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Our capacity to measure diverse aspects of human biology has developed rapidly in the past decades, but the rate at which these techniques have generated insights into the biological correlates of psychopathology has lagged far behind. The slow progress is partly due to the poor sensitivity, specificity and replicability of many findings in the literature, which have in turn been attributed to small effect sizes, small sample sizes and inadequate statistical power. A commonly proposed solution is to focus on large, consortia-sized samples. Yet it is abundantly clear that increasing sample sizes will have a limited impact unless a more fundamental issue is addressed: the precision with which target behavioral phenotypes are measured. Here, we discuss challenges, outline several ways forward and provide worked examples to demonstrate key problems and potential solutions. A precision phenotyping approach can enhance the discovery and replicability of associations between biology and psychopathology.

Background: Hand hygiene (HH) is a fundamental component of infection prevention within all healthcare settings. We implemented a hospital-wide program built on overt HH observation, real-time feedback, and thematic analysis of HH misses., Methods: A robust observer training program was established to include foundational training in the WHO's My Five Moments of HH. Observational data from 2011 to 2019 were analyzed by unit, provider type, and thematic analyses of misses., Results: During the study period, we conducted 160,917 hospital-wide observations on 29 units (monthly average of 1,490 observations). Institutional compliance remained above 95% from 2013 to 2019. Thematic analysis revealed "touching self" and "touching phone" as common, institution-wide reasons for HH misses., Discussion: Overt observations facilitated communication between HH program and healthcare staff to better understand workflow and educate staff on HH opportunities. This program is an integral part of the Infection Prevention team and has been deployed to collect supplemental data during clusters and outbreaks investigations., Conclusions: In addition to having rich HH data, successes of this program, include increased awareness of IPC practices, enhanced communication about patient safety, enriched dialog and feedback around HH misses, and relationship building among program observers, unit staff and leaders., (Copyright © 2022 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.)

Background: Central line-associated bloodstream infections (CLABSI) in ambulatory pediatric populations are difficult to track at an institutional level, especially for complex patients seen by multiple divisions and home health infusion agencies., Methods: A multidisciplinary team comprised of key stakeholders from divisions with the most patients discharged with a central line utilized Lean Six Sigma methodology of Define-Measure-Analyze-Design-Verify (DMADV) to create a standardized data collection process for all ambulatory CLABSIs and infection event reviews., Results: A surveillance workflow was created to track, identify, and confirm ambulatory CLABSIs in all patients with an indwelling central line. Defined surveillance criteria included scope of patients eligible for ambulatory CLABSI surveillance, numerator definitions, and denominator calculations. Additionally, a novel attribution method was created for ambulatory CLABSIs in complex patient populations shared among multiple divisions and home care infusion services., Conclusions: This report is a novel institutional approach to accurately surveil, attribute, and calculate ambulatory CLABSI data in a pediatric healthcare system., (Copyright © 2022 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.)

Introduction: Healthcare-associated viral infections (HAVI) are a common cause of patient harm in the pediatric population. We implemented a HAVI prevention bundle in 2015, which included 6 core elements: caregiver screening, symptom-based isolation, personal protective equipment (PPE), hand hygiene, staff illness procedures, and monitoring of environmental cleanliness. Enhanced bundle elements were introduced at the start of the COVID-19 pandemic, which provided an opportunity to observe the effectiveness of the bundle with optimal adherence to prevention practices, and to measure the impact on respiratory HAVI epidemiology., Methods: Respiratory HAVIs were confirmed through review of medical records and application of the National Health Safety Network (NHSN) surveillance criteria for upper respiratory infections (URIs) with predetermined incubation periods for unit attribution. Descriptive statistics of the study population were examined, and comparative analyses were performed on demographic and process metrics. Data analysis was conducted using R statistical software., Results: We observed an overall decrease in respiratory HAVI of 68%, with prepandemic rates of 0.19 infections per 1,000 patient significantly decreased to a rate of 0.06 per 1,000 patient days in the pandemic period (P < .01). Rhinovirus made up proportionally more of our respiratory HAVI in the pandemic period (64% vs 53%), with respiratory HAVI secondary only to rhinovirus identified during 8 of 16 months in the pandemic period. Compliance with our HAVI prevention bundle significantly improved during pandemic period., Conclusions: Enhancement of our HAVI bundle during the COVID-19 pandemic contributed toward significant reduction in nosocomial transmission of respiratory HAVI. Even with prevention practices optimized, respiratory HAVIs secondary to rhinovirus continued to be reported, likely due to the capacity of rhinovirus to evade bundle elements in hospital, and infection prevention efforts at large in the community, leaving vulnerable patients at continued risk., (Copyright © 2022 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.)

