Rapid Genotyping of Nothobranchius furzeri Embryos, Larvae, and Adult Fish via High-Resolution Melt Analysis (HRMA).

CRISPR-Cas9 has eased the induction of sequence-specific mutations and has therefore become a powerful tool to generate mutant lines for studying the role of specific genes. The cellular repair of Cas9-induced double-stranded DNA breaks by the error-prone nonhomologous end-joining pathway can result in various indel mutations. Having identified and chosen a specific mutation in a target gene, the establishment of a respective mutant line requires a feasible and precise method to differentiate the genotypes of the offspring. Here, we provide a protocol that allows genotyping of large numbers of Nothobranchius furzeri embryos, larvae, and adults harboring a previously identified indel or point mutation in a short time via high-resolution melt analysis (HRMA).

Sex chromosome differentiation via changes in the Y chromosome repeat landscape in African annual killifishes Nothobranchius furzeri and N. kadleci.

Homomorphic sex chromosomes and their turnover are common in teleosts. We investigated the evolution of nascent sex chromosomes in several populations of two sister species of African annual killifishes, Nothobranchius furzeri and N. kadleci, focusing on their under-studied repetitive landscape. We combined bioinformatic analyses of the repeatome with molecular cytogenetic techniques, including comparative genomic hybridization, fluorescence in situ hybridization with satellite sequences, ribosomal RNA genes (rDNA) and bacterial artificial chromosomes (BACs), and immunostaining of SYCP3 and MLH1 proteins to mark lateral elements of synaptonemal complexes and recombination sites, respectively. Both species share the same heteromorphic XY sex chromosome system, which thus evolved prior to their divergence. This was corroborated by sequence analysis of a putative master sex determining (MSD) gene gdf6Y in both species. Based on their divergence, differentiation of the XY sex chromosome pair started approximately 2 million years ago. In all populations, the gdf6Y gene mapped within a region rich in satellite DNA on the Y chromosome long arms. Despite their heteromorphism, X and Y chromosomes mostly pair regularly in meiosis, implying synaptic adjustment. In N. kadleci, Y-linked paracentric inversions like those previously reported in N. furzeri were detected. An inversion involving the MSD gene may suppress occasional recombination in the region, which we otherwise evidenced in the N. furzeri population MZCS-121 of the Limpopo clade lacking this inversion. Y chromosome centromeric repeats were reduced compared with the X chromosome and autosomes, which points to a role of relaxed meiotic drive in shaping the Y chromosome repeat landscape. We speculate that the recombination rate between sex chromosomes was reduced due to heterochiasmy. The observed differences between the repeat accumulations on the X and Y chromosomes probably result from high repeat turnover and may not relate closely to the divergence inferred from earlier SNP analyses.

Molecular Sexing of Nothobranchius furzeri Embryos and Larvae.

Differences between the sexes are of increasing interest in basic and applied research with regard to development, behavior, aging, and diseases. Although the African turquoise killifish Nothobranchius furzeri, a model for aging research well known for its remarkably short life span, develops strong sexual dimorphism in adulthood, there is no visible indicator of its sex in embryonic and juvenile stages. To address this issue, we developed a molecular sexing assay exploiting two large sequence polymorphisms in the minimal sex-determining region (SDR) of N. furzeri These polymorphisms are sequence deletions on the Y chromosome that involve the lack or truncation of one or multiple microsatellites. The simple polymerase chain reaction (PCR) readout of the assays described here allows the sexing of N. furzeri embryos and larvae in a medium- to high-throughput and cost-efficient manner.

TAL Effector DNA-Binding Principles and Specificity.

Transcription activator-like effectors (TALEs) are proteins with a unique DNA-binding domain that confers both a predictable and programmable specificity. The DNA-binding domain consists typically of 34-amino acid near-identical repeats. The repeats form a right-handed superhelical structure that wraps around the DNA double helix and exposes the variable amino acids at position 13 of each repeat to the sense strand DNA bases. Each repeat binds one base in a highly specific, non-overlapping, and comma-free fashion. Although TALE specificities are encoded in a simple way, sophisticated rules can be taken into account to build highly efficient DNA-binding modules for biotechnological use.

