Investigation of euthanasia techniques in four species of cockroaches.

While cockroaches are commonly exhibited in zoos and museums, studied in research laboratories, and even kept as pets, scientifically based guidelines for their euthanasia are lacking. This study assessed euthanasia techniques in four species of cockroaches (Dubia (Blaptica dubia), red runner (Shelfordella lateralis), Madagascar hissing (Gromphadorhina portentosa), and giant cave (Blaberus giganteus)). In an initial pilot study, two hundred fifty adult Dubia cockroaches were exposed in groups of ten to a cotton ball soaked with 2 mL of isoflurane in a 1 L air-tight chamber. Thirty minutes beyond loss of any individual movement, groups were exposed to one of the following secondary treatments: freezing at -18 °C or -80 °C from 0.25 to 24 hours; immersion in 10% neutral buffered formalin, 70% isopropyl alcohol, or reverse osmosis water for 0.25 or 0.5 hours; or intracoelomic injection of potassium chloride (456 mEq/kg) or pentobarbital-based euthanasia solution (3.9 g/kg). A control group remained in the air-tight isoflurane chamber for 24 hours. Following all treatments, cockroaches were monitored for an additional 24 hours for spontaneous movement. Irreversible loss of movement was considered synonymous with irreversible loss of consciousness (death). Across all species, isoflurane anesthesia followed by either 70% isopropyl alcohol immersion for 0.25 or 0.5 hours or isoflurane exposure for 24 hours resulted in euthanasia in 100% of cockroaches. This study is the first evaluation of American Veterinary Medical Association-recommended euthanasia protocols in cockroaches.

Comparison of Corticosterone Concentrations in Dermal Secretions and Urine in Free-Ranging Marine Toads (Rhinella marina) in Human Care.

Corticosterone concentrations have been measured in amphibians by collecting blood or urine samples. However, blood sampling is invasive, and urine can be difficult to collect. A novel method of swabbing the skin of an amphibian has been utilized in numerous species but has not been verified in marine toads (Rhinella marina). This pilot study tested dermal swabs as a noninvasive method for collecting and measuring dermal corticosterone secretions. Swabs were used to collect dermal secretion samples from sixty-six free-ranging marine toads collected on Zoo Miami grounds. The subsequent day the toads were shipped to the North Carolina Zoo where dermal samples were collected again. Additional dermal and urine samples were collected on days 9, 15, 32, and 62 under human care to measure corticosterone concentrations. There was no significant correlation (P ≥ 0.05) noted between corticosterone concentrations reported in dermal swabs and those in urine samples at all four of the euthanasia time points or between the corticosterone concentrations reported in either urine or dermal swabs and the weight of the toads. Dermal swab concentrations (ng/mL) were significantly higher (P ≤ 0.05) on the day of capture (0.64 ± 0.03) and the day of arrival (0.67 ± 0.03) than on day 15 (0.47 ± 0.03). The urine corticosterone concentrations decreased while the toads were in human care with a significant decrease (P ≤ 0.05) between days 9 (0.45 ± 0.07) and 32 (0.21 ± 0.06). This study demonstrated that dermal swabs can be used to collect marine toad corticosterone concentration samples.

Systematic review of marine environmental DNA metabarcoding studies: toward best practices for data usability and accessibility.

The emerging field of environmental DNA (eDNA) research lacks universal guidelines for ensuring data produced are FAIR-findable, accessible, interoperable, and reusable-despite growing awareness of the importance of such practices. In order to better understand these data usability challenges, we systematically reviewed 60 peer reviewed articles conducting a specific subset of eDNA research: metabarcoding studies in marine environments. For each article, we characterized approximately 90 features across several categories: general article attributes and topics, methodological choices, types of metadata included, and availability and storage of sequence data. Analyzing these characteristics, we identified several barriers to data accessibility, including a lack of common context and vocabulary across the articles, missing metadata, supplementary information limitations, and a concentration of both sample collection and analysis in the United States. While some of these barriers require significant effort to address, we also found many instances where small choices made by authors and journals could have an outsized influence on the discoverability and reusability of data. Promisingly, articles also showed consistency and creativity in data storage choices as well as a strong trend toward open access publishing. Our analysis underscores the need to think critically about data accessibility and usability as marine eDNA metabarcoding studies, and eDNA projects more broadly, continue to proliferate.

