Surface Of Plasma Membrane Research Articles

Tumor-oriented and chemo-photothermal nanoplatform capable of sensitizing chemotherapy and ferroptosis against osteosarcoma metastasis

The clinical use of chemotherapy for refractory osteosarcoma (OS) is limited due to its multiorgan toxicity. To overcome this challenge, new dosage forms and combination treatments, such as phototherapy, are being explored to improve targeted delivery and cytocompatibility of chemotherapeutic agents. In addition, inducing ferroptosis in iron-rich tumors could be a promising strategy to enhance OS therapy. In this study, a novel formulation was developed using natural biological H-ferritin (HFn) encapsulating the photosensitizer IR-780 and the chemotherapy drug gemcitabine (Gem) for OS-specific targeted therapy (HFn@Gem/IR-780 NPs). HFn@Gem/IR-780 NPs were designed to specifically bind and internalize into OS cells by interacting with transferrin receptor 1 (TfR1) which is overexpressed on the surface of OS cell membranes. The Gem and IR-780 were then released responsively under mildly acidic conditions in tumors. HFn@Gem/IR-780 NPs achieved cascaded antitumor therapeutic efficacy through the combination of chemotherapy and phototherapy under near-infrared irradiation in vitro and in vivo. Importantly, HFn@Gem/IR-780 NPs demonstrated excellent safety profile with significantly decreased drug exposure to normal organs, indicating its potential for reducing systemic toxicity. Thus, utilizing HFn as a vehicle to encapsulate highly effective antitumor drugs provides a promising approach for the treatment of OS metastasis and relapse.

Red Blood Cell Hemoglobin Oxygen Transfer in Deep-Diving Marine Mammals

Objective: Some marine mammals of the order pinniped and cetacean can dive for long durations to forage for food and travel over distances. During these extended dives both pinnipeds and cetaceans experience extreme tissue hypoxia. Numerous adaptations have evolved to allow these species to effciently deliver and utilize oxygen (O2). One genetic difference, which our laboratory has proposed to play an important role in O2 transport effciency in modern hominids, is the inactivation of the CMP-Neu5AC hydroxylase (CMAH) gene. Pinnipeds inactivated Cmah ~ 40 million years ago but, cetaceans still express this N-glycan modifying enzyme. CMAH converts the sialic acid, N-acetylneuraminic acid (Neu5Ac), to its hydroxylated form, N-glycolylneuraminic acid (Neu5Gc). These sialic acids are located at the end of branched sugar chains on the surface of cell membranes. As a result, a change in composition could affect cell membrane charge and hydrophobicity, as well as potentially alter O2 diffusion laterally or across cell membranes. Hypothesis: In this study we hypothesized that O2 transfer from hemoglobin (Hb) in red blood cells (RBCs) to myoglobin (Mb) would differ between cetaceans (CMAH+) and pinnipeds (CMAH-). Methods: This hypothesis was tested using optical absorption spectroscopy to measure O2 exchange between oxygenated Hb in RBC and deoxygenated Mb collected in several species of mustelids and pinniped (CA sea otter, CA sea lion, and walrus) and cetaceans (Bottlenose Dolphin, Beluga, and Orca). Measurements of cell diameters (diffusion length) were also measured. Results: The mean oxygen transfer (O2D/A410) was 47% lower in pinnipeds than cetaceans (p<0.01). The amount of O2 transferred in cetaceans increased with longer reported maximal dive durations. There was a trend for overall RBC diameters to be larger in cetaceans than pinnipeds (p=0.054). Summary: The RBC O2 transfer data in these hypoxia tolerant marine mammals suggest a transfer mechanism that is more effcient in cetaceans in a normoxic environment. Conclusions: More effcient oxygen transfer in cetaceans, who retained the Cmah gene, compared to pinnipeds and mustelids, who did not retain the Cmah gene, suggests that the gene knockout may not be the only factor that facilitates delivery of O2 in these marine mammals. A trend for larger RBC diameters in cetaceans than pinnipeds also suggests that diffusion length is not a main contributing factor. Future experiments to measure oxygen transfer from RBC O2-Hb to Mb under different O2 environments are needed to more closely model what may occur during long duration dives. NSF1929325. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Heterogeneity of Monocarboxylate Transporter Function in Gastrointestinal Epithelium

