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Bacterial flagella and type IV pili (TFP) are surface appendages that enable motility and mechanosensing through distinct mechanisms. These structures were previously thought to have no components in common. Here, we report that TFP and some flagella share proteins PilO, PilN, and PilM, which we identified as part of the Helicobacter pylori flagellar motor. H. pylori mutants lacking PilO or PilN migrated better than wild type in semisolid agar because they continued swimming rather than aggregated into microcolonies, mimicking the TFP-regulated surface response. Like their TFP homologs, flagellar PilO/PilN heterodimers formed a peripheral cage that encircled the flagellar motor. These results indicate that PilO and PilN act similarly in flagella and TFP by differentially regulating motility and microcolony formation when bacteria encounter surfaces.

Glycoprotein 2 (GP2) is a widely distributed protein in the digestive tract, contributing to mucosal barrier maintenance, immune homeostasis, and antigen-specific immune response, while also being linked to inflammatory bowel disease (IBD) pathogenesis. This review sheds light on the extensive distribution of GP2 within the gastrointestinal tract and its intricate interplay with the immune system. Furthermore, the significance of GP2 autoantibodies in diagnosing and categorizing IBD is underscored, alongside the promising therapeutic avenues for modulating GP2 to regulate immunity and maintain mucosal balance.

By incompletely understood mechanisms, type 2 (T2) inflammation present in the airways of severe asthmatics drives the formation of pathologic mucus which leads to airway mucus plugging. Here we investigate the molecular role and clinical significance of intelectin-1 (ITLN-1) in the development of pathologic airway mucus in asthma. Through analyses of human airway epithelial cells we find that ITLN1 gene expression is highly induced by interleukin-13 (IL-13) in a subset of metaplastic MUC5AC+ mucus secretory cells, and that ITLN-1 protein is a secreted component of IL-13-induced mucus. Additionally, we find ITLN-1 protein binds the C-terminus of the MUC5AC mucin and that its deletion in airway epithelial cells partially reverses IL-13-induced mucostasis. Through analysis of nasal airway epithelial brushings, we find that ITLN1 is highly expressed in T2-high asthmatics, when compared to T2-low children. Furthermore, we demonstrate that both ITLN-1 gene expression and protein levels are significantly reduced by a common genetic variant that is associated with protection from the formation of mucus plugs in T2-high asthma. This work identifies an important biomarker and targetable pathways for the treatment of mucus obstruction in asthma.

HIV-infected individuals have increased risk for cardiovascular disease (CVD) despite suppressive antiretroviral therapy (ART). This is likely a result of persistent immune activation and systemic inflammation. Extracellular vesicles (EVs) have emerged as critical mediators of intercellular communication and may drive inflammation contributing to CVD. EVs were characterized in plasma from 74 HIV-infected individuals on combination antiretroviral therapy (cART) and 64 HIV-uninfected controls with paired carotid intima-media thickness (cIMT) assessment. EVs were profiled with markers reflecting lymphoid, myeloid, and endothelial origin. Seventeen plasma inflammatory biomarkers were also assessed. Human umbilical vein endothelial cell (HUVEC) apoptosis was quantified after EV exposure. A significant correlation was observed in HIV-infected participants between cIMT and EVs expressing CD16, and the monocyte-related markers CD4, CD14, and CX3CR1 showed a similar but nonsignificant association with cIMT. No significant correlation between cIMT measurements from HIV-uninfected individuals and EVs was observed. Levels of serum amyloid A, C-reactive protein, and myeloperoxidase significantly correlated with CD14+, CD16+, and CX3CR1+ EVs. No correlation was noted between cIMT and soluble inflammatory markers. HUVECs showed increased necrosis after exposure to the EV-containing fraction of plasma derived from HIV-infected individuals compared to uninfected controls. Our study reveals that EVs expressing monocyte markers correlated with cIMT in HIV-infected individuals on cART. Moreover, EV fractions derived from HIV-infected individuals lead to greater endothelial cell death via necrotic pathways. Collectively, EVs have potential as biomarkers of and therapeutic targets in the pathogenesis of CVD in the setting of treated HIV disease. IMPORTANCE HIV-infected individuals have a 2-fold-increased risk of cardiovascular disease compared with the general population, yet the mechanisms underlying this comorbidity are unclear. Extracellular vesicles have emerged as important mediators in cell-cell communication and, given what we know of their biology, may drive inflammation contributing to cardiovascular disease in this vulnerable population.

Natural killer (NK) cells can detect antibody-coated cells through recognition by the CD16 Fc receptor. The importance of CD16 in human NK cell biology has long been appreciated, but how CD16 functions in mouse NK cells remains poorly understood. Here, we report drastic differences between human and mouse CD16 functions in NK cells. We demonstrate that one of the adaptor molecules that CD16 associates with and signals through, CD3ζ, plays a critical role in these functional differences. Using a systematic approach, we demonstrate that residues in the transmembrane domain of the mouse CD3ζ molecule prevent efficient complex formation with mouse CD16, thereby dampening receptor function. Mutating these residues in mouse CD3ζ to those encoded by human CD3ζ resulted in rescue of CD16 receptor function. We reveal that the mouse CD3ζ transmembrane domain adopts a tightly packed confirmation, preventing association with CD16, whereas human CD3ζ adopts a versatile configuration that accommodates receptor assembly.

Intelectins are carbohydrate-binding proteins implicated in innate immunity and highly conserved across chordate evolution, including both ascidians and humans. Human intelectin-1 (ITLN1) is highly abundant within the intestinal mucosa and binds microbial but not host glycans. Genome-wide association studies identified SNPs in ITLN1 that are linked to susceptibility for Crohn's disease. Moreover, ITLN1 has been implicated in the pathophysiology of obesity and associated metabolic disease. To gain insight on biological activities of human ITLN1 in vivo, we developed a C57BL/6 mouse model genetically targeting the gene encoding the functional mouse ortholog. In wild-type C57BL/6 mice, both mRNA and protein analysis showed high expression of Itln1 in the small intestine, but manifold lower levels in colon and other extraintestinal tissues. Whereas intestinal expression of human ITLN1 localizes to goblet cells, our data confirm that mouse Itln1 is expressed in Paneth cells. Compared to wild-type littermate controls, mice homozygous for the Itln1 hypomorphic trapping allele had reduced expression levels of Itln1 expression (~10,000-fold). The knockout mice exhibited increased susceptibility in an acute model of experimentally induced colitis with 2% w/v dextran sulfate sodium (DSS). In a model of chronic colitis using a lower dose of DSS (1.5% w/v), which enabled a detailed view of disease activity across a protracted period, no differences were observed in body weight, fecal texture, hemoccult scores, food/water intake, or colon length at necropsy, but there was a statistically significant genotype over time effect for the combined fecal scores of disease activity. In model of diet-induced obesity, using two western-style diets, which varied in amounts of sugar (as sucrose) and saturated fat (as lard), mice with Itln1 expression ablated showed no increased susceptibility, in terms of weight gain, food intake, plasma markers of obesity compared to wildtype littermates. While the mouse genetic knockout model for Itln1 holds promise for elucidating physiological function(s) for mammalian intelectins, results reported here suggest that Itln1, a Paneth cell product in C57BL/6 mice, likely plays a minor role in the pathophysiology of chemically induced colitis or diet-induced obesity.

OBJECTIVES: Alterations in the intestinal microbiota are linked with a wide range of autoimmune and inflammatory conditions, including inflammatory bowel diseases (IBD), where pathobionts penetrate the intestinal barrier and promote inflammatory reactions. In patients with IBD, the ability of intestinal macrophages to efficiently clear invading pathogens is compromised resulting in increased bacterial translocation and excessive immune reactions. Here, we investigated how an IBD-associated loss-of-function variant in the protein tyrosine phosphatase non-receptor type 2 (PTPN2) gene, or loss of PTPN2 expression affected the ability of macrophages to respond to invading bacteria. DESIGN: IBD patient-derived macrophages with wild-type (WT) PTPN2 or carrying the IBD-associated PTPN2 SNP, peritoneal macrophages from WT and constitutive PTPN2-knockout mice, as well as mice specifically lacking PTPN2 in macrophages were infected with non-invasive K12 Escherichia coli, the human adherent-invasive E. coli (AIEC) LF82, or a novel mouse AIEC (mAIEC) strain. RESULTS: Loss of PTPN2 severely compromises the ability of macrophages to clear invading bacteria. Specifically, loss of functional PTPN2 promoted pathobiont invasion/uptake into macrophages and intracellular survival/proliferation by three distinct mechanisms: Increased bacterial uptake was mediated by enhanced expression of carcinoembryonic antigen cellular adhesion molecule (CEACAM)1 and CEACAM6 in PTPN2-deficient cells, while reduced bacterial clearance resulted from defects in autophagy coupled with compromised lysosomal acidification. In vivo, mice lacking PTPN2 in macrophages were more susceptible to mAIEC infection and mAIEC-induced disease. CONCLUSIONS: Our findings reveal a tripartite regulatory mechanism by which PTPN2 preserves macrophage antibacterial function, thus crucially contributing to host defence against invading bacteria.

