Background: In kidney transplant (KT) recipients, CMV infection poses significant morbidity and mortality. Both prophylactic and pre-emptive approaches for preventing CMV infection have been utilized. Objective: To compare the effectiveness of routine prophylaxis vs. pre-emptive treatment for preventing CMV disease after KT.Methods: We conducted a systematic review and meta-analysis comparing the effectiveness of routine prophylaxis vs. pre-emptive treatment for preventing CMV disease after KT. Combining 4 comprehensive search terms ; we searched PubMed, EMBASE, ISI Web of Science, and Cochrane Central Register from inception through January 2011. We also evaluated studies referenced in review articles and abstracts from meetings of major nephrology and transplant societies (2009–2011). Two authors independently extracted data and assessed methodological criteria. The primary outcome was the pooled estimate of the odds ratio (OR) of developing CMV infection. Secondary outcomes included OR of acute rejection, OR of graft loss and OR of death within first year of KT. Comprehensive Meta-analysis V2 software was used for data analysis. Results: Analysis of 9 randomized controlled trials  with CMV infection as an outcome revealed the OR of CMV infection to be 0.34  for the prophylactic vs. the pre-emptive groups. The OR of acute rejection  was 0.52  with prophylactic approach compared to pre-emptive treatment; graft loss  were similar between the two groups.Conclusions: Prophylactic approach is superior to pre-emptive approach in preventing CMV infection within the first year of kidney transplant. The risk of developing acute rejection is also lower with prophylactic approach in the first year of transplant but there is no significant difference in graft loss or mortality with either approach. CMV infection has also been associated with increased risk of acute graft rejection , reported on acute rejection within the first year (1358 patients) . The funp=0.008) . Analysip=0.001) . OR of gWe found that prophylactic approach is superior to pre-emptive treatment in preventing CMV infection within the first year of kidney transplant. We also found that the risk of developing acute graft rejection with universal prophylaxis is lower, but there is no significant difference in the risk of graft loss or mortality in the first year with either approach. With prophylactic approach, there is always a potential concern for development of late-onset CMV disease (occurring >3 months) since the duration of prophylaxis is typically not more than three months. Although mixed results exist, double blind RCTs favor six months prophylaxis for preventing late-onset CMV infection . In our Pre-emptive therapy does have advantages of its own. This approach requires frequent monitoring of CMV replication (by checking CMV DNA PCR in blood), and CMV infection is less likely to be missed or go untreated if asymptomatic viral replication is detected early. However, the exact cut-off of elevated CMV PCR for initiating treatment with anti-viral drugs has not been well established. Different transplant centers use variable cut-offs ranging from 1000 to 2000 copies of CMV PCR. This can lead to overtreatment in patients (if cut-off used is too low) who may otherwise never have developed symptomatic CMV disease. As a result, they may be exposed to unnecessary side-effects of such anti-viral agents and increased cost associated with the therapy. On the other hand, this can also lead to under-treatment (if cut-off used is too high) from failure to detect asymptomatic viremia that may eventually progress to develop symptomatic disease. Asymptomatic CMV viremia has been shown to reduce graft survival and increase mortality (hazard ratio of 2.9) .Using prophylactic approach has some limitations too. The risk of developing symptomatic CMV disease in high-risk groups with D+/R serology without anti-viral prophylaxis is about 60% . TherefoOur review has the following limitations: 1) we did not perform a complete cost-effectiveness analysis between the two approaches; 2) we did not compare the risk of CMV infection in populations that received lymphocyte-depleting agents as opposed to other induction agents (as exposure to lymphocyte-depleting agents is considered to be a known risk factor for development of CMV disease); 3) the outcome data is only for one year; and 4) publication bias, a limitation inherent to most meta-analyses, since negative studies are less likely to be reported. We concluded that prophylaxis is superior to the pre-emptive approach in preventing CMV infection within the first year of kidney transplant and that the risk of developing acute rejection is also lower with prophylaxis than the pre-emptive approach within the first year. Although prophylactic approach favored less risk of acute rejection, there was no difference in graft loss or mortality in one year. However, acute rejection can lead to graft loss earlier than in patients with no rejection. Long-term follow-up data are warranted to evaluate the risk of graft loss and mortality with both prophylactic and pre-emptive approaches. While the two approaches might result in similar costs, since a major driver of the cost in the pre-emptive approach is the cost of CMV PCR testing, if individual transplant centers have the capability of performing the test, the pre-emptive approach might prove to be more cost-effective. We found no significant difference in the risk of graft loss or death in the first year of kidney tranplant with either approach.


Here we present a novel neurofeedback subsystem for the presentation of motivationally relevant visual feedback during the self-regulation of functional brain activation. Our “motivational neurofeedback” approach uses functional magnetic resonance imaging (fMRI) signals elicited by visual cues (pictures) and related to motivational processes such as craving or hunger. The visual feedback subsystem provides simultaneous feedback through these images as their size corresponds to the magnitude of fMRI signal change from a target brain area. During self-regulation of cue-evoked brain responses, decreases and increases in picture size thus provide real motivational consequences in terms of cue approach vs. cue avoidance, which increases face validity of the approach in applied settings. Further, the outlined approach comprises of neurofeedback (regulation) and “mirror” runs that allow to control for non-specific and task-unrelated effects, such as habituation or neural adaptation. The approach was implemented in the Python programming language. Pilot data from 10 volunteers showed that participants were able to successfully down-regulate individually defined target areas, demonstrating feasibility of the approach. The newly developed visual feedback subsystem can be integrated into protocols for imaging-based brain-computer interfaces (BCI) and may facilitate neurofeedback research and applications into healthy and dysfunctional motivational processes, such as food craving or addiction. Recent advancements in real-time functional magnetic resonance imaging (fMRI) have been based on the increased availability of high-field (in excess of 1.5 tesla) MRI scanners, fast data acquisition sequences, improved real-time pre-processing, improved statistical analysis techniques and methods of data visualization and feedback. These developments have made the implementation of fMRI brain-computer interfaces (fMRI-BCI) and neurofeedback possible  with simultaneous visual stimulus presentation. Such approaches are especially interesting for research into neural and behavioral responses that are passively triggered by salient emotional or motivational cues, such as fear responses evoked by emotional pictures or craving elicited by drug-related pictures. The degree of drug cue-elicited neural activation within circuits of the orbitofrontal cortex  may be reduced, confounding the effects of effortful down-regulation. Here, we delineate a novel approach in which feedback-guided self-regulation is based on visual changes in the stimulus responsible for the targeted brain responses itself. Specifically, the present paradigm uses appetitive food pictures to evoke responses in brain circuits related to hunger and food craving. The success of regulating those responses during picture exposure in the neurofeedback session is represented as gradual changes in image size. Compared to the feedback stimuli used by Veit et al. and Brühl et al. our paradigm has the advantage that distracting/dual-task effects associated with monitoring the feedback stimulus during cue exposure are minimized. At the same time, by linking decreases in image size to successful down-regulation and image size increases to failure, respectively, the paradigm provides real motivational consequences of the participant’s regulation effort, i.e., the stimuli mimic avoidance behavior during successful down-regulation and approach behavior during unsuccessful down-regulation. Such behavioral/motivational relevance of the task can increase face validity of the neurofeedback training and may thus facilitate its therapeutic use in pathologies of motivational systems, such as obesity or addiction.Another challenge of visual cue reactivity neurofeedback is that repeated exposure to the same or similar visual stimulus will lead to a decrease of neural responses. Such effects have been well documented in brain imaging tasks using fMRI adaption or “repetition suppression” techniques, in which the reduction of BOLD responses associated with the repeated presentation of identical stimuli is used as a tool to characterize the neural representation of visual objects  and neutral control stimuli , or a fixation baseline (regulation runs). A 3 T whole body scanner  with an 8-channel head coil is used. The parameterisation of the BOLD sensitive echo planar imaging (EPI) sequence is as follows: TR = 2 s, TE = 45 ms, flip angle = 80°, 30 slices, FOV = 192 mm, image matrix 64 × 64, in-plane voxel size = 3 mm × 3 mm, slice thickness = 4 mm and a gap of 1 mm. Image reconstruction, distortion correction and image averaging are performed on the MRI scanner computer and stored in the MRI image pool.The signal analysis subsystem is performed using Turbo-Brainvoyager (TBV) version 3.0 . TBV reThe signal feedback subsystem retrieves the rtp files, processes and analyses the BOLD signal values contained within. Feedback is presented to the participant in real-time as food image sizes corresponding to the percentage change in BOLD signal values of the ROI during food picture presentation relative to fixation baseline using a moving average across three consecutive TRs. Feedback is presented to the participant with a delay that depends on the time for signal acquisition, signal analysis and signal feedback processing. Minimizing the delay is critical for volitional control  leading to a consecutive display of 11 different image sizes in total. Image sizes can vary between 10% and 100% of the original image size (1013 by 760 pixels) that are distributed across the whole size range. The size of the food image displayed at the first TR of each down-regulation block is set to 50% of the maximum image size, which corresponds to the percent signal change (PSC) at the first TR (FPSC) relative to the preceding rest block. Here, the FPSC is taken as the reference scaling point for the calibration of subsequent PSCs. The increase or decrease in image size calibration of the PSC is described using the following calibration algorithm:def PSCDisplay(PSC):if PSC < FPSC - 100:Display 10% of the image sizeelif FPSC - 100 <= PSC < FPSC - 75:Display 15% of the image sizeelif FPSC - 75 <= PSC < FPSC - 50:Display 20% of the image sizeelif FPSC - 50 <= PSC < FPSC - 25:Display 30% of the image sizeelif FPSC - 25 <= PSC < FPSC:Display 40% of the image sizeelif PSC == FPSC:Display 50% of the image sizeelif FPSC + 25 >= PSC > FPSC:Display 60% of the image sizeelif FPSC + 50 >= PSC > FPSC + 25:Display 70% of the image sizeelif FPSC + 75 >= PSC > FPSC + 50:Display 80% of the image sizeelif FPSC + 100 >= PSC > FPSC + 75:Display 90% of the image sizeelif PSC > FPSC + 100:Display 100% of the image sizeretun DisplayThis manner of calibrating the PSCs ensures that the effect of the FPSC is reflected in all the image sizes and adapts the feedback subsystem to peoples’ individual neurophysiological response range. Each neurofeedback block uses as its feedback signal a different food image from a pool of 16 pictures selected at random. By default the range of the response is set to a 1% deviation from baseline (upwards or downwards), which implies that a 1% up-regulation will result in presentation of the full-size image and 1% down-regulation in presentation of the smallest image. Further up- or down-regulation will then not be reflected in further in- or decreases of the image size. However, the gain of the conversion from the extracted brain signal feature  to image size can be set freely to reflect an individual’s response range, and can be adjusted adaptively to reflect dynamic changes in self-regulation ability. The food images were taken from the International Affective Picture System  as it works in the opposite direction. Figures After each neurofeedback run, an associated mirror/control run is presented to the participant. In the mirror run the exact same picture (size) sequence (using the same picture exemplar) is repeated that was shown in the preceding neurofeedback run. The only difference is that during the mirror run participants are instructed NOT to regulate their brain responses during exposure to the food images but should passively watch the image (size) sequences instead. By this means, the mirror run can serve as a perceptual control: when BOLD responses from the mirror run are (offline) subtracted from BOLD responses in the neurofeedback run, any decrease in brain activation (in the target ROI or elsewhere in the brain) Figures M = 21.40 years, Standard Deviation (SD) = 2.27) were acquired to test whether the newly developed motivational neurofeedback paradigm can be successfully used to down-regulate brain activation in response to appetitive food pictures. Participants were asked not to eat for 4 h before the scanning session to increase motivational brain responses related to hunger or food craving. Written informed consent was obtained in accordance with the local ethics committee prior to the start of the study.Data from 10 female participants , resulting in a total run length of 222 s. The sequence of four neurofeedback and mirror runs were presented using the trial/run structure described above. Due to technical problems, for three participants only three neurofeedback/mirror runs could be acquired. For ROI analyses testing the feasibility of down-regulation of the target area in the neurofeedback runs, we used the pre-processed functional images created by the TBV software, which were co-registered to individual high-resolution T1-weighted anatomical images and spatially normalized to Talairach space. Individual volume time courses were spatially smoothed using a kernel with a full width at half maximum (FWHM) of 4 mm and temporally filtered with a high pass filter of 2 cycles/time course. For each run and participant separately, mean beta estimates in the target area were then extracted based on individual whole-brain GLMs and three different predictors for BOLD signal changes during (i) mirror, (ii) regulation (neurofeedback); and (iii) rest blocks. Table Based on a whole brain GLM analysis for the contrast between food and neutral pictures during the localiser run we visually selected for each participant individually a target area (ROI) encompassing the cluster showing the strongest response in the statistical activation maps  = 2.49, p = 0.034, and in run 3, t(9) = 2.77, p = 0.022. Activation levels were not significantly different between regulation and passive viewing in run 1 and run 4, ts < 1.0. However, as indicated in Figure During the neurofeedback run participants were asked to reduce the size of the food images displayed on the projector screen by reducing the average fMRI signal strength in the target ROI. During the mirror run the participants were instructed to passively view the food image sequence repeated from the previous neurofeedback run. Figure Providing motivationally relevant neurofeedback through changes in stimulus size may also provide an avenue to help patients gain control over dysfunctional motivational processes, such as craving elicited by environmental cues in substance dependence. It is well known that visual cue reactivity, and specifically craving responses to drug-related cues  are a major determinant of relapse after treatment of addiction Weiss, . MoreoveOn the other hand, the present paradigm allows participants to directly manipulate and control the visual cue which can be predicted to increase the participant’s sense of agency and perception of self-efficacy Bandura, —factors during exposure to those cues; and (ii) to increase motivational relevance of visual feedback by using decreases (avoidance) or increases (approach) of stimulus size as an indication of successful or unsuccessful down-regulation. Further, the feedback subsystem is able to adapt to peoples’ individual neurophysiological response range. These individual ranges are scaled to fall within a standard range before being mapped to the corresponding image sizes for display. Importantly, the introduction of the mirror run controls for physical/perceptual confounds, allowing separation of BOLD response changes resulting from successful regulation from those related to variations of visual stimulus properties or visual responses, such as habituation. Finally, the present feedback subsystem displays a new food image size every 2 s allowing good performance and providing a minimum delay in line with existing visual feedback subsystems  feedback-guided regulation of visual cue reactivity, i.e., to control brain responses to pictorial cues 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.


To evaluate the effects of diabetic ketoacidosis (DKA) on neurocognitive functions in children and adolescents presenting with new-onset type 1 diabetes.Newly diagnosed patients were divided into two groups: those with DKA and those without DKA (non-DKA). Following metabolic stabilization, the patients took a mini-mental status exam prior to undergoing a baseline battery of cognitive tests that evaluated visual and verbal cognitive tasks. Follow-up testing was performed 8-12 weeks after diagnosis. Patients completed an IQ test at follow-up.There was no statistical difference between the DKA and non-DKA groups neither in alertness at baseline testing nor in an IQ test at follow-up. The DKA group had significantly lower baseline scores than the non-DKA group for the visual cognitive tasks of design recognition, design memory and the composite visual memory index (VMI). At follow-up, Design Recognition remained statistically lower in the DKA group, but the design memory and the VMI tasks returned to statistical parity between the two groups. No significant differences were found in verbal cognitive tasks at baseline or follow-up between the two groups. Direct correlations were present for the admission CO2 and the visual cognitive tasks of VMI, design memory and design recognition. Direct correlations were also present for admission pH and VMI, design memory and picture memory.Pediatric patients presenting with newly diagnosed type 1 diabetes and severe but uncomplicated DKA showed a definite trend for lower cognitive functioning when compared to the age-matched patients without DKA. Type 1 diabetes in children and adolescents is associated with varying degrees of diminished cognitive function, frequently involving memory, but also the subcategories of other neuropsychological domains . StudiesDKA is an acute metabolic and immunologic crisis that occurs in approximately 25 percent of children in the United States with new-onset type 1 diabetes . This liIn addition to a neurotoxic effect of glucocorticoids, support for the physiologic stress of DKA and its treatment is also associated with a systemic increase of CRP  thus supA recent cross-sectional retrospective study using a computer version of published tasks reported that type 1 diabetic children and adolescents who had a history of uncomplicated [no clinical cerebral edema (CE)] DKA scored lower in cognitive memory tasks of both immediate event-spatial position and event-color associations compared to an age-matched group without a history of DKA . In anotIn this study, we aimed to investigate the contribution of DKA to the neurocognitive changes observed in children with type 1 diabetes. To avoid the uncertainties of retrospective information and its potential influence in the pathogenesis of cognitive deficits caused by: 1) the degree and duration of hyperglycemia; 2) the frequency/degree of hypoglycemia; 3) potential contribution of macro-and microangiopathic changes in long-term diabetes; and 4) the comorbidity of depression, we conducted this study in newly diagnosed children and adolescents presenting with and without DKA and prospectively administered a standard and well-validated neurocognitive test battery at two points in time. This test battery evaluated: visual planning and memory, verbal memory and expressive language. The primary outcome was the difference in cognitive function based on this test battery.Patients between the ages of 7 and 18 years presenting to the North Carolina Children’s Hospital with new-onset type 1 diabetes were considered for participation. Exclusion criteria included non-English speaking patients or parents , chronic medication use (except hormone replacement with levothyroxine), developmental delay, learning difficulties or neuropsychological conditions as reported by parents. Fourteen patients were invited to participate between November 2011 and December 2012. DKA was managed by the pediatric intensive care unit per usual protocol, including intravenous insulin and fluid hydration.The study protocol was approved by institutional review board. Patients assented to the study along with parents’ consent. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.The initial cognitive tests were performed prior to hospital discharge. If DKA was present, initial testing occurred at least 24 hours after DKA resolution (defined as pH≥7.32 and CO2≥18 meq/L) and transfer to the pediatric ward. Follow-up testing (T2) was eight to twelve weeks after the diagnosis of type 1 diabetes during a follow-up visit to the Pediatric Diabetes Clinic. This follow-up testing battery consisted of the same cognitive tasks administered at the initial testing as well as a shortened measure of intelligence using the Wechsler Abbreviated Scale of Intelligence (WASI-IQ).Blood chemistries were collected on admission for each patient. Blood glucose was monitored prior to cognitive tests that were administered in the morning when possible. Patients completed a one-hour testing battery administered by a psychologist. The initial testing battery consisted of a Mini-Mental Status Exam (MMSE) to evaluate for alertness prior to other testing. This was followed by standard neurocognitive tests of visual and verbal cognitive tasks. The visual planning and memory tasks used the following instruments: a) Wide range assessment of memory and learning, second edition (WRAML-2) for design memory, design recognition, picture memory and visual memory index (VMI); b) Stanford-Binet for bead memory, c) Wechsler Intelligence Scale for Children-IV (WISC-IV) for forward spatial span and backward spatial span and d) Delis Kaplan Executive Function System (DKEFS) for design fluency. The verbal memory and expressive language tasks used: a) Wide range assessment of memory and learning, second edition for verbal learning, verbal learning recall and verbal learning recognition; b) WISC-IV for digit span; and c) DKEFS for verbal fluency, with subtests of letter fluency and category switching.Standard scores and composite scores were determined for the DKA and the non-DKA groups. A Mann-Whitney U test of group