Short-chain fatty acids and ketone bodies are important energy fuels of GI epithelial cells. Slc16a family encodes proton-coupled monocarboxylate transporters (MCTs) that mediate cellular metabolism of those energy fuels. Increasing evidence demonstrates that adenocarcinomas with overexpression of MCTs are better able to maintain microenvironmental pH and high energy metabolism to grow. However, the precise distribution and function of each MCT subtype in normal epithelial or precancerous cell lineages are mostly unknown. We investigated cell type-specific expressions of MCT subtypes MCT1, MCT2, and MCT4 by immunofluorescence in normal and gastric metaplasia model mice. As total MCT knockout leads to early lethality, we generated inducible, intestine-targeted knockout mouse models for MCT1 and MCT4, respectively. In normal GI tissues, MCT1 was strongly expressed on basolateral membranes of gastric surface cells and enterocytes, with intense staining of proliferating cells and enteroendocrine cells. No staining was observed in Brunner’s glands or Paneth cells. The low affnity subtype, MCT4 expression was localized to the first corpus glands and tuft cells in the stomach, suggesting that these cells may monitor the luminal chemical environment. Intestinal MCT4 was localized on basolateral membranes of mature enterocytes and tuft cells. In contrast, MCT2, which has the highest affnity for lactate, was localized reciprocally to MCT1. Strong MCT2 staining was identified on the basolateral membrane of secretory cells, including gastric parietal and chief cells, Brunner’s glands, and Paneth cells. The distinct MCT2 expression suggests that those secretory cells possess different energy metabolism and MCT2 may contribute to their intracellular pH homeostasis. In gastric metaplasia tissues of Mist1-Kras(G12D) mice, MCT1+ metaplastic cells were increased, and Ki67+ cells were more dependent on MCT1 than normal tissues. MCT2 expression in chief cells was disappeared during metaplasia development. MCT1ΔIEC mice lost MCT1 expression on day 8 after tamoxifen injection and decreased transcriptions of other subtypes, Mct4, Mct7, and Mct10 in intestinal mucosa. MCT1ΔIEC jejunum showed significant increase in crypt length, indicating that MCT1 loss disrupted proliferative cell function. Contrary, MCT4ΔIEC mice had no change in crypt length or Mct1 expression, but increased Mct7, and Mct10. These observations indicate that different cell types require a different MCT subtype. The increase in MCT1-dependency in proliferative metaplastic glands is consistent with the predicted effect of MCT1 inhibitors for cancer treatment. Alterations in MCT subtype expression might be a marker for early diagnosis of metaplastic cells. R37 CA244970 and R01 CA272687 to EC, VDDRC P30-058404 and VICC GI SPORE P50-CA236733 to IK. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Low Expression MCEMP1 Promoting Lung Adenocarcinoma Progression by and its Clinical Value.

Lung cancer is a highly prevalent tumor with a lack of biological markers that reflect its progression. Mast cell surface membrane protein 1 (MCEMP1, also known as C19ORF59) has not been reported in lung adenocarcinoma (LUAD). We aimed to investigate the role of MCEMP1 in LUAD. MCEMP1 expression in LUAD was analyzed using The Cancer Genome Atlas (TCGA) data, and conducted univariate and multivariate Cox regression analyses to evaluate the prognostic significance of MCEMP1 expression in TCGA. Tumor Immune Estimation Resource (TIMER) was used for examining the correlation between MCEMP1 expression and immune cell infiltration in LUAD. Furthermore, proliferation, migration, invasion, and colony-forming ability were investigated using LUAD cell lines. MCEMP1 had low expression in LUAD patient tissues and was correlated with lymph node metastasis, differentiation level, and tumor status. The Area under Curve (AUC) value of MCEMP1 for the Receiver Operating Characteristic (ROC) curve analysis was 0.984. The immune infiltration analysis revealed a correlation between MCEMP1 expression and the extent of macrophages and neutrophil infiltration in LUAD. Additionally, MCEMP1 has low expression in clinical samples, MCEMP1 overexpressed in LUAD cells substantially reduced cell growth, migration, and invasion of malignant cells. Low expression MCEMP1 promotes LUAD progression, which provides new insights and a potential biological target for future LUAD therapies.

