High levels of depressive symptoms often occur among individuals that use or that are dependent on methamphetamine (MA). Thailand is currently experiencing an epidemic of MA use among youth. Understanding the nature of the relationship between depressive symptoms and MA use and identifying those most at risk can further understanding of prevention and treatment options for youth who use MA and present with depressive symptoms.In 2011, we conducted a cross sectional epidemiologic study that examined associations between MA use and high levels of depressive symptoms among adolescents and young adults aged 14–29 living in Chiang Mai province, Thailand. A combination of cluster and systematic sampling was conducted to obtain a study sample of participants actively recruited in Chiang Mai province. Depressive symptoms were measured using a Thai translation of the Centers for Epidemiologic Studies Depression scale (CES-D). The independent variables measured reported lifetime and recent MA use within the past 3 months. Multivariate logistic regression models were used to assess associations between MA use and high levels of depressive symptoms.n = 394) of the sample reported ever having consumed MA and 31 % (n = 124) of lifetime users reported recent MA use within the past 3 months. Recent MA use was associated with high levels of depressive symptoms .Approximately 19 % (This is one of the first studies to examine the association between MA use and high levels of depressive symptoms in a general Thai population. The odds of having high levels of depressive symptoms was significantly greater among recent MA users compared to non-users. These findings support the need for policies, programs and interventions to prevent and treat depressive symptoms presenting among MA using Thai adolescents and young adults in rural Chiang Mai province, Thailand to aid in cessation of MA use. Furthermore, additional research is needed to investigate treatment options for adolescents and young adults in Thailand that use MA and present with high levels of depressive symptoms. This research demonstrates an association between methamphetamine and depressive symptoms, controlling for confounding, among a general population of Thai adolescents and young adults in Chiang Mai Province, Thailand.This research provides evidence to support the need for interventions to target this comorbidity among young people in rural areas of Thailand that are exposed to methamphetamine and serves as a foundation for further research into effective treatment of depressive symptoms and cessation of methamphetamine use among youth in rural Northern Thailand.Methamphetamine (MA) use became ubiquitous as a recreational drug among adolescents and young adults in Thailand beginning in the mid-1990s . The popn = 400) of study participants had a diagnosis of major depression within a year prior to admission, and another 44 % met the criteria for major depression but were excluded because their depressive symptoms were attributed to drug dependence [MA use has been shown to be associated with severe psychological harms and commonly is associated with depression . Studiespendence . In a stpendence . Most stThe nature of the relationship between depressive symptoms and MA use, abuse and dependence  and the prevalence of these comorbid conditions have public health implications for treatment and prevention that have received little attention outside of the United States. Thailand, which is currently experiencing an MA epidemic, warrants further attention in terms of prevention and treatment of these comorbid conditions . PopulatOur research attempts to add to the growing body of knowledge of mental health and substance use research emerging in Thailand using probabilistic sampling methods that improve the generalizability of results to the general population of adolescents and young adults in rural areas of northern Thailand where MA distribution and use is prominent. The primary purpose of this study is to describe the prevalence of depressive symptoms among MA users and to assess the association between MA use and depressive symptoms among Thais 14 – 29 years of age residing in rural areas of northern Thailand in close proximity to the Burma and Lao borders. We hypothesize that there will be an association between MA use and high levels of depressive symptoms.n = 2055). The study has been described in detail elsewhere [This study uses data from a baseline survey of behavioral data administered prior to the start of a community cluster-randomized community mobilization trial in six districts of rural Chiang Mai province in northern Thailand. Baseline data collection took place within a complex combination of nested cluster and systematic random sampling, beginning at the district level and proceeding with cluster sampling of sub-districts, communities and villages. Next, systematic random sampling was conducted to sample from a frame consisting of households and finally individuals, which yielded a representative sample of adolescents and young adults aged 14–29 years of age. The response rate during recruitment procedures was over 90 %  . The CESItems used to measure drug use behaviors were obtained from portions of the Risk Behavior Assessment (RBA) questionnaire . MA use p <0.10 and variables that have been identified in the literature as confounders were included in the final multivariate logistic regression model. Because the data were cross-sectional, prevalence odds ratios (POR) with 95 % confidence intervals were estimated to compare the odds of having high levels of depressive symptoms between reported MA users against a reference group of abstainers. Due to clustering of individuals by district, a sensitivity analysis was performed to account for the clustered nature of the data by incorporating the “xtlogit” command in Stata as an extension of multivariate logistic regression models [Comparisons were made between sociodemographic and substance use variables between those categorized as having high levels of depressive symptoms and those having low levels of depressive symptoms using chi-squared tests. Crude logistic regression was implemented to examine associations between lifetime and recent MA use and high levels of depressive symptoms. Crude logistic regression models were also conducted to assess associations between sociodemographic or drug use variables and high levels of depressive symptoms to assess potential confounders. Covariates that achieved a level of statistical significance of n models . Interacn = 2055) consisted predominately of young adults (n = 1247) with a median age of 20 who were predominantly single, separated, widowed or divorced (79 %), of Thai ethnicity (100 %), and Buddhist (99 %) and reported living primarily at their parent’s house during the past 3 months (77 %), currently attending school (59 %), and currently being unemployed (56 %). Approximately 19 % reported ever having consumed MA (n = 394) with 31 % of lifetime users reported recent MA use within the past 3 months (n = 124). Relative to those without reported baseline lifetime MA use, individuals reporting ever having consumed MA were significantly more likely to be male (p < 0.001).Descriptive statistics can be found in Table N = 2055) met criteria for exhibiting high levels of depressive symptoms. The mean CES-D score estimated for the entire sample was 17.01 (S.D.: 6.39). The range of CES-D scores was 0–57. Among lifetime MA users, the prevalence of high levels of depressive symptoms was approximately 12 % (n = 46) compared to 8 % of abstainers and 16 % among recent MA users compared to 10 % of abstainers. Relative to those who reported no lifetime MA use at baseline (Table p <0.05). Of those who reported a history of lifetime MA use, recent MA users were almost twice as likely to have high levels of depressive symptoms compared to nonusers . Compared to those without high levels of depressive symptoms, those defined as having high levels of depressive symptoms at baseline were more likely to be female (p < 0.001). Table p < 0.01).Approximately 9 % of the sample N = 055 met cWe examined demographic and behavioral variables identified in the literature as confounders. In our study, associations between high levels of depressive symptoms and age, gender and current school attendance were statistically significant Table . In otheHigh levels of depressive symptoms were associated with reported recent MA use. This study quantifies the prevalence of high levels of depressive symptoms comparing MA users to non-MA users in a general population of adolescent and young Thai adults. The study also examines the association between depressive symptoms and MA among Thai youth living in rural areas using a complex sampling strategy consisting of multistage, community-clustered and systematic random sampling. Such a study sampling method was necessary given the ubiquity of MA trafficking and distribution in this region. Consistent with our hypothesis, high levels of depressive symptoms were positively associated with MA use.We observed high rates of depressive symptoms in the entire sample and among MA users. In our study, approximately 12 % of individuals who reported ever using methamphetamine had high levels of depressive symptoms. The prevalence of depression among MA users is comparable with the estimates reported among psychostimulant users in the U.S. . There aThe relationship between MA and depression could potentially be bidirectional. Symptoms indicative of depression could lead to initiation and continuation of MA use. Consequently, affective and motivational dysfunction related to depression can occur as a result of chronic use and withdrawal, which could possibly contribute to the continuation of MA use for the maintenance of depressive symptoms. This maintenance of depressive symptoms is prolonged due to sustained use of drugs in greater amounts, more frequent use or injecting in order to relieve depressive symptoms resulting from chronic use . One lonOf note is the observed independent crude and adjusted association between gender and high levels of depressive symptoms. Of the participants that endorsed high levels of depressive symptoms, 6 % were male compared to 12 % of females. On the other hand, approximately 33 % of males reported recent MA use compared to 23 % of females and explains the lack of effect modification by gender. We had hypothesized a priori that gender would be a confounder since previous literature has documented higher levels of depressive symptoms in females, whether induced by substance use or independent of substance use, and has documented that males are more likely to use illicit substances than females. Other covariates were added to the multivariate model if they met the aforementioned criteria for being included, but had little effect on the estimate describing MA use and high levels of depressive symptoms.Treatment of depressive symptoms presenting with substance use and dependence of MA has been problematic. Several randomized controlled trials conducted to evaluate the efficacy of various antidepressants in relieving depressive symptoms and achieving cessation have found treatment to be inconsistently efficacious among individuals who were dependent on or abused MA or cocaine –30. All There are some limitations inherent in this study that must be considered. First, the study was cross-sectional. A temporal relationship between MA use and high levels of depressive symptoms cannot be established. Second, there could be underreporting of stigmatized behaviors and stigmatization of MA use by Thai communities remains a concern among youth . MisclasThis study presents strengths relevant for drawing public health implications. The large sample size of the study population, the probabilistic sampling methodology, and the extensive coverage of several communities within sub-districts are strong characteristics of this study. Relatively little research has been conducted in Thailand that quantifies the prevalence of high levels of depressive symptoms among adolescents and young adults who use MA, either in clinical samples or the general population. Little mental health or substance abuse research, in general, has been conducted in Thailand, but recent efforts to address these knowledge gaps are leading to a burgeoning field of substance use and mental health research in Thailand, and this study contributes to that research base. Most notably, an assessment of Thailand’s mental health care services by the Thai Ministry of Health and the World Health Organization specified that only 1 % of health-related research published in peer reviewed journals in Thailand is mental health research . To our Additional longitudinal and qualitative studies should be conducted to examine how other individual-level factors and multiple-level factors  predict the onset of depressive symptoms among Thai youth to better understand the individual-level and structural factors driving the onset of depression. Longitudinal studies should also be designed to assess if depressive symptoms precede or are a consequence of substance use.Generally, this study could be used as the basis to begin the evaluation of the effectiveness of treatment and prevention interventions for depressive symptoms associated with MA use in Thailand as well as other low- and middle-income countries. The positive association seen in our study suggests that accessible and culturally acceptable mental health care services should be integrated with drug treatment programs among drug-dependent youth. The results also indicated that policies aimed at maximizing resources in rural Thailand to deliver more humane and evidence-based approaches to care and treatment of depressive symptoms among MA users could contribute to the prevention of MA use and the reduction of depressive symptoms among Thai youth.Associations between MA use and high levels of depressive symptoms were observed. Given the ubiquity of MA use among adolescents and young adults in this region of Thailand, resources should target this population for the provision of culturally acceptable mental health care services for MA-dependent Thai youth.


