An initial HOPS-mediated fusion event is critical for autophagosome transport initiation from the axon terminal.

In neurons, macroautophagy/autophagy is a frequent and critical process. In the axon, autophagy begins in the axon terminal, where most nascent autophagosomes form. After formation, autophagosomes must initiate transport to exit the axon terminal and move toward the cell body via retrograde transport. During retrograde transport these autophagosomes mature through repetitive fusion events. Complete lysosomal cargo degradation occurs largely in the cell body. The precipitating events to stimulate retrograde autophagosome transport have been debated but their importance is clear: disrupting neuronal autophagy or autophagosome transport is detrimental to neuronal health and function. We have identified the HOPS complex as essential for early autophagosome maturation and consequent initiation of retrograde transport from the axon terminal. In yeast and mammalian cells, HOPS controls fusion between autophagosomes and late endosomes with lysosomes. Using zebrafish strains with loss-of-function mutations in vps18 and vps41, core components of the HOPS complex, we found that disruption of HOPS eliminates autophagosome maturation and disrupts retrograde autophagosome transport initiation from the axon terminal. We confirmed this phenotype was due to loss of HOPS complex formation using an endogenous deletion of the HOPS binding domain in Vps18. Finally, using pharmacological inhibition of lysosomal proteases, we show that initiation of autophagosome retrograde transport requires autophagosome maturation. Together, our data demonstrate that HOPS-mediated fusion events are critical for retrograde autophagosome transport initiation through promoting autophagosome maturation. This reveals critical roles for the HOPS complex in neuronal autophagy which deepens our understanding of the cellular pathology of HOPS-complex linked neurodegenerative diseases.Abbreviations: CORVET: Class C core vacuole/endosome tethering; gRNA: guide RNA; HOPS: homotypic fusion and protein sorting; pLL: posterior lateral line; Vps18: VPS18 core subunit of CORVET and HOPS complexes; Vps41: VPS41 subunit of HOPS complex.

Non-vesicular phosphatidylinositol transfer plays critical roles in defining organelle lipid composition.

Phosphatidylinositol (PI) is the precursor lipid for the minor phosphoinositides (PPIns), which are critical for multiple functions in all eukaryotic cells. It is poorly understood how phosphatidylinositol, which is synthesized in the ER, reaches those membranes where PPIns are formed. Here, we used VT01454, a recently identified inhibitor of class I PI transfer proteins (PITPs), to unravel their roles in lipid metabolism, and solved the structure of inhibitor-bound PITPNA to gain insight into the mode of inhibition. We found that class I PITPs not only distribute PI for PPIns production in various organelles such as the plasma membrane (PM) and late endosomes/lysosomes, but that their inhibition also significantly reduced the levels of phosphatidylserine, di- and triacylglycerols, and other lipids, and caused prominent increases in phosphatidic acid. While VT01454 did not inhibit Golgi PI4P formation nor reduce resting PM PI(4,5)P2 levels, the recovery of the PM pool of PI(4,5)P2 after receptor-mediated hydrolysis required both class I and class II PITPs. Overall, these studies show that class I PITPs differentially regulate phosphoinositide pools and affect the overall cellular lipid landscape.

Roles of Phosphatidylinositol 4-Phosphorylation in Non-vesicular Cholesterol Trafficking.

Cholesterol (Chol) is an essential component of all eukaryotic cell membranes that affects the function of numerous peripheral as well as integral membrane proteins. Chol is synthesized in the ER, but it is selectively enriched within the plasma membrane (PM) and other endomembranes, which requires Chol to cross the aqueous phase of the cytoplasm. In addition to the classical vesicular trafficking pathways that are known to facilitate the bulk transport of membrane intermediates, Chol is also transported via non-vesicular lipid transfer proteins that work primarily within specialized membrane contact sites. Some of these transport pathways work against established concentration gradients and hence require energy. Recent studies highlight the unique role of phosphoinositides (PPIns), and phosphatidylinositol 4-phosphate (PI4P) in particular, for the control of non-vesicular Chol transport. In this chapter, we will review the emerging connection between Chol, PPIns, and lipid transfer proteins that include the important family of oxysterol-binding protein related proteins, or ORPs.

Metabolic routing maintains the unique fatty acid composition of phosphoinositides.

