Accumulation Of Vesicles Research Articles

Abstract 1370: Overexpression of the small GTPase Rab27B regulates autophagy flux and contributes to tumor growth in colorectal cancer

Abstract Rab27B is a member of the Rab small GTPases that belong to the RAS-like GTPase superfamily. Rab27B has been shown to regulate endosomal trafficking, exosome secretion, and delivery of secretory granules. However, the role of Rab27B in colorectal cancer (CRC) tumor biology is unknown. Using CRC tissue microarrays, a significant upregulation of Rab27B was detected in staged adenocarcinoma compared to normal colon tissue. Interestingly, microsatellite unstable (MSI) tumors showed a higher increase in Rab27B (2.4-fold) than microsatellite stable (MSS) tumors (1.6-fold), consistent with TCGA data. To study the role of Rab27B in CRC, we targeted Rab27B in MSI HCT116 cell line by CRISPR-Cas9 deletion and siRNA knockdown. In both cases, loss of Rab27B resulted in a dysregulation of cellular autophagy and reduction in extracellular vesicle secretion. Rab27B-depleted cells showed an abnormal accumulation of autophagy vesicles and increase in autophagy markers LC3-II and p62, indicating a defect in autophagy flux. To characterize this autophagy defect, we used eGFP-mCherry-LC3 reporter, lysotracker and Cyto-ID staining to identify that autophagy flux is blocked at the autophagosome/lysosome fusion step when Rab27B is lost. To test whether the GTPase activity of Rab27B is needed for the autophagy role, we added back constitutively active and dominant negative GTPase mutants of Rab27B in Rab27B KO cells and observed autophagy phenotype rescue with only wildtype and constitutively active GTPase forms. As autophagy has been shown to have a pro-survival role in tumor growth and stress response, we hypothesized that this defect in autophagy flux resulting from Rab27B deletion will impact cellular stress response and reduce tumor growth. While Rab27B knockout (KO) cells show similar in vitro 2D-growth characteristics as wildtype cells under normal conditions, loss of Rab27B resulted in a 60% reduction in cell viability in response to starvation conditions. Similarly, a 94% reduction in colony formation was observed when grown in soft agar, along with deficiencies in spheroid formation. In vivo, Rab27B KO cells showed a major reduction (p<0.0001) in tumor growth xenografts compared to wildtype HCT116 cells. Taken together, these results demonstrate a new role of Rab27B in the autophagy trafficking process in CRC. Futures studies will focus on further characterizing the relationship among Rab27B overexpression, microsatellite instability, and autophagy in vitro and in vivo using Rab27B knockout mouse model, as well as investigating whether Rab27B can be targeted as a potential new therapeutic strategy. Citation Format: Sahida Afroz, Ranjan Preet, Vikalp Vishwakarma, Dan A. Dixon. Overexpression of the small GTPase Rab27B regulates autophagy flux and contributes to tumor growth in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1370.

PPARα Inhibits Astrocyte Inflammation Activation by Restoring Autophagic Flux after Transient Brain Ischemia

Astrocyte inflammation activation is an important cause that hinders the recovery of motor function after cerebral ischemia. However, its molecular mechanism has not yet been clearly clarified. The peroxisome proliferator-activated receptor α (PPARα) is a ligand-activated nuclear transcriptional factor. This study aims to further clarify the role of PPARα in astrocyte inflammation activation after cerebral ischemia and to explore the underlying mechanism. Astrocyte activation was induced in an in vivo model by transient middle cerebral artery occlusion (tMCAO) in mice. The in vitro model was induced by an oxygen-glucose deprivation/reoxygenation (OGD/R) in a primary culture of mouse astrocyte. PPARα-deficient mice were used to observe the effects of PPARα on astrocyte activation and autophagic flux. Our results showed that PPARα was mainly expressed in activated astrocytes during the chronic phase of brain ischemia and PPARα dysfunction promoted astrocyte inflammatory activation. After cerebral ischemia, the expressions of LC3-II/I and p62 both increased. Autophagic vesicle accumulation was observed by electron microscopy in astrocytes, and the block of autophagic flux was indicated by an mRFP-GFP-LC3 adenovirus infection assay. A PPARα deficit aggravated the autophagic flux block, while PPARα activation preserved the lysosome function and restored autophagic flux in astrocytes after OGD/R. The autophagic flux blocker bafilomycin A1 and chloroquine antagonized the effect of the PPARα agonist on astrocyte activation inhibition. This study identifies a potentially novel function of PPARα in astrocyte autophagic flux and suggests a therapeutic target for the prevention and treatment of chronic brain ischemic injury.