Increased Scholarly Activity: A Benefit of 4-Year Family Medicine Residencies.

INTRODUCTION: The optimal length of Family Medicine Residency is unknown. As part of the American Board of Family Medicine 4-year Length of Training (LoT) pilot project, Naval Hospital Jacksonville (NHJ) maintained a dual-track 3- and 4-year Family Medicine Residency, graduating seven 4-year residents over consecutive 4 years of the LoT program. One measure of success regarding the impact of 4-year residents on program outcomes is scholarly output during residency. MATERIALS AND METHODS: Cumulative scholarly activity points are tracked for all NHJ residents. Cumulative scholarly activity points, points per year per, and raw percentile USMLE/COMLEX scores from academic years 2016-17 to 2019-20 were compared between PGY3 and PGY4 graduates using one-way ANOVA to 95% confidence with post hoc Tukey honestly significant difference pairwise comparison to evaluate pairwise significance between groups where multi-group differences were found. RESULTS: During the 2016-17 through 2019-20 academic years, NHJ had 28 residents complete 3 years of training without interruption (3 Years), 11 residents complete 3 years of training interrupted by general medical officer tours (Resiterns), and 7 residents complete 4 years of training without interruption (4 Years). There were no significant differences in average raw USMLE and COMLEX scores between 3 Year (71%), Resitern (68%), and 4 Year (76%) residents (P = .335). 4-Year residents had significantly more cumulative scholarly points (103) than 3-Year residents (32.6, P < .001) and Resiterns (18.7, P < .001) and also had more cumulative scholarly points per year of residency (27.8) than 3-Year residents (9.8, P < .001) and Resiterns (7.0, P < .001). CONCLUSIONS: An observed benefit of a 4-year Family Medicine Residency was a marked increase in scholarly output at this program.

Maximizing lifestyle changes to manage type 2 diabetes.

Four dietary plans can reduce A1C levels but may differ in long-term outcomes. Intensive lifestyle interventions may even make remission possible.

Hypertensive Conditions: Essential (Primary) Hypertension in Adults.

Blood pressure (BP) screening using an office-based measurement is recommended for adults 18 years and older without a history of hypertension. If abnormal, the BP measurement should be repeated twice with the average of those final two readings used to determine the BP category. Home BP monitoring and ambulatory BP monitoring are beneficial in patients for whom there is a concern for masked or white-coat hypertension. Guidelines differ regarding the BP cutoff used for the diagnosis of hypertension. Lifestyle modifications are the foundation of hypertension management with the Dietary Approaches to Stop Hypertension (DASH) diet being the most effective dietary modification. First-line pharmacotherapy should include one or more of the following: an angiotensin-converting enzyme inhibitor, an angiotensin receptor blocker, a dihydropyridine calcium channel blocker, and a thiazide or thiazidelike diuretic. Compared with standard BP control, intensive BP control (ie, systolic BP less than 120 mm Hg) leads to a decrease in atherosclerotic cardiovascular disease and all-cause mortality in patients with elevated risk but increases adverse effects, including hypotension, electrolyte abnormalities, acute kidney injury, and syncope.

Hypertensive Conditions: Secondary Causes of Hypertension in Adults.

Secondary hypertension (HTN) refers to high blood pressure (BP) caused by an identifiable and potentially correctable condition or disease. Common causes of secondary HTN include renovascular disease, renal parenchymal disease, primary hyperaldosteronism, drug and substance use, and obstructive sleep apnea; less common etiologies include pheochromocytoma/paraganglioma, Cushing syndrome, thyroid and parathyroid conditions, congenital adrenal hyperplasia, and aortic coarctation. An identifiable secondary cause of HTN is present in approximately 10% of adult patients with HTN. Early recognition of suggestive clinical findings and laboratory results enables the timely diagnosis of specific secondary causes of HTN. Correct diagnosis of a causative underlying condition can lead to more effective, even curative management and subsequent cardiovascular risk reduction. Management involves treating the underlying condition. Some patients may benefit from referral to a specialist with specific expertise in treating the causative condition.

Hypertensive Conditions: Hypertension in Children and Adolescents.

