Design,synthesis and biological evaluation of 3-benzyloxy-linked pyrimidinylphenylamine derivatives as potent HIV-1 NNRTIs

A novel series of 3-benzyloxy-linked pyrimidinylphenylamine derivatives (8a–8s) was designed, synthesized and evaluated for their in vitro anti-HIV activity in MT-4 cell cultures. Most of the compounds inhibited wild-type (wt) HIV-1 replication in the lower micromolar concentration range (EC₅₀ = 0.05–35 μM) with high selectivity index (SI) values (ranged from 10 to >4870). In particular, 8h and 8g displayed excellent antiretroviral activity against wt HIV-1 with low cytotoxicity (EC₅₀ = 0.07 μM, CC₅₀ >347 μM, SI >4870; EC₅₀ = 0.05 μM, CC₅₀ = 42 μM, SI = 777, respectively), comparable to that of the marked drug nevirapine (EC₅₀ = 0.113 μM, CC₅₀ >15 μM, SI >133). In order to confirm the binding target, 8h was selected to perform the anti-HIV-1 RT assay. Additionally, preliminary structure activity relationship (SAR) analysis and molecular docking studies of newly synthesized compounds were also discussed, as well as the predicted physicochemical properties.     

Fibroblast growth factor homologous factors control neuronal excitability through modulation of voltage-gated sodium channels

Neurons integrate and encode complex synaptic inputs into action potential outputs through a process termed "intrinsic excitability." Here, we report the essential contribution of fibroblast growth factor homologous factors (FHFs), a family of voltage-gated sodium channel binding proteins, to this process. Fhf1-/-Fhf4-/- mice suffer from severe ataxia and other neurological deficits. In mouse cerebellar slice recordings, WT granule neurons can be induced to fire action potentials repetitively (approximately 60 Hz), whereas Fhf1-/-Fhf4-/- neurons often fire only once and at an elevated voltage spike threshold. Sodium channels in Fhf1-/-Fhf4-/- granule neurons inactivate at more negative membrane potential, inactivate more rapidly, and are slower to recover from the inactivated state. Altered sodium channel physiology is sufficient to explain excitation deficits, as tested in a granule cell computer model. These findings offer a physiological mechanism underlying human spinocerebellar ataxia induced by Fhf4 mutation and suggest a broad role for FHFs in the control of excitability throughout the CNS.     

Characterization of M. tuberculosis SerB2, an Essential HAD-Family Phosphatase,Reveals Novel Properties

M. tuberculosis harbors an essential phosphoserine phosphatase (MtSerB2, Rv3042c) that contains two small- molecule binding ACT-domains (Pfam 01842) at the N-terminus followed by the phosphoserine phosphatase (PSP) domain. We found that exogenously added MtSerB2 elicits microtubule rearrangements in THP-1 cells. Mutational analysis demonstrates that phosphatase activity is co-related to the elicited rearrangements, while addition of the ACT-domains alone elicits no rearrangements. The enzyme is dimeric, exhibits divalent metal- ion dependency, and is more specific for l- phosphoserine unlike other classical PSPases. Binding of a variety of amino acids to the ACT-domains influences MtSerB2 activity by either acting as activators/inhibitors/have no effects. Additionally, reduced activity of the PSP domain can be enhanced by equimolar addition of the ACT domains. Further, we identified that G18 and G108 of the respective ACT-domains are necessary for ligand-binding and their mutations to G18A and G108A abolish the binding of ligands like l- serine. A specific transition to higher order oligomers is observed upon the addition of l- serine at ∼0.8 molar ratio as supported by Isothermal calorimetry and Size exclusion chromatography experiments. Mutational analysis shows that the transition is dependent on binding of l- serine to the ACT-domains. Furthermore, the higher-order oligomeric form of MtSerB2 is inactive, suggesting that its formation is a mechanism for feedback control of enzyme activity. Inhibition studies involving over eight inhibitors, MtSerB2, and the PSP domain respectively, suggests that targeting the ACT-domains can be an effective strategy for the development of inhibitors.     

Enhanced suicidal erythrocyte death in mice carrying a loss-of-function mutation of the adenomatous polyposis coli gene

