Comparing and contrasting Escherichia coli and Mycobacterium tuberculosis mechanosensitive channels (MscL) - New gain of function mutations in the loop region

Sequence analysis of 35 putative MscL homologues was used to develop an optimal alignment for Escherichia coli and Mycobacterium tuberculosis MscL and to place these homologues into sequence subfamilies. By using this alignment, previously identified E. coli MscL mutants that displayed severe and very severe gain of function phenotypes were mapped onto the M. tuberculosis MscL sequence. Not all of the resulting M. tuberculosis mutants displayed a gain of function phenotype; for instance, normal phenotypes were noted for mutations at Ala20, the analogue of the highly sensitive Gly22 site in E. coli. A previously unnoticed intersubunit hydrogen bond in the extracellular loop region of the M. tuberculosis MscL crystal structure has been analyzed. Cross-linkable residues were substituted for the residues involved in the hydrogen bond, and cross-linking studies indicated that these sites are spatially close under physiological conditions. In general, mutation at these positions results in a gain of function phenotype, which provides strong evidence for the importance of the loop region in MscL channel function. No analogue to this interesting interaction could be found in E. coli MscL by sequence alignment. Taken together, these results indicate that caution should be exercised in using the M. tuberculosis MscL crystal structure to analyze previous functional studies of E. coli MscL

Impact of Gendered Topics in Letters of Recommendation on Perceived Importance for Making a Hiring Decision in Geosciences

Scientific fields are perceived of as more masculine than feminine and stereotypes of scientists are more closely associated with stereotypes of men than of women (Carli et al., 2016). Supporting this point, Elmore, Block, Bowers and Dutt (2019) uncovered 31 topics from letters of recommendation for post-doctoral applicants in the geosciences and found that these topics described male and female applicants differently and, in a manner consistent with gender stereotypes. As the number of women in the geosciences declines further up the academic ladder (National Science Foundation, 2017) and as letters of recommendation play an important role in academic hiring (Abbott et al. 2010), it is important to understand if the use of gendered topics in these letters may reduce the likelihood of female advancement. Thus, in the present study we gathered questionnaire data from 250 geoscience researchers and scientists asking them to rate and rank the topics uncovered by Elmore et al. (2019) in terms of their importance when making hiring decisions. Results showed geoscientists valued research productivity and publishing over being a teacher, student, or department citizen. Topics expressed more in letters for male applicants in Elmore et al. (2019) were rated as significantly more important when making a hiring decision than topics expressed more in letters for female applicants. Further, the male topics identified in Elmore et al. (2019) were ranked more often as the most important topics and less often as least important topics when making a hiring decision compared to the female topics. Finally, 68% of participants indicated they attribute 50% or more of their hiring decision to information found in letters of recommendation, underscoring the importance of letters of recommendation to career advancement in the geosciences

Modular design of heterologous pathways for portability across diverse microorganisms

We utilized a method for the high-throughput assembly of heterologous biochemical pathways, and an integration platform for these pathways into the genomes of diverse, non-model microorganisms. Carbon catabolic pathways have been chosen as the first test cases for the pathway design and assembly method. Catabolic pathway optimization has the inherent advantage that growth selections can be used to separate pathways with differing productivities. As such, it provides a tractable testbed for the development of modular engineering approaches. First, we selected pathway enzymes from a group of evolutionarily-divergent host organisms, to capture a wide diversity of possible enzyme structures and pathway configurations. The coding sequences of each pathway have been synthesized and assembled into individual plasmids with unique barcodes, to be combinatorially constructed into pathways using the CombiGEM DNA assembly method 1. These pathways will be assembled in pooled reactions, resulting in the final generation of ~1M pathways. These pathways will be integrated into the recipient microbial host genome using a phage serine recombinase system 2. A “Landing Pad” comprised of three attB sites will be integrated into the host genome using the Tn7 transposase 3. Finally, the designed pathways will be integrated into the recipient genome, through recombination of the Landing Pad attB site, with the respective attP site on the target pathway’s destination vector. Modified hosts will be grown under the selection of the target carbohydrate substrate. The identification and fitness of each pathway will be measured by sequencing the barcodes of the final versus initial pooled populations. In-depth analysis including metabolomics, transcriptomics and proteomics can illuminate regulatory and metabolic changes that result from introduction of the heterologous pathway. Experimental evolution can also be used with poorly-functioning pathways to select for improved variants. Characterizing the resulting mutants can help to identify factors that were initially limiting activity. Comparing successful, unsuccessful, and evolved pathways will help to explain why species differ in their ability to functionally express various heterologous pathways of interest. Ultimately, the final pathway analysis can be used to design modular metabolic units that are highly active and portable across dissimilar microbes. Understanding the requirements for effective use of various heterologous pathways will allow selection of the best pathway for a particular host, based on its unique genetics and physiology. 1. Wong, A. S. L., Choi, G. C. G., Cheng, A. A., Purcell, O. & Lu, T. K. Massively parallel high-order combinatorial genetics in human cells. Nat. Biotechnol. 33, 952–961 (2015). 2. Elmore, J. R., Furches, A., Wolff, G. N., Gorday, K. & Guss, A. M. Development of a high efficiency integration system and promoter library for rapid modification of Pseudomonas putida KT2440. Metab. Eng. Commun. 5, 1–8 (2017). 3. Choi, K.-H. & Schweizer, H. P. mini-Tn7 insertion in bacteria with single attTn7 sites: example Pseudomonas aeruginosa. Nat. Protoc. 1, 153–161 (2006)

