Studies have shown that transgender women (TGW) are disproportionately affected by HIV, with an estimated HIV prevalence of 19.1% among TGW worldwide. After receiving a diagnosis, HIV-positive TGW have challenges accessing effective HIV treatment, as demonstrated by lower rates of virologic suppression and higher HIV-related mortality. These adverse HIV outcomes have been attributed to the multiple sociocultural and structural barriers that negatively affect their engagement within the HIV care continuum. Guidelines for feminizing hormonal therapy among TGW recommend combinations of oestrogens and androgen blockers. Pharmacokinetic studies have shown that certain antiretroviral therapy (ART) agents, such as protease inhibitors (PIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) and cobicistat, interact with ethinyl estradiol, the key oestrogen component of oral contraceptives (OCPs). The goal of this article is to provide an overview of hormonal regimens used by TGW, to summarize the known drug-drug interactions (DDIs) between feminizing hormonal regimens and ART, and to provide clinical care recommendations.The authors identified English language articles examining DDIs between oestrogen therapy, androgen blockers and ART published between 1995 and 2015 using PubMed, Cumulative Index to Nursing and Allied Health Literature and EBSCOhost.Published articles predominantly addressed interactions between ethinyl estradiol and NNRTIs and PIs. No studies examined interactions between ART and the types and doses of oestrogens found in feminizing regimens. DDIs that may have the potential to result in loss of virologic suppression included ethinyl estradiol and amprenavir, unboosted fosamprenavir and stavudine. No clinically significant DDIs were noted with other anti-retroviral agents or androgen blockersThere are insufficient data to address DDIs between ART and feminizing hormone regimens used by TGW. There is an urgent need for further research in this area, specifically pharmacokinetic studies to study the direction and degree of interactions between oral, injectable and transdermal estradiol and ART. Clinicians need to be vigilant about possible interactions and monitor hormone levels if concerns arise. More research is also needed on the provision of hormone therapy and gender-affirming care on the long-term health outcomes of HIV-positive TGW. Transgender individuals are those whose gender identities and/or gender expression do not fully align with their assigned sex at birth. Many cultures around the world recognize transgender people using varied terms such as “Kathoeys” in Thailand ; “HijrasDespite the fact that few countries use HIV surveillance methods that permit the identification of transgender people, global estimates have demonstrated an increased HIV burden among transgender women (TGW), with a pooled HIV prevalence of 19.1%, and an odds of HIV among transwomen 48 times that of the general population ,11.TGW face a complex array of psychosocial challenges that complicate their access and adherence to HIV care, such as limited access to and avoidance of health care because of stigma and past negative experiences with providers, prioritization of gender-related health care and concerns about adverse interactions between antiretroviral therapy (ART) medications and hormone therapy ,13. Societ al. also showed disparities with ART at one clinical site, with fewer TGW (59%) than non-transgender people (82%) taking ART [Data on the HIV care continuum among TGW are limited because both informational and institutional erasures render many TGW invisible in data collection and research . HIV tesking ART . Even whking ART . A studyking ART . A studyking ART . This laking ART , but theking ART .Hormone therapy is one aspect of gender-affirming care that is utilized as part of medical transition. Gender affirmation in clinical settings goes far beyond hormones and includes the creation of a welcoming environment, including the use of patients’ preferred names and pronouns and providers who are knowledgeable about transgender health issues . The desThe goal of feminizing hormone therapy is to induce the secondary sex characteristics of the affirmed gender and to reduce the sex characteristics of the individual's natal sex. For TGW, feminizing regimens usually involve the use of an androgen blocker in addition to oestrogen, with many regimen options available, as outlined in In the United States, spironolactone, a mineralocorticoid-receptor antagonist with anti-androgen properties, is most frequently used for androgen blockade . CyproteThe proportion of TGW who opt to start hormones varies significantly across countries because of the availability of oestrogens and androgen blockers, cost and whether these are available only through medical sources or without prescriptions. In addition, some TGW opt not to use hormones for personal/identity reasons or medical contraindications. The highest rates of hormone use are in countries where hormones are readily available without prescription, and where a greater level of acceptance exists towards transgender persons, for example, Thailand, where studies report hormone use among TGW of 73 to 94%, mostly obtained without a prescription –40. In tSince the advent of ART, people living with HIV are living longer, healthier lives. There has been an increase in awareness of some of the age-related health conditions that are more prevalent in this population that hold particular considerations for HIV-positive TGW receiving hormone therapy.Low bone mineral density (BMD) has been reported to occur more frequently in HIV-positive individuals ,54 as weCardiovascular disease (CVD) is another area of special concern for TGW living with HIV. A recent prospective study demonstrated increased CVD mortality in this population . FeminizThe goals of HIV treatment are to improve health outcomes for people living with HIV and to reduce HIV transmission. There are five classes of antiretroviral agents that have different mechanisms of action during the HIV life cycle. Effective treatment regimens usually consist of ART with at least three agents. The initial regimen usually includes three agents from two classes, most often two nucleoside reverse transcriptase inhibitors (NRTIs) with either a non-nucleoside reverse transcriptase inhibitor (NNRTI), a protease inhibitor (PI), which is usually “boosted” with ritonavir, or an integrase strand transfer inhibitor (INSTI). Two other antiretroviral agents block entry of HIV into human cells using different mechanisms, the CCR5 receptor antagonists and fusion inhibitors.The recent update in WHO HIV treatment guidelines, expanding ART to all individuals regardless of CD4 cell count, means that an additional 9 million individuals, including many transgender individuals, are now eligible for ART. It is extremely timely therefore to determine what impact, if any, hormone therapy has on the uptake as well as adverse effect profile of ART.The recommendations for initial antiretroviral regimens vary globally based on considerations such as cost, availability of generic agents, adverse effect profile and prevalence of comorbidities. The WHO guidelines recommend an initial regimen of two NRTIs, either lamivudine (3TC) or emtricitabine (FTC) with TDF and the NNRTI efavirenz (EFV), or if not available, nevirapine (NVP) . Other NThe use of emtricitabine and TDF for PrEP is an important biomedical intervention in preventing HIV acquisition. A recent substudy of the iPrEx trial revealed that TGW were less likely to have detectable drug levels compared to non-transgender MSM . FurtherThe current guidelines for transgender care listed in Many antiretroviral agents are metabolized through the cytochrome P450 (CYP) system , allowinTo identify studies investigating antiretroviral agents and the components of feminizing hormone regimens, the authors retrieved English language articles from 1995 to 2015 using PubMed (MEDLINE), Cumulative Index to Nursing and Allied Health Literature (CINAHL) and EBSCOhost. Medical subject headings (MeSH) terms for ART  were cross-referenced with feminizing hormonal agents  and androgen blockers . Androgen blockers were also cross-referenced with “interactions.” The authors reviewed abstracts for relevance, with subsequent full-text review for data abstraction. Inclusion criteria were primary DDI and pharmacokinetic studies that evaluated interactions between antiretroviral agents and oestrogens or androgen blockers. Additional information was obtained from pharmacokinetic data listed in package inserts for antiretroviral agents and named anti-androgens, the University of Liverpool's HIV drug interactions website  and the in vitro study) [in vivo pharmacokinetic DDI studies. All of the available studies evaluated ART interactions with OCPs and not with other feminizing hormones. The studies were all conducted in non-TGW, with 10 to 34 participants. In all but five studies [The authors reviewed 165 unduplicated articles that included references to feminizing hormonal agents. These were reviewed for relevance, resulting in identification of 26 peer-reviewed articles, of which 8 were reviews ,75–81. Oo study)  were in  studies ,95,96,98in vitro study in peripheral blood lymphocytes. It may be prudent however to recommend that these ART drugs be avoided in the treatment of TGW receiving feminizing hormones.There are few studies that have examined interactions between exogenous oestrogens and ART, and these have all investigated effects of OCPs . AlthougThere were no published pharmacokinetic studies that investigated interactions between ART and spironolactone or finasteride. Drug package inserts, the DHHS guidelines and the HIV drug interactions website also did not flag potential drug interactions with these agents. Finasteride is an inhibitor of type II alpha-reductase, blocking conversion of testosterone to 5-alpha-dihydrotestosterone (DHT). Although finasteride is primarily metabolized through the CYP3A4, it has no effects on the cytochrome P450 system ,101; theA special consideration for HIV-positive TGW is that anti-androgens may be unnecessary for medical transition purposes because HIV infection is frequently associated with low testosterone levels. Studies of HIV-positive natal men have shown a prevalence of hypogonadism of approximately 25% . Before This review did not find evidence of clinically significant interactions between ART and feminizing hormones (ethinyl estradiol) for medical transition. Although the possible increase in CVD and osteoporosis among TGW living with HIV is concerning, it underscores the importance of engagement in HIV primary care, which as noted above could be enhanced by provision of hormone therapy and other gender-affirming services. Engagement in primary care services will allow for appropriate health care screenings as well as identification and reduction of modifiable cardiovascular risk factors, such as tobacco use, hyperlipidemia, overweight and obesity.In fact, bundling HIV care with hormone and other aspects of gender-affirming care may result in synergistic enhancements, as has been demonstrated in other medical contexts. A preliminary data analysis, led by MD, of the ongoing HRSA SPNS Transgender Women of Color initiative found associations between current ARV use, undetectable viral load and receipt of HIV primary care in the past six months and receiving a hormone prescription from the participant's HIV primary care provider (as opposed to other sources).Although the data investigating oestrogens and ART come mainly from OCP data, it is likely that, apart from amprenavir, unboosted fosamprenavir and possibly stavudine, no clinically significant interactions exist between feminizing regimens and ART. These data are also important for HIV prevention among at-risk HIV-negative TGW, who may not be inclined to use PrEP until clear information is available about potential interactions between TDF and feminizing hormone regimens .Clinicians should maintain vigilance for perceived or actual interactions between hormone therapy and ART, and take appropriate clinical or patient educational action as indicated. This may include monitoring estradiol levels while on ART in order to assess for elevated or subtherapeutic levels as well as ongoing viral load monitoring.Clinicians should be aware of the possible increase in CVD and osteoporosis among HIV-positive TGW on hormones and seek to identify and reduce modifiable risk factors.There is an urgent need for further research, specifically pharmacokinetic and pharmacodynamics studies to evaluate for interactions between oral, injectable and transdermal estradiol and ARTs. More research is also needed on the impact of hormone therapy on long-term health outcomes of HIV-positive TGW, not only to evaluate adverse effects but also to assess the potential positive impact of hormones on HIV-specific outcomes, like retention and engagement in care and virologic suppression as well as other preventive health needs.The key limitation to this review is the lack of evidence-based data to support recommendations for TGW living with HIV. Meaningful inclusion of sexual orientation and gender identity data in national surveillance systems, as well as prospective studies investigating the long-term health outcomes for transgender people accessing medical transition services, will allow for more robust data upon which to build guidelines for care.


