The room temperature crystal structure of a bacterial phytochrome determined by serial femtosecond crystallography

Phytochromes are a family of photoreceptors that control light responses of plants, fungi and bacteria. A sequence of structural changes, which is not yet fully understood, leads to activation of an output domain. Time-resolved serial femtosecond crystallography (SFX) can potentially shine light on these conformational changes. Here we report the room temperature crystal structure of the chromophore-binding domains of the Deinococcus radiodurans phytochrome at 2.1 angstrom resolution. The structure was obtained by serial femtosecond X-ray crystallography from microcrystals at an X-ray free electron laser. We find overall good agreement compared to a crystal structure at 1.35 angstrom resolution derived from conventional crystallography at cryogenic temperatures, which we also report here. The thioether linkage between chromophore and protein is subject to positional ambiguity at the synchrotron, but is fully resolved with SFX. The study paves the way for time-resolved structural investigations of the phytochrome photocycle with time-resolved SFX.Peer reviewe

Differentiation of Human Embryonic Stem Cells to Regional Specific Neural Precursors in Chemically Defined Medium Conditions

Background: Human embryonic stem cells (hESC) provide a unique model to study early events in human development. The hESC-derived cells can potentially be used to replace or restore different tissues including neuronal that have been damaged by disease or injury. Methodology and Principal Findings: The cells of two different hESC lines were converted to neural rosettes using adherent and chemically defined conditions. The progenitor cells were exposed to retinoic acid (RA) or to human recombinant basic fibroblast growth factor (bFGF) in the late phase of the rosette formation. Exposing the progenitor cells to RA suppressed differentiation to rostral forebrain dopamine neural lineage and promoted that of spinal neural tissue including motor neurons. The functional characteristics of these differentiated neuronal precursors under both, rostral (bFGF) and caudalizing (RA) signals were confirmed by patch clamp analysis. Conclusions/Significance: These findings suggest that our differentiation protocol has the capacity to generate regionspecific and electrophysiologically active neurons under in vitro conditions without embryoid body formation, co-cultur

Twist exome capture allows for lower average sequence coverage in clinical exome sequencing

Background Exome and genome sequencing are the predominant techniques in the diagnosis and research of genetic disorders. Sufficient, uniform and reproducible/consistent sequence coverage is a main determinant for the sensitivity to detect single-nucleotide (SNVs) and copy number variants (CNVs). Here we compared the ability to obtain comprehensive exome coverage for recent exome capture kits and genome sequencing techniques. Results We compared three different widely used enrichment kits (Agilent SureSelect Human All Exon V5, Agilent SureSelect Human All Exon V7 and Twist Bioscience) as well as short-read and long-read WGS. We show that the Twist exome capture significantly improves complete coverage and coverage uniformity across coding regions compared to other exome capture kits. Twist performance is comparable to that of both short- and long-read whole genome sequencing. Additionally, we show that even at a reduced average coverage of 70× there is only minimal loss in sensitivity for SNV and CNV detection. Conclusion We conclude that exome sequencing with Twist represents a significant improvement and could be performed at lower sequence coverage compared to other exome capture techniques

A Solve-RD ClinVar-based reanalysis of 1522 index cases from ERN-ITHACA reveals common pitfalls and misinterpretations in exome sequencing

Purpose Within the Solve-RD project (https://solve-rd.eu/), the European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies aimed to investigate whether a reanalysis of exomes from unsolved cases based on ClinVar annotations could establish additional diagnoses. We present the results of the “ClinVar low-hanging fruit” reanalysis, reasons for the failure of previous analyses, and lessons learned. Methods Data from the first 3576 exomes (1522 probands and 2054 relatives) collected from European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies was reanalyzed by the Solve-RD consortium by evaluating for the presence of single-nucleotide variant, and small insertions and deletions already reported as (likely) pathogenic in ClinVar. Variants were filtered according to frequency, genotype, and mode of inheritance and reinterpreted. Results We identified causal variants in 59 cases (3.9%), 50 of them also raised by other approaches and 9 leading to new diagnoses, highlighting interpretation challenges: variants in genes not known to be involved in human disease at the time of the first analysis, misleading genotypes, or variants undetected by local pipelines (variants in off-target regions, low quality filters, low allelic balance, or high frequency). Conclusion The “ClinVar low-hanging fruit” analysis represents an effective, fast, and easy approach to recover causal variants from exome sequencing data, herewith contributing to the reduction of the diagnostic deadlock

Using therapeutic cloning to fight human disease: A conundrum or reality?

