Delivery of ceramide phosphoethanolamine lipids to the cleavage furrow through the endocytic pathway is essential for male meiotic cytokinesis.

Cell division, wherein 1 cell divides into 2 daughter cells, is fundamental to all living organisms. Cytokinesis, the final step in cell division, begins with the formation of an actomyosin contractile ring, positioned midway between the segregated chromosomes. Constriction of the ring with concomitant membrane deposition in a specified spatiotemporal manner generates a cleavage furrow that physically separates the cytoplasm. Unique lipids with specific biophysical properties have been shown to localize to intercellular bridges (also called midbody) connecting the 2 dividing cells; however, their biological roles and delivery mechanisms remain largely unknown. In this study, we show that ceramide phosphoethanolamine (CPE), the structural analog of sphingomyelin, has unique acyl chain anchors in Drosophila spermatocytes and is essential for meiotic cytokinesis. The head group of CPE is also important for spermatogenesis. We find that aberrant central spindle and contractile ring behavior but not mislocalization of phosphatidylinositol phosphates (PIPs) at the plasma membrane is responsible for the male meiotic cytokinesis defect in CPE-deficient animals. Further, we demonstrate the enrichment of CPE in multivesicular bodies marked by Rab7, which in turn localize to cleavage furrow. Volume electron microscopy analysis using correlative light and focused ion beam scanning electron microscopy shows that CPE-enriched Rab7 positive endosomes are juxtaposed on contractile ring material. Correlative light and transmission electron microscopy reveal Rab7 positive endosomes as a multivesicular body-like organelle that releases its intraluminal vesicles in the vicinity of ingressing furrows. Genetic ablation of Rab7 or Rab35 or expression of dominant negative Rab11 results in significant meiotic cytokinesis defects. Further, we show that Rab11 function is required for localization of CPE positive endosomes to the cleavage furrow. Our results imply that endosomal delivery of CPE to ingressing membranes is crucial for meiotic cytokinesis.

SSSPTA is essential for serine palmitoyltransferase function during development and hematopoiesis.

Serine palmitoyltransferase complex (SPT) mediates the first and rate-limiting step in the de novo sphingolipid biosynthetic pathway. The larger subunits SPTLC1 and SPTLC2/SPTLC3 together form the catalytic core while a smaller third subunit either SSSPTA or SSSPTB has been shown to increase the catalytic efficiency and provide substrate specificity for the fatty acyl-CoA substrates. The in vivo biological significance of these smaller subunits in mammals is still unknown. Here, using two null mutants, a conditional null for ssSPTa and a null mutant for ssSPTb, we show that SSSPTA is essential for embryogenesis and mediates much of the known functions of the SPT complex in mammalian hematopoiesis. The ssSPTa null mutants are embryonic lethal at E6.5 much like the Sptlc1 and Sptlc2 null alleles. Mx1-Cre induced deletion of ssSPTa leads to lethality and myelopoietic defect. Chimeric and competitive bone marrow transplantation experiments show that the defect in myelopoiesis is accompanied by an expansion of the Lin-Sca1+c-Kit+ stem and progenitor compartment. Progenitor cells that fail to differentiate along the myeloid lineage display evidence of endoplasmic reticulum stress. On the other hand, ssSPTb null mice are homozygous viable, and analyses of the bone marrow cells show no significant difference in the proliferation and differentiation of the adult hematopoietic compartment. SPTLC1 is an obligatory subunit for the SPT function, and because Sptlc1-/- and ssSPTa-/- mice display similar defects during development and hematopoiesis, we conclude that an SPT complex that includes SSSPTA mediates much of its developmental and hematopoietic functions in a mammalian model.

Defective cortex glia plasma membrane structure underlies light-induced epilepsy in cpes mutants.

Seizures induced by visual stimulation (photosensitive epilepsy; PSE) represent a common type of epilepsy in humans, but the molecular mechanisms and genetic drivers underlying PSE remain unknown, and no good genetic animal models have been identified as yet. Here, we show an animal model of PSE, in Drosophila, owing to defective cortex glia. The cortex glial membranes are severely compromised in ceramide phosphoethanolamine synthase (cpes)-null mutants and fail to encapsulate the neuronal cell bodies in the Drosophila neuronal cortex. Expression of human sphingomyelin synthase 1, which synthesizes the closely related ceramide phosphocholine (sphingomyelin), rescues the cortex glial abnormalities and PSE, underscoring the evolutionarily conserved role of these lipids in glial membranes. Further, we show the compromise in plasma membrane structure that underlies the glial cell membrane collapse in cpes mutants and leads to the PSE phenotype.

