A tutorial on model informed approaches to cardiovascular safety with focus on cardiac repolarisation.

Drugs can affect the cardiovascular (CV) system either as an intended treatment or as an unwanted side effect. In both cases, drug-induced cardiotoxicities such as arrhythmia and unfavourable hemodynamic effects can occur, and be described using mathematical models; such a model informed approach can provide valuable information during drug development and can aid decision-making. However, in order to develop informative models, it is vital to understand CV physiology. The aims of this tutorial are to present (1) key background biological and medical aspects of the CV system, (2) CV electrophysiology, (3) CV safety concepts, (4) practical aspects of development of CV models and (5) regulatory expectations with a focus on using model informed and quantitative approaches to support nonclinical and clinical drug development. In addition, we share several case studies to provide practical information on project strategy (planning, key questions, assumptions setting, and experimental design) and mathematical models development that support decision-making during drug discovery and development.

Measuring the benefits of using market based approaches to provide water and sanitation in humanitarian contexts.

The use of cash transfers and market based programming (CT/MBP) to increase the efficiency and effectiveness of emergency responses is gaining prominence in the humanitarian sector. However, there is a lack of existing indicators and methodologies to monitor activities designed to strengthen water and sanitation (WaSH) markets. Gender and vulnerability markers to measure the impact of such activities on different stakeholders is also missing. This study identifies parameters to monitor, evaluate and determine the added value of utilising CT/MBP to achieve WaSH objectives in humanitarian response. The results of the work revealed that CT/MBP can be used to support household, community and market level interventions to effectively reduce transmission of faeco-oral diseases. Efficiency, effectiveness, sustainability, appropriateness and equity were identified as useful parameters which correlated to widely accepted frameworks against which to evaluate humanitarian action. The parameters were found to be directly applicable to the case of increasing demand and supply of point of use water treatment technology for a) disaster resilience activities, and b) post-crisis response. The need for peer review of the parameters and indicators and pilot measurement in humanitarian contexts was recognised.

Metabolic dysfunction following weight cycling in male mice.

BACKGROUND: Combatting overweight or obesity can lead to large fluctuations in an individual's body weight, often referred to as weight cycling or 'yo-yo' dieting. Current evidence regarding the potentially damaging effects of these changes is conflicting. METHODS: Here, we assess the metabolic effects of weight cycling in a murine model, comprising three dietary switches to normal or high-fat diets at 6 week intervals; male C57BL/6 mice were fed either a control (C) or high-fat (F) diet for 6 weeks (n=140/group). C and F groups were then either maintained on their initial diet (CC and FF, respectively) or switched to a high-fat (CF) or control (FC) diet (n=35/group). For the final 6 week interval, CC and CF groups were returned to the control diet (CCC and CFC groups), while FC and FF groups were placed on a high-fat diet (FCF and FFF) (n=28/group). RESULTS: For the majority of metabolic outcomes changes aligned with dietary switches; however, assessment of neuropeptides and receptors involved in appetite regulation and reward signalling pathways reveal variable patterns of expression. Furthermore, we demonstrate that multiple cycling events leads to a significant increase in internal fat deposition, even when compared with animals maintained on a high-fat diet (internal fat: FCF: 7.4±0.2 g vs FFF: 5.6±0.2 g; P<0.01). CONCLUSIONS: Increased internal adipose tissue is strongly linked to the development of metabolic syndrome associated conditions such as type 2 diabetes, cardiovascular disease and hypertension. Although further work will be required to elucidate the mechanisms underlying the neuronal control of energy homoeostasis, these studies provide a causative link between weight cycling and adverse health.

Reprogramming of hepatic fat accumulation and 'browning' of adipose tissue by the short-chain fatty acid acetate.

BACKGROUND/OBJECTIVES: Short-chain fatty acids, produced by microbiome fermentation of carbohydrates, have been linked to a reduction in appetite, body weight and adiposity. However, determining the contribution of central and peripheral mechanisms to these effects has not been possible. SUBJECTS/METHODS: C57BL/6 mice fed with either normal or high-fat diet were treated with nanoparticle-delivered acetate, and the effects on metabolism were investigated. RESULTS: In the liver, acetate decreased lipid accumulation and improved hepatic function, as well as increasing mitochondrial efficiency. In white adipose tissue, it inhibited lipolysis and induced 'browning', increasing thermogenic capacity that led to a reduction in body adiposity. CONCLUSIONS: This study provides novel insights into the peripheral mechanism of action of acetate, independent of central action, including 'browning' and enhancement of hepatic mitochondrial function.

