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Chimeric antigen receptors (CARs) are fusion proteins with an extracellular antigen recognition domain and numerous intracellular signaling domains that have been genetically modified. CAR-engineered T lymphocyte-based therapies have shown great success against blood cancers; however, potential fatal toxicity, such as in cytokine release syndrome, and high costs are some shortcomings that limit the clinical application of CAR-engineered T lymphocytes and remain to overcome. Natural killer (NK) cells are the focal point of current immunological research owing to their receptors that prove to be promising immunotherapeutic candidates for treating cancer. However, to date, manipulation of NK cells to treat malignancies has been moderately successful. Recent progress in the biology of NK cell receptors has greatly transformed our understanding of how NK cells recognize and kill tumor and infected cells. CAR-NK cells may serve as an alternative candidate for retargeting cancer because of their unique recognition mechanisms, powerful cytotoxic effects especially on cancer cells in both CAR-dependent and CAR-independent manners and clinical safety. Moreover, NK cells can serve as an 'off-the-shelf product' because NK cells from allogeneic sources can also be used in immunotherapies owing to their reduced risk of alloreactivity. Although ongoing fundamental research is in the beginning stages, this review provides an overview of recent developments implemented to design CAR constructs to stimulate NK activation and manipulate NK receptors for improving the efficiency of immunotherapy against cancer, summarizes the preclinical and clinical advances of CAR-NK cells against both hematological malignancies and solid tumors and confronts current challenges and obstacles of their applications. In addition, this review provides insights into prospective novel approaches that further enhance the efficiency of CAR-NK therapies and highlights potential questions that require to be addressed in the future., 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 © 2021 Khawar and Sun.)

For a long time, proteins with enzymatic activity have not been usually considered to carry out other functions different from catalyzing chemical reactions within or outside the cell. Nevertheless, in the last few years several reports have uncovered the participation of numerous enzymes in other processes, placing them in the category of moonlighting proteins. Some moonlighting enzymes have been shown to participate in complex processes such as cell adhesion. Cell adhesion plays a physiological role in multiple processes: it enables cells to establish close contact with one another, allowing communication; it is a key step during cell migration; it is also involved in tightly binding neighboring cells in tissues, etc. Importantly, cell adhesion is also of great importance in pathophysiological scenarios like migration and metastasis establishment of cancer cells. Cell adhesion is strictly regulated through numerous switches: proteins, glycoproteins and other components of the cell membrane. Recently, several cell membrane enzymes have been reported to participate in distinct steps of the cell adhesion process. Here, we review a variety of examples of membrane bound enzymes participating in adhesion of immune cells., 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 © 2021 López-Cortés, Díaz-Alvarez and Ortega.)

Aim: CD73 overexpression has been reported in several malignancies and is considered to be a novel immune checkpoint. However, the role and significance of CD73 in gastric cancer (GC) still remain obscure. We aim to investigate the role of CD73 expression in predicting prognosis, shaping immune contexture and guiding therapeutic strategy in GC., Methods: The study enrolled four independent cohorts with a total of 902 patients with GC. CD73 expression and immune contexture were examined by immunohistochemistry, single-sample gene set enrichment analysis and flow cytometry. Clinical outcomes of patient subgroups were evaluated using the Kaplan-Meier curves and Cox proportional hazard analysis. All statistical tests were two-sided., Results: CD73 was identified as an independent adverse prognostic factor for survival in GC. CD73 high tumours showed a specific microenvironment with more CD8+ T cell infiltration, but these CD8+ T cells displayed a dysfunctional phenotype. Furthermore, the CD73 (NT5E) mRNA level was associated with the Cancer Genome Atlas molecular subtypes, and NT5E high tumours showed significant fibroblast growth factor receptor 2 activation and vascular endothelial growth factor and receptor enrichment. In addition, CD73 high tumours indicated better chemotherapeutic responsiveness to fluorouracil yet a worse objective response rate to pembrolizumab in GC., Conclusions: High CD73 expression indicated an immunoevasive contexture with CD8+ T cell dysfunction and represented an independent predictor for adverse clinical outcomes. As a potential immunotherapeutic target, CD73 could potentially be a novel biomarker for adjuvant chemotherapy, targeted therapies and immunotherapy. The crucial role of CD73 in the therapeutic landscape of GC needs further validation retrospectively and prospectively., Competing Interests: Conflict of interest statement The authors declare no conflict of interest., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The present study aimed to assess spinal tract formation in neurons originating from cervical (C7), brachial (C14), and thoracic (T4) regions, with the lumbar (LS2) region as a reference, in a chick embryo. For the assessment of the spinal tracts, we introduced a vector expressing human placental alkaline phosphatase into progenitor cells generated after neural tube closure and belonging to the above segments, using in ovo electroporation. The ascending axons took primarily similar paths: dorsal commissural, ventral commissural, and dorsal non-commissural paths, with some variance depending on their originating segments. Some populations of non-commissural neurons later extended their axons following a ventral path. The elongation rates of these axons are primarily constant and tended to increase over time; however, some variations depending on the originating segments were also observed. Some of the dorsally ascending axons entered into the developing cerebellum, and spinocerebellar neurons originating from T4 projected their axons into the cortex of the cerebellum differently from those from LS2. These results unveil an overall picture of early ascending spinal tract formation., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Cripto-1 is an essential protein for human development that plays a key role in the early phase of gastrulation in the differentiation of an embryo as well as assists with wound healing processes. Importantly, Cripto-1 induces epithelial to mesenchymal transition to turn fixed epithelial cells into a more mobile mesenchymal phenotype through the downregulation of epithelial adhesion molecules such as E-cadherin, occludins, and claudins, and the upregulation of mesenchymal, mobile proteins, such as N-cadherin, Snail, and Slug. Consequently, Cripto-1's role in inducing EMT to promote cell motility is beneficial in embryogenesis, but detrimental in the formation, progression and metastasis of malignant tumors. Indeed, Cripto-1 is found to be upregulated in most cancers, such as breast, lung, gastrointestinal, hepatic, renal, cervical, ovarian, prostate, and skin cancers. Through its role in EMT, Cripto-1 can remodel cancer cells to enable them to travel through the extracellular matrix as well as blood and lymphatic vessels to metastasize to different organs. Additionally, Cripto-1 promotes the survival of cancer stem cells, which can lead to relapse in cancer patients.

Over 100 kinds of proteins are expressed as glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) on the cell surface in mammalian cells. GPI-APs possess unique properties in terms of their intracellular trafficking and association with lipid rafts. Although it is clear that GPI-APs play critical roles in various biological phenomena, it is poorly understood how the GPI moiety contributes to these mechanisms. More than 30 genes are involved in the correct biosynthesis of GPI-APs. We here constructed a cell library in which 32 genes involved in GPI biosynthesis were knocked out in human embryonic kidney 293 cells. Using the cell library, the surface expression and sensitivity to phosphatidylinositol-specific phospholipase C of GPI-APs were analyzed. Furthermore, we identified structural motifs of GPIs that are recognized by a GPI-binding toxin, aerolysin. The cell-based GPI-knockout library could be applied not only to basic researches, but also to applications and methodologies related to GPI-APs.

