Large-scale chemoproteomics expedites ligand discovery and predicts ligand behavior in cells.

Chemical modulation of proteins enables a mechanistic understanding of biology and represents the foundation of most therapeutics. However, despite decades of research, 80% of the human proteome lacks functional ligands. Chemical proteomics has advanced fragment-based ligand discovery toward cellular systems, but throughput limitations have stymied the scalable identification of fragment-protein interactions. We report proteome-wide maps of protein-binding propensity for 407 structurally diverse small-molecule fragments. We verified that identified interactions can be advanced to active chemical probes of E3 ubiquitin ligases, transporters, and kinases. Integrating machine learning binary classifiers further enabled interpretable predictions of fragment behavior in cells. The resulting resource of fragment-protein interactions and predictive models will help to elucidate principles of molecular recognition and expedite ligand discovery efforts for hitherto undrugged proteins.

Perspectives on scientific error.

Theoretical arguments and empirical investigations indicate that a high proportion of published findings do not replicate and are likely false. The current position paper provides a broad perspective on scientific error, which may lead to replication failures. This broad perspective focuses on reform history and on opportunities for future reform. We organize our perspective along four main themes: institutional reform, methodological reform, statistical reform and publishing reform. For each theme, we illustrate potential errors by narrating the story of a fictional researcher during the research cycle. We discuss future opportunities for reform. The resulting agenda provides a resource to usher in an era that is marked by a research culture that is less error-prone and a scientific publication landscape with fewer spurious findings.

Amplifying local serotonin signaling prior to dry-off hastens mammary gland involution and redevelopment in dairy cows.

The monoamine serotonin (5-hydroxytryptamine, 5-HT) has been reported to inhibit milk protein gene expression and increase mammary epithelial cell (MEC) tight junction permeability after milk stasis. We hypothesized that increasing serotonin synthesis and signaling within the mammary epithelium before milk stasis would increase systemic and local involution markers, and downregulate the expression of milk protein and tight junction during involution, leading to more efficient tissue growth during the redevelopment phase. Herein, we examined the outcomes of increasing local mammary 5-HT synthesis before milk stasis on involution biomarkers, mammary gland microstructure, and gene and protein expression during the dry period. Multiparous Holstein cows were administered intramammary infusions (via the teat canal) of sterile water (CON, 4 mL/teat, n = 7) or 5-hydroxy-l-tryptophan (5-HTP, serotonin precursor, 20 mg/teat, n = 7) once daily for 5 d before dry-off (d 0). Blood, milk, and mammary secretions were collected and analyzed for components and metabolites. Mammary secretions were collected 12 h after the last milking and on d 1 to 4 during the dry period at 1200 h. Mammary gland biopsies were performed on d 4 (i.e., involution phase) and d 36 (i.e., redevelopment phase) of the dry period for histological and molecular evaluation. Milk protein and tight junction gene expression was quantified via real-time PCR. Hematoxylin and eosin staining, immunohistochemistry (Ki67), and immunofluorescence (serotonin, cleaved caspase 3) were performed to visualize tissue microstructure and to quantify serotonin intensity and cell turnover. Data were analyzed in SAS (SAS Institute Inc.) using 2-way ANOVA. After d 0, mammary secretions of 5-HTP cows had increased concentrations of 5-HT, lactoferrin, and bovine serum albumin. On d 1, 5-HTP cows had greater α-lactalbumin concentrations in plasma relative to CON. Serotonin intensity was increased in the mammary tissue of 5-HTP cows on d 4, relative to CON. On d 4, milk protein and tight junction gene expression was downregulated, MEC number was reduced, and cleaved caspase 3 protein was greater in mammary tissue of 5-HTP cows, relative to CON. On d 36, milk protein genes were upregulated, and the lumen:outer alveolar area and Ki67-positive cells were increased in the mammary tissue of 5-HTP cows, relative to CON. Amplifying serotonin signaling in the mammary epithelium before milk stasis at dry-off achieves greater apoptosis, leading to a reduction in MEC, allowing for greater cell proliferation, which results in more MEC during the redevelopment phase preceding the onset of lactation.

Peripheral serotonin regulates glucose and insulin metabolism in Holstein dairy calves.

