Large Vacuoles Research Articles

Signet Ring Cell Esophageal Adenocarcinoma: A Rare Histologic Variant

A 68-year-old man with a history of heavy smoking presented with 3 weeks of dysphagia to solids. Endoscopy revealed erythema in the lower esophagus with scattered white plaques covering umbilicated nodules (Figure A); on gastric retroflexion, these lesions extended beyond the esophagogastric junction (Figure B). Infectious esophagitis, specifically esophageal candidiasis or herpes esophagitis, was suspected, but surprisingly the biopsies revealed squamous mucosa with infiltrating adenocarcinoma containing copious signet ring cells (SRC) (Figure C: low [left] and high [right] magnification; arrows indicate SRC with large intracellular mucin-containing vacuoles displacing nuclei to the cell periphery). SRC comprised >50% of the tumor mass. Immunostaining detected overexpressed programmed death-ligand-1 (PD-L1) but not human epidermal growth factor receptor-2 (HER-2). Positron emission tomography/computed tomography revealed distal esophageal wall thickening with mild metabolic uptake but no enlarged, metabolically active lymph nodes. Endoscopic-ultrasonography revealed an irregular circumferential mucosal mass extending 36 cm from the incisors to 1 cm distal to the esophagogastric junction, invading the adventitia but not regional lymph nodes (Figure D). The cancer progressed despite neoadjuvant chemoradiotherapy, esophagectomy, and adjuvant immunotherapy; the patient died 8 months after presentation. SRC adenocarcinoma accounts for <10% of esophageal adenocarcinomas and portends a worse outcome. Clearly, the presence of esophageal skip lesions should not exclude the possibility of neoplasia. The prevalence and prognostic value of PD-L1 and HER-2 overexpression in esophageal SRC adenocarcinoma are unknown.

Vampyrophrya pelagica (Chatton and Lwoff 1930), an apostome ciliate infectious on calanoid copepod from the Northeastern Arabian Sea

BackgroundZooplankton provides a dynamic habitat to various kinds of epibionts in the marine ecosystem and also forms a source of food and nutrients to various parasites and parasitoids. The study was conducted to identify the association of ciliates with calanoid copepod of Northeastern Arabian Sea. Zooplankton samples collected were analysed under stereomicroscope for any kind of ciliate attachments. The infective stage and major characteristics of the ciliates were discussed. The present observation is the first report of apostome ciliate Vampyrophrya pelagica on calanoid copepod Bestiolina similis from the open ocean waters off Tarapur, Northeastern Arabian Sea, during the early spring intermonsoon season and also a new host record for the ciliate.ResultsAnalysis of ~ 2530 copepod revealed the presence of 12 copepod specimens infested with apostome ciliate Vampyrophrya pelagica. All the host copepod, identified as Bestiolina similis, were females. Approximately 50–60 ciliates occupied the host endocuticular region. V. pelagica was represented by fully grown trophont stage characterized by the presence of a large food vacuole surrounded by a thin layer of cytoplasm with numerous lipid bodies.ConclusionThe study establishes a new host record, calanoid copepod B. similis for V. pelagica. The report is the first of its kind from the Indian waters and extends the biogeographic boundary of the apostome ciliate.

RETRACTED: Histological, ultrastructural and immunohistochemical studies on the ameliorative role of Cinnamon zeylanicum against high cholesterol diet-induced hypercholesterolemia in the kidney of adult male albino rats

Cholesterol is an important type of lipid as it is involved in the structure of cell membrane, synthesis of steroid hormones, bile acid and vitamin D. Many diseases affect various mammalian organs, including the kidney, as a result of high cholesterol levels (hypercholesterolemia). Cinnamon zeylanicum (C. zeylanicum) proves its efficiency as it has anti-inflammatory and antioxidant prosperities. This study aimed to investigate the possible ameliorative role of C. zeylanicum on hypercholesterolemia-induced the renal toxicity in albino rats. Forty adult male albino rats were equally divided into four groups. The first group served as the control one. The second group was supplemented with C. zeylanicum powder (15% w/w) with the standard diet. The third group was fed high cholesterol diet (HCD) to induce acute hypercholesterolemia. The fourth group was fed HCD provided with C. zeylanicum powder (15% w/w). At the end of the experiment (8th weeks), kidneys were removed and prepared for histological, immunohistochemical and ultrastructure studies. Rats-fed HCD showed degenerated glomeruli and tubular cells with vacuolated or coagulated cytoplasm and pyknotic nuclei. Moreover, the renal cortex ultrastructural examination showed degenerated podocytes, parietal and mesangial cells, as well as the proximal and distal tubular cells appeared with rarified cytoplasm, degenerated mitochondria, large fat vacuoles and complete damaged microvilli. The same group showed a significant increase in the expression of desmin and inducible nitric oxide synthase. On the other hand, animals fed HCD provided with C. zeylanicum showed an obvious improvement in the observed histological, ultrastructural and immunohistochemical changes. The architecture of the renal cortex appeared mostly similar to the control one. This study concluded that C. zeylanicum has a promising role in treating the nephron-toxicity of HCD due to its natural constituent that is responsible for its pharmaceutical effects.