Glycolysis and tumor progression promoted by the m6A writer VIRMA via m6A-dependent upregulation of STRA6 in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal malignancies, highlighting the urgent need to elucidate the underlying oncogenic mechanisms. VIRMA is a classic isoform of methyltransferases that participates in epigenetic transcriptomic modification in eukaryotic mRNAs. However, the exact roles of VIRMA in PDAC remain unclear. Here, we identified that VIRMA is highly expressed in PDAC, and histone modifications of the promoter may partly account for this dysregulation. Moreover, VIRMA is closely related to glycolysis and poor prognosis in PDAC. We further determined that STRA6 is a direct downstream target of VIRMA in PDAC by RNA sequencing (RNA-seq) and m6A sequencing (m6A-seq). VIRMA is involved in gene expression regulation via 3’ UTR targeting of STRA6 mRNA. Furthermore, the m6A reader IGF2BP2 was shown to critically contribute to the stability of STRA6 mRNA. We describe the role of VIRMA in promoting signaling via the STRA6/STAT3 axis, which results in increased levels of HIF-1α, a key activator of glycolysis. In vivo and in vitro experiments reveal that the VIRMA-STRA6-STAT3-HIF-1α axis plays an instrumental role in glycolysis and tumor progression in PDAC. In conclusion, we demonstrate that VIRMA can increase glycolysis in PDAC by upregulating STRA6, a cell surface membrane protein that stimulates the STAT3 pathway, thereby activating HIF-1α and leading to pancreatic cancer malignancy. Overall, our data strongly suggest that the VIRMA-STRA6-STAT3-HIF-1α axis is a viable therapeutic target in PDAC.

Oleoylethanolamide and Palmitoylethanolamide Enhance IFNβ-Induced Apoptosis in Human Neuroblastoma SH-SY5Y Cells.

Oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) are endogenous lipids that act as agonists of the peroxisome proliferator-activated receptor α (PPARα). Recently, an interest in the role of these lipids in malignant tumors has emerged. Nevertheless, the effects of OEA and PEA on human neuroblastoma cells are still not documented. Type I interferons (IFNs) are immunomodulatory cytokines endowed with antiviral and anti-proliferative actions and are used in the treatment of various pathologies such as different cancer forms (i.e., non-Hodgkin's lymphoma, melanoma, leukemia), hepatitis B, hepatitis C, multiple sclerosis, and many others. In this study, we investigated the effect of OEA and PEA on human neuroblastoma SH-SY5Y cells treated with IFNβ. We focused on evaluating cell viability, cell proliferation, and cell signaling. Co-exposure to either OEA or PEA along with IFNβ leads to increased apoptotic cell death marked by the cleavage of caspase 3 and poly-(ADP ribose) polymerase (PARP) alongside a decrease in survivin and IKBα levels. Moreover, we found that OEA and PEA did not affect IFNβ signaling through the JAK-STAT pathway and the STAT1-inducible protein kinase R (PKR). OEA and PEA also increased the phosphorylation of p38 MAP kinase and programmed death-ligand 1 (PD-L1) expression both in full cell lysate and surface membranes. Furthermore, GW6471, a PPARα inhibitor, and the genetic silencing of the receptor were shown to lower PD-L1 and cleaved PARP levels. These results reveal the presence of a novel mechanism, independent of the IFNβ-prompted pathway, by which OEA and PEA can directly impair cell survival, proliferation, and clonogenicity through modulating and potentiating the intrinsic apoptotic pathway in human SH-SY5Y cells.

Perbedaan Derajat Aglutinasi Pemeriksaan Golongan Darah Metode Tabung Berdasarkan Konsentrasi Suspensi Sel 5% Segera Periksan Dengan Lama Penyimpanan 5 Hari

Background: ABO b;ood group examination is an examination carried out to determinate the type of blood group in humans that detects the presence of antigens on the surface of the red blood cell membrane by means of the Tube Method various consentrations of erythrocyte cell suspension,namely 5%, 10% and 15%. Research purpose: To determinate the effect of various consentrations of erythrocyte cell suspension with ready-to-use 0,9% NaCl and 0,9% table salt solvent on the degree of agglutination in blood group examination Tube method. Research method: This research is a pure experimental study with a total sample 41 people, this study was conducted on January 16-20, 2023 at the Muhammadiyah Palembang IKesT Clinical Pathology Laboratory. Results: The results showed that the blood group examination was done by the tube method with a concentration of erythrocyte cell suspension, namely 5%, 10% and 15% with 0,9% NaCl solvent ready-to-use and 0,9% NaCl, table salt solvent resulting in a degree of agglutination of +4. The Kruskal-wallis test result in the Cell Grouping method obtained 0,000 and in the Serum Grouping method obtained 0,051. Conclusion: The conclusion in this study is that there is no effect of various consentrations of erythrocyte cell suspension with ready-to-use 0,9% NaCl and 0,9% NaCl Solvent Kitchen Salt on the degree of agglutination in blood group examination Tube Method so that 0,9% NaCl solvent from table salt can be used as an alternative to ready-to-use 0,9% NaCl reagent in blood group tests by tube method. 
 Keywords: Blood type, Aglutination