medians was used to test for statistical differences between the DKA and non-DKA group’s demographics and admission values for HbA1c, heart rate (HR) for degree of dehydration, pH, bicarbonate (CO2); MMSE at baseline cognitive testing and WASI-IQ at follow-up cognitive testing. Statistical differences between the two groups at baseline and follow-up testing on the cognitive tasks for visual and verbal variables were established using Mann-Whitney U test of group medians. Effect size differences (ES-d) for DKA and non-DKA across the cognitive tasks for the baseline and follow-up testing were also calculated. Chi-square analysis was used to identify association between cognitive task score changes from baseline to follow-up and DKA/non-DKA patient status. Correlations were calculated between cognitive tasks measured at baseline (T1) with chemistries obtained at admission and between cognitive tasks at baseline or follow-up (T2) with pre-test blood glucose.All patients had uneventful recoveries. No patient required mannitol for CE or experienced blood glucose (BG) <70 mg/dL during hospitalization. Three of 14 invited patients declined to participate. Three of the four patients with DKA and five of the seven patients without DKA completed follow-up testing. Patients with DKA underwent baseline testing 29-34 hours after DKA resolution. Patients’ admission parameters and demographics are in At baseline, the DKA group scored lower on several visual tasks compared to the group without DKA.At follow-up, design recognition in the DKA group remained statistically lower than the non-DKA group; all of the other visual tasks achieved or maintained statistical parity between the two groups. There were no significant differences between the DKA and non-DKA groups at baseline or follow-up testing for any of the verbal tasks . The corLast, chi-square analysis for association of the number of cognitive score that increases between baseline and follow-up showed a significant association  for the DKA group. Similarly, the cognitive variables, VMI and digit span, had significant inverse correlations with the DKA group , meaning that lower cognitive scores correlate with the DKA group.This is the first prospective study to report acute cognitive deficits in newly diagnosed children and adolescents with type 1 diabetes following correction of uncomplicated  DKA. Our data support a neuronal insult during the metabolic and immunologic crisis of DKA and/or its treatment that results in acute and possibly long-term cognitive deficits. We recorded statistically lower median baseline (T1) task scores in three of the eight visual cognitive tasks tested in the newly diagnosed DKA group in comparison to an age-matched newly diagnosed non-DKA group. Whether the improvement in design memory and VMI scores three months (T2) after the correction of DKA are final new baselines is unknown, since the follow-up tasks were performed at a time when brain remodeling was possibly occurring . These lThe combined admission CO2 and pH of the DKA and non-DKA groups versus the T1 results of the visual and verbal cognitive tasks  showed sAlthough the pathogenesis of the cognitive deficiencies associated with DKA requires further study, it has been reported that ketone bodies, by-products of metabolic dysregulation, are capable in-vitro of differentially perturbating cerebral capillary endothelial cells (CCEC)  and the Also, 3-deoxyglucosone  and methThese by-products are unlikely to be the sole candidates of neuronal insult/deficit since the milieu in children and adolescents with severe DKA also increases systemic and cerebral oxidative stress ,35, SIR Limitations of this study are the small sample size, the short duration of follow-up and the absence of testing non-diabetic children and adolescents. Strengths of the study are that: 1) It is a prospective study involving patients of comparable ages with newly diagnosed type 1 diabetes with and without DKA; 2) It eliminates the confounding factors of hypoglycemia and microvascular disease; 3) It uses standardized cognitive testing; 4) It reports short-term follow-up testing; 5) It extends our insight into the subtleties of the early cognitive deficits in young patients with new-onset type 1 diabetes and DKA; and 6) It shows the importance of longitudinal testing.Understanding the physiological and functional changes that accompany DKA may contribute to early detections of brain changes associated with DKA, prior to the onset of chronic debilitating effects of type 1 diabetes and may inform about the mechanisms associated with subsequent cognitive decline. Modalities to study the effect of diabetes on the brain have evolved over the years and provide new opportunities not only to examine the impact of DKA on neurocognitive dysfunction but also to understand the underlying mechanisms. Therefore, further studies in larger populations using new static and functional neuroimaging tools are needed. Nevertheless, this study serves to encourage the careful follow-up, preferably in a coordinated care setting, of all children/adolescents with type 1 diabetes, especially those who have had DKA. Whether routine neurocognitive testing can be recommended in children with type 1 diabetes as part of their follow-up is an important question that remains to be answered.


Bleomycin has become an integral part of chemotherapy in patients with germ-cell tumors. One of the most feared side effects is bleomycin-induced pneumonitis. In patients with mild or moderate BIP, radiological signs disappear almost completely within nine months after discontinuation of bleomycin treatment.We present a patient with a history of non seminoma of the testis and bleomycin-induced pneumonitis. During follow-up, regression of the hypothesis of eosinophilic migration to the liver after regression of bleomycin-induced pneumonitis is highly suspicious based on transient eosinophilia and focal eosinophilic liver disease.As follow up may consist of CT scanning in germ-line tumor patients, transient eosinophilic liver lesions reported during regressive bleomycin-induced pneumonitis should not be presumed automatically as metastatic tumor relapse and require further sequential imaging and pathological examination. Streptomyces verticillus. Bleomycin acts as an oncolytic agent by inducing DNA strand breakage and subsequent has become an integral part of chemotherapy in patients with germ-cell tumors[Bleomycin is a glycopeptide antibiotic produced by the bacterium l tumors,2. Bleoml tumors. One of l tumors. BIP is l tumors. Treatmel tumors,5. In pal tumors. In thisA 41-year-old man was diagnosed with stage IIA good risk non seminoma of the left-sided testis and treated with hemiorchidectomy and adjuvant three cycles of bleomycin, etoposide and cisplatin. He received a total dose of 270 mg bleomycin during treatment. After the last course of chemotherapy, a chest and abdominal CT-scan (CT 1) revealed complete remission of the metastatic lesions. However, we unexpectedly discovered fibrosis in both lungs with signs of bronchiolitis obliterans and focally organizing pneumonia, probably induced by bleomycin Figure a and3b.This CT revealed further regression of pulmonary fibrosis, and regression of liver lesions in segment 2 (n = 1) and in segment 8 (n = 3). At 10 months one lesion was further decreased 3 lesions were stable. Interestingly, subsequent blood measurements showed a normalization of the percentage eosinophils in the three months between the first occurrence of liver lesions on CT and second CT with diminishment of these liver lesions and eventually calcification. This leads to the hypothesis that both findings are related to each other Figure .We have seen the concomittant regression of BIP and onset of focal eosinophilic liver disease (FELD) with eosinophilia. By exclusion diagnosis and thorough pathologic examination, this relationship in time led to the hypothesis of eosinophilic migration. Our understanding of the pathogenesis of BIP is mainly based on data derived from animal studies. Endothelial damage of the lung vasculature by bleomycin-induced free radicals is associated with an acquired loss of bleomycin hydrolase activity and followed by an influx of inflammatory cells. There iIn this patient the hypothesis of eosinophilic migration to the liver after regression of BIP is suggestive given the transient eosinophilia and presence of FELD. As follow up may consist of CT scanning in germ-line tumor patients, transient eosinophilic liver lesions reported during regressive BIP should not be presumed automatically as metastatic tumor relapse and require further sequential imaging and pathological examination.Patient has given his consent for publication of case-report.BIP: Bleomycin-induced pneumonitis; CT: Computer tomography; FELD: Focal eosinophilic liver disease; MRI: Magnetic resonance imaging.No funding sources need to be credited. No conflicts of interest should bementioned.IN provided the figures of pathological findings. JF provided the figures of radiological findings. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/13/491/prepub


Studies using the placental transcriptome to identify key molecules relevant for preeclampsia are hampered by a relatively small sample size. In addition, they use a variety of bioinformatics and statistical methods, making comparison of findings challenging. To generate a more robust preeclampsia gene expression signature, we performed a meta-analysis on the original data of 11 placenta RNA microarray experiments, representing 139 normotensive and 116 preeclamptic pregnancies. Microarray data were pre-processed and analyzed using standardized bioinformatics and statistical procedures and the effect sizes were combined using an inverse-variance random-effects model. Interactions between genes in the resulting gene expression signature were identified by pathway analysis  and protein-protein associations (STRING). This approach has resulted in a comprehensive list of differentially expressed genes that led to a 388-gene meta-signature of preeclamptic placenta. Pathway analysis highlights the involvement of the previously identified hypoxia/HIF1A pathway in the establishment of the preeclamptic gene expression profile, while analysis of protein interaction networks indicates CREBBP/EP300 as a novel element central to the preeclamptic placental transcriptome. In addition, there is an apparent high incidence of preeclampsia in women carrying a child with a mutation in CREBBP/EP300 (Rubinstein-Taybi Syndrome). The 388-gene preeclampsia meta-signature offers a vital starting point for further studies into the relevance of these genes (in particular CREBBP/EP300) and their concomitant pathways as biomarkers or functional molecules in preeclampsia. This will result in a better understanding of the molecular basis of this disease and opens up the opportunity to develop rational therapies targeting the placental dysfunction causal to preeclampsia. Preeclampsia, defined as the development of high blood pressure in combination with proteinuria after 20 weeks of gestation, is a common (incidence 2–8%) and potentially severe pregnancy complication for both mother and child . At presThe molecular mechanisms that play a role in the development of preeclampsia are largely unknown. Over the past decades, much research has been dedicated to identify preeclampsia-specific molecules that can either serve as a biomarker for prognostic/diagnostic purposes, or that play a functional role in the disease and hence are potential therapeutic targets. The main approach that has been used for this purpose is the analysis of gene expression in the placenta of normotensive and preeclamptic pregnancies. This has resulted in the identification of factors unquestionably associated with preeclampsia, such as soluble Vascular Endothelial Growth factor receptor 1 (sFLT1)  and soluWe have recently performed a systematic review and meta-analysis on 30 studies reporting lists of differentially expressed genes (DEGs) in relation to preeclamptic placenta . This geFor the current study