Clinico-radiological features, molecular spectrum, and identification of prognostic factors in developmental and epileptic encephalopathy due to inosine triphosphate pyrophosphatase (ITPase) deficiency

Developmental and epileptic encephalopathy 35 (DEE 35) is a severe neurological condition caused by biallelic variants in ITPA, encoding inosine triphosphate pyrophosphatase, an essential enzyme in purine metabolism. We delineate the genotypic and phenotypic spectrum of DEE 35, analyzing possible predictors for adverse clinical outcomes. We investigated a cohort of 28 new patients and reviewed previously described cases, providing a comprehensive characterization of 40 subjects. Exome sequencing was performed to identify underlying ITPA pathogenic variants. Brain MRI (magnetic resonance imaging) scans were systematically analyzed to delineate the neuroradiological spectrum. Survival curves according to the Kaplan–Meier method and log-rank test were used to investigate outcome predictors in different subgroups of patients. We identified 18 distinct ITPA pathogenic variants, including 14 novel variants, and two deletions. All subjects showed profound developmental delay, microcephaly, and refractory epilepsy followed by neurodevelopmental regression. Brain MRI revision revealed a recurrent pattern of delayed myelination and restricted diffusion of early myelinating structures. Congenital microcephaly and cardiac involvement were statistically significant novel clinical predictors of adverse outcomes. We refined the molecular, clinical, and neuroradiological characterization of ITPase deficiency, and identified new clinical predictors which may have a potentially important impact on diagnosis, counseling, and follow-up of affected individuals

International training programmes to support First Responders when attending the scene of a suspected suicide and engaging in postvention activities: A Scoping Review

This Scoping Review aims to systematically review the literature on international training programmes to support First Responders when attending the scene of a suspected suicide and engaging in postvention activities

Optimising expression of the recombinant fusion protein biopesticide ω-hexatoxin-Hv1a /GNA in Pichia pastoris: Sequence modifications and a simple method for the generation of multi-copy strains

Production of recombinant protein bio-insecticides on a commercial scale can only be cost effective if host strains with very high expression levels are available. A recombinant fusion protein containing an arthropod toxin, ω-hexatoxin-Hv1a, (from funnel web spider Hadronyche versuta) linked to snowdrop lectin (Galanthus nivalis agglutinin; GNA) is an effective oral insecticide and candidate biopesticide. However, the fusion protein was vulnerable to proteolysis during production in the yeast Pichia pastoris. To prevent proteolysis, the Hv1a/GNA fusion expression construct was modified by site-directed mutagenesis to remove a potential Kex2 cleavage site at the C-terminus of the Hv1a peptide. To obtain a high expressing clone of P. pastoris to produce recombinant Hv1a/GNA, a straightforward method was used to produce multi-copy expression plasmids, which does not require multiple integrations to give clones of P. pastoris containing high copy numbers of the introduced gene. Removal of the Kex2 site resulted in increased levels of intact fusion protein expressed in wild-type P. pastoris strains, improving levels of intact recombinant protein recoverable. Incorporation of a C-terminal (His)6 tag enabled single step purification of the fusion protein. These modifications did not affect the insecticidal activity of the recombinant toxin towards lepidopteran larvae. Introduction of multiple expression cassettes increased the amount of secreted recombinant fusion protein in a laboratory scale fermentation by almost tenfold on a per litre of culture basis. Simple modifications in the expression construct can be advantageous for the generation of high expressing P. pastoris strains for production of a recombinant protein, without altering its functional properties