Photodynamic therapy and adapalene in dermatology: synergistic effects in immortal human keratinocytes

We compared the effects of methyl levulinate (MAL)/photodynamic therapy (PDT) and adapalene, evaluated singly, versus combination therapy on HaCaT keratinocytes. Our aim was to determine whether the additive/synergistic outcomes of combination treatment were such that doses of each component could be reduced without affecting treatment efficacy. The analysis of data from specific PDT/adapalene combinations results indicating effects ranging from additive to clearly synergistic. We first evaluated the effects on cell viability, cell cycle and protein expression profiles of each individual treatment, then we tried to understand the reasons and the mechanisms whereby cells under combined treatment move more efficiently to death. Although we observed therapeutic strengthening by increasing either drug doses or light fluences, reinforcement was particularly marked in combinations in which PDT was predominant. Thus, if PDT is appropriately tuned, the dose of the drug can be reduced without compromising the therapeutic response. As both photodynamic therapy and adapalene have been and are therapeutic approaches to treat different dermatological pathologies such as acne, actinic keratosis etc., our data suggest that the adapalene-PDT combination may have important spin-off in clinical dermatology as a strategy to tackle this nasty condition

Separase prevents genomic instability by controlling replication fork speed

Proper chromosome segregation is crucial for preserving genomic integrity, and errors in this process cause chromosome mis-segregation, which may contribute to cancer development. Sister chromatid separation is triggered by Separase, an evolutionary conserved protease that cleaves the cohesin complex, allowing the dissolution of sister chromatid cohesion. Here we provide evidence that Separase participates in genomic stability maintenance by controlling replication fork speed. We found that Separase interacted with the replication licensing factors MCM2-7, and genome-wide data showed that Separase co-localized with MCM complex and cohesin. Unexpectedly, the depletion of Separase increased the fork velocity about 1.5-fold and caused a strong acetylation of cohesin's SMC3 subunit and altered checkpoint response. Notably, Separase silencing triggered genomic instability in both HeLa and human primary fibroblast cells. Our results show a novel mechanism for fork progression mediated by Separase and thus the basis for genomic instability associated with tumorigenesis

Separase prevents genomic instability by controlling replication fork speed

Proper chromosome segregation is crucial for preserving genomic integrity, and errors in this process cause chromosome mis-segregation, which may contribute to cancer development. Sister chromatid separation is triggered by Separase, an evolutionary conserved protease that cleaves the cohesin complex, allowing the dissolution of sister chromatid cohesion. Here we provide evidence that Separase participates in genomic stability maintenance by controlling replication fork speed. We found that Separase interacted with the replication licensing factors MCM2-7, and genome-wide data showed that Separase co-localized with MCM complex and cohesin. Unexpectedly, the depletion of Separase increased the fork velocity about 1.5-fold and caused a strong acetylation of cohesin's SMC3 subunit and altered checkpoint response. Notably, Separase silencing triggered genomic instability in both HeLa and human primary fibroblast cells. Our results show a novel mechanism for fork progression mediated by Separase and thus the basis for genomic instability associated with tumorigenesis

Burnout in cardiac anesthesiologists. results from a national survey in italy

Objective: There is increasing burnout incidence among medical disciplines, and physicians working in emergency settings seem at higher risk. Cardiac anesthesiology is a stressful anesthesiology subspecialty dealing with high-risk patients. The authors hypothesized a high risk of burnout in cardiac anesthesiologists. Design: National survey conducted on burnout Setting: Italian cardiac centers. Participants: Cardiac anesthesiologists. Interventions: The authors administered via email an anonymous questionnaire divided into 3 parts. The first 2 parts evaluated workload and private life. The third part consisted of the Maslach Burnout Inventory test with its 3 constituents: high emotional exhaustion, high depersonalization, and low personal accomplishment. Measurements and Main Results: The authors measured the prevalence and risk of burnout through the Maslach Burnout Inventory questionnaire and analyzed factors influencing burnout. Among 670 contacts from 71 centers, 382 cardiac anesthesiologists completed the survey (57%). The authors found the following mean Maslach Burnout Inventory values: 14.5 ± 9.7 (emotional exhaustion), 9.1 ± 7.1 (depersonalization), and 33.7 ± 8.9 (personal accomplishment). A rate of 34%, 54%, and 66% of respondents scored in “high” or “moderate-high” risk of burnout (emotional exhaustion, depersonalization, and personal accomplishment, respectively). The authors found that, if offered to change subspecialty, 76% of respondents would prefer to remain in cardiac anesthesiology. This preference and parenthood were the only 2 investigated factors with a protective effect against all components of burnout. Significantly lower burnout scores were found in more experienced anesthesiologists. Conclusion: A relatively high incidence of burnout was found in cardiac anesthesiologists, especially regarding high depersonalization and low personal accomplishment. Nonetheless, most of the respondents would choose to remain in cardiac anesthesiology

