Central obesity, body mass index, metabolic syndrome and mortality in Mediterranean breast cancer patients.

Obesity and metabolic disorders have been associated with poor outcomes in non-Mediterranean breast cancer (BC) patients. The purpose of this study was to investigate the prognostic potential of anthropometric variables in patients with early BC living in Southern Mediterranean region of Italy. We enrolled 955 consecutive early BC patients treated in hospitals in Naples between 2009 and 2013 (median follow-up 11.8-year ending 15/09/2022). Body mass index (BMI), waist circumference (WC), waist-to-hip ratio (WHR) and metabolic syndrome (MetS) were collected. All-cause and BC-specific mortality were calculated. At the last day of contact 208 (22%) patients had died, 131 (14%) from BC. High WC (≥ 88 cm) or WHR (> 0.85) and the MetS were significantly associated with moderately increased risk of all-cause mortality (HR=1.39, 1.62, 1.61, respectively). A significant increased risk of BC-specific mortality was found in obese patients, in those with high WC, high WHR and those with MetS (HR=1.72, 1.71, 1.80, 1.81, respectively). Central obesity significantly increased total and BC-specific mortality particularly in pre-menopausal women and in luminal subtypes, while in post-menopause MetS was a stronger risk factor. Obesity and MetS may impair the effectiveness of BC therapies hence active lifestyle interventions are encouraged.

Enhancing identification and treatment of patients with concomitant chronic venous insufficiency and diabetes mellitus. A modified Delphi study from the CODAC (ChrOnic venous disease and Diabetes Advisory Council) group.

BACKGROUND: Chronic venous insufficiency (CVI) and diabetes mellitus (DM) pose significant burdens to patients and healthcare systems. While the two diseases share a number of commonalities in risk factors and pathophysiology, they are often assessed and managed separately. This can lead to a worsening of comorbidities and limitations in a patient's quality of life. This project aims to develop recommendations to enhance the identification and treatment of patients with concomitant CVI and DM. METHODS: Using a modified Delphi method, a panel of experts developed 38 Likert Scale and two multiple choice questions across six key themes. These were used to form an online survey which was disseminated through a convenience sampling approach to CVI and DM healthcare professionals across Europe, Central America, South America, and the Middle East. The threshold for consensus was set at ≥75%. RESULTS: A total of 238 responses were received. 27/38 statements attained >90% agreement, nine of 38 attained between 75-90%, and two failed to meet the threshold (<75%). The awareness around the impact of the two diseases was high, but a gap was highlighted in the identification of patients with concomitant CVI and DM. CONCLUSIONS: The high level of agreement shows that healthcare professionals are aware of the gaps in identification and treatment of patients with concomitant CVI and DM, and of the need to approach this as a combined therapy area. An algorithm is proposed to help the identification of at-risk patients and to provide recommendations on the management of patients with concomitant disease.

Management of Cancer-Related Pain With Intrathecal Drug Delivery: A Systematic Review and Meta-Analysis of Clinical Studies.

BACKGROUND: Despite increased attention paid to assessment and management, pain continues to be a prevalent and undertreated symptom in patients with cancer. Intrathecal drug delivery (IDD) is a therapeutic option that allows targeted delivery of analgesics to the intrathecal space. OBJECTIVE: The aim of this review was to examine the efficacy of managing cancer-related pain with IDD. Secondary objectives included the effects of IDD on systemic opioid use and infection rates. EVIDENCE REVIEW: A systematic search of the literature published between 1990 and 2019 was performed to identify studies evaluating the efficacy and/or safety of IDD with external or implanted pumps in patients with cancer-related pain. Data were extracted and meta-analyses performed to determine the mean changes in pain levels at short-, mid-, and long-term intervals; changes in opioid (oral morphine equivalent [OME]) daily dose; and infection rates. Changes were assessed compared with baseline. FINDINGS: Pain levels were decreased from baseline: On a 0 to 10 scale, mean differences were -4.34 (95% CI [-4.93 to -3.75], p < 0.001) at 4 to 5 weeks; -4.34 (95% CI [-5.07 to -3.62], p < 0.001) at 6 to 12 weeks; and -3.32 (95% CI [-4.60 to -2.04], p < 0.001) at >6 months. Weighted mean OME consumption was reduced by 308.24 (SE = 22.72) mg/d. Weighted mean infection rates were ∼3% for external and implanted pumps. CONCLUSIONS: Meta-analyses show a statistically significant and sustained decrease in cancer pain with IDD, compared with baseline. Systemic opioid consumption was reduced on average by >50% after IDD. Infection rates were comparable with other indications.

