Faculty Bibliography

Purpose: Heterozygous pathogenic variants in various FOXP genes cause specific developmental disorders. The phenotype associated with heterozygous variants in FOXP4 has not been previously described.
Method(s): We assembled a cohort of eight individuals with heterozygous and mostly de novo variants in FOXP4: seven individuals with six different missense variants and one individual with a frameshift variant. We collected clinical data to delineate the phenotypic spectrum, and used in silico analyses and functional cell-based assays to assess pathogenicity of the variants.
Result(s): We collected clinical data for six individuals: five individuals with a missense variant in the forkhead box DNA-binding domain of FOXP4, and one individual with a truncating variant. Overlapping features included speech and language delays, growth abnormalities, congenital diaphragmatic hernia, cervical spine abnormalities, and ptosis. Luciferase assays showed loss-of-function effects for all these variants, and aberrant subcellular localization patterns were seen in a subset. The remaining two missense variants were located outside the functional domains of FOXP4, and showed transcriptional repressor capacities and localization patterns similar to the wild-type protein.
Conclusion(s): Collectively, our findings show that heterozygous loss-of-function variants in FOXP4 are associated with an autosomal dominant neurodevelopmental disorder with speech/language delays, growth defects, and variable congenital abnormalities.
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The human GABAB receptor-a member of the class C family of G-protein-coupled receptors (GPCRs)-mediates inhibitory neurotransmission and has been implicated in epilepsy, pain and addiction¹. A unique GPCR that is known to require heterodimerization for function^2-6, the GABAB receptor has two subunits, GABAB1 and GABAB2, that are structurally homologous but perform distinct and complementary functions. GABAB1 recognizes orthosteric ligands^7,8, while GABAB2 couples with G proteins^9-14. Each subunit is characterized by an extracellular Venus flytrap (VFT) module, a descending peptide linker, a seven-helix transmembrane domain and a cytoplasmic tail¹⁵. Although the VFT heterodimer structure has been resolved¹⁶, the structure of the full-length receptor and its transmembrane signalling mechanism remain unknown. Here we present a near full-length structure of the GABAB receptor at atomic resolution, captured in an inactive state by cryo-electron microscopy. Our structure reveals several ligands that preassociate with the receptor, including two large endogenous phospholipids that are embedded within the transmembrane domains to maintain receptor integrity and modulate receptor function. We also identify a previously unknown heterodimer interface between transmembrane helices 3 and 5 of both subunits, which serves as a signature of the inactive conformation. A unique 'intersubunit latch' within this transmembrane interface maintains the inactive state, and its disruption leads to constitutive receptor activity.
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DNA combing technology is a powerful methodology for the study of DNA replication in vivo. This tool can be used to identify origins of replication, assess of directionality of forks, and measure fork speed. Over the years, the method has been used extensively to study nuclear DNA replication. The first step involves the incorporation of thymidine analogs (CldU and IdU) into nascent DNA chains and followed by their visualization with immunofluorescence using antibodies that can distinguish the two analogs. Recently, we adapted and fine-tuned DNA combing technology to the specifics of mitochondrial DNA (Phillips et al., 2017, p. 155). The protocol, which we termed mito-SMARD (mitochondrial single molecule analysis of replication DNA), provides in vivo insight into mitochondrial DNA (mtDNA) replication with high resolution.

An estimated 92% of the world's population live in regions where people are regularly exposed to high levels of anthropogenic air pollution. Historically, research on the effects of air pollution have focused extensively on cardiovascular and pulmonary health. However, emerging evidence from animal and human studies has suggested that chronic exposures to air pollution detrimentally change the functioning of the central nervous system with the result being proteinopathy, neurocognitive impairment, and neurodegenerative disease. Case analyses of aging World Trade Center responders suggests that a single severe exposure may also induce a neuropathologic response. The goal of this report was to explore the neuroscientific support for the hypothesis that inhaled particulate matter might cause an Alzheimer's-like neurodegenerative disease, in order to consider proposed mechanisms and latency periods linking inhaled particulate matter and neurodegeneration, and to propose new directions in this line of research.

[This corrects the article DOI: 10.1016/j.csbj.2018.09.003.].

Mitochondria and their associated membranes actively participate in biosynthesis, trafficking, and degradation of cellular phospholipids. Two crucial lipid biosynthetic activities of mitochondria include (i) the decarboxylation of phosphatidylserine to phosphatidylethanolamine and (ii) the de novo synthesis of cardiolipin. Here we describe protocols to measure these two activities, applying isotope-labeled or exogenous substrates in combination with thin-layer chromatography or mass spectrometry.

An increasing number of technologies combine spatial discrimination with molecule identification, as has been done for decades through immunohistochemistry and in situ hybridization. The difference is that now a sleuth of commercially available platforms promises to significantly increase the number of targets that can be identified on a single sample. They also provide higher throughput in terms of number of samples that can be interrogated, and some offer semiautomation with straightforward sample preparation. All these characteristics set up the new spatial profiling technologies as attractive candidates for biomedical research facilities. However, to be able to discriminate between them it is necessary to share real world experiences using them in our cores. This panel session brings together several researchers with hands on experience using two or more spatial profiling technologies. These will include antibody based (Opal/Vectra Polaris, Ultivue, MIBI, Hyperion) and nucleic acid (Visium, GeoMx) based platforms. Each panelist will give a quick overview of the technology, its biochemical principles, costs, advantages and disadvantages as they have identified them and compared them to each other or a gold standard, if available. At the end, we will have an open discussion where the audience is encouraged to ask questions and provide their experiences

We write to introduce our novel group formed to confront some of the issues raised by the COVID-19 pandemic. Information about the group, which we named "cure COVid for Ever and for All" (RxCOVEA), its dynamic membership (changing regularly), and some of its activities-described in more technical detail for expert perusal and commentary-are available upon request.