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Connexin43 (Cx43) forms gap junctions that couple the granulosa cells of ovarian follicles. In Cx43 knockout mice, follicle growth is restricted as a result of impaired granulosa cell proliferation. We have used these mice to examine the importance of specific Cx43 phosphorylation sites in follicle growth. Serines at residues 255, 262, 279, and 282 are MAP kinase substrates that, when phosphorylated, reduce junctional conductance. Mutant forms of Cx43 were constructed with these serines replaced with amino acids that cannot be phosphorylated. These mutants were transduced into Cx43 knockout ovarian somatic cells that were combined with wild-type oocytes and grafted into immunocompromised female mice permitting follicle growth in vivo. Despite residues 255 or 262 being mutated to prevent their being phosphorylated, recombinant ovaries constructed with these mutants were able to rescue the null phenotype, restoring complete folliculogenesis. In contrast, Cx43 with serine to alanine mutations at both residues 279 and 282 or at all four residues failed to rescue folliculogenesis; the mutant molecules were largely confined to intracellular sites, with few gap junctions. Using an in vitro proliferation assay, we confirmed a decrease in proliferation of granulosa cells expressing the double mutant construct. These results indicate that Cx43 phosphorylation by MAP kinase at serines 279 and 282 occurs in granulosa cells of early follicles and that this is involved in regulating follicle development. [ABSTRACT FROM AUTHOR]

Gap junctions and gap junction communication have long been recognized to play roles in tissue organization and remodeling through both cell autonomous and intercellular means. We hypothesized that these processes become dysregulated during pancreas cancer progression. Molecular and histological characterization of the gap junction protein, connexin43, during progression of pancreatic ductal adenocarcinoma could yield insight into how these events may contribute to or be modulated during carcinogenesis. In a mouse model of pancreatic ductal adenocarcinoma generated through targeted endogenous expression of Kras(G12D) in the murine pancreas, we examined the evolving expression and localization of connexin43. Overall, connexin43 expression increased over time, and its localization became more widespread. At early stages, connexin43 is found almost exclusively in association with the basolateral membrane of duct cells found in invasive lesions. Connexin43 became increasingly associated with the surrounding stroma over time. Connexin43 phosphorylation was also altered during tumorigenesis, as assessed by migrational changes of the protein in immunoblots. These data suggest a potential role for gap junctions and connexin43 in mediating interactions between and amongst the stromal and epithelial cells in pancreatic ductal adenocarcinoma. [ABSTRACT FROM AUTHOR]

BACKGROUND: Soy isoflavones have numerous biological properties that suggest that they may protect against colorectal cancer. Colorectal epithelial cell proliferation has been used extensively as an intermediate endpoint biomarker for colorectal neoplasia. OBJECTIVE: We tested the hypothesis that supplementation with soy protein containing isoflavones decreases colorectal epithelial cell proliferation. DESIGN: A 12-mo randomized intervention was conducted in men and women aged 50-80 y with recently diagnosed adenomatous polyps. One hundred fifty participants were enrolled and randomly assigned to an active treatment group (58 g protein powder/d containing 83 mg isoflavones/d; +ISO) or a control group (ethanol-extracted soy-protein powder containing 3 mg isoflavones; -ISO). Biopsy specimens from the cecum, sigmoid colon, and rectum were collected at baseline and at the 12-mo follow-up. Ki-67 antibody immunohistostaining was used to detect cell proliferation. One hundred twenty-five participants completed the study, and proliferation was measured in the first 91 who completed the study. RESULTS: In the sigmoid colon, cell proliferation increased by 0.9 (95% CI: 0.09, 1.9) labeled nuclei per crypt more (11%) in the +ISO group than in the -ISO group over the 12-mo intervention, which was opposite the direction predicted. The number of labeled nuclei per 100 mum crypt height also increased more in the +ISO than in the -ISO group. In the cecum and sigmoid colon, but not in the rectum, the proliferation count increased as the serum genistein concentration increased. Proliferation distribution and crypt height were not changed by treatment at any site. CONCLUSIONS: Supplementation with soy protein containing isoflavones does not reduce colorectal epithelial cell proliferation or the average height of proliferating cells in the cecum, sigmoid colon, and rectum and increases cell proliferation measures in the sigmoid colon. Copyright © 2005 American Society for Clinical Nutrition [ABSTRACT FROM AUTHOR]

Background and Aims: Clinically validated biomarker of pancreatic ductal adenocarcinoma (PDAC), carbohydrate antigen 19-9 (CA19-9), has limited sensitivity and specificity for early-stage disease. Circulating miRNAs in plasma associated with cancer relevant pathways were developed as early detection biomarkers., Methods: 2083 miRNAs in 15 μl of plasma from multicenter age-matched cohorts ( N  = 203: healthy controls, n  = 46; pancreatitis controls, n  = 36; diagnosed cases: n  = 121) and a prediagnostic Prostate, Lung, Colorectal, and Ovarian age- and gender-matched cohort ( N  = 96; controls, n  = 48; prediagnosed cases, n  = 48) were interrogated. A three-miRNA biomarker signature was developed for early-stage PDAC., Results: The three-miRNA signature (let-7i-5p, miR-130a-3p and miR-221-3p) detected PDAC from healthy controls independently (area under the curve [AUC] of stage I, II, I-IV = 0.970, 0.975, 0.974) and in combination with CA19-9 (AUC of stage I, II, I-IV = 1.000, 0.992, 0.995). It also discriminated chronic pancreatitis (AUC of stage I, II, I-IV = 0.932, 0.931, 0.929), improving performance of CA19-9 alone (AUC of stage I, II, I-IV = 0.763, 0.701, 0.735) in combination (AUC of stage I, II, I-IV = 0.971, 0.943, 0.951). Blinded validation in prediagnostic Prostate, Lung, Colorectal, and Ovarian cohort revealed lead-time trajectory increase in AUC from 0.702 to 0.729 to 0.757 at twelve-, six-, and three-months before PDAC diagnosis, respectively. The signature also helped stratification of patients with different circulating tumor DNA and imaging subtypes., Conclusion: Plasma miRNAs associated with oncogenic pathways may serve as PDAC early detection biomarkers., (© 2024 The Authors.)

Background: Cardiac arrhythmias are the main cause of sudden death due to Chronic Chagasic Cardiomyopathy (CCC). Here we investigated alterations in connexin 43 (Cx43) expression and phosphorylation in cardiomyocytes as well as associations with cardiac arrhythmias in CCC., Methods: C57Bl/6 mice infected with Trypanosoma cruzi underwent cardiac evaluations at 6 and 12 months after infection via treadmill testing and EKG. Histopathology, cytokine gene expression, and distribution of total Cx43 and its phosphorylated forms Cx43 S368 and Cx43 S325/328/330 were investigated. Human heart samples obtained from subjects with CCC were submitted to immunofluorescence analysis. In vitro simulation of a pro-inflammatory microenvironment (IL-1β, TNF, and IFN-γ) was performed in H9c2 cells and iPSC-derived cardiomyocytes to evaluate Cx43 distribution, action potential duration, and Lucifer Yellow dye transfer., Results: Mice chronically infected with T. cruzi exhibited impaired cardiac function associated with increased inflammation, fibrosis and upregulated IL-1β, TNF, and IFN-γ gene expression. Confocal microscopy revealed altered total Cx43, Cx43 S368 and Cx43 S325/328/330 localization and phosphorylation patterns in CCC, with dispersed staining outside the intercalated disc areas, i.e., in lateral membranes and the cytoplasm. Reduced co-localization of total Cx43 and N-cadherin was observed in the intercalated discs of CCC mouse hearts compared to controls. Similar results were obtained in human CCC heart samples, which showed Cx43 distribution outside the intercalated discs. Stimulation of human iPSC-derived cardiomyocytes or H9c2 cells with IL-1β, TNF, and IFN-γ induced alterations in Cx43 localization, reduced action potential duration and dye transfer between adjacent cells., Conclusion: Heart inflammation in CCC affects the distribution and phosphorylation pattern of Cx43, which may contribute to the generation of conduction disturbances in Chagas disease., 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 Barreto, Neves, Cardoso, Meira, Daltro, Figueira, Santos, Silva, Távora, Neto, Macambira, Lampe, Coutinho, Kasai Brunswick, Ribeiro dos Santos, Campos de Carvalho and Soares.)