The midbody at the center of the intercellular bridge connecting dividing cells recruits the machinery essential for the final steps of cytokinesis. 1-5 Successive abscission on both sides of the midbody generates a free midbody remnant (MBR) that can be inherited and accumulated in many cancer, immortalized, and stem cells, both in culture and in vivo. 6-12 Strikingly, this organelle was recently shown to contain information that induces cancer cell proliferation, influences cell polarity, and promotes dorso-ventral axis specification upon interaction with recipient cells. 13-16 Yet the mechanisms by which the MBR is captured by either a daughter cell or a distant cell are poorly described. 10 , 14 Here, we report that BST2/tetherin, a well-established restriction factor that blocks the release of numerous enveloped viruses from the surface of infected cells, 17-20 plays an analogous role in retaining midbody remnants. We found that BST2 is enriched at the midbody during cytokinesis and localizes at the surface of MBRs in a variety of cells. Knocking out BST2 induces the detachment of MBRs from the cell surface, their accumulation in the extracellular medium, and their transfer to distant cells. Mechanistically, the localization of BST2 at the MBR membrane is both necessary and sufficient for the interaction between MBRs and the cell surface. We thus propose that BST2 tethers post-cytokinetic midbody remnants to the cell surface. This finding reveals new parallels between cytokinesis and viral biology 21-26 that unexpectedly extend beyond the ESCRT-dependent abscission step., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In many tumors, CD73 (NT5E), a rate-limiting enzyme in adenosine biosynthesis, is upregulated by TGF-β and drives tumor progression. Conversely, CD73 is downregulated in endometrial carcinomas (EC) despite a TGF-β-rich environment. Through gene expression analyses of normal endometrium samples of the uterine cancer TCGA data set and genetic and pharmacological studies, we discovered CD73 loss shifts TGF-β1 from tumor suppressor to promoter in EC. TGF-β1 upregulated CD73 and epithelial integrity in vivo in the normal endometrium and in vitro in early stage EC cells. With loss of CD73, TGF-β1-mediated epithelial integrity was abrogated. EC cells developed TGF-β1-mediated stress fibers and macromolecule permeability, migration, and invasion increased. In human tumors, CD73 is downregulated in deeply invasive stage I EC. Consistent with shifting TGF-β1 activity, CD73 loss increased TGF-β1-mediated canonical signaling and upregulated cyclin D1 (CCND1) and downregulated p21 expression. This shift was clinically relevant, as CD73 Low /CCND1 High expression associated with poor tumor differentiation, increased myometrial and lymphatic/vascular space invasion, and patient death. Further loss of CD73 in CD73 Low expressing advanced stage EC cells increased TGF-β-mediated stress fibers, signaling, and invasiveness, whereby adenosine A1 receptor agonist, CPA, dampened TGF-β-mediated invasion. These data identify CD73 loss as essential for shifting TGF-β activity in EC., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Matriptase-2, a serine protease expressed in hepatocytes, is a negative regulator of hepcidin expression. The purpose of the study was to investigate the interaction of matriptase-2 with hemojuvelin protein in vivo. Mice lacking the matriptase-2 proteolytic activity ( mask mice) display decreased content of hemojuvelin protein. Vice versa, the absence of hemojuvelin results in decreased liver content of matriptase-2, indicating that the two proteins interact. To further characterize the role of matriptase-2, we investigated iron metabolism in mask mice fed experimental diets. Administration of iron-enriched diet increased liver iron stores as well as hepcidin expression. Treatment of iron-overloaded mask mice with erythropoietin increased hemoglobin and hematocrit, indicating that the response to erythropoietin is intact in mask mice. Feeding of an iron-deficient diet to mask mice significantly increased spleen weight as well as the splenic content of erythroferrone and transferrin receptor proteins, indicating stress erythropoiesis. Liver hepcidin expression was decreased; expression of Id1 was not changed. Overall, the results suggest a complex interaction between matriptase-2 and hemojuvelin, and demonstrate that hepcidin can to some extent be regulated even in the absence of matriptase-2 proteolytic activity.

Background/aim: Successful therapy of EGFR-mutant NSCLC remains a challenging task despite initial benefits with the usage of EGFR tyrosine kinase inhibitors. Cancer immunotherapy has shown promising results in certain tumors, but response rate in EGFR-mutant NCLC is low, because these tumors are thought to have weak immunogenicity., Materials and Methods: We used data from in vivo NSCLC databases as well as from in vitro cell culture experiments to investigate the regulation of CD73 by EGFR., Results: EGFR expression was correlated with CD73 expression in patients' datasets, with EGFR-mutant tumors showing higher expression than their EGFR wildtype counterparts. Treatment of EGFR-mutant NSCLC cell lines with EGFR TKI reduced expression of CD73 at both mRNA and protein level. Among EGFR downstream signaling pathways, the Ras-Raf-ERK pathway was involved in the regulation of CD73 expression directly via ERK1/2 without the engagement of RSKs or MSKs., Conclusion: The results of this study may provide novel therapeutic strategies for the treatment of oncogene-driven NSCLC., (Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Neuritin (Nrn1) is a small highly conserved extracellular membrane protein involved in the process of neural cell survival and differentiation, axonal and dendritic growth, and synapse formation and maturation. Previous studies have demonstrated that intravitreal injection of recombinant Nrn1 as a gene therapy could alleviate retinal ganglion cell (RGC) apoptosis and promote optic nerve axon regeneration after optic nerve crush (ONC). However, the mechanism underlying the repairing effect of Nrn1 against optic never injury remains elusive. In this study, a rAAV2-mediated Nrn1 overexpression vector (AAV2-Nrn1) was applied to treat ONC through intravitreal injection for the purpose of further exploring the effect and mechanism of Nrn1 in repairing the injured optic nerve. The results showed that AAV2-Nrn1 was mainly transfected into RGCs without affecting astrocytes. Nrn1 overexpression effectively reduced RGC apoptosis and promoted optic nerve regeneration and visual function restoration as demonstrated by retinal imaging, histopathological analysis, and physiological function detection in vivo following ONC. Immunoblot assay revealed that functional molecules of Nrn1 activated the Akt1 and Stat3 pathways and inhibited the mitochondrial apoptotic pathway. The results of the present study may provide experimental evidence for further application of Nrn1 to the clinical treatment of optic nerve injury.

Metastasis is the leading cause of death in lung adenocarcinomas. Identifying potential prognostic biomarkers and exploiting regulatory mechanisms could improve the diagnosis and treatment of lung cancer patients. We previously found that cluster of differentiation 109 (CD109) was upregulated in lung tumor tissues, and CD109 overexpression was correlated with the invasive and metastatic capacities of lung adenocarcinoma cells. However, the contribution of CD109 to lung tumorigenesis remains to be elucidated. In the present study, we identified that CD109 was upregulated in metastatic lung adenocarcinoma cells, and elevation of CD109 was correlated with epithelial-to-mesenchymal transition (EMT) traits in patients with lung adenocarcinoma. Functionally, CD109 expression was crucial for EMT gene expressions, tumor invasiveness, and cancer stemness properties. Moreover, elevation of CD109 was accompanied by upregulation of the yes-associated protein (YAP) signature in metastatic lung cancer cells and lung cancer patients, and activation of YAP was demonstrated to participate in CD109-elicited EMT gene expressions and tumor invasiveness. Our study reveals the molecular mechanism underlying CD109 in lung tumor aggressiveness, and CD109 could be a potential diagnostic and therapeutic target for lung cancer patients.

Introduction: Glucose-regulated protein 78 (GRP78) is a stress-inducible molecular chaperone expressed within the endoplasmic reticulum where it acts as a master regulator of the unfolded protein response (UPR) pathway. At times of ER stress, activation of the UPR, a multimolecular pathway, limits proteotoxicity induced by misfolded proteins. In malignancies, including multiple myeloma which is characterized by an accumulation of misfolded immunoglobulins, GRP78 expression is increased, with notable translocation of GRP78 to the cell surface. Studies suggest cell-surface GRP78 (csGRP78) to be of prognostic significance with emerging evidence that it interacts with a myriad of co-ligands to activate signaling pathways promoting cell proliferation and survival or apoptosis., Areas Covered: This review focuses on the role of ER and csGRP78 in physiology and oncogenesis in multiple myeloma, addressing factors that shift the balance in GRP78 signaling from survival to apoptosis. The role of GRP78 as a potential prognostic biomarker is explored and current therapeutics in development aimed at targeting csGRP78 are addressed. We conducted a PubMed literature search using the keywords 'GRP78,' 'multiple myeloma' reviewing studies prior to 2020., Expert Opinion: Cell-surface GRP78 expression is a potential novel prognostic biomarker in myeloma and targeting of csGRP78 is promising and requires further investigation.

