Salmonella to various biocides could act as a driver of antibiotic resistance.Biocides are widely used to prevent infection. We aimed to determine whether exposure of Salmonella enterica serovar Typhimurium was exposed to four biocides with differing modes of action. Antibiotic-resistant mutants were selected during exposure to all biocides and characterized phenotypically and genotypically to identify mechanisms of resistance.rpoA was also selected and contributed to the MDR phenotype. Other mutants were highly resistant to both quinolone antibiotics and the biocide triclosan.All biocides tested selected MDR mutants with decreased antibiotic susceptibility; these occurred randomly throughout the experiments. Mutations that resulted in de-repression of the multidrug efflux pump AcrAB-TolC were seen in MDR mutants. A novel mutation in This study shows that exposure of bacteria to biocides can select for antibiotic-resistant mutants and this is mediated by clinically relevant mechanisms of resistance prevalent in human pathogens. Exposure to low concentrations of drug is significant as the window of time and drug concentrations where selective pressure is exerted is much larger than in conventional experiments, where bacteria are exposed to lethal concentrations of antibiotic and only highly resistant strains can survive. When exposed to low concentrations of antibacterial, the number of potential target genes for which mutation may be beneficial and confer decreased susceptibility to antibiotics is much larger than the complement of genes that may be able to confer high-level resistance if mutated.10 Importantly, such exposure regimes allow multiple generations of bacteria to compete under low selective pressure, and this allows selection of epistatic combinations of mutations in individual strains. Individually, such mutations may have a low impact on antibiotic activity, but in combination they may have greater effects as a result of synergy. This also allows compensatory mutations to evolve in concert with resistance mutations and so minimize any fitness costs resulting from resistance.11Antibiotic resistance is one of the great global challenges facing modern medicine.12 Biocides (disinfectants) are crucial to successful infection control and are widely used in hospitals, on farms, in the food industry and in the home for the control of microorganisms.14 Biocides are often mixtures of active agents that exhibit high toxicity against target organisms. As a result, it is difficult for bacteria to become resistant to the recommended in-use concentrations of many biocides as mutation within a single gene will not usually confer resistance. However, there are several examples where bacteria have become biocide resistant or show decreased susceptibility to biocides; some major mechanisms of antibiotic resistance are also relevant to bacterial survival of biocide challenge.15 Amongst these, MDR efflux systems can export both antibiotics and many biocides. Growth as a biofilm can also dramatically reduce the efficacy of biocide activity.17Prevention of infection is of paramount importance as the outcomes of infection with an antibiotic-resistant bacterial strain are often more severe than those of infections with antibiotic-susceptible strains.20 Many common biocides are now detectable in the environment and some, such as triclosan, have been found in human urine, serum and breast milk.24 Exposure to antibiotics in the environment can result in the selection of antibiotic-resistant bacteria, which are a recognized reservoir of potential infection for humans via contamination of foodstuffs, water and surfaces with resistant pathogenic bacteria.26 The accumulation of biocides in the environment 28 also represents a potentially prolonged, low-level stress, which could exert selective pressure towards selection of bacteria with decreased biocide susceptibility, which could be cross-resistant to antibiotics. The possible results of such selective pressure are currently uncertain.Exposure of bacteria to biocides can select for mutants with decreased biocide susceptibility and, as we and others have seen, these mutants often display a decrease in susceptibility to various antibiotics, indicating that biocides can act as drivers of antibiotic resistance under laboratory conditions.Salmonella enterica serovar Typhimurium. We also used genome sequencing to determine the genetic basis for the observed resistance phenotypes.To identify the consequences of exposure of bacteria to low levels of biocide and whether there are any implications for selection of antibiotic resistance, we investigated the impact of exposure to low levels of four biocides representing different active classes on antibiotic resistance and bacterial fitness of Four biocides representing classes with active ingredients belonging to different functional groups were chosen for use in the study: Superkill  is a mixture of aldehydes and quaternary ammonium compounds; AQAS  is a quaternary ammonium compound; Virkon  is an oxidative compound; and Trigene  is a halogenated tertiary amine compound.S. enterica serovar Typhimurium strain SL1344 was the parental strain used in all experiments, representing both a pathogen and a model Gram-negative bacterium. To determine the impact of repeated, low-level exposure to biocides, SL1344 was exposed to biocides at sub-lethal concentrations that restricted growth rates to ∼70% of those in drug-free media. Therefore, Superkill and AQAS were both added to media at a concentration of 0.0018%, Trigene at 0.0005% and Virkon at 0.2%. Overnight cultures of SL1344 grown in LB broth at 37°C with shaking at 150 rpm were used to inoculate fresh, pre-warmed broth (50 μL into 5 mL) containing biocide. After 12 h of growth, a 1 mL sample was taken and stored in glycerol at −20°C and 50 μL was used to inoculate fresh media containing the same concentration of biocide (the second sub-culture). The bacterial cultures were repeatedly sub-cultured over 4 days  in each biocide. Sampling was every 12 h to allow each culture to reach stationary phase before sub-culture to fresh media. A total of ∼50 generations were passaged in each experiment. In parallel to the biocide exposure experiments, SL1344 was also sub-cultured eight times in LB broth with no biocide as a control. The eight frozen samples from each biocide exposure experiment and the control experiment were serially diluted and inoculated onto antibiotic-free LB agar; after overnight incubation, these plates (with ∼200 colonies each) were replica plated onto LB agar plates, each containing a different antimicrobial at a concentration that inhibited growth of SL1344. Nalidixic acid was used at 8 mg/L, chloramphenicol at 4 mg/L, tetracycline at 4 mg/L, kanamycin at 8 mg/L and triclosan at 0.12 mg/L. After overnight incubation, colonies that grew on one or more of the antimicrobial-containing plates were selected from the original antibiotic-free LB master plate, their identity confirmed using the API 20E test  and retained for further investigation.29 Susceptibility to antibiotics and biocides was also determined after five passages in drug-free media to determine whether selected resistances were stable. The phenotype of the mutants in Biolog Phenomicroarray antimicrobial plates (PM11-20) was determined in duplicate and compared with that of SL1344. Data were analysed for significant differences in both replicates at multiple timepoints and in at least two sequential concentrations of test compound.The antimicrobial susceptibility of mutants resistant to one or more agents after exposure to biocide was determined for five antibiotics and five biocides using a panel of antibiotics and the standardized agar dilution method following the recommendations of the BSAC.30 The fitness costs of any of the selected mutations were evaluated in competitive index experiments by introducing SL1344 and mutants into LB broth in equal numbers and comparing the ratios of mutant to parent over time as previously described.30 To determine whether any mutants had changes in cellular morphology, cells of each mutant were Gram stained and visualized under a microscope.The growth rates of selected mutants were compared with that of the parental strain SL1344 by measuring the OD of cultures at 600 nm in a FLUOstar Optima reader (BMG Labtech) as previously described.® Genomic DNA Purification Kit. The quantity and quality of the DNA was confirmed by agarose gel electrophoresis and by analysis using a NanoDrop Spectrophotometer (Life Technologies) and Bioanalyzer (Life Technologies). Mutants were sequenced using a 454 GS FLX instrument (Roche) with Titanium chemistry and the resulting sequence reads were mapped against the SL1344 reference genome using the gsMapper alignment module of Newbler, run through the xBASE-NG web site. High-confidence SNPs were identified as previously described.31 Mutations detected consistently in all sequenced isolates were deemed to be differences between our WT laboratory strain and the sequenced SL1344 reference strain and were excluded from further analysis. Mutations and SNPs identified were verified by PCR and Sanger sequencing.From each of the biocide exposure experiments, four drug-resistant mutants representing the range of phenotypes were selected for sequencing. Mutants were selected to reflect those that emerged early and late in the selection period. Genomic DNA was extracted from 5 mL overnight cultures grown in drug-free LB media using a Promega WizardrpoA and zur genes were amplified from SL1344 and cloned into a pBADmyc-HisA expression vector (Invitrogen) to create pBAD-rpoA and pBAD-zur, respectively. The plasmid pBAD-zur was transformed into the Trigene-selected mutants and the plasmid pBAD-rpoA was transformed into AQAS-selected mutants. Induction of expression of each gene was with 0.05% arabinose. RT–PCR, using primers internal to the WT gene, confirmed expression of the complements within the mutants. Site-directed mutagenesis was used to recreate the rpoA and zur changes found in the biocide exposure experiments, primers were designed to amplify the mutations found within these genes and mutagenesis was accomplished using the Red/ET recombinase kit  according to the manufacturer's instructions.The coding sequences of the WT Salmonella Typhimurium SL1344 were repeatedly sub-cultured in subinhibitory concentrations of four biocides  with different chemistries and modes of action. Samples were taken regularly from each lineage and mutants with altered antibiotic susceptibility were identified using antibiotic indicator breakpoint plates containing concentrations of antibiotic that restrict growth of SL1344. No mutants that were retained demonstrated increased tolerance to the biocides they were exposed to, but, from as early as the second sub-culture , all biocides selected mutants with altered susceptibility to antibiotics .All colonies identified as having decreased susceptibility to any of the antibiotics tested were retained from the drug-free master plate and the MICs of a panel of drugs were determined  to antibiotics and biocides was similar and showed broad, decreased susceptibility to drugs of different classes  were selected after exposure to multiple biocides. The four AQAS mutants all carried an SNP in rpoA, encoding the RNA polymerase α subunit. Three of the AQAS mutants also had mutations in ramR, which encodes the local repressor of ramA, encoding a transcriptional activator that regulates the AcrAB-TolC MDR efflux system. Two of these mutants carried a 2 bp deletion within ramR and the other carried a point mutation resulting in a substitution within the DNA-binding domain of RamR. The remaining AQAS mutant, AQ1, also had a mutation in hpaA, a gene that codes for a putative AraC family regulatory protein. Of the four Superkill mutants selected for sequencing, three carried a 12 nt deletion in ramR, but for the other (SK7) insufficient depth of sequence was achieved to confidently identify mutations.In the mutants sequenced, seven genes were found to contain mutations Table . Five gefabI . The substitution identified has previously been shown to confer triclosan resistance.32 