Psychopathy, characterized by symptoms of emotional detachment, reduced guilt and empathy and a callous disregard for the rights and welfare of others, is a strong risk factor for immoral behavior. Psychopathy is also marked by abnormal attention with downstream consequences on emotional processing. To examine the influence of task demands on moral evaluation in psychopathy, functional magnetic resonance imaging was used to measure neural response and functional connectivity in 88 incarcerated male subjects (28 with Psychopathy Checklist Revised (PCL-R) scores ⩾30) while they viewed dynamic visual stimuli depicting interpersonal harm and interpersonal assistance in two contexts, implicit and explicit. During the implicit task, high psychopathy was associated with reduced activity in the dorsolateral prefrontal cortex and caudate when viewing harmful compared with helpful social interactions. Functional connectivity seeded in the right amygdala and right temporoparietal junction revealed decreased coupling with the anterior cingulate cortex (ACC), anterior insula, striatum and ventromedial prefrontal cortex. In the explicit task, higher trait psychopathy predicted reduced signal change in ACC and amygdala, accompanied by decreased functional connectivity to temporal pole, insula and striatum, but increased connectivity with dorsal ACC. Psychopathy did not influence behavioral performance in either task, despite differences in neural activity and functional connectivity. These findings provide the first direct evidence that hemodynamic activity and neural coupling within the salience network are disrupted in psychopathy, and that the effects of psychopathy on moral evaluation are influenced by attentional demands. Most of these results fit with previous theoretical and empirical work documenting a key role of emotion in moral reasoning, especially the processing of distress signals and associations with action outcomes,29 and pointing out that cognitive processing alone is insufficient to guide moral judgment.30 Thus, if an individual does not possess a capacity for experiencing affiliative prosocial emotions to accompany or guide their actions and predict their consequences on others, having only an explicit knowledge of moral norms may be insufficient to motivate moral and caring behaviors.32For instance, the literature on morality has been dominated by moral dilemmas, with early neuroscience investigations arguing for separate cognitive and affective processes.34 However, limiting investigations to hypothetical dilemmas cannot provide a full account of atypical moral reasoning in psychopathy,35 and, even in the absence of behavioral differences, this disorder is marked by abnormal neural recruitment and anatomical connectivity.36 Furthermore, some of these dilemmas are problematic and tell us very little about moral decision-making in everyday life.37 Several neuroimaging studies have indeed demonstrated that criminal psychopaths show greater hemodynamic activity in the lateral prefrontal cortex during processing of emotional stimuli than do non-psychopaths, and this finding is usually interpreted as either representing compensatory processes for deficient paralimbic activity or top-down cognitive control of salient stimuli.38 Moreover, a strong distinction between affect and cognition may be a false dichotomy, both at the psychological and neurobiological levels.39 For instance, although it may be possible to assign especially affective or cognitive roles to specific areas of the cortex or limbic system, many 'affective' and 'cognitive' regions such as the hypothalamus, vmPFC, ACC, amygdala and lateral prefrontal cortex have widespread overlapping structural and functional reciprocal connections.40Taken together, these studies are consistent with the notion that psychopathy is characterized by an over-reliance on cognitive deliberation because these individuals lack an intuitive affective aversion to harming others.41 In conjunction with studies of neurological patients and community samples, a complex picture is emerging, wherein moral cognition involves the interaction of automatic processes, which support intuitions that are usually affectively laden, and controlled processes, which support deliberation and reasoning.42 Importantly, these complementary computational systems arise from partially overlapping neural networks that support domain-general processes such as affective arousal, perspective-taking, attention, decision-making and motivational salience.44 Nodes of these networks, which are consistently implicated in functional magnetic resonance imaging studies of moral reasoning, include the amygdala, aINS, ACC and vmPFC, as well as the posterior superior temporal sulcus (pSTS/TPJ), dorsolateral prefrontal cortex (dlPFC) and posterior cingulate.45 The right temporoparietal junction (rTPJ), in particular, has received a great deal of focus, as this region is involved with not only theory of mind46 and moral judgment50 but also with reorienting responses and biological motion perception.51 This region is therefore a primary candidate in processing morally laden information. Also of particular interest for this study is the salience network, which is anchored by the dorsal aspects of the ACC and aINS, and extends across many regions, including the superior temporal pole, supplementary motor area, amygdala, ventral striatopallidum, hypothalamus, dorsomedial thalamus, and periaqueductal gray and ventral tegmental area.10 This network facilitates attention allocation toward personally or motivationally salient information.44 In the context of moral judgments, this salience system is also responsible for dynamically orchestrating shifts between cognitive control and default mode networks.52Taking into account the large-scale nature of neural connections and adopting a network view of brain function has been an important step to understand how the nervous system supports complicated mental and behavioral activities.53 However, in other contexts psychopathy has been linked to greater activity in these regions.54 One appealing resolution to this apparent contradiction proposes that selective attention is also abnormal in psychopathy.55 In non-moral contexts, such as fear-potentiated startle, both behavioral and amygdala activity differences between groups were found to be reduced by focusing attention to threat-relevant information.56 This fits with recent research indicating that individuals with high and low psychopathic traits differ in the way they modulate attention to morally or socially relevant information,57 depending on their current goals and mental states. On this view, both bottom-up automatic and top-down controlled processes are disrupted in psychopathy. However, no study to date has directly manipulated task goals with respect to morally relevant information in the same individuals with different levels of psychopathy.Thus, an important next step in clinical neuroscience is to investigate the extent to which psychopathic traits relate to typical and atypical recruitments of these networks in the context of moral evaluations. Previous neuroimaging studies that investigated the impact of psychopathic traits on the salience network have produced inconsistent findings. Some studies reported that individuals with psychopathy fail to attach the appropriate significance to the distress cues of others, and show decreased hemodynamic response in the aINS and amygdala.62 Implicit moral judgments correspond to decisions focusing on non-relevant aspects of the stimuli  when perceiving morally laden stimuli.59 In healthy individuals, morally relevant information is especially salient and therefore influences processing at multiple stages, even when it is not task-relevant.11 Examining both explicit and implicit processes in psychopathy can help to distinguish between two of the primary competing hypotheses about socioemotional processing in psychopathy, namely, whether psychopathy is marked by a failure to spontaneously encode task-irrelevant moral information as salient, or a failure to appropriately respond to such information.The current study was designed to investigate how psychopathy, both as a clinical taxon and personality dimension, influences neural encoding of moral valence in contexts when moral content is task-relevant or task-irrelevant. As discussed above, a great deal of work has already been performed on the neural networks that support explicit moral reasoning. Implicit moral processing, on the other hand, has received less attention in the neuroimaging literature, although there have been a few investigations.62 and reported an augmentation in hemodynamic response in the vmPFC during the explicit condition, and greater dlPFC activity in the implicit condition. The present study assesses implicit and explicit processing of moral information by having the same participants evaluate the stimuli during two tasks. Two regions of particular interest are the amygdala and right pSTS/TPJ. Historically, the amygdala has been argued to signal and prioritize the affective relevance of stimuli, regardless of whether or not it is task-relevant,63 and to provide a route for this salient information to influence downstream processing in other cortical regions, largely independent of top-down attention control.64 However, recent investigations have shown that the amygdala is not completely immune from top-down influences,56 and this has relevance to atypical affective processing in psychopathy.65 Moreover, because the amygdala is highly interconnected with much of the cortex, functional connectivity seeded in the amygdala is expected to reveal distinct patterns of connectivity in explicit and implicit moral contexts. Functional connectivity seeded in the rTPJ was also assessed because, in addition to its previously discussed role in multiple processes important for sociomoral cognition, several studies have found an association between psychopathy and rTPJ/STS gray matter abnormalities.67To our knowledge, only one neuroimaging study has directly contrasted explicit and implicit moral processing tasks in its design,68 During the explicit moral evaluation task, because psychopaths lack an intuitive aversive response to harm, they are expected to rely more on cognitive (controlled) computations, as evidenced by increased recruitment at the whole-brain level of prefrontal regions, rather than regions that support rapid processing, such as parahippocampus, amygdala, ventral ACC and brainstem. Moreover, this cognitive processing style is expected to require greater reliance on the integrative capacities of ACC, as evidenced by increased functional connectivity to this region from the right TPJ and amygdala.We hypothesized that during implicit moral evaluation, where the presence or absence of harm is not task-relevant, psychopathy will be inversely related to neural activity in nodes of the salience network. Similarly, we predicted that psychopathy would lead to reduced functional connectivity seeded in both right amygdala and right TPJ to widespread cortical and limbic areas, especially to core nodes of the salience network .n=6), transferred to another site before PCL-R scores could be collected (n=8), excessive movement in