Assessing the Impact of Agents with Antiviral Activities on Transmembrane Ionic Currents: Exploring Possible Unintended Actions

As the need for effective antiviral treatment intensifies, such as with the coronavirus disease 19 (COVID-19) infection, it is crucial to understand that while the mechanisms of action of these drugs or compounds seem apparent, they might also interact with unexplored targets, such as cell membrane ion channels in diverse cell types. In this review paper, we demonstrate that many different drugs or compounds, in addition to their known interference with viral infections, may also directly influence various types of ionic currents on the surface membrane of the host cell. These agents include artemisinin, cannabidiol, memantine, mitoxantrone, molnupiravir, remdesivir, SM-102, and sorafenib. If achievable at low concentrations, these regulatory effects on ion channels are highly likely to synergize with the identified initial mechanisms of viral replication interference. Additionally, the immediate regulatory impact of these agents on the ion-channel function may potentially result in unintended adverse effects, including changes in cardiac electrical activity and the prolongation of the QTc interval. Therefore, it is essential for patients receiving these related agents to exercise additional caution to prevent unnecessary complications.

Novel method of measurement of in vitro drug uptake in OATP1B3 overexpressing cells in the presence of dextran.

In predictions about hepatic clearance (CLH), a number of studies explored the role of albumin and transporters in drug uptake by liver cells, challenging the traditional free-drug theory. It was proposed that liver uptake can occur for transporter substrate compounds not only from the drug's unbound form but also directly from the drug-albumin complex, aphenomenon known as uptake facilitated by albumin. In contrast to albumin, dextran does not exhibit binding properties for compounds. However, as a result of its inherent capacity for stabilization, it is widely used to mimic conditions within cells. The uptake of eight known substrates of the organic anion-transporting polypeptide 1B3 (OATP1B3) was assessed using a human embryonic kidney cell line (HEK293), which stably overexpresses this transporter. An inert polymer, dextran, was used to simulate cellular conditions, and the results were compared with experiments involving human plasma and human serum albumin (HSA). This study is the first to demonstrate that dextran increases compound uptake in cells with overexpression of the OATP1B3 transporter. Contrary to the common theory that highly protein-bound ligands interact with hepatocytes to increase drug uptake, the results indicate that dextran's interaction with test compounds does not significantly increase concentrations near the cell membrane surface. We evaluated the effect of dextran on the uptake of known substrates using OATP1B3 overexpressed in the HEK293 cell line, and we suggest that its impact on drug concentrations in liver cells may differ from the traditional role of plasma proteins and albumin.

Lysyl oxidase-like 4 promotes the invasiveness of triple-negative breast cancer cells by orchestrating the invasive machinery formed by annexin A2 and S100A11 on the cell surface.

Our earlier research revealed that the secreted lysyl oxidase-like 4 (LOXL4) that is highly elevated in triple-negative breast cancer (TNBC) acts as a catalyst to lock annexin A2 on the cell membrane surface, which accelerates invasive outgrowth of the cancer through the binding of integrin-β1 on the cell surface. However, whether this machinery is subject to the LOXL4-mediated intrusive regulation remains uncertain. Cell invasion was assessed using a transwell-based assay, protein-protein interactions by an immunoprecipitation-Western blotting technique and immunocytochemistry, and plasmin activity in the cell membrane by gelatin zymography. We revealed that cell surface annexin A2 acts as a receptor of plasminogen via interaction with S100A10, a key cell surface annexin A2-binding factor, and S100A11. We found that the cell surface annexin A2/S100A11 complex leads to mature active plasmin from bound plasminogen, which actively stimulates gelatin digestion, followed by increased invasion. We have refined our understanding of the role of LOXL4 in TNBC cell invasion: namely, LOXL4 mediates the upregulation of annexin A2 at the cell surface, the upregulated annexin 2 binds S100A11 and S100A10, and the resulting annexin A2/S100A11 complex acts as a receptor of plasminogen, readily converting it into active-form plasmin and thereby enhancing invasion.

