Voluntary medical male circumcision (VMMC) reduces HIV acquisition among heterosexual men by approximately 60%. VMMC is a surgical procedure and some adverse events (AEs) are expected. Swaziland’s Ministry of Health established a toll-free hotline to provide general information about VMMC and to manage post-operative clinical AEs through telephone triage.We retrospectively analyzed a dataset of telephone calls logged by the VMMC hotline during a VMMC campaign. The objectives were to determine reasons clients called the VMMC hotline and to ascertain the accuracy of telephone-based triage for VMMC AEs. We then analyzed VMMC service delivery data that included date of surgery, AE type and severity, as diagnosed by a VMMC clinician as part of routine post-operative follow-up. Both datasets were de-identified and did not contain any personal identifiers. Proportions of AEs were calculated from the call data and from VMMC service delivery data recorded by health facilities. Sensitivity analyses were performed to assess the accuracy of phone-based triage compared to clinically confirmed AEs.A total of 17,059 calls were registered by the triage nurses from April to December 2011. Calls requesting VMMC education and counseling totaled 12,492 (73.2%) and were most common. Triage nurses diagnosed 384 clients with 420 (2.5%) AEs. According to the predefined clinical algorithms, all moderate and severe AEs (153) diagnosed through telephone-triage were referred for clinical management at a health facility. Clinicians at the VMMC sites diagnosed 341 (4.1%) total clients as having a mild (46.0%), moderate (47.8%), or severe (6.2%) AE. Eighty-nine (26%) of the 341 clients who were diagnosed with AEs by clinicians at a VMMC site had initially called the VMMC hotline. The telephone-based triage system had a sensitivity of 69%, a positive predictive value of 83%, and a negative predictive value of 48% for screening moderate or severe AEs of all the AEs.The use of a telephone-based triage system may be an appropriate first step to identify life-threatening and urgent complications following VMMC surgery. More than 40 observational studies and three randomized controlled trials have demonstrated that voluntary medical male circumcision (VMMC) reduces HIV acquisition among heterosexual men by approximately 60%–4. ExtenMale circumcision is a minor surgical procedure that involves risk. Some adverse events (AEs) are expected, even when surgery is conducted in sanitary settings by trained clinicians. Several factors can minimize complications following VMMC, including client assessment and history-taking; screening for sexually transmitted infections (STIs), genital anomalies, and bleeding disorders; use of skilled VMMC providers; correct post-operative client instruction and care; client adherence to post-operative instructions; and appropriate monitoring, assessment, and management of AEs. All complications must be closely monitored to ensure that clients receive appropriate post-operative clinical management. Timely identification, proper triage and management, and accurate reporting of AEs following circumcision are critical to maintaining high-quality, safe VMMC service delivery.AE classifications are generally defined as intra-operative if they occur during surgery or before discharge from the clinic, or post-operative if they occur following discharge from the clinic. AEs are also classified by severity . Mild AEs are generally understood to be part of routine surgery and are easily managed . Moderate and severe AEs may require further treatment, such as antibiotics, or re-exploration of the surgical site. Definitions of AE types associated with VMMC vary slightly by country but generally include bleeding, infection, pain, swelling (hematoma), reaction to local anesthesia, wound disruption, damage to penis, sexual or erectile dysfunction, scarring, torsion of penis, excessive or insufficient skin removed, and difficulty urinating. Both intra- and post-operative AEs related to VMMC surgery are uncommon–12.Data on AEs following elective adolescent and adult circumcision are limited. In the three randomized controlled trials on VMMC , the mean combined rate for all AEs was 5.0%, although adherence to post-operative instructions for wound care may have been better in trial settings than in routine service delivery, 13, 14.Swaziland has the highest HIV prevalence in the world, at 31%, and prevalence is highest among females aged 15–25 years and males aged 25–33 years. The HIVSoka Uncobe ("circumcise and conquer" in siSwati), and it aimed to provide VMMC surgery to approximately 150,000 adolescent and adult males aged 15–49 years. The campaign used the country’s existing VMMC sites and established several temporary sites to expand service delivery sites throughout the country.In hopes of controlling its HIV epidemic and reducing new infections, Swaziland planned an ambitious national VMMC campaign to be conducted in 2011 that would condense its five-year strategy into a single year. The campaign was called Given the expected surge of VMMC clients during the campaign, and the corresponding increase in the number of AEs, Swaziland’s Ministry of Health implemented a telephone-based VMMC hotline. The toll-free hotline served two purposes: to give Swazi citizens the opportunity to ask trained nurses general questions about VMMC, and to triage post-operative AEs. The hotline utilized staff nurses from Swaziland’s Emergency Preparedness and Response (EPR) department who routinely handled a variety of disasters and emergencies by telephone.In the VMMC clinics, all clients were given an appointment card that contained the date and location of their next follow-up visit. This card also contained the VMMC hotline number that they could call if they needed information, advice, or assistance regarding post-operative healing and management of any AEs. The hotline number was also included on advertisements and posters and during radio spots promoting the VMMC campaign throughout the country. Prospective clients and others interested in VMMC were recommended to call the hotline with questions about VMMC. The objectives of this analysis were to describe the reasons VMMC clients called the VMMC hotline and to assess the accuracy of telephone-based triage for VMMC in differentiating among mild, moderate, and severe AEs.Soka Uncobe campaign, the EPR was staffed with five trained nurses who provided 24-hour coverage on a rotating basis.The nurses who provided telephone triage for VMMC completed a full course of competency-based clinical training on male circumcision under local anesthesia, which is the same training required of surgical nurses in VMMC facilities. Triage nurses were trained to assess VMMC clients’ concerns without the advantage of visual inspection or face-to-face interaction, by using comprehensive algorithms of AEs developed by a team of experienced VMMC-provider clinicians in Swaziland. They were required to rely on their communication skills and knowledge of the VMMC procedure, routine post-operative healing stages, and common post-operative AEs in order to accurately ascertain the client’s condition and needs. The function of the telephone triage nurse was to determine whether the caller had a VMMC-related complaint or sought information about VMMC. For those seeking information, the triage nurses provided general VMMC-related information and education VMMC service delivery in Swaziland. For those calling with suspected AEs, the triage nurses would use a series of algorithms to determine the cause and severity of the complaint. The triage nurse would then direct the caller to the appropriate emergency services, if necessary, and/or recommend medical follow-up based on established triage protocols Figure . During Treatment algorithms were created for four common AEs that have the potential to become severe: bleeding, voiding difficulty, pain, and infection. Triage nurses utilized the algorithms to guide their telephone-based clinical decision-making Figures ,3 and4Soka Uncobe VMMC campaign. The call data included purpose of the call, AE type and severity for those experiencing complications, and algorithm used for telephone triage. We analyzed service delivery data at VMMC sites/clinics that included VMMC date and AE type and severity, as confirmed by a VMMC clinician. Both datasets were de-identified and did not contain any personal identifiers. Proportions of AEs were calculated from the VMMC hotline call data and from service delivery data recorded by the VMMC health facilities. Sensitivity and specificity analyses were performed to assess the validity of phone-based triage compared to clinically confirmed moderate and severe AEs for those clients with one or more AEs and were assessed by both the triage nurses and the clinicians at VMMC clinics.We retrospectively reviewed a dataset of telephone calls logged by the triage nurses during the Design and implementation of a complete set of tools and procedures to monitor and evaluate a national scale up of MC for Swaziland" 2011-2021.A non-research determination and approval for secondary data analysis was provided by the Ministry of Health of the Kingdom of Swaziland and U.S. Centers for Disease Control and Prevention IRB for the scale-up project entitled: "Soka Uncobe campaign, call data were gathered from April 13, 2011, to December 29, 2011. A total of 17,059 calls were registered by the triage nurses. Calls requesting VMMC education and counseling were most common, totaling 12,492 (73.2%). Five hundred (2.9%) of the calls received were complaints that the triage nurses managed by phone  returned for routine post-operative care within seven days, as recommended. A total of 341 clients had a mild, moderate, or severe AE . The most common AEs were infection (184), swelling (44), and bleeding (39). Most AEs were mild  or moderate , while severe AEs were least frequent , resulting in a post-operative moderate and severe AE rate of 2.2%  among those who returned for follow-up at a VMMC site during the campaign  had initially called the VMMC hotline with complaints following their VMMC. Fifty-two 58.4%) were referred by the triage nurses to a VMMC site as the telephone triage indicated that they had a moderate or severe AE. The triage nurses determined that 37clients (41.6%) did not have a moderate or severe AE according to the algorithms, but these clients went to clinic without referral or were referred to reassure them. Of the 52 clients who called the VMMC hotline and were diagnosed with a moderate or severe AE through telephone triage, 43 clients (82.7%) were confirmed to have a moderate or severe AE by VMMC site clinicians, and 9 clients (17.3%) were clinically diagnosed to not have a moderate or severe AE. The AE algorithms used by the triage nurses therefore had a sensitivity of 69%, a positive predictive value of 83%, and a negative predictive value of 48% .


