Transmembrane Research Articles

CSPG4 involvement in endometrial decidualization contributes to the pathogenesis of preeclampsia.

Preeclampsia (PE) is a condition of pregnancy in which symptoms of hypertension develop after 20weeks of gestation. it can lead to placental dysfunction, maternal and perinatal mortality and morbidity. The incidence of PE is increasing, posing a serious threat to the lives of pregnant women and their unborn children. Currently, most of the research on the pathogenesis of PE has focused on placenta, However, maternal decidualization is the basis for placental formation and growth. CSPG4 (Chondroitin sulfate proteoglycan 4) is a transmembrane protein that plays a role in cell proliferation, invasion, and migration. However, its function during decidualization is not yet understood. In this study, we investigated the role of CSPG4 and found that its expression was significantly down-regulated in the decidual tissue of patients with severe PE compared to normal pregnant women. During artificially induced decidualization, CSPG4 expression was significantly increased. Knockdown of CSPG4 by siRNA inhibited decidualization, which, in turn, inhibited the invasion of trophoblast cells. In both pseudopregnant and pregnant mice, endometrial stromal cells proliferated rapidly and Cspg4 expression increased during decidualization. Therefore, we believe that CSPG4 plays a crucial role in the process of decidualization. The defect in decidualization caused by abnormal CSPG4 expression could lead to insufficient trophoblast invasion, ultimately contributing to the occurrence of PE.

Therapeutic BCG vaccine protects against long COVID: The BATTLE randomized clinical trial.

Bacillus Calmette-Guérin (BCG) injected during the COVID-19 convalescence period was safe and enhanced recovery from anosmia and dysgeusia in the acute phase. To report the long-term results of the BATTLE trial, BCG vaccine in adults with mild COVID-19. Design: Double-blind, placebo-controlled, randomized (1:1) clinical trial. BCG intradermal vaccine and placebo. A total of 157 BCG and 142 placebo recipients participated in the 6-month follow-up, and 97 BCG and 95 placebo recipients participated in the 12-month follow-up. Long COVID symptoms and mechanistic analyses. BCG reduced hearing problems at 6 months (odds ratio [OR]=0.26) and sleeping, concentration, memory, and vision problems at 12 months (OR=0.45, 0.36, 0.38, and 0.36, respectively). Sensitivity analyses confirmed that long COVID-19 symptoms were reduced at the 6- and 12-month follow-ups (p=0.010 and 0.031, respectively). BCG's crossover interaction paradoxically increased hair loss in women and decreased it in men at 6 months (p=0.032). BCG immunomodulation is likely mediated through inhibition of Fas ligand expression in the blood and increased induction of IL6, IL10, interferon-induced transmembrane protein 3, and angiotensin-converting enzyme 2 in cultured human macrophages. Long-term follow-up of the BATTLE trial participants revealed that BCG protects against long COVID development if administered within the COVID-19 convalescence period. The response to BCG was subject-specific, including a paradoxical crossover interaction based on sex. Not tested for previous mycobacterial exposure; loss to follow-up, particularly at 12 months.

A fluorescence and SERS dual-mode biosensor for quantification and imaging of Mucin1 in living cells

Mucin1 (MUC1) is a cell surface transmembrane protein overexpressed in multiple types of tumor cells, which is generally considered as a tumor-associated biomarker. Thus, quantifying and imaging of MUC1 in tumor cells is of great significance for the diagnosis and biological therapy of tumors. Herein, a fluorescence (FL) and surface-enhanced Raman scattering (SERS) dual-mode biosensor was developed for sensitive detection and imaging of MUC1 in living cells. The FL-SERS biosensor was based on self-assembled satellite structures mediated by the competition of MUC1 and complementary DNA modified on Au nanostars (AuNS-cDNA-PEG) with aptamer modified on quantum dots (QD-PDDA-Apt). This biosensor achieved dual-mode quantification and imaging of MUC1 by quenching the FL signal of QD and significantly enhancing the SERS signal of PDDA through the metal FL quenching effect and hotspot effect of AuNS, respectively. The dual-mode biosensor exhibited high sensitivity to MUC1, with a detection limit of 1.19 fg/mL under FL mode and 1.16 fg/mL under SERS mode. Moreover, this biosensor displayed good selectivity, nice biological stability and low cytotoxicity. Importantly, this biosensor possessed an excellent MUC1 dual-mode imaging capability with high specificity in different tumor cells, providing a new idea for clinical diagnosis of tumors.