Tumor cells develop various mechanisms to escape immune surveillance. In this context, oncometabolites secreted by tumor cells due to deregulated metabolic pathways, have been in the spotlight of researchers during the last years. 5'-Deoxy-5'-methylthioadenosine (MTA) phosphorylase (MTAP) deficiency in tumors results in the accumulation of MTA within the tumor microenvironment and thereby negatively influencing immune functions of various immune cells, including T and NK cells. The influence of MTA on T cell activation has been recently described in more detail, while its impact on NK cells is still largely unknown. Therefore, we aimed to illuminate the molecular mechanism of MTA-induced NK cell dysfunction. NK cell cytotoxicity against target cells was reduced in the presence of MTA in a dose-dependent manner, while NK cell viability remained unaffected. Furthermore, we revealed that MTA blocks NK cell degranulation and cytokine production upon target cell engagement as well as upon antibody stimulation. Interestingly, the immune-suppressive effect of MTA was less pronounced in healthy donors harboring an expansion of NKG2C + NK cells. Finally, we demonstrated that MTA interferes with various signaling pathways downstream of the CD16 receptor upon NK cell activation, including the PI3K/AKT/S6, MAPK/ERK, and NF-κB pathways. In summary, we revealed that MTA blocks NK cell functions like cytotoxicity and cytokine production by interfering with the signaling cascade of activating NK cell receptors. Specific targeting of MTA metabolism in MTAP-deficient tumors therefore could offer a promising new strategy to reverse immune dysfunction of NK cells within the tumor microenvironment., (Copyright © 2020 Jacobs, Schlögl, Strobl, Völkl, Stoll, Mougiakakos, Malmberg, Mackensen and Aigner.)

Interferon-induced BST2 (bone marrow stromal cell antigen 2) inhibits viral replication by tethering enveloped virions to the cell surface to restrict viral release and by inducing the NFKB-dependent antiviral immune response. However, the mechanism by which BST2 uses the selective autophagy pathway to inhibit viral replication is poorly understood. In this study, we showed that BST2 expression was significantly increased during porcine epidemic diarrhea virus (PEDV) infection of Vero cells by IRF1 targeting its promoter. We also showed that BST2 suppressed PEDV replication by binding and degrading the PEDV-encoded nucleocapsid (N) protein. The downregulation of N protein was blocked by macroautophagy/autophagy inhibitors but not a proteasome inhibitor, implying that the N protein was degraded via the selective autophagy pathway. Both the BST2 and N protein interacted with the E3 ubiquitin ligase MARCHF8/MARCH8 and the cargo receptor CALCOCO2/NDP52, and the ubiquitination of N protein was necessary for the degradation of N mediated by the BST2-MARCHF8 axis. The knockdown of MARCHF8 or ATG5 with small interfering RNAs blocked the selective autophagy pathway, rescued the protein abundance of PEDV N in 293T cells, and prevented the inhibition of PEDV replication by BST2 in Vero cells. Together, our data demonstrate the novel mechanism of BST2-mediated virus restriction, in which BST2 recruits MARCHF8 to catalyze the ubiquitination of the PEDV N protein. The ubiquitinated N protein is then recognized by CALCOCO2/NDP52, which delivers it to autolysosome for degradation through the selective autophagy pathway. Abbreviations : 3MA: 3-methyladenine; ATG: autophagy-related; Baf A1: bafilomycin A 1 ; BST2: bone marrow stromal cell antigen 2; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CC: coiled-coil; ChIP: chromatin immunoprecipitation; Co-IP: co-immunoprecipitation; CQ: chloroquine; CT: cytoplasmic tail; DAPI: 4',6-diamidino-2-phenylindole; GPI: glycosyl-phosphatidylinositol; hpi: hours post infection; IRF1: interferon regulatory factor 1; ISG: IFN-stimulated gene; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MARCHF8/MARCH8: membrane-associated ring-CH-type finger 8; MOI: multiplicity of infection; N protein: nucleocapsid protein; PED: porcine epidemic diarrhea; PEDV: porcine epidemic diarrhea virus; RT: room temperature; siRNA: small interfering RNA; STAT: signal transducer and activator of transcription; TCID 50 : 50% tissue culture infectious doses; TM: transmembrane.

Background: The prognostic and therapeutic potential of microRNAs (miRNAs) in spinal cord injury (SCI) has aroused increasing concerns. This study aims to research the functions of miR-29a/199B in the neurological function recovery after SCI and the mechanical mechanism., Methods: A rat model with SCI was induced with sham-operated ones as control. The locomotor function and coordination of rat hindlimbs were determined by a Basso, Beattie, and Bresnahan (BBB) locomotor rating scale and a ladder-climbing test, respectively. Expression of a neurofilament protein NF-200 and synaptophysin in gray matter of rats was determined to evaluate neuronal recovery in a cellular perspective. Binding relationships between miR-29a/199B with RGMA were predicted and validated using luciferase assays. Altered expression of miR-29a/199B and RGMA was introduced to explore their functions in rat neurological functions. The protein level and phosphorylation of STAT3 in gray matter were measured by western blot analysis., Results: miR-29a and miR-199B were poorly expressed, while RGMA was abundantly expressed in gray matter at injury sites. Either miR-29a or miR-199B could bind to RGMA. Overexpression of miR-29a/199B or silencing of RGMA led to an increase in BBB locomotor scores, hindlimb coordination ability, and the expression of NF-200 and synaptophysin in gray matter. Further inhibition in miR-29a/199B blocked the promoting roles of RGMA silencing in neurological recovery. Upregulation of miR-29a/199B or downregulation of RGMA suppressed the phosphorylation of STAT3., Conclusion: This study evidenced that miR-29a and miR-199B negatively regulated RGMA to suppress STAT3 phosphorylation, therefore promoting the neurological function recovery in rats following SCI.

Epileptogenesis is a potential process. Mossy fibre sprouting (MFS) and synaptic plasticity promote epileptogenesis. Overexpression of repulsive guidance molecule a (RGMa) prevents epileptogenesis by inhibiting MFS. However, other aspects underlying the RGMa regulatory process of epileptogenesis have not been elucidated. We studied whether RGMa could be modulated by microRNAs and regulated RhoA in epileptogenesis. Using microRNA databases, we selected four miRNAs as potential candidates. We further experimentally confirmed miR-20a-5p as a RGMa upstream regulator. Then, in vitro, by manipulating miR-20a-5p and RGMa, we investigated the regulatory relationship between miR-20a-5p, RGMa and RhoA, and the effects of this pathway on neuronal morphology. Finally, in the epilepsy animal model, we determined whether the miR-20a-5p-RGMa-RhoA pathway influenced MFS and synaptic plasticity and then modified epileptogenesis. Our results showed that miR-20a-5p regulated RGMa and that RGMa regulated RhoA in vitro. Furthermore, in primary hippocampal neurons, the miR-20a-5p-RGMa-RhoA pathway regulated axonal growth and neuronal branching; in the PTZ-induced epilepsy model, silencing miR-20a-5p prevented epileptogenesis through RGMa-RhoA-mediated synaptic plasticity but did not change MFS. Overall, we concluded that silencing miR-20a-5p inhibits axonal growth and neuronal branching and prevents epileptogenesis through RGMa-RhoA-mediated synaptic plasticity in the PTZ-induced epilepsy model, thereby providing a possible strategy to prevent epileptogenesis., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Polymorphonuclear leukocytes (PMNs) are crucial for initial control of Streptococcus pneumoniae (pneumococcus) lung infection; however, as the infection progresses their persistence in the lungs becomes detrimental. Here we explored why the antimicrobial efficacy of PMNs declines over the course of infection. We found that the progressive inability of PMNs to control infection correlated with phenotypic differences characterized by a decrease in CD73 expression, an enzyme required for production of extracellular adenosine (EAD). EAD production by CD73 was crucial for the ability of both murine and human PMNs to kill S. pneumoniae. In exploring the mechanisms by which CD73 controlled PMN function, we found that CD73 mediated its antimicrobial activity by inhibiting IL-10 production. PMNs from wild-type mice did not increase IL-10 production in response to S. pneumoniae; however, CD73 -/- PMNs up-regulated IL-10 production upon pneumococcal infection in vitro and during lung challenge. IL-10 inhibited the ability of WT PMNs to kill pneumococci. Conversely, blocking IL-10 boosted the bactericidal activity of CD73 -/- PMNs as well as host resistance of CD73 -/- mice to pneumococcal pneumonia. CD73/IL-10 did not affect apoptosis, bacterial uptake, and intracellular killing or production of antimicrobial neutrophil elastase and myeloperoxidase. Rather, inhibition of IL-10 production by CD73 was important for optimal reactive oxygen species (ROS) production by PMNs. ROS contributed to PMN antimicrobial function as their removal or detoxification impaired the ability of PMNs to efficiently kill S. pneumoniae. This study demonstrates that CD73 controls PMN antimicrobial phenotype during S. pneumoniae infection., (©2020 Society for Leukocyte Biology.)