Hypertension (HTN) in children and adolescents is a spectrum of disease, ranging from elevated blood pressure (BP) to stage 1 and 2 HTN. The prevalence of elevated BP and HTN in this age group has increased significantly over the past 20 years, particularly in girls. Screening for HTN in asymptomatic children and adolescents is controversial. Primary HTN is now the predominant cause of HTN among the pediatric population in the United States, especially among adolescents. Secondary pediatric HTN is high BP due to an underlying medical condition and is more common among children 6 years and younger. Ambulatory BP monitoring should be considered in pediatric patients with repeatedly elevated office BP measurements. All children with BP greater than the 90th percentile should be encouraged to adopt lifestyle changes, but those with persistent or severe elevations in BP may benefit from pharmacotherapy.

Hypertensive Conditions: Hypertensive Disorders in Pregnancy.

Hypertensive disorders in pregnancy (HDP) represent a spectrum of disease that affect women through pregnancy and the immediate postpartum period. These conditions are associated with significant morbidity and mortality during and after pregnancy and have been linked to cardiovascular disease later in life. The HDP spectrum includes gestational hypertension (HTN), preeclampsia, eclampsia, HELLP (hemolysis, elevated liver enzymes, and low platelet count) syndrome, chronic HTN, and chronic HTN with superimposed preeclampsia. Low-dose aspirin is recommended as a preventive drug after 12 weeks' gestation in women who are at high risk of preeclampsia. In HDP, close blood pressure (BP) monitoring, laboratory evaluation, and fetal assessment are warranted. Labetalol and nifedipine extended release are first-line oral antihypertensives for outpatient BP management of chronic HTN; labetalol, hydralazine, and nifedipine immediate release are used for hospitalized patients. HDP may develop or progress in the postpartum period; continued vigilance is important in the puerperium.

The Spiritual Assessment.

The spiritual assessment offers the opportunity to enhance the patient-physician relationship and incorporate patient views that may have a significant impact on clinical decision-making. Multiple studies have demonstrated that patients' expectations of spiritual discussions in the health care setting are not being met. Perceived barriers reported by physicians include lack of time, training, and experience. There is a variety of physician approaches to assess and incorporate spirituality in the health care setting. A spiritual assessment is recommended when a patient is admitted to the hospital, has a significant clinical decline while in the hospital, is receiving psychosocial services for the treatment of substance use disorder, or when addressing palliative care. Tools for spiritual assessment include the Open Invite mnemonic, which initiates the topic and relies on a conversational approach, and the HOPE questions, which offer a structured approach to determine the relevance of spirituality to the patient's overall health and assist with the development of an individualized care plan. Although physicians should respect the right of patients who do not want to discuss this topic, multiple studies demonstrate significant relationships between spiritual interventions and improved mental and physical health outcomes.

A mechanism for red coloration in vertebrates.

Red coloration is a salient feature of the natural world. Many vertebrates produce red color by converting dietary yellow carotenoids into red ketocarotenoids via an unknown mechanism. Here, we show that two enzymes, cytochrome P450 2J19 (CYP2J19) and 3-hydroxybutyrate dehydrogenase 1-like (BDH1L), are sufficient to catalyze this conversion. In birds, both enzymes are expressed at the sites of ketocarotenoid biosynthesis (feather follicles and red cone photoreceptors), and genetic evidence implicates these enzymes in yellow/red color variation in feathers. In fish, the homologs of CYP2J19 and BDH1L are required for ketocarotenoid production, and we show that these enzymes are sufficient to produce ketocarotenoids in cell culture and when ectopically expressed in fish skin. Finally, we demonstrate that the red-cone-enriched tetratricopeptide repeat protein 39B (TTC39B) enhances ketocarotenoid production when co-expressed with CYP2J19 and BDH1L. The discovery of this mechanism of ketocarotenoid biosynthesis has major implications for understanding the evolution of color diversity in vertebrates.

Methods for extracting and analyzing carotenoids from bird feathers.

Carotenoid pigments serve many endogenous functions in organisms, but some of the more fascinating are the external displays of carotenoids in the colorful red, orange and yellow plumages of birds. Since Darwin, biologists have been curious about the selective advantages (e.g., mate attraction) of having such ornate features, and, more recently, advances in biochemical methods have permitted researchers to explore the composition and characteristics of carotenoid pigments in feathers. Here we review contemporary methods for extracting and analyzing carotenoids in bird feathers, with special attention to the difficulties of removal from the feather keratin matrix, the possibility of feather carotenoid esterification and the strengths and challenges of different analytical methods like high-performance liquid chromatography and Raman spectroscopy. We also add an experimental test of current common extraction methods (e.g., mechanical, thermochemical) and find significant differences in the recovery of specific classes of carotenoids, suggesting that no single approach is best for all pigment or feather types.