Trichuris trichiura is a soil-transmitted helminth which is prevalent in warm, moist,tropical and subtropical regions of the world with poor sanitation. Heavy whipwormcan result either in Trichuris dysenteric syndrome - especially in children - or in achronic colitis. In heavy infections, worms can spread proximally and may causeileitis. Here we provide first microscopic evidence for a T. trichiura adult wormembedded in the rectum of a post-Colonial Brazilian adult mummy. During Colonial andpost-Colonial times, many European chroniclers described a parasitic disease namedMaculo whose symptomatology coincides with heavy helminthiasis. Based on our findingsand on comparison of ancient textual evidence with modern description of heavywhipworm, we feel confident in considering that the two syndromes are expressions ofthe same pathological condition. Trichuris trichiura is a soil-transmitted helminth which is prevalent inwarm, moist, tropical and subtropical regions of the world with poor sanitation  (rescent) . The infTrichuris dysenteric syndrome (TDS) especially in young children; TDSis characterised by mucoid diarrhoea, rectal bleeding and rectal prolapse complicated withsevere bacterial secondary infection  unearthed from thetopsoil of Itacambira's church .Here we report on a case of T. trichiura eggs in its faeces although the exact burdenof the infection could not be established due to the eggs' poor state of preservation. Both nutritional and sanitary conditions amongthe people who partook these expeditions were extremely poor and people were, therefore,exposed to several infections caused by different parasites.T. trichiura eggs have been abundantly found in coprolites ofancient individuals from all continents dating both to prehistoric and historic periods or doença do bicho (disease of the bug); this disease wascharacterised by "rectal inflammation, fetid mucous elimination, ulcerations and bloodydiarrhoea" accompanied, sometimes, by rectal prolapse  were given between the end of the XVI century [Maculo syndrome, which was enhanced by poor nutrition and unsanitaryconditions, is claimed to have caused hundreds of deaths in Colonial and post-ColonialBrazil (The lBrazil . Based o


The importance of microbial activity to ecosystem function in aquatic ecosystems is well established, but microbial diversity has been less frequently addressed. This review and synthesis of 100s of published studies on stream microbial diversity shows that factors known to drive ecosystem processes, such as nutrient availability, hydrology, metal contamination, contrasting land-use and temperature, also cause heterogeneity in bacterial diversity. Temporal heterogeneity in stream bacterial diversity was frequently observed, reflecting the dynamic nature of both stream ecosystems and microbial community composition. However, within-stream spatial differences in stream bacterial diversity were more commonly observed, driven specifically by different organic matter (OM) compartments. Bacterial phyla showed similar patterns in relative abundance with regard to compartment type across different streams. For example, surface water contained the highest relative abundance of Actinobacteria, while epilithon contained the highest relative abundance of Cyanobacteria and Bacteroidetes. This suggests that contrasting physical and/or nutritional habitats characterized by different stream OM compartment types may select for certain bacterial lineages. When comparing the prevalence of physicochemical effects on stream bacterial diversity, effects of changing metal concentrations were most, while effects of differences in nutrient concentrations were least frequently observed. This may indicate that although changing nutrient concentrations do tend to affect microbial diversity, other environmental factors are more likely to alter stream microbial diversity and function. The common observation of connections between ecosystem process drivers and microbial diversity suggests that microbial taxonomic turnover could mediate ecosystem-scale responses to changing environmental conditions, including both microbial habitat distribution and physicochemical factors. Aquatic microbial diversity is well understood to be a key component of aquatic ecosystem functioning , and majStreams and rivers are hotspots of microbially mediated carbon (C) and nutrient processing within landscapes . MicrobiPseudomonas sp. isolates with variation in diversity correlated most strongly with water temperature, storm events and sunshine  AND TS = “diversity” for all full years available (through 2013). I screened search results and kept all papers that (i) reported microbial taxonomic diversity including data collected using molecular, microscopy, and “culture for diversity” methods and (ii) evaluated the effect of some environmental variable on microbial diversity. I accepted papers that reported diversity as richness, as a diversity index incorporating some estimate of relative abundance of taxa, or as the relative abundance of measured taxa (community structure). These search parameters may not catch some relevant studies, however, the resulting synthesis of 294 papers is to my knowledge the most comprehensive to date.To collect as many published, peer-reviewed studies on stream microbial diversity as possible, I searched the Web of Science database using the parameters TS = (“stream” OR “river” OR “lotic”) AND TS = (microbin situ hybridization (FISH), other molecular methods and other non-molecular methods; and (iii) the environmental driver(s) evaluated by the study . All studies were also categorized by the OM compartment sampled coarse particular organic matter , water column, epilithon (biofilm attached to any hard surface), streambed sediment, hyporheic biofilm or water, and other , and by the scale of investigation . Many studies included multiple experiments or comparisons, or comparisons that could be classified under multiple categories .These studies were categorized based on (i) the microbial group of interest ; (ii) the methodology used , terminal restriction fragment length polymorphism (T-RFLP), sequencing of genes from clone libraries, sequencing of genes from next-generation technology libraries, fluorescent It is not valid to compare the values of derived diversity metrics or the abundance of microorganisms based on data collected with different methodologies and taxonomic resolutions, so a fully quantitative metaanalysis, using a response index, was not possible. However, it is valid to accept significant results of a study as informative, no matter the data type. For example, a meta-analysis of heterogeneity in soil microbial communities showed greater-than-random spatial similarity no matter the technique used to measure diversity, but the magnitude of heterogeneity detected was greater if a lower-resolution taxonomic definition was utilized . Therefopost hoc comparisons using R Commander , nor FISH data, which, while based on the 16S rRNA gene sequence, tend to not quantify bacterial phyla such as Acidobacteria, Cyanobacteria, or Verrucomicrobia. While differences in extraction protocol, sequencing primers, sequencing depth, and other technical particulars introduce potentially large sources of non-environmental variation to this analysis, signals that rise above the noise must be particularly strong. I tested the hypothesis that OM compartment type affected bacterial community composition across studies with a one-way analysis of variance (ANOVA) and Bonferonni ommander .Figure 1). Many fungal studies utilized a definition of taxonomic identity based on conidial morphology, making microscopy the most common methodology in the database (26% of studies), followed by the “community fingerprint” techniques of DGGE and T-RFLP  and sequencing of ribosomal genes from clone libraries . Only 4% of studies utilized next-generation sequencing, all published in 2012 or 2013; this reflects a more rapid increase in papers using molecular methods in the past decade . In total, almost half of the comparisons measured spatiotemporal variation in microbial community composition , and the most commonly evaluated environmental drivers were nutrient concentration (11%) and OM quality or quantity . Finally, among compartment types studied, CPOM was best represented , followed by the water column , epilithic biofilms (18%), and benthic sediment .A total of 294 papers published between 1976 and 2013 were included in the analysis; these papers contained 520 comparisons of the effects of spatiotemporal heterogeneity or defined environmental drivers on microbial community composition in streams (Appendix). The majority of papers examined bacterial communities (56%), many examined fungal communities (36%), and the remainder examined archaeal, protozoan, or stramenopile communities .The overall distribution of published significant versus non-significant effects of environmental variation on stream microbial community composition was 88.5% versus 11.5%. The most common source of spatio-temporal heterogeneity was within-stream heterogeneity among different OM compartments (97% significance among published effects), followed by within-stream temporal, among-stream, and within-stream longitudinal heterogeneity . The results of ANOVA post hoc comparisons showed significant among-compartment differentiation in a number of phyla, including the Acidobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria, and other Bacteria (Table 2). Among different stream compartments, water column samples contained the highest relative abundance of Actinobacteria, epilithon samples contained the highest relative abundance of Bacteroidetes and Cyanobacteria, FBOM samples contained the highest relative abundance of Acidobacteria, and sediment samples contained the second highest relative abundance of Acidobacteria and the highest relative abundance of other Bacterial sequences.Because a good number of studies reported the dominant bacterial phyla and subphyla in 16S rRNA gene sequence libraries from defined sample types , it was possible to evaluate which taxonomic groups varied in relative abundance among compartments . The studies evaluating metals effects did not report widely comparable taxonomic data , so it was not possible to perform an evaluation of metals effects on microbial community composition.The category of defined environmental drivers with the most commonly significant effect was metals (100%), followed by temperature, OM quantity or quality and hydrology, land use and nutrient concentrations .This meta-analysis of environmental variation in stream microbial diversity was dominated by within-stream studies ; to contrast with other study scales, the 73 among-stream comparisons showed a total distribution of 86% significant effects, with 100% significance within all categories of variation except land use and nutrient concentrations , and the 11 among-region comparisons showed 100% significance within all categories of variation . This temporal variation includes seasonal changes  than longitudinal (83%) or among-stream (86%) variation in lotic microbiota . Differences in stream microbiota between surface water, rock surfaces, leaves and wood, and streambed sediment – within the same stream – were more common than any other spatiotemporal effect. Only one study found similar microbial diversity between compartment types; this study took place in a contaminated stream where the high metals concentrations were hypothesized to limit fungal diversity . The high relative abundance of Cyanobacteria and Bacteroidetes in epilithic biofilm makes sense, as autotrophic microbes such as Cyanobacteria are more competitive on an inorganic substrate, and Bacteroidetes are known to be characteristic of well-developed stream biofilms , with the exception of the always-significant effect of metals. As noted earlier, the only study that reported no significant differentiation among OM compartments was undertaken in metal-contaminated streams . This may point to a relatively high level of functional diversity and redundancy regarding nutrient kinetics within natural microbial communities: cells may have strategies to operate under a range of nutrient concentrations . The dominant controls on bacterioplankton diversity, such