Calretinin distribution in the octopus brain: an immunohistochemical and in situ hybridization histochemical analysis

The distribution of calretinin containing neurons examined by in situ hybridization mapping was compared with that obtained by immunocytochemistry in the brain of octopus. Results revealed a close correspondence between the two types of investigations. Western blot analysis disclosed a 29 kDa protein immunostained with anti-calretinin antibody. Calretinin containing neurons were localized mainly in the cortex of octopus lobes, including the vertical, frontal, basal, buccal, palliovisceral, pedal and branchial, with variations of staining intensity and density of immunoreactive cells. The amacrine cells surrounding calretinin containing neuronal bodies of the cortex were also labeled unlike the glial cells. The close correspondence of blotting analysis, immunocytochemistry and in situ hybridization indicates with no doubt that calretinin, like other calcium-binding proteins previously studied, is also present in the nervous system of cephalopods. Furthermore, although recent findings localize calretinin also in endocrine glands, the presence of this calcium-binding protein in the brain of octopus indicates that calretinin appeared early in the phylogeny as a neuronal protein already in invertebrates

Cell-to-Cell Adhesion and Neurogenesis in Human Cortical Development: A Study Comparing 2D Monolayers with 3D Organoid Cultures

SUMMARY Organoids (ORGs) are increasingly used as models of cerebral cortical development. Here, we compared transcriptome and cellular phenotypes between telencephalic ORGs and monolayers (MONs) generated in parallel from three biologically distinct induced pluripotent stem cell (iPSC) lines. Multiple readouts revealed increased proliferation in MONs, which was caused by increased integrin signaling. MONs also exhibited altered radial glia (RG) polarity and suppression of Notch signaling, as well as impaired generation of intermediate progenitors, outer RG, and cortical neurons, which were all partially reversed by reaggregation of dissociated cells. Network analyses revealed co-clustering of cell adhesion, Notch-related transcripts and their transcriptional regulators in a module strongly downregulated in MONs. The data suggest that ORGs, with respect to MONs, initiate more efficient Notch signaling in ventricular RG owing to preserved cell adhesion, resulting in subsequent generation of intermediate progenitors and outer RG, in a sequence that recapitulates the cortical ontogenetic process

ATM inhibition blocks glucose metabolism and amplifies the sensitivity of resistant lung cancer cell lines to oncogene driver inhibitors

Background: ATM is a multifunctional serine/threonine kinase that in addition to its well-established role in DNA repair mechanisms is involved in a number of signaling pathways including regulation of oxidative stress response and metabolic diversion of glucose through the pentose phosphate pathway. Oncogene-driven tumorigenesis often implies the metabolic switch from oxidative phosphorylation to glycolysis which provides metabolic intermediates to sustain cell proliferation. The aim of our study is to elucidate the role of ATM in the regulation of glucose metabolism in oncogene-driven cancer cells and to test whether ATM may be a suitable target for anticancer therapy. Methods: Two oncogene-driven NSCLC cell lines, namely H1975 and H1993 cells, were treated with ATM inhibitor, KU55933, alone or in combination with oncogene driver inhibitors, WZ4002 or crizotinib. Key glycolytic enzymes, mitochondrial complex subunits (OXPHOS), cyclin D1, and apoptotic markers were analyzed by Western blotting. Drug-induced toxicity was assessed by MTS assay using stand-alone or combined treatment with KU55933 and driver inhibitors. Glucose consumption, pyruvate, citrate, and succinate levels were also analyzed in response to KU55933 treatment. Both cell lines were transfected with ATM-targeted siRNA or non-targeting siRNA and then exposed to treatment with driver inhibitors. Results: ATM inhibition deregulates and inhibits glucose metabolism by reducing HKII, p-PKM2Tyr105, p-PKM2Ser37, E1α subunit of pyruvate dehydrogenase complex, and all subunits of mitochondrial complexes except ATP synthase. Accordingly, glucose uptake and pyruvate concentrations were reduced in response to ATM inhibition, whereas citrate and succinate levels were increased in both cell lines indicating the supply of alternative metabolic substrates. Silencing of ATM resulted in similar changes in glycolytic cascade and OXPHOS levels. Furthermore, the driver inhibitors amplified the effects of ATM downregulation on glucose metabolism, and the combined treatment with ATM inhibitors enhanced the cytotoxic effect of driver inhibitors alone by increasing the apoptotic response. Conclusions: Inhibition of ATM reduced both glycolytic enzymes and OXPHOS levels in oncogene-driven cancer cells and enhanced apoptosis induced by driver inhibitors thus highlighting the possibility to use ATM and the driver inhibitors in combined regimens of anticancer therapy in vivo

