Changes In Cellular Morphology Research Articles

Inflammatory and deleterious role of gut microbiota-derived trimethylamine on colon cells.

Trimethylamine (TMA) is produced by the intestinal microbiota as a by-product of metabolism of dietary precursors. TMA has been implicated in various chronic health conditions. However, the effect of TMA in the colon and the underlying mechanism was not clear. In this study, TMA exhibited toxic effects in vitro as well as in vivo. TMA-induced oxidative stress causes DNA damage, and compromised cell membrane integrity leading to the release of LDH outside the cells which ultimately leads to cell death. Besides, TMA also exhibited pronounced increase in cell cycle arrest at G2/M phase in both HCT116 and HT29 cell lines. TMA was found to be genotoxic and cytotoxic as the TMA concentration increased from 0.15 mM. A decreased ATP intracellular content was observed after 24h, 48h, and 72h treatment in a time and dose-dependent manner. For in vivo research, TMA (100 mM, i.p. and intra-rectal) once a week for 12 weeks caused significant changes in cellular morphology of colon and rectum epithelium as assessed by H & E staining. TMA also significantly increased the infiltration of inflammatory cells in the colon and rectal epithelium indicating the severity of inflammation. In addition, TMA caused extensive mucosal damage and distortion in the epithelium, decrease in length of small intestine compared to control mice. In conclusion, these results highlight the detrimental effects of TMA in the colon and rectal epithelium.

Silencing FHL2 inhibits bleomycin-induced pulmonary fibrosis through the TGF-β1/Smad signaling pathway

ObjectiveThis study aimed to investigate the inhibiting effects of FHL2 and Arbutin on cell fibrosis and their possible mechanisms. MethodsThe mRNA expression of FHL2 in pulmonary fibrosis tissues was analyzed by bioinformatics. TGF⁃β1 induced fibrosis of mouse lung fibroblast (Mlg) and mouse primary pulmonary fibroblast (PPF) in rat's lung fibroblasts. FHL2 siRNA was transfected into Mlg and mouse PPF cells to inhibit FHL2. FHL2, α-smooth muscle actin (α-SMA), collagen 1 (Col I), and Fibronectin (Fn) were detected by qRT-PCR. Western blot expression levels of Smad3, p-Smad3, Smad2, and p-Smad2 proteins in cells. High-throughput drug screening for FHL2 inhibitors and the inhibitory effect of Arbutin on pulmonary fibrosis were validated in cellular and animal models of pulmonary fibrosis. ResultsThe mRNA expression of FHL2 in lung fiber tissue was increased. Meanwhile, the decrease of FHL2 expression significantly inhibited the cellular fibrosis morphological changes of rat's lung fibroblasts (Mlgs) and primary lung fibroblasts (PPFs). The expression levels of α⁃SMA, Col I, and Fn were decreased. High-throughput screening showed that Arbutin targeted FHL2. Arbutin alleviated bleomycin (BLM)-induced pulmonary fibrosis in rats by inhibiting FHL2 and then the TGF-β1/Smad signaling pathway. ConclusionInhibition of FHL2 can effectively reduce the fibrosis process induced by TGF⁃β1 and bleomycin, and then inhibit the fibrosis.

Pituitary Stem Cell Regulation by Zeb2 and BMP Signaling.

Epithelial to mesenchymal transition (EMT) is important for many developing organs, and for wound healing, fibrosis, and cancer. Pituitary stem cells undergo an EMT-like process as they migrate and initiate differentiation, but little is known about the input of signaling pathways or the genetic hierarchy of the transcriptional cascade. Prop1 mutant stem cells fail to undergo changes in cellular morphology, migration, and transition to the Pou1f1 lineage. We used Prop1 mutant mice to identify the changes in gene expression that are affiliated with EMT-like processes. BMP and TGF-β family gene expression was reduced in Prop1 mutants and Elf5, a transcription factor that characteristically suppresses EMT, had elevated expression. Genes involved in cell-cell contact such as cadherins and claudins were elevated in Prop1 mutants. To establish the genetic hierarchy of control, we manipulated gene expression in pituitary stem cell colonies. We determined that the EMT inducer, Zeb2, is necessary for robust BMP signaling and repression of Elf5. We demonstrated that inhibition of BMP signaling affects expression of target genes in the Id family, but it does not affect expression of other EMT genes. Zeb2 is necessary for expression of the SHH effector gene Gli2. However, knock down of Gli2 has little effect on the EMT-related genes, suggesting that it acts through a separate pathway. Thus, we have established the genetic hierarchy involved in the transition of pituitary stem cells to differentiation.

