Mast cell (MC) degranulation is the foundation of the acute phase of allergic rhinitis (AR). Previously, downregulation of GATA binding protein 3 (GATA-3) was shown to suppress MC activation in an AR mouse model. Binding of microRNA-135a (miR-135a) to GATA-3 was also observed, and overexpression of this miRNA decreased GATA-3 mRNA and protein expression. However, the effects of miR-135a on MCs during AR are currently unknown. In the present study, we utilized a lentiviral (LV) vector to intranasally administer miR-135a to ovalbumin (OVA)-sensitized AR mice. Following miR-135a treatment, the total serum IgE concentration observed during AR was significantly reduced. In the nasal mucosa, the expression of T-box expressed in T cells (T-bet) was higher, whereas that of GATA-3 was lower in the AR mice following miRNA treatment. Notably, during AR, the ratio of type 1 T-helper cells (Th1) to type 2 (Th2) cells in the spleen is unbalanced, favoring Th2. However, administering miR-135a to the AR mice appeared to balance this ratio by increasing and decreasing the percentage of Th1 and Th2 cells, respectively. MiR-135a also appeared to strongly suppress the infiltration of eosinophils and MCs into the nasal mucosa, and it was specifically localized in the MCs, suggesting that its influence is modulated through regulation of GATA-3 in these cells. Additional work identifying the full therapeutic potential of miR-135a in the treatment of AR and diseases involving allergen-induced inflammation is warranted. Allergen-induced inflammation of the upper airway, also known as allergic rhinitis (AR), is characterized by a variety of symptoms, all of which are caused by an inappropriate or exaggerated immune reaction involving numerous cytokines and pro-inflammatory factors. Mast cells (MCs), specifically the granules in their cytoplasm that are known to contain a variety of powerful biologically active inflammatory mediators, have been shown to play a particularly critical role in AR. In fact, MC degranulation is the foundation of the acute phase of AR, whereby the percentage of MC degranulation reflects the degree of the allergic reaction for the patient ,2. NotabUnderstanding the regulatory pathways involved in gene expression during AR-related inflammation, including that of GATA-3, is essential to provide potential therapeutic targets. In the last two decades, microRNAs (miRNAs or miRs) have been shown to suppress gene expression at the post-transcriptional level by base pairing to the 3′-untranslated region (3′-UTR) of their target mRNA, thus inhibiting protein translation and/or inducing degradation –6. FurthIn the present study, we sought to identify the effects of miR-135a-mediated GATA-3 regulation on the MCs of mice with AR to further understand the pathogenesis of this common allergen-induced immune response. To this end, we utilized lentiviral (LV) miR-135a to infect ovalbumin (OVA)-sensitized AR mice, which were then used to determine the localization and effects of miR-135a overexpression on GATA-3 and MC in AR mice.ad libitum. All procedures were approved by the Animal Ethics Committee at Renmin Hospital, Wuhan University .Pathogen-free male BALB/c mice  were purchased from the Wuhan Institute of Biological Products Co., Ltd , and divided into four experimental groups (n = 12 each): normal (control), AR , positive , and negative . All animals were housed in the campus animal facility under standard conditions, including typical light/dark cycles, average temperature (18–22°C), and mild humidity (50–60%), and were given food and water Following a five-day adaptation period, AR was induced in the AR, negative, and positive groups as previously described . BrieflyThe mice were intraperitoneally administered 300 μl of saline containing 100 μg OVA and 1 mg of aluminum hydroxide  on days 0, 7, and 14 to promote primary sensitization. On days 21–35, mice in these three groups were intranasally sensitized with 40 μl of saline containing 400 μg of OVA (20 μl per nostril) for secondary immunization. The normal group was intraperitoneally injected with 300 μl of saline on days 0, 7, and 14, and then treated intranasally with 40 μl of saline on days 21–35 after the initial treatment .6 IFUs of the LV miR-135a were intranasally administered to mice that were fully awake in the positive group within 3 h of AR induction on days 21–35 according to a previously established protocol [6 IFUs of an empty LV vector, whereas the normal group was treated with saline . All of the LV vectors utilized in this study expressed enhanced green fluorescent protein (GFP), which enabled fluorescent localization. Following construction, 2 × 10h saline .Blood was collected from the orbital venous plexus of anesthetized mice 24 h after the final intranasal sensitization. The samples were centrifuged for 10 min at 1000 × rpm to isolate the serum and stored at −80°C until use. Total serum IgE was determined using a Mouse IgE ELISA Kit .TM, NY, USA) according to the manufacturer’s instruction. The expression of miR-135a, T-bet, and GATA-3 was quantified by real-time quantitative polymerase chain reaction (RT-qPCR). We utilized specific target gene primers purchased from Invitrogen Biotechnology Co. (China) for miR-135a (#MIMAT0000147), T-bet (#NM019507.2), and GATA-3 (#NM008091) in conjunction with FastStart Universal SYBR Green Master Mix (Rox) according to the manufacturer’s protocol . A SLAN® Real-Time PCR Detection System  was used with the following cycling conditions: denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 15s and 60°C for 60 s. A final extension was performed at 60°C for 5 min. Data were analyzed using the 2−ΔΔCT method as previously described [Total RNA from the nasal mucosa was extracted using TRIzol reagent . The RNA (2 μg) was then reverse transcribed using a RevertAid First Strand cDNA Synthesis Kit  for 30 min. Unbound antibody was then washed away with flow cytometry staining buffer twice at 1500 × rpm for 5 min. Samples were permeabilized with a fixation/permeabilization working buffer for 1 h and then washed with permeabilization buffer twice at 2500 × rpm for 5 min. Next, the samples were incubated with 1 μl of purified anti-mouse CD16/CD32  as an Fc block for 15 min, and then stained with 3 μl of anti-mouse T-bet-PE  or 5 μl of anti-mouse GATA-3-PE antibody  for 30 min. All incubations and washes were performed at 4°C. Cells were analyzed on a FACScan flow cytometer .In Th cells, the transcription factors T-bet and GATA-3 are specific to Th1 and Th2, respectively –17. Thusanalysis . All reaMice were painlessly sacrificed with an intraperitoneal injection of 1% pentobarbital sodium 24 h after the last intranasal sensitization treatment. The heads of the mice were then removed and fixed with 4% paraformaldehyde overnight, followed by decalcification in an EDTA decalcifying solution for two weeks. The specimens were dehydrated in a series of increasing concentrations of ethanol and embedded in paraffin. Coronal sections (5 μm thick) of the nasal mucosa were obtained and histologically analyzed with hematoxylin and eosin (H&E) and MC staining to visualize eosinophils and MCs. These two cell types were counted in five randomly selected high-power fields (HPFs) at 400× magnification.Laser scanning confocal microscopy was used to visualize the localization of LV-mmu-miR-135a in MCs. Sections of the nasal mucosa obtained above were dried in a 60°C drying oven for 30 min, deparaffinized, and rehydrated using a standard protocol. The specimens were then incubated in an EDTA-Tris antigen retrieval solution at 90°C for 30 min. After recovery at room temperature, specimens were blocked with 10% normal goat serum for 1 h at room temperature and immunostained with a monoclonal rabbit anti-mouse MC tryptase antibody  overnight at 4°C. IgG isotype controls were used for each group of specimens at the same concentration. The sections were then washed in phosphate-buffered saline (PBS) three times and labeled with fluorescently tagged goat anti-rabbit secondary antibody  for 1 h in darkness at room temperature. Sections were also incubated with the fluorescent nuclear dye DAPI  to determine the specific location of LV-mmu-miR-135a expression. The sections were again washed with PBS three times, and then mounted and sealed with antifade mounting medium .The nasal mucosa sections were evaluated within 24 h of staining using a laser scanning confocal microscope . LV-mmu-miR-135a localization was observed in HPFs at 400 × magnification.All data are presented as the mean ± SEM. Differences between groups were compared using the non-parametric Kruskal-Wallis test, followed by the Mann-Whitney U test. Statistical calculations were performed using IBM SPSS Statistics 19.0 software  and P < 0.05 was considered significant.To determine the effect of LV miR-135a on the allergic response observed in the AR mice, we measured the level of serum IgE . The lev12. To further understand the role of miR-135a during AR in the present investigation, we sought to overexpress miR-135a in the nasal mucosa of AR-induced mice via intranasal infection with an LV vector containing miR-135a. Notably, after AR mice were treated with LV miR-135a (positive group), miR-135a was significantly upregulated, indicating successful LV-host gene transduction in the nasal passages of these mice  for the AR group indicates a Th2 bias, as expected, and treatment with LV miR-135a appears to decrease this ratio back to normal physiological levels.Th cell polarization, or the ratio of Th1 cells to Th2 cells, was measured by cytometrically counting the number of CD4ch mouse . As showal group . NotablyIncreased infiltration of leukocytes is a primary index used to diagnose AR. Thus, to evaluate the effect of miR-135a on the histological profile of our OVA-induced AR mouse model, we stained the nasal mucosa with H&E and MC staining solutions. The cytoplasm of the eosinophils was stained red , whereasLaser scanning confocal microscopy was used to determine whether LV miR-135a localized specifically in the MCs of treated AR mice. As shown in In the present study, we investigated the effects of LV miR-135a on the allergic response that occurs during OVA-induced AR-related inflammation in a mouse model. Inflammation in any tissue is known to involve the activation of eosinophils and degranulation of MCs, which in turn release a variety of pro-inflammatory mediators that can cause tissue damage ,19,20 anin vitro that appears to be caused by changes in the targeted transcription of v-Maf musculoaponeurotic fibrosarcoma oncogene homolog (c-Maf), resulting in the upregulation of IL-4 [in vivo is limited.Currently, the treatment options for patients with AR include allergen avoidance, pharmacotherapy, and immunotherapy. Recently, many researchers have indicated that miRNAs are relatively stable compared to many other mRNA moieties and even some proteins. This stability could be exploited to clinically modulate gene expression in a number of diseases, including AR and allergen-induced inflammation ,24. In f of IL-4 ,28. MiR- of IL-4 . AlthougFurthermore, although miRNA toxicity is likely minimal , the delOur decision to focus on the therapeutic potential of miR-135a stems from our previous work indicating that this miR-135a can specifically bind to GATA-3 and that overexpression of miR-135a in AR mice decreases the mRNA and protein expression of both GATA-3 and IL-4 . Here, tIn our study, we found that LV miR-135a significantly decreased the number of MCs in the lamina propria tissue in the positive group, but the number of MCs in the epithelial layer was not significantly different across the AR and positive and negative groups. Amin  indicateLaser scanning confocal microscopy indicated that our vector not only successfully transfected the nasal mucosa but also specifically infected the MCs, as shown by the overlap of miR-135a expression with expression of tryptase, a known marker of mature MCs ,37. TakiOverall, the present study has demonstrated that LV miR-135a can be used to correct Th1/Th2 imbalances in the nasal mucosa and spleen of AR-induced mice. Administration of LV miR-135a also appears to strongly suppress the allergen-induced inflammation commonly observed in AR mice, which includes increased total serum IgE concentrations, infiltration of eosinophils and MCs in the nasal mucosa, and increased GATA-3 expression, all of which returned to normal levels following treatment. It is likely that LV miR-135a-mediated changes in MCs via direct targeting of GATA-3. Although additional work is necessary to fully elucidate the function of miR-135a during AR, this study provides a foundation for the use of intranasally administered miRNA-containing LV vectors for treatment of allergen-induced inflammation.S1 FigA is a representative image (magnification:100×) of a lung in the positive group; B is a representative image (magnification:100×) of a lung in the negative group.(TIF)Click here for additional data file.