Гістологічна характеристика печінки плітки (Rutilus rutilus Linnaeus, 1758) як біоіндикатора якості водного середовища

Purpose. To assess the variability of the histological parameters of the medial zone of roach (Rutilus rutilus) liver. To establish the level of effect of the transformed conditions of existence on fish physiological status. To determine the prospect of using histological monitoring data in ichthyological practice to assess the morphofunctional state of fish. Methodology. Primary materials were obtained in different parts of the Southern Bug River, which differ in their degrees of pollution. Lab processing of ichthyological samples was performed in accordance with generally recognized methods and manuals. Histological studies were carried out using proprietary equipment and original techniques specifically designed for diagnosing fish tissues. The obtained results were processed by the methods of variation statistics using MS Excel with an emphasis on the errors of the average values. Findings. Microanatomical studies of roach hepatic parenchyma allowed establishing that the most significant changes occured in the transformed conditions of existence and were contrastingly recorded in the medial zone of the organ, which could conditionally be considered a bioindicative zone. With the potential effect of toxicants dissolved in water, a shift in cell function was observed in the liver. Visually, this was manifested as a change in the structuring of cytoplasmic inclusions and in the intensity of nucleus staining, which simultaneously occured with a direct shift in cytological characteristics. An 1.20-fold increase in cell area adequately leads to an 1.18-fold increase in the cytoplasm area, as well as the Hertwig constant or nuclear-cytoplasmic ratio (0,25-0,28), which was statistically significant. The obtained data indicate a change in the nature of intracellular metabolism. An increase in the number of intracellular cavities is a direct evidence of the attenuation of the glycogen-accumulative function of hepatocytes. An increase in the nucleolus/nucleus diameter ratio (0.28) indicates a change in cellular function, which occurs through the activation of lipid synthesis. No typical necrosis foci were found in the parenchyma of the liver of fish from the places of concentration of commercial effluents, which are an evidence of a moderate accumulation of fat-soluble pollutants in the liver. The absence of pronounced pathologies in most individuals indicates significant reserve-compensatory potencies of the organ. The presence of individual hepatocytes with two nucleoli in the nuclei in the liver parenchyma allows judging the borderline, i.e. the prepathological state of the organ. In some cases, an increase in the proportion of dystrophic hepatocytes with large vacuoles filled with an optically transparent liquid was established, which allowed identifying an irreversible pathology - liver balloon dystrophy, resulting from a severe impairment of cellular metabolism. Histological analysis of roach liver parenchyma from conditionally clean and polluted waters allowed, in parallel, establishing relatively low levels of intraspecific and individual variability of cell populations. The revealed fact testifies to the plasticity of the liver as an organ with significant reserve potencies, which are contrastingly manifested under conditions of technogenic pressure in waters of various origins and intended purposes. This perspective provides evidence for the evolutionary definition of the liver as the body’s universal detoxification system. Originality. New data are presented, as well as existing data on the normal and altered histological structure of the medial zone of the liver of roach, which live under transformed conditions of existence, were significantly updated. For the first time, a comparative histological and cytological analysis of the structure of the medial zone of the liver of age-1+ roach was carried out. Based on a comparative analysis, the information on the taxonomic features of compensatory changes in liver tissue components in polluted water areas was renewed and updated. Practical walue. The information obtained is of value in morphophysiological studies, as well as in ichthyological practice in connection with the modern problem of lowering the vital status of fish populations, which exists in conditions of degraded aquatic ecosystems. The use of the obtained data allows qualitative assessment of the physiological status of fish in order to predict environmental hazards in water areas of different origin and purpose. Key words: roach, parenchyma, liver, hepatocyte, bioindication, cellular function, water area.