Type I Collagen Suspension Induces Neocollagenesis and Myodifferentiation in Fibroblasts In Vitro

The ability of a collagen-based matrix to support cell proliferation, migration, and infiltration has been reported; however, the direct effect of an aqueous collagen suspension on cell cultures has not been studied yet. In this work, the effects of a high-concentration aqueous suspension of a micronized type I equine collagen (EC-I) have been evaluated on a normal mouse fibroblast cell line. Immunofluorescence analysis showed the ability of EC-I to induce a significant increase of type I and III collagen levels, parallel with overexpression of crucial proteins in collagen biosynthesis, maturation, and secretion, prolyl 4-hydroxylase (P4H) and heat shock protein 47 (HSP47), as demonstrated by western blot experiments. The treatment led, also, to an increase of α-smooth muscle actin (α-SMA) expression, evaluated through western blot analysis, and cytoskeletal reorganization, as assessed by phalloidin staining. Moreover, scanning electron microscopy analysis highlighted the appearance of plasma membrane extensions and blebbing of extracellular vesicles. Altogether, these results strongly suggest that an aqueous collagen type I suspension is able to induce fibroblast myodifferentiation. Moreover, our findings also support in vitro models as a useful tool to evaluate the effects of a collagen suspension and understand the molecular signaling pathways possibly involved in the effects observed following collagen treatment in vivo

Low grade endotoxemia and oxidative stress in offspring of patients with early myocardial infarction

Background and aims: Offspring of patients with early myocardial infarction are at higher cardiovascular risk, but the underlying physio-pathological mechanism is unclear. NADPH oxidase-type 2 (NOX-2) plays a pivotal role as mediator of oxidative stress and could be involved in activating platelets in these patients. Furthermore, altered intestinal permeability and serum lipopolysaccharide (LPS) could be a trigger to promote NOX-2 activation and platelet aggregation. This study aims to evaluate the behavior of low grade endotoxemia, oxidative stress and platelet activation in offspring of patients with early myocardial infarction. Methods: We enrolled, in a cross-sectional study, 46 offspring of patients with early myocardial infarction and 86 healthy subjects (HS). LPS levels and gut permeability (assessed by zonulin), oxidative stress (assessed by serum NOX-2-derived peptide (sNOX2-dp) release, hydrogen peroxide (H2O2) production and isoprostanes), serum nitric oxide (NO) bioavailability and platelet activation (by serum thromboxane B2 (TXB2) and soluble P-Selectin (sP-Selectin)) were analyzed. Results: Compared to HS, offspring of patients with early myocardial infarction had higher values of LPS, zonulin, serum isoprostanes, sNOX2-dp H2O2, TXB2, p-selectin and lower NO bioavailability. Logistic regression analysis showed that the variables associated with offspring of patients with early myocardial infarction were LPS, TXB2 and isoprostanes. The multiple linear regression analysis confirmed that serum NOX-2, isoprostanes, p-selectin and H2O2 levels were significantly associated to LPS. Furthermore, serum LPS, isoprostanes and TXB2 levels were significantly associated with sNOX-2-dp. Conclusions: Offspring of patients with early myocardial infarction have a low grade endotoxemia that could generate oxidative stress and platelet activation increasing their cardiovascular risk. Future studies are needed to understand the role of dysbiosis in this population

Identification of Sclerostin as a Putative New Myokine Involved in the Muscle-to-Bone Crosstalk

