The formation of ubiquitin rich condensates triggers recruitment of the ATG9A lipid transfer complex to initiate basal autophagy.

Autophagy is an essential cellular recycling process that maintains protein and organelle homeostasis. ATG9A vesicle recruitment is a critical early step in autophagy to initiate autophagosome biogenesis. The mechanisms of ATG9A vesicle recruitment are best understood in the context of starvation-induced non-selective autophagy, whereas less is known about the signals driving ATG9A vesicle recruitment to autophagy initiation sites in the absence of nutrient stress. Here we demonstrate that loss of ATG9A or the lipid transfer protein ATG2 leads to the accumulation of phosphorylated p62 aggregates in the context of basal autophagy. Furthermore, we show that p62 degradation requires the lipid scramblase activity of ATG9A. Lastly, we present evidence that poly-ubiquitin is an essential signal that recruits ATG9A and mediates autophagy foci assembly in nutrient replete cells. Together, our data support a ubiquitin-driven model of ATG9A recruitment and autophagosome formation during basal autophagy.

Prone position for acute respiratory distress syndrome and the hazards of meta-analysis.

BACKGROUND: Researchers have tried unsuccessfully for many years using randomized controlled trials to show the efficacy of prone ventilation in treating ARDS. These failed attempts were of use in designing the successful PROSEVA trial, published in 2013. However, the evidence provided by meta-analyses in support of prone ventilation for ARDS was too low to be conclusive. The present study shows that meta-analysis is indeed not the best approach for the assessment of evidence as to the efficacy of prone ventilation. METHODS: We performed a cumulative meta-analysis to prove that only the PROSEVA trial, due to its strong protective effect, has substantially impacted on the outcome. We also replicated nine published meta-analyses including the PROSEVA trial. We performed leave-one-out analyses, removing one trial at a time from each meta-analysis, measuring p values for effect size, and also the Cochran's Q test for heterogeneity assessment. We represented these analyses in a scatter plot to identify outlier studies influencing heterogeneity or overall effect size. We used interaction tests to formally identify and evaluate differences with the PROSEVA trial. RESULTS: The positive effect of the PROSEVA trial accounted for most of the heterogeneity and for the reduction of overall effect size in the meta-analyses. The interaction tests we conducted on the nine meta-analyses formally confirmed the difference in the effectiveness of prone ventilation between the PROSEVA trial the other studies. CONCLUSIONS: The clinical lack of homogeneity between the PROSEVA trial design and the other studies should have discouraged the use of meta-analysis. Statistical considerations support this hypothesis, suggesting that the PROSEVA trial is an independent source of evidence.

Supercomputer-Based Ensemble Docking Drug Discovery Pipeline with Application to Covid-19.

We present a supercomputer-driven pipeline for in silico drug discovery using enhanced sampling molecular dynamics (MD) and ensemble docking. Ensemble docking makes use of MD results by docking compound databases into representative protein binding-site conformations, thus taking into account the dynamic properties of the binding sites. We also describe preliminary results obtained for 24 systems involving eight proteins of the proteome of SARS-CoV-2. The MD involves temperature replica exchange enhanced sampling, making use of massively parallel supercomputing to quickly sample the configurational space of protein drug targets. Using the Summit supercomputer at the Oak Ridge National Laboratory, more than 1 ms of enhanced sampling MD can be generated per day. We have ensemble docked repurposing databases to 10 configurations of each of the 24 SARS-CoV-2 systems using AutoDock Vina. Comparison to experiment demonstrates remarkably high hit rates for the top scoring tranches of compounds identified by our ensemble approach. We also demonstrate that, using Autodock-GPU on Summit, it is possible to perform exhaustive docking of one billion compounds in under 24 h. Finally, we discuss preliminary results and planned improvements to the pipeline, including the use of quantum mechanical (QM), machine learning, and artificial intelligence (AI) methods to cluster MD trajectories and rescore docking poses.

Supercomputer-Based Ensemble Docking Drug Discovery Pipeline with Application to Covid-19.