Multi-gene panel testing increases germline predisposing mutations' detection in a cohort of breast/ovarian cancer patients from Southern Italy.

Breast cancer is the most common neoplasia in females worldwide, about 10% being hereditary/familial and due to DNA variants in cancer-predisposing genes, such as the highly penetrant BRCA1/BRCA2 genes. However, their variants explain up to 25% of the suspected hereditary/familial cases. The availability of NGS methodologies has prompted research in this field. With the aim to improve the diagnostic sensitivity of molecular testing, a custom designed panel of 44 genes, including also non-coding regions and 5' and 3' UTR regions, was set up. Here, are reported the results obtained in a cohort of 64 patients, including also few males, from Southern Italy. All patients had a positive personal and/or familial history for breast and other cancers, but tested negative to routine BRCA analysis. After obtaining their written informed consent, a genomic DNA sample/patient was used to obtain an enriched DNA library, then analyzed by NGS. Sequencing data analysis allowed the identification of pathogenic variants in 12 of tested patients (19%). Interestingly, MUTYH was the most frequently altered gene, followed by RNASEL, ATM, MSH6, MRE11A, and PALB2 genes. The reported resultsreinforce the need for enlarged molecular testing beyond BRCA genes, at least in patients with a personal and familial history, strongly suggestive for a hereditary/familial form. This gives also a hint to pursue more specific precision oncology therapy.

Microbiome composition indicate dysbiosis and lower richness in tumor breast tissues compared to healthy adjacent paired tissue, within the same women.

BACKGROUND: Breast cancer (BC) is the most common malignancy in women, in whom it reaches 20% of the total neoplasia incidence. Most BCs are considered sporadic and a number of factors, including familiarity, age, hormonal cycles and diet, have been reported to be BC risk factors. Also the gut microbiota plays a role in breast cancer development. In fact, its imbalance has been associated to various human diseases including cancer although a consequential cause-effect phenomenon has never been proven. METHODS: The aim of this work was to characterize the breast tissue microbiome in 34 women affected by BC using an NGS-based method, and analyzing the tumoral and the adjacent non-tumoral tissue of each patient. RESULTS: The healthy and tumor tissues differed in bacterial composition and richness: the number of Amplicon Sequence Variants (ASVs) was higher in healthy tissues than in tumor tissues (p = 0.001). Moreover, our analyses, able to investigate from phylum down to species taxa for each sample, revealed major differences in the two richest phyla, namely, Proteobacteria and Actinobacteria. Notably, the levels of Actinobacteria and Proteobacteria were, respectively, higher and lower in healthy with respect to tumor tissues. CONCLUSIONS: Our study provides information about the breast tissue microbial composition, as compared with very closely adjacent healthy tissue (paired samples within the same woman); the differences found are such to have possible diagnostic and therapeutic implications; further studies are necessary to clarify if the differences found in the breast tissue microbiome are simply an association or a concausative pathogenetic effect in BC. A comparison of different results on similar studies seems not to assess a universal microbiome signature, but single ones depending on the environmental cohorts' locations.

Biomarkers in Triple-Negative Breast Cancer: State-of-the-Art and Future Perspectives.

Triple-negative breast cancer (TNBC) is a heterogeneous group of tumors characterized by aggressive behavior, high risk of distant recurrence, and poor survival. Chemotherapy is still the main therapeutic approach for this subgroup of patients, therefore, progress in the treatment of TNBC remains an important challenge. Data derived from molecular technologies have identified TNBCs with different gene expression and mutation profiles that may help developing targeted therapies. So far, however, only a few of these have shown to improve the prognosis and outcomes of TNBC patients. Robust predictive biomarkers to accelerate clinical progress are needed. Herein, we review prognostic and predictive biomarkers in TNBC, discuss the current evidence supporting their use, and look at the future of this research field.

A multi-gene panel beyond BRCA1/BRCA2 to identify new breast cancer-predisposing mutations by a picodroplet PCR followed by a next-generation sequencing strategy: a pilot study.