the magnetic resonance imaging (n=7), poor task performance (n=2) or failure to complete the task (n=1). Thus, the final sample consisted of 88 male subjects (31.2±7.3 years). The study was approved by the Institutional Review Boards from the University of Chicago and the University of New Mexico.Overall, 112 male volunteers, all of whom were incarcerated in medium-security North American correctional facilities, participated in the study. A total of 21 participants were excluded from analysis because of lifetime threshold for either bipolar disorder or major depressive disorder , whereas participants with scores of less than 20 were assigned to the low-psychopathy group . Groups did not differ in terms of age, IQ or accuracy on either the implicit or explicit tasks . Participants provided informed written consent and were compensated with pay consistent with the facility hourly labor wage. Inclusion criteria were IQ greater than 70 and age less than 50 years.Trained research assistants conducted PCL-R assessments, including file review and interview. Intelligence quotient (IQ) was assessed via Wechsler Adult Intelligence Scale. Power analyses indicated that for a medium effect size of 0.5, groups of at least 27 were required to achieve power greater than 0.8.70 Briefly, three static images are extracted from videos and presented in succession to create apparent motion . Blocks consisted of five scenes of the same type, each followed by a one second fixation cross. There were four runs, each containing three blocks of each type presented in pseudo-random order and interspersed with rest blocks of 14 s. In each block, one randomly selected scene was followed by a question screen (2000 ms). Participants indicated their response ('Yes' or 'No') using one of two buttons. The first two runs were intended to assess implicit moral judgment; therefore, rather than explicitly cuing participants to attend to or evaluate the morally relevant content of the stimuli, the question slide asked whether or not the action occurred indoors ('Was this inside?'). The last two runs assessed explicit moral judgment by asking participants whether the action was morally wrong ('Was it wrong?'). Thus, both tasks required participants to make simple Yes–No choices about morally laden stimuli; however, moral information was only relevant in the later task. Before the start of the run participants were given instructions as to what question they would be asked and reminded of the button–response mappings. The button–response mapping was counterbalanced across participants.In the scanner, participants viewed scenes depicting interpersonal harm or interpersonal assistance . Stimuli were presented via the E-Prime 1.0 software .Scans were collected using the Mind Research Network 1.5 Tesla Siemens Magnetom Avanto Mobile unit  equipped with a 12-element head coil and advanced sequence gradients. Echoplanar imaging images were acquired with a gradient echo pulse sequence  in MATLAB . Echoplanar imaging images were realigned, filtered (128-s cutoff), co-registered and normalized to the SPM echoplanar imaging template, and smoothed . General linear models were used for statistical analysis. For each model, Block types (Bad and Good) were modeled beginning at the onset of the first picture in the block and lasting until the end of the block (18 s). Motion parameters and run order were included as nuisance regressors. For each Task type (Implicit or Explicit), individual contrast images were generated for the effect of Block (Bad>Good). At the second level, independent sample x=22, y=-2, z=−16) and right TPJ , based on coordinates taken from a meta-analysis of functional magnetic resonance imaging studies of morality.45 Within each task, the Bad–Good contrast was used as the psychological regressor in separate psychophysiological interaction (PPI) analyses. As before, first-level PPI images were analyzed at the second level with age, IQ, task accuracy and PCL-R scores entered as covariates.To examine functional connectivity, the mean activity was extracted from a 6-mm-radius sphere centered around the right amygdala , but not the explicit task (P>0.4). None of the other correlations reached significance after correction for multiple comparisons. Accuracy rates were higher in the explicit task than in the implicit task (t(87)=5.42, P<0.001, d=0.578).Descriptive statistics and zero-order correlations among dependent variables are shown in x=−22, y=18, z=−6) for the interpersonal harm > interpersonal assistance contrast. PCL-R Factor 2 scores uniquely predicted reduced activity in dorsal ACC .During the implicit task, psychopathy was associated with reduced signal change in the right caudate and dlPFC, but greater signal change in left insula, right temporal pole and subgenual ACC . In the The connectivity analysis revealed that psychopathy scores were generally associated with widespread reductions in functional coupling seeded in the right amygdala across both implicit and explicit moral evaluation tasks . When haA similar pattern of results obtained for the PPI seeded in rTPJ . Total P73To the best of our knowledge, no study to date has examined the neural response and functional connectivity in both implicit and explicit processing of harm-related moral scenarios in forensic psychopaths. By utilizing ecologically valid depictions of interpersonal harm versus interpersonal assistance, when the harm was and was not task-relevant, the results of this study provide the first direct evidence for the influence of task demands on neural processing of moral information in psychopathy. Although high- and low-psychopathy groups did not differ in their behavioral performance on these tasks, there were several striking differences in the neural networks recruited as well as in functional connectivity, even after controlling for age and IQ. When inmates with high psychopathy scores viewed morally laden scenarios, they showed widespread decreases in functional connectivity seeded in the rTPJ and right amygdala, two important computational nodes previously associated with intention understanding and emotional saliency, which contribute to moral cognition.62 In healthy individuals, third-person pain is a salient cue that is critical to elicit empathic concern,74 and the low-psychopathy group likely spontaneously allocated attention to the cues of harm, even though they were not task-relevant. Indeed, psychopathy scores predicted decreased coupling between the right pSTS/TPJ and aINS, a region that is reliably associated with emotional awareness and empathic processing.29 Psychopathy has consistently been associated with an abnormally low aversive response to the distress of others;75 thus, greater activity in the dlPFC in the low-psychopathy group during the implicit moral evaluation task may indicate extra mental effort, or compensatory activity, required to inhibit such an automatic response when viewing harmful interactions.76As expected, during the implicit task , the low77 suggesting that individuals with high Factor 1 scores may perceive harmful interactions as more enjoyable. Psychopathy also predicted reduced response in the amygdala and dACC (62 although PCL-R scores did predict reduced functional connectivity between vmPFC and both right amygdala and right pSTS/TPJ (When interpersonal harm was task-relevant, psychopathy, both categorized as a taxon and as a dimensional variable, was associated with increased hemodynamic response in the dorsal striatum . Interesand dACC , althougand dACC . Unlike pSTS/TPJ  and 4. F78 This overlap across these three domains has been argued to represent a single system whose core function is to determine the optimal response to motivationally relevant situations.78 The dACC has also previously been shown to be an important node for orchestrating interactions between widespread cortical networks in service of moral judgment.52 Thus, decreased signal in dACC, especially in conjunction with the reduced activity in bilateral parahippocampal gyrus and amygdala, indicate that when explicitly evaluating dynamic visual scenes during moral evaluation, individuals high in psychopathy do not encode interpersonal harm as particularly salient. This fits with previous studies linking psychopathy to reduced amygdala response in social and moral contexts.79 However, our results are in contrast with some previous studies suggesting that focusing attention to socioemotional information reduces neuronal differences between controls and psychopaths.56 Moreover, given the increased functional connectivity between pSTS/TPJ and ACC, our results may also suggest that other domain-general aspects of information processing are disrupted in psychopathy.The ACC, because of its unique reciprocal connectivity with subcortical and cortical regions, is an important integrative hub, with dACC in particular exhibiting reliable recruitment during cognitive control, negative affect and nociception.56 However, because group differences in amygdala hemodynamics were only observed in the explicit task, and because these differences occurred without corresponding effects in prefrontal regions, the current results are inconsistent with the hypothesis that psychopathy levels influence amygdala response via increased top-down attentional control,80 at least in the current study.Interestingly, whereas the high-psychopathy group did show greater activity in a cluster in the left superior frontal gyrus, there were only significant group differences in dlPFC during implicit moral reasoning . Convers81 and further supports the argument that disrupted amygdala–prefrontal connections are part of the neurobiological basis of psychopathy.82 Moreover, during the explicit task, PCL-R scores predicted decreased connectivity to right temporal pole, inferior frontal gyrus and dorsomedial PFC, but increased connectivity to the dorsal ACC (83 A similar pattern was observed in the right pSTS/TPJ-seeded connectivity analysis (As indicated by the connectivity analysis , individrsal ACC . Given trsal ACC , which ianalysis , with PCanalysis . Psychopanalysis .Taken together, the findings from the current study provide an initial examination of the influence of task demands on the neural activity in the salience network during moral evaluations in psychopathy. During explicit moral evaluations, psychopathy was marked by reduced activity in several nodes of the salience network, including the amygdala and ACC. Moreover, functional connectivity analyses seeded in the right amygdala and right pSTS/TPJ showed increased coupling to the dorsal ACC. Conversely, when information about interpersonal harm was task-irrelevant, high trait psychopathy predicted reduced activity in dlPFC and decreased connectivity seeded in both the right amygdala and right pSTS/TPJ with ACC, aINS and vmPFC. Overall, functional connectivity analyses can identify patterns of communication between regions that contrast analyses may not detect. Connectivity analyses in our study identified regions whose response covaries with activity in two important computational nodes, amygdala and right pSTS/TPJ, during implicit and explicit conditions, contributing to create a dynamic model of circuits underlying moral evaluation in psychopathy.