Background: Clinical canine massage involves muscle tissue manipulation and fascial release techniques to rehabilitate injured soft tissues. Quantitative efficacy data are lacking. This cross-sectional study aimed to determine how dogs respond to canine massage therapy practiced by Canine Massage Guild UK practitioners., Methods: In 2018, case notes from a convenience sample of 527 dogs were shared, with permission from owners, by a self-selected sample of 65 practitioners. Changes in number and severity of issues for five pain indicators (gait, posture, daily activity, behaviour, performance) and quality of life score, reported by owner and practitioners, were investigated., Results: Significant reductions in reported pain severity scores were recorded for all pain indicators over successive treatments (p < 0.001), with each treatment causing further significant reduction in pain severity. Number of pain indicators recorded over successive treatment sessions remained constant, in keeping with a cohort presenting with degenerative disease and chronic pain. All dogs and diagnostic variables responded similarly. Post-treatment a dog was significantly more likely to have a 'positive' quality of life., Conclusions: This cross-sectional study indicates canine massage therapy may effectively reduce myofascial and musculoskeletal pain severity reported by owners and practitioners associated with gait, posture, behavioural and performance issues and reduction in daily activities. Although this is not a double-blind trial, and there is no control group, this study suggests massage therapy may be a valid treatment for myofascial and musculoskeletal pain typically derived from muscular injuries, arthritis/other orthopaedic conditions., (© 2021 The Authors. Veterinary Record published by John Wiley & Sons Ltd on behalf of British Veterinary Association.)

Background: Influenza vaccination of healthcare workers (HCWs) has been recommended for more than 30 years. In 2009, HCWs were designated as a priority group by the Centers for Disease Control and Prevention. Current HCW vaccination rates are 78% across all settings and reach approximately 92% among those employed in hospital settings. Over the last decade, it has become clear that mandatory vaccine policies result in maximal rates of HCW immunization., Methods: In this observational 10-year study, we describe the implementation of a mandatory influenza vaccination policy in a dedicated quaternary pediatric hospital setting by a multidisciplinary team. We analyzed 10 years of available data from deidentified occupational health records from 2009-2010 through the 2018-2019 influenza seasons. Descriptive statistics were performed using Stata v15 and Excel., Results: Sustained increases in HCW immunization rates above 99% were observed in the 10 years postimplementation, in addition to a reduction in exemption requests and healthcare-associated influenza. In the year of implementation, 145 (1.6%) HCWs were placed on temporary suspension for failure to receive the vaccine without documentation of an exemption, with 9 (0.06%) subsequently being terminated. Since then, between 0 and 3 HCWs are terminated yearly for failure to receive the vaccine., Conclusions: Implementation of our mandatory influenza vaccination program succeeded in successfully increasing the proportion of immunized HCWs at a quaternary care children's hospital, reducing annual exemption requests with a small number of terminations secondary to vaccine refusal. Temporal trends suggest a positive impact on the safety of our patients., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Consumer suspicion of conventional pet foods, along with perceived health benefits of alternative diets, are fuelling development of the latter. These include raw meat diets, in vitro meat products, and diets based on novel protein sources such as terrestrial and marine plants, insects, yeast and fungi. However, some claim vegan diets may be less palatable, or may compromise animal welfare. We surveyed 4,060 dog or cat guardians to determine the importance to them of pet food palatability, and the degree to which their animals displayed specific behavioural indicators of palatability at meal times. Guardians were asked to choose one dog or cat that had been within their household for at least one year, and not on a prescription or therapeutic diet. Of 3,976 respondents who played some role in pet diet decision-making, palatability was the third most important among 12 factors cited as important when choosing pet diets. For 1,585 respondents feeding conventional or raw meat diets, who stated they would realistically consider alternative diets, palatability was the fourth most important among 14 desired attributes. For the 2,308 dogs included, reported observations of 10 behavioural indicators of palatability at meal times reliably indicated significant effects of increased reports of appetitive behaviour by dogs on a raw meat diet, as opposed to a conventional diet. There was no consistent evidence of a difference between vegan diets and either the conventional or raw meat diets. For the 1,135 cats included, reported observations of 15 behavioural indicators indicated that diet made little difference to food-oriented behaviour. Based on these owner-reported behaviours, our results indicate that vegan pet foods are generally at least as palatable to dogs and cats as conventional meat or raw meat diets, and do not compromise their welfare, when other welfare determinants, such as nutritional requirements, are adequately provided., Competing Interests: No authors have competing interests.

