Melanoma central nervous system metastases: An update to approaches,challenges, and opportunities

In February 2018, the Melanoma Research Foundation and the Moffitt Cancer Center hosted the Second Summit on Melanoma Central Nervous System (CNS) Metastases in Tampa, Florida. In this white paper, we outline the current status of basic science, translational, and clinical research into melanoma brain metastasis development and therapeutic management. We further outline the important challenges that remain for the field and the critical barriers that need to be overcome for continued progress to be made in this clinically difficult area.     

Protein kinase A and two phosphatases are components of the inositol 1,4,5-trisphosphate receptor macromolecular signaling complex

The inositol 1,4,5-trisphosphate receptor (IP3R) is a ubiquitously expressed intracellular calcium (Ca(2+)) release channel on the endoplasmic reticulum. IP3Rs play key roles in controlling Ca(2+) signals that activate numerous cellular functions including T cell activation, neurotransmitter release, oocyte fertilization and apoptosis. There are three forms of IP3R, all of which are ligand-gated channels activated by the second messenger inositol 1,4,5-trisphosphate. Channel function is modulated via cross-talk with other signaling pathways including those mediated by kinases and phosphatases. In particular IP3Rs are known to be regulated by cAMP-dependent protein kinase (PKA) phosphorylation. In the present study we show that PKA and the protein phosphatases PP1 and PP2A are components of the IP3R1 macromolecular signaling complex. PKA phosphorylation of IP3R1 increases channel activity in planar lipid bilayers. These studies indicate that regulation of IP3R1 function via PKA phosphorylation involves components of a macromolecular signaling complex.     

SPIN2: Predicting sequence profiles from protein structures using deep neural networks

Designing protein sequences that can fold into a given structure is a well‐known inverse protein‐folding problem. One important characteristic to attain for a protein design program is the ability to recover wild‐type sequences given their native backbone structures. The highest average sequence identity accuracy achieved by current protein‐design programs in this problem is around 30%, achieved by our previous system, SPIN. SPIN is a program that predicts sequences compatible with a provided structure using a neural network with fragment‐based local and energy‐based nonlocal profiles. Our new model, SPIN2, uses a deep neural network and additional structural features to improve on SPIN. SPIN2 achieves over 34% in sequence recovery in 10‐fold cross‐validation and independent tests, a 4% improvement over the previous version. The sequence profiles generated from SPIN2 are expected to be useful for improving existing fold recognition and protein design techniques. SPIN2 is available at http://sparks-lab.org .     

Metallopeptide-promoted inactivation of angiotensin-converting enzyme and endothelin-converting enzyme 1: toward dual-action therapeutics

A series of metallopeptides based on the amino terminal copper/nickel (ATCUN) binding motif have been evaluated as classical inhibitors and catalytic inactivators of both rabbit and human angiotensin-converting enzyme (hACE), and human endothelin-converting enzyme 1 (hECE-1). The cobalt complex [KGHK–Co(NH₃)₂]²⁺, where KGHK is lysylglycylhistidyllysine, displayed similar K _I and IC₅₀ values to those found for [KGHK–Cu]⁺, in spite of the enhanced charge, and so either the influence of charge is offset by the steric influence of the axially coordinated ammine ligands, or binding is dominated by contributions from the amino acid side chains, especially the C-terminal lysine that mimics the binding pattern observed for lisinopril. Moreover, the inhibition observed for [KGHK–Co(NH₃)₂]²⁺ contrasts with the activation of hACE by Co²⁺(aq), reflecting the stimulation of enzyme activity following replacement of the catalytic zinc cofactor by cobalt ion at each of the two active sites. Quantitative analysis of the dose-dependent stimulation of activity by Co²⁺(aq) yielded apparent affinities of 1.3 ± 0.2 and 56 ± 8 μM for the two sites in the presence of saturating Zn²⁺ (10 μM). Catalytic inactivation of hACE by [KGHK–Cu] ⁺ at subsaturating concentrations had previously been characterized, with k _obs = 2.9 ± 0.5 × 10⁻² min⁻¹. Under similar conditions, the same complex is found to catalytically inactivate hECE-1, with k _obs = 2.12 ± 0.16 × 10⁻² min⁻¹, demonstrating the potential for dual-action activity against two key drug targets in cardiovascular disease. Irreversible inactivation of a drug target represents a novel mechanism of drug action that complements existing classical inhibitor strategies that underlie current drug discovery efforts.Electronic Supplementary Material Supplementary material is available to authorized users in the online version of this article at .     

A novel multiplex PCR assay for the identification of Indian crocodiles

Illegal hunting has been a major threat for the survival of wildlife fauna, including the three crocodile species that India harbours: Crocodylus palustris, Crocodylus porosus and Gavialis gangeticus. Although law prevents trade on these species, illicit hunting for trade continues to threaten the survival of these endangered species; conservation strategies therefore require a rapid molecular identification technique for Indian crocodiles. A multiplex polymerase chain reaction (PCR) assay with species-specific primers, considered as one of the most effective molecular techniques, is described herein. The primers were designed to yield species-specific sized amplicons. The assay discriminates the three Indian crocodile species unambiguously within a short time period using only simple agarose gel electrophoresis. We recommend this multiplex PCR assay to be used in the identification of Indian crocodile species.     

