Mapping recurrent mosaic copy number variation in human neurons.

When somatic cells acquire complex karyotypes, they often are removed by the immune system. Mutant somatic cells that evade immune surveillance can lead to cancer. Neurons with complex karyotypes arise during neurotypical brain development, but neurons are almost never the origin of brain cancers. Instead, somatic mutations in neurons can bring about neurodevelopmental disorders, and contribute to the polygenic landscape of neuropsychiatric and neurodegenerative disease. A subset of human neurons harbors idiosyncratic copy number variants (CNVs, "CNV neurons"), but previous analyses of CNV neurons are limited by relatively small sample sizes. Here, we develop an allele-based validation approach, SCOVAL, to corroborate or reject read-depth based CNV calls in single human neurons. We apply this approach to 2,125 frontal cortical neurons from a neurotypical human brain. SCOVAL identifies 226 CNV neurons, which include a subclass of 65 CNV neurons with highly aberrant karyotypes containing whole or substantial losses on multiple chromosomes. Moreover, we find that CNV location appears to be nonrandom. Recurrent regions of neuronal genome rearrangement contain fewer, but longer, genes.

Mapping the Complex Genetic Landscape of Human Neurons.

When somatic cells acquire complex karyotypes, they are removed by the immune system. Mutant somatic cells that evade immune surveillance can lead to cancer. Neurons with complex karyotypes arise during neurotypical brain development, but neurons are almost never the origin of brain cancers. Instead, somatic mutations in neurons can bring about neurodevelopmental disorders, and contribute to the polygenic landscape of neuropsychiatric and neurodegenerative disease. A subset of human neurons harbors idiosyncratic copy number variants (CNVs, "CNV neurons"), but previous analyses of CNV neurons have been limited by relatively small sample sizes. Here, we developed an allele-based validation approach, SCOVAL, to corroborate or reject read-depth based CNV calls in single human neurons. We applied this approach to 2,125 frontal cortical neurons from a neurotypical human brain. This approach identified 226 CNV neurons, as well as a class of CNV neurons with complex karyotypes containing whole or substantial losses on multiple chromosomes. Moreover, we found that CNV location appears to be nonrandom. Recurrent regions of neuronal genome rearrangement contained fewer, but longer, genes.

Transcription-associated DNA DSBs activate p53 during hiPSC-based neurogenesis.

Neurons are overproduced during cerebral cortical development. Neural progenitor cells (NPCs) divide rapidly and incur frequent DNA double-strand breaks (DSBs) throughout cortical neurogenesis. Although half of the neurons born during neurodevelopment die, many neurons with inaccurate DNA repair survive leading to brain somatic mosaicism. Recurrent DNA DSBs during neurodevelopment are associated with both gene expression level and gene length. We used imaging flow cytometry and a genome-wide DNA DSB capture approach to quantify and map DNA DSBs during human induced pluripotent stem cell (hiPSC)-based neurogenesis. Reduced p53 signaling was brought about by knockdown (p53KD); p53KD led to elevated DNA DSB burden in neurons that was associated with gene expression level but not gene length in neural progenitor cells (NPCs). Furthermore, DNA DSBs incurred from transcriptional, but not replicative, stress lead to p53 activation in neurotypical NPCs. In p53KD NPCs, DNA DSBs accumulate at transcription start sites of genes that are associated with neurological and psychiatric disorders. These findings add to a growing understanding of how neuronal genome dynamics are engaged by high transcriptional or replicative burden during neurodevelopment.

New Quinone Antibiotics against Methicillin-Resistant S. aureus.

There is an urgent need for the development of new antibiotics. Here, we describe the inhibitory activity of new quinone compounds against methicillin-resistant Staphylococcus aureus (ATCC® 43300), methicillin-sensitive S. aureus (ATCC® 29213), and two clinical isolates from Chile (ISP-213 and ISP-214). We observed 99.9% reduction in viability within 2 h of exposure without the cultures exhibiting any post-antibiotic effect, which was twice the kinetics to that observed with vancomycin. These clinical isolates did not acquire resistance to these quinone derivatives during the course of our study. We found that these compounds protected larvae of the greater wax moth, sp. Galleria mellonella, from infection by these MRSA clinical strains as effectively as vancomycin. These quinone derivatives are potential drug candidates worth further development.

Neurons with Complex Karyotypes Are Rare in Aged Human Neocortex.

A subset of human neocortical neurons harbors complex karyotypes wherein megabase-scale copy-number variants (CNVs) alter allelic diversity. Divergent levels of neurons with complex karyotypes (CNV neurons) are reported in different individuals, yet genome-wide and familial studies implicitly assume a single brain genome when assessing the genetic risk architecture of neurological disease. We assembled a brain CNV atlas using a robust computational approach applied to a new dataset (>800 neurons from 5 neurotypical individuals) and to published data from 10 additional neurotypical individuals. The atlas reveals that the frequency of neocortical neurons with complex karyotypes varies widely among individuals, but this variability is not readily accounted for by tissue quality or CNV detection approach. Rather, the age of the individual is anti-correlated with CNV neuron frequency. Fewer CNV neurons are observed in aged individuals than in young individuals.

Improved molecular karyotyping in glioblastoma.

Uneven replication creates artifacts during whole genome amplification (WGA) that confound molecular karyotype assignment in single cells. Here, we present an improved WGA recipe that increased coverage and detection of copy number variants (CNVs) in single cells. We examined serial resections of glioblastoma (GBM) tumor from the same patient and found low-abundance clones containing CNVs in clinically relevant loci that were not observable using bulk DNA sequencing. We discovered extensive genomic variability in this class of tumor and provide a practical approach for investigating somatic mosaicism.

Detection of pathogen-specific antibodies by loop-mediated isothermal amplification.

Loop-mediated isothermal amplification (LAMP) is a method for enzymatically replicating DNA that has great utility for clinical diagnosis at the point of care (POC), given its high sensitivity, specificity, speed, and technical requirements (isothermal conditions). Here, we adapted LAMP for measuring protein analytes by creating a protein-DNA fusion (referred to here as a "LAMPole") that attaches oligonucleotides (LAMP templates) to IgG antibodies. This fusion consists of a DNA element covalently bonded to an IgG-binding polypeptide (protein L/G domain). In our platform, LAMP is expected to provide the most suitable means for amplifying LAMPoles for clinical diagnosis at the POC, while quantitative PCR is more suitable for laboratory-based quantification of antigen-specific IgG abundance. As proof of concept, we measured serological responses to a protozoan parasite by quantifying changes in solution turbidity in real time. We observed a >6-log fold difference in signal between sera from vaccinated versus control mice and in a clinical patient sample versus a control. We assert that LAMPoles will be useful for increasing the sensitivity of measuring proteins, whether it be in a clinical laboratory or in a field setting, thereby improving acute diagnosis of a variety of infections.