The limitation of plasticity in the adult brain impedes functional recovery later in life from brain injury or disease. This pressing clinical issue may be resolved by enhancing plasticity in the adult brain. One strategy for triggering robust plasticity in adulthood is to reproduce one of the hallmark physiological events of experience-dependent plasticity observed during the juvenile critical period: to rapidly reduce the activity of parvalbumin (PV)-expressing interneurons and disinhibit local excitatory neurons. This may be achieved through the enhancement of local inhibitory inputs, particularly those of somatostatin (SST)-expressing interneurons. However, to date the means for manipulating SST interneurons for enhancing cortical plasticity in the adult brain are not known. We show that SST interneuron-selective overexpression of Lypd6, an endogenous nicotinic signaling modulator, enhances ocular dominance plasticity in the adult primary visual cortex (V1). Lypd6 overexpression mediates a rapid experience-dependent increase in the visually evoked activity of SST interneurons as well as a simultaneous reduction in PV interneuron activity and disinhibition of excitatory neurons. Recapitulating this transient activation of SST interneurons using chemogenetics similarly enhanced V1 plasticity. Notably, we show that SST-selective Lypd6 overexpression restores visual acuity in amblyopic mice that underwent early long-term monocular deprivation. Our data in both male and female mice reveal selective modulation of SST interneurons and a putative downstream circuit mechanism as an effective method for enhancing experience-dependent cortical plasticity as well as functional recovery in adulthood. SIGNIFICANCE STATEMENT The decline of cortical plasticity after closure of juvenile critical period consolidates neural circuits and behavior, but this limits functional recovery from brain diseases and dysfunctions in later life. Here we show that activation of cortical somatostatin (SST) interneurons by Lypd6, an endogenous modulator of nicotinic acetylcholine receptors, enhances experience-dependent plasticity and recovery from amblyopia in adulthood. This manipulation triggers rapid reduction of PV interneuron activity and disinhibition of excitatory neurons, which are known hallmarks of cortical plasticity during juvenile critical periods. Our study demonstrates modulation of SST interneurons by Lypd6 to achieve robust levels of cortical plasticity in the adult brain and may provide promising targets for restoring brain function in the event of brain trauma or disease., (Copyright © 2020 the authors.)

Testisin (encoded by PRSS21) is a membrane anchored serine protease, which is tethered to the cell surface via a glycosylphosphatidylinositol (GPI)-anchor. While testisin is found in abundance in spermatozoa, it is also expressed in microvascular endothelial cells where its function is unknown. Here we identify testisin as a novel regulator of physiological hormone-induced angiogenesis and microvascular endothelial permeability. Using a murine model of rapid physiological angiogenesis during corpus luteal development in the ovary, we found that mice genetically deficient in testisin (Prss21-/-) show a substantially increased incidence of hemorrhages which are significantly more severe than in littermate control Prss21+/+ mice. This phenotype was associated with increased vascular leakiness, demonstrated by a greater accumulation of extravasated Evans blue dye in Prss21-/- ovaries. Live cell imaging of in vitro cultured microvascular endothelial cells depleted of testisin by siRNA knockdown revealed that loss of testisin markedly impaired reorganization and tubule-like formation on Matrigel basement membranes. Moreover testisin siRNA knockdown increased the paracellular permeability to FITC-albumin across endothelial cell monolayers, which was associated with decreased expression of the adherens junction protein VE-cadherin and increased levels of phospho(Tyr658)-VE-cadherin, without affecting the levels of the tight junction proteins occludin and claudin-5, or ZO-1. Decreased expression of VE-cadherin in the neovasculature of Prss21-/- ovaries was also observed without marked differences in endothelial cell content, vascular claudin-5 expression or pericyte recruitment. Together, these data identify testisin as a novel regulator of VE-cadherin adhesions during angiogenesis and indicate a potential new target for regulating neovascular integrity and associated pathologies., Competing Interests: The authors have declared that no competing interests exist.

As a canonical lymphocyte antigen-6/urokinase-type plasminogen activator receptor Ly6/uPAR family protein, lymphocyte antigen 6 complex, locus E (LY6E), plays important roles in immunological regulation, T cell physiology, and oncogenesis. Emerging evidence indicates that LY6E is also involved in the modulation of viral infection. Consequently, viral infection and associated pathogenesis have been associated with altered LY6E gene expression. The interaction between viruses and the host immune system has offered insights into the biology of LY6E. In this review, we summarize the current knowledge of LY6E in the context of viral infection, particularly viral entry.

Host response to viral infection is a highly regulated process involving engagement of various host factors, cytokines, chemokines, and stimulatory signals that pave the way for an antiviral immune response. The response is manifested in terms of viral sequestration, phagocytosis, and inhibition of genome replication, and, finally, if required, lymphocyte-mediated clearance of virally infected cells. During this process, cross-talk between viral and host factors can shape disease outcomes and immunopathology. Bone marrow stromal antigen 2 ( BST-2 ), also know as tetherin, is induced by type I interferon produced in response to viral infections, as well as in certain cancers. BST-2 has been shown to be a host restriction factor of virus multiplication through its ability to physically tether budding virions and restrict viral spread. However, BST-2 has other roles in the host antiviral response. This review focuses on the diverse functions of BST-2 and its downstream signaling pathways in regulating host immune responses.

Leukemia develops as the result of intrinsic features of the transformed cell, such as gene mutations and derived oncogenic signaling, and extrinsic factors, such as a tumor-friendly, immunosuppressed microenvironment, predominantly in the lymph nodes and the bone marrow. There, high extracellular levels of nucleotides, mainly NAD + and ATP, are catabolized by different ectonucleotidases, which can be divided in two families according to substrate specificity: on one side those that metabolize NAD + , including CD38, CD157, and CD203a; on the other, those that convert ATP, namely CD39 (and other ENTPDases) and CD73. They generate products that modulate intracellular calcium levels and that activate purinergic receptors. They can also converge on adenosine generation with profound effects, both on leukemic cells, enhancing chemoresistance and homing, and on non-malignant immune cells, polarizing them toward tolerance. This review will first provide an overview of ectonucleotidases expression within the immune system, in physiological and pathological conditions. We will then focus on different hematological malignancies, discussing their role as disease markers and possibly pathogenic agents. Lastly, we will describe current efforts aimed at therapeutic targeting of this family of enzymes., (Copyright © 2019 Vaisitti, Arruga, Guerra and Deaglio.)

Extracellular adenine nucleotides participate in cell-to-cell communication and modulate the immune response. The concerted action of ectonucleotidases CD39 and CD73 plays a major role in the local production of anti-inflammatory adenosine, but both ectonucleotidases are rarely co-expressed by human T cells. The expression of CD39 on T cells increases upon T cell activation and is high at sites of inflammation. CD73, in contrast, disappears from the cellular membrane after activation. The possibility that CD73 could act in trans would resolve the conundrum of both enzymes being co-expressed for the degradation of ATP and the generation of adenosine. An enzymatically active soluble form of CD73 has been reported, and AMPase activity has been detected in body fluids of patients with inflammation and cancer. It is not yet clear how CD73, a glycosylphosphatidylinositol (GPI)-anchored protein, is released from the cell membrane, but plausible mechanisms include cleavage by metalloproteinases and shedding mediated by cell-associated phospholipases. Importantly, like many other GPI-anchored proteins, CD73 at the cell membrane is preferentially localized in detergent-resistant domains or lipid rafts, which often contribute to extracellular vesicles (EVs). Indeed, CD73-containing vesicles of different size and origin and with immunomodulatory function have been found in the tumor microenvironment. The occurrence of CD73 as non-cell-bound molecule widens the range of action of this enzyme at sites of inflammation. In this review, we will discuss the generation of non-cell-bound CD73 and its physiological role in inflammation.