Phosphoinositide lipids (PPIn) are enriched in stearic- and arachidonic acids (38:4) but how this enrichment is established and maintained during phospholipase C (PLC) activation is unknown. Here we show that the metabolic fate of newly synthesized phosphatidic acid (PA), the lipid precursor of phosphatidylinositol (PI), is influenced by the fatty acyl-CoA used with preferential routing of the arachidonoyl-enriched species toward PI synthesis. Furthermore, during agonist stimulation the unsaturated forms of PI(4,5P)2 are replenished significantly faster than the more saturated ones, suggesting a favored recycling of the unsaturated forms of the PLC-generated hydrolytic products. Cytidine diphosphate diacylglycerol synthase 2 (CDS2) but not CDS1 was found to contribute to increased PI resynthesis during PLC activation. Lastly, while the lipid transfer protein, Nir2 is found to contribute to rapid PPIn resynthesis during PLC activation, the faster re-synthesis of the 38:4 species does not depend on Nir2. Therefore, the fatty acid side-chain composition of the lipid precursors used for PI synthesis is an important determinant of their metabolic fates, which also contributes to the maintenance of the unique fatty acid profile of PPIn lipids.

Non-Aneurysmal Subarachnoid Hemorrhage in COVID-19: A Case Report and Review of Literature.

BACKGROUND: Hemorrhagic cerebrovascular events, either due to aneurysmal rupture or spontaneous subarachnoid hemorrhage (SAH), are not rare in COVID-19. Several mechanisms such as coagulopathy, cytokine storm, viral endotheliopathy, hypertension, and immune modulation might play a role in the pathogenesis of SAH in COVID-19. This study aimed to report the first case of spontaneous non-aneurysmal SAH associated with SARS-CoV-2 from India. We briefly discussed the possible pathogenetic mechanisms underlying this process and succinctly reviewed the relevant literature. CASE REPORT: We herein report a case of a non-comorbid young woman infected with SARS-CoV-2 presenting with thunderclap headache and eventually non-aneurysmal SAH, who recovered with conservative management. CONCLUSION: Headache, although a very common clinical feature of COVID-19 itself, must be investigated in detail to identify alternate causes that may be life-threatening. This case also incites further enquiry into the possible pathogenic mechanisms of neurovascular complications in COVID-19.

Retrograde Mitochondrial Transport Is Essential for Organelle Distribution and Health in Zebrafish Neurons.

In neurons, mitochondria are transported by molecular motors throughout the cell to form and maintain functional neural connections. These organelles have many critical functions in neurons and are of high interest as their dysfunction is associated with disease. While the mechanics and impact of anterograde mitochondrial movement toward axon terminals are beginning to be understood, the frequency and function of retrograde (cell body directed) mitochondrial transport in neurons are still largely unexplored. While existing evidence indicates that some mitochondria are retrogradely transported for degradation in the cell body, the precise impact of disrupting retrograde transport on the organelles and the axon was unknown. Using long-term, in vivo imaging, we examined mitochondrial motility in zebrafish sensory and motor axons. We show that retrograde transport of mitochondria from axon terminals allows replacement of the axon terminal population within a day. By tracking these organelles, we show that not all mitochondria that leave the axon terminal are degraded; rather, they persist over several days. Disrupting retrograde mitochondrial flux in neurons leads to accumulation of aged organelles in axon terminals and loss of cell body mitochondria. Assays of neural circuit activity demonstrated that disrupting mitochondrial transport and function has no effect on sensory axon terminal activity but does negatively impact motor neuron axons. Taken together, our work supports a previously unappreciated role for retrograde mitochondrial transport in the maintenance of a homeostatic distribution of mitochondria in neurons and illustrates the downstream effects of disrupting this process on sensory and motor circuits.SIGNIFICANCE STATEMENT Disrupted mitochondrial transport has been linked to neurodegenerative disease. Retrograde transport of this organelle has been implicated in turnover of aged organelles through lysosomal degradation in the cell body. Consistent with this, we provide evidence that retrograde mitochondrial transport is important for removing aged organelles from axons; however, we show that these organelles are not solely degraded, rather they persist in neurons for days. Disrupting retrograde mitochondrial transport impacts the homeostatic distribution of mitochondria throughout the neuron and the function of motor, but not sensory, axon synapses. Together, our work shows the conserved reliance on retrograde mitochondrial transport for maintaining a healthy mitochondrial pool in neurons and illustrates the disparate effects of disrupting this process on sensory versus motor circuits.

Axonal Transport and Mitochondrial Function in Neurons.

