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Non-small-cell lung cancer (NSCLC) is a major category of lung cancer, with high incidence and high mortality. Natural antisense long noncoding RNAs (lncRNAs) are involved in the development of NSCLC via their regulation of biological processes. However, the function of the lncRNA Hedgehog-interacting protein antisense RNA 1 (HHIP-AS1) in NSCLC is mostly unknown. In the study discussed here, HHIP-AS1 and HHIP levels were predicted based on the TCGA database, and detected via qRT-PCR or western blotting assays. Cell proliferation, migration, and invasion were measured via CCK-8 and trans-well assays. Related protein levels were measured using western blotting analysis. The results showed that HHIP-AS1 and HHIP levels are downregulated in NSCLC, and that low HHIP-AS1 and HHIP expression is associated with poor outcomes. HHIP-AS1 overexpression represses cell proliferation, migration, and invasion in NSCLC. HHIP-AS1 enhances HHIP expression and stability, and this effect is mediated by CELF2. HHIP silencing attenuates the suppressive roles of HHIP-AS1 in proliferation, migration, and invasion. As a result of these findings, it is concluded that HHIP-AS1 overexpression restrains proliferation, migration, and invasion of NSCLC cells by increasing HHIP stability via its targeting of CELF2.

SARS-CoV-2 spike (S) protein mediates virus attachment to the cells and fusion between viral and cell membranes. Membrane fusion is driven by mutual interaction between the highly conserved heptad-repeat regions 1 and 2 (HR1 and HR2) of the S2 subunit of the spike. For this reason, these S2 regions are interesting therapeutic targets for COVID-19. Although HR1 and HR2 have been described as transiently exposed during the fusion process, no significant antibody responses against these S2 regions have been reported. Here we designed chimeric proteins that imitate highly stable HR1 helical trimers and strongly bind to HR2. The proteins have broad inhibitory activity against WT B.1 and BA.1 viruses. Sera from COVID-19 convalescent donors showed significant levels of reactive antibodies (IgG and IgA) against the HR1 mimetic proteins, whereas these antibody responses were absent in sera from uninfected donors. Moreover, both inhibitory activity and antigenicity of the proteins correlate positively with their structural stability but not with the number of amino acid changes in their HR1 sequences, indicating a conformational and conserved nature of the involved epitopes. Our results reveal previously undetected spike epitopes that may guide the design of new robust COVID-19 vaccines and therapies., This work was supported by grants CV20.26565 from the Consejería de Economía y Conocimiento, Junta de Andalucía (Spain), PID2019.107515RB.C21 from the Spanish State Research Agency (SRA/10.13039/501100011033), and co-funded by ERDF/ESF, “A way to make Europe”/“Investing in your future”. The work performed in C.M.'s laboratory was supported by grants from ANRS (Agence Nationale de Recherches sur le SIDA et les hépatites virales), the Investissements d'Avenir program managed by the ANR under reference ANR-10-LABX-77 and EHVA (No. 681032, Horizon 2020). Work in S.B.'s laboratory was supported by grants from the Agence Nationale de la Recherche (ANR) (ANR-11-LABX-0070_TRANSPLANTEX), the INSERM (UMR_S 1109), the Institut Universitaire de France (IUF), all the University of Strasbourg (IDEX UNISTRA), the European Regional Development Fund (European Union) INTERREG V program (project no. 3.2 TRIDIAG) and MSD-Avenir grant AUTOGEN. We are grateful to the Spanish Radiation Synchrotron Source (ALBA), Barcelona, Spain and the European Synchrotron Radiation Facility (ESRF), Grenoble, France, for the provision of time and staff assistance at XALOC (ALBA) and ID30B and ID23-2 (ESRF) beamlines during diffraction data collection. We thank María Carmen Salinas-García for her assistance in carrying out the crystallization screenings. We also thank Pilar González-García for helping us with the statistical analysis.

Triggering receptor expressed on myeloid cells 2 (TREM2), which is typically expressed in microglia/macrophage, is a crucial receptor modulating the activation of immune cells. Recent single-cell RNA sequencing studies of the tumor microenvironment (TME) have found that TREM2 is highly expressed in several subgroups of tumor-associated macrophages (TAMs) with immunosuppressive activity. TREM2 knockout mice are more resistant to tumor growth than wild-type mice in multiple mice tumor models. But the function of TREM2 expression in TAMs of hepatocellular carcinoma (HCC) is still unclear. Here we used a self-complementary adeno-associated virus (scAAV) with macrophage tropism to efficiently knockdown TREM2 in TAMs in vivo and found that the growth of hepatocellular carcinoma was suppressed by TREM2 knockdown. Moreover, we found that TREM2 knockdown remodeled TAMs to an immune-stimulating status and enhanced the therapeutic effect of PD-1 immune-checkpoint blockade in HCC.

Background: 5XFAD humanized mutant mice and Trem2 knockout (T2KO) mice are two mouse models relevant to the study of Alzheimer’s disease (AD)-related pathology. Objective: To determine hippocampal transcriptomic and polyadenylation site usage alterations caused by genetic mutations engineered in 5XFAD and T2KO mice. Methods: Employing a publicly available single-nucleus RNA sequencing dataset, we used Seurat and Sierra analytic programs to identify differentially expressed genes (DEGs) and differential transcript usage (DTU), respectively, in hippocampal cell types from each of the two mouse models. We analyzed cell type-specific DEGs further using Ingenuity Pathway Analysis (IPA). Results: We identified several DEGs in both neuronal and glial cell subtypes in comparisons of wild type (WT) versus 5XFAD and WT versus T2KO mice, including Ttr, Fth1, Pcsk1n, Malat1, Rpl37, Rtn1, Sepw1, Uba52, Mbp, Arl6ip5, Gm26917, Vwa1, and Pgrmc1. We also observed DTU in common between the two comparisons in neuronal and glial subtypes, specifically in the genes Prnp, Rbm4b, Pnisr, Opcml, Cpne7, Adgrb1, Gabarapl2, Ubb, Ndfip1, Car11, and Stmn4. IPA identified three statistically significant canonical pathways that appeared in multiple cell types and that overlapped between 5XFAD and T2KO comparisons to WT, including ‘FXR/RXR Activation’, ‘LXR/RXR Activation’, and ‘Acute Phase Response Signaling’. Conclusion: DEG, DTU, and IPA findings, derived from two different mouse models of AD, highlight the importance of energy imbalance and inflammatory processes in specific hippocampal cell types, including subtypes of neurons and glial cells, in the development of AD-related pathology. Additional studies are needed to further characterize these findings.

Aim. The aim of this study is to identify cuproptosis-related lncRNAs and construct a prognostic model for pancreatic cancer patients for clinical use. Methods. The expression profile of lncRNAs was downloaded from The Cancer Genome Atlas database, and cuproptosis-related lncRNAs were identified. The prognostic cuproptosis-related lncRNAs were obtained and used to establish and validate a prognostic risk score model in pancreatic cancer. Results. In total, 181 cuproptosis-related lncRNAs were obtained. The prognostic risk score model was constructed based on five lncRNAs (AC025257.1, TRAM2-AS1, AC091057.1, LINC01963, and MALAT1). Patients were assigned to two groups according to the median risk score. Kaplan–Meier survival curves showed that the difference in the prognosis between the high- and low-risk groups was statistically significant. Multivariate Cox analysis showed that our risk score was an independent risk factor for pancreatic cancer patients. Receiver operator characteristic curves revealed that the cuproptosis-related lncRNA model can effectively predict the prognosis of pancreatic cancer. The principal component analysis showed a difference between the high- and low-risk groups intuitively. Functional enrichment analysis showed that different genes were involved in cancer-related pathways in patients in the high- and low-risk groups. Conclusion. The risk model based on five prognostic cuproptosis-related lncRNAs can well predict the prognosis of pancreatic cancer patients. Cuproptosis-related lncRNAs could be potential biomarkers for pancreatic cancer diagnosis and treatment.