as light and algal abundance, predation, temperature and salinity  may be related in part to autochthonous versus allochtonous OM source, as noted earlier. While it is possible that the structure and function of microbiota on allochthonous substrates responds differently to environmental changes than those associated with autochthonous substrates (While most work on aquatic microbial diversity has focused on planktonic communities , in lotisalinity , may difsalinity . A predobstrates , like prbstrates . The topbstrates , and hypbstrates .In addition to the organic substrate heterogeneity presented by the benthos-dominated lotic ecosystem, the level of interaction between benthic and pelagic habitats affects aquatic diversity and function . BacteriSurface-attached and bed sediment-entrained cells are not always accounted for in broad views of microbial life in aquatic ecosystems , yet theLZ collected and synthesized the data and wrote the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Epileptic seizures of focal origin are classically considered to arise from a focal epileptogenic zone and then spread to other brain regions. This is a key concept for semiological electro-clinical correlations, localization of relevant structural lesions, and selection of patients for epilepsy surgery. Recent development in neuro-imaging and electro-physiology and combinations, thereof, have been validated as contributory tools for focus localization. In parallel, these techniques have revealed that widespread networks of brain regions, rather than a single epileptogenic region, are implicated in focal epileptic activity. Sophisticated multimodal imaging and analysis strategies of brain connectivity patterns have been developed to characterize the spatio-temporal relationships within these networks by combining the strength of both techniques to optimize spatial and temporal resolution with whole-brain coverage and directional connectivity. In this paper, we review the potential clinical contribution of these functional mapping techniques as well as invasive electrophysiology in human beings and animal models for characterizing network connectivity. Epilepsy is one of the most frequent chronic neurological disorder, with an incidence of 50/100,000/year and a prevalence of 0.5–1% . One thifocal” as indicating seizures arising primarily “within networks limited to one hemisphere and that may be discrete or more widely distributed”  , 16. A RIn this paper, we review the converging evidence from different brain mapping techniques in human and animal models that epilepsy is related to the dysfunction of a large-scale brain networks, with alterations of physiological brain networks. We will particularly focus on the clinical impact of this new view of epilepsy as a network disease.An electronic literature search was conducted for articles on this topic regarding human and animal subjects. Sources searched included PubMed and relevant books. Words used in the search included the text words and subject headings of epilep*, functional connect*, resting-state functional network*, temporal epilepsy, extra-temporal epilepsy, electroencephalogram (or EEG), simultaneous functional MRI (fMRI) and EEG (or EEG-fMRI), electric and magnetic source imaging , intracranial EEG (or iEEG or sEEG), cortico-cortical evoked potential, and single-pulse electrical stimulation. The words were searched independently and in combination. For each citation considered, the abstract was read (when available), and articles were excluded if they were outside the scope of the review. Studies published only in abstract form, letters, and technical reports were excluded. The bibliography of each of the retrieved papers was examined to identify relevant references that could have been missed by electronic search. The findings were described taking into account the limit of words and the critical insight of the authors.Functional interactions between brain regions activity, can be characterized in several ways. On the one hand, functional connectivity (FC), the most widely used metrics, measures the statistical dependency between different signals obtained by correlation analysis. However, such strategy does not account for the direction of the information flow and cannot therefore infer causality relationships. On the other hand, effective or directed connectivity investigates directional relationships and aims at describing causal influences. Effective connectivity can be investigated using model-driven techniques such as structural equation modeling  and dynaThe concept of brain networks originated, and has largely benefited, from the use of resting-state fMRI. fMRI detects blood oxygen level dependent (BOLD) signal change reflecting metabolically active brain areas not only in relation to a specific physiologic or pathologic event  but alsoBiswal and colleagues demonstrated for the first time (1995) that brain regions that are functionally related, show temporal correlations in the low frequency component of the BOLD signal. In other words, fMRI FC detects zones that exhibit correlated BOLD fluctuations and, as a result, belong to the same functional network . StudiesFunctional connectivity can be measured while the subject is performing a behavioral and cognitive task (task-related FC), or while the subject is not performing any specific task (RS-FC). The RSN that is mainly activated in condition of resting wakefulness and deactivated in task performing is called default-mode network (DMN) . This pha priori hypothesis,” like seed-based approach  are reliable techniques for estimating the localization of the cortical generators of epileptic activity –55 and tThe analysis of interictal epileptic discharges has principally aimed at localizing epileptic generators in the context of pre-surgical evaluations rather than studying large brain networks. Case reports or small MEG series showed promising results for the localizing value of the regions with high information outflow, estimated by connectivity analysis –63. In aIn generalized epilepsies, connectivity studies have highlighted a network of hyperconnected anterior regions in photosensitive patients . NetworkAnother study used co-occurrence of MEG interictal spikes to build graphs of connectivity between the estimated sources of these spikes. In seven patients also investigated with stereotactic iEEG, the connections revealed by MEG were confirmed by iEEG .Similarly to fMRI studies, future work will need to distinguish between transient connectivity alterations related to interictal discharges, that are known to be associated with subtle cognitive impairment , and deeSimultaneous EEG and fMRI (EEG-fMRI) detects hemodynamic changes in the brain related to events of interest identified in the EEG . CombiniBOLD responses to a neural event are usually detected with a delay of 4–6 s . NeverthThe combination of ESI with EEG-fMRI can offer complementary information for improving each single technique Figure . AlthougEEG-fMRI studies can give insights about epileptogenesis. Interictal spikes of different types of epilepsy , are associated with deactivation in the precuneus and posterior cingulate cortex , regionsA specific area, localized in the medial orbito-frontal gyrus (piriform cortex), called “area tempestas”, seems to be involved in the genesis or propagation of epileptic activity , 88 in fFrom a methodological point of view, multimodal combination between EEG/ESI, fMRI, and diffusion imaging tractography will allow exploring functional and structural connectivity at a finer spatio-temporal scale. Some initial small studies have highlighted the potential of these combinations –96.Unfortunately, only few of the many studies on RS-FC have been done with the simultaneous recording of EEG. Spikes cause a transient cognitive impairment, by affecting, e.g., memory retrieval in rats , and memThe majority of RS-fMRI studies in focal epilepsies have focused on TLE, which is the most common form of focal epilepsy in adults and offers the advantage of being one of the most homogeneous groups within the focal epilepsy syndromes.Temporal lobe epilepsy has been the first epileptic syndrome to be considered as epileptogenic network  with relIn unilateral mesial TLE (MTLE), the affected amygdala and hippocampus  are less connected between them and also with other consistent RSNs, such as the mesolimbic and the DMN, suggesting that these functional interictal changes explain cognitive and psychiatric impairments often found in patients with this type of epilepsy . SeveralDefault-mode network. Laufs and colleagues  in attention tasks. Cataldi et al.  showed that focal spikes are associated with networks of widespread metabolic changes, specific for each type of epilepsy .Negishi et al.  revealedDifferent theories have been proposed about the patho-physiology of “generalized seizures”. Meeren et al.  reviewedSeveral EEG-fMRI studies showed that during short GSWD , 148–150Concerning the role of subcortical structures, in patients with IGE, it has been shown that both the anterior nucleus of thalamus (ANT) and the centromedian/parafascicular (Cm/Pf) nucleus (which provides diffuse inputs to the cortex) are activated during GSWD; the activity of the cortico-reticular Cm/Pf preceded that of the ANT, suggesting that the Cm/Pf is involved in GSWD initiation or early propagation, while the ANT in its maintenance .Recent studies have used fMRI to investigate whether resting-state FC between thalamus, basal ganglia, and DMN areas is altered in patients with IGE, even during GSWD-free periods of brain activity Figure . Wang etOther RSNs can be affected in patients with IGE, reflecting specific cognitive impairment when compared to controls. A seed-based RS-FC study in 14 patients with IGE showed that attention network is impaired even in interictal periods, and that this impairment is related with the disease duration .One study in 60 IGE patients specifically addressed the question whether pharmacoresistance was correlated with RS-FC changes in the DMN . DMN conThe most frequent IGE syndrome is juvenile myoclonic epilepsy (JME), where seizures are characterized by myoclonic jerks of the upper limbs, often triggered by cognitive inputs. Several RS and task-related functional studies have shown an impairment of connectivity among supplementary motor area and the rest of the brain –170, sugNumerous EEG-fMRI studies have been conducted on pediatric syndromes . SeveraConcerning idiopathic focal epilepsies of childhood, these comprise a broad spectrum of phenotypes showing an overlap with each other, from benign childhood epilepsy with centro-temporal spikes (BECTS) to more severe seizure and cognitive disorders, like atypical benign partial epilepsy (ABPE), continuous spikes and waves during slow sleep (CSWS), and Landau-Kleffner syndrome. In patients with BECTS, EEG-fMRI studies have revealed focal spike-associated BOLD signal changes in the sensorimotor cortex, well corresponding to spikes localization, and typical seizure semiology –180. In A single-subject and group-analysis study  on patieAn EEG-fMRI study in thirteen patients with ring 20 chromosome syndrome  shows spThe indication for video-iEEG monitoring is the absence of a unique focal hypothesis regarding the source of the patient’s seizures (obtained with non-invasive investigation), or the need for cortical mapping of the epileptogenic cortex vs. eloquent cortex . TherefoThe concept of the seizure-onset zone as a single, circumscribed brain region implies that, assuming that intracranial electrodes sample this region, ictal iEEG activity should invariably start there. Careful observation of ictal iEEG recordings, however, reveals that this is not always the case. Rather, there are patients in whom clinically indistinguishable seizures seem to start at any of two or more distinct brain areas . ObservaThe same authors analyzed the neurophysiological correlates of alterations of consciousness in TLE . They foMeasures of directed connectivity in seizure networks are starting to reveal the internal organization of the individual nodes that make up the network. To date, most iEEG studies use mathematical approaches to determine the direction of connections. For instance, using focal cortical dysplasia