Survival response to increased ceramide involves metabolic adaptation through novel regulators of glycolysis and lipolysis.

The sphingolipid ceramide elicits several stress responses, however, organisms survive despite increased ceramide but how they do so is poorly understood. We demonstrate here that the AKT/FOXO pathway regulates survival in increased ceramide environment by metabolic adaptation involving changes in glycolysis and lipolysis through novel downstream targets. We show that ceramide kinase mutants accumulate ceramide and this leads to reduction in energy levels due to compromised oxidative phosphorylation. Mutants show increased activation of Akt and a consequent decrease in FOXO levels. These changes lead to enhanced glycolysis by upregulating the activity of phosphoglyceromutase, enolase, pyruvate kinase, and lactate dehydrogenase to provide energy. A second major consequence of AKT/FOXO reprogramming in the mutants is the increased mobilization of lipid from the gut through novel lipase targets, CG8093 and CG6277 for energy contribution. Ubiquitous reduction of these targets by knockdown experiments results in semi or total lethality of the mutants, demonstrating the importance of activating them. The efficiency of these adaptive mechanisms decreases with age and leads to reduction in adult life span of the mutants. In particular, mutants develop cardiac dysfunction with age, likely reflecting the high energy requirement of a well-functioning heart. The lipases also regulate physiological triacylglycerol homeostasis and are important for energy metabolism since midgut specific reduction of them in wild type flies results in increased sensitivity to starvation and accumulation of triglycerides leading to cardiac defects. The central findings of increased AKT activation, decreased FOXO level and activation of phosphoglyceromutase and pyruvate kinase are also observed in mice heterozygous for ceramide transfer protein suggesting a conserved role of this pathway in mammals. These data reveal novel glycolytic and non-autonomous lipolytic pathways in response to increased ceramide for sustenance of high energy demanding organ functions like the heart.

Sphingolipid biosynthesis is essential for metabolic rewiring during TH17 cell differentiation.

T helper 17 (TH17) cells are implicated in autoimmune diseases, and several metabolic processes are shown to be important for their development and function. In this study, we report an essential role for sphingolipids synthesized through the de novo pathway in TH17 cell development. Deficiency of SPTLC1, a major subunit of serine palmitoyl transferase enzyme complex that catalyzes the first and rate-limiting step of de novo sphingolipid synthesis, impaired glycolysis in differentiating TH17 cells by increasing intracellular reactive oxygen species (ROS) through enhancement of nicotinamide adenine dinucleotide phosphate oxidase 2 activity. Increased ROS leads to impaired activation of mammalian target of rapamycin C1 and reduced expression of hypoxia-inducible factor 1-alpha and c-Myc-induced glycolytic genes. SPTLCI deficiency protected mice from developing experimental autoimmune encephalomyelitis and experimental T cell transfer colitis. Our results thus show a critical role for de novo sphingolipid biosynthetic pathway in shaping adaptive immune responses with implications in autoimmune diseases.

A nutrient responsive lipase mediates gut-brain communication to regulate insulin secretion in Drosophila.

Pancreatic ß cells secrete insulin in response to glucose elevation to maintain glucose homeostasis. A complex network of inter-organ communication operates to modulate insulin secretion and regulate glucose levels after a meal. Lipids obtained from diet or generated intracellularly are known to amplify glucose-stimulated insulin secretion, however, the underlying mechanisms are not completely understood. Here, we show that a Drosophila secretory lipase, Vaha (CG8093), is synthesized in the midgut and moves to the brain where it concentrates in the insulin-producing cells in a process requiring Lipid Transfer Particle, a lipoprotein originating in the fat body. In response to dietary fat, Vaha stimulates insulin-like peptide release (ILP), and Vaha deficiency results in reduced circulatory ILP and diabetic features including hyperglycemia and hyperlipidemia. Our findings suggest Vaha functions as a diacylglycerol lipase physiologically, by being a molecular link between dietary fat and lipid amplified insulin secretion in a gut-brain axis.