We present a supercomputer-driven pipeline for in-silico drug discovery using enhanced sampling molecular dynamics (MD) and ensemble docking. We also describe preliminary results obtained for 23 systems involving eight protein targets of the proteome of SARS CoV-2. THe MD performed is temperature replica-exchange enhanced sampling, making use of the massively parallel supercomputing on the SUMMIT supercomputer at Oak Ridge National Laboratory, with which more than 1ms of enhanced sampling MD can be generated per day. We have ensemble docked repurposing databases to ten configurations of each of the 23 SARS CoV-2 systems using AutoDock Vina. We also demonstrate that using Autodock-GPU on SUMMIT, it is possible to perform exhaustive docking of one billion compounds in under 24 hours. Finally, we discuss preliminary results and planned improvements to the pipeline, including the use of quantum mechanical (QM), machine learning, and AI methods to cluster MD trajectories and rescore docking poses.

Distal determinants of mental health conditions (MHC) for asylum-seeking children in Greece: A health system enhancement study.

Mental health conditions (MHC) of asylum-seeking children in Greece are dire, but still recovering from the financial crisis, Greece cannot afford the cost of mental health treatments. We were motivated to understand the root causes of these mental health problems to explore the possibilities for prevention. We developed our inferences in four ways: (1) secondary analyses of thirty-nine semi-structured informational interviews conducted with national and international aid organizations and healthcare providers; (2) secondary analyses of nine interviews with asylum seekers; (3) direct observation during six refugee camp visits from June 1 to July 28, 2017; and (4) a literature review to develop a diagnostic tree of causal outcomes. Results revealed eight proximal causes: chronic stress, trauma, at-risk population without protection and assistance, the large number of vulnerable groups, feeling of insecurity, feeling of lacking control, a lack of autonomy, and feeling helpless and hopeless. We identified sixty-nine distal determinants of adverse MHC beneath the proximal causes. Too numerous and too diverse to treat effecvively with limited resources, these root causes of MHC were thematically grouped into: laws and regulations, capacity and resources, accountability and standards, prioritization, bias and stigma, and displacement. Using a common health systems framework, we developed strategic policy approaches to target the root causes, which could prevent ill-health while saving time and resources.

Progesterone inhibits cytokine/TNF-α production by porcine CL macrophages via the genomic progesterone receptor.

In pigs, luteolytic sensitivity to PGF-2α (=LS) is delayed until d 13 of the estrous cycle. While the control of LS is unknown, it is temporally associated with macrophage (MAC; which secretes tumor necrosis factor [TNF]-α) infiltration into the corpora lutea (CL), and previous studies have shown that TNF-α induces LS in porcine luteal cells (LCs) in culture. This study was designed to explore the control of LS by CL macrophage (CL MAC)/TNF-α by progesterone (P4), and to examine the hypothesis that P4 acting via the genomic P4 receptor (PGR) inhibits CL MAC TNF-α and thus plays a key role in regulating LS during the pig estrous cycle. In experiment 1, the effects of LCs on CL MAC cytokine/TNF-α mRNA expression in co-culture were examined (MID cycle; ~d 7-12; no LS); results showed that LC was inhibitory to cytokine/TNF-α. In experiment 2, the effects of P4 or R5020 (PGR-agonist) on CL MAC cytokine/TNF-α mRNA expression were examined (MID cycle; ~d 7-12; no LS); results showed that both P4 and R5020 dose-dependently inhibited TNF-α. In experiment 3, CL MACs were isolated from CL at MID (~d 7-12; no LS) and LATE (~d 13-18; + LS) cycle, and TNF-α/PGR mRNA measured. Results indicated that while TNF-α mRNA was 4.2-fold greater in CL MACs from LATE vs MID cycle, PGR mRNA was 4.5-fold greater in CL MACs from MID vs LATE cycle. These data support our hypothesis and suggest that progesterone, acting via PGR, plays a critical physiological role in the control of TNF-α production by CL MACs and LS during the pig estrous cycle.