Peripheral serotonin regulates energy metabolism in several mammalian species, however, the potential contribution of serotonergic mechanisms as metabolic and endocrine regulators in growing dairy calves remain unexplored. Objectives were to characterize the role of serotonin in glucose and insulin metabolism in dairy calves with increased serotonin bioavailability. Milk replacer was supplemented with saline, 5-hydroxytryptophan (90 mg/d), or fluoxetine (40 mg/d) for 10-d (n = 8/treatment). Blood was collected daily during supplementation and on days 2, 7, and 14 during withdrawal. Calves were euthanized after 10-d supplementation or 14-d withdrawal periods to harvest liver and pancreas tissue. 5-hydroxytryptophan increased circulating insulin concentrations during the supplementation period, whereas both treatments increased circulating glucose concentration during the withdrawal period. The liver and pancreas of preweaned calves express serotonin factors (ie, TPH1, SERT, and cell surface receptors), indicating their ability to synthesize, uptake, and respond to serotonin. Supplementation of 5-hydroxytryptophan increased hepatic and pancreatic serotonin concentrations. After the withdrawal period, fluoxetine cleared from the pancreas but not liver tissue. Supplementation of 5-hydroxytryptophan upregulated hepatic mRNA expression of serotonin receptors (ie, 5-HTR1B, -1D, -2A, and -2B), and downregulated pancreatic 5-HTR1F mRNA and insulin-related proteins (ie, Akt and pAkt). Fluoxetine-supplemented calves had fewer pancreatic islets per microscopic field with reduced insulin intensity, whereas 5-hydroxytryptophan supplemented calves had increased islet number and area with greater insulin and serotonin and less glucagon intensities. After the 14-d withdrawal of 5-hydroxytryptophan, hepatic mRNA expression of glycolytic and gluconeogenic enzymes were simultaneously downregulated. Improving serotonin bioavailability could serve as a potent regulator of endocrine and metabolic processes in dairy calves.

Increasing serotonin bioavailability alters gene expression in peripheral leukocytes and lymphoid tissues of dairy calves.

Dairy calves are born with a naïve immune system, making the pre-weaning phase a critical window for immune development. In the U.S., 40-60% of dairy farms feed milk replacer to pre-weaned calves, which are devoid of bioactive factors with immunological roles. Serotonin is a bioactive factor with immunoregulatory properties naturally produced by the calf and present in milk. Human and rodent immune cells express the serotonin machinery, but little is known about the role of serotonin in the bovine immune system. Supplementing milk replacer with 5-hydroxytryptophan (serotonin precursor) or fluoxetine (reuptake inhibitor) increases serotonin bioavailability. We hypothesized that increased serotonin bioavailability promotes serotonergic signaling and modulates the expression of immune related genes in peripheral leukocytes and immune-related tissues of dairy calves. The present experiment targeted candidate genes involved in serotonin production, metabolism, transport, signaling and immune regulation. We established that bovine peripheral leukocytes express all known serotonin receptors, and can synthesize, uptake and degrade serotonin due to the expression of serotonin metabolism-related genes. Indeed, we showed that increasing serotonin bioavailability alters gene expression of serotonin receptors and immune-related genes. Further research will determine whether manipulation of the serotonin pathway could be a feasible approach to bolster dairy calves' immune system.

Fluorophosphonate-Based Degrader Identifies Degradable Serine Hydrolases by Quantitative Proteomics.

Novel chemical biology probes linking a serine hydrolase-directed fluorophosphonate warhead and cereblon-binding pomalidomide were assessed for the degradation of serine hydrolases. A quantitative proteomics approach to detect degraded proteins revealed that, despite the engagement of ∼40 serine hydrolases, degradation was achieved for only a single serine hydrolase, lysophospholipase II (LYPLA2).

Pre- and postnatal heat stress abatement affects dairy calf thermoregulation and performance.

