Machine learning-based phenotypic imaging to characterise the targetable biology of Plasmodium falciparum male gametocytes for the development of transmission-blocking antimalarials.

Preventing parasite transmission from humans to mosquitoes is recognised to be critical for achieving elimination and eradication of malaria. Consequently developing new antimalarial drugs with transmission-blocking properties is a priority. Large screening campaigns have identified many new transmission-blocking molecules, however little is known about how they target the mosquito-transmissible Plasmodium falciparum stage V gametocytes, or how they affect their underlying cell biology. To respond to this knowledge gap, we have developed a machine learning image analysis pipeline to characterise and compare the cellular phenotypes generated by transmission-blocking molecules during male gametogenesis. Using this approach, we studied 40 molecules, categorising their activity based upon timing of action and visual effects on the organisation of tubulin and DNA within the cell. Our data both proposes new modes of action and corroborates existing modes of action of identified transmission-blocking molecules. Furthermore, the characterised molecules provide a new armoury of tool compounds to probe gametocyte cell biology and the generated imaging dataset provides a new reference for researchers to correlate molecular target or gene deletion to specific cellular phenotype. Our analysis pipeline is not optimised for a specific organism and could be applied to any fluorescence microscopy dataset containing cells delineated by bounding boxes, and so is potentially extendible to any disease model.

Developing a tool to assess the health-related quality of life in calves with respiratory disease: content validation.

Bovine respiratory disease (BRD) is a major welfare and productivity issue for calves. Despite the extensive negative impacts on calf welfare and performance, BRD remains challenging to detect and treat effectively. However, the clinical signs of disease are only one aspect of the disease that is experienced by the individual. The assessment of emotional experience in animals is not straightforward, but it is increasingly recognised that the quality of behaviour and demeanour of an individual is a reflection of their internal emotional state. The aim of the present study was to complete the content validation stage of the development process for a health-related quality of life (HRQOL) tool. This was based around indicators from an existing conceptual framework containing twenty-three indicators in two domains (clinical signs and behavioural expression). The content validation stage involves engaging with key stakeholders. For this study, this took the form of a survey and discussions with focus groups, which are standard methods in this field. A survey and stakeholder focus groups were conducted to assess the usefulness of each indicator and its relevance for inclusion within a HRQOL tool. In the survey, participants were asked to rate the usefulness of each of the indicators using a 4-point scale which were then dichotomised into 'useful' and 'less useful'. Based on the 'useful' result, each indicator within the domains was ranked. A similar approach was taken with the responses from the focus groups. Focus group participants were asked to select indicators that they felt were of use and the result of this was used to rank each of the indicators. The ranks of the indicators from both the survey and the focus groups along with the transcripts from the focus groups were used to determine the indicators from each domain to include within the HRQOL tool. Indicators within the clinical signs domain that were included were nasal discharge, cough, respiratory effort, ocular appearance (discharge and vibrancy), body and head posture and ear carriage. For the domain of behavioural expression, the indicators included were movement to feed, responsiveness, spatial proximity, volume of feed intake, motivation at feed and vigour. The next stage will be to validate the construction of the HRQOL tool through its use in practice. The inclusion of indicators that allow the experiential aspects of disease to be recorded in health assessments will likely increase the ability of farmers and others to detect respiratory disease in calves.

Cell-Type-Specific Circadian Bioluminescence Rhythms in Dbp Reporter Mice.

Circadian rhythms are endogenously generated physiological and molecular rhythms with a cycle length of about 24 h. Bioluminescent reporters have been exceptionally useful for studying circadian rhythms in numerous species. Here, we report development of a reporter mouse generated by modification of a widely expressed and highly rhythmic gene encoding D-site albumin promoter binding protein (Dbp). In this line of mice, firefly luciferase is expressed from the Dbp locus in a Cre recombinase-dependent manner, allowing assessment of bioluminescence rhythms in specific cellular populations. A mouse line in which luciferase expression was Cre-independent was also generated. The Dbp reporter alleles do not alter Dbp gene expression rhythms in liver or circadian locomotor activity rhythms. In vivo and ex vivo studies show the utility of the reporter alleles for monitoring rhythmicity. Our studies reveal cell-type-specific characteristics of rhythms among neuronal populations within the suprachiasmatic nuclei ex vivo. In vivo studies show Dbp-driven bioluminescence rhythms in the liver of Albumin-Cre;DbpKI/+ "liver reporter" mice. After a shift of the lighting schedule, locomotor activity achieved the proper phase relationship with the new lighting cycle more rapidly than hepatic bioluminescence did. As previously shown, restricting food access to the daytime altered the phase of hepatic rhythmicity. Our model allowed assessment of the rate of recovery from misalignment once animals were provided with food ad libitum. These studies confirm the previously demonstrated circadian misalignment following environmental perturbations and reveal the utility of this model for minimally invasive, longitudinal monitoring of rhythmicity from specific mouse tissues.