Loss-of-function mutations in human adenomatous polyposis coli (APC) lead to multiple colonic adenomatous polyps eventually resulting in colonic carcinoma. Similarly, heterozygous mice carrying defective APC (apc(Min/+)) suffer from intestinal tumours. The animals further suffer from anaemia, which in theory could result from accelerated eryptosis, a suicidal erythrocyte death triggered by enhanced cytosolic Ca(2+) activity and characterized by cell membrane scrambling and cell shrinkage. To explore, whether APC-deficiency enhances eryptosis, we estimated cell membrane scrambling from annexin V binding, cell size from forward scatter and cytosolic ATP utilizing luciferin-luciferase in isolated erythrocytes from apc(Min/+) mice and wild-type mice (apc(+/+)). Clearance of circulating erythrocytes was estimated by carboxyfluorescein-diacetate-succinimidyl-ester labelling. As a result, apc(Min/+) mice were anaemic despite reticulocytosis. Cytosolic ATP was significantly lower and annexin V binding significantly higher in apc(Min/+) erythrocytes than in apc(+/+) erythrocytes. Glucose depletion enhanced annexin V binding, an effect significantly more pronounced in apc(Min/+) erythrocytes than in apc(+/+) erythrocytes. Extracellular Ca(2+) removal or inhibition of Ca(2+) entry with amiloride (1 mM) blunted the increase but did not abrogate the genotype differences of annexin V binding following glucose depletion. Stimulation of Ca(2+) -entry by treatment with Ca(2+) -ionophore ionomycin (10 μM) increased annexin V binding, an effect again significantly more pronounced in apc(Min/+) erythrocytes than in apc(+/+) erythrocytes. Following retrieval and injection into the circulation of the same mice, apc(Min/+) erythrocytes were more rapidly cleared from circulating blood than apc(+/+) erythrocytes. Most labelled erythrocytes were trapped in the spleen, which was significantly enlarged in apc(Min/+) mice. The observations point to accelerated eryptosis and subsequent clearance of apc(Min/+) erythrocytes, which contributes to or even accounts for the enhanced erythrocyte turnover, anaemia and splenomegaly in those mice.     

Role played by disabled-2 in albumin induced MAP Kinase signalling

Albumin has been shown to activate the mitogen activated protein kinase (MAPK) pathway in proximal tubular cells (PTECs) of the kidney. Megalin, the putative receptor for albumin has potential signalling properties. However, the mechanisms by which megalin signals are unclear. The adaptor phosphoprotein Disabled-2 (Dab2) is known to interact with the cytoplasmic tail of megalin and may be involved in albumin-mediated MAPK signalling. In this study, we investigated the role of Dab2 in albumin-mediated MAPK signalling and further studied the role of Dab2 in albumin-induced TGFβ-1 secretion, a MAPK dependent event. We used RNA interference to knockdown Dab2 protein abundance in HKC-8 cells a model of human PTECs. Albumin activated ERK1,2 and Elk-1 in a MEK-1 dependent manner and resulted in secretion of TGFβ-1. In the absence of albumin, knockdown of Dab2 resulted in a trend towards increase in pERK1,2 consistent with its putative role as an inhibitor of cell proliferation. However albumin-induced ERK1,2 activation was completely abolished by Dab2 knockdown. Dab2 knockdown did not however result in inhibition of albumin-induced TGFβ-1 secretion. These results suggest that Dab2 is a ligand dependent bi-directional regulator of ERK1,2 activity by demonstrating that in addition to its more traditional role as an inhibitor of ERK1,2 it may also activate ERK1,2.     

Models for first-pass FMS investment analysis

Consider a firm that could choose either an inexpensive product-specific (dedicated) facility or a costly flexible facility, or a combination of the two, in order to satisfy demand for two product groups. Flexible technology offers benefits of scale and scope economies. However, it may have added operational costs due to the need to have excess capacity (to permit changeovers) and to maintain cycle stocks. As a consequence, the economic viability of flexible technology is significantly affected by the choice of the operating doctrine. This article presents first-pass decision models to help the firm choose the optimum sizes of facilities and the degree of flexibility for the flexible facility such that the contingent operational costs are simultaneously optimized. Two variations of the problem are considered. Whereas, the first applies to a situation in which the demand rates are constant and predictable, the second considers random demands. In each case, this study provides a formulation of the problem, structural results, and the sensitivity of the results to cost parameters. These results have modest data and computational requirements, making them suitable for first-cut attempts at narrowing available choices.     

Can intact liposomes be absorbed in the gut?

Inulin, EDTA, or maltose were given intragastrically to mice, either free or entrapped in mono-shell liposomes of egg phosphatidylcholine or the non-digestible diether and dialkyl analog of phosphatidylcholine. Results indicate that liposomes may protect a drug from premature digestion but cannot carry it through the intestinal wall.     

Oviposition behaviour of Phlebotomus argentipes - A laboratory-based study

The breeding habitat of sandflies is a little studied and poorly understood phenomenon. More importantly, oviposition behaviour is a largely neglected aspect of sandfly biology and this knowledge gap further undermines our understanding of the biology of sandflies. Pheromones released by the eggs play an important role in identifying good sites for oviposition by female insects. Several recent studies have examined the oviposition pheromone. The present study provides a preliminary report on the oviposition behaviour of Phlebotomus argentipes, the only vector of kala-azar (or visceral leishmaniasis) on the Indian sub-continent. Sandflies prefer to oviposit their eggs on surfaces that contain organic substances, especially substances with an odour of decaying animal products and the remains of conspecific eggs. The results presented here suggest that the odour released by the organic substances of old sandfly colony remains that contain dead flies, old unhatched eggs, larval food containing vertebrate faeces, frass and other organic matter serves as an attractant for the ovipositing females of P. argentipes and hence greatly increases the number of oviposited eggs compared to eggs deposited in controlled oviposition pots. This result will be helpful in maintaining an efficient colony of P. argentipes and may be a promising tool for monitoring and controlling the target insect as part of a synergistic approach.