Notch: a mastermind of vascular morphogenesis

The way in which multiple cell types organize themselves into a carefully sculpted, 3D labyrinth of vessels that regulate blood flow throughout the body has been a longstanding mystery. Clinicians familiar with congenital cardiovascular disease recognize how genetic variants and modest perturbations in this complex set of spatiotemporal interactions and stochastic processes can result in life-threatening anomalies. Although the mystery is not yet fully solved, we are poised at an exciting juncture, as insights from murine disease models are converging with advances in human genetics to shed new light on puzzling clinical phenotypes of vascular disease. The study by High et al. in this issue of the JCI establishes a model system that mimics clinical features of congenital cardiovascular disease and further defines the role of the Notch signaling pathway in the neural crest as an essential determinant of cardiovascular structure (see the related article beginning on page 353).     

The impact of the SARS-CoV-2 pandemic on the management of chronic limb-threatening ischemia and wound care

In the wake of the coronavirus pandemic, the critical limb ischemia (CLI) Global Society aims to develop improved clinical guidance that will inform better care standards to reduce tissue loss and amputations during and following the new SARS-CoV-2 era. This will include developing standards of practice, improve gaps in care, and design improved research protocols to study new chronic limb-threatening ischemia treatment and diagnostic options. Following a round table discussion that identified hypotheses and suppositions the wound care community had during the SARS-CoV-2 pandemic, the CLI Global Society undertook a critical review of literature using PubMed to confirm or rebut these hypotheses, identify knowledge gaps, and analyse the findings in terms of what in wound care has changed due to the pandemic and what wound care providers need to do differently as a result of these changes. Evidence was graded using the Oxford Centre for Evidence-Based Medicine scheme. The majority of hypotheses and related suppositions were confirmed, but there is noticeable heterogeneity, so the experiences reported herein are not universal for wound care providers and centres. Moreover, the effects of the dynamic pandemic vary over time in geographic areas. Wound care will unlikely return to prepandemic practices. Importantly, Levels 2–5 evidence reveals a paradigm shift in wound care towards a hybrid telemedicine and home healthcare model to keep patients at home to minimize the number of in-person visits at clinics and hospitalizations, with the exception of severe cases such as chronic limb-threatening ischemia. The use of telemedicine and home care will likely continue and improve in the postpandemic era.     

A PCR procedure to determine the sequence of large polypeptides by rapid walking through a cDNA library.

A procedure that uses the PCR to make rapid successive steps through a random-primed cDNA library has been developed to provide a method for sequencing very long genes that are difficult to obtain as a single clone. In each successive step, the portions of partial clones that extend out from the region of known DNA sequence are amplified by two stages of PCR with nested, outward-directed primers designed approximately 50 bases in from the end of the known sequence, together with a general primer based on the sequence of the vector. This procedure has been used to determine the coding sequence of the cDNA for the beta heavy chain of axonemal dynein from embryos of the sea urchin Tripneustes gratilla. By starting from a single parent clone, whose translated amino acid sequence overlapped the microsequence of a tryptic peptide of the beta heavy chain, and making 3 such walk steps downstream and 14 walk steps upstream, we obtained a sequence of 13,799 base pairs that had an open reading frame of 13,398 base pairs. This sequence encodes a polypeptide with 4466 residues of Mr 511,804 that is believed to correspond to the complete beta heavy chain of ciliary outer arm dynein.     

Heart rate control in patients with atrial fibrillation referred for exercise testing

Clinical practice guidelines for patients with atrial fibrillation (AF) recommended a heart rate (HR) of 60 to 80 beats/min at rest and 90 to 115 at moderate exercise. The degree to which HR control at rest and with exercise in patients with AF complies with these recommendations is unknown. HR at rest and at peak exercise was retrospectively examined in 1,097 consecutive patients with AF referred for exercise myocardial perfusion imaging. In a subgroup of 195 patients, HR was also measured at an intermediate "moderate" level. Median HR at rest was 80 beats/min, at the upper end of the recommended range of 60 to 80. Only patients administered a beta blocker (BB; 31%) had lower (p <0.001) median HRs at rest. Median HR at moderate exercise was 128 beats/min, higher than the range of 90 to 115 recommended by the guidelines. Only patients administered a BB had significantly reduced HRs (p <0.003) at moderate exercise. Median peak exercise HR was 147 beats/min. Forty-five percent of patients exceeded their age-predicted maximal HR. Patients administered BBs were significantly less likely (p <0.01) to exceed their age-predicted maximal HR. In conclusion, in patients with AF, HR control at rest and during exercise often did not comply with guideline recommendations. Regimens including a BB were more effective in achieving HR control.     