Knockout of TMEM206 in mice associated with a loss of corneal transparency.

To investigate the role of transmembrane protein 206 (TMEM206) in corneal edema in mice by knockout the TMEM206 gene using CRISPR/Cas9 editing technology. TMEM206-knockout mice were generated using the CRISPR-Cas9 system. Variations in ophthalmic pathology were observed using slit lamp microscope and optical coherence tomography (OCT), intraocular pressure (IOP) was measured using a TonoLab Rebound Tonometer, and the ultrastructure of the corneal was observed using a transmission electron microscope. Corneal opacity was observed in 4/18 homozygous TMEM206-/- mice whereas a similar change was not observed in heterozygous TMEM206+/- mice and wild-type littermates. OCT examination showed that the mean central cornea thickness was 125±5.4 µm in 4 homozygous TMEM206-/- mice developed corneal edema and 115±1.2 µm in wild-type mice (t=3.468, P<0.05) at 43wk. The mean IOP was 12.08±0.07 mm Hg in four right eyes with corneal edema and 12.03±0.03 mm Hg in three normal left eyes (P>0.05). Transmission electron microscopy revealed a disruption in the organization of the collagen fibrils in the central part of the cornea in homozygous TMEM206-/- mice. TMEM206 is associated with corneal edema which caused organizational disruption of collagen fibrils in corneas of mice.

Nicotinamide Riboside Ameliorates Fructose-Induced Lipid Metabolism Disorders in Mice by Activating Browning of WAT, and May Be Also Related to the Regulation of Gut Microbiota.

This study aims to observe the preventive effect of nicotinamide riboside (NR) on fructose-induced lipid metabolism disorders and explore its mechanism. Male C57BL/6J mice were fed a 20% fructose solution and given 400 mg/kg NR daily by gavage for 10 weeks. The results indicated that NR supplementation significantly reduced the body weight, liver weight, white adipose tissue (WAT) weight, serum, and hepatic lipid levels. NR upregulated the protein expression levels of sirtuin-1 (SIRT1), AMP-activated protein kinase (AMPK), PR domain containing 16 (PRDM16), uncoupling protein 1 (UCP1), peroxisome proliferator-activated receptor-gamma coactiva-tor-1-alpha (PGC-1α), nuclear respiratory factor 1-encoding gene (NRF1), mitochondrial transcription factor A (TFAM), cluster of differentiation 137 (CD137), transmembrane protein 26 (TMEM26), and T-box 1 (TBX1). Moreover, NR enhanced the Actinobacteria and Enterorhabdus abundance. Spearman's correlation analysis revealed that significant correlations exist between Firmicutes, Bacteroidetes, and Erysipelotrichaceae with browning-related indicators. In conclusion, NR could alleviate lipid metabolic abnormalities induced by fructose through activating SIRT1/AMPK-mediated browning of WAT. The mechanism by which NR improves fructose-induced lipid metabolism disorders may also be associated with the modulation of intestinal flora.

Abstract A042: Desmosome mutations in the microenvironment regulate melanoma proliferation

