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.

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.

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.

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.

Using direct antiglobulin test results to reduce unnecessary cold agglutinin testing.

BACKGROUND: Cold agglutinin disease (CAD) is a rare autoimmune hemolytic anemia mediated by autoantibodies that preferentially react at 4°C. Laboratory testing for cold-reactive autoantibodies is laborious and may not be ordered judiciously, particularly in patients with a negative direct antiglobulin test (DAT). We sought to determine whether a negative DAT using anti-human complement (anti-C3) rules out elevated cold agglutinin (CA) titers and the diagnosis of CAD. STUDY DESIGN AND METHODS: We performed a retrospective study of patients with a CA test performed at three major academic medical centers: Barnes-Jewish Hospital (2003-2014), Vanderbilt University Medical Center (2007-2009), and Massachusetts General Hospital (2009-2014). RESULTS: This study included 801 patients, of whom 51% (n = 410) had a DAT within the 7 days before CA testing. A total of 98% of patients with a negative DAT using anti-C3 had a negative CA titer (<64). Only five subjects had a negative DAT using anti-C3 and an elevated CA titer. CONCLUSIONS: Overutilization of CA testing could be reduced by establishing laboratory acceptance criteria based on a positive DAT using anti-C3. Such acceptance criteria would have reduced CA testing by 68% for those with an available DAT result.

School nurses and care coordination for children with complex needs: an integrative review.

Health care for students with chronic needs can be complex and specialized, resulting in fragmentation, duplication, and inefficiencies. Students who miss school due to chronic conditions lose valuable educational exposure that contributes to academic success. As health-related disabilities increase in prevalence so does the need for the coordination of care within the school and between the school and service providing agencies. This integrative literature review provides a synthesis of published evidence identifying and describing the core concepts associated with the role of school nurses in providing care coordination/case management to students with complex needs. Six core essentials of nurse-provided care coordination were identified: collaboration, communication, care planning and the nursing process, continuous coordination, clinical expertise, and complementary components. Recommendations for improving care coordination were elucidated in the review. Analysis of the literature can help assure application of best practice methods for the coordination of care for students in the school setting.

Control of highly pathogenic avian influenza in Quang Tri province, Vietnam: voices from the human-animal interface.

INTRODUCTION: Highly pathogenic avian influenza (HPAI) is caused by the haemagglutinin 5, neuraminidase 1 (H5N1) influenza A virus. Around 80% of households in rural Vietnam raise poultry, which provides food security and nutrition to their households and beyond. Of these, around 15-20% are semi-commercial producers, producing at least 28% of the country's chicken. Through learning the experiences of these semi-commercial farmers, this study aimed to explore the local understandings and sociocultural aspects of HPAI's impact, particularly the aetiology, diagnosis, and the prevention and control methods in one Vietnamese rural province. METHODS: This study was conducted in Quang Tri province, Vietnam. Quang Tri province has eight districts. Five of these districts were at high risk of HPAI during the study period, of which three were selected for the present study. Within these three districts, six communes were randomly selected for the study from the list of intervention communes in Quang Tri province. Six out of the 26 intervention communes in Quang Tri were therefore selected. Participants were randomly selected and recruited from lists of semi-commercial farmers, village animal health workers, village human health workers and local authorities so that the study population (representative population) included an amount of variability similar to that of the wider population. RESULTS: A key benefit of this village-level control program was the residential proximity of animal and human health professionals. Participants were well aware of the typical clinical signs for avian influenza and of the reporting process for suspect cases. However there was extensive room for improvement in Quang Tri province regarding access to the HPAI vaccine, essential medical equipment for animal use, and available financial support. CONCLUSIONS: This qualitative research study provided an important insight for in-country policy makers and international stakeholders. It is vital that there are continued efforts to prevent and control highly pathogenic avian influenza through support programs that provide locally appropriate information and resources to those at the human-animal interface.

The Pfam protein families database.

Pfam is a widely used database of protein families, currently containing more than 13,000 manually curated protein families as of release 26.0. Pfam is available via servers in the UK (http://pfam.sanger.ac.uk/), the USA (http://pfam.janelia.org/) and Sweden (http://pfam.sbc.su.se/). Here, we report on changes that have occurred since our 2010 NAR paper (release 24.0). Over the last 2 years, we have generated 1840 new families and increased coverage of the UniProt Knowledgebase (UniProtKB) to nearly 80%. Notably, we have taken the step of opening up the annotation of our families to the Wikipedia community, by linking Pfam families to relevant Wikipedia pages and encouraging the Pfam and Wikipedia communities to improve and expand those pages. We continue to improve the Pfam website and add new visualizations, such as the 'sunburst' representation of taxonomic distribution of families. In this work we additionally address two topics that will be of particular interest to the Pfam community. First, we explain the definition and use of family-specific, manually curated gathering thresholds. Second, we discuss some of the features of domains of unknown function (also known as DUFs), which constitute a rapidly growing class of families within Pfam.