The Trigene mutants also contained a 2 bp insertion within zur, which encodes a putative regulatory protein controlling zinc uptake systems. The Trigene mutants also contained a point mutation within gyrA encoding the α-subunit of DNA gyrase; the resulting substitution is known to confer quinolone resistance.33 The same gyrA mutation was also seen in both mutants selected after Virkon exposure that were quinolone resistant. The other two mutants, V4 and V8, both carried the same mutation in fabI as seen in the Trigene-selected mutants. Finally, one Virkon-selected mutant, V4, also carried a deletion within avtA encoding an alanine–valine transaminase.All four Trigene mutants contained identical mutations in ramR, gyrA and fabI have all been extensively characterized by ourselves and others.34 However, there were also some novel mutations in genes not previously associated with susceptibility to antimicrobials; these included the rpoA mutation, seen in all the AQAS mutants, and the mutation within zur, seen in all the Trigene mutants.The mutations identified in rpoA and zur mutations on antibiotic susceptibility in those mutants with changes in these genes were investigated both by recreating these mutations in SL1344 and by restoring the WT alleles of these genes in trans. A WT copy of rpoA was introduced into mutants AQ1, AQ2, AQ4 and AQ5 on plasmid pBADmyc-HisA. In the same way, a WT copy of zur was introduced into T2, T3, T6 and T9. Transcription of both genes was confirmed by RT–PCR and found to be similar to WT levels. When mutations were recreated in SL1344, the mutation within zur had no impact on antibiotic susceptibility and the rpoA mutation resulted in an MIC profile identical to that of AQ1 . There was no statistically significant difference between the other values for mutants compared with SL1344 , conferred decreased susceptibility to the quinolones enoxacin and nalidixic acid , but had increased susceptibility to 12 other compounds (Table S2). Inactivation of ramR  resulted in significantly increased respiration in the presence of seven compounds, including DNA gyrase and protein synthesis inhibitors, consistent with increased expression of MDR efflux pumps.35 Mutation in fabI was seen in mutants recovered after exposure to both Trigene and Virkon and in conjunction with mutations in gyrA and zur, or avtA, respectively. Mutants with either combination were better able to grow (measured by determining respiration rates) in the presence of enoxacin and, in combination with gyrA, this was extended to other quinolones. The fabI mutation in concert with avtA did not provide a benefit when tested with any of the other compounds in the Biolog.To further examine the possible impact of the key genotypes recovered after biocide exposure, a Biolog Phenomicroarray was used to examine the ability of each strain to respire in various concentrations of a wide panel of drugs. These experiments showed that mutation in o AQAS AQ, conferrOverall, these data indicate that exposure to biocides can select mutant bacteria with altered susceptibility to a wide range of antimicrobial agents.25 here we showed that exposure to sub-lethal concentrations of all the biocides evaluated resulted in the selection of antibiotic-resistant mutants. The antibiotic susceptibility phenotypes of the mutants essentially fell into three major groups: those that were MDR; those resistant to both quinolones and triclosan; and those resistant to single agents. Surprisingly, irrespective of the biocide, exposure resulted in mutations in the same seven genes with mutations in certain genes selected for by each biocide. Both AQAS and Superkill selected for MDR mutants due to loss-of-function mutations in ramR that confer de-repression of the AcrAB-TolC MDR efflux pump.35 In addition, mutants with a substitution within GyrA at codon 87 were also selected after exposure to two of the biocides (Trigene and Virkon). This may initially seem surprising as this mutation is classically associated with resistance to quinolone antibiotics. However, we recently described a broad and generic benefit in antimicrobial survival resulting from this mutation in Salmonella.30 This also explains why it has been favoured in separate biocide exposure experiments, and this study extends the range of conditions under which this mutation is beneficial.Many antimicrobial products are present in the environment at low levels;ramR and gyrA mutations recovered after biocide exposure in vitro are commonly seen in clinical isolates of Salmonella and other Enterobacteriaceae and therefore their selection by biocide exposure is a concern. The mutations identified after exposure to diverse biocides with different mechanisms of action indicate that there are convergent pathways to the survival of antimicrobial stress present in Enterobacteriaceae, and both antibiotics and biocides can favour selection of identical mutations in the same genes . Mutants emerged readily after exposures to different biocides and were present after only two exposures (sub-cultures) for three of the four biocides tested. Once selected, mutants were stable; there was no evidence of accumulation of multiple mutations after further biocide exposure. The mutations selected had no great fitness costs when tested in competitive index experiments against the parental strain, and the occurrence of the ramR, gyrA and fabI mutations in clinical and veterinary isolates confirms that they carry no prohibitive fitness cost.37Both the 15 It has been demonstrated that exposure to sub-lethal concentrations of antibiotics can exert strong selective pressure and promote the rapid emergence of mutants with better ability to grow under stress than WT strains.10 Importantly, repeated exposure allows epistatic interactions to be subjected to selective pressure; this may have occurred in this study in the case of the rpoA and zur mutations. The zur mutation alone contributed no major discernible phenotype when complemented or recreated in isolation in the parental strain, but the rpoA mutation did result in decreased susceptibility to nalidixic acid and increased susceptibility to triclosan; however, this phenotype disappeared when complemented, suggesting dominance of the parental allele. In Escherichia coli, RpoA is known to interact with MarA and modulate its activity.38 In this study rpoA mutations analogous to those described in E. coli were found in concert with de-repression of ramA, encoding RamA, a close homologue of MarA , so RpoA may also modulate the activity of RamA. The mutation in zur resulted in loss of function of Zur, a predicted transcriptional repressor. The zur system is involved in zinc uptake and mutations in zur were found in mutants with changes in both fabI and gyrA; therefore, it is not immediately clear how the resulting altered proteins may interact. In the absence of a phenotype it is possible that the zur mutation may be coincident and occurred in the relevant lineage before the other mutations described appeared.The experimental design used maintained constant concentrations of biocides in each selection experiment , and none of the mutants identified on the basis of antibiotic tolerance showed any change in biocide MICs. These data indicate that selection of resistance to the biocides themselves did not occur under these conditions. The biocides used in this study are highly toxic to bacteria; developing resistance to these compounds would require multiple mutations and potentially a much longer selective window would be needed to allow adaptation to these compounds. However, it was evident that exposure to sub-lethal concentrations of biocide exerted selective pressure and mutations that promote bacterial survival also influenced antibiotic susceptibility. Consistent with the findings after low-level exposure in this study, we previously showed that bacteria overexpressing efflux pumps are better able to survive high concentrations of biocide.Legislation concerning biocides requires proof of efficacy against target species at in-use concentrations; our data presented here indicate that sub-optimal concentrations of biocides can select common mutations that confer clinically relevant antibiotic resistance. The increasing accumulation of antimicrobial compounds, including biocides, in the environment, food and people represents a selective environment where microbes can be exposed to persistent low levels of biocides. Mutants such as those described in this manuscript could be selected in the absence of, or prior to, antibiotic treatment. Therefore, the role of environmental selection of antibiotic-resistant bacteria by biocides should be considered by government agencies as policies to control antibiotic-resistant bacteria are formulated and implemented.BBSRC grant BB/GO12016/1.This study was supported by None to declare.JAC Online .Tables S1 and S2 and Figures S1 and S2 are available as Supplementary data at


These factors affect not only individual genes or bio-molecules but also the entire biological networks that in turn increase or decrease the risk of illness or affect illness severity. This review focuses on recent developments in the field of systems biology which use multidimensional data to discover biological networks affected by combat exposure and post-deployment disease states. By integrating large-scale, high-dimensional molecular, physiological, clinical, and behavioral data, the molecular networks that directly respond to perturbations that can lead to PTSD can be identified and causally associated with PTSD, providing a path to identify key drivers. Reprogrammed neural progenitor cells from fibroblasts from PTSD patients could be established as an in vitro assay for high throughput screening of approved drugs to determine which drugs reverse the abnormal expression of the pathogenic biomarkers or neuronal properties.Posttraumatic stress disorder  Current understanding of pathophysiology and treatment of deployment-related medical and psychiatric disorders such as PTSD is hampered by the complexity of the human system in which these syndromes are manifested, and by the lack of knowledge regarding how exposures lead to symptoms that cause deployment-related psychological injury , prognosis (assessed post-trauma), and symptom severity. Genome-wide association studies (GWAS) are conducted to provide relevant information regarding individual responses to deployment; variations in the DNA can induce changes in molecular states driving different physiological states or symptoms. Genes may also be uniquely linked to networks that are perturbed by environmental exposures that are more proximally associated with increased disease risk  are needed . Conditional manipulations of candidate molecules with viral gene transfer and optogenetics enable examination of highly specific and nuanced alterations of biological activity . Viral vectors can be used in a relatively rapid and high-throughput manner, to over-express genes of interest or silence them in a site-specific, inducible fashion, with validated high-titer viruses provided to each of the sites to examine genetic manipulations across our several convergent animal models . Similarly, specific brain circuits can be activated or silenced using optogenetic mechanisms to understand the neural mechanisms through which the targeted genes control complex behavior. The effects of these manipulations on animal behavior and neural molecular networks inform the relevance of blood markers to brain. It also permits identification of biological networks that must be targeted for treatment.Biomarkers and gene networks observed in blood and multiple brain regions can be compared . This approach will allow identification of blood-based bio-markers in animals that parallel the blood-based bio-markers in humans . Through the knowledge of which blood-based markers best represent brain changes, we can identify the gene networks measureable in blood that are most relevant for the prevention and treatment of PTSD.Biological networks associated with short-term responses to trauma exposure may also predict longer-term post-deployment symptom trajectory and long term risk for