Prenatal heat stress during late gestation exerts long-term effects on growth and productivity of the dairy calf. Further, direct exposure to heat stress during the preweaning period impairs calf thermoregulation and performance. We examined the effects of heat stress abatement during the prenatal period, postnatal period, or both on calf performance. We hypothesized that calves exposed to pre- and postnatal heat stress abatement would perform most optimally in terms of thermoregulation, growth, and health responses when compared with calves that are heat-stressed at any time in the pre- or postnatal periods. Holstein calves born to heat-stressed (HT) or cooled (CL) dams during late gestation (44 ± 5 d; prenatal HT or CL) were exposed to heat stress or cooling postnatally for 56 d (postnatal HT or CL), resulting in 4 treatments: HT-HT, HT-CL, CL-HT, and CL-CL; n = 12/treatment. Calves were administered 4 L of pooled colostrum and after 2 d of age allotted 10 L/d milk replacer and up to 3 kg/d concentrate in automatic feeder group pens (n = 6/pen). Postnatal cooling was achieved by 2 fans (average wind speed 2 m/s). Thermoregulatory responses (respiration rate and heart rate; rectal, body, and skin temperature), feed intake, growth parameters including average daily gain and medication events were recorded, and blood samples were collected weekly. Thermoregulatory responses were lower in postnatal CL calves compared with postnatal HT. In the afternoon, HT-HT calves had the highest respiration rate and rectal temperature, HT-CL calves had the lowest respiration rate, and CL-HT calves had the lowest heart rate compared with the other treatment groups. Prenatal CL calves weighed more at birth and weaning with a tendency for greater average daily gain compared with prenatal HT calves, whereas postnatal CL calves had increased milk replacer and concentrate intake and a tendency for reduced fever, infection, and total medication events relative to postnatal HT. Prenatal HT calves were esophageal tube fed more often than prenatal CL. Blood hematocrit and 24-h serum IgG concentration were greater in prenatal CL calves relative to prenatal HT. Prenatal heat stress abatement improves weight gain, hematocrit, and immunoglobulin transfer, whereas postnatal heat stress abatement modulates thermoregulatory responses, feed intake, and calf health. This study is the first to characterize the combined effects of pre- and postnatal heat stress or active cooling on the dairy calf.

The development of an ultra-short, maternal mental health screening tool in South Africa.

PURPOSE: The burden of common perinatal mental disorders (CPMD) in low-and-middle-income countries is substantially higher than high-income countries, with low levels of detection, service provision and treatment in resource-constrained settings. We describe the development of an ultra-short screening tool to detect antenatal depression, anxiety disorders and maternal suicidal ideation. METHODS: A sample of 376 women was recruited at a primary-level obstetric clinic. Five depression and anxiety symptom-screening questionnaires, demographics and psychosocial risk questionnaires were administered. All participants were assessed with the Mini-International Neuropsychiatric Interview (MINI), a structured, diagnostic interview. Screening tool items were analysed against diagnostic data using multiple logistic regression and receiver operating curve (ROC) analysis. RESULTS: The prevalence of MINI-defined major depressive episode (MDE) and/or anxiety disorders was 33%. Overall, 18% of participants expressed suicidal ideation and behaviour, 54% of these had no depression or anxiety diagnosis. Multiple logistic regression identified four screening items that were independently predictive of MDE and anxiety disorders, investigating depressed mood, anhedonia, anxiety symptoms and suicidal ideation. ROC analysis of these combined items yielded an area under the curve of 0.83 (95% CI 0.78-0.88). A cut-off score of 2 or more offered a sensitivity of 78% and specificity of 82%. CONCLUSION: This novel screening tool is the first measure of CPMD developed in South Africa to include depressed mood, anxiety symptoms and suicidal ideation. While the tool requires further investigation, it may be useful for the early identification of mental health symptoms and morbidity in the perinatal period.

Increasing serotonin bioavailability in preweaned dairy calves impacts hematology, growth, and behavior.

