Structure of the space of folding protein sequences defined by large language models.

Proteins populate a manifold in the high-dimensional sequence space whose geometrical structure guides their natural evolution. Leveraging recently-developed structure prediction tools based on transformer models, we first examine the protein sequence landscape as defined by an effective energy that is a proxy of sequence foldability. This landscape shares characteristics with optimization challenges encountered in machine learning and constraint satisfaction problems. Our analysis reveals that natural proteins predominantly reside in wide, flat minima within this energy landscape. To investigate further, we employ statistical mechanics algorithms specifically designed to explore regions with high local entropy in relatively flat landscapes. Our findings indicate that these specialized algorithms can identify valleys with higher entropy compared to those found using traditional methods such as Monte Carlo Markov Chains. In a proof-of-concept case, we find that these highly entropic minima exhibit significant similarities to natural sequences, especially in critical key sites and local entropy. Additionally, evaluations through Molecular Dynamics suggests that the stability of these sequences closely resembles that of natural proteins. Our tool combines advancements in machine learning and statistical physics, providing new insights into the exploration of sequence landscapes where wide, flat minima coexist alongside a majority of narrower minima.

Which protocol for prostate biopsies in patients with a positive MRI? Interest of systematic biopsies by sectors.

BACKGROUND: Current prostate biopsy (PBx) protocol for prostate cancer (PCa) diagnosis is to perform systematic biopsies (SBx) combined with targeted biopsies (TBx) in case of positive MRI (i.e. PI-RADS ≥ 3). To assess the utility of performing SBx in combination with TBx, we determined the added value of SBx brought to the diagnosis of PCa according to their sextant location and MRI target characteristics. METHODS: In our local prospectively collected database, we conducted a single-center retrospective study including all patients with a suspicion of PCa, who underwent transrectal ultrasound-guided (TRUS) prostate biopsies (PBx) with a prior MRI and a single lesion classified as PI-RADS ≥ 3. We have characterized the SBx according to their location on MRI: same sextant (S-SBx), adjacent sextant (A-SBx), ipsilateral side (I-SBx) and contralateral side (C-SBx). The added value of SBx and TBx was defined as any upgrading to significant PCa (csPCa) (ISUP ≥2). RESULTS: 371 patients were included in the study. The added value of SBx was 10% overall. Regarding the lesion location and the SBx sextant, the added value of SBx was: 5.1% for S-SBx, 5.4% for A-SBx, 4.9% for I-SBx and 1.9% for C-SBx. The overall added value of SBx was 6.8% for PI-RADS 3 lesions, 14% for PI-RADS 4 lesions and 6.7% for PI-RADS 5 lesions (p = 0.063). The added value of SBx for contralateral side was 1.9% (2/103), 3.1% (5/163) and 0% (0/105) for PI-RADS 3, PI-RADS 4 and PI-RADS 5 lesions, respectively (p = 0,4). The added value of SBx was lower when the number of TBx was higher (OR 0.57; CI 95% 0.37-0.85; p = 0.007). CONCLUSIONS: Our results suggest that the utility of performing SBx in the contralateral lobe toward the MRI lesion was very low, supporting that they might be avoided.

The paradox-breaking panRAF plus SRC family kinase inhibitor, CCT3833, is effective in mutant KRAS-driven cancers.

BACKGROUND: KRAS is mutated in ∼90% of pancreatic ductal adenocarcinomas, ∼35% of colorectal cancers and ∼20% of non-small-cell lung cancers. There has been recent progress in targeting G12CKRAS specifically, but therapeutic options for other mutant forms of KRAS are limited, largely because the complexity of downstream signaling and feedback mechanisms mean that targeting individual pathway components is ineffective. DESIGN: The protein kinases RAF and SRC are validated therapeutic targets in KRAS-mutant pancreatic ductal adenocarcinomas, colorectal cancers and non-small-cell lung cancers and we show that both must be inhibited to block growth of these cancers. We describe CCT3833, a new drug that inhibits both RAF and SRC, which may be effective in KRAS-mutant cancers. RESULTS: We show that CCT3833 inhibits RAF and SRC in KRAS-mutant tumors in vitro and in vivo, and that it inhibits tumor growth at well-tolerated doses in mice. CCT3833 has been evaluated in a phase I clinical trial (NCT02437227) and we report here that it significantly prolongs progression-free survival of a patient with a G12VKRAS spindle cell sarcoma who did not respond to a multikinase inhibitor and therefore had limited treatment options. CONCLUSIONS: New drug CCT3833 elicits significant preclinical therapeutic efficacy in KRAS-mutant colorectal, lung and pancreatic tumor xenografts, demonstrating a treatment option for several areas of unmet clinical need. Based on these preclinical data and the phase I clinical unconfirmed response in a patient with KRAS-mutant spindle cell sarcoma, CCT3833 requires further evaluation in patients with other KRAS-mutant cancers.