Comparison of the exposure-response relationship of dapagliflozin in adult and paediatric patients with type 2 diabetes mellitus.

AIMS: To quantitatively compare the exposure-response relationship of dapagliflozin in adult and paediatric patients with type 2 diabetes mellitus (T2DM) and to assess the potential impact of covariate effects. METHODS: Data from three clinical studies of single-dose (2.5, 5 and 10 mg), orally administered dapagliflozin in adult (NCT00162305, NCT00538174) and paediatric (NCT01525238) patients with T2DM were analysed to examine the relationship between dapagliflozin exposure (area under concentration-time curve) and response [24-h urinary glucose excretion (UGE)] using a sigmoidal maximum effect model. Baseline fasting plasma glucose (FPG), estimated glomerular filtration rate (eGFR), baseline 24-h UGE, sex and race were evaluated as covariates. RESULTS: Data from 63 predominantly white or Asian (92.4%) adult and 20 paediatric (45.8% white; 45.8% black) patients were included. The model appeared robust, with predictions fitting well with observed data. Baseline eGFR, FPG and sex were significant covariates in both populations; race was a significant covariate in the paediatric population only. Model-predicted UGE response was higher in paediatric (47.4, 67.5 and 85.9 g/24 h for 2.5, 5 and 10 mg) than in adult (31.2, 43.5 and 54.3 g/24 h for 2.5, 5 and 10 mg) patients, which may be associated with the higher eGFR values in paediatric patients. CONCLUSIONS: After a single oral dose of dapagliflozin, adult and paediatric patients with T2DM had similar exposure-response relationships after accounting for significant covariates. These results support the planned dosage strategy for a phase III dapagliflozin safety and efficacy study in paediatric patients with T2DM, for whom treatment options are currently limited.

Hygienic food to reduce pathogen risk to bumblebees.

Bumblebees are ecologically and economically important pollinators, and the value of bumblebees for crop pollination has led to the commercial production and exportation/importation of colonies on a global scale. Commercially produced bumblebee colonies can carry with them infectious parasites, which can both reduce the health of the colonies and spillover to wild bees, with potentially serious consequences. The presence of parasites in commercially produced bumblebee colonies is in part because colonies are reared on pollen collected from honey bees, which often contains a diversity of microbial parasites. In response to this threat, part of the industry has started to irradiate pollen used for bumblebee rearing. However, to date there is limited data published on the efficacy of this treatment. Here we examine the effect of gamma irradiation and an experimental ozone treatment on the presence and viability of parasites in honey bee pollen. While untreated pollen contained numerous viable parasites, we find that gamma irradiation reduced the viability of parasites in pollen, but did not eliminate parasites entirely. Ozone treatment appeared to be less effective than gamma irradiation, while an artificial pollen substitute was, as expected, entirely free of parasites. The results suggest that the irradiation of pollen before using it to rear bumblebee colonies is a sensible method which will help reduce the incidence of parasite infections in commercially produced bumblebee colonies, but that further optimisation, or the use of a nutritionally equivalent artificial pollen substitute, may be needed to fully eliminate this route of disease entry into factories.

Explaining trends in alcohol-related harms in Scotland 1991-2011 (II): policy, social norms, the alcohol market, clinical changes and a synthesis.

OBJECTIVE: To provide a basis for evaluating post-2007 alcohol policy in Scotland, this paper tests the extent to which pre-2007 policy, the alcohol market, culture or clinical changes might explain differences in the magnitude and trends in alcohol-related mortality outcomes in Scotland compared to England & Wales (E&W). STUDY DESIGN: Rapid literature reviews, descriptive analysis of routine data and narrative synthesis. METHODS: We assessed the impact of pre-2007 Scottish policy and policy in the comparison areas in relation to the literature on effective alcohol policy. Rapid literature reviews were conducted to assess cultural changes and the potential role of substitution effects between alcohol and illicit drugs. The availability of alcohol was assessed by examining the trends in the number of alcohol outlets over time. The impact of clinical changes was assessed in consultation with key informants. The impact of all the identified factors were then summarised and synthesised narratively. RESULTS: The companion paper showed that part of the rise and fall in alcohol-related mortality in Scotland, and part of the differing trend to E&W, were predicted by a model linking income trends and alcohol-related mortality. Lagged effects from historical deindustrialisation and socio-economic changes exposures also remain plausible from the available data. This paper shows that policy differences or changes prior to 2007 are unlikely to have been important in explaining the trends. There is some evidence that aspects of alcohol culture in Scotland may be different (more concentrated and home drinking) but it seems unlikely that this has been an important driver of the trends or the differences with E&W other than through interaction with changing incomes and lagged socio-economic effects. Substitution effects with illicit drugs and clinical changes are unlikely to have substantially changed alcohol-related harms: however, the increase in alcohol availability across the UK is likely to partly explain the rise in alcohol-related mortality during the 1990s. CONCLUSIONS: Future policy should ensure that alcohol affordability and availability, as well as socio-economic inequality, are reduced, in order to maintain downward trends in alcohol-related mortality in Scotland.