Novel quantitative TaqMan real-time PCR assays for detection of Cryptosporidium at the genus level and genotyping of major human and cattle-infecting species.

AIMS: Development of TaqMan MGB real-time PCR assays for quantitative typing of major cattle and human-pathogenic Cryptosporidium species. METHODS AND RESULTS: Three specific TaqMan MGB real-time PCRs, based on the SSU rRNA gene, were directed towards livestock-restricted Cryptosporidium andersoni and Cryptosporidium bovis as well as both human-pathogenic Cryptosporidium parvum and Cryptosporidium hominis. A generic TaqMan assay further identified all known Cryptosporidium species and simultaneously monitored PCR inhibition through an external amplification control. The generic and specific assays were highly reproducible, and all displayed a detection limit of one oocyst per reaction. The specific TaqMan protocols also proved valuable for specifically detecting and quantifying target DNA in the presence of non-target DNA in environmental samples. CONCLUSIONS: All TaqMan MGB real-time PCR assays fulfilled the required specificity and sensitivity criteria, both on laboratory strains and on a surface water matrix. SIGNIFICANCE AND IMPACT OF THE STUDY: No molecular-based method was yet available for the quantitative detection of C. andersoni and the cluster formed by C. bovis, Cryptosporidium ryanae and Cryptosporidium xiaoi. This work provides a novel tool to evaluate the parasite load from domestic ruminants and humans, and to improve assessment and management of microbial risk through better appraisal of the origin and fate of faecal pollutions.

School asthma screening and case management: attendance and learning outcomes.

Asthma is related to school absenteeism and underperformance in elementary students. This pilot study assessed whether school nurse case management (CM) in children identified with asthma impacts academic performance and school absenteeism in one school. A validated questionnaire was used to identify children at risk for asthma and CM was provided to link these students to medical care and assure asthma action plans at school. In the 40 children with confirmed diagnosis who received CM, academic performance on standardized testing postintervention was similar to the 76 children who were low risk for asthma. Average days absent due to illness in the CM group were reduced from 5.8 to 3.7 days in the postintervention school year. School nurse screening, CM, and collaboration with a medical provider resulted in early identification, referral, and subsequent treatment of students at risk for asthma and may have contributed to reduced illness absences.

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.

Socio-ecological factors shape the distribution of a cultural keystone species in Malaysian Borneo.

Biophysical and socio-cultural factors have jointly shaped the distribution of global biodiversity, yet relatively few studies have quantitatively assessed the influence of social and ecological landscapes on wildlife distributions. We sought to determine whether social and ecological covariates shape the distribution of a cultural keystone species, the bearded pig (Sus barbatus). Drawing on a dataset of 295 total camera trap locations and 25,755 trap days across 18 field sites and three years in Sabah and Sarawak, Malaysian Borneo, we fitted occupancy models that incorporated socio-cultural covariates and ecological covariates hypothesized to influence bearded pig occupancy. We found that all competitive occupancy models included both socio-cultural and ecological covariates. Moreover, we found quantitative evidence supporting Indigenous pig hunting rights: predicted pig occupancy was positively associated with predicted high levels of Indigenous pig-hunting groups in low-accessibility areas, and predicted pig occupancy was positively associated with predicted medium and low levels of Indigenous pig-hunting groups in high-accessibility areas. These results suggest that bearded pig populations in Malaysian Borneo should be managed with context-specific strategies, promoting Indigenous pig hunting rights. We also provide important baseline information on bearded pig occupancy levels prior to the 2020-2021 outbreak of African Swine Fever (ASF), which caused social and ecological concerns after mass dieoffs of bearded pigs in Borneo. The abstract provided in Malay is in the Supplementary file.

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.

Pharmacology of GPR55 in yeast and identification of GSK494581A as a mixed-activity glycine transporter subtype 1 inhibitor and GPR55 agonist.