Left-sided pulmonary artery agenesis is a rare malformation that commonly requires childhood intervention secondary to associated congenital cardiovascular anomalies. We present an uncommon case of left-sided agenesis with an associated right-sided aortic arch and significant hypoplasia of the ipsilateral lung. Additionally, there is radiographic evidence of emphysema and pulmonary artery hypertension. Pulmonary artery agenesis is not a common entity, but should be considered in adult patients presenting with recurrent pneumonias and radiographic evidence suggestive of pulmonary hypoplasia. A prompt diagnosis is beneficial for affected individuals who may be candidates for a revascularization procedure or embolization of collaterals. Earlier diagnosis also allows for proper management and follow-up care, considering pulmonary artery hypertension is a severe complication of pulmonary artery agenesis. Unilateral absence of pulmonary artery (UAPA) is a rare congenital malformation that may develop in isolation or in association with congenital cardiovascular anomalies -4. In caUAPA is believed to result from the involution of the proximal sixth aortic arch, thereby forgoing expected embryological fusion with the pulmonary trunk ,4. Most A 69-year-old female with a greater than 100-pack-year smoking history and known emphysema presented to a pulmonologist for worsening shortness of breath. The patient reported a remote history of pneuomonia, increased shortness of breath with mild physical activity, audible wheezing, and chest tightness. She denied lower extremity swelling, paroxysmal nocturnal dyspnea, or palpitations. There was no history of cardiac disease or cardiac surgery.The patient underwent initial chest imaging at an outside facility and the imaging was not available at the time of presentation at our institution. A chest radiograph revealed hyperinflation of the right lung with leftward shift of the mediastinum. The interpreting radiologist noted left lower lobe volume loss and recommended further evaluation to exclude an obstructing endobronchial mass. Subsequent contrast-enhanced computed tomography (CT) of the chest Figures , Video 1Left-sided agenesis is a rare entity that occurs half as frequently as right-sided agenesis. The development of left-sided agenesis has been described in two different subtypes . ApproxiThe typical radiographic findings include: absence of an ipsilateral pulmonary artery shadow with enlargement of the contralateral shadow, volume loss of the affected ipsilateral lung and ipsilateral mediastinal shift with hyperinflation of the contralateral lung. The findings are nonspecific in an adult patient, but may represent bronchial obstruction from mucus plugging or endobronchial malignancy, prior lobectomy, or congenital pulmonary hypoplasia.This unique case illustrates a rather uncommon adult cause of mediastinal shift with volume loss of the ipsilateral lung. While there are many causes for lung volume loss on chest radiography, malignancy causing endobronchial obstruction must be excluded in an older patient with a smoking history. In this case, the follow-up contrast enhanced CT of the chest revealed a diagnosis of UAPA, an associated vascular anomaly, development of centrilobular emphysema, and pulmonary artery hypertension.The treatment for UAPA is patient specific and will vary depending on age of presentation, associated cardiovascular anomalies, and symptomatology. The options in young patients include revascularization of the distal affected pulmonary artery. The procedure results in a systemic-to-pulmonary artery shunt, considering intrapulmonary arteries are still formed during embryological development ,5. LobecLeft-sided pulmonary artery agenesis is a rare malformation that commonly requires childhood intervention secondary to associated congenital cardiovascular anomalies. Our patient presented and was diagnosed with a known variant, left-sided agenesis with a right-sided aortic arch, which remained undiagnosed until adulthood. Reaching adulthood in this population is uncommon and initial radiographic findings may mimic malignancy. Most importantly, correct diagnosis is essential for appropriate clinical management and follow-up care, considering pulmonary artery hypertension remains a serious complication.


Objective. Bilateral internal iliac artery aneurysms constitute the utmost configuration of infrarenal aortoiliac disease. We detail characteristic aortoiliac disease patterns and reconstructive techniques we have used, along with a visualized decision-making chart and a short review of the literature. Material and Methods. A retrospective, observational study of twelve clinical cases of patients with aortoiliac disease are described. Two patients had a common iliac artery aneurysm and were managed by the application of inversed stent-grafts; another case was repaired by the insertion of a standard bifurcated stent-graft flared in the right common iliac artery and with an iliac branched device in the left iliac arterial axis. Open approach was used in 5 cases and in 4 cases a combination of aortouniliac stent-grafting with femoral-femoral bypass was applied. Results. Technical success was 100%. One endoleak type Ib in a flared iliac limb was observed and corrected by internal iliac embolism and use of an iliac limb stent-graft extension. We report 100% patency rate during 26.3 months of followup. Conclusion. Individualized techniques for the management of isolated iliac or aortoiliac aneurismal desease with special concern in maintaining internal iliac artery perfusion lead to elimination of perioperative complications and long-term durability and patency rates. Iliac artery aneurysms (IAAs) participate in various aortoiliac aneurismal (AIA) patterns that frequently necessitate sophisticated reconstructions. An IAA represents a 50% arterial diameter increase, compared to normal; in absolute terms, common iliac arteries (CIAs) are considered aneurismal with diameter > 18.5 mm for men  and >15 mm for women ; internal iliac artery's (IIA) diameter, in both genders, is 0.54 ± 0.15 cm ; isolateIn this paper we present complex aortoiliac aneurismal patterns focusing on technical details and visualize solutions performed by us and reported in the literature.Twelve characteristic cases of patients with aortoiliac disease treated in our department (except from the last case) by the same surgeon, between 2004 and 2010, are presented , having  1st case the patient had a large isolated left CIAA; one inverted stent-graft and a second extension  stent-graft were used; the inverted graft method has been described in previous report [In thes report  , the 8 mm graft was anastomosed with distal right IIA and reimplanted to the Y-graft  with AAA and left CIA occlusion was repaired with a right aortouniliac graft (A complex case (ac graft  followed7th case) with the migrated stent-graft following EVAR and highly angulated graft limbs, a right aortouniliac graft was deployed followed by FFBP; both IIAs were preserved (In the patient (reserved .8th case) with AAA and bilateral CIAAs, we used a bifurcated aortic stent-graft with a flared right leg and a left iliac branched device (IBD) (In the woman (ce (IBD) . The folce (IBD) . Embolisce (IBD) . 9th case of the woman with the dissecting AAA and the right renal and right CIA malperfusion , following distal aortic ligation at bifurcation, left IIA was antegrade reperfused via left CIA, as proximal left EIA was reimplanted to the aortobifemoral graft left limb (In theeft limb .12th case) with bilateral CIAAs and right IIAA, a combination of left aortouniliac stent-graft, left IIA embolization, reversed U-stenting of the right EIA to distal IIA, and a FFBP was performed; this case has been detailed in a previous report [Finally, in the patient . There was no further secondary operation for these patients.Perioperative and hospital mortality was nil. No patient suffered from renal impairment. Mean ICU stay was 2 days. During a mean follow-up (FU) of 26.3 months  one patient suffered a fatal cardiac arrest at 3rd postoperative year; patency rate of all grafts was 100%. In one patient, an incomplete right leg ischemia was present on 1st postoperative day, which was assisted by a femoral-femoral bypass; in another patient, a CFAA was noticed in the FU CT almost 3 years postoperatively, and another presented postoperative uncomplicated left pelvic pain and an abdominal hernia. One endoleak type 1b was observed due to fixation loss of the flared stent-graft's right limb  Y-graft with distal anastomoses or reimplantation of healthy iliac arteries' segments ; howeverThe IIA reimplantation to the graft is commonly used; Millite et al.  reported 2nd case, The exception for the rule, concerning open repair, is the isolated CIIA, like in the first two cases of patients we present, where anatomy  allowed minimal invasive pure endovascular repair; even if stent-grafting fails, secondary operations in iliacs are less complicated and mostly can be reaccomplished by catheters. In the first of the aforementioned cases, the stent-to-stent technique provided the necessary columnar support and solitary stent-graft collapse into the large aneurysm was obviated . By impl 4th case, In open repair, sigmoid surveillance is needed , 25. In When IIA preservation through median laparotomy and endotracheal anaesthesia is unsafe, pure endovascular repair is attractive for high risk patients; however, EVAR application with standard stent-grafts is limited due to anatomic proximal aortic morphology or due to concomitant CIAAs or IIAAs which lead to unavoidable IIA occlusion due to lack of favorable distal landing zone; clinical EUROSTAR data also showed that EVAR in aortoiliac aneurysms using conventional stent-grafts was associated with higher type I endoleak incidence, secondary interventions, and delayed rupture . CombinaParodi and Ferreira  suggestePatients with lower life expectancy, comorbidities, and hostile abdomen, and as stated those with isolated CIAAs, are candidates for pure endovascular repair. Stent-grafting anatomical suitability is based on preoperative CT studies; excessive iliac tortuosity along with circumferential vessel wall calcification and significant arterial occlusive disease through which endoluminal access is planned consist hostile anatomy for endovascular intervention , 34. In In case of insufficient CIA distal landing zone for aortoiliac aneurysms, bell-bottom technique with a flared-cuff to the unfavorable landing zone has been proposed ; this cuIsolated IIAAs pose a demanding challenge for endovascular repair. Parsons et al.  reported 8th case, Recently, Iliac Branched Devices (IBDs) have been proposed to partially solve IIA orifice engagement; the IBD was used for the preservation of the left IIA in the case of the woman with aortoiliac aneurysmosis (Theoretically, IBDs may be used for bilateral CIAAs for preserving both IIAs. Preoperative high resolution CT-angiography study is required along with minimal IIA tortuosity and long landing zone. In case of IIAA and inadequate length of distal IIA landing zone for the IBD, an IIA branch may serve as IBD's landing zone with deliberate other branch occlusion. In a meta-analysis from Naik  nine repIn this study we aimed at providing technical considerations concerning aortoiliac aneurysms' management reviewing most of current options emphasizing on the individualization of treatment. By presenting these characteristic cases, our intention was to enlighten the diverse aspects of aortoiliac repair underlining that they are combined in a complimentary and not antagonizing association; we presume that patients' best outcome arises rather from optimum preoperative matching and not by adherence to a particular method.Improper strategy concerning IIA reperfusion may promptly result in hindgut ischemia , 25 or i