By analyzing multiple gene panels, next-generation sequencing is more effective than conventional procedures in identifying disease-related mutations that are useful for clinical decision-making. Here, we aimed to test the efficacy of an 84 genes customized-panel in BRCA1 and BRCA2 mutation-negative patients. Twenty-four patients were enrolled in this study. DNA libraries were prepared using a picodroplet PCR-based approach and sequenced with the MiSeq System. Highly putative pathogenic mutations were identified in genes other than the commonly tested BRCA1/2: 2 pathogenic mutations one in TP53 and one in MUTYH; 2 missense variants in MSH6 and ATM, respectively; 2 frameshift variants in KLLN, and ATAD2, respectively; an intronic variant in ANPEP, and 3 not functionally known variants (a frameshift variant in ATM a nonsense variant in ATM and a missense variant in NFE2L2). Our results show that this molecular screening will increase diagnostic sensitivity leading to a better risk assessment in breast cancer patients and their families. This strategy could also reveal genes that have a higher penetrance for breast and ovarian cancers by matching gene mutation with familial and clinical data, thereby increasing information about hereditary breast and ovarian cancer genetics and improving cancer prevention measures or therapeutic approaches.

Effect of Intrathecal Baclofen on Pain and Quality of Life in Poststroke Spasticity.

Background and Purpose- Intrathecal baclofen (ITB) is an effective treatment for managing patients with severe poststroke spasticity, who can experience continued pain and decline in their quality of life (QoL). SISTERS (Spasticity In Stroke-Randomized Study) was a randomized, controlled, open-label, multicenter, phase 4 study to evaluate ITB therapy versus conventional medical management (CMM) with oral antispastic medications for treatment of poststroke spasticity. Methods- Poststroke patients with spasticity in ≥2 extremities and an Ashworth Scale score of ≥3 in ≥2 affected lower extremity muscle groups were randomized (1:1) to ITB (N=31) or CMM (N=29). Both treatment arms received physiotherapy throughout. The primary outcome was the change in average Ashworth Scale score in the lower extremities of the affected side from baseline to month 6. Here, we report results for secondary outcomes: pain via the Numeric Pain Rating Scale, health-related QoL by the EuroQol-5 dimensional 3 level utility score and health status visual analog scale score, stroke-specific QoL, and patient satisfaction. Analyses were performed on an intention-to-treat basis. Results- We observed significant treatment effects in favor of ITB over CMM for changes from baseline to month 6 in Numeric Pain Rating Scale scores for actual pain (ITB versus CMM: mean, -1.17 [SD, 3.17] versus 0.00 [3.29]; median, -1.00 versus 0.00; P=0.0380) and least pain (mean, -1.61 [2.29] versus 0.24 [3.07]; median, -1.00 versus 0.00; P=0.0136), and EuroQol-5 dimensional 3 level utility scores (mean, +0.09 [0.26] versus +0.01 [0.16]; median, +0.07 versus 0.00; P=0.0197). Between-group differences were not statistically significant for EuroQol-5 dimensional 3 level visual analog scale, stroke-specific QoL summary, or Numeric Pain Rating Scale worst pain scores, although ITB patients showed greater numeric improvements from baseline during follow-up. More ITB patients than CMM patients (73% versus 48%) were satisfied with the spasticity reduction at month 6. Conclusions- These data support that ITB therapy is associated with improvements in pain and QoL in poststroke patients. Clinical Trial Registration- URL: https://www.clinicaltrials.gov . Unique identifier: NCT01032239.

Intrathecal baclofen therapy versus conventional medical management for severe poststroke spasticity: results from a multicentre, randomised, controlled, open-label trial (SISTERS).

BACKGROUND: Intrathecal baclofen (ITB) is a treatment option for patients with severe poststroke spasticity (PSS) who have not reached their therapy goal with other interventions. METHODS: 'Spasticity In Stroke-Randomised Study' (SISTERS) was a randomised, controlled, open-label, multicentre phase IV study to evaluate the efficacy and safety of ITB therapy versus conventional medical management (CMM) with oral antispastic medications for treatment of PSS. Patients with chronic stroke with spasticity in ≥2 extremities and an Ashworth Scale (AS) score ≥3 in at least two affected muscle groups in the lower extremities (LE) were randomised (1:1) to ITB or CMM. Both treatment arms received physiotherapy throughout. The primary outcome was the change in the average AS score in the LE of the affected body side from baseline to month 6. Analyses were performed for all patients as randomised (primary analysis) and all randomised patients as treated (safety analysis). RESULTS: Of 60 patients randomised to ITB (n=31) or CMM (n=29), 48 patients (24 per arm) completed the study. The primary analysis showed a significant effect of ITB therapy over CMM (mean AS score reduction, -0.99 (ITB) vs -0.43 (CMM); Hodges-Lehmann estimate, -0.667(95.1%CI -1.0000 to -0.1667); P=0.0140). More patients reported adverse events while receiving ITB (24/25 patients, 96%; 149 events) compared with CMM (22/35, 63%; 77 events), although events were generally consistent with the known safety profile of ITB therapy. CONCLUSIONS: These data support the use of ITB therapy as an alternative to CMM for treatment of generalised PSS in adults. TRIAL REGISTRATION NUMBER: NCT01032239; Results.

