Developmental and cell cycle progression defects in Drosophila hybrid males

Matings between D. melanogaster females and males of sibling species in the D. melanogaster complex yield hybrid males that die prior to pupal differentiation. We have reexamined a previous report suggesting that the developmental defects in these lethal hybrid males reflect a failure in cell proliferation that may be the consequence of problems in mitotic chromosome condensation. We also observed a failure in cell proliferation, but find in contrast that the frequencies of mitotic figures and of nuclei staining for the mitotic marker phosphohistone H3 in the brains of hybrid male larvae are extremely low. We also found that very few of these brain cells in male hybrids are in S phase, as determined by BrdU incorporation. These data suggest that cells in hybrid males are arrested in either the G(1) or G(2) phases of the cell cycle. The cells in hybrid male brains appear to be particularly sensitive to environmental stress; our results indicate that certain in vitro incubation conditions induce widespread cellular necrosis in these brains, causing an abnormal nuclear morphology noted by previous investigators. We also document that hybrid larvae develop very slowly, particularly during the second larval instar. Finally, we found that the frequency of mitotic figures in hybrid male larvae mutant for Hybrid male rescue (Hmr) is increased relative to lethal hybrid males, although not to wild-type levels, and that chromosome morphology in Hmr(-) hybrid males is also not completely normal.     

LoCoH: nonparameteric kernel methods for constructing home ranges and utilization distributions

Parametric kernel methods currently dominate the literature regarding the construction of animal home ranges (HRs) and utilization distributions (UDs). These methods frequently fail to capture the kinds of hard boundaries common to many natural systems. Recently a local convex hull (LoCoH) nonparametric kernel method, which generalizes the minimum convex polygon (MCP) method, was shown to be more appropriate than parametric kernel methods for constructing HRs and UDs, because of its ability to identify hard boundaries (e.g., rivers, cliff edges) and convergence to the true distribution as sample size increases. Here we extend the LoCoH in two ways: "fixed sphere-of-influence," or r-LoCoH (kernels constructed from all points within a fixed radius r of each reference point), and an "adaptive sphere-of-influence," or a-LoCoH (kernels constructed from all points within a radius a such that the distances of all points within the radius to the reference point sum to a value less than or equal to a), and compare them to the original "fixed-number-of-points," or k-LoCoH (all kernels constructed from k-1 nearest neighbors of root points). We also compare these nonparametric LoCoH to parametric kernel methods using manufactured data and data collected from GPS collars on African buffalo in the Kruger National Park, South Africa. Our results demonstrate that LoCoH methods are superior to parametric kernel methods in estimating areas used by animals, excluding unused areas (holes) and, generally, in constructing UDs and HRs arising from the movement of animals influenced by hard boundaries and irregular structures (e.g., rocky outcrops). We also demonstrate that a-LoCoH is generally superior to k- and r-LoCoH (with software for all three methods available at http://locoh.cnr.berkeley.edu).     

Comparative expression of endogenous retrotransposons in adult mouse mammary gland during normal development and differentiation

Amplified expression of the endogenous retrotransposons, intracisternal A particles (IAPs) and murine leukemia virus-related elements (MLVEs), along with decreased expression of VL30 elements frequently occurs during mouse mammary tumorigenesis. We have now analyzed the expression of these retroelements during the normal developmental and differentiation cycle of the mammary gland as found in virgin, pregnant, lactating, and post-lactation adult female BALB/c mice. Retrotransposon expression was either unchanged or decreased during the progressive stages of the cycle compared to virgin tissue. Likewise, growth of mammary epithelial cells in primary culture had little or no effect on expression of IAPs, MLVEs and VL30 sequences. Thus, the dramatic changes involving these retrotransposons in many mouse mammary tumors appear unrelated to any normal state.     