Selective photodynamic effects on cervical adenocarcinoma cells provided by F127 Pluronic®-based micelles modulating hypericin delivery

Cervical cancer is a leading cause of death among women. The endocervical adenocarcinoma (ECA) represents an aggressive and metastatic type of cancer with no effective treatment options currently available. We evaluated the antitumoral and anti-migratory effects of hypericin (HYP) encapsulated on Pluronic F127 (F127/HYP) photodynamic therapy (PDT) against a human cell line derived from invasive cervical adenocarcinoma (HeLa) compared to a human epithelial cell line (HaCaT). The phototoxicity and cytotoxicity of F127/HYP were evaluated by the following assays: colorimetric assay, MTT, cellular morphological changes by microscopy and long-term cytotoxicity by clonogenic assay. In addition, we performed fluorescence microscopy to analyze cell uptake and subcellular distribution of F127/HYP, cell death pathway and reactive oxygen species (ROS) production. The PDT mechanism was determined with sodium azide and D-mannitol and cell migration by wound-healing assay. The treatment with F127/HYP promoted a phototoxic result in the HeLa cells in a dose-dependent and selective form. Internalization of F127/HYP was observed mainly in the mitochondria, causing cell death by necrosis and ROS production especially by the type II PDT mechanism. Furthermore, F127/HYP reduced the long-term proliferation and migration capacity of HeLa cells. Overall, our results indicate a potentially application of F127/HYP micelles as a novel approach for PDT with HYP delivery to more specifically treat ECA.

Live-Cell Labeling and Artificial Intelligence Approaches for High-Resolution XYZT Imaging of Cytoskeletal Dynamics During Collective Cell Migration.

Collective cell migration is crucial for a variety of pathophysiological processes including embryonic development, wound healing, carcinoma invasion, and sprouting angiogenesis. The behavior of leading and following cells during migration is highly dynamic and involves extensive cellular morphological changes mediated by the actin cytoskeleton. Imaging these rapid and dynamic changes over time requires expression of fluorescent proteins and/or live labeling with fluorescent probes, followed by acquiring series of image stacks at short intervals. This presents significant challenges related to dye cytotoxicity, signal loss, and in particular phototoxicity resulting from repeated irradiation, especially when using separate channels for multiple dyes and when imaging large z-stacks at short time intervals. In this chapter, we present methods for multicolor live-cell labeling of primary human endothelial cell populations, followed by multi-position time-lapse imaging in 2D and in 3D protein matrices. These approaches can be performed in combination with RNA interference to suppress the expression of specific proteins, as well as in mosaic assays using mixtures of differentially labeled cell populations. Finally, we present a protocol for long-term imaging at low laser intensity to minimize laser-induced cell damage, followed by post-imaging signal enhancement using artificial intelligence.

Significance of Oct‐4 transcription factor as a pivotal therapeutic target for CD44+/24− mammary tumor initiating cells: Aiming at the root of the recurrence

Breast cancer (BC) remains one of the deadliest and frequently diagnosed metastatic cancers worldwide. Cancer stem cells (CSCs) are the cell population within the tumor niche, having an epithelial to mesenchymal (EMT) transition phenotype, high self-renewal, vigorous metastatic capacity, drug resistance, and tumor relapse. Identification of targets for induction of apoptosis is essential to provide novel therapeutic approaches in BC. Our earlier studies showed that Vitamin C induces apoptotic cell death by losing redox balance in TNBC CSCs. In this study, we have attempted to identify previously unrecognized CSC survival factors that can be used as druggable targets for bCSCs apoptosis regulators isolated from the TNBC line, MDA MB 468. After a thorough literature review, Oct-4 was identified as the most promising marker for its unique abundance in cancer and absence in normal cells and the contribution of Oct-4 to the sustenance of cancer cells. We then validated a very high expression of Oct-4 in the MDA MB 468 bCSCs population using flow-cytometry. The loss of Oct-4 was carried out using small interfering RNA(siRNA)-mediated knockdown in the bCSCs, followed by assessing for cellular apoptosis. Our results indicated that Oct-4 knockdown induced cell death, changes in cellular morphology, inhibited mammosphere formation, and positive for Annexin-V expression, thereby indicating the role of Oct-4 in bCSC survival. Moreover, our findings also suggest the direct interaction between Oct-4 and Vitamin C using in silico docking. This data, hence, contributes towards novel information about Oct-4 highlighting this molecule as a novel survival factor in bCSCs.