we retrieved the original microarray data from 11 independent placental mRNA microarray experiments. Microarray data were subjected to quality analysis, pre-processed and analyzed using standardized bioinformatics and statistical procedures. This resulted in a comprehensive preeclampsia gene signature, which upon further analysis led to the identification of a novel pathway associated with preeclampsia.Datasets were identified by systematic review , searchiThe meta-analysis was performed according to the stepwise approach described in . In brieSTRING 9.1  was usedKEGG Pathway analysis was performed using Graphite . The 388GSEAPreranked was used for gene set enrichment analysis . All 861Ingenuity Pathway Analysis (Qiagen) Upstream Regulator Analysis was performed to identify upstream regulators that can explain the observed 388 gene meta-signature. Analysis was performed using standard settings. The resulting activation z-score was used to infer the activation states of the predicted regulator. A z-score of > 2 (activated) or <-2 (inhibited) was considered significant.Gene Expression Barcode: RNA microarray data for human placentas of different gestational ages was obtained from the NCBI Gene Expression Omnibus (accession GSE9984). Samples were pre-processed and barcoded as described by the Gene Expression Barcode 3.0 using the R package frma . ProbeseFor the current meta-analysis, we included 11 different studies representing a total of 151 normotensive and 132 preeclamptic placenta samples . Nine stQuality analysis of the microarray data resulted in the removal of 28 samples. One dataset  consisteINHBA, CGB, F5, HTRA4, PVRL4, RDH13). Calculation of the standardized mean difference (mu) based on the studies that did contain a gene-specific probe for these six genes shows that all are differentially expressed. Two of the 12 genes  were among the 688 differentially expressed genes in the initial meta-signature, but were removed in the leave-one-out-analysis, and four genes  were not significantly differentially expressed in our meta-analysis on the original microarray data. For those genes in the gene list-driven signature that were also differentially expressed in the current analysis, the direction of differential expression was identical except for SOD1, SPP1 and VIM. SOD1 and VIM, which have been described in the literature both as being upregulated and downregulated in the preeclamptic placenta, are definitely downregulated in the current analysis. In fact, SOD1 is one of the most strongly downregulated genes in the preeclamptic placenta, as shown in SPP1. Although reported as being upregulated in the preeclamptic placenta in three different publications, our current meta-analysis identifies SPP1 as a gene downregulated in preeclampsia.Comparison of the current 388-gene meta-signature with the gene list-driven signature of 40 genes we previously obtained from published gene lists reported as being differentially expressed in preeclamptic placenta [Two recent studies also performed a meta-analysis on microarray data exclusively. Vaiman et al  analyzedTo further identify preeclampsia-related pathways or networks within our 388-gene meta-signature, the STRING network of both physical and functional interactions between proteins was used . By emplAs shown in Parallel approaches using pathway analysis resulted in a large number of pathways that are enriched in the 388-gene meta-signature with a consistent enrichment for the hypoxia/HIF1A pathway in Ingenuity Pathway Analysis, Graphite, and Gene Set Enrichment Analysis .st and 2nd trimester placentas [nd, and even 1st trimester placenta.To obtain information if the genes listed in the meta-signature are also expressed earlier during gestation, we performed a Gene Expression Barcode analysis on a microarray data set including 1lacentas . As showWe established a robust 388-gene meta-signature in what is presently the most complete meta-analysis of the preeclamptic placental transcriptome. Our meta-analysis not only greatly increases the number of samples included as compared to the individual studies, but also largely resolves the between-study variation in bioinformatics and statistical analysis, which was one of the major inconsistencies reported in our previous gene list-driven meta-analysis .Recently, several other approaches to summarize placental mRNA expression data in relation to preeclampsia have been reported. As mentioned above, we previously performed a systematic review and vote-counting-based meta-analysis on published preeclampsia lists of DEGs from both genome-wide microarray and smaller scale mRNA expression studies . Most ge−16, Fisher’s exact test) with the genes in the meta-signature of Vaiman and colleagues, and with the meta-signature reported by Moslehi and colleagues . However, our current approach has generated many more DEGs. Possible reasons for this are multiple and include (i) an increase in statistical power by the larger number of studies included in our meta-analysis, (ii) renormalization of the original data using an uniform set of state-of-the-art methods whenever possible, and (iii) use of stringent quality control criteria to detect and discard low-quality arrays. Differences in meta-analysis techniques used also impact the meta-signatures obtained. Combining effect sizes using an inverse-variance random-effects model has several advantages compared to the meta-analysis techniques used by the other two studies [The 388-gene meta-signature we currently present shows considerable overlap  Of the 20 suitable studies identified, only seven had submitted their data to a publicly available repository; the data of four additional studies were obtained directly from the authors. Although the acquired original data represents the majority of samples, it is disappointing that from almost half of the studies data did not become available.(ii) In order to obtain summary statistics and p-values based on data from all studies, only the genes present on all platforms were taken into account. This results in a large decrease of the gene universe in the current analysis  compared to the more than 20,000 genes located on most of the microarray platforms included. In particular, the inclusion of the Operon platforms  dramatically decreases the number of genes in the intersection.st and 2nd trimester. CREBBP shows clear placental expression from 1st trimester onwards, but the level of EP300 mRNA expression in the early placenta is less pronounced. Novel developments allowing the quantification of placental RNAs in maternal blood offers the enticing opportunity to study placental gene expression in ongoing pregnancy [A shortcoming of the current meta-analysis  in relation to both function of gene products and their potential role as predictors in preeclampsia is the timing of the gene expression pattern. All mRNA microarray data included in the current meta-analysis are generated from placentas at delivery, while the initial defects leading to preeclampsia occur earlier during pregnancy , 36. Basregnancy .A further asset would be the coupling of individual gene expression data to more extensive clinical parameters. The method and time of delivery and the use of antihypertensive drugs, among other things, are likely to affect placental gene expression. Preeclampsia, as a heterogeneous condition consists of several subtypes with different etiology and degree of placental contribution . UnfortuCREBBP/EP300 were not identified as DEGs in the other two published meta-analyses, their involvement in preeclampsia was suggested based on association with differentially expressed transcription factors by Vaiman and colleagues [Identification of functionally relevant pathways from the large number of genes in the preeclampsia-placental signature is a major challenge. Building networks based on protein-protein interactions can help to identify these functionally relevant pathways. Even when using a rather conservative approach, a protein-protein interaction network within our preeclampsia meta-signature emerges with CREBBP as key protein within this network. CREBBP interacts with several proteins well known to be associated with preeclampsia, such as NFE2L2 , 40, LEPlleagues .CREBBP and EP300 are causative for Rubinstein-Taybi syndrome . RSTS is a rare  autosomal dominant congenital disorder characterized by mental retardation, dysmorphic facial features, postnatal growth deficiency, and skeletal abnormalities [Mutations in malities . Almost malities . Interesmalities –51. ThusAnalysis of canonical pathways and gene expression patterns clearly indicated a prominent role for hypoxia in the establishment of the preeclamptic gene meta-signature, a conclusion that was also drawn by Moslehi et al, based on the DEGs from their meta-analysis .Preeclamptic placentas are compromised by hypoxia/ischemia, and the effect on gene expression is largely mediated via HIF-1α . More thThe robust 388-gene signature of differentially expressed genes in the preeclamptic placenta serves as a starting point to investigate their function in the development of preeclampsia. This will result in a better understanding of the molecular basis of this disease and opens up the opportunity to develop rational therapies targeting the placental dysfunction causal to preeclampsia.S1 FigForest plots of the 388 differentially expressed genes in the preeclamptic placenta ordered on the absolute value of the standardized mean difference see . Squares(PDF)Click here for additional data file.S2 FigAll 388 genes from the meta‐signature were used as input for STRING analysis and a network was built based on high confidence (0.8) evidence from experimental protein‐protein interaction (blue lines) and curated (purple lines) databases. Proteins are indicated by nodes labeled with the encoding gene symbol. Two genes (CEBPA and SEPP1) are not present in the used STRING version 9.1. The network is enriched in interactions (p = 0.007) using the intersection of 8,612 genes present on all platforms as background. Additional cluster analysis was used to color the nodes of the interaction networks. Yellow nodes represent proteins that have no protein‐protein interaction at this confidence setting.(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 TableIndicated microarray samples were analyzed by the Gene Expression Barcode 3.0 and the resulting z-scores are shown. The z-score is the number of standard deviations above the mean expression signal of the unexpressed null distribution. An average z-score of ≥ 3 for a gene-specific probe has been labeled as being expressed in that trimester.(XLSX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


Trigeminal neuralgia (TN) is a chronic disorder characterized by paroxysmal facial pain. Adequate prophylactic drug therapy is often limited by the lack of efficacy and intolerance due to central nervous system side effects.Subcutaneous injections of botuinum toxin type A (BT-A) are a promising treatment option for patients with insatisfactory response to drug therapy or neurosurgical intervention.This is the study protocoll of a prospective, placebo-controlled, double blind clinical trial investigating the add-on therapy of subcutaneous of BT-A injections to standard treatment.BoTN is a prospective, randomized, double-blind, placebo-controlled trial with a randomized withdrawal design. Eligible patients with classic TN who are otherwise refractory to medical and neurosurgical treatment will receive subcutaneous injections of BT-A into injection sites of the affected trigeminal branch.In the first phase phase all patients will receive verum in a single blinded intervention and twelve weeks later therapy responders will be 1:1 allocated to the verum or placebo  arm. This time the intervention is double blind and injections will be done at the same sites.This trial will be conducted in a tertiary outpatient clinic specialized in the treatment of headache and facial pain.BoTN is designed to assess the efficacy and safety of local BT-A injections in additon to standard prophylactic treatment.No conflict of interest.