The diversity of regulatory genetic variants and their mechanisms of action reflect the complexity and context-specificity of gene regulation. Regulatory variants are important in human disease and defining such variants and establishing mechanism is crucial to the interpretation of disease-association studies. This review describes approaches for identifying and functionally characterizing regulatory variants, illustrated using examples from common diseases. Insights from recent advances in resolving the functional epigenomic regulatory landscape in which variants act are highlighted, showing how this has enabled functional annotation of variants and the generation of hypotheses about mechanism of action. The utility of quantitative trait mapping at the transcript, protein and metabolite level to define association of specific genes with particular variants and further inform disease associations are reviewed. Establishing mechanism of action is an essential step in resolving functional regulatory variants, and this review describes how this is being facilitated by new methods for analyzing allele-specific expression, mapping chromatin interactions and advances in genome editing. Finally, integrative approaches are discussed together with examples highlighting how defining the mechanism of action of regulatory variants and identifying specific modulated genes can maximize the translational utility of genome-wide association studies to understand the pathogenesis of diseases and discover new drug targets or opportunities to repurpose existing drugs to treat them. Regulatory genetic variation is important in human disease. The application of genome-wide association studies (GWAS) to common multifactorial human traits has revealed that most associations arise in non-coding DNA and implicate regulatory variants that modulate gene expression . Gene exIdentifying regulatory variants and establishing their function is of significant current research interest as we seek to use GWAS for drug discovery and clinical benefit ,7. GWAS Here I review approaches to understanding regulatory variation, from the viewpoint of both researchers needing to identify and establish the function of variants underlying a particular disease association, and those seeking to define the extent of regulatory variants and their mechanism of action at a genome-wide scale. I describe the importance of understanding context-specificity in resolving regulatory variants, including defining the disease-relevant epigenomic landscape in which variants operate, to enable functional annotation. I highlight the utility of eQTL studies for linking variants with altered expression of genes and the experimental approaches for establishing function, including descriptions of recent techniques that can help. I provide a strategic view, illustrated by examples from human disease, that is relevant to variants occurring at any genomic location, whether in classical enhancer elements or other locations where there is the potential to modulate gene regulation.Regulatory variation most commonly involves single-nucleotide variants (SNVs) but also encompasses a range of larger structural genomic variants that can affect gene expression, including copy number variation . Gene reFunctional variants can occur at both genic and intergenic sites, with consequences that include both up- and down-regulation of expression, differences in the kinetics of response or altered specificity. The effect of regulatory variants depends on the sequences that they modulate  and the functional regulatory epigenomic landscape in which they occur. This makes regulatory variants particularly challenging to resolve, as this landscape is typically dynamic and context specific. Defining which sequences are modulated by variants has been facilitated by several approaches: analysis of signatures of evolutionary selection and sequence conservation; experimental identification of regulatory elements; and epigenomic profiling in model organisms, and more recently in humans, for diverse cell and tissue types and conditions ,20.The understanding of the consequences of genetic variation for gene expression provides a more tractable intermediate molecular phenotype than a whole-organism phenotype, where confounding by other factors increases heterogeneity. This more direct relationship with underlying genetic diversity might account in part for the success of approaches resolving association with transcription of sequence variants, such as eQTL mapping ,5.The heritable contribution to common polygenic disease remains challenging to resolve, but GWAS have now mapped many loci with high statistical confidence. Over 90% of trait-associated variants are found to be located in non-coding DNA, and they are significantly enriched in chromatin regulatory features, notably DNase I hypersensitive sites . MoreoveCCND1 [SOX9 [MYC [Several studies have identified functional variants involving enhancer elements and altered transcription factor binding. These include a GWAS variant associated with renal cell carcinoma that results in impaired binding and function of hypoxia inducible factor at a novel enhancer of CCND1 ; a commoCCND1 ; and gerD1 [SOX9  and MYC OX9 [MYC , respectOX9 [MYC .IRGM that alters binding by the microRNA mir-196, enhancing mRNA transcript stability and altering the efficacy of autophagy, thus affecting the anti-bacterial activity of intestinal epithelial cells [TNFRSF1A associated with multiple sclerosis, which encodes a novel form of TNFR1 that can block tumor necrosis factor [HNF1B promoter [Functional variants in promoters have also been identified that are associated with disease. These include the extreme situation in which a gain-of-function regulatory SNV created a new promoter-like element that recruits GATA1 and interferes with expression of downstream α-globin-like genes, resulting in α-thalassemia . Other eal cells . Some SNal cells , which ms factor . Diseasepromoter .To identify functional variants, fine mapping of GWAS signals is vital. This can be achieved by using large sample sizes, incorporating imputation or sequence-level information, and involving diverse populations to maximize statistical confidence and resolve LD structure. Interrogation of available functional genomic datasets to enable functional annotation of identified variants and association with genes based on eQTL mapping is an important early step in prioritization and hypothesis generation. However, such analysis must take note of what is known of the pathophysiology of the disease, because the most appropriate cell or tissue type needs to be considered given the context-specificity of gene regulation and functional variants. Two case studies (Box 1) illustrate many of the different approaches that can be used to investigate the role of regulatory variants in loci identified by GWAS. These provide context for a more detailed discussion of techniques and approaches in the remainder of this review.This section describes approaches and tools for functional annotation of variants, considering in particular the usefulness of resolving the context-specific regulatory epigenomic landscape and of mapping gene expression as a quantitative trait of transcription, protein or metabolites.chromatin accessibility using DNase I hypersensitivity (DNase-seq) mapping ,33 and pchromatin conformation capture (3C), which can be scaled using HTS to enable mapping of genome-wide interactions for all loci (Hi-C)  or for stargeted arrays or genome-wide HTS to define differential DNA methylation ; the nonHigh-resolution epigenomic profiling at genome-wide scale using high-throughput sequencing (HTS) has enabled annotation of the regulatory landscape in which genetic variants are found and may act. This includes mapping regulatory features based on:The ENCyclopedia Of DNA Elements (ENCODE) Project  has genein vivo in diverse cell types and tissues [The FANTOM5 project  has recently published work complementing and extending ENCODE by using cap analysis of gene expression (CAGE) and single-molecule sequencing to define comprehensive atlases of transcripts, transcription factors, promoters, enhancers and transcriptional regulatory networks ,46. This tissues . It buil tissues . Enhance tissues .Publicly accessible data available through genome browsers significantly enhances the utility to investigators of ENCODE, FANTOM5 and other datasets that allow functional annotation and interpretation of regulatory variants, while tools integrating datasets in a searchable format further enable hypothesis generation and identification of putative regulatory variants Table  39,47,4,447,48. cis-regulatory modules. They found that the regulation involving such sequences was combinatorial and depended on complex patterns of co-occurring binding sites [PPARG gene locus that altered binding of the homeodomain transcription factor PRRX1. This was experimentally validated using allele-specific approaches and effects on lipid metabolism and glucose homeostasis were demonstrated.Determining which variants are located in regulatory regions is further helped by analysis of conservation of DNA sequences across species (phylogenetic conservation) to define functional elements. Lunter and colleagues  recentlyng sites . Applicacis-acting and those at a distance are likely to be trans-acting. Resolution of trans-eQTLs is challenging, requiring large sample sizes owing to the number of comparisons performed, because all genotyped variants in the genome can be considered for association. However, this resolution is important given how informative eQTLs can be for defining networks, pathways and disease mechanism [cis-eQTL mapping, trans-eQTL analysis allows discovery of previously unappreciated relationships between genes, as a variant showing local cis association with expression of a gene might also be found to show trans association with one or more other genes  or provide new biological insights [Mapping gene expression as a quantitative trait is a powerful way to define the regions and markers associated with differential expression between individuals . Applicaechanism . When cos Figure . For exar Figure c. This cpression . Trans-eet genes . For othinsights .Figure 1eQTLs are typically context specific, dependent for example on cell type -60 and sThe majority of published eQTL studies have quantified gene expression using microarrays. Application of RNA-seq enables high-resolution eQTL mapping, including association with abundance of alternatively spliced transcripts and quantification of allele-specific expression ,67. The In theory, eQTLs defined at the transcript level might not be reflected at the protein level. However, recent work by Kruglyak