Morphometric Changes of Osteocyte Lacunar in Diabetic Pig Mandibular Cancellous Bone.

Osteocytes play an important role in bone metabolism. The interactions of osteocytes with the surrounding microenvironment can alter cellular and lacunar morphological changes. However, objective quantification of osteocyte lacunae is challenging due to their deep location in the bone matrix. This project established a novel method for the analytical study of osteocytes/lacunae, which was then used to evaluate the osteocyte morphological changes in diabetic pig mandibular bone. Eight miniature pigs were sourced, and diabetes was randomly induced in four animals using streptozotocin (STZ) administration. The mandibular tissues were collected and processed. The jawbone density was evaluated with micro-CT. Osteocyte lacunae were effectively acquired and identified using backscattered electron scanning microscopy (BSE). A significantly decreased osteocyte lacunae size was found in the diabetic group. Using the acid etching method, it was demonstrated that the area of osteocyte and lacunae, and the pericellular areas were both significantly reduced in the diabetes group. In conclusion, a standard and relatively reliable method for analyzing osteocyte/lacunae morphological changes under compromised conditions has been successfully established. This method demonstrates that diabetes can significantly decrease osteocyte/lacunae size in a pig's mandibular cancellous bone.

SCARA5 induced ferroptosis to effect ESCC proliferation and metastasis by combining with Ferritin light chain

BackgroundEsophageal squamous cell carcinoma (ESCC) remains one of the most lethal cancers worldwide accompany with an extremely poor prognosis. Therefore, this study aims to screen for new molecules affecting ESCC and explore their mechanisms of action to provide ideas for targeted therapies for ESCC.MethodsFirstly, we screened out the membrane protein SCARA5 by high-throughput sequencing of the ESCC patient tissues, and RT-qPCR and WB were used to verify the differential expression of SCARA5 in esophageal cell lines, and IHC analyzed the expression localization of SCARA5 in ESCC tissue. Then, flow cytometry, wound healing assay, Transwell assay and CCK-8 assay were used to explore the effects of SCARA5 on cell cycle, migration and invasion as well as cell proliferation activity of esophageal squamous carcinoma cells. Meanwhile, transmission electron microscopy was used to detect changes in cellular mitochondrial morphology, and flow cytometry were used to detect changes in intracellular reactive oxygen metabolism, and immunofluorescence and flow cytometry were used to detect changes in intracellular Fe2+. Mechanistically, co-immunoprecipitation was used to detect whether SCARA5 binds to ferritin light chain, and ferroptosis-related protein expression was detected by WB. Finally, the tumor xenograft model was applied to validation the role of SCARA5 tumor growth inhibition in vivo.ResultsWe found that SCARA5 was aberrantly decreased in ESCC tissues and cell lines. Furthermore, we confirmed that SCARA5 suppressed the cell cycle, metastasis and invasion of ESCC cells. Meanwhile, we also found that overexpression of SCARA5 caused changes in mitochondrial morphology, accumulation of intracellular reactive oxygen species and increased intracellular Fe2+ in ESCC cells, which induced ferroptosis in ESCC cells. Mechanically, we validated that SCARA5 combined with ferritin light chain and increased intracellular Fe2+. As well as, overexpression SCARA5 induced ferroptosis by increasing ferritin light chain in nude mice subcutaneous tumors and inhibited the growth of nude mice subcutaneous tumors.ConclusionCollectively, our findings demonstrated that SCARA5 suppressed the proliferation and metastasis of ESCC by triggering ferroptosis through combining with ferritin light chain.

