A CAG repeat threshold for therapeutics targeting somatic instability in Huntington's disease.

The Huntington's disease mutation is a CAG repeat expansion in the huntingtin gene that results in an expanded polyglutamine tract in the huntingtin protein. The CAG repeat is unstable and expansions of hundreds of CAGs have been detected in Huntington's disease post-mortem brains. The age of disease onset can be predicted partially from the length of the CAG repeat as measured in blood. Onset age is also determined by genetic modifiers, which in six cases involve variation in DNA mismatch repair pathways genes. Knocking-out specific mismatch repair genes in mouse models of Huntington's disease prevents somatic CAG repeat expansion. Taken together, these results have led to the hypothesis that somatic CAG repeat expansion in Huntington's disease brains is required for pathogenesis. Therefore, the pathogenic repeat threshold in brain is longer than (CAG)40, as measured in blood, and is currently unknown. The mismatch repair gene MSH3 has become a major focus for therapeutic development, as unlike other mismatch repair genes, nullizygosity for MSH3 does not cause malignancies associated with mismatch repair deficiency. Potential treatments targeting MSH3 currently under development include gene therapy, biologics and small molecules, which will be assessed for efficacy in mouse models of Huntington's disease. The zQ175 knock-in model carries a mutation of approximately (CAG)185 and develops early molecular and pathological phenotypes that have been extensively characterized. Therefore, we crossed the mutant huntingtin allele onto heterozygous and homozygous Msh3 knockout backgrounds to determine the maximum benefit of targeting Msh3 in this model. Ablation of Msh3 prevented somatic expansion throughout the brain and periphery, and reduction of Msh3 by 50% decreased the rate of expansion. This had no effect on the deposition of huntingtin aggregation in the nuclei of striatal neurons, nor on the dysregulated striatal transcriptional profile. This contrasts with ablating Msh3 in knock-in models with shorter CAG repeat expansions. Therefore, further expansion of a (CAG)185 repeat in striatal neurons does not accelerate the onset of molecular and neuropathological phenotypes. It is striking that highly expanded CAG repeats of a similar size in humans cause disease onset before 2 years of age, indicating that somatic CAG repeat expansion in the brain is not required for pathogenesis. Given that the trajectory for somatic CAG expansion in the brains of Huntington's disease mutation carriers is unknown, our study underlines the importance of administering treatments targeting somatic instability as early as possible.

Multiple cereblon genetic changes are associated with acquired resistance to lenalidomide or pomalidomide in multiple myeloma.

Emergence of drug resistance to all available therapies is the major challenge to improving survival in myeloma. Cereblon (CRBN) is the essential binding protein of the widely used immunomodulatory drugs (IMiDs) and novel CRBN E3 ligase modulator drugs (CELMoDs) in myeloma, as well as certain proteolysis targeting chimeras (PROTACs), in development for a range of diseases. Using whole-genome sequencing (WGS) data from 455 patients and RNA sequencing (RNASeq) data from 655 patients, including newly diagnosed (WGS, n = 198; RNASeq, n = 437), lenalidomide (LEN)-refractory (WGS, n = 203; RNASeq, n = 176), and pomalidomide (POM)-refractory cohorts (WGS, n = 54; RNASeq, n = 42), we found incremental increases in the frequency of 3 CRBN aberrations, namely point mutations, copy losses/structural variations, and a specific variant transcript (exon 10 spliced), with progressive IMiD exposure, until almost one-third of patients had CBRN alterations by the time they were POM refractory. We found all 3 CRBN aberrations were associated with inferior outcomes to POM in those already refractory to LEN, including those with gene copy losses and structural variations, a finding not previously described. This represents the first comprehensive analysis and largest data set of CBRN alterations in myeloma patients as they progress through therapy. It will help inform patient selection for sequential therapies with CRBN-targeting drugs.

The Food Web of Boiling Springs Lake Appears Dominated by the Heterolobosean Tetramitus thermacidophilus Strain BSL.

We studied the protist grazers of Boiling Springs Lake (BSL), an acid geothermal feature in Lassen Volcanic National Park, using a combination of culture and genetic approaches. The major predator in BSL is a vahlkampfiid ameba closely related (95% 18S+ITS rRNA identity) to Tetramitus thermacidophilus, a heterolobose ameboflagellate recently isolated from volcanic geothermal acidic sites in Europe and Russia, as well as an uncultured heterolobosean from the nearby Iron Mountain acid mine drainage site. Tetramitus thermacidophilus strain BSL is capable of surviving the physical extremes of BSL, with optimal growth at 38-50 °C and pH 2-5. This bacterivore also ingested conidiospores of the ascomycete Phialophora sp., but ultrastructural observations reveal the latter may not be readily digested, and conidia were not separable from the ameoboflagellate culture, suggesting a possible symbiosis. DGGE fingerprint transects studies showed the organism is restricted to near-lake environs, and we detected an average of ~500 viable cysts/cm(3) sediment on the shoreline. Other grazing protists were isolated from lakeshore environments, including the lobose amebae Acanthamoeba sp. and Hartmannella sp., and the kinetoplastid flagellate Bodo sp., but none could tolerate both low pH and high temperature. These appear to be restricted to cooler near lake geothermal features, which also contain other potential grazer morphotypes observed but not successfully cultured, including ciliates, euglenids, testate amebae, and possible cercozoans. We compare the food web of BSL with other acidic or geothermal sites, and discuss the impact of protists in this unique environment.