Evaluating the effects of polices on nursing care as promoted by the Veneto region: the research protocol and its implications for public health.

Study design: Multi-centre mixed-method study design organised into several phases. Background: The Veneto region has recently defined a set of policies on nursing care by determining the needed amount of daily care in minutes and by initiating a systematic measurement of nursing outcomes; also, with a more recent policy, missed nursing care (MNC) has been established as a process measure of interest. To measure the effect of these policies, a research protocol - aimed at evaluating several end points - has been designed, involving a large target population and hospital units. The aim of this manuscript is to briefly present the research protocol and to discuss the public health implications of its expected end-points. Methods: The endpoints of the protocol are: (a) to describe the frequency of MNC as perceived by nurses; (b) to identify contributing factors; (c) to identify practices adopted in low-occurrence MNC units and to assess the effectiveness of implementing them in units with higher levels of MNC; (d) to explore the relationship between the amount of nursing care provided, MNC, and patient outcomes; and (e) to validate a tool that measures MNC as perceived by patients/caregivers. A total of 3,460 nurses, 5,000 patient/day and 160 nursing coordinators of the medical and surgical units of public hospitals in the Veneto Region will be included. Conclusions: Measuring the association between the amount of nursing care and patient outcomes, as well as evaluating the role of MNC as perceived by nurses and patients in hindering or increasing the risk of some patient outcomes can provide a body of evidence capable of further informing policies in the field, both at the national and at the international level. Moreover, emerging good practices capable of preventing or minimising MNC, sharing and implementing them in other units where high levels of missed care are reported and evaluating their effectiveness, can also inform public health policies.

Reported muscle symptoms during statin treatment amongst Italian dyslipidaemic patients in the real-life setting: the PROSISA Study.

AIM: Statin-associated muscle symptoms (SAMS) are a major determinant of poor treatment adherence and/or discontinuation, but a definitive diagnosis of SAMS is challenging. The PROSISA study was an observational retrospective study aimed to assess the prevalence of reported SAMS in a cohort of dyslipidaemic patients. METHODS: Demographic/anamnestic data, biochemical values and occurrence of SAMS were collected by 23 Italian Lipid Clinics. Adjusted logistic regression was performed to estimate odds ratio (OR) and 95% confidence intervals for association between probability of reporting SAMS and several factors. RESULTS: Analyses were carried out on 16 717 statin-treated patients (mean ± SD, age 60.5 ± 12.0 years; 52.1% men). During statin therapy, 9.6% (N = 1599) of patients reported SAMS. Women and physically active subjects were more likely to report SAMS (OR 1.23 [1.10-1.37] and OR 1.35 [1.14-1.60], respectively), whist age ≥ 65 (OR 0.79 [0.70-0.89]), presence of type 2 diabetes mellitus (OR 0.62 [0.51-0.74]), use of concomitant nonstatin lipid-lowering drugs (OR 0.87 [0.76-0.99]), use of high-intensity statins (OR 0.79 [0.69-0.90]) and use of potential interacting drugs (OR 0.63 [0.48-0.84]) were associated with lower probability of reporting SAMS. Amongst patients reporting SAMS, 82.2% underwent dechallenge (treatment interruption) and/or rechallenge (change or restart of statin therapy), with reappearance of muscular symptoms in 38.4% (3.01% of the whole cohort). CONCLUSIONS: The reported prevalence of SAMS was 9.6% of the whole PROSISA cohort, but only a third of patients still reported SAMS after dechallenge/rechallenge. These results emphasize the need for a better management of SAMS to implement a more accurate diagnosis and treatment re-evaluation.