The COVID-19 pandemic caused lifestyle changes, with unknown effect on pets' quality of life (QoL). Between May and July 2020, we distributed an online survey to investigate the role of several factors on feline and canine QoL, including lockdown-related factors. We used existing scales to measure human and pets' personalities (Reinforcement Sensitivity Theory Personality Questionnaire, RST-PQ; RST-Dog; RST-Cat) and the human-animal relationship (Lexington Attachment to Pets Scale, LAPS) and the Milan Pet Quality of Life instrument (MPQL). Overall, 235 participants reported about 242 adult pets (N cats = 78, N dogs = 164). Factor analysis confirmed the structure and internal reliability of the existing scales (RST-PQ, RST-Dog, RST-Cat, LAPS) and suggested a four-factor structure for the MPQL (physical, psychological, social, environmental). The results indicate that the pets' psysical QoL was largely explained by pet-related elements (pets' demographics and life experience, and pets' personality). Conversely, the pets' psychological QoL was explained mostly by owner-related elements, such as the owners' demographics, COVID-19-related changes, and the owners' personality. Predictably, the pets' environmental QoL is mostly explained by environmental factors, such as the outdoor access in the home environment and the country. Finally, the pets' social QoL was explained by the larger combination of models: pets' characteristics and personality, environment and COVID-19-related changes, and the pet-human relationship. These findings can be explained by two non-mutually exclusive mechanisms. The reported changes may be a by-product of the COVID-19 pandemic's psychological and lifestyle effects on the owners, which in turn alter the way the owners interact with their pets and look after them. However, the owners' characteristics and mood may bias their answers regarding their pets.

The application of alibi witness scenarios to deception detection has been overlooked. Experiment 1 was a study of the verifiability approach in which truth-telling pairs completed a mission together, whereas in lying pairs one individual completed this mission alone and the other individual committed a mock theft. All pairs were instructed to convince the interviewer that they completed the mission together by writing individual statements on their own followed by a collective statement together as a pair. In the individual statements, truth-telling pairs provided more checkable details that demonstrated they completed the mission together than lying pairs, whereas lying pairs provided more uncheckable details than truth-telling pairs. The collective statements made truth-telling pairs provide significantly more checkable details that demonstrated they were together in comparison to the individual statements, whereas no effect was obtained for lying pairs. Receiver Operating Characteristic curves revealed high accuracy rates for discriminating between truths and lies using the verifiability approach across all statement types. Experiment 2 was a lie detection study whereby observers' abilities to discriminate between truths and lies using the verifiability approach were examined. This revealed that applying the verifiability approach to collective statements improved observers' ability to accurately detect deceit. We suggest that the verifiability approach could be used as a lie detection technique and that law enforcement policies should consider implementing collective interviewing., (Copyright © 2020 Elsevier B.V. All rights reserved.)