Abstract Desmosomes are transmembrane protein complexes critical for cell-cell adhesion in epithelial tissues and other cell types. In skin cancers like melanoma, epithelial cells called keratinocytes are the predominant cells in the primary melanoma microenvironment, yet it remains largely unknown whether this cell type is subject to genetic alterations during melanoma development and its role in early melanoma progression. In this study, using an integrative analysis of tumor mutations and protein biophysical interactions, we identify a high frequency of genetic alterations in desmosome-related genes in human cancers, with the highest occurrences in cutaneous melanoma. Notably, over 70% of cutaneous melanoma cases exhibit mutations in desmosome genes and desmosome gene mutations are associated with lowered expression of the desmosome complex in melanoma. Using spatial transcriptomics and immunohistochemistry analyses, we find that the decrease in desmosome gene expression predominantly occurs in keratinocytes within the tumor microenvironment rather than in the melanoma cells themselves. To study the functional significance of this alteration in melanoma development, we used a human melanoma/keratinocyte co-culture system using primary melanoma cell lines. Our studies show that knockdown of desmosome genes in keratinocytes markedly increased proliferation of adjacent melanoma cells in melanoma/keratinocyte co-cultures. Additionally, melanoma cell proliferation is enhanced when exposed to media preconditioned by desmosome-deficient keratinocytes. These observations suggest that the gradual accumulation of mutations in desmosome genes within neighboring keratinocytes may create a conducive environment that primes melanoma cells for neoplastic transformation. This study underscores the critical role of genetic alterations in the tumor microenvironment in melanoma progression and highlights the potential for targeting desmosome-related pathways in therapeutic strategies against melanoma. Citation Format: Mohita Tagore. Desmosome mutations in the microenvironment regulate melanoma proliferation [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A042.

Gene expression patterns of the LDL receptor and its inhibitor Pcsk9 in the adult zebrafish brain suggest a possible role in neurogenesis

AbstractThe low‐density lipoprotein receptor (LDLr) is the first member of a closely related transmembrane protein family. It is known for its involvement in various physiological processes, mainly in the regulation of lipid metabolism, especially in the brains of mammals and zebrafish. In zebrafish, two ldlr genes (ldlra and b) have been identified and their distribution in the brain is not well documented. Recently, the roles of ldlr and its inhibitor pcsk9 in regenerative process after telencephalic brain injury have been discussed. In this study, we explored the expression patterns of these genes during zebrafish development. We found that ldlra expression was detected at the end of the pharyngula period (48 hpf) and increased during the larval stage. Conversely, ldlrb expression was observed from zygotic to larval stages. Using techniques like in situ hybridization and taking advantage of transgenic fish, we demonstrated the widespread distribution of ldlra, ldlrb and pcsk9 in the brain of adult zebrafish. Specifically, these genes were expressed in neurons and neural stem cells and also at lower levels in endothelial cells. As expected, intraperitoneal injection of fluorescent‐labelled LDLs resulted in their uptake by cerebral endothelial cells in a homeostatic context, whereas they diffused within the brain parenchyma after telencephalic injury. However, after intracerebroventricular injections into animals, LDL particles were not taken up by neural stem cells. In conclusion, our results provide additional evidence for LDLr expression in the brain of adult zebrafish. These results raise the question of the role of LDLr in the cholesterol/lipid imbalance in cerebral complications.

SLITRK2 as a prognostic and immunological biomarker in gastric cancer

BackgroundSLIT and NTRK-like protein 2 (SLITRK2) encodes a transmembrane protein that regulates neurite outgrowth. Some studies have demonstrated that SLITRK2 overexpressed in glioma. But the expression pattern, prognostic value and immunologic function of SLITRK2 in tumors remains unknown.MethodsThe expression pattern of SLITRK2 among pan-cancers was examined through the Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx). We analyzed the association between SLITRK2 expression level and tumor stage among pan-cancers. Kaplan–Meier survival analysis was utilized to investigate the prognostic relevance of SLITRK2 across 33 different types of cancers. Moreover, the correlations among SLITRK2 expression, immune cell infiltration, immunomodulatory related genes, tumor mutation burden (TMB), microsatellite instability (MSI) were evaluated. The relationship between SLITRK2 expression and crucial genes mutations was also illustrated. By using tissue microarray (TMA), the expression of SLITRK2 in 89 paired gastric cancer (GC) tissues was investigated.ResultsOur study indicated that SLITRK2 expression varied across cancers. Elevated SLITRK2 expression was positively related to advanced tumor stage, poor overall survival (OS) and reduced disease-free survival (DFS). Bioinformatic analyses underscore SLITRK2’s role in immune response, with its expression significantly tied to immune cell infiltration and marker expression. Based on TMA data, SLITRK2 expression level was positively associated with differentiation, lymph node metastasis, AJCC stage, TNM stage, and poor survival outcome in GC patients.ConclusionOur findings provided that SLITRK2 may function as a biomarker by regulating immune cell infiltration. In addition, we verified that high SLITRK2 expression was correlated with poor prognosis in GC.