Feasibility of perinatal mood screening and text messaging on patients’ personal smartphones

Archives of Women's Mental Health - Screens and adjunctive treatments for perinatal mood are available, but barriers prevent many women from receiving them. Mobile technology may help bypass...     

UBQLN2 mutations are not a frequent cause of amyotrophic lateral sclerosis in Ireland

Mutations in UBQLN2 have been shown to be a cause of dominant X-linked amyotrophic lateral sclerosis (ALS). Occurrences of mutations in this gene vary across ALS populations. We screened UBQLN2 for mutations in a final cohort of 150 Irish ALS patients. Individuals who were from families with male-to-male transmission or who carried pathogenic hexanucleotide repeat expansions in C9orf72 were excluded. Apart from common synonymous variation, no sequence variants in UBQLN2 were observed. Mutations in UBQLN2 are therefore not a frequent cause of ALS in the Irish population.     

Preservation of genetic and regulatory robustness in ancient gene duplicates of Saccharomyces cerevisiae

Biological systems remain robust against certain genetic and environmental challenges. Robustness allows the exploration of ecological adaptations. It is unclear what factors contribute to increasing robustness. Gene duplication has been considered to increase genetic robustness through functional redundancy, accelerating the evolution of novel functions. However, recent findings have questioned the link between duplication and robustness. In particular, it remains elusive whether ancient duplicates still bear potential for innovation through preserved redundancy and robustness. Here we have investigated this question by evolving the yeast Saccharomyces cerevisiae for 2200 generations under conditions allowing the accumulation of deleterious mutations, and we put mechanisms of mutational robustness to a test. S. cerevisiae declined in fitness along the evolution experiment, but this decline decelerated in later passages, suggesting functional compensation of mutated genes. We resequenced 28 genomes from experimentally evolved S. cerevisiae lines and found more mutations in duplicates—mainly small-scale duplicates—than in singletons. Genetically interacting duplicates evolved similarly and fixed more amino acid–replacing mutations than expected. Regulatory robustness of the duplicates was supported by a larger enrichment for mutations at the promoters of duplicates than at those of singletons. Analyses of yeast gene expression conditions showed a larger variation in the duplicates’ expression than that of singletons under a range of stress conditions, sparking the idea that regulatory robustness allowed a wider range of phenotypic responses to environmental stresses, hence faster adaptations. Our data support the persistence of genetic and regulatory robustness in ancient duplicates and its role in adaptations to stresses.Biological systems are inherently robust to perturbations, maintaining the same phenotypes in the face of environmental and genetic challenges (Gu et al. 2003; Stelling et al. 2004; Wagner 2005b). Robustness is key to the emergence of biological complexity and diversification as more robust systems can explore a larger set of phenotypes, allowing greater potential for evolving novel adaptations (Draghi et al. 2010; Payne and Wagner 2014). Determining the factors that provide systems with robustness would pave the way for a more complete understanding of the origin of adaptations and biological complexity. However, despite major efforts in understanding robustness (Wagner 2012), the factors that increase robustness of biological systems and their characterization remain to be determined.Gene duplication has been considered to have a major role in genetic robustness (Lynch and Conery 2000), as the presence of two copies performing identical or overlapping functions confers immunity to the deleterious effects of mutations occurring in one of the gene copies. Additionally, gene duplication has been credited with great importance in generating evolutionary novelties (Ohno 1999) because the selection-free exploration of the genotype space, due to genetic redundancy, allows one gene copy to probe a wider range of phenotypes (Payne and Wagner 2014). Arguably, gene duplication provides an invaluable opportunity to explore the link between genetic robustness and evolvability. Indeed, a number of studies have shown that major gene duplication events, such as whole-genome duplication (WGD) in angiosperms (Wendel 2000; Blanc and Wolfe 2004a) and animals (Otto and Whitton 2000; Hoegg et al. 2004), are concomitant with the emergence of morphological, metabolic, and physiological innovations (Otto and Whitton 2000; Holub 2001; Lespinet et al. 2002; Hoegg et al. 2004; Kim et