FOXO (Forkhead box O) transcription factors as important determinants in aging and longevity. FOXO proteins represent a subfamily of transcription factors conserved from Caenorhabditis elegans to mammals that act as key regulators of longevity downstream of insulin and insulin‐like growth factor signaling. Invertebrate genomes have one FOXO gene, while mammals have four FOXO genes: FOXO1, FOXO3, FOXO4, and FOXO6. In mammals, this subfamily is involved in a wide range of crucial cellular processes regulating stress resistance, metabolism, cell cycle arrest, and apoptosis. Their role in longevity determination is complex and remains to be fully elucidated. Throughout this review, the mechanisms by which FOXO factors contribute to longevity will be discussed in diverse animal models, from Hydra to mammals. Moreover, compelling evidence of FOXOs as contributors for extreme longevity and health span in humans will be addressed.Aging constitutes the key risk factor for age‐related diseases such as cancer and cardiovascular and neurodegenerative disorders. Human longevity and healthy aging are complex phenotypes influenced by both environmental and genetic factors. The fact that genetic contribution to lifespan strongly increases with greater age provides basis for research on which “protective genes” are carried by long‐lived individuals. Studies have consistently revealed  This decline constitutes the key risk factor for age‐related diseases such as cancer and cardiovascular and neurodegenerative disorders. It is therefore not surprising that humankind has put considerable efforts in understanding the processes of aging (and how to delay them) since ancient times.et al., et al., et al., et al., et al., Human longevity and healthy aging are complex phenotypes influenced by environmental  and genetic factors  later in life, thereby confirming the compression of morbidity hypothesis proposed by Fries in 1980  Kenyon, . IGF‐1 iet al., et al., et al., et al., et al., C. elegans to mammals but, while invertebrates have only one FOXO gene, mammals have four FOXO genes: FOXO1 (FKHR), FOXO3 (FKHRL1), FOXO4 (AFX), and FOXO6  was postulated to be causative for aging, it has been hypothesized that FOXO factors influence aging and age‐related diseases by increasing the antioxidant capacity of cells , and adulthood. If the environmental conditions are not favorable , L1 worms develop into alternative larval form called the dauer larva , a hormone receptor similar to insulin and IGF‐1 receptors, doubled the lifespan of the worm .DAF‐16 functions as the major target of the IIS pathway , which is regulated by DAF‐16/FoxO activity in the intestine, and that this regulation allows DAF‐16 activity in the intestine to influence DAF‐16 activity in other tissues. But insulin‐like peptides may not be the only signals that act downstream of DAF‐16/FoxO to influence lifespan. In daf‐16; daf‐2 double mutants, the expression of DAF‐16 specifically in one tissue (intestine/adipose tissue) can increase the worm lifespan by 60%  homologous to mammalian insulin receptors yields dwarf females with up to an 85% extension of adult longevity and dwarf males with reduced late age‐specific mortality  encodes an insulin receptor substrate that functions in the IIS pathway. Mutation of this gene extends fruit fly median lifespan by up to 48% in homozygotes and 36% in heterozygotes , and ablation of the cells expressing only three of the seven dilps in neuroendocrine cells of the brain is sufficient to increase longevity , delaying muscle functional decay and extending lifespan  will enlarge our knowledge of how FOXO factors affect cellular/organismal lifespan. To further comprehend how FOXOs affect longevity, it is of high importance to understand how human FOXO sequence variants (namely FOXO3A) affect protein expression, its structure, or transcriptional activity. In order to see how these variants translate into physiological profiles, future investigations should address how these variants affect the level of FOXO proteins and their downstream effectors in serum. This approach has been used successfully in patients with vitiligo, in which FOXO3A levels were shown to be decreased when compared with the control group (Ozel Turkcu Compression of morbidity relates to both extended lifespan and delayed onset of age‐related diseases, such as cancer and cardiovascular disorders. The development of molecules targeting aging mechanisms that underlie a number of age‐related diseases is an exciting field that is nowadays in its first steps. It is noteworthy that clinical trials to test lifespan extension in humans would be challenging and require markers that can detect difference in aging rate across a short time frame. But given the potential of FOXO proteins to impact on numerous disorders such as cancer, diabetes, neurodegeneration, or immune system dysfunction, novel therapeutic modalities based on FOXOs will most likely take place in the near future.None declared.


Prostate cancer (PCa) is the major cause of cancer-related death among the male population of Western society, and androgen-deprivation therapy (ADT) represents the first line in PCa treatment. However, although androgen receptor (AR) expression is maintained throughout the various stages of PCa, ADT frequently fails. Clinical studies have demonstrated that different androgen/AR signaling pathways operate in target tissues. AR stimulates growth and transformation of target cells, but under certain conditions slows down their proliferation. In this review, we discuss the role of AR in controlling different functions of mesenchymal and transformed mesenchymal cells. Findings here presented support the role of AR in suppressing proliferation and stimulating migration of stromal cells, with implications for current approaches to cancer therapy. Prostate cells are the primary targets of androgens, which regulate development, growth, and function of prostate. In this organ, androgens act through the androgen receptor (AR), which is expressed in both epithelium and stroma, and studies with recombinant tissues indicate that prostatic development is mediated by stromal, but not epithelial AR . Sex steThe role of epithelial AR in PCa has been extensively studied in the last few decades. There are, however, many other AR target tissues throughout the body , though AR paradoxically fosters or inhibits proliferation of target cells depending on cell type, microenvironment, and hormone levels . ReciproThe dichotomous response (migration/proliferation) of fibroblasts to different androgen concentrations mimics that of growth factors, which trigger motility or proliferation [reviewed in Ref. ]. A flurHere, we discuss the opposite (proliferative or migratory) functions of stromal AR and the role of the upstream AR/FlnA complex in stromal cell decision to “go or grow.” Since the AR/FlnA complex drives androgen signaling toward migration and halts cell cycle, this complex might be specifically targeted, with implications in the therapy of AR-related human diseases. The potential role of non-genomic signaling activated by androgen/AR axis in cancer-associated fibroblasts (CAFs) is also discussed.AR mRNA levels are very similar in the stroma and epithelium, although AR-mediated transcription is differentially regulated in each, with a more active co-regulator recruitment by epithelial AR.In the early 2000s, we made the exciting finding that NIH3T3 cells and mouse embryo fibroblasts (MEFs) harbor a transcriptionally incompetent AR , 11, 20.A few years ago, by recombination of prostate stromal WPMY-1 cells with PCa epithelial PC-3 cells in a mouse model, Niu and colleagues showed that stromal but not epithelial AR promotes tumor proliferation at very early stage . NoteworProstate CAFs express AR, although the role of this receptor in prostate tumorigenesis is still unclear. Loss of AR expression in the tumor stroma, but not in the surrounding normal prostatic stroma tissue, increases the risk of relapse following radical prostatectomy , 24. StrWe have dissected in recent years the pathways activated by different androgen concentrations in fibroblasts, and this approach has revealed some fascinating aspects. Low androgen concentration 1 pM) induces proliferation in NIH3T3 fibroblasts as a consequence of AR association with Src and p85, the regulatory subunit of PI3-K . This co pM inducOn this basis, we may hypothesize that AR-mediated activation of MAPK induces the release of chemokines and growth factors by CAFs when androgen levels are low. Indeed, MAPK activation increases growth factor release in various cancer cell types . Byin vitro and in vivo . These events lead to cell motility and simultaneous cell cycle arrest in mesenchymal and transformed mesenchymal cells. Interference in AR/FlnA complex assembly by new molecules, such as AR-derived peptides , may repThe Review Editor Daniela Pasquali declares that, despite being affiliated to the same institution as the authors, the review process was handled objectively and no conflict of interest exists. 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.