The cellular protein bone marrow stromal antigen-2 (BST-2)/tetherin acts against a variety of enveloped viruses by restricting their release from the plasma membrane. The HIV-1 accessory protein Vpu counteracts BST-2 by downregulating it from the cell surface and displacing it from virion assembly sites. Previous comparisons of Vpus from transmitted/founder viruses and between viruses isolated during acute and chronic infection led to the identification of a tryptophan at position 76 in Vpu (W76) as a key determinant for the displacement of BST-2 from virion assembly sites. Although present in Vpus from clades B, D, and G, W76 is absent from Vpus from clades A, C, and H. Mutagenesis of the C-terminal region of Vpu from two clade C viruses led to the identification of a conserved LL sequence that is functionally analogous to W76 of clade B. Alanine substitution of these leucines partially impaired virion release. This impairment was even greater when the mutations were combined with mutations of the Vpu β-TrCP binding site, resulting in Vpu proteins that induced high surface levels of BST-2 and reduced the efficiency of virion release to less than that of virus lacking vpu Microscopy confirmed that these C-terminal leucines in clade C Vpu, like W76 in clade B, contribute to virion release by supporting the displacement of BST-2 from virion assembly sites. These results suggest that although encoded differently, the ability of Vpu to displace BST-2 from sites of virion assembly on the plasma membrane is evolutionarily conserved among clade B and C HIV-1 isolates. IMPORTANCE Although targeted by a variety of restriction mechanisms, HIV-1 establishes chronic infection in most cases, in part due to the counteraction of these host defenses by viral accessory proteins. Using conserved motifs, the accessory proteins exploit the cellular machinery to degrade or mistraffic host restriction factors, thereby counteracting them. The Vpu protein counteracts the virion-tethering factor BST-2 in part by displacing it from virion assembly sites along the plasma membrane, but a previously identified determinant of that activity is clade specific at the level of protein sequence and not found in the clade C viruses that dominate the pandemic. Here, we show that clade C Vpu provides this activity via a leucine-containing sequence rather than the tryptophan-containing sequence found in clade B Vpu. This difference seems likely to reflect the different evolutionary paths taken by clade B and clade C HIV-1 in human populations., (Copyright © 2019 American Society for Microbiology.)

Cell migration is essential for proper development and the defense against pathogens. Our previous work detailed a pathway of REversion-inducing-Cysteine-rich protein with Kazal motifs (RECK) isoform-mediated invasion in which a shorter RECK protein competes with MMP9 for interaction with the canonical RECK protein on the cell surface. Here we demonstrate that the mechanism through which RECK isoforms affect cell migration is mediated through changes in the levels of post-translational modifications (PTM) of α-tubulin. We show that both the canonical and short RECK isoforms modulate levels of tubulin acetylation and detyrosination. We demonstrate that these changes are sufficient to modulate the rate of fibroblast migration. If these tubulin PTMs are not altered, the effects of the canonical RECK isoform on cell migration are reversed. In defining the molecular pathway linking RECK and tubulin PTMs, we found that MMP9 and integrin activity both act as upstream regulators of tubulin acetylation and detyrosination. Overall, we propose a mechanism in which RECK isoforms on the cell surface have opposing effects on cell migration through MMP9-modulated changes to integrin-extracellular matrix (ECM) interactions that, in turn, affect microtubule PTMs., (Copyright © 2019 Elsevier Inc. All rights reserved.)

As vascular disease is complex and the various manifestations are influenced by differences in vascular bed architecture, exposure to shear and mechanical forces, cell types involved, and inflammatory responses, in vivo models are necessary to recapitulate the complex physiology and dynamic cellular interactions during pathogenesis. Murine knockout models are commonly used tools for investigators to study the role of a specific gene or pathway in multifaceted disease traits. Although valuable, these models are not perfect, and this is particularly true in regard to CD73 (cluster of differentiation 73), the extracellular enzyme that generates adenosine from AMP. At baseline, CD73-deficient mice do not present with an overt phenotype, whereas CD73-deficient humans present with the complex phenotype of vascular calcification, arteriomegaly and tortuosity, and calcification in small joints. In this review, we highlight the differences between the mouse and human systems and discuss the potential to leverage findings in mice to inform us on the human conditions.

Ectonucleotidase-mediated ATP catabolism provides a powerful mechanism to control the levels of extracellular adenosine. While increased adenosine A2A receptor (A2AR) signaling has been well-documented in both Parkinson's disease models and patients, the source of this enhanced adenosine signalling remains unclear. Here, we show that the ecto-5'-nucleotidase (CD73)-mediated adenosine formation provides an important input to activate A2AR, and upregulated CD73 and A2AR in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease models coordinatively contribute to the elevated adenosine signalling. Importantly, we demonstrate that CD73-derived adenosine-A2AR signalling modulates microglial immunoresponses and morphological dynamics. CD73 inactivation significantly attenuated lipopolysaccharide-induced pro-inflammatory responses in microglia, but enhanced microglia process extension, movement and morphological transformation in the laser injury and acute MPTP-induced Parkinson's disease models. Limiting CD73-derived adenosine substantially suppressed microglia-mediated neuroinflammation and improved the viability of dopaminergic neurons and motor behaviours in Parkinson's disease models. Moreover, CD73 inactivation suppressed A2AR induction and A2AR-mediated pro-inflammatory responses, whereas replenishment of adenosine analogues restored these effects, suggesting that CD73 produces a self-regulating feed-forward adenosine formation to activate A2AR and promote neuroinflammation. We further provide the first evidence that A2A enhanced inflammation by antagonizing dopamine-mediated anti-inflammation, suggesting that the homeostatic balance between adenosine and dopamine signalling is key to microglia immunoresponses. Our study thus reveals a novel role for CD73-mediated nucleotide metabolism in regulating neuroinflammation and provides the proof-of-principle that targeting nucleotide metabolic pathways to limit adenosine production and neuroinflammation in Parkinson's disease might be a promising therapeutic strategy., (© The Author(s) (2019). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Elucidating the mechanisms of recurrence of embryonic signaling pathways in tumorigenesis has led to the discovery of onco-fetal players which have physiological roles during normal development but result aberrantly re-activated in tumors. In this context, Nodal and Cripto-1 are recognized as onco-developmental factors, which are absent in normal tissues but are overexpressed in several solid tumors where they can serve as theranostic agents., Objective: To collect, review and discuss the most relevant papers related to the involvement of Nodal and Cripto-1 in the development, progression, recurrence and metastasis of several tumors where they are over-expressed, with a particular attention to their occurrence on the surface of the corresponding sub-populations of cancer stem cells (CSC)., Results: We have gathered, rationalized and discussed the most interesting findings extracted from some 370 papers related to the involvement of Cripto-1 and Nodal in all tumor types where they have been detected. Data demonstrate the clear connection between Nodal and Cripto-1 presence and their multiple oncogenic activities across different tumors. We have also reviewed and highlighted the potential of targeting Nodal, Cripto-1 and the complexes that they form on the surface of tumor cells, especially of CSC, as an innovative approach to detect and suppress tumors with molecules that block one or more mechanisms that they regulate., Conclusion: Overall, Nodal and Cripto-1 represent two innovative and effective biomarkers for developing potential theranostic anti-tumor agents that target normal as well as CSC subpopulations and overcome both pharmacological resistance and tumor relapse., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: In this study, we evaluate the role of RGMa (Repulsive Guidance Molecule a) during peripheral nerve regeneration using the mouse median nerve model., Methods: By real-time PCR and Western Blot analysis, we examined expression changes of RGMa mRNA and RGMa protein in neural tissue after transection and microsurgical repair of the mouse median nerve distal to the transection site. We evaluated histomorphometrical changes in neural tissue distal to the injury site and functional recovery of the grasping force after median nerve transection and repair in wild-type mice and RGMa+/- heterozygous mice., Results: RT-PCR revealed a 1,8 fold increase of RGMa mRNA two weeks and a 4,4 fold increase of RGMa mRNA 3 weeks after nerve transection and repair in the nerve segment distal to the injury site. In Western blot analysis, we could show a high increase of RGMa in the nerve segment distal to the injury site at day 14. Histomorphometrical analysis showed significant differences between wild-type animals and heterozygous animals. The absolute number of myelinated fibres was significantly higher in operated heterozygous RGMa+/- animals compared to operated wildtye animals. Using the functional grasping test, we could demonstrate that peripheral nerve regeneration is significantly diminished in heterozygous RGMa+/- mice., Conclusions: Employing the mouse median nerve model in transgenic animals, we demonstrate that RGMa plays an important role during peripheral nerve regeneration.