First Nations status and emergency department triage scores in Alberta: a retrospective cohort study

Background:Previous studies have found that race is associated with emergency department triage scores, raising concerns about potential health care inequity. As part of a project on quality of care for First Nations people in Alberta, we sought to understand the relation between First Nations status and triage scores.Methods:We conducted a population-based retrospective cohort study of health administrative data from April 2012 to March 2017 to evaluate acuity of triage scores, categorized as a binary outcome of higher or lower acuity score. We developed multivariable multilevel logistic mixed-effects regression models using the levels of emergency department visit, patient (for patients with multiple visits) and facility. We further evaluated the triage of visits related to 5 disease categories and 5 specific diagnoses to better compare triage outcomes of First Nations and non–First Nations patients.Results:First Nations status was associated with lower odds of receiving higher acuity triage scores (odds ratio [OR] 0.93, 95% confidence interval [CI] 0.92–0.94) compared with non–First Nations patients in adjusted models. First Nations patients had lower odds of acute triage for all 5 disease categories and for 3 of 5 diagnoses, including long bone fractures (OR 0.82, 95% CI 0.76–0.88), acute upper respiratory infection (OR 0.90, 95% CI 0.84–0.98) and anxiety disorder (OR 0.67, 95% CI 0.60–0.74).Interpretation:First Nations status was associated with lower odds of higher acuity triage scores across a number of conditions and diagnoses. This may reflect systemic racism, stereotyping and potentially other factors that affected triage assessments.

Health outcomes are markedly worse for First Nations than non–First Nations people. Although this is largely because of inequities in the social determinants of health,^1–4 inequities in the provision of health care also exist.^5,6 Emergency departments serve as a point of accessible health care. Status First Nations patients make up 4.8% of unique patients and 9.4% of emergency visits in Alberta,⁷ and Canadian studies describe First Nations patients’ experiences with racism when seeking emergency care.^8,9Evaluating triage contributes empirically to understanding the health care of First Nations patients insofar as triage is a quantifiable, intermediate process by which systemic racism¹⁰ may influence patient outcomes. The Canadian Triage Acuity Scale¹¹ is a 5-level scale used to classify the severity of patient symptoms. Triage nurses use a brief assessment, medical history, and presenting signs and symptoms to assign each patient a triage score that determines the priority in which the patient should be seen by a provider. Therefore, accurate triage is important for patient health outcomes.¹² In practice, triage is a social interaction where local practice, biases, stereotypes and communication barriers come into play. Studies have found that women receive less acute triage scores than men,^13,14 and that racial minority^13,15–17 and Indigenous^18–20 patients receive less acute triage scores than white or non-Indigenous patients. Indeed, Indigenous patients in Canada have described a perception “of social triaging in the [emergency department], whereby decisions about who is seen first seemed to them [to be] based less on triaged clinical priorities but on the social positioning of the patient.”²¹ Differential triage scores for minority populations raise health equity concerns.As part of a larger mixed-methods project evaluating the quality of emergency care for First Nations people in Alberta, we sought to evaluate quantitative differences in emergency visit characteristics and outcomes of First Nations and non–First Nations people in Alberta. Specifically, we aimed to estimate the relation between First Nations status and acuity of triage, and to evaluate whether predictors of acuity differ by First Nations status.     

Validation of the isothermal Schistosoma haematobium Recombinase Polymerase Amplification (RPA) assay,coupled with simplified sample preparation,for diagnosing female genital schistosomiasis using cervicovaginal lavage and vaginal self-swab samples

