Venomic Characterization of the Terebridae and Novel Terebrid Neuropeptides

Unravelling the complex mixture of neuropeptides produced by the terebrid venom duct holds the promise of discovering tomorrow\u27s therapeutics. Cone snails have already demonstrated the potential found in the venom of these unusual marine organisms, through the commercial approval of drugs for pain and other indications. Terebrids, as the sister family to the cone snails, have been much less investigated, but have a species richness that makes them very attractive in the search for novel neuropeptides. The venomics research described in this work encompasses the first comprehensive characterization of the terebrid venom duct transcriptomes of two species, Cinguloterebra anilis and Terebra subulata. De novo assembly and analysis were performed using next generation sequencing technology and state of the art bioinformatics tools to identify the cocktail of peptides, teretoxins, produced by the venom duct. These disulfide rich peptides often show a remarkable specificity for subtypes of ion channels and ligand-gated receptors, giving them therapeutic potential, but they are only available in vanishingly small amounts from the natural source. As a result, identification of teretoxins using next generation sequencing is a prelude to synthesizing them in sufficient quantities to test for bioactivity. Here recombinant expression and solid phase peptide synthesis have both been utilized for the synthesis of four different teretoxins, with a special focus on recombinant synthesis to design a reproducible strategy for synthesizing disulfide rich peptides greater than 40 amino acids in length. Preliminary characterization of bioactivity was performed by injecting synthesized toxin into the polychaete N. virens. A teretoxin identified from Terebra guttata, Tg77, has produced promising results in this assay, with repeated trials showing the effects of uncoordinated movement and rigid paralysis. Further testing on Tg77 and other teretoxins will be carried forward, with an evident need for high throughput assays to provide an efficient means for the testing of novel compounds with a variety of potential molecular targets

Literature search – Exploring in silico protein toxicity prediction methods to support the food and feed risk assessment

This report is the outcome of an EFSA procurement (NP/EFSA/GMO/2018/01) reviewing relevant scientific information on in silico prediction methods for protein toxicity, that could support the food and feed risk assessment. Several proteins are associated with adverse (toxic) effects in humans and animals, by a variety of mechanisms. These are produced by plants, animals and bacteria to prevail in hostile environments. In the present report, we present an integrated pipeline to perform a comprehensive literature and database search applied to proteins with toxic effects. \u201cToxin activity\u201d and \u201ctoxin-antitoxin system\u201d strings were used as inputs for this pipeline. UniProtKB was considered as the reference database, and only the UniProtKB curator-reviewed proteins were considered in the pipeline. Experimentally- determined structures and homology-based in silico 3D models were retrieved from protein structures repositories; family-, domain-, motif- and other molecular signature-related information was also obtained from specific databases which are part of the InterPro consortium. Protein aggregation associated with adverse effects was also investigated using different search strategies. This work can serve as the basis for further exploring novel risk assessment strategies for new proteins using in silico predictive methods

Slow transit constipation: clinical and aetiological studies.

PhDConstipation is the second most commonly self-reported gastrointestinal symptom. On the basis of anorectal physiological investigations and colonic transit studies, a subgroup of patients with several intractable symptoms, but without organic disease will be found to have slow transit constipation (STC). STC is a condition of gut dysmotility which predominantly affects young women, and may result in surgical intervention with variable, often unsatisfactory results. The aetiology remains elusive. New aetiological hypotheses for STC were examined following full clinical and pathophysiological characterisation of a large cohort of 130 patients referred to our institution over the last 10 years. Aspects of nerve and muscle dysfunction were studied. A new scoring system demonstrated some ability of multiple symptoms to discriminate STC from other forms of constipation. Detailed clinical and gastrointestinal physiological studies confirmed the heterogeneity of STC patients. Some significant physiological differences were detectable between clinically defined sub-groups of patients and refuted previous assumptions based on smaller numbers. Detailed neurophysiological studies, including quantitative peripheral sensory and autonomic testing, provided evidence of a small fibre neuropathy in a proportion of patients with STC. Mutational screening of some early-onset cases for a possible congenital pathogenetic mechanism, based on the observation that some STC patients had relatives with Hirschsprung's disease demonstrated that mutation of 2 important genes now implicated in this disorder were not a frequent cause of STC. Serum immunoprecipitation assays showed that anti-neuronal ion channel autoantibodies may have an as yet unrecognised role in the development of STC in a small proportion of acquired cases. An inclusion body myopathy was identifiable in colonic tissue of patients with STC, and this appeared to arise secondary to denervation. Further knowledge of the single or multiple pathogenetic mechanisms leading to this clinical condition may allow more rational or directed therapies aimed at the correction of the disease process or processes themselves