Whether transgene flow from crops to cross‐compatible weedy relatives will result in negative environmental consequences has been the topic of discussion for decades. An important component of environmental risk assessment depends on whether an introgressed transgene is associated with a fitness change in weedy populations. Several crop‐weed pairs have received experimental attention. Perhaps, the most worrisome example is transgene flow from genetically engineered cultivated rice, a staple for billions globally, to its conspecific weed, weedy rice. China's cultivated/weedy rice system is one of the best experimentally studied systems under field conditions for assessing how the presence of transgenes alters the weed's fitness and the likely impacts of that fitness change. Here, we present the cultivated/weedy rice system as a case study on the consequences of introgressed transgenes in unmanaged populations. The experimental work on this system reveals considerable variation in fitness outcomes ‐ increased, decreased, and none ‐ based on the transgenic trait, its introgressed genomic background, and the environment. A review of similar research from a sample of other crop‐wild pairs suggests such variation is the rule. We conclude such variation in fitness correlates supports the case‐by‐case method of biosafety regulation is sound. Weeds are great challenges for crop production, particularly those that are the same biological species (conspecific) as the crop they infest. For example, weed beets infest sugarbeet fields, and weedy rice infests cultivated rice fields. The phenotypic similarity of such conspecific weeds to the related crops often frustrates visually based hand weeding. Also, genetic similarity means that the crop and the weed are so physiologically similar that herbicide must be applied on the weed with great precision to prevent application on the crop, again requiring visual discrimination Barrett . Becausevia pollen‐mediated gene flow  crop species has stimulated discussion about whether crop‐to‐weed and crop‐to‐wild transgene flow might have an ecological or environmental impact . These wild taxa not only occur as weeds of rice but also as weeds for some other crops . A recent study suggested that the application of hybrid rice may also result in the origin and evolution of some weedy rice populations in China .Broad definitions of weedy rice involve “plants of the genus et al.'s  definitiThe traits of weedy rice relative to its crop ancestor enumerated above enable it reproduce and persist in rice fields for years. The close evolutionary relationship of weedy rice with its cultivated and wild progenitors results in sporadic multiway gene flow in sympatry or peripatry Fig. . Thus, wThis trend reflects the adaptive evolution of weedy rice in human‐managed agricultural environments. Pre‐existing variation and that provided by gene flow has enabled the evolution of crop mimicry; that is, weedy rice typically mimics the phenotypic and physiological characters of its locally co‐occurring rice cultivars. Crop mimicry is an evolutionary response to selection under farmers’ visually guided hand weeding Barrett . Obvious® herbicide‐tolerant rice variety, a product of “mutation breeding” is grown in USA, South and Central Americas, southern Europe, and Southeast Asia  the intended expressed transgene phenotype; (ii) the typical gene flow rates from the crop to the weed; (iii) the phenotype of weed with an introgressed transgene in terms of their ability to reproduce and increase relative to the nonintrogressed weed; and (iv) the introgressed weed's relative ability to reduce the yield of their sympatric crop cultivar. To address these factors, we examine the state of the art in China, is the primary site of GE rice development, for the various intended GE phenotypes being field released and in line for commercialization.China has invested substantially on developing biotechnology  in China has effectively controlled cotton's major target insect pest, cotton bollworm (Helicoverpa armigera Hubner)  in 2009, after nearly 15 years of assessment for food safety and environmental safety (Lu http://www.stee.agri.gov.cn/biosafety/spxx/t20091022_819217.htm). In addition, five other GE lines were also approved for field experiments, including Kemingdao (KMD) (expressing a cry1Ab transgene), T1c‐9 (cry1C transgene), T2A‐1 (cry2A transgene), and Kefeng‐6 and Kefeng‐8 (both expressing tightly linked cry1Ac/CpTI transgenes) . The transgene expresses the enzyme phosphinothricin‐Acetyl transferase which converts PPT into a nonphytotoxic metabolite.The development of herbicide‐tolerant GE rice in China has also developed rapidly in the last two decades, motivated by China's recent shift from manual or mechanical weed management to chemical control. As direct seeding rapidly replaces seedling transplantation, highly efficient herbicide‐tolerant GE rice lines are expected to reduce rice production costs. The first herbicide GE rice line containing a Agrobacterium sp. strain CP4)  expression by adding the strong ubiquitin promotor from maize, or/and the induced mutation epsps 102 created via the error‐prone polymerase chain reaction   traits. Transgene stacking can be achieved by various methods such as cotransformation with different transgenes, hybridization between GE lines with different transgenic events, linked transgenes or multigene cassette transformation, and retransformation Halpin . Many weBy the end of 2010, more than 170 GE rice lines created in China had entered biosafety assessment trials authorized by Chinese government Zhu . NonetheSpontaneous pollen‐mediated gene flow between domesticated crops and spatially adjacent wild/weedy taxa is not uncommon  and herbicide tolerance genes are used as selectable markers in GE rice transformation. They also have been utilized as markers in various experiments to detect the frequencies of rice transgene flow   with their weedy (or wild) relatives as illustrated in Fig. The fitness effects of a transgene are expected to be largely determined by the type of transgenes (e.g. insect resistance and herbicide resistance) incorporated in wild/weedy population the type of anticipated selection pressure in the environment, and the amount of admixture in the transgenic hybrid lineages  to cross with weedy rice populations from different geographic locations. Bt and CpTI transgenic rice lines were developed to deter lepidopteran pests, such as rice stem borers and rice leaf‐folders. Several generations of crop‐weed hybrid progeny (F1–F7) with transgene‐present and transgene‐absent lineages were produced to study the long‐term fitness effect of the insect‐resistance transgenes. We compared an array of fitness‐related traits  for these hybrid lineages, as well as their crop and weedy progenitor under both natural‐insect and low‐insect pressure. The multiyear experiment found the following general trends:We have conducted multiyear common‐garden experiments to estimate the fitness effect of insect‐resistance transgenes that transformed into cultivated rice . In a glyphosate‐free environment, they found that the transgenic hybrid lineage had higher seed production, greater EPSPS protein levels, tryptophan concentrations, photosynthetic rates, and percent seed germination compared with nontransgenic controls. These results suggest that phenotypic changes associated with such an epsps transgene could result in faster establishment and increased competitive ability of the transgene‐bearing individuals compared to nontransgenic weedy rice and cultivated rice.With regard to herbicide‐resistance transgenes, Oard et al.  conducteg et al.  carried Other field studies of weedy rice individuals with introgressed non‐GE crop herbicide resistance have shown fitness advantages related with faster germination  traits to battle the complex of insect pests of rice and other yield constraints in rice  In contrast, herbicide‐resistance transgenes should deserve more attention since the herbicide pressure in GE rice field will always favor the spread and fixation of any herbicide‐resistance transgene in weedy rice population, unless effective mitigation strategy to be developed to minimize the occurrence of gene flow or reduce the fitness of crop‐weed hybrids.The environmental biosafety assessment of the consequences of crop transgene flow to weedy rice populations through artificially created crop‐weed hybrid lineages and subsequent fitness testing has proven costly and time consuming gene flow in rice occurs at a relatively low frequency (0.001–2.3% per generation), transgene introgression into weedy population is essentially inevitable because weedy rice and cultivated rice co‐occur and year‐after‐year in the same fields. Transgene flow from GE rice to weedy rice can result in diverse fitness effects, depending on the type of transgenes and the selective pressure to which the GE crop‐weed hybrid descendants are exposed. In addition, other factors such as the genetic background of weedy rice populations that have obtained the transgenes may also influence the fitness effects of a particular transgene  confer a fitness benefit for crop‐weed rice hybrid progeny under high insect pressure  to weedy rice populations appears to considerably change the fitness of the crop‐weed hybrid progeny, both with and without the application of glyphosate herbicide sprays, and possibly the evolutionary potential of the hybrid progeny by altering their rate of biosynthesis and photosynthesis  through genetic engineering in China.Click here for additional data file.