Purpose of Review: Luminal chemosensing is a term used to describe how small molecules in the gut lumen interact with the host through surface receptors or via transport into the submucosa. In this review, we have summarized recent advances of understanding luminal chemosensing in the gastroduodenal mucosa, with a particular emphasis on how chemosensing affects mucosal protective responses and the metabolic syndrome., Recent Findings: In the past decade, data have supported the hypothesis that gut luminal chemosensing not only is important for the local or remote regulation of gut function but also contributes to the systemic regulation of metabolism, energy balance and food intake. We have provided examples of how luminal nutrients such as long-chain fatty acids (LCFAs), endogenous compounds such as bile acids, bacterial metabolites such as short-chain fatty acids (SCFAs) and bacterial components such as lipopolysaccharide (LPS) activate cognate receptors expressed on key effector cells such as enteroendocrine cells and inflammatory cells in order to profoundly affect organ function through the initiation or suppression of inflammatory pathways, altering gut barrier function and nutrient uptake, altering gut motility and visceral pain pathways, and preventing mucosal injury., Summary: These recent discoveries in this area have provided new possibilities for identifying novel molecular targets for the treatment of mucosal injury, metabolic disorders and abnormal visceral sensation. Understanding luminal chemosensory mechanisms may help to identify novel molecular targets for the treatment and prevention of mucosal injury, metabolic disorders and abnormal visceral sensation.

We reported previously that microglia decreased the growth of human brain tumor-initiating cells (BTICs). Through microarray analyses of BTICs exposed in vitro to microglia, we found the induction of several genes ascribed to have roles in cell cycle arrest, reduced cell proliferation and differentiation. Herein, we tested the hypothesis that one of these genes, growth arrest specific 1 (Gas1), is a novel growth reduction factor that is induced in BTICs by microglia. We found that microglia increased the expression of Gas1 transcript and protein in glioblastoma patient-derived BTIC lines. Using neurosphere assay we show that RNAi-induced reduction of Gas1 expression in BTICs blunted the microglia-mediated BTIC growth reduction. The role of Gas1 in mediating BTIC growth arrest was further validated using orthotopic brain xenografts in mice. When microglia-induced Gas1-expressing BTIC cells (mGas1-BTICs) were implanted intra-cranially in mice, tumor growth was markedly decreased; this was mirrored in the remarkable increase in survival of mGas1-BT025 and mGas1-BT048 implanted mice, compared to mice implanted with non-microglia-exposed BTIC cells. In conclusion, this study has identified Gas1 as a novel factor and mechanism through which microglia arrest the growth of BTICs for anti-tumor property.

Recent findings regarding the cellular biology and immunology of BST-2 (also known as tetherin) indicate that its function could be exploited as a universal replication inhibitor of enveloped respiratory viruses (e.g., influenza, respiratory syncytial virus, etc.). BST-2 inhibits viral replication by preventing virus budding from the plasma membrane and by inducing an antiviral state in cells adjacent to infection via unique inflammatory signaling mechanisms. This review presents the first comprehensive summary of what is currently known about BST-2 anti-viral function against respiratory viruses, how these viruses construct countermeasures to antagonize BST-2, and how BST-2 function might be targeted to develop therapies to treat respiratory virus infections. The authors address the current gaps in knowledge, including the need for mechanistic understanding of BST-2 antagonism by respiratory viruses, that should be bridged to achieve that goal., (© 2018 WILEY Periodicals, Inc.)

Neuritin is a member of the neurotrophic factor family, which is activated by neural activity and neurotrophins, and promotes neurite growth and branching. It has shown to play an important role in neuronal plasticity and regeneration. It is also involved in other biological processes such as angiogenesis, tumorigenesis and immunomodulation. Thus far, however, the primary mechanisms of neuritin, including whether or not it acts through a receptor or which downstream signals might be activated following binding, are not fully understood. Recent evidence suggests that neuritin may be a potential therapeutic target in several neurodegenerative diseases. This review focuses on the recent advances in studies regarding the newly identified functions of neuritin and the signaling pathways related to these functions. We also discuss current hot topics and difficulties in neuritin research.

Introduction: COPD is a chronic inflammatory disease of lung. The inflammatory response in COPD is associated with neutrophils, macrophages, T lymphocytes, and bronchial epithelial cells, and occurs mainly in the small airway, leading to irreversible airflow limitation., Methods: In order to investigate the microRNA-mRNA interaction in the microenvironment of the COPD airway, we used next-generation sequencing and bioinformatics in this study., Results: We identified four genes with microRNA-mRNA interactions involved in COPD small-airway bronchial epithelial cells: NT5E , SDK1 , TNS1 , and PCDH7 . Furthermore, miR6511a-5p- NT5E interaction was found to be involved in small-airway bronchial epithelial cells, large-airway bronchial epithelial cells, and alveolar macrophages., Conclusion: Our results showed that miR6511a-5p- NT5E interaction plays an important role in COPD, which might be associated with cell-cell contact, activation of leukocytes, activation of T lymphocytes, and cellular homeostasis. These findings provide new information for further investigations of the COPD microenvironment, and may help to develop new diagnostic or therapeutic strategies targeting the bronchial epithelium for COPD., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Circulating late endothelial progenitor cells (LEPCs) home to injured vessels, initiating blood vessel regeneration. This process requires the initial adhesion of LEPCs to endothelial cells within the wounded site. In this study, treating LEPCs with IFN-γ enhanced wound healing through BST2-mediated adhesion to endothelial cells. We found that IFN-γ significantly upregulated BST2 expression in both LEPCs and ECs and increased tube formation in LEPCs. Upregulated BST2 increased LEPC adhesion to ECs through a tight homophilic interaction of its extracellular domain. Finally, when the IFN-γ-treated LEPCs were injected into the wounded mouse tail vein, superior therapeutic effects of wound closure were observed. This study provides a useful application to enhance the adhesion of LEPCs for vessel regeneration and wound closure., (© 2018 Federation of European Biochemical Societies.)

Flaviviruses enter host cells through the process of clathrin-mediated endocytosis, and the spectrum of host factors required for this process are incompletely understood. Here we found that lymphocyte antigen 6 locus E (LY6E) promotes the internalization of multiple flaviviruses, including West Nile virus, Zika virus, and dengue virus. Perhaps surprisingly, LY6E is dispensable for the internalization of the endogenous cargo transferrin, which is also dependent on clathrin-mediated endocytosis for uptake. Since viruses are substantially larger than transferrin, we reasoned that LY6E may be required for uptake of larger cargoes and tested this using transferrin-coated beads of similar size as flaviviruses. LY6E was indeed required for the internalization of transferrin-coated beads, suggesting that LY6E is selectively required for large cargo. Cell biological studies found that LY6E forms tubules upon viral infection and bead internalization, and we found that tubule formation was dependent on RNASEK, which is also required for flavivirus internalization, but not transferrin uptake. Indeed, we found that RNASEK is also required for the internalization of transferrin-coated beads, suggesting it functions upstream of LY6E. These LY6E tubules resembled microtubules, and we found that microtubule assembly was required for their formation and flavivirus uptake. Since microtubule end-binding proteins link microtubules to downstream activities, we screened the three end-binding proteins and found that EB3 promotes virus uptake and LY6E tubularization. Taken together, these results highlight a specialized pathway required for the uptake of large clathrin-dependent endocytosis cargoes, including flaviviruses., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Purpose: Trimodal therapy is a reasonable bladder-preserving option to radical cystectomy. However, many tumors are radioresistive. In this sense, the identification of new prognostic and predictive biomarkers that allow the selection of patients with better responses to radiation therapy would improve outcomes. With the aim of using ecto-5'-nucleotidase/CD73 as a predictive biomarker, the role of this enzyme in the context of radiotherapy in T24 human bladder cancer cell line was investigated., Methods: T24 cell line was exposure to a single dose of radiation (4 Gray) and trypan blue assay (pharmacological assays of viability/cumulative population doubling), flow cytometry (cell cycle/cell death/active caspase-3/ecto-5'-nucleotidase/CD73 protein staining), DAPI staining (nuclear morphometric assay), RT-PCR and real-time PCR, malachite green method (ectonucleotidase enzymatic assay), and HPLC (analysis of AMP metabolism) were carried out. T24 cell line in which ecto-5'-nucleotidase/CD73 has been completely silenced (5'KO) was also used., Results: The exposure of T24 cell line to a single dose (4 Gray) of radiation-induced cell death and triggered a transitory increase in ecto-5'-nucleotidase/CD73 expression, increased ectonucleotidase activity, and led to adenosine and inosine accumulation in the extracellular medium. Pharmacological inhibition or knocking out ecto-5'-nucleotidase/CD73 rescued cells' proliferative capacity, reducing their sensitivity to radiation., Conclusion: Our findings show that the induction of ecto-5'-nucleotidase/CD73 by radiation contributes to the radiosensitivity of T24 cell line.

