressed exclusively in testes by alternative splicing and localizes to giant mitochondria in spermatids. In cell culture, CnnT targets to the mitochondrial surface, recruits the MT nucleator γ-tubulin ring complex (γ-TuRC), and is sufficient to convert mitochondria to MTOCs independent of core pericentriolar proteins that regulate MT assembly at centrosomes. We mapped two separate domains in CnnT: one that is necessary and sufficient to target it to mitochondria and another that is necessary and sufficient to recruit γ-TuRCs and nucleate MTs. In elongating spermatids, CnnT forms speckles on the giant mitochondria that are required to recruit γ-TuRCs to organize MTs and support spermiogenesis. This molecular characterization of the mitochondrial MTOC defines a minimal molecular requirement for MTOC generation and implicates the potent role of Cnn (or its related) proteins in the direct regulation of MT assembly and organization of non-centrosomal MTOCs.

La-related protein 6 controls ciliated cell differentiation.

BACKGROUND: La-related protein 6 (LARP6) is an evolutionally conserved RNA-binding protein. Vertebrate LARP6 binds the 5' stem-loop found in mRNAs encoding type I collagen to regulate their translation, but other target mRNAs and additional functions for LARP6 are unknown. The aim of this study was to elucidate an additional function of LARP6 and to evaluate the importance of its function during development. METHODS: To uncover the role of LARP6 in development, we utilized Morpholino Oligos to deplete LARP6 protein in Xenopus embryos. Then, embryonic phenotypes and ciliary structures of LAPR6 morphants were examined. To identify the molecular mechanism underlying ciliogenesis regulated by LARP6, we tested the expression level of cilia-related genes, which play important roles in ciliogenesis, by RT-PCR or whole mount in situ hybridization (WISH). RESULTS: We knocked down LARP6 in Xenopus embryos and found neural tube closure defects. LARP6 mutant, which compromises the collagen synthesis, could rescue these defects. Neural tube closure defects are coincident with lack of cilia, antenna-like cellular organelles with motility- or sensory-related functions, in the neural tube. The absence of cilia at the epidermis was also observed in LARP6 morphants, and this defect was due to the absence of basal bodies which are formed from centrioles and required for ciliary assembly. In the process of multi-ciliated cell (MCC) differentiation, mcidas, which activates the transcription of genes required for centriole formation during ciliogenesis, could partially restore MCCs in LARP6 morphants. In addition, LARP6 likely controls the expression of mcidas in a Notch-independent manner. CONCLUSIONS: La-related protein 6 is involved in ciliated cell differentiation during development by controlling the expression of cilia-related genes including mcidas. This LARP6 function involves a mechanism that is distinct from its established role in binding to collagen mRNAs and regulating their translation.

Methods to Establish Drosophila Cell Lines.

Hundreds of Drosophila cell lines have been established in the labs of many researchers over the last decades and have been important tools for research. Although these cells often deviate from normal cell physiology and genetic composition, such systems nonetheless are powerful models for biochemical, cell biological, and genetics studies that are experimentally difficult in vivo. While published descriptions of cell line generation are available in the literature, how to generate new Drosophila cell lines can be challenging for beginners. Here, we describe a detailed, simple protocol to establish new Drosophila cell lines.

Drosophila melanogaster as a model for basal body research.

The fruit fly, Drosophila melanogaster, is one of the most extensively studied organisms in biological research and has centrioles/basal bodies and cilia that can be modelled to investigate their functions in animals generally. Centrioles are nine-fold symmetrical microtubule-based cylindrical structures required to form centrosomes and also to nucleate the formation of cilia and flagella. When they function to template cilia, centrioles transition into basal bodies. The fruit fly has various types of basal bodies and cilia, which are needed for sensory neuron and sperm function. Genetics, cell biology and behaviour studies in the fruit fly have unveiled new basal body components and revealed different modes of assembly and functions of basal bodies that are conserved in many other organisms, including human, green algae and plasmodium. Here we describe the various basal bodies of Drosophila, what is known about their composition, structure and function.

The Seckel syndrome and centrosomal protein Ninein localizes asymmetrically to stem cell centrosomes but is not required for normal development, behavior, or DNA damage response in Drosophila.

Ninein (Nin) is a centrosomal protein whose gene is mutated in Seckel syndrome (SCKL, MIM 210600), an inherited recessive disease that results in primordial dwarfism, cognitive deficiencies, and increased sensitivity to genotoxic stress. Nin regulates neural stem cell self-renewal, interkinetic nuclear migration, and microtubule assembly in mammals. Nin is evolutionarily conserved, yet its role in cell division and development has not been investigated in a model organism. Here we characterize the single Nin orthologue in Drosophila Drosophila Nin localizes to the periphery of the centrosome but not at centriolar structures as in mammals. However, Nin shares the property of its mammalian orthologue of promoting microtubule assembly. In neural and germline stem cells, Nin localizes asymmetrically to the younger (daughter) centrosome, yet it is not required for the asymmetric division of stem cells. In wing epithelia and muscle, Nin localizes to noncentrosomal microtubule-organizing centers. Surprisingly, loss of nin expression from a nin mutant does not significantly affect embryonic and brain development, fertility, or locomotor performance of mutant flies or their survival upon exposure to DNA-damaging agents. Although it is not essential, our data suggest that Nin plays a supportive role in centrosomal and extracentrosomal microtubule organization and asymmetric stem cell division.

Assessment of Chicken-Egg Membrane as a Dressing for Wound Healing.

OBJECTIVES: To examine the efficacy of the folk remedy of chicken-egg membrane dressing on wound healing. DESIGN: Full-thickness excisional wounds were created on 14 male Sprague-Dawley rats in 2 separate trials. Each animal received 2 wounds on the upper back. One wound was untreated, and the other was dressed with chicken-egg membrane to assess its impact on wound healing. Half of the rats received egg membrane treatment on the inferior wound, whereas the other half received egg membrane treatment on the superior wound. Membrane replacement, wound debridement, and imaging were done on days 5, 8, and 10 and then imaging continued on days 12, 14, 16, 18, and 20 of the experiment. Healing rate was measured based on the wound area over the 20 days of the experiment. RESULTS: The wounds dressed with chicken-egg membrane had a significantly (P < .01) faster rate of healing compared with the control at the early stages of healing between days 0 and 5. This group healed 21% faster during this early phase, compared with the control group. Overall, however, wound healing rates were indistinguishable from days 5 to 20. CONCLUSION: Chicken-egg membrane dressing significantly improves healing of cutaneous wounds in the early stages of wound healing.