Fluid therapy in neurointensive care patients: ESICM consensus and clinical practice recommendations

Objective To report the ESICM consensus and clinical practice recommendations on fluid therapy in neurointensive care patients. Design A consensus committee comprising 22 international experts met in October 2016 during ESICM LIVES2016. Teleconferences and electronic-based discussions between the members of the committee subsequently served to discuss and develop the consensus process. Methods Population, intervention, comparison, and outcomes (PICO) questions were reviewed and updated as needed, and evidence profiles generated. The consensus focused on three main topics: (1) general fluid resuscitation and maintenance in neurointensive care patients, (2) hyperosmolar fluids for intracranial pressure control, (3) fluid management in delayed cerebral ischemia after subarachnoid haemorrhage. After an extensive literature search, the principles of the Grading of Recommendations Assessment, Development and Evaluation (GRADE) system were applied to assess the quality of evidence (from high to very low), to formulate treatment recommendations as strong or weak, and to issue best practice statements when applicable. A modified Delphi process based on the integration of evidence provided by the literature and expert opinions­using a sequential approach to avoid biases and misinterpretations­was used to generate the final consensus statement. Results The final consensus comprises a total of 32 statements, including 13 strong recommendations and 17 weak recommendations. No recommendations were provided for two statements. Conclusions We present a consensus statement and clinical practice recommendations on fluid therapy for neurointensive care patients.

Toll-like receptor and related cytokine mRNA expression in bovine corpora lutea during the oestrous cycle and pregnancy.

Improving our understanding of the mechanisms controlling the corpus luteum (CL) and its role in regulating the reproductive cycle should lead to improvements in the sustainability of today's global animal industry. The corpus luteum (CL) is a transient endocrine organ composed of a heterogeneous mixture steroidogenic, endothelial and immune cells, and it is becoming clear that immune mechanisms play a key role in CL regulation especially in luteolysis. Toll-like receptors (TLR) mediate innate immune mechanisms via the production of pro-inflammatory cytokines, especially within various tissues, although the role of TLR within CL remains unknown. Thus, the objectives of this study were to characterize TLR mRNA expression in the CL during the oestrous cycle and in pregnancy (day 30-50), and to examine the role of TLR signalling in luteal cells. Corpora lutea were collected at various stages of the cycle and pregnancy and analysed for TLR and cytokine mRNA expression. In addition, luteal cells were cultured with the TLR4 ligand (lipopolysaccharide, LPS) for 24 h to evaluate the role of TLR4 in regulating luteal function. Toll-like receptors 1, 2, 4, 6, tumour necrosis factor alpha (TNF), interferon gamma (IFN-G), and interleukin (IL)-12, mRNA expressions were greatest in regressing CL compared with earlier stages (p < .05), whereas no change was observed for IL-6 mRNA expression. Cytokine mRNA expression in cultured luteal cells was not altered by LPS. Based on these data, one or more of the TLRs found within the CL may play a role in luteolysis, perhaps via pro-inflammatory cytokine mRNA expression.

Site of PGF2α injection does not alter effectiveness of the Select Synch + controlled internal drug release and timed artificial insemination protocol.

Beef Quality Assurance programs have contributed to significant improvements in the wholesomeness of beef available for consumption. Injection site blemishes in the round have declined since the promotion of administering intramuscular injections in the neck. Unfortunately, many producers continue to administer estrus synchronization (ES) drugs in the rump. The objective of this study was to compare the effectiveness of injection site of PGF2α, in ES protocols, on steroid hormone concentrations and pregnancy rates. A Select Synch + 7-day controlled internal drug release ES protocol was conducted with the site of PGF2α injection alternated between neck and rump in beef cattle (n = 312) at the Ohio State University Agricultural Technical Institute and North Carolina State University. Blood samples (n = 75) were collected at controlled internal drug release insertion and at the time of artificial insemination (AI) to determine if progesterone (P4) and estrogen (E2) concentrations varied due to PGF2α injection site. All cattle were confirmed pregnant by ultrasonography at approximately 30 and 90 days after insemination in North Carolina and approximately 70 days after insemination in Ohio. Data were analyzed as randomized complete block designs in PROC GLIMMIX with animal as the experimental unit. Differences were declared significant at P < 0.05. Site of PGF2α injection, in either the neck or rump, did not affect (P > 0.05) overall conception rates in response to AI (58.4% and 55.6%, respectively). Altering PGF2α injection site did not impact P4, E2 concentrations, or the P4:E2 ratio at AI (P > 0.05). However, cattle inseminated after displaying estrus had greater (P < 0.05) pregnancy rates than timed AI (67.8 vs. 47.5%, respectively). First service conception rates and pregnancy rates were consistent with previous reports. Overall, altering the location of the PGF2α injection during ES did not change circulating hormone concentrations at AI or pregnancy rates; therefore, cattle producers should follow Beef Quality Assurance guidelines when administering ES protocols.