BackgroundFemale genital schistosomiasis (FGS) is a neglected and disabling gynecological disease that can result from infection with the parasitic trematode Schistosoma haematobium. Accurate diagnosis of FGS is crucial for effective case management, surveillance and control. However, current methods for diagnosis and morbidity assessment can be inaccessible to those at need, labour intensive, costly and unreliable. Molecular techniques such as PCR can be used to reliably diagnose FGS via the detection of Schistosoma DNA using cervicovaginal lavage (CVL) samples as well as lesser-invasive vaginal self-swab (VSS) and cervical self-swab samples. PCR is, however, currently unsuited for use in most endemic settings. As such, in this study, we assessed the use of a rapid and portable S. haematobium recombinase polymerase amplification (Sh-RPA) isothermal molecular diagnostic assay, coupled with simplified sample preparation methodologies, to detect S. haematobium DNA using CVL and VSS samples provided by patients in Zambia.Methodology/Principal findingsVSS and CVL samples were screened for FGS using a previously developed Sh-RPA assay. DNA was isolated from VSS and CVL samples using the QIAamp Mini kit (n = 603 and 527, respectively). DNA was also isolated from CVL samples using two rapid and portable DNA extraction methods: 1) the SpeedXtract Nucleic Acid Kit (n = 223) and 2) the Extracta DNA Tissue Prep Kit (n = 136). Diagnostic performance of the Sh-RPA using VSS DNA extacts (QIAamp Mini kit) as well as CVL DNA extracts (QIAamp Mini kit, SpeedXtract Nucleic Acid Kit and Extracta DNA Tissue Prep Kit) was then compared to a real-time PCR reference test.Results suggest that optimal performance may be achieved when the Sh-RPA is used with PuVSS samples (sensitivity 93.3%; specificity 96.6%), however no comparisons between different DNA extraction methods using VSS samples could be carried out within this study. When using CVL samples, sensitivity of the Sh-RPA ranged between 71.4 and 85.7 across all three DNA extraction methods when compared to real-time PCR using CVL samples prepared using the QIAamp Mini kit. Interestingly, of these three DNA extraction methods, the rapid and portable SpeedXtract method had the greatest sensitivity and specificity (85.7% and 98.1%, respectively). Specificity of the Sh-RPA was >91% across all comparisons.Conclusions/SignificanceThese results supplement previous findings, highlighting that the use of genital self-swab sampling for diagnosing FGS should be explored further whilst also demonstrating that rapid and portable DNA isolation methods can be used to detect S. haematobium DNA within clinical samples using RPA. Although further development and assessment is needed, it was concluded that the Sh-RPA, coupled with simplified sample preparation, shows excellent promise as a rapid and sensitive diagnostic tool capable of diagnosing FGS at the point-of-care in resource-poor schistosomiasis-endemic settings.     

Divided we stand: tracking cell proliferation with carboxyfluorescein diacetate succinimidyl ester

Most techniques for assessing cell division can either detect limited numbers of cell divisions (bromodeoxyuridine incorporation) or only quantify overall proliferation (tritiated thymidine incorporation). In the majority of cases, viable cells of known division history cannot subsequently be obtained for functional studies. The cells of the immune system undergo marked proliferation and differentiation during the course of an immune response. The relative lack of an organized structure of the lymphohaemopoietic system, in contrast with other organ systems, makes lineage interrelationships difficult to study. Coupled with the remarkable degree of mobility engendered by recirculation, the differentiation occurring along with cell division in the immune system has not been readily accessible for investigation. The present article reviews the development of a cell division analysis procedure based on the quantitative serial halving of the membrane permeant, stably incorporating fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE or CFDA, SE). The technique can be used both in vitro and in vivo, allowing eight to 10 successive divisions to be resolved by flow cytometry. Furthermore, viable cells from defined generation numbers can be sorted by flow cytometry for functional analysis.     

A human genome map of comparative anchor tagged sequences.

Effective comparative mapping inference utilizing developing gene maps of animal species requires the inclusion of anchored reference loci that are homologous to genes mapped in the more "gene-dense" mouse and human maps. Nominated anchor loci, termed comparative anchor tagged sequences (CATS), have been ordered in the mouse linkage map, but due to the dearth of common polymorphisms among human coding genes have not been well represented in human linkage maps. We present here an ordered framework map of 314 comparative anchor markers in humans based on mapping analysis in the Genebridge 4 panel of radiation hybrid cell lines, plus empirically optimized CATS PCR primers which detect these markers. The ordering of these homologous gene markers in human and mouse maps provides a framework for comparative gene mapping of representative mammalian species.