Graph-based autoencoder integrates spatial transcriptomics with chromatin images and identifies joint biomarkers for Alzheimer’s disease

Tissue development and disease lead to changes in cellular organization, nuclear morphology, and gene expression, which can be jointly measured by spatial transcriptomic technologies. However, methods for jointly analyzing the different spatial data modalities in 3D are still lacking. We present a computational framework to integrate Spatial Transcriptomic data using over-parameterized graph-based Autoencoders with Chromatin Imaging data (STACI) to identify molecular and functional alterations in tissues. STACI incorporates multiple modalities in a single representation for downstream tasks, enables the prediction of spatial transcriptomic data from nuclear images in unseen tissue sections, and provides built-in batch correction of gene expression and tissue morphology through over-parameterization. We apply STACI to analyze the spatio-temporal progression of Alzheimer’s disease and identify the associated nuclear morphometric and coupled gene expression features. Collectively, we demonstrate the importance of characterizing disease progression by integrating multiple data modalities and its potential for the discovery of disease biomarkers.

Osteogenic Potential of Monosodium Urate Crystals in Synovial Mesenchymal Stem Cells.

Background and Objectives: Deposits of monosodium urate (MSU) crystals due to increased levels of uric acid (UA) have been associated with bone formation and erosion, mainly in patients with chronic gout. The synovial membrane (SM) comprises several types of cells, including mesenchymal stem cells (SM-MSCs); however, it is unknown whether UA and MSU induce osteogenesis through SM-MSCs. Materials and Methods: Cultures of SM were immunotyped with CD44, CD69, CD90, CD166, CD105, CD34, and CD45 to identify MSCs. CD90+ cells were isolated by immunomagnetic separation (MACS), colony-forming units (CFU) were identified, and the cells were exposed to UA (3, 6.8, and 9 mg/dL) and MSU crystals (1, 5, and 10 μg/mL) for 3 weeks, and cellular morphological changes were evaluated. IL-1β and IL-6 were determined by ELISA, mineralization was assessed by alizarin red, and the expression of Runx2 was assessed by Western blot. Results: Cells derived from SM and after immunomagnetic separation were positive for CD90 (53 ± 8%) and CD105 (52 ± 18%) antigens, with 53 ± 5 CFU identified. Long-term exposure to SM-MSCs by UA and MSU crystals did not cause morphological damage or affect cell viability, nor were indicators of inflammation detected. Mineralization was observed at doses of 6.8 mg/dL UA and 5 μg/mL MSU crystals; however, the differences were not significant with respect to the control. The highest dose of MSU crystals (10 μg/mL) induced significant Runx2 expression with respect to the control (1.4 times greater) and SM-MSCs cultured in the osteogenic medium. Conclusions: MSU crystals may modulate osteogenic differentiation of SM-MSCs through an increase in Runx2.

Dexmedetomidine Reduce Multiple Organ Injury Induced by Lung Ischemia/ Reperfusion of Mice Through Inhibiting Endoplasmic Reticulum Stress Response