Background: The biology of Hepatitis E Virus (HEV), a common cause of epidemic and sporadic hepatitis, is still being explored. HEV exits liver through bile, a process which is essential for its natural transmission by feco-oral route. Though the process of this polarised HEV egress is not known in detail, HEV pORF3 and hepatocyte actin cytoskeleton have been shown to play a role., Methods: Our transcriptome analysis in Hepatitis E virus (HEV) replicon transfected Huh7 cells at 24 and 72 hrs indicated that at 24hrs, both LncBISPR and BST2, expressed by a bidirectional promoter were highly upregulated whereas at 72 hrs, BST2 expression was comparatively reduced accompanied by normal levels of BISPR. These findings were confirmed by qPCR analysis. Co-localisation of BST2 and HEV pORF2 was confirmed in HEV transfected Huh7 by confocal microscopy. To investigate the role of BISPR/BST2 in HEV life cycle, particularly virus egress, we generated Huh7 cells with ~8kb deletion in BISPR gene using Crispr-Cas9 system. The deletion was confirmed by PCR screening, Sanger sequencing and Real time PCR. Virus egress in ΔBISPR Huh7 and Huh7 cells was compared by measuring HEV positive strand RNA copy numbers in cell lysates and culture supernatants at 24 and 72 hrs post HEV replicon transfection and further validated by western blot for HEV pORF2 capsid protein., Results: ΔBISPR Huh7 cells showed ~8 fold increase in virus egress at 24 hrs compared to Huh7 cells. No significant difference in virus egress was observed at 72hrs. Immunohistochemistry in histologically normal liver and HEV associated acute liver failure revealed BST2 overexpression in HEV infected hepatocytes and a dominant canalicular BST2 distribution in normal liver in addition to the cytoplasmic localisation reported in literature., Conclusions: These findings lead us to believe that BISPR and BST2 may regulate egress of HEV virions into bile in vivo. This system may also be used to scale up virus production in vitro.

Reck, a GPI-anchored membrane protein, and Gpr124, an orphan GPCR, have been implicated in Wnt7a/Wnt7b signaling in the CNS vasculature. We show here that vascular endothelial cell (EC)-specific reduction in Reck impairs CNS angiogenesis and that EC-specific postnatal loss of Reck, combined with loss of Norrin, impairs blood-brain barrier (BBB) maintenance. The most N-terminal domain of Reck binds to the leucine-rich repeat (LRR) and immunoglobulin (Ig) domains of Gpr124, and weakening this interaction by targeted mutagenesis reduces Reck/Gpr124 stimulation of Wnt7a signaling in cell culture and impairs CNS angiogenesis. Finally, a soluble Gpr124(LRR-Ig) probe binds to cells expressing Frizzled, Wnt7a or Wnt7b, and Reck, and a soluble Reck(CC1-5) probe binds to cells expressing Frizzled, Wnt7a or Wnt7b, and Gpr124. These experiments indicate that Reck and Gpr124 are part of the cell surface protein complex that transduces Wnt7a- and Wnt7b-specific signals in mammalian CNS ECs to promote angiogenesis and regulate the BBB., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The aim of our study was to analyze polymorphonuclear neutrophil (PMN) functions in elderly individuals compared with those in healthy young participants, directly in whole blood to avoid issues with data interpretation related to cell isolation procedures. Despite the presence of increased circulating levels of proinflammatory cytokines, resting PMNs from the elderly individuals were not activated as shown by normal CD62L and CD11b expression at the PMN surface and normal constitutive reactive oxygen species (ROS) production. However, suboptimal stimulation induced modulations of CD62L and CD11b expression, which positively correlated with the interleukin-6 circulating level, suggesting a possible in vivo preactivation of old PMNs by this cytokine. In addition, PMN phagocytosis of opsonized Escherichia Coli was decreased in elderly individuals. Furthermore, upon preincubation of elderly whole-blood samples with tumor necrosis factor-α or Toll Receptor agonists, we observed a reduced PMN oxidative burst in response to formyl peptides. Elderly participants also exhibited an increased percentage of the immunosuppressive CD16 bright /CD62L dim PMN subpopulation, which was characterized by a lower phagocytic index and a reduced ROS production compared with the CD16 bright /CD62L bright subset. Thus, the reduced phagocytosis and ROS production associated with an expansion of immunosuppressive CD16 bright /CD62L dim PMN subpopulation might be involved in the increased susceptibility to bacterial and fungal infections with old age., (© The Author 2016. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Most components of the cholinergic system are detected in skeletogenic cell types in vitro, yet the function of this system in skeletogenesis remains unclear. Here, we analyzed endochondral ossification in mutant murine fetuses, in which genes of the rate-limiting cholinergic enzymes acetyl- (AChE), or butyrylcholinesterase (BChE), or both were deleted (called here A-B+, A+B-, A-B-, respectively). In all mutant embryos bone growth and cartilage remodeling into mineralizing bone were accelerated, as revealed by Alcian blue (A-blu) and Alizarin red (A-red) staining. In A+B- and A-B- onset of mineralization was observed before E13.5, about 2 days earlier than in wild type and A-B+ mice. In all mutants between E18.5 to birth A-blu staining disappeared from epiphyses prematurely. Instead, A-blu+ cells were dislocated into diaphyses, most pronounced so in A-B- mutants, indicating additive effects of both missing ChEs in A-B- mutant mice. The remodeling effects were supported by in situ hybridization (ISH) experiments performed on cryosections from A-B- mice, in which Ihh, Runx2, MMP-13, ALP, Col-II and Col-X were considerably decreased, or had disappeared between E18.5 and P0. With a second approach, we applied an improved in vitro micromass model from chicken limb buds that allowed histological distinction between areas of cartilage, apoptosis and mineralization. When treated with the AChE inhibitor BW284c51, or with nicotine, there was decrease in cartilage and accelerated mineralization, suggesting that these effects were mediated through nicotinic receptors (α7-nAChR). We conclude that due to absence of either one or both cholinesterases in KO mice, or inhibition of AChE in chicken micromass cultures, there is increase in cholinergic signalling, which leads to increased chondroblast production and premature mineralization, at the expense of incomplete chondrogenic differentiation. This emphasizes the importance of cholinergic signalling in cartilage and bone formation., Competing Interests: The authors have declared that no competing interests exist.

Purpose: Obesity is known as a chronic inflammatory state whereby anti-inflammatory adipokines, such as omentin-1 levels, are decreased. The present study aims to determine omentin-1 levels in relation to dietary intake, inflammation, and immune response in healthy obese individuals., Method: A total of 170 obese participants (body mass index [BMI] ≥ 30) were recruited in this cross-sectional study. Body composition was evaluated by a body composition analyzer, and inflammatory markers (high-sensitivity C-reactive protein [hs-CRP], interleukin (IL)-4, IL-6, IL-1β, IL-17, IL-10, IL-13, and tumor necrosis factor-alpha [TNF-α]) were measured by enzyme-linked immunosorbent assay (ELISA) kits., Results: We observed associations between higher serum levels of omentin-1 and lower levels of fasting insulin, glucose, total cholesterol, IL-6, and TNF-α concentrations; higher levels of IL-13, IL-4, and IL-1β were associated with higher serum levels of omentin-1 (all p < 0.05). Omentin-1 levels were not associated with IL-10, hs-CRP, and IL-17 concentrations. We also observed associations between higher intake of saturated fatty acid (SFA) and omentin-1 levels, even after adjustment for total energy intake (p = 0.04). Women with low intake of SFA had higher levels of omentin-1 (p = 0.03); a similar relation was not observed in men., Conclusion: Omentin-1 has an anti-inflammatory role in obesity and exerts its effects probably by inducing an increase in Th-2 cytokines comprising IL-13 and IL-4. Omentin-1 is not related to IL-17, a regulatory T cell cytokine, which modulates T helper balance. Levels of inflammatory cytokines are decreased in higher concentrations of omentin-1, except IL-1β. Lower intake of SFA may modify omentin-1 levels in women. Our study demonstrated the probable protective role of omentin-1 in obesity-related inflammation and insulin resistance.

