Comparison of microarray-predicted closest genomes to sequencing for poliovirus vaccine strain similarity and influenza A phylogeny.

We evaluate sequence data from the PathChip high-density hybridization array for epidemiological interpretation of detected pathogens. For influenza A, we derive similar relative outbreak clustering in phylogenetic trees from PathChip-derived compared to classical Sanger-derived sequences. For a positive polio detection, recent infection could be excluded based on vaccine strain similarity.

Pathogen chip for respiratory tract infections.

Determining the viral etiology of respiratory tract infections (RTI) has been limited for the most part to specific primer PCR-based methods due to their increased sensitivity and specificity compared to other methods, such as tissue culture. However, specific primer approaches have limited the ability to fully understand the diversity of infecting pathogens. A pathogen chip system (PathChip), developed at the Genome Institute of Singapore (GIS), using a random-tagged PCR coupled to a chip with over 170,000 probes, has the potential to recognize all known human viral pathogens. We tested 290 nasal wash specimens from Filipino children <2 years of age with respiratory tract infections using culture and 3 PCR methods-EraGen, Luminex, and the GIS PathChip. The PathChip had good diagnostic accuracy, ranging from 85.9% (95% confidence interval [CI], 81.3 to 89.7%) for rhinovirus/enteroviruses to 98.6% (95% CI, 96.5 to 99.6%) for PIV 2, compared to the other methods and additionally identified a number of viruses not detected by these methods.

Over expression of wild type or a catalytically dead mutant of Sirtuin 6 does not influence NFκB responses.

SIRT6 is involved in inflammation, aging and metabolism potentially by modulating the functions of both NFκB and HIF1α. Since it is possible to make small molecule activators and inhibitors of Sirtuins we wished to establish biochemical and cellular assays both to assist in drug discovery efforts and to validate whether SIRT6 represents a valid drug target for these indications. We confirmed in cellular assays that SIRT6 can deacetylate acetylated-histone H3 lysine 9 (H3K9Ac), however this deacetylase activity is unusually low in biochemical assays. In an effort to develop alternative assay formats we observed that SIRT6 overexpression had no influence on TNFα induced nuclear translocation of NFκB, nor did it have an effect on nuclear mobility of RelA/p65. In an effort to identify a gene expression profile that could be used to identify a SIRT6 readout we conducted genome-wide expression studies. We observed that overexpression of SIRT6 had little influence on NFκB-dependent genes, but overexpression of the catalytically inactive mutant affected gene expression in developmental pathways.

Differential effects of taurine treatment and taurine deficiency on the outcome of renal ischemia reperfusion injury.

Taurine possesses membrane stabilization, osmoregulatory and antioxidant properties, aspects of relevance to ischemic injury. We tested the hypothesis that body taurine status is a determinant of renal ischemic injury. Accordingly, renal function and structure were examined in control (C), taurine-treated (TT) and taurine deficient (TD) rats that were subjected to bilateral renal ischemia (60 min) followed by reperfusion (IR); sham operated rats served as controls. Baseline urine osmolality was greater in the TD group than in the control and the TT groups, an effect associated with increased renal aquaporin 2 level. The IR insult reduced urine osmolality (i.e., day-1 post insult); the TD/IR group displayed a more marked recovery in urine osmolality by day-6 post insult than the other two groups. Fluid and sodium excretions were lower in the TD/IR group, suggesting propensity to retention. Histopathological examination revealed the presence of tubular necrotic foci in the C/IR group than sham controls. While renal architecture of the TD/IR group showed features resembling sham controls, the TT/IR group showed dilated tubules, which lacked immunostaining for aquaporin 2, but not 1, suggestive of proximal tubule origin. Finally, assessment of cell proliferation and apoptosis revealed lower proliferation but higher apoptotic foci in the TT/IR group than other IR groups. Collectively, the results indicate that body taurine status is a major determinant of renal IR injury.

Antithrombotic potential of GW813893: a novel, orally active, active-site directed factor Xa inhibitor.