The mechanisms involved in such reconfiguration have been extensively investigated by cell-focused studies, but the actual changes at the network level remain elusive. Since a failure to generate gasping has been linked to Sudden Infant Death Syndrome (SIDS), the study of gasping generation and pharmacological approaches to promote it may have clinical relevance. Here, we study the changes in network dynamics and circuit reconfiguration that occur during the transition to fictive-gasping generation in the brainstem slice preparation by recording the preBötC with multi-electrode arrays and assessing correlated firing among respiratory neurons or clusters of respiratory neurons (multiunits). We studied whether the respiratory network reconfiguration in hypoxia involves changes in either the number of active respiratory elements, the number of functional connections among elements, or the strength of these connections. Moreover, we tested the influence of isocitrate, a Krebs cycle intermediate that has recently been shown to promote breathing, on the configuration of the preBötC circuit during normoxia and on its reconfiguration during hypoxia. We found that, in contrast to previous suggestions based on cell-focused studies, the number and the overall activity of respiratory neurons change only slightly during hypoxia. However, hypoxia induces a reduction in the strength of functional connectivity within the circuit without reducing the number of connections. Isocitrate prevented this reduction during hypoxia while increasing the strength of network connectivity. In conclusion, we provide an overview of the configuration of the respiratory network under control conditions and how it is reconfigured during fictive-gasping. Additionally, our data support the use of isocitrate to favor respiratory rhythm generation under normoxia and to prevent some of the changes in the respiratory network under hypoxic conditions.The neuronal circuit in charge of generating the respiratory rhythms, localized in the pre-Bötzinger complex (preBötC), is configured to produce fictive-eupnea during normoxia and reconfigures to produce fictive-gasping during hypoxic conditions  Neuronal assemblies are embedded in complex networks that act in concert to generate the function of a given brain region  and those arising from groups of neurons [multiunitary activity (MUA)] . All experimental protocols were approved by the local Committee on Ethics of Animal Experimentation (INB-UNAM). Experiments were performed according to the Mexican Official Norm for the Use and Care of Laboratory Animals (NOM-062-ZOO-1999).Experiments were performed using 6–9-day-old (P6–P9) CD-1 mice  of the selected units or multiunits  were discarded . We calculated the mean frequency of recordings for both units and multiunits. The reciprocal of the median inter-spike intervals (ISI) was taken to obtain an approximation of intraburst frequency of inspiratory neurons ; correlations that did not reach this threshold received a value of zero . To compare the strength of the correlations in the presence of hypoxia and/or isocitrate, we subtracted the control correlation matrix from the correlation matrices in any given experimental conditions and used the resulting values as the change in correlation (ΔCorrelation) . Similarly, the correlation matrices in the presence of isocitrate were subtracted from the control matrices in hypoxic conditions in the presence of isocitrate. The graphic representation of the network in any given condition was made with the open access software Cystoscape (Cytoscape Consortium).To evaluate the population burst pattern of preBötC activity we measured the peak-to-peak amplitude of the raw signal every 45 ms. By averaging the amplitude signals for several bursts, it is possible to clearly distinguish the changes in burst pattern occurring during the transition from fictive-eupnea to fictive-gasping Figure  Recordind Figure . The sord Figure , as a chd Figure . Timestad Figure , which wd Figure , 2A. Thed Figure  with a ld Figure . Autocord Figure . Cross-c Figures , 5A. To  Figures , 5B. Simt-test or Repeated Measures ANOVA followed by a post-hoc pair-wise Tukey's or Dunnett's test as needed using GraphPad Prism and, in a few cases, (as for median frequency) Kruskal-Wallis ANOVA followed by the Dunn's test. Statistical significance was accepted at p-values <0.05.All quantifications were grouped by unitary, multi-unitary, or the merge of both. Representative data are given as mean ± s.e.m., and the median frequency is reported with the interquartile range. Statistical differences among groups were tested using either a paired or unpaired Student's SD above the correlation noise  and within a lag period of ± 5 ms. As expected, respiratory multiunitary recordings exhibited inspiratory activity. Of the unitary recordings, 87% were also identified as inspiratory units (range: 73–100%) . Figure Next, we proceeded to characterize network interactions by means of cross-correlation analysis among respiratory elements Figure . Cross-ce Figure . Correlae Figure . Moreovee Figure . While te Figure , suggeste Figure , 4A. TheAs previously reported . Figure Correlation matrices show that isocitrate increases the strength of most of the functional links among respiratory elements Figures . Thus, tThe reconfiguration of the respiratory CPG that transforms the preBötC network from a control configuration that generates fictive-eupnea in normoxia to a hypoxic configuration that generates fictive-gasping involves complex changes at single-cell and network levels (Ramirez et al., Changes in respiratory network configuration leading to gasping generation have both basic and clinical implications. On the one hand, it might represent the best example of an acute and reversible network reconfiguration in a mammalian CPG (Ramirez et al., Since the seminal study by Lieske et al. , it has Here we considered as “respiratory recording nodes” all electrodes in the array capable of recording both unitary and multi-unitary activity (Bedenbaugh and Gerstein, In this study we have determined the global changes occurring in the respiratory network configuration under hypoxic conditions by analyzing the changes in cross-correlation among the elements recorded by the “recording respiratory nodes” of a MEA, as previously done in different neural networks (Galan et al., in vivo and in vitro (Hülsmann et al., The other major finding of the present study is that isocitrate can change the preBötC configuration, which would explain why isocitrate promotes eupnea and gasping generation both 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.