IKKγ/NEMO Localization into Multivesicular Bodies

The NF-κB pathway is central pathway for inflammatory and immune responses, and IKKγ/NEMO is essential for NF-κB activation. In a previous report, we identified the role of glycogen synthase kinase-3β (GSK-3β) in NF-κB activation by regulating IKKγ/NEMO. Here, we show that NEMO phosphorylation by GSK-3β leads to NEMO localization into multivesicular bodies (MVBs). Using the endosome marker Rab5, we observed localization into endosomes. Using siRNA, we identified the AAA-ATPase Vps4A, which is involved in recycling the ESCRT machinery by facilitating its dissociation from endosomal membranes, which is necessary for NEMO stability and NF-κB activation. Co-immunoprecipitation studies of NEMO and mutated NEMO demonstrated its direct interaction with Vps4A, which requires NEMO phosphorylation. The transfection of cells by a mutated and constitutively active form of Vps4A, Vps4A-E233Q, resulted in the formation of large vacuoles and strong augmentation in NEMO expression compared to GFP-Vps4-WT. In addition, the overexpression of the mutated form of Vps4A led to increased NF-κB activation. The treatment of cells with the pharmacologic V-ATPase inhibitor bafilomycin A led to a dramatic downregulation of NEMO and, in this way, inhibited NF-κB signal transduction. These results reveal an unexpected role for GSK-3β and V-ATPase in NF-κB signaling activation.

Morphological and Immunophenotypical Changes of Human Bone Marrow Adipocytes in Marrow Metastasis and Myelofibrosis.

The bone marrow adipose tissue constitutes more than two-thirds of the bone marrow volume in adult life and is known to have unique metabolic and functional properties. In neoplastic disorders, bone marrow adipocytes (BMAds) contribute to create a favorable microenvironment to survival and proliferation of cancer cells. Many studies explored the molecular crosstalk between BMAds and neoplastic cells, predominantly in ex-vivo experimental systems or in animal models. However, little is known on the features of BMAds in the human neoplastic marrow. The aim of our study was to analyze the in situ changes in morphology and immunophenotype of BMAds in two different types of neoplastic marrow conditions. We selected a series of archival iliac crest and vertebral bone biopsies from patients with bone marrow metastasis (MET), patients with myeloproliferative neoplasia with grade-3 myelofibrosis (MPN-MF) and age-matched controls (CTR). We observed a significant reduction in the number of BMAds in MET and MPN-MF compared to CTR. Accordingly, in the same groups, we also detected a significant reduction in the mean cell diameter and area. Immunolocalization of different adipocyte markers showed that, compared to CTR, in both MET and MPN-MF the percentages of adiponectin- and phosphorylated hormone sensitive lipase-positive BMAds were significantly reduced and increased respectively. No statistically significant difference was found between MET and MPN-MF. Interestingly, in one MET sample, “remodeled” BMAds containing a large lipid vacuole and multiple, smaller and polarized lipid droplets were identified. In conclusion, our data show that in different types of marrow cancers, BMAds undergo significant quantitative and qualitative changes, which need to be further investigated in future studies.

A Satellite dsRNA Attenuates the Induction of Helper Virus-Mediated Symptoms in Aspergillus flavus.

Aspergillus flavus is an important fungal pathogen of animals and plants. Previously, we reported a novel partitivirus, Aspergillus flavus partitivirus 1 (AfPV1), infecting A. flavus. In this study, we obtained a small double-stranded (ds) RNA segment (734 bp), which is a satellite RNA of the helper virus, AfPV1. The presence of AfPV1 altered the colony morphology, decreased the number of conidiophores, created significantly larger vacuoles, and caused more sensitivity to osmotic, oxidative, and UV stresses in A. flavus, but the small RNA segment could attenuate the above symptoms caused by the helper virus AfPV1 in A. flavus. Moreover, AfPV1 infection reduced the pathogenicity of A. flavus in corn (Zea mays), honeycomb moth (Galleria mellonella), mice (Mus musculus), and the adhesion of conidia to host epithelial cells, and increased conidial death by macrophages. However, the small RNA segment could also attenuate the above symptoms caused by the helper virus AfPV1 in A. flavus, perhaps by reducing the genomic accumulation of the helper virus AfPV1 in A. flavus. We used this model to investigate transcriptional genes regulated by AfPV1 and the small RNA segment in A. flavus, and their role in generating different phenotypes. We found that the pathways of the genes regulated by AfPV1 in its host were similar to those of retroviral viruses. Therefore, some pathways may be of benefit to non-retroviral viral integration or endogenization into the genomes of its host. Moreover, some potential antiviral substances were also found in A. flavus using this system.

Human Sperm Morphology as a Marker of Its Nuclear Quality and Epigenetic Pattern.