and colleagues  in largecis-regulated protein expression in plasma [Mapping protein abundance as a quantitative trait (pQTL mapping) is important in ongoing efforts to understand regulatory variants and the functional follow-up of GWAS. However, a major limitation has been availability of appropriate high-throughput methods for quantification. A highly multiplexed proteomic platform involving modified aptamers was used to map n plasma , and micn plasma . The appn plasma .There are multiple ways in which genetic variation can modulate the nature, abundance and function of proteins, including effects of non-coding variants on transcription, regulation of translation and RNA editing, and alternative splicing. In coding sequences, non-synonymous variants can also affect regulation of splicing and transcript stability. An estimated 15% of codons have been proposed by Stergachis and colleagues  to speciTHEM4 and the cytochrome P450 CYP3A5 genes [The role of genetic variation in modulating human blood metabolites was highlighted by a recent large study by Shin and colleagues  of 7,824A5 genes .Finally, analysis of epigenetic phenotypes as quantitative traits has proved very informative. Degner and colleagues  analyzedIn this section I review different approaches and methodologies that can help establish mechanism for regulatory variants. These tools can be used to test hypotheses that have been generated from functional annotation of variants and eQTL mapping. In some instances, data will be publicly available through repositories or accessible through genome browsers to enable analysis Table , for exaCis-acting regulatory variants modulate gene expression on the same chromosome. Resolution of allele-specific differences in transcription can be achieved using transcribed SNVs to establish the allelic origin of transcripts in individuals heterozygous for those variants [cis-regulatory variants rather than genotype-independent allele-specific epigenetic effects. Rare regulatory variants were found to account for the majority of identified allele-specific expression events [cis-eQTL mapping and can provide mechanistic evidence of regulatory variants acting in cis.variants . Alternavariants . Early gvariants . Mappingvariants ,81. Lappvariants  analyzedn events . Battle in vitro approaches such as electrophoretic mobility shift assays, while ex vivo approaches such as ChIP applied to heterozygous cell lines or individuals can provide direct evidence of relative occupancy by allele [Allele-specific transcription factor recruitment provides further mechanistic evidence for how regulatory variants act. Genome-wide analyses - for example, of binding of the NF-κB transcription factor family by ChIP-seq  - have py allele . A furthy allele .cis-regulatory elements and gene promoters can be identified by chromatin conformation capture methods, which provide mechanistic evidence to support hypotheses regarding the role of distal regulatory elements in modulating expression of particular genes and how this may be modulated by specific regulatory genetic variants. For some loci and target regions, 3C remains an informative approach, but typically investigators following up GWAS have several associated loci of interest to interrogate. Here, use of the Capture-C approach [Physical interactions between approach   for human cell lines and primary cells is now more tractable following advances in genome editing technologies such as transcription activator-like effector nucleases (TALENs) [SLFN5 and used CRISPR-Cas9 to demonstrate loss of inducibility by IFNβ on conversion from the heterozygous to homozygous  state in a human embryonic kidney cell line. Claussnitzer and colleagues [PPARG2 gene; they replaced the endogenous risk allele in a human pre-adipocyte cell strain with the non-risk allele and showed increased expression of the transcript.Model organisms have been very important in advancing our understanding of regulatory variants and modulated genes (Box 1). Analysis of variants and putative regulatory elements in an (TALENs)  and in p(TALENs) -92. The (TALENs) ) to dire(TALENs) ,95 and t(TALENs) ,61. Lee lleagues  used CRITNFSF11, IL27 and ICOSLG as potential repurposing opportunities for Crohn’s disease, given mismatches between GWAS associations with Crohn’s involving these genes and current drug indications. To maximize the potential of GWAS for therapeutics, and in particular for drug repurposing, it is important to have better resolution of the identity of genes modulated by GWAS variants so that associations can be established between genes and traits. When an existing drug is known to be effective in a given trait, it can then be considered for use in a further trait that shows association with the same target gene.Genomics-led research has significant potential to enhance drug discovery and enable more targeted use of therapeutics by implicating particular genes and pathways ,96. Thiscis-eQTL mapping, overlap with genes identified as causing rare Mendelian traits  and molecular pathway enrichment analysis, could help prioritize and interpret results of GWAS for rheumatoid arthritis with a view to guiding drug discovery. Fugger and colleagues [TNFR1 that can mimic effects of TNF-blocking drugs. The functional variant was associated by GWAS with multiple sclerosis, but not with other autoimmune diseases, and mechanistically it was found to result in a novel soluble form of TNFR1 that can block TNF. The genetic data parallel clinical experience with anti-TNF therapy, which in general is highly effective in autoimmune disease but in multiple sclerosis can promote onset or exacerbations. This work shows how knowing the mechanism and spectrum of disease association across different traits can help in developing and using therapeutics.Two examples illustrate how knowledge of functional regulatory variants and association with specific traits can guide likely utility and application. Okada and colleagues  recentlylleagues  identifiThe quest for regulatory genetic variants remains challenging but is facilitated by a number of recent developments, notably in terms of functional annotation and tools for genome editing, mapping chromatin interactions and identifying QTLs involving different intermediate phenotypes such as gene expression at the transcript and protein level. Integrative genomic approaches will further enable such work by allowing investigators to effectively combine and interrogate complex and disparate genomic datasets ,99. A reKey bottlenecks that need to be overcome include the generation of functional genomics data in a broad range of cell and tissue types relevant to disease (for other key issues that remain to be resolved see Box 2). Cell numbers can be limiting for some technologies, and a range of environmental contexts need to be considered. Moving to patient samples is challenging given heterogeneity related, for example, to stage of disease and therapy, but will be an essential component of further progress in this area. QTL mapping has proven highly informative but similarly requires large collections of samples, for diverse cell types, in disease-relevant conditions. The widespread adoption of new genome editing techniques and ongoing refinement of these remarkable tools will considerably advance our ability to generate mechanistic insights into regulatory variants, but at present these lack easy scalability for higher-throughput application. It is also essential to consider the translational relevance of this work, in particular how knowledge of regulatory variants can inform drug discovery and repurposing, and how academia and pharma can work together to inform and maximize the utility of genetic studies.SORT1, LDL cholesterol and myocardial infarctionSORT1 , and the effects were seen in liver but not subcutaneous and omental intestinal fat. The minor allele created a predicted binding site for C/EBP transcription factors, and allele-specific differences were seen using electrophoretic mobility shift assays and ChIP. Manipulating levels of C/EBP resulted in loss or gain of allelic effects on reporter gene expression and, in cells of different genotypic background, effects could be seen on SORT1 expression; human embryonic stem cells were used to show that this was specific to hepatocyte differentiation. Small interfering (siRNA) knockdown and viral overexpression studies of hepatic Sort1 in humanized mice with different genetic backgrounds demonstrated a function for Sort1 in altering LDL-C and very-low-density lipoprotein (VLDL) levels by modulating hepatic VLDL secretion. A genomic approach thus identified SORT1 as a novel lipid-regulating gene and the sortilin pathway as a target for potential therapeutic intervention [A pioneering study by Musunuru and colleagues published in 2010  demonstrrvention .FTO,RFX5and obesity: effects at a distanceFTO [FTO gene shows highly significant association with obesity by GWAS [FTO was found to encode an enzyme involved in control of body weight and metabolism based on evidence from FTO-deficient mice [FTO expression or function. Smemo and colleagues [FTO and mapped the regulatory interactions between genomic loci using 3C. Strikingly, their initial studies in mouse embryos revealed that the intronic GWAS locus showed physical interactions not only with the Fto promoter but also with the Irx3 gene (encoding a homeodomain transcription factor gene expressed in the brain) over 500 kb away. The interaction with Irx3 was confirmed in adult mouse brains and also human cell lines and zebrafish embryos. Data from the ENCODE project showed that the intronic FTO GWAS region is conserved, and its chromatin landscape suggested multiple regulatory features based on chromatin marks, accessibility and transcription factor binding. Smemo et al. [Irx3 depends on long-range elements. Strikingly, the GWAS variants associated with obesity showed association with levels of expression of IRX3 but not of FTO in human brain samples. Moreover, Irx3 knockout mice showed up to 30% reduction in body weight through loss of fat mass and increased basal metabolic rate, revealing a previously unrecognized role for IRX3 in regulating body weight. The multifaceted approach adopted by Smemo and colleagues [Regulatory variants may modulate expression of the most proximal gene, but they can have effects at a significant distance  making resolution of the functional basis of GWAS signals of association difficult . Recent FTO  highligh by GWAS -105. Folent mice  and froment mice . There wlleagues  considero et al.  then estlleagues  illustraWhat are the modulated genes underlying GWAS loci?By what specific mechanisms do particular disease-associated regulatory variants act?How can we resolve regulatory variants in a disease context?Can epigenomic profiling of chromatin accessibility and modifications be applied to small numbers of cells?Are genome editing techniques amenable to throughput experiments?How can we use knowledge of disease association integrated with functional evidence to repurpose existing therapeutics?Can knowledge of disease-associated regulatory variants and modulated genes provide new drug targets for development?Will regulatory variants, in particular those acting in trans, provide new insights into biological pathways and networks?


