A C-terminal cysteine residue is required for peptide-based inhibition of the NGF/TrkA interaction at nM concentrations:implications for peptide-based analgesics

Inhibition of the NGF/TrkA interaction presents an interesting alternative to the use of non-steroidal anti-inflammatories and/or opioids for the control of inflammatory, chronic and neuropathic pain. Most prominent of the current approaches to this therapy is the antibody Tanezumab, which is a late-stage development humanized monoclonal antibody that targets NGF. We sought to determine whether peptides might similarly inhibit the NGF/TrkA interaction and so serve as future therapeutic leads. Starting from two peptides that inhibit the NGF/TrkA interaction, we sought to eliminate a cysteine residue close to the C-terminal of both sequences, by an approach of mutagenic analysis and saturation mutagenesis of mutable residues. Elimination of cysteine from a therapeutic lead is desirable to circumvent manufacturing difficulties resulting from oxidation. Our analyses determined that the cysteine residue is not required for NGF binding, but is essential for inhibition of the NGF/TrkA interaction at pharmacologically relevant peptide concentrations. We conclude that a cysteine residue is required within potential peptide-based therapeutic leads and hypothesise that these peptides likely act as dimers, mirroring the dimeric structure of the TrkA receptor

Anti-nociceptive and desensitizing effects of olvanil on capsaicin-induced thermal hyperalgesia in the rat

Background: Olvanil (NE 19550) is a non-pungent synthetic analogue of capsaicin, the natural pungent ingredient of capsicum which activates the transient receptor potential vanilloid type-1 (TRPV1) channel and was developed as a potential analgesic compound. Olvanil has potent anti-hyperalgesic effects in several experimental models of chronic pain. Here we report the inhibitory effects of olvanil on nociceptive processing using cultured dorsal root ganglion (DRG) neurons and compare the effects of capsaicin and olvanil on thermal nociceptive processing in vivo; potential contributions of the cannabinoid CB1 receptor to olvanil’s anti-hyperalgesic effects were also investigated. Methods: A hot plate analgesia meter was used to evaluate the anti-nociceptive effects of olvanil on capsaicin-induced thermal hyperalgesia and the role played by CB1 receptors in mediating these effects. Single cell calcium imaging studies of DRG neurons were employed to determine the desensitizing effects of olvanil on capsaicin-evoked calcium responses. Statistical analysis used Student’s t test or one way ANOVA followed by Dunnett’s post-hoctest as appropriate. Results: Both olvanil (100 nM) and capsaicin (100 nM) produced significant increases in intracellular calcium concentrations [Ca2+]I in cultured DRG neurons. Olvanil was able to des ensitise TRPV1 responses to further capsaicin exposure more effectively than capsaicin. Intra plantar injection of capsaicin (0.1, 0.3 and 1μg) produced a robust TRPV1-dependant thermal hyperalgesia in rats, whilst olvanil (0.1, 0.3 and 1μg) produced no hyperalgesia, emphasizing its lack of pungency. The highest dose of olvanil significantly reduced the hyperalgesic effects of capsaicin in vivo. Intraplantar injection of the selective cannabinoid CB1 receptor antagonist rimonabant (1μg) altered neither capsaicin-induced thermal hyperalgesia nor the desensitizing properties of olvanil, indicating a lack of involvement of CB1receptors. Conclusions: Olvanil is effective in reducing capsaicin-induced thermal hyperalgesia, probably via directly desensitizingTRPV1 channels in a CB 1 receptor-independent fashion. The results presented clearly support the potential for olvanil in the development of new topical analgesic preparations for treating chronic pain conditions while avoiding the unwanted side effects of capsaicin treatments

Eye-Hand Coordination during Dynamic Visuomotor Rotations

Background for many technology-driven visuomotor tasks such as tele-surgery, human operators face situations in which the frames of reference for vision and action are misaligned and need to be compensated in order to perform the tasks with the necessary precision. The cognitive mechanisms for the selection of appropriate frames of reference are still not fully understood. This study investigated the effect of changing visual and kinesthetic frames of reference during wrist pointing, simulating activities typical for tele-operations. Methods using a robotic manipulandum, subjects had to perform center-out pointing movements to visual targets presented on a computer screen, by coordinating wrist flexion/extension with abduction/adduction. We compared movements in which the frames of reference were aligned (unperturbed condition) with movements performed under different combinations of visual/kinesthetic dynamic perturbations. The visual frame of reference was centered to the computer screen, while the kinesthetic frame was centered around the wrist joint. Both frames changed their orientation dynamically (angular velocity\u200a=\u200a36\ub0/s) with respect to the head-centered frame of reference (the eyes). Perturbations were either unimodal (visual or kinesthetic), or bimodal (visual+kinesthetic). As expected, pointing performance was best in the unperturbed condition. The spatial pointing error dramatically worsened during both unimodal and most bimodal conditions. However, in the bimodal condition, in which both disturbances were in phase, adaptation was very fast and kinematic performance indicators approached the values of the unperturbed condition. Conclusions this result suggests that subjects learned to exploit an \u201caffordance\u201d made available by the invariant phase relation between the visual and kinesthetic frames. It seems that after detecting such invariance, subjects used the kinesthetic input as an informative signal rather than a disturbance, in order to compensate the visual rotation without going through the lengthy process of building an internal adaptation model. Practical implications are discussed as regards the design of advanced, high-performance man-machine interfaces