Methods for Detecting PER2:LUCIFERASE Bioluminescence Rhythms in Freely Moving Mice.

Circadian rhythms are driven by daily oscillations of gene expression. An important tool for studying cellular and tissue circadian rhythms is the use of a gene reporter, such as bioluminescence from the reporter gene luciferase controlled by a rhythmically expressed gene of interest. Here we describe methods that allow measurement of circadian bioluminescence from a freely moving mouse housed in a standard cage. Using a LumiCycle In Vivo (Actimetrics), we determined conditions that allow detection of circadian rhythms of bioluminescence from the PER2 reporter, PER2::LUC, in freely behaving mice. The LumiCycle In Vivo applies a background subtraction that corrects for effects of room temperature on photomultiplier tube (PMT) output. We tested delivery of d-luciferin via a subcutaneous minipump and in the drinking water. We demonstrate spikes in bioluminescence associated with drinking bouts. Further, we demonstrate that a synthetic luciferase substrate, CycLuc1, can support circadian rhythms of bioluminescence, even when delivered at a lower concentration than d-luciferin, and can support longer-term studies. A small difference in phase of the PER2::LUC bioluminescence rhythms, with females phase leading males, can be detected with this technique. We share our analysis scripts and suggestions for further improvements in this method. This approach will be straightforward to apply to mice with tissue-specific reporters, allowing insights into responses of specific peripheral clocks to perturbations such as environmental or pharmacological manipulations.

Effects of COVID-19 on Health Care Workers.

COVID-19 continues to spread across the United States with a continued increase in reported infections and deaths. How this virus effects pregnancy, particularly mothers and their infants around and after delivery, is of particular concern for health care workers. Moreover, concerns for compassion fatigue in the health care worker, as they attempt to provide comprehensive care to this population, is a documented concern that could have long-term effects on workers' ability to provide care. This article will describe the current concerns for the transmission of COVID-19 from the mother to the infant and how that has affected recommendations from several national and international organizations around maternal/infant testing, isolation, breastfeeding, and the infant requiring neonatal intensive care. Effects that changing recommendations may have on health care workers and care delivery, and how these may contribute to compassion fatigue, will also be discussed.

Building coalitions: A statewide nursing organization's role in changing nursing education regulation during the COVID-19 pandemic.

The California Association of Colleges of Nursing (CACN), representing California's baccalaureate and higher degree nursing education programs, has raised concerns for over two-years about the number, relevance, and legitimacy of nursing education regulations. Formal CACN letters to state regulators did not affect change. While California nursing education regulations require 75% direct patient contact for all clinical courses, meeting this requirement became impossible as clinical agencies closed to nursing students during the beginning of the COVID-19 pandemic. Nursing regulatory change was urgently needed to provide greater flexibility in meeting clinical course objectives using simulation and other online learning modalities. At stake was the graduation of over 14,971 RN students from public and private nursing programs. While state regulators opposed a legislative approach, CACN collaborated with stakeholders to support legislation that led to a reduction in direct patient care hours, allowing nursing students to progress and graduate. This longstanding advocacy work was accelerated by the pandemic and required leadership and knowledge about the legislative process for nurse educators to succeed. The ultimate goal for CACN is to forge a more respectful relationship and greater collaboration between educators and regulators to enhance quality, reduce costs, and redundancies in nursing education in this state.

Use of Licensed Vocational Nurses in California Schools: A Descriptive Study.

The demand for schools to provide complex health-care services for students with chronic conditions has induced districts to hire licensed vocational nurses (LVNs). Questions remain about how overlapping responsibilities and skills of nursing staff works to facilitate care. The purpose of this mixed-methods descriptive study was to examine the use of LVNs to identify factors related to the supports and impediments to school nurse (SN) practice. The sample consisted of members of the California School Nurse Organization, and methodologies consisted of an online survey and one-on-one interviews. LVNs perform duties within their scope of practice, allowing SNs time for activities related to the Framework for 21st Century School Nursing PracticeTM, confirming appropriate use, and alignment with National Association of School Nurses guidance on the utilization of LVNs. Identified areas of concern included role definition and orientation and supervision of the LVN. Insights into best practices for the effective addition of the LVN to the team are provided.