SEQUIN is an R/Shiny framework for rapid and reproducible analysis of RNA-seq data.

SEQUIN is a web-based application (app) that allows fast and intuitive analysis of RNA sequencing data derived for model organisms, tissues, and single cells. Integrated app functions enable uploading datasets, quality control, gene set enrichment, data visualization, and differential gene expression analysis. We also developed the iPSC Profiler, a practical gene module scoring tool that helps measure and compare pluripotent and differentiated cell types. Benchmarking to other commercial and non-commercial products underscored several advantages of SEQUIN. Freely available to the public, SEQUIN empowers scientists using interdisciplinary methods to investigate and present transcriptome data firsthand with state-of-the-art statistical methods. Hence, SEQUIN helps democratize and increase the throughput of interrogating biological questions using next-generation sequencing data with single-cell resolution.

A defined roadmap of radial glia and astrocyte differentiation from human pluripotent stem cells.

Human gliogenesis remains poorly understood, and derivation of astrocytes from human pluripotent stem cells (hPSCs) is inefficient and cumbersome. Here, we report controlled glial differentiation from hPSCs that bypasses neurogenesis, which otherwise precedes astrogliogenesis during brain development and in vitro differentiation. hPSCs were first differentiated into radial glial cells (RGCs) resembling resident RGCs of the fetal telencephalon, and modulation of specific cell signaling pathways resulted in direct and stepwise induction of key astroglial markers (NFIA, NFIB, SOX9, CD44, S100B, glial fibrillary acidic protein [GFAP]). Transcriptomic and genome-wide epigenetic mapping and single-cell analysis confirmed RGC-to-astrocyte differentiation, obviating neurogenesis and the gliogenic switch. Detailed molecular and cellular characterization experiments uncovered new mechanisms and markers for human RGCs and astrocytes. In summary, establishment of a glia-exclusive neural lineage progression model serves as a unique serum-free platform of manufacturing large numbers of RGCs and astrocytes for neuroscience, disease modeling (e.g., Alexander disease), and regenerative medicine.

Stress-free cell aggregation by using the CEPT cocktail enhances embryoid body and organoid fitness.

Embryoid bodies (EBs) and self-organizing organoids derived from human pluripotent stem cells (hPSCs) recapitulate tissue development in a dish and hold great promise for disease modeling and drug development. However, current protocols are hampered by cellular stress and apoptosis during cell aggregation, resulting in variability and impaired cell differentiation. Here, we demonstrate that EBs and various organoid models (e.g., brain, gut, kidney) can be optimized by using the small molecule cocktail named CEPT (chroman 1, emricasan, polyamines, trans-ISRIB), a polypharmacological approach that ensures cytoprotection and cell survival. Application of CEPT for just 24 h during cell aggregation has long-lasting consequences affecting morphogenesis, gene expression, cellular differentiation, and organoid function. Various qualification methods confirmed that CEPT treatment enhanced experimental reproducibility and consistently improved EB and organoid fitness as compared to the widely used ROCK inhibitor Y-27632. Collectively, we discovered that stress-free cell aggregation and superior cell survival in the presence of CEPT are critical quality control determinants that establish a robust foundation for bioengineering complex tissue and organ models.

Identification of MET10-932 and characterization as an allele reducing hydrogen sulfide formation in wine strains of Saccharomyces cerevisiae.

A vineyard isolate of the yeast Saccharomyces cerevisiae, UCD932, was identified as a strain producing little or no detectable hydrogen sulfide during wine fermentation. Genetic analysis revealed that this trait segregated as a single genetic determinant. The gene also conferred a white colony phenotype on BiGGY agar (bismuth-glucose-glycine-yeast agar), which is thought to indicate low basal levels of sulfite reductase activity. However, this isolate does not display a requirement for S-containing amino acids, indicating that the sulfate reduction pathway is fully operational. Genetic crosses against known mutations conferring white colony color on BiGGY agar identified the gene leading to reduced H(2)S formation as an allele of MET10 (MET10-932), which encodes a catalytic subunit of sulfite reductase. Sequence analysis of MET10-932 revealed several corresponding amino acid differences in relation to laboratory strain S288C. Allele differences for other genes of the sulfate reduction pathway were also detected in UCD932. The MET10 allele of UCD932 was found to be unique in comparison to the sequences of several other vineyard isolates with differing levels of production of H(2)S. Replacing the MET10 allele of high-H(2)S-producing strains with MET10-932 prevented H(2)S formation by those strains. A single mutative change, corresponding to T662K, in MET10-932 resulted in a loss of H(2)S production. The role of site 662 in sulfide reduction was further analyzed by changing the encoded amino acid at this position. A change back to threonine or to the conservative serine fully restored the H(2)S formation conferred by this allele. In addition to T662K, arginine, tryptophan, and glutamic acid substitutions similarly reduced sulfide formation.

