Bizonal cardiac engineered tissues with differential maturation features in a mid-throughput multimodal bioreactor

Functional three-dimensional (3D) engineered cardiac tissue (ECT) models are essential for effective drug screening and biological studies. Application of physiological cues mimicking those typical of the native myocardium is known to promote the cardiac maturation and functionality in vitro. Commercially available bioreactors can apply one physical force type at a time and often in a restricted loading range. To overcome these limitations, a millimetric-scalemicroscope-integrated bioreactor was developed to deliver multiple biophysical stimuli to ECTs. In this study, we showed that the single application of auxotonic loading (passive) generated a bizonal ECT with a unique cardiac maturation pattern. Throughout the statically cultured constructs and in the ECT region exposed to high passive loading, cardiomyocytes predominantly displayed a round morphology and poor contractility ability. The ECT region with a low passive mechanical stimulation instead showed both rat- and human-origin cardiac cell maturation and organization, as well as increased ECT functionality

Combination of antibodies directed against different ErbB3 surface epitopes prevents the establishment of resistance to BRAF/MEK inhibitors in melanoma

Patients with metastatic melanoma bearing V600 mutations in BRAF oncogene clinically benefit from the treatment with BRAF inhibitors alone or in combination with MEK inhibitors. However, a limitation to such treatment is the occurrence of resistance. Tackling the adaptive changes helping cells survive from drug treatment may offer new therapeutic opportunities. Very recently the ErbB3 receptor has been shown to act as a central node promoting survival of BRAF mutated melanoma. In this paper we first demonstrate that ErbB3/AKT hyperphosphorylation occurs in BRAF mutated melanoma cell lines following exposure to BRAF and/or MEK inhibitors. This strongly correlates with increased transcriptional activation of its ligand neuregulin. Anti-ErbB3 antibodies impair the establishment of de novo cell resistance to BRAF inhibition in vitro. In order to more potently ablate ErbB3 activity we used a combination of two anti-ErbB3 antibodies directed against distinct epitopes of its extracellular domain. These two antibodies in combo with BRAF/MEK inhibitors potently inhibit in vitro cell growth and tumor regrowth after drug withdrawal in an in vivo xenograft model. Importantly, residual tumor masses from mice treated by the antibodies and BRAF/ERK inhibitors combo are characterized almost exclusively by large necrotic areas with limited residual areas of tumor growth. Taken together, our findings support the concept that triple therapy directed against BRAF/MEK/ErbB3 may be able to provide durable control of BRAF mutated metastatic melanoma

Activation of an early feedback survival loop involving phospho-ErbB3 is a general response of melanoma cells to RAF/MEK inhibition and is abrogated by anti-ErbB3 antibodies

BACKGROUND: Treatment of advanced melanoma has been improved with the advent of the BRAF inhibitors. However, a limitation to such treatment is the occurrence of resistance. Several mechanisms have been identified to be responsible for the development of resistance, either MEK-dependent or MEK-independent. In order to overcome resistance due to reactivation of MEK signaling, MEK inhibitors are being clinically developed with promising results. However, also in this case resistance inevitably occurs. It has been recently reported that ErbB3, a member of the EGFR receptor family, may be involved in the establishment of drug resistance. METHODS: Three melanoma cell lines were tested: LOX IMVI (BRAF V600E), MST-L (BRAF V600R) and WM266 (BRAF V600D). Phosphorylation of Receptor Tyrosine Kinases (RTKs) was assessed by an RTK array. Western blot analysis was performed on total protein extracts using anti-ErbB3, anti-AKT and anti-ERK 1/2 antibodies. The expression of neuregulin after vemurafenib treatment was assessed by Real Time PCR and Western blotting. The growth inhibitory effects of vemurafenib, GSK1120212b and/or anti-ErbB3 mAbs were evaluated by in vitro colony formation assays. RESULTS: In the present study we demonstrate that ErbB3 is the main RTK undergoing rapidly hyperphosphorylation upon either treatment with a BRAF inhibitor or with a MEK inhibitor in a panel of melanoma cell lines harboring a variety of V600BRAF mutations and that this results in a strong activation of phospho-AKT. Importantly, ErbB3 activation is fully abrogated by the simultaneous use of anti-ErbB3 monoclonal antibodies, which are also shown to potently synergize with BRAF inhibitors in the inactivation of both AKT and ERK pathways and in the inhibition of melanoma cell growth. We show that upregulation of phospho-ErbB3 is due to an autocrine loop involving increased transcription and production of neuregulin by melanoma cells. CONCLUSIONS: On the basis of these results, we propose that initial co-treatment with BRAF and/or MEK inhibitors and anti-ErbB3 antibodies should be pursued as a strategy to reduce the ErbB3-dependent feedback survival mechanism and enhance duration of clinical response