In this study, we examined regions in the left and right hemisphere language network that were altered in terms of the underlying neural activation and effective connectivity subsequent to language rehabilitation. Eight persons with chronic post-stroke aphasia and eight normal controls participated in the current study. Patients received a 10 week semantic feature-based rehabilitation program to improve their skills. Therapy was provided on atypical examples of one trained category while two control categories were monitored; the categories were counterbalanced across patients. In each fMRI session, two experimental tasks were conducted: (a) picture naming and (b) semantic feature verification of trained and untrained categories. Analysis of treatment effect sizes revealed that all patients showed greater improvements on the trained category relative to untrained categories. Results from this study show remarkable patterns of consistency despite the inherent variability in lesion size and activation patterns across patients. Across patients, activation that emerged as a function of rehabilitation on the trained category included bilateral IFG, bilateral SFG, LMFG, and LPCG for picture naming; and bilateral IFG, bilateral MFG, LSFG, and bilateral MTG for semantic feature verification. Analysis of effective connectivity using Dynamic Causal Modeling (DCM) indicated that LIFG was the consistently significantly modulated region after rehabilitation across participants. These results indicate that language networks in patients with aphasia resemble normal language control networks and that this similarity is accentuated by rehabilitation. Most studies of language recovery have examined the recovery process in the chronic stage and have found that the recovery of language function in aphasia is a more complex process than a simple reversal of normal left hemisphere lateralization or exclusive recruitment of left perilesional and other left language areas, and likely reflects a combination of the two  as well as the cognitive mechanism targeted in therapy (semantic processing during the semantic feature verification). Thus, in addition to the fact that both the picture naming and semantic feature tasks have been well-examined in fMRI experiments across both patients with aphasia and normal controls, these tasks are well-tailored to the behavioral language rehabilitation implemented in the study and compensate for the modest number of participants by providing within-subject replication across similar tasks. The following were the research aims and hypotheses proposed in the present study:To examine behavioral changes in patients with aphasia who receive a semantic feature-based naming therapy. Our previous work has shown that a theory based rehabilitation aimed at improving lexical retrieval for atypical examples results in improvement of trained items as well as generalization to untrained items within the same category , inferior parietal regions , precentral gyrus (PCG) and the dorsolateral , and inferior frontal regions (IFG), in patients with aphasia who have residual tissue in these regions. Furthermore, we expect changes in activation in these regions in patients who show improvements in language function after rehabilitation.To explore changes in effective connectivity using DCM. Given the current robust evidence for regions involved in the two tasks examined in this experiment, DCM is the ideal methodology as it is hypothesis-driven and allows researchers to examine whether rehabilitation changes the nature and strength of connectivity between language regions in patients. Importantly, DCM has been used to examine the nature of the damaged or reorganized network in clinical populations  or BNT scores  , all of whom had a single stroke, participated in the study. All participants had infarcts in the left hemisphere with the exception of #5 who had a stroke in the right hemispherer = 0.65, p < 0.05) indicating that the naming impairment was consistent with their overall language impairment profile. All participants gave consent according to BU IRB protocol.In addition, six tests assessing semantic and phonological processing were administered to examine the nature of phonological processing, semantic processing and naming. While performance varied across participants, semantic processing scores were higher than phonological processing scores in general  with no history of brain damage were recruited to obtain normative data on task-specific activation for the two language tasks. Exclusionary criteria included neurological disorders such as stroke, transient ischemic attacks, Parkinson's disease, Alzheimer's disease, psychological illness, learning disability, seizures, and attention deficit disorders. The controls did not receive language therapy and were scanned at only one time point. Half the controls were scanned as part of a different ongoing experiment, the details of which are provided in Supplementary Table birds, vegetables, furniture, clothing, and musical instruments) were used to study naming and semantic processing during the rehabilitation tasks across the eight patients. Twenty-four items were selected for each category that comprised typical and atypical examples . Patients were exposed to three of the five categories: a trained category (probed weekly), an assessed category (only tested before and after rehabilitation) and a monitored category (probed at the same frequency as the trained category). The assignment of trained, untrained, and monitored categories was counterbalanced across participants and constrained by their initial naming accuracy. Hence if the patient named more than 70% of the items in the category at baseline, that category was eliminated from potential set of stimuli for that patient. Each category contained 12 typical and 12 atypical examples that were selected from our previous rehabilitation studies   items accurately across two consecutive sessions. Subsequently, three post-rehabilitation naming probes, using the same procedures as the baseline and rehabilitation probes, were administered to calculate the efficacy of rehabilitation. Effect sizesBoth patients and control participants participated in the fMRI experiment. As noted before, the controls completed the fMRI experiment once in order to identify regions of activation that are normally engaged for the two tasks. All patients completed two fMRI scans, one during the baseline testing phase (pre-treatment scan) and one during the post-rehabilitation phase (post-treatment scan).For both picture naming and semantic feature tasks . Four different runs of the picture naming task were used, each containing 20 experimental stimuli (pictures to name) and 20 control stimuli (scrambled pictures). Two runs consisted of stimuli from the trained category and the other two runs consisted of stimuli from the untrained categories. Participants were required to name each picture aloud or say “SKIP” for pictures they could not name. The control stimuli were pixelated, scrambled versions of the experimental stimuli. Participants were required to say “SKIP” for each control item. The experimental pictures were presented for 5 s while the scrambled control stimuli were presented for 3 s.four categories. In both the experimental and control conditions, each trial consisted of a picture prime (presented for 1 s) followed by the target stimulus (presented for 4 s). Half of these prime stimuli and their corresponding target stimuli were presented in black and white and the other half were presented in color. The target stimulus was the prime picture repeated with a written phrase below. This design was used to provide enough time for patients with aphasia to process the visual attributes of the picture prior to making a decision. In the experimental condition, the written phrase was a semantic feature, and participants indicated by button press whether the written semantic feature applied to the pictured item. The control stimuli contained a scrambled picture (presented in either black-and-white or color) and a statement regarding the color of the scrambled picture (either “black and white” or “color”). Participants indicated by button press whether the statement regarding the color of the picture was true or false  sensitive functional images were collected using the following parameters: 31 axial slices, 3 mm thick, 0.3 mm interslice gap, 80 × 78 matrix, FOV = 240 mm, flip angle = 90, fold-over direction = AP, TR = 2000 ms, TE = 35 ms. Picture naming responses were recorded in software OptiMRI 2.4  with live noise cancelation. Semantic feature responses were recorded using a left hand button response box for both groups of participants.Magnetic resonance images were acquired at Boston University Center for Biomedical Imaging on a 3 Tesla Philips Acheiva MRI scanner. High-resolution T1-weighted images were acquired with the following parameters: 140 sagittal slices, 1 mmhttp://www.cabiatl.com/mricro/) . Slice timing correction was applied with reference to the middle slice. Structural scans were coregistered to a mean functional image obtained from realignment performed on functional scans for motion correction. For each patient, a lesion map was drawn on their T1 image using MRIcron  was utilized. All fMRI images/volumes were registered to mean slice. Also, the realignment parameters (motion correction parameters) were used as regressors in the first level GLM analysis. In addition, for specific individuals , volumes with large variations (>0.05 mm) in scan-to-scan motion were repaired via linear interpolation using the ArtRepair toolbox in SPM8 ]-[pre-rehabilitation (picture-scrambled)] for each task. Activations maps were thresholded at a family wise error threshold (FWE) < 0.05. Uncorrected p < 0.001 activations were examined when FWE thresholds were not significant. Coordinates for activated voxels were entered into the Anatomy