Bone and muscle have been recognized as endocrine organs since they produce and secrete “hormone-like factors” that can mutually influence each other and other tissues, giving rise to a “bone–muscle crosstalk”. In our study, we made use of myogenic (C2C12 cells) and osteogenic (2T3 cells) cell lines to investigate the effects of muscle cell-produced factors on the maturation process of osteoblasts. We found that the myogenic medium has inhibitory effects on bone cell differentiation and we identified sclerostin as one of the myokines produced by muscle cells. Sclerostin is a secreted glycoprotein reportedly expressed by bone/cartilage cells and is considered a negative regulator of bone growth due to its role as an antagonist of the Wnt/β-catenin pathway. Given the inhibitory role of sclerostin in bone, we analyzed its expression by muscle cells and how it affects bone formation and homeostasis. Firstly, we characterized and quantified sclerostin synthesis by a myoblast cell line (C2C12) and by murine primary muscle cells by Western blotting, real-time PCR, immunofluorescence, and ELISA assay. Next, we investigated in vivo production of sclerostin in distinct muscle groups with different metabolic and mechanical loading characteristics. This analysis was done in mice of different ages (6 weeks, 5 and 18 months after birth) and revealed that sclerostin expression is dynamically modulated in a muscle-specific way during the lifespan. Finally, we transiently expressed sclerostin in the hind limb muscles of young mice (2 weeks of age) via in vivo electro-transfer of a plasmid containing the SOST gene in order to investigate the effects of muscle-specific overproduction of the protein. Our data disclosed an inhibitory role of the muscular sclerostin on the bones adjacent to the electroporated muscles. This observation suggests that sclerostin released by skeletal muscle might synergistically interact with osseous sclerostin and potentiate negative regulation of osteogenesis possibly by acting in a paracrine/local fashion. Our data point out a role for muscle as a new source of sclerostin.Bone and muscle have been recognized as endocrine organs since they produce and secrete “hormone-like factors” that can mutually influence each other and other tissues, giving rise to a “bone–muscle crosstalk”. In our study, we made use of myogenic (C2C12 cells) and osteogenic (2T3 cells) cell lines to investigate the effects of muscle cell-produced factors on the maturation process of osteoblasts. We found that the myogenic medium has inhibitory effects on bone cell differentiation and we identified sclerostin as one of the myokines produced by muscle cells. Sclerostin is a secreted glycoprotein reportedly expressed by bone/cartilage cells and is considered a negative regulator of bone growth due to its role as an antagonist of the Wnt/β-catenin pathway. Given the inhibitory role of sclerostin in bone, we analyzed its expression by muscle cells and how it affects bone formation and homeostasis. Firstly, we characterized and quantified sclerostin synthesis by a myoblast cell line (C2C12) and by murine primary muscle cells by Western blotting, real-time PCR, immunofluorescence, and ELISA assay. Next, we investigated in vivo production of sclerostin in distinct muscle groups with different metabolic and mechanical loading characteristics. This analysis was done in mice of different ages (6 weeks, 5 and 18 months after birth) and revealed that sclerostin expression is dynamically modulated in a muscle-specific way during the lifespan. Finally, we transiently expressed sclerostin in the hind limb muscles of young mice (2 weeks of age) via in vivo electro-transfer of a plasmid containing the SOST gene in order to investigate the effects of muscle-specific overproduction of the protein. Our data disclosed an inhibitory role of the muscular sclerostin on the bones adjacent to the electroporated muscles. This observation suggests that sclerostin released by skeletal muscle might synergistically interact with osseous sclerostin and potentiate negative regulation of osteogenesis possibly by acting in a paracrine/local fashion. Our data point out a role for muscle as a new source of sclerostin

Association between PaO2/FiO2 ratio and thrombotic events in COVID-19 patients

PaO2/FiO(2) (P/F ratio) is considered a marker of hypoxia/hypoxemia and mortality. Several prothrombotic changes are associated with the decrease of P/F ratio. The role of P/F ratio in patients with arterial and venous thrombosis remains unclear. The aim of this study was to assess in patients with coronavirus disease 2019 (COVID-19), the association between P/F ratio and arterial/venous thrombosis. One thousand and four hundred and six COVID-19 patients were recruited; 289 (21%) patients had P/F ratio < 200 and 1117 (79%) >= 200. Compared to the patients with P/F ratio >= 200, those with P/F ratio < 200 were older and with higher levels of glycemia, D-dimer and lower levels of albumin. Multiple linear regression analysis showed that albumin (standardized coefficient beta: 0.156; SE: 0.001; p = 0.0001) and D-dimer (standardized coefficient beta: -0.135; SE: 0.0001; p = 0.0001) were associated with P/F ratio. During the hospitalization 159 patients were transferred in intensive care unit (ICU), 253 patients died, 156 patients had arterial or venous thrombotic events. A bivariate logistic analysis was performed to analyze the predictors of thrombosis in COVID-19 patients; P/F ratio < 200 (Odds Ratio: [OR] 1.718, 95% Confidence Interval [CI] 1.085-2.718, p = 0.021), albumin (OR 1.693, 95% CI 1.055-2.716, p = 0.029), D-dimer (OR 3.469, 95% CI 2.110-5.703, p < 0.0001), coronary artery disease (CAD) (OR 1.800, 95% CI 1.086-2.984, p = 0.023) and heart failure (OR 2.410 95% CI 1.385-4.193, p = 0.002) independently predicted thrombotic events in this population. This study suggests that the P/F ratio is associated with thrombotic events by promoting a hypercoagulation state in patients hospitalized for COVID-19