When in a vulnerable situation (such as walking alone at night), an approaching person may be seen as 'threatening'. Here, we are interested in how well participants' judgments of threat reflected the trait aggression of approaching target people. We use two similar experiments to demonstrate and replicate the relationship between judgments of threat and target aggression. In both studies participants judged how threatening they found 22 approaching people (presented in videos). In Study One, participants judged the targets whilst sitting at a computer. In Study Two, participants were standing and were either oriented facing the videos, or oriented away from the videos so they had to look over their shoulder. This was to emulate a potentially threatening person approaching from behind. Across both studies, there was strong evidence that the average judgments of the threat posed by the approaching targets accurately reflected the targets' trait aggression. It was also found that there was noteworthy variability in individual participants' ability to detect aggression, with a few participants even having an inverse relationship between threat and the target's aggression. This research demonstrates that judgments of how 'threatening' a person is can be used to accurately index trait aggression at a distance., Competing Interests: All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.Informed consent was obtained from all individual participants included in the study.All of the authors declare that they have no conflicts of interest.

Behavioral observation techniques which relate action to personality have long been neglected (Furr and Funder in Handbook of research methods in personality psychology, The Guilford Press, New York, 2007) and, when employed, often use human judges to code behavior. In the current study we used an alternative to human coding (biomechanical research techniques) to investigate how personality traits are manifest in gait. We used motion capture technology to record 29 participants walking on a treadmill at their natural speed. We analyzed their thorax and pelvis movements, as well as speed of gait. Participants completed personality questionnaires, including a Big Five measure and a trait aggression questionnaire. We found that gait related to several of our personality measures. The magnitude of upper body movement, lower body movement, and walking speed, were related to Big Five personality traits and aggression. Here, we present evidence that some gait measures can relate to Big Five and aggressive personalities. We know of no other examples of research where gait has been shown to correlate with self-reported measures of personality and suggest that more research should be conducted between largely automatic movement and personality.

The detection of potential danger is an important factor in avoiding harm that is even more important for vulnerable populations such as children. This study explores whether children can recognise the potential for a dangerous encounter from observing the gait of an approaching individual. The participants are divided into three age groups: 13- to 15-year-olds, 16- to 17-year-olds, and over 18s. Participants made judgments of nine, point light presentations of people walking on a treadmill. Ratings of intimidation made by participants were used to assess their ability to detect the walkers' trait aggression. The ability to detect trait aggression accurately was found to increase with age, as does the consistency in ratings between individuals within the same age group. The importance of experiential learning in the acquisition of aggression detection skills is highlighted., Competing Interests: No potential conflict of interest was reported by the authors., (© 2016 The Australian and New Zealand Association of Psychiatry, Psychology and Law.)

Defining the constituent regulatory molecules in tendon is critical to understanding the process of tendon repair and instructive to the development of novel treatment modalities. The purpose of this study is to define the structural, expressional, and mechanical changes in the tendon injury response, and elucidate the roles of two class I small leucine-rich proteoglycans (SLRPs). We utilized biglycan-null, decorin-null and wild type mice with an established patellar tendon injury model. Mechanical testing demonstrated functional changes associated with injury and the incomplete recapitulation of mechanical properties after 6 weeks. In addition, SLRP deficiency influenced the mechanical properties with a marked lack of improvement between 3 and 6 weeks in decorin-null tendons. Morphological analyses of the injury response and role of SLRPs demonstrated alterations in cell density and shape as well as collagen alignment and fibril structure resulting from injury. SLRP gene expression was studied using RT-qPCR with alterations in expression associated with the injured tendons. Our results show that in the absence of biglycan initial healing may be impaired while in the absence of decorin later healing is clearly diminished. This suggests that biglycan and decorin may have sequential roles in the tendon response to injury.