Tooth Mobility among Patients visiting a Tertiary Care Centre: A Descriptive Cross-sectional Study.

Introduction: Periodontitis is a chronic inflammatory disease that results in the destruction of supporting tissue and bone leading to tooth mobility. Tooth mobility if untreated can lead to tooth loss. However, very few studies exist for its assessment. The aim of this study was to find out the prevalence of tooth mobility among patients visiting a tertiary care centre. Methods: This descriptive cross-sectional study was conducted among individuals visiting a tertiary care dental hospital from 1st April to 30th June 2022 after obtaining ethical clearance from the Institutional Review Committee (Reference number: 2202202202). Individuals more than 13 years who gave consent and fulfilled the study criteria were enrolled. Tooth mobility was assessed using Lindhe and Nyman's classification. Proforma also included demographics, simplified oral hygiene index, gingival index, body mass index, and smoking status. Convenience sampling was done. Point estimate and 95% Confidence Interval were calculated. Results: Among 163 patients, 65 (39.88%) patients (32.36-47.40, 95% Confidence Interval) had tooth mobility. Conclusions: The prevalence of tooth mobility was higher than in studies done in similar settings. Keywords: periodontitis; prevalence; tooth mobility.

In Silico Characterization of Intracellular Localization Signals and Structural Features of Mosquito Densovirus (MDV) Viral Proteins.

As entomopathogenic viruses, mosquito densoviruses (MDVs) are widely studied for their potential as biocontrol agents and molecular laboratory tools for mosquito manipulation. The nucleus of the mosquito cell is the site for MDV genome replication and capsid assembly, however the nuclear localization signals (NLSs) and nuclear export signals (NES) for MDV proteins have not yet been identified. We carried out an in silico analysis to identify putative NLSs and NESs in the viral proteins of densoviruses that infect diverse mosquito genera (Aedes, Anopheles, and Culex) and identified putative phosphorylation and glycosylation sites on these proteins. These analyses lead to a more comprehensive understanding of how MDVs are transported into and out of the nucleus and lay the foundation for the potential use of densoviruses in mosquito control and basic research.

Cell-nonautonomous function of ceramidase in photoreceptor homeostasis.

Neutral ceramidase, a key enzyme of sphingolipid metabolism, hydrolyzes ceramide to sphingosine. These sphingolipids are critical structural components of cell membranes and act as second messengers in diverse signal transduction cascades. Here, we have isolated and characterized functional null mutants of Drosophila ceramidase. We show that secreted ceramidase functions in a cell-nonautonomous manner to maintain photoreceptor homeostasis. In the absence of ceramidase, photoreceptors degenerate in a light-dependent manner, are defective in normal endocytic turnover of rhodopsin, and do not respond to light stimulus. Consistent with a cell-nonautonomous function, overexpression of ceramidase in tissues distant from photoreceptors suppresses photoreceptor degeneration in an arrestin mutant and facilitates membrane turnover in a rhodopsin null mutant. Furthermore, our results show that secreted ceramidase is internalized and localizes to endosomes. Our findings establish a role for a secreted sphingolipid enzyme in the regulation of photoreceptor structure and function.

Ceramide kinase regulates phospholipase C and phosphatidylinositol 4, 5, bisphosphate in phototransduction.

Phosphoinositide-specific phospholipase C (PLC) is a central effector for many biological responses regulated by G-protein-coupled receptors including Drosophila phototransduction where light sensitive channels are activated downstream of NORPA, a PLCbeta homolog. Here we show that the sphingolipid biosynthetic enzyme, ceramide kinase, is a novel regulator of PLC signaling and photoreceptor homeostasis. A mutation in ceramide kinase specifically leads to proteolysis of NORPA, consequent loss of PLC activity, and failure in light signal transduction. The mutant photoreceptors also undergo activity-dependent degeneration. Furthermore, we show that a significant increase in ceramide, resulting from lack of ceramide kinase, perturbs the membrane microenvironment of phosphatidylinositol 4, 5, bisphosphate (PIP(2)), altering its distribution. Fluorescence image correlation spectroscopic studies on model membranes suggest that an increase in ceramide decreases clustering of PIP(2) and its partitioning into ordered membrane domains. Thus ceramide kinase-mediated maintenance of ceramide level is important for the local regulation of PIP(2) and PLC during phototransduction.

