VEGF modulates erythropoiesis through regulation of adult hepatic erythropoietin synthesis

Vascular endothelial growth factor (VEGF) exerts crucial functions during pathological angiogenesis and normal physiology. We observed increased hematocrit (60-75%) after high-grade inhibition of VEGF by diverse methods, including adenoviral expression of soluble VEGF receptor (VEGFR) ectodomains, recombinant VEGF Trap protein and the VEGFR2-selective antibody DC101. Increased production of red blood cells (erythrocytosis) occurred in both mouse and primate models, and was associated with near-complete neutralization of VEGF corneal micropocket angiogenesis. High-grade inhibition of VEGF induced hepatic synthesis of erythropoietin (Epo, encoded by Epo) >40-fold through a HIF-1alpha-independent mechanism, in parallel with suppression of renal Epo mRNA. Studies using hepatocyte-specific deletion of the Vegfa gene and hepatocyte-endothelial cell cocultures indicated that blockade of VEGF induced hepatic Epo by interfering with homeostatic VEGFR2-dependent paracrine signaling involving interactions between hepatocytes and endothelial cells. These data indicate that VEGF is a previously unsuspected negative regulator of hepatic Epo synthesis and erythropoiesis and suggest that levels of Epo and erythrocytosis could represent noninvasive surrogate markers for stringent blockade of VEGF in vivo.     

Kinase gene fusions in defined subsets of melanoma

Genomic rearrangements resulting in activating kinase fusions have been increasingly described in a number of cancers including malignant melanoma, but their frequency in specific melanoma subtypes has not been reported. We used break‐apart fluorescence in situ hybridization (FISH) to identify genomic rearrangements in tissues from 59 patients with various types of malignant melanoma including acral lentiginous, mucosal, superficial spreading, and nodular. We identified four genomic rearrangements involving the genes BRAF, RET, and ROS1. Of these, three were confirmed by Immunohistochemistry (IHC) or sequencing and one was found to be an ARMC10‐BRAF fusion that has not been previously reported in melanoma. These fusions occurred in different subtypes of melanoma but all in tumors lacking known driver mutations. Our data suggest gene fusions are more common than previously thought and should be further explored particularly in melanomas lacking known driver mutations.     

Anatomical variant of the meniscus related to posterior junction: a case report

Background

There are several reports on anatomical differences of the meniscus. However, there are only a few reports on abnormalities in both menisci and anatomical differences in anterior cruciate ligament insertions.

Case presentation

This is a case report of a 36-year-old Hispanic man presenting symptoms, including knee pain, locking, and effusion, with an anatomical abnormality of the menisci corresponding to the fusion of the posterior horns of the menisci in tandem with the insertion of the posterior meniscus fibers in the anterior cruciate ligament.

Conclusions

This is the first study describing a meniscus anatomical variant with isolated posterior junction of the posterior horn with an anomalous insertion to the anterior cruciate ligament. The recognition of meniscus variants is important as they can be misinterpreted for more significant pathology on magnetic resonance images.

     

Electrostatic mutations in iberiotoxin as a unique tool for probing the electrostatic structure of the maxi-K channel outer vestibule

Iberiotoxin (IbTX or alpha-KTx 1.3), a selective, high-affinity blocker of the large-conductance, calcium-activated (maxi-K) channel, exhibits a unique, asymmetric distribution of charge. To test how these charges control kinetics of IbTX binding, we generated five mutants at two positions, K27 and R34, that are highly conserved among other isotoxins. The dissociation and association rate constants, koff and kon, were determined from toxin-blocked and -unblocked durations of single maxi-K channels incorporated into planar lipid bilayers. Equilibrium dissociation constant (Kd) values were calculated from koff/kon. The IbTX mutants K27N, K27Q, and R34N caused large increases in Kd values compared to wild-type, suggesting that the IbTX interaction surface encompasses these residues. A well-established pore-blocking mechanism for IbTX predicts a voltage dependence of toxin-blocked times following occupancy of a potassium binding site in the channel pore. Time constants for block by K27R were approximately 5-fold slower at -20 mV versus +40 mV, while neutralization of K27 relieved the voltage dependence of block. This suggests that K27 in IbTX interacts with a potassium binding site in the pore. Neutralized mutants of K27 and R34, with zero net charge, displayed toxin association rate constants approximately 10-fold slower than wild-type. Association rates for R34N diminished approximately 19-fold when external potassium was increased from 30 to 300 mM. These findings suggest that simple net charge and diffusional processes do not control ingress of IbTX into the channel vestibule.     

Identification and biochemical characterization of a novel nortriterpene inhibitor of the human lymphocyte voltage-gated potassium channel, Kv1.3

A novel nortriterpene, termed correolide, purified from the tree Spachea correae, inhibits Kv1.3, a Shaker-type delayed rectifier potassium channel present in human T lymphocytes. Correolide inhibits 86Rb+ efflux through Kv1.3 channels expressed in CHO cells (IC50 86 nM; Hill coefficient 1) and displays a defined structure-activity relationship. Potency in this assay increases with preincubation time and with time after channel opening. Correolide displays marked selectivity against numerous receptors and voltage- and ligand-gated ion channels. Although correolide is most potent as a Kv1.3 inhibitor, it blocks all other members of the Kv1 family with 4-14-fold lower potency. C20-29-[3H]dihydrocorreolide (diTC) was prepared and shown to bind in a specific, saturable, and reversible fashion (Kd = 11 nM) to a single class of sites in membranes prepared from CHO/Kv1.3 cells. The molecular pharmacology and stoichiometry of this binding reaction suggest that one diTC site is present per Kv1.3 channel tetramer. This site is allosterically coupled to peptide and potassium binding sites in the pore of the channel. DiTC binding to human brain synaptic membranes identifies channels composed of other Kv1 family members. Correolide depolarizes human T cells to the same extent as peptidyl inhibitors of Kv1.3, suggesting that it is a candidate for development as an immunosuppressant. Correolide is the first potent, small molecule inhibitor of Kv1 series channels to be identified from a natural product source and will be useful as a probe for studying potassium channel structure and the physiological role of such channels in target tissues of interest.     