The complex and elaborate architecture of a neuron poses a great challenge to the cellular machinery which localizes proteins and organelles, such as mitochondria, to necessary locations. Proper mitochondrial localization in neurons is particularly important as this organelle provides energy and metabolites essential to form and maintain functional neural connections. Consequently, maintenance of a healthy pool of mitochondria and removal of damaged organelles are essential for neuronal homeostasis. Long distance transport of the organelle itself as well as components necessary for maintaining mitochondria in distal compartments are important for a constant supply of healthy mitochondria at the right time and place. Accordingly, many neurodegenerative diseases have been associated with mitochondrial abnormalities. Here, we review our current understanding on transport-dependent mechanisms that regulate mitochondrial replenishment. We focus on axonal transport and import of mRNAs and proteins destined for mitochondria as well as mitochondrial fusion and fission to maintain mitochondrial homeostasis in distal compartments of the neuron.

Stem cell proliferation is induced by apoptotic bodies from dying cells during epithelial tissue maintenance.

Epithelial tissues require the removal and replacement of damaged cells to sustain a functional barrier. Dying cells provide instructive cues that can influence surrounding cells to proliferate, but how these signals are transmitted to their healthy neighbors to control cellular behaviors during tissue homeostasis remains poorly understood. Here we show that dying stem cells facilitate communication with adjacent stem cells by caspase-dependent production of Wnt8a-containing apoptotic bodies to drive cellular turnover in living epithelia. Basal stem cells engulf apoptotic bodies, activate Wnt signaling, and are stimulated to divide to maintain tissue-wide cell numbers. Inhibition of either cell death or Wnt signaling eliminated the apoptosis-induced cell division, while overexpression of Wnt8a signaling combined with induced cell death led to an expansion of the stem cell population. We conclude that ingestion of apoptotic bodies represents a regulatory mechanism linking death and division to maintain overall stem cell numbers and epithelial tissue homeostasis.

Adipofascial perforator flaps: Its role in reconstruction of soft-tissue defects of lower leg and ankle.

BACKGROUND: Despite advances in surgical skills, adipofascial flaps are still less preferred option for coverage of leg defect. We evaluate the use of perforator-based adipofascial flap in small-to-medium-sized soft-tissue defects in the lower limb. PATIENTS AND METHODS: After localisation of perforators along the major axial vessels in the lower limb using handheld Doppler, adipofascial flap based on the nearest best perforator of anterior tibial artery, posterior tibial artery and peroneal artery was raised to cover soft-tissue defect in 21 patients. The flap was transposed over the defect and covered by split-skin graft. Donor site was primarily closed. Flap complications, functional and aesthetic outcomes are noted in follow-up. RESULTS: There was partial loss of flap in five patients. After debridement and dressings, split-skin grafting was done for four patients and one patient was managed with local flap. Scar over the flap was stable with no reports of recurrent ulceration or breakdown of wound in 6-month follow-up. Four of five patients reported adequate healing of the fractured bone. Average visual analogue score was 8.24/10 for appearance of donor site as evaluated by the patient. CONCLUSIONS: Perforator-based adipofascial flap is a good alternative for coverage of small-to-medium-sized soft-tissue defect of the leg, particularly over the malleolus and lower part of the leg. Use of adipofascial tissue and primary closure of the donor site causes minimal donor-site morbidity. Adipofascial perforator flap provides aesthetically superior recipient-site scar with satisfactory functional outcome.

Analyzing Neuronal Mitochondria in vivo Using Fluorescent Reporters in Zebrafish.

Despite their importance for cellular viability, the actual life history and properties of mitochondria in neurons are still unclear. These organelles are distributed throughout the entirety of the neuron and serve many functions, including: energy production (ATP), iron homeostasis and processing, calcium buffering, and metabolite production, as well as many other lesser known activities. Given their importance, understanding how these organelles are positioned and how their health and function is maintained is critical for many aspects of cell biology. This is best illustrated by the diverse disease literature which demonstrates that abnormal mitochondrial movement, localization, size, or function often correlates with neural pathology. In the following methods article, we will describe the techniques and tools we have optimized to directly visualize mitochondria and analyze mitochondrial lifetime, health, and function in neurons in vivo using fluorescent reporters in the zebrafish. The zebrafish system is ideal for in vivo studies of mitochondrial biology as: (1) neuronal circuits develop rapidly, within days; (2) it is genetically accessible; and (3) embryos and larvae are translucent allowing imaging in a completely intact vertebrate nervous system. Using these tools and techniques, the field is poised to answer questions of mitochondrial biology in the context of neuronal health and function in normal and disease states.

Wnt signaling balances specification of the cardiac and pharyngeal muscle fields.