Abstract 5089: Therapeutically targeting endometrial cancer by ICAM1 antibody-drug conjugates

Abstract Background: Endometrial cancer is the most frequently occurring gynecologic cancer in developed countries and the third leading cause of cancer deaths among women. It remains one of the few malignant tumors with rapidly increasing mortality and morbidity. Patients with recurrent or metastatic endometrial cancer continue to experience poor outcomes. Antibody-drug conjugates (ADCs) represent a class of targeted therapeutic modalities that offer biomarker-based therapeutic options for patients with endometrial cancer. In this study, we proposed to identify a novel molecular target for the preclinical development of an ADC for endometrial cancer-targeted therapy. Methods: Firstly, to determine the level of target expression within endometrial cancer tissue components as well as differences, we performed immunohistochemical staining of human endometrial cancer and paracancerous tissue. Secondly, the expression of the target on the cell membrane surface was quantitated and qualitatively verified using flow cytometry and single-photon confocal microscopy. In the third step, an endocytosis assay was used to verify the endocytosis of the target antibody by EC cells. Confocal imaging can directly observe the endocytosis of the target antibody, and flow cytometry can be analyzed and quantified. Co-localization experiments with antibody fluorescence can verify the specific site where the target antibody is endocytosed into cancer cells. We then designed and characterized two targeted ADCs. Next, the in vitro inhibitory activities of these ADCs against endometrial cancer cell lines and normal cells were evaluated using the CCK8 assay. Finally, we established a cell line-derived xenograft (CDX) model of endometrial cancer to validate the in vivo efficacy and bio-safety of the ADCs. Results: We have determined a correlation between ICAM1 overexpression and EC, and IHC staining of patient tissues showed significantly higher levels of ICAM1 expression in endometrial cancer tissues than in paracancerous tissues. The high cell surface expression of ICAM1 was verified in a panel of various human endometrial cancer cell lines. Moreover, two ICAM1 ADCs were established using clinically-approved ADC linker and payload combinations. It was visually confirmed that ICAM1 ADCs selectively recognize endometrial tumors and efficiently enter endometrial cells via antigen-mediated endocytosis. We compared the anti-tumor activity of ICAM1 ADCs with standard-of-care chemodrugs, and ICAM1 ADCs outperformed chemodrugs in the CDX model of endometrial cancer without toxicity. Conclusion: We identified that ICAM1 is a promising ADC target for endometrial cancer and confirmed the potent anti-tumor activity and biosafety of ICAM1 ADCs in vitro and in vivo, warranting further pre-clinical and clinical investigations. Citation Format: Hanfei Xie, Lu Sun, ShiLi Yao, Peng Guo, YingLi Zhang. Therapeutically targeting endometrial cancer by ICAM1 antibody-drug conjugates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5089.

BK ZERO isoform HEK293 stably transfected cell lines differing 3'UTRs to assess miR-9 regulation.

Research has identified the large conductance voltage- and calcium-activated potassium channel (BK) as a key regulator of neuronal excitability genetically associated to behavioral alcohol tolerance. Sensitivity to ethanol at the molecular level is characterized by acute potentiation of channel activity. BK isoforms show variations in alcohol sensitivity and are differentially distributed on the plasma membrane surface in response to prolonged exposure. MicroRNA (MiRNA) targeting of alcohol-sensitive isoforms coupled with active internalization of BK channels in response to ethanol are believed to be key in establishing homeostatic adaptations that produce persistent changes within the plasma membrane of neurons. In fact, microRNA 9 (miR-9) upregulated expression is a key event in persistent alcohol tolerance mediating acute EtOH desensitization of BK channels. The exact nature of these interactions remains a current topic of discussion. To further study the effects of miR-9 on the expression and distribution of BK channel isoforms we designed an experimental model by transfecting human BK channel isoforms ZERO heterologous constructs in human embryonic kidney cells 293 (HEK293) cells respectively expressing 2.1 (miR-9 responsive), 2.2 (unresponsive) and control (no sequence) 3'untranslated region (3'UTR) miRNA recognition sites. We used imaging techniques to characterize the stably transfected monoclonal cell lines, and electrophysiology to validate channel activity. Finally, we used immunocytochemistry to validate isoform responsiveness to miR-9. Our findings suggest the cell lines were successfully transfected to express either the 2.1 or 2.2 version of ZERO. Patch clamp recordings confirm that these channels retain their functionality and immunohistochemistry shows differential responses to miR-9, making these cells viable for use in future alcohol dependence studies.