Intrahepatic cholangiocarcinoma (ICC) is an aggressive bile duct malignancy that frequently exhibits isocitrate dehydrogenase ( IDH1/IDH2 ) mutations. Mutant IDH (IDHm) ICC is dependent on SRC kinase for growth and survival and is hypersensitive to inhibition by dasatinib, but the molecular mechanism underlying this sensitivity is unclear. We found that dasatinib reduced p70 S6 kinase (S6K) and ribosomal protein S6 (S6), leading to substantial reductions in cell size and de novo protein synthesis. Using an unbiased phosphoproteomic screen, we identified membrane-associated guanylate kinase, WW, and PDZ domain containing 1 (MAGI1) as an SRC substrate in IDHm ICC. Biochemical and functional assays further showed that SRC inhibits a latent tumor-suppressing function of the MAGI1-protein phosphatase 2A (PP2A) complex to activate S6K/S6 signaling in IDHm ICC. Inhibiting SRC led to activation and increased access of PP2A to dephosphorylate S6K, resulting in cell death. Evidence from patient tissue and cell line models revealed that both intrinsic and extrinsic resistance to dasatinib is due to increased phospho-S6 (pS6). To block pS6, we paired dasatinib with the S6K/AKT inhibitor M2698, which led to a marked reduction in pS6 in IDHm ICC cell lines and patient-derived organoids in vitro and substantial growth inhibition in ICC patient-derived xenografts in vivo. Together, these results elucidated the mechanism of action of dasatinib in IDHm ICC, revealed a signaling complex regulating S6K phosphorylation independent of mTOR, suggested markers for dasatinib sensitivity, and described a combination therapy for IDHm ICC that may be actionable in the clinic.

Despite decades of work, small-cell lung cancer (SCLC) remains a frustratingly recalcitrant disease. Both diagnosis and treatment are challenges: low-dose computed tomography (the approved method used for lung cancer screening) is unable to reliably detect early SCLC, and the malignancy's 5 year survival rate stands at a paltry 7%. Clearly, the development of novel diagnostic and therapeutic tools for SCLC is an urgent, unmet need. CD133 is a transmembrane protein that is expressed at low levels in normal tissue but is overexpressed by a variety of tumors, including SCLC. We previously explored CD133 as a biomarker for a novel autoantibody-to-immunopositron emission tomography (PET) strategy for the diagnosis of SCLC, work that first suggested the promise of the antigen as a radiotheranostic target in the disease. Herein, we report the in vivo validation of a pair of CD133-targeted radioimmunoconjugates for the PET imaging and radioimmunotherapy of SCLC. To this end, [ 89 Zr]Zr-DFO-αCD133 was first interrogated in a trio of advanced murine models of SCLC─i.e., orthotopic, metastatic, and patient-derived xenografts─with the PET probe consistently producing high activity concentrations (>%ID/g) in tumor lesions combined with low uptake in healthy tissues. Subsequently, a variant of αCD133 labeled with the β-emitting radiometal 177 Lu─[ 177 Lu]Lu-DTPA-A″-CHX-αCD133─was synthesized and evaluated in a longitudinal therapy study in a subcutaneous xenograft model of SCLC, ultimately revealing that treatment with a dose of 9.6 MBq of the radioimmunoconjugate produced a significant increase in median survival compared to a control cohort. Taken together, these data establish CD133 as a viable target for the nuclear imaging and radiopharmaceutical therapy of SCLC.

Autoantibodies (AAb) are an immunological resource ripe for exploitation in cancer detection and treatment. Key to this translation is a better understanding of the self-epitope that AAb target in tumor tissue, but do not bind to in normal tissue. Posttranslational modifications (PTMs) on self-proteins are known to break tolerance in many autoimmune diseases and have also recently been described in cancer. This scope of possible autoantigens is quite broad and new high-dimensional and -throughput technologies to probe this repertoire will be necessary to fully exploit their potential. Here, we discuss the strengths and weaknesses of existing high-throughput platforms to detect AAb, review the current methods for characterizing immunogenic PTMs, describe the main challenges to identifying disease-relevant antigens and suggest the properties of future technologies that may be able to address these challenges. We conclude that exploiting the evolutionary power of the immune system to distinguish between self and nonself has great potential to be translated into antibody-based clinical applications., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The transplantation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) has shown promise in preclinical models of myocardial infarction, but graft myocardium exhibits incomplete host-graft electromechanical integration and a propensity for pro-arrhythmic behavior. Perhaps contributing to this situation, hPSC-CM grafts show low expression of connexin 43 (Cx43), the major gap junction (GJ) protein, in ventricular myocardia. We hypothesized that Cx43 expression and function could be rescued by engineering Cx43 in hPSC-CMs with a series of phosphatase-resistant mutations at three casein kinase 1 phosphorylation sites (Cx43-S3E) that have been previously reported to stabilize Cx43 GJs and reduce arrhythmias in transgenic mice. However, contrary to our predictions, transgenic Cx43-S3E hPSC-CMs exhibited reduced Cx43 expression relative to wild-type cells, both at baseline and following ischemic challenge. Cx43-S3E hPSC-CMs showed correspondingly slower conduction velocities, increased automaticity, and differential expression of other connexin isoforms and various genes involved in cardiac excitation-contraction coupling. Cx43-S3E hPSC-CMs also had phosphorylation marks associated with Cx43 GJ internalization, a finding that may account for their impaired GJ localization. Taken collectively, our data indicate that the Cx43-S3E mutation behaves differently in hPSC-CMs than in adult mouse ventricular myocytes and that multiple biological factors likely need to be addressed synchronously to ensure proper Cx43 expression, localization, and function.

The clinical management of patients with indeterminate pulmonary nodules is associated with unintended harm to patients and better methods are required to more precisely quantify lung cancer risk in this group. Here, we combine multiple noninvasive approaches to more accurately identify lung cancer in indeterminate pulmonary nodules. We analyzed 94 quantitative radiomic imaging features and 41 qualitative semantic imaging variables with molecular biomarkers from blood derived from an antibody-based microarray platform that determines protein, cancer-specific glycan, and autoantibody-antigen complex content with high sensitivity. From these datasets, we created a PSR (plasma, semantic, radiomic) risk prediction model comprising nine blood-based and imaging biomarkers with an area under the receiver operating curve (AUROC) of 0.964 that when tested in a second, independent cohort yielded an AUROC of 0.846. Incorporating known clinical risk factors (age, gender, and smoking pack years) for lung cancer into the PSR model improved the AUROC to 0.897 in the second cohort and was more accurate than a well-characterized clinical risk prediction model (AUROC = 0.802). Our findings support the use of a multi-omics approach to guide the clinical management of indeterminate pulmonary nodules.