CSF angiogenin levels in amyotrophic lateral Sclerosis-Frontotemporal dementia spectrum.

Objective: Angiogenin (ANG) is a pro-angiogenic and neurotrophic factor with an important role in stress-induced injury, by promoting neovascularization and neuronal survival. Identification of loss-of-function mutations and evidence of beneficial effect of ANG administration in transgenic SOD1G93A mice have linked ANG to the pathogenesis of Amyotrophic Lateral Sclerosis (ALS), stimulating interest in considering circulating ANG levels as an ALS disease biomarker although robust evidence is still lacking. Aim of our study was to assess differences of ANG levels in the cerebrospinal fluid (CSF) of a large cohort of patients with ALS and frontotemporal dementia (FTD) compared to controls and to explore correlations between ANG content and disease-related clinical variables. Methods: ANG levels were measured in CSF samples using a commercially available ELISA kit in 88 patients affected with ALS and/or FTD and 46 unrelated individuals (control group). Results: ANG levels didn't differ significantly between cases and controls. Patients with FTD or ALS-FTD showed significantly increased CSF concentration of ANG compared to ALS patients without dementia and controls in a multivariate regression model (p < 0.001). No correlations were found in ALS/FTD patients between ANG levels and clinical parameters, including age, presence of C9orf72 repeat expansion, body mass index (BMI). Conclusions: our findings highlight a role of ANG as CSF biomarker useful to identify ALS patients with concurrent FTD and suggest that it should be further explored as potential biomarker for FTD.

Arthropathy in acromegaly: a questionnaire-based estimation of motor disability and its relation with quality of life and work productivity.

PURPOSE: Arthropathy is a common and disabling complication of acromegaly. Since in this condition radiological findings rarely correspond to functional impairment, we elected to quantify in a large cohort of acromegalic patients: the degree of motor disability compared with data from general population, the impact of joint involvement on quality of life and work productivity, and to look for associated factors. METHODS: In 211 acromegalic patients, 131 with controlled disease and 80 with active disease, eight validated scales were used to evaluate the (i) prevalence and distribution of arthropathy, (ii) degree of motor disability and joint symptoms (VAS, AIMS symptoms and WOMAC), (iii) quality of life (AcroQoL and PASQ) and work capability (WPAI:GH) as consequences of joint complications. RESULTS: Using the WOMAC questionnaire, for which population based normative values are available, a significantly higher prevalence and severity of motor disability was detected in acromegalics compared to the general population from literature. The results provided by the different questionnaires turned out to be highly concordant. All measures of motor disability correlated both with impaired quality of life and motor disability and were worse in females and in patients with higher BMI. CONCLUSIONS: The questionnaires VAS, AIMS symptoms, and WOMAC (this latter both as a whole and with its functionality subscale), with their scores, proved to be the most adequate tools to evaluate motor disability and its consequences on both quality of life and work productivity in acromegaly. Female gender and higher BMI are associated with worse articular symptoms.

Prevalence of Polysubstance Abuse and Dual Diagnosis in Patients Admitted to Alcohol Rehabilitation Units for Alcohol-Related Problems in Italy: Changes in 15 Years.

