Anti-tumor effect of CT-322 as an adnectin inhibitor of vascular endothelial growth factor receptor-2

CT-322 is a new anti-angiogenic therapeutic agent based on an engineered variant of the tenth type III domain of human fibronectin, i.e., an Adnectin™, designed to inhibit vascular endothelial growth factor receptor (VEGFR)-2. This PE Gylated Adnectin was developed using an mRNA display technology. CT-322 bound human VEGFR-2 with high affinity (K_D, 11 nM), but did not bind VEGFR-1 or VEGFR-3 at concentrations up to 100 nM, as determined by surface plasmon resonance studies. Western blot analysis showed that CT-322 blocked VEGF-induced phosphorylation of VEGFR-2 and mitogen-activated protein kinase in human umbilical vascular endothelial cells. CT-322 significantly inhibited the growth of human tumor xenograft models of colon carcinoma and glioblastoma at doses of 15–60 mg/kg administered 3 times/week. Anti-tumor effects of CT-322 were comparable to those of sorafenib or sunitinib, which inhibit multiple kinases, in a colon carcinoma xenograft model, although CT-322 caused less overt adverse effects than the kinase inhibitors. CT-322 also enhanced the anti-tumor activity of the chemotherapeutic agent temsirolimus in the colon carcinoma model. The high affinity and specificity of CT-322 binding to VEGFR-2 and its anti-tumor activities establish CT-322 as a promising anti-angiogenic therapeutic agent. Our results further suggest that Adnectins are an important new class of targeted biologics that can be developed as potential treatments for a wide variety of diseases.Key words: CT-322, adnectin, VEGFR-2, tumor angiogenesis, angiogenesis inhibitor, biologics, targeted therapy     

Effects of glucose-dependent insulinotropic peptide on behavior

Glucose-dependent insulinotropic peptide (GIP) is an incretin hormone that rises rapidly in response to nutrient ingestion. The GIP receptor is widely expressed in the brain including the brain stem, telencephalon, diencephalon, olfactory bulb, pituitary, and cerebellum. Until recently it was not clear what the endogenous ligand for this receptor was because no GIP expression had been demonstrated in the brain. GIP synthesis has now been documented in the dentate gyrus of the hippocampus. To define GIP effects on behavior we utilized a mouse model a GIP-overexpressing transgenic mouse (GIP Tg). Specifically, anxiety-related behavior, exploration, memory, and nociception were examined. Compared to age-matched adult male C57BI/6 controls GIP Tg mice displayed enhanced exploratory behavior in the open-field locomotor activity test. GIP Tg mice also demonstrated increased performance in some of the motor function tests. These data suggest that the GIP receptor plays a role in the regulation of locomotor activity and exploration. To our knowledge, this is the first report of effects of GIP on behavior.     

Prediction of Hematopoietic Stem Cell Transplantation Related Mortality- Lessons Learned from the In-Silico Approach: A European Society for Blood and Marrow Transplantation Acute Leukemia Working Party Data Mining Study

Models for prediction of allogeneic hematopoietic stem transplantation (HSCT) related mortality partially account for transplant risk. Improving predictive accuracy requires understating of prediction limiting factors, such as the statistical methodology used, number and quality of features collected, or simply the population size. Using an in-silico approach (i.e., iterative computerized simulations), based on machine learning (ML) algorithms, we set out to analyze these factors. A cohort of 25,923 adult acute leukemia patients from the European Society for Blood and Marrow Transplantation (EBMT) registry was analyzed. Predictive objective was non-relapse mortality (NRM) 100 days following HSCT. Thousands of prediction models were developed under varying conditions: increasing sample size, specific subpopulations and an increasing number of variables, which were selected and ranked by separate feature selection algorithms. Depending on the algorithm, predictive performance plateaued on a population size of 6,611–8,814 patients, reaching a maximal area under the receiver operator characteristic curve (AUC) of 0.67. AUCs’ of models developed on specific subpopulation ranged from 0.59 to 0.67 for patients in second complete remission and receiving reduced intensity conditioning, respectively. Only 3–5 variables were necessary to achieve near maximal AUCs. The top 3 ranking variables, shared by all algorithms were disease stage, donor type, and conditioning regimen. Our findings empirically demonstrate that with regards to NRM prediction, few variables “carry the weight” and that traditional HSCT data has been “worn out”. “Breaking through” the predictive boundaries will likely require additional types of inputs.     