Prognostic or predictive value of circulating cytokines and angiogenic factors for initial treatment of multiple myeloma in the GIMEMA MM0305 randomized controlled trial

Abstract Background Several new drugs are approved for treatment of patients with multiple myeloma (MM), but no validated biomarkers are available for the prediction of a clinical outcome. We aimed to establish whether pretreatment blood and bone marrow plasma concentrations of major cytokines and angiogenic factors (CAFs) of patients from a phase 3 trial of a MM treatment could have a prognostic and predictive value in terms of response to therapy and progression-free and overall survival and whether these patients could be stratified for their prognosis. Methods Blood and bone marrow plasma levels of Ang-2, FGF-2, HGF, VEGF, PDGF-β, IL-8, TNF-α, TIMP-1, and TIMP-2 were determined at diagnosis in MM patients enrolled in the GIMEMA MM0305 randomized controlled trial by an enzyme-linked immunosorbent assay (ELISA). These levels were correlated both reciprocally and with the type of therapy and patients’ characteristics and with a group of non-MM patients as controls. Results No significant differences were detected between the blood and bone marrow plasma levels of angiogenic cytokines. A cutoff for each CAF was established. The therapeutic response of patients with blood plasma levels of CAFs lower than the cutoff was better than the response of those with higher levels in terms of percentage of responding patients and quality of response. Conclusion FGF-2, HGF, VEGF, and PDGF-β plasma levels at diagnosis have predictive significance for response to treatment. The stratification of patients based on the levels of CAFs at diagnosis and their variations after therapy is useful to characterize different risk groups concerning outcome and response to therapy. Trial registration Clinical trial information can be found at the following link: NCT0106317

The Impact of the COVID-19 Pandemic on Women's Perinatal Mental Health: Preliminary Data on the Risk of Perinatal Depression/Anxiety from a National Survey in Italy

Increasing evidence suggests that during the COVID-19 pandemic, anxiety and depression during the perinatal period increased. The aim of the study is to estimate the prevalence of risk for both maternal depression and anxiety among women attending 18 healthcare centres in Italy during the SARS-COV-2 pandemic and to investigate the psychosocial risks and protective factors associated. It was divided into a retrospective phase (2019, 2020, and the first nine months of 2021) and a prospective phase (which began in November 2021 and it is still ongoing), which screened 12,479 and 2349 women, respectively, for a total of 14,828 women in the perinatal period. To evaluate the risk of anxiety and depression, the General Anxiety Disorder-7 (GAD-7), the Edinburgh Postnatal Depression Scale (EPDS), and an ad hoc form were used to collect sociodemographic variables. In the prospective study, the average age of the women is 31 (range 18-52) years. Results showed that the percentage of women who had EPDS score ≥9 increased from 11.6% in 2019 to 25.5% in the period ranging from November 2021 to April 2022. In logistic regression models, the variables associated with the risk of depression at a level ≤0.01 include having economic problems (OR 2.16) and not being able to rely on support from relatives or friends (OR 2.36). Having the professional status of the housewife is a lower risk (OR 0.52). Those associated with the risk of anxiety include being Italian (OR 2.97), having an education below secondary school level (OR 0.47), having some or many economic problems (OR 2.87), being unable to rely on support from relatives or friends (OR 2.48), and not having attended an antenatal course (OR 1.41). The data from this survey could be useful to determine the impact of the SARS-COV-2 pandemic on women and to establish a screening program with common and uniformly applied criteria which are consistent with national and international women's mental health programs