GPM6A is a glycoprotein in endothelial cells, and its biological function in the development of hepatocellular carcinoma (HCC) is unknown. Through Affymetrix gene expression microarray and bioinformatic analysis, very low GPM6A expression was found in HCC tissue. The present study aims to explore the function and regulatory mechanism of GPM6A in HCC development and progression. Levels of GPM6A expression in HCC specimens from different disorders and various hepatoma cell lines were determined, and its role on cell proliferation was evaluated in hepatoma cells stably overexpressing GPM6A. Modulation of a specific microRNA (miRNA) on its expression and function was evaluated with miRNA mimetic transfection. Herein, it is reported that much lower GPM6A levels were found in HCC tissues than pericancerous liver tissues and correlated to a poor prognosis. GPM6A overexpression inhibited cell proliferation, suppressed colony formation, migration and invasion in two hepatoma cell types. Available evidence does not support that genetic and epigenetic dysregulation contributes significantly to GPM6A inactivation in HCC. Additional findings demonstrated that miR-96-5p acted directly on the 3'-UTR of the GPM6A gene and significantly decreased its mRNA and protein levels. MiR-96-5p transfection promoted proliferation, migration and invasion of SMMC-7721 and MHCC-97H hepatoma cells; whereas the function of oncogenic microRNA-96 was significantly inhibited in GPM6A-overexpressed hepatoma cells. In conclusion, GPM6A expression in HCC is commonly suppressed regardless its base disease types, and its low expression in HCC tissues is most likely attributed to upregulated miR-96-5p. GPM6A may function as a valuable biomarker for HCC progression and prognosis.

Primary dental pulp cells can be differentiated into odontoblasts, which are responsible for dentin formation and mineralization. Successful differentiation has been verified using a few markers. However, odontoblast-specific cell surface markers have not been fully studied yet. Previously, we have constructed monoclonal antibodies to identify cell surface molecules of odontoblasts by decoy immunization. Mass spectrometry confirmed that one of these antibodies, the anti-OD40 antibody, recognized LEucine PRoline-Enriched Proteoglycan 1 (LEPRE1) as a specific antigen. Among three alternative splicing isoforms of LEPRE1, this new antibody showed the most stable antigenic affinity for LEPRE1a, a canonical transcript. Anti-OD40 antibody could strongly bind to the cell surface as odontogenic differentiation progressed. Intracellular and cell surface accumulation of this protein completely disappeared during dentin maturation and mineralization. Overexpression and knock-down experiments showed that intracellular LEPRE1/OD40 should not accumulate too much and that it should move from ER to the cell surface for efficient odontogenic differentiation. Indeed, when LEPRE1/OD40 already located on the cell surface was blocked with antibody, odontogenic differentiation of human dental pulp cells was inhibited. In addition to its role in differentiation, our new antibody which only recognizes LEPRE1a isoform specifically will be useful in studying differentiated functions between LEPRE1a and other isoforms found in osteogenesis imperfecta and dentinogenesis imperfecta.

Interleukin 11 (IL-11) was discovered in 1990 in fibrocyte-like stromal cells of the bone marrow, but there has recently been an increased interest in the cytokine. Understanding the physiological roles of cytokines will allow their use as pharmacological agents in clinical practice. Studies have indicated that IL-11 affects the mechanism for the development of a number of pathologies of the nervous system. IL-11 plays a significant role in the central nervous system. The local expression of this cytokine by nerve cells has been observed. The current work summarizes the results of studies which found that the cytokine affects the mechanism of development of pathologies of the central nervous system. In the near future, this cytokine may be used clinically to fix the mechanisms that are involved in the development of pathological conditions of the nervous system.

Ambient air pollution aggravates the process of Alzheimer's disease (AD) pathology. Currently, the exact inflammatory mechanisms underlying these links from clinical research remain largely unclear.This study included 1,131 cognitively intact individuals from the Chinese Alzheimer's Biomarker and LifestylE database with data provided on cerebrospinal fluid (CSF) AD biomarkers (amyloid beta-peptide 42 [Aβ42], total tau [t-tau], and phosphorylated tau [p-tau]), neuroinflammatory (CSF sTREM2), and systemic inflammatory markers (high sensitivity C-reactive protein and peripheral immune cells). The 2-year averaged levels of ambient fine particulate matter with diameter 2.5 μm (PMAmbient 2-year averaged exposure of PMThese findings demonstrated a strong link between PM

The COVID-19 pandemic has boosted significant research in developing monoclonal antibodies (mAbs) to treat and prevent SARS-CoV-2 infection. Clinical trials have shown that mAbs are safe and effective in preventing hospitalization and death in patients with mild to moderate COVID-19 risk factors for progression. mAbs have also been effective for treating severe disease in seronegative patients and preventing COVID-19. So far, studies have been carried out in a largely unvaccinated population at a time when the omicron variant was not described. Future research should address these limitations and provide information on specific population groups, including immunosuppressed and previously infected individuals.

New drugs that precisely target the immune mechanisms critical for cytotoxic T lymphocyte (CTL) and natural killer (NK) cell driven pathologies are desperately needed. In this perspective, we explore the cytolytic protein perforin as a target for therapeutic intervention. Perforin plays an indispensable role in CTL/NK killing and controls a range of immune pathologies, while being encoded by a single copy gene with no redundancy of function. An immunosuppressant targeting this protein would provide the first-ever therapy focused specifically on one of the principal cell death pathways contributing to allotransplant rejection and underpinning multiple autoimmune and postinfectious diseases. No drugs that selectively block perforin-dependent cell death are currently in clinical use, so this perspective will review published novel small molecule inhibitors, concluding with

The SARS-CoV-2 Omicron variant (B.1.1.529 lineage) escapes antibodies that neutralize the ancestral virus. We tested human serum panels from participants with differing infection and vaccination status using a multiplex surrogate virus neutralization assay targeting 20 sarbecoviruses. We found that bat and pangolin sarbecoviruses showed significantly less neutralization escape than the Omicron variant. We propose that SARS-CoV-2 variants have emerged under immune selection pressure and are evolving differently from animal sarbecoviruses.

Myocarditis is a complex disease characterized by myocardial inflammatory cell infiltration. The purpose of our study was to investigate the gene and single-cell signature to explore the involvement of immune cells in myocarditis. Gene expressions merged from GSE35182 and GSE35182 datasets were subjected to differential expression gene (DEG) analysis and PPI network construction. The correlation analysis of DEGs with immune cell infiltration was performed. Single-cell RNA sequencing (scRNA-seq) was downloaded from GSE174458. A total of 58 DEGs were identified, including 51 DEGs upregulated and 7 DEGs downregulated in the myocarditis group compared with the control group. GO and KEGG enrichment analyses revealed that myocarditis triggered DEGs mainly involved in immune-related processes and pathways. PPI network analysis identified 20 central hub genes. Occurrence of myocarditis induced significant enrichment of conventional dendritic cell 2 (cDC2), plasmacytoid DC, and plasma cell in myocardial tissue. Mmp12, Gpnmb, and Atp6v0d2 expressions were positively correlated with cDC abundance, of which only Mmp1 and Gpnmb were shared with hub gene list. A total of 20972 cells in scRNA-seq yielded 26 cell clusters and annotated 9 cell types, including fibroblasts, neutrophils, stromal cells, monocytes, basophils, B cells, natural killer T cells, innate lymphoid cells, and T cells, and only proportion of natural killer T cells and monocytes were higher in the myocarditis than in control. Monocytes annotated 3 subclusters including DC, macrophage, and monocytes. Hub genes of Ctss, Mpeg1, Cybb, H2-Ab1, Ly86, CD74, and Lgals3 were highly expressed in monocytes cluster. Among DC-correlated DEGs, Mmp12 was mainly expressed in monocyte cluster, and Gpnmb was mainly expressed in fibroblast cluster, whereas Atp6v0d2 expression has a weaker signal and weaker cell preference. In conclusion, DC infiltration and its associated pivotal genes may be responsible for progression of myocarditis. Our study expands and provides novel information on the immune cell engagement of myocarditis.