Explaining trends in alcohol-related harms in Scotland, 1991-2011 (I): the role of incomes, effects of socio-economic and political adversity and demographic change.

OBJECTIVE: This paper tests the extent to which differing trends in income, demographic change and the consequences of an earlier period of social, economic and political change might explain differences in the magnitude and trends in alcohol-related mortality between 1991 and 2011 in Scotland compared to England & Wales (E&W). STUDY DESIGN: Comparative time trend analyses and arithmetic modelling. METHODS: Three approaches were utilised to compare Scotland with E&W: 1. We modelled the impact of changes in income on alcohol-related deaths between 1991-2001 and 2001-2011 by applying plausible assumptions of the effect size through an arithmetic model. 2. We used contour plots, graphical exploration of age-period-cohort interactions and calculation of Intrinsic Estimator coefficients to investigate the effect of earlier exposure to social, economic and political adversity on alcohol-related mortality. 3. We recalculated the trends in alcohol-related deaths using the white population only to make a crude approximation of the maximal impact of changes in ethnic diversity. RESULTS: Real incomes increased during the 1990s but declined from around 2004 in the poorest 30% of the population of Great Britain. The decline in incomes for the poorest decile, the proportion of the population in the most deprived decile, and the inequality in alcohol-related deaths, were all greater in Scotland than in E&W. The model predicted less of the observed rise in Scotland (18% of the rise in men and 29% of the rise in women) than that in E&W (where 60% and 68% of the rise in men and women respectively was explained). One-third of the decline observed in alcohol-related mortality in Scottish men between 2001 and 2011 was predicted by the model, and the model was broadly consistent with the observed trends in E&W and amongst women in Scotland. An age-period interaction in alcohol-related mortality was evident for men and women during the 1990s and 2000s who were aged 40-70 years and who experienced rapidly increasing alcohol-related mortality rates. Ethnicity is unlikely to be important in explaining the trends or differences between Scotland and E&W. CONCLUSIONS: The decline in alcohol-related mortality in Scotland since the early 2000s and the differing trend to E&W were partly described by a model predicting the impact of declining incomes. Lagged effects from historical social, economic and political change remain plausible from the available data.

Sexual dimorphism in relation to adipose tissue and intrahepatocellular lipid deposition in early infancy.

Sexual dimorphism in adiposity is well described in adults, but the age at which differences first manifest is uncertain. Using a prospective cohort, we describe longitudinal changes in directly measured adiposity and intrahepatocellular lipid (IHCL) in relation to sex in healthy term infants. At median ages of 13 and 63 days, infants underwent quantification of adipose tissue depots by whole-body magnetic resonance imaging and measurement of IHCL by in vivo proton magnetic resonance spectroscopy. Longitudinal data were obtained from 70 infants (40 boys and 30 girls). In the neonatal period girls are more adipose in relation to body size than boys. At follow-up (median age 63 days), girls remained significantly more adipose. The greater relative adiposity that characterises girls is explained by more subcutaneous adipose tissue and this becomes increasingly apparent by follow-up. No significant sex differences were seen in IHCL. Sex-specific differences in infant adipose tissue distribution are in keeping with those described in later life, and suggest that sexual dimorphism in adiposity is established in early infancy.

The effects of different feeding strategies providing different levels of vitamin A on animal performance, carcass traits, and the conversion rate of subcutaneous fat color in cull-cows.