as a model of a circumscribed seizure-onset zone and applying partial directed coherence analysis, Varotto et al.  found thAnother potential breakthrough in the investigation of epileptic networks could stem, in a somewhat paradoxical fashion, from micro-electrode array recordings, which revealed new insights on the pathophysiology of epilepsy. Schevon et al.  insertedfibers de passage, an important caveat to keep in mind when interpreting the directionality information provided by these data  in epileptic patients consists of administering electrical currents to the brain tissue in order to transiently influence or perturb its function. A technique almost as old as epilepsy surgery, it has mostly been used to probe the function of the cortex directly underlying or surrounding the stimulation site –205. In ese data . This hiEvoked effective connectivity has revealed strong intralobar connectivity in the temporal and frontal lobes, as well as connections between the frontal and temporal lobes that are more prominent in the frontal-to-temporal than in the temporal-to-frontal direction , 211. AnDirect electrical stimulation has also been used to specifically evaluate epileptic networks, the general idea being that the responses of remote sites to stimulation of epileptogenic cortex  or the rRecording the activity of any node suspected to be determinant in the disease is not feasible in human beings, contrarily to animal research. Moreover, animal-related technologies offer the possibility to desiccate and manipulate cellular and molecular components of such networks, as well as scrutinizing the associated structural and functional alterations. A great perspective in pathological networks study is detecting features associated with the risk of recurrence after a first seizure as well as predicting the evolution toward pharmaco-resistance.Animal models allow studying networks connectivity and recording the underlying brain activity with high spatial coverage and resolution , and addIn animals and human beings, focal onsets have been identified in generalized epilepsy, and complex large-scale network involvement has been shown in focal epilepsies , 11, 14.Models of focally induced epileptic disorders might avoid this limitation. One of them, electrical kindling, triggers focal epileptic activity using focal electrical stimulation in accordance with standard stimulation parameters, e.g., duration of the stimulation, frequency, and intensity of the stimulus , 236. Thin vitro Mg2+-free model of hippocampal epilepsy, Gong et al.  signal in a rat model of TLE. They revealed the decreased correlation of several pairs of brain structures, most of them included left amygdala and left entorhinal cortex  stopped the ongoing hippocampal paroxysmal discharges (HPD), while higher current intensities were needed to stop the HPD if DBS was applied to neighboring areas  considered epilepsy as a systemic disease, centered in the brain, due to an altered “defluxion of cold phlegm” through the body. In more recent times, the concept of epilepsy as “focus disease” has been largely developed –282, wheConcerning “generalized” epilepsy, neuro-imaging, and especially connectivity studies have allowed considering them as focal brain disorders with fast bilateral discharge propagation. This concept leads to the idea that focal and generalized epilepsies are the two extremes of a single spectrum and to a possible new way of studying mechanisms of AED: do they have an effect on particular nodes of a network where receptors are more expressed? Is it possible to detect an anatomical target to avoid generation/propagation of seizures, using disconnection or stimulation? For all these reasons, translational research in light of network analysis, based on fundamental science through animal experiments and clinical perspectives through human research, opens new opportunities to better understand the complexity of epilepsy and define new and more effective treatments for patients.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Brucella is a facultative intracellular pathogen that causes zoonotic infection known as brucellosis which results in abortion and infertility in natural host. Humans, especially in low income countries, can acquire infection by direct contact with infected animal or by consumption of animal products and show high morbidity, severe economic losses and public health problems. However for survival, host cells develop complex immune mechanisms to defeat and battle against attacking pathogens and maintain a balance between host resistance and Brucella virulence. On the other hand as a successful intracellular pathogen, Brucella has evolved multiple strategies to evade immune response mechanisms to establish persistent infection and replication within host. In this review, we mainly summarize the “Stealth” strategies employed by Brucella to modulate innate and the adaptive immune systems, autophagy, apoptosis and possible role of small noncoding RNA in the establishment of chronic infection. The purpose of this review is to give an overview for recent understanding how this pathogen evades immune response mechanisms of host, which will facilitate to understanding the pathogenesis of brucellosis and the development of novel, more effective therapeutic approaches to treat brucellosis. Brucella species are the causative agent of brucellosis, among one of major bacterial zoonotic diseases. Brucella is Gram-negative, non-spore-forming, non-motile, facultative anaerobe and intracellular in nature which mainly affect the reproductive tract and cause abortion and infertility in natural host , B. abortus , B. suis , B. canis , B. ovis , B. neotomae , B. cetacea , B. pinnipedia , B. microti , and B. inopinata  and certain host parameters including breed, genetic background, age, sex, and physiological status  to avoid this degradation  but result in inhibition of vacuolar attachment of the proteolytic enzyme, cathepsin D  . Autophagy proteins play an important role in the production of (autophagic Brucella-containing vacuole aBCVs) and pathogen observed in aBCVs contain LAMP1 at the later stage after infection (48–72 h). Finally through the lytic and non-lytic mechanisms, the pathogen is released from the cell  and reactive nitrogen intermediates (RNI) which ultimately help this pathogen during transportation to lymphoid tissues facilitated by neutrophils , the pathogen contains longer fatty acid residue (C28) and due to this modification in LPS structure which reduces its endotoxic properties by decreasing TLR4 agonist action , it competes with myeloid differentiation response gene 88 (MyD88) that ultimately facilitates ubiquitination and degradation of Mal and inhibits both TLR4 and TLR2 signaling  shows sequence resemblances with Toll/IL-1 recptors (TIR) domain family. Different studies investigated the role of Btp1 in DC maturation due to significance of TIR domain in the TLR signaling  fusion of endoplasmic reticulum with endocytic compartment occurs in the Sar1- and Rab2- dependent manner. In macrophages and epithelioid cells during early stages of brucellosis, rBCV convert into a specialized property compartment known as autophagic BCV (aBCV). Recent evidence supports the theory that autophagy-associated proteins play a key role in the biogenesis of rBCV and contribute to completion of intracellular life cycle, Brucella subverts the host cell membrane trafficking pathways  into rBCV  and autophagy in V Figure .Brucella-infected macrophages via TcpB facilitates protein folding that affects Brucella intracellular growth (Smith et al., Brucella replication and rBCV biogenesis. Hence, this is an essential strategy used by Brucella to combat with host immune response for which further study is required to investigate the involvement of autophagy in Brucella pathogenesis as well as in innate and adaptive immune mechanisms.It is also observed that UPR activation in the Brucella to maintain intracellular niche for its replication. Brucella inhibits apoptotic mechanism in infected macrophages with the induction of chemical stimuli (Gross et al., B. melitensis infection there is down regulation of genes involved in apoptotic pathway in mitochondria (He et al., Brucella-infected macrophages treated with Nedd4 decrease this activity that in turn stops calpain 2 degradation and leads to macrophages apoptosis (Cui et al., Apoptosis is an extremely well-regulated manner of programmed cell death, which is generally facilitated by the initiation of caspases, and excellent host defense response against intracellular bacteria. However, inhibition of apoptosis is another strategy of Brucella-macrophage to enhance virulence by inhibiting macrophage cell death. After the invasion of smooth Brucella into macrophages for replication, the pathogen automatically converts into rough mutants that can results in macrophage cytotoxicity that favors bacterial egress and dissemination (Pei et al., B. abortus 2308 rough mutants that lack of surface LPS exhibited failure in the inhibition of apoptosis (Pei et al., Another important strategy that is employed involves the interaction of Brucella lipoprotein increases T cells apoptosis which depends upon TNF-α secretion, sheding light on mechanisms that Brucella uses to directly inhibit T cells responses, involved in adaptive immune evasion (Figure Brucella- infected neutrophils and monocytes have obvious upregulation of various adhesion molecules such as CD106 and CD54 that result in inhibition of apoptosis in brucellosis (Scian et al., Brucella for intracellular replication that in turn facilitates evasion of the immune response.n Figure . AdditioMicroRNAs are part of small noncoding RNA involved in regulation of gene expression and in cellular mechanisms, apoptosis and signal transduction (He and Hannon, Brucella adapt diverse environmental conditions and use multiple strategies to evade host cell defense. It would seem that Brucella sRNA may play a significant role in bacterial responses to stress. Hfq protein mediates most of the sRNA-mRNA interactions which is required for virulence and to control bacterial stress response in pathogen–host interactions (Papenfort and Vogel, Brucella stress response (Robertson and Roop, B.melitensis hfq mutant (Cui et al., B. abortus sRNAs, abcR1 and abcR2 play essential roles in pathogenicity and in chronic infection, resulting in a significant decrease in intracellular survival in a mouse model and in macrophages as well (Caswell et al., Brucella virulence. However, their inhibited expression within macrophages led to decreased Brucella intracellular survival indicating that miRNAs are essential for the adaptation to stress conditions and ultimately cause modulation of Brucella intracellular survival (Wang et al., Casewell et al. reported that Brucella (Castaneda-Ramirez et al., B. melitensis-infected cells and found several miRNAs such as miR-92a, miR-93, let-7b, miR-1981, and miR-181b differentially expressed compared to mock-infected cells, and purposed that these miRNAs might be involved in immune response mechanisms, autophagy and apoptosis (Zheng et al., Brucella for immune evasion.Inhibited expression of syntaxin mRNA with small interfering RNAs modulated initial phagocytosis and intracellular survival of Brucella spp. shows deliberate release through direct discharge to poses risk to public health. Brucella has remarkable strategies enabling avoidance of the host immune response and facilitating the establishment of chronic infections. However, for survival the host cells have developed complex immune mechanisms to defeat and battle against pathogens and maintain a balance between host resistance and Brucella virulence. During the intracellular replication Brucella shows typical tissue tropism for lymphoreticular and reproductive systems that helps in evasion of innate and adaptive immune mechanism of host to establish clinical disease manifestations and pathogenesis. At the early stage of infection symptoms observed in humans includes, fatigue, pyrexia, anorexia, myalgia and diaphoresis. However, during chronic stage of brucellosis, persistence occurs in the tissues of mononuclear phagocyte system including bone marrow, lymph nodes, liver and spleen. Likewise it also persists in