IENFD) is useful in the evaluation of small‐fiber neuropathy (SFN). Recent guidelines recommend extending the spectrum of controls for IENFD assessment by evaluation of patients whose clinical picture mimics that of SFN. The aim of this study was to broaden the spectrum of IENFD controls by the assessment of patients with cervical spondylotic myelopathy (CSM) and painful feet.Intraepidermal nerve fiber density (IENFD from skin biopsy samples and quantitative sensory testing (QST) were performed in a cohort of 14 CSM patients , with painful feet, exhibiting no clinical or electrophysiological signs of large‐fiber polyneuropathy, and no risk factors for peripheral neuropathies.Evaluation of CSM patients (86%), while the IENFD values were within reference range. The mean IENFD value (6.87 ± 2.78 fibers/mm) did not differ from that of an age‐ and sex‐matched cohort of healthy volunteers .Quantitative sensory testing abnormalities were found in all but two of the CSM as one of the clinical conditions mimicking SFN and provided further support for the use of IENFD assessment in case of suspicion of SFN.The study confirmed normal skin biopsy findings in patients with  In recent years, skin biopsy with assessment of intraepidermal nerve fiber density (IENFD) has increasingly been used in the evaluation of small‐fiber neuropathy (SFN)  abnormities in this distribution, may also be found in certain other clinical conditions, particularly those affecting central somatosensory pathways; these may be confused with SFN. Recently, this emerged in a study of patients with multiple sclerosis and restless legs syndrome  pain intensity of at least 3 as assessed by a numerical rating scale ranging from 0 to 10, in which 0 represented “no pain” and 10 “the worst pain I can imagine”; (2) no risk factors for peripheral neuropathies in the medical history ; (3) normal or increased deep tendon reflexes in the lower legs; (4) normal nerve conduction studies in the lower extremities, to exclude large nerve‐fiber involvement in this distribution; and (5) absence of other diseases or conditions leading to foot pain . Finally, only 14 patients fulfilled inclusion criteria and were willing to participate  Emery . SeveritStandard short‐latency SEPs from the median and the tibial nerves were elicited with electrical stimulation of mixed nerves at the wrist and the ankle, and recorded using a Nicolet four‐channel Viking II unit.Motor evoked potencials (MEPs) were elicited using a MAGSTIM 200 magnetic stimulator and circular 90‐mm (type 9784) stimulating coil with a peak magnetic field strength of 2.0 T. On‐line data acquisition was performed using a Dantec Keypoint electromyograph. MEPs were elicited by means of transcranial and root magnetic stimulation, and recorded from abductor digiti minimi and abductor hallucis muscles on both sides.The details of the testing procedures of both SEPs and MEPs were previously published in detail   as primary antibody, and goat anti‐rabbit IgG labeled with fluorescein probe as secondary antibody . The intraepidermal nerve fibers were counted manually at ×630 magnification using a Leica DMLB microscope . The epidermal length of the skin section was measured with calibrated software . The average intraepidermal nerve fiber density (IENFD) per millimeter of epidermal length was calculated according to current guidelines  which corresponds with high values of mJOA score in all the cases Table . ClinicaCentral conduction abnormalities in somatosensory or motor‐evoked potentials were found in a small proportion of the cases due to associated lesions of the corticospinal tract and dorsal columns at the cervical level.Quantitative sensory testing protocol revealed numerous sensory abnormities in the lower extremities in most of the participants Table . Only twx‐2 SD and x‐2.5 SD of the reference data set, while all the other values did not exceed normal range (Table P > 0.05)  pattern of involvement mimicking SFN, which is supposed to result from spinothalamic tract dysfunction and thus is not reflected by any abnormality in SEP examination. Central conduction abnormality of somatosensory‐evoked potentials results from the lesion of dorsal spinal column, while dysfunction of the dorsal horn in the lower cervical spine leads to segmental abnormality of N13 wave (only).The main limitation of this study is sample size of CSM patients, which proved impossible to expand despite considerable effort, primarily as a result of the strict inclusion criteria. At the beginning of the study, quite a large cohort of CSM patients being followed up in our department were screened for the presence of symptoms and signs similar to those seen in SFN. The prevalence of CSM, however, is significantly higher in older patients, among whom there are only a few individuals with a complete absence of risk factors for peripheral neuropathies together with normal large nerve‐fiber function according to nerve conduction studies. On the other hand, the inclusion criteria were strict enough to exclude as many patients with suspected coincidence of CSM and peripheral neuropathy (as a confounding factor for such a type of study) as possible.Two of the CSM patients in this study showed borderline IENFD values according to our own reference data. Despite very strict inclusion criteria, the possible association of CSM and SFN cannot be excluded in these individuals. In a recent study, an overall minimum prevalence of SFN, about 52.95 per 100,000 individuals, was revealed, with an increasing incidence and prevalence in elderly patients compared with younger ones (Peters et al. None declared.


Australian farmers and their workers are exposed to a wide variety of pesticides. Organophosphate (OP) insecticides are a widely used class of pesticide used for animal husbandry practices , crop production for pest control (Dimethoate) and in public health . Acute poisonings with this class of insecticide are reported among agricultural workers and children around the globe, due to the inhibition of acetylcholinesterase (AChE). Less is known about chronic exposures. Regular monitoring of erythrocyte AChE will enable farmers to identify potential exposure to organophosphate insecticides and take action to reduce exposures and improve their health and safety practices. This study aims to assess and improve the integration of AChE monitoring into routine point of care health clinics, and provide farming and non-farming people with a link between their AChE activity and their household chemical and agrichemical use.n = 50) and non-farmers (n = 30). Baseline data are collected regarding demographic, health conditions and behaviours, Kessler 10 (K10) scores, chemical use and personal protection. Baseline anthropometric measures include height, weight, hip and waist circumference, body fat analysis and, biochemical analysis of fasted total serum cholesterol, triglycerides, low-density cholesterol (LDL), high-density cholesterol (HDL) and blood glucose. Analysis of erythrocyte cholinesterase (EAChE) activity is also conducted using a finger prick test. Testing of EAChE is then repeated in all participants every 3 weeks for a maximum of three times over a period 10 weeks. Participants are provided with full feedback and counselling about their EAChE activity after each reading and a detailed summary provided to all participants at the completion of the study. Data will be analysed using repeated measures within a general linear model.The research will target individuals who work on mixed farming enterprises and routinely using OPs (This work will provide an evidence base and recommendations for the integration of EAChE monitoring into Australian rural health clinics, leading to research which will further quantify pesticide exposure both on the farm and in the home, highlighting the importance of sustaining and providing a safe work and home environment for farming communities.ACTRN12613001256763 Australian farmers and their workers are exposed to a wide variety of pesticides . PesticiThe acute effect of OP exposure is well documented in Australia and overseas for humans, pests and animals , 6, 9. HUnderstanding the mode of action of a chemical is a critical component of the risk assessment process. Cholinesterase activity is a direct link to exposure and this pilot study will produce valuable data on possible exposures for farmers routinely using OP’s for animal husbandry or crop production.Monitoring cholinesterase levels during agricultural health screening and assessment is a key objective of Agrisafe™ clinics in Australia. However, its incorporation into such clinics will depend on health professional’s skills and knowledge. This study will further prepare health care providers for integration of correct cholinesterase testing procedures into point of care (POC) health checks. This work will further highlight pesticide exposure as a risk for farmers, workers and their families, leading to an easily adopted method that will quantify the environmental exposures providing early detection. Research has also shown links between cholinesterase activity and vascular complications in diabetic patients . DiabeteThe Cholinesterase Research Outreach Project (CROP) will use clinical sampling to determine the level of exposure associated with the routine use of OPs by Victorian farmers and/or their workers. This preliminary study will examine the methodology of testing for EAChE activity at POC and provide evidence for future work by identifying the exposure patterns and pathways of individuals most at risk in farming communities. It is anticipated that CROP will inform future research studying pesticide exposure in farming communities.To evaluate if a difference in cholinesterase activity was present in farmers before and after exposure, sample size is determined at the power of 80 % (β = 0.2) with α set at 0.05 for an effect size of one standard deviation. This requires the sample size to be a minimum of 30 farming individuals exposed to pesticides. By considering the probable retention rate (≥85 %) for a 7-month study, based on previous research in similar populations,  the targConsent will be obtained from 80 participants recruited via existing contacts and industry groups, letterbox drops and newspaper articles from a number of farming and community groups, specifically;n = 50) who are using OPs and a convenience group of non-farming individuals (n = 30) from the rural community of Hamilton, Australia.Mixed farming enterprises and sheep dipping/spraying contractors  will be obtained from participants with baseline physical assessments of blood pressure measurement (repeated twice), pulse rate, body fat percentage , respiratory function test (PiKo meter) also measured  . The typA validated questionnaire, the Kessler 10 (K10) will be used to measure psychological distress. The K10 is a 10-item questionnaire intended to yield a global measure of distress based on questions about anxiety and depressive symptoms that a person has experienced in the most recent 4 week period . This suOccurrence of illness or injury experienced within the last 3 months will be measured using a questionnaire describing 33 symptoms of chemical exposure, in random order of severity. The illness and injury data will be standardised and analysed accordingly.In addition to completion of the agrichemical use survey of 13 questions, self-reported chemical use is recorded during each visit. Participants are provided with counselling regarding their EAChE levels in accordance with AgriSafe guidelines Fig. . The USAp < 0.05.All data will be managed and analysed within the statistical program SPSS . SPSS is a statistical package widely used in quantitative social science research and is suitable for multivariate analysis. Data will be analysed using repeated measures (subjects as own controls) within the General Linear Model. Correlation coefficients between pesticide exposure and metabolic variables will be calculated using Pearson’s and Spearman’s correlation as appropriate. P values will be considered statistically significant at All adults participating in the study are to be provided with a plain language statement and will provide informed written consent. Ethics approval has been granted to the project by Deakin University Human Research Ethics Committee (HREC 2013-100 dated 18/06/2013). All researchers involved in data collection have a Victorian Police check undertaken.Organophosphates are still used widely with the actions and attitudes of farmers and workers using these pesticides is vital to their wellbeing and that of their families. It is common for mild to chronic depression of EAChE activity to be reported as ‘normal’ due to the wide reference range for EAChE activity . It is i