medical comorbidities. Subjects with PTSD are at substantially increased risk of cardiovascular disease, and cardiovascular and metabolic risk factors, including hypertension, dyslipidemia, obesity, diabetes  and determine causal relationships among them. For example, once the basic analyses confirm which gene expression traits are most strongly modulated by genetic variants, the possible molecular network structures to consider given the data can be constrained by forcing the genes affected by the causal genetic variants observed to serve as more upstream regulators of the downstream effects that result from this genetic perturbation , and time point.To more fully elucidate the complexity of PTSD using network-based approaches, network level analyses can be used to construct interaction networks in which the edges between the nodes reflect association-based relationships or direct physical interactions, and probabilistic causal networks in which the edges between the nodes are directed, reflecting statistically inferred or known causal relationships. As many experimental and computational studies have uncovered, many functional modules (sub-networks of coherent molecular activity that resides within a larger network) are highly conserved across tissues, populations, and species. Therefore, the resulting validated module-based biomarkers are more robust than the putative single gene biomarkers extracted from individual datasets. In addition to constructing different modular networks using weighted gene co-expression network analysis, candidate modules from data generated in one condition can be identified, and then these modules can be tested across other conditions to identify core network modules that are common across a disease spectrum. There are a number of advantages to this network approach: 1) The resulting modules are more compact than a disease signature and thus serve to minimize unrelated downstream signals; 2) The modules are distinct and more reasonably well-defined with respect to conditions/tissues/species that invoke them; 3) This method provides multiple robust discriminative biomarkers co-validated across experimental conditions; and 4) The modular network can reduce the overall dimensionality in the molecular space leaving open the possibility of describing it by relatively small-scale ordinary differential equation (ODE) models  can be considered as a functionally validated set of variants that have been shown to be enriched for associating with disease traits, given they reflect perturbations in the molecular networks. Given variants like eSNPs associate with a biologically relevant phenotype like gene expression, such variants identified in longitudinal datasets can be used as a more restricted set to test for associations to PTSD .While eSNPs on their own can be a useful filter to apply in a genetic analysis, different constellations of eSNPs affect different networks that drive disease processes independently Schadt, . OrganizAlthough animals and humans share genes, pathways, and networks, the biological context and integration of these genes with other systems may be different in different species. This can result in failures in drug development for CNS disorders. For example, many Alzheimer's disease drugs can show efficacy in animals but not translate to novel therapeutics in patients. To maximally exploit data from animal models toward understanding PTSD in humans, it is necessary to have an understanding of the genetic and molecular pathways from relevant human cells, in this case, neural cells .The ability to focus on human nerve cells relies on recently developed methods in mammals to take cells from other parts of the body (most frequently skin cells/fibroblasts) and convert them to pluripotent stem cells , from which any cell can be generated, or directly into either neural progenitor cells (iNPC) or neurons (iN). The revolutionary nature of these approaches led to a Nobel Prize in October of 2012, just 6 years after Dr. Shinya Yamanaka demonstrated that with the addition of just four factors, mammalian cells can be reprogrammed into iPSCs  to determine which drugs reverse the abnormal expression of the pathogenic biomarkers or neuronal properties  . That is


The use of oxidative status markers in animal conservation is dual-faceted, as they reflect the effects of environmentally-induced stress on animal populations and they predict individuals' fitness prospects. Paradoxically, conservation studies rarely include them. Here, we raise awareness of their use, and show how they can valuably complement conservation studies. a priori individual perspectives of reproduction and survival as well as to assess a posteriori the effect of human activities on the fitness of species of conservation concern and wildlife in general. Markers of oxidative status may therefore help conservation practitioners to identify conservation threats to animal populations and to maximize the success of wildlife management. Despite these potential benefits for animal conservation programmes, up to now markers of oxidative status have only been reported anecdotally in conservation studies. The aim of this review is therefore to raise awareness by conservation practitioners of the use of markers of oxidative status. Towards this end, we first describe how environmental disruptions due to human activities can translate into variation in oxidative status. Second, we show how individual and population variation in oxidative status may contribute to the success or the failure of reintroduction or translocation programmes. Finally, we emphasize the technical features specific to the measurement of markers of oxidative status in conservation programmes, which may help investigators with the interpretation of their results. Such prior knowledge about markers of oxidative status may encourage conservation physiologists to use them in order to enhance the success of conservation programmes and wildlife management.Recent ecological studies have shown that oxidative status could have a significant impact on fitness components in wild animals. Not only can oxidative status reflect the environmental conditions that animals experience, but it can also predict their chances of reproduction and survival in the future in their natural habitat. Such important characteristics make markers of oxidative status informative tools to evaluate  In the last 50 years, environmental conditions have changed at an unprecedented rate, impacting heavily on ecological processes . It is iPresumably because the hypothalamic–pituitary–adrenal axis first integrates environmental disruptions in vertebrates, stress hormones (glucocorticoids) have become key biomarkers to assess the physiological response of animals to these environmental disruptions  and haveMethods and interpretation’). Measuring levels of oxidative stress is highly fitness relevant, because high oxidative stress levels may compromise survival and reproduction (Geothlypis trichas) that do not survive from one year to another exhibit higher levels of oxidative damage than individuals that are able to survive . Classical research on glucocorticoids has also made a clear distinction between acute and chronic exposure, because of the different consequences for individual behaviour and physiology . Reactive oxygen species are byproducts of the aerobic metabolism of mitochondria or of other functions within the organism, such as immune cell activity. If left uncontrolled by antioxidant mechanisms, ROS can generate oxidative damage to biological compounds. Such an imbalance between ROS production and antioxidant defences resulting in oxidative damage has been termed ‘oxidative stress’ . Differe survive . Importaysiology . Such a The dual-faceted aspect of oxidative stress (antioxidant defences vs. oxidative damage) makes it a unique physiological marker to assess the protective response that animals are able to mount in order to cope with environmental stressors, as well as the deleterious effects they undergo when facing adverse conditions. For instance, an elevation of antioxidant defences coupled with stable levels of oxidative damage suggests that animals are able to protect themselves against the new oxidative conditions they face. In contrast, decreased antioxidant defences coupled with increased levels of oxidative damage suggest that they are unable to respond to new conditions, leading to an exhaustion of antioxidant defences and a rise in oxidative damage. Obviously, any intermediate situation between these two extremes is possible Fig. . We shouAnother appealing aspect of markers of oxidative stress for conservationists is their universality. Indeed, while measurements of glucocorticoids are confined to vertebrates (corticosterone or cortisol according to the taxon considered), the measurement of markers of oxidative stress can, in principle, be applied to any species with aerobic metabolism and even to anaerobic organisms exposed to oxygen , making Finally and importantly for conservation purposes, markers of oxidative status cannot only be used to describe the individual physiological status, but also to make predictions about individual perspectives of survival or reproduction. For instance, it has been found that markers of oxidative status can predict recruitment, fledging success or survival in different bird species . DiscrepDespite the clear advantages associated with the measurement of markers of oxidative status to assess how animals cope with new environmental conditions , they have only been reported recently and anecdotally in conservation studies if compared with the vast conservation literature on glucocorticoids and immune parameters Fig. . As suchHuman activities can impinge on individual oxidative status in a myriad of ways Fig. . ConservArgopecten ventricosus) exposed to blue crabs  exposed to visual or chemical cues from larvae of dragonflies  exposed to frequent experimental nest predation  from the Cayman Islands occupying habitats with high tourist pressure had decreased levels of serum antioxidant capacity and increased levels of serum oxidative damage  from this region still show signs of contamination, such as high levels of plasma oxidative damage  can only protect themselves partly against heat-induced oxidative stress, as illustrated by an increase in antioxidant enzymes accompanied by an increase in oxidative damage levels (Coturnix coturnix japonica) do not appear to be able to protect themselves against heat-induced oxidative stress at all, as suggested by a decrease in antioxidant enzymes and a concomitant increase in oxidative damage , which show increased antioxidant enzymes while dehydrated, thereby maintaining oxidative damage stable (Camelus dromaderius), found different results. Indeed, contrary to amphibians, camels exposed to 20 days of water deprivation are only partly able to compensate for the effects of dehydration on oxidative status, as suggested by increased levels of glutathione (an endogenously synthesized antioxidant) associated with an increase in oxidative damage  in the Southern ocean (Pygoscelis adeliae) from the Antarctic Peninsula show both lower plasma antioxidant capacity and lower levels of oxidative damage than Adélie penguins from other regions (Anas platyrhynchos) after fasting for several days  inhabiting poor-quality territories (with low insect density) show stable plasma antioxidant capacity but increased oxidative damage (Dentex dentex), prolonged starvation is associated with increased oxidative damage together with an increase in the activity of antioxidant enzymes  inhabiting forests with frequent natural tree-fall gap openings in the canopy have higher oxidative damage than birds inhabiting forests with undisturbed canopy (Apis mellifera) transferred to greenhouses where only strawberries or eggplants occur show a down-regulation of antioxidant system genes coupled to an increase in oxidative damage compared with bees from locations with greater plant diversity . This high exposure to new infectious diseases may be reflected by changes in the oxidative status of infected animals. This is because an organism first responds to