The T4bSS of Legionella features a two-step secretion pathway with an inner membrane intermediate for secretion of transmembrane effectors.

To promote intracellular survival and infection, Legionella spp. translocate hundreds of effector proteins into eukaryotic host cells using a type IV b protein secretion system (T4bSS). T4bSS are well known to translocate soluble as well as transmembrane domain-containing effector proteins (TMD-effectors) but the mechanisms of secretion are still poorly understood. Herein we investigated the secretion of hydrophobic TMD-effectors, of which about 80 were previously reported to be encoded by L. pneumophila. A proteomic analysis of fractionated membranes revealed that TMD-effectors are targeted to and inserted into the bacterial inner membranes of L. pneumophila independent of the presence of a functional T4bSS. While the T4bSS chaperones IcmS and IcmW were critical for secretion of all tested TMD-effectors, they did not influence inner membrane targeting of these proteins. As for soluble effector proteins, translocation of all investigated TMD-effectors depended on a C-terminal secretion signal. A deeper analysis of the TMD-effector SidF showed that this signal needed to be presented towards the cytoplasmic side of the inner membrane and that a small periplasmic loop was required for efficient translocation. We propose that strongly hydrophobic TMD-effectors are secreted in a two-step secretion process: Initially, an inner membrane intermediate is formed, that is extracted towards the cytoplasmic side, possibly by the help of the type IV coupling protein complex and subsequently secreted into eukaryotic host cells by the T4bSS core complex. Overall, our study highlights the amazing versatility of T4bSS to secrete soluble and TMD-effectors from different subcellular locations of the bacterial cell.

The acyltransferase transmembrane protein 68 regulates breast cancer cell proliferation by modulating triacylglycerol metabolism

BackgroundCellular carcinogenesis is often marked by the accumulation of lipid droplets (LDs) due to reprogrammed lipid metabolism. LDs are dynamic organelles that primarily store intracellular triacylglycerol (TAG) and cholesteryl esters (CEs). Transmembrane protein 68 (TMEM68), a potential modifier of human breast cancer risk and outcomes, functions as a diacylglycerol acyltransferase, synthesizing TAG. However, the specific roles of TMEM68 in breast cancer cells remain unclear.MethodsGene expression profiling interactive analysis and survival analysis were conducted. TMEM68 was overexpressed or knockdown in breast cancer cells to assess its impact on cell proliferation, migration and invasion. Targeted quantitative lipidomic analysis and quantitative polymerase chain reaction were used to profile lipid alterations and examine gene expression related to lipid metabolism following changes in TMEM68 levels.ResultsTMEM68 gene was upregulated in breast cancer patients and higher TMEM68 levels were associated with poorer survival outcomes. Overexpression of TMEM68 increased breast cancer cell proliferation and invasion, whereas knockdown had minimal or no impact on reducing proliferation and invasion. Altering TMEM68 levels resulted in corresponding changes in TAG levels and cytoplasmic LDs, with overexpression increasing both and knockdown decreasing them. Lipidomic analysis revealed that TMEM68 regulated TAG levels and altered diacylglycerol content in breast cancer cells. Additionally, TMEM68 influenced the metabolism of glycerophospholipids, CEs and acylcarnitines. TMEM68 also modified the expression of key genes encoding enzymes related to neutral lipid metabolism, including TAG and CEs.ConclusionsTMEM68 is highly expressed in breast cancer and negatively correlated with survival. Its overexpression promotes breast cancer cell proliferation while knockdown has varied effects depending on TMEM68 levels. TMEM68 regulates intracellular TAG and LDs contents along with alterations in glycerophospholipids. These findings suggest that TMEM68 may drive breast cancer cells proliferation by modulating TAG and LD content.

The Dystrophin-Dystroglycan complex ensures cytokinesis efficiency in Drosophila epithelia.

