Metagenomic Exploration of Viruses throughout the Indian Ocean

The characterization of global marine microbial taxonomic and functional diversity is a primary goal of the Global Ocean Sampling Expedition. As part of this study, 19 water samples were collected aboard the Sorcerer II sailing vessel from the southern Indian Ocean in an effort to more thoroughly understand the lifestyle strategies of the microbial inhabitants of this ultra-oligotrophic region. No investigations of whole virioplankton assemblages have been conducted on waters collected from the Indian Ocean or across multiple size fractions thus far. Therefore, the goals of this study were to examine the effect of size fractionation on viral consortia structure and function and understand the diversity and functional potential of the Indian Ocean virome. Five samples were selected for comprehensive metagenomic exploration; and sequencing was performed on the microbes captured on 3.0-, 0.8- and 0.1 µm membrane filters as well as the viral fraction (<0.1 µm). Phylogenetic approaches were also used to identify predicted proteins of viral origin in the larger fractions of data from all Indian Ocean samples, which were included in subsequent metagenomic analyses. Taxonomic profiling of viral sequences suggested that size fractionation of marine microbial communities enriches for specific groups of viruses within the different size classes and functional characterization further substantiated this observation. Functional analyses also revealed a relative enrichment for metabolic proteins of viral origin that potentially reflect the physiological condition of host cells in the Indian Ocean including those involved in nitrogen metabolism and oxidative phosphorylation. A novel classification method, MGTAXA, was used to assess virus-host relationships in the Indian Ocean by predicting the taxonomy of putative host genera, with Prochlorococcus, Acanthochlois and members of the SAR86 cluster comprising the most abundant predictions. This is the first study to holistically explore virioplankton dynamics across multiple size classes and provides unprecedented insight into virus diversity, metabolic potential and virus-host interactions.     

Intrasteric control of AMPK via the gamma1 subunit AMP allosteric regulatory site

AMP-activated protein kinase (AMPK) is a alphabetagamma heterotrimer that is activated in response to both hormones and intracellular metabolic stress signals. AMPK is regulated by phosphorylation on the alpha subunit and by AMP allosteric control previously thought to be mediated by both alpha and gamma subunits. Here we present evidence that adjacent gamma subunit pairs of CBS repeat sequences (after Cystathionine Beta Synthase) form an AMP binding site related to, but distinct from the classical AMP binding site in phosphorylase, that can also bind ATP. The AMP binding site of the gamma(1) CBS1/CBS2 pair, modeled on the structures of the CBS sequences present in the inosine monophosphate dehydrogenase crystal structure, contains three arginine residues 70, 152, and 171 and His151. The yeast gamma homolog, snf4 contains a His151Gly substitution, and when this is introduced into gamma(1), AMP allosteric control is substantially lost and explains why the yeast snf1p/snf4p complex is insensitive to AMP. Arg70 in gamma(1) corresponds to the site of mutation in human gamma(2) and pig gamma(3) genes previously identified to cause an unusual cardiac phenotype and glycogen storage disease, respectively. Mutation of any of AMP binding site Arg residues to Gln substantially abolishes AMP allosteric control in expressed AMPK holoenzyme. The Arg/Gln mutations also suppress the previously described inhibitory properties of ATP and render the enzyme constitutively active. We propose that ATP acts as an intrasteric inhibitor by bridging the alpha and gamma subunits and that AMP functions to derepress AMPK activity.     

Epsilon substituted lysinol derivatives as HIV-1 protease inhibitors

A series of HIV-1 protease inhibitors containing an epsilon substituted lysinol backbone was synthesized. Two novel synthetic routes using N-boc-l-glutamic acid alpha-benzyl ester and 2,6-diaminopimelic acid were developed. Incorporation of this epsilon substituent enabled access to the S2 pocket of the enzyme, affording high potency inhibitors. Modeling studies and synthetic efforts suggest the potency increase is due to both conformational bias and van der Waals interactions with the S2 pocket.     

Neurotrophic peptide aldehydes: solid phase synthesis of fellutamide B and a simplified analog

A combination of solid phase and solution phase synthetic methods have been used to complete the total synthesis of the neurotrophic lipopeptide aldehyde fellutamide B (2). The beta-hydroxy aliphatic tail was prepared by regioselective reductive opening of a cyclic sulfate, and later coupled to a solid phase resin. The synthetic compound was then examined in cytotoxicity and nerve growth factor (NGF) induction assays. A simplified analog of fellutamide B also showed activity.     