Attempts to enrich or identify DNA with cytosine methylation have been commonly carried out using anti-5-methylcytosine or anti-MBD2 (methyl-CpG binding domain protein 2) antibody in immunoprecipitation (IP) assays. However, a careful and systematic control experiment to examine the sensitivity and specificity of this approach has not been reported. It is of critical importance to understand the potential pitfalls of this approach and to avoid potential misinterpretation of findings.We found that increased concentration of antibody used in the assay increased the amount of overall DNA captured as expected. The increased number of methylated cytosines in/on the DNA fragment also increased the amount of DNA captured by the antibody. Importantly, the antibody can bind to some fully unmethylated DNA fragments, even when fully methylated DNA is present in the same experiment.The sensitivity of anti-5-methylcytosine antibody and anti-MBD2 antibody/MBD2 binding varies with the number of methylated cytosines on the DNA target. The specificity of these antibodies can also vary for different DNA target sequences. DNA fragments with fewer CpG sites may not bind to these antibodies even when all are methylated while DNA fragments with more CpG sites may bind to the antibodies when only some of these sites are methylated. More importantly, binding of DNA to these antibodies does not always indicate the presence of DNA methylation. It is clear that false positive and false negative findings can be easily reached even though it does not nullify these convenient and simple methods completely. Great caution should be taken for the interpretation of IP results using these antibodies and rigorous confirmation by sodium bisulfite sequencing is essential. The feasibility of using antibodies in immunoprecipitation (IP) to pull down DNA harboring 5-methylcytosine is evaluated in this study. DNA fragments from a plasmid with different numbers of CpG sites that can be methylated in vitro to different levels are used as targets in the IP. Plasmid DNA fragments with a single CpG methylation pattern and a mixture of plasmid DNA with different CpG methylation patterns were tested in the IP experiments. After IP, the precipitated DNA is analyzed quantitatively and qualitatively to determine the sensitivity and specificity of the assay.in vitro with SssI, HhaI, and HpaII methylases individually according to the manufacturer’s instruction (NEB). The unmethylated and methylated plasmid DNA were digested with restriction enzyme MseI, followed by phenol/chloroform extraction and ethanol precipitation, and resuspended in TE  to generate DNA fragments harboring different numbers of methylated CpG sites  that include unmethylated and HhaI-methylated input DNA since there are no HhaI sites within the PCR amplicon for sodium bisulfite sequencing  than unmethylated, HhaI-methylated, or HpaII-methylated DNA (Table Both anti-5-methyl-C and anti-MBD2 antibodies can pull down DNA fragments with multiple methylated CpG sites. The capability of both of these antibodies to pull down methylated DNA increases with the number of CpG sites on the DNA fragment. Anti-5-methyl-C antibody can pull down unmethylated DNA at a non-negligible level around 2%, and it can pull down DNA fragments with more than 30 CpG sites reliably and consistently. While anti-MBD2 appears to have good specificity for pulling down highly methylated DNA with 12 or more CpG sites indirectly through the binding of MBD2 protein to the DNA, it may be affected by DNA sequence or conformation. Fully unmethylated DNA can be captured by both antibodies and create false positive findings. Also, some DNA fragments may bind to the beads used in the assay and create false positive finding. These types of false positive findings without rigorous confirmation had led to the report of 5-methyl-C presence in human mitochondrial DNA . Also, D


Changes in cis-regulatory element composition that result in novel patterns of gene expression are thought to be a major contributor to the evolution of lineage-specific traits. Although transcription factor binding events show substantial variation across species, most computational approaches to study regulatory elements focus primarily upon highly conserved sites, and rely heavily upon multiple sequence alignments. However, sequence conservation based approaches have limited ability to detect lineage-specific elements that could contribute to species-specific traits. In this paper, we describe a novel framework that utilizes a birth-death model to trace the evolution of lineage-specific binding sites without relying on detailed base-by-base cross-species alignments. Our model was applied to analyze the evolution of binding sites based on the ChIP-seq data for six transcription factors  along the lineage toward human after human-mouse common ancestor. We estimate that a substantial fraction of binding sites (∼58–79% for each factor) in humans have origins since the divergence with mouse. Over 15% of all binding sites are unique to hominids. Such elements are often enriched near genes associated with specific pathways, and harbor more common SNPs than older binding sites in the human genome. These results support the ability of our method to identify lineage-specific regulatory elements and help understand their roles in shaping variation in gene regulation across species. Recent experimental studies showed that the evolution of transcription factor binding sites (TFBS) is highly dynamic, with sites differing a great deal even between closely related mammalian species. Despite the substantial experimental evidence for rapid divergence of regulatory protein-binding events across species, computational methods designed to analyze regulatory elements evolution have focused primarily on phylogenetic footprinting approaches, in which putative functional regulatory elements are identified according to strong sequence conservation. Cross-species comparisons of non-coding sequences are limited in their ability to fully understand the evolution of regulatory sequences, particularly in cases where the elements are selected for novelty or species-specific. We have developed a novel framework to reconstruct the history of lineage-specific TFBS and showed that large amount of TFBS in human were born after human-mouse divergence. These elements also have distinct biological implications as compared to more ancient ones. This method can help understand the roles of lineage-specific TFBS in shaping gene regulation across different species. Changes in gene regulation play a key role in the evolution of morphological traits Despite substantial experimental evidence for rapid divergence of regulatory protein-binding events across species, computational models designed to analyze regulatory elements using cross-species comparisons have focused primarily upon ‘phylogenetic footprinting’ approaches, in which putatively functional regulatory elements are identified according to sequence conservation In this work, we introduce a novel evolutionary framework through which lineage-specific TFBSs can be inferred on a genome-wide scale. In contrast to conservation-based approaches Our goal is to detect lineage-specific rates of TFBS evolution and the branch of origin for individual TFBS. Here, lineage means any ancestral branch in the phylogeny or a branch leading toward any modern species. Our approach is to model TFBS evolution using a birth-death framework, in which individual TFBSs can be gained, lost, or conserved within a given lineage during evolution. The rate of TFBS creation (birth rate) and loss (death rate) are first estimated from a set of orthologous sequences, and are subsequently used to trace the evolutionary origin of individual TFBSs at the sequence level. The birth rate  is obtained. Every node in the phylogeny is then associated with a (random) variable gion see . EvolutiThe model framework and its motivations are illustrated in We note that in addition to the robustness of the parameter estimates of in vivo TF occupancy genome-wide In this work, we applied our method to ChIP-seq data, which is now commonly used to map To determine differences in the rate of motif evolution along specific lineages, we first assume a simple (null) model in which the birth and death rates  region relative to the summit of the peaks. Orthologous regions were then determined using 46-way multiz alignments from the UCSC Genome Browser Using our approach, we sought to determine the branch of origin for each human binding site for the six TFs. Each binding site was thus either inferred to be present in the common human-mouse ancestor, or a more recent lineage leading to human using the phylogeny shown in In addition to estimating the fraction of ancestral binding sites present in the human-mouse common ancestor, our approach allows us to estimate the age distribution across all binding site occurrences. As shown in Since the total number of protein-bound sites for each factor is quite large, these fractions represent a considerable number of sites unique to humans or closely-related primate lineages. The number of human binding sites originating since the common hominid ancestor ranged from 1,084 to 3,931 for each factor, including 440 to 1,409 human-specific binding sites. The rate of appearance for new sites along different ancestral lineages showed a relatively stable rate of creation for new binding sites for most TFs up through the common Simian-primate ancestor (Recent works have emphasized the emergence of new regulatory elements via transposable elements (TEs) To assess the accuracy of the age estimates, we first compared our results to ChIP-seq data from human and mouse. Using analogous cell types across species, we determined the amount of overlap between human ChIP-seq peaks and ChIP-seq peaks in the orthologous regions in mouse. A human ChIP-seq peak was considered to be ‘shared’ with mouse if its summit was within 200 bp of a mouse ChIP-seq peak summit in the orthologous region . The amount of overlap was assessed separately for regions containing a human binding site present in the common human-mouse ancestor and for regions that are not ancestral.We emphasize that, as illustrated previously in Although a relatively sizeable portion of shared ChIP-seq peaks contained only TFBSs predicted to be lineage-specific, in the majority of cases (>90%) the mouse TFBS did not occur within in sequence region orthologous to the human peak region used, but was instead offset to a non-overlapping region within a mouse peak. Very few of these TFBSs actually aligned across the two species, compared with those with predicted ancestral origin.X) in each ChIP-seq peak and then calculated the average score (M) as well as standard deviation (SD) across all peaks in the genome. We then grouped the binding sites according to their branch of origin  and calculated the average PhyloP score (X). Finally, we calculated the Z-score, i.e. (X-M)/SD). As expected, older binding regions show higher sequence level conservation than younger ones , embryonic stem cells (H1hESC), cerebellum (HAC), and kidney cells (HRE). Interestingly, we observed substantial differences in the age distribution of cell-type specific and ubiquitously-bound sites. These results are consistent with previous observations that cell-type specific CTCF binding sites are less conserved across mammalian species than ubiquitously-bound sites in vivo verified enhancers listed in the VISTA Enhancer Browser in vivo transgenic mouse embryos essay based on VISTA Enhancer Browser, which confirms that the predicted enhancer also functions as an enhancer in mouse. It certainly remains to be solved whether this enhancer regulates the same genes in human and mouse and why the ChIP-seq signal on ancient MAX/MYC binding site is much weaker than the younger one in human. Nevertheless, this example demonstrates the ability of our method to compare functional level dynamics with sequence level difference in an evolutionary framework.Using our framework, we then utilized genome-wide chromatin data to search for potential functional consequences driven by birth or death of specific lineage-specific TFBS. We intersected the lineage-specific TFBSs with predicted human enhancer regions marked by ChromHMM model in silico methods without relying on accurate cross-species alignments. Studies regarding the evolution of TFBSs are largely separable into those that emphasize cross-species conservation of regulatory elements in silico and experiment-based studies. Although computational approaches have had some success in identifying cis-regulatory alterations responsible for changes in phenotype The approach presented here represents an initial step towards understanding regulatory element evolution using in vivo were originally absent in human-mouse common ancestor, which is consistent with previous cross-species comparisons noting substantial divergence in ChIP-seq protein-binding events across the two species Drosophila using alternative approaches In this context, the high fraction of TFBSs that have recent origins after human-mouse divergence is particularly notable. For all six factors analyzed, the majority of human TFBSs bound Among TFBSs in humans, a considerable amount of them are unique to hominids and are even human-specific. Since there are an estimated ∼1700–1900 TFs in the human genome The functional categories of genes close to recently derived binding sites may serve to shed new light upon adaptive traits obtained along the lineage leading to human. It is interesting that, despite substantial differences in the biological functions generally associated with the six TFs analyzed, genes near binding sites with recent origins are enriched for several sensory and neural related pathways and processes. We note that since the ChIP-seq data we used in this study were not derived from neuronal cells, further study is needed to more comprehensively understand the roles of hominid-specific sites in specific cell types. Nevertheless, identifying such lineage-specific regulatory elements not only provides potential insight on human biology, but may also provide new knowledge on the molecular mechanisms of human diseases.A natural future direction for this work would be to determine the specific regulatory effects of the recently derived TFBSs identified using this method. For instance, enrichment for within-species variation among recently derived binding sites raises the intriguing possibility that recently derived TFBSs most responsible for phenotypic differences across species are also the elements responsible for within-species variation. Future work will be necessary to demonstrate whether this is the case and the differences in traits brought about by this variation. Also, our current model needs to be integrated with gene expression data to understand the interplay between cis-regulatory element evolution  and gene expression differences across different species Our approach is designed to estimate genome-wide rates of evolution for a given motif according to a birth-death framework  (y-axis), where binding sites are separated according to the branch of origin (x-axis). (A) Each colored plot corresponds to a single consensus motif; the fraction of binding sites overlapping TEs documented by RepeatMasker (TIF)Click here for additional data file.Figure S2PhyloP conservation vs. TFBS with different branch of origins. We used the PhyloP mammalian conservation scores available at the UCSC Genome Browser to determine the sequence conservation level for TFBS with different branch of origins in human. X-axis shows TFBS with different branch of origins for four different window sizes surrounding the peak summit. Y-axis shows the Z-score distribution for each group. For a specific TF, we first computed the average PhyloP score (X) in each ChIP-seq peak and then calculated the average score (M) as well as standard deviation (SD) across all peaks in the genome. We then grouped the binding sites according to their branch of origin  and calculated the average PhyloP score (X). Finally, we calculated the Z-score, i.e. (X-M)/SD. t-statistic from t-test between the youngest and the oldest for each group is also shown.