AIMS: Alcohol abuse has long been known as a disease with social and economic burden to society. Given the complex nature of alcohol treatment, it is worthwhile to examine the change over time of patients admitted to residential alcohol abuse rehabilitation units. METHODS: The data were collected from two Italian projects on alcoholics performed in the mid-1990s (ASSALT) and in 2009 (CORRAL), respectively. Categorical variables were considered in terms of absolute and relative frequencies. Comparisons of relative frequencies between groups were assessed by means of Fisher's exact test. Mixed logistic regression models were fitted to CORRAL data to identify the predictors of the probability of being a polysubstance abuser or having a dual diagnosis. The association estimates were reported as adjusted odds ratios and relative 95% confidence intervals. RESULTS: Compared to the mid-1990s, in 2009 patients were older (P= 0.0003), with a higher level of education (P= 0.0204), with fewer comorbidities (liver disease except cirrhosis, P < 0.0001; polyneuropathy, P= 0.0001), more frequently polysubstance abusers (P < 0.0001), affected by dual diagnosis (P < 0.0001). In 2009, the probability of being a polysubstance abuser was higher in younger and in patients with dual psychiatric diagnosis. Female gender and polysubstance abuse were positively associated to the probability of being affected by dual psychiatric diagnosis. CONCLUSIONS: The increment of patients admitted to residential programs for alcohol dependence with polysubstance abuse and/or dual psychiatric diagnoses suggests the need to pay more attention to both psychological/psychiatric interventions and internal medicine/physical rehabilitation. SHORT SUMMARY: The results of this study suggest that further research is needed to identify the best treatment strategy that is safe and effective for the new population of alcoholics.

s-Heptazine oligomers: promising structural models for graphitic carbon nitride.

Graphitic carbon nitride (g-CN) has interesting catalytic properties but is difficult to study due to its structure and how it is produced. In this study, linear s-heptazine oligomers were synthesized to serve as well-defined molecular models for g-CN. Cyclic voltammetry, absorption and emission spectroscopies showed a clear shift of properties towards those of g-CN as the number of heptazine units increased. DFT calculations supported the characterizations, and helped refine the properties observed.

A novel protocol for the one-pot borylation/Suzuki reaction provides easy access to hinge-binding groups for kinase inhibitors.

The one-pot borylation/Suzuki reaction is a very efficient means of accessing cross-coupling products of two aryl-halide partners that generally requires the use of specific catalysts or ligands and/or relatively long reaction times. This new microwave-assisted method provides a quick one-pot borylation/Suzuki reaction protocol that we applied to the synthesis of various bi- or poly-aryl scaffolds, including a variety of aryl and heteroaryl ring systems and the core frameworks of kinase inhibitors vemurafenib and GDC-0879.

Risk of atrial fibrillation among bisphosphonate users: a multicenter, population-based, Italian study.

UNLABELLED: Bisphosphonate treatment is used to prevent bone fractures. A controversial association of bisphosphonate use and risk of atrial fibrillation has been reported. In our study, current alendronate users were associated with a higher risk of atrial fibrillation as compared with those who had stopped bisphosphonate (BP) therapy for more than 1 year. INTRODUCTION: Bisphosphonates are widely used to prevent bone fractures. Controversial findings regarding the association between bisphosphonate use and the risk of atrial fibrillation (AF) have been reported. The aim of this study was to evaluate the risk of AF in association with BP exposure. METHODS: We performed a nested case-control study using the databases of drug-dispensing and hospital discharge diagnoses from five Italian regions. The data cover a period ranging from July 1, 2003 to December 31, 2006. The study population comprised new users of bisphosphonates aged 55 years and older. Patients were followed from the first BP prescription until an occurrence of an AF diagnosis (index date, i.e., ID), cancer, death, or the end of the study period, whichever came first. For the risk estimation, any AF case was matched by age and sex to up to 10 controls from the same source population. A conditional logistic regression was performed to obtain the odds ratio with 95% confidence intervals (CI). The BP exposure was classified into current (<90 days prior to ID), recent (91-180), past (181-364), and distant past (≥365) use, with the latter category being used as a reference point. A subgroup analysis by individual BP was then carried out. RESULTS: In comparison with distant past users of BP, current users of BP showed an almost twofold increased risk of AF: odds ratio (OR) = 1.78 and 95% CI = 1.46-2.16. Specifically, alendronate users were mostly associated with AF as compared with distant past use of BP (OR, 1.97; 95% CI, 1.59-2.43). CONCLUSION: In our nested case-control study, current users of BP are associated with a higher risk of atrial fibrillation as compared with those who had stopped BP treatment for more than 1 year.

Screening for gestational diabetes in the Lombardy region: A population-based study.