Human α-N-acetylglucosaminidase: cDNA cryptic site removal and native secretion signal addition significantly enhance enzyme expression and secretion

Human α-N-acetylglucosaminidase (Naglu) is a lysosomal acid hydrolase implicated in Mucopolysaccharidosis type IIIB (MPS IIIB). We utilize a Spodoptera frugiperda (Sf9) system to express Naglu fused to a synthetic protein transduction domain in hopes to facilitate delivery of Naglu across the blood–brain barrier, thus allowing enzyme replacement therapy to treat neurological symptoms.Although human recombinant Naglu was previously produced in Sf9, low expression levels suggested degradation via cryptic mRNA splicing. Two cryptic splice sites discovered within Naglu cDNA were altered by site-directed mutagenesis, reducing Naglu mRNA degradation. A native Naglu secretion-signaling peptide was efficiently recognized by the Sf9 system. Significantly higher enzyme activity was seen from multiple adherent Sf9 cultures stably expressing mutagenized Naglu over those expressing wildtype Naglu (P = 0.000; 3.4-fold average increased specific activity). Suspension cultures demonstrated a 4.0-fold increase in overall enzyme activity secreted post-mutagenesis. Thus elimination of cryptic splicing directly resulted in higher Naglu expression.     

Formation and morphology of epidermal sclerites from a deep-sea hydrothermal vent solenogaster (Helicoradomenia sp., Solenogastres, Mollusca)

The deep-sea hydrothermal vent solenogaster Helicoradomenia is covered with calcium carbonate sclerites. Light and electron microscopy reveal varying morphologies of these sclerites. Many sclerites have hollow tips and/or are pitted and etched. Bacteria are found on and in sclerites. Initial sclerite formation occurs in an extracellular crystalline chamber formed by the invagination of a cuboidal basal cell of the columnar microvillus mantle epithelium. As the sclerite grows, it fills the crystalline chamber resulting in direct contact with the microvilli of both the basal cell and neighboring secondary sclerite-forming cells. These cells shape a collar around the base of the growing sclerite. As growth continues, the sclerite-forming cells stretch around the sclerite forming a sheath in which the base of the sclerite resides. Mature sclerites grow through the cuticle into the external environment. The erosion pattern of sclerites reveals a less stable inner medullary region and a harder outer cortical region. This points to a secondary character state, where foremost hollow acicular sclerites develop into solid sclerites. This is in agreement with the systematic position of the genus Helicoradomenia within Simrothiellidae, a taxon typically with hollow sclerites.     

Effect of Acute Hyperglycemia on Left Ventricular Contractile Function in Diabetic Patients with and without Heart Failure: Two Randomized Cross-Over Studies

Background

It is unknown whether changes in circulating glucose levels due to short-term insulin discontinuation affect left ventricular contractile function in type 2 diabetic patients with (T2D-HF) and without (T2D-nonHF) heart failure.

Materials and Methods

In two randomized cross-over-designed trials, 18 insulin-treated type 2 diabetic patients with (Ejection Fraction (EF) 36±6%, n = 10) (trial 2) and without systolic heart failure (EF 60±3%, n = 8) (trial 1) were subjected to hyper- and normoglycemia for 9–12 hours on two different occasions. Advanced echocardiography, bicycle exercise tests and 6-minute hall walk distance were applied.