Innate immune response constitutes the first line of defense against pathogens. Inflammatory responses involve close contact between different populations of cells. These adhesive interactions mediate migration of cells to sites of infection leading the effective action of cells within the lesions. Cell adhesion molecules are critical to controlling immune response mediating cell adhesion or chemotaxis, as well as coordinating actin-based cell motility during phagocytosis and chemotaxis. Recently, a newly discovered neuroplastin (Np) adhesion molecule is found to play an important role in the nervous system. However, there is limited information on Np functions in immune response. To understand how Np is involved in innate immune response, a mouse model of intraperitoneal infection was established to investigate the effect of Np on macrophage-mediated clearance of E. coli infection and its possible molecular mechanisms.Specific deficiency mice with Nptn gene controlling Np65 isoform were employed in this study. The expression levels of mRNA and proteins were detected by qPCR and western blot, or evaluated by flow cytometry. The expression level of NO and ROS were measured with their specific indicators. Cell cycle and apoptosis were detected by specific detection kits. Acid phosphatase activity was measured by flow cytometry after labelling with LysoRed fluorescent probe. Bone marrow derived macrophages (BMDMs) were isolated from bone marrow of mice hind legs. Cell proliferation was detected by CCK8 assay. Cell migration was measured by wound healing assay or transwell assay.The lethal dose of E. coli infection in Np65This study for the first time demonstrates that Np is involved in multi-function of phagocytes during bacterial infection, proposing that Np adhesion molecule plays a critical role in clearing pathogen infection in innate immunity.

Cystatin A (CSTA) is a cysteine protease inhibitor that is expressed highly during osteoporosis. However, the exact role of CSTA in osteoporosis remains unknown. In this study, we examined the role of CSTA in the formation, differentiation, and bone resorption of osteoclasts. We extracted bone marrow cells from 8-week-old wildtype mice to obtain RANKL and M-CSF-induced osteoclasts. We performed CSTA overexpression and knockdown experiments in the cells. We analyzed the role of CSTA in the process of osteoclasts by trap staining. In addition, we studied the contribution of CSTA to osteogenesis through the DAP12/TREM2 (DNAX-activating protein of 12 kDa/Triggering receptor expressed on myeloid cells-2) complex. We analyzed the role of CSTA in postmenopausal osteoporosis using OVX mouse models. We found that the silencing of CSTA inhibited the differentiation and formation of osteoclasts. The loss of CSTA weakened the expression of osteoclast marker genes. In contrast, overexpression of CSTA significantly increased differentiation and formation of osteoclasts and enhanced bone resorption. Immunofluorescence staining indicated that CSTA and DAP12 are co-expressed in osteoclasts, and the loss of either DAP12 or TREM2 inhibited osteoclast differentiation and bone resorption. Suppression of CSTA decreased DAP12 and TREM2 expression, whereas overexpression of CSTA rescued the loss of TREM2 expression caused by DAP12 knockdown. Co-immunoprecipitation and co-localization experiments indicated that CSTA interacted with DAP12. In addition, we found that injection of si-CSTA into OVX mice significantly improved bone parameters. Our research indicates that CSTA interacts with the DAP12/TREM2 complex and could be a potential targeted therapy for osteoporosis management.

Backgroundand aims: inflammation, particularly that mediated by bacterial components translocating from the gut to the liver and binding to toll-like receptors (TLRs), is central to cholestatic liver injury. The triggering receptor expressed on myeloid cells-2 (TREM-2) inhibits TLR-mediated signaling and exerts a protective role in hepatocellular injury and carcinogenesis. This study aims to evaluate the role of TREM-2 in cholestasis.Methods: TREM-2 expression was analyzed in the livers of pa-tients with primary biliary cholangitis (PBC) or primary scle-rosing cholangitis (PSC), and in mouse models of cholestasis. Wild-type (WT) and Trem-2 deficient (Trem-2-/-) mice were subjected to experimental cholestasis and gut sterilization. Pri-mary cultured Kupffer cells were incubated with lipopolysac-charide and/or ursodeoxycholic acid (UDCA) and inflammatory responses were analyzed.Results: TREM-2 expression was upregulated in the livers of patients with PBC or PSC, and in murine models of cholestasis. Compared to WT, the response to bile duct ligation (BDL)-induced obstructive cholestasis or alpha-naphtylisothiocyanate (ANIT)-induced cholestasis was exacerbated in Trem-2-/-mice. This was characterized by enhanced necroptotic cell death, in-flammatory responses and biliary expansion. Antibiotic treat-ment partially abrogated the effects observed in Trem-2-/-mice after BDL. Experimental overexpression of TREM-2 in the liver of WT mice downregulated ANIT-induced IL-33 expression and neutrophil recruitment. UDCA regulated Trem-1 and Trem-2 expression in primary cultured mouse Kupffer cells and damp-ened inflammatory gene transcription via a TREM-2-dependent mechanism.Conclusions: TREM-2 acts as a negative regulator of inflamma-tion during cholestasis, representing a novel potential thera-peutic target.Lay summary: Cholestasis (the reduction or cessation of bile flow) causes liver injury. This injury is exacerbated when gut-derived bacterial components interact with receptors (spe-cifically Toll-like receptors or TLRs) on liver-resident immune cells, promoting inflammation. Herein, we show that the anti-inflammatory receptor TREM-2 dampens TLR-mediated signaling and hence protects against cholestasis-induced liver injury. Thus, TREM-2 could be a potential therapeutic target in cholestasis., European Union (EU); Horizon 2020; European Research Council (ERC); Spanish Carlos III Health Institute (ISCIII); Fondo Europeo de Desarrollo Regional (FEDER); Diputación Foral de Gipuzkoa; Department of Health of the Basque Country; Euskadi RIS3; Department of Industry of the Basque Country; Junta de Castilla y Leon; La Caixa Scientific Foundation; Centro Internacional sobre el Envejecimiento; Fundació Marato TV3; Austrian Science Fund; German Research Foundation (DFG); German Ministry of Health (BMG); DEEP LIVER; Università Politecnica delle Marche; CRUK; MRC; Spanish Ministry of Economy and Competitiveness; MINECO; “Ramón y Cajal” Programme; Ministry of Universities ; University of the Basque Country; Instituto de Salud Carlos III (CIBERehd); Fundación Científica de la Asociación Española Contra el Cáncer (AECC Scientific Foundation); SFB-CRC 1382-Project A01

Familial British dementia (FBD) and familial Danish dementia (FDD) are autosomal dominant forms of dementia caused by mutations in the integral membrane protein 2B (ITM2B, also known as BRI2) gene. Secretase processing of mutant BRI2 leads to secretion and deposition of BRI2-derived amyloidogenic peptides, ABri and ADan that resemble APP/β-amyloid (Aβ) pathology, which is characteristic of Alzheimer's disease (AD). Amyloid pathology in FBD/FDD manifests itself predominantly in the microvasculature by ABri/ADan containing cerebral amyloid angiopathy (CAA). While ABri and ADan peptide sequences differ only in a few C-terminal amino acids, CAA in FDD is characterized by co-aggregation of ADan with Aβ, while in contrast no Aβ deposition is observed in FBD. The fact that FDD patients display an earlier and more severe disease onset than FBD suggests a potential role of ADan and Aβ co-aggregation that promotes a more rapid disease progression in FDD compared to FBD. It is therefore critical to delineate the chemical signatures of amyloid aggregation in these two vascular dementias. This in turn will increase the knowledge on the pathophysiology of these diseases and the pathogenic role of heterogenous amyloid peptide interactions and deposition, respectively. Herein, we used matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) in combination with hyperspectral, confocal microscopy based on luminescent conjugated oligothiophene probes (LCO) to delineate the structural traits and associated amyloid peptide patterns of single CAA in postmortem brain tissue of patients with FBD, FDD as well as sporadic CAA without AD (CAA+) that show pronounced CAA without parenchymal plaques. The results show that CAA in both FBD and FDD consist of N-terminally truncated- and pyroglutamate-modified amyloid peptide species (ADan and ABri), but that ADan peptides in FDD are also extensively C-terminally truncated as compared to ABri in FBD, which contributes to hydrophobicity of ADan species. Further, CAA in FDD showed co-deposition with Aβ x-42 and Aβ x-40 species. CAA+ vessels were structurally more mature than FDD/FBD CAA and contained significant amounts of pyroglutamated Aβ. When compared with FDD, Aβ in CAA+ showed more C-terminal and less N-terminally truncations. In FDD, ADan showed spatial co-localization with Aβ3pE-40 and Aβ3-40 but not with Aβx-42 species. This suggests an increased aggregation propensity of Aβ in FDD that promotes co-aggregation of both Aβ and ADan. Further, CAA maturity appears to be mainly governed by Aβ content based on the significantly higher 500/580 patterns observed in CAA+ than in FDD and FBD, respectively. Together this is the first study of its kind on comprehensive delineation of Bri2 and APP-derived amyloid peptides in single vascular plaques in both FDD/FBD and sporadic CAA that provides new insight in non-AD-related vascular amyloid pathology. Cover Image for this issue: https://doi.org/10.1111/jnc.15424.