LMO2 expression reflects the different stages of blast maturation and genetic features in B-cell acute lymphoblastic leukemia and predicts clinical outcome

BACKGROUND: LMO2 is highly expressed at the most immature stages of lymphopoiesis. In T-lymphocytes, aberrant LMO2 expression beyond those stages leads to T-cell acute lymphoblastic leukemia, while in B cells LMO2 is also expressed in germinal center lymphocytes and diffuse large B-cell lymphomas, where it predicts better clinical outcome. The implication of LMO2 in B-cell acute lymphoblastic leukemia must still be explored. DESIGN AND METHODS: We measured LMO2 expression by real time RT-PCR in 247 acute lymphoblastic leukemia patient samples with cytogenetic data (144 of them also with survival and immunophenotypical data) and in normal hematopoietic and lymphoid cells. RESULTS: B-cell acute lymphoblastic leukemia cases expressed variable levels of LMO2 depending on immunophenotypical and cytogenetic features. Thus, the most immature subtype, pro-B cells, displayed three-fold higher LMO2 expression than pre-B cells, common-CD10+ or mature subtypes. Additionally, cases with TEL-AML1 or MLL rearrangements exhibited two-fold higher LMO2 expression compared to cases with BCR-ABL rearrangements or hyperdyploid karyotype. Clinically, high LMO2 expression correlated with better overall survival in adult patients (5-year survival rate 64.8% (42.5%-87.1%) vs. 25.8% (10.9%-40.7%), P= 0.001) and constituted a favorable independent prognostic factor in B-ALL with normal karyotype: 5-year survival rate 80.3% (66.4%-94.2%) vs. 63.0% (46.1%-79.9%) (P= 0.043). CONCLUSIONS: Our data indicate that LMO2 expression depends on the molecular features and the differentiation stage of B-cell acute lymphoblastic leukemia cells. Furthermore, assessment of LMO2 expression in adult patients with a normal karyotype, a group which lacks molecular prognostic factors, could be of clinical relevance

Albiglutide and cardiovascular outcomes in patients with type 2 diabetes and cardiovascular disease (Harmony Outcomes): a double-blind, randomised placebo-controlled trial

Background: Glucagon-like peptide 1 receptor agonists differ in chemical structure, duration of action, and in their effects on clinical outcomes. The cardiovascular effects of once-weekly albiglutide in type 2 diabetes are unknown. We aimed to determine the safety and efficacy of albiglutide in preventing cardiovascular death, myocardial infarction, or stroke. Methods: We did a double-blind, randomised, placebo-controlled trial in 610 sites across 28 countries. We randomly assigned patients aged 40 years and older with type 2 diabetes and cardiovascular disease (at a 1:1 ratio) to groups that either received a subcutaneous injection of albiglutide (30–50 mg, based on glycaemic response and tolerability) or of a matched volume of placebo once a week, in addition to their standard care. Investigators used an interactive voice or web response system to obtain treatment assignment, and patients and all study investigators were masked to their treatment allocation. We hypothesised that albiglutide would be non-inferior to placebo for the primary outcome of the first occurrence of cardiovascular death, myocardial infarction, or stroke, which was assessed in the intention-to-treat population. If non-inferiority was confirmed by an upper limit of the 95% CI for a hazard ratio of less than 1·30, closed testing for superiority was prespecified. This study is registered with ClinicalTrials.gov, number NCT02465515. Findings: Patients were screened between July 1, 2015, and Nov 24, 2016. 10 793 patients were screened and 9463 participants were enrolled and randomly assigned to groups: 4731 patients were assigned to receive albiglutide and 4732 patients to receive placebo. On Nov 8, 2017, it was determined that 611 primary endpoints and a median follow-up of at least 1·5 years had accrued, and participants returned for a final visit and discontinuation from study treatment; the last patient visit was on March 12, 2018. These 9463 patients, the intention-to-treat population, were evaluated for a median duration of 1·6 years and were assessed for the primary outcome. The primary composite outcome occurred in 338 (7%) of 4731 patients at an incidence rate of 4·6 events per 100 person-years in the albiglutide group and in 428 (9%) of 4732 patients at an incidence rate of 5·9 events per 100 person-years in the placebo group (hazard ratio 0·78, 95% CI 0·68–0·90), which indicated that albiglutide was superior to placebo (p<0·0001 for non-inferiority; p=0·0006 for superiority). The incidence of acute pancreatitis (ten patients in the albiglutide group and seven patients in the placebo group), pancreatic cancer (six patients in the albiglutide group and five patients in the placebo group), medullary thyroid carcinoma (zero patients in both groups), and other serious adverse events did not differ between the two groups. There were three (<1%) deaths in the placebo group that were assessed by investigators, who were masked to study drug assignment, to be treatment-related and two (<1%) deaths in the albiglutide group. Interpretation: In patients with type 2 diabetes and cardiovascular disease, albiglutide was superior to placebo with respect to major adverse cardiovascular events. Evidence-based glucagon-like peptide 1 receptor agonists should therefore be considered as part of a comprehensive strategy to reduce the risk of cardiovascular events in patients with type 2 diabetes. Funding: GlaxoSmithKline