Background: Human sperm chromatin condensation is a sum of epigenetic events that allows for the near-complete replacement of histones with protamines. Under high-magnification microscopy, nuclear vacuoles have been described as thumbprints with poor chromatin condensation. The objective of this study is to examine whether vacuolated spermatozoa carry specific epigenetic marks, which may influence embryo development. Methods: The presence and three-dimensional distribution of ten epigenetic marks (protamine-P2, histone-H3, H3K4me1/me2/me3, H3K9me1/me2/me3, H3K27me3, H4k20me2) were evaluated and compared in morphometrically normal spermatozoa according to the presence or absence of a large vacuole occupying more than 15% of the head surface (n = 4193). Results: Vacuolated spermatozoa were significantly more frequently labelled with H3 and H3K4me3 than normal spermatozoa (88.1% ± 2.7 and 78.5% ± 5.2 vs. 74.8% ± 4.8 and 49.1% ± 7.4, respectively; p = 0.009 and p < 0.001) and significantly less marked by P2 and H3K27me3 (50.2% ± 6.2 and 63.9% ± 6.3 vs. 82.1% ± 4.4 and 73.6% ± 5.1, respectively; p < 0.001 and p = 0.028). In three dimensions, vacuoles are nuclear concavities filled with DNA carrying the H3K4me3 marker. Conclusion: High-magnification microscopy is a simple tool to estimate in real time the sperm epigenetic profile. The selection of normal spermatozoa without vacuoles and the deselection of spermatozoa with vacuoles appear to be epigenetically favorable to embryo development and safe offspring.

Proton-gated anion transport governs macropinosome shrinkage

Intracellular organelles change their size during trafficking and maturation. This requires the transport of ions and water across their membranes. Macropinocytosis, a ubiquitous form of endocytosis of particular importance for immune and cancer cells, generates large vacuoles that can be followed optically. Shrinkage of macrophage macropinosomes depends on TPC-mediated Na+ efflux and Cl− exit through unknown channels. Relieving osmotic pressure facilitates vesicle budding, positioning osmotic shrinkage upstream of vesicular sorting and trafficking. Here we identify the missing macrophage Cl− channel as the proton-activated Cl− channel ASOR/TMEM206. ASOR activation requires Na+-mediated depolarization and luminal acidification by redundant transporters including H+-ATPases and CLC 2Cl−/H+ exchangers. As corroborated by mathematical modelling, feedback loops requiring the steep voltage and pH dependencies of ASOR and CLCs render vacuole resolution resilient towards transporter copy numbers. TMEM206 disruption increased albumin-dependent survival of cancer cells. Our work suggests a function for the voltage and pH dependence of ASOR and CLCs, provides a comprehensive model for ion-transport-dependent vacuole maturation and reveals biological roles of ASOR.

Bacillus-Based Products to Control Meloidogyne incognita Races 3 and 4 in Cotton and Compared Histopathology Using B. methylotrophicus

Cotton meloidoginosis caused by Meloidogyne incognita races 3 and 4 is an important disease and can cause intense damage. The objective of this work was to test the effectiveness of commercial Bacillus-based products in the control of M. incognita races 3 and 4 of cotton in a greenhouse. Plants with and without nematode inoculation were used and subsequently evaluated for 200 days after the application of treatments. The products with Bacillus subtilis, B. amyloliquefasciens and B. subtilis + B. lincheniformis showed the best results in terms of shoot fresh matter weight. In relation to shoot dry weight, treatments with B. methylotrophicus (87 g, with nematode) and B. subtilis (80 g, with nematode) were superior, with emphasis on B. methylotrophicus. In the evaluation of root fresh weight, only the treatment with B. methylotrophicus (148.8 g, with nematode) provided statistically higher weight than the control. In item dry weight of root without nematode and nematode reproduction factor, the treatment with B. methylotrophicus stood out from the other treatments, making this the selected product to conduct the subsequent tests. With the acid fuchsin staining method, it was possible to verify that there was reduction in the penetration of J2 in the first days for plants treated with the bacteria. Upon adoption of the toluidine blue staining method, it was possible to observe abnormalities in giant cells with formation of vacuoles, thinner cell wall and females with large vacuoles inside. Thus, there is evidence that the use of biological products can be effective in controlling M. incognita.