Triggering receptor expressed on myeloid cells 2 (Trem2) is highly expressed on myeloid cells and is involved in cellular lipid homeostasis and inflammatory processes. Trem2 deletion in mice (Trem2

Messenger RNA (mRNA) holds great potential as a disease-modifying treatment for a wide array of monogenic disorders. Niemann-Pick disease type C1 (NP-C1) is an ultra-rare monogenic disease that arises due to loss-of-function mutations in the NPC1 gene, resulting in the entrapment of unesterified cholesterol in the lysosomes of affected cells and a subsequent reduction in their capacity for cholesterol esterification. This causes severe damage to various organs including the brain, liver, and spleen. In this work, we describe the use of NPC1-encoded mRNA to rescue the protein insufficiency and pathogenic phenotype caused by biallelic NPC1 mutations in cultured fibroblasts derived from an NP-C1 patient. We first evaluated engineering strategies for the generation of potent mRNAs capable of eliciting high protein expression across multiple cell types. We observed that “GC3” codon optimization, coupled with N1-methylpseudouridine base modification, yielded an mRNA that was approximately a thousand-fold more potent than wildtype, unmodified mRNA in a luciferase reporter assay, and consistently superior to other mRNA variants. Our data suggest that the improved expression associated with this design strategy was due in large part to the increased secondary structure of the designed mRNAs. Both codon optimization and base modification appear to contribute to increased secondary structure. Applying these principles to the engineering of NPC1-encoded mRNA, we observed a normalization in NPC1 protein levels after mRNA treatment, as well as a rescue of the mutant phenotype. Specifically, mRNA treatment restored the cholesterol esterification capacity of patient cells to wildtype levels, and induced a significant reduction in both unesterified cholesterol levels (>57% reduction compared to Lipofectamine-treated control in a cholesterol esterification assay) and lysosome size (157 μm2 reduction compared to lipofectamine-treated control). These findings show that engineered mRNA can correct the deficit caused by NPC1 mutations. More broadly, they also serve to further validate the potential of this technology to correct diseases associated with loss-of-function mutations in genes coding for large, complex, intracellular proteins.

Alzheimer's disease (AD) has multiple clinically and pathologically defined subtypes where the underlying causes of such heterogeneity are not well established. Rare TREM2 variants confer significantly increased risk for clinical AD in addition to other neurodegenerative disease clinical phenotypes. Whether TREM2 variants are associated with atypical clinical or pathologically defined subtypes of AD is not known. We studied here the clinical and pathological features associated with TREM2 risk variants in an autopsy-confirmed cohort. TREM2 variant cases were more frequently associated with non-amnestic clinical syndromes. Pathologically, TREM2 variant cases were associated with an atypical distribution of neurofibrillary tangle density with significantly lower hippocampal NFT burden relative to neocortical NFT accumulation. In addition, NFT density but not amyloid burden was associated with an increase of dystrophic microglia. TREM2 variant cases were not associated with an increased prevalence, extent, or severity of co-pathologies. These clinicopathological features suggest that TREM2 variants contribute to clinical and pathologic AD heterogeneity by altering the distribution of neurofibrillary degeneration and tau-dependent microglial dystrophy, resulting in hippocampal-sparing and non-amnestic AD phenotypes.

Patients with end-stage kidney disease (ESKD) exhibit an elevated cardiovascular risk. Chronic inflammation is one of the main mechanisms of cardiovascular disease (CVD). Lipopolysaccharide has been proposed as a link between systemic inflammation and CVD. Herein, we evaluated whether lipopolysaccharide-binding protein (LBP), a surrogate marker of lipopolysaccharide and consequent inflammation, is associated with cardiovascular events in ESKD.We performed a prospective cohort study of maintenance haemodialysis patients. Baseline serum LBP levels were categorized into tertiles and also modelled continuously for analyses. Cox regression methods were used to evaluate the association of serum LBP levels with cardiovascular events.A total of 360 haemodialysis patients were included in this analysis. During a median follow-up of 3.1 years, 90 (25.0%) patients had cardiovascular events. Patients in the upper tertile of serum LBP levels had a significantly greater risk of cardiovascular events [hazard ratio (HR) 4.87; 95% confidence intervals (CI), 2.12-11.15] than those in the lower tertile, independent of age, sex, hypertension, diabetes, CVD, dialysis vintage, body mass index, non-high-density lipoprotein cholesterol, albumin, phosphorus, high-sensitivity C-reactive protein, and interleukin-6. The association was consistent regardless of whether competing risk of death was accounted for (subdistribution HR 4.87; 95% CI, 1.96-12.11 for upper versus lower tertiles) or serum LBP was analysed as a continuous variable (HR 1.30; 95% CI, 1.02-1.66 per 1 SD increment).Serum LBP levels were independently associated with cardiovascular events in heomodialysis patients. LBP might serve as a novel biomarker for CVD in ESKD.

Variants of triggering receptor expressed on myeloid cells 2 (TREM2) are associated with an increased incidence of Alzheimer’s disease, as well as other neurodegenerative disorders. TREM2 is glycosylated in vitro and in vivo, but the significance of the modification is unknown. We previously established a sensitive and specific reporter cell model involving cultured Jurkat cells stably expressing a luciferase reporter gene and a gene encoding a TREM2DAP12 fusion protein to monitor TREM2-dependent signalling. In the present study, we prepared modified reporter cells to investigate the role of the N-glycans at N20 and N79. We show that the N-glycans at N79 have a requisite role in translocation of TREM2 to the cell surface, while the N-glycans at both N20 and N79 have a critical role in intracellular signal transduction. Our results indicate that structural changes to the TREM2 N-glycans may cause microglial dysfunction that contributes to the pathogenesis of neurodegenerative disorders and that maintaining the integrity of TREM2 N-glycosylation and the responsible glycosyltransferases may be a novel therapeutic strategy to treat these disorders.

Synaptic vesicle glycoprotein 2A (SV2A) is a transmembrane protein that binds levetiracetam and is involved in neurotransmission via an unknown mechanism. SV2A-immunoreactivity is reduced in animal models of epilepsy, and in postmortem hippocampi from patients with temporal lobe epilepsy. It is not known if other regions outside the hippocampus are affected in epilepsy, and whether SV2A expression is permanently reduced or regulated over time. In this study, we induced a generalized status epilepticus (SE) by systemic administration of lithium-pilocarpine to adult female rats. The brains from all animals experiencing SE were collected at different time points after the treatment. The radiotracer, [11C]-UCB-J, binds to SV2A with high affinity, and has been used for in vivo imaging as an a-proxy marker for synaptic density. Here we determined the level of tritiated UCB-J binding by semiquantitative autoradiography in the cerebral cortex, hippocampus, thalamus, and hypothalamus, and in cortical subregions. A prominent and highly significant reduction in SV2A binding capacity was observed over the first days after SE in the cerebral cortex and the hippocampus, but not in the thalamus and hypothalamus. The magnitude in reduction was larger and occurred earlier in the hippocampus and the piriform cortex, than in other cortical subregions. Interestingly, in all areas examined, the binding capacity returned to control levels 12 weeks after the SE comparable to the chronic epileptic phase. These data indicate that lithium-pilocarpine-induced epileptogenesis involves both loss and gain of synapses in the in a time-dependent manner.