infection through the production of non-specific toxic compounds (such as ROS) to eliminate infectious agents. Importantly, because of their non-specificity, ROS are also toxic to the host's tissues  that have been experimentally immunostimulated exhibit lower plasma antioxidant capacity and increased oxidative damage  in a dose-dependent manner , it was found that different individual coping styles may be associated with differences in pre-restraint or acute stress-induced blood oxidative status in a natural setting  showed that neophobic individuals had lower plasma non-enzymatic antioxidant capacity and higher plasma oxidative damage than neophilic ones; and that fast-exploring individuals had higher plasma non-enzymatic antioxidant capacity and lower plasma oxidative damage than slow-exploring ones (Egernia whitii), Links between personality and oxidative status were also found in birds and reptiles using wild-caught animals tested in captive conditions. A report on greenfinches  from various islands of the archipelago show differences in plasma oxidative damage, plasma non-enzymatic antioxidant capacity or plasma carotenoids (Turdus merula in Passer domesticus in Uta stansburiana) showed higher levels of stress hormones, which were associated with higher oxidative damage and suppressed immunity. Moreover, urban populations had higher reproductive output and decreased survival  are also translated at the physiological level. Compared with the short-lived phenotype, offspring snakes from the long-lived ecotype consumed equal amounts of oxygen, produced more energy for a given amount of consumed oxygen, had lower mitochondrial production of ROS and had DNA in red blood cells that was damaged more readily but was also repaired more efficiently. Divergences between ecotypes in the mechanisms regulating oxidative status are not limited to vertebrates, but have a general applicability. For example, Arctica islandica .Inter-population differences in markers of oxidative status can also be of genetic origin. For instance, The importance of considering markers of oxidative status in reintroduction and translocation programmes is further strengthened by the fact that genetic inbreeding (increase in homozygosity) may make individuals more sensitive to damage. Inbreeding depression results in reduced evolutionary fitness e.g.  that makTheoretically, markers of oxidative status can be measured in any biological samples. Various methods have been described in several recent publications . Hence, Capra hircus; Meles meles; In species of conservation concern and in reintroduction programmes, physiological measurements necessarily need to be restricted to biological matrices for which sampling is as non-invasive as possible and which does not lead to the death of animals. We have summarized in Tables Measurement of markers of oxidative status in a single biological matrix may not be optimal, because it may not necessarily reflect the oxidative status of the whole organism or the oxidative status of tissues that mostly impinge on fitness. For example, a combination of matrices such as blood (both plasma and red blood cells), urine (for those species that urinate), tissue biopsies and sperm might provide a good picture of oxidative stress to both soma and germ cells (see Tables S-transferase). Likewise, Although differential sensitivity of markers of oxidative status can be expected, two meta-analyses showed that the sensitivity to strong chronic stressors did not differ significantly. A posteriori effects of environmental disruptions on markers of oxidative status’). Towards this end, it is important to analyse the variation of markers of oxidative status in populations subject to different environmental conditions and characterized by different outputs in terms of body condition, breeding success, survival or demographic trends. This approach can first be conducted in populations geographically separated and that experience different environmental conditions. For instance, we previously found that declining populations of Pygoscelis penguins experiencing climate warming exhibit lower antioxidant defences than other populations, and that demographic trends and antioxidant defences were so tightly bound that antioxidant defences might be used by conservationists as an index to estimate the population health of populations of unknown demographic status  antioxidant defences, reflecting the protective response that animals are able to mount when facing adverse conditions; and (ii) oxidative damage, reflecting the deleterious effects they undergo Fig. . Howevera priori assessments of the oxidative status of the individuals to reintroduce and of the host population may increase the chances of success, because some individuals are likely to be a better match for the oxidative status phenotype of the host population.Prior knowledge of the oxidative status of individuals within a potential host population may also be useful for conservation programmes aiming to reintroduce animals into new habitats. As illustrated in the above paragraphs, it is essential for reintroduction programmes to maximize the chances of survival of the individuals being reintroduced. Towards this end, It is important to emphasize that, within a species, markers of oxidative status do not fluctuate only because of exposure to stress but also because of natural variation throughout life. For instance, oxidative damage can increase during growth, reproduction or senescence , the effAs stated in the aforementioned paragraphs, quantification of the variation of markers of oxidative status in relation to individual life stages and attributes and to environmental conditions would be a first step to define reference values for the population or species target. Another relevant step would be to assess whether certain diseases or parasitic infections typical of the species under study result in increased levels of oxidative damage or reductions in antioxidant defences. Such an assessment would provide relevant information about the diagnostic value of a given marker of oxidative status. Another important preliminary step in the application of oxidative status markers in wildlife management would be to complement baseline values with reference values collected from captive or wild animals (when possible) subjected to different stressful conditions. This would provide information about stress-induced values of markers of oxidative status and about the magnitude (e.g. maximum peak) and the timing of these changes .Finally, we highlight the fact that markers of oxidative status change in concert with other physiological end-points that are often used in conservation studies. Challenging conditions are, for example, known to affect glucocorticoid levels and the activity of immune cells, which are also functionally related to oxidative status. Hence, we make the point that merging information about stress hormones, immunocompetence and oxidative status would be important in order to provide better characterization of the response of natural animal populations to environmental perturbations and assess the relative importance of each physiological parameter in predicting the responses of populations to changes in environmental conditions. For example, if one physiological parameter stands out from the others in terms of predictive power, it could then be selected as the most relevant tool to be used in conservation of a given species. Principal component analysis can be a valuable tool to identify which parameters provide redundant information about the oxidative status and which ones are most sensitive to a certain environmental challenge . Hence, Conservation physiology is emerging as an essential component of conservation biology (


Thyroid nodules are common, occurring in almost two-thirds of some populations; among these only about 7% are malignant . The mosFeatures that raise suspicion include a nodule in males, one that is solitary, growing, hard, fixed and associated with hoarseness . While t99mTc) was the monopoly of nuclear medicine for over a quarter of a century with hot nodules interpreted to be benign and cold possibly malignant, Thyroid imaging with radioactive iodine and Technetium 9mTc was The practice of oncology has changed with the introduction of PET scanning, and PET is used in the initial diagnosis, staging, follow-up, re-staging and prognostic assessment in many cancers. Well differentiated thyroid cancers (WDTC) unfortunately do not lend themselves to an initial PET assessment due to wide variability of results  Figure , 3. TherThe level of anatomical detail in computerized tomography (CT) and MR have made these the investigation of choice for most macroscopic pathological processes A consensus that has emerged over the last few years is that ultrasound currently offers the most valuable tool in the early assessment of thyroid cancer . The advThe ability to identify non-palpable nodulesAccurately measure nodule and detect any interval change in sizeDifferentiate thyroid from non-thyroid nodules  , 20Identify cervical lymphadenopathy and characterize enlarged nodes into benign and malignant Stratify thyroid nodules according to probability of malignancy -24In MNG select nodules for FNACEvaluate residual thyroid tissue after surgeryEvaluate diffuse thyroid changes Guide needle tip placement for FNACGiven the ubiquity of thyroid nodules, it would be impossible to biopsy all nodules for selection for surgery and ultrasound criteria have been used to try and divide nodules into those that are suspicious or look malignant and those in whom biopsy can be deferred , 6. All Several benign and malignant ultrasound gray scale and Doppler features have emerged over the last few years , 26 Tab. These cThere are several methods of characterization of ultrasound features. The original TIRADS papers , 27 descThere has been extensive work on determining the statistical significance of each ultrasound feature in predicting malignancy , 22, 23.Sensitivities and specificity for the various schemes have been worked out . SensitiThe later schemes, especially of Kwak, are simple enough to be practically implementable by even less experienced radiologists , 30.A recently published retrospective study with 8806 patients  used onlWork has been done on trying to identify, with certainty, benign nodules on ultrasound -35 and cThe management of thyroid nodules has become increasingly complex. In almost all other cases of focal disease, the aim of early detection is to identify cases that might be malignant, so that an appropriate intervention can be planned. Managing cases of early thyroid cancer is not so straight forward. Most patients of thyroid cancer will die with thyroid cancer but not of thyroid cancer . Tools fThis raises two important questions in patients with palpable or impalpable thyroid nodules; is this likely to be malignant? And, if malignant, does this warrant aggressive treatment? There are no easy answers to these questions, especially the second, but due to resource constraints some stratification strategy that will obviate biopsy as a first step is needed.The whole discussion of thyroid imaging boils down to establishing thresholds at which to decide if biopsy needs to be done or not. The American Thyroid Association has laid down very detailed criteria of biopsy thresholds . These cMany groups have demonstrated sufficient confidence in their ultrasound findings to use the FNAC results only to confirm their initial ultrasound impression. In cases of discordant FNACs , they suggest a second FNAC reading .Ultrasound imaging has been used, though with less-than-perfect sensitivity, for identifying patients who do not need an FNACC but more work is needed to ensure that the patients who are not biopsied do not develop cancer at a later stage. The development of new applications and new methods of extracting information from ultrasound data like elastography and contrast will bring in more sensitivity and specificity to the diagnostic process. At present we can confidently state that ultrasound imaging offers a powerful tool to help in making management decisions in thyroid nodular disease.