male reproductive organs, placenta and fetus. At early process of infectious pathogenesis, Brucella modulate the immune response mechanism of host to quickly translocate through mucosal immune barrier and is endocytosed by mucosal macrophages and DCs. Stealthy brucellae use different type of strategies to establish and maintain chronic infection by fusion of type IV secretary system dependent BCVs with lysosome to evade intracellular destruction. Brucella infects the host cell and protects itself by limiting PRRs including complement system and TLR signaling pathways. Brucella LPS shows non-cononical structural differences that inhibit antigen presentation to T cells and dampens innate and adaptive immune mechanisms. Moreover, Brucella LPS inhibits activation of DCs to subvert the immune response and establish protective Th1 immune response by secreting IL-12 and preventing T-cell stimulatory action. Brucella modulates MHC-I and MHC-II expression prompted by IFN-γ which depend upon cytokine regulation. In addition, Brucella uses multiple strategies such as cloaking to avoid recognition by the autophagy mechanism and development of replicative niches. Furthermore, inhibition of apoptosis is another strategy of Brucella to evade the immune response to establish chronic infection.Although in brucellosis humans are infected as incidental host, approximately 500,000 new cases reported annually, yet no patient-friendly treatment or effective vaccines available for humans. Additionally, Brucella adapted to the intra-macrophage environment it extends intracellular replication to reach other systems preferably to target cells and tissues such as skeletal tissues, male genitalia, placental trophoblasts, reticuloendothelial system and endothelium. Newly identified protein UPR and Btp1/TcpB are also important in innate immune evasion during early stages of infection. Recent evidence suggests that Yip1A and IRE1α are essential for Brucella replication and rBCV biogenesis that ultimately supports nutrient acquisition and pathogen cell-to-cell spread. Even though numerous strategies employed by Brucella to evade the immune response have been identified, there are many questions that need to be answered in respect to other “stealth” mechanisms. What mechanism is involved in apoptosis inhibition by Brucella in infected host cells remains to be clarified? How do miRNAs interfere with innate and adaptive immune response mechanisms? How miRNAs cause modulation in Brucella intracellular survival? What is the role of miRNAs in autophagy and apoptosis mechanism will be an open question for the next few years. In depth understanding of the “stealth” strategies used by this pathogen will facilitate focus on the pathogenicity of bacterium and development of novel effective therapeutic approaches to treat brucellosis in the future.Once WA and ZL designed the research and wrote the manuscript. KZ helped to prepared the figures. ZL revised the manuscript critically for relevant intellectual content. All authors have read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Malaria currently causes more harm to human beings than any other parasitic disease, and disproportionally affects low-income populations. The ethical issues raised by efforts to control or eliminate malaria have received little explicit analysis, in comparison with other major diseases of poverty. While some ethical issues associated with malaria are similar to those that have been the subject of debate in the context of other infectious diseases, malaria also raises distinct ethical issues in virtue of its unique history, epidemiology, and biology. This paper provides preliminary ethical analyses of the especially salient issues of: (i) global health justice, (ii) universal access to malaria control initiatives, (iii) multidrug resistance, including artemisinin-based combination therapy (ACT) resistance, (iv) mandatory screening, (v) mass drug administration, (vi) benefits and risks of primaquine, and (vii) malaria in the context of blood donation and transfusion. Several ethical issues are also raised by past, present and future malaria research initiatives, in particular: (i) controlled infection studies, (ii) human landing catches, (iii) transmission-blocking vaccines, and (iv) genetically-modified mosquitoes. This article maps the terrain of these major ethical issues surrounding malaria control and elimination. Its objective is to motivate further research and discussion of ethical issues associated with malaria—and to assist health workers, researchers, and policy makers in pursuit of ethically sound malaria control practice and policy. Malaria morbidity and mortality are largely preventable via existing interventions . There has been a significant increase in financial aid directed at malaria control in the last decade, resulting in a halving of global malaria burden since 2004 , 2. HoweProgrammes combatting other similarly important infectious diseases, such as HIV and tuberculosis, raise many ethical issues that have been identified and addressed to some extent –8, yet mThe prevalence of malaria is inversely proportional to GDP per capita. Sachs and Malaney aptly note that “where malaria prospers most, human societies have prospered least” . VulneraInter alia, justice requires both improvement of access to existing interventions and increased research and development of new tools for malaria control, both of which require an influx of funding from wealthy nations and donor organisations. Malaria burden is a major example of global inequities, which are receiving heightened attention at the international level as reflected by the Sustainable Millennium Development Goals.International efforts towards malaria control thus represent a major opportunity to remedy some of the past and present injustices manifested by global disparities in wealth and freedom from preventable disease , 12. IntAnopheles mosquitoes, and by displacing human populations [Anthropogenic climate change affects human health in many ways, and has the potential to alter malaria disease patterns by changing the habitats of ulations –15. Areaulations  and poteulations . While fA core ethical issue for malaria policy is the urgent need for universal access to control interventions , 5. WithThe cost of anti-malarial medications poses similar challenges, and lack of access to adequate health services commonly leads to treatment failure and death. Counterfeit drugs, for example, may be more widely available in areas with limited healthcare infrastructure . In addiOverall the lack of access to prevention and treatment for the most impoverished or isolated will only increase the injustices associated with malaria. Universal coverage that is free or genuinely affordable will be the only way to sustainably control or eradicate the disease. It will also have secondary benefits such as decreasing pressure on hospitals by reducing the total incidence of severe malaria, and by preventing the emergence and spread of resistance.Anti-malarial resistance, especially to artemisinin-based combination therapy (ACT), is a major threat to malaria control efforts. Resistance generally results from inappropriate, incomplete or inadequate courses of treatment. This most commonly occurs in the context of poverty, counterfeit medications, and weak healthcare infrastructure , 23. TheThe current ethical priorities to address drug resistance are therefore: universal availability of (i) diagnostic support (including resistance testing), (ii) targeted, locally effective treatments, before multi-resistant strains emerge, and (iii) increased research and development of new drugs (to combat multi-resistant strains). Universal access to these resources would significantly address current injustices, and help to prevent major future harms and increased costs due to resistance.Several malaria control strategies have specific ethical implications. The following sections address some examples that require the balancing of individual liberties against public health goals.Mandatory screening has been implemented in many countries targeting or maintaining malaria elimination. This involves the compulsory testing of asymptomatic travellers or immigrants from malaria endemic zones , 27. TheResistant malaria creates a further justification for mandatory screening e.g., to prevent the spread of ACT-resistance from South East Asia to India and Africa : a recenAt present, the circumstances in which mass drug administration (MDA) would provide a cost-effective long-term benefit have yet to be clearly defined, but it could play an important role in eradication efforts . EthicalIn all cases, communities should be closely involved in programme design. If mass screening is used prior to MDA, the test should be chosen based on programme goals and community acceptability where feasible, some form of informed consent at the community and/or individual level should be coupled with well-designed education and social awareness campaigns to ensure population-wide effectiveness .Plasmodium falciparum, primaquine is safe and largely effective [Plasmodium vivax carry higher risks. While treatment-related deaths are rare, the risks of haemolysis conferred by different genotypes and phenotypes in different populations have not been well defined, and no point-of-care test can determine individual risk. This poses practical as well as ethical challenges for clinicians and policy makers, especially where primaquine is considered in P. vivax elimination MDA including aparasitaemic individuals, for whom the ratio of risks to benefits would be highest [The aminoquinolones (primarily primaquine) present a unique ethical case in malaria control. The use of such drugs is one of the few ethical issues specific to malaria that has received explicit ethical analysis . When usffective , yet hig highest .P. vivax. Meanwhile, the moral priorities to adequately inform patients and reduce overall harms where possible should guide further research aimed at improving treatment programmes. Thus, ultimate goals of research in this area should be development of (i) accurate point-of-care testing for risk of haemolysis (G6PD deficiency) and (ii) less toxic alternatives to primaquine. Current efforts should meanwhile include (i) epidemiological mapping of G6PD deficiency traits in regions aiming at elimination, (ii) further elucidation of phenotypic risk in different genotypes of G6PD deficiency, and (iii) primaquine dose-finding studies [Alternative agents are needed for those who cannot take primaquine, including pregnant women . In the  studies , 36–38.Malaria was one of the first recorded transfusion-transmitted infections . Althouglasting symptoms among participants [acute symptoms—including fever, headache, joint and muscle pains, and even cardiac events—which go beyond many definitions of ‘minimal harm’ that are commonly employed in the ethical assessment of non-therapeutic research [Controlled infection studies involve intentionally exposing human beings to pathogens in order to observe the natural history of disease, develop research models of infection, or test the efficacy of drugs and vaccines . Modern icipants . Howeverresearch . Furtherresearch , which pYet such studies remain important in testing new vaccines or treatments, and it may be more useful to perform them in malaria endemic populations. Risks of controlled infection studies may be somewhat less for participants with substantial immunity from prior infection, and the benefits of participation may also be greater . On the other hand, such studies should only occur in contexts with access to affordable quality healthcare, high standards of research conduct, and robust ethical oversight so that the safety of participants is ensured .Malaria research to measure mosquito or malaria density has often involved the use of human landing catches, where human participants act as mosquito ‘traps’. In some cases, people working as human landing catches may receive 50–100 mosquito bites in one nightshift , 47. TheEthical issues related to vaccination have been discussed for other diseases , 54, andThe avenue of transmission-blocking vaccines (TBVs) is a novel case study in vaccine ethics, especially since they aim only at preventing transmission