Influence of Climate on Long-Term Recovery of Adirondack Mountain Lakewater Chemistry from Atmospheric Deposition of Sulfur and Nitrogen

In this study, we assessed temporal patterns and long-term trends in nitrate (NO3-), two forms of aluminum (inorganic, Ali, and organic, Alo), and dissolved organic carbon (DOC) concentrations in the water of 29 Adirondack Mountain, New York lakes, and the potential effects of ambient weather conditions (i.e., climatic variation) on these patterns and trends

A detailed study of the diastereoselective catalytic hydrogenation of 6-hydroxytetrahydroisoquinoline-(3R)-carboxylic ester intermediates

A key step towards a highly-selective antagonist of ionotropic glutamate receptors entails the diastereoselective arene hydrogenation of an enantiopure tetrahydroisoquinoline. An extensive screen using parallel reactors was conducted and led to the discovery of several Pd/C catalysts giving high yield and improved diastereoselectivity from 75 : 25 to 95 : 5. A detailed kinetic study of the best system was performed and supports the reduction occuring in two-steps.

Integrated Screening for Arginase Inhibitors

Arginase is an enzyme that catalyzes the formation of L-ornithine and urea from L-arginine. L-arginine is also a substrate for nitric oxide synthase (NOS), resulting in the formation of nitric oxide (NO) which is a key vasodilator. Not surprisingly, arginase inhibitors are being studied to treat various diseases, including hypertension, erectile dysfunction, atherosclerosis, wound healing and myocardial reperfusion injury. Recently, the use of virtual screening and docking to identify and characterize novel arginase inhibitors as potential therapeutics to treat leshmania infections has been reported in the literature. Hence, there is interest in the development of new and improved arginase inhibitors. Here, we describe the use of an iterative in silico and in vitro work-flow for identifying novel arginase inhibitors. The first in silico arm of the work-flow involves the use of library design, virtual screening, docking, and consensus scoring to identify predicted hit compounds. The in vitro arm involves rapid assaying of predicted hits in an optimized arginase assay. Confirmed hits are passed into the second in silico arm which involves ligand-based screening, docking, and consensus scoring. The crank is turned on the in silico – in vitro – in silico cycle until a promising candidate for hit-to-lead optimization has been identified. Preliminary results appear encouraging, providing hope that a novel arginase drug candidate will be identified and that our computational work-flow will prove useful on other targets

Complex maps without invariant densities

We consider complex polynomials $f(z) = z^\ell+c_1$ for $\ell \in 2\N$ and $c_1 \in \R$, and find some combinatorial types and values of $\ell$ such that there is no invariant probability measure equivalent to conformal measure on the Julia set. This holds for particular Fibonacci-like and Feigenbaum combinatorial types when $\ell$ sufficiently large and also for a class of `long-branched' maps of any critical order.Comment: Typos corrected, minor changes, principally to Section

Computational Design of Novel Insulin Degrading Enzyme Inhibitors

Human insulin degrading enzyme (IDE) plays a role in the proteolytic cleavage of insulin, glucagon, and other short, hydrophobic peptides with roles in glucose and cellular metabolism. Because of IDE’s role in insulin clearance, IDE inhibitors may hold promise as therapies for potentiating insulin signaling in patients suffering from type 2 diabetes mellitus. IDE is a large (~100 kDa) chambered protease of the conserved M16A subfamily of zinc metalloproteases. The enzyme adopts a structure that is analogous to a clamshell formed by the joining of the N terminal and C terminal domains. The characteristic zinc binding and catalytic motif (HXXEH) is positioned within the enzyme’s N terminus, while C terminal residues also play important roles in substrate binding and catalysis. Here, we describe the use of a computational work-flow for identifying novel IDE inhibitors. The work flow integrates mutation-based active site structural analysis, virtual screening, docking and fragment-based design. Initial computational results appear promising and should lead to assay testing in the near future