Microsatellite Instability assessment in Black South African Colorectal Cancer patients reveal an increased incidence of suspected Lynch syndrome.

Microsatellite Instability (MSI) is a hallmark of colorectal cancer (CRC) and occurs in 15-16% of CRC. Molecular biological information of CRC in South Africa (SA) is largely unrecorded. This study was undertaken to determine the frequency of MSI, with particular reference to Lynch syndrome (LS) with a view to improve surveillance and prevention strategies. This was a retrospective study on CRC samples diagnosed between 2011-2015 at Charlotte Maxeke Johannesburg Academic Hospital (CMJAH). Samples diagnosed between 2011-2012 were screened for MSI by PCR and mismatch repair (MMR) immunohistochemistry (IHC), and additional BRAFV600E mutational analysis performed. T-tests, Fischer's exact and Chi square statistical tests were applied. Twelve percent of patients displayed MSI, with increased frequency in black (15%) versus other ethnic group (OEG) (8%) patients. MSI patients were significantly younger than microsatellite stable (MSS) patients, however when stratified by ethnicity, black patients were predominantly younger (median age: 47), with increased MSH2/6 loss, and no BRAF mutations. These findings suggest a large proportion of young black SA CRC patients develop via the LS pathway due to earlier age onset and predominant MSH2/6 protein loss. SA patients of other ethnicities appear to follow the more well established sporadic MSI pathway.

The direct labor cost for providing life-saving blood to air medical transport for pre-hospital transfusion.

An IRB approved 2-year review and cost analysis of all packed red blood cells (pRBCs) issued, transfused and returned to the blood bank by air medical transport services for pre-hospital transfusion was performed. The cost to the blood bank for issuing and returning pRBCs that were not transfused in the pre-hospital setting was $3.24 per unit. Over the study period, there were 334 pRBCs not transfused by air medical services and returned to the blood bank totaling $1082.16 in direct labor costs, a trivial amount for providing possibly life-saving blood for pre-hospital transfusion.

Addressing Illness-Related Chronic Absences.

School attendance is a predictor of academic achievement. Chronic absenteeism, defined as missing 10% or more school days affects 14% of all students nationwide. District attendance processes, policies, and data were analyzed in a demographically diverse southern California high school. A review of the attendance history of 117 ninth and tenth graders, who missed at least 10% of days in school, showed that 66% of the absences were due to illness. Prior to the project, these students were not referred for nursing intervention. Results of this quality improvement project supported the adoption of a specific code for absences due to illness. A district procedure for illness chronic absence was adopted to allow early nursing intervention for students with chronic absences prior to the development of illness-related academic underperformance. This project demonstrates nursing roles in the quality improvement and care coordination aspects of the NASN's Framework for 21st Century School Nursing Practice™.

Storage Age of RBCs Transfused by a Prehospital Patient Transport Program vs the Hospital Emergency Department.

OBJECTIVES: Recently published data suggest that transfusion of RBCs stored for 22 days or longer was associated with increased mortality among massively transfused trauma patients. METHODS: We performed a 24-month retrospective review of medical transport service transfusion records and a 2-month, overlapping review of transfusions of uncrossmatched RBCs in the emergency department. RESULTS: RBC units issued to the transport program were older than RBC units issued to the emergency department trauma refrigerator (10.6 vs 8.7 days, P < .001). Similarly, RBC units were older at the time of transfusion during transport compared with the emergency department (20.3 vs 14.3 days, P < .001). CONCLUSIONS: Transport programs and blood banks should jointly review their RBC inventory management programs. Issuing RBCs to a medical transport program is a logistical challenge that, unless actively managed, may not ensure access to blood that is as fresh as blood in the emergency department.

Protecting participants in health research: The South African Material Transfer Agreement.