Efficacy and safety of oral baclofen in the management of spasticity: A rationale for intrathecal baclofen.

Oral baclofen has long been a mainstay in the management of spasticity. This review looks at the clinical evidence for the efficacy and safety of oral baclofen in patients with spasticity of any origin or severity, to determine whether there is a rationale for the use of intrathecal baclofen. Results suggest that oral baclofen may be effective in many patients with spasticity, regardless of the underlying disease or severity, and that it is at least comparable with other antispasmodic agents. However, adverse effects, such as muscle weakness, nausea, somnolence and paraesthesia, are common with oral baclofen, affecting between 25% and 75% of patients, and limiting its usefulness. Intrathecal baclofen may be an effective alternative as the drug is delivered directly into the cerebrospinal fluid, thus bypassing the blood-brain barrier and thereby optimizing the efficacy of baclofen while minimizing drug-related side-effects. Intrathecal baclofen is a viable option in patients who experience intolerable side-effects or who fail to respond to the maximum recommended dose of oral baclofen.

A Novel Pathogenic BRCA1 Splicing Variant Produces Partial Intron Retention in the Mature Messenger RNA.

About 10% of all breast cancers arise from hereditary mutations that increase the risk of breast and ovarian cancers; and about 25% of these are associated with the BRCA1 or BRCA2 genes. The identification of BRCA1/BRCA2 mutations can enable physicians to better tailor the clinical management of patients; and to initiate preventive measures in healthy carriers. The pathophysiological significance of newly identified variants poses challenges for genetic counseling. We characterized a new BRCA1 variant discovered in a breast cancer patient during BRCA1/2 screening by next-generation sequencing. Bioinformatic predictions; indicating that the variant is probably pathogenetic; were verified using retro-transcription of the patient's RNA followed by PCR amplifications performed on the resulting cDNA. The variant causes the loss of a canonic donor splice site at position +2 in BRCA1 intron 21; and consequently the partial retention of 156 bp of intron 21 in the patient's transcript; which demonstrates that this novel BRCA1 mutation plays a pathogenetic role in breast cancer. These findings enabled us to initiate appropriate counseling and to tailor the clinical management of this family. Lastly; these data reinforce the importance of studying the effects of sequence variants at the RNA level to verify their potential role in disease onset.

The molecular analysis of BRCA1 and BRCA2: Next-generation sequencing supersedes conventional approaches.

BACKGROUND: Accurate and sensitive detection of BRCA1/2 germ-line mutations is crucial for the clinical management of women affected by breast cancer, for prevention and, notably, also for the identification of at-risk healthy relatives. The most widely used methods for BRCA1/2 molecular analysis are Sanger sequencing, and denaturing high performance liquid chromatography (dHPLC) followed by the Sanger method. However, recent findings suggest that next-generation sequencing (NGS)-based approaches may be an efficient tool for diagnostic purposes. In this context, we evaluated the effectiveness of NGS for BRCA gene analysis compared with dHPLC/Sanger sequencing. METHODS: Seventy women were screened for BRCA1/2 mutations by both dHPLC/Sanger sequencing and NGS, and the data were analyzed using a bioinformatic pipeline. RESULTS: Sequence data analysis showed that NGS is more sensitive in detecting BRCA1/2 variants than the conventional procedure, namely, dHPLC/Sanger. CONCLUSION: Next-generation sequencing is more sensitive, faster, easier to use and less expensive than the conventional Sanger method. Consequently, it is a reliable procedure for the routine molecular screening of the BRCA1/2 genes.

Nuclear factor I-C regulates TGF-{beta}-dependent hair follicle cycling.