Within both dietary groups, some of the rats had free access to a running wheel , whereas the controls  had no possibility of extra physical activity. At the end of the four weeks, we measured the effects of VR on various metabolic syndrome-associated parameters: (i) biochemical parameters, (ii) the content and composition of triacylglycerols (TAG), diacylglycerols (DAG), ceramides and membrane phospholipids, and (iii) substrate utilization in brown adipose tissue. In both dietary groups, VR led to various positive effects: reduced epididymal and perirenal fat depots; increased epididymal adipose tissue lipolysis; decreased amounts of serum TAG, non-esterified fatty acids and insulin; a higher insulin sensitivity index. While tissue ceramide content was not affected, decreased TAG accumulation resulted in reduced and modified liver, heart and skeletal muscle DAG. VR also had a beneficial effect on muscle membrane phospholipid composition. In addition, compared with the CD group, the CD+VR rats exhibited increased fatty acid oxidation and protein content in brown adipose tissue. Our results confirm that physical activity in a non-obese model of severe dyslipidemia has many beneficial effects and can even counteract the negative effects of sucrose consumption. Furthermore, they suggest that the mechanism by which these effects are modulated involves a combination of several positive changes in lipid metabolism.While metabolic syndrome is often associated with obesity, 25% of humans suffering from it are not obese and the effect of physical activity remains unclear in such cases. Therefore, we used hereditary hypertriaclyglycerolemic (HHTg) rats as a unique model for studying the effect of spontaneous physical activity [voluntary running (VR)] on metabolic syndrome-related disorders, such as dyslipidemia, in non-obese subjects. Adult HHTg males were fed standard (CD) or high-sucrose (HSD) diets  A sedentary lifestyle and the increased consumption of sugar, high-fructose corn syrup and energetically-rich food have contributed to a rapid increase in the prevalence of obesity, metabolic syndrome and type 2 diabetes –3. For tde novo lipogenesis, which results in increased liver lipid content and increased very-low-density lipoprotein (VLDL) secretion  on metabolic syndrome-associated disorders, we used non-obese hereditary hypertriaclyglycerolemic (HHTg) rats as a unique model of dyslipidemia and metabolic syndrome. Various researchers have shown that forced and voluntary exercise produce very different effects on tested animals ,49. AnimOur results suggest that VR, even in the absence of obesity, has a positive effect on lipid metabolism and reduces the negative effects of sucrose consumption. While separately VR and a high-sucrose diet had only a slight effect on total body weight, they had a significant effect on body composition and metabolism. Depending on the intensity of physical exercise, VR reduced the weight of perirenal and epididymal fat depots whilst also increasing lipolysis and protein content. Apart from these positive changes in adipose tissue, positive changes in the lipid metabolism of the VR rats resulted in lower concentrations of serum TAG and NEFA; these reductions were also correlated with VR intensity. Furthermore, the positive effect of VR on insulin sensitivity was confirmed by a higher ISI and by a reduced non-fasting insulin concentration. Collectively, these positive changes were associated with the following beneficial effects: lower sensitivity to oxidative stress; reduced lipid deposition in the liver, muscles, aorta and kidney; reduced content and changed composition of diacylglycerols (DAG) in the measured tissues; modified fatty acid composition in muscle membrane phospholipids; enhanced brown adipose tissue parameters.It is believed that both fructose overfeeding and exercise lead to the increased production of reactive oxygen species (ROS) ,51. In aOne of the most serious complications associated with metabolic syndrome is increased lipid deposition in non-adipose tissues. Increased lipid deposition is associated with a variety of complications: in the liver, it leads to insulin resistance ; in the Increased amounts of lipids in muscles have been linked to muscle insulin resistance . Our datOne of the mechanisms by which increased lipid content in the liver and muscles mediates insulin resistance is through the increased production of lipotoxic intermediates, primarily DAG and ceramides ,17. Hencet al. [in vitro [DAGs are signal molecules capable of increasing the activity of some species of protein kinase C (PKC). Once activated, PKC phosphorylates the insulin receptor substrate (IRS), thereby inhibiting insulin signal transduction . In our et al. , but alsin vitro . Conversin vitro . Furtheret al. reported that endurance-trained athletes had a lower muscle ceramide content than obese humans [Ceramides are signal molecules that inhibit insulin signal transduction in muscles via the inhibition of protein kinase B (PKB). They are also associated with inflammation, oxidative stress, mitochondrial dysfunction and other metabolic disturbances ,64. We fe humans . Such die humans ). On thee humans .From the above, it seems that the total amount of lipids and lipotoxic intermediates in tissue may be a key factor in the development of insulin resistance and metabolic syndrome. However, our results, together with evidence from the literature , suggestex vivo oxidation of exogenous albumin-bound fatty acids. We found that VR increased BAT activity in the CD+VR group, but not in the HSD+VR group. Our results for CD+VR support the hypothesis that after physical exercise BAT metabolic activity increases through the sympathetic nervous system. However, the results for HSD+VR indicate that high-sucrose feeding could interfere with this positive mechanism.The mechanisms described above suggest that physical activity positively affects lipid metabolism, but it also appears to have beneficial effects for brown adipose tissue (BAT) metabolism ,24. BAT Our results suggest that physical activity, even in the absence of obesity, can counteract the negative effects of sucrose consumption and the factors involved in the pathogenesis of metabolic syndrome-related disorders. In our study, voluntary running was associated with various beneficial changes, including lower concentrations of serum triacylglycerols and non-esterified fatty acids, reduced oxidative stress, and, in particular, decreased insulin resistance. All of these changes were to some extent connected to positive changes in lipid metabolism. Reduced lipid deposition in the liver, muscles, kidney and aorta was accompanied by the lower content and altered composition of diacylglycerols in the liver, heart and soleus muscle, as well as by fatty acid remodeling in muscle membrane phospholipids. We have suggested that the mechanism through which physical activity positively modulates lipid metabolism involves increased fatty acid oxidation in brown adipose tissue. These data provide important new findings about the positive effects of voluntary exercise and demonstrate its applicability to the treatment of dyslipidemia and metabolic syndrome.


Objective. To compare early empiric antifungal treatment with culture-directed treatment in critically ill patients with intra-abdominal sepsis. Methods. A prospective observational cohort study was conducted between August 2010 and July 2011, on SICU patients admitted after surgery for gastrointestinal perforation, bowel obstruction or ischemia, malignancy and anastomotic leakages. Patients who received antifungal treatment within two days of sepsis onset were compared to patients who received culture-directed antifungal treatment in terms of mortality rate and clinical improvement. Patients' demographics, comorbidities, severity-of-illness scores, and laboratory results were systematically collected and analysed. Results. Thirty-three patients received early empiric and 19 received culture-directed therapy. Of these, 30 from the early empiric group and 18 from culture-directed group were evaluable and analysed. Both groups had similar baseline characteristics and illness severity. Patients on empiric antifungal use had significantly lower 30-day mortality (P = 0.03) as well as shorter median time to clinical improvement (P = 0.025). Early empiric antifungal therapy was independently associated with survival beyond 30 days . Conclusion. Early empiric antifungal therapy in surgical patients with intra-abdominal sepsis was associated with reduced mortality and warrants further evaluation in randomised controlled trials. Candida, which was also ranked as the fourth most common cause of nosocomial bloodstream infections in United States [The incidence of nosocomial invasive mycoses has increased markedly over recent years , particud States , 4, 7, 8d States , 6, 7, 9d States , 5–7. Candida species in blood cultures and a slow turnaround time of 2-3 days [ prophylactic, preemptive, and empiric therapy, for high-risk patients have been explored, in place of culture-directed antifungal therapy [Definitive diagnosis of IC relies on conventional culture technique, given its nonspecific clinical signs and symptoms. Unfortunately, culture-based diagnoses are hampered by the low yield of2-3 days , 11, lea2-3 days , 13, alt therapy , 15. prophylaxis is not recommended by the latest guidelines from the Infectious Disease of America (IDSA) in ICU patients as the cost-effectiveness and potential ecologic effect of such an approach have not been fully elucidated [Antifungal prophylaxis with fluconazole had been assessed in several trials –18, invoucidated . preemptive therapy where treatment is started in response to increased Candida riskscores, for example, Candida colonization index (CCI), or levels of Candida biomarkers such as (1-3)-β-D-glucan [ Candida biomarkers display higher positive predictive values; however, they lack sensitivity and are thus not able to identify all cases of invasive candidiasis [An alternative strategy would beD-glucan , 21, 22.didiasis . FurtherEarly empiric therapy may be the most practical strategy for centers with limited resources. With this approach, there is a need to clearly identify which patient groups will benefit the most from early antifungal treatment. This is because the indiscriminate initiation of empiric antifungal agents may render them ineffective over time with the progression of fungal resistance. Patients who receive antifungal agents are also at risk of developing adverse effects due to these drugs. As such, the benefit of starting antifungal treatment must always outweigh patients' risk for developing drug-related serious adverse effects.Known risk factors for IC include the use of broad-spectrum antibacterial agents, use of central venous catheters, receipt of parenteral nutrition, receipt of renal replacement therapy by patients in ICUs, neutropenia, use of implantable prosthetic devices, and receipt of immunosuppressive agents or gastrointestinal surgery . PatientIn our hospital, there was no consensus among the ICU and infectious diseases physicians on which was the best strategy to adopt. This led to varied practices on the treatment of fungal infections in the ICU, with some physicians subscribing to early empiric approach while others using the culture-directed approach. The preemptive strategy was not employed due to limited funds and manpower constraints.A prospective observational cohort study was thus performed on our patients at high risk for intra-abdominal sepsis and IC, to evaluate if the early empiric use of antifungal treatment led to better survival than culture-directed treatment. Secondary objectives were to determine if early empiric antifungal therapy could shorten length of SICU and hospital stay, as well as time to clinical improvement.This study was approved by the SingHealth Centralized Institutional Review Board (CIRB number: 2009/698/D) and a waiver of informed consent was obtained.to determine if the use of early empiric antifungal treatment was associated with lower mortality when compared to culture-directed treatment;to evaluate if early empiric antifungal treatment was associated with shorter ICU stay, shorter time to clinical improvement, and length of hospitalization for survivors.The objectives of the study were as follows:This was a single-centre, prospective observational cohort study conducted at a tertiary hospital between July 2011 and January 2012. All patients admitted to the SICU after surgery within the study period were identified from the electronic hospital records and screened for study eligibility.Patients were included in the study if they (1) were at least 21 years old, (2) underwent recent abdominal surgery for gastrointestinal perforation, malignancy, pancreatitis, intestinal obstruction, severe peritonitis, or liver transplant, and (3) had persistent postsurgical presentation of a systemic inflammatory response syndrome (SIRS) despite receiving broad spectrum antibiotics with intra-abdominal infection as the cause of sepsis, for example, peritonitis, colitis, and abscesses.Patients who (1) were less than 21 years of age, (2) had received any