Cull cows represent a significant percentage of revenue received from the U.S. beef industry; however, cull cows are heavily price discounted at time of slaughter. This experiment's objective is to evaluate different feeding strategies and their effects on body condition score, subcutaneous fat color, and carcass yield and quality traits in cull cows. The central hypothesis is feeding a high-energy diet, with low levels of vitamin A, for 56 d will improve animal performance, carcass yield, and quality traits in addition to capturing the point (rate) of the conversion of yellow to white subcutaneous fat. In the present experiment 98 Angus crossbreed cows were utilized. Cows were fed either low vitamin A (LVA) diet consisting of whole shelled corn, soybean hulls, soybean meal, and a mineral-vitamin supplement or high vitamin A (HVA) diet, formulated using whole shelled corn, fescue hay, dry distiller grains with soluble, and a mineral-vitamin supplement for 56 d. During the 56 d feeding period, body weights and condition scores, and subcutaneous adipose samples were collected every 14 d. On day 56, cattle were slaughtered; 48 h postmortem carcass characteristics and objective color scores (subcutaneous adipose tissue) were recorded and a sample of the longissimus dorsi lumborum was collected. Subcutaneous adipose tissue samples were utilized to record subjective color scores and then ground to be analyzed for ß-carotene concentration. The longissimus dorsi lumborum samples (2.54 cm slices) were removed for Warner-Bratzler shear force (WBSF) and pH testing. Data were analyzed using the MIXED procedure of SAS. Feeding cull cows LVA resulted in differences in subcutaneous carcass fat color (P = 0.01) as well as b* values (P < 0.01) on day 56 compared with HVA. Subjective fat color scores were not different (P > 0.10) on day 0 or 14 but were different (P ≤ 0.05) on days 28, 42, and 56. Additionally, 9-cis-ß-carotene concentration on day 56 were different (P = 0.05) between treatments. A trend was noticed for all-trans-ß-carotene concentration (P = 0.10) on day 56 as well. Cull cow body weights were greater (P ≤ 0.04) when fed the LVA diet starting on days 14, 28, and 42; and a trend was noticed on day 56 (P = 0.09). Overall, cows fed the LVA treatment for 56 d exhibited decreased adipose yellowness and ß-carotene concentrations as well as increased live weights.

Involvement of proteoglycans in tendinopathy.

A major feature of chronic tendinopathy is a change in the nature and organisation of the extracellular matrix of tendon. Increased levels of proteoglycans have been shown in the extracellular matrix of tendinopathic tendons and these appear to influence the increased hydration and swelling of the tissue that is a feature of this condition. There is a paucity of knowledge about proteoglycans in normal and tendinopathic tendons. This review sets out to describe the nature, function and metabolism of proteoglycans present in normal tendon and in tendinopathy and outlines how changes in proteoglycan metabolism may contribute to the development and progression of this disease.

Excessive appetitive arousal in Prader-Willi syndrome.

This study focused on genetic and behavioural aspects of one important component of the motivation to eat - how appetitive arousal is elicited through the presentation of food-associated stimuli. Individuals with Prader-Willi syndrome, a genetic disorder associated with hyperphagia, and control participants completed a computerised response task in the presence of motivational stimuli. In controls, appetitive arousal was specific to particular stimuli. In contrast, individuals with PWS showed a non-specific pattern of arousal. Over-activation of the anticipatory motivation system may be one consequence of the genetic disorder in PWS.

Hypofractionated adjuvant surgical cavity radiotherapy following resection of limited brain metastasis.

BACKGROUND: Following surgical resection of oligometastatic disease to the brain there is a high rate of local relapse which is reduced by the addition of focal radiation therapy, often delivered as single fraction stereotactic radiosurgery (SRS) to the surgical cavity. This study audited the outcomes of an alternative approach using hypofractionated radiation therapy (HFRT) to the surgical resection cavity. METHODS AND MATERIALS: Seventy-nine patients who received surgical resection and focal radiation therapy to the surgical cavity using HFRT with intensity modulated radiation therapy with or without stereotactic radiotherapy were identified. Doses were delivered in five fractions every second day for 10 days. Follow-up involved MRI surveillance with three-monthly MRI scans post resection. The major endpoints were local control at the surgical cavity site, and presence of radiation necrosis at the treated site. RESULTS: Seventy-nine patients were included for the analysis with a median follow-up of 10.8 months. Of the cohort, 56% experienced intracranial progression, with all patients progressing distant to the resection cavity, and 7% progressing locally in addition. The one-year local control rate was 89.8%. The median progression-free survival was 10.0 months and median overall survival was 14.3 months. There was one CTCAE grade 3 toxicity of symptomatic radiation necrosis with no grade 4-5 toxicities seen. CONCLUSIONS: The rate of local relapse following HFRT to the surgical cavity is low with minimal risk of radiation necrosis. HFRT can be considered as an alternative to SRS for focal radiotherapy after brain metastasis resection.

Coordinated changes in energy intake and expenditure following hypothalamic administration of neuropeptides involved in energy balance.

