Lambpatterson2155

DNA sequence variants with allele fractions below 1% are difficult to detect and quantify by sequencing owing to intrinsic errors in sequencing-by-synthesis methods. Although molecular-identifier barcodes can detect mutations with a variant-allele frequency (VAF) as low as 0.1% using next-generation sequencing (NGS), sequencing depths of over 25,000× are required, thus hampering the detection of mutations at high sensitivity in patient samples and in most samples used in research. Here we show that low-frequency DNA variants can be detected via low-depth multiplexed NGS after their amplification, by a median of 300-fold, using polymerase chain reaction and rationally designed 'blocker' oligonucleotides that bind to the variants. Using an 80-plex NGS panel and a sequencing depth of 250×, we detected single nucleotide polymorphisms with a VAF of 0.019% and contamination in human cell lines at a VAF as low as 0.07%. With a 16-plex NGS panel covering 145 mutations across 9 genes involved in melanoma, we detected low-VAF mutations (0.2-5%) in 7 out of the 19 samples of freshly frozen tumour biopsies, suggesting that tumour heterogeneity could be notably higher than previously recognized.Cell therapies for the treatment of skin disorders could benefit from simple, safe and efficient technology for the transdermal delivery of therapeutic cells. Conventional cell delivery by hypodermic-needle injection is associated with poor patient compliance, requires trained personnel, generates waste and has non-negligible risks of injury and infection. Here, we report the design and proof-of-concept application of cryogenic microneedle patches for the transdermal delivery of living cells. The microneedles are fabricated by stepwise cryogenic micromoulding of cryogenic medium with pre-suspended cells, and can be easily inserted into porcine skin and dissolve after deployment of the cells. In mice, cells delivered by the cryomicroneedles retained their viability and proliferative capability. In mice with subcutaneous melanoma tumours, the delivery of ovalbumin-pulsed dendritic cells via the cryomicroneedles elicited higher antigen-specific immune responses and led to slower tumour growth than intravenous and subcutaneous injections of the cells. Biocompatible cryomicroneedles may facilitate minimally invasive cell delivery for a range of cell therapies.Direct cardiac reprogramming of fibroblasts to cardiomyocytes presents an attractive therapeutic strategy to restore cardiac function following injury. Cardiac reprogramming was initially achieved through overexpression of the transcription factors Gata4, Mef2c and Tbx5; later, Hand2 and Akt1 were found to further enhance this process1-5. Yet, staunch epigenetic barriers severely limit the ability of these cocktails to reprogramme adult fibroblasts6,7. We undertook a screen of mammalian gene regulatory factors to discover novel regulators of cardiac reprogramming in adult fibroblasts and identified the histone reader PHF7 as the most potent activating factor8. Mechanistically, PHF7 localizes to cardiac super enhancers in fibroblasts, and through cooperation with the SWI/SNF complex, it increases chromatin accessibility and transcription factor binding at these sites. Furthermore, PHF7 recruits cardiac transcription factors to activate a positive transcriptional autoregulatory circuit in reprogramming. Importantly, PHF7 achieves efficient reprogramming in the absence of Gata4. Here, we highlight the underexplored necessity of cardiac epigenetic readers, such as PHF7, in harnessing chromatin remodelling and transcriptional complexes to overcome critical barriers to direct cardiac reprogramming.Ecological theory suggests that habitat disturbance differentially influences distributions of habitat generalist and specialist species. While well-established for macroorganisms, this theory has rarely been explored for microorganisms. Here we tested these principles in permeable (sandy) sediments, ecosystems with much spatiotemporal variation in resource availability and physicochemical conditions. Microbial community composition and function were profiled in intertidal and subtidal sediments using 16S rRNA gene amplicon sequencing and metagenomics, yielding 135 metagenome-assembled genomes. Community composition and metabolic traits modestly varied with sediment depth and sampling date. Several taxa were highly abundant and prevalent in all samples, including within the orders Woeseiales and Flavobacteriales, and classified as habitat generalists; genome reconstructions indicate these taxa are highly metabolically flexible facultative anaerobes and adapt to resource variability by using different electron donors and acceptors. In contrast, obligately anaerobic taxa such as sulfate reducers and candidate lineage MBNT15 were less abundant overall and only thrived in more stable deeper sediments. selleck We substantiated these findings by measuring three metabolic processes in these sediments; whereas the habitat generalist-associated processes of sulfide oxidation and fermentation occurred rapidly at all depths, the specialist-associated process of sulfate reduction was restricted to deeper sediments. A manipulative experiment also confirmed habitat generalists outcompete specialist taxa during simulated habitat disturbance. Together, these findings show metabolically flexible habitat generalists become dominant in highly dynamic environments, whereas metabolically constrained specialists are restricted to narrower niches. Thus, an ecological theory describing distribution patterns for macroorganisms likely extends to microorganisms. Such findings have broad ecological and biogeochemical ramifications.Patterns of species diversity provide fundamental insights into the underlying mechanisms and processes that regulate biodiversity. The species-time relationship (STR) has the potential to be one such pattern; in a comparable manner to its more extensively studied spatial analogue, the species-area relationship (SAR), which has been pivotal in the development of ecological models and theories. We sought to determine the mechanisms and processes that underpin STR patterns of temporal turnover by sampling bacterial communities within ten water-filled tree-holes on the same European beech tree through the course of a year. We took this natural model system to represent an archipelago of islands of varying sizes and with shared common immigration sources. We observed an inverse relationship between STR-derived turnover rates and island size. Further, turnover was related to island size and not island isolation within the study system as indicated by a low frequency of dispersal limitation and high homogenizing dispersal.