Understanding barriers to men's support for family planning in rural Ethiopia-findings from the USAID Transform: Primary Health Care Project Gender Analysis.

BACKGROUND: Evidence suggests that supportive male engagement in health care services, including family planning, remains low in many countries, despite known benefits for female partners. In 2017-2018, the United States Agency for International Development Transform: Primary Health Care Project conducted a participatory gender analysis, collecting relevant data to better understand Ethiopian men's lack of support for the uptake of family planning services. METHODS: Qualitative data were collected through 96 unique participatory group discussions with community members via a semistructured discussion guide and participatory activity; data were disaggregated by sex, age, and marital status. In-depth interviews (91) conducted with service providers, health system managers, and health extension workers used semistructured guides. Discussants and interviewees were selected purposefully, drawn from 16 rural woredas in four project regions: Amhara; Oromia; Tigray; and Southern Nations, Nationalities, and Peoples' Region. Data collectors took notes and transcribed audio recordings. The research team deductively and inductively coded transcripts to develop preliminary findings later validated by key technical project staff and stakeholders. RESULTS: Findings reinforce existing knowledge on the dominant role of men in health care-related decision making in rural Ethiopia, although such decision making is not always unilateral in practice. Barriers at the societal level impede men's support for family planning; these include norms, values, and beliefs around childbearing; religious beliefs rooted in scriptural narratives; and perceived adverse health impacts of family planning. Lack of efforts to engage men in health care facilities, as well as the perception that health care facilities do not meet men's needs, highlight systems-level barriers to men's use of family planning services. CONCLUSIONS: Findings indicate several opportunities for stakeholders to increase men's support for family planning in rural Ethiopia, including systems-wide approaches to shape decision making, social and behavior change communication efforts, and additional research and assessment of men's experiences in accessing health care services.

Evidence suggests that in instances where men participate when their partners access health care services, their partners experience positive health benefits. Regardless, men tend not to participate. During 2017­2018, the United States Agency for International Development Transform: Primary Health Care Project conducted research to identify gender-related issues that hinder the delivery of primary health care services in Ethiopia. The research team conducted 96 group discussions with male and female community members, as well as 91 in-depth interviews with health care service providers, health system managers, and health extension workers. Participants were specifically selected from 16 rural districts, or woredas, in four regions where the project is active. The researchers then categorized information in the resulting transcripts by common themes, and the data analysis team met to draw out the main findings. Later, a meeting was held with key project staff and stakeholders in Addis Ababa to verify the findings. Findings reinforce existing knowledge on the dominant role of men in health care­related decision making for households in rural Ethiopia, although women often play an important role as well. The research also identified widespread male opposition to family planning due to norms, desires, and societal perceptions around childbearing; religious beliefs; and concerns about the perceived health risks of family planning methods. Further, findings showed that the promotion of family planning methods and services do not explicitly target men, and men believe that current services do not respond to their needs. Respondents suggested opportunities for stakeholders to mitigate these barriers.

Development of a Real-Time Pectic Oligosaccharide-Detecting Biosensor Using the Rapid and Flexible Computational Identification of Non-Disruptive Conjugation Sites (CINC) Biosensor Design Platform.

Fluorescently labeled, solute-binding proteins that change their fluorescent output in response to ligand binding are frequently used as biosensors for a wide range of applications. We have previously developed a "Computational Identification of Non-disruptive Conjugation sites" (CINC) approach, an in silico pipeline utilizing molecular dynamics simulations for the rapid design and construction of novel protein-fluorophore conjugate-type biosensors. Here, we report an improved in silico scoring algorithm for use in CINC and its use in the construction of an oligogalacturonide-detecting biosensor set. Using both 4,5-unsaturated and saturated oligogalacturonides, we demonstrate that signal transmission from the ligand-binding pocket of the starting protein scaffold to the CINC-selected reporter positions is effective for multiple different ligands. The utility of an oligogalacturonide-detecting biosensor is shown in Carbohydrate Active Enzyme (CAZyme) activity assays, where the biosensor is used to follow product release upon polygalacturonic acid (PGA) depolymerization in real time. The oligogalacturonide-detecting biosensor set represents a novel enabling tool integral to our rapidly expanding platform for biosensor-based carbohydrate detection, and moving forward, the CINC pipeline will continue to enable the rational design of biomolecular tools to detect additional chemically distinct oligosaccharides and other solutes.

