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Somatic mutations accumulate in healthy tissues as we age, giving rise to cancer and potentially contributing to ageing. To study somatic mutations in non-neoplastic tissues, we developed a series of protocols to sequence the genomes of small populations of cells isolated from histological sections. Here, we describe a complete workflow that combines laser-capture microdissection (LCM) with low-input genome sequencing, while circumventing the use of whole-genome amplification (WGA). The protocol is subdivided broadly into four steps: tissue processing, LCM, low-input library generation and mutation calling and filtering. The tissue processing and LCM steps are provided as general guidelines that might require tailoring based on the specific requirements of the study at hand. Our protocol for low-input library generation uses enzymatic rather than acoustic fragmentation to generate WGA-free whole-genome libraries. Finally, the mutation calling and filtering strategy has been adapted from previously published protocols to account for artifacts introduced via library creation. To date, we have used this workflow to perform targeted and whole-genome sequencing of small populations of cells (typically 100–1,000 cells) in thousands of microbiopsies from a wide range of human tissues. The low-input DNA protocol is designed to be compatible with liquid handling platforms and make use of equipment and expertise standard to any core sequencing facility. However, obtaining low-input DNA material via LCM requires specialized equipment and expertise. The entire protocol from tissue reception through whole-genome library generation can be accomplished in as little as 1 week, although 2–3 weeks would be a more typical turnaround time. This protocol describes a workflow that combines laser-capture microdissection with low-input genome sequencing, while circumventing the use of whole-genome amplification, for accurate detection of somatic mutations in non-neoplastic tissues.

Coronavirus disease 2019 (COVID-19) has seen a shift in remote work from being a discretionary flexible work policy to a mandatory requirement for several workplaces. This ‘forced flexibility’ has meant that organisations and their employees have had to adapt swiftly to new requirements and arrangements for how work is done. Working remotely, often at home in ‘virtual workspaces’, has become commonplace for many employees across Australia and globally. Drawing on findings from two qualitative phases of research in an Australian resources company, we use conservation of resources theory to explore the factors influencing processes related to wellbeing and productivity for employees working remotely in the COVID-19 environment. We identify challenges associated with working remotely, as well as important resources for wellbeing and productivity. Practical implications are also discussed.

Purpose The purpose of this paper is to use conservation of resources (COR) theory as a lens for comparing the impact of line management on Bangladeshi public and private nurses’ perception of work harassment, well-being and turnover intentions where Anglo-American and European management models have been super-imposed on an existing different culture. Design/methodology/approach Survey data were collected from 317 Bangladeshi nurses’ (131 from the public sector and 186 from the private sector). Structural equation modelling was used for analysis. Findings High work harassment was associated with low-being, and together with management practices, it explained approximately a quarter of private sector nurses’ well-being. In total, management, work harassment and employee well-being explained approximately a third of the turnover intentions of public sector nurses, whereas only work harassment explained approximately a third of private sector nurses’ turnover intentions. The findings suggest a differential impact of management on work harassment across the public and private sector. Research limitations/implications Cross-sectional data are susceptible to common method bias. A common latent factor was included, and several items that were explained by common method variance were controlled. Further, the findings are limited by the sample size from one sector and the use of only one developing country. Practical implications It is a waste of resources to transplant Anglo-American and European management models to developing countries without understanding the impact on nurses’ outcomes. Originality/value Anglo-American and European management models are not easily transferable to the Bangladesh context probably because of the impact of ties and corruption. Line management is a positive resource that builds employee well-being for public sector employees only.

Neutrophils play fundamental roles in innate immune response, shape adaptive immunity, and are a potentially causal cell type underpinning genetic associations with immune system traits and diseases. Here, we profile the binding of myeloid master regulator PU.1 in primary neutrophils across nearly a hundred volunteers. We show that variants associated with differential PU.1 binding underlie genetically-driven differences in cell count and susceptibility to autoimmune and inflammatory diseases. We integrate these results with other multi-individual genomic readouts, revealing coordinated effects of PU.1 binding variants on the local chromatin state, enhancer-promoter contacts and downstream gene expression, and providing a functional interpretation for 27 genes underlying immune traits. Collectively, these results demonstrate the functional role of PU.1 and its target enhancers in neutrophil transcriptional control and immune disease susceptibility. PU.1 is a master regulator of myeloid development but its role in disease-relevant neutrophils is not well known. Here, the authors look at primary neutrophils from a human population and find that genetic variants affecting binding of PU.1 are associated with cell count and disease susceptibility.