Cytokinesis physically separates daughter cells at the end of cell division. This step is particularly challenging for epithelial cells, which are connected to their neighbors and to the extracellular matrix by transmembrane protein complexes. To systematically evaluate the impact of the cell adhesion machinery on epithelial cytokinesis efficiency, we performed an RNAi-based modifier screen in the Drosophila follicular epithelium. Strikingly, this unveiled adhesion molecules and transmembrane receptors that facilitate cytokinesis completion. Among these is Dystroglycan, which connects the extracellular matrix to the cytoskeleton via Dystrophin. Live imaging revealed that Dystrophin and Dystroglycan become enriched in the ingressing membrane, below the cytokinetic ring, during and after ring constriction. Using multiple alleles, including Dystrophin isoform-specific mutants, we show that Dystrophin/Dystroglycan localization is linked with unanticipated roles in regulating cytokinetic ring contraction and in preventing membrane regression during the abscission period. Altogether, we provide evidence that, rather than opposing cytokinesis completion, the machinery involved in cell-cell and cell-matrix interactions has also evolved functions to ensure cytokinesis efficiency in epithelial tissues.

Associations Between Urinary Phthalate Metabolites and Decreased Serum α-Klotho Level: A Cross-Sectional Study Among US Adults in Middle and Old Age.

Phthalates are widely used chemicals with ubiquitous human exposure. Evidence indicated that phthalate exposure was associated with an increased risk of aging-related diseases. Klotho is a transmembrane protein with anti-aging functions, and its association with phthalates remains unknown. To find the association between phthalate exposure and serum α-Klotho, a cross-sectional study was performed in 4482 adults (40-79 years old) who completed the National Health and Nutrition Examination Survey (NHANES) (2007-2016). As shown in the results of multivariable linear regression analyses, mono(carboxynonyl) phthalate (MCNP) and mono-n-butyl phthalate (MBP) were inversely associated with α-Klotho, and the regression coefficients of MCNP and MBP were -1.14 (95% confidence interval (CI): -2.00, -0.27) and -0.08 (95% CI: -0.14, -0.02). Subgroup analyses based on the quartiles of each phthalate metabolite showed that both MCNP and MBP were only inversely associated with α-Klotho in the subgroups of the highest levels. For mono-isobutyl phthalate (MIBP), the inverse association with α-Klotho was only statistically significant in the subgroup of the lowest level, and the regression coefficient was -26.87 (95% CI: -52.53, -1.21). Our findings suggest that α-Klotho might be involved in the association of phthalate exposure with aging-related diseases. Future research investigating the causality between phthalates and α-Klotho and its underlying mechanisms is encouraged.

Structure of scavenger receptor SCARF1 and its interaction with lipoproteins.

SCARF1 (scavenger receptor class F member 1, SREC-1 or SR-F1) is a type I transmembrane protein that recognizes multiple endogenous and exogenous ligands such as modified low-density lipoproteins (LDLs) and is important for maintaining homeostasis and immunity. But the structural information and the mechanisms of ligand recognition of SCARF1 are largely unavailable. Here, we solve the crystal structures of the N-terminal fragments of human SCARF1, which show that SCARF1 forms homodimers and its epidermal growth factor (EGF)-like domains adopt a long-curved conformation. Then, we examine the interactions of SCARF1 with lipoproteins and are able to identify a region on SCARF1 for recognizing modified LDLs. The mutagenesis data show that the positively charged residues in the region are crucial for the interaction of SCARF1 with modified LDLs, which is confirmed by making chimeric molecules of SCARF1 and SCARF2. In addition, teichoic acids, a cell wall polymer expressed on the surface of gram-positive bacteria, are able to inhibit the interactions of modified LDLs with SCARF1, suggesting the ligand binding sites of SCARF1 might be shared for some of its scavenging targets. Overall, these results provide mechanistic insights into SCARF1 and its interactions with the ligands, which are important for understanding its physiological roles in homeostasis and the related diseases.

Ion channels in acinar cells in acute pancreatitis: crosstalk of calcium, iron, and copper signals.