antifungal agents prior to SICU admission, and (3) had existing positive fungal cultures prior to ICU admission were excluded from the study.As this was a noninterventional study, the choice, dose, and duration of antifungal and time to initiation of therapy were decided upon by patients' primary physicians. Depending on whether their antifungal treatment was empirical or culture-directed,patients who fulfilled the inclusion criteria were retrospectively allocated to two separate arms for analysis. Empiric antifungal therapy was defined as the receipt of any systemic antifungal drug, such as intravenous or oral azoles, intravenous echinocandins, or intravenous amphotericin B, prior to the first reporting of results for fungal cultures taken at the onset of sepsis. Culture-directed antifungal therapy was defined as the initiation of antifungal therapy after positive fungal culture results .Patients' sociodemographics, comorbidities, underlying surgical condition, Acute Physiology and Chronic Health Evaluation II (APACHE II) and Sequential Organ Failure Assessment (SOFA) scores, routine laboratory values, microorganism culture results, and pertinent information from the case notes or patients' electronic medical records were systemically recorded on a standardized data collection form. All patients were followed up until death or discharge from hospital, whichever event occurred earlier. An infectious diseases physician blinded to patients' treatment group assessed patients' clinical outcomes to determine infectious-related and fungal-related mortality.Cultures of blood, urine, and intra-abdominal specimens , respiratory tract , and skin and soft tissue (deep wound swabs) were performed whenever clinically indicated. Microorganisms were identified according to standard techniques in our hospital laboratory. Candida was isolated from the blood, intra-abdominal fluid, intra-abdominal wound tissue, or urine specimens, with accompanying signs and symptoms of infection. Isolates of Candida krusei were assumed to be intrinsically resistant to fluconazole. A positive fungal urine culture was deemed significant if there are more than 105 colonies of Candida per milliliter of urine with accompanying urine full examination microscopy elements (UFEME) analysis exhibiting elevated total white blood cells of more than 6 cells/mL, that is, pyuria and epithelial cells of less than 1 cell/mL. Candida isolated from the sputum was not considered significant as Candida pneumonia and lung abscess are very uncommon in nonneutropenic patients [A positive fungal culture was considered significant ifpatients , 24. Candida isolated at any site (except sputum) upon death; or in the absence of positive fungal cultures, as on-going sepsis despite adequate antibiotics coverage for any coexisting bacterial infection and source control.Mortality was evaluated as 30-day overall mortality, which was defined as death due to all causes within 30 days from the start of sepsis. Fungal-related mortality was defined as on-going sepsis withLength of SICU stay and hospitalization were measured from the time of SICU admission till death or discharge. Days to overall clinical improvement were calculated from the day of sepsis onset to the date when the subject showed an improvement in clinical parameters , accompanied with either wound healing  and/or improved gut function .Assuming a mortality rate of 40% in high-risk surgical patients  with del in situ. One patient was still on antifungal treatment at the point of analysis and all clinical outcomes could not be evaluated yet.Out of 233 patients screened during the study period, 52 patients were started on antifungal therapy and included into the study. Of which, only 48 were eventually analyzed for study outcomes. Three patients were lost to follow-up as they defaulted further treatment and opted for hospital discharge whilst on antifungal therapy, total parenteral nutrition, and abdominal drainsThe sociodemographics, comorbidities, and severity of illness were fairly similar in both groups of patients, except that there were more males in the empiric arm. There were more patients with gastrointestinal perforations in the culture-directed group . Candida. Distribution of antifungal agents used and duration of therapy prescribed were fairly similar between both patient groups (P < 0.001).Two-thirds of the patients were given fluconazole (66.67%) and one-third was given echinocandins (31.25%). Amphotericin was used in a previous transplant recipient with blastoconidia in her blood cultures and the primary physician wanted to cover for Cryptococcus and Trichosporon in addition tot groups , while t Candida isolated was unspeciated (42.9%), likely because they were from the sputum or urine, which made up more than one-third of the total number of samples cultured. For speciated Candida, C. albicans was the most common species (26.8%), followed by C. tropicalis (12.5%), and C. glabrata (10.7%). Overall, there were significantly more patients with positive fungal cultures in the culture-directed arm from sterile and nonsterile sites. On the contrary, there was no difference in the proportion of patients with concomitant bacterial infections between both arms (The majority of theoth arms .P = 0.03). Early empiric treatment also reduced fungal-related mortality but it did not significantly reduce length of hospital or ICU stay . This observation was borne out in the analysis of the individual clinical parameters  and patients who underwent surgery for gastrointestinal malignancy  were found to have lower likelihood of 30-day overall mortality.After adjusting for possible confounders using multivariate logistic regression, patients who received early empiric antifungal therapy  via the gastrointestinal tract mucosal barrier; (b) via an intravascular catheter; and (c) from a localized focus of infection [ Candida. This in turn restricts widespread invasive candidiasis and decreases mortality risk. This concept is echoed in the existing body of evidence in the critically ill population which suggests that early antifungal therapy be used for patients with high risk (10% to 15%) for IC [ Candida diseases in nonneutropenic adult patients, in which the initiation of fluconazole or echinocandin for ICU patients who are persistently febrile but without microbiological evidence is marginally supported based on existing evidence [There are three main routes of entry by whichnfection . The ear) for IC . The IDS) for IC . More reevidence . Candida in patients with candidemia was an independent predictor of hospital mortality [In this study, the patient population selected was identified to be at particular risk for IC and most likely to benefit from early empiric antifungal therapy  or fungaortality . Their oortality . The allIt was also noted that patients who started on early empiric antifungal treatment showed faster time to overall clinical improvement. However, there was no difference in the length of ICU stay or hospitalization. This was likely because patients had multiple factors affecting their ICU and hospital discharge such as need for mechanical ventilation or continuous renal replacement therapy.Interestingly, from the regression analysis, patients who underwent abdominal surgery for malignancy were also more likely to survive beyond 30 days. This is biologically plausible as these surgeries were elective in nature and generally clean with little or no contamination of the intra-abdominal space. On the other hand, surgeries due to perforated gut and intestinal obstruction were often associated with gross contamination of the intra-abdominal space with the stomach contents. Patients who underwent recurrent operations for anastomotic leaks were also predisposed to intra-abdominal sepsis as a result of gut space contamination. The extent of gut contamination, as opposed to the reason for abdominal surgery, is probably the crucial factor affecting patients' survival in intra-abdominal sepsis as it indirectly reflects the bacterial and fungal burden in the site of infection. Candida species involved. Zilberberg et al. evaluated the effect on inappropriate antifungal treatment in patients with candidal bloodstream infections and found that treatment delay of more than 24 hours or inadequate antifungal dose was independently associated with hospital mortality [Other than timing of antifungal therapy, the adequacy of antifungal agent used has been shown to have a significant impact on treatment efficacy. Adequacy of antifungal therapy is dependent on the choice, dose of the antifungal agent used, and types and susceptibility patterns of theortality . This obortality , who rep Candida species while azoles are fungistatic. A neutropenic murine invasive candidiasis model found that echinocandins reduce fungal burden in tissues more rapidly than fluconazole [Despite little clinical data demonstrating clear superiority of echinocandins to other antifungal agents in invasive candidiasis, the IDSA guidelines for the management of candidiasis recommended that preference be given to an echinocandin when empirically treating a hemodynamically unstable or moderately-to-severely ill nonneutropenic patient for suspected candidiasis . The ratconazole , thus suAs this study was noninterventional, the choice of antifungal agent prescribed was left to the primary physicians. Although not statistically significant, echinocandins were prescribed more often for empiric treatment while azoles were favored in culture-directed therapy. This reflected the primary physicians' general preference to cover more broadly for azole-non-susceptible species as well as for rapid reduction of fungal load early on in severe sepsis, in line with IDSA's recommendations. Fluconazole was most frequently used for culture-directed therapy as physicians generally felt more comfortable prescribing it based on susceptibility results. Furthermore, patients who were still alive when fungal culture results were back tended to be more stable clinically thus diminishing the need for rapid fungicidal activity. Candida were sufficiently implicated. Furthermore, the heavy fungal burden at the site of infection necessitates the use of fungicidal agents.Based on our study observations, using an echinocandin for empiric therapy may be a better option in patients with intra-abdominal sepsis with suspected IC as non-albicansWe were unable to recruit the target sample size within the study period due to the slow accrual rate in the 10-bedded SICU. Interestingly, we observed that the number of patients who received early empiric treatment increased over the study period. This pattern of use was probably due to increasing physician awareness and application of the empiric treatment approach recommended by the IDSA. As the number of patients who received culture-directed treatment declined, this study could not be feasibly continued.Given the nonrandomized treatment allocation in this study, it was possible that treatment bias may have occurred when patients who received early empiric antifungal therapy were more aggressively treated than patients who were treated according to culture results. This may have confounded the reduced mortality observed in the empiric group, making it seem more significant than it really was.The study outcomes may also be confounded by the difference in the number of positive fungal cultures between both groups. Unfortunately, further stratification by status of fungal cultures could not be reliably performed due to small sample size. The international classification system for fungal infections published by the European Organisation for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group also could not be consistently applied to our study population . Apart fNevertheless, the diagnostic limitations of our study were minimized by ensuring that our patient cohort had the highest likelihood of invasive candidiasis according to the presence of risk factors such as use of total parenteral nutrition and broad-spectrum antibiotics, high APACHE scores, and gastrointestinal tract perforations , 34. PurSecondary outcomes such as clinical improvement also had subjective elements which may have caused data misclassification. This was minimized by blinding the ID physician who evaluated patients' clinical outcomes.Despite the absence of strong evidence, the high mortality rates associated with delayed antifungal treatment supports early initiation of empiric antifungals. In our study, we observed that the patients with intra-abdominal sepsis, in whom empiric antifungal therapy was started earlier, had lower mortality and faster clinical recovery. Large-scale randomized controlled studies are warranted to determine the true effect of this approach on patients' survival. Future studies should also address the optimal duration of empiric antifungal therapy as well as the long-term effects of increased empiric antifungal use as current studies do not address potential emergence of less azole-susceptible strains. Until more data is available, the use of empiric antifungal treatment should be reserved for patients at the highest risk for IC where the benefit outweighs the risk of antifungal use.


