The human channel gating–modifying A749G CACNA1D (Cav1.3) variant induces a neurodevelopmental syndrome–like phenotype in mice

Germline de novo missense variants of the CACNA1D gene, encoding the pore-forming α1 subunit of Cav1.3 L-type Ca2+ channels (LTCCs), have been found in patients with neurodevelopmental and endocrine dysfunction, but their disease-causing potential is unproven. These variants alter channel gating, enabling enhanced Cav1.3 activity, suggesting Cav1.3 inhibition as a potential therapeutic option. Here we provide proof of the disease-causing nature of such gating-modifying CACNA1D variants using mice (Cav1.3AG) containing the A749G variant reported de novo in a patient with autism spectrum disorder (ASD) and intellectual impairment. In heterozygous mutants, native LTCC currents in adrenal chromaffin cells exhibited gating changes as predicted from heterologous expression. The A749G mutation induced aberrant excitability of dorsomedial striatum–projecting substantia nigra dopamine neurons and medium spiny neurons in the dorsal striatum. The phenotype observed in heterozygous mutants reproduced many of the abnormalities described within the human disease spectrum, including developmental delay, social deficit, and pronounced hyperactivity without major changes in gross neuroanatomy. Despite an approximately 7-fold higher sensitivity of A749G-containing channels to the LTCC inhibitor isradipine, oral pretreatment over 2 days did not rescue the hyperlocomotion. Cav1.3AG mice confirm the pathogenicity of the A749G variant and point toward a pathogenetic role of altered signaling in the dopamine midbrain system

Potential of poly(styrene-co-divinylbenzene) monolithic columns for the LC-MS analysis of protein digests

Two polystyrene-based capillary monolithic columns of different length (50 and 250 mm) were used to evaluate the effects of column length on gradient separation of protein digests. A tryptic digest of a 9-protein mixture was used as a test sample. Peak capacities were determined from selected extracted ion chromatograms, and tandem mass spectrometry data were used for database matching using the MASCOT search engine. Peak capacities and protein identification scores were higher for the long column with all gradients. Peak capacities appear to approach a plateau for longer gradient times; maximum peak capacity was estimated to be 294 for the short column and 370 for the long column. Analyses with similar gradient slope produced a ratio of the peak capacities of 3.36 for the long and the short column, which is slightly higher than the expected value of the square root of the column length ratio. The use of a longer monolith improves peptide separation, as reflected by higher peak capacity, and also increases protein identification, as observed from higher identification scores and a larger number of identified peptides. Attention has also been paid to the peak production rate (PPR, peak capacity per unit time). For short analysis times, the short column produces a higher PPR, while for analysis times longer than 40 min, the PPR of the 250-mm column is higher

Multi-year interlaboratory exercises for the analysis of illicit drugs and metabolites in wastewater:development of a quality control system

Thirty-seven laboratories from 25 countries present the development of an inter-laboratory testing scheme for the analysis of seven illicit drug residues in standard solutions, tap- and wastewater. Almost 10 000 concentration values were evaluated: triplicates of up to five samples and 26 laboratories per year. The setup was substantially improved with experiences gained across the six repetitions (e.g. matrix type, sample conditions, spiking levels). From this, (pre-)analytical issues (e.g. pH adjustment, filtration) were revealed for specific analytes which resulted in formulation of best-practice protocols for inter-laboratory setup and analytical procedures. The results illustrate the effectiveness of the inter-laboratory setup to assess laboratory performance in the framework of wastewater-based epidemiology. The exercise proved that measurements of laboratories were of high quality (>80% satisfactory results for six out of seven analytes) and that analytical follow-up is important to assist laboratories in improving robustness of wastewater-based epidemiology results