(TIF)Click here for additional data file.Figure S3Background SNP density for TFBSs with different branch of origin. TFBSs were grouped into different branches of origin (X-axis). To calculate the background SNP density surrounding these TFBSs, we extended 1 kb, 500 bp, or 125 bp to both directions  and counted the number of common SNPs in this 2 kb window. The figure shows that there are no significant differences of SNP density surrounding the TFBSs with different branches of origin.(TIF)Click here for additional data file.Figure S4Positive enhancer rate based on the VISTA enhancer database. The positive rate is the percentage of human enhancers that also show enhancer activity in mouse. The expected rate is based on all the enhancers overlapping with TFBS used in our six TF data sets. ‘More ancestral enhancer’ has higher positive enhancer rate compared with ‘more lineage-specific enhancers’ or ‘neutral enhancer’, which is generally consistent with our computational prediction that the ancestral TFBS are more functionally conserved than lineage-specific TFBS, even though this comparison dataset is not ideal. See Supplementary Results in (TIF)Click here for additional data file.Figure S5Comparison between our method and MotifMap. A receiver operating characteristic (ROC) curve shows the prediction power between our method and MotifMap. ROC curves for MotifMap were generated using different BBLS thresholds  to call a TFBS as a conserved one. In our method, we tested two shift sizes, +/−15 bp (light blue) and +/−30 bp (dark blue). The results from MotifMap were based on +/−15 bp shift size (magenta). See Supplementary Results in (TIF)Click here for additional data file.Figure S6Sensitivity of our method when we add noisy sites in leaf nodes. Sensitivity of our method on the uncertainty of number of binding sites in leaf nodes was determined by randomly deleting/adding 5% sites in +/−100 bp of peak summit in all the species except human. Predictions were compared with original results and the changes of branch of origin for each binding site were counted. Y-axis shows the percentage of binding sites with various branch change. 0 means the prediction of branch of origins remain same before and after we randomly delete or insert binding sites.(TIF)Click here for additional data file.Figure S7Sensitivity of our method when we change the branch lengths. Sensitivity of our method on the branch length of phylogenetic tree was characterized by randomly changing the branch lengths to a certain extent. The length of each branch in phylogenetic tree can be varied in certain range relative to its original length (shown on X-axis). Y-axis shows the percentage of binding sites that have different branch of origin before and after we randomly change branch lengths in the phylogenetic tree.(TIF)Click here for additional data file.Table S1Comparison of consensus motifs.(PDF)Click here for additional data file.Table S2Gene functions and pathways associated with hominid-specific TFBS.(PDF)Click here for additional data file.Table S3Performance comparison with MotifMap and PReMod.(PDF)Click here for additional data file.Table S4Gene functions and pathways associated with simian-specific TFBS.(PDF)Click here for additional data file.Table S5Gene functions and pathways associated with ancestral TFBS.(PDF)Click here for additional data file.Text S1Supplementary methods and supplementary results.(PDF)Click here for additional data file.


Renal disease is a common complication of HIV-infected patients, associated with increased risk of cardiovascular events, progression to AIDS, AIDS-defining illness, and mortality. Early and accurate identification of renal disease is therefore crucial to improve patient outcomes. The use of serum creatinine, along with proteinuria, to detect renal involvement is essentially to screen for markers of glomerular disease and may not be effective in detecting earlier stages of renal injury. Therefore, more sensitive and specific markers are needed in order to early identify HIV-infected patients at risk of renal disease. This review article summarizes some new and important urinary markers of tubular injury in HIV-infected patients and their clinical usefulness in the renal safety follow-up of TDF-treated patients. Renal disease is a common complication of HIV-infected patients, associated with increased risk of cardiovascular events, progression to AIDS, AIDS-defining illness, and mortality  –4. MoreoCurrent guidelines for the management of HIV-infected patients recommended the assessment of renal disease by using proteinuria and creatinine-based estimated glomerular filtration rate (eGFR) . HoweverRenal tubular abnormalities are common and a substantial proportion of HIV-infected patients on HAART could have prevalent renal tubular injury in the absence of glomerular defects, probably resulting in a near future decrease in glomerular function and a higher incidence of urinary protein –18. MoreNeutrophil gelatinase-associated lipocalin (NGAL) is a small, 25-kDa protein that belongs to the lipocalin protein family released from neutrophils and many epithelial cell types including kidney tubular cells. It is representative of the functioning tubular mass and is secreted into the urine as a response to tubular injury . In HIV-Urinary NGAL was significantly higher in patients with HIVAN compared with HIV-infected patients without renal disease and HIV-negative controls , 31. The3 and history of AIDS, were also associated with higher urinary A1M levels [Alpha-1 microglobulin (A1M) is a small molecular weight protein (27 kDa) present in various body fluids. In the healthy kidney, it passes freely through the glomerular membranes, and about 99% is reabsorbed and catabolized by the proximal tubular cells. Increased A1M in urine can therefore be an early sign of renal damage, primarily on the proximal tubules . UrinaryM levels . Higher M levels .Urinary A1M has been suggested to be the most sensitive parameter to detect kidney tubular dysfunction in HIV-infected patients . Among HAs tubular proteinuria defined by A1M is associated with exposure to TDF , urinaryβ) 2-microglobulin (B2-M) is an 11.8 kDa protein produced by cells expressing major histocompatibility complex (MHC) class I. It is filtered freely through the glomeruli of the kidney, and a majority of it is reabsorbed and catabolized by renal proximal tubular cells; thus, measurement of urinary B2-M can serve as a useful biomarker to evaluate renal tubular injury [Beta  is a hydrolytic lysosomal enzyme found predominantly in proximal tubule. It has been demonstrated as a useful marker of renal tubular impairment in various conditions involved with renal injury or dysfunction . UrinaryIn HIV-infected patients, urinary excretion of NAG was significantly higher in those with rather than without tubular damage . In one Kidney injury molecule-1 (KIM-1) is a transmembrane protein and its expression is not measurable in normal proximal tubule cells but is markedly upregulated with injury/dedifferentiation . It has Higher concentration of KIM-1/Cr was observed in HIV-infected patients receiving antiretroviral therapy as compared to healthy individuals, although the difference was not statistically significant . CumulatRetinol-binding protein (RBP) is a low molecular weight protein (21 kDa) that transports retinol  from the liver to peripheral tissues. It passes freely through glomerular membranes and is reabsorbed by renal proximal tubules cells where it is catabolized, so an increase in the urinary excretion of RBP indicates proximal tubule injury and/or impaired proximal tubular function , 58. HigUrinary excretion of RBP was significantly higher in HIV-infected patients than in the HIV-negative controls, even in patients without renal disease . In thisRBP is also a promising biomarker for monitoring renal tubular function in patients receiving TDF and for distinguishing patients with normal tubular function or mild tubular dysfunction from those with severe renal tubular disease and Fanconi syndrome . UrinaryLiver-type fatty acid binding protein (L-FABP) is a low molecular weight (15 kDa) intracellular carrier protein that is expressed in the renal proximal tubule and liver. In renal disease L-FABP gene expression in the kidney was upregulated and its urinary excretion was found to correlate with the severity of tubulointerstitial injury, reflecting stresses on the proximal tubules . Higher Higher concentrations of urinary L-FABP/Cr were also found in HIV/HCV coinfected patients as compared to the HIV-monoinfected individuals and to healthy subjects. Furthermore, patients receiving the regimen based on EFV had higher levels of L-FABP/Cr compared to individuals treated with other regimens and to healthy controls . All theCystatin C (CysC), a cysteine protease inhibitor constantly produced by all nucleated cells, is a low molecular weight (13 kD) basic protein freely filtered by the kidney glomerulus and is reabsorbed by the tubules, where it is almost totally catabolized . IndividHigher levels of urinary CysC were found in patients with established HIV-associated renal diseases than in patients without proteinuria or trace proteinuria . CysC leThe use of creatinine-based estimates of renal function and proteinuria to detect renal involvement is essentially to screen for markers of glomerular disease and may not detect abnormalities in the tubular function. Urinary excretions of the tubular proteins are significantly higher in HIV-infected patients compared with the uninfected controls and in the patients with rather than without tubular injury. The urinary excretion of these tubular proteins increase even before the diagnosis of proteinuria and/or a decrease in glomerular function, indicating that renal tubular injury is an early event in patients with HIV and tubular proteins could be useful for evaluating early renal involvement . Exposur


Very high levels of one or more classes of immunoglobulins can be found in children with various clinical conditions. Diagnostic protocols have been developed for defined forms of IgG, IgM or IgE hypergammaglobulinemia, which could be the expression of both chronic inflammatory disease and primary immunodeficiency syndromes. In contrast, except for well described conditions such as IgA nephropathy and Henoch Schönlein Purpura, much less is known about hyper-IgA.We analyzed and discussed the diagnostic significance of very-high IgA levels in the clinical practice of a tertiary care children hospital.We collected all IgA determinations performed on children aged less than 16 years at the laboratories of Immunopathology of the Institute for Maternal and Child Health, IRCCS Burlo Garofolo, from 2009 to 2013. IgA values greater than three standard deviations above the mean, based on the local reference values for three age groups, were considered as “hyper-IgA” . For subjects with repeated hyper-IgA measures on different determinations, only the first value was recorded. To estimate the burden of diseases associated with hyper-IgA, a control age-matched group of 200 subjects with normal IgA values was randomly selected from the same laboratory series. Physicians who cared for each patient were asked to fill out a questionnaire regarding the clinical diagnosis and the interpretation of increased values of IgA. In addition, the levels of IgG and IgM greater than 2 standard deviations above the mean were recorded as well, where available. Subjects whose clinical records were not available were excluded from the analysis.A total of 12650 measurements of serum IgA were performed during the period of the study on 6364 subjects. Ninety-one subjects  had one or more hyper-IgA values . Clinical records were available for 83/91 subjects with hyper-IgA and 173/200 age-matched controls with normal IgA. Most subjects with hyper-IgA (73.5%) suffered from a severe immune defect, a chronic rheumatic disease or an inflammatory bowel disease, while these conditions were very rare in a control group with normal IgA values (8%).The potential role of hyper-IgA in pediatric diagnostics may be greater than usually recognized, even if this high prevalence of serious disease with high IgA may not apply to general population. We suggested that hyper-IgA in children should always rise the suspicion of a serious immune defect, a chronic rheumatic disease or an inflammatory gastrointestinal disorder.None declared.