of malaria from the vaccinated person to healthy individuals . Though Multiple strategies for genetic modification of mosquitoes are under study, aiming to contribute to transmission interruption for malaria (and other mosquito-borne diseases) –59. ImplEthical issues in malaria control and research have hitherto received little explicit analysis in the published literature. This review provides an initial summary of the main issues at hand, but further work is required in many of the areas discussed above, especially since the field is likely to evolve with future changes in malaria science, epidemiology and policy.The epidemiology of malaria is a stark reminder of global injustice in relation to disease risk and health outcomes. The recent improvements in funding and control initiatives have in part been driven by an awareness of these injustices: that those least able to access malaria control mechanisms are often those who need them most, and that there are significant research gaps for malaria and other diseases of poverty. Similarly, ethics can help to guide policy and treatment in areas such as the control of anti-malarial resistance, mass screening and treatment programmes, and the use of primaquine. In malaria research, specific ethics guidelines should be developed to ensure valuable research programmes continue without imposing excessive risks on participants. Looking toward future possibilities, prospective consideration of ethical issues may assist in the appropriate and successful implementation of novel techniques such as malaria vaccines or genetically-modified mosquitoes. Recent capacity building efforts for local ethics expertise in endemic regions have been a positive development.The preliminary analyses offered here are intended to motivate further research on ethical issues associated with malaria, inform ethics review processes related to control interventions, and assist health workers, researchers, and policy-makers in pursuit of ethically sound malaria control efforts. The ultimate beneficiaries of such work will include patients and residents of malaria endemic areas who are in the greatest need of ethically designed malaria programmes now and in the coming decades.


Due in part to the increasing incidence of obesity in developed nations, recent research aims to elucidate neural circuits that motivate humans to overeat. Earlier research has described how the nucleus accumbens shell (AcbSh) motivates organisms to feed by activating neuronal populations in the lateral hypothalamus (LH). However, more recent research suggests that the LH may in turn communicate with the AcbSh, both directly and indirectly, to re-tune the motivation to consume foods with homeostatic and food-related sensory signals. Here, we discuss the functional and anatomical evidence for an LH to AcbSh connection and its role in eating behaviors. The LH appears to modulate Acb activity directly, using neurotransmitters such as hypocretin/orexin or melanin concentrating hormone (MCH). The LH also indirectly regulates AcbSh activity through certain subcortical “relay” regions, such as the lateral septum (LS), ventral pallidum (VP), and paraventricular thalamus, using a variety of neurotransmitters. This review aims to summarize studies on these topics and outline a model by which LH circuits processing energy balance can modulate AcbSh neural activity to regulate feeding behavior. The rise in obesity rates in the U.S.  neurons, are distributed throughout the LH Hahn,  and mediLH innervation of mesolimbic circuitry serves to integrate homeostatic information with motivation to procure foods and the valuation of food tastes. Indeed, food deprivation increases “liking” responses while caloric satiety decreases them  subregion. Lesioning the AcbSh increases food intake, while AcbSh stimulation decreases it . Activation of GABAIn a complimentary manner, AMPA receptor blockade in the aAcbSh increases food intake while in the pAcbSh such blockade decreases intake, though no effects on “liking” or “disliking” are observed from this manipulation  in most of the AcbSh suppresses food intake and hedonic reactions to sucrose while increasing defensive treading and aversive reactions to sucrose  as an indirect route for AcbSh to LH signaling in the control of food intake. Anterograde tracing studies have shown that the AcbSh projects to the medial VP , nesfatin-1, corticotropin releasing factor (CRF), neurotensin, and galanin , hippocampus, and amygdala -Met-enkephalinamide (DALA) into the caudal LS increases in food intake in satiated rats, but LS administration of the broad spectrum opioid receptor agonist MR-2034  into the VP (Fallon and Leslie, In the rat, the PVT is the most dorsomedial region of the midline thalamus (Paxinos and Watson, Activation studies examining c-fos expression demonstrate how the PVT, particularly its anterior part, is activated by multiple food-associated and metabolic signals. The PVT tracks the time of day; c-fos expression changes within the aPVT and pPVT based on time of day (Novak and Nunez, Lesion studies further associate the PVT with food anticipation, food intake, and metabolism. Lesions of the aPVT reduce food anticipatory-mediated increases in blood corticosterone and locomotion without affecting food intake (Nakahara et al., Neuropharmacological evidence examining PVT orexin and GABA input also implicates the PVT in food intake regulation. Orexin 1 receptor knockdown in the pPVT decreases high fat diet consumption without affecting progressive ratio breakpoint for high fat foods in either satiated or food-deprived conditions (Choi et al., The LH innervates the PVT, as confirmed by anterograde (Goto et al., In turn, the PVT projects to the AcbSh (Brog et al., Although it is likely that multiple glutamatergic inputs, not solely the PVT, regulate AcbSh-mediated feeding, the PVT likely has at least a modulatory role on food intake. Electrophysiological data indicates that although PVT input to the AcbSh produces EPSPs, it does not produce action potentials as other glutamatergic inputs do (O’Donnell and Grace, A receptors induces eating (Stratford and Wirtshafter, The concept of an LH to PVT to AcbSh circuit is supported by the effects of GABA in the PVT and glutamate effects in the AcbSh. Activating pPVT GABAHowever, excitation of pPVT neurons, via orexin receptor activation, also induces intake (Barson et al., An alternate explanation is the existence of a presynaptic “brake” on glutamatergic inputs to the AcbSh. Most AcbSh glutamatergic afferents, including the PVT, express the inhibitory mGluR2 presynaptically (Conn and Pin, In summary, LH input informs PVT subregions of bodily nutrient states and palatable food availability. Then, this input modulates aPVT-mediated palatable food anticipation and metabolic signaling as well as modifying the pPVT’s motivational signals to procure foods. Finally, these PVT divisions can collectively potentiate food-directed behaviors through the aAcbSh and pAcbSh, particularly in non-stressful familiar environments. Further anatomical study can clarify whether specific PVT neuron subsets, such as those governing GABA-mediated food intake vs. others governing orexin-mediated intake, selectively innervate AcbSh projection neurons or AcbSh interneurons.Here we have described multiple ways that the LH can regulate food intake via the AcbSh both directly and indirectly, and can do so by altering various aspects of food-seeking behavior. Multiple subdivisions of the LH differentially innervate the AcbSh and utilize different combinations of neurotransmitters. Neurotransmitters localized in the LH are implicated in food intake and effort to procure foods, and administration of LH-originating neuropeptides or their antagonists into the AcbSh impact such behaviors. Also, the LH projects to numerous other brain regions, some of which project to the AcbSh, regulate its activity, and subsequently influence food-directed behaviors. A collection of the pathways we have described can be found in Table The AcbSh and the LH have a direct bidirectional anatomical connection (Usuda et al., The LH sends projections to the LS (Deller et al., The plVP receives orexin input from the LH that enhances liking of foods (Ho and Berridge, A receptors induces food intake (Stratford and Wirtshafter, LH input can excite the aPVT and pPVT through orexin innervation (Ishibashi et al., Here we have also proposed or added to three potential indirect pathways by which the LH can regulate AcbSh activity, either by modulating the motivation to feed or by changing the reward value of foods. These routes are summarized in Figure A few issues arise when interpretting the wealth of aforementioned anatomical data. One issue is that many tracing studies do not specify whether LH projections innervate LS/VP/PVT projection neurons or interneurons. Also, inputs to these brain regions could be en passant, allowing the LH to regulate not only these but other regions receiving collaterals. Further, some anatomical studies using light microscopy rely on visualizing varicosities to suggest innervation of an efferent target, though such innervation may not actually occur in the observed brain region. Visualization of these circuits with electron microscopy can resolve these issues, and further investigation with other methods can properly define the purpose of specific projections in these described circuits. Such data will paint a more complete picture of how energy state integrators impact the motivation to feed and the rewarding value of foods.Considering the various avenues by which homeostatic and sensory inputs can inform AcbSh-mediated motivational and reward-related processing, how then is such processing executed into action? We have mentioned that AcbSh output is routed back through the LH in order to initiate feeding. Indeed, the LH is required for AcbSh-mediated feeding behavior (Stratford and Wirtshafter, Drawing conclusions about brain structure functions from lesion and microinjection studies can be difficult if careful scrutiny is not paid to the specific anatomical spaces involved. Overlaying “empirical” functional spaces with native chemoarchitecture and connectivity maps in future studies is absolutely essential for delineating the exact neural substrates by which certain aspects of a behavior, such as food intake, are regulated Khan, . TowardsThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Fusarium oxysporum (Fo) species complex consists of ubiquitous soil inhabiting fungi that can infect and cause disease in over 120 different plant species including tomato, banana, cotton, and Arabidopsis. However, in many cases Fo colonization remains symptomless or even has beneficial effects on plant growth and/or stress tolerance. Also in pathogenic interactions a lengthy asymptomatic phase usually precedes disease development. All this indicates a sophisticated and fine-tuned interaction between Fo and its host. The molecular mechanisms underlying this balance are poorly understood. Plant hormone signaling networks emerge as key regulators of plant-microbe interactions in general. In this review we summarize the effects of the major phytohormones on the interaction between Fo and its diverse hosts. Generally, Salicylic Acid (SA) signaling reduces plant susceptibility, whereas Jasmonic Acid (JA), Ethylene (ET), Abscisic Acid (ABA), and auxin have complex effects, and are potentially hijacked by Fo for host manipulation. Finally, we discuss how plant hormones and Fo effectors balance the interaction from beneficial to pathogenic and vice versa.Plants interact with a huge variety of soil microbes, ranging from pathogenic to mutualistic. The  Fusarium oxysporum (Fo), one of the most relevant plant pathogens in global agriculture, is a widespread soil-borne fungus that invades roots and causes vascular wilt disease through colonization of xylem tissue , which refers to a specific plant host, as a particular isolate typically produces disease only within a limited range of host species  protein. For several Six proteins a contribution to virulence has been demonstrated, designating them as bona fide effectors , but not to Fo f.sp. raphani (Forp) pointing to an isolate-specific role of SA-dependent defense responses  