Lipid Polarization during Cytokinesis.

The plasma membrane of eukaryotic cells is composed of a large number of lipid species that are laterally segregated into functional domains as well as asymmetrically distributed between the outer and inner leaflets. Additionally, the spatial distribution and organization of these lipids dramatically change in response to various cellular states, such as cell division, differentiation, and apoptosis. Division of one cell into two daughter cells is one of the most fundamental requirements for the sustenance of growth in all living organisms. The successful completion of cytokinesis, the final stage of cell division, is critically dependent on the spatial distribution and organization of specific lipids. In this review, we discuss the properties of various lipid species associated with cytokinesis and the mechanisms involved in their polarization, including forward trafficking, endocytic recycling, local synthesis, and cortical flow models. The differences in lipid species requirements and distribution in mitotic vs. male meiotic cells will be discussed. We will concentrate on sphingolipids and phosphatidylinositols because their transbilayer organization and movement may be linked via the cytoskeleton and thus critically regulate various steps of cytokinesis.

Drosophila melanogaster Scramblases modulate synaptic transmission.

Scramblases are a family of single-pass plasma membrane proteins, identified by their purported ability to scramble phospholipids across the two layers of plasma membrane isolated from platelets and red blood cells. However, their true in vivo role has yet to be elucidated. We report the generation and isolation of null mutants of two Scramblases identified in Drosophila melanogaster. We demonstrate that flies lacking either or both of these Scramblases are not compromised in vivo in processes requiring scrambling of phospholipids. Instead, we show that D. melanogaster lacking both Scramblases have more vesicles and display enhanced recruitment from a reserve pool of vesicles and increased neurotransmitter secretion at the larval neuromuscular synapses. These defects are corrected by the introduction of a genomic copy of the Scramb 1 gene. The lack of phenotypes related to failure of scrambling and the neurophysiological analysis lead us to propose that Scramblases play a modulatory role in the process of neurotransmission.

dagLogo: An R/Bioconductor package for identifying and visualizing differential amino acid group usage in proteomics data.

Sequence logos have been widely used as graphical representations of conserved nucleic acid and protein motifs. Due to the complexity of the amino acid (AA) alphabet, rich post-translational modification, and diverse subcellular localization of proteins, few versatile tools are available for effective identification and visualization of protein motifs. In addition, various reduced AA alphabets based on physicochemical, structural, or functional properties have been valuable in the study of protein alignment, folding, structure prediction, and evolution. However, there is lack of tools for applying reduced AA alphabets to the identification and visualization of statistically significant motifs. To fill this gap, we developed an R/Bioconductor package dagLogo, which has several advantages over existing tools. First, dagLogo allows various formats for input sets and provides comprehensive options to build optimal background models. It implements different reduced AA alphabets to group AAs of similar properties. Furthermore, dagLogo provides statistical and visual solutions for differential AA (or AA group) usage analysis of both large and small data sets. Case studies showed that dagLogo can better identify and visualize conserved protein sequence patterns from different types of inputs and can potentially reveal the biological patterns that could be missed by other logo generators.

Sptlc1 is essential for myeloid differentiation and hematopoietic homeostasis.

Serine palmitoyltransferase (SPT) long-chain base subunit 1 (SPTLC1) is 1 of the 2 main catalytic subunits of the SPT complex, which catalyzes the first and rate-limiting step of sphingolipid biosynthesis. Here, we show that Sptlc1 deletion in adult bone marrow (BM) cells results in defective myeloid differentiation. In chimeric mice from noncompetitive BM transplant assays, there was an expansion of the Lin- c-Kit+ Sca-1+ compartment due to increased multipotent progenitor production, but myeloid differentiation was severely compromised. We also show that defective biogenesis of sphingolipids in the endoplasmic reticulum (ER) leads to ER stress that affects myeloid differentiation. Furthermore, we demonstrate that transient accumulation of fatty acid, a substrate for sphingolipid biosynthesis, could be partially responsible for the ER stress. Independently, we find that ER stress in general, such as that induced by the chemical thapsigargin or the fatty acid palmitic acid, compromises myeloid differentiation in culture. These results identify perturbed sphingolipid metabolism as a source of ER stress, which may produce diverse pathological effects related to differential cell-type sensitivity.