Antagonism of the proinflammatory and pronociceptive actions of canonical and biased agonists of protease-activated receptor-2.

BACKGROUND AND PURPOSE: Diverse proteases cleave protease-activated receptor-2 (PAR2) on primary sensory neurons and epithelial cells to evoke pain and inflammation. Trypsin and tryptase activate PAR2 by a canonical mechanism that entails cleavage within the extracellular N-terminus revealing a tethered ligand that activates the cleaved receptor. Cathepsin-S and elastase are biased agonists that cleave PAR2 at different sites to activate distinct signalling pathways. Although PAR2 is a therapeutic target for inflammatory and painful diseases, the divergent mechanisms of proteolytic activation complicate the development of therapeutically useful antagonists. EXPERIMENTAL APPROACH: We investigated whether the PAR2 antagonist GB88 inhibits protease-evoked activation of nociceptors and protease-stimulated oedema and hyperalgesia in rodents. KEY RESULTS: Intraplantar injection of trypsin, cathespsin-S or elastase stimulated mechanical and thermal hyperalgesia and oedema in mice. Oral GB88 or par2 deletion inhibited the algesic and proinflammatory actions of all three proteases, but did not affect basal responses. GB88 also prevented pronociceptive and proinflammatory effects of the PAR2-selective agonists 2-furoyl-LIGRLO-NH2 and AC264613. GB88 did not affect capsaicin-evoked hyperalgesia or inflammation. Trypsin, cathepsin-S and elastase increased [Ca(2+) ]i in rat nociceptors, which expressed PAR2. GB88 inhibited this activation of nociceptors by all three proteases, but did not affect capsaicin-evoked activation of nociceptors or inhibit the catalytic activity of the three proteases. CONCLUSIONS AND IMPLICATIONS: GB88 inhibits the capacity of canonical and biased protease agonists of PAR2 to cause nociception and inflammation.

Effect of protein supplementation and forage allowance on the growth and reproduction of beef heifers grazing stockpiled tall fescue.

Stockpiled tall fescue can provide adequate winter forage for beef cattle, although unsupplemented replacement heifers may display marginal performance before breeding. The objective of this study was to determine if protein supplementation and/or additional forage improves growth and reproductive performance of replacement heifers grazing stockpiled fescue. Cattle averaging 272 ± 1.59 kg were stratified by BW and then randomly assigned to 1 of 4 plots within a pasture replication. Treatment combinations were assigned in a 2 × 2 factorial arrangement and included 1) a conservative forage allocation ("normal," targeting 85% forage use) and mineral supplement (normal forage allocation with mineral supplement [FM]), 2) normal forage allocation with protein tub (FT), 3) more liberal forage allocation ("extra," targeting 70% forage use) and mineral supplement (extra forage allocation with mineral supplement [EM]), and 4) "extra forage allocation with protein tub (ET). Treatments were administered for 8 wk from early November to early January. Heifers were fed fescue hay for 1 wk before breeding in late January. Heifers were synchronized with the 7-d CO-Synch + controlled internal drug release device protocol and inseminated in late January. Heifers were checked for pregnancy by ultrasonography at 35 and 90 d after AI. Main and interaction effects between the 2 treatments were determined. Total supplement intake was greater for protein tub than mineral supplement (0.36 vs. 0.11 kg·heifer·d, respectively; < 0.0001), and the additional dietary protein in the tub groups resulted in greater serum urea N concentrations ( < 0.0001; 8.15 vs. 10.4 mg/dL for mineral and protein tub, respectively). Forage utilization efficiency was greater for normal than extra forage allocation (74.7 vs. 65.8%, respectively; < 0.0001). Main effects of both treatments on ADG were significant ( < 0.0001; 0.28, 0.43, 0.43, and 0.51 kg·heifer·d for FM, FT, EM, and ET, respectively). There was an interaction effect of the 2 treatments on change in BCS ( < 0.05; 0.12, 0.10, 0.18, and 0.31 for FM, FT, EM, and ET, respectively). Reproductive tract scores, pelvic area, and AI pregnancy rates were not different between treatments ( > 0.05). Overall, feeding a protein supplement or providing extra forage increased gain and interacted to increase BCS but did not have an effect on reproductive performance. Supplementing with protein and providing extra forage are strategies that can increase gain in heifers, which could aid heifers in reaching puberty before estrous synchronization.