BACKGROUND: Factor Xa (FXa) has been a target of considerable interest for drug development efforts aimed at suppressing thrombosis. In this report, a new orally active, small molecule, active-site directed FXa inhibitor, GW813893, has been profiled in a succession of in vitro and in vivo assays involved in its preclinical characterization as a potential antithrombotic therapeutic. METHODS: In vitro profiling of GW813893 consisted of assessing its inhibitory potential against FXa and a broad panel of related and unrelated enzymes and receptors. Additionally, the FXa inhibition potential of GW813893 was assessed in prothrombinase and plasma-based clotting assays. In vivo characterization of GW813893 consisted of thrombosis studies in a rat inferior vena cava model, a rat carotid artery thrombosis model, and a rabbit jugular thrombosis model. Bleeding studies were conducted in a rat tail transection model. Ex vivo determinations of compound effects on FX and clotting activity were also undertaken. RESULTS: GW813893 was more than 90-fold selective over all enzymes tested, and it inhibited FXa and prothrombinase activity with a Ki of 4.0 nM and 9.7 nM, respectively. In vivo, GW813893 concentration-dependently suppressed thrombotic activity in all models tested. The antithrombotic activity correlated with the suppression of plasma-based clotting activity and the inhibition of plasma FX activity (P < 0.02). Over the antithrombotic dose-range, an increased bleeding diathesis was not observed. CONCLUSION: These experiments demonstrate that GW813893 is a potent, selective, orally active inhibitor of FXa. The data suggest that GW813893 has robust antithrombotic potential at doses that have no detectable hemostasis liability. Collectively, the profile suggests that GW813893 has the preclinical pharmacology underpinnings of an oral antithrombotic therapeutic.

The discovery of 2-amino-3,5-diarylbenzamide inhibitors of IKK-alpha and IKK-beta kinases.

A potent and selective series of 2-amino-3,5-diarylbenzamide inhibitors of IKK-alpha and IKK-beta is described. The most potent compounds are 8h, 8r and 8v, with IKK-beta inhibitory potencies of pIC(50) 7.0, 6.8 and 6.8, respectively. The series has excellent selectivity, both within the IKK family over IKK-epsilon, and across a wide variety of kinase assays. The potency of 8h in the IKK-beta enzyme assay translates to significant cellular activity (pIC(50) 5.7-6.1) in assays of functional and mechanistic relevance.

Factor Xa inhibitors: S1 binding interactions of a series of N-{(3S)-1-[(1S)-1-methyl-2-morpholin-4-yl-2-oxoethyl]-2-oxopyrrolidin-3-yl}sulfonamides.

Factor Xa inhibitory activities for a series of N-{(3S)-1-[(1S)-1-methyl-2-morpholin-4-yl-2-oxoethyl]-2-oxopyrrolidin-3-yl}sulfonamides with different P1 groups are described. These data provide insight into binding interactions within the S1 primary specificity pocket; rationales are presented for the derived SAR on the basis of electronic interactions through crystal structures of fXa-ligand complexes and molecular modeling studies. A good correlation between in vitro anticoagulant activities with lipophilicity and the extent of human serum albumin binding is observed within this series of potent fXa inhibitors. Pharmacokinetic profiles in rat and dog, together with selectivity over other trypsin-like serine proteases, identified 1f as a candidate for further evaluation.

Construction of a Saccharomyces cerevisiae strain expressing the Leishmania major nucleoside hydrolase gene.

Nucleoside hydrolase (NH) (EC 3.2.2.3) is an essential enzyme in the purine-pyrimidine salvage pathway utilised by many protozoan parasites and may be a useful drug target. However, the search for NH inhibitors has been hampered by the lack of suitable in vitro screens. We have constructed a Saccharomyces cerevisiae strain that requires expression of the Leishmania major nucleoside hydrolase (LmNH) enzyme for growth and that may be suitable as a screen for NH inhibitors. The gene encoding LmNH was amplified using polymerase chain reaction with L. major genomic DNA as the template, cloned into an expression vector and used to transform a yeast mutant unable to grow on uridine as the sole source of uracil. Expression of LmNH yielded an ca. 35.6kDa protein, which was shown to be functional as the mutant strain was able to grow on medium containing uridine as the sole source of uracil. Importantly, this work has resulted in a strain that can be used to screen compounds as potential inhibitors of this essential Leishmania enzyme.