Chronic kidney disease is a progressive disease that may lead to end-stage renal disease. Interstitial fibrosis develops as the disease progresses. Therapies that focus on fibrosis to delay or reverse progressive renal failure are limited. We and others showed that sphingosine kinase 2-deficient mice (Sphk2–/–) develop less fibrosis in mouse models of kidney fibrosis. Sphingosine kinase2 (SphK2), one of two sphingosine kinases that produce sphingosine 1-phosphate (S1P), is primarily located in the nucleus. S1P produced by SphK2 inhibits histone deacetylase (HDAC) and changes histone acetylation status, which can lead to altered target gene expression. We hypothesized that Sphk2 epigenetically regulates downstream genes to induce fibrosis, and we performed a comprehensive analysis using the combination of RNA-seq and ChIP-seq. Bst1/CD157 was identified as a gene that is regulated by SphK2 through a change in histone acetylation level, and Bst1–/– mice were found to develop less renal fibrosis after unilateral ischemia-reperfusion injury, a mouse model of kidney fibrosis. Although Bst1 is a cell-surface molecule that has a wide variety of functions through its varied enzymatic activities and downstream intracellular signaling pathways, no studies on the role of Bst1 in kidney diseases have been reported previously. In the current study, we demonstrated that Bst1 is a gene that is regulated by SphK2 through epigenetic change and is critical in kidney fibrosis.

Doxorubicin (DOX), a commonly used anticancer agent, causes cardiotoxicity that begins with the first dose and may progress to heart failure years after treatment. An inflammatory response associated with neutrophil recruitment has been recognized as a mechanism of DOX-induced cardiotoxicity. This study aimed to validate mRNA expression of the previously identified biomarkers of DOX-induced cardiotoxicity, PGLYRP1, CAMP, MMP9, and CEACAM8, and to assay their protein expression in the peripheral blood of breast cancer patients. Blood samples from 40 breast cancer patients treated with DOX-based chemotherapy were collected before and after the first chemotherapy cycle and > 2 years after treatment. The protein and gene expression of PGLYRP1/Tag7, CAMP/LL37, MMP9/gelatinase B, and CEACAM8/CD66b were determined using ELISA and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Receiver operating characteristic (ROC) curve analysis was used to determine the diagnostic value of each candidate biomarker. Patients with cardiotoxicity (n = 20) had significantly elevated levels of PGLYRP1, CAMP, MMP9, and CEACAM8 at baseline, after the first dose of DOX-based chemotherapy, and at > 2 years after treatment relative to patients without cardiotoxicity (n = 20). The first dose of DOX induced significantly higher levels of all examined biomarkers in both groups of patients. At > 2 years post treatment, the levels of all but MMP9 dropped below the baseline. There was a good correlation between the expression of mRNA and the target proteins. We demonstrate that circulating levels of PGLYRP1, CAMP, MMP9, and CEACAM8 can predict the cardiotoxicity of DOX. This novel finding may be of value in the early identification of patients at risk for cardiotoxicity.

Human dental tissue mesenchymal stem cells (DT-MSCs) constitute an attractive alternative to bone marrow-derived mesenchymal stem cells (BM-MSCs) for potential clinical applications because of their accessibility and anti-inflammatory capacity. We previously demonstrated that DT-MSCs from dental pulp (DP-MSCs), periodontal ligaments (PDL-MSCs), and gingival tissue (G-MSCs) show immunosuppressive effects similar to those of BM, but to date, the DT-MSC-mediated immunoregulation of T lymphocytes through the purinergic pathway remains unknown. In the present study, we compared DP-MSCs, PDL-MSCs, and G-MSCs in terms of CD26, CD39, and CD73 expression; their ability to generate adenosine (ADO) from ATP and AMP; and whether the concentrations of ADO that they generate induce an immunomodulatory effect on T lymphocytes. BM-MSCs were included as the gold standard. Our results show that DT-MSCs present similar characteristics among the different sources analyzed in terms of the properties evaluated; however, interestingly, they express more CD39 than BM-MSCs; therefore, they generate more ADO from ATP. In contrast to those produced by BM-MSCs, the concentrations of ADO produced by DT-MSCs from ATP inhibited the proliferation of CD3 + T cells and promoted the generation of CD4 + CD25 + FoxP3 + CD39 + CD73 + Tregs and Th17 + CD39 + lymphocytes. Our data suggest that DT-MSCs utilize the adenosinergic pathway as an immunomodulatory mechanism and that this mechanism is more efficient than that of BM-MSCs.

Synthesis of novel unnatural amino acids (UAAs) from 4-oxo-4-phenylbut-2-enoic acid derivatives with intramolecular aza-Michael addition reaction in the presence of chlorosulfonyl isocyanate (CSI) was reported in soft conditions without any metal catalyst. Acids and base as a catalyst, and solvents effects were investigated for the synthesis of novel UAAs. This novel method provides inexpensive, practicable, and efficient approach to generate UAAs. The use of UAAs has attracted great interest in the development of therapeutic agents and drug discovery to improve their properties. In this context, in addition to the synthesis of new UAAs, their inhibition effects on important metabolic enzymes of acetylcholinesterase (AChE) and carbonic anhydrases I and II (hCA I and II) enzymes were investigated. The compound 2g showed the best inhibition for CA I and AChE enzymes, while compound 2i exhibited the best inhibition profile against CA II isoenzyme. The inhibition values of these compounds were found as 1.85 ± 0.64 for AChE, 0.53 ± 0.07 for hCA I, 0.44 ± 0.15 µM for hCA II, respectively, and they showed a stronger inhibitory property than acetazolamide (standard inhibitor for hCA I and II) and tacrine (standard inhibitor for AChE) molecules. The activity of the studied molecule against different proteins that are hCA I (PDB ID: 2CAB), hCA II (PDB ID: 5AML), and AChE (PDB ID: 1OCE) was examined. Finally, the drug properties of the studied molecule were examined by performing absorption, distribution, metabolism, excretion, and toxicity analysis., (© 2024 The Author(s). Journal of Biochemical and Molecular Toxicology published by Wiley Periodicals LLC.)

Background: Lymphocyte antigen 9 (LY9) participates in the development of several tumors and diseases but has not been reported yet in lung adenocarcinoma (LUAD)., Methods: First, we analyzed the expression and prognostic value of LY9 in pan-cancer, including LUAD. Additionally, we conducted a correlation analysis of LY9 expression in LUAD with immune cell infiltration using the TIMER database and the CIBERSORT algorithm, and with immune checkpoints using the GEPIA database. Also, we constructed a potential ceRNA network for LY9. Furthermore, we explored LY9-related pathways by Gene Set Enrichment Analysis (GSEA). Finally, validation of differential expression at the mRNA level was obtained from the GEO database. We collected LUAD tissues for Quantitative Real-time PCR (qRT-PCR) to verify the expression of LY9, CD8, and CD4 and calculated the correlation between them. We also conducted immunohistochemistry (IHC) to verify the protein expression of LY9., Results: Results showed that LY9 was highly expressed in various tumors, including LUAD. Besides, patients with high LY9 expression presented longer overall survival (OS) and more multiple lymphocyte infiltrations. The expression of LY9 in LUAD strongly and positively correlates with multiple immune cell infiltration and immune checkpoints. The functional enrichment analysis indicated that LY9 was involved in multiple immune-related pathways and non-small cell lung cancer. Moreover, a ceRNA regulatory network of LINC00943-hsa-miR-141-3p-LY9 might be involved. Finally, GSE68465 dataset confirmed differential expression of LY9 mRNA levels in LUAD and the qRT-PCR results verified LY9 had a strong and positive correlation with CD4 and CD8 T cells. Unfortunately, IHC did not detect the expression of LY9 protein level in tumor tissues and WB experiments validated the protein expression of LY9 in the OCI-AML-2 cell line., Conclusions: Therefore, we hypothesized that LY9 could serve as a potential, novel prognostic biomarker for LUAD and could predict immunotherapy efficacy at the mRNA level., Competing Interests: The authors declare there are no competing interests., (©2024 Deng et al.)