The synthetic TRPML1 agonist ML-SA1 rescues Alzheimer-related alterations of the endosomal-autophagic-lysosomal system.

Abnormalities in the endosomal-autophagic-lysosomal (EAL) system are an early event in Alzheimer's disease (AD) pathogenesis. However, the mechanisms underlying these abnormalities are unclear. The transient receptor potential channel mucolipin 1(TRPML1, also known as MCOLN1), a vital endosomal-lysosomal Ca2+ channel whose loss of function leads to neurodegeneration, has not been investigated with respect to EAL pathogenesis in late-onset AD (LOAD). Here, we identify pathological hallmarks of TRPML1 dysregulation in LOAD neurons, including increased perinuclear clustering and vacuolation of endolysosomes. We reveal that induced pluripotent stem cell (iPSC)-derived human cortical neurons expressing APOE ε4, the strongest genetic risk factor for LOAD, have significantly diminished TRPML1-induced endolysosomal Ca2+ release. Furthermore, we found that blocking TRPML1 function in primary neurons by depleting the TRPML1 agonist PI(3,5)P2 via PIKfyve inhibition, recreated multiple features of EAL neuropathology evident in LOAD. This included increased endolysosomal Ca2+ content, enlargement and perinuclear clustering of endolysosomes, autophagic vesicle accumulation and early endosomal enlargement. Strikingly, these AD-like neuronal EAL defects were rescued by TRPML1 reactivation using its synthetic agonist ML-SA1. These findings implicate defects in TRPML1 in LOAD EAL pathogenesis and present TRPML1 as a potential therapeutic target.

STING signalling is terminated through ESCRT-dependent microautophagy of vesicles originating from recycling endosomes

Stimulator of interferon genes (STING) is essential for the type I interferon response against a variety of DNA pathogens. Upon emergence of cytosolic DNA, STING translocates from the endoplasmic reticulum to the Golgi where STING activates the downstream kinase TBK1, then to lysosome through recycling endosomes (REs) for its degradation. Although the molecular machinery of STING activation is extensively studied and defined, the one underlying STING degradation and inactivation has not yet been fully elucidated. Here we show that STING is degraded by the endosomal sorting complexes required for transport (ESCRT)-driven microautophagy. Airyscan super-resolution microscopy and correlative light/electron microscopy suggest that STING-positive vesicles of an RE origin are directly encapsulated into Lamp1-positive compartments. Screening of mammalian Vps genes, the yeast homologues of which regulate Golgi-to-vacuole transport, shows that ESCRT proteins are essential for the STING encapsulation into Lamp1-positive compartments. Knockdown of Tsg101 and Vps4, components of ESCRT, results in the accumulation of STING vesicles in the cytosol, leading to the sustained type I interferon response. Knockdown of Tsg101 in human primary T cells leads to an increase the expression of interferon-stimulated genes. STING undergoes K63-linked ubiquitination at lysine 288 during its transit through the Golgi/REs, and this ubiquitination is required for STING degradation. Our results reveal a molecular mechanism that prevents hyperactivation of innate immune signalling, which operates at REs.

Human filaggrin monomer does not seem to be a proteasome target.