To investigate the effect of Dexmedetomidine reduce brain, myocardium and kidney injury induced by lung ischemia/reperfusion of mice through inhibiting endoplasmic reticulum stress response. SPF C57BL/6J male mice were randomly divided into 5 groups (n = 10): the sham operation group (group sham), the ischemia reperfusion Group (Group I/R), the Atipamezole Group (Group Atip), dexmedetomidine Group (Group-DEX) and dexmedetomidine+AtipamezoleGroup (Group DA). Lung ischemia reperfusion models were established with the method of 30min in-vivo left hilus occlusion followed by 180min reperfusion. In addition to the steps same as Group I/R, the mice in Groups Atip, Dex and DA were given Atip(250μg/kg), Dex(20μg/kg), and Dex+ Atip(20μg/kg+250μg/kg) respectively by intraperitoneal injection 30min before hilar occlusion. After reperfusion, the blood was taken from the eye-orbit to determine the activity of serum CK-MB and LDH and the levels of serum creatinine and urea nitrogen. The brain tissue was taken to determine the brain water content. The tissue of myocardium, brain and kidney was taken to observe the cellular morphology change under the light microscope. The expression of Caspase3 activity in heart, brain and kidney was determined with Thermos Scientific Microplate Reader. The TUNEL method was used to detect the apoptotic index of heart, brain and kidney. The Western blot and RT-PCR were used to determine the expression of proteins and mRNA of JNK, Caspase12, CHOP and GRP78. Compared with Group Sham, obvious injury in the tissue of myocardium, brain and kidney was observed in the other four groups under the light microscope. The activity of CKMB and LDH, and the levels of serum creatinine, urea nitrogen and brain water content were significantly increased as well. The apoptotic index, Caspase3 activity, and the expression of the proteins and mRNA of JNK, Caspase12, CHOP and GRP78 were also significantly elevated. Compared with Group I/R, Atip and DA, the mitigated injury in the myocardium, brain and kidney was observed in Group DEX under the light microscope. The activity of CK-MB and LDH, and the levels of serum creatinine, urea nitrogen and brain water content were decreased and the apoptotic index, Caspase3 activity, and the expression of the proteins and mRNAs of JNK, Caspase12 and CHOP were also significantly decreased in Group DEX, but the proteins and mRNA expression of GRP78 were significantly elevated. For the comparison among theGroup I/R, Atip and DA, there is no significant difference was found.(grp78).The pre-administration of DEX can mitigate the heart, brain and kidney injury induced by lung ischemia reperfusion. The mechanism may be related to the activation of α2-adrenergic receptor and inhibition of endoplasmic reticulum over-response.

PMON52 Use of PDE5 Inhibitors as Potential Treatment for Isolated Growth Hormone Deficiency Caused by Alternate Splicing of GH1 Gene

Abstract Mutations in the GH1 gene cause isolated growth hormone deficiency (IGHD) by affecting production, secretion, and stability of growth hormone as well as its binding to GHR. Genetic mutations resulting from splicing errors are known to increase the production of a small 17.5 kD isoform of human growth hormone. This smaller isoform of GH1 is inactive and is linked to IGHD. While studying the impact of small GH isoform on GH production and secretion, we noticed that cells transfect with sequences carrying the mutated GH gene that resulted in splicing defects produce the short 17.5 kD version of GH, looked very different from the control cells with WT GH gene. We found that production of short GH isoform distorts the cell morphology, contributing to detrimental effects seen in the patients with mutations in GH1 gene causing alternate splicing. We hypothesized that cellular distortion caused by small GH isoform and overall GH secretion can be a target and a tool to experimental approaches for reversing the damaging effects of small GH protein, thereby providing potential treatments for IGHD caused by splicing errors. It is known for some time that increasing cAMP enhances 26S proteasome activity and the degradation of cell proteins, including the selective breakdown of misfolded proteins, and recently increase in cGMP levels has also been shown to regulate protein degradation. Inhibitors of phosphodiesterase 5 (PDE5; e.g., Viagra (sildenafil)), which raise cGMP by inhibiting its hydrolysis to GMP, are widely used for the treatment of erectile dysfunction and pulmonary hypertension, and stimulators of soluble guanylyl cyclases (riociguat), which stimulate cGMP synthesis, are used as treatments for pulmonary hypertension and heart failure. Using our model of cellular morphology changes, have investigated the impact of chemicals that enhance cGMP levels by inhibiting PDE5 or activating guanyl cyclases. The cells expressing small isoform of GH consistently show distorted rounded morphology. Treatment with BAY 41-2272, a cGMP inducer slightly improved the cell morphology in small isoform GH expressing cells. BAY 41-2272 did not show any adverse effects of normal and WT GH expressing cells. Treatment of sildenafil to cells expressing 17.5 kD isoform of GH markedly improved the cell morphology compared to untreated cells, especially after 48h when a larger percentage of cells that were distorted (rounder shapes) regained the shape of normal cells expressing WT GH and no damage to cells expressing WT-GH was observed. Another PDE5 inhibitor tadalafil also caused a marked improvement in cell morphology caused by expression of small GH isoform. Overall, it seems the strategy to regulate the cGMP levels in cells transfected in small GH isoform worked and needs further investigation. A detailed analysis of the impact of PDE5 inhibitors on GH expression and secretion will be presented. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Pulse-Picking Multimodal Nonlinear Optical Microscopy.