Skin appendages such as teeth and hair share several common signaling pathways. The nuclear factor I C (NFI-C) transcription factor has been implicated in tooth development, but a potential role in hair growth had not been assessed. In this study we found that NFI-C regulates the onset of the hair growth cycle. NFI-C(-/-) mice were delayed in the transition from the telogen to anagen phase of the hair follicle cycle after either experimental depilation or spontaneous hair loss. Lack of NFI-C resulted in delayed induction of the sonic hedgehog, Wnt5a, and Lef1 gene expression, which are key regulators of the hair follicle growth initiation. NFI-C(-/-) mice also showed elevated levels of transforming growth factor ß1 (TGF-ß1), an inhibitor of keratinocyte proliferation, and of the cell cycle inhibitor p21 at telogen. Reduced expression of Ki67, a marker of cell proliferation, was noted at the onset of anagen, indicating impaired activation of the hair progenitor cells. These findings implicate NFI-C in the repression of TGF-ß1 signaling during telogen stage, resulting in the delay of progenitor cell proliferation and hair follicle regeneration in NFI-C-deficient mice. Taken together with prior observations, these findings also designate NFI-C as a regulator of adult progenitor cell proliferation and of postnatal tissue growth or regeneration.

Nuclear factor I-C links platelet-derived growth factor and transforming growth factor beta1 signaling to skin wound healing progression.

Transforming growth factor beta (TGF-beta) and platelet-derived growth factor A (PDGFAlpha) play a central role in tissue morphogenesis and repair, but their interplay remain poorly understood. The nuclear factor I C (NFI-C) transcription factor has been implicated in TGF-beta signaling, extracellular matrix deposition, and skin appendage pathologies, but a potential role in skin morphogenesis or healing had not been assessed. To evaluate this possibility, we performed a global gene expression analysis in NFI-C(-/-) and wild-type embryonic primary murine fibroblasts. This indicated that NFI-C acts mostly to repress gene expression in response to TGF-beta1. Misregulated genes were prominently overrepresented by regulators of connective tissue inflammation and repair. In vivo skin healing revealed a faster inflammatory stage and wound closure in NFI-C(-/-) mice. Expression of PDGFA and PDGF-receptor alpha were increased in wounds of NFI-C(-/-) mice, explaining the early recruitment of macrophages and fibroblasts. Differentiation of fibroblasts to contractile myofibroblasts was also elevated, providing a rationale for faster wound closure. Taken together with the role of TGF-beta in myofibroblast differentiation, our results imply a central role of NFI-C in the interplay of the two signaling pathways and in regulation of the progression of tissue regeneration.

Small-molecule inhibitor of USP7/HAUSP ubiquitin protease stabilizes and activates p53 in cells.

Deregulation of the ubiquitin/proteasome system has been implicated in the pathogenesis of many human diseases, including cancer. Ubiquitin-specific proteases (USP) are cysteine proteases involved in the deubiquitination of protein substrates. Functional connections between USP7 and essential viral proteins and oncogenic pathways, such as the p53/Mdm2 and phosphatidylinositol 3-kinase/protein kinase B networks, strongly suggest that the targeting of USP7 with small-molecule inhibitors may be useful for the treatment of cancers and viral diseases. Using high-throughput screening, we have discovered HBX 41,108, a small-molecule compound that inhibits USP7 deubiquitinating activity with an IC(50) in the submicromolar range. Kinetics data indicate an uncompetitive reversible inhibition mechanism. HBX 41,108 was shown to affect USP7-mediated p53 deubiquitination in vitro and in cells. As RNA interference-mediated USP7 silencing in cancer cells, HBX 41,108 treatment stabilized p53, activated the transcription of a p53 target gene without inducing genotoxic stress, and inhibited cancer cell growth. Finally, HBX 41,108 induced p53-dependent apoptosis as shown in p53 wild-type and null isogenic cancer cell lines. We thus report the identification of the first lead-like inhibitor against USP7, providing a structural basis for the development of new anticancer drugs.

Combination of active and inactive siRNA targeting the mitotic kinesin Eg5 impairs silencing efficiency in several cancer cell lines.