Relative abundance profiles of bacterial populations measured by sequencing DNA or RNA of marker genes can widely differ. These differences, made apparent when calculating ribosomal RNA:DNA ratios, have been interpreted as variable activities of bacterial populations. However, inconsistent correlations between ribosomal RNA:DNA ratios and metabolic activity or growth rates have led to a more conservative interpretation of this metric as the cellular protein synthesis potential (PSP). Little is known, particularly in freshwater systems, about how PSP varies for specific taxa across temporal and spatial environmental gradients and how conserved PSP is across bacterial phylogeny. Here, we generated 16S rRNA gene sequencing data using simultaneously extracted DNA and RNA from fractionated (free-living and particulate) water samples taken seasonally along a eutrophic freshwater estuary to oligotrophic pelagic transect in Lake Michigan. In contrast to previous reports, we observed frequent clustering of DNA and RNA data from the same sample. Analysis of the overlap in taxa detected at the RNA and DNA level indicated that microbial dormancy may be more common in the estuary, the particulate fraction, and during the stratified period. Across spatiotemporal gradients, PSP was often conserved at the phylum and class levels. PSPs for specific taxa were more similar across habitats in spring than in summer and fall. This was most notable for PSPs of the same taxa when located in the free-living or particulate fractions, but also when contrasting surface to deep, and estuary to Lake Michigan communities. Our results show that community composition assessed by RNA and DNA measurements are more similar than previously assumed in freshwater systems. However, the similarity between RNA and DNA measurements and taxa-specific PSPs that drive community-level similarities are conditional on spatiotemporal factors. High-throughput sequencing of conserved marker genes, particularly the small subunit ribosomal RNA gene, allows for rapid and affordable censuses of microbial life , a eutrophic drowned river mouth freshwater estuary  and the SILVA release 119 taxonomy as previously described . Mothur output files, classification files and procedure are available at https://github.com/DenefLab/Frontiers2016Denef and the procedure is also included as an R markdown document   to test if season, filter fraction, lake, station, depth, and day/night could significantly explain variation in the bacterial community composition. We also performed a partial Mantel test  to determine whether environmental factors explained additional variation in RNA level Bray-Curtis dissimilarities after DNA-level variation had been taken into account.To determine differences in bacterial community composition, we merged replicate samples by averaging, rounding down, and scaling each OTU read count to the smallest merged library size   as well as for the top 200 most abundant OTUs. To test for significant differences in OTU overlap based on season, sampling location, filter fraction, or depth, we performed a Kruskall-Wallis test  ordination using the pcoa function (vegan). Similarly, we calculated the Bray-Curtis dissimilarity among RNA samples and among DNA samples and visualized these in a PCoA ordination. A Kruskall-Wallis test was performed as described above to compare whether sample dissimilarity in ribosomal RNA:DNA ratios between PA and FL, Muskegon Lake and Lake Michigan, and surface and deep water was a function of season. We performed a PERMANOVA analysis as described above to determine if season, filter fraction, lake, station, and day/night could significantly explain variation in log2 ribosomal RNA:DNA ratios between samples. Finally, we performed a Procrustes analysis (procrustes and protest functions in vegan) to determine whether the ordination patterns were significantly different between DNA, RNA, and RNA:DNA ratio data.We calculated the Euclidean distance between samples based on the logP > 0.10), but did have significant differences in ribosomal RNA:DNA ratio between summer and fall Lake Michigan PA and FL fractions .To identify the taxa that were drivers of community-level similarities in ribosomal RNA:DNA ratios between FL and PA fraction in spring, and differences in summer and fall, we identified OTUs that did not have differences in ribosomal RNA:DNA ratio between spring Lake Michigan PA and FL fractions . However, the correlation was weaker relative to the correlation between the RNA level Bray-Curtis dissimilarity and the same environmental factors without accounting for the patterns already observed at the DNA level . When repeating the latter analysis one environmental factor at a time, only “Season” and “Lake” resulted in significant partial Mantel test statistics (p < 0.05).Groups of samples clustered primarily by filter fraction [free-living (FL) vs. particulate (PA)], time, and station [particularly Lake Michigan (M15 and M110) vs. Muskegon Lake estuary (MLB) samples; Figure We did not observe a difference in the proportion of OTUs that were identified by both DNA and RNA assays between seasons, filter fractions, stations, or depths Figure . HoweverActinobacteria and to a lesser extent particulate fraction Planctomycetes contributed significantly less to the community RNA pool than to the DNA pool , free-living Betaproteobacteria, and most Cyanobacteria tended to contribute more to the community RNA pool than to the DNA pool. Particulate fraction Betaproteobacteria, and several Bacteroidetes classes showed either limited differential representation between observed RNA and DNA community composition, or displayed a mixed signal . The most over- and/or underrepresented OTUs in both fractions were listed in Table Among abundantly represented phyla, l Figure . Alphaprl Figure . As Actip = 0.001). Most noticeably, while the RNA and DNA spring samples clustered separately from the summer and fall samples and PA and FL communities formed separate clusters, both PA and FL spring RNA:DNA ratio data clustered together with FL Lake Michigan summer and fall communities. In an alternate approach to analyze these patterns, we observed that the distance between community-level RNA:DNA ratios in PA relative to FL fractions, in Lake Michigan relative to Muskegon Lake, and in surface relative to deep water was generally lower in spring relative to summer and fall . In spring, we observed higher between-habitat similarities in RNA:DNA ratios than in summer and fall. Finally, we contribute to the expanding work on analyzing the phylogenetic conservation of traits by revealing how PSP, as measured by ribosomal RNA:DNA ratios, is conserved for some phyla, most likely as a function of cell size.Our observation that sample community composition varies more across spatiotemporal gradients than between RNA and DNA measurements opposes previous analyses by Jones and Lennon . They shIn Muskegon Lake there was less overlap between OTUs observed in the RNA and DNA measurements than between replicate DNA or replicate RNA samples. This result may be explained by higher bacterial richness in urban estuaries due to urban runoff and influx of riverine and soil bacteria . This allowed us to evaluate the impact of stochastic effects associated with sampling and sequencing on OTU overlap. Subsequently, we contrasted DNA and RNA OTU overlap to replicate sample overlap. This allowed us to identify significant impacts of habitat on DNA and RNA sample OTU overlap that we could not have made without the availability of replicate measurements. We also reduced the impacts of stochasticity by averaging OTU counts from replicates and scaling the read depth to the smallest merged library size to remove the impact of many rare and spurious OTUs . Similarly, RNA:DNA ratios in spring for the offshore FL samples resembles all other spring RNA:DNA ratios, despite the higher similarity of DNA and RNA community composition to lake Michigan samples taken at other times of the year Figures , 5. The While we cannot confidently infer bacterial contributions to ecosystem processes from ribosomal RNA:DNA ratios, our analyses identified several intriguing patterns that deserve follow-up. In particular, the seasonal changes in habitat-dependent ribosomal RNA:DNA ratios pose the question: to what extent are bacterially mediated ecosystem processes more similar across all habitats in spring? This is particularly pertinent for communities occupying the particulate and free-living fractions of freshwater lakes, as many studies in recent years have described large differences in community composition across these habitats. Future studies will need to extend our findings beyond the marker gene level, either through activity assays or by characterization of the transcriptome of the populations driving the seasonal shifts in ribosomal RNA:DNA ratios observed here.VD designed the study, analyzed data, and was the primary author of the manuscript; MF analyzed data and contributed to the writing of the paper; MB and MS provided new analytical tools, analyzed data, and contributed to the writing of the manuscript.VD was supported by the Community Sequencing Program  and funds from the Fred A. and Barbara M. Erb Family Foundation and the University of Michigan Water Center. MS was supported by the National Science Foundation Graduate Research Fellowship (GRFP) program.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Rapid biological changes are expected to occur on tropical elevational gradients as species migrate upslope or go extinct in the face of global warming. We established a series of 9 1-ha plots in old-growth tropical rainforest in Costa Rica along a 2700 m relief elevational gradient to carry out long-term monitoring of tropical rain forest structure, dynamics and tree growth. Within each plot we mapped, identified, and annually measured diameter for all woody individuals with stem diameters >10 cm for periods of 3-10 years. Wood species diversity peaked at 400-600 m and decreased substantially at higher elevations. Basal area and stem number varied by less than two-fold, with the exception of the 2800 m cloud forest summit, where basal area and stem number were approximately double that of lower sites. Canopy gaps extending to the forest floor accounted for <3% of microsites at all elevations. Height of highest crowns and the coefficient of variation of crown height both decreased with increasing elevation. Rates of turnover of individuals and of stand basal area decreased with elevation, but rates of diameter growth and stand basal area showed no simple relation to elevation. We discuss issues encountered in the design and implementation of this network of plots, including biased sampling, missing key meteorological and biomass data, and strategies for improving species-level research. Taking full advantage of the major research potential of tropical forest elevational transects will require sustaining and extending ground based studies, incorporation of new remotely-sensed data and data-acquisition platforms, and new funding models to support decadal research on these rapidly-changing systems. A notable feature of the continental tropics is the very flat gradient in temperature decrease with