al. 2004; Maere et al. 2005).Despite the apparent causal link between gene duplication and evolutionary innovation, the neutral exploration of genotype space by a duplicated gene requires the persistence of both gene copies for long periods. This clashes with the evolutionary instability of genetic redundancy, illustrated by the fact that 92% of duplicates in Saccharomyces cerevisiae, originated through WGD roughly 100 million years ago (Mya) (Wolfe and Shields 1997), have returned to single gene copies in extant S. cerevisiae. The rate of preservation of genes in duplicate varies, however, among organisms, with some exhibiting up to 30% of their genes in duplicate (Blanc and Wolfe 2004b; Cui et al. 2006). Genetic robustness, along with other factors such as selection for increasing gene dosage (Conant and Wolfe 2008) and gene balance (Birchler et al. 2005; Freeling and Thomas 2006), has been proposed to allow the persistence of genes in duplicate for longer periods of time, thereby providing opportunity for innovation through mutation (Gu et al. 2003; Fares et al. 2013). This claim is supported by larger fitness effects associated with the deletion of singletons compared to duplicates in yeast (Gu et al. 2003), functional compensation of deleted gene copies (VanderSluis et al. 2010), higher robustness of duplicates to transient gene knockdowns in Caenorhabditis elegans (Conant and Wagner 2004), and the contribution of gene duplicates to provide functional back-up against deleterious human mutations (Hsiao and Vitkup 2008). Recent studies have challenged, however, the link between gene duplication and genetic robustness, revealing a more complex relationship between duplicate preservation, genetic redundancy, and robustness. For example, using synthetic lethality genetic maps, Ihmels et al. (2007) found that duplicates, although exhibiting functional compensation, account for only 25% of the mutational robustness of a system. Furthermore, Wagner (2000) analyzed a number of duplicated genes and found no evidence of compensatory effects for null mutations between gene copies with high sequence or expression similarities. Moreover, a recent study has shown that in natural populations of yeast, close duplicates are unlikely to provide substantial functional compensation (Plata and Vitkup 2013). Thus, it is unresolved whether gene duplication provides mutational robustness through genetic redundancy. Since genetic redundancy and robustness are directly linked to evolvability, finding whether or not gene duplication provides sufficient genetic robustness to overcome the energetic and metabolic cost of maintaining additional genetic material is crucial to link gene duplication with the evolution of novel traits. Also, finding appreciable genetic redundancy between the copies of ancient duplicates would support their potential for future biological innovations.The studies conducted so far to probe the link between gene duplication, genetic redundancy, and mutational robustness have been obscured by the complex mixture of the genomic signatures of natural selection and genetic drift. These mixed signatures make it difficult to disentangle the role of genetic redundancy and mutational robustness in the emergence of novel functions from that of selection favoring adaptive mutations. Moreover, most studies ignore the role of the mechanism of duplication, WGD versus small-scale duplication (SSD), in providing mutational robustness and thus opportunity for innovation (Carretero-Paulet and Fares 2012; Fares et al. 2013). It is expected that the present genetic robustness and incomplete functional compensation of today’s duplicates are the remainders of an ancient larger genetic robustness that emerged at the time of gene duplication. Owing to the functional diversification of duplicates, quantification of preserved genetic robustness is complex and requires a direct test of the robustness of current, long-term preserved duplicates to deleterious mutations. Therefore, a definitive resolution of the controversy of whether ancient gene duplicates provide genetic robustness must come from testing the impact of deleterious mutations on duplicates in comparison with singletons. In this study, we resolved the controversy by conducting an experiment that allows the accumulation of deleterious mutations in the genome of S. cerevisiae. Using experimental evolution allows disentangling adaptive mutations from deleterious and neutral mutations and testing hypotheses under tightly controlled experimental conditions, which are not possible in comparative genomics studies. We test, for the first time, whether duplicates tolerate more deleterious mutations in their coding and regulatory regions than expected under the assumption of no genetic robustness.