Insulin-like peptide 3 (INSL3), a major product of testicular Leydig cells, is also expressed by the ovary, but its functional role remains poorly understood. Here, we quantified expression of INSL3 and its receptor RXFP2 in theca interna cell (TIC) and granulosa cell compartments of developing bovine antral follicles and in corpora lutea (CL). INSL3 and RXFP2 mRNA levels were much higher in TIC than granulosa cell and increased progressively during follicle maturation with INSL3 peaking in large (11-18 mm) estrogen-active follicles and RXFP2 peaking in 9- to 10-mm follicles before declining in larger (11-18 mm) follicles. Expression of both INSL3 and RXFP2 in CL was much lower than in TIC. In situ hybridization and immunohistochemistry confirmed abundant expression of INSL3 mRNA and protein in TIC. These observations indicate follicular TIC rather than CL as the primary site of both INSL3 production and action, implying a predominantly autocrine/paracrine role in TIC. To corroborate the above findings, we showed that in vitro exposure of TIC to a luteinizing concentration of LH greatly attenuated expression of both INSL3 and its receptor while increasing progesterone secretion and expression of STAR and CYP11A1. Moreover, in vivo, a significant cyclic variation in plasma INSL3 was observed during synchronized estrous cycles. INSL3 and estradiol-17β followed a similar pattern, both increasing after luteolysis, before falling sharply after the LH surge. Thus, theca-derived INSL3, likely from the dominant preovulatory follicle, is detectable in peripheral blood of cattle, and expression is down-regulated during luteinization induced by the preovulatory LH surge. Collectively, these findings underscore the likely role of INSL3 as an important intrafollicular modulator of TIC function/steroidogenesis, while raising doubts about its potential contribution to CL function.

Bone morphogenetic proteins (BMPs) are firmly implicated as intra-ovarian regulators of follicle development and steroidogenesis. Here we report a microarray analysis showing that treatment of cultured bovine theca cells (TC) with BMP6 significantly (>twofold; P < 0.01) up- or down-regulated expression of 445 genes. Insulin-like peptide 3 (INSL3) was the most heavily down-regulated gene (-43-fold) with cytochrome P450, subfamily XVII (CYP17A1) and other key steroidogenic transcripts including steroidogenic acute regulatory protein (STAR), cytochrome P450 family 11, subfamily A1 (CYP11A1) and 3 beta-hydroxysteroid dehydrogenase type 1 (HSD3B1) also down-regulated. BMP6 also reduced expression of nuclear receptor subfamily 5A1 (NR5A1) known to target the promoter regions of the aforementioned genes. Real-time PCR confirmed these findings and also revealed a marked reduction in expression of INSL3 receptor, relaxin/insulin-like family peptide receptor 2 (RXFP2). Secretion of INSL3 protein and androstenedione were also suppressed suggesting a functional link between BMP and INSL3 pathways in controlling androgen synthesis. RNAi-mediated knockdown of INSL3 reduced INSL3 mRNA (75%) and protein (94%) level and elicited a 77% reduction in CYP17A1 mRNA and 83% reduction in androstenedione secretion. Knockdown of RXFP2 also reduced CYP17A1 expression (81%) and androstenedione secretion (88%). Conversely, treatment with exogenous (human) INSL3 increased androstenedione secretion ∼twofold. The CYP17A1 inhibitor abiraterone abolished androgen secretion and reduced expression of both INSL3 and RXFP2. Collectively, these findings indicate a positive autoregulatory role for INSL3 signaling in maintaining thecal androgen production, and visa versa. Moreover, BMP6-induced suppression of thecal androgen synthesis may be mediated, at least in part, by reduced INSL3-RXFP2 signaling.