Nonlinear optical microscopy techniques can map chemical compositions in biological samples in a label-free manner. Commonly used nonlinear optical processes for imaging include multiphoton excitation fluorescence (MPEF), second harmonic generation (SHG), and coherent Raman scattering (CRS). Femtosecond lasers are typically used for MPEF and SHG due to the requirement of high peak power for excitation, while picosecond lasers are preferred for CRS due to the need for high spectral resolution. Therefore, it is challenging to integrate CRS with MPEF and SHG for chemical imaging. We develop a pulse-picking strategy based on an acousto-optic modulator that can program the duty cycle of the laser pulse train, significantly increasing the pulse peak power at low input average power. This approach offers strong enhancement of nonlinear optical signals and makes hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy compatible with MPEF and SHG for multimodal imaging at low laser average power. The pulse-picking method also enables the evaluation and comparison of phototoxicity of laser pulses at different average and peak power levels. The photo-perturbations to biological samples are evaluated using cellular dynamics and sample morphological changes, allowing the selection of optimal laser power for the best sensitivity and minimal phototoxicity.

Deciphering the Binding Insights of Novel Disubstituted Anthraquinone Derivatives with G-Quadruplex DNA to Exhibit Selective Cancer Cell Cytotoxicity.

Anthraquinone-based compounds are well-known as duplex DNA as well as G-quadruplex DNA binders. Implications of various anthraquinone derivatives for specific recognition of G-quadruplex DNA over duplex DNA is a 'challenging' research work that requires adequate experience with molecular design. To address this important issue, we designed and synthesized ten new 2,6-disubstituted anthraquinone-based derivatives with different functionalized piperazinyl side-chains. Among these, particular compounds with certain distant groups have shown selective and significant binding affinities toward the c-MYC and c-KIT G-quadruplex DNA over the duplex DNA, as noticed from various biophysical experiments. The structural difference of quadruplex and duplex DNA was utilized to probe these derivatives for the end-stacking mode of binding with G-quadruplex DNA. The ability of the ligands to halt DNA synthesis by stabilizing G-quadruplex structures is one of the crucial points to further apply them for quadruplex-mediated anti-cancer therapeutics. Interestingly, these ligands trigger apoptosis to exhibit selective cytotoxicity toward cancer cells over normal cells. This was further evidenced by ligand-induced cell cycle arrest as well as cellular apoptotic morphological changes. These blood-compatible ligands provided detailed structure-activity relationship approaches for the molecular design of anthraquinone-based G-quadruplex binders.

Antimicrobial resistance of microorganisms present in periodontal diseases: A systematic review and meta-analysis.

ObjectiveThe aim of this study was to estimate the antimicrobial resistance in microorganisms present in periodontal diseases.MethodsA systematic review was conducted according to the PRISMA statement. The MEDLINE (PubMed/Ovid), EMBASE, BVS, CINAHL, and Web of Science databases were searched from January 2011 to December 2021 for observational studies which evaluated the antimicrobial resistance in periodontal diseases in permanent dentition. Studies that allowed the antimicrobial consumption until the time of sample collection, studies that used laboratory acquired strains, studies that only characterized the microbial strain present, assessment of cellular morphological changes, sequencing system validation, and time series were excluded. Six reviewers, working in pairs and independently, selected titles, abstracts, and full texts extracting data from all studies that met the eligibility criteria: characteristics of patients, diagnosis of infection, microbial species assessed, antimicrobial assessed, identification of resistance genes, and virulence factors. “The Joanna Briggs Institute” critical appraisal for case series was adapted to assess the risk of bias in the included studies.ResultsTwenty-four studies (N = 2.039 patients) were included. Prevotella and Porphyromonas species were the most cited microorganisms in the included studies, and the virulence factors were related to Staphylococcus aureus. The antimicrobial reported with the highest frequency of resistance in the included studies was ampicillin (39.5%) and ciprofloxacin showed the lowest frequency of resistance (3.4%). The most cited genes were related to macrolides. The quality of the included studies was considered critically low.ConclusionNo evidence was found regarding the profile of antimicrobial resistance in periodontal diseases, requiring further research that should focus on regional population studies to address this issue in the era of increasing antimicrobial resistance.Clinical relevanceThe knowledge about the present microorganism in periodontal diseases and their respective antimicrobial resistance profiles should guide dentists in prescribing complementary therapy for these infections.Systematic review registration[http://dx.doi.org/10.1097/MD.0000000000013158], identifier [CRD42018077810].