toolbox, v.17, to obtain the label for each active region. For normal controls, all four runs (two from each category) were combined into one GLM for each task as no treatment was provided.First level analysis was performed based on the General Linear Model (GLM) in SPM8. Task timings (stimulus onsets and durations) were convolved with the canonical hemodynamic response function (HRF) and its temporal derivative. Conditions included pictures, scrambled pictures, and fixations, and were modeled in the GLM separately for each scan. Motion parameters were included in the model as regressors. Serial correlations were accounted for using an AR (1) model. The model was estimated using a restricted maximum likelihood approach (ReML). For the patients, pre-rehabilitation and post-rehabilitation scan activation maps were calculated based on Effective connectivity analysis was applied using the DCM toolbox in SPM8. DCM uses differential equations to model inter-regional interactions to infer their directionality and context-dependent modulations . This resulted in 12 VOIs for the picture naming task and 12 VOIs for the semantic feature task. Within an active cluster, the voxel with the highest T-value was selected. Subject-specific eigenvariates were extracted as spheres of 5 mm around the MNI coordinate of the peak voxel and adjusted for the effect of interest.All normal healthy controls showed overlapping activation in language areas for each task; thus, a common model space was constructed for this group  was extracted for each of the two tasks using the same procedure as that described for normal controls. All DCM models were deterministic, bilinear, two-state with mean-centered inputs. The DCM models were set up based on guidelines from previous studies  present at both the pre- and post-rehabilitation scan (at t-tests) to understand changes after rehabilitation  with random-effects (rfx) was initially performed on each model space to find the best fit model. Due to the variability in activation within patients, the best fit model was not uniform across patients and, hence, was not consequently pursued for controls or patients. Instead, for both groups, BPA was computed to investigate connectivity parameters for each connection (Ep.B) and region (Ep.C). For controls, these connectivity parameters were plotted to create a normal language network for each task that then served as a reference framework to evaluate the patient rehabilitated networks. For patients, BPA parameters were computed for trained and untrained categories separately. These subject-specific parameter estimates were used for second-level analysis  = 17.3, p < 0.0001] and percent change ; post-hoc tests show a significantly larger effect size and percent change for the trained category relative to the untrained assessed category (p < 0.0001) and the untrained monitored category (p < 0.00001). Therefore, a clear effect of rehabilitation on the trained category was observed.As can be seen in Table Activation patterns for normal controls are provided for each individual control across regions that are broadly involved in language processing. Table F = 4.00, p < 0.001]. Figure p values significant at least <0.05).Each of the VOIs identified in Tables F = 11.3, p < 0.0001]. Figure p values significant at least <0.05). Due to the high number of different connections and limited number of participants, neither of the ANOVAs performed on the Ep.B  values for the two tasks was significant. To summarize, the goal of the control analysis was to use the normal control VOIs and corresponding DCM analysis (which includes a broader set of VOIs than is available for patients) as a reference for the interpretation of patient DCM connectivity changes.Likewise, a one-way ANOVA using Ep.C estimates as the dependent measure and input to regions as the independent measure across the eight participants for the semantic feature verification task was found to be significant , the interaction between rehabilitation and the effect size covariate was also not significant  and finally, the interaction between rehabilitation and category also was not significant . Second, when estimates on regions (Ep. C) were examined, the effect of rehabilitation was significant ; while the interaction between rehabilitation and effect size covariate was not significant , the interaction between rehabilitation and category was significant . Post-hoc LSD tests showed lower Ep.C values after rehabilitation for the trained category but not for the untrained category .For the picture naming task, when estimates on connections (Ep.B) were examined, the effect of rehabilitation was not significant , the interaction between rehabilitation and effect size covariate also was significant , and finally, the interaction between rehabilitation and category was significant . Post-hoc LSD tests showed higher Ep.B values after rehabilitation for the untrained category than the trained category . When examining the estimates on regions (Ep.C), the effect of rehabilitation was not significant , and the interaction between rehabilitation and the effect size covariate was not significant . Finally, the interaction between rehabilitation and category also was not significant . These results, when significant, point to lower BPA values for the trained relative to the untrained category. They, however, do not explain which regions change their connectivity as a function of rehabilitation and if the network changes are consistent from patient to patient.Similar analyses were performed for the semantic feature task. First, when estimates on connections (Ep.B) were examined, the effect of rehabilitation modulation was significant [One likely reason for the lack of significance in some of the analyses is inter-participant variability. Therefore, individual repeated measures ANOVAs on pre- and post-estimates as dependent measures for both Ep.B and Ep.C and participants as the independent variable were performed for the trained category for the picture naming and semantic feature tasks (see Supplementary Section). All the analyses showed a significant patient-by-rehabilitation interaction, indicating that certain patients showed greater differences between their pre- and post-treatment Ep.B and Ep.C estimate values for both tasks. Therefore, it is likely that inter-subject differences overshadowed any group level differences.t-tests for individual participants. For each patient, paired t-tests were performed on pre-rehabilitation and post-rehabilitation averaged BPA Ep.B  over the entire model space with a significance criterion set at p < 0.05. As displayed in Figure Therefore, as in the fMRI analysis, individual patient data were analyzed before and after rehabilitation as paired Likewise, for the semantic feature verification task  all patients improved as a function of rehabilitation, and improvements across patients were greater for the trained categories than the untrained categories, (b) in terms of the fMRI changes in activation, there were several regions such as LIFG, RIFG, LPCG, LMFG, RMFG, LMTG, and RMTG that were consistently active in normal controls and in several of post > pre-rehabilitation comparisons for patients, and (c) in terms of connectivity changes across patients, LIFG was the most modulated region, independent of task and as a function of rehabilitation. Each of these results will be addressed in greater depth below.As noted in the introduction, naming deficits are pervasive in individuals with aphasia and a semantic feature-based treatment that emphasizes semantic feature analysis and phonological access was effective in improving word retrieval even in individuals with chronic aphasia. While we have examined generalization to untrained items in our previous work, in this study we instead chose to examine the changes on the trained category (including both trained and untrained items) only using two tasks (picture naming and semantic feature verification) that were germane to the rehabilitation. Results showed that a semantic-feature based naming intervention resulted in improvements in naming function in all patients on the trained category. While the efficacy of this intervention has been examined before  look qualitatively different from the normal language network (captured at one time point). These results can be interpreted as follows. First, task-specific modulation for normal controls indicated a bilateral network for picture naming and a left-lateralized network for semantic feature verification. These results are consistent with findings of an fMRI study that examined semantic categorization (predominantly semantic processing) and rhyme detection  in normal individuals  and the corresponding modulations of regions indicative of their relative contributions within the network. The individual patient networks, in contrast, are indicative of reorganized or altered networks that have not completely returned to their normal-like function  indicates that there is a systematic way the network reorganizes for language recovery as a function of rehabilitation.The small number of healthy controls and patients involved in the study limits the conclusions that can be drawn from the study. Also, the inherent variability in lesion and behavioral profiles across the participants warranted all the behavioral, neuroimaging and connectivity data to be analyzed at the individual participant level. While this type of analysis , required several modifications to the traditional group level analysis, the explicit attempt to account for and detail the inherent variability across the patients with aphasia is a clear strength of this study. Within the patient group, there are several levels of controls and replications that have been incorporated in the experimental design which allow meaningful interpretations to be drawn from the study (Kiran et al., 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.