Absence of a functional proteasome in the suprabasal layers of the epidermis is responsible for keratosis linearis with ichthyosis congenital and sclerosing keratoderma syndrome. Patient epidermis shows hypergranulosis associated with abnormally shaped keratohyalin granules and abnormal distribution of filaggrin in the Stratum granulosum and Stratum corneum. This suggests that the proteasome is involved in the degradation of filaggrin. To test this hypothesis, the proteasome proteolytic activity was inhibited in 3D reconstructed human epidermis (RHE) with the specific clasto-lactacystin β-lactone inhibitor. Confirming the efficacy of inhibition, ubiquitinated proteins accumulated in treated RHEs as compared to controls. Levels of urocanic acid (UCA) and pyrrolidone carboxylic acid (PCA), the end products of filaggrin degradation, were reduced. However, neither filaggrin accumulation nor appearance of filaggrin-derived peptides were observed. On the contrary, the amount of filaggrin was shown to decrease, and a similar tendency was observed for profilaggrin, its precursor. Accumulation of small cytoplasmic vesicles associated with a significant increase in autophagy markers indicated activation of the autophagy process upon proteasome inhibition. Taken together, these results suggest that the perturbation of UCA and PCA production after proteasome inhibition was probably due to down-regulation of filaggrin expression rather than to blocking of filaggrin proteolysis.

Lysosomal dysfunction in Schwann cells is involved in bortezomib-induced peripheral neurotoxicity.

Bortezomib (BTZ) is a proteasome inhibitor serves as a first-line drug for multiple myeloma treatment. BTZ-induced peripheral neuropathy (BIPN) is the most common adverse effect of BTZ with an incidence as high as 40-60%. However, the pathological mechanisms underlying BIPN remain largely unclear. BTZ leads to dramatic Schwann cell demyelination in sciatic nerves. Previous studies implied that myelin debris was predominantly degraded via autophagy-lysosome pathway in Schwann cells. However, the association of autophagy with BIPN has not been made. Mice were treated with BTZ (2mg/kg, i.v.) on Day1 and Day4 each week for continuous 4weeks. BTZ-treated mice showed enhanced mechanical hyperalgesia, decreased tail nerve conduction and sciatic nerve demyelination. Unexpectedly, BTZ led to the accumulation of autophagic vesicles, LC3-II and p62 in the sciatic nerve. Moreover, BTZ blocked autophagic flux in RSC96 Schwann cells as determined by mcherry-GFP-LC3 assay, suggesting BTZ may impair lysosomal function rather than inducing autophagy in Schwann cells. BTZ significantly reduced the lysosomal activity in Schwann cells as determined by reduced LysoTracker Red and DQ-Red-BSA staining and increased the level of immature Cathepsin B (CTSB). Remarkably, lysosomal activators PP242 and Torin1, significantly reversed the blockage of autophagic flux by BTZ. We further verified that Torin1 rescued the demyelination, nerve conduction and reduced the mechanical hyperalgesia in BIPN mice. Additionally, Torin1 did not compromise the efficacy of BTZ in suppressing multiple myeloma RPMI8226 cell. Taken together, we identified that lysosomal dysfunction in Schwann cells caused by BTZ is involved in the BIPN pathology. Improved lysosomal function in Schwann cells can be a promising strategy for BIPN treatment.

Deficiency of (n-6) but Not (n-3) Polyunsaturated Fatty Acids Inhibits the Secretagogue Effect of Prolactin in Lactating Rat Mammary Epithelial Cells

The repercussions of various kinds of dietary polyunsaturated fatty acid (PUFA) deficiencies on the fatty acid composition of membranes and on the secretory activity of lactating female rat mammary epithelial cells were investigated. Primiparous female rats were fed different PUFA diets from weaning: adequate (n-6) and (n-3) PUFA supply; overall PUFA deficiency; specific (n-6) PUFA deficiency or specific (n-3) PUFA deficiency. Mammary gland phospholipids contained very low amount of (n-3) PUFA in control rats, and only 1% docosahexaenoic acid. The fatty acid composition of membrane phospholipids reflected the type of diet received by the animals, i.e., the diets deficient in the (n-3) or (n-6) PUFA series resulted in lower (n-3) or (n-6) PUFA, and the (n-3) + (n-6) deficient diet caused a true overall PUFA deficiency in the membranes. The morphology of cells from overall PUFA- or (n-6) PUFA-deficient rats showed an accumulation of secretory vesicles in the cytoplasm. Basal casein secretion was independent of the diet and of the composition of membrane phospholipids. However, prolactin did not have a secretagogue effect on cells from (n-6) PUFA- or overall PUFA-deficient rats but retained this effect on cells from (n-3)-deficient rats. These results emphasize the specific role of (n-6) PUFA in the functioning of the lactating mammary epithelial cell.