Construction of a highly selective and sensitive carbohydrate-detecting biosensor utilizing Computational Identification of Non-disruptive Conjugation sites (CINC) for flexible and streamlined biosensor design.

Fluorescently-labeled solute-binding proteins that alter their fluorescence output in response to ligand binding have been utilized as biosensors for a variety of applications. Coupling protein ligand binding to altered fluorescence output often requires trial and error-based testing of both multiple labeling positions and fluorophores to produce a functional biosensor with the desired properties. This approach is laborious and can lead to reduced ligand binding affinity or altered ligand specificity. Here we report the Computational Identification of Non-disruptive Conjugation sites (CINC) for streamlined identification of fluorophore conjugation sites. By exploiting the structural dynamics properties of proteins, CINC identifies positions where conjugation of a fluorophore results in a fluorescence change upon ligand binding without disrupting protein function. We show that a CINC-developed maltooligosaccharide (MOS)-detecting biosensor is capable of rapid (kon = 20 µM-1s-1), sensitive (sub-µM KD) and selective MOS detection. The MOS-detecting biosensor is modular with respect to the spectroscopic properties and demonstrates portability to detecting MOS released via α-amylase-catalyzed depolymerization of starch using both a stopped-flow and a microplate reader assay. Our MOS-detecting biosensor represents a first-in-class probe whose design was guided by changes in localized dynamics of individual amino acid positions, supporting expansion of the CINC pipeline as an indispensable tool for a wide range of protein engineering applications.

Calmodulin binding to the dehydrogenase domain of NADPH oxidase 5 alters its oligomeric state.

Superoxide generated by NADPH Oxidase 5 (Nox5) is regulated by Ca2+ through the interaction of its self-contained Ca2+ binding domain and dehydrogenase domain (DH). Recently, calmodulin (CaM) has been reported to enhance the Ca2+ sensitivity of Nox5 by binding to the CaM-binding domain sequence (CMBD), in which the interaction between CaM and Nox5 is largely unclear. Here, we used the CMBD peptide and truncated DH constructs, and separately studied their interaction with CaM by fluorescence, calorimetry, and dynamic light scattering. Our results revealed that each half-domain of CaM binds one CMBD peptide with a binding constant near 106 M-1 and a binding enthalpy change of -3.81 kcal/mol, consistent with an extended 1:2 CaM:CMBD structure. However, the recombinant truncated DH proteins exist as oligomers, possibly trimer and tetramer. The oligomeric states are concentration and salt dependent. CaM binding appears to stabilize the DH dimer complexed with CaM. The thermodynamics of CaM binding to the DH is comparable to the peptide-based study except that the near unity binding stoichiometry and a large conformational change were observed. Our result suggests that the oligomeric states of Nox5, mediated by its DH domain and CaM, may be important for its superoxide-generating activity.

Comparative nutrient analysis of four species of cockroaches used as food for insectivores by life stage, species, and sex.

A variety of insects are fed to insectivorous animals; however, nutritional analyses are often limited to adult life stages. Four species of nymph and adult female and male cockroaches (Blaberus giganteus, Blaptica dubia, Blatta lateralis, and Gromphadorhina portentosa) were analyzed for moisture, crude protein (CP), acid detergent fiber (ADF), fat, ash, and mineral content. The small sample size of this study precluded statistical analyses, however comparatively, CP in adult B. lateralis and B. dubia was greater than the CP in nymphs of the same species. Adult and nymph B. dubia had the greatest CP (96.6% and 65.3%, respectively) compared to the other three species. Inversely, fat content in adult B. lateralis and B. dubia was lower than that of nymphs of the same species. All adults contained similar levels of ADF, yet adult B. giganteus had greater ADF than nymphs; conversely, B. lateralis and B. dubia adults had less ADF than nymphs. There were differences noted in mineral parameters among the four species and life stages. Adult B. giganteus had less Ca than G. portentosa, and the lowest Ca content of the four species of nymphs and adults analyzed. This study underscores the differences in nutrient content with respect to life stage and species in previously understudied cockroaches to improve nutrient intake in captive insectivores.