Pre-treatment of cultured cells with (R)-(−)-2-oxothiazolidine-4-carboxylic acid (OTC), a cysteine analog rescued the cytosolic FoxO. At these concentrations, MAPK phosphorylation, in particular p38 and ERK, and PPAR gamma coactivator-1 (PGC-1) levels were increased, while AKT phosphorylation remained unchanged. FoxO phosphorylation level was markedly reduced with the use of SB203580 (a p38 MAPK inhibitor) and PD98059 (an ERK inhibitor). We conclude that FoxO phosphorylation after Mn exposure occurs in parallel with, and independent of the insulin-signaling pathway. FoxO levels and its translocation into the nucleus are part of early events compensating for Mn-induced neurotoxicity and may serve as valuable targets for neuroprotection in the setting of Mn-induced neurodegeneration.Environmental exposure to manganese (Mn) leads to a neurodegenerative disease that has shared clinical characteristics with Parkinson's disease (PD). Mn-induced neurotoxicity is time- and dose-dependent, due in part to oxidative stress. We ascertained the molecular targets involved in Mn-induced neurodegeneration using astrocyte culture as: (1) Astrocytes are vital for information processing within the brain, (2) their redox potential is essential in mitigating reactive oxygen species (ROS) levels, and (3) they are targeted early in the course of Mn toxicity. We first tested protein levels of Mn superoxide dismutase -2 (SOD-2) and glutathione peroxidase (GPx-1) as surrogates of astrocytic oxidative stress response. We assessed levels of the forkhead winged-helix transcription factor O (FoxO) in response to Mn exposure. FoxO is highly regulated by the insulin-signaling pathway. FoxO mediates cellular responses to toxic stress and modulates adaptive responses. We hypothesized that FoxO is fundamental in mediating oxidative stress response upon Mn treatment, and may be a biomarker of Mn-induced neurodegeneration. Our results indicate that 100 or 500 µM of MnCl Environmental exposure to manganese (Mn) leads to manganism, characterized by dopaminergic (DAergic) neurodegeneration, with clinical findings in humans that are reminiscent of idiopathic Parkinson's disease (PD) The present study is related to the possible implications of Mn effects in brain cells, as Mn-neurotoxicity may have a role in neurodegenerative processes and disorders of the basal ganglia. The prevalence of PD and other neurodegenerative diseases are on the rise, and prevalence and incidence of PD in the general population is perhaps well underestimated Most cells, including astrocytes, have protective mechanisms against ROS R)-(−)-2-oxothiazolidine-4-carboxylic acid (OTC), a cysteine analog and a precursor of GSH, the major intracellular antioxidant, attenuated the levels of Mn induced F2-IsoP, and further increased total FoxO protein expression. OTC also led to changes in the subcellular localization of the FoxO proteins, reversing the subcellular changes induced by Mn exposure. These data suggest that regulation of FoxO may serve as potential therapeutic targets for Mn-induced neurodegenerative processes.Herein, we set out to determine FoxO-related biological targets associated with Mn exposure in cultured neonatal astrocytes. We show that Mn treatment (both at 6 and at 24 hrs) led to induction of mitochondrial superoxide dismutase 2 (SOD2), but no significant changes in glutathione peroxidase . We also show that F2-isoprostane (F2-IsoP) levels, biomarkers of ROS generation and lipid peroxidation, were elevated in response to Mn exposure. Both FoxO 3a  and PGC-1 protein levels were coordinately elevated at 6 and at 24 hrs after Mn exposure. In this paper, we explore a Mn-induced phosphorylation mechanism of FoxO in astrocytes. We tested whether it occurs through the insulin-signaling pathway and AKT or MAPK activation. Our data lead us to propose a protective and compensatory role for FoxO as a biomarker of neurodegeneration. We also propose a role for MAPK activation in FoxO signaling as a contributing effector in Mn-induced neurodegeneration. Pre-treatment of cultured cells with . MnCl2 was obtained from Sigma  and cells were exposed for 6 or 24 hrs. The following two Mn concentrations were used, 100 and 500 µM (as MnCl2), in all the experiments. In other sets of experiments, cells were pre-treated with (R)-(−)-2-oxothiazolidine-4-carboxylic acid (OTC) for 24 hrs at a concentration of 1 mM, (referred to as OTC pretreated cells). OTC was obtained from Sigma . Upon completion of the timed Mn exposures, the cells were rinsed with PBS and harvested for protein isolation and purification.Cultured astrocytes were exposed to MnWhole cell lysates were prepared from astrocyte cultures after Mn treatment with RIPA lysis buffer, as previously described 2-IsoPs levels were measured with an isotope dilution method and detected with gas chromatography/mass spectrometry (GC/MS), following standard protocols for purification and analysis, as previously described After Mn exposure, astrocytes were harvested and placed at −80°C until use. F2 treatment alone for 6 hrs, (3) OTC pretreatment for 24 hrs prior to exposure to MnCl2 for 6 hrs, and (4) OTC alone. Astrocytes were fixed in cold methanol and incubated on ice for 5 minutes. They were then placed at room temperature. Blocking was performed for 2 hrs at room temperature, using Power Block Universal Blocking Reagent . The primary antibodies, FoxO  and p-FoxO , were used at a dilution of 1∶50. All primary antibody dilutions were prepared in a diluent from BioGenex . Incubation of all primary antibodies was carried out for 2 hrs at room temperature. Secondary antibodies (Alexa Fluor 568 goat anti-rabbit IgGH+L) were prepared in the above-mentioned BioGenex diluent at a concentration of 2 µg/µl. Incubation of all secondary antibodies was also carried out at room temperature for 2 hrs. Finally nuclear staining was performed at room temperature for 20 minutes, using 1 µM of DAPI . After washing in DDH2O, coverslips were mounted onto glass-slides with mounting medium . High-resolution imaging was performed in the Cell Imaging Shared Resource Center at Vanderbilt University Medical Center.For the immunocytochemistry staining, we assessed expression of pFoxO and total FoxO in four different experimental groups of cultured astrocytes: (1) untreated (control cells), (2) MnClpost hoc test was performed for analyzing differences between treatment groups. Statistical significance was considered for P values <0.05.Data presented in this paper are reported as Mean ± S.D. of the mean. One-way analysis of variance (ANOVA) followed by Bonferroni's 2-IsoP levels. We found that F2-IsoP levels increased in response to Mn exposure. IsoP levels were 171.5±23.19 pg/mg protein for control cells, compared with 269.9±23.19 pg/mg protein after 6 hrs in Mn exposed astrocytes  and SB 203580 (SB) to inhibit MAPK/ERK and MAPK/p38 pathways, respectively. Astrocytes were pre-treated with each inhibitor for 30 min prior to Mn exposure. Pre-treated astrocytes with LY, SB or PD alone did not lead to changes in pFoxO levels . Upon MnGiven recent reports that cooperation between FoxO and PGC-1 are required for regulation of oxidative protective genes 2 (as described above). The histological findings were identical for all treated arms of the study. Cell imaging showed that phosphorylated FoxO levels in the cytosol were significantly reduced upon Mn exposure  and elevated levels of the antioxidant enzyme SOD-2. These results are in agreement with data previously published by our laboratory Gadd45), cell-cycle arrest (p27kip1), and anti-apoptotic effects (Bim and Fas ligand) Mn toxicity leads to elevated levels of FoxO . Downstream of the insulin signaling pathway lays a group of Forkhead subfamily of transcription factors, referred to as the FoxO group. Under conditions of low  protein kinase B (PKB) activity, FoxO protein is predominantly nuclear, where it interacts with essential cofactors for transcriptional activation of genes with context-dependent effects on cellular stress In our studies, both total levels and phosphorylated FoxO levels were elevated after exposure to Mn . DephospOur data suggest that this level of Mn-induced FoxO phosphorylation is accomplished in parallel with the insulin-signaling pathway. We found that Mn led to marked increase in MAPK/ERK phosphorylation levels, while the levels of phosphorylated AKT remained relatively unchanged . Taking Our data suggests that Mn exposure leads to elevated levels of phosphorylated MAPK/ERK and MAPK/p38 , which iOur data also suggest that Mn increases the levels of PGC-1α in astrocytes. PGC-1 protein levels were increased parallel with elevated levels of FoxO , and 7. Environmental exposure to Mn leads to a neurodegenerative illness that has shared pathophysiology and clinical findings with PD. It is hypothesized that increased oxidative stress and elevated ROS levels are at the basis of Mn-induced neuronal toxicity. The molecular intermediates leading to neuronal cell death remain unknown. Given their critical role in maintaining brain homeostasis, using astrocytes for the experiments is relevant to study FoxO and FoxO targets in Mn-induced neurotoxicity. We found that FoxO, MAPK, and PGC-1 were key intermediary molecules in Mn-induced oxidative stress response. Mn treatment induced elevated levels of total FoxO, with reduction of cytosolic pFoxO levels as seen by immunocytochemistry. Mn exposure also triggered MAPK/ERK and MAPK/p38 activations, leading us to believe that the observed phosphorylation or de-activation of FoxO occurred through MAPK, in parallel with the insulin-signaling pathway. Interestingly, phosphorylated MAPK induction was readily corrected with the use of antioxidants, showing simultaneous reduction in levels of phosphorylated FoxO but marked increase in total FoxO . Mn also led to elevated levels of PGC-1, a molecule hypothesized to co-regulate oxidant protective genes with FoxO