Hydroalcoholic extract of Buxus sempervirens shows antiproliferative effect on melanoma, colorectal carcinoma and prostate cancer cells by affecting the autophagic flow.

Buxus sempervirens (European Box, Buxaceae, boxwood) has been used in folk medicine to treat rheumatism, arthritis, fever, malaria and skin ulceration while, in recent years, interest has grown on possible employment of boxwood extracts in cancer therapy. We studied the effect of hydroalcoholic extract from dried leaves of Buxus sempervirens (BSHE) on four human cell lines (BMel melanoma cells, HCT116 colorectal carcinoma cells, PC3 prostate cancer cells, and HS27 skin fibroblasts) to ascertain its possible antineoplastic activity. This extract inhibited proliferation of all cell lines in different degree as shown, after 48h-exposure and MTS assay, by the values of GR50 (normalized growth rate inhibition50) that were 72, 48, 38, and 32μg/mL for HS27, HCT116, PC3 and BMel cells, respectively. At the above GR50 concentrations, 99% of all studied cells remained vital showing accumulation of acidic vesicles in the cytoplasm, mainly around nuclei, whereas a higher extract concentration (125μg/mL) was cytotoxic causing, after 48h-exposure, death of all BMel and HCT116 cells. Immunofluorescence showed microtubule-associated light chain three protein (LC3, a marker for autophagy) to be localized on the above acidic vesicles when cells were treated for 48h with BSHE (GR50 concentrations). Western blot analysis revealed, in all treated cells, a significant increase (2.2-3.3 times at 24h) of LC3II, i.e., the phosphatidylethanolamine conjugate of the cytoplasmic form LC3I that is recruited in autophagosome membranes during autophagy. Such increase was accompanied, in all cell lines treated for 24h or 48h with BSHE, by a significant increment (2.5-3.4 times at 24h) of p62, an autophagic cargo protein undergoing degradation during the autophagic process. Therefore, BSHE appeared to promote autophagic flow with its following blockade and consequent accumulation of autophagosome or autolysosomes. The antiproliferative effects of BSHE also involved cell cycle regulators such as p21 (HS27, BMel and HCT116 cells) and cyclin B1 (HCT116, BMel and PC3 cells) whereas, among apoptosis markers, BSHE only decreased (30%-40% at 48h) the expression of the antiapoptotic protein survivin. It was concluded that BSHE impairs autophagic flow with arrest of proliferation and death in both fibroblasts and cancer cells, being the latter much more sensitive to these effects.

Rab GTPases, tethers, and SNAREs work together to regulate Arabidopsis cell plate formation.

Cell plates are transient structures formed by the fusion of vesicles at the center of the dividing plane; furthermore, these are precursors to new cell walls and are essential for cytokinesis. Cell plate formation requires a highly coordinated process of cytoskeletal rearrangement, vesicle accumulation and fusion, and membrane maturation. Tethering factors have been shown to interact with the Ras superfamily of small GTP binding proteins (Rab GTPases) and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), which are essential for cell plate formation during cytokinesis and are fundamental for maintaining normal plant growth and development. In Arabidopsis thaliana, members of the Rab GTPases, tethers, and SNAREs are localized in cell plates, and mutations in the genes encoding these proteins result in typical cytokinesis-defective phenotypes, such as the formation of abnormal cell plates, multinucleated cells, and incomplete cell walls. This review highlights recent findings on vesicle trafficking during cell plate formation mediated by Rab GTPases, tethers, and SNAREs.