Establishing a Clinically Relevant Large Animal Model Platform for TBI Therapy Development: Using Cyclosporin A as a Case Study

We have developed the first immature large animal translational treatment trial of a pharmacologic intervention for traumatic brain injury (TBI) in children. The preclinical trial design includes multiple doses of the intervention in two different injury types (focal and diffuse) to bracket the range seen in clinical injury and uses two post-TBI delays to drug administration. Cyclosporin A (CsA) was used as a case study in our first implementation of the platform because of its success in multiple preclinical adult rodent TBI models and its current use in children for other indications. Tier 1 of the therapy development platform assessed the short-term treatment efficacy after 24 h of agent administration. Positive responses to treatment were compared with injured controls using an objective effect threshold established prior to the study. Effective CsA doses were identified to study in Tier 2. In the Tier 2 paradigm, agent is administered in a porcine intensive care unit utilizing neurological monitoring and clinically relevant management strategies, and intervention efficacy is defined as improvement in longer term behavioral endpoints above untreated injured animals. In summary, this innovative large animal preclinical study design can be applied to future evaluations of other agents that promote recovery or repair after TBI

PKMζ is essential for spinal plasticity underlying the maintenance of persistent pain

<p>Abstract</p> <p>Background</p> <p>Chronic pain occurs when normally protective acute pain becomes pathologically persistent. We examined here whether an isoform of protein kinase C (PKC), PKMζ, that underlies long-term memory storage in various brain regions, also sustains nociceptive plasticity in spinal cord dorsal horn (SCDH) mediating persistent pain.</p> <p>Results</p> <p>Cutaneous injury or spinal stimulation produced persistent increases of PKMζ, but not other atypical PKCs in SCDH. Inhibiting spinal PKMζ, but not full-length PKCs, reversed plasticity-dependent persistent painful responses to hind paw formalin and secondary mechanical hypersensitivity and SCDH neuron sensitization after hind paw capsaicin, without affecting peripheral sensitization-dependent primary heat hypersensitivity after hind paw capsaicin. Inhibiting spinal PKMζ, but not full-length PKCs, also reversed mechanical hypersensitivity in the rat hind paw induced by spinal stimulation with intrathecal dihydroxyphenylglycine. Spinal PKMζ inhibition also alleviated allodynia 3 weeks after ischemic injury in rats with chronic post-ischemia pain (CPIP), at a point when allodynia depends on spinal changes. In contrast, spinal PKMζ inhibition did not affect allodynia in rats with chronic contriction injury (CCI) of the sciatic nerve, or CPIP rats early after ischemic injury, when allodynia depends on ongoing peripheral inputs.</p> <p>Conclusions</p> <p>These results suggest spinal PKMζ is essential for the maintenance of persistent pain by sustaining spinal nociceptive plasticity.</p

Next-generation sequencing identifies the natural killer cell microRNA transcriptome

Natural killer (NK) cells are innate lymphocytes important for early host defense against infectious pathogens and surveillance against malignant transformation. Resting murine NK cells regulate the translation of effector molecule mRNAs (e.g., granzyme B, GzmB) through unclear molecular mechanisms. MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally regulate the translation of their mRNA targets, and are therefore candidates for mediating this control process. While the expression and importance of miRNAs in T and B lymphocytes have been established, little is known about miRNAs in NK cells. Here, we used two next-generation sequencing (NGS) platforms to define the miRNA transcriptomes of resting and cytokine-activated primary murine NK cells, with confirmation by quantitative real-time PCR (qRT-PCR) and microarrays. We delineate a bioinformatics analysis pipeline that identified 302 known and 21 novel mature miRNAs from sequences obtained from NK cell small RNA libraries. These miRNAs are expressed over a broad range and exhibit isomiR complexity, and a subset is differentially expressed following cytokine activation. Using these miRNA NGS data, miR-223 was identified as a mature miRNA present in resting NK cells with decreased expression following cytokine activation. Furthermore, we demonstrate that miR-223 specifically targets the 3′ untranslated region of murine GzmB in vitro, indicating that this miRNA may contribute to control of GzmB translation in resting NK cells. Thus, the sequenced NK cell miRNA transcriptome provides a valuable framework for further elucidation of miRNA expression and function in NK cell biology