Small cell lung cancer (SCLC) elicits the generation of autoantibodies that result in unique paraneoplastic neurological syndromes. The mechanistic basis for the formation of such autoantibodies is largely unknown but is key to understanding their etiology. We developed a high-dimensional technique that enables detection of autoantibodies in complex with native antigens directly from patient plasma. Here, we used our platform to screen 1009 human plasma samples for 3600 autoantibody-antigen complexes, finding that plasma from patients with SCLC harbors, on average, fourfold higher disease-specific autoantibody signals compared with plasma from patients with other cancers. Across three independent SCLC cohorts, we identified a set of common but previously unknown autoantibodies that are produced in response to both intracellular and extracellular tumor antigens. We further characterized several disease-specific posttranslational modifications within extracellular proteins targeted by these autoantibodies, including citrullination, isoaspartylation, and cancer-specific glycosylation. Because most patients with SCLC have metastatic disease at diagnosis, we queried whether these autoantibodies could be used for SCLC early detection. We created a risk prediction model using five autoantibodies with an average area under the curve of 0.84 for the three cohorts that improved to 0.96 by incorporating cigarette smoke consumption in pack years. Together, our findings provide an innovative approach to identify circulating autoantibodies in SCLC with mechanistic insight into disease-specific immunogenicity and clinical utility.

Small cell lung cancer (SCLC) is a deadly neuroendocrine tumor for which there are no screening methods sensitive enough to facilitate early, effective intervention. We propose targeting the neuroendocrine tumor neoantigen CD133 via antibody-based early detection and PET (immunoPET) to facilitate earlier and more accurate detection of SCLC. Methods: RNA sequencing datasets, immunohistochemistry, flow cytometry, and Western blots were used to quantify CD133 expression in healthy and SCLC patients. CD133 was imaged in vivo using near-infrared fluorescence (NIRF) immunoimaging, and 89 Zr immunoPET. Anti(α)-CD133 autoantibody levels were measured in SCLC patient plasma using antibody microarrays. Results: Across 6 publicly available datasets, CD133 messenger RNA was found to be higher in SCLC tumors than in other tissues, including healthy or normal adjacent lung and non-SCLC samples. Critically, the upregulation of CD133 messenger RNA in SCLC was associated with a significant increase (hazard ratio, 2.62) in death. CD133 protein was expressed in primary human SCLC, in SCLC patient-derived xenografts, and in both SCLC cell lines tested (H82 and H69). Using an H82 xenograft mouse model, we first imaged CD133 expression with NIRF. Both in vivo and ex vivo NIRF clearly showed that a fluorophore-tagged αCD133 homed to lung tumors. Next, we validated the noninvasive visualization of subcutaneous and orthotopic H82 xenografts via immunoPET. An αCD133 antibody labeled with the positron-emitting radiometal 89 Zr demonstrated significant accumulation in tumor tissue while producing minimal uptake in healthy organs. Finally, plasma αCD133 autoantibodies were found in subjects from cohort studies up to 1 year before SCLC diagnosis. Conclusion: In light of these findings, we conclude that the presence of αCD133 autoantibodies in a blood sample followed by CD133-targeted 89 Zr-immunoPET could be an effective early detection screening strategy for SCLC., (© 2022 by the Society of Nuclear Medicine and Molecular Imaging.)

Connexins are assembled into dodecamer intercellular channels, a collection of which is termed a gap junction, and their canonical function allowing direct exchange of ions and metabolites has been unequivocally established. When initially assembled into undocked cell surface connexin hemichannels, healthy cells may also engage in cell signaling via a regulated small-molecule release. Recent advances in the field have led to an expanded view of the functional roles of intercellular channels and hemichannels in both physiology and pathology. As more of the 21-member human connexin family is intensely interrogated, mounting evidence points to the biological uniqueness of each member, and no longer can we confidently refer to all connexins engaging in the same cellular processes. Innovations in high-resolution cryo-electron microscopy have revealed important insights into the structure of functionally important domains of both hemichannels and channels. These and other studies have established a foundation of knowledge that should allow inhibitory smart drug design for situations where enhanced intercellular or hemichannel activity is at the root of a connexin-linked disease. Assessment of the connexin interactome, which varies widely for each connexin subtype, continues to provide regulatory insights into the assembly and function of connexins that exhibit a short half-life. As the most intensely studied, Cx43 is found in about 50% of all human cell types and is extensively regulated by multiple inhibitory and enhancing phosphorylation events that have direct implications on tissue function and outcomes of disease, including cancer. Here, we briefly discuss these advances and give our thoughts on where the field is headed., Competing Interests: PDL notes that he is an unpaid member of the Scientific Advisory Board of FirstString Research LLC but states that this service has not influenced this presentation. DWL declares that he has no competing interests.No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed., (Copyright: © 2022 Lampe PD et al.)

The 21-member connexin gene family exhibits distinct tissue expression patterns that can cause a diverse array of over 30 inherited connexin-linked diseases ranging from deafness to skin defects and blindness. Intriguingly, germline mutations can cause disease in one tissue while other tissues that abundantly express the mutant connexin remain disease free, highlighting the importance of the cellular context of mutant expression. Modeling connexin pathologies in genetically modified mice and tissue-relevant cells has informed extensively on no less than a dozen gain- and loss-of-function mechanisms that underpin disease. This review focuses on how a deeper molecular understanding of the over 930 mutations in 11 connexin-encoding genes is foundational for creating a framework for therapeutic interventions., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Purpose: We sought to examine the prognostic value of fluorodeoxyglucose-positron emission tomography (PET) imaging during chemoradiation for unresectable non-small cell lung cancer for survival and hypothesized that tumor PET response is correlated with peripheral T-cell function., Methods and Materials: Forty-five patients with American Joint Committee on Cancer version 7 stage IIB-IIIB non-small cell lung cancer enrolled in a phase II trial and received platinum-doublet chemotherapy concurrent with 6 weeks of radiation (NCT02773238). Fluorodeoxyglucose-PET was performed before treatment start and after 24 Gy of radiation (week 3). PET response status was prospectively defined by multifactorial radiologic interpretation. PET responders received 60 Gy in 30 fractions, while nonresponders received concomitant boosts to 74 Gy in 30 fractions. Peripheral blood was drawn synchronously with PET imaging, from which germline DNA sequencing, T-cell receptor sequencing, and plasma cytokine analysis were performed., Results: Median follow-up was 18.8 months, 1-year overall survival (OS) 82%, 1-year progression-free survival 53%, and 1-year locoregional control 88%. Higher midtreatment PET total lesion glycolysis was detrimental to OS (1 year 87% vs 63%, P < .001), progression-free survival (1 year 60% vs 26%, P  = .044), and locoregional control (1 year 94% vs 65%, P  = .012), even after adjustment for clinical/treatment factors. Twenty-nine of 45 patients (64%) were classified as PET responders based on a priori definition. Higher tumor programmed death-ligand 1 expression was correlated with response on PET ( P  = .017). Higher T-cell receptor richness and clone distribution slope were associated with improved OS ( P  = .018-0.035); clone distribution slope was correlated with PET response ( P  = .031)., Conclusions: Midchemoradiation PET imaging is prognostic for survival; PET response may be linked to tumor and peripheral T-cell biomarkers., (© 2021 The Authors.)