The Unusual Aggregation and Fusion Activity of the Antimicrobial Peptide W-BP100 in Anionic Vesicles

Cationic antimicrobial peptides (CAMPs) offer a promising strategy to counteract bacterial resistance, mostly due to their membrane-targeting activity. W-BP100 is a potent broad-spectrum cecropin-melittin CAMP bearing a single N-terminal Trp, which was previously found to improve its antibacterial activity. W-BP100 has high affinity toward anionic membranes, inducing membrane saturation at low peptide-to-lipid (P/L) ratios and membrane permeabilization, with the unique property of promoting the aggregation of anionic vesicles only at specific P/L ratios. Herein, we aimed to investigate this unusual behavior of W-BP100 by studying its aggregation and fusion properties with negatively-charged large (LUVs) or giant (GUVs) unilamellar vesicles using biophysical tools. Circular dichroism (CD) showed that W-BP100 adopted an α-helical conformation in anionic LUVs, neutralizing its surface charge at the aggregation P/L ratio. Its fusion activity, assessed by Förster resonance energy transfer (FRET) using steady-state fluorescence spectroscopy, occurred mainly at the membrane saturation/aggregation P/L ratio. Confocal microscopy studies confirmed that W-BP100 displays aggregation and detergent-like effects at a critical P/L ratio, above which it induces the formation of new lipid aggregates. Our data suggest that W-BP100 promotes the aggregation and fusion of anionic vesicles at specific P/L ratios, being able to reshape the morphology of GUVs into new lipid structures.

Optimization of staging algorithm for newly detected prostate cancer by using PET/CT with <sup>68</sup>Ga-PSMA: a retrospective study

INTRODUCTION: Prostate cancer (PC) is the most common malignancy in men worldwide and ranks third in mortality. Improvement of the results of staging of newly detected prostate cancer is rightfully associated with the active use in clinical practice of positron emission tomography combined with computed tomography (PET/CT) with radiotracers based on prostate-specific membrane antigen (PSMA) ligands.OBJECTIVE: The aim of the study was to determine the capabilities of PET/CT with 68Ga-PSMA in evaluating the prevalence of newly diagnosed breast cancer in comparison with traditional imaging techniques (computed tomography, magnetic resonance imaging and bone scan) and to determine the role of this technology in the choice of treatment algorithm.MATERIALS AND METHODS: 120 patients aged 46 to 74 years (median age 62.5 years) with histologically verified prostate cancer underwent PET/CT with 68Ga-PSMA in our center to assess disease prevalence. The selection criteria for the study were: prostate-specific antigen (PSA) level of 5 ng/ml and above, presence of newly detected, histologically verified prostate cancer, no treatment, suspected metastatic lesion of pelvic and skeletal lymph nodes according to CT, MRI and OSG. All patients were divided into groups according to PSA level and Gleason score. Statistics: Statistical processing of the results was performed by methods of variance statistics using Statistica 10.0, GraphPad Prism 9.3.1. Wilcoxon-Mann-Whitney and Friedman tests for ANOVA were used to determine the significance of differences between comparison groups. The chi-square test with Yates correction was used to determine the reliability of intergroup differences for nominal measures (such as the presence of previous recurrences). The level of correlation was assessed using Spearman criterion.RESULTS: PET/CT imaging analysis in addition to MRI, CT, and OSG data showed TNM staging changes in 63 of 120 patients (52.3%) due to the detection of additional foci of metastatic lesions. Change of data about local spread of tumor with increasing of TNM stage due to detection of pathological RFP accumulation in seminal vesicles in 10 of 120 patients (8.3%), without structural changes using conventional imaging methods. In 20 of 64 patients (31.3%), PET/CT revealed lesions of regional lymph nodes (N) that were not visualized by conventional imaging methods due to their small size. Metastatic lesions of distant lymph nodes (M1a) and bones (M1b) undetected by CT, MRI, and OSG were found in 27 (22.5%) and 32 (26.7%) of 120 patients, respectively. At the same time, foci of pathological accumulation of 68Ga-PSMA in the bones without structural changes on CT were detected in 7 and 32 patients (21.8%).DISCUSSION: One of the objectives of this study was to compare the diagnostic capabilities of standard diagnostic imaging techniques, in particular MRI, CT and Bone scan, with the hybrid technology of PET/CT with 68Ga-PSMA to improve the accuracy of PCa staging in order to determine treatment tactics. The results demonstrate minimal superiority of PET/CT with 68Ga-PSMA over MRI in terms of sensitivity (96% and 94%, respectively) in assessing local disease prevalence. Tumor invasion of the seminal vesicles, in most cases, was detected in patients with a Gleason score greater than 8. At the same time there was a tendency for an increase in the level of radiotracer accumulation in the tumor tissue of the seminal vesicles depending on the differentiation group of PCa. Analysis of the histological material obtained after the prostatectomy demonstrated tumorous invasion of the seminal vesicles in 26 (37.1%) out of 70 operated patients. Coincidence of histological examination results with PET/CT data was found in 22 patients, with MRI data — in 20 patients. The sensitivity, specificity, positive predictive value, and negative predictive value of PET/CT were 85%, 92%, 85%, and 92%, while the corresponding values from MRI were as follows: 77%, 88%, 77%, and 88%. Analyzing the levels of 68Ga-PSMA uptake in the tumor tissue, we found that with increasing prostate differentiation group, there was a persistent increase in radiotracer accumulation in the prostate tumor tissue. We also evaluated the interrelation of RFP accumulation with PSA level. The patients with PCa with PSA level ≥10.0 ng/ml demonstrated high accumulation of radiotracer accumulation compared to those with PSA <10.0 ng/ml (p<0.001).CONCLUSIONS: The use of 68Ga-PSMA PET/CT in the staging of newly diagnosed and untreated cancers provides comprehensive information on the local, regional, and distant extent of the disease, and in some cases contributes to a change in TNM stage of the disease in a single study. The use of this method before planned surgical treatment of PCa can significantly reduce the risk of early postoperative relapse, especially in patients with a Gleason score of more than 7 and a PSA level greater than 20 ng/ml.