The aging population is at an increased risk of tendon injury and tendinopathy. Elucidating the molecular basis of tendon aging is crucial to understanding the age-related changes in structure and function in this vulnerable tissue. In this study, the structural and functional features of tendon aging are investigated. In addition, the roles of decorin and biglycan in the aging process were analyzed using transgenic mice at both mature and aged time points. Our hypothesis is that the increase in tendon injuries in the aging population is the result of altered structural properties that reduce the biomechanical function of the tendon and consequently increase susceptibility to injury. Decorin and biglycan are important regulators of tendon structure and therefore, we further hypothesized that decreased function in aged tendons is partly the result of altered decorin and biglycan expression. Biomechanical analyses of mature (day 150) and aged (day 570) patellar tendons revealed deteriorating viscoelastic properties with age. Histology and polarized light microscopy demonstrated decreased cellularity, alterations in tenocyte shape, and reduced collagen fiber alignment in the aged tendons. Ultrastructural analysis of fibril diameter distributions indicated an altered distribution in aged tendons with an increase of large diameter fibrils. Aged wild type tendons maintained expression of decorin which was associated with the structural and functional changes seen in aged tendons. Aged patellar tendons exhibited altered and generally inferior properties across multiple assays. However, decorin-null tendons exhibited significantly decreased effects of aging compared to the other genotypes. The amelioration of the functional deficits seen in the absence of decorin in aged tendons was associated with altered tendon fibril structure. Fibril diameter distributions in the decorin-null aged tendons were comparable to those observed in the mature wild type tendon with the absence of the subpopulation containing large diameter fibrils. Collectively, our findings provide evidence for age-dependent alterations in tendon architecture and functional activity, and further show that lack of stromal decorin attenuates these changes., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Granulosa cells are the main ovarian source of inhibins, activins and activin-binding protein (follistatin) while germ (oogonia, oocytes) and somatic (theca, granulosa, luteal) cells express activin receptors, signaling components and inhibin co-receptor (betaglycan). Activins are implicated in various intra-ovarian roles including germ cell survival and primordial follicle assembly; follicle growth from preantral to mid-antral stages; suppression of thecal androgen production; promotion of granulosa cell proliferation, FSHR and CYP19A1 expression; enhancement of oocyte developmental competence; retardation of follicle luteinization and/or atresia and involvement in luteolysis. Inhibins (primarily inhibin A) are produced in greatest amounts by preovulatory follicles (and corpus luteum in primates) and suppress FSH secretion through endocrine negative feedback. Together with follistatin, inhibins act locally to oppose auto-/paracrine activin (and BMP) signaling thus modulating many of the above processes. The balance between activin-inhibin shifts during follicle development with activin signalling prevailing at earlier stages but declining as inhibin and betaglycan expression rise., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Low-density lipoprotein (LDL) has recently been shown to be oxidized by iron within the lysosomes of macrophages, and this is a novel potential mechanism for LDL oxidation in atherosclerosis. Our aim was to characterize the chemical and physical changes induced in LDL by iron at lysosomal pH and to investigate the effects of iron chelators and α-tocopherol on this process. LDL was oxidized by iron at pH 4.5 and 37 °C and its oxidation monitored by spectrophotometry and high-performance liquid chromatography. LDL was oxidized effectively by FeSO(4) (5-50 μM) and became highly aggregated at pH 4.5, but not at pH 7.4. The level of cholesteryl esters decreased, and after a pronounced lag, the level of 7-ketocholesterol increased greatly. The total level of hydroperoxides (measured by the triiodide assay) increased up to 24 h and then decreased only slowly. The lipid composition after 12 h at pH 4.5 and 37 °C was similar to that of LDL oxidized by copper at pH 7.4 and 4 °C, i.e., rich in hydroperoxides but low in oxysterols. Previously oxidized LDL aggregated rapidly and spontaneously at pH 4.5, but not at pH 7.4. Ferrous iron was much more effective than ferric iron at oxidizing LDL when added after the oxidation was already underway. The iron chelators diethylenetriaminepentaacetic acid and, to a lesser extent, desferrioxamine inhibited LDL oxidation when added during its initial stages but were unable to prevent aggregation of LDL after it had been partially oxidized. Surprisingly, desferrioxamine increased the rate of LDL modification when added late in the oxidation process. α-Tocopherol enrichment of LDL initially increased the rate of oxidation of LDL but decreased it later. The presence of oxidized and highly aggregated lipid within lysosomes has the potential to perturb the function of these organelles and to promote atherosclerosis.