Magnetic colloidal nanoformulations to remotely trigger mechanotransduction for osteogenic differentiation

Nowadays, diseases associated with an ageing population, such as osteoporosis, require the development of new biomedical approaches to bone regeneration. In this regard, mechanotransduction has emerged as a discipline within the field of bone tissue engineering. Herein, we have tested the efficacy of superparamagnetic iron oxide nanoparticles (SPIONs), obtained by the thermal decomposition method, with an average size of 13 nm, when exposed to the application of an external magnetic field for mechanotransduction in human bone marrow-derived mesenchymal stem cells (hBM-MSCs). The SPIONs were functionalized with an Arg-Gly-Asp (RGD) peptide as ligand to target integrin receptors on cell membrane and used in colloidal state. Then, a comprehensive and comparative bioanalytical characterization of non-targeted versus targeted SPIONs was performed in terms of biocompatibility, cell uptake pathways and mechanotransduction effect, demonstrating the osteogenic differentiation of hBM-MSCs. A key conclusion derived from this research is that when the magnetic stimulus is applied in the first 30 min of the in vitro assay, i.e., when the nanoparticles come into contact with the cell membrane surface to initiate endocytic pathways, a successful mechanotransduction effect is observed. Thus, under the application of a magnetic field, there was a significant increase in runt-related transcription factor 2 (Runx2) and alkaline phosphatase (ALP) gene expression as well as ALP activity, when cells were exposed to RGD-functionalized SPIONs, demonstrating osteogenic differentiation. These findings open new expectations for the use of remotely activated mechanotransduction using targeted magnetic colloidal nanoformulations for osteogenic differentiation by drug-free cell therapy using minimally invasive techniques in cases of bone loss.

PIGN c.776T>C (p.Phe259Ser) variant present in trans with a pathogenic variant for PIGN-congenital disorder of glycosylation: Bella-Noah syndrome

Glycosylation is the most common protein and lipid post-translational modification in humans. Congenital disorders of glycosylation (CDG) are characterized by both genetic and clinical heterogeneity, presenting multisystemic manifestations, and in most cases are autosomal recessive in inheritance. The PIGN gene is responsible for the addition of phosphoethanolamine to the first mannose in the glycosylphosphatidylinositol (GPI)-anchor biosynthesis pathway, a highly conserved process that enables proteins to bind to the cell surface membrane. Here, we report a family with two siblings pediatric cases with the exact same compound heterozygous variants in PIGN. The (c.776T > C) variant of uncertain significance (VUS) together with a known pathogenic variant (c.932T > G), resulting in clinical features compatible with PIGN-related conditions, more specific the CDG. This is the first time that PIGN variant c.776T > C is reported in literature in individuals with PIGN-congenital disorder of glycosylation (PIGN-CDG), and the current submission in ClinVar by Invitae® is specifically of our case. Detailed clinical information and molecular analyses are presented. Here, we show for the first time two affected siblings with one pathogenic variant (c.932T > G) and the c.776T > C VUS in trans. In honor of the family, we propose the name Bella-Noah Syndrome for disorder.

The effect of triclosan on intergeneric horizontal transmission of plasmid-mediated tigecycline resistance gene tet(X4) from Citrobacter freundii isolated from grass carp gut