A wide range of adipokines identified over the past years has allowed considering the white adipose tissue as a secretory organ closely integrated into overall physiological and metabolic control. Apelin, a ubiquitously expressed peptide was known to exert different physiological effects mainly on the cardiovascular system and the regulation of fluid homeostasis prior to its characterization as an adipokine. This has broadened its range of action and apelin now appears clearly as a new player in energy metabolism in addition to leptin and adiponectin. Apelin has been shown to act on glucose and lipid metabolism but also to modulate insulin secretion. Moreover, different studies in both animals and humans have shown that plasma apelin concentrations are usually increased during obesity and type 2 diabetes. This mini-review will focus on the various systemic apelin effects on energy metabolism by addressing its mechanisms of action. The advances concerning the role of apelin in metabolic diseases in relation with the recent reports on apelin concentrations in obese and/or diabetic subjects will also be discussed. Nutrient metabolism and energy homeostasis are tightly regulated by endocrine, paracrine, and autocrine factors. Moreover, skeletal muscle, liver, adipose tissue and pancreatic β-cells play a major role in the maintenance of energy balance. A rapid modification of energy balance leads to obesity that in turn is a crucial cause of insulin resistance. Several mechanisms linking obesity to insulin resistance have been proposed. Among them, adipocyte-secreted factors or adipokines have been shown to play an important role. Alteration of their production (excess or deficit) could directly promote or delay the onset of insulin resistance. The role of leptin and adiponectin has been extensively studied  respectively. Later on, the study of Yue et al. also reported that apelin was able to stimulate glucose transport in vitro in C2C12 muscle cells through a pathway involving AMPK but not eNOS  mice exhibiting hyperinsulinemia and insulin resistance  fed mice. Indeed, Higuchi et al. showed that daily ip apelin injections during 2 weeks decrease the triglycerides content in adipose tissue and the weight of different fat depots in standard mice and in HFD fed mice  -induced lipolysis through a pathway involving Gq, Gi, and AMPK  observed in brown adipose tissue (Higuchi et al., 2 consumption and body temperature were also increased in HFD fed Tg-apelin mice (Yamamoto et al., Energy expenditure in response to apelin treatment has also been studied via thermogenesis. Higuchi et al. reported that rectal temperature and OAll together, these studies clearly show that apelin, by itself, exerts metabolic functions such as glucose uptake but also improves insulin sensitivity since, for example, at the end of chronic apelin treatment, insulin-induced glucose transport was increased in skeletal muscles and there is an overall better insulin and glucose tolerance (Attane et al., Numerous studies have reported increased plasma apelin concentrations in obese and/or diabetic subjects (for review see Castan-Laurell et al., The metabolic effects of apelin Figure  describeThe 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.


Harnessing the impressive therapeutic potential of Nitric oxide (NO) remains an ongoing challenge. This paper describes several of the current strategies both with respect to the underlying chemistry and physics and to the applications where they have shown promise. Included in this overview are molecular systems such as NONOates that release NO through chemical reactions and delivery vehicles such as nanoparticles that can generate, store, transport and deliver NO and related bioactive forms of NO such as nitrosothiols. Although there has been much positive movement, it is clear that we are only at the early stages of knowing how to precisely produce, transport and deliver to targeted sites therapeutic levels of NO and related molecules. Nitric oxide (NO) is a free radical that is naturally produced in mammals. It has been well known to play a key role in a wide variety of physiological and pathophysiological important processes, such as neurotransmission, neuronal communication, platelet adhesion, thrombosis, vasodilation, inflammation and wound healing . Over thOrganic nitrates and nitrites are the NO donor drugs currently in use to treat coronary artery disease. Nitroglycerin (glyceryl trinitrate), isosorbide dinitrate, isosorbide mononitrate and isoamyl nitrite are the examples of this class . These pSodium nitroprusside (SNP) is another NO-based drug that is in use for vasodilation in emergency setting to treat acute hypertension . SNP is Diazeniumdiolates (NONOates) are another class of NO donors that are getting great attention recently for the ease of preparation and generating predictable amounts of NO from these products ,12. NONOet al. (PF6)3 has been synthesized and bound to lipid carriers for topical administration . The potet al.  demonstrThe presented platforms all offer various benefits supported by several levels of evidence. From a translational standpoint, safety and practicality are the two greatest challenges. Even the clinically available NO-generating compounds, such as the organic nitrites/nitrates, have some limitations. Organic nitrates only release NO in tissues expressing the enzyme mitochondrial aldehyde dehydrogenase 2; however, this enzyme is irreversibly inactivated with use thus resulting in the emergence of tachyphylaxis , thereby limiting the utility of this class of materials. Furthermore, continued use has been shown to upregulate PDE5, resulting in increased vasoconstriction; the complete opposite of the desired clinical outcome .N-nitrosopyrrolidine, an extraordinarily potent experimental hepatocarcinogen  revealed no evidence of toxicity. Most recently, as part of large animal study (pig) examining the efficacy of these nanoparticles to prevent and treat hemorrhagic shock, organ histology showed no evidence of damage due to the nanoparticles including a detailed histological examination of kidney . Overall the emerging picture is one in which silane-derived nanoparticles are nontoxic. The exact mechanism of elimination for both topically applied nanoparticles and systemically applied nanoparticles still needs to be explored.NO delivery via nanoparticles raises the added safety issue associated with the nanoparticles themselves. Most of the toxicity studies to date relating to NO delivery involve the silane- or silica-derived nanoparticles. No major toxicity has been observed to date. For the hybrid hydrogel nanoparticles both topical and systemic studies have been conducted. Topically applied hybrid hydrogel nanoparticles were shown not to cause any detectable inflammatory response in the surrounding skin in a rat model . SystemiThe results to date indicate that the above-described NO delivery platforms have the potential to impact both topical and systemic clinical applications. Potential topical applications include uses that cover enhanced rates of wound healing and treatment of a wide variety of topical infections by most pathogens including ESKAPE organisms, fungus and parasites such as those associated with cutaneous leishmaniasis. The ability of some nanoparticle formulations to penetrate skin and release NO in a slow sustained manner opens the door for a topical treatment of erectile dysfunction. Systemic applications could include treatment of vascular inflammatory conditions arising from hemorrhage, hemoglobinopathies, hemolytic disorders and diseases caused by NO-sensitive organisms such as Chagas disease and African sleeping sickness. In all cases, the dosing and timing will prove critical since an inflammatory cascade once fully underway is fed by NO generated by macrophages. The emerging picture is one in which NO delivered at an early enough stage within the vasculature can short circuit the inflammatory cascade.S-nitrosothiols and nitrite is a challenging but important biomedical objective.Harnessing the therapeutic potential of nitric oxide and related bioactive forms of nitric oxide such as Controlled targeted nitric oxide delivery is therapeutically desirable for both systemic and topical applications.There are several promising strategies that are currently being developed and tested.Promising approaches include nanoparticle delivery platforms that can ultimately result in both controlled delivery and tissue-specific targeting.Toxicity and pharmacokinetic studies are still required before clinical translation is fully possible.