Comparison of pregnancy rates in beef cattle after a fixed-time AI with once- or twice-used controlled internal drug release devices.

The use of fixed-time artificial insemination (FTAI) provides producers with numerous benefits including the use of superior genetics, shorter breeding and calving seasons, and a more uniform calf crop. However, the cost of implementing FTAI protocols is one of the several drawbacks hindering their use in the beef industry. Potential injection-site lesions from intramuscular injections of the hormones necessary for estrus synchronization are also a cause of concern for carcass quality. The objectives of this experiment were to (1) determine whether or not a twice-used controlled internal drug release (CIDR) device would be effective in an FTAI protocol without adversely affecting pregnancy rate and (2) whether or not the subcutaneous administration of PGF2α affects pregnancy rate. Nulliparous females (n = 99) between 13 and 27 months of age and multiparous cows (n = 43) between 48 and 74 months of age were synchronized for estrus using the 7-day CO-Synch + CIDR protocol. The females were randomly assigned to one of the two treatments: (1) a once-used CIDR (control) or (2) a twice-used CIDR device (treatment) incorporated into their synchronization protocol. The females were also randomly assigned to have their injection of PGF2α administered either intramuscularly or subcutaneously. Blood was taken in a random subset of nulliparous females (n = 52) just before device removal and assayed for concentration of progesterone. The concentration of progesterone was higher (P = 0.01) in the animals that received once-used CIDR devices than that in those received twice-used CIDR devices (3.4 ± 0.5 and 1.4 ± 0.5 ng/mL, respectively). There was no significant effect of parity (P = 0.82), artificial insemination technician (P = 0.60), PGF2α administration (P = 0.83), or treatment (P = 0.67) on pregnancy rates to artificial insemination which were 75.4 ± 6.0% and 71.7 ± 6.4%, for animals that received once- and twice-used CIDR devices, respectively. This study provides evidence that although concentration of progesterone is decreased in animals treated with a twice-used CIDR device, there is still a sufficient release of progesterone from the device to effectively synchronize estrus without adversely affecting the fertility of a herd.

The role of the intensive care unit in real-time surveillance of emerging pandemics: the Italian GiViTI experience.

The prompt availability of reliable epidemiological information on emerging pandemics is crucial for public health policy-makers. Early in 2013, a possible new H1N1 epidemic notified by an intensive care unit (ICU) to GiViTI, the Italian ICU network, prompted the re-activation of the real-time monitoring system developed during the 2009-2010 pandemic. Based on data from 216 ICUs, we were able to detect and monitor an outbreak of severe H1N1 infection, and to compare the situation with previous years. The timely and correct assessment of the severity of an epidemic can be obtained by investigating ICU admissions, especially when historical comparisons can be made.

Transient receptor potential vanilloid 4 inhibits mouse colonic motility by activating NO-dependent enteric neurotransmission.

UNLABELLED: Recent studies implicate TRPV4 receptors in visceral pain signaling and intestinal inflammation. Our aim was to evaluate the role of TRPV4 in the control of gastrointestinal (GI) motility and to establish the underlying mechanisms. We used immunohistochemistry and PCR to study TRPV4 expression in the GI tract. The effect of TRPV4 activation on GI motility was characterized using in vitro and in vivo motility assays. Calcium and nitric oxide (NO) imaging were performed to study the intracellular signaling pathways. Finally, TRPV4 expression was examined in the colon of healthy human subjects. We demonstrated that TRPV4 can be found on myenteric neurons of the colon and is co-localized with NO synthase (NOS-1). In vitro, the TRPV4 agonist GSK1016790A reduced colonic contractility and increased inhibitory neurotransmission. In vivo, TRPV4 activation slowed GI motility and reduced stool production in mouse models mimicking pathophysiological conditions. We also showed that TRPV4 activation inhibited GI motility by reducing NO-dependent Ca(2+) release from enteric neurons. In conclusion, TRPV4 is involved in the regulation of GI motility in health and disease. KEY MESSAGES: • Recent studies implicate TRPV4 in pain signaling and intestinal inflammation. • Our aim was to characterize the role of TRPV4 in the control of GI motility. • We found that TRPV4 activation reduced colonic contractility. • Our studies also showed altered TRPV4 mRNA expression in IBS-C patients. • TRPV4 may be a novel pharmacological target in functional GI diseases.