increasing latitude . In contIn this paper we describe the establishment of a network of forest inventory plots designed to study the long-term performance of tropical forest vegetation on a steep elevational transect. Forest structure and to a less extent dynamics along elevational transects have been described in several areas around the tropics  old-growth forest, although there are significant areas of secondary forest resulting from natural disturbances such as landslides and earthquakes and from previous deforestation at some elevations. Previous work on the geology, soils, and soil litter chemistry is reviewed in , , , 11], , , 13], , , 15]. I. I15]. IFrom this perspective, we do not expect generality in comparing tropical elevational transects. Over any significant elevational range temperature will always decrease. How that decrease affects forest structure and function however will vary idiosyncratically among elevational transects depending on the factors mentioned above. Tree diameter growth rates in particular, and hence stand-level dynamics in old-growth forest, will be strongly determined by precipitation and insolation. A transect that spans relatively hot wet lowland sites to cool dry montane sites will likely be very different from a transect from hot dry lowland sites to cool moist montane sites. And a transect such as the Volcán Barva transect, which is humid to perhumid at every site with moderate temperatures, would be yet again different, even without considering conditions of disturbance, edaphic factors and human influences.The most interesting questions for ecosystem science on any given transect are therefore "What are the relative roles of climate, soil, disturbance, and human influence on the physical and biological structure of forests on this transect?" Resolving some of these issues, for example the relative roles of different nutrients in limiting productivity, can only be addressed through experimental manipulations . DisturbAt this point in our research we can summarize the Volcán Barva Transect (VBT) in relation to other tropical elevational transects as follows:The VBT is completely humid to perhumid; lack of water is not a major restriction on tree growth, although excess water and associated cloudiness likely is.≥10 cm diameter at all elevations.Species diversity is moderate by continental tropical rain forests standards , and folForest physical structure is characterized by (a) a low frequency at all elevations of canopy gaps extending to the forest floor ("Brokaw gaps" ), (b) taStem number and plot basal area, as well as rates of diameter growth, mortality and recruitment are generally intermediate by tropical rain forest standards .Forest above-ground biomass residence time increases substantially with elevation.The sections of the landscape selected for study cf  were notForest soils are well developed and extensive at every site, and non-soil microsites such as rock outcrops account for minimal area.In summary, the VBT plot network is characterized by a large elevational gradient with closed-canopy forest at all elevations, rugged terrain, high rainfall and forest soil coverage, intermediate levels of species diversity, dynamics and tree diameter growth rates, and minimal direct impacts of human intervention. In the following section we address how this description applies to the larger Volcán Barva landscape outside the plots, as well as a variety of issues related to elevational transect studies using permanent forest inventory plots.As we designed and implemented the network of forestry inventory plots along this tropical elevational transect we faced a number of theoretical and practical issues. While similar research efforts on other tropical elevational transects Click here for additional data file.S1 Dataset(TXT)Click here for additional data file.S2 Dataset(TXT)Click here for additional data file.


Streptomyces, has found application in human therapy, in the food and beverage industries and as pesticide. It displays a broad spectrum of activity, targeting ergosterol but bearing a particular mode of action different to other polyene macrolides. The biosynthesis of this only antifungal agent with a GRAS status has been thoroughly studied, which has permitted the manipulation of producers to engineer the biosynthetic gene clusters in order to generate several analogues. Regulation of its production has been largely unveiled, constituting a model for other polyenes and setting the leads for optimizing the production of these valuable compounds. This review describes and discusses the molecular genetics, uses, mode of action, analogue generation, regulation and strategies for increasing pimaricin production yields.Pimaricin (natamycin) is a small polyene macrolide antibiotic used worldwide. This efficient antimycotic and antiprotozoal agent, produced by several soil bacterial species of the genus  Streptomyces, a class of filamentous soil-dwelling bacteria that undergo a complex life cycle involving differentiation and sporulation. It belongs to the polyene class of macrolide polyketides, and displays a strong and broad spectrum mould inhibition activity, yet being safe and effective at very low concentrations. Because its molecular target is ergosterol, an essential constituent of fungal membranes, and bacteria do not contain sterols in their membranes, it is inactive against bacteria. For the same reason, it is extremely reluctant to microbial resistance since the only way fungi would have to evade its action would be to change sterols from their membranes. For these reasons, and because of its low toxicity to mammalian cells, this molecule has become one of the major mould inhibitors used in the food industry. Its use was approved in 1967 as a cheese preservative, and since then, it has been extended to a wide variety of foods and beverages. In addition, PIM has been regarded as the most important agent in the management of fungal keratitis, a leading cause of blindness in corneal diseases, which is relatively common in warm climates and developing countries. Furthermore, it is also used as a crop protection agent to prevent mould contamination. Besides its antifungal action, it is also active against protozoa having ergosterol in their membranes.Pimaricin (PIM), also called natamycin, tennecetin, natacyn and E235, is a natural product produced by given members of the genus Useful reviews about polyene macrolide biosynthesis and analogue generation are available in the literature , where the first producing strain, Its structural core is a 26-membered macrolactone ring with four conjugated double bonds (chromophore) Fig. . Its tetBecause of its amphiphilic nature, it is poorly soluble in water and almost insoluble in non-polar solvents. As a powder, it is stable in the dark, with no loss of activity, but it is light sensitive in aqueous suspensions. Its CAS number is 7681-93-8.PIM has a strong antifungal activity on most fungi . For many years, it was thought that its mode of action on fungal membranes was to act as a permeabilising agent, as occurs with other polyenes Bolard . HoweverPenicillium discolor without causing extensive cell damage (i.e. without membrane permeabilisation), and Breukink and his group have also found that PIM impairs vacuole fusion via perturbation of ergosterol-dependent priming reactions that precede membrane fusion  or whey protein cheese (e.g. ricotta), also in dried uncooked meats and in iberico and prosciutto hams. PIM can also be used to extend the shelf life of different fruit and vegetable preparations, salad mixes, baking products, sauces, fish, poultry, etc. Fig. . RecentlBesides its direct application on foods, PIM has been successfully incorporated into different packaging films/coatings   mass-rearing facilities has been reported   from 12 acetate and one propionate units has been thoroughly studied  harbouring 13 homologous sets of enzyme activities (modules) and a total of 60 catalytic domains . This discrete thioesterase plays an important editing role in the selection of starter acyl units and in the removal of aberrant extender units during PIM biosynthesis  Fig. . The fidcin Fig.  . Finally, the glycosyltransferase pimK catalyses the attachment of mycosamine to the pimaricinolide -mannose derived from fructose-6-phosphate  transporter proteins, which combine both ATPase and transmembrane domains within the same polypeptide   proposed to be responsible for the formation of the carboxyl exocyclic group  acts before the glycosylation step in the PIM biosynthetic pathway  DNA-binding domain with a C-terminal half homologous to guanylate cyclases and large ATP-binding regulators of the LuxR family (LAL) , DNaseI protection studies, reverse transcription quantitative PCR (RT-qPCR) and gene promoter replacement experiments and determined that it binds a single operator that contains three heptameric direct repeats of the consensus CGGCAAG with 4-bp spacers  is exactly conserved in the intergenic region between scnRII and scnRI in the scn gene cluster of S. chattanoogensis  and polyoxins (polR)  , nysRIV (nystatin) or pteF (filipin) into the strain . These binding sites were located inside or upstream from genes involved in different aspects of both primary and secondary metabolism, including genetic information processing, DNA replication and repair, energy metabolism, carbohydrate metabolism, lipid metabolism, morphological differentiation, transcriptional regulation and secondary metabolite biosynthesis, amongst others  takes place following phosphate depletion. The phoU-phoR-phoP region of S. natalensis has been characterised  , although in this case indirectly  that can damage cell components. The redox-based regulation network triggered by an imbalance of the intracellular ROS homeostasis, in particular intracellular HStreptomyces sp., both morphological differentiation and secondary metabolite biosynthesis are controlled by quorum-sensing signals that act at nanomolar concentrations.Quorum sensing is a communication mechanism that allows bacteria to detect a high density of population and react by different mechanisms of adaptation. Growing cells produce extracellular signals  that are detected by the remaining cells of the culture, which in turn respond to this stimulus by the transcription or repression of given genes. In Streptomyces use γ-butyrolactones  (GBL) as autoinducers     -1,4-butanediol)  also triggers PIM production in the mutants  Fig.  (Recio eol) Fig. . PI factS. natalensis although at higher concentrations than PI factor , S. rimosus (rimocidin), S. griseus (candicidin), S. filipinensis (filipin), S. albulus (tetrafungin), S. eurocidicus (eurocidin) and S. cinnamoneum (fungichromin). Although the exact mechanism remains unknown, the action of glycerol seems to be independent of PI factor-inducing effect  as a carbon and energy source for growth of different bacteria but had no obvious role in antifungal production  into the chromosome of a given polyene producer boosts the production of the corresponding polyene , have also been described with improved results   will certainly constitute a very valuable tool in the efforts to improve PIM production.Thanks to the advances in the genetic manipulation of PIM producers and the considerable progress in the knowledge about the regulation of PIM production; several PIM derivatives with improved properties have been developed, and many of the bottlenecks hampering PIM production at high titres have been overcome. The future application of this knowledge at the industrial scale will hopefully permit to satisfy the growing PIM commercial demands. In this sense, the present availability of the genome sequences of PIM producers such as