This case study features a woman who presented with clinical and laboratory findings consistent with hyperosmolar hyperglycemic non-ketotic syndrome (HHNS), systemic inflammatory response syndrome (SIRS), and non-thyroidal illness syndrome (NTIS) who was noted to have a transient decrease in myocardial function. To our knowledge, this is the first case discussing the overlapping pathophysiological mechanisms could increase susceptibility to SIRS-induced cardiomyopathy. It is imperative that this clinical question be investigated further as such a relationship may have significant clinical implications for prevention and future treatments, particularly in patients similar to the one presented in this clinical case.A 53-year old Caucasian female presented to the Emergency Department for cough, nausea, vomiting and “feeling sick for 3 weeks.” Labs were indicative of diabetic ketoacidosis. Initial electrocardiograms were suggestive of possible myocardial infarction and follow-up echocardiogram showed severely depressed left ventricular systolic function which resolved upon treatment of ketoacidosis.We suggest that her cardiomyopathy could have three synergistic sources: SIRS, HHNS and NTIS. Overlapping mechanisms suggest uncontrolled diabetes mellitus and NTIS could increase susceptibility to SIRS-induced cardiomyopathy as seen in this case. HHNS and SIRS cause cardiac tissue injury through mechanisms including impairment of fatty acid oxidation and formation of reactive oxygen species, as well as modifying the function of membrane calcium channels. As a result, it is conceivable that diabetes may amplify the deleterious effects of inflammatory stressors on cardiac myocytes. This novel case report offers a path for future research into prevention and treatment of SIRS-induced cardiomyopathy in, but not exclusive to, the setting of diabetes. This case study features a woman who presented with clinical and laboratory findings consistent with hyperosmolar hyperglycemic non-ketotic syndrome (HHNS), systemic inflammatory response syndrome (SIRS), and non-thyroidal illness syndrome (NTIS) who was noted to have a transient decrease in myocardial function. We suggest that her cardiomyopathy could have three synergistic sources: SIRS, HHNS and NTIS. Overlapping mechanisms suggest uncontrolled diabetes mellitus and NTIS could increase susceptibility to SIRS-induced cardiomyopathy as seen in this case.A 53-year old female presented to the Emergency Department for cough, nausea, vomiting and “feeling sick for 3 weeks.” She reported an allergy to penicillin but no other significant past medical history. On initial assessment, patient was afebrile, tachycardic (125 beats/minute), tachypneic (22 breaths/minute), with blood pressure of 109/74 mmHg, and oxygen saturation of 72 % on room air. Physical exam showed no other abnormalities.Initial laboratories showed leukocytosis , hyperglycemia (glucose 796 mg/dl), hyponatremia , a hemoglobin A1c of 17.2 %, and an elevated troponin T (0.19 ng/mL) and BNP (2137 pg/mL). Measured osmolarity was 349 mOsm/L with only small ketones in the blood and an arterial lactate of 5.62 mmol/L. Arterial blood gas suggested metabolic acidosis (pH = 7.264 | PCO2 = 25.9 mmHg | Bicarbonate = 13 mmol/L). Calculated anion gap was 30 mEq/l. Chest X-ray showed bilateral reticular opacities without cardiomegaly , free T4 1.01 ng/dl (nr: 0.93–1.70), free T3 2.89 pg/ml (nr:2.30–4.20). BNP was 42 pg/ml .Our patient presented a case of severe reversible myocardial dysfunction with HHNS as debut of diabetes mellitus, NTIS, and meeting SIRS criteria. Although sepsis of pulmonary origin was initially suspected, the clinical course  suggest that SIRS was secondary to HHNS . The obsMyocardial dysfunction is a common complication in patients with SIRS secondary to sepsis and is associated with an increased risk of mortality of up to 70–90 % , 3. SystA common mechanism among these clinical situations is a high level of pro-inflammatory cytokines such as tumor necrosis alpha  and interleukin 6 (IL-6). Bacterial products and pro-inflammatory cytokines increase production of nitric oxide –7 and re2+ entry into cells and release from sarcoplasmic reticulum (SR) decreases and sarcomere shortening is reduced [SERCA2a gene expression [2+ [The sarcoendoplasmic reticulum adenosine triphosphatase 2a (SERCA2a) and sarcolemmal voltage-gated L-type calcium channels are often affected. As a consequence, Ca reduced , 12. Bacpression , reduce sion [2+ , and dowsion [2+ , 16.. Low ATP levels and a decrease in the phosphocreatine/ATP ratio have been found in deadly cases of septic shock [SIRS can cause myocardial energy deficiencyic shock –19. Bothic shock . Pyruvatic shock  and phosic shock  decreaseFactors responsible for FAO inhibition during septic shock are a decrease in mitochondrial membrane carnitine shuttle activity and a decrease in peroxisome proliferator-activated receptor alpha and peroxisome proliferator-activated receptor gamma co-activator-1α gene expression , 24. AlsAcute hyperglycemic crisis can cause SIRS, and both hyperosmolar hyperglycemic non-ketotic syndrome and diabetic ketoacidosis are associated with a severe inflammatory state . HoweverDiabetic cardiomyopathy refers to ventricular dysfunction in the absence of coronary artery disease and hypertension . DiastolOur patient also presented with low thyroid hormone levels with normal TSH, a condition known as NTIS or sick euthyroid syndrome. Although NTIS is probably the most common cause of hypothyroidism, the current consensus advises against the administration of thyroid hormones to patients with NTIS as thyroid hormones increase respiratory rate, oxygen consumption, energy expenditure and heat production, NTIS is considered an adaptive response to counteract catabolism during illness , and thiAs we have explained, both HHNS and SIRS cause cardiac tissue injury through similar mechanisms including impairment of fatty acid oxidation and formation of reactive oxygen species, as well as modifying the function of membrane calcium channels. As a result, it is conceivable that diabetes may amplify the deleterious effects of inflammatory stressors on cardiac myocytes. It is imperative that this clinical question be investigated further as such a relationship may have significant clinical implications for prevention and future treatments, particularly in patients similar to the one presented in this clinical case.Written informed consent was obtained from the patient for publication of this Case Report and any accompanying images through standard institutional protocol. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


The hyperglycemic condition from dysregulated glucose homeostasis has been defined stress hyperglycemia. Following burn trauma it persists in a hypermetabolic flow phase as a response of burn itself and sepsis. Serial Sequential Organ Failure Assesment (SOFA) score is recommended from American Burn Association for evaluation of organ dysfunction/failure.The aim of this study is to test the relation of serial SOFA score evaluation and probability of critical hyperglycemia during burn injury.-rd,7-th,14-th and 21 -st day after burn injury.This is an observational prospective cohort study. Population is composed of adults hospitalized in ICU of the Service of Burns near University Hospital Center, Tirana, Albania for 5 years(2010-2015).Patients are grouped according glucose values on three categories: Patients with Euglycemia ( 80-120 mg/dL), Moderate Hyperglycemia (121-180 mg/dL) and Critical Hyperglycemia (> 180 mg/dL ).Test characteristics and performance as well as AUC are calculated for SOFA score on the 3The prevalence of Critical Hyperglycemia in adult burn patients is 6.9%. Using the value 6 as the cutoff SOFA scoring for dysfunction/ failure, serial SOFA score evaluation and presence of critical Hyperglycemia have a good correlation. On the 14-th day after burn the values of Area Under the Curve, Positive Likelihood Ratios and Positive Predicted Values (PPV) are the better values as complementary information to clinical assessment Figure .Figure 1In patients with clinical sepsis after severe burns the probability for Critical hyperglycemia is higher after the first week  This tells us that 2 of 5 patients with sepsis after the first week of the illness will demonstrate critical hyperglycemia. Hyperglycemia is a sign of sepsis in severely burned adult patients.


Objectives. To evaluate alterations in sexual function and genital sensitivity after anastomotic repair (AR) and free graft urethroplasty (FGU) for bulbar urethral strictures. Methods. Patients treated with AR (n = 31) or FGU (n = 16) were prospectively evaluated before, 6 weeks and 6 months after urethroplasty. Evaluation included International Prostate Symptom Score (IPSS), 5-Item International Index of Erectile Function (IIEF-5), Ejaculation/Orgasm Score (EOS), and 3 questions on genital sensitivity. Results. At 6 weeks, there was a significant decline of IIEF-5 for AR , whereas there was no significant change for FGU . After 6 months, differences with baseline were not significant overall and among subgroups. At 6 weeks, there was a significant decline in EOS for AR . In the FGU group there was no significant change . Overall and among subgroups, EOS normalized at 6 months. After 6 weeks and 6 months, respectively, 62.2 and 52% of patients reported alterations in penile sensitivity with no significant differences among subgroups. Conclusions. AR is associated with a transient decline in erectile and ejaculatory function. This was not observed with FGU. Bulbar AR and FGU are likely to alter genital sensitivity. Although a short bulbar stricture can be treated by dilation or endoscopic urethrotomy, longer or recurrent strictures are best treated by urethroplasty as it provides the best chance of success –3. Anastn = 31) versus FGU (n = 16) ((i)Qmax) and the International Prostate Symptom Score (IPSS) questionnaire; the IPSS ranges from 0 (no lower urinary tract symptoms) to 35 (severe lower urinary tract symptoms);urinary symptoms: maximum urinary flow ((ii)erectile function: the abridged 5-item version of the International Index of Erectile Function (IIEF-5) ; this sc(iii)ejaculation/orgasm: the sum of questions 9 and 10 from IIEF (long version) [(iv)postoperative genital sensitivity: a nonvalidated in-house questionnaire containing 3 dichotomous questions on glans tumescence, alterations in genital sensitivity, and cold feeling in the glans; further analysis of glans tumescence was only done in patients reporting normal erectile function (IIEF-5 ≥ 20) in order to avoid contamination of diminished glans tumescence due to globally diminished penile tumescence.Patients were evaluated preoperatively, after 6 weeks and 6 months. In the first six months, no phosphodiesterase-5 inhibitors were prescribed to stimulate sexual rehabilitation. In case of suspicion of stricture recurrence (Qmax < 15 mL/s and/or IPSS > 19), retrograde urethrography and