Effects of dietary salt and fat on taurine excretion in healthy and diseased rats.

Taurine modulates renal and cardiovascular function. Although the kidney regulates body taurine status, the impact of renal and cardiovascular risk factors, such as dietary intake of excess NaCl and saturated fat, on renal handling of taurine is less clear. One would predict that the kidney would modulate taurine excretion during dietary NaCl excess to insure adequate osmotic homeostasis. Similarly, fat feeding would be expected to affect taurine homeostasis, as taurine is involved in bile acid conjugation and therefore fat emulsification. To examine these aspects, male rats were divided into four groups: basal fat diet (control), high fat diet (FAT), basal fat and high salt diet (SALT) and a combination of a high fat and salt diet (FATSALT). While the control, FAT and SALT groups excreted similar amounts of taurine; the SALTFAT group excreted significantly more taurine than the other 3 groups. Although all of the dietary regimens increased renal tissue content of taurine, the increases were greatest in the two SALT groups. In a subsequent study, we examined the effect of excess dietary fat on taurine handling by the hypertensive (H) and hypertensive-glucose intolerant (HGI) rat. When fed a basal fat diet, the HGI group excreted more taurine than the H group, an effect likely related to increased endogenous taurine biosynthesis, alterations in renal function or a combination of the two effects. While excess fat intake increased urinary taurine excretion in the H group, it reduced taurine excretion in the HGI group. Nonetheless, kidney taurine content was similar in the 4 groups. Taken together, the data suggest that dietary constituents and preexisting systemic disorders are important modulators of renal handling of taurine.

Structure- and property-based design of factor Xa inhibitors: pyrrolidin-2-ones with acyclic alanyl amides as P4 motifs.

Structure-based drug design was exploited in the synthesis of 3-(6-chloronaphth-2-ylsulfonyl)aminopyrrolidin-2-one-based factor Xa (fXa) inhibitors, incorporating an alanylamide P4 group with acyclic tertiary amide termini. Optimized hydrophobic contacts of one amide substituent in P4 were complemented by hydrophobicity-modulating features in the second, producing potent fXa inhibitors including examples with excellent anticoagulant properties.

5-(1H-Benzimidazol-1-yl)-3-alkoxy-2-thiophenecarbonitriles as potent, selective, inhibitors of IKK-epsilon kinase.

The identification and hit-to-lead exploration of a novel, potent and selective series of substituted benzimidazole-thiophene carbonitrile inhibitors of IKK-epsilon kinase is described. Compound 12e was identified with an IKK-epsilon enzyme potency of pIC(50) 7.4, and has a highly encouraging wider selectivity profile, including selectivity within the IKK kinase family.

Design and synthesis of orally active pyrrolidin-2-one-based factor Xa inhibitors.

A series of novel, non-basic 3-(6-chloronaphth-2-ylsulfonyl)aminopyrrolidin-2-one-based factor Xa (fXa) inhibitors, incorporating an alanylamide P4 group, was designed and synthesised. Within this series, the N-2-(morpholin-4-yl)-2-oxoethyl derivative 24 was shown to be a potent, selective fXa inhibitor with good anticoagulant activity. Moreover, 24 possessed highly encouraging rat and dog pharmacokinetic profiles with excellent oral bioavailabilities in both species.

Mechanisms underlying afterload-induced exacerbation of myocardial infarct size: role of T-type Ca2+ channel.