TRPML1-Induced Lysosomal Ca2+ Signals Activate AQP2 Translocation and Water Flux in Renal Collecting Duct Cells.

Lysosomes are acidic Ca2+ storage organelles that actively generate local Ca2+ signaling events to regulate a plethora of cell functions. Here, we characterized lysosomal Ca2+ signals in mouse renal collecting duct (CD) cells and we assessed their putative role in aquaporin 2 (AQP2)-dependent water reabsorption. Bafilomycin A1 and ML-SA1 triggered similar Ca2+ oscillations, in the absence of extracellular Ca2+, by alkalizing the acidic lysosomal pH or activating the lysosomal cation channel mucolipin 1 (TRPML1), respectively. TRPML1-dependent Ca2+ signals were blocked either pharmacologically or by lysosomes' osmotic permeabilization, thus indicating these organelles as primary sources of Ca2+ release. Lysosome-induced Ca2+ oscillations were sustained by endoplasmic reticulum (ER) Ca2+ content, while bafilomycin A1 and ML-SA1 did not directly interfere with ER Ca2+ homeostasis per se. TRPML1 activation strongly increased AQP2 apical expression and depolymerized the actin cytoskeleton, thereby boosting water flux in response to an hypoosmotic stimulus. These effects were strictly dependent on the activation of the Ca2+/calcineurin pathway. Conversely, bafilomycin A1 led to perinuclear accumulation of AQP2 vesicles without affecting water permeability. Overall, lysosomal Ca2+ signaling events can be differently decoded to modulate Ca2+-dependent cellular functions related to the dock/fusion of AQP2-transporting vesicles in principal cells of the CD.

Mechanistic Insights and Potential Therapeutic Approaches in PolyQ Diseases via Autophagy.