KwaZulu-Natal (KZN) has the highest burden of notified multidrug-resistant tuberculosis (MDR TB) and extensively drug-resistant (XDR) TB cases in South Africa. A better understanding of spatial heterogeneity in the risk of drug-resistance may help to prioritize local responses.Between July 2012 and June 2013, we conducted a two-way Lot Quality Assurance Sampling (LQAS) study to classify the burden of rifampicin (RIF)-resistant TB among incident TB cases notified within the catchment areas of seven laboratories in two northern and one southern district of KZN. Decision rules for classification of areas as having either a high- or low-risk of RIF resistant TB  were based on consultation with local policy makers.We classified five areas as high-risk and two as low-risk. High-risk areas were identified in both Southern and Northern districts, with the greatest proportion of RIF resistance observed in the northernmost area, the Manguzi community situated on the Mozambique border.Our study revealed heterogeneity in the risk of RIF resistant disease among incident TB cases in KZN. This study demonstrates the potential for LQAS to detect geographic heterogeneity in areas where access to drug susceptibility testing is limited. In South Africa, the latest published estimates from a nationally representative tuberculosis (TB) drug resistance survey date back to 2002. At that time, 1.8% of new (treatment-naïve) TB cases and 6.7% of previously treated cases  were infected with multidrug-resistant (MDR) TB, defined by resistance to first-line drugs isoniazid and rifampicin [An epidemic of extensively drug resistant (XDR) TB in Tugela Ferry, in the KwaZulu-Natal (KZN) province of South Africa, captured global attention in 2006–7  and commDue primarily to variable access to DST, routine surveillance data in South Africa has not historically been used to investigate local heterogeneity in the burden or risk of drug-resistant TB. Population-representative drug resistance surveys, such as the one performed in 2002, can provide overall average estimates of the risk of drug resistant TB disease. However, these studies are resource-intensive (and thus infrequently performed) and do not provide information at a level of geographic granularity that may be valuable for programmatic decision making ,7.Lot quality assurance sampling (LQAS) is an alternative survey approach that allows for the classification of an area’s risk of resistance among newly diagnosed TB cases; this approach prioritizes the desire to identify areas that exceed pre-specified thresholds over a precise estimate of the proportion of drug resistance among new TB cases ,9. This We applied LQAS in several jurisdictions of KZN to explore its potential to assess spatial variation in the risk of rifampicin (RIF)-resistant TB among new TB cases in KZN. This evaluation was undertaken to gain a better understanding of the prevalence of drug-resistant TB in specific geographic areas and help inform local programs on activities and allocation of resources to prevent ongoing transmission.® MTB/RIF (GeneXpert) assay, facilitating routine collection of data describing RIF resistance, was initiated in South Africa in 2011 [This study was conducted in three health districts of KZN: Sisonke, uMkhanyakude, and Zululand. Health districts were chosen through stakeholder consultation based on their perceived high  and low (Sisonke) drug-resistant TB burden. Seven sites across the three health districts were selected for inclusion; each site included a district hospital and all of the health facilities in the hospital catchment area. In each site, sputum samples are routinely collected from individuals with presumptive TB presenting at community clinics and hospitals and sent to a central district hospital laboratory for testing. Systematic surveillance of drug-resistance has not been conducted in this region. Rollout of the Xpert in 2011 ; howeverTwo of the seven sites selected for inclusion in the study were not using GeneXpert at the time of recruitment; LQAS was conducted prospectively in these sites to allow us to collect and process samples with GeneXpert at the time of treatment initiation. The remaining five laboratories had transitioned from smear microscopy to GeneXpert as the primary TB diagnostic tool, and LQAS was thus conducted retrospectively as additional patient samples were not required.The study population comprised newly-diagnosed, bacteriologically-confirmed patients with TB who were ≥18 years of age and who had never previously received more than one month of treatment for TB. Patients who were unable to provide informed consent, had received more than two doses of TB treatment for current TB disease at the time of GeneXpert diagnostic testing, or were incarcerated at the time of recruitment were ineligible.Stakeholder guidance and previous drug resistance surveys informed our sample size and decision rule calculations. We determined that a sample size of 82 isolates per site and a corresponding decision rule of four drug-resistant cases would allow us to achieve our desired LQAS performance requirements: less than 10% probability of erroneously classifying an area as “high risk” if the true proportion of RIF resistance was 2% or lower, and less than 10% probability of erroneously classifying an area as “low risk” if the true proportion of RIF resistance was greater than 8% . AccordiIn sites where GeneXpert was not yet available, TB diagnosis was determined by sputum smear microscopy. Consecutively-tested patients with smear-positive disease were identified daily at each central hospital laboratory and assessed for eligibility by study personnel. Patients whose laboratory records indicated that they were <18 years of age or incarcerated were excluded immediately; study staff continued eligibility assessment of all remaining patients at the clinic where their sputum sample(s) had been collected. Date of birth and treatment history were reviewed in each patient’s clinic record, and patients who were <18 years of age, classified as retreatment patients, or who had received more than two doses of TB treatment for the current infection were excluded. (Patients who had received more than two doses of treatment prior to collection of a specimen for GeneXpert testing were excluded due to concerns about the impact that longer treatment duration might have on laboratory results). Study staff arranged to meet eligible patients at the clinic–often when the patient returned to receive TB results–or at their home. Eligible patients were asked to provide written informed consent, complete a short questionnaire, and provide a sputum sample. Previous treatment status was based on record review and verbal report. Specimens were transported to a research laboratory in Durban where they underwent GeneXpert testing, (liquid—MGIT) culture, and, when culture positive, drug susceptibility testing (DST). When RIF resistance was detected, study personnel communicated these results to each jurisdiction’s TB coordinator and primary health care supervisor. Recruitment continued until 82 GeneXpert-positive isolates had been collected at each site.GeneXpert was used as the primary TB diagnostic tool in the remaining five jurisdictions. This avoided the need for prospective recruitment and collection of a second sputum specimen. Lab and clinic records were reviewed to determine eligibility; patients who appeared to be eligible were contacted telephonically by study staff. Upon giving verbal informed consent, patients were asked to confirm whether or not they had been treated for TB in the past. Patients who had received more than one month of TB treatment at any point in the past or more than 2 doses of TB treatment prior to the date of the diagnostic specimen or who could not be reached were excluded. We recruited all eligible patients consecutively at these sites to mimic the approach used for the prospective enrollment until 82 patients with GeneXpert-positive results had been identified in each site.All analyses were performed using Stata version 13 . We evaluated differences in demographic and clinical characteristics between cases that were eligible and ineligible for inclusion, and between patient cohorts at study sites using Pearson’s chi-square test and Fisher’s exact test . Differences in median age between sites were assessed using the Wilcoxon rank-sum test.Approval for this study was granted by the South African Medical Research Council’s Ethics Committee, KZN’s Department of Health, the Institutional Review Boards at Brigham and Women’s Hospital, and the U.S. Centers for Disease Control and Prevention. Formal support was also obtained from each health district in which the study was conducted.Between July 2012 and March 2013, 513 patients with positive sputum smears were assessed for eligibility at two sites in Zululand Health District: Vryheid Hospital and Nkonjeni Hospital. Twenty-three weeks of recruitment were required to reach our desired sample size in Vryheid and 22 weeks were required in Nkonjeni.Among patients assessed for eligibility, a total of 349 were deemed ineligible or were excluded from the analysis because their sputum sample did not yield a positive GeneXpert result; 164 patients were included in the analytic sample . Fifty-fp = 0.01), and smear grades assigned to the diagnostic sputum samples of eligible patients tended to be higher than those assigned to ineligible patients (p = 0.04). There were no significant differences in age or smear grade between eligible and ineligible patients in Vryheid (data not shown).Eligible and ineligible patients did not differ with respect to sex in either site, but enrolled participants in Nkonjeni had a younger median age  than ineligible patients   of the 82 isolates collected in Vryheid, and in Nkonjeni, RIF resistance was detected in five (6.1%) of 82 isolates . The forGeneXpert and culture results were available for 162 isolates across both sites; we found 93% agreement between methods. GeneXpert positive/culture negative results comprised the majority of discordant pairs . MTB wasThe laboratory records of 847 patients with positive GeneXpert results were retrospectively reviewed in order to achieve our desired sample size in five sites across three health districts: Benedictine Hospital in Zululand; Manguzi Hospital in uMkhanyakude; and St. Apollinaris, EG Usher Memorial, and Christ the King Hospitals in Sisonke. The period over which test results were reviewed varied by site and ranged from four to seven months.Of 817 patients who met eligibility criteria based on laboratory records, an additional 270 were excluded: 119 were not eligible ; 122 had missing or inadequate health records; 27 had died prior to the time of the assessment; and 2 were excluded for other reasons . An addiWe observed no significant differences between eligible and ineligible patients with respect to sex, age or GeneXpert classification in any of the retrospective sites. There was no significant difference in the proportion of RIF-resistant cases among enrolled patients and those who we could not trace, with the exception of one site, Benedictine. Characteristics of enrolled patients did not differ significantly between sites .Four or more cases of RIF resistance were detected in four sites in the retrospective component of this study; these were thus classified as high-risk areas for RIF resistance .Lot quality assurance sampling allowed us to detect heterogeneity in risk of RIF-resistant TB across geographic regions of KZN. While the prospective and retrospective components used slightly different inclusion criteria and recruitment approaches, limiting direct comparison of results, we do note that variability in RIF resistance risk among new cases was observed across sites using both study designs. One of two sites in the prospective component was classified as a high-burden area, as were four of five sites in the retrospective component.In the Manguzi site, located in Umkhanyakude, the northernmost district of KZN, we found a substantially higher risk of RIF resistance than in other sites surveyed. In this site we found that 13.4% new patients (i.e. not previously treated) had RIF-resistant TB, but the small sample size associated with the LQAS survey means that the 95% confidence interval for this sample spans from 6.9% to 22.7%. While the LQAS approach aims to classify the level of risk based on a pre-determined threshold, since our sample was drawn from a consecutive