Atherosclerosis has become prevalent not only in Western industrialized countries but all over the globe. Gpnmb, a transmembrane protein expressed by macrophages, has been detected in aortic lesions. We created an ApoE/Gpnmb-double knockout mouse using Crispr-Cas9 to examine the effect of Gpnmb deficiency on the development of atherosclerotic plaques. Feeding female mice a high cholesterol diet for 8 and 12 weeks, we detected an increased plaque size in aortic root sections of Gpnmb-deficient compared to control mice. However, the plaque area in whole thoracic and abdominal aorta was not different. Despite its strong expression in macrophages in aortic plaques, Gpnmb exerts only a minor effect on the growth of the atherosclerotic plaques in female mice. Future studies should examine plaque stability and include both sexes to elucidate the sex-specific function of Gpnmb in atherosclerosis.

Acne affects 1 in 10 people globally, often resulting in disfigurement. The disease involves excess production of lipids, particularly squalene, increased growth ofCutibacterium acnes, and a host inflammatory response with foamy macrophages. By combining single-cell and spatial RNA sequencing as well as ultrahigh-resolution Seq-Scope analyses of early acne lesions on back skin, we identified TREM2 macrophages expressing lipid metabolism and proinflammatory gene programs in proximity to hair follicle epithelium expressing squalene epoxidase. We established that the addition of squalene induced differentiation of TREM2 macrophages in vitro, which were unable to killC. acnes. The addition of squalene to macrophages inhibited induction of oxidative enzymes and scavenged oxygen free radicals, providing an explanation for the efficacy of topical benzoyl peroxide in the clinical treatment of acne. The present work has elucidated the mechanisms by which TREM2 macrophages and unsaturated lipids, similar to their involvement in atherosclerosis, may contribute to the pathogenesis of acne.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces cancer cell death and contributes to tumor rejection by cytotoxic lymphocytes in cancer immunosurveillance and immunotherapy. TRAIL and TRAIL receptor agonists have garnered wide popularity as promising agents for cancer therapy. We previously demonstrated that the loss of fucosylation in cancer cells impairs TRAIL sensitivity; however, the precise structures of the fucosylated glycans that regulate TRAIL sensitivity and their carrier molecules remain elusive. Herein, we observed that Lewis glycans among various fucosylated glycans positively regulate TRAIL-induced cell death. Specifically, Lewis glycans on lacto/neolacto glycosphingolipids, but not glycoproteins including TRAIL receptors, enhanced TRAIL-induced formation of the cytosolic caspase 8 complex, without affecting the formation of the membranous receptor complex. Furthermore, type I Lewis glycan expression in colon cancer cell lines and patient-derived cancer organoids was positively correlated with TRAIL sensitivity. These findings provide novel insights into the regulatory mechanism of TRAIL-induced cell death and facilitate the identification of novel predictive biomarkers for TRAIL-related cancer therapies in future.

Sporadic early-onset Alzheimer's disease (EOAD) and autosomal dominant Alzheimer's disease (ADAD) provide the opportunity to investigate the physiopathological mechanisms in the absence of aging, present in late-onset forms. Frontotemporal dementia (FTD) causes early-onset dementia associated to tau or TDP43 protein deposits. A 15% of FTD cases are caused by mutations in C9orf72, GRN, or MAPT genes. Lymphoblastoid cell lines (LCLs) have been proposed as an alternative to brain tissue for studying earlier phases of neurodegenerative diseases. The aim of this study is to investigate the expression profile in EOAD, ADAD, and sporadic and genetic FTD (sFTD and gFTD, respectively), using brain tissue and LCLs. Sixty subjects of the following groups were included: EOAD, ADAD, sFTD, gFTD, and controls. Gene expression was analyzed with Clariom D microarray (Affymetrix). Brain tissue pairwise comparisons revealed six common differentially expressed genes (DEG) for all the patients' groups compared with controls: RGS20, WIF1, HSPB1, EMP3, S100A11 and GFAP. Common up-regulated biological pathways were identified both in brain and LCLs (including inflammation and glial cell differentiation), while down-regulated pathways were detected mainly in brain tissue (including synaptic signaling, metabolism and mitochondrial dysfunction). CD163, ADAMTS9 and LIN7A gene expression disruption was validated by qPCR in brain tissue and NrCAM in LCLs in their respective group comparisons. In conclusion, our study highlights neuroinflammation, metabolism and synaptic signaling disturbances as common altered pathways in different AD and FTD forms. The use of LCLs might be appropriate for studying early immune system and inflammation, and some neural features in neurodegenerative dementias.

Lung injury caused by pulmonary inflammation is one of the main manifestations of respiratory diseases. Vasorin (VASN) is a cell-surface glycoprotein encoded by the VASN gene and is expressed in the lungs of developing mouse foetuses. Previous research has revealed that VASN is associated with many diseases. However, its exact function in the lungs and the underlying mechanism remain poorly understood.To investigate the molecular mechanisms involved in lung disease caused by VASN deficiency, a VASN gene knockout (VASNWe believe that these data provide molecular evidence for the regulatory role of VASN in inflammation in the context of lung injury.

Colorectal cancer (CRC) is a major cause of cancer-related deaths. Metastasis is enhanced through epithelial-mesenchymal transition (EMT), a process primarily induced by the transforming growth factor beta (TGF-β)-mediated canonical Smad pathway. This study focused on plexin D1 (PLXND1), a chemoreceptor for the ligand SEMA3E to mechanosensory, showing that PLXND1 induces EMT via activation of the PI3K/AKT pathway in CRC cells. The findings showed that PLXND1-knockdown decreases cell migration and invasion significantly, and that the binding of p61-SEMA3E to the PLXND1 enhances the invasiveness and migration through EMT. Furin inhibitor suppresses EMT, decreasing cell migration and invasion. Furin cleaves full-length SEMA3E and converts it to p61-SEMA3E, suggesting that furin inhibitors block PLXND1 and p61-SEMA3E binding. Furin is a potential therapeutic target for the purpose of suppressing EMT by inhibiting the binding of p61-SEMA3E to PLXND1. In vivo experiments have shown that PLXND1-knockdown suppresses EMT. Mesenchymal cells labeled with ZEB1 showed heterogeneity depending on PLXND1 expression status. The high-expression group of PLXND1 in 182 CRC samples was significantly associated with poor overall survival compared with the low-expression group (P = 0.0352, median follow-up period of 60.7 months) using quantitative real-time polymerase chain reaction analysis. Further research is needed to determine whether cell fractions with a different expression of PLXND1 have different functions.

We employed the co-culture of CD34Mature HMC and osteoclast precursors were cultured from monocytes isolated from human buffy coat. The osteoclast precursors were incubated with HMC or receptor activator of nuclear factor kappa-B ligand (RANKL) for a week prior to determination of osteoclast maturation through characterization by their morphology and tartrate resistant acid phosphatase (TRAP) expression. The bone absorption activity was determined by pit formation on osteo-assay plate.Mature osteoclasts were identified following co-culture of osteoclast precursors with HMC for one week in the absence of RANKL and they were capable of bone resorption. These actions of HMC on osteoclasts were not affected by mast cell activators such anti-IgE or substance P but could be reversed by osteoprotegerin (OPG) in the co-culture system suggesting the involvement of RANKL. The expression of RANKL on the cell surface of HMC was confirmed by flow cytometry and the density was not affected by activation of HMC.Our study provided direct evidence confirming the initiation of osteoclast proliferation and activation by mast cells through cell surface RANKL suggesting that mast cells may contribute to bone destruction in pathological conditions such as osteoporosis.

Podoplanin (PDPN) is a marker of lung type I alveolar cells, kidney podocytes, and lymphatic endothelial cells. The overexpression of PDPN contributes to the malignant progression of tumors. Therefore, the development of anti-PDPN monoclonal antibodies (mAbs) to animals is essential to evaluate the pathogenesis and cellular functions. Using peptide immunization, we previously developed an anti-elephant PDPN (elePDPN) mAb, PMab-295, which is useful for flow cytometry, Western blotting, and immunohistochemistry. In this study, we determined the critical epitope of PMab-295 by enzyme-linked immunosorbent assay (ELISA). We performed ELISA with the alanine-substituted peptides of elePDPN extracellular domain (amino acids 38-51), and found that PMab-295 did not recognize the alanine-substituted peptides of M41A, P44A, and E47A. Furthermore, these peptides could not inhibit the recognition of PMab-295 to elePDPN-expressing cells by flow cytometry and immunohistochemistry. The results indicate that the binding epitope of PMab-295 includes Met41, Pro44, and Glu47 of elePDPN.