Intrinsic genetic characteristics determine tumor-modifying capacity of fibroblasts: matrix metalloproteinase-3 5A/5A genotype enhances breast cancer cell invasion

Background Stromal fibroblasts can contribute to tumor invasion through the release of matrix metalloproteinases (MMPs). Population studies have suggested that single nucleotide polymorphisms (SNPs) in MMP genes influence levels of expression and may be associated with breast cancer risk and with disease progression. This study directly examined the impact of MMP SNP genotype on the ability of host fibroblasts to promote tumor cell invasion. Methods Primary breast fibroblasts were isolated from patients with (n = 13) or without (n = 19) breast cancer, and their ability to promote breast cancer cell invasion was measured in in vitro invasion assays. Fibroblast invasion-promoting capacity (IPC) was analyzed in relation to donor type (tumor or non-tumor patient), MMP-1, MMP-3, and MMP-9 SNP genotype and MMP activity using independent samples t test and analysis of variance. All statistical tests were two-sided. Results Tumor-derived fibroblasts promoted higher levels of invasion than normal fibroblasts (p = 0.041). When IPC was related to genotype, higher levels of IPC were generated by tumor fibroblasts with the high-expressing MMP-3 5A/5A genotype compared with the 5A/6A and 6A/6A genotypes (p = 0.05 and 0.07, respectively), and this was associated with enhanced MMP-3 release. The functional importance of MMP-3 was demonstrated by enhanced invasion in the presence of recombinant MMP-3, whereas reduction occurred in the presence of a specific MMP-3 inhibitor. An inverse relationship was demonstrated between fibroblast IPC and the high-expressing MMP-1 genotype (p = 0.031), but no relationship was seen with MMP-9 SNP status. In contrast, normal fibroblasts showed no variation in IPC in relation to MMP genotype, with MMP-3 5A/5A fibroblasts exhibiting significantly lower levels of IPC than their tumor-derived counterparts (p = 0.04). Conclusion This study has shown that tumor-derived fibroblasts exhibit higher levels of IPC than normal fibroblasts and that the MMP-3 5A/5A genotype contributes to this through enhanced MMP-3 release. Despite a high-expressing genotype, normal fibroblasts do not exhibit higher IPC or enhanced MMP release. This suggests that more complex changes occur in tumor-derived fibroblasts, enabling full expression of the MMP SNP genotype and these possibly are epigenetic in nature. The results do suggest that, in women with breast cancer, a high-expressing MMP-3 genotype may promote tumor progression more effectively

Selective Targeting of TRPV1 Expressing Sensory Nerve Terminals in the Spinal Cord for Long Lasting Analgesia

Chronic pain is a major clinical problem and opiates are often the only treatment, but they cause significant problems ranging from sedation to deadly respiratory depression. Resiniferatoxin (RTX), a potent agonist of Transient Receptor Potential Vanilloid 1 (TRPV1), causes a slow, sustained and irreversible activation of TRPV1 and increases the frequency of spontaneous excitatory postsynaptic currents, but causes significant depression of evoked EPSCs due to nerve terminal depolarization block. Intrathecal administration of RTX to rats in the short-term inhibits nociceptive synaptic transmission, and in the long-term causes a localized, selective ablation of TRPV1-expressing central sensory nerve terminals leading to long lasting analgesia in behavioral models. Since RTX actions are selective for central sensory nerve terminals, other efferent functions of dorsal root ganglion neurons can be preserved. Preventing nociceptive transmission at the level of the spinal cord can be a useful strategy to treat chronic, debilitating and intractable pain

Correlating Global Gene Regulation to Angiogenesis in the Developing Chick Extra-Embryonic Vascular System

International audienceBACKGROUND: Formation of blood vessels requires the concerted regulation of an unknown number of genes in a spatial-, time- and dosage-dependent manner. Determining genes, which drive vascular maturation is crucial for the identification of new therapeutic targets against pathological angiogenesis. METHOLOGY/PRINCIPAL FINDINGS: We accessed global gene regulation throughout maturation of the chick chorio-allantoic membrane (CAM), a highly vascularized tissue, using pan genomic microarrays. Seven percent of analyzed genes showed a significant change in expression (>2-fold, FDR<5%) with a peak occurring from E7 to E10, when key morphogenetic and angiogenic genes such as BMP4, SMO, HOXA3, EPAS1 and FGFR2 were upregulated, reflecting the state of an activated endothelium. At later stages, a general decrease in gene expression occurs, including genes encoding mitotic factors or angiogenic mediators such as CYR61, EPAS1, MDK and MYC. We identified putative human orthologs for 77% of significantly regulated genes and determined endothelial cell enrichment for 20% of the orthologs in silico. Vascular expression of several genes including ENC1, FSTL1, JAM2, LDB2, LIMS1, PARVB, PDE3A, PRCP, PTRF and ST6GAL1 was demonstrated by in situ hybridization. Up to 9% of the CAM genes were also overexpressed in human organs with related functions, such as placenta and lung or the thyroid. 21-66% of CAM genes enriched in endothelial cells were deregulated in several human cancer types (P<.0001). Interfering with PARVB (encoding parvin, beta) function profoundly changed human endothelial cell shape, motility and tubulogenesis, suggesting an important role of this gene in the angiogenic process. CONCLUSIONS/SIGNIFICANCE: Our study underlines the complexity of gene regulation in a highly vascularized organ during development. We identified a restricted number of novel genes enriched in the endothelium of different species and tissues, which may play crucial roles in normal and pathological angiogenesis

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030