Background: Glycoprotein-2 (GP2) IgA is a predictor of disease severity in primary sclerosing cholangitis (PSC). We examined GP2's occurrence in the biliary tract, the site of inflammation., Methods: GP2 was analyzed using ELISA, immunoblotting, mass spectrometry, and immunohistochemistry. The samples included: 20 bile and 30 serum samples from PSC patients, 23 bile and 11 serum samples from patients with gallstone disease (GD), 15 bile samples from healthy individuals undergoing liver-donation surgery (HILD), 20 extracts of gallstones (GE) obtained during cholecystectomy, and 101 blood-donor sera., Results: Biliary GP2 concentrations were significantly higher in patients with PSC and GD than in HILD (p < 0.0001). Serum GP2 levels were similar in PSC and GD patients, and controls, but lower than in bile (p < 0.0001). GP2 was detected in all 20 GEs. Mass spectrometry identified GP2 in the bile of 2 randomly selected GD and 2 PSC patients, and in none of 2 HILD samples. GP2 was found in peribiliary glands in 8 out of 12 PSC patients, showing morphological changes in acinar cells, but not in GD-gallbladders., Conclusions: GP2 is present in bile of PSC and GD patients. It is synthesized in the peribiliary glands of PSC patients, supporting a pathogenic role for biliary GP2 in PSC., Competing Interests: Declaration of competing interest PM served as a speaker supported by Alfa Wasserman and Chiesi. CS has served as a speaker for the Falk Foundation, as a consultant for BiomX and Pliant and has received research funding from Galapagos and BiomX. SL is an employee at Medipan GmbH. KG is an employee of GA Generic Assays. DR is an employee at GA Generic Assays and Medipan and owns stocks and shares of both companies., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Cell surface marker expression is one of the criteria for defining human mesenchymal stem or stromal cells (MSC) in vitro. However, it is unclear if expression of markers including CD73 and CD90 reflects the in vivo origin of cultured cells. We evaluated expression of 15 putative MSC markers in primary cultured cells from periosteum and cartilage to determine whether expression of these markers reflects either the differentiation state of cultured cells or the self-renewal of in vivo populations. Cultured cells had universal and consistent expression of various putative stem cell markers including > 95% expression CD73, CD90 and PDPN in both periosteal and cartilage cultures. Altering the culture surface with extracellular matrix coatings had minimal effect on cell surface marker expression. Osteogenic differentiation led to loss of CD106 and CD146 expression, however CD73 and CD90 were retained in > 90% of cells. We sorted freshly isolated periosteal populations capable of CFU-F formation on the basis of CD90 expression in combination with CD34, CD73 and CD26. All primary cultures universally expressed CD73 and CD90 and lacked CD34, irrespective of the expression of these markers ex vivo indicating phenotypic convergence in vitro. We conclude that markers including CD73 and CD90 are acquired in vitro in most 'mesenchymal' cells capable of expansion. Overall, we demonstrate that in vitro expression of many cell surface markers in plastic-adherent cultures is unrelated to their expression prior to culture., (© 2024. The Author(s).)

Objectives: The use of infrapatellar fat pad adipose stem cells (IPFP-ASCs) shows an age-independent proliferation and differentiation potential. In addition, the pronounced chondrogenic potential of IPFP-ASCs makes them promising candidates for research for use in other methods of regenerative therapy. The purpose of this study was to ascertain the presence and compare the relative abundance of cells exhibiting an immunohistochemical profile characteristic of adipose-derived mesenchymal stem cells in selected samples of the stromal vascular fraction (SVF) obtained from the IPFP and subcutaneous fat tissue., Methods: A direct immunohistochemical study was carried out in serial paraffin sections of the SVF of the infrapatellar fat pad (IPFP) and subcutaneous tissue, using monoclonal antibodies. The minimum criteria were established by the International Society for Cell Therapy to ensure the identity of mesenchymal stem cells use CD73, CD90, and CD105 as positive markers and CD34, CD31, and CD45 as a negative., Results: According to the results of histological, immunohistochemical, morphometric, and statistical studies, it was found that in the SVF of IPFP and subcutaneous adipose tissue, the relative number of cells with the profile CD105+, CD73+, CD34+, CD31-, CD45- in the standard field of view (×200), the SVF of IPFP was 1.58%, whereas the SVF of subcutaneous adipose tissue was 6.92 %, which was statistically significantly greater by 4.38 times (p ​< ​0.05)., Conclusion: The presence of a sufficient number of mesenchymal stromal cells in IPFP in combination with their topographic relationship with the structures of the joint determines the use of the SVF of the IPFP for the treatment of diseases of the knee joint., Level of Evidence: III., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

CD73, a cell surface-bound nucleotidase, serves as a crucial metabolic and immune checkpoint. Several studies have shown that CD73 is widely expressed on immune cells and plays a critical role in immune escape, cell adhesion and migration as a costimulatory molecule for T cells and a factor in adenosine production. However, recent studies have revealed that the protumour effects of CD73 are not limited to merely inhibiting the antitumour immune response. Nicotinamide adenine dinucleotide (NAD+) is a vital bioactive molecule in organisms that plays essential regulatory roles in diverse biological processes within tumours. Accumulating evidence has demonstrated that CD73 is involved in the transport and metabolism of NAD, thereby regulating tumour biological processes to promote growth and proliferation. This review provides a holistic view of CD73-regulated NAD + metabolism as a complex network and further highlights the emerging roles of CD73 as a novel target for cancer therapies., (© 2024. The Author(s).)

Background: This study aims to analyze the pathogenic gene in a Chinese family with non-syndromic hearing loss and identify a novel mutation site in the TNC gene., Methods: A five-generation Chinese family from Anhui Province, presenting with autosomal dominant non-syndromic hearing loss, was recruited for this study. By analyzing the family history, conducting clinical examinations, and performing genetic analysis, we have thoroughly investigated potential pathogenic factors in this family. The peripheral blood samples were obtained from 20 family members, and the pathogenic genes were identified through whole exome sequencing. Subsequently, the mutation of gene locus was confirmed using Sanger sequencing. The conservation of TNC mutation sites was assessed using Clustal Omega software. We utilized functional prediction software including dbscSNV&#95;AdaBoost, dbscSNV&#95;RandomForest, NNSplice, NetGene2, and Mutation Taster to accurately predict the pathogenicity of these mutations. Furthermore, exon deletions were validated through RT-PCR analysis., Results: The family exhibited autosomal dominant, progressive, post-lingual, non-syndromic hearing loss. A novel synonymous variant (c.5247A > T, p.Gly1749Gly) in TNC was identified in affected members. This variant is situated at the exon-intron junction boundary towards the end of exon 18. Notably, glycine residue at position 1749 is highly conserved across various species. Bioinformatics analysis indicates that this synonymous mutation leads to the disruption of the 5' end donor splicing site in the 18th intron of the TNC gene. Meanwhile, verification experiments have demonstrated that this synonymous mutation disrupts the splicing process of exon 18, leading to complete exon 18 skipping and direct splicing between exons 17 and 19., Conclusion: This novel splice-altering variant (c.5247A > T, p.Gly1749Gly) in exon 18 of the TNC gene disrupts normal gene splicing and causes hearing loss among HBD families., (© 2024. The Author(s).)

Background: Myocardial inflammation and apoptosis induced by cirrhosis are among the primary mechanisms of cirrhotic cardiomyopathy. CD73, a common extracellular nucleotidase also known as 5'-nucleotidase, is associated with the progression of inflammation and immunity in multiple organs. However, the mechanism by which CD73 contributes to myocardial inflammation and apoptosis in cirrhosis remains unclear., Methods: In this study, a cirrhotic cardiomyopathy model in mice was established by bile duct ligation. Myocardial-specific overexpression of CD73 was achieved by tail vein injection of AAV9 (adeno-associated virus)-cTNT-NT5E-mCherry, and cardiac function in mice was assessed using echocardiography. Myocardial inflammation infiltration and apoptosis were evaluated through pathological observation and ELISA assays. The expression of CD73, A2AR, apoptotic markers, and proteins related to the NF-κB pathway in myocardial tissue were measured., Results: In the myocardial tissue of the cirrhotic cardiomyopathy mouse model, the expression of CD73 and A2AR increased. Overexpression of CD73 in the myocardium via AAV9 injection and stimulation of A2AR with CGS 21680 inhibited myocardial inflammation and cardiomyocyte apoptosis induced by cirrhosis. Additionally, overexpression of CD73 suppressed the activation of the NF-κB pathway by upregulating the expression of the adenosine receptor A2A., Conclusion: Our study reveals that the CD73/A2AR signaling axis mitigates myocardial inflammation and apoptosis induced by cirrhosis through negative feedback regulation of the NF-κB pathway., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zhao, Shao, Xia, Liu, Zhang, Zhao, Dang, Qian, Xu, Yu and Wang.)

In addition to their immunosuppressive effect, cytostatics conditioning prior to adoptive therapy such as chimeric antigen receptor (CAR) T cells may play a role in debulking and remodeling the tumor microenvironment. We investigated in vitro the killing efficacy and impact of treosulfan and fludarabine on ovarian cancer cells expressing mesothelin (MSLN) and effect on MSLN-targeting CAR T cells. Treosulfan and fludarabine had a synergetic effect on killing of SKOV3 and OVCAR4 cells. Sensitivity to the combination of treosulfan and fludarabine was increased when SKOV3 cells expressed MSLN and when OVCAR4 cells were tested in hypoxia, while MSLN cells surface expression by SKOV3 and OVCAR4 cells was not altered after treosulfan or fludarabine exposure. Exposure to treosulfan or fludarabine (10 µM) neither impacted MSLN-CAR T cells degranulation, cytokines production upon challenge with MSLN + OVCAR3 cells, nor induced mitochondrial defects. Combination of treosulfan and fludarabine decreased MSLN-CAR T cells anti-tumor killing in normoxia but not hypoxia. In conclusion, treosulfan and fludarabine killed MSLN + ovarian cancer cells without altering MSLN-CAR T cells functions (at low cytostatics concentration) even in hypoxic conditions, and our data support the use of treosulfan and fludarabine as conditioning drugs prior to MSLN-CAR T cell therapy., (© 2024. The Author(s).)