urethroscopy were done. A functional definition of failure was used which includes the need for any additional urethral manipulation (including dilation) [Out of 258 male patients who underwent urethroplasty between October 2010 and February 2014, 90 patients with a bulbar stricture only were planned to be treated with AR or FGU and eligible to participate in this prospective study. Only native Dutch speaking patients who signed the informed consent  and who filled in the preoperative questionnaires and at least one postoperative questionnaire (at 6 weeks and/or 6 months) were included in this analysis. Finally, 47 patients were included for further analysis and divided into two groups: AR ((n = 16) . Prepuceilation) .Patients were operated on in a single center (GUH) by two surgeons (Nicolaas Lumen and Willem Oosterlinck). AR was preferred whenever a tension-free anastomosis could be made (stricture length < 3 cm on urethrography and/or peroperative findings). For longer strictures, FGU was performed. For both techniques, a midline perineal incision is made; the bulbospongiosus muscle is incised at the midline and dissected away from the corpus spongiosum. In case of AR, the corpus spongiosum is circumferentially freed at the level of the stricture. The corpus spongiosum and urethra are transected at this site. The fibrotic urethra and spongiosus edges are resected until healthy urethra is present at both the distal and proximal ends. The urethra is then spatulated in order to obtain a broad oblique anastomosis, which is finalized by 8–10 interrupted resorbable 4.0 sutures. In case of FGU, the stricture is opened ventrally on the tip of the catheter. The stricture length is measured and a graft is taken accordingly. The graft is sutured into the urethra in a ventral onlay fashion. The corpus spongiosum is closed over the graft for vascular supply and mechanical support (spongioplasty). The urethral catheter is maintained for 14 days and a voiding cystourethrogram is made upon removal.t-test or the Welch modified t-test for, respectively, equal and unequal distributions. Categorical variables were evaluated by chi-square or Fischer's exact test. The 2-year recurrence-free survival was estimated by Kaplan-Meier statistics and groups were compared by log rank statistics. To evaluate changes in IPSS, IIEF-5 score, and EOS between baseline and at 6 weeks and 6 months, mean differences were calculated by paired-samples t-test.Descriptive statistics were performed to evaluate the whole population and both subgroups. To compare both groups, continuous variables were evaluated by independent-samples p = 0.018) and strictures were shorter with AR compared to FGU . Both groups were comparable for follow-up duration, stricture etiology, previous interventions, and presence of suprapubic catheter and for preoperative urinary flow, IPSS, IIEF-5, and EOS (p = 0.395). Estimated 2-year recurrence-free survival rate was 93% and 72%, respectively, for AR and FGU (p = 0.347). Overall and in both groups, there was a significant improvement of the urinary flow at latest follow-up. Accordingly, there was a significant improvement in IPSS after 6 weeks and 6 months overall and in both groups . This decline remained significant for AR . However, for FGU, there was no significant change in IIEF-5 score . After 6 months, there were no longer significant changes in IIEF-5 score overall , for AR  and for FGU .Thirty-three patients, respectively, 19 and 14 patients in the AR- and FGU-group, reported to have sexual intercourse and filled in the IIEF-5 . Overallp = 0.111). However, in the AR-group there was a significant decline in EOS . This was not the case in the FGU-group . After 6 months, EOS returned to baseline. The decline for AR  was no longer significant.Thirty-seven patients, respectively, 23 and 14 patients in the AR- and FGU-group, tried to have ejaculation/orgasm  and completed the EOS . Overallp = 0.517). Only one patient, treated by AR, had a cold feeling in the glans. At 6 months, 13 patients (52%) reported to have altered genital sensitivity. Again, this proportion was not significantly different with AR compared to FGU . At 6 months, no one reported a cold feeling in the glans. Of 20 patients with IIEF-5 ≥ 20 at 6 weeks 1/10 (10%) and 4/10 (40%) of patients in, respectively, the AR- and FGU-groups reported no glans tumescence (p = 0.303). At 6 months, 1/6 (16.7%) and 3/5 (60%) patients with IIEF-5 ≥ 20, respectively, treated by AR and FGU reported no glans tumescence (p = 0.242). Of the 4 patients treated with oral mucosa, 2 had altered genital sensitivity and no glans tumescence at 6 weeks and 6 months.At 6 weeks and 6 months, respectively, 45 and 25 patients filled in the questionnaire on genital sensitivity and on cold feeling in the glans. At 6 weeks, 28 patients (62.2%) reported to have altered genital sensitivity. This proportion was not significantly different between AR and FGU  of erectile nerves penetrating the corporal bodies at that location ; this hycomplete transection of the corpus spongiosum that might be associated with a higher risk of bleeding and with postoperative haematoma and inflammation; this needs some time to recover; this might withhold patients to have satisfactory sexual activity or might provoke psychological problems.In this series, ventral FGU was performed, with no significant decrease in sexual functioning at 6 weeks and 6 months. It would be interesting to know whether dorsal FGU affects sexual functioning. One would expect a higher incidence of sexual dysfunction if the hypothesis of more extensive and circumferential dissection of the bulbar corpus spongiosum is (in part) responsible for sexual dysfunction.We speculate that the observed transient decline in erectile function with AR might be related to the following:In this series, a transient decline in EOS was seen with AR, whereas there was no significant difference observed with FGU. Erickson et al. found an improvement of ejaculatory function after urethroplasty (mix of AR and FGU) , but a lIn this series, postoperative changes in genital sensitivity were present in approximately 2 out of 3 and 1 out of 2 patients after, respectively, 6 weeks and 6 months. Changes in genital sensitivity were not significantly different among subgroups. Palminteri et al. found a change in genital sensitivity after FGU in 50% of patients . This isThis series again underlines the concern of possible alterations in sexual functioning and genital sensitivity after bulbar urethroplasty. Therefore it should be part of the evaluation of patients treated by urethroplasty. Jackson et al. recently validated patient reported outcome measures (PROMs) for urethroplasty . HoweverFurthermore, it would be interesting to evaluate whether modifications in urethroplasty techniques such as muscle- and nerve-sparing bulbar urethroplasty  and vessImportant limitations of the present series are the small sample size and the missing data in the postoperative questionnaires.AR is associated with a transient decline in erectile and ejaculatory function. This was not observed with FGU. Bulbar urethroplasty is likely to provoke changes in genital sensitivity. Further prospective studies with validated and internationally accepted patient reported outcome measures (PROMs) are needed for further confirmation.


Importantly, this was associated with a novel JQ1 action of down-regulating AR-V transcript and protein expression. Overall, this study demonstrates that AR-Vs broadly restore AR chromatin binding events that are otherwise suppressed during endocrine therapy, and provides pre-clinical rationale for BET inhibition as a strategy for inhibiting expression and chromatin binding of AR-Vs in PCa.Androgen receptor (AR) variants (AR-Vs) expressed in prostate cancer (PCa) lack the AR ligand binding domain (LBD) and function as constitutively active transcription factors. AR-V expression in patient tissues or circulating tumor cells is associated with resistance to AR-targeting endocrine therapies and poor outcomes. Here, we investigated the mechanisms governing chromatin binding of AR-Vs with the goal of identifying therapeutic vulnerabilities. By chromatin immunoprecipitation and sequencing (ChIP-seq) and complementary biochemical experiments, we show that AR-Vs display a binding preference for the same canonical high-affinity androgen response elements (AREs) that are preferentially engaged by AR, albeit with lower affinity. Dimerization was an absolute requirement for constitutive AR-V DNA binding and transcriptional activation. Treatment with the bromodomain and extraterminal (BET) inhibitor JQ1 resulted in inhibition of AR-V chromatin binding and impaired AR-V driven PCa cell growth  The androgen receptor (AR) is a steroid receptor transcription factor that regulates the expression of genes required for development and physiologic function of the prostate gland . AdditioOne mechanism proposed to support persistent AR transcriptional activity in CRPC is synthesis of constitutively active AR variant (AR-V) proteins lacking the AR LBD –13. SeveThe absence of a LBD presents a formidable challenge for direct inhibition of AR-Vs in CRPC because alternative sites on the AR protein appropriate for specific and high-affinity drug targeting are not evident. An indirect approach may be through inhibition of AR-V transcriptional activation mechanisms. However, there is a paucity of information regarding the molecular activity of AR-Vs in CRPC. To address this limitation, we used chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq) and complementary biochemical approaches to understand genome-wide AR-V DNA binding. We find that the mechanisms governing AR-V chromatin interaction resemble those of the prototypical androgen-activated AR: AR-Vs bind DNA as a dimer and utilize canonical inverted repeat androgen response elements (AREs). Moreover, we demonstrate the bromodomain and extraterminal (BET) family inhibitor JQ1 inhibits AR-V mRNA and protein expression, thereby disrupting the AR-V:DNA interaction and repressing the androgen-independent cell growth program driven by AR-Vs.2 for no longer than 15 passages after resuscitation of frozen stocks.R1-AD1 and R1-D567 cells have been described . LNCaP, siRNAs targeted to AR exon 1 (siAR1) and exon 3 (siAR2) were purchased from Dharmacon. Antibodies were specific for the AR NTD , ERK-2 , HA tag , BRD2 , the TSC2 COOH-terminal domain  or AR-V7 .Plasmid and lentivirus