Early-onset colorectal cancer has been on the rise in Western populations. Here, we compare patient characteristics between those with early- (<50 years) vs. late-onset (≥50 years) disease in a large multinational cohort of colorectal cancer patients ( n = 2193). We calculated descriptive statistics and assessed associations of clinicodemographic factors with age of onset using mutually-adjusted logistic regression models. Patients were on average 60 years old, with BMI of 29 kg/m 2 , 52% colon cancers, 21% early-onset, and presented with stage II or III (60%) disease. Early-onset patients presented with more advanced disease (stages III-IV: 63% vs. 51%, respectively), and received more neo and adjuvant treatment compared to late-onset patients, after controlling for stage (odds ratio (OR) (95% confidence interval (CI)) = 2.30 (1.82-3.83) and 2.00 (1.43-2.81), respectively). Early-onset rectal cancer patients across all stages more commonly received neoadjuvant treatment, even when not indicated as the standard of care, e.g., during stage I disease. The odds of early-onset disease were higher among never smokers and lower among overweight patients (1.55 (1.21-1.98) and 0.56 (0.41-0.76), respectively). Patients with early-onset colorectal cancer were more likely to be diagnosed with advanced stage disease, to have received systemic treatments regardless of stage at diagnosis, and were less likely to be ever smokers or overweight.

Spatial patterns of gene expression manifest at scales ranging from local (e.g., cell-cell interactions) to global (e.g., body axis patterning). However, current spatial transcriptomics methods either average local contexts or are restricted to limited fields of view. Here, we introduce sci-Space, which retains single-cell resolution while resolving spatial heterogeneity at larger scales. Applying sci-Space to developing mouse embryos, we captured approximate spatial coordinates and whole transcriptomes of about 120,000 nuclei. We identify thousands of genes exhibiting anatomically patterned expression, leverage spatial information to annotate cellular subtypes, show that cell types vary substantially in their extent of spatial patterning, and reveal correlations between pseudotime and the migratory patterns of differentiating neurons. Looking forward, we anticipate that sci-Space will facilitate the construction of spatially resolved single-cell atlases of mammalian development., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Myocardial connexin 43 (Cx43) forms gap junctions and hemichannels, and is also present within subsarcolemmal mitochondria. The protein is phosphorylated by several kinases including mitogen-activated protein kinase (MAPK), protein kinase C (PKC), and casein kinase 1 (CK1). A reduction in Cx43 content abrogates myocardial infarct size reduction by ischemic preconditioning (IPC). The present study characterizes the contribution of Cx43 phosphorylation towards mitochondrial function, hemichannel activity, and the cardioprotection by IPC in wild-type (WT) mice and in mice in which Cx43-phosphorylation sites targeted by above kinases are mutated to non-phosphorylatable residues (Cx43 MAPKmut , Cx43 PKCmut , and Cx43 CK1mut mice). The amount of Cx43 in the left ventricle and in mitochondria was reduced in all mutant strains compared to WT mice and Cx43 phosphorylation was altered at residues not directly targeted by the mutations. Whereas complex 1 respiration was reduced in all strains, complex 2 respiration was decreased in Cx43 CK1mut mice only. In Cx43 epitope-mutated mice, formation of reactive oxygen species and opening of the mitochondrial permeability transition pore were not affected. The hemichannel open probability was reduced in Cx43 PKCmut and Cx43 CK1mut but not in Cx43 MAPKmut cardiomyocytes. Infarct size in isolated saline-perfused hearts after ischemia/reperfusion (45 min/120 min) was comparable between genotypes and was significantly reduced by IPC (3 × 3 min ischemia/5 min reperfusion) in WT, Cx43 MAPKmut , and Cx43 PKCmut , but not in Cx43 CK1mut mice, an effect independent from the amount of Cx43 and the probability of hemichannel opening. Taken together, our study shows that alterations of Cx43 phosphorylation affect specific cellular functions and highlights the importance of Cx43 phosphorylation by CK1 for IPC's cardioprotection.

Pancreatic ductal adenocarcinoma (PDA) is aggressive, highly metastatic and characterized by a robust desmoplasia. Connexin proteins that form gap junctions have been implicated in tumor suppression for over 30 years. Cx43, the most widely expressed connexin, regulates cell behaviors, including migration and proliferation. Thus, we hypothesized that Cx43 could regulate PDA progression. Phosphorylation of Cx43 by Casein Kinase 1 (CK1) regulates gap junction assembly. We interbred the well-established Kras LSL-G12D/+;p48Cre/+ (KC) mouse model of PDA with homozygous "knock-in" mutant Cx43 mice bearing amino acid substitution at CK1 sites (Cx43 CK1A ) and found profound and surprising effects on cancer progression. Crossing the Cx43 CK1A mouse onto the KC background (termed KC;Cx CK1A ) led to significant extension of lifespan, from a median of 370 to 486 days (p = 0.03) and a decreased incidence of metastasis (p = 0.045). However, when we examined early stages of disease, we found more rapid onset of tissue remodeling in the KC;Cx CK1A mouse followed by divergence to a cystic phenotype. During tumorigenesis, gap junctions are increasingly present in stromal cells of the KC mice but are absent from the KC;Cx43 CK1A mice. Tail vein metastasis assays with cells derived from KC or KC;Cx CK1A tumors showed that KC;Cx CK1A cells could efficiently colonize the lung and downregulate Cx43 expression, arguing that inhibition of metastasis was not occurring at the distal site. Instead, stromal gap junctions, their associated signaling events or other unknown Cx43-dependent events facilitate metastatic capacity in the primary tumor.

Early detection by screening significantly reduces mortality from colorectal cancer, but 40% of guideline-eligible patients are not screened as recommended in the United States. Novel strategies to improve screening uptake overall and efforts to deploy best practices to underserved populations are a high priority for health care. This review focuses on existing biomarkers in practice and those in development with clinical relevance to early detection of colorectal neoplasia, with an emphasis on those developed by investigators of the NCI's Early Detection Research Network. Aberrantly methylated DNA markers (blood and stool), stool-based markers (including fecal immunochemical test-DNA), and a variety of blood-based marker assays in development (protein markers, glycoproteins including mucins, and cell-free DNA tests) are reviewed. Individual markers and biomarker panels, sample resources, and barriers to translating biomarkers to clinical practice are discussed. See all articles in this CEBP Focus section, "NCI Early Detection Research Network: Making Cancer Detection Possible.", (©2020 American Association for Cancer Research.)

The gap junction protein Connexin43 (Cx43) is highly regulated by phosphorylation at over a dozen sites by probably at least as many kinases. This Cx43 "kinome" plays an important role in gap junction assembly and turnover. We sought to gain a better understanding of the interrelationship of these phosphorylation events particularly related to src activation and Cx43 turnover. Using state-of-the-art live imaging methods, specific inhibitors and many phosphorylation-status specific antibodies, we found phospho-specific domains in gap junction plaques and show evidence that multiple pathways of disassembly exist and can be regulated at the cellular and subcellular level. We found Src activation promotes formation of connexisomes (internalized gap junctions) in a process involving ERK-mediated phosphorylation of S279/282. Proteasome inhibition dramatically and rapidly restored gap junctions in the presence of Src and led to dramatic changes in the Cx43 phospho-profile including to increased Y247, Y265, S279/282, S365, and S373 phosphorylation. Lysosomal inhibition, on the other hand, nearly eliminated phosphorylation on Y247 and Y265 and reduced S368 and S373 while increasing S279/282 phosphorylation levels. We present a model of gap junction disassembly where multiple modes of disassembly are regulated by phosphorylation and can have differential effects on cellular signaling.