Distinct cortical substrates thus mediate the vestibular percepts of spatial orientation and self-motion.The cardinal features of vestibular dysfunction are illusory self-motion (vertigo) and spatial disorientation. Testing 18 acute focal cortical lesion patients, Kaski   When we walk in our environment, we readily determine our travelled distance and location using visual cues. In the dark, estimating travelled distance uses a combination of somatosensory and vestibular  cues. The observed inability of patients with complete peripheral vestibular failure to update their angular travelled distance during active or passive turns in the dark implies a privileged role for vestibular cues during human angular orientation. As vestibular signals only provide inertial cues of self-motion , the brain must convert motion information to distance information  to maintain our spatial orientation during self-motion in the dark. It is unknown, however, what brain areas are involved in converting vestibular-motion signals to those that enable such vestibular-spatial orientation. Hence, using voxel-based lesion–symptom mapping techniques, we explored the effect of acute right hemisphere lesions in 18 patients on perceived angular position, velocity and motion duration during whole-body angular rotations in the dark. First, compared to healthy controls’ spatial orientation performance, we found that of the 18 acute stroke patients tested, only the four patients with damage to the temporoparietal junction showed impaired spatial orientation performance for leftward  compared to rightward  rotations. Second, only patients with temporoparietal junction damage showed a congruent underestimation in both their travelled distance (perceived as shorter) and motion duration (perceived as briefer) for leftward compared to rightward rotations. All 18 lesion patients tested showed normal self-motion perception. These data suggest that the cerebral cortical regions mediating vestibular-motion (‘am I moving?’) and vestibular-spatial perception (‘where am I?’) are distinct. Furthermore, the congruent contralesional deficit in time (motion duration) and position perception, seen only in temporoparietal junction patients, may reflect a common neural substrate in the temporoparietal junction that mediates the encoding of motion duration and travelled distance during vestibular-guided navigation. Alternatively, the deficits in timing and spatial orientation with temporoparietal junction lesions could be functionally linked, implying that the temporoparietal junction may act as a cortical temporal integrator, combining estimates of self-motion velocity over time to derive an estimate of travelled distance. This intriguing possibility predicts that timing abnormalities could lead to spatial disorientation. The perception of self-motion lies at the heart of everyday human life. In the light visual cues dominate self-motion perception whereas vestibular cues are critically important when moving in the dark . Both ty. Indeed, primate recordings of thalamocortical pathways relaying vestibular signals of angular motion to the cerebral cortex have so far only yielded vestibular signals of head velocity .A variety of cerebral cortical areas have been associated with vestibular motion perception (i.e. illusory self-motion) as demonstrated by percepts of illusory rotational self-motion elicited by direct electrocortical stimulation during awake neurosurgery, including the superior temporal gyrus . ConsistMuch less studied is how the vestibular motion signal is transformed to derive vestibular spatial perception required for spatial orientation (‘where am I?’). Primate studies have identified vestibular-position signals in the posterior parietal cortex ; howeverThus, to identify the neural substrates of vestibular perception (and their underlying mechanisms), we assessed performance in a series of simple vestibular reorientation tasks in the dark in 18 patients with acute hemisphere brain lesions and an age-matched group. We used three different tasks to assess vestibular-spatial perception, vestibular-motion perception, and motion duration perception. We therefore evaluated: (i) whether focal cortical lesions influence self-motion perception and/or vestibular-guided spatial orientation; and (ii) whether the brain regions that mediate the vestibular percepts of self-motion versus spatial-orientation are distinct or overlapping. We used a voxel-based lesion–symptom mapping (VLSM) analysis to determine the relationship between lesion location and performance on the behavioural tasks. Identification of these areas would be important for understanding the neuro-anatomical basis of the vestibular symptoms of vertigo (vestibular-motion perception) and spatial disorientation .We tested 18 patients with focal right hemispheric cortical strokes between 3 and 12 days after stroke . Data weAll clinical and experimental testing was conducted within a 24-h epoch to minimize the possibility of spontaneous recovery between testing sessions. Stroke patients were on anti-platelet, anti-coagulation, anti-hypertensive and cholesterol lowering drugs, but none were administered acute psychoactive medication. Fourteen age-matched controls with no history of neurological or peripheral vestibular disease were also tested. Throughout the behavioural testing, fatigue was avoided in the patients by careful monitoring and allowing short breaks when necessary.Two patients with known idiopathic bilateral peripheral vestibular failure confirmed with laboratory testing  were recruited from our neuro-otology clinics . Both patients with peripheral vestibular failure had no other neurological impairment including no history of cerebrovascular disease. These two patients performed the behavioural tasks to confirm the dependence of these tasks upon vestibular functioning. Written informed consent was obtained from all participants for all experimental procedures as approved by the local research ethics committee.http://www.cabiatl.com/mricro/mricro/index.html) on a graphics tablet (WACOM). A T1-weighted template consisting of 12 axial slices was used to demarcate the lesions for all patients. Lesion overlap and subtraction were performed in MRIcro. Lesion subtraction was performed by directly comparing those patients who fell outside the 95% confidence intervals (CI) of healthy controls with those who did not, with the latter comprising negative values. This method allows for direct comparison of two groups of patients with one acting as a control, but therefore treats the deficit as a binary phenomenon (http://www.mccauslandcenter.sc.edu/mricro/npm/), and examined all the stroke patients as a single group, treating position bias as a continuous measure. A t-statistic was generated for each voxel (Brain lesions in stroke patients were imaged by MRI or CT (Patients S11 and S13) and plotted using MRIcro software  at which vestibular nystagmus was first seen (criteria as for 2 every 3 s) from a stationary start, either to the right or to the left. They were asked to depress one of two buttons (right or left) as soon as they perceived the movement and its direction (Using a modified version of the technique described (irection B. The tiPRight>Left) should be 0.5. Subjects’ temporal bias was thus calculated as the probability of the subject indicating that rightward rotations were longer than leftward when the durations of leftward and rightward rotations were equal (termed PRight>Left).This task assessed subjects’ ability to discriminate the duration of self-motion. Given that updating our spatial orientation is vestibular-dependent when we turn in the dark, we assessed whether this updating of perceived position from velocity cues involved estimates of motion duration. Subjects were seated in a motorized rotating chair in total darkness, with white noise played through headphones C. SubjecAll signals were recorded at a sampling rate of 250 Hz for off-line analysis. Eye movements were recorded during the motion perception task using DC-coupled horizontal electro-oculography (EOG) with EOG signals filtered at 30 Hz. Eye movement traces showed no evidence of spontaneous nystagmus in the light or dark, and no vestibular oculomotor asymmetries were detectable in any patient. A chair tachometer, which was used to record chair velocity for all tasks and angular chair displacement, was read from an off-axis angular encoder whose signal was recorded with an accuracy of <1° for rightward and leftward rotations. Specifically each degree of angular rotation was represented by four computer units during digital sampling of the chair position signal. Psychometric probability curves were plotted to display the time perception data using Sigmaplot .t-test, one-way ANOVA and repeated measures ANOVA were used to compare between group responses in the experiments.Parametric statistics including The group ‘position biases’  are shown in The patients’ lesion distributions are shown in We performed a VLSM analysis, including all 18 stroke patients and examined position bias as a continuous measure throughout the whole group. This analysis showed that the most significant regions C, red asP > 0.1). In addition the patients’ motion perceptual thresholds were within the normal values previously reported in the literature . Given the evidence suggesting that the insula is involved in processing vestibular signals of head motion, we also compared motion perceptual thresholds for contralesional and ipsilesional rotations between eight insular and 10 non-insular lesion patients and 14 healthy controls. However, we found no difference in motion perception between these groups .Patients S1–S18 showed normal ocular motor (vestibular-ocular reflex) and motion perception thresholds  should be 0.5. For the control healthy aged-match group, the average PRight>Left was 0.48, which was significantly different from the spatial-deficit patient group’s average PRight>Left of 0.76 .An unbiased subject’s probability of indicating that rightward rotations were longer than leftward when the durations of leftward and rightward rotations were equal (https://www.google.com/maps/views/streetview), she reliably described all landmarks on the left and right sides of the street when describing it from opposite viewpoints, ruling out any significant degree of representational neglect.Clinical data for the neglect testing are shown in Our data show that: (i) deficits in vestibular-guided spatial orientation (the Position task) were manifest only in those patients whose lesions involved the TPJ; (ii) patients with spatial disorientation on the Position task showed a temporal bias (in the Time comparison task) congruent to the direction and magnitude of their spatial disorientation; (iii) vestibular-sensed self-motion perception was not affected by focal brain lesions; and (iv) vestibular-guided spatial orientation deficits were not secondary to spatial neglect. Put simply, patients whose lesion involved the TPJ underestimated both their travelled distance and motion duration during vestibular-guided leftward turns in the dark when compared to rightward rotations.We consider two main possibilities to explain the apparent relationship between deficits in time comparison and spatial orientation in patients with damage to the TPJ.First, it may be that the mechanisms mediating the perception of motion duration (Time task) and spatial orientation (Position task) are not functionally linked but that their apparent linkage (shown in our results) could arise simply because their neural correlates occupy an overlapping brain location in the TPJ. As such, that Time task deficits manifest in TPJ lesion patients would reflect a general timing role of the TPJ rather than any specific role in spatial orientation. Supporting its general role in timing, the TPJ has been linked with estimating and perceiving duration  timing deficits, without spatial deficits, could be isolated in at least some patients; however, we did not observe isolated deficits in either spatial orientation or temporal estimation; and (ii) common deficits in time and spatial performance could arise as a result of deficits in cognitive processes that could simultaneously influence spatial and/or temporal perceptual performance, e.g. attention mechanisms and the mental number line deviation . NeglectAlternatively, a mechanistic hypothesis dictates that the neural processes underlying motion duration perception and spatial orientation are functionally linked, predicting a tight relationship between vestibular spatial perception and motion duration perception, with congruent deficits in both. In line with this, we found that patients with worse spatial performance showed a worse temporal performance, suggesting a tight overlapping in neural correlates between these two functions. A link between spatial and temporal estimates may be of particular relevance for spatial orientation and ‘path integration’—the process by which the distance travelled within the environment is derived from motion cues  .The use of timing estimates to derive a spatial estimate is suggestive of an internal model , thought to be the main cortical locus involved in processing vestibular signals of head motion . A recenOur data show that the TPJ is critically involved in vestibular spatial perception (‘where am I?’), but not vestibular motion perception (‘am I moving?’). It follows that these two faculties are separately encoded in the brain. This predicts that deficits in spatial disorientation arising from cortical disturbances could occur separately from deficits in self-motion perception. Our finding that deficits in vestibular spatial function were congruent with deficits in motion duration perception is intriguing, and leads us to speculate that the TPJ may act as a cortical temporal integrator that combines estimates of self-motion velocity over time to mediate the updating of travelled distance when navigating in the dark and under vestibular guidance.