Valproic acid (VPA) is used widely to treat epilepsy and bipolar disorder. Women undergoing VPA treatment reportedly have an increased incidence of polycystic ovarian syndrome (PCOS)-like symptoms including hyperandrogenism and oligo- or amenorrhoea. To investigate potential direct effects of VPA on ovarian steroidogenesis we used primary bovine theca (TC) and granulosa (GC) cells maintained under conditions that preserve their 'follicular' phenotype. Effects of VPA (7.8-500 µg/ml) on TC were tested with/without LH. Effects of VPA on GC were tested with/without FSH or IGF analogue. VPA reduced (P<0.0001) both basal (70% suppression; IC(50) 67±10 µg/ml) and LH-induced (93% suppression; IC(50) 58±10 µg/ml) androstenedione secretion by TC. VPA reduced CYP17A1 mRNA abundance (>99% decrease; P<0.0001) with lesser effects on LHR, STAR, CYP11A1 and HSD3B1 mRNA (<90% decrease; P<0.05). VPA only reduced TC progesterone secretion induced by the highest (luteinizing) LH dose tested; TC number was unaffected by VPA. At higher concentrations (125-500 µg/ml) VPA inhibited basal, FSH- and IGF-stimulated estradiol secretion (P<0.0001) by GC without affecting progesterone secretion or cell number. VPA reversed FSH-induced upregulation of CYP19A1 and HSD17B1 mRNA abundance (P<0.001). The potent histone deacetylase (HDAC) inhibitors trichostatin A and scriptaid also suppressed TC androstenedione secretion and granulosal cell oestrogen secretion suggesting that the action of VPA reflects its HDAC inhibitory properties. In conclusion, these findings refute the hypothesis that VPA has a direct stimulatory action on TC androgen output. On the contrary, VPA inhibits both LH-dependent androgen production and FSH/IGF-dependent estradiol production in this in vitro bovine model, likely by inhibition of HDAC.

Evidence supports local roles for transforming growth factor β superfamily members including activins and bone morphogenetic proteins (BMP) in follicle development. Access of these ligands to signalling receptors is likely modulated by extracellular binding proteins (BP). In this study, we compared ex vivo expression of four BPs (chordin, gremlin, noggin and follistatin) in granulosal (GC) and theca interna (TC) compartments of developing bovine antral follicles (1-18  mm). Effects of FSH and IGF on BMP and BP expression by cultured GC, and effects of LH and BMPs on BP expression by cultured TC were also examined. Follicular expression of all four BP transcripts was higher in GC than TC compartments (P < 0.001) a finding confirmed by immunohistochemistry. Follicle category affected (P < 0.01) gremlin and follistatin mRNA abundance, with a significant cell-type × follicle category interaction for chordin, follistatin and noggin. Noggin transcript abundance was lower (P < 0.05) in GC of large 'E-active' than 'E-inactive' follicles while follistatin mRNA level was higher (P < 0.01). FSH enhanced CYP19, FSHR, INHBA and follistatin by GC without affecting BMP or BMP-BP expression. IGF increased CYP19 and follistatin, reduced BMP4, noggin and gremlin but did not affect chordin or FSHR mRNA levels. LH increased TC androgen secretion but had no effect on BMP or BP expression. BMPs uniformly suppressed TC androgen production whilst increasing chordin, noggin and gremlin mRNA levels up to 20-fold (P < 0.01). These findings support the hypothesis that extracellular BP, mostly from GC, contribute to the regulation of intrafollicular BMP/activin signalling. Enhancement of thecal BP expression by BMP implies an autoregulatory feedback role to prevent excessive signalling.