Background: A high-altitude environment has inhibitory effects on obesity. Tibetans are not a high-risk population for obesity, but there are still obese individuals within that population. Obesity has become a worldwide health problem, and previous studies have found that obesity is closely associated with hereditary factors. Few studies have investigated obesity in Tibetans, and the association between gene polymorphisms and obesity in Tibetans remains unclear. Methods: Our study investigated the fat mass of 140 native Tibetan individuals (70 men and 70 women) from Lhasa and analyzed the associations between polymorphisms of melanocortin 4 receptor (MC4R), Src homology 2B adapter protein 1 (SH2B1), and neuronal growth regulator 1 (NEGR1) and obesity. Result: Among Tibetan individuals, there were differences in genotype and allele frequencies between those in the obesity group and those in the healthy group at MC4R (rs17782313) and SH2B1 (rs7359397). The polymorphisms of MC4R (rs17782313) were associated with fat mass and obesity in Tibetan men and women, and there was an association between SH2B1 (rs7359397) polymorphisms and fat mass and obesity in Tibetan men. However, polymorphisms of NEGR1 (rs3101336) were not associated with fat mass or obesity in Tibetan individuals. Conclusion: Among Tibetan individuals, polymorphisms of MC4R (rs17782313) and SH2B1 (rs7359397) were associated with obesity, but NEGR1 (rs3101336) polymorphisms were not associated with obesity.

Hyaluronan (HA) is a high-molecular-weight (HMW) glycosaminoglycan, which is a fundamental component of the extracellular matrix that is involved in a variety of biological processes. We previously showed that the HYBID/KIAA1199/CEMIP axis plays a key role in the depolymerization of HMW-HA in normal human dermal fibroblasts (NHDFs). However, its roles in normal human epidermal keratinocytes (NHEKs) remained unclear. HYBID mRNA expression in NHEKs was lower than that in NHDFs, and NHEKs showed no depolymerization of extracellular HMW-HA in culture, indicating that HYBID does not contribute to extracellular HA degradation. In this study, we found that the cell-free conditioned medium of NHEKs degraded HMW-HA under weakly acidic conditions (pH 4.8). This degrading activity was abolished by hyaluronidase 1 (HYAL1) knockdown but not by HYAL2 knockdown. Newly synthesized HYAL1 was mainly secreted extracellularly, and the secretion of HYAL1 was increased during differentiation, suggesting that epidermal interspace HA is physiologically degraded by HYAL1 according to pH decrease during stratum corneum formation. In HA synthesis, hyaluronan synthase 3 (HAS3) knockdown reduced HA production by NHEKs, and interferon-γ-dependent HA synthesis was correlated with increased HAS3 expression. Furthermore, HA production was increased by TMEM2 knockdown through enhanced HAS3 expression. These results indicate that NHEKs regulate HA metabolism via HYAL1 and HAS3, and TMEM2 is a regulator of HAS3-dependent HA production., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Currently utilized serum tumor markers and fecal immunochemical tests do not have sufficient diagnostic power for colorectal cancer (CRC) due to their low sensitivities. To establish non-invasive urinary protein biomarkers for early CRC diagnosis, we performed stepwise analyses employing urine samples from CRCs and healthy controls (HCs)., Methods: Among 474 urine samples, 363 age- and sex-matched participants (188 HCs, 175 stage 0-III CRCs) were randomly divided into discovery (16 HCs, 16 CRCs), training (110 HCs, 110 CRCs), and validation (62 HCs, 49 CRCs) cohorts., Results: Of the 23 urinary protein candidates comprehensively identified from mass spectrometry in the discovery cohort, urinary levels of dipeptidase 1 (uDPEP1) and Trefoil factor1 (uTFF1) were the two most significant diagnostic biomarkers for CRC in both training and validation cohorts using enzyme-linked immunosorbent assays. A urinary biomarker panel comprising uDPEP1 and uTFF1 significantly distinguished CRCs from HCs, showing area under the curves of 0.825-0.956 for stage 0-III CRC and 0.792-0.852 for stage 0/I CRC. uDPEP1 and uTFF1 also significantly distinguished colorectal adenoma (CRA) patients from HCs, with uDPEP1 and uTFF1 increasing significantly in the order of HCs, CRA patients, and CRC patients. Moreover, expression levels of DPEP1 and TFF1 were also significantly higher in the serum and tumor tissues of CRC, compared to HCs and normal tissues, respectively., Conclusions: This study established a promising and non-invasive urinary protein biomarker panel, which enables the early detection of CRC with high sensitivity., (© 2024. Japanese Society of Gastroenterology.)

Head and neck squamous cell carcinoma (HNSCC) is a global cancer burden with a 5-year overall survival rate of around 50%, stagnant for decades. A tumour-induced immunosuppressive microenvironment contributes to HNSCC progression, with the adenosine (ADO) pathway and an upregulated expression of inhibitory immune checkpoint regulators playing a key role in this context. The correlation between high neutrophil-to-lymphocyte ratio (NLR) with advanced tumour staging suggests involvement of neutrophils (NØ) in cancer progression. Interestingly, we associated a high NLR with an increased intracellular PD-L1 localization in primary HNSCC samples, potentially mediating more aggressive tumour characteristics and therefore synergistically favouring tumour progression. Still, further research is needed to harness this knowledge for effective treatments and overcome resistance. Since it is hypothesized that the tumour microenvironment (TME) may be influenced by small extracellular vesicles (sEVs) secreted by tumours (TEX), this study aims to investigate the impact of HNSCC-derived TEX on NØ and blockade of ADO receptors as a potential strategy to reverse the pro-tumour phenotype of NØ. UMSCC47-TEX exhibited CD73 enzymatic activity involved in ADO signalling, as well as the immune checkpoint inhibitor PD-L1. Data revealed that TEX induce chemotaxis of NØ and the sustained interaction promotes a shift into a pro-tumour phenotype, dependent on ADO receptors (P1R), increasing CD170 high subpopulation, CD73 and PD-L1 expression, followed by an immunosuppressive secretome. Blocking A3R reduced CD73 and PD-L1 expression. Co-culture experiments with HNSCC cells demonstrated that TEX-modulated NØ increase the CD73/PD-L1 axis, through Cyclin D-CDK4/6 signalling. To support these findings, the CAM model with primary tumour was treated with NØ supernatant. Moreover, these NØ promoted an increase in migration, invasion, and reduced cell death. Targeting P1R on NØ, particularly A3R, exhibited potential therapeutic strategy to counteract immunosuppression in HNSCC. Understanding the TEX-mediated crosstalk between tumours and NØ offers insights into immunomodulation for improving cancer therapies., (© 2024 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

The Hedgehog (HH) pathway regulates embryonic development of anterior tongue taste fungiform papilla (FP) and the posterior circumvallate (CVP) and foliate (FOP) taste papillae. HH signaling also mediates taste organ maintenance and regeneration in adults. However, there are knowledge gaps in HH pathway component expression during postnatal taste organ differentiation and maturation. Importantly, the HH transcriptional effectors GLI1, GLI2 and GLI3 have not been investigated in early postnatal stages; the HH receptors PTCH1, GAS1, CDON and HHIP, required to either drive HH pathway activation or antagonism, also remain unexplored. Using lacZ reporter mouse models, we mapped expression of the HH ligand SHH, HH receptors, and GLI transcription factors in FP, CVP and FOP in early and late postnatal and adult stages. In adults we also studied the soft palate, and the geniculate and trigeminal ganglia, which extend afferent fibers to the anterior tongue. Shh and Gas1 are the only components that were consistently expressed within taste buds of all three papillae and the soft palate. In the first postnatal week, we observed broad expression of HH signaling components in FP and adjacent, non-taste filiform (FILIF) papillae in epithelium or stroma and tongue muscles. Notably, we observed elimination of Gli1 in FILIF and Gas1 in muscles, and downregulation of Ptch1 in lingual epithelium and of Cdon, Gas1 and Hhip in stroma from late postnatal stages. Further, HH receptor expression patterns in CVP and FOP epithelium differed from anterior FP. Among all the components, only known positive regulators of HH signaling, SHH, Ptch1, Gli1 and Gli2, were expressed in the ganglia. Our studies emphasize differential regulation of HH signaling in distinct postnatal developmental periods and in anterior versus posterior taste organs, and lay the foundation for functional studies to understand the roles of numerous HH signaling components in postnatal tongue development., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Kumari et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Plasmacytoid dendritic cells (pDCs) are vital players in antiviral immune responses because of their high levels of IFN-α secretion. However, this attribute has also implicated them as critical factors behind the immunopathogenesis of inflammatory diseases, and no currently available therapy can efficiently inhibit pDCs' aberrant activation. Mesenchymal stromal cells (MSCs) possess stromal immunomodulatory functionality, regulating immune cell activation through several mechanisms, including the adenosinergic (CD39/CD73/adenosine) pathway. The IFN-γ preconditioning of bone marrow MSCs improves their inhibitory properties for therapy applications; however, isolating human gingival tissue-derived MSCs (hGMSCs) is more accessible. These cells have shown better immunomodulatory effects, yet the outcome of IFN-γ preconditioning and its impact on the adenosinergic pathway has not been evaluated. This study first validated the immunoregulatory properties of primary-cultured hGMSCs, and the results showed that IFN-γ preconditioning strengthens CD39/CD73 coexpression, adenosine production, and the regulatory properties of hGMSC, which were confirmed by describing for the first time their ability to reduce pDC activation and their IFN-α secretion and to increase the frequency of CD73+ pDC. In addition, when CD73's enzymatic activity was neutralized in hGMSCs, adenosine production and the IFN-γ preconditioning effect were restrained. This evidence might be applied to design hGMSCs- and adenosine-based immunotherapeutic strategies for treating inflammatory disorders that are associated with pDC overactivation.