Escherichia coli (UPEC). Advanced drug delivery systems aim at exploiting a glycan-mediated targeting mechanism, similar to the UPEC invasion pathway, to increase bioavailability. This may be realized by conjugation of intravesically applied drugs or drug carriers to chosen plant lectins. Higher local drug concentrations in or nearby bacterial reservoirs may be gained, with higher chances for complete eradication. In this study, preliminary parameters to clarify the potential of this biorecognitive approach were evaluated. Glycan-triggered interaction cascades and uptake processes of several plant lectins with distinct carbohydrate specificities were characterized, and wheat germ agglutinin (WGA) could be identified as the most promising targeter for crossing the urothelial membrane barrier. In partially differentiated primary cells, intracellular accumulation sites were largely identical for GlcNAc- and Mannose-specific lectins. This indicates that WGA-mediated delivery may also enter host cells via the FimH-dependent uptake pathway.Urinary tract infections (UTIs) are among the most common bacterial infections. In an increasing number of cases, pathogen (multi-)resistance hampers durable treatment success via the standard therapies. On the functional level, the activity of urinary excreted antibiotics is compromized by the efficient tissue colonization mechanism of uropathogenic  Escherichia coli (UPEC) is the mannose-mediated adhesion of the bacterial FimH lectin domains to membrane proteins and lipids. This FimH/carbohydrate interaction can trigger active uptake processes, allowing the bacteria to cross the rigid membrane barrier and proliferate in cytosolic or membrane-bound compartments, where they are protected from antibiotics and the host immune system. Release from these reservoirs back into the bladder lumen after a certain lag time is considered a main cause for recurrent infections [Urinary tract infections (UTIs) rank among the most common bacterial infections and represent a severe burden to the society, with significant impact on patient quality of life and overall health care costs . Despitefections ,3.The increasing prevalence of problematic cases calls for the development of novel therapeutic strategies that allow for a more efficient delivery of the antibiotic drug to the bacterial reservoirs. Today, intravesical instillations of antibiotics are mainly adopted as salvage therapy in complex (recurrent/multiresistant) cases, and offer the advantage of providing direct access to the diseased tissue . HoweverIn this study, we aimed to elucidate the potential of this biorecognitive approach in a well-defined and easy-to-monitor tissue culture model. Due to broad availability and a (relatively) close analogy to human systems , primaryLens culinaris agglutinin; UEA: Ulex europaeus agglutinin; PNA: Peanut agglutinin; GNL: Galanthus nivalis lectin; DBA: Dolichus biflorus agglutinin). Competitive inhibition studies with complementary carbohydrates were carried out to confirm the specificity of the binding process and gain information on the average binding affinity.In screening assays, WGA was identified as the most promising targeter due to extensive and stable cell binding. The glycan-mediated targeting approach was shown to bear advantages as compared to non-specific protein/cell interactions, e.g., observed for albumin. Overall, the experimentally determined lectin binding capacity decreased in order: WGA >> LCA > UEA > PNA > GNL > DBA  and LCA (fLCA) were compared in detail, since WGA harbours the highest binding potential (mainly via GlcNAc) and LCA targets glycan epitopes similar to those recognized by FimH (mannose) . Quenchi® 594/647 conjugate of WGA (aWGA) and fLCA resulted in a near-complete overlap of the individual channels, indicating a high parallelism in the processing pathway of both lectins. In a metabolically quiescent state (4 °C), mere staining of the cell surface was visible, whereas at 37 °C intracellular clustering pointed to endocytotic uptake . The suggested administration of the prepared delivery vehicles via intravesical instillation facilitates a localized administration to the site of disease, exposing pathogens to high concentrations of the active agent. Hence, the use of lectins for biorecognitive targeting strategies might indeed increase the chances for good response. The stable bioadhesion of the applied vehicle might not only counteract the harsh washout conditions in the urinary tract, but may also facilitate a continuous release of the drug and hence lead to a better concentration-over-time profile.Moreover, a direct lectin/API conjugation can be realized, as demonstrated in other areas of application . InternaWGA-decorated, biodegradable nano- or microparticulate drug carriers may, in addition to facilitating stable binding, allow fragile compounds to withstand the hostile environmental conditions in the urinary tract. The use of such advanced delivery systems could greatly increase therapeutic outcome in the treatment of UTIs.Wheat germ agglutinin (fWGA) and Lens culinaris agglutinin (fLCA) were purchased from Vector Laboratories . All other chemicals were obtained from Life Technologies  or Sigma . Porcine bladders obtained from a local abattoir and were processed according to an established protocol [Fluorescein-labelled protocol  with sli5 cells, 50 µL) for 30 min at 4 °C (surface loading). After washing with PBS, cells were either kept at 4 °C or warmed to 37 °C to initiate energy-dependent uptake. Endosomal/lysosomal accumulation was followed via flow cytometry using the restored fluorescence signal  as a control.The energy-dependent internalization of fWGA and fLCA was studied by assessing the acidic fluorescein quench upon uptake in intracellular compartments. Single cells were incubated with the respective lectin  594/647 conjugate of WGA (aWGA) and fLCA were conducted to compare their uptake and intracellular localization. Confluent cell monolayers on glass coverslips were washed and surface-loaded with 50 µL of aWGA/fLCA solution (12.5 nM each) for 30 min at 4 °C. After thorough washing, cells were further incubated for 60 min at 37 °C or 4 °C. Imaging studies were carried out using a deconvolution microscopy system.The results of this study indicate that plant lectins may indeed be utilizable as mediators of a FimH-like uptake stimulation. Prolonged residence times and enhanced intracellular urothelial accumulation can be achieved via the specific targeting of surface-accessible carbohydrates. This may ultimately allow for counteracting limitations in existing UTI treatment schedules, such as the loss of drug by urinary excretion. Advanced delivery systems that follow the UPEC invasion pathway may in future constitute a potent strategy to eradicate multiresistant UPEC in their hard-to-reach reservoirs. Currently, surface-functionalized microparticulate carrier systems are being tested for lectin-targeted administration of antibiotics in complex cases of disease.


The need to obtain food is a critical proximate driver of an organism’s movement that shapes the foraging and survival of individual animals. Consequently, the relationship between hunger and foraging has received considerable attention, leading to the common conception that hunger primarily enhances a “food-intake maximization” (FIMax) strategy and intensive search. A complementary explanation, however, suggests a trade-off with precautions taken to reduce the risk of physiological collapse from starvation, under a strategy we denote as “energy-expenditure minimization” (EEMin). The FImax-EEmin trade-off may interact with the forager’s hunger level to shape a complex (non-monotonic) response pattern to increasing hunger. Yet, this important trade-off has rarely been investigated, particularly in free-ranging wild animals.Gyps fulvus) in southern Israel. Transmitters combining GPS and accelerometers provided high-resolution data on vultures’ movements and behavior, enabling the identification of feeding events and the estimation of food deprivation periods , which is used as a proxy for hunger.We explored how hunger affects the movements of adult griffon vultures , the vultures’ FIMax response was less intensive and resulted in longer starvation periods, while, in summer, more intensive FIMax response to hunger resulted in shorter FDPs.Our results show a flexible, non-monotonic response of free-ranging wild animals to increasing hunger levels, reflecting a trade-off between increasing motivation to find food and the risk of starvation. The proposed trade-off offers a unifying perspective to apparently contradictory or case-specific empirical findings.The online version of this article (doi:10.1186/2051-3933-1-5) contains supplementary material, which is available to authorized users. The movement of individual organisms is a key component of many ecological and evolutionary processes across a broad range of spatiotemporal scales, reflecting a dynamic interplay of factors that are both internal and external to the focal organism . InternaThe need to obtain food or other essential resources is a major internal driver of various ranging movements referred to as foraging. For an animal with a given set of internal states  and constraints (motion and navigation capacities), foraging movements are generally subject to a basic trade-off between food intake rate and locomotion costs such as energetic expenditure, time allocation and predation risk. The degree of satiation (or hunger) an animal experiences at a given time can profoundly impact the internal state and the constraints, as well as the outcomes of these trade-offs –11. For food-intake maximization” (FIMax). This strategy may be reflected in either longer search time and distance  and cross through regions where food might be scarcer (without SFSs), forcing them to reduce feeding intensity during this season. Few individuals also exhibit long-range forays in autumn [Why do vultures display these two distinct foraging strategies? Foraging strategies may be age- and sex-dependent . The firn autumn , furthern autumn . During n autumn . Summer In contrast to experimental manipulation, our methodology does not provide information on the quantity and quality of consumed food. Vultures’ hunger levels may vary substantially after a feeding event. Yet, our reliable data on the lack of feeding enable addressing the main hypothesis of this study; regardless of the additional uncertainty associated with meal size, hunger levels invariably increase as FDP lengthen. Additional data on exact hunger levels (or at least consumed food) could improve our ability to explain the remaining observed variance in the response of the vultures. Although we found no association between the length of the current and previous FDPs, hunger might also vary due to recent feeding history rather than exclusively depending on the timing of the last feeding event.Alternative explanations for the observed differences in vulture movement characteristics between the two FDP sequence types could be partly attributed to our indirect proxy for hunger. What appears to be an intense FIMax strategy may actually reflect a hungry vulture whose current hunger level corresponds to a longer FDP than that estimated by our proxy. The difference in travel distances for day zero agrees with this alternative explanation: While distances are very short for the long sequences, they are substantially higher for short ones, possibly suggesting that vultures were hungrier already at this phase. Nevertheless, such a directional bias, whereby stops classified as feeding events are consistently a product of false-positive classification errors, is extremely unlikely considering the high accuracy of the classification algorithms, the conservative estimation of feeding events based on “eating” ACC measurements, and the consistency of the hump-shaped response for long FDPs.Most previous studies of the effect of FDP on movement were indoor experiments on a limited set of small-bodied animals. Our study presents one of the first attempts to address this challenge in free-ranging animals that move over large areas on a daily basis. Our results reveal a broad flexibility in the behavioral mechanisms used by griffon vultures to cope with increasing hunger levels. The initial response to food deprivation is increased activity and movement, indicative of greater searching efforts and attempt to maximize food intake. A switch to energy-conserving strategy is evident in extended FDPs. These results agree with previous empirical evidence, and offer a unifying perspective to apparently contradictory (or case-specific) findings. Another level of flexibility in the vultures’ response to increasing hunger level is reflected in their ability to vary the intensity of a food-intake maximization strategy. Less intensive efforts resulted in longer FDPs and were more common during the winter and autumn, reflecting seasonal breeding cycle and environmental conditions. Vultures’ flexibility might reflect an adaptive response to unpredictable resources and should be considered by managers when applying supplementary feeding programs. For instance, a feeding regime during spring should be more intensive since vultures’ feeding intensity and activity levels during this period are higher.Additional file Additional file Additional file Comparing hungry and satiated vultures. (DOCX 101 KB)Additional file 1: FDP effects on vulture movement associated with feeding on occasional carcasses. (DOCX 136 KB)Additional file 2: The effect of FDP length on vulture movement characteristics (Tables S2-S3). Three alternative models were considered: No effect, a linear effect, and a hump-shaped effect. The tables present the best fitting model for each variable using AICc and adjusted R2 estimation of goodness of fit. Table S2 presents the effect for the whole dataset and Table S3 for a subset of long FDPs (>=6). (DOCX 16 KB)Additional file 3:


Unraveling the machineries that deal with threats during DNA replication is of critical importance to understand how cells maintain genome stability. Faithful duplication of the genome is fundamental for the propagation of life, yet DNA is particularly vulnerable during the replication process. DNA replication forks encounter a large number of obstacles in the template DNA in each cell cycle. These perturbations lead to fork stalling and if not quickly resolved the fork can collapse through disassembly of the replication machinery. In case of widespread stalling of forks cells enter a state commonly referred to as ‘replicative stress’ that challenge genome stability. Cellular mechanisms have evolved to monitor this process and ensure completion of genome replication by different means. Following replication stress, cell cycle checkpoint pathways are activated to stabilize stalled replication forks and halt cell cycle progression. Genome duplication is then completed by employing several pathways, which include the activation of nearby dormant replication origins, the repriming of DNA replication downstream of the damage site, and tolerance to the damage thanks to lesion bypass mediated by specialized DNA polymerases .in vivo in higher eukaryotes [in vitro supporting that FBH1 is a bona fide fork regression enzyme in mammalian cells [The mechanisms underlying critical processes at the DNA replication fork still remain to be fully elucidated, and new important factors are emerging. The FBH1 helicase is a less investigated regulator of the response to stalled replication forks. FBH1 is a member of the conserved UvrD family of 3′-5′ DNA helicases  that haskaryotes . Importaper se may be insufficient to activate the checkpoint, as fork regression and replication stress do not always co-occur [What is the role of FBH1-mediated replication fork regression? This process can either be an end product or intermediate in fork processing reactions. As an end product, regression may stabilize the fork for example by shielding vulnerable single-stranded DNA regions. This can provide additional genomic safety and time for neighboring forks to complete DNA replication. The data at hand also suggest that regressed forks may elicit signals to signify that severe replication stress is present. Notably, the very end of the regressed arm of the four-way junction is a DNA double-stranded end, which has obvious similarity to the DNA double-strand break (DSB) generated for example after ionizing radition. In line with this notion, whereas replication fork stalling does not induce detectable DNA DSB formation immediately after HU treatment, we observed that HU nonetheless induces a marked FBH1-dependent DNA DSB-like signaling response, which is already evident 1-2 hours following HU treatment. This response can be measured by the marked phosphorylation of DNA DSB-related ATM targets before detectable DSBs were apparent . Thus, oco-occur ,5. It isStrikingly, the response to long-term fork stalling also requires FBH1. When replication forks are stalled more than 12-16 hours with HU, there is measurable cleavage of the fork catalyzed by the structure-specific endonuclease MUS81, which requires FBH1 helicase activity. This reaction generates double-stranded DNA ends that can be used for recombination-dependent replication fork restart. However, the MUS81-induced DNA breakage triggers p53 activation that ultimately also serves to suppress cell proliferation untill replication stress has been eliminated .Based on our understanding so far, FBH1-mediated DNA replication fork processing is emerging as a new pathway for genome protection in response to replication stress. Future studies will aim to unravel how this pathway interacts with other fork processing and signaling pathways.


Many families worldwide have at least one member with a behavioral or mental disorder, and yet the majority of the public fails to correctly recognize symptoms of mental illness. Previous research has found that Mental Health Literacy (MHL)—the knowledge and positive beliefs about mental disorders—tends to be higher in European and North American cultures, compared to Asian and African cultures. Nonetheless quantitative research examining the variables that explain this cultural difference remains limited. The purpose of our study was fourfold: (a) to validate measures of MHL cross-culturally, (b) to examine the MHL model quantitatively, (c) to investigate cultural differences in the MHL model, and (d) to examine collectivism as a predictor of MHL. We validated measures of MHL in European American and Indian samples. The results lend strong quantitative support to the MHL model. Recognition of symptoms of mental illness was a central variable: greater recognition predicted greater endorsement of social causes of mental illness and endorsement of professional help-seeking as well as lesser endorsement of lay help-seeking. The MHL model also showed an overwhelming cultural difference; namely, lay help-seeking beliefs played a central role in the Indian sample, and a negligible role in the European American sample. Further, collectivism was positively associated with causal beliefs of mental illness in the European American sample, and with lay help-seeking beliefs in the Indian sample. These findings demonstrate the importance of understanding cultural differences in beliefs about mental illness, particularly in relation to help-seeking beliefs. They didn't take people out in the sunshine, where you begin to feel better…They didn't involve the whole community…Instead what they did was they took people one at a time into dingy little rooms and had them talk for an hour about bad things that had happened to them.”Rwandan man Solomon, The Rwandan man's confusion over Western mental health care illustrates how beliefs about mental illness and their treatment can differ across cultures Jorm, . The WorMental health literacy (MHL) refers to the “knowledge and beliefs about mental disorders which aid their recognition, management or prevention” : The professional help-seeking beliefs measure developed in the current study will be valid (i) across cultures and (ii) between mental disorders.Collectivist individuals tend to value in-group relations and seeking advice from the in-group : Greater collectivism will be associated with more positive professional help-seeking beliefs.As only a minority of individuals with symptoms of mental illness seek professional help, it is even more important for them to seek help from other sources Jorm, . Indeed,H1(b): The lay help-seeking beliefs measure developed in the current study will be valid (i) across cultures and (ii) between mental disorders.Jorm et al.  comparedH3: More positive lay help-seeking beliefs will significantly predict more positive professional help-seeking beliefs.Van Hook  further H2(b): Greater collectivism will be associated with more positive lay help-seeking beliefs.Furthermore, individuals faced with symptoms of mental illness seek support from a range of informal sources before seeking professional help : The MHL model will display indirect effects between collectivism and professional help-seeking beliefs through lay help-seeking beliefs.Another aspect of MHL is recognition of mental disorders, which tends to be poor amongst members of the public  than their Asian and African counterparts  more positive professional help-seeking beliefs, and (ii) more negative lay help-seeking beliefs.Also recall that the literature has indicated that there is a strong positive relationship between lay and professional help-seeking beliefs : The MHL model will display indirect effects between recognition and professional help-seeking beliefs through lay help-seeking beliefs.A further facet of MHL concerns beliefs about the causes of symptoms of mental disorders. Psychopathological models draw on social and biological factors when explaining causes of mental disorders. In the present study we developed a social and biological causal beliefs measure and hypothesized that:H1(c): The social and biological causal beliefs measure developed in the current study will be valid (i) across cultures and (ii) between mental disorders.Greater endorsement of both biological and social causes of mental illness reflects better knowledge of mental illness and therefore identification of the need to seek help from a professional : Individuals who more strongly endorsed social causal beliefs would hold more positive (i) lay and (ii) professional help-seeking beliefs.H6(b): Individuals who more strongly endorsed biological causal beliefs would hold more positive professional help-seeking beliefs.Further, Jorm and associates  found anH7(a): The MHL model will show significant indirect effects between recognition and (i) lay and (ii) professional help-seeking beliefs through causal beliefs.Endorsement of causal beliefs also varies between cultures  social and less endorsement of (ii) biological causal beliefs.Recall that we predicted that social causal beliefs would be significantly positively associated with lay and professional help-seeking beliefs, while biological causal beliefs more would be significantly positively related to professional help-seeking beliefs and significantly negatively related to lay help-seeking beliefs. We therefore also hypothesized that:H7(b): The MHL model will show significant indirect effects between collectivism and (i) lay and (ii) professional help-seeking beliefs through causal beliefs.Finally, also recall that the literature indicated a strong positive relationship between lay and professional help-seeking beliefs .N = 100) and Indians currently living in India (N = 108) to participate in this study. Ipeiriotis  scored “2,” and naming the particular mental disorder displayed in the vignette scored “3” . Reliabilities were α = 0.75 for all mental disorders and α = 0.89, 0.68, and 0.49 for schizophrenia, depression and GAD, respectively.After reading the vignettes, participants were also presented the causal beliefs, lay help-seeking beliefs and professional help-seeking beliefs measures developed in the present study. We chose the items for these measures from studies that had examined MHL using qualitative approaches  to 5 (Does not explain the behavior). Items of the causal beliefs measure were reverse-coded such that higher scores represent greater agreement that causes explain the person's behavior.The lay help-seeking beliefs and professional help-seeking beliefs measures posed the following question: “To what extent do you think it would be helpful or harmful for your friend to… ?.” Participants were asked to rate items of both the lay and professional help-seeking beliefs measures along a 7-point Likert scale that ranged from 