Peptide aptamers: tools for biology and drug discovery.

Peptide aptamer technology is relatively youthful. It has the advantage over existing techniques that the reagents identified are designed for expression in eukaryotic cells. This allows the construction of molecular tools that allow the logic of genetics, from knockouts to extragenic suppressors, to be applied to studies of proteins in tissue culture cells. Until recently, the available tools have limited our understanding of cell biology. The same limitation restricts out ability to validate the numerous candidate drug targets emerging from genome-wide approaches to cellular biology. Peptide aptamers represent a stride forwards in the evolution of a modular, molecular tool kit for cell biology and for drug target validation. The authors predict that they will also play a role in the transition from genomic to proteomic microarray technology.     

Effects of inhibitors of eicosanoid synthesis on insulin release by neonatal pancreatic islets

In the pancreatic islet, eicosanoids may arise from both cyclooxygenase- and lipoxygenase-dependent metabolism of arachidonic acid. The inclusion of inhibitors of selective steps in these pathways indicated that in cultured neonatal rat islets, arachidonic acid may be metabolised through both pathways, concurrent with insulin release stimulated by D-glucose, D-glyceraldehyde and 2-ketoisocaproate. The effects of the inhibitors suggested that the products of the lipoxygenase pathway were necessary for the stimulatory effects of nutrients to be observed. In contrast to glucose, where insulin release was stimulated in the presence of inhibitors of cyclooxygenase, the stimulatory action of D-glyceraldehyde, 2-ketoisocaproate and melittin was only minimally affected by these inhibitors, although it was inhibited by lipoxygenase inhibition. These findings support a major stimulatory role for products of the lipoxygenase pathway of arachidonic acid metabolism in nutrient-induced secretion, and a negative or modulatory role of cyclooxygenase pathway products on glucose-stimulated insulin release in the neonatal islet.     

Data-driven method to infer the seizure propagation patterns in an epileptic brain from intracranial electroencephalography

Surgical interventions in epileptic patients aimed at the removal of the epileptogenic zone have success rates at only 60-70%. This failure can be partly attributed to the insufficient spatial sampling by the implanted intracranial electrodes during the clinical evaluation, leading to an incomplete picture of spatio-temporal seizure organization in the regions that are not directly observed. Utilizing the partial observations of the seizure spreading through the brain network, complemented by the assumption that the epileptic seizures spread along the structural connections, we infer if and when are the unobserved regions recruited in the seizure. To this end we introduce a data-driven model of seizure recruitment and propagation across a weighted network, which we invert using the Bayesian inference framework. Using a leave-one-out cross-validation scheme on a cohort of 45 patients we demonstrate that the method can improve the predictions of the states of the unobserved regions compared to an empirical estimate that does not use the structural information, yet it is on the same level as the estimate that takes the structure into account. Furthermore, a comparison with the performed surgical resection and the surgery outcome indicates a link between the inferred excitable regions and the actual epileptogenic zone. The results emphasize the importance of the structural connectome in the large-scale spatio-temporal organization of epileptic seizures and introduce a novel way to integrate the patient-specific connectome and intracranial seizure recordings in a whole-brain computational model of seizure spread.     

Interleukin-10 Mediated Autoregulation of Murine B-1 B-Cells and Its Role in Borrelia hermsii Infection

B cells are typically characterized as positive regulators of the immune response, primarily by producing antibodies. However, recent studies indicate that various subsets of B cells can perform regulatory functions mainly through IL-10 secretion. Here we discovered that peritoneal B-1 (B-1P) cells produce high levels of IL-10 upon stimulation with several Toll-like receptor (TLR) ligands. High levels of IL-10 suppressed B-1P cell proliferation and differentiation response to all TLR ligands studied in an autocrine manner in vitro and in vivo. IL-10 that accumulated in cultures inhibited B-1P cells at second and subsequent cell divisions mainly at the G1/S interphase. IL-10 inhibits TLR induced B-1P cell activation by blocking the classical NF-κB pathway. Co-stimulation with CD40 or BAFF abrogated the IL-10 inhibitory effect on B-1P cells during TLR stimulation. Finally, B-1P cells adoptively transferred from the peritoneal cavity of IL-10^−/− mice showed better clearance of Borrelia hermsii than wild-type B-1P cells. This study described a novel autoregulatory property of B-1P cells mediated by B-1P cell derived IL-10, which may affect the function of B-1P cells in infection and autoimmunity.     