AIM: As the treatment of hyperglycaemia during pregnancy with diet or insulin reduces the risk of adverse maternal outcomes and perinatal complications, screening for gestational diabetes mellitus (GDM) is included, albeit to variable extents, in all guidelines of care for pregnant women. The aim of the present investigation was to estimate the proportion of pregnancies screened for GDM in Lombardy between 2007 and 2010, and to identify predictors of screening. METHODS: A retrospective cross-sectional study using regional healthcare utilization databases of Lombardy was conducted. The study included all residents of Lombardy without pregestational diabetes who delivered between 1 January 2007 and 31 December 2010. The proportion of pregnancies with at least one screening test for GDM was calculated, along with the odds ratios and 95% confidence intervals associated with selected covariates for GDM screening. RESULTS: Of the 362,818 pregnancies included in the sample, 30% were screened for GDM. The proportion of pregnancies screened increased slightly from 2007 (27%) to 2010 (33%) and with maternal age (from 28% among women<25 years to 32% among those ≥35 years), and varied widely across local health management organizations (HMOs) of residence (range: 20% to 68%). Socioeconomic indicators (education, immigrant status), obstetric history and prepregnancy hypertension were independent predictors of GDM screening. CONCLUSION: The study finding of a low rate of pregnant women screened for GDM among residents of Lombardy supports the need for programmes to improve training of healthcare professionals, to raise women's awareness of GDM and to eliminate barriers to GDM screening.

Cyclic AMP/PKA-promoted apoptosis: insights from studies of S49 lymphoma cells.

Increases in cyclic AMP (cAMP) are pro-apoptotic in numerous cell types, but the mechanisms of cAMP-promoted apoptosis are poorly defined. We have used murine S49 T-lymphoma cells as a model to provide insight into these mechanisms. Increases in cAMP in wild-type (WT) S49 cells were first noted to kill these cells in the 1970 s, but only in recent years, it was shown that this occurs by the intrinsic (mitochondria-dependent) apoptotic pathway. The apoptotic response does not occur in protein kinase A-null (kin-) clonal variants of WT S49 cells and thus is mediated by protein kinase A (PKA). A second S49 clonal variant, cAMP-Deathless (D-), has PKA activity but lacks cAMP-promoted apoptosis. Apoptosis in WT S49 cells occurs many hours after cAMP/PKA-promoted G1 cell cycle arrest and involves increased expression of Bim, a pro-apoptotic member of the Bcl-2 (B-cell lymphoma-2) family. This increase in Bim expression does not occur in kin- or D- S49 cells and knockdown of Bim blunts cAMP-mediated apoptosis in WT cells. Cytotoxic T lymphocyte antigen-2 also appears to contribute to cAMP/PKA-promoted apoptosis of S49 cells. Based on time-dependent differences in gene expression between WT, D- and kin- S49 cells following incubation with 8-(4-chlorophenylthio)-cAMP, additional genes and proteins are likely involved in this apoptosis. Studies with S49 cells should reveal further insight regarding the mechanisms of cAMP/PKA-promoted cell death, including the identification of proteins that are targets to enhance (e. g., in cancer) or inhibit (e. g., cardiac failure) apoptosis in response to hormones, neurotransmitters, and drugs.

[Alcohol-addiction inpatient: characteristics of patients and rehabilitation program]. / La residenzialità alcologica ospedaliera: caratteristiche dei pazienti e dei percorsi terapeutici/riabilitativi.