1 (Very helpful) to 7 (Very harmful). Items of the lay and professional help-seeking beliefs measures were reverse-coded such that higher scores represent beliefs of greater helpfulness. See Table For our newly developed causal beliefs, lay help-seeking beliefs, and professional help-seeking beliefs measures, we examined measurement equivalence or invariance . Then the algorithm computes maximum likelihood estimates. We used SPSS 20 to input items into the expectation-maximization algorithm. To increase the power of our data we input items associated with one particular subscale at time, as this increases the correlations between items  cultures and (ii) mental disorders. We performed multi-group CFA with culture—European Americans vs. Indians—as the group-variable, and tested this measure separately for each mental disorder. The model displayed in Figure ps > 0.05). Yet seeing a psychologist loaded more strongly in the European American sample while going to counseling or therapy loaded more strongly in the Indian sample  = 22.09, p = 0.002]. On closer, inspection several items did not load invariantly , however it proved to be a poor fit  cultures and (ii) mental disorders  = 19.45, p = 0.04]. The item fresh air did not load equally between groups (see Schizophrenia in Table ps > 0.05). The refined model held an adequate model fit and had significantly improved  cross-culturally and (ii) between mental disorders. See Figure First we tested the measure in regards to the depression data. The measurement model was an adequate fit (ii) – establishing validity between mental disorders—was only partially supported, as items of the causal beliefs scale were only valid in relation to the depression and schizophrenia data.As the social causal beliefs and the lay help-seeking beliefs measures held poor model fit in regards to the GAD data, only the depression and schizophrenia data was used in the following analyses. In all following analyses we employed social causal beliefs, lay help-seeking beliefs and professional help-seeking beliefs as the latent variables, with both the depression and schizophrenia items loading on the respective constructs.p > 0.05) and inspection of the histograms showed that the professional help-seeking beliefs, lay help-seeking beliefs and social causal beliefs constructs were normally distributed in both cultural samples. Recognition showed negative kurtosis in the Indian sample (z = −3.67). However, inspection of the frequencies showed that 50% of the Indian participants recognized the symptoms represented in the vignette as a mental disorder and there was variation in the recognition scores that would allow for valid correlations, which does not support a flooring effect.Normality tests . Thus we tested further models removing any paths between latent variables that were non-significant in both cultures, until all remaining paths were significant in either cultural group. See Table We used multi-group structural equation modeling to test the hypothesized moderated-mediation model  – and also significantly negatively predicted lay help-seeking beliefs in the Indian sample—lending partial support to H5(ii). This indicates that, on the one hand, both European American and Indians who were better at recognizing mental disorders were more likely to endorse seeking professional help and, on the other hand, Indians who demonstrated better recognition were less likely to endorse seeking lay help. Thus cultural group significantly moderated the association between recognition and lay help-seeking beliefs, lending further support to the research question—how culture will moderate the MHL model.All aspects of d Figure . First, Hypothesis 6—associations of social causal beliefs within the MHL model (i) social causal beliefs were significantly positively associated with lay help-seeking beliefs in the Indian sample, while the association trended toward significance in the European American sample. This indicates that Indian participants who reported greater agreement with social causes were also more likely to endorse seeking lay help. Further, this lends greater insight into the RQ—how the MHL model is moderated by culture. Third, H6(a)(ii) was refuted as the association between social causal beliefs and professional help-seeking beliefs was non-significant in both cultural groups (p > 0.05). Finally, H6(b) related to biological causal beliefs and because we did not find cross-cultural validity for the biological causal beliefs subscale we were not able to examine this hypothesis.Next we examined l Figure . First, H8(i), collectivism significantly positively predicted social causal beliefs in the European American sample, while the association trended toward significance in the Indian sample. This indicates that European American participants who reported greater collectivism were more likely to believe symptoms described in the vignette were due to social causes. Finally, H8(ii) related to biological causal beliefs, thus we were not able to examine these hypotheses.Next, lending support to Moreover, the association between lay and professional help-seeking beliefs was significant and positive in the Indian sample but non-significant in the European American sample Figure . A chi dIn the final step we tested the indirect effects within the MHL literacy model—examining Hypotheses 4 and 7. The indirect effects were tested via a bootstrapping procedure  and 9 we found significant indirect effects between collectivism as well as social causal beliefs and professional help-seeking beliefs through lay help-seeking beliefs in the Indian sample  was refuted, as the indirect effect between recognition and professional help-seeking beliefs via lay help-seeking beliefs was non-significant in both cultural groups (Table ps Table .Hypothesis 7(a)(i) was partially supported—recognition was indirectly associated with greater lay help-seeking beliefs via greater social causal beliefs in the European American sample and the association trended toward significance in the Indian sample (Table Hypothesis 7(b) (i) was partially confirmed as collectivism was indirectly associated with lay help-seeking beliefs through social causal beliefs in the European American sample, while this was not the case in the Indian sample (Table Hypothesis 7(a) (ii) and 7(b) (ii) were refuted, as the association between social causal beliefs and professional help-seeking beliefs was non-significant in both cultural groups (p > 0.05).RQ, confirming that culture acts as a moderator to the MHL model.Finally, the findings of associations between variables and indirect effects within the MHL model give further insight into our The present findings showed strong associations between the elements—recognition, causal, lay and professional help-seeking beliefs—of the proposed MHL model . Jorm (traumatic event and stress), while items that did not hold equivalence may have been too situation- or person-specific .For all three of our MHL measures, we retained items that held cross-cultural invariance. In respect to the causal beliefs measure, the items relating to biological causes did not hold cross-cultural invariance (e). Jorm  proposedtake medication and go to psychiatric clinic). This may be due to perceived accessibility and availability of mental health facilities across cultures. Facilities for mental health issues are scarce in India (World Health Organisation, In relation to the professional help-seeking beliefs measure, the retained items reflect a biological model of mental illness (e.g., Furthermore, most items of the lay help-seeking beliefs measure were retained, which is in line with previous findings that across cultures individuals with mental health issues draw support from a wide range of informal sources (Cooper-Patrick et al., On the one hand, we aimed to establish cross-cultural validity for the novel MHL measures, and on the other hand we intended to examine three of the most prevalent mental disorders worldwide—depression, schizophrenia, and GAD. Unfortunately, the GAD data did not yield strong results and we were not able to use the GAD data in our analyses. This may be because compared to GAD, depression and schizophrenia receive greater attention in books, TV shows, movies, and awareness campaigns (Tartakovsky, After establishing validity across cultures and mental disorders of the developed MHL measures, we examined the MHL model itself. Overall, we found strong quantitative support for the MHL model. We found that recognition of symptoms of mental illness was the strongest predictor of all other aspects of the MHL model. In both cultures greater recognition was associated with more positive professional help-seeking beliefs, while in the Indian sample it was also positively associated with lay help-seeking beliefs. This gives support to the literature that stresses the link between knowing about symptoms of mental illness and endorsement of seeking appropriate help for these symptoms (Jorm et al., Our findings showed an overwhelming cultural difference in the MHL model. The main cultural difference was that in the Indian sample, lay help-seeking beliefs was a major variable, while in the European American sample it did not have any significant associations with other aspects of the model. From our findings it appears that the European American sample did not view lay help as a relevant source of help in relation to mental health issues. Contrary to our findings, previous literature stressed that across cultures the public appears to hold a preference for seeking help from lay as opposed to professional sources when faced with issues of mental health (Cooper-Patrick et al., Furthermore, the majority of the MHL literature has examined professional help-seeking beliefs (Tata and Leong, Moreover, we found an indirect effect between collectivism and professional help-seeking beliefs through lay help-seeking beliefs in the Indian sample, with greater collectivism being related to more positive beliefs about seeking lay help and in turn greater endorsement of seeking professional help. Interestingly, contrary to previous findings (Tata and Leong, Further, contrary to previous research (Sheikh and Furnham, Finally, in the European American sample greater collectivism was associated with greater endorsement of social causal beliefs. The literature suggested that collectivists would be more likely to attribute causes of mental illness to the community (Narikiyo and Kameoka, One of the main strengths of the present study was that it adopted a quantitative approach to examine the MHL model. This is in contrast to many studies that either employed a qualitative approach (Jorm et al., A further strength of the present study was that we examined professional as well as lay help-seeking beliefs. Most studies investigating MHL have solely focused on professional help-seeking (Tata and Leong, The present approach necessarily had limitations. First, presentation of the symptoms of mental illness may vary between cultures (Williams and Healy, Another limitation stems from the use of the expectation-maximization method to infer missing values, and therefore findings need to be interpreted with caution. However, the alleged shortcomings of the expectation-maximization method—e.g., multiple modes, saddlepoints, ridges—should be interpreted as inherent features of the algorithm, because expectation-maximization fares well in comparison to other computational methods Schafer, .A further limitation stems from the recognition scale we used, which was a reflection of knowledge of mental disorders. Future research may want to use a more detailed knowledge scale to be able to discriminate between categorizing someone as ill as opposed to knowing what is going on with the person.In conclusion, our study revealed significant cultural differences in knowledge as well as beliefs about causes and help-seeking for mental illness. This underlines the importance of understanding beliefs of mental illness in different cultures in order to develop more effective, approachable, and culturally sensitive mental health care systems. In doing so individuals like the Rwandan man, who was puzzled by the cultural differences in mental health care, do not need to wonder what “bizarre things” mental health workers are providing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.