colonization of the stem  strains also failed to detect activation of typical SA marker genes, at least at the relatively early time points analyzed , an essential component of JA perception, strongly increased resistance to Focn biotrophic pathogens and often involves suppression of SA-dependent defense responses  produce JA-isoleucin and JA-leucin conjugates in culture filtrates that induce senescence-like symptoms on Arabidopsis leaves in a COI1-dependent manner  even showed markedly enhanced susceptibility to Focn under sterile conditions  resulted in enhanced disease symptoms upon ly Table . It is wly Table . MoreoveFo strains revealed a massive induction of ET/JA-responsive genes such as Plant Defensins (PDFs) and Pathogenesis-Related (PR) genes as well as genes encoding ethylene biosynthesis enzymes  mutant is impaired in ethylene perception and shows reduced disease symptoms upon Fol inoculation  colonization but have greatly reduced disease symptoms  colonization during the biotrophic phase of infection, whereas secondary metabolites with hormonal- or toxic-activity trigger plant damage/death during necrotrophic growth.How does e Hasan, . Fo alsoFo strains survive in soil, in the rhizosphere or within plant tissues without causing disease symptoms, and some strains even confer extensive beneficial effects (Alabouvette et al., Fo isolate can employ diverse interaction/colonization strategies whose outcome possibly depends on the “compatibility” of a putative host plant. Thus, the “infection tools” of a Fo strain, likely comprised of a combination of effectors, enzymes, and secondary metabolites, determine the outcome of an interaction: either endophytic with potential beneficial effects for the host or pathogenic with various levels of disease and in extreme cases plant death. This idea is supported by the observation that transfer of one specific Fol chromosome, which contains most of its effector genes plus a secondary metabolite cluster, conferred pathogenicity to an endophytic strain (Ma et al., A growing body of evidence suggests that the majority of Fo manages to trick the hormonal network of its hosts, and how these interactions can have opposite outcomes ranging from pathogenesis to mutualism.Clearly more research in the areas of genomics and effector biology is required to understand how Fo strains. However, their roles vary depending on the host plant and the fungal strain involved, suggesting that the manipulation of the host hormonal network differs between individual Fo strains. Genetic interference with hormone regulators mostly reduced disease symptoms, as seen for JA, ABA, and auxin, indicating that the ability to hijack plant hormone pathways is a requirement for pathogenesis. This scenario implies a strong adaptation to a particular host plant, potentially leading to the narrow host range observed in the Fo species complex. How exactly the manipulation of phytohormone signaling differs between Fo strains—and if this is indeed the key difference between pathogenic and endophytic interactions—remains an intriguing question for future research. Comparison of the respective effector repertoires as well as a better understanding of their mode of action will help answering these questions and may furthermore reveal novel approaches for plant protection, either by breeding or by optimizing Fo strains for biocontrol.Phytohormones determine colonization and disease symptom development during interactions with pathogenic XD and NT wrote the manuscript together with input from FT. The scope and the topic were developed by XD, NT, and FT.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Compared to the diversity of other floral organs, the steps in anther ontogeny, final cell types, and overall organ shape are remarkably conserved among Angiosperms. Defects in pre-meiotic anthers that alter cellular composition or function typically result in male-sterility. Given the ease of identifying male-sterile mutants, dozens of genes with key roles in early anther development have been identified and cloned in model species, ordered by time of action and spatiotemporal expression, and used to propose explanatory models for critical steps in cell fate specification. Despite rapid progress, fundamental issues in anther development remain unresolved, and it is unclear if insights from one species can be applied to others. Here we construct a comparison of Arabidopsis, rice, and maize immature anthers to pinpoint distinctions in developmental pace. We analyze the mechanisms by which archesporial (pre-meiotic) cells are specified distinct from the soma, discuss what constitutes meiotic preparation, and review what is known about the secondary parietal layer and its terminal periclinal division that generates the tapetal and middle layers. Finally, roles for small RNAs are examined, focusing on the grass-specific phasiRNAs. Successful anther development results in pollen dispersal. Steps required to achieve this are conveniently divided into three phases: organ patterning and initial cell differentiation, meiosis, and post-meiotic gametophyte development. Historically most studies have focused on meiosis and pollen biogenesis, with the assumption that a simple lineage model explained how the typical four somatic wall layers with central archesporial (AR) cells arose from a stamen primordium Figure . StartinThe most detailed description of cellular numbers, shapes, and volumes during fate acquisition is available for maize utilizing 3-D reconstruction from confocal microscopy . In maize a rapid elongation phase featuring exclusively anticlinal divisions prolongs stage 4 Figure , prior tmac1 mutant  many anther-specific mutants Ma, ; (2) a cSPOROCYTELESS (SPL) which was shown to be essential to AR cell differentiation and meiotic entry in both anthers and ovules , in which AR cells failed to form and were replaced by vascular bundles. When MSCA1 was identified as a glutaredoxin    and divide periclinally forming the EN and SPL , that through subsequent periclinal divisions will differentiate as EN, ML, and TAP . As LRR-RLKs are known to dimerize, hetero-dimerization utilizing different isoforms or family members may contribute to differential signal interpretation in peripheral L2-d and presumptive AR cells during column formation, or between other cell types at later stages  mutants, stamen, and lemma development are severely compromised , which are secondary siRNAs derived from Recently two size classes of phased siRNAs (phasiRNAs) were found to be preferentially expressed in grass inflorescences, particularly in anthers  filed by Stanford University entitled “Method for Modulating the Number of Archesporial Cells in a Developing Anther.” The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We compare cortisol levels in monkeys at two sites with varying habitat disturbance within Kibale National Park, Uganda. Both species have higher cortisol levels at the less disturbed of the two sites. Factors such as social dynamics or predation may be responsible, illustrating the subtleties of wild primate ecophysiology. Lophocebus albigena) were higher at a highly disturbed site (Mainaro) in Kibale National Park, Uganda compared with a minimally disturbed site (Ngogo) in the same habitat. Here, we expand on this research, reporting on cortisol concentrations in two other cercopithecid monkeys (Cercopithecus ascanius and Piliocolobus rufomitratus) at the same two sites. We hypothesized that C. ascanius would show no difference between sites, given its preference for secondary forests, while P. rufomitratus would have higher cortisol concentrations at the disturbed site. Contrary to expectations, both species exhibited significantly higher cortisol concentrations at Ngogo  compared with Mainaro (disturbed). We suggest that these results may be caused by inter- or intragroup social dynamics, intersite differences in predation rate, fruit/food availability and chemistry, or a combination of these factors. These initial evaluations of urinary cortisol concentrations provide testable hypotheses on habitat disturbance and Kibale primate ecophysiology.Non-invasive measurement of urinary cortisol is a proven method of evaluating the impact of environmental stressors on wild primates. Variation in cortisol concentrations can reflect physiological stress, and prolonged elevation of circulating cortisol can significantly affect individual and population-level health. In a previous study, we found that urinary cortisol concentrations in grey-cheeked mangabeys ( Deterioration of environmental quality due to factors such as drought, increased predation and habitat disturbance can affect wild animal population health. Social factors, including competition for food and mating opportunities and instability in social relationships, can also influence well-being. Ecological or social perturbations may disrupt homeostasis and force immediate physiological adjustments, changing the hormonal milieu that regulates growth, immune function and reproduction . The devCortisol is a glucocorticoid produced by the adrenal cortex under stimulation by adrenocorticotrophic hormone. It is responsible for classic stress responses to environmental challenges. It acts as an anti-inflammatory agent, facilitates the cellular uptake of glucose and acts as a catabolic agent. Its molecular structure and function are conserved across most vertebrates and all mammals, making it a viable and robust agent of investigation for conservation biology . MultiplLophocebus albigena johnstoni) at a highly disturbed site than in conspecifics at a relatively undisturbed site in Kibale National Park, Uganda, presumably due to the physiological stress of living in the disturbed habitat , sympatric with mangabeys at both sites, show similar disturbance-related differences in cortisol concentrations. We predicted that red colobus monkeys would show such differences because, like mangabeys, they preferentially use old-growth forest. In contrast, we predict that between-site differences would not occur in redtail monkeys, because they prefer young successional forest, like that covering much of the disturbed site.We previously found that urinary cortisol concentrations were higher in grey-cheeked mangabeys (2) is a moist, evergreen medium-altitude forest with a mosaic of old-growth forest, swamp, grassland and regenerating forest of varying ages , reflecting its lack of recent human disturbance .Kibale National Park, Uganda . This assay has previously been deployed successfully in non-human primates . The value of α was set at 0.05.Results showed heteroscedastic distribution, so we logarithmically transformed raw data. Runs tests and residual plotting confirmed normality, with all results having a This research adhered to the legal requirements of Uganda and to the American Society of Primatologists Principles for the Ethical treatment of Nonhuman Primates. The Yale University Institutional Animal Care and Use Committee issued a waiver for the research because of its strictly non-invasive methods.n = 26; and Ngogo, n = 24) and 30 red colobus samples . Redtails and red colobus at both sites followed the expected circadian pattern of decreasing cortisol from morning to evening. For redtails, cortisol concentrations did not decline significantly in relationship to time of day at either site . Diurnal variation in cortisol concentrations was not significant for red colobus at either site, nor did the slopes differ significantly  showed elevated cortisol concentrations 2–5 days afterwards  are also major primate predators in Kibale  may result from biogenic (intra- or interspecific factors) rather than anthropogenic causes. Future studies at Kibale and for other primate species must combine physiology, socioecology, habitat structure, ecological community and nutritional data collection. Monitoring these variables will provide a more nuanced and cohesive evaluation of glucocorticoid responses in wild primates and provide measures of individual/community health and potential stressors.We received funding support from the Yale Institute for Biospheric Studies Program in Reproductive Ecology, the L.S.B. Leakey Foundation, the Great Ape Trust of Iowa and the Yale University Department of Anthropology.