The role of caldesmon in the regulation of endothelial cytoskeleton and migration

The actin- and myosin-binding protein, caldesmon (CaD) is an essential component of the cytoskeleton in smooth muscle and non-muscle cells and is involved in the regulation of cell contractility, division, and assembly of actin filaments. CaD is abundantly present in endothelial cells (EC); however, the contribution of CaD in endothelial cytoskeletal arrangement is unclear. To examine this contribution, we generated expression constructs of l-CaD cloned from bovine endothelium. Wild-type CaD (WT-CaD) and truncated mutants lacking either the N-terminal myosin-binding site or the C-terminal domain 4b (containing actin- and calmodulin-binding sites) were transfected into human pulmonary artery EC. Cell fractionation experiments and an actin overlay assay demonstrated that deleting domain 4b, but not the N-terminal myosin-binding site, resulted in decreased affinity to both the detergent-insoluble cytoskeleton and soluble actin. Recombinant WT-CaD co-localized with acto-myosin filaments in vivo, but neither of CaD mutants did. Thus both domain 4b and the myosin-binding site are essential for proper localization of CaD in EC. Overexpression of WT-CaD led to cell rounding and formation of a thick peripheral subcortical actin rim in quiescent EC, which correlated with decreased cellular migration. Pharmacological inhibition of p38 MAPK, but not ERK MAPK, caused disassembly of this peripheral actin rim in CaD-transfected cells and decreased CaD phosphorylation at Ser531 (Ser789 in human h-CaD). These results suggest that CaD is critically involved in the regulation of the actin cytoskeleton and migration in EC, and that p38 MAPK-mediated CaD phosphorylation may be involved in endothelial cytoskeletal remodeling.     

Reduced productivity in adult yellowfever mosquito (Diptera: Culicidae) populations

Male and female Aedes aegypti (L.) mosquitoes of the laboratory strain ROCK were irradiated with 130 mw of argon 514.5 nm laser microbeams for 0.04, 0.25, 0.4, and 0.5 s, respectively. Egg production, percentage hatch, and productivity (average number of adults surviving after 3 wk) were used to assess mutagenic effects. Mortality was high for males in all laser radiation groups and increased with time of exposure. Except for the group treated for 0.25 s, significant reductions in total F1 progeny also were demonstrated for all other experimentals when male parents were exposed to laser radiation. Females showed a high mortality when subjected to 0.4- and 0.5-s laser radiation. No F1 progeny were produced when parental females were exposed for 0.25, 0.4, and 0.5 s. Numbers of F1 progeny from females exposed to 0.04 s of laser radiation were significantly reduced. A comparison of weekly mean number of progeny showed that the important differences in productivity occurred during the first and second week, respectively, when either male or female adult parents were subjected to laser radiation.     

Effects of sodium nitroprusside in aortic stenosis associated with severe heart failure: pressure-volume loop analysis using a numerical model

In the recently published clinical study [Use of Nitroprusside in Left Ventricular Dysfunction and Obstructive Aortic Valve Disease (UNLOAD)], sodium nitroprusside (SNP) improved cardiac function in patients with severe aortic stenosis (AS) and left ventricular (LV) systolic dysfunction. We explored the possible mechanisms of these findings using a series of numerical simulations. A closed-loop lumped parameters model that consists of 24 differential equations relating pressure and flow throughout the circulation was used to analyze the effects of varying hemodynamic conditions in AS. Hemodynamic data from UNLOAD study subjects were used to construct the initial simulation. Systemic vascular resistance (SVR), heart rate, and aortic valve area were directly entered into the model while end-systolic and end-diastolic pressure-volume (P-V) relationships were adjusted using previously published data to match modeled and observed end-systolic and end-diastolic pressures and volumes. Initial simulation of SNP treatment by a reduction of SVR was not adequate. To obtain realistic model hemodynamics that reliably reproduce SNP treatment effects, we performed a series of simulations while simultaneously changing end-systolic elastance (E(es)), end-systolic volume at zero pressure (V(0)), and diastolic P-V shift. Our data indicate that either an E(es) increase or V(0) decrease is necessary to obtain realistic model hemodynamics. In five patients, we corroborated our findings by using the model to duplicate individual P-V loops obtained before and during SNP treatment. In conclusion, using a numerical model, we identified ventricular function parameters that are responsible for improved hemodynamics during SNP infusion in AS with LV dysfunction.     

Effects of intermittent aeration periods on a structured-bed reactor continuously fed on the post-treatment of sewage anaerobic effluent

Bioprocess and Biosystems Engineering - This study assessed the simultaneous nitrification and denitrification processes and remaining organic matter removal from anaerobic reactor effluent...