CO-dependent H2 evolution by Rhodospirillum rubrum: role of CODH:CooF complex.

Upon exposure to CO during anaerobic growth, the purple phototrophic bacterium Rhodospirillum rubrum expresses a CO-oxidizing H(2) evolving enzymatic system. The CO-oxidizing enzyme, carbon monoxide dehydrogenase (CODH), has been purified and extensively characterized. However the electron transfer pathway from CODH to the CO-induced hydrogenase that evolves H(2) is not well understood. CooF is an Fe-S protein that is the proposed mediator of electron transfer between CODH and the CO-induced hydrogenase. Here we present the spectroscopic and biochemical properties of the CODH:CooF complex. The characteristic EPR signals observed for CODH are largely insensitive to CooF complexation. Metal analysis and EPR spectroscopy show that CooF contains 2 Fe(4)S(4) clusters. The observation of 2 Fe(4)S(4) clusters for CooF contradicts the prediction of 4 Fe(4)S(4) clusters based on analysis of the amino acid sequence of CooF and structural studies of CooF homologs. Comparison of in vivo and in vitro CO-dependent H(2) evolution indicates that approximately 90% of the activity is lost upon cell lysis. We propose that the loss of two labile Fe-S clusters from CooF during cell lysis may be responsible for the low in vitro CO-dependent H(2) evolution activity. During the course of these studies, a new assay for CODH:CooF was developed using membranes from an R. rubrum mutant that did not express CODH:CooF, but expressed high levels of the CO-induced hydrogenase. The assay revealed that the CO-induced hydrogenase requires the presence of CODH:CooF for optimal H(2) evolution activity.

Uncultivated microbial eukaryotic diversity: a method to link ssu rRNA gene sequences with morphology.

Protists have traditionally been identified by cultivation and classified taxonomically based on their cellular morphologies and behavior. In the past decade, however, many novel protist taxa have been identified using cultivation independent ssu rRNA sequence surveys. New rRNA "phylotypes" from uncultivated eukaryotes have no connection to the wealth of prior morphological descriptions of protists. To link phylogenetically informative sequences with taxonomically informative morphological descriptions, we demonstrate several methods for combining whole cell rRNA-targeted fluorescent in situ hybridization (FISH) with cytoskeletal or organellar immunostaining. Either eukaryote or ciliate-specific ssu rRNA probes were combined with an anti-α-tubulin antibody or phalloidin, a common actin stain, to define cytoskeletal features of uncultivated protists in several environmental samples. The eukaryote ssu rRNA probe was also combined with Mitotracker® or a hydrogenosomal-specific anti-Hsp70 antibody to localize mitochondria and hydrogenosomes, respectively, in uncultivated protists from different environments. Using rRNA probes in combination with immunostaining, we linked ssu rRNA phylotypes with microtubule structure to describe flagellate and ciliate morphology in three diverse environments, and linked Naegleria spp. to their amoeboid morphology using actin staining in hay infusion samples. We also linked uncultivated ciliates to morphologically similar Colpoda-like ciliates using tubulin immunostaining with a ciliate-specific rRNA probe. Combining rRNA-targeted FISH with cytoskeletal immunostaining or stains targeting specific organelles provides a fast, efficient, high throughput method for linking genetic sequences with morphological features in uncultivated protists. When linked to phylotype, morphological descriptions of protists can both complement and vet the increasing number of sequences from uncultivated protists, including those of novel lineages, identified in diverse environments.

Gene expression normalization in a dual-compartment system: a real-time quantitative polymerase chain reaction protocol for symbiotic anthozoans.

Traditional real-time quantitative polymerase chain reaction protocols cannot be used accurately with symbiotic organisms unless the relative contribution of each symbiotic compartment to the total nucleic acid pool is known. A modified 'universal reference gene' protocol was created for reef-building corals and sea anemones, anthozoans that harbour endosymbiotic dinoflagellates belonging to the genus Symbiodinium. Gene expression values are first normalized to an RNA spike and then to a symbiont molecular proxy that represents the number of Symbiodinium cells extracted and present in the RNA. The latter is quantified using the number of genome copies of heat shock protein-70 (HSP70) amplified in the real-time quantitative polymerase chain reaction. Gene expression values are then normalized to the total concentration of RNA to account for differences in the amount of live tissue extracted among experimental treatments and replicates. The molecular quantification of symbiont cells and effect of increasing symbiont contributions to the nucleic acid pool on gene expression were tested in vivo using differentially infected sea anemones Aiptasia pulchella. This protocol has broad application to researchers who seek to measure gene expression in mixed organism assemblages.