OBJECTIVE: The hypothalamic control of energy balance is regulated by a complex network of neuropeptide-releasing neurons. Although the effect of these neuropeptides on individual aspects of energy homoeostasis has been studied, the coordinated response of these effects has not been comprehensively investigated. We have simultaneously monitored a number of metabolic parameters following intracerebroventricular (ICV) administration of 1 and 3 nmol of neuropeptides with established roles in the regulation of feeding, activity and metabolism. Ad libitum- fed rats received the orexigenic neuropeptides neuropeptide Y (NPY), agouti-related protein (AgRP), melanin-concentrating hormone (MCH) or orexin-A. Overnight-food-deprived rats received an ICV injection of the anorectic peptides alpha-melanocyte-stimulating hormone (MSH), corticotrophin-releasing factor (CRF) or neuromedin U (NMU). RESULTS: Our results reveal the temporal sequence of the effects of these neuropeptides on both energy intake and expenditure, highlighting key differences in their function as mediators of energy balance. NPY and AgRP increased feeding and decreased oxygen consumption, with the effects of AgRP being more prolonged. In contrast, orexin-A increased both feeding and oxygen consumption, consistent with an observed increase in activity. The potent anorexigenic effects of CRF were accompanied by a prolonged increase in activity, whereas NMU injection resulted in significant but short-lasting inhibition of food intake, ambulatory activity and oxygen consumption. alpha-MSH injection resulted in significant increases in both ambulatory activity and oxygen consumption, and reduced food intake following administration of 3 nmol of the peptide. CONCLUSION: We have for the first time, simultaneously measured several metabolic parameters following hypothalamic administration of a number of neuropeptides within the same experimental system. This work has shown the interrelated effects of these neuropeotides on activity, energy expenditure and food intake, thus facilitating comparison between the different hypothalamic systems.

Bacterial motility: membrane topology of the Escherichia coli MotB protein.

The MotB protein of Escherichia coli is an essential component of the force generators that couple proton movement across the cytoplasmic membrane to rotation of the flagellar motors. The membrane topology of MotB was examined to explore the possibility that it might form a proton channel. MotB--alkaline phosphatase fusion proteins were constructed to identify likely periplasmic domains of the MotB molecule. Fusions distal to a putative membrane-spanning segment near the amino terminus of MotB exhibited alkaline phosphatase activity, indicating that an extensive carboxyl-terminal portion of MotB may be located on the periplasmic side of the membrane. Protease treatment of MotB in spheroplasts confirmed this view. The simple transmembrane organization of MotB is difficult to reconcile with a role as a proton conductor.

Evaluating drug toxicity signals: is a hierarchical classification of evidence useful or a hindrance?

PURPOSE: Only randomized clinical trials (RCTs) are deemed suitable to determine causality, as epidemiological studies are considered not 'robust'. The objective of this study was to evaluate whether causality should remain the only consideration with drug toxicity signals. Cyclooxygenase-2 (Cox-2) inhibitors provided an example. METHODS: Our study population included patients aged 40+ years prescribed Cox-2 inhibitors in the General Practice Research Database (GPRD) (N = 150 000). We estimated the risks of upper gastrointestinal (GI) events and myocardial infarction (MI). Attributable risks were estimated using simulation methodology based on various hypothetical scenarios. RESULTS: The risk-benefit profile was strongly related to the rate of GI events. With a RCT incidence, the GI benefits would exceed MI risks. With a 'real-life' GI incidence, the benefits did not exceed the risks substantially. The onset and offset of drug effects also predicted the magnitude of both risks and benefits. If risks and benefits occurred in different sub-groups, the risk-benefit profile varied substantially. Also, it was found that any restriction of use to patients at high risk of drug toxicity may not improve the risk-benefit profile when this restriction affected patients who would benefit most. CONCLUSIONS: We conclude that rigid classification of evidence is not appropriate in the monitoring of risks and benefits and all valid study evidence--not only that derived from a RCT--needs to be included. The first priority should be to consider the potential impact of a drug toxicity signal.

Increased versican content is associated with tendinosis pathology in the patellar tendon of athletes with jumper's knee.