This research examines the impact of lecturer-student exchange (student-LMX) on engagement, course satisfaction, achievement, and intention to leave university prematurely for 363 students in one Australian university. Survey and grade point average (GPA) data were collected from domestic undergraduate first- and second-year students and analysed using structural equation modelling. The results indicated that student's levels of engagement and course satisfaction fully mediated the relationship between student-LMX and intention to leave university, when demographic and socio-economic factors were controlled for. In an era when low student engagement and attrition is often attributed to individual demographic factors, and lecturers are under increasing threat of being replaced by technology, this research offers compelling evidence regarding the role of lecturer-student relationships in enhancing tertiary student outcomes.(HRK / Abstract übernommen).

The spins of the black holes involved in each of the four mergers that have been detected in gravitational waves so far were either small or not aligned with the binary orbit. Distinguishing black hole spin alignment with gravitational waves One of the best-measured parameters from the gravitational wave chirps caused by merging binary black holes is the effective spin of the binary—a combination of the spins of the individual black holes. If the black holes came from a pre-existing binary star system, then the expectation is that the spins will be aligned. On the other hand, if the binary black hole systems were formed through dynamical interactions, the spins will be randomly aligned. William Farr et al . examine the spin parameters for the four mergers reported so far and find at 2.4 σ significance that the spins were not aligned. Only ten more merger events will be needed to raise this to 5 σ if most of the spins are not aligned. The direct detection of gravitational waves 1 , 2 , 3 , 4 from merging binary black holes opens up a window into the environments in which binary black holes form. One signature of such environments is the angular distribution of the black hole spins. Binary systems that formed through dynamical interactions between already-compact objects are expected to have isotropic spin orientations 5 , 6 , 7 , 8 , 9 (that is, the spins of the black holes are randomly oriented with respect to the orbit of the binary system), whereas those that formed from pairs of stars born together are more likely to have spins that are preferentially aligned with the orbit 10 , 11 , 12 , 13 , 14 . The best-measured combination of spin parameters 3 , 4 for each of the four likely binary black hole detections GW150914, LVT151012, GW151226 and GW170104 is the ‘effective’ spin. Here we report that, if the magnitudes of the black hole spins are allowed to extend to high values, the effective spins for these systems indicate a 0.015 odds ratio against an aligned angular distribution compared to an isotropic one. When considering the effect of ten additional detections 15 , this odds ratio decreases to 2.9 × 10 −7 against alignment. The existing preference for either an isotropic spin distribution or low spin magnitudes for the observed systems will be confirmed (or overturned) confidently in the near future.

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for virus infection through the engagement of the human ACE2 protein 1 and is a major antibody target. Here we show that chronic infection with SARS-CoV-2 leads to viral evolution and reduced sensitivity to neutralizing antibodies in an immunosuppressed individual treated with convalescent plasma, by generating whole-genome ultra-deep sequences for 23 time points that span 101 days and using in vitro techniques to characterize the mutations revealed by sequencing. There was little change in the overall structure of the viral population after two courses of remdesivir during the first 57 days. However, after convalescent plasma therapy, we observed large, dynamic shifts in the viral population, with the emergence of a dominant viral strain that contained a substitution (D796H) in the S2 subunit and a deletion (ΔH69/ΔV70) in the S1 N-terminal domain of the spike protein. As passively transferred serum antibodies diminished, viruses with the escape genotype were reduced in frequency, before returning during a final, unsuccessful course of convalescent plasma treatment. In vitro, the spike double mutant bearing both ΔH69/ΔV70 and D796H conferred modestly decreased sensitivity to convalescent plasma, while maintaining infectivity levels that were similar to the wild-type virus.The spike substitution mutant D796H appeared to be the main contributor to the decreased susceptibility to neutralizing antibodies, but this mutation resulted in an infectivity defect. The spike deletion mutant ΔH69/ΔV70 had a twofold higher level of infectivity than wild-type SARS-CoV-2, possibly compensating for the reduced infectivity of the D796H mutation. These data reveal strong selection on SARS-CoV-2 during convalescent plasma therapy, which is associated with the emergence of viral variants that show evidence of reduced susceptibility to neutralizing antibodies in immunosuppressed individuals. Chronic infection with SARS-CoV-2 leads to the emergence of viral variants that show reduced susceptibility to neutralizing antibodies in an immunosuppressed individual treated with convalescent plasma.