Demonstration of elevated levels of active cathepsin S in dextran sulfate sodium colitis using a new activatable probe.

BACKGROUND: Proteases play a major role in inflammatory diseases of the gastrointestinal tract. Activatable probes are a major technological advance, enabling sensitive detection of active proteases in tissue samples. Our aim was to synthesize an activatable probe for cathepsin S and validate its use in a mouse model of colitis. METHODS: We designed and synthesized a new fluorescent activatable probe, NB200, for the detection of active cathepsin S. Colitis was induced in C57BL/6 mice by the administration of 3% dextran sulfate sodium (DSS). Homogenized mouse colons, with or without the addition of the specific cathepsin S inhibitor MV026031, were incubated with NB200 in a fluorescent plate reader. KEY RESULTS: NB200 selectively detected purified cathepsin S and not other common inflammatory proteases. Homogenates of colon from mice with DSS colitis induced a significant fluorescent increase when compared to control animals (control vs DSS: p < 0.05 at 200 min and p < 0.01 at 220-240 min), indicating cathepsin S activation. The cathepsin S inhibitor abolished this increase in fluorescence (DSS vs DSS + MV026031: p < 0.05 at 140 min, p < 0.01 at 180 min, p < 0.001 at 200-240 min), which confirms cathepsin S activation. Cathepsin S activity correlated with the disease activity index (Spearman r = 0.77, p = 0.017). CONCLUSIONS & INFERENCES: Our investigation has demonstrated the utility of activatable probes for detecting protease activity in intestinal inflammation. Panels of such probes may allow 'signature' protease profiles to be established for a range of inflammatory diseases and disorders.

Attitudinal barriers to participation in oncology clinical trials: factor analysis and correlates of barriers.

Patient participation in cancer clinical trials is low. Little is known about attitudinal barriers to participation, particularly among patients who may be offered a trial during an imminent initial oncology consult. The aims of the present study were to confirm the presence of proposed subscales of a recently developed cancer clinical trial attitudinal barriers measure, describe the most common cancer clinical trials attitudinal barriers, and evaluate socio-demographic, medical and financial factors associated with attitudinal barriers. A total of 1256 patients completed a survey assessing demographic factors, perceived financial burden, prior trial participation and attitudinal barriers to clinical trials participation. Results of a factor analysis did not confirm the presence of the proposed four attitudinal barriers subscale/factors. Rather, a single factor represented the best fit to the data. The most highly-rated barriers were fear of side-effects, worry about health insurance and efficacy concerns. Results suggested that less educated patients, patients with non-metastatic disease, patients with no previous oncology clinical trial participation, and patients reporting greater perceived financial burden from cancer care were associated with higher barriers. These patients may need extra attention in terms of decisional support. Overall, patients with fewer personal resources (education, financial issues) report more attitudinal barriers and should be targeted for additional decisional support.

Effects of service sire on prenatal mortality and prolificacy in ewes.