Background: Dietary patterns low in glycemic load are associated with reduced risk of cardiometabolic diseases. Improvements in serum lipid concentrations may play a role in these observed associations., Objective: We investigated how dietary patterns differing in glycemic load affect clinical lipid panel measures and plasma lipidomics profiles., Methods: In a crossover, controlled feeding study, 80 healthy participants (n = 40 men, n = 40 women), 18-45 y were randomized to receive low-glycemic load (LGL) or high glycemic load (HGL) diets for 28 days each with at least a 28-day washout period between controlled diets. Fasting plasma samples were collected at baseline and end of each diet period. Lipids on a clinical panel including total-, VLDL-, LDL-, and HDL-cholesterol and triglycerides were measured using an auto-analyzer. Lipidomics analysis using mass-spectrometry provided the concentrations of 863 species. Linear mixed models and lipid ontology enrichment analysis were implemented., Results: Lipids from the clinical panel were not significantly different between diets. Univariate analysis showed that 67 species on the lipidomics panel, predominantly in the triacylglycerol class, were higher after the LGL diet compared to the HGL (FDR < 0.05). Three species with FA 17:0 were lower after LGL diet with enrichment analysis (FDR < 0.05)., Conclusion: In the context of controlled eucaloric diets with similar macronutrient distribution, these results suggest that there are relative shifts in lipid species, but the overall pool does not change. Further studies are needed to better understand in which compartment the different lipid species are transported in blood, and how these shifts are related to health outcomes. This trial was registered at clinicaltrials.gov as NCT00622661.

Background: Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) show tremendous promise for cardiac regeneration, but the successful development of hESC-CM-based therapies requires improved tools to investigate their electrical behavior in recipient hearts. While optical voltage mapping is a powerful technique for studying myocardial electrical activity ex vivo, we have previously shown that intra-cardiac hESC-CM grafts are not labeled by conventional voltage-sensitive fluorescent dyes. We hypothesized that the water-soluble voltage-sensitive dye di-2-ANEPEQ would label engrafted hESC-CMs and thereby facilitate characterization of graft electrical function and integration., Methods: We developed and validated a novel optical voltage mapping strategy based on the simultaneous imaging of the calcium-sensitive fluorescent protein GCaMP3, a graft-autonomous reporter of graft activation, and optical action potentials (oAPs) derived from di-2-ANEPEQ, which labels both graft and host myocardium. Cardiomyocytes from three different GCaMP3+ hESC lines (H7, RUES2, or ESI-17) were transplanted into guinea pig models of subacute and chronic infarction, followed by optical mapping at 2 weeks post-transplantation., Results: Use of a water-soluble voltage-sensitive dye revealed pro-arrhythmic properties of GCaMP3+ hESC-CM grafts from all three lines including slow conduction velocity, incomplete host-graft coupling, and spatially heterogeneous patterns of activation that varied beat-to-beat. GCaMP3+ hESC-CMs from the RUES2 and ESI-17 lines both showed prolonged oAP durations both in vitro and in vivo. Although hESC-CMs partially remuscularize the injured hearts, histological evaluation revealed immature graft structure and impaired gap junction expression at this early timepoint., Conclusion: Simultaneous imaging of GCaMP3 and di-2-ANEPEQ allowed us to acquire the first unambiguously graft-derived oAPs from hESC-CM-engrafted hearts and yielded critical insights into their arrhythmogenic potential and line-to-line variation.

Connexin-43 (Cx43) gap junctions provide intercellular coupling, which ensures rapid action potential propagation and synchronized heart contraction. Alterations in Cx43 localization and reductions in gap junction coupling occur in failing hearts, contributing to ventricular arrhythmias and sudden cardiac death. Recent reports have found that an internally translated Cx43 isoform, GJA1-20k, is an auxiliary subunit for the trafficking of Cx43 in heterologous expression systems. Here, we have created a mouse model by using CRISPR technology to mutate a single internal translation initiation site in Cx43 (M213L mutation), which generates full-length Cx43, but not GJA1-20k. We found that GJA1M213L/M213L mice had severely abnormal electrocardiograms despite preserved contractile function, reduced total Cx43, and reduced gap junctions, and they died suddenly at 2 to 4 weeks of age. Heterozygous GJA1M213L/WT mice survived to adulthood with increased ventricular ectopy. Biochemical experiments indicated that cytoplasmic Cx43 had a half-life that was 50% shorter than membrane-associated Cx43. Without GJA1-20k, poorly trafficked Cx43 was degraded. The data support that GJA1-20k, an endogenous entity translated independently of Cx43, is critical for Cx43 gap junction trafficking, maintenance of Cx43 protein, and normal electrical function of the mammalian heart.

Introduction: Blood-based, cancer-associated biomarkers are susceptible to a variety of well-known preanalytical factors. The influence of bowel preparation before a diagnostic colonoscopy on biomarker levels is, however, poorly investigated. The present study assessed the influence of bowel preparation on colorectal cancer-associated biomarkers. In addition, the effect of single versus double centrifugation of plasma biomarkers was assessed., Methods: Blood samples were collected pre- and post-bowel preparation from 125 subjects scheduled for first time diagnostic colonoscopy due to symptoms attributable to CRC. The samples were separated into serum and EDTA plasma, and analyzed by four independent collaborators for: 1) the proteins AFP, CA19-9, CEA, hs-CRP, CyFra21-1, Ferritin, Galectin-3 and TIMP-1, 2) the proteins BAG4, IL6ST, vWF, CD44 and EGFR, 3) the glycoprotein Galectin-3 ligand, and 4) cell-free DNA (cfDNA). Statistical analysis of biomarker data has been performed using mixed modelling, including repeated measures., Results: The biomarkers generally showed negligible variation between pre- and post-bowel preparation except for CyFra21-1, Ferritin, BAG4 and cfDNA. CyFra21-1 levels were systematically reduced with 29% (95% CI 21-36%) by bowel preparation (p ≤ 0.0001). Ferritin was not significantly different between pre- and post-bowel preparation (p = 0.07), however the estimated difference (increase) was 18%. BAG4 was systematically reduced by 12% (95% CI 1-22%, p = 0.04), while cfDNA showed a significant increase of 28% (95% CI 17-39%, p < 0.0001). Double centrifugation compared to single centrifugation showed reduced vWF (ratio 0.86, p ≤ 0.0001) and CD44 (ratio 0.85, p = 0.016), but increased IL6ST levels (ratio 1.18, p = 0.014)., Conclusions: Results of the present study demonstrated systematic, statistically significant differences between pre-bowel and post-bowel preparation levels for three independent blood-based biomarkers (BAG4, CyFra21-1, cfDNA), illustrating the importance of timing of sample collection for biomarker analyses., 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 © 2020 Elsevier B.V. All rights reserved.)

Aberrant expression of the cardiac gap junction protein connexin-43 (Cx43) has been suggested as playing a role in the development of cardiac disease in the mdx mouse model of Duchenne muscular dystrophy (DMD); however, a mechanistic understanding of this association is lacking. Here, we identified a reduction of phosphorylation of Cx43 serines S325/S328/S330 in human and mouse DMD hearts. We hypothesized that hypophosphorylation of Cx43 serine-triplet triggers pathological Cx43 redistribution to the lateral sides of cardiomyocytes (remodeling). Therefore, we generated knockin mdx mice in which the Cx43 serine-triplet was replaced with either phospho-mimicking glutamic acids (mdxS3E) or nonphosphorylatable alanines (mdxS3A). The mdxS3E, but not mdxS3A, mice were resistant to Cx43 remodeling, with a corresponding reduction of Cx43 hemichannel activity. MdxS3E cardiomyocytes displayed improved intracellular Ca2+ signaling and a reduction of NADPH oxidase 2 (NOX2)/ROS production. Furthermore, mdxS3E mice were protected against inducible arrhythmias, related lethality, and the development of cardiomyopathy. Inhibition of microtubule polymerization by colchicine reduced both NOX2/ROS and oxidized CaMKII, increased S325/S328/S330 phosphorylation, and prevented Cx43 remodeling in mdx hearts. Together, these results demonstrate a mechanism of dystrophic Cx43 remodeling and suggest that targeting Cx43 may be a therapeutic strategy for preventing heart dysfunction and arrhythmias in DMD patients.