The expression of Neuritin-1 (NRN1), a neurotrophic factor crucial for neurodevelopment and synaptic plasticity, is enhanced by the Brain Derived Neurotrophic Factor (BDNF). Although the receptor of NRN1 remains unclear, it is suggested that NRN1's activation of the insulin receptor (IR) pathway promotes the transcription of the calcium voltage-gated channel subunit alpha1 C (CACNA1C). These three genes have been independently associated with schizophrenia (SZ) risk, symptomatology, and brain differences. However, research on how they synergistically modulate these phenotypes is scarce. We aimed to study whether the genetic epistasis between these genes affects the risk and clinical presentation of the disorder via its effect on brain structure. First, we tested the epistatic effect of NRN1 and BDNF or CACNA1C on (i) the risk for SZ, (ii) clinical symptoms severity and functionality (onset, PANSS, CGI and GAF), and (iii) brain cortical structure (thickness, surface area and volume measures estimated using FreeSurfer) in a sample of 86 SZ patients and 89 healthy subjects. Second, we explored whether those brain clusters influenced by epistatic effects mediate the clinical profiles. Although we did not find a direct epistatic impact on the risk, our data unveiled significant effects on the disorder's clinical presentation. Specifically, the NRN1-rs10484320 x BDNF-rs6265 interplay influenced PANSS general psychopathology, and the NRN1-rs4960155 x CACNA1C-rs1006737 interaction affected GAF scores. Moreover, several interactions between NRN1 SNPs and BDNF-rs6265 significantly influenced the surface area and cortical volume of the frontal, parietal, and temporal brain regions within patients. The NRN1-rs10484320 x BDNF-rs6265 epistasis in the left lateral orbitofrontal cortex fully mediated the effect on PANSS general psychopathology. Our study not only adds clinical significance to the well-described molecular relationship between NRN1 and BDNF but also underscores the utility of deconstructing SZ into biologically validated brain-imaging markers to explore their mediation role in the path from genetics to complex clinical manifestation., (© 2024. The Author(s).)

The GPR124/RECK/WNT7 pathway is an essential regulator of CNS angiogenesis and blood-brain barrier (BBB) function. GPR124, a brain endothelial adhesion seven-pass transmembrane protein, associates with RECK, which binds and stabilizes newly synthesized WNT7 that is transferred to frizzled (FZD) to initiate canonical β-catenin signaling. GPR124 remains enigmatic: although its extracellular domain (ECD) is essential, the poorly conserved intracellular domain (ICD) appears to be variably required in mammals versus zebrafish, potentially via adaptor protein bridging of GPR124 and FZD ICDs. GPR124 ICD deletion impairs zebrafish angiogenesis, but paradoxically retains WNT7 signaling upon mammalian transfection. We thus investigated GPR124 ICD function using the mouse deletion mutant Gpr124ΔC. Despite inefficiently expressed GPR124ΔC protein, Gpr124ΔC/ΔC mice could be born with normal cerebral cortex angiogenesis, in comparison with Gpr124-/- embryonic lethality, forebrain avascularity and hemorrhage. Gpr124ΔC/ΔC vascular phenotypes were restricted to sporadic ganglionic eminence angiogenic defects, attributable to impaired GPR124ΔC protein expression. Furthermore, Gpr124ΔC and the recombinant GPR124 ECD rescued WNT7 signaling in culture upon brain endothelial Gpr124 knockdown. Thus, in mice, GPR124-regulated CNS forebrain angiogenesis and BBB function are exerted by ICD-independent functionality, extending the signaling mechanisms used by adhesion seven-pass transmembrane receptors., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Multiple cellular pathways are affected by HPV E6 and E7 oncoproteins, including endocytic and cellular trafficking. HPV-16 E7 can target the adaptor protein (AP) complex, which contains proteins important during endocytosis transport. To further investigate the role of HPV E7 during this process, we analysed the expression of cell surface proteins in NIKS cells expressing HPV-16 E7. We show that different cell surface proteins are regulated by HPV-16 E7 via interaction with AP2. We observed that the expression of MET and CD109 membrane protein seems to be upregulated in cells expressing E7. Moreover, the interaction of MET and CD109 with AP2 proteins is disrupted by HPV-16 E7. In addition, in the absence of HPV-16 E7, there is a downregulation of the cell membrane expression of MET and CD109 in HPV-positive cell lines. These results expand our knowledge of the functions of E7 and open new potential cellular pathways affected by this oncoprotein., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The author is an Editorial Board Member/Editor-in-Chief/Associate Editor/Guest Editor for [Tumour Virus Research] and was not involved in the editorial review or the decision to publish this article., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Orbital connective tissue changes are contributors to the pathogenesis in thyroid eye disease (TED). Activated fibroblasts respond to immune stimuli with proliferation and increased hyaluronan (HA) production. Cyclosporin A (CsA) was reported to be beneficial in the treatment of TED. PDGF isoforms are increased in orbital tissue of TED patients and enhance HA production. We aimed to study the effect of CsA on HA production and hyaluronan synthase (HAS1, 2 and 3) and hyaluronidase (HYAL1 and 2) mRNA expressions in orbital fibroblasts (OFs). Measurements were performed in the presence or absence of CsA (10 μM) in unstimulated or PDGF-BB (10 ng/ml) stimulated OFs. The HA production of TED OFs (n = 7) and NON-TED OFs (n = 6) were measured by ELISA. The levels of mRNA expressions were examined using RT-PCR. The proliferation rate and metabolic activity were measured by BrdU incorporation and MTT assays, respectively. Treatment with CsA resulted in an average 42% decrease in HA production of OFs (p < 0.0001). CsA decreased the expression levels of HAS2, HAS3 and HYAL2 (p = 0.005, p = 0.005 and p = 0.002, respectively.) PDGF-BB increased HA production (p < 0.001) and HAS2 expression (p = 0.004). CsA could reduce the PDGF-BB-stimulated HA production (p < 0.001) and HAS2 expression (p = 0.005) below the untreated level. In addition, CsA treatment caused a decrease in proliferation potential (p = 0.002) and metabolic activity (p < 0.0001). These findings point to the fact that CsA affects HA metabolism via HAS2, HAS3 and HYAL2 inhibition in OFs. In addition to its well characterized immunosuppressant properties, CsA's beneficial effect in TED may be related to its direct inhibitory effect on basal and growth factor stimulated HA production., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

A 52-year-old male developed right knee pain after hiking in Guatemala. On his return he underwent a knee MRI for an indication of medial knee pain, which demonstrated a medial meniscal tear. However, the MRI demonstrated marked tortuosity and dense calcification of the popliteal artery, confirmed on subsequent radiographs. Review of previous CT studies of the abdomen and lower extremities showed severe ectasia and arterial calcification in the femoral and popliteal arteries bilaterally, but no calcifications in the aorta and common iliac arteries. Dual energy CT studies of the extremities demonstrated extensive periarticular soft tissue calcification throughout the wrists, hands, ankle and feet without evidence of uric acid. Review of the electronic medical records revealed a diagnosis of Arterial Calcification due to Deficiency of CD73 (ACDC), a rare genetic disorder presenting with debilitating pain in the wrists and hands, claudication of the calves, thighs and buttocks, progressing to chronic ischemia of the feet which may be limb-threatening. The patient was enrolled in an NIH trial of bisphosphonates and dual-antiplatelet therapy with stabilization of symptoms. This case discusses the imaging findings of this rare condition, differential diagnosis to consider, and current management., Competing Interests: Disclosures: None, (Copyright Journal of Radiology Case Reports.)