The development and transplantation of autologous cells derived from nuclear transfer embryonic stem cell (NT-ESC) lines to treat patients suffering from disease has been termed therapeutic cloning. Human NT is still a developing field, with further research required to improve somatic cell NT and human embryonic stem cell differentiation to deliver safe and effective cell replacement therapies. Furthermore, the implications of transferring mitochondrial heteroplasmic cells, which may harbor aberrant epigenetic gene expression profiles, are of concern. The production of human NT-ESC lines also remains plagued by ethical dilemmas, societal concerns, and controversies. Recently, a number of alternate therapeutic strategies have been proposed to circumvent the moral implications surrounding human nuclear transfer. It will be critical to overcome these biological, legislative, and moral restraints to maximize the potential of this therapeutic strategy and to alleviate human disease

Stem cell based therapy for spinal cord injury

Stem cells (SC) represent a new therapeutic approach for spinal cord injury (SCI) by enabling improved sensory and motor functions in animal models. The main goal of SC based therapy for SCI is replacement of neurons and glial cells that undergo cell death soon after injury. Stem cells are able to promote remyelination via oligodendroglia cell replacement, to produce trophic factors enhancing neurite outgrowth, axonal elongation and fibre density and to activate resident or transplanted progenitor cells across the lesion cavity. While several SC transplantation strategies have shown promising yet partial efficacy, mechanistic proof is generally lacking and is arguably the largest impediment toward faster progress and clinical application. The main challenge ahead is to spurn cooperation between clinicians, researchers and patients in order to define and optimize mechanisms of SC function and to establish the ideal source/s of SC that produce efficient and also safe therapeutic approaches.This study was supported by Serbian Ministry of Science (project numbers ON 175069 and ON175103) and Andalusian Council of Health (PI-0113-2010).Peer Reviewe

Platform to study intracellular polystyrene nanoplastic pollution and clinical outcomes

Increased pollution by plastics has become a serious global environmental problem, but the concerns for human health have been raised after reported presence of microplastics (MPs) and nanoplastics (NPs) in food and beverages. Unfortunately, few studies have investigate the potentially harmful effects of MPs/NPs on early human development and human health. Therefore, we used a new platform to study possible effects of polystyrene NPs (PSNPs) on the transcription profile of preimplantation human embryos and human induced pluripotent stem cells (hiPSCs). Two pluripotency genes, LEFTY1 and LEFTY2, which encode secreted ligands of the transforming growth factor‐beta, were downregulated, while CA4 and OCLM, which are related to eye development, were upregulated in both samples. The gene set enrichment analysis showed that the development of atrioventricular heart valves and the dysfunction of cellular components, including extracellular matrix, were significantly affected after exposure of hiPSCs to PSNPs. Finally, using the HiPathia method, which uncovers disease mechanisms and predicts clinical outcomes, we determined the APOC3 circuit, which is responsible for increased risk for ischemic cardiovascular disease. These results clearly demonstrate that better understanding of NPs bioactivities and its implications for human health is of extreme importance. Thus, the presented platform opens further aspects to study interactions between different environmental and intracellular pollutions with the aim to decipher the mechanism and origin of human diseases.Serbian Ministry of Education, Science and Technology, Grant/Award Number: ON175103; European Regional Development Funds; Spanish Ministry of Economy and Competitiveness, Grant/Award Number: SAF2017‐88908‐R; Engineering and Physical Sciences Research Council of the United Kingdom, Grant/Award Number: EP/S001433/1; ERC consolidator, Grant/Award Number: 614629