B-lymphocytes recognize antigen via B-cell antigen receptors (BCRs). This binding induces signaling, leading to B-cell activation, proliferation and differentiation. Early events of BCR signaling include reorganization of actin and membrane spreading, which facilitates increased antigen gathering. We have previously shown that the gap junction protein connexin43 (Cx43; also known as GJA1) is phosphorylated upon BCR signaling, and its carboxyl tail (CT) is important for BCR-mediated spreading. Here, specific serine residues in the Cx43 CT that are phosphorylated following BCR stimulation were identified. A chimeric protein containing the extracellular and transmembrane domains of CD8 fused to the Cx43 CT was sufficient to support cell spreading. Cx43 CT truncations showed that the region between amino acids 246-307 is necessary for B-cell spreading. Site-specific serine-to-alanine mutations (S255A, S262A, S279A and S282A) resulted in differential effects on both BCR signaling and BCR-mediated spreading. These serine residues can serve as potential binding sites for actin remodeling mediators and/or BCR signaling effectors; therefore, our results may reflect unique roles for each of these serines in terms of linking the Cx43 CT to actin remodeling., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Although the gut microbiome has been associated with dietary patterns linked to health, microbial metabolism is not well characterized. This ancillary study was a proof of principle analysis for a novel application of metaproteomics to study microbial protein expression in a controlled dietary intervention. We measured the response of the microbiome to diet in a randomized crossover dietary intervention of a whole-grain, low glycemic load diet (WG) and a refined-grain, high glycemic load diet (RG). Total proteins in stools from 9 participants at the end of each diet period ( n = 18) were analyzed by LC MS/MS and proteins were identified using the Human Microbiome Project (HMP) human gut microbiome database and UniProt human protein databases. T-tests, controlling for false discovery rate (FDR) <10%, were used to compare the Gene Ontology (GO) biological processes and bacterial enzymes between the two interventions. Using shotgun proteomics, more than 53,000 unique peptides were identified including microbial (89%) and human peptides (11%). Forty-eight bacterial enzymes were statistically different between the diets, including those implicated in SCFA production and degradation of fatty acids. Enzymes associated with degradation of human mucin were significantly enriched in the RG diet. These results illustrate that the metaproteomic approach is a valuable tool to study the microbial metabolism of diets that may influence host health.

Background: Non-steroidal anti-inflammatory drugs, e.g., celecoxib, are commonly used for inflammatory conditions, but can be associated with adverse effects. Combined glucosamine hydrochloride plus chondroitin sulfate (GH+CS) are commonly used for joint pain and have no known adverse effects. Evidence from in vitro, animal and human studies suggest that GH+CS have anti-inflammatory activity, among other mechanisms of action., Objective: We evaluated the effects of GH+CS versus celecoxib on a panel of 20 serum proteins involved in inflammation and other metabolic pathways., Methods: Samples were from a randomized, parallel, double-blind trial of pharmaceutical grade 1500 mg GH + 1200 mg CS (n=96) versus 200 mg celecoxib daily (n=93) for 6- months in knee osteoarthritis (OA) patients. Linear mixed models adjusted for age, sex, body mass index, baseline serum protein values, and rescue medicine use assessed the intervention effects of each treatment arm adjusting for multiple testing., Results: All serum proteins except WNT16 were lower after treatment with GH+CS, while about half increased after celecoxib. Serum IL-6 was significantly reduced (by 9%, P=0.001) after GH+CS, and satisfied the FDR<0.05 threshold. CCL20, CSF3, and WNT16 increased after celecoxib (by 7%, 9% and 9%, respectively, P<0.05), but these serum proteins were no longer statistically significant after controlling for multiple testing., Conclusion: The results of this study using samples from a previously conducted trial in OA patients, demonstrate that GH+CS reduces circulating IL-6, an inflammatory cytokine, but is otherwise comparable to celecoxib with regard to effects on other circulating protein biomarkers., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Purpose: To compare the ability of radiological semantic and quantitative texture features in lung cancer diagnosis of pulmonary nodules., Materials and Methods: A total of N = 121 subjects with confirmed non-small-cell lung cancer were matched with 117 controls based on age and gender. Radiological semantic and quantitative texture features were extracted from CT images with or without contrast enhancement. Three different models were compared using LASSO logistic regression: "CS" using clinical and semantic variables, "T" using texture features, and "CST" using clinical, semantic, and texture variables. For each model, we performed 100 trials of fivefold cross-validation and the average receiver operating curve was accessed. The AUC of the cross-validation study (AUC CV ) was calculated together with its 95% confidence interval., Results: The AUC CV (and 95% confidence interval) for models T, CS, and CST was 0.85 (0.71-0.96), 0.88 (0.77-0.96), and 0.88 (0.77-0.97), respectively. After separating the data into two groups with or without contrast enhancement, the AUC (without cross-validation) of the model T was 0.86 both for images with and without contrast enhancement, suggesting that contrast enhancement did not impact the utility of texture analysis., Conclusions: The models with semantic and texture features provided cross-validated AUCs of 0.85-0.88 for classification of benign versus cancerous nodules, showing potential in aiding the management of patients., Key Points: • Pretest probability of cancer can aid and direct the physician in the diagnosis and management of pulmonary nodules in a cost-effective way. • Semantic features (qualitative features reported by radiologists to characterize lung lesions) and radiomic (e.g., texture) features can be extracted from CT images. • Input of these variables into a model can generate a pretest likelihood of cancer to aid clinical decision and management of pulmonary nodules.

Connexin (Cx) mimetic peptides derived from extracellular loop II sequences (e.g., Gap27: SRPTEKTIFII; Peptide5: VDCFLSRPTEKT) have been used as reversible, Cx-specific blockers of hemichannel (HCh) and gap junction channel (GJCh) function. These blockers typically require high concentrations (~5 µM, <1 h for HCh; ~100 µM, >1 h for GJCh) to achieve inhibition. We have shown that addition of a hexadecyl ( Hdc ) lipid tail to the conserved SRPTEKT peptide sequence (SRPTEKT- Hdc ) results in a novel, highly efficacious, and potent inhibitor of mechanically induced Ca 2+ -wave propagation (IC 50 64.8 pM) and HCh-mediated dye uptake (IC 50 45.0 pM) in Madin-Darby canine kidney cells expressing rat Cx43 (MDCK43). The lack of similar effect on dye coupling (NBD-MTMA) suggested channel conformation-specific inhibition. Here we report that SRPTEKT- Hdc inhibition of Ca 2+ -wave propagation, dye coupling, and dye uptake depended on the functional configuration of Cx43 as determined by phosphorylation at serine 368 (S368). Ca 2+ -wave propagation was enhanced in MDCK cells expressing single-site mutants of Cx43 that mimicked (MDCK43-S368D) or favored (MDCK43-S365A) phosphorylation at S368. Furthermore, SRPTEKT- Hdc potently inhibited GJCh-mediated Ca 2+ -wave propagation (IC 50 230.4 pM), dye coupling, and HCh-mediated dye uptake in MDCK43-S368D and -S365A cells. In contrast, Ca 2+ -wave propagation, dye coupling, and dye uptake were largely unaffected (IC 50 12.3 μM) by SRPTEKT- Hdc in MDCK43-S368A and -S365D cells, mutations that mimic or favor dephosphorylation at S368. Together, these data indicate that SRPTEKT- Hdc is a potent inhibitor of physiological Ca 2+ -wave signaling mediated specifically by the pS368 phosphorylated form of Cx43.