To investigate associations of pain intensity in those with long‐term back pain, with their partners' rating of key constructs of relationship quality: cohesion (activities together), consensus , satisfaction .CI) are reported.Self‐report questionnaires on relationship quality (partner‐rated), depression (partner‐rated), relationship length, and pain intensity (patient‐rated) were collected from back pain patients and their partners (N = 71). Linear regression was carried out to test for associations, standardized coefficients (β) and 95% confidence intervals . Furthermore lower ratings of consensus were reported where patient pain intensity interacted with partner depression .There was no main effect between patient pain intensity and partner rating of relationship quality. However, partner ratings of relationship quality were lower if the partner reported increasing depressive symptoms. Adjusting for the effects of partner depression show that ratings of consensus  from partners were actually higher with increasing levels of pain intensity in patients (β 0.54, 95% These findings illustrate the association of pain outcomes beyond the patient within a primary care sample. Moderators of the responses about the relationship construct of consensus generated by partners appear to be partners' own level of depressive symptoms and whether their depressive symptoms are associated with the patients' pain intensity. Consultations should consider the social context of patients with pain. Long‐term back pain is a common and major health concern, with a lifetime prevalence estimate of over 70% in industrialized nations It is generally accepted that the experience of back pain is shaped at a biopsychosocial level However, more recent research has revealed a more nuanced understanding of a complex relationship between partners where one has pain. For example Cano et al. In this current study, we consider the association between patients' reported pain intensity and their partners' rating of relationship quality  to ascertain whether patient pain intensity associates with judgements of the relationship quality in partners. Consistent with previous literature This is a cross‐sectional study nested within a larger longitudinal follow‐up study of patients seeking primary health care for low back pain (LBP).Full details of the original study can be found elsewhere Ethical approval was obtained from the North Staffordshire and Central Cheshire Research Ethics Committees for all stages of this study.Pain intensity was measured by calculating the mean of three numerical rating scales  for the patient's least, usual, and current (at the time of filling in the questionnaire) levels of LBP over the previous 2 weeks. Higher scores indicate higher levels of reported pain intensity The Hospital Anxiety and Depression Scale (HADS) was used to assess the partner's level of depressive symptoms. The HADS contained seven items of depressive symptoms; item scores ranged from 0 to 3; scale scores ranged from 0 to 21, and higher scores indicated greater levels of depressive symptoms The Revised Dyadic Adjustment Scale (RDAS) Information on gender, age, and length of relationship (years/months) were collected from both patients and their partners.Pearson's correlations were first used to describe the associations between the partner‐rated constructs of relationship quality, patient‐rated pain intensity, partner's depressive symptoms and other variables. The association of patient‐rated pain intensity and partner‐rated relationship constructs were tested using three separate hierarchical linear regression models, one for each of the relationship constructs . Each regression model contains four hierarchical analysis steps. Step one tested the direct unadjusted association of patients' rated pain intensity with the partner‐rated relationship quality construct. Step two adjusted the step one analysis for partner depressive symptoms. Step three adjusted for any interaction between partner depressive symptoms and patient's pain intensity. Finally step four carried out adjustment for partner age, gender, and the length of time (in years) of the relationship between patient and partner. This stepped model was chosen in order to consider direct associations between patient‐rated pain intensity and partner‐rated relationship quality constructs, to adjust for the potential influence of depressive symptoms on judgements of relationship quality  t‐tests showed no significant differences between patients and partners on their age , and no significant differences between patients and partners on their judgements of relationship length . Table In total, 299 patients indicated they had a current partner, and 71 (24%) partners responded. We have no information on how many patients passed on partner questionnaires to their partner. Patients with partners who responded were more likely to be female (67% vs 61%), were significantly older (57 years vs 53 years), reported a significant, longer length of relationship with their current partner (32 years vs 25 years), and reported significantly higher levels of pain intensity (mean 3.2 vs 2.4). The mean ages of partners (58.9 years) and patients (58.1 years) were similar. Gender distribution showed that 69% of patients were female, this corresponded to 69% of the partners being male; there was no same‐sex relationships reported within this cohort. Partners indicated that they had been in a relationship with the patient for a mean of 32 years. Paired sample Table P = 0.54, consensus β −0.09, P = 0.47, satisfaction β 0.14, P = 0.28). Adjustment for partner‐rated depressive symptoms at step two showed no change in the nonsignificant associations between pain intensity and the relationship quality constructs, with partner depressive symptoms being the only consistent variable to be associated with all partners' rated relationship quality variables . Results at step three  show for the cohesion model, no association of patient pain intensity and partner ratings of cohesion, and no association for the interaction between patient pain intensity and partner depressive symptoms. Only partner‐rated depressive symptoms associated with cohesion, with higher levels of depressive symptoms being associated with lower ratings of cohesion. Similar results were found for the satisfaction model. However, results for consensus indicated a significant association between patient pain intensity and partner ratings of consensus, with the direction being positive . As with the models for cohesion and satisfaction, partner depressive symptoms were negatively associated with consensus. There was also a significant association between the interaction term (combined effects of patient pain intensity and partner depressive symptoms) and consensus, the direction was negative . The inclusion of confounders in the final step of the regression models  did not significantly alter the reported associations. Model fit testing (ANOVA F tests for regression models) showed that all models were not significant at step 1 (inclusion of pain intensity), but all models were significant at all subsequent steps. Table Results at step one  found no direct unadjusted association between patient pain intensity and any of the partner‐rated relationship constructs  can be found for all relationship quality constructs within Table The theoretical model by Cano and Leong Even though this study has shown that relationship length did not play a significant role within the regression analyses, it may still be an important factor. This study included partners with a mean average relationship length of 32 years, the minimum relationship length being 7 years. Added to that is this cohort of patients reported the presence of LBP over 5 years previously, which is suggestive that both themselves (patients) and their partners have been exposed to, and to some extent are still exposed to the influence of pain. Research suggests that couples, where one has a chronic illness, can over time adapt and accommodate to the illness and that this can have a beneficial effect on the relationship A key strength of this study is the use of data provided by both the patient and their partner as this gives a broader view point of the potential cross‐over between patient‐rated variables and partner‐rated variables. Many previous studies have reported only patient's perspectives on relationship quality which may be influenced by the patient's mood state and level of pain A further strength of this study is the inclusion of patients and their partners from a primary care sample. Many previous studies, which have included partners, have included patients from secondary care settings  or via media advertisements which may have limited generalizibility to community samples A weakness of the study is the low response rate for partners (24%). We have no way of ascertaining how many partners actually refused to take part as we relied on the patient passing a partner questionnaire over to their partner. It may be that partners with lower relationship quality did not wish to participate. Examining the differences of patient outcomes between those who had a partner who responded and those who did not respond showed that included patients were older, had lived with their partner longer, and reported a greater level of pain intensity. This may have reduced the generalizibility of the results, and further research within different cohorts would be needed to establish if such effects are similar for couples who are younger and/or have less relationship time with their partner. This study is also cross‐sectional, and therefore, we cannot make any assumptions about cause and effect. While stratification is a valid way of investigating interaction analyses Increasing consideration has been given to the inclusion of family members  in the treatment of those with chronic illnesses In conclusion, this study has shown that patient pain intensity is associated with the relationship quality rating of their partners, significantly an effect is found for the consensus aspect  of their relationship quality. The findings show this effect is elicited and moderated by the partner's level of depressive symptoms, but when this depression is statistically controlled, the relationship between patients' pain intensity and partners' ratings of consensus is positive. The results of this study demonstrate the associations of patients' pain intensity beyond the patient, and that social context should be considered when evaluating the impact of pain.