Renal tubular injury is an important pathological change associated with diabetic nephropathy (DN), in which ferroptosis of renal tubular epithelial cells is critical to its pathogenesis. Inhibition of the glutathione/glutathione peroxidase 4 (GSH/GPX4) axis is the most important mechanism in DN tubular epithelial cell ferroptosis, but the underlying reason for this is unclear. Our biogenic analysis showed that a zinc-dependent metalloproteinase, dipeptidase 1 (DPEP1), is associated with DN ferroptosis. Here, we investigated the role and mechanism of DPEP1 in DN tubular epithelial cell ferroptosis. DPEP1 upregulation was observed in the renal tubular epithelial cells of DN patients and model mice, as well as in HK-2 cells stimulated with high glucose. Furthermore, the level of DPEP1 upregulation was associated with the degree of tubular injury in DN patients and HK-2 cell ferroptosis. Mechanistically, knocking down DPEP1 expression could alleviate the inhibition of GSH/GPX4 axis and reduce HK-2 cell ferroptosis levels in a high glucose environment. HK-2 cells with stable DPEP1 overexpression also showed GSH/GPX4 axis inhibition and ferroptosis, but blocking the GSH/GPX4 axis could mitigate these effects. Additionally, treatment with cilastatin, a DPEP1 inhibitor, could ameliorate GSH/GPX4 axis inhibition and relieve ferroptosis and DN progression in DN mice. These results revealed that DPEP1 can promote ferroptosis in DN renal tubular epithelial cells via inhibition of the GSH/GPX4 axis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The pathogenesis of osteoporosis (OP) is closely associated with the disrupted balance between osteogenesis and adipogenesis in bone marrow-derived mesenchymal stem cells (BMSCs). We analyzed published single-cell RNA sequencing (scRNA-seq) data to dissect the transcriptomic profiles of bone marrow-derived cells in OP, reviewing 56 377 cells across eight scRNA-seq datasets from femoral heads (osteoporosis or osteopenia n = 5, osteoarthritis n = 3). Seventeen genes, including carboxypeptidase M (CPM), were identified as key osteogenesis-adipogenesis regulators through comprehensive gene set enrichment, differential expression, regulon activity, and pseudotime analyses. In vitro, CPM knockdown reduced osteogenesis and promoted adipogenesis in BMSCs, while adenovirus-mediated CPM overexpression had the reverse effects. In vivo, intraosseous injection of CPM-overexpressing BMSCs mitigated bone loss in ovariectomized mice. Integrated scRNA-seq and bulk RNA sequencing analyses provided insight into the MAPK/ERK pathway's role in the CPM-mediated regulation of BMSC osteogenesis and adipogenesis; specifically, CPM overexpression enhanced MAPK/ERK signaling and osteogenesis. In contrast, the ERK1/2 inhibitor binimetinib negated the effects of CPM overexpression. Overall, our findings identify CPM as a pivotal regulator of BMSC differentiation, which provides new clues for the mechanistic study of OP., (© 2024 Federation of American Societies for Experimental Biology.)

Placenta Accreta Spectrum (PAS) is a life-threatening condition in which placental trophoblastic cells abnormally invade the uterus, often up to the uterine serosa and, in extreme cases, tissues beyond the uterine wall. Currently, there is no clinical assay for the non-invasive detection of PAS, and only ultrasound and MRI can be used for its diagnosis. Considering the subjectivity of visual assessment, the detection of PAS necessitates a high degree of expertise and, in some instances, can lead to its misdiagnosis. In clinical practice, up to 50% of pregnancies with PAS remain undiagnosed until delivery, and it is associated with increased risk of morbidity/mortality. Although many studies have evaluated the potential of fetal biomarkers circulating in maternal blood, very few studies have evaluated the potential of circulating placental extracellular vesicles (EVs) and their miRNA contents for molecular detection of PAS. Thus, to purify placental EVs from maternal blood, we customized our robust ultra-sensitive immuno-purification assay, termed EV-CATCHER, with a monoclonal antibody targeting the membrane Placental Alkaline Phosphatase (PLAP) protein, which is unique to the placenta and present on the surface of placental EVs. Then, as a pilot evaluation, we compared the miRNA expression profiles of placental EVs purified from the maternal plasma of women diagnosed with placenta previa (controls, n = 16); placenta lying low in uterus but not invasive) to those of placental EVs purified from the plasma of women with placenta percreta (cases, n = 16), PAS with the highest level of invasiveness. Our analyses reveal that miRNA profiling of PLAP + EVs purified from maternal plasma identified 40 differentially expressed miRNAs when comparing these two placental pathologies. Preliminary miRNA pathway enrichment and gene ontology analysis of the top 14 upregulated and top nine downregulated miRNAs in PLAP + EVs, purified from the plasma of women diagnosed with placenta percreta versus those diagnosed with placenta previa, suggests a potential role in control of cellular invasion and motility that will require further investigation.

Elite controllers (ECs) are an exceptional group of people living with HIV (PLWH) that control HIV replication without therapy. Among the mechanisms involved in this ability, natural killer (NK)-cells have recently gained much attention. We performed an in-deep phenotypic analysis of NK-cells to search for surrogate markers associated with the long term spontaneous control of HIV. Forty-seven PLWH (22 long-term EC [PLWH-long-term elite controllers (LTECs)], 15 noncontrollers receiving antiretroviral treatment [ART] [PLWH-onART], and 10 noncontrollers cART-naïve [PLWH-offART]), and 20 uninfected controls were included. NK-cells homeostasis was analyzed by spectral flow cytometry using a panel of 15 different markers. Data were analyzed using FCSExpress and R software for unsupervised multidimensional analysis. Six different subsets of NK-cells were defined on the basis of CD16 and CD56 expression, and the multidimensional analysis revealed the existence of 68 different NK-cells clusters based on the expression levels of the 15 different markers. PLWH-offART presented the highest disturbance of NK-cells homeostasis and this was not completely restored by long-term ART. Interestingly, long term spontaneous control of HIV (PLWH-LTEC group) was associated with a specific profile of NK-cells homeostasis disturbance, characterized by an increase of CD16 dim CD56 dim subset when compared to uninfected controls (UC) group and also to offART and onART groups (p < 0.0001 for the global comparison), an increase of clusters C16 and C26 when compared to UC and onART groups (adjusted p-value < 0.05 for both comparisons), and a decrease of clusters C10 and C20 when compared to all the other groups (adjusted p-value < 0.05 for all comparisons). These findings may provide clues to elucidate markers of innate immunity with a relevant role in the long-term control of HIV., (© 2024 Wiley Periodicals LLC.)

The detection of trace cancer markers in body fluids such as blood/serum is crucial for cancer diseases screening and treatment, which requires high sensitivity and specificity of biosensors. In this study, a peanut structure cascaded lasso (PSCL) shaped fiber sensing probe based on fiber laser demodulation method was proposed to specifically detect the carcinoembryonic antigen related cell adhesion molecules 5 (CEACAM5) protein in serum. Thanks for the narrow linewidth and high signal-to-noise ratio (SNR) of the laser spectrum, it is easier to distinguish small spectral changes than interference spectrum. Adding the antibody modified magnetic microspheres (MMS) to form the sandwich structure of "antibody-antigen-antibody-MMS", and amplified the response caused by biomolecular binding. The limit of detection (LOD) for CEACAM5 in buffer could reach 0.11 ng/mL. Considering the common threshold of 5 ng/mL for CEA during medical screening and the cut off limit of 2.5 ng/mL for some kits, the LOD of proposed biosensor meets the actual needs. Human serum samples from a hospital were used to validate the real sensing capability of proposed biosensor. The deviation between the measured value in various serum samples and the clinical value ranged from 1.9 to 9.8 %. This sensing scheme holds great potential to serve as a point of care testing (POCT) device and extend to more biosensing applications., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Kawasaki disease (KD) is a pediatric systemic vasculitis characterized by endothelial cell dysfunction. Semaphorin 7A (Sema7A) has been reported to regulate endothelial phenotypes associated with cardiovascular diseases, while its role in KD remains unknown. This study aims to investigate the effect of Sema7A on endothelial permeability and inflammatory response in KD conditions., Methods: Blood samples were collected from 68 KD patients and 25 healthy children (HC). The levels of Sema7A and A Disintegrin and Metalloprotease 17 (ADAM17) in serum were measured by enzyme-linked immunosorbent assay (ELISA), and Sema7A expression in blood cells was analyzed by flow cytometry. Ex vivo monocytes were used for Sema7A shedding assays. In vitro human coronary artery endothelial cells (HCAECs) were cultured in KD sera and stimulated with Sema7A, and TNF-α, IL-1β, IL-6, and IL-18 of HCAECs were measured by ELISA and qRT-PCR. HCAECs monolayer permeability was measured by FITC-dextran., Results: The serum level of Sema7A was significantly higher in KD patients than in HC and correlated with disease severity. Monocytes were identified as one of the source of elevated serum Sema7A, which implicates a process of ADAM17-dependent shedding. Sera from KD patients induced upregulation of plexin C1 and integrin β1 in HCAECs compared to sera from HC. Sema7A mediated the proinflammatory cytokine production of HCAECs in an integrin β1-dependent manner, while both plexin C1 and integrin β1 contributed to Sema7A-induced HCAEC hyperpermeability., Conclusions: Sema7A is involved in the progression of KD vasculitis by promoting endothelial permeability and inflammation through a plexin C1 and integrin β1-dependent pathway. Sema7A may serve as a potential biomarker and therapeutic target in the prognosis and treatment of KD., (© 2024. The Author(s).)

