Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib

The vascular endothelial growth factor receptor‐1 (VEGFR‐1) is a tyrosine kinase receptor frequently expressed in melanoma. Its activation by VEGF‐A or placental growth factor (PlGF) promotes tumour cell survival, migration and invasiveness. Moreover, VEGFR‐1 stimulation contributes to pathological angiogenesis and induces recruitment of tumour‐associated macrophages. Since melanoma acquired resistance to BRAF inhibitors (BRAFi) has been associated with activation of pro‐angiogenic pathways, we have investigated VEGFR‐1 involvement in vemurafenib resistance. Results indicate that human melanoma cells rendered resistant to vemurafenib secrete greater amounts of VEGF‐A and express higher VEGFR‐1 levels compared with their BRAFi‐sensitive counterparts. Transient VEGFR‐1 silencing in susceptible melanoma cells delays resistance development, whereas in resistant cells it increases sensitivity to the BRAFi. Consistently, enforced VEGFR‐1 expression, by stable gene transfection in receptor‐negative melanoma cells, markedly reduces sensitivity to vemurafenib. Moreover, melanoma cells expressing VEGFR‐1 are more invasive than VEGFR‐1 deficient cells and receptor blockade by a specific monoclonal antibody (D16F7 mAb) reduces extracellular matrix invasion triggered by VEGF‐A and PlGF. These data suggest that VEGFR‐1 up‐regulation might contribute to melanoma progression and spreading after acquisition of a drug‐resistant phenotype. Thus, VEGFR‐1 inhibition with D16F7 mAb might be a suitable adjunct therapy for VEGFR‐1 positive tumours with acquired resistance to vemurafenib.     

A new formulation for the project scheduling problem under limited resources

In this paper we propose a new formulation for the Resource Constrained Project Scheduling Problem (RCPSP) and minimum makespan objective. The new formulation exploits three variables, one associated with the start time of an activity, one associated with the finish time of an activity, and the last one associated with the amount of an activity in progress at a given time. We provide an extensive experimentation, and a comparison with known mathematical formulations for the RCPSP in the literature.     

Weight Loss Expectations in Obese Patients Seeking Treatment at Medical Centers

Objective: To investigate weight loss expectations (expected 1‐year BMI loss, dream BMI, and maximum acceptable BMI) in obese patients seeking treatment and to examine whether expectations differ by sex, weight, diet and weight history, age, psychological factors, and primary motivations for weight loss. Research Methods and Procedures: 1891 obese patients seeking treatment in 25 Italian medical centers (1473 women; age, 44.7 ± 11.0 years; BMI, 38.2 ± 6.5 kg/m²) were evaluated. Diet and weight history, weight loss expectations, and primary motivation for seeking treatment (health or improving appearance) were systematically recorded. Psychiatric distress, binge eating, and body image dissatisfaction were tested by self‐administered questionnaires (Symptom CheckList‐90, Binge Eating Scale, and Body Uneasiness Test). Results: In 1011 cases (53.4%), 1‐year expected BMI loss was ≥9 kg/m², dream BMI was 26.0 ± 3.4 kg/m² (corresponding to a 32% loss), and maximum acceptable BMI was 29.3 ± 4.4 kg/m² (?23%). BMI and age were the strongest predictors of weight goals. Weight loss necessary to reach the desired targets was largely in excess of weight loss observed during previous dieting. Psychiatric distress, body dissatisfaction, and binge eating did not predict weight loss expectations. The primary motivation for weight loss was concern for future or present health; women seeking treatment to improve appearance had a lower grade of obesity, were younger, and had first attempted weight loss at a younger age. Discussion: Obese Italian patients had unrealistic weight loss expectations. There were significant disparities between patients’ perceptions and physicians’ weight loss recommendations of desirable treatment outcome.     