Modern resource management faces trade-offs in the provision of various ecosystem goods and services to humanity. For fisheries management to develop into an ecosystem-based approach, the goal is not only to maximize economic profits, but to consider equally important conservation and social equity goals. We introduce such a triple-bottom line approach to the management of multi-species fisheries using the Baltic Sea as a case study. We apply a coupled ecological-economic optimization model to address the actual fisheries management challenge of trading-off the recovery of collapsed cod stocks versus the health of ecologically important forage fish populations. Management strategies based on profit maximization would rebuild the cod stock to high levels but may cause the risk of stock collapse for forage species with low market value, such as Baltic sprat . Economi A central issue in ecosystem-based management (EBM) is to identify potential trade-offs among multiple ecosystem goods and services A challenge of EBM lies in balancing a number of potentially conflicting interests related to resource use, their equitable distribution and conservation. Such “triple-bottom line” solutions are commonly seen as the ideal outcome of conservation and management Gadus morhua) versus the health of ecologically important forage fish stocks in the Baltic Sea. Many of the cod stocks in the North Atlantic have suffered from overfishing and population collapse Here, we provide a practical example on how to advance fisheries management towards an EBM approach by analyzing social-ecological trade-offs in a multi-species fisheries system. As an illustrative case study, we address the trade-off between recovery of Atlantic cod  and sprat (Sprattus sprattus). Then, we explore two different management approaches for protecting the sprat stock for its ecological value, one based on profit maximization only, and an alternative considering equity between demersal and pelagic fishing sectors. Our work suggests that recovery strategies for cod  may be very different when based on profit maximization alone, or when taking into account additional ecological and societal objectives, such as interacting species and fisheries rights (i.e. equity) during the planning process.Using a coupled ecological-economic optimization model framework we first derive the profit maximizing management solution for the entire multi-species fishery, including cod and the major Baltic Sea forage species herring (G. morhua (cod) and the two forage fish species C. harengus (herring) and S. sprattus (sprat). Our model is an extension of a single-species age-structured fishery model We developed and applied a combined three-species, age-structured ecological-economic model, including the predatory Residual (M1) and predation (M2) mortality estimates for the different age-classes of herring and sprat are based on regression analysis, using the output of a stochastic multi-species model SMS For modeling profits of the cod fishery, we use the specification from For the multi-species setting, the objective is to maximizeThe first part of this objective is the intertemporal utility of fishing income; where fishing income is a generalized mean of fishing incomes from the cod, sprat, and herring fisheries,The parameter per se is a measure of inequality, we use 1 – the Gini coefficient to obtain a measure of equity. Ranging from 0 to 1, a value of 1 represents perfect equality, a value of zeo maximum inequality. Details on the calculation are given in the Supporting Information  as well as an equity measure. The equity measure is based on the Gini index and is calculated as (1-Gini coefficient)*100. Pie slices give information on species-specific outcome. The size of each pie slice is calculated relative to status quo values 2008–2010 pa) and the minimum biological acceptable limit (Blim) for parent stock biomass  Profit-maximizing multi-species harvesting would also result in a healthy and sustainable population size of herring , as the pa and Blim , respectively, causing potential sprat profit losses of 13 of 48%. At the same time, cod fishing mortalities are less affected and would need to be increased only to 0.39 and 0.41 for Blim and Bpa, respectively (from 0.38 in the profit optimum). The cod fishing sector would only loose 1.2 or 2.6% of its potential profit. This result indicates that under an economic optimization, as performed here, the economically efficient solution to protect the sprat stock is a pronounced direct reduction of the fishing pressure on this lower market value forage fish species, in combination with a minor increase in fishing pressure directed towards its predator.While this management strategy would only marginally affect cod and herring profits , the relClearly, the conservation strategy of increasing the sprat stock by directly decreasing the sprat fishing mortality would be ecologically and economically efficient, since it requires only a minor reduction of the cod stock and hence has only a minor effect on the highly profitable cod fishing sector. But, while the pelagic herring fishery sector would benefit from a slight increase in profits , the sprlim and Bpa would require a strong reduction of optimal cod stock sizes to c. 474 and 346 thousand tons, respectively  relative to the profit-maximizing multi-species solution.An alternative to apply an increasing value to the conservation of the sprat stock is to explore the consequences of an increasing equitable resource distribution between fishing sectors. We defined equity based on relative profits of the three interacting species using the Gini-Index see , and optectively . Howeverlim and Bpa, respectively. The sprat fishing sector would achieve c. 92% or 172% higher profits at sprat Blim and Bpa, respectively, compared to the profit-maximizing multi-species solution, while the effect on herring profits would be negligible.Naturally reduced profits of the high value cod fishery make up for most the conservation costs inherent in the management strategy considering equity . Cod prolim or Bpa levels would need an increase in cod F to 0.54 and 0.67 (from 0.38 in the profit maximizing scenario). Historical evidence suggests that the cod stock biomass would still be sustainably conserved under such fishing pressure.Increased equity between fishing sectors can only be achieved by a lowered predation pressure on sprat and hence a reduction of the cod stock due to a stronger fishing pressure . AchieviOur case study from the Baltic Sea revealed that EBM approaches to fisheries require model systems that account for multi-species trophic interactions and have the ability to link ecology and economy Our study area has been shown to be well suited for addressing applied ecological questions relevant to EBM pa while at the same time maintaining the equity level, may provide a reasonable compromise, i.e., a zone of ‘new consensus’ Our results confirm that triple-bottom-line management solutions are usually costly Last but not least, operationally applying ecological-economic models systems in a way demonstrated in our study will facilitate coordinated management decisions among interacting use sectors as well as stakeholder involvement, both critical components in EBM approaches leading to increased societal values of exploited ecosystems Materials S1Ecological-economic model, equity, and programming code.(DOC)Click here for additional data file.Materials S2Sensitivity analysis. Including Figure S1 (Relationship between the parameter of the welfare function [measured in Euros per kg of sprat spawning stock biomass] and the resulting steady-state spawning stock biomass of sprat), as well as Figure S2 (Relationship between the parameter of the welfare function and the resulting steady-state spawning stock biomass of sprat (a), and between the parameter of the welfare function and the resulting steady-state equity, as measured by 1 – the GINI coefficient.(DOCX)Click here for additional data file.Materials S3Single versus multi-species management. Including Figure S3  in terms of spawning stock size (full line) and profit (dotted line) versus optimal multispecies management (blue lines); data from profit maximizing model run 2010-2040; trade-offs between cod and sprat (left panel), cod and herring (middle) and herring and sprat (right).(DOCX)Click here for additional data file.Table S1Parameters of the multi-species ecological-economic model. Subscripts C, S, H refer to cod, sprat and herring, respectively.(DOCX)Click here for additional data file.Table S2Biomass limits using the precautionairy approach (Bpa) and limit biomass levels (Blim) as well as different target fishing mortality rates depending on availability.(DOCX)Click here for additional data file.