GPR55 is a G protein-coupled receptor activated by L-α-lysophosphatidylinositol and suggested to have roles in pain signaling, bone morphogenesis, and possibly in vascular endothelial cells. It has affinity for certain cannabinoids (molecules that interact with the cannabinoid CB(1) and CB(2) receptors), but investigation of its functional role in cell-based systems and in tissue has been limited by a lack of selective pharmacological tools. Here, we present our characterization of GPR55 in the yeast Saccharomyces cerevisiae and in human embryonic kidney (HEK293) cells. We describe GSK494581A (1-{2-fluoro-4-[1-(methyloxy)ethyl]phenyl}-4-{[4'-fluoro-4-(methylsulfonyl)-2-biphenylyl]carbonyl}piperazine), a selective small-molecule ligand of GPR55 identified through diversity screening. GSK494581A is one of a series of benzoylpiperazines originally identified and patented as inhibitors of the glycine transporter subtype 1 (GlyT1). The structure-activity relationship between GPR55 and GlyT1 is divergent across this series. The most GPR55-selective example is GSK575594A (3-fluoro-4-(4-{[4'-fluoro-4-(methylsulfonyl)-2-biphenylyl]carbonyl}-1-piperazinyl)aniline), which is approximately 60-fold selective for GPR55 (pEC(50) = 6.8) over GlyT1 (pIC(50) = 5.0). Several exemplars with activity at GPR55 and GlyT1 have been profiled at a broad range of other molecular targets and are inactive at cannabinoid receptors and all other targets tested. The benzoylpiperazine agonists activate human GPR55 but not rodent GPR55, suggesting that the relatively low level of sequence identity between these orthologs (75%) translates to important functional differences in the ligand-binding site.

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.

The Pfam protein families database.

Pfam is a comprehensive collection of protein domains and families, represented as multiple sequence alignments and as profile hidden Markov models. The current release of Pfam (22.0) contains 9318 protein families. Pfam is now based not only on the UniProtKB sequence database, but also on NCBI GenPept and on sequences from selected metagenomics projects. Pfam is available on the web from the consortium members using a new, consistent and improved website design in the UK (http://pfam.sanger.ac.uk/), the USA (http://pfam.janelia.org/) and Sweden (http://pfam.sbc.su.se/), as well as from mirror sites in France (http://pfam.jouy.inra.fr/) and South Korea (http://pfam.ccbb.re.kr/).

Visualizing jet lag in the mouse suprachiasmatic nucleus and peripheral circadian timing system.

Circadian rhythms regulate most physiological processes. Adjustments to circadian time, called phase shifts, are necessary following international travel and on a more frequent basis for individuals who work non-traditional schedules such as rotating shifts. As the disruption that results from frequent phase shifts is deleterious to both animals and humans, we sought to better understand the kinetics of resynchronization of the mouse circadian system to one of the most disruptive phase shifts, a 6-h phase advance. Mice bearing a luciferase reporter gene for mPer2 were subjected to a 6-h advance of the light cycle and molecular rhythms in suprachiasmatic nuclei (SCN), thymus, spleen, lung and esophagus were measured periodically for 2 weeks following the shift. For the SCN, the master pacemaker in the brain, we employed high-resolution imaging of the brain slice to describe the resynchronization of rhythms in single SCN neurons during adjustment to the new light cycle. We observed significant differences in shifting kinetics among mice, among organs such as the spleen and lung, and importantly among neurons in the SCN. The phase distribution among all Period2-expressing SCN neurons widened on the day following a shift of the light cycle, which was partially due to cells in the ventral SCN exhibiting a larger initial phase shift than cells in the dorsal SCN. There was no clear delineation of ventral and dorsal regions, however, as the SCN appear to have a population of fast-shifting cells whose anatomical distribution is organized in a ventral-dorsal gradient. Full resynchronization of the SCN and peripheral timing system, as measured by a circadian reporter gene, did not occur until after 8 days in the advanced light cycle.

The cytoplasmic 60 kDa progesterone receptor isoform predominates in the human amniochorion and placenta at term.

BACKGROUND: The mechanism that initiates human parturition has been proposed to be 'functional progesterone withdrawal' whereby the 116 kDa B-isoform of the progesterone receptor (PR-B) switches in favour of the 94 kDa A-isoform (PR-A) in reproductive tissues. Recently, other PR isoforms, PR-S, PR-C and PR-M generated from the same gene have been identified and partially characterised. METHODS AND RESULTS: Using immunohistochemical, western blotting and RT-PCR techniques, evidence is provided that indicates the major PR isoform present in human term fetal membranes (amnion and chorion) and syncytiotrophoblast of the placenta is neither of the classical nuclear PR-B or PR-A isoforms but is the N-terminally truncated 60 kDa PR-C isoform. Evidence is also provided that this 60 kDa isoform resides in the cytoplasm of the expressing cell types. Data are also presented to show that PR-B, PR-A and PR-S isoforms are essentially absent from the amnion and chorion, whereas PR isoforms A, B, C and S are all present in the decidua, with PR-A being the major isoform. The syncytiotrophoblast of the placenta contains the cytoplasmic 60 kDa isoform, but not isoforms PR-A, PR-B or PR-S. CONCLUSION: The major PR isoform in the amnion, chorion and placenta is a 60 kDa protein that could be PR-C, suggesting that the cytoplasmic isoform has a specific role in extra-embryonic tissues and may be involved in the regulation of human parturition.