High-resolution molecular phenotyping of human IPSC organoids using CLARITY and 2-photon microscopy

High-resolution molecular phenotyping of human IPSC organoids using CLARITY and 2-photon microscopy Simone Tomasi, Soraya Scuderi, Anahita Amiri, Giovanna G. Altobelli, Jessica Mariani, Cheryl Dambrot, Gianfilippo Coppola, Flora M. Vaccarino To understand the role of gene regulation in human brain development and neuropsychiatric disorders, it is essential to develop cellular models of the human brain. Induced pluripotent stem cells (iPSC)-derived brain organoids can be used to investigate the role of gene regulatory elements, noncoding RNA, and in general, noncoding disease associated gene variants in brain development and function. Organoids enable gradients of morphogenes and other extracellular cues to build up in the intercellular milieu and to interact with the genetic and epigenetic background of a given progenitor cell during the course of brain development. We have developed an iPSC-derived organoid model of the early human forebrain, where differentiation of cortical excitatory and inhibitory neurons can be studied in a reproducible fashion, enabling a more precise identification of molecular events crucially involved in the specification of distinct neuronal subtypes. However, a precise assessment of protein and RNA expression in intact organoids is hampered by the limited penetration of molecular probes, therefore requiring the preparation of thin sections and greatly limiting the capacity of exploring molecular and cellular features in a 3D environment. Here, we labeled telencephalic excitatory and inhibitory lineages in using pLenti-CAMKII-GFP and pLenti-DlxI12b-BG-DsRed vectors, then used two-photon microscopy to image the genetically-encoded fluorescence at higher resolution in live forebrain organoids. Next, we used CLARITY to clear the organoids and perform immunostainings on the intact cell aggregates. Our current protocol enables a 3D reconstruction of GFP/TdTomato filled cells allowing the analysis of axonal and dendritic arborization, dendrite length, synapse and spine distribution, as well as stereological counts of structures labeled by specific markers. Using these combined approaches, we aim at comparing intra-organoid layer cytoarchitecture and its emerging connectivity with parallel data from RNA-seq and ChIP-seq experiments, and develop new tools for linking the molecular and cellular features of organoids derived from different individuals

beta-1 adrenergic receptor blokade and cardiac beta-2 overexpression stimulates angiogenesis in the failing heart

Angiogenesis in the failing heart is stimulated beta-1 AR blockade and cardiac beta-2 AR overexpression

Myocardial β2-adrenoceptor gene delivery promotes coordinated cardiac adaptive remodelling and angiogenesis in heart failure

Background and purpose: We investigated whether β(2) -adrenergic receptor (β(2) AR) overexpression could promote angiogenesis and improve blood perfusion and left ventricular (LV) remodeling of the failing heart. Experimental approach: At this aim, we explored the angiogenic effects of β(2) AR overexpression in a rat model of post-myocardial infarction (MI) heart failure (HF). Cardiac adenoviral-mediated β(2) AR overexpression has been obtained via direct intramyocardial injection 4-weeks post-MI. Adenovirus(Ad)-GFP and saline injected rats served as controls. Furthermore, we extended our observation to β(2) AR-/- mice undergoing MI. Key results: Transgenes were robustly expressed in the LV at 2-weeks post gene therapy, whereas their expression was minimal at 4-weeks post-gene delivery. In HF rats, cardiac β(2) AR overexpression resulted in enhanced basal and isoproterenol-stimulated cardiac contractility at 2-weeks post-gene delivery. At 4-weeks post-gene transfer Ad-β(2) AR HF rats showed improved LV remodeling and cardiac function. Importantly, β(2) AR overexpression was associated with markedly increased capillary and arteriolar length density, and enhanced in vivo myocardial blood flow and coronary reserve. At the molecular level, cardiac β(2) AR gene transfer induced the activation of VEGF/Akt/eNOS proangiogenic pathway. In β(2) AR-/- mice, we found a ∼25% reduction of cardiac capillary density compared to β(2) AR+/+ mice. The lack of β(2) AR was associated with higher 30-day mortality rate and LV dilatation, and worse global cardiac contractility compared to controls. Conclusion(s) and implications:β(2) AR plays an important role in the regulation of the angiogenic response in HF. The activation of VEGF/Akt/eNOS pathway seems to be strongly involved in this mechanism