Chinese herbal medicine for psoriasis: evaluation of clinical evidence and investigation of the anti-psoriatic effects of specific Chinese medicinal herbs

Psoriasis is a chronic and recurrent skin disease that affects 1-5% of the population. Conventional medical treatments can have significant side effects. There is, as yet, no curative regimen for the disease. Around half of psoriasis patients use complementary and alternative medicine including herbal medicine (HM). Consequently, the efficacy and safety of these HMs needs to be systematically evaluated. The project aimed to evaluate the clinical efficacy and safety of HM for psoriasis, identify the promising herbs for psoriasis, investigate the anti-psoriatic actions of the most promising herbs, and develop an in silico method for investigating their biological targets and pathways. Study quality assessments, systematic reviews and meta-analyses were undertaken in the first component of the project. This component firstly analysed clinical trials of HMs used internally and the second part focussed on topical HMs. Due to the diversity of topical HMs, the second part was further divided into three sections based on their intervention types: single herb, multi-ingredient herbal formula, and HM plus anti-psoriatic pharmacotherapy (APP). Based on 39 studies, 12 herbs were selected as promising for psoriasis: Oldenlandia, Rehmannia, Salvia, Aloe, Indigo, Camptotheca, Mahonia, Sophora, Lithospermum, Cnidium, Dictamnus and borneol. The second component of the study focussed on the likely mechanisms of action of the identified herbs. Firstly, for each of the main herbs, their relevant biological properties were reviewed. This identified evidence for anti-inflammatory, anti-proliferative, anti-angiogenic, wound healing/skin repair and/or anti-pruritic actions for extracts of the plants and/or their bioactive constituents. Then, 482 compounds contained in the short-listed herbs and their associated species were identified using Encyclopaedia of TCM and other sources. Then 350 biological targets were located with HIT database for these compounds. 70 targets of APPs were identified from the DrugBank database from 20 APPs approved by FDA. They mainly included NR3C1, COX (1, 2), RAR (α, β, γ-1), RXR-α & RXR-β, CD2, TNF and VDR. The 350 biological targets were filtered by the 70 APP targets. Ten targets were common to the APPs and the herbs. After excluding the Cytochrome family of enzymes, 9 APP-like herbs were identified: Oldenlandia, Rehmannia, Salvia, Aloe, Indigo, Camptotheca, Mahonia, Sophora, Lithospermum. The database PANTHER was used to identify biological processes and pathways that involved targets of the four identified herbs: Salvia, Rehmannia, Indigo and Camptotheca. The main pathways were Apoptosis signalling pathway, Angiogenesis, Gonadotropin releasing hormone receptor pathway, Inflammation mediated by chemokine and cytokine signalling pathway, and Interleukin signalling pathway, which are primarily related to inflammation, proliferation and angiogenesis. The project identified promising herbs for psoriasis which showed potential APP-like actions and identified their likely mechanisms of action. The in silico rapid identification approach which was based on the results of meta-analyses of clinical trial outcomes was proposed as a more general method for adding value to the results of systematic reviews of herbal medicines and as an large-scale analysis solution to identifying directions for clinical trials and drug discovery