Global change drivers are rapidly altering resource availability and biodiversity. While there is consensus that greater biodiversity increases the functioning of ecosystems, the extent to which biodiversity buffers ecosystem productivity in response to changes in resource availability remains unclear. We use data from 16 grassland experiments across North America and Europe that manipulated plant species richness and one of two essential resources—soil nutrients or water—to assess the direction and strength of the interaction between plant diversity and resource alteration on above-ground productivity and net biodiversity, complementarity, and selection effects. Despite strong increases in productivity with nutrient addition and decreases in productivity with drought, we found that resource alterations did not alter biodiversity–ecosystem functioning relationships. Our results suggest that these relationships are largely determined by increases in complementarity effects along plant species richness gradients. Although nutrient addition reduced complementarity effects at high diversity, this appears to be due to high biomass in monocultures under nutrient enrichment. Our results indicate that diversity and the complementarity of species are important regulators of grassland ecosystem productivity, regardless of changes in other drivers of ecosystem function. These i2  or water2 . Further2 –25, poteWhere there is a reduction in resource availability, biodiversity can buffer productivity declines via a number of mechanisms. These include the greater likelihood of tolerant species being present , which Given this wide range of possible interactions between biodiversity and resource availability on productivity, we examined the net balance of these mechanisms by measuring the strength and direction of the interaction between plant species richness and resource availability. This was achieved by performing a meta-level analysis, which is distinct from meta-analysis in that we used raw data and did not calculate effect sizes ,33. This2.(a)2 enrichment or temperature, we further narrowed our selection criteria to only include studies that increased resource availability by adding nutrients or decreased resource availability by reducing water availability, thus ensuring comparability of studies. We obtained datasets from 16 experiments . Hector et al. unpublished was treated as two independent studies because sown plant species richness was independently crossed with nutrient addition and drought treatments. In the selected experiments, nutrients were added once per year as NPK fertilizer or ammonium nitrate (NH4NO3) and water availability was generally reduced using rain-out shelters .We created our database by consulting recently published meta-analyses on biodiversity–ecosystem function relationships ,35,36. Wn = 4032), and the drought dataset consists of 788 plots (n = 2150). All observations are of above-ground plant productivity (g m−2) for each plot in each year of each experiment . For experiments that increased nutrient availability, we used peak biomass (a proxy for ANPP), while for experiments that reduced water availability, we used post-drought biomass harvested immediately  following the termination of experimental drought treatments.In total, the nutrient addition dataset comprises observations from 1199 plots , following Loreau & Hector . This su to zero .(b)For nutrient addition and drought studies, we fitted separate linear mixed-effects models that test for the effects of plant species richness, treatment and the interaction of the two on above-ground productivity. Plant species richness was the natural log of sown plant species richness and treatment was a binary variable . Random effects were included for a study factor, interactions of study with plant species richness, treatment, time (year of experiment) and their interactions, and a plot (within study) term. We accounted for repeated measurements within plots by using a first-order autoregressive covariance structure, which fitted the data better than a compound symmetry covariance structure based on the Akaike information criterion. To test for the effects of plant species richness, treatment and the interaction of the two on net biodiversity, complementarity and selection effects, we fitted separate linear mixed-effects models for nutrient addition and drought studies. For nutrient addition studies, we used the same model structure described above; for drought studies, we simplified the aforementioned model due to model convergence issues. The simplified model contained the same fixed- and random-effects structure as the previous models, but did not include a temporal correlation covariance structure. Above-ground productivity was square-root transformed to meet model assumptions, which were checked by visually inspecting residual plots for homogeneity and quantile–quantile plots for normality. Models were fitted using the asreml function in the asreml package in R, and Wald tests and variance components were extracted using the test.asreml function in the pascal package .4NO3) by fitting a linear mixed effects model where treatment changed to a variable with three levels . Additionally, we assessed the robustness of our results to covariates: for nutrient addition experiments, the amount of nutrients added , and for water reduction experiments, drought duration. The latter was used as a proxy for drought severity because of its significantly negative correlation with plant productivity across biomes [We performed sensitivity analyses to test whether results differed when excluding either low- (sown plant species richness = 1) or high-diversity communities (sown plant species richness greater than 20). To do so, we fitted the linear mixed-effects models to both subsets for each type of resource alteration as described above. For nutrient addition studies, we also tested the sensitivity of our results to the type of fertilizer used (NPK or NHs biomes . Droughts biomes .3.(a)For both types of resource alteration, above-ground productivity varied significantly by plant species richness and treatment but there was no significant interaction between the two . Product4NO3), we found similar results to the main analysis  and treatment  but there was no significant interaction between the two fixed effects (p > 0.10). Lastly, including the amount of nutrients added or drought duration as covariates did not explain significant amounts of variation in above-ground productivity (p > 0.10), nor did they alter the significant effects of either plant species richness or treatment on above-ground productivity.Results from the sensitivity analyses, in which low- or high-diversity communities were excluded, were in agreement with the results derived from full datasets for both types of resource alteration . In all cases, productivity increased significantly with increasing plant species richness and varied significantly by treatment; but the interaction of plant species richness and treatment did not have significant effects on productivity. When accounting for the type of fertilizer (NPK or NH(b)With increasing plant species richness, net biodiversity and complementarity effects increased significantly in both nutrient addition and drought studies (4.(a)In our meta-level study, we found that plant diversity increased ecosystem productivity irrespective of nutrient or water availability. Our results suggest that nutrient enrichment and drought did not alter the strength or the direction of biodiversity–ecosystem function relationships in grassland plant communities despite significantly increasing and reducing productivity, respectively. This finding is consistent with previous studies that have found that positive biodiversity–ecosystem function relationships are robust to intensive management activities of grasslands, such as fertilization and mowing frequency ,41, alth(b)Contrary to expectations that high-diversity communities may exploit additional resources to a greater extent than low-diversity communities , we foun(c)Irrespective of water availability, above-ground productivity increased significantly with plant diversity. This result is unexpected because drought is usually expected to strongly reduce above-ground productivity of high-diversity communities due to their greater overall biomass ,51, as g(d)In this study, we found that the effect of the interaction between diversity and both types of resource alterations—nutrient enrichment and drought—on above-ground productivity was not significant. Non-significant interaction effects do not indicate that the hypothesized mechanisms were not operating , but rather that they produced a response that was neutral across studies. The strong variation of site-level responses, as evidenced by the large variation in the effect of the interaction between plant diversity and treatment on above-ground productivity among studies, suggests that the relative balance of these mechanisms varied between studies, possibly due to differences in experimental design, site-level variation in resource limitations, and the adaptation of the local species pool to these limitations.Lastly, environmental conditions, such as soil nutrient pools and precipitation, also varied over time and could have altered the realized magnitude of experimental treatments. Future research should therefore investigate the cause and importance of between-site variation in determining the interaction between resource availability and diversity, as it is possible that interactions could be stronger in certain ecosystems. This question may be best addressed using standardized methodology, e.g. by using global networks of standardized experiments that enable sources of across-site variation in environmental conditions to be quantified precisely ,58,59.5.The present results show that the positive effects of biodiversity on above-ground productivity are robust to resource alterations. This finding is consistent with other recent meta-analyses, which have found that the interaction between diversity and other drivers of ecosystem function is surprisingly weak in determining a range of ecosystem functions and properties, such as decomposition and soil microbial biomass carbon ,12. Impo