The initial stages of acute pancreatitis (AP) are characterized by a significant event - acinar ductal metaplasia (ADM). This process is a crucial feature of both acute and chronic pancreatitis, serving as the first step in the development of pancreatic cancer. Ion channels are integral transmembrane proteins that play a pivotal role in numerous biological processes by modulating ion flux. In many diseases, the expression and activity of ion channels are often dysregulated. Metal ions, including calcium ions (Ca2+), ferrous ions (Fe2+), and Copper ions (Cu2+), assume a distinctive role in cellular metabolism. These ions possess specific biological properties relevant to cellular function. However, the interactions among these ions exacerbate the imbalance within the intracellular environment, resulting in cellular damage and influencing the progression of AP. A more in-depth investigation into the mechanisms by which these ions interact with acinar cells is essential for elucidating AP's pathogenesis and identifying novel therapeutic strategies. Currently, treatment for AP primarily focuses on pain relief, complications prevention, and prognosis improvement. There are limited specific treatments targeting acinous cell dedifferentiation or ion imbalance. This study aims to investigate potential therapeutic strategies by examining ion crosstalk within acinar cells in the context of acute pancreatitis.

The CLEC3B inhibits cellular proliferation and metastasis of cholangiocarcinoma through Wnt/β-catenin pathway.

Cholangiocarcinoma (CCA) is a cancer of the biliary system, including intrahepatic and extrahepatic cholangiocarcinoma, and is highly aggressive. C-type lectins family member 3b (CLEC3B) is a Ca2+ binding transmembrane protein with different biological functions in a variety of cancers. The objective of this study was to explore the biological function of CLEC3B in CCA. The CLEC3B gene was identified using the TCGA database and survival analysis of the cholangiocarcinoma clinical cohort. The expression CLEC3B cholangiocarcinoma and correlation with prognosis was investigated in our patient cohort. The effects of CLEC3B on proliferation, apoptosis, migration and invasion were verified in QBC939 and HUCCT1 cells. The effect of CLEC3B on the tumor formation was proved by xenograft tumor model in nude mice. The signaling pathway of CLEC3B in regulating CCA progression was further analyzed RNA sequencing and western blot. CLEC3B was decreased in the cholangiocarcinoma in the database. The mRNA and protein expression level of CLEC3B were significantly lower and correlated with poor overall survival in cholangiocarcinoma of our patient cohort. In vitro experiments proved that overexpression of CLEC3B can inhibit proliferation, migration and invasion in bile duct cancer cells. The CLEC3B was correlated with epithelial-mesenchymal transition and apoptosis. The calcium could promote the biological function of CLEC3B. The vivo study indicated that CLEC3B inhibited tumor formation. RNA sequencing indicating CLEC3B may transduce signal through e Wnt/β-catenin signaling pathway. The CLEC3B inhibits cellular proliferation and migration of cholangiocarcinoma through the Wnt/β-catenin pathway.

The Antiviral Effects of Heat-Killed Lactococcus lactis Strain Plasma Against Dengue, Chikungunya, and Zika Viruses in Humans by Upregulating the IFN-α Signaling Pathway.

The growing risk of contracting viral infections due to high-density populations and ecological disruptions, such as climate change and increased population mobility, has highlighted the necessity for effective antiviral treatment and preventive measures against Dengue virus (DENV), Chikungunya virus (CHIKV), and Zika virus (ZIKV). Recently, there has been increasing attention on the use of probiotics as a potential antiviral option to reduce virus infections. The present study aimed to assess the immunomodulatory effects of heat-killed Lactococcus lactis strain plasma (LC-Plasma) on peripheral blood mononuclear cells (PBMCs) and its subsequent antiviral response against DENV, CHIKV, and ZIKV. To evaluate the immunomodulatory effects of LC-Plasma on PBMCs isolated from healthy individuals, PBMCs were cultured at a density of 2 × 105 cells/well and stimulated with 10 µg/mL of LC-Plasma. LC-plasma-stimulated PBMCs demonstrated elevated interferon-alpha (IFN-α) production and cluster of differentiation 86 (CD86) and human leukocyte antigen-DR isotype (HLA-DR) upregulation, potentially linked to plasmacytoid dendritic cell (pDC) activation. The replication of DENV, CHIKV, and ZIKV was dose-dependently inhibited when Huh-7 cells were stimulated with LC-Plasma-stimulated PBMC supernatant (LCP Sup). IFN-stimulated gene (ISG) expression, including IFN-stimulated gene 15 (ISG15), IFN-stimulated exonuclease gene 20 (ISG20), IFN-induced transmembrane protein 1 (IFITM-1), myxovirus resistance protein A (MxA), and radical S-adenosyl methionine domain-containing protein 2 (RSAD2), was significantly upregulated in LCP Sup-stimulated Huh-7 cells. Findings from this study indicate that LC-Plasma has the potential to induce IFN-α production, leading to an enhancement in the expression of ISGs and contributing to a broad-spectrum antiviral response. Thus, LC-Plasma may serve as a rational adjunctive treatment to ameliorate viral diseases, warranting future clinical trials.