The receptor Triggering Receptor Expressed on Myeloid cells 2 (TREM2) is associated with several neurodegenerative diseases including Alzheimer's Disease and TREM2 stimulation represents a novel therapeutic opportunity. TREM2 can be activated by antibodies targeting the stalk region, most likely through receptor dimerization. Endogenous ligands of TREM2 are suggested to be negatively charged apoptotic bodies, mimicked by phosphatidylserine incorporated in liposomes and other polyanionic molecules likely binding to TREM2 IgV fold. However, there has been much discrepancy in the literature on the nature of phospholipids (PLs) that can activate TREM2 and on the stability of the corresponding liposomes over time. We describe optimized liposomes as robust agonists selective for TREM2 over TREM1 in cellular system. The detailed structure/activity relationship studies of lipid polar heads indicate that negatively charged lipid heads are required for activity and we identified the shortest maximally active PL sidechain. Optimized liposomes are active on both TREM2 common variant and TREM2 R47H mutant. Activity and selectivity were further confirmed in different native TREM2 expressing cell types including on integrated cellular responses such as stimulation of phagocytic activity. Such tool agonists will be useful in further studies of TREM2 biology in cellular systems alongside antibodies, and in the design of small molecule synthetic TREM2 agonists.

Leukocyte Immunoglobulin (Ig)-like Receptors (LILR) LILRB1 and LILRB2 play a pivotal role in maintaining self-tolerance and modulating the immune response through interaction with classical and non-classical Human Leukocyte Antigen (HLA) molecules. Although both diversity and natural selection patterns over HLA genes have been extensively evaluated, little information is available concerning the genetic diversity and selection signatures on the LIRB1/2 regions. Therefore, we identified the LILRB1/2 genetic diversity using next-generation sequencing in a population sample comprising 528 healthy control individuals from São Paulo State, Brazil. We identified 58 LILRB1 Single Nucleotide Variants (SNVs), which gave rise to 13 haplotypes with at least 1% of frequency. For LILRB2, we identified 41 SNVs arranged into 11 haplotypes with frequencies above 1%. We found evidence of either positive or purifying selection on LILRB1/2 coding regions. Some residues in both proteins showed to be under the effect of positive selection, suggesting that amino acid replacements in these proteins resulted in beneficial functional changes. Finally, we have shown that allelic variation (six and five amino acid exchanges in LILRB1 and LILRB2, respectively) affects the structure and/or stability of both molecules. Nonetheless, LILRB2 has shown higher average stability, with no D1/D2 residue affecting protein structure. Taken together, our findings demonstrate that LILRB1 and LILRB2 are highly polymorphic and provide strong evidence supporting the directional selection regime hypothesis.

Thymic epithelial tumors (TETs) mainly consist of thymoma and thymic carcinoma. Complete surgical resection is vital for the successful management of these TETs, and adjuvant therapy such as systematic chemotherapy and/or radiotherapy plays important roles in the management of recurrent or metastasized disease. However, there is still a lack of a standard treatment after the failure of these adjuvant therapies. There is thus a need to develop molecular targeted therapies for advanced malignant TETs. In the present study, we evaluated the biological significance of brain-derived neurotrophic factor (BDNF)/tropomyosin receptor kinase B (TrkB) signaling for TETs.The expression of TrkB in 48 formalin-fixed, paraffin-embedded TET specimens (43 thymoma and 5 thymic carcinoma) collected by surgical resection was evaluated immunohistochemically. A thymic carcinoma cell line was evaluated for the role of BDNF/TrkB signaling pathway in an in vitro assay.High TrkB expression was related to significantly poor prognosis in patients with TETs. In vitro experiments showed that BDNF/TrkB signaling was involved in the proliferation of Ty-82 cells, but not their invasion and migration.TrkB expression is a biomarker of the prognosis for TETs and the BDNF/TrkB signaling pathway is potentially a new therapeutic target for mTETs.

SARS-CoV-2 variants may threaten the effectiveness of vaccines and antivirals to mitigate serious COVID-19 disease. This is of most concern in clinically vulnerable groups such as older adults. We analysed 72 sera samples from 37 individuals, aged 70–89 years, vaccinated with two doses of BNT162b2 (Pfizer–BioNTech) 3 weeks apart, for neutralizing antibody responses to wildtype SARS-CoV-2. Between 3 and 20 weeks after the second vaccine dose, neutralizing antibody titres fell 4.9-fold to a median titre of 21.3 (neutralization dose 80%), with 21.6% of individuals having no detectable neutralizing antibodies at the later time point. Next, we examined neutralization of 21 distinct SARS-CoV-2 variant spike proteins with these sera, and confirmed substantial antigenic escape, especially for the Omicron (B.1.1.529, BA.1/BA.2), Beta (B.1.351), Delta (B.1.617.2), Theta (P.3), C.1.2 and B.1.638 spike variants. By combining pseudotype neutralization with specific receptor-binding domain (RBD) enzyme-linked immunosorbent assays, we showed that changes to position 484 in the spike RBD were mainly responsible for SARS-CoV-2 neutralizing antibody escape. Nineteen sera from the same individuals boosted with a third dose of BNT162b2 contained higher neutralizing antibody titres, providing cross-protection against Omicron BA.1 and BA.2. Despite SARS-CoV-2 immunity waning over time in older adults, booster vaccines can elicit broad neutralizing antibodies against a large number of SARS-CoV-2 variants in this clinically vulnerable cohort.

Background and Aims: Reliable noninvasive biomarkers are an unmet clinical need for the diagnosis of NASH. This study investigates the diagnostic accuracy of the circulating triggering receptor expressed on myeloid cells 2 (plasma TREM2) as a biomarker for NASH in patients with NAFLD and elevated liver stiffness. Approach and Results: We collected cross-sectional, clinical data including liver biopsies from a derivation (n = 48) and a validation cohort (n = 170) of patients with elevated liver stiffness measurement (LSM ≥ 8.0 kPa). Patients with NAFLD activity scores (NAS) ≥4 were defined as having NASH. Plasma TREM2 levels were significantly elevated in patients with NASH of the derivation cohort, with an area under the receiver operating characteristics curve (AUROC) of 0.92 (95% confidence interval [CI], 0.84–0.99). In the validation cohort, plasma TREM2 level increased approximately two-fold in patients with NASH, and a strong diagnostic accuracy was confirmed (AUROC, 0.83; 95% CI, 0.77–0.89; p < 0.0001). Plasma TREM2 levels were associated with the individual histologic features of NAS: steatosis, lobular inflammation, and ballooning (p < 0.0001), but only weakly with fibrosis stages. Dual cutoffs for rule-in and rule-out were explored: a plasma TREM2 level of ≤38 ng/ml was found to be an optimal NASH rule-out cutoff (sensitivity 90%; specificity 52%), whereas a plasma TREM2 level of ≥65 ng/ml was an optimal NASH rule-in cutoff (specificity 89%; sensitivity 54%). Conclusions: Plasma TREM2 is a plausible individual biomarker that can rule-in or rule-out the presence of NASH with high accuracy and thus has the potential to reduce the need for liver biopsies and to identify patients who are eligible for clinical trials in NASH.