CRX) gene, a key transcription factor in retinal development. Zinc finger nucleases (ZFNs) designed to cleave in the 3′ UTR of CRX were transfected into hESCs along with a donor construct containing homology to the target region, eGFP reporter, and a puromycin selection cassette. Following selection, polymerase chain reaction (PCR) and sequencing analysis of antibiotic resistant clones indicated targeted integration of the reporter cassette at the 3′ of the CRX gene, generating a CRX‐GFP fusion. Further analysis of a clone exhibiting homozygote integration of the GFP reporter was conducted suggesting genomic stability was preserved and no other copies of the targeting cassette were inserted elsewhere within the genome. This clone was selected for differentiation towards the retinal lineage. Immunocytochemistry of sections obtained from embryoid bodies and quantitative reverse transcriptase PCR of GFP positive and negative subpopulations purified by fluorescence activated cell sorting during the differentiation indicated a significant correlation between GFP and endogenous CRX expression. Furthermore, GFP expression was found in photoreceptor precursors emerging during hESC differentiation, but not in the retinal pigmented epithelium, retinal ganglion cells, or neurons of the developing inner nuclear layer. Together our data demonstrate the successful application of ZFN technology to generate CRX‐GFP labeled hESC lines, which can be used to study and isolate photoreceptor precursors during hESC differentiation. Stem Cells2016;34:311–321The purpose of this study was to generate human embryonic stem cell (hESC) lines harboring the green fluorescent protein (GFP) reporter at the endogenous loci of the Cone‐Rod Homeobox ( Degeneration of retinal pigmented epithelium (RPE) and photoreceptors are a frequent cause of vision impairment and blindness associated with many inherited retinal diseases and age related macular degeneration (AMD). Replacement of these cells with stem cell derived equivalents provides an excellent approach to preserve retinal structure, function and vision. Two clinical trials are at the moment focusing on replenishment of degenerate retina in Stargard't disease and AMD with human pluripotent stem cell derived RPE. However the same momentum has not been achieved for replacement of photoreceptor cells mainly due to our incomplete understanding of the best stage of achieving pluripotent stem cell derived photoreceptor maturation and integration and lack of cell surface markers to purify these cells at ease. For this reason, we have used zinc finger nuclease technology to introduce a reporter gene into the endogenous locus a key transcription factor, CRX, known to be expressed in postmitotic retinal photoreceptor precursors and to play a key role in photoreceptor genesis and maturation. We have generated and fully characterised this reporter labelled human pluripotent stem cell and have shown that genetic manipulation occurs only at the chosen locus and does not interfere with the maintenance of pluripotency. Furthermore, this approach has enabled us to mimic the expression of the CRX gene and to assess these cells during pluripotent stem cell differentiation. Our data strongly suggest that CRX is expressed in photoreceptor precursors during pluripotent stem cell differentiation, thus providing us with a tool to study and purify the molecular expression profile and engraftment capacity of these cells. To the best of our knowledge, this is the first fully characterised reporter stem cell line of endogenous CRX in emerging photoreceptor precursors and for this reason and others stated above should be of wide interest to the readers of Stem Cells.FokI endonuclease domain , a DNA cleaving domain which operates upon dimerization Zinc finger nucleases (ZFNs) are designer nucleases which can be engineered to target a specific DNA sequence, offering huge potential for genetically modifying cells with complex genomes, such as mammalian cells −6One important application is genetic modification of human pluripotent stem cells. Gene targeting of mouse embryonic stem cells using classical homologous recombination‐based methods has been widely used, however, such conventional gene targeting approaches are not easily transferred to human embryonic stem cells (hESCs), mainly due to a substantial reduction in efficiency during early postnatal development display a rod phenotype upon transplantation, while those isolated during the embryonic peak of cone genesis and which express Crx (cone‐rod homeobox gene), display a cone phenotype following transplantation with a shift towards the rod phenotype if isolated from postnatal retina Nrl‐ and Crx‐expressing cells isolated from postnatal murine retina. To begin to answer some of these questions, we set out to generate hESC lines that harbor reporter genes under the control of key transcription factors known to play an important role in retinal photoreceptor cell commitment. We chose CRX as a candidate for the following reasons: (a) CRX expression is localized to post‐mitotic photoreceptor precursors before the development of outer segments Crx plays an essential role in cone and rod genesis CRX gene 3′ UTR and enable the introduction of a GFP reporter at the 3′ terminus of this gene. Furthermore, expression of the GFP reporter mimics the expression of endogenous CRX during the differentiation of hESCs towards a retinal lineage, enabling the fluorescent labeling of CRX‐expressing cells with GFP during the differentiation process. Immunocytochemistry with various retinal markers up to day 90 of hESC differentiation indicates that CRX expression is observed in hESC‐derived photoreceptor precursors which have exited the cell cycle and lack the expression of well‐established markers characterizing the early eye field, retinal ganglion cells, and the inner nuclear layer. Together, these data suggest that CRX‐GFP labeled hESC lines created using ZFNs provide an excellent tool with which to study retinal development in vitro and photoreceptor precursors at various stages of differentiation, which can be utilized to help define the integration potential of hESC‐derived photoreceptor precursors in the intact and diseased retina.To date, reporter‐based studies for the selection of retinal progenitor cells have been very successful in the mouse The H9 hESC line from WiCell Inc. was used in this study. Expansion of hESCs was performed on feeder cells as described previously 5 cells then underwent nucleofection with 2 µg of each ZFN mRNA using the Human Stem Cell Nucleofector Kit 1 (Lonza) with a Nuclefector 2b device (Lonza). Following nucleofection cells were cultured as before, with the addition of Y‐27632 for 24 hours, and genomic DNA extracted  48 hours later. The frequency of cleavage products was then assessed using the SURVEYOR (CEL‐I) Mutation Detection Kit (Transgenomic), following the manufacturer's instructions) Supporting Information Fig. 1B). Briefly, polymerase chain reaction (PCR) of the region harboring the target site of the ZFN pair was performed from the genomic DNA (for primers see Supporting Information Table 1), the PCR products were denatured and then cooled to anneal oligonucleotides, followed by incubation with the CEL‐I enzyme at 42 °C for 40 minutes. The digests were then analysed by polyacrylamide gel electrophoresis and the fraction of cleaved products assessed.The ZFN pair used was designed, produced, and activity tested in K562 cells by Sigma‐Aldrich , following manufacturer's guidelines. Briefly, colonies were fixed in 4% paraformaldehyde, permeabilized with 1% Saponin, blocked with 3% bovine serum albumin (BSA), incubated with antibodies against OCT4 and SSEA4 (see Supporting Information Table 3 for details) for 3 hours, washed in phosphate‐buffered saline (PBS), incubated with appropriate secondary antibodies for 1 hour, washed, incubated with 4′,6‐diamidino‐2‐phenylindole, and imaged on a Nikon A1R confocal microscope. At least 10 colonies from each clone were assessed for pluripotency.Pluripotency of the H9 CRX‐GFP clones was then tested by spontaneous differentiation and assessment of the expression of germ layer markers identifying ectoderm (beta‐III tubulin), endoderm , and mesoderm (smooth muscle actin). Colonies were dissociated by collagenase IV (Life Technologies) and allowed to form embryoid bodies (EBs) in suspension in low‐attachment plates containing a general differentiation medium . Medium was changed daily for 7 days, EBs were then transferred onto gelatin‐coated chamber slides and cultured for a further 7 days. Presence for markers of the germ layers were then assessed using the 3‐Germ Layer Immunocytochemistry Kit (Life Technologies) following manufacturers guidelines. Briefly, colonies were fixed in 4% paraformaldehyde, permeabilized with 1% Saponin, blocked with 3% BSA, incubated with beta‐III tubulin, alpha‐fetoprotein, and smooth muscle actin antibodies (see Supporting Information Table 3 for details) for 3 hours, washed in PBS, incubated with appropriate secondary antibodies for 1 hour, washed, incubated with 4′,6‐diamidino‐2‐phenylindole, and imaged on a Nikon A1R confocal microscope. At least five EBs from each clone were assessed for markers of the three germ layers.CRX homology arm sequences (see Supporting Information Table 1) and genomic DNA extracted  from puromycin‐resistant colonies. PCR products were resolved on a 1% agarose gel. Sequencing was performed by Eurofins Genomics on genomic DNA extracted from puromycin‐resistant colonies and using primers flanking and residing across the donor construct (for primer details see Supporting Information Table 1).To detect the presence or absence of correctly targeted insertion of the GFP reporter cassette, PCR was performed using GoTaq Long PCR Master Mix (Promega), primers flanking the 5′ and 3′Copy number analysis of GFP puromycin‐resistant clone 1 genomic DNA was carried out using a quantitative PCR (qPCR) approach with TaqMan Copy Number Assays and Genotyping Master Mix (Life Technologies) following manufacturers guidelines. Briefly, a duplex qPCR reaction was performed with an RNase P reference and GFP assay (Supporting Information Table 1) to quantify the copy number of GFP using clone 1 genomic DNA alongside untreated wild‐type H9 hESC genomic DNA. Four technical replicates were performed for each sample. Reactions were run on a 7500 Fast Real‐Time PCR System (Life Technologies) and data analyzed in Copy Caller software (Life Technologies).For the prediction of potential off‐target binding sites for the 3′ CRX ZFN pair, the PROGNOS online tool EBs resulting from the directed differentiation of hESCs towards a retinal phenotype were collected at days 30, 60, and 90 of differentiation and immunocytochemistry analysis performed on cryostat sections as described previously t‐test.EBs were disassociated into single cells using 0.05% Trypsin for 30 minutes. Trypsin was inactivated with serum‐containing media and the cells washed with PBS. Cells were sorted on a FACS Aria (BD) into GFP positive and negative fractions.RNA was extracted from each fraction , cDNA synthesized , preamplified , and qPCR performed  using a QuantStudio7 system and software (Life Technologies). For TaqMan assays used see Supporting Information Table 1. Statistical analysis was performed using GraphPad Prism software (Version 6.05) with statistical significance tested using Student's Karyotypes were determined by standard G‐banding procedure as described previously 5 hESC were injected subcutaneously into the right flank of in adult SCID mice and maintained for 8–12 weeks Approximately 5–10 × 10CRX gene to avoid disruption of the coding sequence, preventing the loss of CRX function and maximizing the maintenance of protein localization and function. For this reason, we mutated the stop codon of CRX introduced in the targeting cassette, which was followed by the eGFP sequence , indicating that CRX is not expressed with RAX or PAX6 during the early stages of optic cup formation. As