Identification of linear B cell epitopes on the E146L protein of African swine fever virus with monoclonal antibodies

The outbreak and spread of African swine fever virus (ASFV) have caused considerable economic losses to the pig industry worldwide. Currently, to promote the development of effective ASF vaccines, especially subunit vaccines, more antigenic protein targets are urgently needed. In this work, six transmembrane proteins (I329L, E146L, C257L, EP153R, I177L, and F165R) were expressed in mammalian cell lines and screened with pig anti-ASFV serum. It was found that the E146L protein was an immunodominant protein antigen among the six selected proteins. Moreover, the E146L protein induced antibody responses in all immunized pigs. To gain insight into the antigenic characteristics of the E146L protein, three monoclonal antibodies (mAbs; 12H12, 15G1, and 15H10) were generated by immunizing BALB/c mice with the purified E146L protein. The epitopes of the mAbs were further finely mapped through a peptide fusion protein expression strategy. Finally, the epitopes of the mAbs were identified as 48PDESSIAYMRFRN61 of the mAb 12H12, 138TLTGLQRII146 of the mAb 15G1, and 30GWSPFKYSKGNT41 of the mAb 15H10. Furthermore, the epitope of mAb 15H10 was validated as the immunodominant epitope with ASFV-infected pig sera. The chemically synthesized mAb 15H10 epitope peptide (EP1) exhibited the most extensive immunoreactivity with artificially or naturally ASFV-infected pig sera. The epitope 15H10 is located on the surface of the E146L protein and is highly conserved. These findings provide insight into the structure and function of the E146L protein of ASFV.

Abstract 4123639: Tumor Endothelial Marker 1 is Upregulated in Atrial Cardiac Fibroblasts in Atrial Fibrillation and Correlates With Atrial Fibrosis

Background: Tumor endothelial marker 1 (TEM1 or CD248) is a transmembrane protein found in activated mesenchymal cells during embryo development. Its expression level becomes very low or undetectable in adult tissue. Re-expression of TEM1 in fibroblasts has been reported in hepatic, renal, and pulmonary fibrosis. We evaluated atrial TEM1 expression in atrial fibrillation (AF) and its relation with atrial fibrosis. Methods and Results: All data were presented as mean±standard error. Left atrial (LA) tissues were collected from 30 patients (pts) (mean age 64.0±1.6 yrs, male 76.7%) with AF underwent cardiac surgery (mitral and/or tricuspid valve surgery 96.7%) and maze procedure with LA appendage excision. Immunofluorescence (IF) staining showed TEM1 expressed in atrial cardiac fibroblasts (CFs) in the LA tissue of AF pts, but not in normal LA tissue (US Biomax). Primary human CFs were directly isolated from the LA tissue. IF staining showed TEM1 expression in the isolated CFs from AF pts but not in normal human CFs (ScienCell). Western blot could detect TEM1 expression in the LA tissues of all 30 AF pts. Sirius Red staining was used to visualize collagen and atrial fibrosis was quantified using a pixel-based method (Adobe Photoshop) by calculating the total numbers of red pixels and the percentage (%) of red pixels to total pixels. Atrial fibrosis was increased in AF pts compared with normal control (total number of red pixels: 160740±13066 vs. 50863±6357, % of red pixels to total pixels: 11.4±0.8 vs. 3.8±0.2%, all p&lt;0.001). There was a positive correlation between the expression level of TEM1 (ratio of TEM1/actin band density in western blot) with the severity of atrial fibrosis, including the total numbers of red pixels (r = 0.47, p = 0.01) and the % of red pixels to total pixels (r = 0.48, p = 0.01). TEM1 and galectin expression levels had positive correlation (r = 0.60, p &lt; 0.01). There were no correlations of atrial TEM1 expression with the LA diameter and CHA2DS2-VASc score. In vitro study showed TEM1 knockdown in cultured CFs with siRNA reduced cell proliferation (scrambled- vs TEM1-siRNA, BrdU absorbance: 1.84±0.05 vs 1.50±0.07, p&lt;0.05), increased starvation-induced cell apoptosis (cell death ELISA absorbance: 0.69±0.11 vs 2.88±0.05, p&lt;0.05) and decreased cell migration. Conclusions: There is increased TEM1 expression in atrial CFs in AF and its expression level is associated with atrial fibrosis. TEM1 expression changes the cell behaviors of CFs.