Discovery of imidazo[1,2-a]-, [1,2,4]triazolo[4,3-a]-, and [1,2,4]triazolo[1,5-a]pyridine-8-carboxamide negative allosteric modulators of metabotropic glutamate receptor subtype 5

Based on a hypothesis that an intramolecular hydrogen bond was present in our lead series of picolinamide mGlu₅ NAMs, we reasoned that an inactive nicotinamide series could be modified through introduction of a fused heterocyclic core to generate potent mGlu₅ NAMs. In this Letter, we describe the synthesis and evaluation of compounds that demonstrate the viability of that approach. Selected analogs were profiled in a variety of in vitro assays, and two compounds were evaluated in rat pharmacokinetic studies and a mouse model of obsessive-compulsive disorder. Ancillary pharmacology screening revealed that members of this series exhibited moderate inhibition of the dopamine transporter (DAT), and SAR was developed that expanded the selectivity for mGlu₅ versus DAT.     

Discovery and optimization of 3-(4-aryl/heteroarylsulfonyl)piperazin-1-yl)-6-(piperidin-1-yl)pyridazines as novel,CNS penetrant pan-muscarinic antagonists

This letter describes the synthesis and structure activity relationship (SAR) studies of structurally novel M₄ antagonists, based on a 3-(4-aryl/heteroarylsulfonyl)piperazin-1-yl)-6-(piperidin-1-yl)pyridazine core, identified from a high-throughput screening campaign. A multi-dimensional optimization effort enhanced potency at human M₄ (hM₄ IC₅₀s < 200 nM), with only moderate species differences noted, and with enantioselective inhibition. Moreover, CNS penetration proved attractive for this series (rat brain:plasma K_p = 2.1, K_p,uu = 1.1). Despite the absence of the prototypical mAChR antagonist basic or quaternary amine moiety, this series displayed pan-muscarinic antagonist activity across M_1-5 (with 9- to 16-fold functional selectivity at best). This series further expands the chemical diversity of mAChR antagonists.     

Effects of Beta-Alanine Supplementation on Brain Homocarnosine/Carnosine Signal and Cognitive Function: An Exploratory Study

Objectives

Two independent studies were conducted to examine the effects of 28 d of beta-alanine supplementation at 6.4 g d^-1 on brain homocarnosine/carnosine signal in omnivores and vegetarians (Study 1) and on cognitive function before and after exercise in trained cyclists (Study 2).

Methods

In Study 1, seven healthy vegetarians (3 women and 4 men) and seven age- and sex-matched omnivores undertook a brain 1H-MRS exam at baseline and after beta-alanine supplementation. In study 2, nineteen trained male cyclists completed four 20-Km cycling time trials (two pre supplementation and two post supplementation), with a battery of cognitive function tests (Stroop test, Sternberg paradigm, Rapid Visual Information Processing task) being performed before and after exercise on each occasion.

Results

In Study 1, there were no within-group effects of beta-alanine supplementation on brain homocarnosine/carnosine signal in either vegetarians (p = 0.99) or omnivores (p = 0.27); nor was there any effect when data from both groups were pooled (p = 0.19). Similarly, there was no group by time interaction for brain homocarnosine/carnosine signal (p = 0.27). In study 2, exercise improved cognitive function across all tests (P<0.05), although there was no effect (P>0.05) of beta-alanine supplementation on response times or accuracy for the Stroop test, Sternberg paradigm or RVIP task at rest or after exercise.

Conclusion

28 d of beta-alanine supplementation at 6.4g d^-1 appeared not to influence brain homocarnosine/carnosine signal in either omnivores or vegetarians; nor did it influence cognitive function before or after exercise in trained cyclists.     

Courtship behavior of male white perch, Morone americana: evidence for control by androgens

Courtship behaviors are androgen-dependent in many vertebrates and castration often decreases courtship. We examined the effectiveness of castration in reducing courtship behaviors and 11-ketotestosterone (KT) and testosterone (T) in restoring them in male white perch. Castrates were given implants containing KT, T or no hormone. Sham-operated males received implants without hormone. Three weeks later, males were exposed to an ovulated female for 1 h and two courtship behaviors were quantified. Attending behavior involves close and continuous following of a female with occasional contact. Circling involves rapid transits around the female in a circular pattern or back and forth in front of her. In plasma samples taken immediately after observations, KT and T were below detectable levels in castrated males but at high physiological levels in males implanted with KT or T. Castrated males given KT attended females more than castrated males given T implants or implants containing no hormone, but not more than sham-operated males. Circling was eliminated by castration but restored by implantation with T or 11-KT to values exhibited by sham-operated males. This is one of the few demonstrations that KT can regulate courtship behavior in a non-territorial and economically important fish species.     

Potent inhibitors of farnesyltransferase and geranylgeranyltransferase-I

Compound 1 has been shown to be a dual prenylation inhibitor with FPTase (IC50=2 nM) and GGPTase-I (IC50=95 nM). Analogues of 1, which replaced the cyanophenyl group with various biaryls, led to the discovery of highly potent dual FPTase/GGPTase-I inhibitors. 4-trifluoromethylphenyl, trifluoropentynyl, and trifluoropentyl were identified as good p-cyano replacements.