Background: Blocking the CD47 "don't eat me"-signal on tumor cells with monoclonal antibodies or fusion proteins has shown limited clinical activity in hematologic malignancies and solid tumors thus far. Main side effects are associated with non-tumor targeted binding to CD47 particularly on blood cells., Methods: We present here the generation and preclinical development of NILK-2401, a CEACAM5×CD47 bispecific antibody (BsAb) composed of a common heavy chain and two different light chains, one kappa and one lambda, determining specificity (so-called κλ body format)., Results: NILK-2401 is a fully human BsAb binding the CEACAM5 N-terminal domain on tumor cells by its lambda light chain arm with an affinity of ≈4 nM and CD47 with its kappa chain arm with an intendedly low affinity of ≈500 nM to enabling tumor-specific blockade of the CD47-SIRPα interaction. For increased activity, NILK-2401 features a functional IgG1 Fc-part. NILK-2401 eliminates CEACAM5-positive tumor cell lines (3/3 colorectal, 2/2 gastric, 2/2 lung) with EC 50 for antibody-dependent cellular phagocytosis and antibody-dependent cellular cytotoxicity ranging from 0.38 to 25.84 nM and 0.04 to 0.25 nM, respectively. NILK-2401 binds neither CD47-positive/CEACAM5-negative cell lines nor primary epithelial cells. No erythrophagocytosis or platelet activation is observed. Quantification of the pre-existing NILK-2401-reactive T-cell repertoire in the blood of 14 healthy donors with diverse HLA molecules shows a low immunogenic potential. In vivo , NILK-2401 significantly delayed tumor growth in a NOD-SCID colon cancer model and a syngeneic mouse model using human CD47/human SIRPα transgenic mice and prolonged survival. In cynomolgus monkeys, single doses of 0.5 and 20 mg/kg were well tolerated; PK linked to anti-CD47 and Fc-binding seemed to be more than dose-proportional for C max and AUC 0-inf . Data were validated in human FcRn TG32 mice. Combination of a CEACAM5-targeting T-cell engager (NILK-2301) with NILK-2401 can either boost NILK-2301 activity (Emax) up to 2.5-fold or allows reaching equal NILK-2301 activity at >600-fold (LS174T) to >3,000-fold (MKN-45) lower doses., Conclusion: NILK-2401 combines promising preclinical activity with limited potential side effects due to the tumor-targeted blockade of CD47 and low immunogenicity and is planned to enter clinical testing., Competing Interests: AS and DH declare employment at LamKap Bio Group. VB, VM, BD, FR, LC, AV, NB, ER, KM, CB, CM, FJ-B, and YP declare employment at Light Chain Bioscience - Novimmune SA. SS-P declares consultancy work for Light Chain Bioscience - Novimmune SA and LamKap Bio. The authors declare that this study received funding from LamKap Bio beta. The funder or its employees had the following involvement in the study: study design, data analysis, decision to publish, and preparation of the manuscript. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Seckinger, Buatois, Moine, Daubeuf, Richard, Chatel, Viandier, Bosson, Rousset, Masternak, Salgado-Pires, Batista, Mougin, Juan-Bégeot, Poitevin and Hose.)

Background: Patients with a stroke often cannot care for themselves after hospital discharge. Assessment of their self-care ability is the first step in planning post-discharge home care. This study aimed to design and validate a measure of perceived self-care ability (PSCA) in stroke patients., Methods: A sequential-exploratory mixed method was conducted in Tehran, Iran, in 2020-2021. The qualitative phase involved in-depth semi-structured interviews with 12 participants. Transcripts were content analyzed. The results guided the development of 81 items. psychometric properties such as face validity (Impact Score > 1.5), content validity ratio (CVR > 0.63), content validity index (Item Content Validity Index: ICVI > 0.78, Scale Content Validity Index/Average: SCVI/Ave > 0.8) and Kappa value (Kappa > 0.7), internal consistency (Cronbach's alpha > 0.7), relative reliability (ICC: inter class correlation coefficient), absolute reliability (Standard Error of Measurement: SEM and Minimal Detectable Changes: MDC), convergent validity (Correlation Coefficient between 0.4-0.7), interpretability, responsiveness, feasibility, and ceiling and floor effects were assessed., Results: Content analysis of the qualitative interviews yielded 5 major categories and 9 subcategories that reflected "Perceptual stability", "Cognitive fluctuations", "Sensory, Motor and Physical health"," The subjective nature" and "The dynamic nature" of PSCA. Results of face and content validity reduced the number of items to 32, capturing three dimensions of PSCA in chronic stroke patients; these dimensions included perceptual ability, threatened health status, and sensory, motor, and cognitive ability. The findings supported the reliability and validity of the measure., Conclusions: The PSCA questionnaire was developed and validated within the Iranian culture. It is useful in assessing the self-care of patients with stroke and in informing practice., (© 2024. The Author(s).)

Background: Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), as a typical tumor marker, has been found to exert immunomodulatory effects in many diseases. We previously reported the clinical and molecular evidences supporting that SARS-Cov-2 infected the gastrointestinal (GI) tract and found a reduction of CEACAM5 in COVID-19 patients' feces which associated with gut dysbiosis. Yet the role of CEACAM5 in GI infection is ill-defined., Methods: Mice models were established through intraperitoneally injecting with recombinant viral spike-Fc to mimic the intestinal inflammation. We collected duodenum, jejunum, ileum and colon samples after 6h, 2 days, 4 days and 7 days of spike-Fc or control-Fc injection to perform proteomic analysis. Blood was collected from healthy donors and peripheral blood mononuclear cells (PBMC) were separated by density gradient centrifugation, then CD4+ T cells were isolated with magnetic beads and co-cultured with Caco-2 cells., Results: In addition to intestinal CEACAM5, the expression of tight junction and the percent of CD4+ T lymphocytes were significantly decreased in spike-Fc group compared to control (p < 0.05), accompanied with increased level of inflammatory factors. The KEGG analysis revealed differentially expressed proteins were mainly enriched in the coronavirus disease (COVID-19), tight junction, focal adhesion, adherens junction and PI3K-Akt signaling pathway. Protein-protein interaction (PPI) network analysis identified the interaction between CEACAM5 and Galectin-9 that was also verified by molecular docking and co-IP assay. We further confirmed a reduction of CEACAM5 in SARS-CoV-2 spike stimulated enterocytes could promote the expression of Galectin-9 protein in CD4+T cells. Then it gave rise to the increasing release of inflammatory factors and increased apoptosis of CD4+T cells by inhibition of PI3K/AKT/mTOR pathway. Ultimately intestinal barrier dysfunction happened., Conclusion: Our results indicated that CEACAM5 overexpression and Galectin-9 knockdown played a protective role in intestinal barrier injury upon spike-Fc stimulation. Collectively, our findings identified firstly that SARS-CoV-2 spike induced intestinal barrier dysfunction through the interaction between CEACAM5 and Galectin-9. The result provides potential therapeutic targets in intestinal barrier dysfunction for treating severe COVID patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Luo, Zhang, Ren, Dou, Wen, Yang, Li, Yan and Han.)

To explore the molecular biological characteristics of lung cancer associated with cystic airspaces (LCCA) and its potential roles on prognosis. A total of 165 LCCAs and 201 non-LCCAs were enrolled in this study. Bulk RNA sequencing was implemented in eight LCCAs and nine non-LCCAs to explore the differentially expressed genes. TCGA data were used to analyze LCCA-specific genes that associated with overall survival (OS). The median age was 60 (IQR 53 to 65) years in LCCA cohort. We found LCCA were predominant in men and had less visceral pleura invasion (VPI) or lympho-vascular invasion (LVI). Moreover, LCCA presented with higher histological heterogeneity. Kaplan-Meier analysis showed that patients of age more than 60 and positive VPI had significantly less PFS in LCCA. Cox regression suggested that LCCA, micropapillary subtype proportion and VPI were the independent risk factors for PFS. LCCA had up-regulated pathways associated with EMT, angiogenesis and cell migration. In addition, LCCA displayed higher levels of immunosuppressor infiltration (M2 macrophages, CAFs and MDSCs) and distinct cell death and metabolic patterns. BCR/TCR repertoire analysis revealed less BCR richness, clonality and high-abundance shared clonotypes in LCCA. Finally, Cox regression analysis identified that four cystic-specific genes, KCNK3, NRN1, PARVB and TRHDE-AS1, were associated with OS of lung adenocarcinoma (LUAD). And cystic-specific risk scores (CSRSs) were calculated to construct a nomogram, which performance well. Our study for the first time indicated significantly distinct molecular biological and immune characteristics between LCCA and non-LCCA, which provide complementary prognostic values in early-stage non-small cell lung cancer (NSCLC)., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2024. Published by Elsevier Ltd.)