Plant diseases are among the major factors limiting crop productivity. A first step towards managing a plant disease under greenhouse and field conditions is to correctly identify the pathogen. Current technologies, such as quantitative polymerase chain reaction (Q-PCR), require a relatively large amount of target tissue and rely on multiple assays to accurately identify distinct plant pathogens. The common disadvantage of the traditional diagnostic methods is that they are time consuming and lack high sensitivity. Consequently, developing low-cost methods to improve the accuracy and rapidity of plant pathogens diagnosis is needed. Nanotechnology, nano particles and quantum dots (QDs) have emerged as essential tools for fast detection of a particular biological marker with extreme accuracy. Biosensor, QDs, nanostructured platforms, nanoimaging and nanopore DNA sequencing tools have the potential to raise sensitivity, specificity and speed of the pathogen detection, facilitate high-throughput analysis, and to be used for high-quality monitoring and crop protection. Furthermore, nanodiagnostic kit equipment can easily and quickly detect potential serious plant pathogens, allowing experts to help farmers in the prevention of epidemic diseases. The current review deals with the application of nanotechnology for quicker, more cost-effective and precise diagnostic procedures of plant diseases. Such an accurate technology may help to design a proper integrated disease management system which may modify crop environments to adversely affect crop pathogens. AuMNPs:magnetic gold nanoparticlesCdS:cadmium sulphideCdTe:cadmium tellurideDON:deoxynivalenolELISA:enzyme-linked immunosorbent assayFD:flavescence doréeFSNP:fluorescent silica nanoparticlesLAMP:loop-mediated isothermal amplificationMRLs:maximum residue limitsNGS:next-generation sequenceOTA:ochratoxin AQD:quantum dotQPCR:quantitative polymerase chain reactionRPA:polymerase Amplification reactionsSAED:selected-area electron diffractionTEM:transmission electron microscopyZEA:zearalenoneSpread of plant diseases has internationally increased, while pathogen identification and control costs are still limited . WorldwidTraditional molecular diagnostic methods are widely used in laboratories all over the world to identify plant pathogenic organisms with high degree of sensitivity and specificity. Still, most of these procedures cannot be applied in the field (on-site detection) or in developing countries where incomes are small. Furthermore, the high price and short shelf half-life of some molecular biology reagents, such as enzymes and primers, limit the application of traditional molecular methods in developing countries. Nanotechnology may have actual solutions against many agriculture problems like plant disease control. Nano-based materials will be presented which will increase the efficacy of fungicides and pesticides, allowing only minor doses to be used. MoreoverThe extension/integration of molecular diagnostics on a nanoscale is a promising technology for identifying pathogens. Nanomolecular diagnostic is the use of nanobiotechnology to diagnose plant diseases and this can be termed as nanodiagnostics. In partiIt is noteworthy that new detection techniques involving nano-biosensors for pathogen identification will likely be a cornerstone to this trend. During tNewly developed nanomaterials with special nanoscale characteristics could present a tremendous breakthrough in pathogen and contaminant detection. Nanotechnology is also driving the development of lab-on-a-chip systems for detecting toxicity in waters, observing nutrients in irrigation water and controlling the quality in food production.,9Nano-phytopathology is a cutting-edge science which uses nanotechnology for detecting, diagnosing and controlling plant disease and their pathogens at an early stage, owing to crop protection from epidemic diseases. The modern plant pathologist strives to apply his knowledge in nanophytopathology to enable understanding of controlling factors of plant diseases and to develop and/or evaluate eco-friendly diagnostic measurements. Modern nanomolecular techniques are used for monitoring or understanding of pathogen population genetics, plant-microbe interactions and gene transfer between pathogens and even the host. Furthermore, nanoparticles such as nanosized silica-silver have recently been applied as antimicrobial and antifungal agents. Additionally, nanomaterials can be used for mycotoxin detection and detoxication, increasing plant resistance, plant disease forecasting and nano-molecular diagnostics of plant pathogens. Potential applications of nanophytopathology are shown in The present review discusses the various applications of nanotechnology in plant pathogen detection.The capability to produce a fast PCR amplicon in a completely moveable set-up will not only allow plant pathologists to achieve more effective and predictable laboratory trials, but also considerably simplify other downstream applications such as those requiring in situ genomic tools for detection.Palm PCR, developed by Ahram Biosystems Company in Korea, is a manageable thermal cycler designed for easy use, accuracy and high efficiency. Despite its tiny size, this controlling device delivers extremely well-organized and quick amplification for different types of DNA samples including the plant genome. The DNA Compared to other diagnostic testing systems, Twista quantitative and portable real-time fluorometer is a customized device developed for examining recombinase polymerase amplification (RPA) reactions, and represents a new revolution in DNA diagnostics, as well as, combining advantage in speed, portability and ease of use with good sensitivity and specificity. Twista RPA fluorometer will supply immediate fast diagnosis, allowing an on-time suitable treatment compared with traditional microbiological assays, which require at least hours and molecular assays that typically need centralized equipment.Many companies are exploring the idea of using nanopore technology. There arPlant pathologists plan to study this ‘lab in a box’ very soon. This refers to packing sophisticated measuring devices, reagents, power supply and other features that now take up laboratory space into a parcel no larger or heavier than a briefcase.A briefcase-sized kit is transferred to a field where crops are growing to search for pathogens that could infect and reduce the yield. This is a quick and precise procedure. Nanodiagnostic kit equipment can easily and quickly detect potential serious plant pathogens, allowing experts to help farmers in prevention of disease epidemics from breaking out. For examGeobacillus stearothermophilus for DNA amplification. There are several detection devices varying from gold nanoparticles tagged with short fragments of DNA to multicolour optical coding for biological tests that have been achieved by embedding different-sized QDs into polymeric microbeads.[Loop-mediated isothermal amplification (LAMP) of DNA is a simple, cost effective, and rapid method for the specific on-site detection method of genomic DNA using a set of six oligonucleotide primers with eight binding sites hybridizing specifically to different regions of a target gene, and a thermophilic DNA polymerase from crobeads. The thiocrobeads. LAMP hasG. stearothermophilus for DNA amplification of Fusarium graminearum.[For instance, LAMP-PCR of DNA is an easy, commercial and quick method for the specific detection of genomic DNA using a set of six oligo primers with eight binding sites hybridizing, specifically to various regions of a target gene , and a tminearum. ThereforF. graminearum isolates, while all reactions with other Fusarium and fungal species included for specificity remained negative, with a response identical to the negative control. This methodology is used to detect plant pathogenic fungi to rhizospheric microorganisms such as Phytophthora sojae,[P. ramorum and P. kernoviae,[Aspergillus flavus and A. parasiticus,[Fusarium.[Positive results were obtained with ra sojae, P. ramorernoviae, Aspergilasiticus, and toxiFusarium.,26,27Utilization of nanomaterials or nanoparticles in biosensors allows the development and use of some novel signal detection procedures and equipment. Different strategies such as antibody–antigen, adhesion–receptor, antibiotic and complementary DNA sequence recognitions have been discovered for a specific detection between target phytopathogenic cells and bio-functionalized nanomaterials.,28Gold nanoparticles are excellent markers to be used in biosensors as several optical or electrochemical procedures can be modified to identify pathogens. A number of nanoparticle-based experiments have been performed to develop biomolecular detection with DNA- or protein-functionalized gold nanoparticles, which are used as the target-specific probes.,29 SeverF. oxysporum when incubated with a mixture of CdCl2 and SeCl4 at room temperature.[F. oxysporum isolates when reacted with a mixture of CdCl2 and TeCl2 at ambient conditions.[QDs are semiconductor nanoparticles that fluoresce when stimulated by an excitation light source. Furthermore, QDs are inorganic fluorophores presenting major advantages over traditional organic fluorophores used as markers on nucleic acids or proteins for visual detection.,36 The mperature. A proficnditions.An effective biosynthesis method to prepare easily harvested biocompatible cadmium telluride (CdTe) QDs with tunable fluorescence emission using yeast cells was developed. The confBiocompatible semiconductor crystals are composed of a nucleus and a shell allowing the binding of ligands and thus the attachment of this fluorescent marker to the target pathogen. A hybridization-based biosensor known as the quartz-crystal microbalance biosensor is a good example. The quartz-crystal microbalance vibrates under electric stimulus, and a change in mass due to the attachment of any compound to its surface can be detected through a reduction in the frequency of its vibration. When a nucleic acid probe is attached to the surface of a quartz-crystal microbalance biosensor and then exposed to complementary PCR product, a hybrid forms and causes the resonance frequency of the quartz-crystal microbalance biosensor to decrease dramatically. This system can be combined with fast PCR protocols to decrease the time for specific environmental detection of plant pathogens.The bio-barcode assay is an ultrasensitive method of amplification and detection of proteins or nucleic acids. DNA bio-barcoded tests employ oligonucleotide-modified magnetic gold nanoparticles (AuMNPs) for signal amplification and for simple separation of a target protein from the sample. The large b-DNA-to-recognition agent ratio affords a means of substantial signal