Patient-adapted, specific activation of HIV-1 by customized TAL effectors (TALEs), a proof of principle study.

The major obstacle to cure infections with human immunodeficiency virus (HIV-1) is integrated proviral genomes, which are not eliminated by antiretroviral therapies (ART). Treatment approaches with latency-reversing agents (LRAs) aim at inducing provirus expression to tag latently-infected cells for clearance through viral cytopathic effects or cytotoxic T cell (CTL) responses. However, the currently tested LRAs reveal evident drawbacks as gene expression is globally induced and viral outgrowth is insecure. Here, we present transcription activator-like effector (TALE) proteins as potent tools to activate HIV-1 specifically. The large variety of circulating HIV-1 strains and, accordingly, integrated proviruses was addressed by the programmable DNA-specificity of TALEs. Using customized engineered TALEs, a substantial transcription activation and viral outgrowth was achieved with cells obtained from different HIV-1 patients. Our data suggest that TALEs may be useful tools in future strategies aimed at removing HIV-1 reservoirs.

A TAL effector repeat architecture for frameshift binding.

Transcription activator-like effectors (TALEs) are important Xanthomonas virulence factors that bind DNA via a unique tandem 34-amino-acid repeat domain to induce expression of plant genes. So far, TALE repeats are described to bind as a consecutive array to a consecutive DNA sequence, in which each repeat independently recognizes a single DNA base. This modular protein architecture enables the design of any desired DNA-binding specificity for biotechnology applications. Here we report that natural TALE repeats of unusual amino-acid sequence length break the strict one repeat-to-one base pair binding mode and introduce a local flexibility to TALE-DNA binding. This flexibility allows TALEs and TALE nucleases to recognize target sequence variants with single nucleotide deletions. The flexibility also allows TALEs to activate transcription at allelic promoters that otherwise confer resistance to the host plant.

Selective slow wave sleep but not rapid eye movement sleep suppression impairs morning glucose tolerance in healthy men.

Shortened nocturnal sleep impairs morning glucose tolerance. The underlying mechanism of this effect is supposed to involve a reduced fraction of slow wave sleep (SWS). However, it remains unanswered if impaired glucose tolerance occurs due to specific SWS reduction or a general disturbance of sleep. Sixteen healthy men participated in three experimental conditions in a crossover design: SWS suppression, rapid eye movement (REM)-sleep disturbance, and regular sleep. Selective sleep stage disturbance was performed by means of an acoustic tone (532Hz) with gradually rising sound intensity. Blood concentrations of glucoregulatory parameters were measured upon an oral glucose tolerance test the next morning. Our data show that morning plasma glucose and serum insulin responses were significantly increased after selective SWS suppression. Moreover, SWS suppression reduced postprandial insulin sensitivity up to 20%, as determined by Matsuda Index. Contrastingly, disturbed REM-sleep did not affect glucose homeostasis. We conclude that specifically SWS reduction is critically involved in the impairment of glucose tolerance associated with disturbed sleep. Therefore, glucose metabolism in subjects predisposed to reduced SWS (e.g. depression, aging, obstructive sleep apnea, pharmacological treatment) should be thoroughly monitored.

Novel prevention program for trichinellosis in inuit communities.

Repeated outbreaks of trichinellosis caused by the consumption of Trichinella-infected walrus (Odobenus rosmarus) meat, which have sometimes led to serious morbidity, have stimulated Inuit communities in Nunavik (northern Quebec), Canada, to develop an innovative trichinellosis prevention program. The program involves preconsumption testing of meat samples from harvested walrus at a regional laboratory and the rapid dissemination of the results of such testing to communities. Local health authorities in Inukjuak conducted an epidemiological investigation after testing identified Trichinella-positive walrus meat in September 1997. This report describes the events that occurred before, during, and after the trichinellosis outbreak and also documents how the prevention program contributed to successful resolution of the outbreak.