Results

Plasma glucose levels differed between study arms (6.5±0.8 mM vs 14.1±2.6 mM (T2D-HF), 5.8±0.4 mM vs 9.9±2.1 mM (T2D-nonHF), p<0.001). Hyperglycemia was associated with an increase in several parameters: maximal global systolic tissue velocity (Vmax) (p<0.001), maximal mitral annulus velocity (S''max) (p<0.001), strain rate (p = 0.02) and strain (p = 0.05). Indices of increased myocardial systolic contractile function were significant in both T2D-HF (Vmax: 14%, p = 0.02; S''max: 10%, p = 0.04), T2D-nonHF (Vmax: 12%, p<0.01; S''max: 9%, p<0.001) and in post exercise S''max (7%, p = 0.049) during hyperglycemia as opposed to normoglycemia. LVEF did not differ between normo- and hyperglycemia (p = 0.17), and neither did peak exercise capacity nor catecholamine levels. Type 2 diabetic heart failure patients'' 6-minute hall walk distance improved by 7% (p = 0.02) during hyperglycemia as compared with normoglycemia.

Conclusions

Short-term hyperglycemia by insulin discontinuation is associated with an increase in myocardial systolic contractile function in type 2 diabetic patients with and without heart failure and with a slightly prolonged walking distance in type 2 diabetic heart failure patients. (Clinicaltrials.gov identifier {"type":"clinical-trial","attrs":{"text":"NCT00653510","term_id":"NCT00653510"}}NCT00653510)     

Migration by soaring or flapping: numerical atmospheric simulations reveal that turbulence kinetic energy dictates bee-eater flight mode

Aerial migrants commonly face atmospheric dynamics that may affect their movement and behaviour. Specifically, bird flight mode has been suggested to depend on convective updraught availability and tailwind assistance. However, this has not been tested thus far since both bird tracks and meteorological conditions are difficult to measure in detail throughout extended migratory flyways. Here, we applied, to our knowledge, the first comprehensive numerical atmospheric simulations by mean of the Regional Atmospheric Modeling System (RAMS) to study how meteorological processes affect the flight behaviour of migrating birds. We followed European bee-eaters (Merops apiaster) over southern Israel using radio telemetry and contrasted bird flight mode (flapping, soaring-gliding or mixed flight) against explanatory meteorological variables estimated by RAMS simulations at a spatial grid resolution of 250 × 250 m(2). We found that temperature and especially turbulence kinetic energy (TKE) determine bee-eater flight mode, whereas, unexpectedly, no effect of tailwind assistance was found. TKE during soaring-gliding was significantly higher and distinct from TKE during flapping. We propose that applying detailed atmospheric simulations over extended migratory flyways can elucidate the highly dynamic behaviour of air-borne organisms, help predict the abundance and distribution of migrating birds, and aid in mitigating hazardous implications of bird migration.     

Development of twelve novel polymorphic microsatellite DNA markers for the Boeseman’s rainbowfish (Melanotaenia boesemani) and tests for their cross-utility in 21 rainbowfish species from West Papua (Indonesia)

We developed 12 microsatellite markers for the endangered Boeseman’s rainbowfish (Melanotaenia boesemani). Twenty-eight individuals from the type locality at Ayamaru Lake were examined, and all loci were polymorphic with a number of alleles per locus varying from 3 to 18. Average observed and expected heterozygosities were 0.681 and 0.678, respectively. Cross-species amplification was successfully obtained for 21 Melanotaenia species, with a number of alleles per locus ranging between 1 and 20. Average observed and expected heterozygosities varied between 0.105 and 0.708 and 0.118–0.755, respectively. Only 21 inbreeding coefficient (Fis) values presented a significant homozygote excess among the 264 locus-by-locus calculated values. Tests for genotyping errors revealed that four of these 21 significant Fis values could be explained by the presence of null alleles. These new microsatellite markers appear highly reliable for further conservation purposes or population genetic studies of the many rainbowfish endangered species.