sample of eligible patients, this estimate should be unbiased for the population from which this sample was drawn. We note that our finding of higher risk of resistance than previously reported estimates supports earlier observations of high rates of MDR TB case notifications in this district dating back to 2007. The high risk was noted despite the fact that clinicians in this district may not have been requesting DST for a large fraction of patients with tuberculosis [Striving to rapidly evaluate risk of drug resistance through LQAS–prospectively as well as retrospectively–we based our analyses on GeneXpert results which consequently limited our assessment to rifampicin resistance. LQAS could be applied to assess distribution of risk of MDR TB in the study region, but due to inconsistent DST practices in South Africa this type of evaluation would need to be conducted prospectively with laboratory procedures performed independently of routine health services. Given the strong association of isoniazid resistance with rifampicin resistance, we do not believe that an LQAS evaluation of the risk MDR TB would yield a different assessment than what we have reported based on GeneXpert results. Classifications of other resistance patterns (e.g. XDR) could also be examined through LQAS, but samples sizes and decision rules would need to re-calculated based on stakeholder guidance.Owing to a number of factors, LQAS was not as rapid an exercise as anticipated when applied prospectively in our study population. A relatively large proportion of patients with smear-positive TB assessed for eligibility were retreatment patients and not eligible, resulting in fewer eligible patients than anticipated. Further, because a second sputum sample was required to perform GeneXpert, culture, and drug susceptibility testing, many patients who would have met inclusion criteria initially were deemed ineligible by the time of recruitment because study staff had been unable to reach them before they had received two doses of treatment. In Nkonjeni, eligible patients were significantly younger and had higher sputum grades than patients who were ineligible; the higher sputum grade is likely a reflection of the high number of patients who were ineligible due to already having initiated anti-TB treatment. Additionally, 45 of the initial 209 (21.5%) enrolled patients were excluded from analysis because their second sample did not yield a positive GeneXpert result. The impact of some of these issues may have been mitigated had LQAS been conducted as a programmatic exercise rather than a research initiative, reducing the time required to reach the desired sample size. Enrollment (including confirmation of treatment history) and secondary sample collection could have been conducted by program staff at the first clinical interaction with every person suspected of having TB; upon availability of microscopy results, GeneXpert testing could be performed on samples produced by patients with positive sputum smear results. Accounting for the proportion of retreatment patients expected among those with unknown status, and assuming that a sample collected on the same day and under the same conditions as a smear-positive sample would have produced a GeneXpert-positive result, we estimate that 92 additional patients would have been eligible for inclusion in Vryheid, and 44 in Nkonjeni, resulting in data collection windows that were reduced by twelve and eight weeks respectively.In contrast, retrospective LQAS was a rapid exercise but was also subject to the quality of existing clinical records. In order to be confident that we were excluding patients being retreated for TB, we had to obtain additional confirmation of treatment history directly from patients through telephone interviews. A sizable proportion of patients who we were unable to contact had to be excluded from the analysis.rpoB mutations or the presence of multiple strains respectively, suggesting RIF resistance. Conversely, the GeneXpert assay is not 100% specific and one or both of these results may have been false (RIF resistant) positives. Even if this were the case, however, the classification of each site as high or low risk would not differ from those based on GeneXpert results alone. Further, due to regional differences in the timing of implementation of GeneXpert as a primary diagnostic test for TB, the retrospective component included persons who were both smear positive and negative, but we required an initial bacteriologic confirmation of smear positivity at our prospective sites to determine eligibility to obtain and test a second sputum sample with GeneXpert. It is possible that this discrepancy may interfere with our ability to compare the two approaches. However, the LQAS decision rules classifying sites as high or low risk within a local geographic area were based on a proportion, and the definitions of the numerator and denominator were consistent across all sites within the local area. Lastly, the clinical data collected were not originally intended for research use. While we were able to counter known discrepancies with respect to treatment history with patient verification, there may have been other data subject to poor record-keeping that could have impacted study observations.Study limitations included poor patient data availability at two clinics in Benedictine’s jurisdiction; they were insufficient for study purposes and these clinics were excluded from the study. Unmeasured drug resistance among patients who could not be traced in the prospective component of the study may have impacted whether or not the decision rule was reached and thus each area’s assigned LQAS classification. However, these individuals comprised a small proportion of all excluded patients. At Benedictine, a site included in the retrospective component of the study, patients that were not able to be traced were more likely to be RIF resistant than enrolled patients. Since this site was classified as a high-risk area, however, if the untraced patients were able to be contacted it would have increased the proportion estimate of RIF resistance, but would not have altered the study classification of this site. Though overall test agreement was high, we did observe some discordance between GeneXpert and culture results, particularly with respect to detection of RIF resistance. Of the three GeneXpert-identified RIF-resistant cases that were not confirmed by culture, one showed growth failure for DST, which we do not consider a truly discrepancy, and the remaining two demonstrated RIF susceptibility. It is not clear why these results were discordant. Resource limitations precluded gene sequencing and genotyping that may have identified Several considerations should be made when planning to apply LQAS in a programmatic context. While we continued sampling until the full sample size had been achieved to allow a comprehensive assessment of LQAS, allowing for early stopping when (and if) the decision rule has been reached ,5 can faOver the past five years, improved access to drug susceptibility testing  and cartridge based nucleic acid amplification tests (e.g. GeneXpert) have increased the detection of MDR cases throughout South Africa. GeneXpert is now used as a routine first-line diagnostic tool, which means that highly detailed spatial mapping of RIF-resistant TB will be possible in the near future. While universal access to DST may transform South Africa’s response to MDR TB if access to effective treatment can be scaled up to meet the burden which is being revealed by GeneXpert, universal DST is not yet the reality in most high burden TB settings. In such settings, new approaches to investigate the spatial distribution of drug resistance, such as LQAS, may help to prioritize the use of limited resources for MDR TB prevention and control.


Lung cancer remains one of the most complex and challenging cancers, being responsible for almost a third of all cancer deaths. This grim picture seems however to be changing, for at least a subset of lung cancers. The number of patients who can benefit from targeted therapies is steadily increasing thanks to the progress made in identifying actionable driver lesions in lung tumours. The success of the latest generation of EGFR and ALK inhibitors in the clinic not only illustrates the value of targeted therapies, but also shows how almost inevitably drug resistance develops. Therefore, more sophisticated approaches are needed to achieve long-term remissions. Although there are still significant barriers to be overcome, technological advances in early detection of relevant mutations and the opportunity to test new drugs in predictive preclinical models justify the hope that we will overcome these obstacles. The identification of patient-specific, targetable genetic alterations on which tumours depend for their survival is redefining the way patients can be treated . AlthougAt the moment, the most prominent benefits of targeted therapies are limited to a small percentage of patients mostly identified by biomarkers that have been correlated with therapy response. Such markers are typically the mutations affecting the gene encoding the drug’s target itself and usually result in a deregulated, always-on growth signal. Although these ‘driver’ mutations are in principle excellent targets/biomarkers, most of the impressive clinical responses to single-agent targeted therapies are limited in time as resistance to the drug almost inevitably ensues. This scenario is a common phenomenon in many different tumour types .Nonetheless, the remarkable regressions achieved by imatinib in CML , gefitinThis increased capacity to identify ‘druggable’ cancer mutations allowed for an increasing number of patients to profit from targeted therapies concomitant with a steady progress in understanding the underlying mechanisms of drug resistance. This knowledge is extremely valuable, in that it enables the rational design of combination therapies hitting simultaneously a driver oncogene and a secondary target responsible for the resistance. While this is a promising approach, multiple pathways conveying drug resistance and increased toxicity limit the use of multiple drugs simultaneously , 9.Often tumours escape therapy by restoring the same signalling pathway that is targeted by the drug. It appears that tumours show remarkable addiction to deregulation of a particular pathway and therefore using multiple drugs acting in the same pathway can be more effective without increasing toxicity to normal cells to unacceptable levels. Vemurafenib resistance in melanoma provides a successful example of such an approach: only four years after the first landmark study showing the benefits of vemurafenib in BRAFV600E-mutant melanoma , a combiImproved PFS and OS is the ultimate test for a rationally designed, biomarker-based therapy, and the rapid approval of this drug combination illustrates the importance of deciphering the precise mechanisms of drug action and the escape mechanisms which cause it to become ineffective.The concept of synthetic lethality (a gain or loss of function in one gene makes a tumour cell fully dependent on another gene) offers intriguing possibilities for interventions, as in principle it also allows the treatment of loss-of-function (e.g. tumour suppressor) lesions and limits toxicity to normal cells. An appealing example is the effect of Parp inhibitors in BRCA-deficient tumours . AlthougThere are other therapeutic options besides targeted therapies that deserve mentioning. Immunotherapy, for example, has finally come to the fore resulting in unprecedented long-lasting remissions in patients with advanced disease . Once agIn this review we want to focus on targeted therapies in lung cancer, more specifically non-small cell lung cancer (NSCLC) as targeted therapies and immunotherapy have been most promising with these tumours. We will minimally discuss small cell lung cancer (SCLC), as we recently published an extensive review on SCLC . FurtherEGFR, KRAS, and other molecules resulting in sustained activation of the MAPK pathway. SCLC is instead characterised by the combined loss of RB, P53, and frequent amplification of MYC oncogenes, absence of activation of the MAPK pathway, and a more prominent dependency on the PI3K pathway. SCC often carries mutations in P53, CDKN2A, genes in the PI3K pathway, and amplification of SOX2, NFE2L2, PDGFRA, and FGFR1. Many other recurrent genetic alterations have been identified