Abstract 4125419: Very-Low-Density Lipoprotein Induces Neuronal Growth with Increased Tyrosine Hydroxylase Expression through Cardiomyocyte-Secreted Exosomes in Metabolic Syndrome

Introduction: Metabolic syndrome (MS) is an important predisposing factor for atrial fibrillation, and is highly related to the autonomic nervous system. Triglyceride-rich very-low-density lipoprotein (VLDL) has specific VLDL receptors (VLDLR), which are abundantly expressed in cardiomyocytes. Hypothesis: VLDL affects neuronal growth through cardiomyocyte-secreted exosomes in metabolic syndrome. Goals/Aims: To determine whether exosomes from VLDL-incubated cardiomyocytes can alter neuronal proliferation. Methods/ Approach: HL-1 cardiomyocytes were incubated with VLDL isolated from human subjects with MS, and exosomes were extracted at the end of the incubation period. VLDLR knockdown HL-1 cells were used to determine the role of VLDLR in exosome secretion. The molecular contents of exosomes were determined by mass spectrometry, which identified neurotropic proteins only detected in the exosomes of MS VLDL-incubated cardiomyocytes. We selected transmembrane protein 14B (TMEM14B) to investigate its effect on neuronal proliferation in P19 cells. Results: Regardless of the knockdown or overexpression of VLDLR in HL-1 cardiomyocytes, exosomes isolated from after VLDL incubation were shown to cause 2.062-fold and 2.084-fold expression of tyrosine hydroxylase (TH) in P19 cells, relative to P19 cells induced by non-VLDL-treated HL-1 cardiomyocyte-secreted exosomes. A similar trend could be seen with HL-1 cardiomyocytes without VLDLR editing, as VLDL-incubated HL-1 cardiomyocytes produced exosomes that could cause 1.926-fold increase in P19 TH expression, relative to treatment by exosomes from HL-1 cardiomyocytes without VLDL-incubation. The P19 cells incubated with TMEM14B demonstrated increased neurite length, with an average of 2.342-fold neurite length when compared with that of control cells. Conclusions: VLDL in MS induced HL-1 cardiomyocytes to secrete exosomes containing neurotrophic molecules and increased dendritic growth of neuronal cells. This effect was VLDLR-independent and was partially caused by TMEM14B in the exosomes.

Aquaporins in Biliary Function: Pathophysiological Implications and Therapeutic Targeting.

Aquaporins (AQPs) are transmembrane proteins permeable to water and a series of small solutes. AQPs play a key role in pathways of hepatobiliary secretion at the level of the liver, bile ducts, and gallbladder. AQP8 and -9 are pivotal in facilitating the osmotic water movement of hepatic bile, which is composed of 95% water. In the biliary tract, AQP1 and -4 are involved in the rearrangement of bile composition by mechanisms of reabsorption/secretion of water. In the gallbladder, AQP1 and -8 are also involved in trans-epithelial bidirectional water flow with the ultimate goal of bile concentration. Pathophysiologically, AQPs have been indicated as players in several hepatobiliary disorders, including cholestatic diseases and cholesterol cholelithiasis. Research on AQP function and the modulation of AQP expression is in progress, with the identification of potent and homolog-specific compounds modulating the expression or inhibiting these membrane channels with promising pharmacological developments. This review summarizes the contribution of AQPs in physiological and pathophysiological stages related to hepatobiliary function.