Testimonies and experiences of patients awaiting transplant and transplanted patients in Italy: a survey aiming to understand their needs

Introduction. Yersinia enterocolitica (Ye) species is divided into 6 biotypes (BT), 1A, 1B, 2, 3, 4, 5 classified based on biochemical reactions and about 70 serotypes, classified based on the structure of the lipopolysaccharide O-antigen. The BT1A is considered non-pathogenic, while the BT 1B-5 are considered pathogenic. Methods. Evaluate the distribution of eleven chromosomal and plasmid virulence genes, ail, ystA, ystB, myfA, hreP, fes, fepD, ymoA, sat, virF and yadA, in 87 Ye strains isolated from food, animals and humans, using two SYBR Green real-time PCR platforms. Results. The main results showed the presence of the ail and ystA genes in all the pathogenic bioserotypes analyzed. The ystB, on the other hand, was identified in all non-pathogenic  strains biotype 1A. The target fes, fepD, sat and hreP were found in both pathogenic biotypes and in BT1A strains. The myfA gene was found in all pathogenic biotype and in some Ye BT1A strains. The virF and yadA plasmid genes were mainly detected in bioserotype 4/O:3 and 2/O:9, while ymoA was identified in all strains. Conclusions. The two molecular platforms could be used to better define some specific molecular targets for the characterization and rapid detection of Ye in different sources which important implications for food safety and animal and human health

Bioreactor Platform for Biomimetic Culture and in situ Monitoring of the Mechanical Response of in vitro Engineered Models of Cardiac Tissue

In the past two decades, relevant advances have been made in the generation of engineered cardiac constructs to be used as functional in vitro models for cardiac research or drug testing, and with the ultimate but still challenging goal of repairing the damaged myocardium. To support cardiac tissue generation and maturation in vitro, the application of biomimetic physical stimuli within dedicated bioreactors is crucial. In particular, cardiac-like mechanical stimulation has been demonstrated to promote development and maturation of cardiac tissue models. Here, we developed an automated bioreactor platform for tunable cyclic stretch and in situ monitoring of the mechanical response of in vitro engineered cardiac tissues. To demonstrate the bioreactor platform performance and to investigate the effects of cyclic stretch on construct maturation and contractility, we developed 3D annular cardiac tissue models based on neonatal rat cardiac cells embedded in fibrin hydrogel. The constructs were statically pre-cultured for 5 days and then exposed to 4 days of uniaxial cyclic stretch (sinusoidal waveform, 10% strain, 1 Hz) within the bioreactor. Explanatory biological tests showed that cyclic stretch promoted cardiomyocyte alignment, maintenance, and maturation, with enhanced expression of typical mature cardiac markers compared to static controls. Moreover, in situ monitoring showed increasing passive force of the constructs along the dynamic culture. Finally, only the stretched constructs were responsive to external electrical pacing with synchronous and regular contractile activity, further confirming that cyclic stretching was instrumental for their functional maturation. This study shows that the proposed bioreactor platform is a reliable device for cyclic stretch culture and in situ monitoring of the passive mechanical response of the cultured constructs. The innovative feature of acquiring passive force measurements in situ and along the culture allows monitoring the construct maturation trend without interrupting the culture, making the proposed device a powerful tool for in vitro investigation and ultimately production of functional engineered cardiac constructs

Characterisation of age and polarity at onset in bipolar disorder

Background Studying phenotypic and genetic characteristics of age at onset (AAO) and polarity at onset (PAO) in bipolar disorder can provide new insights into disease pathology and facilitate the development of screening tools. Aims To examine the genetic architecture of AAO and PAO and their association with bipolar disorder disease characteristics. Method Genome-wide association studies (GWASs) and polygenic score (PGS) analyses of AAO (n = 12 977) and PAO (n = 6773) were conducted in patients with bipolar disorder from 34 cohorts and a replication sample (n = 2237). The association of onset with disease characteristics was investigated in two of these cohorts. Results Earlier AAO was associated with a higher probability of psychotic symptoms, suicidality, lower educational attainment, not living together and fewer episodes. Depressive onset correlated with suicidality and manic onset correlated with delusions and manic episodes. Systematic differences in AAO between cohorts and continents of origin were observed. This was also reflected in single-nucleotide variant-based heritability estimates, with higher heritabilities for stricter onset definitions. Increased PGS for autism spectrum disorder (β = −0.34 years, s.e. = 0.08), major depression (β = −0.34 years, s.e. = 0.08), schizophrenia (β = −0.39 years, s.e. = 0.08), and educational attainment (β = −0.31 years, s.e. = 0.08) were associated with an earlier AAO. The AAO GWAS identified one significant locus, but this finding did not replicate. Neither GWAS nor PGS analyses yielded significant associations with PAO. Conclusions AAO and PAO are associated with indicators of bipolar disorder severity. Individuals with an earlier onset show an increased polygenic liability for a broad spectrum of psychiatric traits. Systematic differences in AAO across cohorts, continents and phenotype definitions introduce significant heterogeneity, affecting analyses