AIM: The treatment of alcohol addiction in Italy has had a progressive evolution of therapeutic structures for in-and outpatients. During the last 20 years there had been a crescent presence of short residential treatment facilities (1-6 months) characterized by a high level of medical and psychotherapeutic intervention. About two years ago 12 of them jointed together in an association called CORRAL (COordinamento of Residenzialità Riabilitive Alcologiche). The aim of this study was to describe the socio-demographic medical and other characteristics of the patients coming for this type of treatment and to describe the characteristics of the residential treatment itself including referring and aftercare. METHODS: Two thousand sixty-one hospitalized patients of the 12 rehabilitative alcohol units were examined by using a questionnaire and collecting various sociodemographic variables and clinical diagnosis of the patients. Even it was asked who had referred the patients, the characteristics of the residential treatment and of the planned aftercare. RESULTS: The present residential facilities are mainly distributed in the North of Italy. The typical patient is male, with a high school instruction and with a comorbidity regarding psychiatric and liver disorders. The majority of the patients were referred by the public ambulatory services for addictions (SerT/SerD). The characteristics of the residential treatment were medical, pharmacological and psychotherapeutic interventions with a high intensity and the presence of a general-purpose staff. CONCLUSIONS: This study outlined a model of residential rehabilitation of alcohol disorders characterized by short duration and a complex, intense therapeutic intervention mainly addressed to patients with a severe clinical condition and a low level of social problems. Further research should be useful to understand better which sort of patient characteristics obtain a better clinical result and for that even economically a major effectiveness from this type of residential treatment.

Dietary folates and cancer risk in a network of case-control studies.

BACKGROUND: Folate deficiency leads to DNA damage and inadequate repair, caused by a decreased synthesis of thymidylate and purines. We analyzed the relationship between dietary folate intake and the risk of several cancers. PATIENTS AND METHODS: The study is based on a network of case-control studies conducted in Italy and Switzerland in 1991-2009. The odds ratios (ORs) for dietary folate intake were estimated by multiple logistic regression models, adjusted for major identified confounding factors. RESULTS: For a few cancer sites, we found a significant inverse relation, with ORs for an increment of 100 µg/day of dietary folate of 0.65 for oropharyngeal (1467 cases), 0.58 for esophageal (505 cases), 0.83 for colorectal (2390 cases), 0.72 for pancreatic (326 cases), 0.67 for laryngeal (851 cases) and 0.87 for breast (3034 cases) cancers. The risk estimates were below unity, although not significantly, for cancers of the endometrium (OR = 0.87, 454 cases), ovary (OR = 0.86, 1031 cases), prostate (OR = 0.91, 1468 cases) and kidney (OR = 0.88, 767 cases), and was 1.00 for stomach cancer (230 cases). No material heterogeneity was found in strata of sex, age, smoking and alcohol drinking. CONCLUSIONS: Our data support a real inverse association of dietary folate intake with the risk of several common cancers.

Passively transmitted gp41 antibodies in babies born from HIV-1 subtype C-seropositive women: correlation between fine specificity and protection.

HIV-exposed, uninfected (EUN) babies born to HIV-infected mothers are examples of natural resistance to HIV infection. In this study, we evaluated the titer and neutralizing potential of gp41-specific maternal antibodies and their correlation with HIV transmission in HIV-infected mother-child pairs. Specific gp41-binding and -neutralizing antibodies were determined in a cohort of 74 first-time mother-child pairs, of whom 40 mothers were infected with HIV subtype C. Within the infected mother cohort, 16 babies were born infected and 24 were PCR negative and uninfected at birth (i.e., exposed but uninfected). Thirty-four HIV-uninfected and HIV-unexposed mother-child pairs were included as controls. All HIV-positive mothers and their newborns showed high IgG titers to linear epitopes within the HR1 region and to the membrane-proximal (MPER) domain of gp41; most sera also recognized the disulfide loop immunodominant epitope (IDE). Antibody titers to the gp41 epitopes were significantly lower in nontransmitting mothers (P < 0.01) and in the EUN babies (P < 0.005) than in HIV-positive mother-child pairs. Three domains of gp41, HR1, IDE, and MPER, elicited antibodies that were effectively transmitted to EUN babies. Moreover, in EUN babies, epitopes overlapping the 2F5 epitope (ELDKWAS), but not the 4E10 epitope, were neutralization targets in two out of four viruses tested. Our findings highlight important epitopes in gp41 that appear to be associated with exposure without infection and would be important to consider for vaccine design.

Baclofen promotes alcohol abstinence in alcohol dependent cirrhotic patients with hepatitis C virus (HCV) infection.