Polyglutamine diseases are a group of congenital neurodegenerative diseases categorized with genomic abnormalities in the expansion of CAG triplet repeats in coding regions of specific disease-related genes. Protein aggregates are the toxic hallmark for polyQ diseases and initiate neuronal death. Autophagy is a catabolic process that aids in the removal of damaged organelles or toxic protein aggregates, a process required to maintain cellular homeostasis that has the potential to fight against neurodegenerative diseases, but this pathway gets affected under diseased conditions, as there is a direct impact on autophagy-related gene expression. The increase in the accumulation of autophagy vesicles reported in neurodegenerative diseases was due to an increase in autophagy or may have been due to a decrease in autophagy flux. These reports suggested that there is a contribution of autophagy in the pathology of diseases and regulation in the process of autophagy. It was demonstrated in various disease models of polyQ diseases that autophagy upregulation by using modulators can enhance the dissolution of toxic aggregates and delay disease progression. In this review, interaction of the autophagy pathway with polyQ diseases was analyzed, and a therapeutic approach with autophagy inducing drugs was established for disease pathogenesis.

Isolation and Purification of Chromaffin Granules from Adrenal Glands and Cultured Neuroendocrine Cells.

Chromaffin granules isolated from adrenal glands constitute a powerful experimental tool to the study ofsecretory vesicle components and their participation in fusion and docking processes, vesicle aggregation, and interactions with cytosolic components. Although it is possible to isolate and purify chromaffin granules from adrenal glands of different species, bovine adrenal glands are the most used tissue source due to its easy handling and the large amount of granules that can be obtained from this tissue. In this chapter, we describe an easy-to-use and short-term protocol for efficiently obtaining highly purified chromaffin granules from bovine adrenal medulla. We additionally include protocols to isolate granules from cultured bovine chromaffin cells and PC12 cells, as well as a section to obtain chromaffin granules from mouse adrenal glands.

Molecular interaction of black tea components with phospholipid vesicles

The molecular interaction of black tea components with phospholipid vesicles has been investigated to elucidate the various functions of black tea infusions, such as astringency, inhibition of cholesterol absorption, and inactivation of viruses. We have developed a simple method to analyze the turbidity of phospholipid vesicle solutions reacted with black tea infusions or components as an indicator of the molecular interaction. Using a portable visible spectrophotometer, the dose-dependency of catechins, theaflavins, thearubigins, and black tea infusions on turbidity was analyzed. The results indicate that authentic catechins and theaflavins independently caused aggregation of the phospholipid vesicles; however, their concentrations in the tea infusions were insufficient. Meanwhile, thearubigins prepared from black tea leaves caused aggregation at the concentration found in the tea infusions. It is concluded that thearubigins, instead of theaflavins, mainly contribute to the interaction with phospholipid vesicles.

An ultrastructural and genomic study on the SARS-CoV-2 variant B.1.210 circulating during the first wave of COVID-19 pandemic in India

PurposeThe study aims to isolate and understand cytopathogenesis, ultrastructure, genomic characteristics and phylogenetic analysis of SARS-CoV-2 virus of B.1.210 lineage, that circulated in India during first wave of the pandemic. MethodsClinical specimen from an interstate traveller from Maharashtra to Karnataka, in May 2020, who was positive by RT PCR for SARS-CoV-2 infection was subjected to virus isolation and Whole Genome Sequencing. Vero cells were used to study cytopathogenesis and ultrastructural features by Transmission Electron Microscopy (TEM). Phylogenetic analysis of the whole genome sequences of several SARS-CoV-2 variants downloaded from GISAID was performed in comparison with the B.1.210 variant identified in this study. ResultsThe virus was isolated in Vero cells and identified by immunofluorescence assay and RT PCR. The growth kinetics in infected Vero cells revealed a peak viral titre at 24 ​h post-infection. Ultrastructural studies revealed distinct morphological changes with accumulation of membrane-bound vesicles containing pleomorphic virions in the cytoplasm, with single or multiple intranuclear filamentous inclusions and dilated rough endoplasmic reticulum with viral particles. Whole genome sequence of the clinical specimen as well as the isolated virus revealed the virus to be of lineage B.1.210 with the D614G mutation in the spike protein. Phylogenetic analysis of the whole genome sequence in comparison with other variants reported globally revealed that the isolated SARS-CoV-2 virus of lineage B.1.210 is closely related to the original Wuhan virus reference sequence. ConclusionsThe SARS-CoV-2 variant B.1.210 virus isolated here showed ultrastructural features and cytopathogenesis similar to that of the virus reported during early phase of pandemic. Phylogenetic analysis showed that the isolated virus is closely related to the original Wuhan virus, thereby suggesting that the SARS-CoV-2 lineage B.1.210 that was circulating in India during the early phase of pandemic is likely to have evolved from the original Wuhan strain.