constructs encoding full-length AR, ARv567es, and AR-V7 (AR 1/2/3/CE3) have been described . LuciferControl shRNA, shAR#1 or shAR#2 were expressed using the lentiviral vector pLV-MSV40 . To geneThe pLV-MSV40-HA vector was derived from pLV-MSV40 by adding a HA tag downstream of SV40 promoter. This was accomplished by PCR of the HA tag fragment from a CMV5 vector with an N-terminal HA tag  using a Construction of transcription activator-like effector nuclease (TALEN) expression vectors targeting the FASN AREI site  was performed by Golden Gate cloning using the mammalian expression vector pC-GoldyTALEN (Addgene #38143) as described previously .MLPA assays were performed as described previously .Western blots were performed as described . Briefly6 cells/plate on 10 cm plates in RMPI 1640 + 10% CSS, allowed to settle for 72 h, then re-fed for 4h with RMPI 1640 + 5% CSS containing vehicle (ETH) or 1 nM DHT prior to fixation. Nuclear pellets were sonicated on ice for eight cycles at 40% amplitude using a 450 Sonifer (Branson). Each cycle consisted of 10 s pulse/10 s rests for 1 min, with 2 min rests between cycles. Lysates were immunoprecipitated with Protein A/G Plus agarose beads (Santa Cruz Biotechnology) pre-blocked with tRNA (Sigma). DNA was purified using a PCR purification kit (Qiagen). Alternatively, immunoprecipitated complexes were boiled in sodium dodecyl sulphate (SDS) loading buffer and subjected to western blot using a mouse monoclonal antibody specific for the AR NTD . For ChIP-seq, 10 ng of DNA (ChIP-enriched or input) was used for library creation with a TruSeq ChIP Sample Preparation Kit (Illumina cat. #IP-202-1012) and sequenced at the U of M Genomics Center using an Illumina HiSeq2000 at 1× 50 bp. Mapping and processing of fastq files were performed in Galaxy .Chromatin immunoprecipitation (ChIP) with an antibody specific for the AR NTD  was performed for three independent biological replicate experiments as described  with then Galaxy . Reads wn Galaxy  using den Galaxy . Peaks wn Galaxy  and CisGn Galaxy , using dCompressed SRA files from ChIP-seq studies assessing BRD2/3/4 chromatin occupancy in VCaP , GSE5506, GSE5506de novo sequence motifs in the peak sets was performed using the Gibbs Motif Sampling approach implemented in CisGenome , with enrichment and significance calculated as above. The diffReps tool  and tdfBRD2 ChIP-PCR was performed exactly as AR ChIP-seq and AR ChIP-PCR, with the exception that cells were fixed with 1.5 mM of freshly prepared ethylene glycolbis(succinimidylsuccinate)  in 10 ml PBS at room temperature for 30 min. For all ChIP-PCR experiments, DNA (ChIP-enriched or input) was subjected to quantitative PCR using PerfeCTa SYBR Green FastMix (Quanta Biosciences) and gene-specific primers listed in Supplemental Table S1.Renilla luciferase activity,R1-AD1, R1-D567 and LNCaP cells were electroporated with 200 pmol siRNA or 12μg plasmid DNA as described ,19. ElecCells were electroporated with 6μg of left and right TALENs and seeded on 6-well plates in RMPI 1640 + 10% CSS for 96 h. Genomic DNA was extracted using a Nucleospin Tissue Kit (Machery-Nagel #740952). The TALEN-targeted region was amplified by PCR using FASN-specific primers , gel purified, and cloned for Sanger sequencing or subjected to T7E1 endonuclease assays. T7E1 assays were performed by incubating 400 ng of PCR product in 1× NEBuffer 2 (New England Biolabs) at 98°C for 5 min, slowly cooling to room temperature, digesting with 1 unit of T7 endonuclease 1 (NEB #M0302S) at 37°C for 1 h, and resolving products by electrophoresis in 2% agarose gels.Conditions for lentivirus virus packaging and cell transduction have been described .Total RNA was isolated using TRIzol (Invitrogen) and reverse-transcribed . Quantitative PCR was performed with cDNA using PerfeCTa SYBR Green FastMix (Quanta Biosciences) with primer sets specific for FASN, FKBP5, LIMA1, PSA, hK2 and GAPDH . Fold changes in mRNA expression levels were calculated using the comparative Ct method as described .Kd) defined as the concentration of IRD700-labeled FASN AREI duplex required to achieve half-maximum binding.IRD700-labled and competitor duplexes were prepared by annealing complementary synthetic DNA oligonucleotides . COS-7 cells growing on 10 cm plates were transfected with empty vector or plasmids encoding AR, ARv567es or AR-V7 (AR 1/2/3/CE3) using Lipofectamine 3000 (Invitrogen) according to the manufacturer's recommendations. COS-7 cells transfected with AR were stimulated with 1 nM mibolerone for 1hr prior to nuclear extraction as described . BindingCells were cultured 72 h on 10 cm plates in RMPI 1640 + 10% CSS and stimulated with 1 nM DHT (R1-AD1 cells) or vehicle  in RMPI 1640 + 5% CSS for 4 h. Cells were lysed, cleared, and 500 μg of protein lysates were used for DNA duplex pull-downs as described  with 1 μGSEA was performed using GSEA v2.07  and 100 μl was injected subcutaneously in the right flank of male nude mice aged 6–7 weeks (Harlan Laboratories). Tumor size was monitored twice a week by measuring with calipers and calculating tumor volume using the formula length (mm) × width (mm) × height (mm). After tumors reached 100 mm3, mice were anesthetized with ketamine/xylazine (100 mg/10 mg/kg), a first tumor biopsy was performed using a 1 mm biopsy punch , and then mice underwent surgical castration. Mice were treated with enzalutamide  by daily oral gavage at 30 mg/kg/day for a total of 7 days. Subsequent tumor biopsies were performed on day 8  and day 14 (two consecutive tumor measurements displaying increased tumor size). On day 16, the pre-defined study endpoint had been reached (tumor volume ≥ 1000 mm3), at which point mice were sacrificed and tumor tissue harvested.All protocols for mouse experiments were approved by the U of M Institutional Care and Use Committee (IACUC). For enzalutamide treatments, R1-AD1/R1-D567 cell mixtures were prepared by counting R1-AD1 and R1-D567 cells and mixing to achieve final cell number ratios of 90% R1-AD1 and 10% R1-D567. Freshly-prepared admixtures were suspended at 1 × 107 cells/ml in 50% RPMI 1640 supplemented with 10% FBS and 50% Matrigel (BD Biosciences) was injected subcutaneously in the right flank of 14 male nude mice aged 6–7 weeks (Harlan Laboratories). After tumors reached 100 mm3, mice were randomized by alternating assignments to control  or treatment  groups, with 250 μl DMSO or 50 mg/kg JQ1 administered by intraperitoneal injection 5 days per week for 4 consecutive weeks. Tumor size was monitored twice a week and mice were euthanized when tumor volume reached 1000 mm3, which was the pre-defined ‘survival’ endpoint for this study. Tumor growth rate and survival analyses were performed using GraphPad Prism 5.0 software .For JQ1 treatments, 100 μl of R1-D567 cell suspension containing 1 × 10AR-V-expressing tumor cells are common features of the subclonal architecture of heterogeneous CRPC cell lines and tissues, and have emerged as an important component of resistance to endocrine therapies –18,42–45in vivo model highlights the importance of understanding the function and regulation of AR-Vs in CRPC, as this may reveal opportunities for novel therapeutic interventions that inhibit AR-V activity and overcome therapeutic resistance.Western blot analysis of biopsied tumor tissue that had been established in intact mice revealed that full-length AR remained the predominant protein species Figure  and D. H2-terminal domain (NTD). Peak calling with data from a single ChIP-seq experiment resulted in the identification of 12 030 AR binding sites in R1-AD1 cells and 3554 ARv567es binding sites in R1-D567 cells . Inspection of a subset of these sites where enrichment was highest for ARv567es-specific signal revealed that most were located in repetitive genomic elements, suggesting that at least some were false positives . In line with this, ARv567es-specific occupancy at these sites was not observed in two independent biological replicate ChIP-seq experiments . Further inspection of ChIP-seq data at the gene-track level and directed ChIP-PCR at representative ARv567es ‘unique’ sites that were not within repetitive elements could not confirm ARv567es binding . Additionally, the putative ARv567es ‘unique’ sites lacked sequence conservation, unlike common or AR ‘unique’ sites . In aggregate, these findings strongly support the concept that the genome-wide binding preference of constitutively active ARv567es closely resembles that of androgen-activated AR.6 mapped reads per condition), and used these consolidated datasets to manually search for an ARv567es-specific signal at 27 genes reported to be unique targets of AR-Vs but not AR  2- and 4-fold lower than full-length AR  with lysates from COS-7 cells expressing ectopic AR and AR-Vs Figure . InteresR Figure  and C. TR Figure  and D. FR Figure . From thBased on these mechanistic findings, we hypothesized that disruption of AR-V:ARE interactions may represent a viable strategy for overcoming AR-V-mediated therapeutic resistance in CRPC. To develop preclinical support for this chromatin targeting concept, we evaluated JQ1, an inhibitor of the BET family of chromatin readers  shown toTo understand the basis for JQ1-mediated down-regulation of AR expression, we analyzed publicly-available ChIP-seq data from studies assessing genome-wide H3K27Ac marks and the effects of JQ1 on BRD2, 3 and 4 binding in LNCaP and VCaP cells ,29. InteIn a previous study, the inhibitory effects of JQ1 on AR chromatin binding were attributed to a direct interaction between AR and BRD4 . We hypoNext, we considered the possibility that the observed reduction in AR mRNA and protein levels could be a non-selective result of the progressive, global decline in cellular mRNA expression that has been shown to ensue following BET inhibition . To test3)  Figure . CollectAR-Vs are expressed widely in CRPC, and have been shown to drive resistance to AR-targeted therapies in diverse cell- and animal-based models . HoweverA recent study of tumor cells circulating in the blood of patients with CRPC found that mRNA expression of the AR-V7 splice variant was associated with resistance to abiraterone or enzalutamide . When quin vitro binding assays, we found that ARv567es and AR-V7 engagement with these AREs was dimerization-dependent, requiring both ARE half-sites and D-box residues in the second zinc finger of the AR DBD, which is also analogous to androgen-activated AR .Supplementary Data are available at NAR Online.