The transmission of antibiotic resistance genes (ARGs) in pathogenic bacteria affects culture animal health, endangers food safety, and thus gravely threatens public health. However, information about the effect of disinfectants - triclosan (TCS) on ARGs dissemination of bacterial pathogens in aquatic animals is still limited. One Citrobacter freundii (C. freundii) strain harboring tet(X4)-resistant plasmid was isolated from farmed grass carp guts, and subsequently conjugative transfer frequency from C. freundii to Escherichia coli C600 (E. coli C600) was analyzed under different mating time, temperature, and ratio. The effect of different concentrations of TCS (0.02, 0.2, 2, 20, 200 and 2000 μg/L) on the conjugative transfer was detected. The optimum conditions for conjugative transfer were at 37 °C for 8h with mating ratio of 2:1 or 1:1 (C. freundii: E. coli C600). The conjugative transfer frequency was significantly promoted under TCS treatment and reached the maximum value under 2.00 μg/L TCS with 18.39 times that of the control group. Reactive oxygen species (ROS), superoxide dismutase (SOD) and catalase (CAT) activities, cell membrane permeability of C. freundii and E. coli C600 were obviously increased under TCS stress. Scanning electron microscope showed that the cell membrane surface of the conjugative strains was wrinkled and pitted, even broken at 2.00 μg/L TCS, while lysed or even ruptured at 200.00 μg/L TCS. In addition, TCS up-regulated expression levels of oxidative stress genes (katE, hemF, bcp, hemA, katG, ahpF, and ahpC) and cell membrane-related genes (fimC, bamE and ompA) of donor and recipient bacteria. Gene Ontology (GO) enrichment demonstrated significant changes in categories relevant to pilus, porin activity, transmembrane transporter activity, transferase activity, hydrolase activity, material transport and metabolism. Taken together, a tet(X4)-resistant plasmid could horizontal transmission among different pathogens, while TCS can promote the propagation of the resistant plasmid.

Synergistic Binding of SARS-CoV-2 to ACE2 and Gangliosides in Native Lipid Membranes.

Viruses utilize cell surface glycans and plasma membrane receptors to attain an adequate attachment strength for initiating cellular entry. We show that SARS-CoV-2 particles bind to endogenous ACE2 receptors and added sialylated gangliosides in near-native membranes. This was explored using supported membrane bilayers (SMBs) that were formed using plasma membrane vesicles having endogenous ACE2 and GD1a gangliosides reconstituted in lipid vesicles. The virus binding rate to the SMBs is influenced by GD1a and inhibition of the ganglioside reduces the extent of virus binding to the membrane receptors. Using combinations of inhibition assays, we confirm that added GD1a in lipid membranes increases the availability of the endogenous ACE2 receptor and results in the synergistic binding of SARS-CoV-2 to the membrane receptors in SMBs.

Morphological analysis for two types of viral particles in vacuoles of SARS-CoV-2-infected cells.

In this study, we analyzed the morphological structure of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in human cells. We identified the two types of viral particles present within the vacuoles of infected cells. Using transmission electron microscopy, we observed that SARS-CoV-2 particles exhibited both low- and high-electron-density structures, which was further confirmed through three-dimensional reconstruction using electron tomography. The budding of these particles was exclusively observed within these vacuoles. Intriguingly, viral particles with low-electron-density structures were confined to vacuoles, whereas those with high-electron-density structures were found in vacuoles and on the cell membrane surface of infected cells. Notably, high-electron-density particles found within vacuoles exhibited the same morphology as those outside the infected cells. This observation suggests that the two types of viral particles identified in this study had different maturation status. Our findings provide valuable insights into the molecular details of SARS-CoV-2 particles, contributing to our understanding of the virus.

Suberin deficiency and its effect on the transport physiology of young poplar roots.

The precise functions of suberized apoplastic barriers in root water and nutrient transport physiology have not fully been elucidated. While lots of research has been performed with mutants of Arabidopsis, little to no data are available for mutants of agricultural crop or tree species. By employing a combined set of physiological, histochemical, analytical, and transport physiological methods as well as RNA-sequencing, this study investigated the implications of remarkable CRISPR/Cas9-induced suberization defects in young roots of the economically important gray poplar. While barely affecting overall plant development, contrary to literature-based expectations significant root suberin reductions of up to 80-95% in four independent mutants were shown to not evidently affect the root hydraulic conductivity during non-stress conditions. In addition, subliminal iron deficiency symptoms and increased translocation of a photosynthesis inhibitor as well as NaCl highlight the involvement of suberin in nutrient transport physiology. The multifaceted nature of the root hydraulic conductivity does not allow drawing simplified conclusions such as that the suberin amount must always be correlated with the water transport properties of roots. However, the decreased masking of plasma membrane surface area could facilitate the uptake but also leakage of beneficial and harmful solutes.

The effect of human PBMCs immobilization on their Аβ42 aggregates-dependent proinflammatory state on a cellular model of Alzheimer's disease.