Transmission of SARS-CoV-2 is uncontrolled in many parts of the world; control is compounded in some areas by the higher transmission potential of the B.1.1.7 variant 1 , which has now been reported in 94 countries. It is unclear whether the response of the virus to vaccines against SARS-CoV-2 on the basis of the prototypic strain will be affected by the mutations found in B.1.1.7. Here we assess the immune responses of individuals after vaccination with the mRNA-based vaccine BNT162b2 2 . We measured neutralizing antibody responses after the first and second immunizations using pseudoviruses that expressed the wild-type spike protein or a mutated spike protein that contained the eight amino acid changes found in the B.1.1.7 variant. The sera from individuals who received the vaccine exhibited a broad range of neutralizing titres against the wild-type pseudoviruses that were modestly reduced against the B.1.1.7 variant. This reduction was also evident in sera from some patients who had recovered from COVID-19. Decreased neutralization of the B.1.1.7 variant was also observed for monoclonal antibodies that target the N-terminal domain (9 out of 10) and the receptor-binding motif (5 out of 31), but not for monoclonal antibodies that recognize the receptor-binding domain that bind outside the receptor-binding motif. Introduction of the mutation that encodes the E484K substitution in the B.1.1.7 background to reflect a newly emerged variant of concern (VOC 202102/02) led to a more-substantial loss of neutralizing activity by vaccine-elicited antibodies and monoclonal antibodies (19 out of 31) compared with the loss of neutralizing activity conferred by the mutations in B.1.1.7 alone. The emergence of the E484K substitution in a B.1.1.7 background represents a threat to the efficacy of the BNT162b2 vaccine. Sera from vaccinated individuals and some monoclonal antibodies show a modest reduction in neutralizing activity against the B.1.1.7 variant of SARS-CoV-2; but the E484K substitution leads to a considerable loss of neutralizing activity.

Vaccines based on the spike protein of SARS-CoV-2 are a cornerstone of the public health response to COVID-19. The emergence of hypermutated, increasingly transmissible variants of concern (VOCs) threaten this strategy. Omicron (B.1.1.529), the fifth VOC to be described, harbours multiple amino acid mutations in spike, half of which lie within the receptor-binding domain. Here we demonstrate substantial evasion of neutralization by Omicron BA.1 and BA.2 variants in vitro using sera from individuals vaccinated with ChAdOx1, BNT162b2 and mRNA-1273. These data were mirrored by a substantial reduction in real-world vaccine effectiveness that was partially restored by booster vaccination. The Omicron variants BA.1 and BA.2 did not induce cell syncytia in vitro and favoured a TMPRSS2-independent endosomal entry pathway, these phenotypes mapping to distinct regions of the spike protein. Impaired cell fusion was determined by the receptor-binding domain, while endosomal entry mapped to the S2 domain. Such marked changes in antigenicity and replicative biology may underlie the rapid global spread and altered pathogenicity of the Omicron variant. The Omicron variant evades vaccine-induced neutralization but also fails to form syncytia, shows reduced replication in human lung cells and preferentially uses a TMPRSS2-independent cell entry pathway, which may contribute to enhanced replication in cells of the upper airway. Altered fusion and cell entry characteristics are linked to distinct regions of the Omicron spike protein.

The evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus leads to new variants that warrant timely epidemiological characterization. Here we use the dense genomic surveillance data generated by the COVID-19 Genomics UK Consortium to reconstruct the dynamics of 71 different lineages in each of 315 English local authorities between September 2020 and June 2021. This analysis reveals a series of subepidemics that peaked in early autumn 2020, followed by a jump in transmissibility of the B.1.1.7/Alpha lineage. The Alpha variant grew when other lineages declined during the second national lockdown and regionally tiered restrictions between November and December 2020. A third more stringent national lockdown suppressed the Alpha variant and eliminated nearly all other lineages in early 2021. Yet a series of variants (most of which contained the spike E484K mutation) defied these trends and persisted at moderately increasing proportions. However, by accounting for sustained introductions, we found that the transmissibility of these variants is unlikely to have exceeded the transmissibility of the Alpha variant. Finally, B.1.617.2/Delta was repeatedly introduced in England and grew rapidly in early summer 2021, constituting approximately 98% of sampled SARS-CoV-2 genomes on 26 June 2021. A study of the evolution of the SARS-CoV-2 virus in England between September 2020 and June 2021 finds that interventions capable of containing previous variants were insufficient to stop the more transmissible Alpha and Delta variants.