Peripheral serotonin has been shown to regulate important physiological functions such as energy homeostasis and immunity, particularly in rodent and humans, but its role is poorly understood in livestock species. Herein, we tested the safety and effectiveness of increasing serotonin bioavailability in preweaned dairy calves by oral supplementation of a serotonin precursor (5-hydroxytryptophan, 5-HTP) or a serotonin reuptake inhibitor (fluoxetine, FLX). Bull Holstein calves (21 ± 2 d old; N = 24) were fed milk replacer (8 L/d) supplemented with either saline as control (CON, 8 mL/d, n = 8), FLX (40 mg/d, approx. 0.8 mg/kg; n = 8), or 5-HTP (90 mg/d, approx. 1.8 mg/kg; n = 8) for 10 consecutive days in a complete randomized block design. Heart rate (HR), respiration rate, rectal temperature, and health scores were recorded daily. Hip height and body weight were measured at d 1, 5, and 10 relative to initiation of supplementation. Blood samples were collected once before the supplementation period (d 1), during the 10-d supplementation period (daily), and during a 14-d withdrawal period (d 2, 3, 4, 7, and 14 relative to initiation of withdrawal). Cerebrospinal fluid and muscle tissue were collected from a subset of calves (n = 12) that were euthanized after the 10-d supplementation or 14-d withdrawal period. Whole blood serotonin concentrations increased in 5-HTP calves and decreased in FLX calves compared with CON (P < 0.001), indicating that serotonin bioavailability was increased in both groups. Whole blood serotonin concentrations of 5-HTP and FLX calves returned to CON levels after 7 d of withdrawal. All calves grew and were considered healthy throughout the study. In fact, calves fed 5-HTP had higher average daily gain compared with CON (0.87 vs 0.66 ± 0.12 kg/d, P = 0.05). Calves fed FLX had lower HR (P = 0.02) and greater red blood cells and hemoglobin counts on d 10 of supplementation compared with CON (P < 0.01). After the 14-d withdrawal period, FLX was not detected in circulation of FLX calves, but was still present in the muscle tissue. Our results demonstrate that manipulation of the serotonin pathway by supplementing FLX or 5-HTP is a feasible and safe approach in preweaned dairy calves; however, it takes more than 14 d for FLX to be completely withdrawn from the body.

Accuracy of a history of blood donation from surrogate witnesses: data from the UK TMER study.

Look-back studies of blood transfusion in Creutzfeldt-Jakob disease commonly rely on reported history from surrogate witnesses. Data from the UK Transfusion Medicine Epidemiology Review have been analysed to determine the accuracy of the blood donation history provided by the relatives of cases. Our results show that only a small percentage of cases were found to be registered as donors on UK Blood Service (UKBS) databases when there was no family report of blood donation. In contrast, a history of reported donation was less accurate.

Selective Downregulation of JAK2 and JAK3 by an ATP-Competitive pan-JAK Inhibitor.

PF-956980 has been used previously as a JAK3-selective chemical probe in numerous cell-based experiments. Here, we report that not only is PF-956980 a pan-JAK ATP-competitive inhibitor but it also causes selective reduction of endogenous JAK2 and JAK3 protein levels in human primary immune cells (in a time-dependent manner), leaving the other JAK family members (JAK1 and TYK2) unchanged. We found that PF-956980 selectively downregulated JAK2 and JAK3 mRNA, corresponding to changes observed at the protein level. This work highlights therapeutic opportunities for the development of pharmacological inhibitors that also modulate the expression of their cognate binding proteins.

Assessing coral health and disease from digital photographs and in situ surveys.

Methods for monitoring the status of marine communities are increasingly adopting the use of images captured in the field. However, it is not always clear how data collected from photographic images relate to historic data collected using traditional underwater visual census methods. Here, we compare coral health and disease data collected in situ by scuba divers with photographic images collected simultaneously at 12 coral reef sites. Five globally relevant coral diseases were detected on 194 colonies from in situ surveys and 79 colonies from photos, whilst 698 colonies from in situ surveys and 535 colonies from photos exhibited signs of compromised health other than disease. Comparisons of in situ surveys with photographic analyses indicated that the number of disease cases occurring in the examined coral populations (prevalence) was six times higher (4.5 vs. 0.8% of colonies), whilst compromised health was three times higher (14 vs. 4% of colonies) from in situ surveys. Skeletal eroding band disease, sponge overgrowth and presence of Waminoa flatworms were not detected in photographs, though they were identified in situ. Estimates of black band disease and abnormally pigmented coral tissues were similar between the two methods. Estimates of the bleached and healthy colonies were also similar between methods and photographic analyses were a strong predictor of bleached (r 2 = 0.8) and healthy (r 2 = 0.5) colony prevalence from in situ surveys. Moreover, when data on disease and compromised health states resulting in white or pale coral colony appearance were pooled, the prevalence of 'white' colonies from in situ (14%) and photographic analyses (11%) were statistically similar. Our results indicate that information on coral disease and health collected by in situ surveys and photographic analyses are not directly comparable, with in situ surveys generally providing higher estimates of prevalence and greater ability to identify some diseases and compromised states. Careful sampling of photographs can however identify signs of coral stress, including some coral diseases, which may be used to trigger early-warning management interventions.