Toxic form of steatosis in dairy cows (clinical, hematological and histological data)

Steatosis is a common disease in dairy cattle, especially during the transit period. Usually, the subclinical form of hepatosis is evenly distributed in the population throughout the year: 30‐70% of cows can be affected. However, severe (toxic) steatosis is rare. The study was carried out on 505 dairy cows in the North‐West of Russia in July‐August. In the blood serum, the concentrations of the main substances were investigated, and the morphological examination of the blood was carried out. Liver samples for histological examination were collected posthumously. 47% of cows had subclinical hepatopathy, 1.78% (9 cows) ‐ a toxic form of steatosis. Clinical signs of severe hepatopathy were observed from 1 to 10 days after calving: anorexia, icterus, ventroflexia, excitability, convulsions, exophthalmos. In the blood: total protein 59.8±0.9 g/l, albumin ‐ 35.1±2.5%, total bilirubin ‐ 23.8±0.8 μmol/l, triglycerides ‐ 0.329±0.01 mmol/l, cholesterol ‐ 8.7±1.0 mmol/l, RBC 3.9±0.1*1012/l, PLT ‐ 190.1±39.8*109/l, WBC ‐ 8.36±0.75*109/l, HCT – 29.1±0.5%. Autopsy: icterus of the mucous membranes, petechiae in the kidneys, intestines, brain membrane, injection of blood vessels, enlargement of the liver and kidneys, the liver capsule breaks easily, the parenchyma is loose. Liver’s parenchyma imprint smears: staining with Saathof solution ‐ much neutral fat. Rumen: detachment of the mucous membrane. Liver: large and small vacuole diffuse fatty infiltration of hepatocytes, thickening of the portal tracts with expressed mononuclear infiltration. The liver has an ordered lobular structure, is moderately full‐blooded, the sinusoids of the liver and the Disse spaces are moderately dilated, staining with Schiff‐iodic acid according to McManus ‐ the absence of glycogen in hepatocytes. Kidneys: hyaline‐drop dystrophy of the epithelium of the renal tubules, fibrosing interstitial glomerulonephritis. Rumen: the mucous membrane forms convex structures of the main plate of various sizes and shapes, covered with multilayer squamous epithelium; there are areas of degeneration. The muscle plate was formed by separate bundles of smooth myocytes lying at the base of the papillae; individual muscle cells were found in the main plate of the papilla. The plate consisted of an inner annular and an outer longitudinal layer of smooth muscle cells, among which there were individual fibers of striated muscle tissue. The serous membrane of the rumen had a typical structure; it was represented by loose connective tissue covered with mesothelium. According to the results of this study, it was revealed that the toxic form of hepatosis is accompanied by multiple organ failure: an expressed violation of the nervous system, hypoprotein‐, dysprotein‐, hyperbilirubin‐ and anemia, large and small‐drop fatty infiltration of hepatocytes, glomerulonephritis, lesions of the rumen and intestinal wall.

Comparative Analysis of the Metabolome and Transcriptome between the Green and Yellow-Green Regions of Variegated Leaves in a Mutant Variety of the Tree Species Pteroceltis tatarinowii.

In nature, many different factors cause plants to develop variegated leaves. To explore the mechanism of variegated leaf formation in Pteroceltis tatarinowii, a mutant variety (‘Jinyuyuan’), which was induced by ethylmethylsulfone, was selected, and its morphological structure, physiology, biochemistry, transcription and metabolism were analysed. According to differences in colour values, the colours were divided into two regions: a green region and a yellow-green region. The chlorophyll content of the two regions was significantly different. Moreover, the yellow-green regions of the leaves were significantly thinner than the green regions. The chloroplast ultrastructure in the yellow-green region revealed small chloroplasts, large vacuoles, small starch grains, obviously increased numbers of osmophilic grains, loose lamellae of the inner capsule and thin lamellae. Moreover, the yellow-green region was accompanied by oxidative stress, and the activity of the oxidative phosphorylation pathway related to oxidative activity in the transcriptome showed an upward trend. Vitamin B6 and proline contents also increased, indicating that the antioxidant activity of cells in the yellow-green region increased. Transcriptomic and metabolomic analysis showed that the differentially expressed genes (DEGs) related to chlorophyll synthesis and metabolism led to a decrease in the photosynthesis and then a decrease in the assimilation ability and contents of sucrose, starch and other assimilates. Amino acid synthesis and metabolism, lipid synthesis and the activity of metabolic pathways were obviously downregulated, and the contents of differentially accumulated metabolites associated with amino acids and lipids were also reduced. At the same time, 31 out of 32 DEGs involved in the flavonoid synthesis pathway were downregulated, which affected leaf colour. We hypothesized that the variegated leaves of P. tatarinowii ‘Jinyuyuan’ are caused by transcriptional and post-transcriptional regulation. Mutations in pigment and flavonoid synthesis pathway genes and transcription factor genes directly affect both pigment and flavonoid synthesis and degradation rate, which in turn affect carbon assimilation, carbon fixation, related protein synthesis and enzyme activity, lipid synthesis and degradation and the activity of other metabolic pathways, eventually leading to the formation of different colour regions.