Interstitial lung disease (ILD) is the main cause of mortality in systemic sclerosis (SSc), and current therapies available are of low efficacy or high toxicity. Thus, the identification of innovative less toxic and high efficacy therapeutic approaches to ILD treatment is an urgent need. The interaction of P-selectin glycoprotein ligand-1 (PSGL-1) with P-selectin initiates leukocyte extravasation and deletion of the corresponding gene (Selplg) induces a SSc-like syndrome with high incidence of ILD in aged mice.Aged PSGL-1 KO (SelplgPSGL-1 KO mice had increased numbers of CD45+ and CD45- cells, including alveolar and interstitial macrophages, eosinophils, granulocytes and NK cells, and myofibroblasts in bronchoalveolar lavage (BAL). CD45+ and CD45- cells expressing pro-inflammatory and pro-fibrotic cytokines were also increased. Lungs from PSGL-1 KO mice showed increased immune cell infiltration and apoptosis and exacerbated interstitial and peribronchial fibrosis. Targeted nanotherapy with LipHA+Ev decreased the myofibroblasts in BAL, cells producing proinflammatory and profibrotic cytokines, and the degree of lung inflammation at histology. LipHA+Ev treatment also decreased the severity of peribronchial and interstitial lung fibrosis, from moderate to mild levels.In PSGL-1 KO mice, targeted nanotherapy with LipHA+Ev was an effective treatment for SSc-ILD, reducing the number of inflammatory and fibrotic cells in BAL and reducing inflammation and fibrosis in lungs.

The transmembrane receptor podoplanin (PDPN) is a platelet aggregation-inducing factor, which is widely expressed in various malignant tumors such as squamous cell carcinomas, mesotheliomas, glioblastomas. Podoplanin regulates a pathway leading to cell invasion and migration. Glioblastoma multiforme (GBM) is the most aggressive and invasive tumor of the central nervous system. A high level of PDPN expression has been reported to be associated with reduced survival, cancer aggression and migration. Aim of study to determine the effect of anti-podoplanin antibody on cell-platelet aggregation, cell invasion and viability in Uppsala 87 malignant glioma (U87MG) cell lines.The expression of podoplanin on U87MG cells was measured using flowcytometry. The dimethyl-thiazol diphenyl-tetrazolium bromide (MTT) assay was used to measure U87MG cell proliferation after treatment by anti-podoplanin monoclonal antibody (NZ-1.3). The invasion of cancer cells was assessed by using a novel microfluidic based assay.The results show reduction of cell viability and cell migration after treatment with anti-podoplanin antibody. After co- treatment of U87MG cells and platelets with and without anti-podoplanin antibody, the cell-platelet aggregation was significantly reduced in anti-podoplanin treated cell.Podoplanin is involved in aggregation of gliobastoma cells, and their viability and invasion and its neutralizing antibody can inhibit this process. So, blocking of podoplanin may be representing a promising therapeutic approach to glioblastoma multiform cancer therapy.

LILRB4 (B4, also known as ILT3/CD85k) is an immune checkpoint of myeloid lineage cells, albeit its mode of function remains obscure. Our recent identification of a common ligand for both human B4 and its murine ortholog gp49B as the fibronectin (FN) N-terminal 30 kDa domain poses the question of how B4/gp49B regulate cellular activity upon recognition of FN in the plasma and/or the extracellular matrix. Since FN in the extracellular matrix is tethered by FN-binding integrins, we hypothesized that B4/gp49B would tether FN in cooperation with integrins on the cell surface, thus they should be in close vicinity to integrins spatially. This scenario suggests a mode of function of B4/gp49B by which the FN-induced signal is regulated. The FN pull-down complex was found to contain gp49B and integrin β 1 in bone marrow-derived macrophages. The confocal fluorescent signals of the three molecules on the intrinsically FN-tethering macrophages were correlated to each other. When FN-poor macrophages adhered to culture plates, the gp49–integrin β 1 signal correlation increased at the focal adhesion, supporting the notion that gp49B and integrin β 1 become spatially closer to each other there. Adherence of RAW264.7 and THP-1 cells to immobilized FN induced phosphorylation of spleen tyrosine kinase, whose level was augmented under B4/gp49B deficiency. Thus, we concluded that B4/gp49B can co-tether FN in cooperation with integrin in the cis configuration on the same cell, forming a B4/gp49B–FN–integrin triplet as a regulatory unit of a focal adhesion-dependent pro-inflammatory signal in macrophages.

Ventilator-associated pneumonia (VAP) is one of the most common causes of nosocomial pneumonia in ventilated neonates. Nevertheless, its diagnosis is challenging due to the nonspecific clinical parameters and the lack of sensitive biomarkers. The main objective of this study was to compare soluble triggering receptors expressed on myeloid cells-1 (sTREM-1) and 25-hydroxy vitamin D as early predictors of neonatal VAP.This prospective cohort study included 85 ventilated neonates divided into the VAP group (n = 33) and the non-VAP group (n = 52). sTREM-1 levels in the endotracheal aspirate (ETA) and serum 25-hydroxy vitamin D levels were measured on the third and seventh days following mechanical ventilation. The Ethical and Research Committee approved the study at Tanta University Hospitals, Egypt (with the Approval code: 32751/12/18).The sTREM-1 cutoff value of0.46 and0.44 ng/ml at 3 and 7 days had a sensitivity of 93.94% and 96.97%, a specificity of 92.31% and 100%, and an area under the receiver operating characteristic curve (AUC) of 0.963 and 0.993, respectively, to predict the development of neonatal VAP. A serum 25-hydroxy vitamin D cutoff value of ≤17.5 ng/ml at 3 and 7 days had a sensitivity of 90.91% and 81.82%, a specificity of 75% and 78.85%, and area under the curve of 0.877 and 0.939, respectively.Both sTREM-1 in ETA and serum 25-hydroxy vitamin D could be used as early predictors of neonatal VAP, but sTREM-1 appears more useful.

Epitope mapping of the interactions between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Abs is challenging because of complexity in protein three-dimensional structures. Protein structure fingerprint technology was applied for epitope mapping of 44 SARS-CoV-2 Abs with three-dimensional structure complexes. The results defined how the epitopes were distributed on SARS-CoV-2 and how the patterns of six CDRs from Abs participated in neutralization. Also, the residue–residue recognition revealed that certain residues had higher frequencies on the interfaces between SARS-CoV-2 and Abs, and the activity correlated with the physicochemical properties of the residues at the interface. Thus, epitope mapping provides significant lead information for development of epitope-based designs for Abs, vaccines, and diagnostic reagents. This is a bioinformatics project of structural data analysis; no animals or cells were used.

Background: Cholesterol loaded macrophage foam cells are a prominent feature of atherosclerotic plaques. Single-cell RNA sequencing has identified foam cells as TREM2 (triggering receptor expressed on myeloid cells 2) positive populations with low expression of inflammatory genes, resembling the TREM2 positive microglia of neurodegenerative diseases. Cholesterol loading of macrophages in vitro results in activation of LXR (liver X receptor) transcription factors and suppression of inflammatory genes. Methods: To test the hypothesis that LXRs mediate anti-inflammatory effects in Trem2 expressing atherosclerotic plaque foam cells, we performed RNA profiling on plaque cells from hypercholesterolemic mice with myeloid LXR deficiency. Results: Myeloid LXR deficiency led to a dramatic increase in atherosclerosis with increased monocyte entry, foam cell formation, and plaque inflammation. Bulk cell-RNA profiling of plaque myeloid cells showed prominent upregulation of inflammatory mediators including oxidative, chemokine, and chemotactic genes. Single-cell RNA sequencing revealed increased numbers of foamy TREM2-expressing macrophages; however, these cells had reduced expression of the Trem2 gene expression module, including phagocytic and cholesterol efflux genes, and had switched to a proinflammatory and proliferative phenotype. Expression of Trem2 was suppressed by inflammatory signals but not directly affected by LXR activation in bone marrow-derived macrophages. Conclusions: Our current studies reveal the key role of macrophage LXRs in promoting the Trem2 gene expression program and in suppressing inflammation in foam cells of atherosclerotic plaques.