Hepatitis C virus (HCV) and alcoholic liver disease (ALD), either alone or in combination, count for more than two thirds of all liver diseases in the Western world. There is no safe level of drinking in HCV-infected patients and the most effective goal for these patients is total abstinence. Baclofen, a GABA(B) receptor agonist, represents a promising pharmacotherapy for alcohol dependence (AD). Previously, we performed a randomized clinical trial (RCT), which demonstrated the safety and efficacy of baclofen in patients affected by AD and cirrhosis. The goal of this post-hoc analysis was to explore baclofen's effect in a subgroup of alcohol-dependent HCV-infected cirrhotic patients. Any patient with HCV infection was selected for this analysis. Among the 84 subjects randomized in the main trial, 24 alcohol-dependent cirrhotic patients had a HCV infection; 12 received baclofen 10mg t.i.d. and 12 received placebo for 12-weeks. With respect to the placebo group (3/12, 25.0%), a significantly higher number of patients who achieved and maintained total alcohol abstinence was found in the baclofen group (10/12, 83.3%; p=0.0123). Furthermore, in the baclofen group, compared to placebo, there was a significantly higher increase in albumin values from baseline (p=0.0132) and a trend toward a significant reduction in INR levels from baseline (p=0.0716). In conclusion, baclofen was safe and significantly more effective than placebo in promoting alcohol abstinence, and improving some Liver Function Tests (LFTs) (i.e. albumin, INR) in alcohol-dependent HCV-infected cirrhotic patients. Baclofen may represent a clinically relevant alcohol pharmacotherapy for these patients.

Reversible alteration of calcium dynamics in cardiomyocytes during acute hypoxia transient in a microfluidic platform.

Heart disease is the leading cause of mortality in western countries. Apart from congenital and anatomical alterations, ischemia is the most common agent causing myocardial damage. During ischemia, a sudden decrease in oxygen concentration alters cardiomyocyte function and compromises cell survival. The calcium handling machinery, which regulates the main functional features of a cardiomyocyte, is heavily compromised during acute hypoxic events. Alterations in calcium dynamics have been linked to both short- and long-term consequences of ischemia, ranging from arrhythmias to heart failure. In this perspective, we aimed at investigating the calcium dynamics in functional cardiomyocytes during the early phase of a hypoxic event. For this purpose, we developed a microfluidic system specifically designed for controlling fast oxygen concentration dynamics through a gas micro-exchanger allowing in line analysis of intracellular calcium concentration by confocal microscopy. Experimental results show that exposure of Fluo-4 loaded neonatal rat cardiomyocytes to hypoxic conditions induced changes in intracellular Ca(2+) transients. Such behavior was reversible and was detected for hypoxic levels below 5% of oxygen partial pressure. The observed changes in Ca(2+) dynamics were mimicked using specific L-type Ca(2+) channel antagonists, suggesting that alterations in calcium channel function occur at low oxygen levels. Reversible alteration in ion channel function, that takes place in response to changes in cellular oxygen, might represent an adaptive mechanism of cardiopreservation during ischemia.

Cyclic AMP is both a pro-apoptotic and anti-apoptotic second messenger.

The second messenger cyclic AMP (cAMP) can either stimulate or inhibit programmed cell death (apoptosis). Here, we review examples of cell types that show pro-apoptotic or anti-apoptotic responses to increases in cAMP. We also show that cells can have both such responses, although predominantly having one or the other. Protein kinase A (PKA)-promoted changes in phosphorylation and gene expression can mediate pro-apoptotic responses, such as in murine S49 lymphoma cells, based on evidence that mutants lacking PKA fail to undergo cAMP-promoted, mitochondria-dependent apoptosis. Mechanisms for the anti-apoptotic response to cAMP likely involve Epac (Exchange protein activated by cAMP), a cAMP-regulated effector that is a guanine nucleotide exchange factor (GEF) for the low molecular weight G-protein, Rap1. Therapeutic approaches that activate PKA-mediated pro-apoptosis or block Epac-mediated anti-apoptotisis may provide a means to enhance cell killing, such as in certain cancers. In contrast, efforts to block PKA or stimulate Epac have the potential to be useful in diseases settings (such as heart failure) associated with cAMP-promoted apoptosis.