High Content Screening of a Kinase-Focused Library Reveals Compounds Broadly-Active against Dengue Viruses

Dengue virus is a mosquito-borne flavivirus that has a large impact in global health. It is considered as one of the medically important arboviruses, and developing a preventive or therapeutic solution remains a top priority in the medical and scientific community. Drug discovery programs for potential dengue antivirals have increased dramatically over the last decade, largely in part to the introduction of high-throughput assays. In this study, we have developed an image-based dengue high-throughput/high-content assay (HT/HCA) using an innovative computer vision approach to screen a kinase-focused library for anti-dengue compounds. Using this dengue HT/HCA, we identified a group of compounds with a 4-(1-aminoethyl)-N-methylthiazol-2-amine as a common core structure that inhibits dengue viral infection in a human liver-derived cell line (Huh-7.5 cells). Compounds CND1201, CND1203 and CND1243 exhibited strong antiviral activities against all four dengue serotypes. Plaque reduction and time-of-addition assays suggests that these compounds interfere with the late stage of viral infection cycle. These findings demonstrate that our image-based dengue HT/HCA is a reliable tool that can be used to screen various chemical libraries for potential dengue antiviral candidates.     

Functional connectivity models for decoding of spatial representations from hippocampal CA1 recordings

Hippocampus stores spatial representations, or maps, which are recalled each time a subject is placed in the corresponding environment. Across different environments of similar geometry, these representations show strong orthogonality in CA3 of hippocampus, whereas in the CA1 subfield a considerable overlap between the maps can be seen. The lower orthogonality decreases reliability of various decoders developed in an attempt to identify which of the stored maps is active at the moment. Especially, the problem with decoding emerges with a need to analyze data at high temporal resolution. Here, we introduce a functional-connectivity-based decoder, which accounts for the pairwise correlations between the spiking activities of neurons in each map and does not require any positional information, i.e. any knowledge about place fields. We first show, on recordings of hippocampal activity in constant environmental conditions, that our decoder outperforms existing decoding methods in CA1. Our decoder is then applied to data from teleportation experiments, in which an instantaneous switch between the environment identity triggers a recall of the corresponding spatial representation . We test the sensitivity of our approach on the transition dynamics between the respective memory states (maps). We find that the rate of spontaneous state shifts (flickering) after a teleportation event is increased not only within the first few seconds as already reported, but this instability is sustained across much longer (> 1 min.) periods.     

Molecular characterization of the human PLC β1 gene

Inositide-specific phospholipase C (PLC) signaling constitutes a central intermediate in a number of cellular functions among which the control of cell growth raises a particular interest. Indeed, we have previously shown that nuclear phospholipase C β1 (PLC β1) is central for the regulation of mitogen-induced cell growth. We have also assigned by fluorescence in situ hybridization (FISH) analysis the PLC β1 to human chromosome 20p12. In this study, we have carried out a detailed analysis of the human gene, showing the existence of alternative splicing, which gives rise, besides the two forms (1a and 1b) already shown in rodents, to a new 600 bp smaller form coding for a 110 kDa protein. We have also identified a new exon at the 5′, showing no homology with the rodent sequence. Here we provide the complete determination of the exon/intron structure of the gene spanning 250 kb of DNA. We found that the exons are quite small, ranging from 49 to 222 bp, while the introns vary between 108 bp and 34,400 bp. The availability of the understanding of the genome organization of this inositide-specific PLC, which represents a key step of the cell cycle related signaling, could actually pave the way for further genetic analysis of p12 region of human chromosome 20 in diseases involving alterations of the control of cell growth such as malignancies.     

NADH oxidase from the extreme thermophile Thermus aquaticus YT-1. Purification and characterisation

A protein with NADH oxidase activity from the extreme thermophile Thermus aquaticus YT-1 was purified and characterised. The enzyme was found to have a relative molecular mass of 110,000 and be composed of two subunits of identical size. FAD was found to be present at a concentration of 0.7 mol/mol dimer and was required for activity. During the oxidation of NADH, oxygen uptake takes place with the production of hydrogen peroxide. The enzyme had, with the exception of a higher glutamic acid and tryptophan content, a similar amino acid composition as the NADH oxidase isolated from the mesophile Bacillus megaterium. Purified NADH oxidase was found to have a Km of 39 microM for beta-NADH and a Vmax of 4.68 mumol NADH mg-1 min-1 and was still active at 95 degrees C. Enzymatic activity was found to be independent of pH between 5.0 and 10.5.