A Clinical Analysis and Literature Review of Six Cases with Primary Pulmonary Lymphoepithelioma-Like Carcinoma.

Methods The clinical data of six patients with primary pulmonary lymphoepithelioma-like carcinoma treated in Zhejiang Taizhou Hospital of Taizhou Enze Medical Center (Group) from May 2014 to December 2018 were summarized and analyzed. Combined with the relevant literature, the primary pulmonary lymphoepithelioma-like carcinoma was analyzed retrospectively. Results The main manifestations of six patients were respiratory symptoms, and cough was the most common. The imaging features of six patients were mainly round-like high-density mass shadow or nodule shadow. All patients were diagnosed by pathology. Microscopically, the cancer cells were nested, with large nuclei and vacuoles and abundant lymphocyte infiltration in the tumor stroma. The positive rates of EBER, p63, CK5/6, and Ki-67 were high, and TTF-1 was negative. Five patients received surgical treatment. One patient developed brain metastasis 12 months after operation and received craniocerebral radiotherapy. The other patients did not receive radiotherapy and chemotherapy, and one patient did not receive treatment. After follow-up, four patients survived so far, the longest survival time was 82 months, one patient lost follow-up, and one patient died of lung metastasis 24 months after operation. Conclusion Primary pulmonary lymphoepitheliomatoid-like carcinoma is a rare lung malignant tumor, whose pathogenesis is related to Epstein-Barr virus infection. The clinical manifestations are nonspecific, but with unique pathological characteristics. Surgical resection is the proper treatment for early-stage patients, and comprehensive treatment with surgery as the main treatment is suitable for late-stage patients. The prognosis is good.

Morphogenesis, ultrastructure, and chemical profiling of trichomes in Artemisia argyi H. Lév. &amp; Vaniot (Asteraceae)

Glandular trichomes of Artemisia argyi H. Lév. & Vaniot are the key tissues for the production of flavonoid and terpenoid metabolites. Artemisia argyi H. Lév. & Vaniot is an herbaceous perennial plant that has been widely used in traditional medicine for thousands of years. Glandular trichomes (GTs) and nonglandular trichomes (NGTs) have been reported on the leaf surface of A. argyi. The aim of this study was to elucidate the morphogenetic process and to analyze the metabolites of trichomes in A. argyi. The morphogenesis of GTs and NGTs was characterized using light, scanning, and transmission electron microscopy. The constituents of GTs were analyzed using laser microdissection combined with gas and liquid chromatography-mass spectrometry. Five developmental stages of two types of GTs and four developmental stages of one type of NGTs were observed. Two types of mature GT and one type of NGT were composed of 10, 5, and 4-6 cells, respectively. A large storage cavity was detected between the cuticle and cell walls in the first type of mature GT. Large nuclei, nucleoli, and mitochondria were observed in the basal and intermediate cells of the second type of GT. In addition, large vacuoles were observed in the basal and apical cells, and large nuclei were observed in the middle cells of NGTs. One monoterpene and seven flavonoids were identified in GTs of A. argyi. We suggest that GTs are the key tissues for the production of bioactive metabolites in A. argyi. This study provides an important theoretical basis and technical approach for clarifying the regulatory mechanisms for trichome development and bioactive metabolite biosynthesis in A. argyi.

A Kinesin Vdkin2 Required for Vacuole Formation, Mycelium Growth, and Penetration Structure Formation of Verticillium dahliae.

The soil-borne vascular fungus Verticillium dahliae infects hundreds of dicotyledonous plants, causing severe wilt diseases. During the initial colonization, V. dahliae develops a penetration peg to enable infection of cotton roots. In some phytopathogenic fungi, vacuoles play a critical role in normal formation of the infection structure. Kinesin 2 protein is associated with vacuole formation in Ustilago maydis. To identify the function of vacuoles in the V. dahliae infection structure, we identified VdKin2, an ortholog of kinesin 2, in V. dahliae and investigated its function through gene knockout. VdKin2 mutants showed severe defects in virulence and were suppressed during initial infection and root colonization based on observation of green fluorescent protein-labeled V. dahliae. We also found that deletion of VdKin2 compromised penetration peg formation and the derived septin neck. Disruption strains were viable and showed normal microsclerotia formation, whereas mycelium growth and conidial production were reduced, with shorter and more branched hyphae. Furthermore, the VdKin2 mutant, unlike wild-type V. dahliae, lacked a large basal vacuole, accompanied by a failure to generate concentrated lipid droplets. Taken together, VdKin2 regulates vacuole formation by V. dahliae, which is required for conidiation, mycelium growth, and penetration structure formation during initial plant root infection.