The small GTPase RhoB regulates endocytic trafficking of receptor tyrosine kinases (RTKs) and the non-receptor kinases Src and Akt. While receptor-mediated endocytosis is critical for signaling processes driving cell migration, mechanisms that coordinate endocytosis with the propagation of migratory signals remain relatively poorly understood. In this study, we show that RhoB is essential for activation and trafficking of the key migratory effectors Cdc42 and Rac in mediating the ability of platelet-derived growth factor (PDGF) to stimulate cell movement. Stimulation of the PDGF receptor-β on primary vascular smooth muscle cells (VSMCs) results in RhoB-dependent trafficking of endosome-bound Cdc42 from the perinuclear region to the cell periphery, where the RhoGEF Vav2 and Rac are also recruited to drive formation of circular dorsal and peripheral ruffles necessary for cell migration. Our findings identify a novel RhoB-dependent endosomal trafficking pathway that integrates RTK endocytosis with Cdc42/Rac localization and cell movement., (Copyright © 2011 Wiley-Liss, Inc.)

We reported previously that bone morphogenetic proteins (BMPs) potently suppress CYP17 expression and androgen production by bovine theca interna cells (TC) in vitro. In this study, real-time PCR was used to analyse gene expression in TC and granulosa cell (GC) layers from developing bovine antral follicles (1-18 mm). Abundance of mRNA transcripts for four BMPs (BMP2, BMP4, BMP6, and BMP7) and associated type I (BMPR1A, BMPR1B, ACVR1 and ACVR1B) and type II (BMPR2, ACVR2A and ACVR2B) receptors showed relatively modest, though significant, changes during follicle development. BMP2 was selectively expressed in GC, while BMP6, BMP7 and betaglycan (TGFBR3) were more abundant in TC. Abundance of betaglycan mRNA (inhibin co-receptor) in TC increased progressively (fivefold; P<0.001) as follicles grew from 1-2 to 9-10 mm. This suggests a shift in thecal responsiveness to GC-derived inhibin, produced in increasing amounts as follicles achieve dominance. This prompted us to investigate whether inhibin can function as a physiological antagonist of BMP action on bovine TC in vitro, in a manner comparable to that for activin signalling. BMP4, BMP6 and BMP7 abolished LH-induced androstenedione secretion and suppressed CYP17 mRNA >200-fold (P<0.001), while co-treatment with inhibin-A reversed the suppressive action of BMP in each case (P<0.001). Results support a physiological role for granulosa-derived inhibin as an antagonist of BMP action on thecal androgen synthesis. A shift in intrafollicular balance between thecal BMP signalling (inhibitory for androgen synthesis) and betaglycan-dependent inhibin signalling (stimulatory for androgen synthesis) accords with the physiological requirement to deliver an adequate supply of aromatase substrate to GC of developing follicles.

LDL oxidation may be important in atherosclerosis. Extensive oxidation of LDL by copper induces increased uptake by macrophages, but results in decomposition of hydroperoxides, making it more difficult to investigate the effects of hydroperoxides in oxidised LDL on cell function. We describe here a simple method of oxidising LDL by dialysis against copper ions at 4 degrees C, which inhibits the decomposition of hydroperoxides, and allows the production of LDL rich in hydroperoxides (626+/-98 nmol/mg LDL protein) but low in oxysterols (3+/-1 nmol 7-ketocholesterol/mg LDL protein), whilst allowing sufficient modification (2.6+/-0.5 relative electrophoretic mobility) for rapid uptake by macrophages (5.49+/-0.75 microg (125)I-labelled hydroperoxide-rich LDL vs. 0.46+/-0.04 microg protein/mg cell protein in 18 h for native LDL). By dialysing under the same conditions, but at 37 degrees C, the hydroperoxides are decomposed extensively and the LDL becomes rich in oxysterols. This novel method of oxidising LDL with high yield to either a hydroperoxide- or oxysterol-rich form by simply altering the temperature of dialysis may provide a useful tool for determining the effects of these different oxidation products on cell function.