A Qualitative Study of the Impact of the COVID-19 Pandemic on Metastatic Breast Cancer Care

The COVID-19 pandemic substantially impacted the delivery of oncology care, particularly for individuals with metastatic cancers. The objective of this study was to qualitatively evaluate the impacts of COVID-19 on metastatic breast cancer (MBC) care among patients. This study consisted of 36 semi-structured qualitative interviews conducted virtually with people living with MBC, who were members of a patient support organization called Project Life. Project Life is an MBC patient-led, web-based wellness community. Responses were analyzed using Phronetic Iterative Analysis. Interviews were conducted from March 14, 2022, to May 31, 2022. Analysis from 36 individual in-depth qualitative interviews revealed the following themes during COVID-19: (1) variable preferences for telehealth (2) disruptions to care, (3) virtualization of social care. Wide variations existed in preferences surrounding telehealth, centered around ideas of convenience. Disruptions to care included delays to diagnostic care, isolation from caregivers, and interruptions associated with COVID-19 infection. These results call for adaptability in oncology care given wide-ranging preferences on telehealth and the shifting of available support services

Coaxial Compound Helicopter for Confined Urban Operations

A rotorcraft was designed for military operations in a confined urban environment. The specifications included major increases in useful load, range, and speed relative current aircraft capabilities, with a size constraint based on the dimensions of urban streets and intersections. Analysis showed that this combination of requirements is best satisfied by a coaxial main-rotor configuration, with lift compounding to off-load the rotors at high speed, and ducted fans under the rotor disk for propulsion. The baseline design is described, and the aircraft performance is summarized for utility, attack, MEDEVAC, and cargo delivery missions. The impact on size and performance is examined for a number of excursions, including lift-offset main rotors. Technology development required to achieve this advance in capability is recommended

Specificity of Clinical Breast Examination in Community Practice

BACKGROUND: Millions of women receive clinical breast examination (CBE) each year, as either a breast cancer screening test or a diagnostic test for breast symptoms. While screening CBE had moderately high specificity (∼94%) in clinical trials, community clinicians may be comparatively inexperienced and may conduct relatively brief examinations, resulting in even higher specificity but lower sensitivity. OBJECTIVE: To estimate the specificity of screening and diagnostic CBE in clinical practice and identify patient factors associated with specificity. DESIGN: Retrospective cohort study. SUBJECTS: Breast-cancer-free female health plan enrollees in 5 states (WA, OR, CA, MA, and MN) who received CBE (N = 1,484). MEASUREMENTS: Medical charts were abstracted to ascertain breast cancer risk factors, examination purpose (screening vs diagnostic), and results (true-negative vs false-positive). Women were considered “average-risk” if they had neither a family history of breast cancer nor a prior breast biopsy and “increased-risk” otherwise. RESULTS: Among average- and increased-risk women, respectively, the specificity (true-negative proportion) of screening CBE was 99.4% [95% confidence interval (CI): 98.8–99.7%] and 97.1% (95% CI: 95.7–98.0%), and the specificity of diagnostic CBE was 68.7% (95% CI: 59.7–76.5%) and 57.1% (95% CI: 51.1–63.0%). The odds of a true-negative screening CBE (specificity) were significantly lower among women at increased risk of breast cancer (adjusted odds ratio 0.21; 95% CI: 0.10–0.46). CONCLUSIONS: Screening CBE likely has higher specificity among community clinicians compared to examiners in clinical trials of breast cancer screening, even among women at increased breast cancer risk. Highly specific examinations, however, may have relatively low sensitivity for breast cancer. Diagnostic CBE, meanwhile, is relatively nonspecific

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

Virus genomes reveal factors that spread and sustained the Ebola epidemic.