differentiation proceeded, abundant RECOVERIN expression was observed at days 60 and 90 Figs. A, 3B, 4AExpression of cone photoreceptor markers (OPN1SW and OPN1MW+OPN1LW) was tested at days 60 and 90 of differentiation and a punctate cytoplasmic expression pattern was observed Figs. D, 4B, 4C was found in the GFP positive fraction  , and cone photoreceptors  were found in the GFP negative fraction  Fig. H, matureion Fig. I. To evaion Fig. H. Converion Fig. H. TogethCRX expression is confined to two cell types during the first 90 days of hESC differentiation, namely RECOVERIN‐expressing photoreceptor precursors situated in the developing outer nuclear layer of the optic cup and in a subpopulation of retinal progenitors that appear to be nonproliferative but do not yet express any of the markers characterizing the mature retinal phenotypes that we have tested herein. Detection of CRX expression as well as genes activated by CRX  indicates that CRX remains functional following the ZFN‐mediated tagging with GFP.In summary, our data suggest that CRX gene, a well described marker of postmitotic photoreceptor precursors. The reason for targeting 3′ of CRX was based on previous findings in the mouse system which indicated that the 5′ UTR (up to 12 kb) was not sufficient to direct high level reporter gene expression in photoreceptor cells compared with a mixture of 5′ and 3′ UTR regions in murine transgenic lines Reporter lines that facilitate the detection of emerging photoreceptors are a useful tool for monitoring and improving hESC/hiPSC differentiation protocols towards retinal lineages and also enable the selection of specific cell types, analysis of their molecular and cell surface profile and engraftment capacity. Using plasmid and lentiviral based approaches, a few reporter hESC and hiPSC lines have been generated to monitor the emergence of retinal ganglion like cells CRX is not expressed at the pluripotent stem cell stage, we subjected our targeted clones to our recently described 3D retinal differentiation protocol CRX during hESC differentiation. Importantly, we were also able to detect GFP expression without the need for immunocytochemistry indicating that GFP confers sufficient reporter activity to monitor the emergence of CRX expressing cells during hESC differentiation. The ability to detect CRX expression also indicated that expression of CRX had not been adversely hampered by ZFN‐mediated tagging with GFP. Furthermore, CRX has been shown to be required for the transactivation of its own gene expression and its maintenance CRX mRNA and protein expression throughout our differentiation time course further reinforces the notion that ZFN‐mediated GFP tagging has not interfered with CRX function.Since CRX is expressed in photoreceptors and in the inner nuclear layer CRX expression in bipolar cells in addition to photoreceptors CRX during hESC differentiation we performed immunocytochemistry with retinal precursor and retinal lineage markers. We further validated this analysis by performing quantitative RT‐PCR of GFP positive and negative populations isolated by FACS. We found that the GFP‐positive population displayed increased expression of photoreceptor markers  when compared with the GFP negative population or unsorted cells. In addition to transactivating its own expression, CRX has been shown to be important for activating and maintaining the expression of several key photoreceptor genes CONE ARRESTIN gene harbors several CRX binding sites in its promoter region which are important for its activation CONE ARRESTIN in the CRX‐GFP‐positive population indicates that the CRX protein remains functional and able to activate downstream targets after ZFN‐mediated GFP tagging.Expression studies in human fetal retina have suggested that RHODOPSIN, a mature rod photoreceptor marker, was greater in the GFP negative subpopulation, suggesting confined expression of the GFP reporter to photoreceptor precursors emerging during 90 days of hESC differentiation. While we could detect expression of cone opsins in a punctate pattern that may be typical of an immature phenotype, we were unable to observe mature rod markers by immunocytochemistry, which indicates either a very low frequency of rod differentiation at day 90, or that the ontogenic stage of day 90 hESC‐derived retinal tissue precedes the developmental peak of rod genesis, akin to the pattern of retinal cell emergence during human development. These data, however, should be interpreted with caution, as the expression of opsins in our study was low or of an immature nature. These findings are corroborated by a recent publication CONE ARRESTIN expression was observed in the CRX‐GFP‐positive population, perhaps indicating a more advanced stage of cone genesis in the CRX‐positive population isolated from hESC differentiation, although this needs to be further investigated using comparative analysis with retina obtained at additional developmental periods. Intriguingly NRL expression is observed in the fetal retina but is absent from the CRX‐GFP‐positive population. This together with the expression of CONE ARRESTIN may mark these cells as cone‐specific progenitors; however, the heterogeneous nature of the hESC differentiation could equally yield populations of varying maturity with some cells beginning to acquire a cone photoreceptor fate while other cells are at an earlier post‐mitotic photoreceptor progenitor phase and rod genesis is yet to occur.It was interesting to observe that the expression of RECOVERIN expression were situated towards the apical surface, so it is possible that they move towards this rim to complete mitosis before acquiring RECOVERIN expression and committing to a photoreceptor fate. If this were the case, these cells should also express Ki67. However, our analysis indicated that all CRX positive cells lack Ki67 expression leaving open the question of what these cells may be. Earlier studies in mouse transgenic models have shown that CRX expression can also be detected in bipolar cells As outlined by previous studies in the mouse model In conclusion we have successfully generated CRX‐GFP reporter hESC lines, which can be used to study the molecular and cell surface profile of human photoreceptor precursors and enable their isolation for transplantation studies. We hope that these hESC lines will provide a universally useful tool with which to monitor and improve differentiation protocols, discover useful cell surface markers and develop clinically applicable strategies for the purification of hESC‐derived retinal photoreceptor precursors for transplantation.J.C.: designed and performed research, data analysis, figure preparation, manuscript writing and final approval of manuscript; C.M.: performed research, figure preparation, data analysis and final approval of manuscript; B.D.: performed data analysis, figure preparation and final approval of manuscript; S.P. and I.M.‐G.: performed data analysis and final approval of manuscript; M.L.: designed and performed research, data analysis, figure preparation, manuscript writing, fund raising and final approval of manuscript.The authors indicate no potential conflicts of interest.Note added in proof: Whilst this paper was under revision, Swaroop and colleagues Supporting Information Figure 1Click here for additional data file.Supporting Information Figure 2Click here for additional data file.Supporting Information Figure 3Click here for additional data file.Supporting Information Figure 4Click here for additional data file.Supporting Information Table 1Click here for additional data file.Supporting Information Table 2Click here for additional data file.Supporting Information Table 3Click here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.


To assess the influence of storage dynamics and nonlinearities in hydrological connectivity on time‐variant stream water ages, we used a new long‐term record of daily isotope measurements in precipitation and streamflow to calibrate and test a parsimonious tracer‐aided runoff model. This can track tracers and the ages of water fluxes through and between conceptual stores in steeper hillslopes, dynamically saturated riparian peatlands, and deeper groundwater; these represent the main landscape units involved in runoff generation. Storage volumes are largest in groundwater and on the hillslopes, though most dynamic mixing occurs in the smaller stores in riparian peat. Both streamflow and isotope variations are generally well captured by the model, and the simulated storage and tracer dynamics in the main landscape units are consistent with independent measurements. The model predicts that the average age of stream water is ∼1.8 years. On a daily basis, this varies between ∼1 month in storm events, when younger waters draining the hillslope and riparian peatland dominates, to around 4 years in dry periods when groundwater sustains flow. This variability reflects the integration of differently aged water fluxes from the main landscape units and their mixing in riparian wetlands. The connectivity between these spatial units varies in a nonlinear way with storage that depends upon precipitation characteristics and antecedent conditions. This, in turn, determines the spatial distribution of flow paths and the integration of their contrasting nonstationary ages. This approach is well suited for constraining process‐based modeling in a range of northern temperate and boreal environments. Stream water age is estimated by modeling high‐resolution isotope time seriesStorage dynamics drive nonlinear spatial patterns of hydrologic connectivityConnectivity variations determine nonstationary stream water ages Isotope measurements for daily and subdaily precipitation and streamflow samples (rather than weekly) is becoming more readily available for multiyear periods at a number of research sites [Tetzlaff et al., Ali et al., Tetzlaff et al., Birkel et al., Birkel et al., Birkel et al., Birkel et al., Birkel et al., Dick et al., Here we report advances in integrating new field insights in models, motivated by new, high‐resolution tracer data from a wet, peatland‐dominated experimental site in Scotland. Previous work has related nonlinear runoff responses to the connectivity of different hydrological response units [2) catchment where extensive tracer data are also available for soil waters and groundwaters, as well as soil moisture content and groundwater level data. Integrating these new data with the model allows us, for the first time, to examine the interaction between nonstationary catchment transit times, water age distributions of different internal fluxes, and nonlinearity in storage‐driven hydrological connectivity. The aim of the paper is to use a coupled flow and tracer model to address the following questions:How well can we simulate daily runoff and tracer dynamics using a parsimonious model in a way that is consistent with measurements of state variables?How do the ages of water fluxes from different conceptual stores  vary and then integrate at the catchment scale to control stream water age?How does this integration vary under different hydroclimatic conditions and what are the associated storage dynamics that control nonlinearities in hydrological connectivity?Here we test the modeling approach with a new, unique multiyear data set of daily tracer measurements in precipitation and streamflow. These data have been collected in a small  in the Scottish Highlands , which is fairly evenly distributed throughout the year, though the period November–February usually has the highest precipitation and runoff events. Annual potential evapotranspiration is around 400 mm and annual runoff is around 700 mm. Runoff event coefficients vary between around 10% with dry antecedent conditions  and ∼40% in wetter periods.The Bruntland Burn is a 3.2 kms Figure . It is d−6 to 10−4 m s−1 [Malcolm et al., Tetzlaff et al., Spagnum spp. mosses and grasses like Molinia caerulea dominating the peatland communities with a canopy ∼0.2 m high and heather  scrubs ∼0.3–0.4 m high dominating the podzols. The heather roots create a moderate amount macroporosity in the upper 0.3 m of the profile. This mainly facilitates vertical drainage, but in the wettest conditions (when the soil is saturated) can cause lateral flow downslope toward the riparian peatland. Trees, mainly Scots pine (Pinus sylvestris) cover about 10% of the catchment mainly on steeper slopes or on plantations.The solid geology is characterized by low permeability, with granite to the north and metamorphic rocks to the south. The stream occupies a glaciated valley with steep slopes and a flat valley bottom. Around 70% of the catchment is covered by glacial drift deposits; mainly undifferentiated till which comprises pebble to boulder sized clasts in a silty‐sand matrix. The depth ranges from around 5 m on the steeper hillslopes, to around 40 m in the valley bottom. The till is the main aquifer for groundwater storage and has a porosity of around 20%, but low permeability with tentative hydraulic conductivity estimates ranging from 10Birkel et al., Dunne et al., Ali et al., Tetzlaff et al., Soulsby et al., Blumstock et al., Tetzlaff et al., Geris et al., Previous work has shown that the hydrological response of the Bruntland Burn is strongly dependent on the connectivity between the steeper podzols and peats in the riparian wetland [2H ranging from ∼−150‰ in winter to ∼−10‰ in summer [Tetzlaff et al., Birkel et al., Tetzlaff et al., Previous work has shown that isotopes in precipitation have a distinct seasonal pattern with δ33.1Capell et al. [Geris et al., The hydrometric and isotope data available for the Bruntland Burn are as follows ; ±0.4 ‰ for deuterium (δ2H)). Isotope signatures are reported in the δ‐notion (‰) after calibration using Vienna Standard Mean Ocean Water (VSMOW) standards. Because of the higher relative precision, we only used δ2H in the modeling though δ18O had a similar variation.For stable isotopes, weekly precipitation sampling started at the Bruntland Burn in October 2003. However, from October 2007, precipitation samples have been collected daily. Occasional gaps in the precipitation record reflect occurrences of sampler failure or site inaccessibility in bad weather. Stream water samples were collected on a weekly basis from October 2007, and then daily between October 2008 and the end of 2009 using an ISCO 3700 automatic water sample. After an 18 month hiatus (due to a funding gap), daily stream sampling restarted on 1 June 2011 and has continued since. Additionally, weekly or fortnightly soil isotope (using Rhizon soil samplers) sampling in three soil profiles  of a representative hillslope transect (encompassing the catena transition from podzols to peat) started in June 2011 and continued until October 2013 [2H and δ18O in precipitation. The best fit (adjR2 = 0.47) and most parsimonious model used air temperature and relative humidity as explanatory variables. This procedure mimics the observed natural variability in precipitation isotopes and avoids the previously applied gap filling using the same isotopic value of the neighboring measurement.To complete the precipitation daily isotope time series, a multiple linear regression (MLR) gap filling procedure was developed and used, though this accounted for <5% of the time series. MLR models based on meteorological variables  were constructed to simulate δ3.2Birkel et al., Birkel et al., Birkel et al., Birkel et al., The model presented here was developed in previous work in the Bruntland Burn catchment that has used tracer data and other empirical measurements in addition to stream discharge to constrain model structures, improve parameterization, and aid calibration [Ali et al., Birkel et al., up) and saturation area (Ssat) model structure. Consequently, catchment precipitation P and potential evapotranspiration PET were distributed into hillslope  and saturation area  according to the extent of dSAT (only the equations for precipitation are given for illustration here):Central to all these models was capturing the nonlinear streamflow response by successfully conceptualizing the hydrological connectivity of the catchment which links the hillslopes (Up) to the riparian zone (Sat) and underlying groundwater (Low) Figure . This coBirkel et al. [Tetzlaff et al., up was often in deficit, but in wetter periods would fill and spill into Ssat, which usually has low or no deficit and usually generated streamflow. The storages S are model state variables and we describe the following fluxes and calibrated parameters. The unsaturated hillslope reservoir upS is drained (flux Q1 in mm d−1) by the linear rate parameter a (day−1) and directly contributes to the saturation area store satS; the recharge rate R (mm d−1) to the groundwater reservoir lowS is linearly calculated using the parameter r (day−1); the lowS generates runoff lowQ (mm d−1) contributing to total streamflow Q (mm d−1) using the linear rate parameter b (day−1). The runoff component satQ (mm d−1) generated nonlinearly from satS conceptualizes saturation overland flow using the rate parameter k (day−1) and the nonlinearity parameter α in a power function‐type equation  shown in blue in Figure up does not contribute directly to streamflow as the steeper hillslopes in the catchment are separated from the channel network by the riparian zone represented by Ssat. Further Ssat is not conceptualized to drain into Slow as the peat soils are saturated with lower subsoil permeability, limiting vertical drainage, and promoting lateral flow [Tetzlaff et al., 2), we did not need to include a channel routing parameter.Thus, the basic rainfall‐runoff model uses only five calibrated parameters , transient snow packs can accumulate, though these typically melt in a week or so, and snowmelt can occasionally generate important stormflow responses in winter and spring. Therefore, we adopted a simple snowmelt routine based on a “master snow depth recession curve” taken from recorded daily snow depth at Balmoral (5 km from the BB) similar to k et al. . The resupS, satS, and lowS in mm) incorporates an additional calibrated storage parameter  for isotope transport simulations  in j storage inflows jI  and k outflow kO components  which characterizes the catchment storage S dynamics  and associated isotope signature c. Additional storage parameters were subsequently converted into time‐variable mixing volumes (MV). This was achieved using the nonlinear expansion and contraction of the saturation zone as a means of relaxing the assumption of complete mixing by varying the contributions from the hillslope and saturation area stores at the catchment scale without introducing new parameters. Thus, as the rainfall‐runoff model dynamically varies the extent of the satS and upS, the complete mixing in individual reservoirs is integrated at the catchment scale in a nonlinear manner. The MVs, therefore, represent a partial mixing mechanism and were calculated according to the catchment wetness (dSAT) state assuming that greater wetness results in greater saturation area extent but also greater potential for mixing (satMV). In turn, it is assumed that the hillslope mixing volume (upMV) decreased as the saturation area expands pF,Qa, b, r, k, α, pupS, psatS, and plowS) using a multiobjective (considering discharge and tracer) nondominated sorting genetic algorithm (NSGA2) for optimization [Deb et al., Deb et al. [Andrews et al. [2H signature in streamwater (KGE_D) [Kling et al., Schaefli and Gupta, The model was calibrated  and the saturation zone (satS). In large precipitation events, positive storage in the latter generates fluxes during higher streamflows which correspond to the maximum expansion of the saturation zone. In smaller events, runoff is generated from the saturation zone which has a more limited extent. In contrast, the large groundwater store (Slow) responds much more gradually to recharge events and subsequent dry‐period discharge, when groundwater becomes the main source of runoff such as in the summers of 2013 and 2014  with similar decreases in groundwater storage as recharge stops. However, the effects of the summer 2013 drought are particularly striking in the plot of the storage dynamics  that subsequently accumulates and is not replenished until the end of the year. It is also apparent in the prolonged, gradual drawdown of groundwater storage, although this probably partly reflects oversimplification in the model structure as some groundwater recharge in summer and early autumn was detected in some groundwater wells [Geris et al., In wetter periods, there are no storage deficits in s Figure . Groundws Figure . This is2H in the saturated area in summer 2013 shows the effect of this evaporative fractionation algorithm, albeit with an apparent overestimation of fluxes from the saturated area in a summer event .A qualitative check on the internal isotope states produced in the three model units was available from measured isotope values Figure . The iso4.2The fluxes modeled from the dynamic storage changes in the different landscape units allows the nonstationarity in water ages in fluxes to be tracked. Though there was considerable uncertainty in the estimates derived from the water ages from the 500 best parameter sets  is summarized in terms of the upper and lower quartiles of the flow distribution in Figure s Figure . In drie75 is 1989 mm (±122 mm SD). For catchment states of >Q25, the mean storage is 1941 mm (±116 mm SD). This apparent decline in effective storage at the higher flows is also evident in the time series  is inad55.1www.ceh.ac.uk/data/nrfa/nhmp/monthly_hs.html). The period included the wettest summer for 5 years in 2012, the coldest spring in 50 years and the warmest, driest summer for 10 years in 2013, and the wettest December/January for 11 years in 2013/2014. It also gave a sufficiently long time series for independent calibration and satisfactory testing of the model structure and parameter sets  are generally close and in the uncertainty range of previous estimates in the study catchment, such as the 1.9 (±0.9) years produced by lumped parameter models [n et al.  showed t5.3Birkel et al. [Harman [Heidbuechel et al., Hrachowitz et al., Benettin et al., The nonstationarity in stream water age distributions could be clearly illustrated for the upper and lower quartiles of the flow distributions Figure . In the l et al.  and the  [Harman . In dry 5.4Tetzlaff et al., Capell et al., The modeling approach used has the advantage of remaining relatively simple, but spatially explicit. It is, thus, a useful tool for applying in data rich catchments where dynamic saturation areas are important runoff sources as a framework for learning about catchment function and for hypothesis testing. In particular, the approach provides insight into spatial variability in storage dynamics, hydrological connectivity, and associated mixing processes that influence the age distribution of different source waters and ultimately stream water. The findings and approach are also particularly relevant to a wide range of northern temperate and boreal watersheds where dynamic activation of flow paths in peat is an important influence on the hydrological response [Frei et al., Geris et al., However, the model can be improved to move beyond its current simplistic semidistributed partitioning of landscape units. In particular, empirical data from the site suggests that improved spatial resolution of the landscape features could improve the hydrological component of the model, particularly in terms of capturing the catchment rewetting after dry periods. Key areas of potential include the influences of microtopography in the valley bottom to distinguish connected and disconnected areas at the small scale [e.g., Blumstock et al., Rodhe, Beven, More direct issues to be addressed for improved tracer modeling revolve around the nonconservative behavior of isotopes. Of particular significance are the influences of evaporative fractionation in summer and fractionation in snowmelt. In the case