Background: Low-glycemic load dietary patterns, characterized by consumption of whole grains, legumes, fruits, and vegetables, are associated with reduced risk of several chronic diseases., Methods: Using samples from a randomized, controlled, crossover feeding trial, we evaluated the effects on metabolic profiles of a low-glycemic whole-grain dietary pattern (WG) compared with a dietary pattern high in refined grains and added sugars (RG) for 28 d. LC-MS-based targeted metabolomics analysis was performed on fasting plasma samples from 80 healthy participants (n = 40 men, n = 40 women) aged 18-45 y. Linear mixed models were used to evaluate differences in response between diets for individual metabolites. Kyoto Encyclopedia of Genes and Genomes (KEGG)-defined pathways and 2 novel data-driven analyses were conducted to consider differences at the pathway level., Results: There were 121 metabolites with detectable signal in >98% of all plasma samples. Eighteen metabolites were significantly different between diets at day 28 [false discovery rate (FDR) < 0.05]. Inositol, hydroxyphenylpyruvate, citrulline, ornithine, 13-hydroxyoctadecadienoic acid, glutamine, and oxaloacetate were higher after the WG diet than after the RG diet, whereas melatonin, betaine, creatine, acetylcholine, aspartate, hydroxyproline, methylhistidine, tryptophan, cystamine, carnitine, and trimethylamine were lower. Analyses using KEGG-defined pathways revealed statistically significant differences in tryptophan metabolism between diets, with kynurenine and melatonin positively associated with serum C-reactive protein concentrations. Novel data-driven methods at the metabolite and network levels found correlations among metabolites involved in branched-chain amino acid (BCAA) degradation, trimethylamine-N-oxide production, and β oxidation of fatty acids (FDR < 0.1) that differed between diets, with more favorable metabolic profiles detected after the WG diet. Higher BCAAs and trimethylamine were positively associated with homeostasis model assessment-insulin resistance., Conclusions: These exploratory metabolomics results support beneficial effects of a low-glycemic load dietary pattern characterized by whole grains, legumes, fruits, and vegetables, compared with a diet high in refined grains and added sugars on inflammation and energy metabolism pathways. This trial was registered at clinicaltrials.gov as NCT00622661., (Copyright © American Society for Nutrition 2019.)

Efficient stem cell differentiation into pancreatic islet cells is of critical importance for the development of cell replacement therapies for diabetes. Here, we identify the expression pattern of connexin 43 (Cx43), a gap junction (GJ) channel protein, in human embryonic stem cell (hESC)-derived definitive endoderm (DE) and primitive gut tube cells, representing early lineages for posterior foregut (PF), pancreatic progenitors (PP), pancreatic endocrine progenitors (PE), and islet cells. As the function of GJ channels is dependent on their gating status, we tested the impact of supplementing hESC-derived PP cell cultures with AAP10, a peptide that promotes Cx43 GJ channel opening. We found that this treatment promotes the expression of DE markers FoxA2 and Sox17, leads to a more efficient derivation of DE, and improves the yield of PF, PP, and PE cells. These results demonstrate a functional involvement of GJ channels in the differentiation of embryonic stem cells into pancreatic cell lineages., (Copyright © 2019. Published by Elsevier Inc.)

The gap junction protein connexin 43 (Cx43) is a key player in wound healing, and inhibitors of Cx43, which speed epidermal wound healing, are currently in clinical trials. Here, we provide direct in vivo evidence that specific phosphorylation events on Cx43 change the physiological response during wound healing. Blocking phosphorylation, through mutation of serine residues in Cx43 at the protein kinase C (PKC) or casein kinase 1 (CK1) sites, significantly slowed the rate of wound closure in vivo and in vitro and resulted in a thicker epidermal layer after reepithelialization. Conversely, preventing Cx43 phosphorylation by mitogen-activated protein kinases (MAPKs) through mutation significantly increased the rate of wound closure in vivo Defects in migration, but not proliferation, in all mutants were partially rescued in vitro by changing serine residues to aspartic or glutamic acid . These data prove that specific Cx43 phosphorylation events play an important role at different stages of wound healing. Thus, a clear physiological understanding of the spatiotemporal regulation of kinase activation and consequent effects on gap junctions could lead to a more targeted approach to modulating Cx43 expression during wound healing., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Increased adiposity and diets high in glycemic load (GL) are associated with increased risk of many chronic diseases including cancer. Using plasma from 80 healthy individuals [40 men/40 women, 29 with DXA-derived low fat mass (FM) and 51 with high FM] in a randomized cross-over-controlled feeding trial and arrays populated with 3,504 antibodies, we measured plasma proteins collected at baseline and end of each of two 28-day controlled diets: a low GL diet high in whole grains, legumes, fruits, and vegetables (WG) and a high GL diet high in refined grains and added sugars (RG). Following univariate testing for proteins differing by diet, we evaluated pathway-level involvement. Among all 80 participants, 172 proteins were identified as differing between diets. Stratifying participants by high and low FM identified 221 and 266 proteins, respectively, as differing between diets (unadjusted P < 0.05). These candidate proteins were tested for overrepresentation in Reactome pathways, corresponding to 142 (of 291) pathways in the high-FM group and 72 (of 274) pathways in the low-FM group. We observed that the cancer-related pathways, DNA Repair, DNA Replication , and Cell Cycle , were overrepresented in the high-FM participants while pathways involved in post-translational protein modification were overrepresented in participants with either FM. Although high-GL diets are associated with increased risk of some cancers, our study further suggests that biology associated with consumption of GL diets is variable depending on an individual's adiposity and dietary recommendations related to cancer prevention be made with the additional consideration of an individual's FM., (©2019 American Association for Cancer Research.)

Gap junctions contain intercellular channels that enable intercellular communication of small molecules while also serving as a signaling scaffold. Connexins, the proteins that form gap junctions in vertebrates, are highly regulated and typically have short (<2 h) half-lives. Connexin43 (Cx43), the predominate connexin in the myocardium and epithelial tissues, is phosphorylated on more than a dozen serine residues and interacts with a variety of protein kinases. These interactions regulate Cx43 and gap junction formation and stability. Casein kinase 1 (CK1)-mediated phosphorylation of Cx43 promotes gap junction assembly. Using murine knock-in technology and quantitative PCR, immunoblotting, and immunoprecipitation assays, we show here that mutation of the CK1 phosphorylation sites in Cx43 reduces the levels of total Cx43 in the myocardium and increases Cx43 phosphorylation on sites phosphorylated by extracellular signal-regulated kinase (ERK). In aged myocardium, we found that, compared with WT Cx43, mutant Cx43 expression increases ERK activation, phosphorylation of Akt substrates, and protection from ischemia-induced injury. Our findings also uncovered that Cx43 interacts with the hypoxia-inducible protein N-Myc downstream-regulated gene 1 protein (NDRG1) and that Cx43 phosphorylation status controls this interaction and dramatically affects NDRG1 stability. We propose that, in addition to altering gap junction stability, Cx43 phosphorylation directly and dynamically regulates cellular signaling through ERK and Akt in response to ischemic injury. We conclude that gap junction-dependent NDRG1 regulation might explain some cellular responses to hypoxia., (© 2019 Solan et al.)