The Ruegeria pomeroyi acuI Gene Has a Role in DMSP Catabolism and Resembles yhdH of E. coli and Other Bacteria in Conferring Resistance to Acrylate

The Escherichia coli YhdH polypeptide is in the MDR012 sub-group of medium chain reductase/dehydrogenases, but its biological function was unknown and no phenotypes of YhdH− mutants had been described. We found that an E. coli strain with an insertional mutation in yhdH was hyper-sensitive to inhibitory effects of acrylate, and, to a lesser extent, to those of 3-hydroxypropionate. Close homologues of YhdH occur in many Bacterial taxa and at least two animals. The acrylate sensitivity of YhdH− mutants was corrected by the corresponding, cloned homologues from several bacteria. One such homologue is acuI, which has a role in acrylate degradation in marine bacteria that catabolise dimethylsulfoniopropionate (DMSP) an abundant anti-stress compound made by marine phytoplankton. The acuI genes of such bacteria are often linked to ddd genes that encode enzymes that cleave DMSP into acrylate plus dimethyl sulfide (DMS), even though these are in different polypeptide families, in unrelated bacteria. Furthermore, most strains of Roseobacters, a clade of abundant marine bacteria, cleave DMSP into acrylate plus DMS, and can also demethylate it, using DMSP demethylase. In most Roseobacters, the corresponding gene, dmdA, lies immediately upstream of acuI and in the model Roseobacter strain Ruegeria pomeroyi DSS-3, dmdA-acuI were co-regulated in response to the co-inducer, acrylate. These observations, together with findings by others that AcuI has acryloyl-CoA reductase activity, lead us to suggest that YdhH/AcuI enzymes protect cells against damaging effects of intracellular acryloyl-CoA, formed endogenously, and/or via catabolising exogenous acrylate. To provide “added protection” for bacteria that form acrylate from DMSP, acuI was recruited into clusters of genes involved in this conversion and, in the case of acuI and dmdA in the Roseobacters, their co-expression may underpin an interaction between the two routes of DMSP catabolism, whereby the acrylate product of DMSP lyases is a co-inducer for the demethylation pathway

Unusual Regulation of a Leaderless Operon Involved in the Catabolism of Dimethylsulfoniopropionate in Rhodobacter sphaeroides

Rhodobacter sphaeroides strain 2.4.1 is a widely studied bacterium that has recently been shown to cleave the abundant marine anti-stress molecule dimethylsulfoniopropionate (DMSP) into acrylate plus gaseous dimethyl sulfide. It does so by using a lyase encoded by dddL, the promoter-distal gene of a three-gene operon, acuR-acuI-dddL. Transcription of the operon was enhanced when cells were pre-grown with the substrate DMSP, but this induction is indirect, and requires the conversion of DMSP to the product acrylate, the bona fide co-inducer. This regulation is mediated by the product of the promoter-proximal gene acuR, a transcriptional regulator in the TetR family. AcuR represses the operon in the absence of acrylate, but this is relieved by the presence of the co-inducer. Another unusual regulatory feature is that the acuR-acuI-dddL mRNA transcript is leaderless, such that acuR lacks a Shine-Dalgarno ribosomal binding site and 5′-UTR, and is translated at a lower level compared to the downstream genes. This regulatory unit may be quite widespread in bacteria, since several other taxonomically diverse lineages have adjacent acuR-like and acuI-like genes; these operons also have no 5′ leader sequences or ribosomal binding sites and their predicted cis-acting regulatory sequences resemble those of R. sphaeroides acuR-acuI-dddL