of the former, there is evidence that the effects of summer fractionation are quite localized in quasi‐permanently connected pools and water tracks in the valley bottom saturation zone [62 upland catchment in the Scottish Highlands. This model conceptualized three main hydrological response units: a dynamically saturated riparian zone of peat soils, freely draining hillslopes, and groundwater in extensive glacial drifts. The flux tracking was simulated with reasonable success over a 40 month period, which included some wet and dry spells that had return periods of around 10 years. The results showed that the estimated average age of stream water (1.8 years) was controlled by nonlinear connectivity between landscape units which determined the relative fluxes of younger water from saturation overland flow (∼10 days) in riparian peatlands and older, deeper groundwaters in the drift (∼4 years). Stream waters were youngest (∼1 month) in larger events when the extent of riparian saturation covered >40% of the catchment and connected fluxes from steeper hillslopes. In drier conditions, the age of stream water approaches that of groundwater. Fluxes between the main landscape units were driven by changes in storage. Relatively small (∼100 mm) changes in dynamic storage accounted for much of the water balance variation that controlled runoff generation. However, much larger total storage volumes (>1500 mm) were needed to account for the mixing processes that damped isotope signals in precipitation. In wet periods, this mixing primarily occurred in riparian wetlands. In drier periods, the hillslopes and groundwater provided the major mixing zones. While both the water and tracer flux dynamics were captured reasonably well, the simple model structure used had limitations in terms of simulating the rewetting of the catchment following dry periods and the effects of evaporative fractionation in the riparian wetlands. More complex spatially distributed models may help to capture these processes.We used a tracer‐aided conceptual runoff model to track daily water and isotope fluxes through a 3.2 km


In particular, maternal stress during pregnancy has been linked to adverse behavioural and emotional outcomes in children. Data from human cohort studies and experimental animal models suggest that modulation of the developing epigenome in the foetus by maternal stress may contribute to the foetal programming of disease. Here, we summarise insights gained from recent studies that may advance our understanding of the role of the placenta in mediating the association between maternal mood disorders and offspring outcomes. First, the placenta provides a record of exposures during pregnancy, as indicated by changes in the placental trancriptome and epigenome. Second, prenatal maternal mood may alter placental function to adversely impact foetal and child development. Finally, we discuss the less well established but interesting possibility that altered placental function, more specifically changes in placental hormones, may adversely affect maternal mood and later maternal behaviour, which can also have consequence for offspring well‐being.Substantial data demonstrate that the early‐life environment, including  Pregnancy can be a challenging time in a woman's life, during which she may experience many changes in her circumstances. Pregnancy can also be a time of vulnerability to perinatal mental illnesses, including depression, stress and postnatal psychosis in utero by the maternal emotional state, in least in part BDNF) COMT) , during the development of emotional and cognitive outcomes, respectively.Prenatal stress at its broadest level includes major life event stress, catastrophic disasters, chronic stress, daily hassles, perceived stress or pregnancy‐specific anxiety along with related symptoms of depression or general anxiety during pregnancy HSD11B2NR3C1 and NR3C2 expression is higher among depressed compared to nondepressed new mothers, providing another mechanism for the programming of adverse offspring outcomes pCRH; which stimulates the production of cortisol via the hypothalamic‐pituitary‐adrenal (HPA) axis], monoamine oxidase A  and placental serotonin transporter  and P‐glycoprotein have also been linked to prenatal stress Placental function is important both for optimal foetal growth and maternal health Hsd11B2N‐acetylglucosamine transferase (Ogt) HSD11B2, whereas maternal depression (but not anxiety) has been associated with increased placental methylation of NR3C1CRH, CRHBP, NR3C1 and FKBP5et al. (12), the effects at individual CpG sites differed between tissues. The majority of CpG sites identified were situated in transcription factor binding regions, and several were associated with offspring birth weight BDNF methylation Although it is clear that there are changes in the expression of genes in the placenta in relation to prenatal stress exposure, the mechanisms underpinning these changes are unclear. One possibility is the epigenetic deregulation of gene expression. Epigenetics describes the marks or tags that are added or removed from DNA sequence and the histones that regulate gene expression in a manner heritable through cell division. Such marks may be altered by exposure to stress during prenatal development, a period when the epigenome is rapidly changing NR3C1 has been associated with prenatal maternal anxiety NR3C1 and CRH is associated with maternal experiences of war trauma and chronic stress NR3C1 and CRH CpG sites identified in these studies are the same as those linked to pre‐eclampsia in cord blood NR3C1 sites affects NGFI‐A binding BDNF methylation in cord blood at birth BDNF promoter region in buccal cells at 2 months of age BDNF DNA methylation may be an important target for future investigation. Maternal cortisol and self‐reported depressive and anxiety symptoms have also been associated with altered DNA methylation of the imprinted genes IGF2 and GNASXLExperimental animal models have demonstrated that early‐life stress can leave a mark on the offspring epigenome, with alterations reported in offspring exposed to prenatal stress Although considerable data from both animal models and human studies support changes in the placenta and foetus in response to maternal stress, which may then contribute to the programming of neurodevelopmental changes in offspring, few studies have explored a placental origin for maternal mood disorders. Pituitary prolactin and the placental lactogens are a group of evolutionarily and functionally‐related hormones important in pregnancy. Human placental lactogen (hPL) is produced by the placental syncytiotrophoblast and secreted into the maternal circulation, replacing prolactin as the main lactogenic hormone during pregnancy et al., in preparation), comprising data that support a causal relationship.Imprinted genes are expressed from one parental allele through epigenetic marking in the germline In summary, the placenta plays a key role in supporting a successful healthy pregnancy. The placenta can provide a record of prenatal adversity, including maternal stress. Changes in placental function, potentially driven by epigenetic processes, may impact neurodevelopmental outcomes for children and both mental and metabolic health into adulthood. Importantly, placental dysfunction may also contribute to maternal mood disorders, either during pregnancy and/or in the immediate postnatal period, by exposing the mother to abnormal levels of placental hormones. At present, most women experiencing heightened levels of prenatal depression, stress and anxiety are undetected and untreated. The potential clinical implications of maternal prenatal mood on foetal and child neurodevelopment are substantial. The provision of better emotional care for all pregnant women and enhanced identification and support for women at particularly high risk of maternal mood disorders will help not only them, but also their children, and potentially subsequent generations.


Mycobacterium tuberculosis (Mtb) infection can be cleared by the innate immune system before the initiation of an adaptive immune response. This innate protection requires a variety of robust cell autonomous responses from many different host immune cell types. However, Mtb has evolved strategies to circumvent some of these defences. In this mini‐review, we discuss these host–pathogen interactions with a focus on studies performed in human cells and/or supported by human genetics studies (such as genome‐wide association studies). Mycobacterium tuberculosis (Mtb) is particularly effective at subverting many of the host immune defences, and this is one reason why it is such a successful human pathogen that has been particularly hard to eradicate. The outcome of infection by Mtb and therefore the clinical manifestation of tuberculosis (TB) depend on many combined factors, such as host genetics, bacterial genetics (virulence factors), the health and nutritional status of the host and whether there has been any prior exposure/immunity and vaccination history. Around half of individuals exposed to Mtb do not exhibit a positive tuberculin skin test  present on Mtb surfaces as they constitutively express pattern recognition receptors such as Toll‐like receptors, Dectin‐1, C‐type lectin receptors (CLRs), nucleotide‐binding oligomerization domain‐containing protein 2 (NOD2), dendritic cell (DC)‐specific intercellular adhesion molecule‐3‐grabbing non‐integrin and mannose receptor , but they live for around 3 months in humans  populations  are also involved in Mtb infection. These innate immune cells are recruited to the site of infection early on and play a role in amplifying the anti‐microbial defence to TB. This is via recognition of infected macrophages through receptor molecules such as NKp44, NKp46 and NKG2D  (Hmama In vitro studies using electron microscopy (EM) and a fluorescence resonance energy transfer‐based method showed that Mtb also localizes in the cytosol in THP‐1 macrophages, primary human macrophages and primary human DCs , the human genome contains only three IRG genes, encoding IRGC, IRGQ and IRGM, but these are not inducible by IFN‐γ. Polymorphisms in the IRGM gene, which is functional in humans, are associated with susceptibility to TB among African‐Americans  and by the 65 kDa guanylate binding protein family. Compared with the mouse genome  and RNS for the control of intracellular mycobacterial replication and disease is well established  gene modulates host cell ROS generation  as well as toxic metals. In the murine model of et al., ctpC, which regulates the intra‐bacterial levels of Zn2+ through efflux of the metal ion (Botella et al., Heavy metal poisoning is emerging as a very effective cell autonomous mechanism of bacterial elimination. Transcriptional studies show that during infection of primary human macrophages, Mtb faces a burst of free zinc, which accumulates within the mycobacterial phagosome (Botella The host innate immune response to TB requires a variety of different host cell types to successfully protect the host from infection. Physical barriers and anti‐microbial substances are just as important as immune cells for protection. There are many different factors that influence the outcome of the initial battle between host and pathogen, including a variety of mechanisms that Mtb has evolved to subvert host defences. If Mtb is not killed by the innate immune response, it will replicate and disseminate and the host adaptive immune response will become critical for control. This review focuses solely on studies performed in humans or in human cells. The majority of the known innate mechanisms involved in Mtb infection have been studied in murine cells. Considering that there are key differences in host cell responses between humans and other animal models, one of the major challenges for the future will be to confirm the relevance of these findings in humans. Better understanding of the mechanisms involved in innate immunity in humans will enable us to develop improved treatments for TB.