Gene silencing by RNA interference (RNAi) has proven to be a powerful tool for investigating gene function in mammalian cells. Combination of several short interfering RNA (siRNA) targeting the same gene is commonly used to improve RNA interference. However, in contrary to the well-described mechanism of RNAi, efficiency of single siRNA compared to pool remains poorly documented. We addressed this issue using several active and inactive siRNA targeting Eg5, a kinesin-related motor involved in mitotic spindle assembly. These siRNA, used alone or in combination, were tested for their silencing efficiency in several cancer cell lines. Here we show that presence of inactive Eg5 siRNA in a pool dramatically decreases knockdown efficacy in a cell line- and dose-dependent manner. Lack of inhibition by unrelated siRNA suggests that a competition may occur during siRNA incorporation into RNA-induced silencing complexes (RISCs) along with the target mRNA. Altogether, our results, which need to be confirmed with additional inactive siRNA, indicate that combination of siRNA may not increase but instead decrease silencing efficiency.

Differentiating the effects of Cx36 and E-cadherin for proper insulin secretion of MIN6 cells.

Connexins have been implicated in many cell functions, even though in most cases it is still unclear whether these functions may actually be mediated by other proteins that are secondarily affected by connexin changes. Secretory systems provide useful models in which to tackle this central question. Primary pancreatic beta-cells and insulin-producing lines are connected by gap junction channels made of Cx36. Using stable transfection of an antisense Cx36 cDNA, we have previously obtained clones of MIN6 cells that featured a markedly reduced expression of Cx36 and impaired insulin secretion. Here, we first show that this change also resulted in loss of E-cadherin and occludin expression, thus preventing the attribution of the secretory defects to a specific type of membrane protein. To investigate this question, we have now restored the expression of either Cx36 or E-cadherin in the Cx36 antisense-transfected cells. We show that a lentivirus-mediated transduction efficiently restored Cx36 expression in MIN6 cells and allowed for expression of variable levels of this protein. We further document that adequate but not excessive levels of Cx36 allowed for recover of normal insulin secretion in response to various secretagogues. Finally, we demonstrate that restoration of normal E-cadherin expression was not able to achieve the same secretory correction. The data demonstrate that Cx36, but not E-cadherin, is necessary to control specific steps of beta-cell secretion.

Lentivirus-mediated transduction of connexin cDNAs shows level- and isoform-specific alterations in insulin secretion of primary pancreatic beta-cells.

We have generated novel lentiviral vectors to integrate various connexin cDNAs into primary, non-dividing cells. We have used these vectors to test whether proper control of insulin secretion depends on a specific connexin isoform and/or on its level of expression. We have observed that transduced connexin32, connexin36 and connexin43 were expressed by primary adult beta-cells at membrane interfaces, were packed into typical gap junction plaques and formed functional channels that allowed a variable coupling, depending on the type and level of connexin expressed. The infected cells spontaneously reaggregated into three-dimensional pseudo-islet organs that could be maintained in culture. We have found that pseudo-islets made by cells transduced with either GFP- or connexin43-expressing lentivirus released insulin in response to various secretagogues similarly to controls. By contrast, pseudo-islets made by cells expressing connexin32, a connexin exogenous to pancreatic islets, or over-expressing connexin36, the endogenous islet connexin, featured a marked decrease in the secretory response to glucose. The data show: (1) that lentiviral vectors allow stable modulation of various connexin in primary, non-proliferating cells; (2) that specific connexin isoforms affect insulin secretion differently; and (3) that adequate levels of coupling via connexin36 channels are required for proper beta-cell function.

Connexin 36 controls synchronization of Ca2+ oscillations and insulin secretion in MIN6 cells.

Cx36 is the predominant connexin isoform expressed by pancreatic beta-cells. However, little is known about the role of this protein in the functioning of insulin-secreting cells. To address this question, we searched for a cell line expressing Cx36 and having glucose-induced insulin secretion comparable to that of primary beta-cells. By evaluating Cx36 expression in MIN6, betaTC3, RIN2A, INS1, and HIT cell lines, which differ in their sensitivity to glucose, we found that wild-type MIN6 cells fit these requirements. Therefore, we stably transfected MIN6 cells with a cDNA coding for a Cx36 antisense sequence to study the role of Cx36 in these cells. Independent clones of MIN6 cells were obtained that had a markedly reduced Cx36 expression. Loss of Cx36 decreased functional gap junctional conductance in these clones. This alteration impaired the synchronization of glucose-induced [Ca(2+)](i) oscillations and insulin secretion in response to glucose, to secretagogues that increase [cAMP](i), and to depolarizing conditions. These data provide the first evidence that Cx36-made channels 1) mediate functional coupling in MIN6 cells, 2) provide for synchronous [Ca(2+)](i) oscillations, and 3) are necessary for proper insulin secretion in response to metabolizable and nonmetabolizable secretagogues.