Canonical Wnt/ß-catenin (Wnt) signaling plays multiple conserved roles during fate specification of cardiac progenitors in developing vertebrate embryos. Although lineage analysis in ascidians and mice has indicated there is a close relationship between the cardiac second heart field (SHF) and pharyngeal muscle (PM) progenitors, the signals underlying directional fate decisions of the cells within the cardio-pharyngeal muscle field in vertebrates are not yet understood. Here, we examined the temporal requirements of Wnt signaling in cardiac and PM development. In contrast to a previous report in chicken embryos that suggested Wnt inhibits PM development during somitogenesis, we find that in zebrafish embryos Wnt signaling is sufficient to repress PM development during anterior-posterior patterning. Importantly, the temporal sensitivity of dorso-anterior PMs to increased Wnt signaling largely overlaps with when Wnt signaling promotes specification of the adjacent cardiac progenitors. Furthermore, we find that excess early Wnt signaling can cell autonomously promote expansion of the first heart field (FHF) progenitors at the expense of PM and SHF within the anterior lateral plate mesoderm (ALPM). Our study provides insight into an antagonistic developmental mechanism that balances the sizes of the adjacent cardiac and PM progenitor fields in early vertebrate embryos.

Retinoic acid negatively regulates dact3b expression in the hindbrain of zebrafish embryos.

Wnt signaling plays important roles in normal development as well as pathophysiological conditions. The Dapper antagonist of ß-catenin (Dact) proteins are modulators of both canonical and non-canonical Wnt signaling via direct interactions with Dishevelled (Dvl) and Van Gogh like-2 (Vangl2). Here, we report the dynamic expression patterns of two zebrafish dact3 paralogs during early embryonic development. Our whole mount in situ hybridization (WISH) analysis indicates that specific dact3a expression starts by the tailbud stage in adaxial cells. Later, it is expressed in the anterior lateral plate mesoderm, somites, migrating cranial neural crest, and hindbrain neurons. By comparison, dact3b expression initiates on the dorsal side at the dome stage and soon after is expressed in the dorsal forerunner cells (DFCs) during gastrulation. At later stages, dact3b expression becomes restricted to the branchial neurons of the hindbrain and to the second pharyngeal arch. To investigate how zebrafish dact3 gene expression is regulated, we manipulated retinoic acid (RA) signaling during development and found that it negatively regulates dact3b in the hindbrain. Our study is the first to document the expression of the paralogous zebrafish dact3 genes during early development and demonstrate dact3b can be regulated by RA signaling. Therefore, our study opens up new avenues to study Dact3 function in the development of multiple tissues and suggests a previously unappreciated cross regulation of Wnt signaling by RA signaling in the developing vertebrate hindbrain.

Depletion of retinoic acid receptors initiates a novel positive feedback mechanism that promotes teratogenic increases in retinoic acid.

Normal embryonic development and tissue homeostasis require precise levels of retinoic acid (RA) signaling. Despite the importance of appropriate embryonic RA signaling levels, the mechanisms underlying congenital defects due to perturbations of RA signaling are not completely understood. Here, we report that zebrafish embryos deficient for RA receptor αb1 (RARαb1), a conserved RAR splice variant, have enlarged hearts with increased cardiomyocyte (CM) specification, which are surprisingly the consequence of increased RA signaling. Importantly, depletion of RARαb2 or concurrent depletion of RARαb1 and RARαb2 also results in increased RA signaling, suggesting this effect is a broader consequence of RAR depletion. Concurrent depletion of RARαb1 and Cyp26a1, an enzyme that facilitates degradation of RA, and employment of a novel transgenic RA sensor line support the hypothesis that the increases in RA signaling in RAR deficient embryos are the result of increased embryonic RA coupled with compensatory RAR expression. Our results support an intriguing novel mechanism by which depletion of RARs elicits a previously unrecognized positive feedback loop that can result in developmental defects due to teratogenic increases in embryonic RA.

Transgenic retinoic acid sensor lines in zebrafish indicate regions of available embryonic retinoic acid.

BACKGROUND: Retinoic acid (RA) signaling plays a critical role in vertebrate development. Transcriptional reporters of RA signaling in zebrafish, thus far, have not reflected the broader availability of embryonic RA, necessitating additional tools to enhance our understanding of the spatial and temporal activity of RA signaling in vivo. RESULTS: We have generated novel transgenic RA sensors in which a RA receptor (RAR) ligand-binding domain (RLBD) is fused to the Gal4 DNA-binding domain (GDBD) or a VP16-GDBD (VPBD) construct. Stable transgenic lines expressing these proteins when crossed with UAS reporter lines are responsive to RA. Interestingly, the VPBD RA sensor is significantly more sensitive than the GDBD sensor and demonstrates there may be almost ubiquitous availability of RA within the early embryo. Using confocal microscopy to compare the expression of the GDBD RA sensor to our previously established RA signaling transcriptional reporter line, Tg(12XRARE:EGFP), illustrates these reporters have significant overlap, but that expression from the RA sensor is much broader. We also identify previously unreported domains of expression for the Tg(12XRARE:EGFP) line. CONCLUSIONS: Our novel RA sensor lines will be useful and complementary tools for studying RA signaling during development and anatomical structures independent of RA signaling.