Reactive morphology of dividing microglia following kainic acid administration.

The microglial response to a pathological microenvironment is hallmarked by a change in cellular morphology. Following a pathological stimulus, microglia become reactive and simultaneously divide to create daughter cells. Although a wide array of microglial morphologies has been observed, the exact functions of these distinct morphologies are unknown, as are the morphology and reactivity status of dividing microglia. In this study, we used kainic acid to trigger microglial activation and cell division. Following a cortical kainic acid injection, microglial morphology and proliferation were examined at 3 days post-injection using immunohistochemistry for ionized calcium binding adapter molecule 1 (Iba1) to stain for microglia, and KI67 as a marker of cell division. Individual microglial cells were isolated from photomicrographs and skeletal and fractal analyses were used to examine cell size and spatial complexity. We examined the morphology of microglia in both wildtype and microglia-specific tumor necrosis factor (TNF)-α knockout mice. Data were analyzed using generalized linear mixed models or a two-way ANOVA. We found that dividing microglia had a more reactive morphology (larger cell body area, longer cell perimeter, and less ramification) compared to microglia that were not dividing, regardless of microglial release of TNF-α. However, we also observed dividing microglia with a complex, more ramified morphology. Changes in microglial morphology and division were greatest near the kainic acid injection site. This study uses robust and quantitative techniques to better understand microglial cell division, morphology, and population dynamics, which are essential for the development of novel therapeutics that target microglia.

WDR73 Depletion Destabilizes PIP4K2C Activity and Impairs Focal Adhesion Formation in Galloway-Mowat Syndrome.

Simple SummaryGalloway–Mowat syndrome is a rare genetic disease, classically characterized by a combination of various neurological symptoms and nephrotic syndrome. WDR73 is the pathogenic gene responsible for Galloway–Mowat syndrome. However, the pathological and molecular mechanisms of Galloway–Mowat syndrome, especially nephrotic syndrome caused by WDR73 deficiency, remains unknown. In this study, we knocked out the WDR73 in human embryonic kidney 293 cells to observe the morphological characteristics of the cells and elucidate the functions of WDR73. Additionally, we used a combination of proteomics, transcriptomics, and biochemical assays to identify the regulated targets of WDR73. We aimed to discover directly interacting molecules and the regulatory pathway of WDR73 and to illustrate the molecular mechanism between the WDR73 pathway and nephrotic disease in Galloway–Mowat syndrome. From the molecular mechanism we found in vitro, we draw a hypothesis that the damage to focal adhesion of podocytes caused by WDR73 defect is the key issue of kidney dysfunction. Finally, we verified the hypothesis in a podocyte-specific conditional knockout Wdr73 mouse model.(1) Background: Galloway–Mowat syndrome (GAMOS) is a rare genetic disease, classically characterized by a combination of various neurological symptoms and nephrotic syndrome. WDR73 is the pathogenic gene responsible for GAMOS1. However, the pathological and molecular mechanisms of GAMOS1, especially nephrotic syndrome caused by WDR73 deficiency, remain unknown. (2) Methods and Results: In this study, we first observed remarkable cellular morphological changes including impaired cell adhesion, decreased pseudopodia, and G2/M phase arrest in WDR73 knockout (KO) HEK 293 cells. The differentially expressed genes in WDR73 KO cells were enriched in the focal adhesion (FA) pathway. Additionally, PIP4K2C, a phospholipid kinase also involved in the FA pathway, was subsequently validated to interact with WDR73 via protein microarray and GST pulldown. WDR73 regulates PIP4K2C protein stability through the autophagy–lysosomal pathway. The stability of PIP4K2C was significantly disrupted by WDR73 KO, leading to a remarkable reduction in PIP2 and thus weakening the FA formation. In addition, we found that podocyte-specific conditional knockout (Wdr73 CKO) mice showed high levels of albuminuria and podocyte foot process injury in the ADR-induced model. FA formation was impaired in primary podocytes derived from Wdr73 CKO mice. (3) Conclusions: Since FA has been well known for its critical roles in maintaining podocyte structures and function, our study indicated that nephrotic syndrome in GAMOS1 is associated with disruption of FA caused by WDR73 deficiency.