Lynx1 regulates synaptic plasticity in the brain by regulating nicotinic acetylcholine receptors (nAChRs). It is not known to which extent Lynx1 can bind to endogenous nAChR subunits in the brain or how this interaction is affected by Alzheimer's disease pathology. We apply affinity purification to demonstrate that a water-soluble variant of human Lynx1 (Ws-Lynx1) isolates α3, α4, α5, α6, α7, β2, and β4 nAChR subunits from human and rat cortical extracts, and rat midbrain and olfactory bulb extracts, suggesting that Lynx1 forms complexes with multiple nAChR subtypes in the human and rodent brain. Incubation with Ws-Lynx1 decreases nicotine-mediated extracellular signal-regulated kinase phosphorylation in PC12 cells and striatal neurons, indicating that binding of Ws-Lynx1 is sufficient to inhibit signaling downstream of nAChRs. The effect of nicotine in PC12 cells is independent of α7 or α4β2 nAChRs, suggesting that Lynx1 can affect the function of native non-α7, non-α4β2 nAChR subtypes. We further show that Lynx1 and oligomeric β-amyloid1-42 compete for binding to several nAChR subunits, that Ws-Lynx1 prevents β-amyloid1-42-induced cytotoxicity in cortical neurons, and that cortical Lynx1 levels are decreased in a transgenic mouse model with concomitant β-amyloid and tau pathology. Our data suggest that Lynx1 binds to multiple nAChR subtypes in the brain and that this interaction might have functional and pathophysiological implications in relation to Alzheimer's disease., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mutations in a synaptic organizing pathway contribute to autism. Autism-associated mutations in MDGA2 (MAM domain containing glycosylphosphatidylinositol anchor 2) are thought to reduce excitatory/inhibitory transmission. However, we show that mutation of Mdga2 elevates excitatory transmission, and that MDGA2 blocks neuroligin-1 interaction with neurexins and suppresses excitatory synapse development. Mdga2(+/-) mice, modeling autism mutations, demonstrated increased asymmetric synapse density, mEPSC frequency and amplitude, and altered LTP, with no change in measures of inhibitory synapses. Behavioral assays revealed an autism-like phenotype including stereotypy, aberrant social interactions, and impaired memory. In vivo voltage-sensitive dye imaging, facilitating comparison with fMRI studies in autism, revealed widespread increases in cortical spontaneous activity and intracortical functional connectivity. These results suggest that mutations in MDGA2 contribute to altered cortical processing through the dual disadvantages of elevated excitation and hyperconnectivity, and indicate that perturbations of the NRXN-NLGN pathway in either direction from the norm increase risk for autism., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Carbon nanotubes (CNTs) have been likened to asbestos in terms of morphology and toxicity. CNT exposure can lead to pulmonary fibrosis and promotion of tumorigenesis. However, the mechanisms underlying CNT-induced carcinogenesis are not well defined. Mesothelin (MSLN) is overexpressed in many human tumors, including mesotheliomas and pancreatic and ovarian carcinomas. In this study, the role of MSLN in the carcinogenic transformation of human bronchial epithelial cells chronically exposed to single-walled CNT (BSW) was investigated. MSLN overexpression was found in human lung tumors, lung cancer cell lines, and BSW cells. The functional role of MSLN in the BSW cells was then investigated by using stably transfected MSLN knockdown (BSW shMSLN) cells. MSLN knockdown resulted in significantly decreased invasion, migration, colonies on soft agar, and tumor sphere formation. In vivo, BSW shMSLN cells formed smaller primary tumors and less metastases. The mechanism by which MSLN contributes to these more aggressive behaviors was investigated by using ingenuity pathway analysis, which predicted that increased MSLN could induce cyclin E expression. We found that BSW shMSLN cells had decreased cyclin E, and their proliferation rate was reverted to nearly that of untransformed cells. Cell cycle analysis showed that the BSW shMSLN cells had an increased G2 population and a decreased S phase population, which is consistent with the decreased rate of proliferation. Together, our results indicate a novel role of MSLN in the malignant transformation of bronchial epithelial cells following CNT exposure, suggesting its utility as a potential biomarker and drug target for CNT-induced malignancies., (Copyright © 2016 the American Physiological Society.)

Background and Aims: The recently identified adipokine omentin-1 is inversely associated with body fatness, metabolic syndrome and cardiovascular disease (CVD) in cross-sectional analyses. However, prospective data on the association between plasma omentin-1 levels and future risk of CVD are lacking. The aim of the study was to investigate the relationship between omentin-1 and incident myocardial infarction (MI) and stroke., Methods: We conducted a case-cohort study nested within the European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam cohort comprising a subsample of 2084 participants, including 50 CVD cases and 350 external incident CVD cases (mean follow-up of 8.2 ± 1.6 years). Prentice modified Cox regression adjusted for established CVD risk factors was used to estimate associations between omentin-1 and risk of MI and stroke, interactions were tested with cross-product terms., Results: After multivariable adjustment, omentin-1 was not significantly associated with risk of MI (HR per doubling omentin-1:1.17; 95%-CI:0.79-1.72; p = 0.43), but with higher risk of stroke (HR per doubling omentin-1:2.22; 95%-CI:1.52-3.22; p < 0.0001). In subgroup analyses, associations between omentin-1 and stroke risk were generally stronger in lower versus higher CVD risk groups. For example, risk of stroke was stronger in participants without metabolic syndrome (HR per doubling omentin-1:2.58; 95%-CI:1.64-4.07; p < 0.0001) compared to those with metabolic syndrome (HR per doubling omentin-1:1.21; 95%-CI:0.59-2.50; p = 0.60) (p for interaction = 0.05). Similar interactions were observed when participants were classified in low or high risk groups according to waist circumference, triglyceride, hsCRP or adiponectin levels., Conclusions: Omentin-1 concentrations may be related to increased stroke risk. This association is stronger in metabolically healthy individuals., (Copyright © 2016 The Author(s). Published by Elsevier Ireland Ltd.. All rights reserved.)

Accumulated evidence shows that vanin-1 (VNN1) plays a key part in glucose metabolism. We explored the effect of VNN1 on cholesterol metabolism, inflammation, apoptosis in vitro, and progression of atherosclerotic plaques in apoE(-/-) mice. Oxidized LDL (Ox-LDL) significantly induced VNN1 expression through an ERK1/2/cyclooxygenase-2/PPARα signaling pathway. VNN1 significantly increased cellular cholesterol content and decreased apoAI and HDL-cholesterol (HDL-C)-mediated efflux by 25.16% and 23.13%, respectively, in THP-1 macrophage-derived foam cells (P < 0.05). In addition, VNN1 attenuated Ox-LDL-induced apoptosis through upregulation of expression of p53 by 59.15% and downregulation of expression of B-cell lymphoma-2 127.13% in THP-1 macrophage (P < 0.05). In vivo, apoE(-/-) mice were divided randomly into two groups and transduced with lentivirus (LV)-Mock or LV-VNN1 for 12 weeks. VNN1-treated mice showed increased liver lipid content and plasma levels of TG (124.48%), LDL-cholesterol (119.64%), TNF-α (148.74%), interleukin (IL)-1β (131.81%), and IL-6 (156.51%), whereas plasma levels of HDL-C (25.75%) were decreased significantly (P < 0.05). Consistent with these data, development of atherosclerotic lesions was increased significantly upon infection of apoE(-/-) mice with LV-VNN1. These observations suggest that VNN1 may be a promising therapeutic candidate against atherosclerosis., (Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.)