Recent advancements in bioengineering have introduced potential alternatives to liver transplantation via the development of self-assembled liver organoids, derived from human-induced pluripotent stem cells (hiPSCs). However, the limited maturity of the tissue makes it challenging to implement this technology on a large scale in clinical settings. In this study, we developed a highly efficient method for generating functional liver organoids from hiPSC-derived carboxypeptidase M liver progenitor cells (CPM+ LPCs), using a microwell structure, and enhanced maturation through direct oxygenation in oxygen-permeable culture plates. We compared the morphology, gene expression profile, and function of the liver organoid with those of cells cultured under conventional conditions using either monolayer or spheroid culture systems. Our results revealed that liver organoids generated using polydimethylsiloxane-based honeycomb microwells significantly exhibited enhanced albumin secretion, hepatic marker expression, and cytochrome P450-mediated metabolism. Additionally, the oxygenated organoids consisted of both hepatocytes and cholangiocytes, which showed increased expression of bile transporter-related genes as well as enhanced bile transport function. Oxygen-permeable polydimethylsiloxane membranes may offer an efficient approach to generating highly mature liver organoids consisting of diverse cell populations., (© 2024 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Regulatory T (Treg) cells are critical in shaping an immunosuppressive microenvironment to favor tumor progression and resistance to therapies. However, the heterogeneity and function of Treg cells in esophageal squamous cell carcinoma (ESCC) remain underexplored. We identified CD177 as a tumor-infiltrating Treg cell marker in ESCC. Interestingly, expression levels of CD177 and PD-1 were mutually exclusive in tumor Treg cells. CD177 + Treg cells expressed high levels of IL35, in association with CD8 + T cell exhaustion, whereas PD-1 + Treg cells expressed high levels of IL10. Pan-cancer analysis revealed that CD177 + Treg cells display increased clonal expansion compared to PD-1 + and double-negative (DN) Treg cells, and CD177 + and PD-1 + Treg cells develop from the same DN Treg cell origin. Importantly, we found CD177 + Treg cell infiltration to be associated with poor overall survival and poor response to anti-PD-1 immunotherapy plus chemotherapy in ESCC patients. Finally, we found that lymphatic endothelial cells are associated with CD177 + Treg cell accumulation in ESCC tumors, which are also decreased after anti-PD-1 immunotherapy plus chemotherapy. Our work identifies CD177 + Treg cell as a tumor-specific Treg cell subset and highlights their potential value as a prognostic marker of survival and response to immunotherapy and a therapeutic target in ESCC., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2024 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis of all cancers. To improve PDAC therapy, we establish screening systems based on organoid and co-culture technologies and find a payload of antibody-drug conjugate (ADC), a bromodomain and extra-terminal (BET) protein degrader named EBET. We select CEACAM6/CD66c as an ADC target and developed an antibody, #84.7, with minimal reactivity to CEACAM6-expressing normal cells. EBET-conjugated #84.7 (84-EBET) has lethal effects on various PDAC organoids and bystander efficacy on CEACAM6-negative PDAC cells and cancer-associated fibroblasts. In mouse studies, a single injection of 84-EBET induces marked tumor regression in various PDAC-patient-derived xenografts, with a decrease in the inflammatory phenotype of stromal cells and without significant body weight loss. Combination with standard chemotherapy or PD-1 antibody induces more profound and sustained regression without toxicity enhancement. Our preclinical evidence demonstrates potential efficacy by delivering BET protein degrader to PDAC and its microenvironment via CEACAM6-targeted ADC., (© 2024. The Author(s).)

Leveraging the specificity of antibody to deliver cytotoxic agent into tumor, antibody-drug conjugates (ADCs) have become one of the hotspots in the development of anticancer therapies. Although significant progress has been achieved, there remain challenges to overcome, including limited penetration into solid tumors and potential immunogenicity. Fully human single-domain antibodies (UdAbs), with their small size and human nature, represent a promising approach for addressing these challenges. Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) is a glycosylated cell surface protein that rarely expressed in normal adult tissues but overexpressed in diverse cancers, taking part in tumorigenesis, progression, and metastasis. In this study, we investigated the therapeutic potential of UdADC targeting CEACAM5. We performed biopanning in our library and obtained an antibody candidate B9, which bound potently and specifically to CEACAM5 protein (K D  = 4.84 nM) and possessed excellent biophysical properties (low aggregation tendency, high homogeneity, and thermal stability). The conjugation of B9 with a potent cytotoxic agent, monomethyl auristatin E (MMAE), exhibited superior antitumor efficacy against CEACAM5-expressing human gastric cancer cell line MKN-45, human pancreatic carcinoma cell line BxPC-3 and human colorectal cancer cell line LS174T with IC 50 values of 38.14, 25.60, and 101.4 nM, respectively. In BxPC-3 and MKN-45 xenograft mice, administration of UdADC B9-MMAE (5 mg/kg, i.v.) every 2 days for 4 times markedly inhibited the tumor growth without significant change in body weight. This study may have significant implications for the design of next-generation ADCs., (© 2023. The Author(s), under exclusive licence to Shanghai Institute of Materia Medica, Chinese Academy of Sciences and Chinese Pharmacological Society.)

Endoplasmic reticulum-associated degradation (ERAD) plays indispensable roles in many physiological processes; however, the nature of endogenous substrates remains largely elusive. Here we report a proteomics strategy based on the intrinsic property of the SEL1L-HRD1 ERAD complex to identify endogenous ERAD substrates both in vitro and in vivo. Following stringent filtering using a machine learning algorithm, over 100 high-confidence potential substrates are identified in human HEK293T and mouse brown adipose tissue, among which ~88% are cell type-specific. One of the top shared hits is the catalytic subunit of the glycosylphosphatidylinositol (GPI)-transamidase complex, PIGK. Indeed, SEL1L-HRD1 ERAD attenuates the biogenesis of GPI-anchored proteins by specifically targeting PIGK for proteasomal degradation. Lastly, several PIGK disease variants in inherited GPI deficiency disorders are also SEL1L-HRD1 ERAD substrates. This study provides a platform and resources for future effort to identify proteome-wide endogenous substrates in vivo, and implicates SEL1L-HRD1 ERAD in many cellular processes including the biogenesis of GPI-anchored proteins., (© 2024. The Author(s).)

Carcinoembryonic antigen (CEACAM5), as a broad-spectrum tumor biomarker, plays a crucial role in analyzing the therapeutic efficacy and progression of cancer. Herein, we propose a novel biosensor based on specklegrams of tapered multimode fiber (MMF) and two-dimensional convolutional neural networks (2D-CNNs) for the detection of CEACAM5. The microfiber is modified with CEA antibodies to specifically recognize antigens. The biosensor utilizes the interference effect of tapered MMF to generate highly sensitive specklegrams in response to different CEACAM5 concentrations. A zero mean normalized cross-correlation (ZNCC) function is explored to calculate the image matching degree of the specklegrams. Profiting from the extremely high detection limit of the speckle sensor, variations in the specklegrams of antibody concentrations from 1 to 1000 ng/mL are measured in the experiment. The surface sensitivity of the biosensor is 0.0012 (ng/mL) -1 within a range of 1 to 50 ng/mL. Moreover, a 2D-CNN was introduced to solve the problem of nonlinear detection surface sensitivity variation in a large dynamic range, and in the search for image features to improve evaluation accuracy, achieving more accurate CEACAM5 monitoring, with a maximum detection error of 0.358%. The proposed fiber specklegram biosensing scheme is easy to implement and has great potential in analyzing the postoperative condition of patients.

A glycosylphosphatidylinositol (GPI) derivative with biotin linked to its mannose III 6-O-position was prepared by a convergent strategy. This biotinylated GPI was demonstrated to bind avidinated proteins readily through biotin-avidin interaction and, therefore, can serve as a universal platform to access various biologically significant GPI-anchored protein analogues.

Background and Objectives: Prior work suggests that cognitive resilience may contribute to the heterogeneity of cognitive decline. This study examined whether distinct cortical proteins provide resilience for different cognitive abilities., Methods: Participants were from the Religious Orders Study or the Rush Memory and Aging Project who had undergone annual assessments of 5 cognitive abilities and postmortem assessment of 9 Alzheimer disease and related dementia (ADRD) pathologies. Proteome-wide examination of the dorsolateral prefrontal cortex using tandem mass tag and liquid chromatography-mass spectrometry yielded 8,425 high-abundance proteins. We applied linear mixed-effect models to quantify residual cognitive change (cognitive resilience) of 5 cognitive abilities by regressing out cognitive decline related to age, sex, education, and indices of ADRD pathologies. Then we added terms for each of the individual proteins to identify cognitive resilience proteins associated with the different cognitive abilities., Results: We included 604 decedents (69% female; mean age at death = 89 years) with proteomic data. A total of 47 cortical proteins that provide cognitive resilience were identified: 22 were associated with specific cognitive abilities, and 25 were common to at least 2 cognitive abilities. NRN1 was the only protein that was associated with more than 2 cognitive abilities (semantic memory: estimate = 0.020, SE = 0.004, p = 2.2 × 10 -6 ; episodic memory: estimate = 0.029, SE = 0.004, p = 5.8 × 10 -1 ; and working memory: estimate = 0.021, SE = 0.004, p = 1.2 × 10 -7 ). Exploratory gene ontology analysis suggested that among top molecular pathways, mitochondrial translation was a molecular mechanism providing resilience in episodic memory, while nuclear-transcribed messenger RNA catabolic processes provided resilience in working memory., Discussion: This study identified cortical proteins associated with various cognitive abilities. Differential associations across abilities may reflect distinct underlying biological pathways. These data provide potential high-value targets for further mechanistic and drug discovery studies to develop targeted treatments to prevent loss of cognition.