Role of MicroRNA-Like RNAs in the Regulation of Spore Morphological Differences in the Entomopathogenic Fungus Metarhizium acridum.

Metarhizium acridum is an important microbial pesticide. Conidia (CO) and blastospores (BS) are two types of spores that occur in different patterns in the M. acridum life cycle and exhibit significant differences in cell morphology, structure, and activity. It may suggest that the fungus has a complex gene regulation mechanism. While previous studies on the differences between CO and BS have mainly focused on cell structure and application, little is known regarding the differences between CO and BS in fungi on the transcriptome levels. MicroRNAs (miRNAs) are small noncoding RNAs crucial to gene regulation and cell function. Understanding the miRNA-like RNAs (milRNA) and mRNA expression profiles related to cell growth and cellular morphological changes would elucidate the roles of miRNAs in spore morphological differences. In this study, 4,646 differentially expressed genes (DEGs) were identified and mainly classified in the GO terms cell, cell part, biological process, and catalytic activity. The KEGG annotation suggested that they were enriched in amino acid biosynthesis, carbohydrate metabolism, ribosome, and oxidative phosphorylation and might be involved in cell activity and structure. There were 113 differentially expressed milRNAs (DEMs), targeting 493 DEGs. Target gene functional analysis revealed that the target genes were mainly enriched in RNA transport, purine metabolism, and the cell cycle. In addition, we identified essential genes from milRNA-mRNA pairs that might participate in cell budding growth and cell membrane and wall integrity, including adenosine deaminase, glycosyl hydrolase, and G-patch domain protein (dno-miR-328-3p), WD repeat-containing protein pop1 (age-miR-127), and GPI-anchored wall transfer protein (cgr-miR-598). MilRNAs might therefore play a crucial role in cell growth and cellular morphological changes as transcriptional and post-transcriptional regulators.

Configuration of the surfaces of epithelial cells infected by human papillomavirus

Human papillomavirus (HPV) types were investigated hy iri situ hybridization (ISH) in biopsiesfroin J8patients with diverse pathologies ofcervix uteri (7 condylomas, 1 CINI +HPV, 3 CIN II, 4 CIN II + HPV and 3 CIN III). Cervical sniears ofthe same patients were processed by Scanning Electron Mycroscopy (SEM) in order to study the configuration of sitrfaces ofcells infected hy different HPV types. Seven condylomas, I CIN I + HPV, 3 CIN II and 3 CIN II + HPV showed alinost identical surface configurations to those of normal cells (short, thin, homogeneous microvi lli o r typicai microridgesj. This group presented low, moderate and high risk HPV types in a episomal form, while I CINII + HPV and3 CINIII with integratedHPV 16/]8 exfoliatedcells covered with gross microvilli, with variahle length and uneven distribution, oran amorphous surface. The presence of HPV 16/18 cannot be sufficient for the development of an anormal configuration, hut the integrated State ofthis viral type coiild he related to the morphological changes in cellular surface morphology. The results of this paper underline the importance of viral integration in the genesis of cervical carcinoma.

Development of chitosan towards the self-healing and mechanically stronger biocompatible hydrogel

Nontoxic, self-healing and biocompatible Chitosan grafted poly (maleic anhydride) based hydrogel (CSHG) have been developed by using Chitosan (CS) and maleic anhydride (MAH) having reasonable mechanical strength with biocompatibility. The self-healing arises due to the presence of free primary amine group and carboxyl group in grafted chitosan either dynamic imine covalent interaction or non-covalent (H-bonding) interactions. In addition to this, the hydrogel showed tremendous swelling at various pH values viz. - 2, 7 and 9, and morphological study reveals the porous nature of the synthesized CSHG hydrogel. The cell viability (MTT) of CS and CSHG was performed on MCF-7 (breast cancer cell line), and phase contrast (cell imaging) of CS and CSHG was taken at 1:5, 1:10, 1:50 and 1:100 ratios in both cases which do not induce any significant changes in cellular morphology thus makes it biocompatible and can be used as biological applications.