Exercise training improves femoral artery blood flow responses to endothelium-dependent dilators in hypercholesterolemic pigs

We tested two hypotheses: 1) that the effects of hypercholesterolemia on endothelial function in femoral arteries exceed those reported in brachial arteries and 2) that exercise (Ex) training enhances endothelium-dependent dilation and improves femoral artery blood flow (FABF) in hypercholesterolemic pigs. Adult male pigs were fed a normal fat (NF) or high-fat/cholesterol (HF) diet for 20 wk. Four weeks after the diet was initiated, pigs were Ex trained or remained sedentary (Sed) for 16 wk, thus yielding four groups: NF-Sed, NF-Ex, HF-Sed, and HF-Ex. Endothelium-dependent vasodilator responses were assessed in vivo by measuring changes in FABF after intra-arterial injections of ADP and bradykinin (BK). Endothelium-dependent and -independent relaxation was assessed in vitro by measuring relaxation responses to BK and sodium nitroprusside (SNP). FABF increased in response to ADP and BK in all groups. FABF responses to ADP and BK were not impaired by HF but were improved by Ex in HF pigs. BK- and SNP-induced relaxation of femoral artery rings was not altered by HF or Ex. To determine whether the mechanism(s) for vasorelaxation of femoral arteries was altered by HF or Ex, BK-induced relaxation was assessed in vitro in the absence or presence of N(G)-nitro-l-arginine methyl ester [l-NAME; to inhibit nitric oxide synthase (NOS)], indomethacin (Indo; to inhibit cyclooxygenase), or l-NAME + Indo. BK-induced relaxation was inhibited by l-NAME and l-NAME + Indo in all groups of femoral arteries. Ex increased the NOS-dependent component of endothelium-dependent relaxation in NF (not HF) arteries. Indo did not inhibit BK-induced relaxation. Collectively, these results indicate that hypercholesterolemia does not alter endothelial function in femoral arteries and that Ex training improves FABF responses to ADP and BK; however, the improvement cannot be attributed to enhanced endothelial function in HF femoral arteries. These data suggest that Ex-induced improvements in FABF in HF arteries are mediated by vascular adaptations in arteries/arterioles downstream from the femoral artery.     

Virological and immunological factors associated with HIV-1 differential disease progression in HLA-B 58:01-positive individuals

Molecular epidemiology studies have identified HLA-B 58:01 as a protective HIV allele. However, not all B 58:01-expressing persons exhibit slow HIV disease progression. We followed six HLA-B 58:01-positive, HIV subtype C-infected individuals for up to 31 months from the onset of infection and observed substantial variability in their clinical progression despite comparable total breadths of T cell responses. We therefore investigated additional immunological and virological factors that could explain their different disease trajectories. Cytotoxic T-lymphocyte (CTL) responses during acute infection predominantly targeted the TW10 and KF9 epitopes in p24(Gag) and Nef, respectively. Failure to target the TW10 epitope in one B 58:01-positive individual was associated with low CD4(+) counts and rapid disease progression. Among those targeting TW10, escape mutations arose within 2 to 15 weeks of infection. Rapid escape was associated with preexisting compensatory mutations in the transmitted viruses, which were present at a high frequency (69%) in the study population. At 1 year postinfection, B 58:01-positive individuals who targeted and developed escape mutations in the TW10 epitope (n = 5) retained significantly higher CD4(+) counts (P = 0.04), but not lower viral loads, than non-B 58:01-positive individuals (n = 17). The high population-level frequency of these compensatory mutations may be limiting the protective effect of the B 58:01 allele.     

Synthesis of a hypoxia-targeted conjugate of the cardioprotective agent 3',4'-dihydroxyflavonol and evaluation of its ability to reduce ischaemia/reperfusion injury

3′,4′-Dihydroxyflavonol (DiOHF) is a cardioprotective flavonol that reduces injury associated with myocardial ischaemia and reperfusion. We hypothesized that the efficacy of DiOHF could be enhanced through its targeting to hypoxic regions of partial reperfusion. Copper(I)-catalyzed ligation of an azide-modified DiOHF analogue to 2-propargyl-nitroimidazole afforded a DiOHF-nitroimidazole conjugate (DiOHF-NIm). When incubated with Con8 cells under normoxic conditions DiOHF-NIm could be detected in both the culture supernatant and cell lysate, whereas under hypoxic conditions it was present in substantially reduced amounts consistent with its selective metabolism under hypoxia. DiOHF-NIm possessed antioxidant activity comparable to DiOHF through scavenging of superoxide produced by NADPH/NADPH oxidase, but had significantly attenuated vasorelaxant activity. DiOHF-NIm treatment significantly reduced lactate dehydrogenase release following ischaemia/reperfusion in hindlimbs of anaesthetized rats (p <0.05), to a level similar to DiOHF treatment but also at earlier time points. DiOHF-NIm significantly reduced levels of myeloperoxidase (p <0.05), a biomarker of neutrophil accumulation, whereas the reduction afforded by DiOHF was not significant. DiOHF-NIm therefore represents a promising potential therapeutic for ischaemia/reperfusion injury.