Phosphatidylinositol 3,5-bisphosphate increases intracellular free Ca2+ in arterial smooth muscle cells and elicits vasocontraction

Phosphoinositide (3,5)-bisphosphate [PI(3,5)P(2)] is a newly identified phosphoinositide that modulates intracellular Ca(2+) by activating ryanodine receptors (RyRs). Since the contractile state of arterial smooth muscle depends on the concentration of intracellular Ca(2+), we hypothesized that by mobilizing sarcoplasmic reticulum (SR) Ca(2+) stores PI(3,5)P(2) would increase intracellular Ca(2+) in arterial smooth muscle cells and cause vasocontraction. Using immunohistochemistry, we found that PI(3,5)P(2) was present in the mouse aorta and that exogenously applied PI(3,5)P(2) readily entered aortic smooth muscle cells. In isolated aortic smooth muscle cells, exogenous PI(3,5)P(2) elevated intracellular Ca(2+), and it also contracted aortic rings. Both the rise in intracellular Ca(2+) and the contraction caused by PI(3,5)P(2) were prevented by antagonizing RyRs, while the majority of the PI(3,5)P(2) response was intact after blockade of inositol (1,4,5)-trisphosphate receptors. Depletion of SR Ca(2+) stores with thapsigargin or caffeine and/or ryanodine blunted the Ca(2+) response and greatly attenuated the contraction elicited by PI(3,5)P(2). The removal of extracellular Ca(2+) or addition of verapamil to inhibit voltage-dependent Ca(2+) channels reduced but did not eliminate the Ca(2+) or contractile responses to PI(3,5)P(2). We also found that PI(3,5)P(2) depolarized aortic smooth muscle cells and that LaCl(3) inhibited those aspects of the PI(3,5)P(2) response attributable to extracellular Ca(2+). Thus, full and sustained aortic contractions to PI(3,5)P(2) required the release of SR Ca(2+), probably via the activation of RyR, and also extracellular Ca(2+) entry via voltage-dependent Ca(2+) channels.     

An essential role for diet in exercise-mediated protection against dyslipidemia, inflammation and atherosclerosis in ApoE⁻/⁻ mice

Background

Diet and exercise promote cardiovascular health but their relative contributions to atherosclerosis are not fully known. The transition from a sedentary to active lifestyle requires increased caloric intake to achieve energy balance. Using atherosclerosis-prone ApoE-null mice we sought to determine whether the benefits of exercise for arterial disease are dependent on the food source of the additional calories.

Methods and Results

Mice were fed a high-fat diet (HF) for 4.5 months to initiate atherosclerosis after which time half were continued on HF while the other half were switched to a high protein/fish oil diet (HP). Half of each group underwent voluntary running. Food intake, running distance, body weight, lipids, inflammation markers, and atherosclerotic plaque were quantified. Two-way ANOVA tests were used to assess differences and interactions between groups. Exercised mice ran approximately 6-km per day with no difference between groups. Both groups increased food intake during exercise and there was a significant main effect of exercise F((1,44) = 9.86, p<0.01) without interaction. Diet or exercise produced significant independent effects on body weight (diet: F(1,52) = 6.85, p = 0.012; exercise: F(1,52) = 9.52, p<0.01) with no significant interaction. The combination of HP diet and exercise produced a greater decrease in total cholesterol (F(1, 46) = 7.9, p<0.01) and LDL (F(1, 46) = 7.33, p<0.01) with a large effect on the size of the interaction. HP diet and exercise independently reduced TGL and VLDL (p<0.05 and 0.001 respectively). Interleukin 6 and C-reactive protein were highest in the HF-sedentary group and were significantly reduced by exercise only in this group. Plaque accumulation in the aortic arch, a marker of cardiovascular events was reduced by the HP diet and the effect was significantly potentiated by exercise only in this group resulting in significant plaque regression (F1, 49 = 4.77, p<0.05).

Conclusion

In this model exercise is beneficial to combat dyslipidemia and protect from atherosclerosis only when combined with diet.     

Controlled Release of Ropinirole Hydrochloride from a Multiple Barrier Layer Tablet Dosage Form: Effect of Polymer Type on Pharmacokinetics and IVIVC

The purpose of the present study was to control in vitro burst effect of the highly water-soluble drug, ropinirole hydrochloride to reduce in vivo dose dumping and to establish in vitro–in vivo correlation. The pharmacokinetics of two entirely different tablet formulation technologies is also explored in this study. For pharmacokinetics study, FDA recommends at least 10% difference in drug release for formulations to be studied but here a different approach was adopted. The formulations F8A and F9A having similar dissolution profiles among themselves and with Requip® XL™ (f₂ value 72, 77, 71 respectively) were evaluated. The C_max of formulation F8A comprising hypromellose 100,000 cP was 1005.16 pg/ml as compared to 973.70 pg/ml of formulation F9A comprising hypromellose 4000 cP irrespective of T_max of 5 and 5.75 h, respectively. The difference in release and extent of absorption in vivo was due to synergistic effect of complex RH release mechanism; however, AUC_0–t and AUC_0–∞ values were comparable. The level A correlation using the Wagner–Nelson method supported the findings where R² was 0.7597 and 0.9675 respectively for formulation F8A and F9A. Thus, in vivo studies are required for proving the therapeutic equivalency of different formulation technologies even though f₂ ≥ 50. The technology was demonstrated effectively at industrial manufacturing scale of 200,000 tablets.KEY WORDS: controlled release polymer, in vitro–in vivo correlation (IVIVC), multiple barrier layer tablets, pharmacokinetics, ropinirole hydrochloride (RH)