The need to transfer human biological materials (HBMs) across national boundaries has become increasingly important in view of increased biobank and commercial activities globally. In light of South Africa (SA)'s history of colonisation and racial discrimination, coupled with well-known instances of exploitation of research participants in the developing world, it is critical that the management of HBMs from and to other jurisdictions is explored and regulated. Material transfer agreements (MTAs) represent an important point of departure in such a process. This article explores the need for a uniform MTA in SA and discusses some aspects of the recently gazetted national MTA, which provides a framework that can serve as a safeguard for cross-border transfer of HBMs in the absence of the National Health Act's chapter 8 regulations in this regard.

Author Correction: An atlas of genetic influences on osteoporosis in humans and mice.

In the version of this article initially published, in Fig. 5a, the data in the right column of 'DAAM2 gRNA1' were incorrectly plotted as circles indicating 'untreated' rather than as squares indicating 'treated'. The error has been corrected in the HTML and PDF versions of the article.

Detection of Nicotine and Nicotine Metabolites in Units of Banked Blood.

OBJECTIVES: To determine the concentrations of nicotine and nicotine metabolites in RBC units as a means to estimate the point prevalence of exposure within the healthy donor pool. METHODS: Segments from 105 RBC units were tested for the presence of nicotine, cotinine, or trans-3'-hydroxycotinine by liquid chromatography-tandem mass spectrometry. RESULTS: Of the 20 (19%) units that contained detectable concentrations of nicotine, cotinine, or trans-3'-hydroxycotinine, 19 (18.1%) contained concentrations consistent with the use of a nicotine-containing product within 48 hours of specimen collection. One RBC unit contained nicotine concentrations consistent with passive exposure. CONCLUSIONS: Chemicals from nicotine-containing products are detectable within the US RBC supply. Further investigation is needed to determine the risks of transfusion-associated exposure to nicotine and other tobacco-associated chemicals among vulnerable patient populations such as neonates.

An atlas of genetic influences on osteoporosis in humans and mice.

Osteoporosis is a common aging-related disease diagnosed primarily using bone mineral density (BMD). We assessed genetic determinants of BMD as estimated by heel quantitative ultrasound in 426,824 individuals, identifying 518 genome-wide significant loci (301 novel), explaining 20% of its variance. We identified 13 bone fracture loci, all associated with estimated BMD (eBMD), in ~1.2 million individuals. We then identified target genes enriched for genes known to influence bone density and strength (maximum odds ratio (OR) = 58, P = 1 × 10-75) from cell-specific features, including chromatin conformation and accessible chromatin sites. We next performed rapid-throughput skeletal phenotyping of 126 knockout mice with disruptions in predicted target genes and found an increased abnormal skeletal phenotype frequency compared to 526 unselected lines (P < 0.0001). In-depth analysis of one gene, DAAM2, showed a disproportionate decrease in bone strength relative to mineralization. This genetic atlas provides evidence linking associated SNPs to causal genes, offers new insight into osteoporosis pathophysiology, and highlights opportunities for drug development.

Diagnostic and treatment preferences for developmental dysplasia of the hip: a survey of EPOS and POSNA members.

PURPOSE: The aim of this study was to elucidate developmental dysplasia of the hip (DDH) diagnosis and treatment preferences among members of the Pediatric Orthopaedic Society of North America (POSNA) and European Paediatric Orthopaedic Society (EPOS). METHODS: A 54-question survey on DDH diagnosis and treatment preferences was distributed to POSNA and EPOS members. Descriptive statistics were performed. RESULTS: A total of 459 responses were analyzed. Ultrasound was the preferred modality for diagnosing DDH in infants less than six months old; few surgeons preferred radiographs. In all, 57% of POSNA members had radiology technicians perform ultrasounds, only 7% of EPOS members did. The percent coverage defining a dislocated hip varied greatly, the most frequent response being < 20% for POSNA and < 40% for EPOS members. Pavlik harnesses were the most popular harness/brace, used by 90% of POSNA and 71% of EPOS members. POSNA members were more likely than EPOS members to use a rigid abduction brace following initial harness/brace failure. For residual acetabular dysplasia, POSNA members were twice as likely as EPOS members to institute hip abduction bracing. Most surgeons would not perform closed reduction at less than three months of age or open reduction at less than six months of age. Most EPOS -members used traction prior to reduction; few POSNA members did. Few POSNA and EPOS members believed that reduction should be delayed until the ossific nucleus was visible. CONCLUSION: There is great variation in the preferred methods for diagnosing and treating DDH. This survey is the largest transcontinental survey to compile diagnostic and treatment preferences for DDH. With wide variations in practice, there is room for quality improvement.