in each lung cancer type, although their frequency is typically below 5%.Thanks to several studies , 20, 21 How can we use this knowledge to design effective therapies? We can view the genetic landscape of lung cancer essentially as a comprehensive collection of recurrent genetic events that influence tumour initiation and progression. This gives us the opportunity to identify on which of these events tumour cells depend for their growth and/or survival, try to link different mutations to a common function, or signalling pathway and finally select a target for intervention on that basis. The target chosen might be a mutated driver or a wild-type (wt), non-mutated protein-activated downstream from several of the mutations found in the tumour. In the case of NSCLC we see frequent lesions in the MAPK pathway. These include gain-of-function mutations in EGFR, K-RAS, BRAF, and EML4-ALK, all signifying the importance of the MAPK pathway for this tumour type.A large body of preclinical  and clinIn the case of EGFR, much progress has been made since two landmark studies on NSCLC patients demonstrated that EGFR inhibitors were effective only in tumours harbouring activating mutations in the EGFR gene , 24. ScrEGFR-mutant lung cancer appears to be a distinct clinical entity and is primarily found in non-smokers . A signiThe question of whether sporadic T790M mutations are already present at the start of the treatment or are acquired following treatment has been around for a long time , and theWhen prior knowledge of a resistance mechanism is available, like in the case of the T790M mutation, having a sufficiently sensitive and robust clinical-grade diagnostic test can help to make a rational decision about a treatment. Digital PCR (ddPCR) is a significant advance in this direction. ddPCR is an increasingly popular and robust methodology that can detect tumour DNA mutations with high sensitivity and specificity compared to other similar techniques. The potential of this technique was first shown in the non-invasive detection of Kras mutations in stool samples of colorectal cancer patients  and moreMouse studies of the T790M escape mutant have illustrated another phenomenon that may be of significant importance for how we treat patients over time. The T790M mutant allows for escape from inhibitors that target the L858R mutation in both mouse  and humaNot surprisingly, in the last decade ample resources were invested in developing better and safer inhibitors capable of inhibiting the various EGFR mutants. Afatinib, for example, belongs to the second generation of inhibitors  designedFortunately, third-generation inhibitors such as AZD9291  and rociThe important lesson we have learned from clinical trials and drug resistance experiments is that the combination between the unique biochemical characteristics of each EGFR inhibitor and the genetic heterogeneity of a tumour is the major determinant of response. Given the importance of MAPK pathway to which a substantial fraction of NSCLC is so profoundly addicted, it remains worthwhile to continue developing new inhibitors for EGFR mutants.The inhibition of ALK is another successful example of molecular therapy in lung cancer. A rearrangement in the genes encoding ALK kinase and EML4 yields an ‘addictive’ oncogenic fusion capable of driving the progression of about 4% of NSCLC . These fThere is luckily a second generation of inhibitors which are able to overcome this problem. Ceritinib  and alecWhich ALK inhibitor to administer as first-line or in the resistant diseases is still an open question, and we do not yet have a full patient stratification scheme based on multiple biomarkers, such as ALK mutation type, HGF plasma levels, and MET amplification status. It is clear however, that treating ALK and ROS1 mutant tumours with the appropriate targeted agents substantially improves patient’s outcome. Although it is still a relatively new field  models , 65, 66,molecule . Still gG12VKRAS-driven mouse model of NSCLC [G12VKRAS-driven mouse model of NSCLC and the outcome of many experiments all point to a pivotal importance of the MAPK [of KRAS) nodes in the MAPK pathway whose inhibition is not toxic per se, but blocks proliferation only in KRAS mutant tumour cells. Puyol et al provided another compelling case of how mouse models can be used to precisely validate a target [The of NSCLC  recapituof NSCLC . Researcof NSCLC . This apthe MAPK , 72 and the MAPK , 74 in tthe MAPK  and c-Raa target  by showia target  and suggHere mouse models provided key evidence that we can identify and validate targets in NSCLC whose inhibition is likely to be maximally effective on tumour and minimally toxic on normal tissue. Future trials will need to show whether these findings can be translated to human lung cancer.L858R+T790MEGFR mouse model, resistance because of EGFR T790M could be prevented by a combination of afatinib and cetuximab [GEMM models of lung cancer are in principle also suited to directly test targeted therapies. A systematic review of pharmacological interventions performed on GEMMs of lung cancer  suggest etuximab . GEMMs aFinally, a systematic investigation of the plasma proteome of GEMMs of NSCLC and SCLC  showed aAnother remarkable advance regards PDX models. Given the access to NSCLC surgical specimens and their propensity to engraft in immunodeficient mice, several groups have been able to establish a sufficiently large collection of PDXs representative of the major molecular subtypes  (EGFR, Aet al reported anovel promising PDX technology [Recently Hodgkinson chnology  which prHuman CTCs engrafted and grew as tumours matching the donor’s histology and response to cisplatin, the standard treatment for SCLC . Technolin vitro and in vivo screening techniques and the validation of novel intervention protocols in GEMMs and PDXs, we can now quickly gain insight into the mechanisms of drug resistance and how to overcome these. This should translate into clinical trials in which longitudinal monitoring of tumour evolution with concomitant adaptation of intervention strategies would receive increased emphasis (The identification of actionable mutations in lung cancer is significantly changing the way patients are treated. Already the first generation of EGFR inhibitors showed how a biomarker-based therapy, when possible, resulted in an overall better patient outcome. While there is a constant improvement in finding eligible patients by deep sequencing of tumour biopsies and testing a wider panel of markers, the most significant challenge we face at the moment is that therapy-resistant tumours quickly develop with complex genetic profiles thereby making a rational treatment decision very difficult. Even using the latest inhibitors, resistant tumours will remain a moving target and this requires a highly individual approach. Here basic research is invaluable. Thanks to the advances of emphasis .


Placental invasiveness—the number of maternal tissue layers separating fetal tissues from maternal blood—is variable across mammalian species. Although this diversity is likely to be functionally important, variation in placental invasiveness remains unexplained. Here we test the hypothesis that increased risk of transplacental transmission of pathogens from the mother to the fetus promotes the evolution of non-invasive placentation, the most likely derived condition in eutherian mammals. Specifically, we predict that non-invasive placentation is associated with increased microparasite species richness relative to more invasive placental types, based on the assumption that higher numbers of microparasites in a population reflects greater risk of transplacental transmission to fetuses. As predicted, higher bacteria species richness is associated with non-invasive placentation. Protozoa species richness, however, shows the opposite pattern. Because invasive placentae facilitate the transfer of maternal antibodies to the fetus, we propose that the ancestral condition of invasive placentation is retained under selection for protection of newborns from higher risk of postnatal protozoan infection. Hence, our findings suggest that a tradeoff exists between protection against bacterial infection prenatally and protozoan infection postnatally. Future studies are needed to investigate how maternal prevalence of infection and the relative pre- versus postnatal risk of fetal infection by different microparasite groups vary among mammalian hosts in relation to placental invasiveness. The placenta is intrinsic to mammalian reproduction and fetal development; it nourishes the developing fetus by transferring nutrients and oxygen from the mother’s bloodstream to the fetus, eliminates the waste products of fetal metabolism, and protects the embryo within its fluid filled cavity ,2. DespiPlacental invasiveness is defined by the number of maternal tissue layers at the areas of exchange separating the maternal blood from the external tissues of fetal origin (the ‘trophoblast’) –4. In noIncreased placental invasiveness was long believed to facilitate fetal nutrition because the direct contact with maternal blood should allow easier fetal access to maternal resources and ultimately lead to enhanced fetal growth rates –7. HowevCytomegalovirus, HIV), protozoa , and bacteria  . For each predictor in the model we report the t-value with degrees of freedom (tdf) and p-value; for each model we report the model log-likelihood (Lh), the estimated value of the phylogenetic signal in the model residuals as quantified by λ (ML λ), and the amount of variance in microparasite species richness explained by the model (R2).For virus species richness, a PGLS polynomial model does not improve the fit to the data relative to a linear model for citation count )Plasmodium, Toxoplasma and Trypanosoma is degraded [Trypanosoma appears to secrete proteases that help to degrade collagen in the syncytium and subsequently binds to the receptors of the mononucleate layer to become internalized and transferred to the fetal side of the placenta [Counter to predictions, protozoa species richness is greater in mammals with invasive hemochorial placentation. The observation that the syncytium of hemochorial human and mice placentae infected by degraded ,55, suggplacenta . As for We propose that the need to pass maternal antibodies to the offspring prior to birth when postnatal risk of protozoan infection is high might explain our result that hemochorial placentation is associated with high protozoa species richness, and represents an evolutionary selective pressure for retaining invasive placentation. Maternal antibodies can increase neonatal survival and growth rates, and influence the development of the offspring’s immune system . MaternaOryctolagus cuniculus) and rodents, all mammals with hemochorial placentation, but is reduced and non-functional in species with non-invasive placentae, such as artiodactyls [The yolk sac placenta is also responsible for IgG transfer to the fetus ,59. Inteodactyls ,57. Thusodactyls ,57,58,60Finally we find no association between placental invasion and virus richness. Why this is the case is unclear but our results indicate that viral transmission may be unaffected by placental morphology.To conclude, we find mixed support for the hypothesis that non-invasive placentation provides a barrier to transplacental transmission of microparasites. Conversely, our result that invasive placentation is associated with greater protozoa richness–and independent evidence that antibodies transfer is high in hemochorial placentae–suggest a new hypothesis that invasive placentation represents an evolutionary adaptation to increase neonatal protection when postnatal risk of infection is high. To fully understand how placental morphology interacts with parasite pressure, future studies could examine the prevalence of infection pre- and postnatally across species with different placental morphology, and investigate how transmission mechanisms are affected by diversity in placental invasiveness. Based on our findings, we expect that higher prevalence of bacterial infection during gestation is more likely to favor the evolution of non-invasive placentation, while greater postnatal risk of protozoan infection promotes the evolution of invasive placentation. In addition, because host behavioral and ecological traits are known to influence levels of parasitism ,35, theyS1 Data(XLSX)Click here for additional data file.S1 File(DOCX)Click here for additional data file.