Colorectal Cancer Stage at Diagnosis Before vs During the COVID-19 Pandemic in Italy

IMPORTANCE Delays in screening programs and the reluctance of patients to seek medical attention because of the outbreak of SARS-CoV-2 could be associated with the risk of more advanced colorectal cancers at diagnosis. OBJECTIVE To evaluate whether the SARS-CoV-2 pandemic was associated with more advanced oncologic stage and change in clinical presentation for patients with colorectal cancer. DESIGN, SETTING, AND PARTICIPANTS This retrospective, multicenter cohort study included all 17 938 adult patients who underwent surgery for colorectal cancer from March 1, 2020, to December 31, 2021 (pandemic period), and from January 1, 2018, to February 29, 2020 (prepandemic period), in 81 participating centers in Italy, including tertiary centers and community hospitals. Follow-up was 30 days from surgery. EXPOSURES Any type of surgical procedure for colorectal cancer, including explorative surgery, palliative procedures, and atypical or segmental resections. MAIN OUTCOMES AND MEASURES The primary outcome was advanced stage of colorectal cancer at diagnosis. Secondary outcomes were distant metastasis, T4 stage, aggressive biology (defined as cancer with at least 1 of the following characteristics: signet ring cells, mucinous tumor, budding, lymphovascular invasion, perineural invasion, and lymphangitis), stenotic lesion, emergency surgery, and palliative surgery. The independent association between the pandemic period and the outcomes was assessed using multivariate random-effects logistic regression, with hospital as the cluster variable. RESULTS A total of 17 938 patients (10 007 men [55.8%]; mean [SD] age, 70.6 [12.2] years) underwent surgery for colorectal cancer: 7796 (43.5%) during the pandemic period and 10 142 (56.5%) during the prepandemic period. Logistic regression indicated that the pandemic period was significantly associated with an increased rate of advanced-stage colorectal cancer (odds ratio [OR], 1.07; 95%CI, 1.01-1.13; P = .03), aggressive biology (OR, 1.32; 95%CI, 1.15-1.53; P < .001), and stenotic lesions (OR, 1.15; 95%CI, 1.01-1.31; P = .03). CONCLUSIONS AND RELEVANCE This cohort study suggests a significant association between the SARS-CoV-2 pandemic and the risk of a more advanced oncologic stage at diagnosis among patients undergoing surgery for colorectal cancer and might indicate a potential reduction of survival for these patients

Infected pancreatic necrosis: outcomes and clinical predictors of mortality. A post hoc analysis of the MANCTRA-1 international study

: The identification of high-risk patients in the early stages of infected pancreatic necrosis (IPN) is critical, because it could help the clinicians to adopt more effective management strategies. We conducted a post hoc analysis of the MANCTRA-1 international study to assess the association between clinical risk factors and mortality among adult patients with IPN. Univariable and multivariable logistic regression models were used to identify prognostic factors of mortality. We identified 247 consecutive patients with IPN hospitalised between January 2019 and December 2020. History of uncontrolled arterial hypertension (p = 0.032; 95% CI 1.135-15.882; aOR 4.245), qSOFA (p = 0.005; 95% CI 1.359-5.879; aOR 2.828), renal failure (p = 0.022; 95% CI 1.138-5.442; aOR 2.489), and haemodynamic failure (p = 0.018; 95% CI 1.184-5.978; aOR 2.661), were identified as independent predictors of mortality in IPN patients. Cholangitis (p = 0.003; 95% CI 1.598-9.930; aOR 3.983), abdominal compartment syndrome (p = 0.032; 95% CI 1.090-6.967; aOR 2.735), and gastrointestinal/intra-abdominal bleeding (p = 0.009; 95% CI 1.286-5.712; aOR 2.710) were independently associated with the risk of mortality. Upfront open surgical necrosectomy was strongly associated with the risk of mortality (p < 0.001; 95% CI 1.912-7.442; aOR 3.772), whereas endoscopic drainage of pancreatic necrosis (p = 0.018; 95% CI 0.138-0.834; aOR 0.339) and enteral nutrition (p = 0.003; 95% CI 0.143-0.716; aOR 0.320) were found as protective factors. Organ failure, acute cholangitis, and upfront open surgical necrosectomy were the most significant predictors of mortality. Our study confirmed that, even in a subgroup of particularly ill patients such as those with IPN, upfront open surgery should be avoided as much as possible. Study protocol registered in ClinicalTrials.Gov (I.D. Number NCT04747990)

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research