The 2013-2016 West African epidemic caused by the Ebola virus was of unprecedented magnitude, duration and impact. Here we reconstruct the dispersal, proliferation and decline of Ebola virus throughout the region by analysing 1,610 Ebola virus genomes, which represent over 5% of the known cases. We test the association of geography, climate and demography with viral movement among administrative regions, inferring a classic 'gravity' model, with intense dispersal between larger and closer populations. Despite attenuation of international dispersal after border closures, cross-border transmission had already sown the seeds for an international epidemic, rendering these measures ineffective at curbing the epidemic. We address why the epidemic did not spread into neighbouring countries, showing that these countries were susceptible to substantial outbreaks but at lower risk of introductions. Finally, we reveal that this large epidemic was a heterogeneous and spatially dissociated collection of transmission clusters of varying size, duration and connectivity. These insights will help to inform interventions in future epidemics

Large-scale exome-wide association analysis identifies loci for White Blood Cell Traits and Pleiotropy with Immune-Mediated Diseases

White blood cells play diverse roles in innate and adaptive immunity. Genetic association analyses of phenotypic variation in circulating white blood cell (WBC) counts from large samples of otherwise healthy individuals can provide insights into genes and biologic pathways involved in production, differentiation, or clearance of particular WBC lineages (myeloid, lymphoid) and also potentially inform the genetic basis of autoimmune, allergic, and blood diseases. We performed an exome array-based meta-analysis of total WBC and subtype counts (neutrophils, monocytes, lymphocytes, basophils, and eosinophils) in a multi-ancestry discovery and replication sample of ∼157,622 individuals from 25 studies. We identified 16 common variants (8 of which were coding variants) associated with one or more WBC traits, the majority of which are pleiotropically associated with autoimmune diseases. Based on functional annotation, these loci included genes encoding surface markers of myeloid, lymphoid, or hematopoietic stem cell differentiation (CD69, CD33, CD87), transcription factors regulating lineage specification during hematopoiesis (ASXL1, IRF8, IKZF1, JMJD1C, ETS2-PSMG1), and molecules involved in neutrophil clearance/apoptosis (C10orf54, LTA), adhesion (TNXB), or centrosome and microtubule structure/function (KIF9, TUBD1). Together with recent reports of somatic ASXL1 mutations among individuals with idiopathic cytopenias or clonal hematopoiesis of undetermined significance, the identification of a common regulatory 3 UTR variant of ASXL1 suggests that both germline and somatic ASXL1 mutations contribute to lower blood counts in otherwise asymptomatic individuals. These association results shed light on genetic mechanisms that regulate circulating WBC counts and suggest a prominent shared genetic architecture with inflammatory and autoimmune diseases

Gender, Animacy, and Variable Constraints: An OT Analysis of Leísmo, Loísmo, and Laísmo

This paper examines the Spanish dialectal phenomena of leísmo, loísmo, and laísmo. I argue that a speaker’s ability to transition towards a more uniform use of [le] or [lo/la] is governed by a set of underlying restrictions on the dative and accusative cases. These restrictions are based on a speaker’s preference towards masculine over feminine gender, as well as a preference towards human over nonhuman animacy. These preferences limit an object NP’s accessibility to the dative and accusative cases. The specific constrictions each speaker employs within their grammar, as well as how they prioritize them, determine how dative and accusative case are assigned. The resulting case assignment patterns produce the various permutations of leísmo and lo(la)ísmo. To represent the relevant case restrictions, I ground my framework within Optimality Theory, which uses constraint rankings, to provide the basis for this model. Using dative and accusative case constraints with respect to gender and animacy, along with markedness and faithfulness constraints, I account for both inter-speaker and intra-speaker variation within leísmo and lo(la)ísmo