Effect of vitamins C and E on spermatogenesis in mice exposed to cadmium.

Cadmium (Cd) is a potential pollutant of the environment. It manifests cyto-toxic effects in different organs in animals. In the present study, intraperitoneal injection of CdCl(2) (1mg/kg body weight) increased lipid peroxidation in Swiss mice testes indicating oxidative stress during 5th to 8th week of post-treatment . The enzymatic activity of superoxide dismutase (SOD), catalase (CT) and peroxidase (PD) were significantly decreased over the post-treatment phase in Cd-treated mice testes compared to vehicle controls. Further, ascorbic acid content also declined significantly in Cd-exposed mice testes. Following Cd treatment, a marked increase in sperm abnormality percentage and significant decrease in sperm count was observed. The purpose of the present study was to evaluate the effect of vitamins C and E supplementation on Cd-treated mice testes. Therefore, Cd-treated mice groups were injected with vitamins C and E, separately, to assess the effect of the vitamins in combating Cd-induced cytotoxicity and other manifestations. Supplementation of vitamin C (10mg/kg body weight) and vitamin E (100mg/kg body weight) to Cd-induced mice groups declined lipid peroxidation, increased sperm count profile, depressed the percentage of sperm abnormality, increased the activity of antioxidant enzymes mentioned above and also increased the concentration of ascorbic acid to a measurable extent. The role of vitamins in reducing oxidative stress-related effects on spermatogenesis in Cd-treated Swiss mice testes have been reported.

Morphological and genetic analyses of the first record of longrakered trevally, Ulua mentalis (Perciformes: Carangidae) and of the pinjalo snapper, Pinjalo pinjalo (Perciformes: Lutjanidae) in the Odisha coast, Bay of Bengal.

Identification of fish species have so far been carried out mostly by classical morpho-taxonomy. In the present study, however, an attempt has been taken to identify two species of fishes Ulua mentalis and Pinjalo pinjalo of order Perciformes which happens to be the first record in Odisha coast Bay of Bengal, India during the year 2015, using DNA barcoding technique for reconfirmation over conventional morpho-taxonomy. During recent past, study of molecular-taxonomical profile of mitochondrial DNA in general and Cytochrome Oxidase subunit I (COI) gene in particular has gained enormous importance for accurate identification of species. In the present study, the partial COI sequence of Ulua mentalis and Pinjalo pinjalo were generated. Analysis using the COI gene produced phylogenetic trees in concurrence with other multi gene studies and we came across the identical phylogenetic relationship considering Neighbor-Joining and Maximum Likelihood tree. Moreover, these molecular data set further testified in Bayesian framework to reevaluate the exact taxonomic groupings within the family. Surprisingly, Ulua mentalis and Pinjalo pinjalo seems to be closely related to their sister taxa.

Testicular dysfunction and antioxidative defense system of Swiss mice after chromic acid exposure.

Chromic acid (CrO(3)), a hexavalent compound of chromium (Cr) that is widely used in different industries, has been associated with reproductive abnormalities in male Swiss mice. The mechanism of male reproductive toxicity is not clear. Metal-induced reactive oxygen species (ROS) may impair spermatogenesis or alter sperm morphology. Therefore, in the present study we investigated the induction of oxidative stress in the testes of mice over time (5th-8th weeks) after a single intraperitoneal dose (1mg/kg body weight) of CrO(3). Exposed animals showed significantly decreased sperm counts and markedly increased rates of sperm abnormality. Oxidative stress was measured in terms of malondialdehyde content, the activities of superoxide dismutase, peroxidase, and catalase, and non-enzymatic antioxidants such as ascorbic acid. The significant changes in exposed groups relative to controls suggest that CrO(3) exposure suppressed antioxidant enzymes and ascorbic acid with a concomitant increase in the level of lipid peroxidation and H(2)O(2) to adversely affect testicular function.

Ceramidase regulates synaptic vesicle exocytosis and trafficking.