amplification. It is also promising by allowing the quick detection of numerous protein targets at low-attomolar concentrations  and nuclNanostructured platform for mycotoxin detection and detoxification mycotoxins are secondary metabolites produced by pathogenic filamentous fungi causing disease and death in humans and other animals. Mycotoxicoses are diseases caused by the ingestion of mycotoxins by humans and animals, mostly through the consumption of polluted food. They can induce various different biological effects, i.e. carcinogenic, mutagenic, teratogenic, estrogenic, immunotoxic, nephratoxic and neurotoxic effects.A variety of biosensors have been developed and described in the recent literature for mycotoxin analysis some of which have the potential of multi-array mycotoxins identification. Prieto-Simon et al.  reviewedNanosensors could be placed directly into the packaging material, where they would serve as ‘electronic noses’ which can detect chemicals released during food spoilage. Some nanosensors are based on microfluidic devices, and theyAn immunosensor technique coupled with a flow injection system can be used for the quick, sensitive and discriminatory quantification of zearalenol in corn silage samples. HorseradPlant disease forecasting is a management method used to forecast the chances or severity of plant diseases and to help farmers make cost-effective decisions for controlling diseases. PresentlNano-sensors can be linked to a GPS for real-time monitoring of disease and distributed throughout the field to monitor soil conditions and crop health.,13 The uEscherichia coli using a flow-injection system. Electrochemical measurement of K3Fe (CN)6, reduced by microbial metabolism, allowed the quantitative determination of bacteria and fungi in 20 minutes. Hashimoto et al. [The nanobiosensor is the product of a combined approach of biology and nanotechnology. These seo et al.  developeXanthomonas axonopodis pv. vesicatoria which causes bacterial spot disease in tomatoes and peppers.[A. niger fungi.[Taking into consideration the particular optical properties of silver nanoparticles, the interaction between silver nanoparticles and sulphurazon-ethyl herbicide was investigated. They fou peppers. Copper oer fungi.Diagnostic imaging refers to a broad slew of technologies used to look inside or outside plant tissues in order to diagnose various plant pathogens. The rapidly expanding use of diagnostic imaging technologies continues to push the boundaries of plant pathologist diagnostic capabilities. Using these techniques, plant doctors are able to diagnose crop diseases earlier and more precisely. NanotechColletotrichum graminicola crawling across the nanofabricated surface assisted the researchers to determine that the fungus needs to make a minimum contact of at least 4.5 μm before it starts to develop appressoria (For example, electron beam and photolithography techniques were used to fabricate topographies that mimic leaf surface features as well as the internal plumbing of plants, and then nano-imaging technologies were used to study how bacteria and fungi invade and colonize the leaf. Lithograressoria . To deveThe application of carbon-coated magnetic nanoparticles and microscopy methods at different levels of resolution to visualize and path the transport and deposition of nanoparticles inside the plant host was reported by González-Melendi et al. .The portable diagnostic equipment, nanoparticle-based, bio-barcoded DNA sensor, and the QD have potential applications in the multiple detection of plant pathogens and toxigenic fungi. To date, mobile diagnostic assays have been developed to rapidly detect plant disease and may be used to prevent epidemics. These nano-based diagnostic kits not only increase the speed of pathogen detection but also increase the accuracy of the diagnosis. Additionally, the combination of nanotechnology with microfluidic systems has been effectively applied in molecular plant pathology and can be adapted to detect specific pathogens and toxins. A good example is the micro-PCR where 40 cycles of PCR can be performed in less than 6 minutes. In the near future, nano-scale devices with novel properties could be used to make smart agricultural systems. For instance, these nanodevices could be used to identify plant health issues before these become observable to the grower. Such devices may be capable of responding to special situations, identifying the problem and taking an appropriate disease management action. In this way, nanosmart devices will act as both a protective and an early warning system. During the next decade, nanodevices, which can make thousands of measurements speedily and very cheaply, will become available. Future prospects in plant disease diagnostic will continue in miniaturization of biochip technology to the nanoscale range. Nanophytopathology can be applied as a tool to understand plant–pathogen interactions, providing new methods for crop protection. Specific nanodevices and DNA nanodevices could enable accurate tracking, detection and diagnosis of plant pathogens in the early stages of plant disease.Quick response within integrated disease management system via external and implanted nanosensor systems.Improvement of rapid laboratory biosensors to detect plant pathogens in the field or post-harvest.Development of retrieval nanosystems for a specific sampling from soil, air and plant samples.Provide rapid and reliable NANO methods for detection of mycotoxins and toxigenic fungi.Detection of pesticide residues in food and feeds.


Contemporary spatial modelling tools can help examine how environmental exposures such as climate and land use together with socio-economic factors sustain infectious disease transmission in humans. Spatial methods can account for interactions across global and local scales, geographic clustering and continuity of the exposure surface, key characteristics of many environmental influences. Using cryptosporidiosis as an example, this review illustrates how, in resource rich settings, spatial tools have been used to inform targeted intervention strategies and forecast future disease risk with scenarios of environmental change. When used in conjunction with molecular studies, they have helped determine location-specific infection sources and environmental transmission pathways. There is considerable scope for such methods to be used to identify data/infrastructure gaps and establish a baseline of disease burden in resource-limited settings. Spatial methods can help integrate public health and environmental science by identifying the linkages between the physical and socio-economic environment and health outcomes. Understanding the environmental and social context for disease spread is important for assessing the public health implications of projected environmental change. Global environmental changes, especially climate change and human exploitation of productive ecosystems have become increasingly important drivers of infectious disease spread ,2. For hRegional variations in disease risk may be due to area-level characteristics as well as the characteristics of the individuals who live in these areas. Spatial approaches are useful for analyzing the associations between area-level environmental exposures, such as climate and proximity to livestock, and individual-level risk factors, such as age, susceptibility and disease risk. Studies that focus on individual risk factors only provide a snapshot of specific infection reservoirs and potential exposures. Further, they are not always financially viable. The increasing availability of large scale, environmental, socio-economic and demographic data sources and freely available software makes an integrated approach using spatial methods possible .Spatial models have become an important tool to examine scale-specific influences. While changes in climate and livestock production practices can drive disease emergence at a global scale, socio-economic factors are more likely to influence disease spread locally. Spatial models explicitly take into account such hierarchical structure in the data. Such models have frequently been applied to model the distribution of vector-borne diseases such as, dengue and malaria ,6, but avs. remote) may also to be characterized by higher exposures to environmental risk factors. Examining disease patterns after modelling out the clustering due to location  can help to identify which factors underlie the observed disease patterns. Moran’s I and the inversely related Geary’s C [Geographical location can influence the risk of infection and confound the association between disease risk and environmental factors. Areas characterized by some populations  and by some location characteristics . In such instances, analyses that do not account for spatial dependence in exposures may lead to overstating the significance of the results. Non-spatial methods applied to such data typically give smaller standard errors for point estimates, compared to spatial models [Environmental exposures such as climate and land use are spatially dependent  cases were associated with higher population density, while high ruminant density and rural living were associated with increased risk in C. parvum (livestock strain) cases [Cryptosporidium strains have been reported from England and Wales [Infection is common in developed countries, where children <5 years old bear the greatest burden of cryptosporidiosis ,41,42. Pn) cases . Similarnd Wales  and New nd Wales ,51. In and Wales ,53,54 anClimate change is likely to influence cryptosporidiosis incidence patterns, as the parasite is easily transmitted through the environment, particularly through water. Historical time-series modelling of cryptosporidiosis incidence with rainfall have reported positive associations in a global meta-analysis of cryptosporidiosis seasonality . High raSocio-economic disadvantage may amplify disease risk in some populations. For example, cryptosporidiosis incidence in Brisbane, Australia was positively associated with the proportion of residents with low education attainment, after correcting for spatial dependency . A recenCryptosporidium spp. was significantly more prevalent in malnourished children 2–5 years old with diarrhoea compared to non-malnourished children [Cryptosporidium infections at the start of the rainy season [In low income countries, cryptosporidiosis is associated with an increased risk of death in children between 1–2 years of age, as estimated by the recent Global Enteric Multi-Centre Study . A recenchildren . Despitechildren  and no schildren . As has children  to produy season  has beeny season . In Indiy season  and the y season  show thaWell-designed spatial statistical models can aid our understanding of how wider socio-environmental interactions may mediate infectious disease spread and account for disparities in disease risk. A significant opportunity exists in using open access spatial environmental and socio-economic data to examine these relationships in resource poor settings, where disease burden is disproportionately high. Knowledge gained could strengthen global public health surveillance and help develop interventions for disease prevention, with future environmental change.