HEMATOLOGICAL AND HISTOLOGICAL STUDY OF ALUMINIUM TOXICITY ON ALBINO RAT

This investigation was carried out to observe the toxic effect of aluminium chloride on selected tissues and the blood of the albino rat (Rattus novergicus). Aluminium toxicity is a rare condition which occurs when the aluminium metal accumulates in the body either by direct inhalation or from drinking water contaminated by this metal. Analysis of the kidney, liver and stomach tissues of rats administered increasing doses of the toxicant showed varying degrees of changes such as the presence of patches of blood stains which indicated possibility of haemorrhage, the presence of large vacuoles of triglyceride indicating a possibility of fatty liver disease while the presence of dark spots indicated the presence of dilated and congested blood vessels in these tissues. Analysis of the blood revealed an increase in neutrophil, platelet, MCHC, haemoglobin, pack cell volume, MCV, MCH and MPV PL. However, there was a decrease in red blood cell, lymphocyte, mixed (eosinophil and basophil), RDW_CV, PDW PL and P_LCR PL all of which were statistically significant at p&lt;0.05, showing a negative effect of the toxicant. This study thus confirms the toxicity of aluminium chloride and as such its consumption should be completely discouraged

Eosinophilic vacuolated tumor (EVT) of kidney demonstrates sporadic TSC/MTOR mutations: next-generation sequencing multi-institutional study of 19 cases

A distinct renal tumor has recently been described as “high-grade oncocytic renal tumor” and “sporadic renal cell carcinoma with eosinophilic and vacuolated cytoplasm”. The Genitourinary Pathology Society (GUPS) consensus proposed a unifying name “eosinophilic vacuolated tumor” (EVT) for this emerging entity. In this multi-institutional study, we evaluated 19 EVTs, particularly their molecular features and mutation profile, using next-generation sequencing. All cases were sporadic and none of the patients had a tuberous sclerosis complex. There were 8 men and 11 women, with a mean age of 47 years (median 50; range 15–72 years). Average tumor size was 4.3 cm (median 3.8 cm; range 1.5–11.5 cm). All patients with available follow-up data (18/19) were alive and without evidence of disease recurrence or progression during the follow-up, ranging from 12 to 198 months (mean 56.3, median 41.5 months). The tumors were well circumscribed, but lacked a well-formed capsule, had nested to solid growth, focal tubular architecture, and showed ubiquitous, large intracytoplasmic vacuoles, round to oval nuclei, and prominent nucleoli. Immunohistochemically, cathepsin K, CD117, CD10, and antimitochondrial antigen were expressed in all cases. Other positive stains included: PAX8, AE1/AE3 and CK18. CK7 was typically restricted only to rare scattered cells. Vimentin, HMB45, melan-A, and TFE3 were negative in all cases. All tumors showed retained SDHB. All cases (19/19) showed non-overlapping mutations of the mTOR pathway genes: TSC1 (4), TSC2 (7), and MTOR (8); one case with MTOR mutation showed a coexistent RICTOR missense mutation. Low mutational rates were found in all samples (ranged from 0 to 6 mutations/Mbp). Microsatellite instability and copy number variations were not found in any of the 17 analyzable cases. EVT represents an emerging renal entity that shows a characteristic and readily identifiable morphology, consistent immunohistochemical profile, indolent behavior, and mutations in either TSC1, TSC2, or MTOR genes.