Investigation of antimicrobial physiology of orthorhombic and monoclinic nanoallotropes of sulfur at the interface of transcriptome and metabolome.

Nanosized elemental sulfur (ES) is already reported to exert superior antimicrobial efficacy than micron-sized ES, which encourages their use in drugs and therapeutics. The aim of the present study is to explore the possible route and mode of antimicrobial action of orthorhombic (α-SNPs) and monoclinic (ß-SNPs) allotropes of sulfur, respectively, at their nano-dimensions. The antimicrobial efficacy of α- and ß-SNPs was determined against both the conventionally ES-resistant and ES-susceptible fungi and bacteria. Both the SNPs inhibited the microbial growth, irrespective of their resistance profile to ES and caused significant deformities on the microbial cell surfaces. However, the extent of antimicrobial efficacy was found to be optimum for α-SNPs, which can be attributed to their size, shape, and surface modification. Subsequent transcript profiling, metabolite profiling, and enzymatic analyses revealed that α- and ß-SNPs impaired a cluster of mitochondrial enzymes involved in cellular respiration and oxidative phosphorylation. ES and SNPs stress were found to elicit the NADPH-dependent glutathione reductase mediated ES-detoxification response in fungi and caused them to undertake the glyoxylate shunt in favor of energy conservation. A simultaneous study was also undertaken to assess the biocompatible or bio-adverse properties of SNPs in terms of their cytotoxic and genotoxic effects against the human derived lung fibroblast cell line (MRC-5). The present study hence explores the antimicrobial physiology of two novel functional materials and demonstrates their compatibility as a future putative antimicrobial drug.

Influence of BCL11A, HBS1L-MYB, HBBP1 single nucleotide polymorphisms and the HBG2 XmnI polymorphism On Hb F levels.

In search of genetic alterations responsible for high fetal hemoglobin (Hb F) phenotypes in the population of eastern India, 91 probands were screened for four polymorphisms by sequencing and/or restriction fragment length polymorphism (RFLP) analysis. These are the A>G allele on the rs4895441 locus in the intergenic region between HBS1L and MYB on chromosome 6, the G>A allele on the rs4671393 locus on chromosome 2 (BCL11A gene), the A>C allele on the rs2071348 (HBBP1 gene) and the XmnI polymorphism (rs7482144, -158 position of HBG2) on chromosome 11. We found a significant association (p = 0.002 and 0.0013) of Hb F levels with rs2071348 and rs4895441, respectively. However, the polymorphism rs4671393 gene did not show significant association with Hb F levels (p = 0.0655). As is well known, the XmnI polymorphism (p <0.0001) showed the strongest association.

Surface-modified sulfur nanoparticles: an effective antifungal agent against Aspergillus niger and Fusarium oxysporum.

Surface-modified sulfur nanoparticles (SNPs) of two different sizes were prepared via a modified liquid-phase precipitation method, using sodium polysulfide and ammonium polysulfide as starting material and polyethylene glycol-400 (PEG-400) as the surface stabilizing agent. Surface topology, size distribution, surface modification of SNPs with PEG-400, quantitative analysis for the presence of sulfur in nanoformulations, and thermal stability of SNPs were determined by atomic force microscopy (AFM), dynamic light scattering (DLS) plus high-resolution transmission electron microscopy (HR-TEM), fourier transform infrared (FT-IR) spectroscopy, energy dispersive X-ray (EDX) spectroscopy, and thermogravimetric analysis (TGA), respectively. A simultaneous study with micron-sized sulfur (S(0)) and SNPs was carried out to evaluate their fungicidal efficacy against Aspergillus niger and Fusarium oxysporum in terms of radial growth, sporulation, ultrastructural modifications, and phospholipid content of the fungal strains using a modified poisoned food technique, spore-germination slide bioassay, environmental scanning electron microscopy (ESEM), and spectrometry. SNPs expressed promising inhibitory effect on fungal growth and sporulation and also significantly reduced phospholipid content.