The leading pathological mechanisms of Alzheimer's disease are amyloidosis and inflammation. The presented work was aimed to study the effect of human peripheral blood mononuclear cells (hPBMcs) cells-matrix adhesion on their pro-inflammatory state in vitro. Although direct interaction of Аβ42 to PBMC is not a cellular model of Alzheimer's disease, PBMCs may serve as test cells to detect Аβ42-dependent molecular effects in monitoring disease progression. Peripheral blood mononuclear cells (PBMCs) are used to assess changes in cytokines released in response to diseases or Alzheimer's disease-specific cytotoxic molecules such as Aβ42. The effect of recombinant amyloid β-peptide rАβ42 on the concentration of endogenous amyloid β-peptide Aβ40 and pro-inflammatory cytokines TNFα and IL-1β in human peripheral blood mononuclear cells that were cultured in suspension and immobilized in alginate microcarriers for 24 h were investigated. The localization and accumulation of Aβ40 and rAβ42 peptides in cells, as well as quantitative determination of the concentration of Aβ40 peptide, TNFα and IL-1β cytokines, was performed by intravital fluorescence imaging. The results were qualitatively similar for both cell models. It was determined that the content of TNFα and Aβ40 in the absence of rAβ42 in the incubation medium did not change for 24 h after incubation, and the content of IL-1β was lower compared to the cells that were not incubated. Incubation of cells in vitro with exogenous rAβ42 led to an increase in the intracellular content of TNFα and Aβ40, and no accumulation of IL-1β in cells was observed. The accumulation of Aβ40 in the cytoplasm was accompanied by the aggregation of rAβ42 on the outer surface of the cell plasma membrane. It was shown that the basic levels of indicators and the intensity of the response of immobilized cells to an exogenous stimulus were significantly greater than those of cells in suspension. To explore whether non-neuronal cells effects in alginate microcarriers were cell-matrix adhesion mediated, we tested the effect of blocking β1 integrins on proamyloidogenic and proinflammation cellular state. Immobilization within alginate hydrogels after incubation with the β1 integrins blocking antibodies showed a remarkable inhibition of TNFα and Aβ40 accumulation in rAβ42-treated cells. It can be concluded that activation of signal transduction and synthesizing activity of a portion of mononuclear cells of human peripheral blood is possible (can significantly increase) in the presence of cell-matrix adhesion.

Plectin plays a role in the migration and volume regulation of astrocytes: a potential biomarker of glioblastoma

BackgroundThe expression of aquaporin 4 (AQP4) and intermediate filament (IF) proteins is altered in malignant glioblastoma (GBM), yet the expression of the major IF-based cytolinker, plectin (PLEC), and its contribution to GBM migration and invasiveness, are unknown. Here, we assessed the contribution of plectin in affecting the distribution of plasmalemmal AQP4 aggregates, migratory properties, and regulation of cell volume in astrocytes.MethodsIn human GBM, the expression of glial fibrillary acidic protein (GFAP), AQP4 and PLEC transcripts was analyzed using publicly available datasets, and the colocalization of PLEC with AQP4 and with GFAP was determined by immunohistochemistry. We performed experiments on wild-type and plectin-deficient primary and immortalized mouse astrocytes, human astrocytes and permanent cell lines (U-251 MG and T98G) derived from a human malignant GBM. The expression of plectin isoforms in mouse astrocytes was assessed by quantitative real-time PCR. Transfection, immunolabeling and confocal microscopy were used to assess plectin-induced alterations in the distribution of the cytoskeleton, the influence of plectin and its isoforms on the abundance and size of plasmalemmal AQP4 aggregates, and the presence of plectin at the plasma membrane. The release of plectin from cells was measured by ELISA. The migration and dynamics of cell volume regulation of immortalized astrocytes were assessed by the wound-healing assay and calcein labeling, respectively.ResultsA positive correlation was found between plectin and AQP4 at the level of gene expression and protein localization in tumorous brain samples. Deficiency of plectin led to a decrease in the abundance and size of plasmalemmal AQP4 aggregates and altered distribution and bundling of the cytoskeleton. Astrocytes predominantly expressed P1c, P1e, and P1g plectin isoforms. The predominant plectin isoform associated with plasmalemmal AQP4 aggregates was P1c, which also affected the mobility of astrocytes most prominently. In the absence of plectin, the collective migration of astrocytes was impaired and the dynamics of cytoplasmic volume changes in peripheral cell regions decreased. Plectin’s abundance on the plasma membrane surface and its release from cells were increased in the GBM cell lines.ConclusionsPlectin affects cellular properties that contribute to the pathology of GBM. The observed increase in both cell surface and released plectin levels represents a potential biomarker and therapeutic target in the diagnostics and treatment of GBMs.