Spatial and temporal behavioural responses of wild cattle to tropical forest degradation.

Identifying the consequences of tropical forest degradation is essential to mitigate its effects upon forest fauna. Large forest-dwelling mammals are often highly sensitive to environmental perturbation through processes such as fragmentation, simplification of habitat structure, and abiotic changes including increased temperatures where the canopy is cleared. Whilst previous work has focused upon species richness and rarity in logged forest, few look at spatial and temporal behavioural responses to forest degradation. Using camera traps, we explored the relationships between diel activity, behavioural expression, habitat use and ambient temperature to understand how the wild free-ranging Bornean banteng (Bos javanicus lowi) respond to logging and regeneration. Three secondary forests in Sabah, Malaysian Borneo were studied, varying in the time since last logging (6-23 years). A combination of generalised linear mixed models and generalised linear models were constructed using >36,000 trap-nights. Temperature had no significant effect on activity, however it varied markedly between forests, with the period of intense heat shortening as forest regeneration increased over the years. Bantengs regulated activity, with a reduction during the wet season in the most degraded forest (z = -2.6, Std. Error = 0.13, p = 0.01), and reductions during midday hours in forest with limited regeneration, however after >20 years of regrowth, activity was more consistent throughout the day. Foraging and use of open canopy areas dominated the activity budget when regeneration was limited. As regeneration advanced, this was replaced by greater investment in travelling and using a closed canopy. Forest degradation modifies the ambient temperature, and positively influences flooding and habitat availability during the wet season. Retention of a mosaic of mature forest patches within commercial forests could minimise these effects and also provide refuge, which is key to heat dissipation and the prevention of thermal stress, whilst retention of degraded forest could provide forage.

Alterations in glutamatergic signaling contribute to the decline of circadian photoentrainment in aged mice.

Robust physiological circadian rhythms form an integral part of well-being. The aging process has been found to negatively impact systems that drive circadian physiology, typically manifesting as symptoms associated with abnormal/disrupted sleeping patterns. Here, we investigated the age-related decline in light-driven circadian entrainment in male C57BL/6J mice. We compared light-driven resetting of circadian behavioral activity in young (1-2 months) and old (14-18 months) mice and explored alterations in the glutamatergic pathway at the level of the circadian pacemaker, the suprachiasmatic nucleus (SCN). Aged animals showed a significant reduction in sensitivity to behavioral phase resetting by light. We show that this change was through alterations in N-Methyl-D-aspartate (NMDA) signaling at the SCN, where NMDA, a glutamatergic agonist, was less potent in inducing clock resetting. Finally, we show that this shift in NMDA sensitivity was through the reduced SCN expression of this receptor's NR2B subunit. Only in young animals did an NR2B antagonist attenuate behavioral resetting. These results can help target treatments that aim to improve both physiological and behavioral circadian entrainment in aged populations.

Duplicate Type and Screen Testing: Waste in the Clinical Laboratory.

CONTEXT: - In the United States, approximately $65 billion dollars is spent per year on clinical laboratory testing, of which 20% to 30% of all testing is deemed inappropriate. There have been multiple studies in the field of transfusion medicine regarding evidence-based transfusion practices, but limited data exist regarding inappropriate pretransfusion testing and its financial and clinical implications. OBJECTIVE: - To assess duplicative testing practices in the transfusion medicine service. DESIGN: - A 24-month retrospective review was performed at a 1025-bed tertiary care center, identifying all duplicate type and screen (TS) tests performed within 72 hours of the previous TS. Duplicative testing was classified as appropriate or inappropriate by predetermined criteria. The level of underordering was analyzed through a query of the electronic event reporting system. A cost analysis was performed to determine the financial impact of inappropriate duplicative TS. RESULTS: - The mean rate of inappropriate, duplicative TS orders was 4.13% (standard deviation ± 4.09%). Rates of inappropriate ordering ranged from 0.01% to 15.5% depending on the clinical service and did not correlate with volume of tests ordered. There were 8 reported cases of delayed blood delivery due to lack of a valid TS during the study period, demonstrating that underordering is also a harmful practice. The laboratory cost of inappropriate testing for the study period was $80,434, and phlebotomy costs were $45,469. CONCLUSIONS: - Our study demonstrates that inappropriate TS ordering is costly, both financially and clinically. By evaluating the percentage of inappropriate TS tests by clinical services, we have identified services that may benefit from additional education and technologic intervention.