Unraveling the antiviral activity of plitidepsin against SARS-CoV-2 by subcellular and morphological analysis

The pandemic caused by the new coronavirus SARS-CoV-2 has made evident the need for broad-spectrum, efficient antiviral treatments to combat emerging and re-emerging viruses. Plitidepsin is an antitumor agent of marine origin that has also shown a potent pre-clinical efficacy against SARS-CoV-2. Plitidepsin targets the host protein eEF1A (eukaryotic translation elongation factor 1 alpha) and affects viral infection at an early, post-entry step. Because electron microscopy is a valuable tool to study virus-cell interactions and the mechanism of action of antiviral drugs, in this work we have used transmission electron microscopy (TEM) to evaluate the effects of plitidepsin in SARS-CoV-2 infection in cultured Vero E6 cells 24 and 48h post-infection. In the absence of plitidepsin, TEM morphological analysis showed double-membrane vesicles (DMVs), organelles that support coronavirus genome replication, single-membrane vesicles with viral particles, large vacuoles with groups of viruses and numerous extracellular virions attached to the plasma membrane. When treated with plitidepsin, no viral structures were found in SARS-CoV-2-infected Vero E6 cells. Immunogold detection of SARS-CoV-2 nucleocapsid (N) protein and double-stranded RNA (dsRNA) provided clear signals in cells infected in the absence of plitidepsin, but complete absence in cells infected and treated with plitidepsin. The present study shows that plitidepsin blocks the biogenesis of viral replication organelles and the morphogenesis of virus progeny. Electron microscopy morphological analysis coupled to immunogold labeling of SARS-CoV-2 products offers a unique approach to understand how antivirals such as plitidepsin work.

Placental Macrophages Following Maternal SARS-CoV-2 Infection in Relation to Placental Pathology

IntroductionIn December 2019, a novel coronavirus, SARS-CoV-2, was identified. Whilst pregnant women appear to be at risk of severe infection, pre-term birth, and stillbirth, it is unclear whether placental dysfunction is a consistent feature of maternal SARS-CoV-2 infection during pregnancy. We aim to describe the immune response in placentas of women who had COVID-19 infection during pregnancy and investigate whether there are any associated morphological changes.MethodsThe placentas of women testing positive for COVID-19 during their pregnancy were compared to contemporaneous controls who were not known to have had COVID-19 during pregnancy. Samples of each placenta were sent for histopathological analysis or underwent immunohistochemical staining for CD163, CD20, CD3, CD31, and SARS-CoV-2 spike protein. A subset of samples were sent for transmission electron microscopy.ResultsThere was a significant increase in the number of CD163+ macrophages in the Post COVID group (p = 0.0020). There was no difference in the percentage of CD3+, CD20+ cells, but there was an increase in placental vascularity in the Post COVID group compared to controls (p = 0.026).There were no structural differences observed between the samples sent for EM analysis. However, one of the placentas from the Post COVID group was seen to have several large sub-apical vacuoles in the syncytiotrophoblast. We did not observe any virions within the vacuoles and SARS-CoV-2 spike protein staining was negative for the sample. Histopathological investigations indicated that there was no specific placental pathology caused by maternal COVID-19 infection in this cohort of samples.ConclusionsThis study did not confirm previous studies which describe a possible increase in cases of both maternal and fetal vascular malperfusion, and placentitis in women who had COVID-19, which were seen in association with adverse pregnancy outcomes. It remains unclear whether observed abnormalities are caused by maternal infection, or whether maternal infection exacerbates existing placental pathology; understanding why some placentas generate these abnormalities is a key goal.

Causative Agents of American Tegumentary Leishmaniasis Are Able to Infect 3T3-L1 Adipocytes In Vitro

Although macrophages have long been considered key players in the course of Leishmania infections, other non-professional phagocytes have lately been shown to maintain low levels of the parasite in safe intracellular niches. Recently, it was demonstrated that the adipose tissue is capable of harboring Old World L. (L.) infantum in mice. However, there is no evidence of experimental adipocyte infection with New World Leishmania species so far. In addition, it was not known whether adipocytes would be permissive for formation of the unique, large and communal parasitophorous vacuoles that are typical of L. (L.) amazonensis in macrophages. Here we evaluated the ability of L. (L.) amazonensis and L. (V.) braziliensis promastigotes and amastigotes to infect 3T3-L1 fibroblast-derived adipocytes (3T3-Ad) using light and transmission electron microscopy. Our results indicate that amastigotes and promastigotes of both species were capable of infecting and surviving inside pre- and fully differentiated 3T3-Ad for up to 144 h. Importantly, L. (L.) amazonensis amastigotes resided in large communal parasitophorous vacuoles in pre-adipocytes, which appeared to be compressed between large lipid droplets in mature adipocytes. In parallel, individual L. (V.) braziliensis amastigotes were detected in single vacuoles 144 h post-infection. We conclude that 3T3-Ad may constitute an environment that supports low loads of viable parasites perhaps contributing to parasite maintenance, since amastigotes of both species recovered from these cells differentiated into replicative promastigotes. Our findings shed light on the potential of a new host cell model that can be relevant to the persistence of New World Leishmania species.