Expansion of the extracellular matrix is a prominent but poorly characterized feature of tendinosis. The present study aimed to characterize the extent and distribution of the large aggregating proteoglycan versican in patients with patellar tendinosis. We obtained tendon from tendinopathy patients undergoing debridement of the patellar tendon and from controls undergoing intramedullary tibial nailing. Versican content was investigated by Western blotting and immunohistochemistry. Microvessel thickness and density were determined using computer-assisted image analysis. Markers for smooth muscle actin, endothelial cells (CD31) and proliferating cells (Ki67) were examined immunohistochemically. Western blot analysis and immunohistochemical staining revealed elevated versican content in the proximal patellar tendon of tendinosis patients (P=0.042). Versican content was enriched in regions of fibrocartilage metaplasia and fibroblast proliferation, as well as in the perivascular matrix of proliferating microvessels and within the media and intima of arterioles. Microvessel density was higher in tendinosis tissue compared with control tissue. Versican deposition is a prominent feature of patellar tendinosis. Because this molecule is not only a component of normal fibrocartilagenous matrices but also implicated in a variety of soft tissue pathologies, future studies should further detail both pathological and adaptive roles of versican in tendons.

Gut microbiota modulates type I interferon and antibody-mediated immune responses in chickens infected with influenza virus subtype H9N2.

Commensal gut microbes play a critical role in shaping host defences against pathogens, including influenza viruses. The current study was conducted to assess the role and mechanisms of action of commensal gut microbiota on the innate and antibody-mediated responses of layer chickens against influenza virus subtype H9N2. A total of 104 one-day-old specific pathogen free chickens were assigned to either of the four treatments, which included two levels of antibiotics treatment (ABX- and ABX+) and two levels of H9N2 virus infection (H9N2- and H9N2+). At day 17 of age, chickens in the H9N2+ group were infected via the oral-nasal route with 400 µl of 107 TCID50/ml (200 µl/each route). Oropharyngeal and cloacal swabs at days 1, 3, 5, 7 and 9 post-infection (p.i.) for virus shedding, tissue samples at 12 h, 24 h and 36 h p.i. for mRNA measurement, and serum samples at days 7 and 14 p.i. for hemagglutination inhibition (HI) assay and IgG antibodies were collected. Virus shedding analysis showed that antibiotic treated (depleted)-H9N2 virus infected chickens showed a significantly higher oropharyngeal virus shedding at all time points, and cloacal shedding at days 3 and 5 p.i. compared to control treated (undepleted)-H9N2 infected chickens. Analysis of mRNA expression showed that infection of depleted chickens with H9N2 virus resulted in significantly down-regulated type I interferon responses both in the respiratory and gastrointestinal tracts compared to undepleted-H9N2 infected chickens. However, antibody-mediated immune response analysis showed a significantly higher HI antibody titre and IgG levels in the serum of chickens depleted with antibiotics and infected with H9N2 virus compared to undepleted-H9N2 infected chickens. In conclusion, findings from the current study suggest that the gut microbiota of chickens plays an important role in the initiation of innate responses against influenza virus infection, while the antibody-mediated immune response remains unaffected.

Genetic analysis of the bacterial flagellum.

Escherichia coli and Salmonella typhimurium invest considerable resources in making flagella, motor organelles that function much like the propellers on a ship. Both classical and molecular genetic studies have begun to reveal how flagellar genes are regulated and how their products build and operate these remarkable devices.

Identification of the bud emergence gene BEM4 and its interactions with rho-type GTPases in Saccharomyces cerevisiae.

The Rho-type GTPase Cdc42p is required for cell polarization and bud emergence in Saccharomyces cerevisiae. To identify genes whose functions are linked to CDC42, we screened for (i) multicopy suppressors of a Ts- cdc42 mutant, (ii) mutants that require multiple copies of CDC42 for survival, and (iii) mutations that display synthetic lethality with a partial-loss-of-function allele of CDC24, which encodes a guanine nucleotide exchange factor for Cdc42p. In all three screens, we identified a new gene, BEM4. Cells from which BEM4 was deleted were inviable at 37 degrees C. These cells became unbudded, large, and round, consistent with a model in which Bem4p acts together with Cdc42p in polarity establishment and bud emergence. In some strains, the ability of CDC42 to serve as a multicopy suppressor of the Ts- growth defect of deltabem4 cells required co-overexpression of Rho1p, which is an essential Rho-type GTPase necessary for cell wall integrity. This finding suggests that Bem4p also affects Rho1p function. Bem4p displayed two-hybrid interactions with Cdc42p, Rho1p, and two of the three other known yeast Rho-type GTPases, suggesting that Bem4p can interact with multiple Rho-type GTPases. Models for the role of Bem4p include that it serves as a chaperone or modulates the interaction of these GTPases with one or more of their targets or regulators.