Rationale: Screening for non-small cell lung cancer is associated with earlier diagnosis and reduced mortality but also increased harm caused by invasive follow-up of benign pulmonary nodules. Lung tumorigenesis activates the immune system, components of which could serve as tumor-specific biomarkers. Objectives: To profile tumor-derived autoantibodies as peripheral biomarkers of malignant pulmonary nodules. Methods: High-density protein arrays were used to define the specificity of autoantibodies isolated from B cells of 10 resected lung tumors. These tumor-derived autoantibodies were also examined as free or complexed to antigen in the plasma of the same 10 patients and matched benign nodule control subjects. Promising autoantibodies were further analyzed in an independent cohort of 250 nodule-positive patients. Measurements and Main Results: Thirteen tumor B-cell-derived autoantibodies isolated ex vivo showed greater than or equal to 50% sensitivity and greater than or equal to 70% specificity for lung cancer. In plasma, 11 of 13 autoantibodies were present both complexed to and free from antigen. In the larger validation cohort, 5 of 13 tumor-derived autoantibodies remained significantly elevated in cancers. A combination of four of these autoantibodies could detect malignant nodules with an area under the curve of 0.74 and had an area under the curve of 0.78 in a subcohort of indeterminate (8-20 mm in the longest diameter) pulmonary nodules. Conclusions: Our novel pipeline identifies tumor-derived autoantibodies that could effectively serve as blood biomarkers for malignant pulmonary nodule diagnosis. This approach has future implications for both a cost-effective and noninvasive approach to determine nodule malignancy for widespread low-dose computed tomography screening.

Pannexin 1 (PANX1)-mediated ATP release in vascular smooth muscle coordinates α1-adrenergic receptor (α1-AR) vasoconstriction and blood pressure homeostasis. We recently identified amino acids 198-200 (YLK) on the PANX1 intracellular loop that are critical for α1-AR-mediated vasoconstriction and PANX1 channel function. We report herein that the YLK motif is contained within an SRC homology 2 domain and is directly phosphorylated by SRC proto-oncogene, nonreceptor tyrosine kinase (SRC) at Tyr 198 We demonstrate that PANX1-mediated ATP release occurs independently of intracellular calcium but is sensitive to SRC family kinase (SFK) inhibition, suggestive of channel regulation by tyrosine phosphorylation. Using a PANX1 Tyr 198 -specific antibody, SFK inhibitors, SRC knockdown, temperature-dependent SRC cells, and kinase assays, we found that PANX1-mediated ATP release and vasoconstriction involves constitutive phosphorylation of PANX1 Tyr 198 by SRC. We specifically detected SRC-mediated Tyr 198 phosphorylation at the plasma membrane and observed that it is not enhanced or induced by α1-AR activation. Last, we show that PANX1 immunostaining is enriched in the smooth muscle layer of arteries from hypertensive humans and that Tyr 198 phosphorylation is detectable in these samples, indicative of a role for membrane-associated PANX1 in small arteries of hypertensive humans. Our discovery adds insight into the regulation of PANX1 by post-translational modifications and connects a significant purinergic vasoconstriction pathway with a previously identified, yet unexplored, tyrosine kinase-based α1-AR constriction mechanism. This work implicates SRC-mediated PANX1 function in normal vascular hemodynamics and suggests that Tyr 198 -phosphorylated PANX1 is involved in hypertensive vascular pathology., (© 2019 DeLalio et al.)

Connexin43 (Cx43) function is influenced by kinases that phosphorylate specific serine sites located near its C-terminus. Stroke is a powerful inducer of kinase activity, but its effect on Cx43 is unknown. We investigated the impact of wild-type (WT) and knock-in Cx43 with serine to alanine mutations at the protein kinase C (PKC) site Cx43 S368A , the casein kinase 1 (CK1) sites Cx43 S325A/328Y/330A , and the mitogen-activated protein kinase (MAPK) sites Cx43 S255/262/279/282A (MK4) on a permanent middle cerebral artery occlusion (pMCAO) stroke model. We demonstrate that MK4 transgenic animals exhibit a significant decrease in infarct volume that was associated with improvement in behavioral performance. An increase in astrocyte reactivity with a concomitant decrease in microglial reactivity was observed in MK4 mice. In contrast to WT, MK4 astrocytes displayed reduced Cx43 hemichannel activity. Pharmacological blockade of Cx43 hemichannels with TAT-Gap19 also significantly decreased infarct volume in WT animals. This study provides novel molecular insights and charts new avenues for therapeutic intervention associated with Cx43 function., (© 2019 Freitas-Andrade et al.)

Many normal tissues undergo age-related drift in DNA methylation, providing a quantitative measure of tissue age. Here, we identify and validate 781 CpG islands (CGI) that undergo significant methylomic drift in 232 normal colorectal tissues and show that these CGI continue to drift in neoplasia while retaining significant correlations across samples. However, compared with normal colon, this drift advanced (∼3-4-fold) faster in neoplasia, consistent with increased cell proliferation during neoplastic progression. The observed drift patterns were broadly consistent with modeled adenoma-to-carcinoma sojourn time distributions from colorectal cancer incidence data. These results support the hypothesis that, beginning with the founder premalignant cell, cancer precursors frequently sojourn for decades before turning into cancer, implying that the founder cell typically arises early in life. At least 77% to 89% of the observed drift variance in distal and rectal tumors was explained by stochastic variability associated with neoplastic progression, whereas only 55% of the variance was explained for proximal tumors. However, gene-CGI pairs in the proximal colon that underwent drift were significantly and primarily negatively correlated with cancer gene expression, suggesting that methylomic drift participates in the clonal evolution of colorectal cancer. Methylomic drift advanced in colorectal neoplasia, consistent with extended sojourn time distributions, which accounts for a significant fraction of epigenetic heterogeneity in colorectal cancer. Importantly, these estimated long-duration premalignant sojourn times suggest that early dietary and lifestyle interventions may be more effective than later changes in reducing colorectal cancer incidence. SIGNIFICANCE: These findings present age-related methylomic drift in colorectal neoplasia as evidence that premalignant cells can persist for decades before becoming cancerous. See related commentary by Sapienza, p. 437 ., (©2018 American Association for Cancer Research.)

The connexin family of channel-forming proteins is present in every tissue type in the human anatomy. Connexins are best known for forming clustered intercellular channels, structurally known as gap junctions, where they serve to exchange members of the metabolome between adjacent cells. In their single-membrane hemichannel form, connexins can act as conduits for the passage of small molecules in autocrine and paracrine signalling. Here, we review the roles of connexins in health and disease, focusing on the potential of connexins as therapeutic targets in acquired and inherited diseases as well as wound repair, while highlighting the associated clinical challenges.

Desmoplakin (DP) is an obligate component of desmosomes, intercellular adhesive junctions that maintain the integrity of the epidermis and myocardium. Mutations in DP can cause cardiac and cutaneous disease, including arrhythmogenic cardiomyopathy (ACM), an inherited disorder that frequently results in deadly arrhythmias. Conduction defects in ACM are linked to the remodeling and functional interference with Cx43-based gap junctions that electrically and chemically couple cells. How DP loss impairs gap junctions is poorly understood. We show that DP prevents lysosomal-mediated degradation of Cx43. DP loss triggered robust activation of ERK1/2-MAPK and increased phosphorylation of S279/282 of Cx43, which signals clathrin-mediated internalization and subsequent lysosomal degradation of Cx43. RNA sequencing revealed Ras-GTPases as candidates for the aberrant activation of ERK1/2 upon loss of DP. Using a novel Ras inhibitor, Ras/Rap1-specific peptidase (RRSP), or K-Ras knockdown, we demonstrate restoration of Cx43 in DP-deficient cardiomyocytes. Collectively, our results reveal a novel mechanism for the regulation of the Cx43 life cycle by DP in cardiocutaneous models., (© 2018 Kam et al.)