Ability to select service sires that minimize partial or complete losses of pregnancy could have major economic impacts in sheep production systems. This study tested the null hypothesis that survival of potential progeny did not vary with breed type of service sire or among individual rams. Data included 980 ewes on 10 farms; each ewe was pregnant to 1 of 67 rams of 12 breeds. Number of conceptuses was estimated once during pregnancy by ultrasonography, either transrectal (embryos) or transabdominal (fetuses), and was compared with number of lambs born to estimate losses. Data were examined first for number of lambs born and second for documented losses. Individual service sires affected number born (P < 0.001), which varied from 0.70 to 2.45 lambs per pregnant ewe. The main effects of breed type on lambs born were not significant, but breed types of both service sires (P < 0.0002) and ewes (P < 0.001) interacted with diagnosed number of conceptuses. Lambs born varied with ewe age (P < 0.0001) and among farms (P < 0.0001), and statistically, farms interacted with number of diagnosed conceptuses (P < 0.0001); season had no effect. In documented losses, there were both main effects of individual service sire and a service sire × number of diagnosed embryos interaction (P < 0.005). Thus, ewes bred to some rams were more apt to lose single pregnancies, whereas ewes bred to other rams were more apt to lose 1 or more embryos or fetuses from multiple pregnancies. Breed type of service sire affected (P < 0.05) prenatal death. Complete losses of single conceptuses tended to be greater in ewes bred to black-faced or hair-type rams (service sire breed type × number of diagnosed conceptuses; P < 0.09). Breed type of ewes also varied in incidence of complete losses (P < 0.05); hair-type ewes (46%) lost more (P < 0.02) documented conceptuses from examination to birth than black-faced (27%), white-faced (20%), or dairy-type (25%) ewes. Greater losses of singles than of multiples occurred in black-faced (37% vs. 18%) and hair-type (64% vs. 27%) ewes than in other breeds (ewe breed type × number of conceptuses; P < 0.03) per ewe. Surprisingly, purebred conceptuses were lost less often (24%) than crossbreds (36.4%; P < 0.002). Selection of rams based on records of prenatal losses in ewes they serviced may be a method to decrease embryonic and fetal wastage. However, further study to determine repeatability of differences among service sires from year to year will be required.

A single test for the determination of parameters of the speed-time relationship for running.

A validated expeditious method is needed to determine critical speed (CS) and the finite distance that can be covered above CS (D'). We tested the hypothesis that a single all-out 3-min running test would accurately determine CS and D'. Seven healthy subjects completed three constant-speed runs on a treadmill for the determination of CS and D', as well as an all-out 3-min test on a track for the determination of end-test speed (ES) and the distance above end-test speed (DES). ES (13.4 ± 2.8 km h(-1)) was not significantly different from the speed-1/time model CS (13.3 ± 2.8 km h(-1)). While DES (141 ± 34 m) was not significantly different from D' (204 ± 103 m), it underestimated D' in 5 of 7 subjects. Thus, the speed-1/time model CS can be accurately determined using a single 3-min test, while caution should be used in relating DES to D'.

Bone morphogenetic protein 6 promotes FSH receptor and anti-Müllerian hormone mRNA expression in granulosa cells from hen prehierarchal follicles.

A growing body of literature provides evidence of a prominent role for bone morphogenetic proteins (BMPs) in regulating various stages of ovarian follicle development. Several actions for BMP6 have been previously reported in the hen ovary, yet only within postselection (preovulatory) follicles. The initial hypothesis tested herein is that BMP6 increases FSH receptor (FSHR) mRNA expression within the granulosa layer of prehierarchal (6-8 mm) follicles (6-8 GC). BMP6 mRNA is expressed at higher levels within undifferentiated (1-8 mm) follicles compared with selected (≥9 mm) follicles. Recombinant human (rh) BMP6 initiates SMAD1, 5, 8 signaling in cultured 6-8 GC and promotes FSHR mRNA expression in a dose-related fashion. In addition, a 21 h preculture with rhBMP6 followed by a 3 h challenge with FSH increases cAMP accumulation, STAR (StAR) expression, and progesterone production. Interestingly, rhBMP6 also increases expression of anti-Müllerian hormone (AMH) mRNA in cultured 6-8 GC. This related BMP family member has previously been implicated in negatively regulating FSH responsiveness during follicle development. Considering these data, we propose that among the paracrine and/or autocrine actions of BMP6 within prehierarchal follicles is the maintenance of both FSHR and AMH mRNA expression. We predict that before follicle selection, one action of AMH within granulosa cells from 6 to 8 mm follicles is to help suppress FSHR signaling and prevent premature granulosa cell differentiation. At the time of selection, we speculate that the yet undefined signal directly responsible for selection initiates FSH responsiveness. As a result, FSH signaling suppresses AMH expression and initiates the differentiation of granulosa within the selected follicle.