PET technology has produced many radiopharmaceuticals that target specific brain proteins and other measures of brain function. Recently, a new approach has emerged to image synaptic density by targeting the synaptic vesicle protein 2A (SV2A), an integral glycoprotein in the membrane of synaptic vesicles and widely distributed throughout the brain. Multiple SV2A ligands have been developed and translated to human use. The most successful of these to date is

Mucins are heavily glycosylated proteins secreted by various cell types, to protect the epithelial surface of the gastrointestinal tract from damage. Currently, increasing studies provided evidence to suggest that mucins play an essential role in regulating tumor progression. However, the role of mucins and the underpinning mechanism of how mucins drive melanoma progression remains elusive. In this study, we first demonstrated that mucin 21 (MUC21) expression was significantly upregulated in metastatic melanoma tissues, and a higher MUC21 expression resulted in poor overall survival in melanoma patients by The Cancer Genome Atlas database analysis. In vitro, MUC21 overexpression markedly promoted proliferative properties and aggressive behavior of melanoma cell A375 and A875, as assessed by Cell Counting Kit-8 and transwell assay. In mechanism, we proved that MUC21 suppressed expression of SLITRK5, an integral membrane protein, leading to activation of prosurvival hedgehog pathway and sustained melanoma development. More importantly, we found that combination of hedgehog pathway inhibitor cyclopamine and chemotherapy revealed an improved anticancer effect in MUC21 overexpression xenograft model. Altogether, our study described a novel role of MUC21 in regulating tumor progression, which offers a promising target for melanoma diagnosis and therapy.

Cancer-associated fibroblasts (CAF) constitute a major component of the tumor microenvironment. The effects of CAFs on the progression of colorectal cancer remain controversial. In this study, we found the ectopic overexpression of Fibronectin leucine-rich transmembrane protein 3 (FLRT3) inhibited the process of epithelial–mesenchymal transition (EMT), as well as the proliferation, migration, invasion, and promote apoptosis of colorectal cancer cells, whereas silencing FLRT3 expression resulted in the opposite phenomenon. FLRT3 downregulation was associated with a poor prognosis in colorectal cancer. Also, FLRT3 expression was significantly related to some clinicopathologic factors, including T stage (P = 0.037), N stage (P = 0.042), and E-cadherin (P = 0.002) level. Via univariate and multivariate analyses, M stage (P < 0.0001), FLRT3 (P = 0.044), and E-cadherin (P = 0.003) were associated with overall survival and were independent prognostic factors for it. Mechanistically, CAFs secreted TGF-β, which downregulated FLRT3 expression by activating SMAD4 to promote aggressive phenotypes in colorectal cancer cells. Moreover, FLRT3 repressed tumorigenesis and lung metastasis, which could be reversed by LY2109761, a dual inhibitor of TGF-β receptor type I and II. Treatment with LY2109761 increased IFN-γ expression in CD8+ T cells and reduced the number of regulatory T cells in the tumor microenvironment. Taken together, we revealed the metastasis-suppressive function of FLRT3, which was attenuated during the CAFs-mediated activation of the TGF-β/SMAD4 signaling pathway to promote EMT in colorectal cancer. LY2109761 that significantly inhibited metastasis could be a new treatment option for advanced colorectal cancer. Implications: CAFs enhance colorectal cancer aggressiveness by reducing FLRT3 expression through activating TGF-β/SMAD4 signaling pathway. CAF-targeted therapy and/or LY2109761 were promising treatments for colorectal cancer.

Purpose: Paclitaxel (PTX) is one of the most potent and commonly used chemotherapies for breast and pancreatic cancer. Several ongoing clinical trials are investigating means of enhancing delivery of PTX across the blood–brain barrier for glioblastomas. Despite the widespread use of PTX for breast cancer, and the initiative to repurpose this drug for gliomas, there are no predictive biomarkers to inform which patients will likely benefit from this therapy. Experimental Design: To identify predictive biomarkers for susceptibility to PTX, we performed a genome-wide CRISPR knockout (KO) screen using human glioma cells. The genes whose KO was most enriched in the CRISPR screen underwent further selection based on their correlation with survival in the breast cancer patient cohorts treated with PTX and not in patients treated with other chemotherapies, a finding that was validated on a second independent patient cohort using progression-free survival. Results: Combination of CRISPR screen results with outcomes from patients with taxane-treated breast cancer led to the discovery of endoplasmic reticulum (ER) protein SSR3 as a putative predictive biomarker for PTX. SSR3 protein levels showed positive correlation with susceptibility to PTX in breast cancer cells, glioma cells, and in multiple intracranial glioma xenografts models. KO of SSR3 turned the cells resistant to PTX while its overexpression sensitized the cells to PTX. Mechanistically, SSR3 confers susceptibility to PTX through regulation of phosphorylation of ER stress sensor IRE1α. Conclusions: Our hypothesis generating study showed SSR3 as a putative biomarker for susceptibility to PTX, warranting its prospective clinical validation.

In the central nervous system, most neurons co-express TrkB and TrkC, the tyrosine kinase receptors for brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3). As NT3 can also activate TrkB, it has been difficult to understand how NT3 and TrkC can exert unique roles in the assembly of neuronal circuits. Using neurons differentiated from human embryonic stem cells expressing both TrkB and TrkC, we compared Trk activation by BDNF and NT3. To avoid the complications resulting from TrkB activation by NT3, we also generated neurons from stem cells engineered to lack TrkB. We found that NT3 activates TrkC at concentrations lower than those of BDNF needed to activate TrkB. Downstream of Trk activation, the changes in gene expression caused by TrkC activation were found to be similar to those resulting from TrkB activation by BDNF, including a number of genes involved in synaptic plasticity. At high NT3 concentrations, receptor selectivity was lost as a result of TrkB activation. In addition, TrkC was down-regulated, as was also the case with TrkB at high BDNF concentrations. By contrast, receptor selectivity as well as reactivation were preserved when neurons were exposed to low neurotrophin concentrations. These results indicate that the selectivity of NT3/TrkC signalling can be explained by the ability of NT3 to activate TrkC at concentrations lower than those needed to activate TrkB. They also suggest that in a therapeutic perspective, the dosage of Trk receptor agonists will need to be taken into account if prolonged receptor activation is to be achieved.

Chronic kidney disease (CKD) is a global public health issue. CKD is caused by the infiltration of various myeloid cell types into renal tissue, resulting in renal fibrosis and tubular atrophy. Unilateral ureteral obstruction (UUO) surgery in mice is a model of CKD and characterized by high expression of the anti-inflammatory receptor, Triggering receptor expressed on myeloid cells 2 (TREM-2), on myeloid cells in affected kidneys. Here, we show that iNOS expression and nitric oxide (NO) induction were decreased in Trem-2

The target sorB gene, related to sorbicillinoid production, and the free expression element, AMA1, were used to verify the methodological approach in Acremonium chrysogenum.CRISPR-Cas9 episomal expression system was used to introduce a point mutation into the sorB gene and the addition of sorB donor DNA achieved complete knockout of target genes. Four BSSS (yeast bud site selection system)-related genes, axl1, axl2, bud3, and bud4 were knocked out without impact on yield, dry weight, or pH. Relationships between morphology and stress tolerance in knockout strains were analyzed.The gene-editing system used in the current study exceeded 80% efficiency and arthrospores development was found to differ from that in wild-type strain.

Objectives Diagnosis of high-grade B-cell lymphoma with MYC and BCL2 or BCL6 rearrangements (double-/triple-hit lymphoma [DTHL]) appears to mandate fluorescence in situ hybridization (FISH) testing for all large B-cell lymphoma (LBCL). Given the low incidence of DTHL, we aimed to identify flow cytometry (FC) and immunohistochemistry (IHC) features of DTHL that could be used to develop an optimal screening strategy. This combined FC-IHC approach has not yet been studied. Methods We compared features of 40 cases of DTHL and 39 cases of diffuse LBCL (DLBCL) without MYC rearrangement. Results Bright CD38 expression (CD38bright) by FC, high MYC expression (≥55%), and double-expressor phenotype by IHC were significantly associated with DTHL. The biomarker combining FC and IHC, CD38bright and/or MYC ≥55%, was superior to FC and IHC markers alone in predicting DTHL. Restricting FISH testing to approximately 25% of LBCL based on CD38brightand/or MYC ≥55% would detect approximately 95% of DTHL-BCL2 and approximately 75% of DHL-BCL6. Conclusions Our study demonstrated that the novel biomarker of CD38bright and/or MYC ≥55% is highly predictive of DTHL. Awareness of the advantages and limitations of this screening strategy would facilitate development of a rational diagnostic workflow to provide high-quality patient care.