Modeling of a Novel Patient-Based MYO5B Point Mutation Reveals Insights Into MVID Pathogenesis

Modeling of a Novel Patient-Based MYO5B Point Mutation Reveals Insights Into MVID Pathogenesis

Axonal plasticity in response to active forces generated through magnetic nano-pulling

Mechanical force is crucial in guiding axon outgrowth before and after synapse formation. This process is referred to as "stretch growth." However, how neurons transduce mechanical input into signaling pathways remains poorly understood. Another open question is how stretch growth is coupled in time with the intercalated addition of new mass along the entire axon. Here, we demonstrate that active mechanical force generated by magnetic nano-pulling induces remodeling of the axonal cytoskeleton. Specifically, the increase in the axonal density of microtubules induced by nano-pulling leads to an accumulation of organelles and signaling vesicles, which, in turn, promotes local translation by increasing the probability of assembly of the "translation factories." Modulation of axonal transport and local translation sustains enhanced axon outgrowth and synapse maturation.

Manipulation of PD-L1 Endosomal Trafficking Promotes Anticancer Immunity.

The aberrant regulation of PD-L1 in tumor cells remains poorly understood. Here, the authorssystematically investigate the endosomal trafficking of plasma membrane PD-L1 in tumor cells.They show that plasma membrane PD-L1 is continuously internalized, and then trafficked from early endosomes to multivesicular bodies/late endosomes, recycling endosomes, lysosomes, and/or extracellular vesicles (EVs). This constitutive endocytic trafficking of PD-L1 is Rab5- and clathrin-dependent. Triazine compound 6J1 blocks the endosomal trafficking of PD-L1 and induces its accumulation in endocytic vesicles by activating Rab5. 6J1 also promotes exosomal PD-L1 secretion by activating Rab27. Together, these effects result in a decrease in the membrane level of PD-L1 in 6J1-treated tumor cells and enables tumor cells to be more susceptible to the tumor-killing activity of T cells in vitro. 6J1 also increases tumor-infiltrating cytotoxic T cells and promotes chemokines secretion in the tumor microenvironment. Rab27 knockdown abolishes 6J1-induced PD-L1 secretion in EVs and revokes the exhausted tumor-infiltrating T cells in tumors, thereby improving the anticancer efficacy of 6J1. Furthermore, a combination of 6J1 and an anti-PD-1 antibody significantly improves the anticancer immune response. Therefore, manipulating PD-L1 endosomal trafficking provides a promising means to promote an anticancer immune response in addition to the immune checkpoint-blocking antibody therapy.

ZmBET5L1 inhibits primary root growth and decreases osmotic stress tolerance by mediating vesicle aggregation and tethering in maize.

Improving osmotic stress tolerance is critical to help crops to thrive and maintain high yields in adverse environments. Here, we characterized a core subunit of the transport protein particle (TRAPP) complex, ZmBET5L1, in maize using knowledge-driven data mining and genome editing. We found that ZmBET5L1 can interact with TRAPP I complex subunits and act as a tethering factor to mediate vesicle aggregation and targeting from the endoplasmic reticulum to the Golgi apparatus. ZmBET5L1 knock-out increased the primary root elongation rate under 20% polyethylene glycol-simulated osmotic stress and the survival rate under drought stress compared to wild-type seedlings. In addition, we found that ZmBET5L1 moderates PIN1 polar localization and auxin flow to maintain normal root growth. ZmBET5L1 knock-out optimized auxin flow to the lateral side of the root and promoted its growth to generate a robust root, which may be related to improved osmotic stress tolerance. Together, these findings demonstrate that ZmBET5L1 inhibits primary root growth and decreases osmotic stress tolerance by regulating vesicle transport and auxin distribution. This study has improved our understanding of the role of tethering factors in response to abiotic stresses and identified desirable variants for breeding osmotic stress tolerance in maize.