Epithelial and stromal-specific immune pathway activation in the murine endometrium post-coitum.

The endometrium is a dynamic tissue, demonstrating cyclical growth/remodelling in preparation for implantation. In mice, seminal constituents trigger mechanisms to prepare the endometrium, a process dubbed 'seminal priming' that modifies immune system components and mediates endometrial remodelling in preparation for pregnancy. An array of cytokines has been reported to mediate this interaction, although much of the literature relates to in vitro studies on isolated endometrial epithelial cells. This study measured changes in immune-related gene expression in endometrial epithelial and stromal cells in vivo following natural mating. CD1 mice were naturally mated and sacrificed over the first 4 days post-coitum (n=3 each day). Endometrial epithelial and stromal compartments were isolated by laser capture microdissection. Labelled cRNA was generated and hybridised to genome-wide expression microarrays. Pathway analysis identified several immune-related pathways active within epithelial and stromal compartments, in particular relating to cytokine networks, matrix metalloproteinases and prostaglandin synthesis. Cluster analysis demonstrated that the expression of factors involved in immunomodulation/endometrial remodelling differed between the epithelial and stromal compartments in a temporal fashion. This study is the first to examine the disparate responses of the endometrial epithelial and stromal compartments to seminal plasma in vivo in mice, and demonstrates the complexity of the interactions between these two compartments needed to create a permissive environment for implantation.

Unrelated adult stem cell donor medical suitability: recommendations from the World Marrow Donor Association Clinical Working Group Committee.

The World Marrow Donor Association (WMDA) fosters collaboration between international registries to facilitate the exchange of hematopoietic stem cell products for unrelated stem cell donor transplantation. As indications for hematopoietic SCT grow, the movement of products across the world will increase. Although competent authorities may regulate products within their country, there is a need to protect the best interests of donors and recipients by identifying universal donor medical suitability criteria. Within this report the WMDA provides a background to unrelated adult donor and recipient safety, recommends a common framework for assessing the health of unrelated adult donors at each stage of the donation pathway and presents a novel mechanism for sharing international consensus criteria for individual medical and lifestyle conditions. Wherever possible, these criteria are evidence-based. By establishing a donor medical suitability working group, the WMDA has developed a process through which donor centers and registries may request a consensus opinion on conditions not already listed, as well as challenge existing criteria. Guidance from the WMDA is intended to complement, not supersede, guidance from national competent authorities and international regulatory bodies.

Serum leptin and loss of control eating in children and adolescents.

BACKGROUND: Both insufficiency and resistance to the actions of the adipocyte-derived hormone leptin promote hunger, increased food intake and greater body weight. Some studies suggest that adults reporting binge eating have increased serum leptin compared with those without binge eating, even after adjusting for the greater adiposity that characterizes binge eaters. Pediatric binge or loss of control (LOC) eating are prospective risk factors for excessive weight gain and may predict development of metabolic abnormalities, but whether LOC eating is associated with higher leptin among children is unknown. We therefore examined leptin and LOC eating in a pediatric cohort. METHODS: A convenience sample of 506 lean and obese youth (7-18 years) was recruited from Washington, DC and its suburbs. Serum leptin was collected after an overnight fast. Adiposity was measured by dual-energy X-ray absorptiometry or air displacement plethysmography. LOC eating was assessed by interview methodology. RESULTS: Leptin was strongly associated with fat mass (r=0.79, P<0.001). However, even after adjusting for adiposity and other relevant covariates, youth with LOC eating had higher serum leptin compared with those without LOC episodes (15.42±1.05 vs 12.36±1.04 ng ml(-1), P<0.001). Neither reported amount of food consumed during a recent LOC episode nor number of LOC episodes in the previous month accounted for differences in leptin (P>0.05). The relationship between LOC eating and leptin appeared to be significant for females only (P=0.002). CONCLUSIONS: Reports of LOC eating were associated with higher fasting leptin in youth, beyond the contributions of body weight. Prospective studies are required to elucidate whether LOC eating promotes greater leptin or whether greater leptin resistance may promote LOC eating.