One consequence of elevated afterload pressure is the activation of the angiotensin II type 1 receptor and nonspecific cation channels with subsequent Ca2+ accumulation via the Na+/H(+)-Na+/Ca2+ exchanger combination and the T-type or L-type Ca2+ channels. Intracellular Ca2+ overload is cytotoxic, in part, by inducing the mitochondrial permeability transition (MPT) pore. Therefore, we tested the hypotheses that: (1) increased afterload pressure worsens myocardial ischemia-reperfusion injury in healthy heart, (2) the Na+/H(+)-Na+/Ca2+ exchanger combination and both the T-type and L-type Ca2+ channels are involved in the exacerbating impact of high afterload pressure on infarct size, and (3) elevated afterload enhances infarct size in part via the MPT pore. Accordingly, the effect of candesartan (angiotensin II type 1 receptor antagonist), cariporide (inhibitor of the Na+/H+ exchanger), mibefradil (T-type Ca2+ channel blocker), diltiazem (L-type Ca2+ channel blocker), or cyclosporine A (inhibitor of MPT pore) were examined. The elevation in afterload pressure from 80 to 160 cmH2O increased baseline myocardial performance but caused larger infarcts and worsened recovery of mechanical function after ischemia reperfusion. Whereas mibefradil abrogated the effect of high afterload pressure on infarct size, the other agents reduced infarct size at both afterload pressures. Hearts exposed to mibefradil, diltiazem, or cariporide displayed greater functional recovery than those exposed to candesartan or cyclosporine A, revealing that an uncoupling exists between reduced cell death and recovery of mechanical function of the viable portions of the myocardium. The data also uncovered an important link between pressure-mediated exacerbation of infarct size and T-type Ca2+ channel activity.

Effects of excess salt and fat intake on myocardial function and infarct size in rat.

Important risk factors for cardiovascular disease include excess dietary intake of saturated fat and (or) salt. This study tested the hypothesis that excess intakes of saturated fat (e.g., beef tallow) and salt cause greater myocardial cell death following ischemia-reperfusion injury than each risk factor alone. Male rats were divided into four groups: basal fat diet (4.5% as calories; control), high fat diet (40% as calories; FAT), basal fat diet and high salt (1% NaCl solution; SALT) and high fat diet and high salt (FATSALT). The gain in body weight was significantly higher for FAT and FATSALT groups than those of either the control or the SALT group. Five weeks of exposure to the dietary regimens did not significantly affect the coronary flow rate and except for the salt-fed group, had no effect on the rate-pressure-product of the isolated heart perfused in Langendorff mode. Although infarct size was not affected by the high fat diet, it was reduced by the high salt regimen relative to the high fat diet or the control groups. When rats were fed the FAT and SALT combination, the effect of salt feeding on infarct size was not observed. In addition, the FATSALT group displayed a more marked deterioration in contractile function following ischemia-reperfusion injury than the other groups. In conclusion, short-term intake of a high fat diet, which significantly increases body weight, does not worsen ischemia-reperfusion injury although the treatment prevents the reduction of infarct size associated with high salt feeding.

Effects of chronic chromium picolinate treatment in uninephrectomized rat.

Chromium picolinate [Cr(pic)3] is a nutritional supplement that is advocated as an adjuvant therapy for impaired glucose tolerance/type 2 diabetes because it improves glucose homeostasis. Because renal dysfunction is a major complication of type 2 diabetes, the potential impact of Cr(pic)3 on kidney function requires due consideration. This investigation takes added importance because the kidney is not only the principal route of elimination for chromium but also an organ that preferentially accumulates it. To avoid the confounding influence of chronic hyperglycemia, and its associated complications, we used the unilaterally nephrectomized (UNX) rat that shows impaired kidney function with age. We tested the hypothesis that chronic treatment of the UNX rat with Cr(pic)3 exacerbates the age-related decline in renal function. Accordingly, UNX rats were fed a diet lacking (eg, control; n=5) or containing 5 mg/kg of Cr(pic)3 (n=7) for 60 days. The treatment did not affect glucose tolerance as reflected by lack of any effect on changes in blood glucose concentration during glucose tolerance testing. Although nonfasting blood glucose concentrations were similar between the 2 groups, plasma insulin concentration was lower in the Cr(pic)3-treated group (P<.05), suggesting improved insulin sensitivity. Body weight, blood pressure, heart rate, daily food and fluid consumption, daily urinary fluid and electrolyte excretions, urine osmolality, and daily protein excretion were similar between the 2 groups before and during Cr(pic)3 treatment. Although renal excretory responses to acute administration of a 5% isotonic saline volume load were similar between the 2 groups, the Cr(pic)3-treated group displayed a more robust ability to excrete a 10% isotonic saline volume load, an effect primarily related to reduced tubular reabsorption of the filtered fluid and sodium loads. In conclusion, chronic Cr(pic)3 did not adversely affect renal function. Rather, the treatment improved the ability of the animal to dispose of an acute isotonic saline volume load, suggesting preservation of renal function in the UNX rat.