A screen for Drosophila synaptic dysfunction mutants identified slug-a-bed (slab). The slab gene encodes ceramidase, a central enzyme in sphingolipid metabolism and regulation. Sphingolipids are major constituents of lipid rafts, membrane domains with roles in vesicle trafficking, and signaling pathways. Null slab mutants arrest as fully developed embryos with severely reduced movement. The SLAB protein is widely expressed in different tissues but enriched in neurons at all stages of development. Targeted neuronal expression of slab rescues mutant lethality, demonstrating the essential neuronal function of the protein. C(5)-ceramide applied to living preparations is rapidly accumulated at neuromuscular junction (NMJ) synapses dependent on the SLAB expression level, indicating that synaptic sphingolipid trafficking and distribution is regulated by SLAB function. Evoked synaptic currents at slab mutant NMJs are reduced by 50-70%, whereas postsynaptic glutamate-gated currents are normal, demonstrating a specific presynaptic impairment. Hypertonic saline-evoked synaptic vesicle fusion is similarly impaired by 50-70%, demonstrating a loss of readily releasable vesicles. In addition, FM1-43 dye uptake is reduced in slab mutant presynaptic terminals, indicating a smaller cycling vesicle pool. Ultrastructural analyses of mutants reveal a normal vesicle distribution clustered and docked at active zones, but fewer vesicles in reserve regions, and a twofold to threefold increased incidence of vesicles linked together and tethered at the plasma membrane. These results indicate that SLAB ceramidase function controls presynaptic terminal sphingolipid composition to regulate vesicle fusion and trafficking, and thus the strength and reliability of synaptic transmission.

Protective action of vitamins on the spermatogenesis in lead-treated Swiss mice.

The protective action of vitamins C and E against lead acetate-induced reduced sperm count and sperm abnormalities in Swiss mice has been studied. Intraperitoneal injection of lead acetate (10mg/kg body weight) in the present study stimulates lipid peroxidation in the testicular tissue, indicated by a significant increase in malondialdehyde content in the experimental mice group. This is associated with an increased generation of noxious reactive oxygen species (ROS). Significantly reduced sperm count associated with increased sperm abnormality percentage in the lead-injected mice group compared to controls substantially proves the ongoing damaging effects of lead-induced ROS on developing germ cells. However, intraperitoneal administration of vitamin C (Vit C) at a concentration equivalent to the human therapeutic dose (10 mg/kg body weight) was able to minimize significantly the testicular malondialdehyde content with a concomitant increase in sperm count and significant decrease in the percentage of abnormal sperm population. Vitamin E (Vit E) (100 mg/kg body weight) treatment of a batch of lead-injected mice had a similar effect as Vit C but with a comparatively lower efficacy. On the other hand, coadministration of both vitamins (Vit C + Vit E) at the above mentioned doses to lead-treated mice led to the most significant decline in malondialdehyde content along with elevated sperm count and reduction in the percentage of abnormal sperm population. The protective action and the synergistic action of both vitamins (C and E) against lead-induced genotoxicity are discussed.

Status of antioxidant defense system in chromium-induced Swiss mice tissues.

Based on epidemiological studies, chromium(VI) compounds are considered as more toxic and carcinogenic than chromium(III) compounds. The deleterious effects of chromium(VI) compounds are diversified affecting almost all the organ systems in a wide variety of animals. The present study, describes the cytotoxic effects of chromium trioxide, a well-known chromium(VI) compound in three tissues (liver, kidney, lungs) of male Swiss mice during post-treatment phase (5th-8th week after treatment). Lipid peroxidation, an index of oxidative stress, was determined as thiobarbituric acid-reactive substances (TBA-Rs) in mice tissues dosed with a single intraperitoneal injection of chromium trioxide (1mg/kg body weight). Tissue specific and statistically significant increases in TBA-Rs was observed in chromium-treated mice groups compared to controls in all the weeks of post-treatment. Endogenous ascorbic acid (vit-C) content of tissues which happens to be one of the stable antioxidants, declined significantly due to chromium-induction. Activity of antioxidative enzymes like superoxide dismutase (SOD), catalase (CT) and peroxidase (PD) was significantly inhibited among chromium-injected mice groups compared to respective controls. Protective role of ascorbic acid and the antioxidative enzymes in chromium-induced cytotoxicity in mice is discussed.