Facial synkinesis is a severe sequelae of facial nerve malfunction. Once the synkinesis is established, it is extremely difficult for patients to recover. Given that the restoration of motor or sensory function after peripheral nerve injury was closely related with cortical plasticity, we investigated cortical plasticity in facial synkinesis patients by the frequency-specific data which remains largely uncharacterized.  Resting-state fMRI was conducted in 20 facial synkinesis patients and 19 healthy controls, and the amplitude of low-frequency fluctuation (ALFF) in five different frequency bands  was calculated, respectively. And the relationship between ALFF and clinical outcomes was also analyzed.  Comparing with the healthy controls, facial synkinesis patients showed significantly different ALFF values, mainly in the sensorimotor areas. Furthermore, increased ALFF of the ipsilateral insula in the slow-6 band was significantly related with better facial nerve function.  Increased ALFF values in the ipsilateral insula might reflect an abnormal state of hypercompensation in motor control of facial synkinesis patients. It provided valuable spatial information about the functionally aberrant regions, which implied the possible involvement of motor control system in facial synkinesis. Facial synkinesis, one of the most troublesome sequelae of facial nerve paralysis, is characterized by synchronous and involuntary contractions of the muscles during volitional facial movements , 2. CurrIn our previous studies, we investigated the cortical functional alterations in patients with unilateral facial synkinesis using the task-designed fMRI , 9. ResuALFF is an effective measurement for detecting low-frequency oscillation (LFO) activities at resting state, which has been widely applied in functional neuroimaging . Recent A total of 20 facial synkinesis patients and 19 healthy controls were enrolled in this study. Diagnosis of facial synkinesis was made by two experienced plastic surgeons from the Department of Plastic Surgery at The Ninth People's Hospital, School of Medicine, Shanghai Jiaotong University, before the fMRI scans. We recruited patients with unilateral facial synkinesis, absence of nerve transposition, and with no other medical or psychological disorders. Participants were excluded if the facial palsy was recurrent or with concurrent peripheral neuropathy and with contraindications to investigation by MRI. The study was approved by the local ethics committee, and all patients provided written informed consent according to the Helsinki Declaration.The Sunnybrook facial grading system was applied for the clinical assessment of peripheral facial nerve palsy . This gr3), and number of acquisitions = 240.The duration from facial palsy to fMRI scanning is provided in http://www.fil.ion.ucl.ac.uk/spm/) on MATLAB 2014a platform. The brain images of right-sided facial synkinesis patients were flipped along the y-axis prior to analysis to ensure the consistency of sides among patients. The first 10 images of each functional session were removed for the signal equilibrium. The acquired functional images were preprocessed as follows: (1) slice scan time correction, (2) head movement correction , (3) spatial normalization of the functional images via standard EPI template, (4) regressing nuisance variables including the white matter and cerebral spinal fluid blood oxygen level-dependent (BOLD) signal and effects of head motion using six head motion profiles, (5) spatial smoothing using a Gaussian kernel of 4 mm full width at half maximum, and (6) removal of linear trends.fMRI data were preprocessed in accordance with pipelines recommended by Yu-Feng et al.  using thNo temporal filtering during preprocessing assures that the entire frequency band below the Nyquist frequency (0.25 Hz) can be examined in subsequent analyses of ALFF.Subsequent to data preprocessing, the time series for each voxel was transformed to the frequency domain using the fast Fourier transform. The power spectrum was obtained in different frequency bands at each voxel. The low-frequency range was subdivided into five bands as previously defined: slow-6 (0-0.01 Hz), slow-5 (0.01-0.027 Hz), slow-4 (0.027-0.073 Hz), slow-3 (0.073-0.198 Hz), and slow-2 (0.198-0.25 Hz) .Z score to allow further comparison between groups.As the power of a given frequency was proportional to the square of the amplitude of this frequency component, the square root of the power spectrum was calculated at each frequency. Then, the averaged square root  of the power spectrum in different frequency bands was calculated . Finallyt-tests were performed for group comparison of each frequency band within the previously generated mask. All significance tests were conducted at a false discovery rate (FDR) threshold of P = 0.05.To investigate the effects of disease on different frequency bands, we performed an ANOVA  using the SPM8 software within a standard  grey matter mask. The group was considered to be a between-subject factor with two levels . The frequency band was regarded as a correlated repeated measure factor with five levels. The interactions of group and frequency were considered as effects in the model. After the interaction effect analysis, significant clusters were generated within a binary mask. Further, post hoc two-sample t-tests between the two groups in each frequency band. Spearman's correlative analyses were used to examine relationships between abnormal ALFF values and Sunnybrook scores in facial synkinesis patients using SPSS 22 software .The correlation between ALFF values of the ROIs in each frequency band and Sunnybrook scores was also analyzed. The ROIs for correlation analysis were extracted from the largest significant cluster revealed by post hoc There were no significant differences in age or gender between the facial synkinesis patients and the healthy controls. Among the facial synkinesis patients, 9 were with right-sided palsy and 11 were left-sided. The duration of facial palsy from onset to fMRI scanning was 26.05 ± 14.20 months. All patients showed a unilateral loss of facial function, with mean Sunnybrook score of 42.45 ± 19.24 points .Significant interaction effects of disease × frequency were distributed over the left insula, right middle temporal cortex, left inferior temporal cortex, right cerebellum, bilateral superior temporal cortex, bilateral orbit frontal cortex, right postcentral cortex, right thalamus, and left anterior cingulate cortex (ACC) .t-tests were performed . However, ALFF values of the other significantly different clusters between groups in the slow-2 to slow-5 bands  failed to show significant correlation with the Sunnybrook scores  in the slow-6 band, ALFF values of facial synkinesis patients' insula were most significantly related with facial synkinesis patients' Sunnybrook scores.In the present study, we investigated frequency-specific ALFF alteration between facial synkinesis patients and healthy controls in the distribution of intrinsic brain hemodynamic oscillations across five frequency bands (slow-2 to slow-6). We also investigated the capability of the frequency-specific alterations in providing predictive information with the frequency-specific alterations. The main findings were concluded as follows: (1) a significant interaction effect of the group × frequency band was observed mainly in the sensorimotor regions; (2) post hoc two-sample ALFF was reported to describe the local spontaneous neuronal activity . PreviouAlthough the slow-2 and slow-3 bands were typically discarded as physiological noises , they wet-test, ALFF values of the left insula showed the most significant difference between the two groups in these three frequency bands. And the ALFF value in the slow-6 band was most significantly correlated with Sunnybrook scores. Previous studies demonstrated that the slow-6 band was ascribed to low-frequency drifts [Of note, the slow-4 and slow-5 bands were thought to be linked with neurophysiological processes, which were typically utilized in resting-state fMRI studies (below 0.1 Hz) , 21. In y drifts , 19. AltThis study demonstrated that facial synkinesis was mostly affected in areas of higher order processing sensorimotor information, including the postcentral cortex, insula, thalamus, ACC, hippocampus, and cerebellum. However, the patterns of ALFF alteration in the ACC or cerebellum areas were not fixed. It suggested that these changes were very likely to be caused by the motor-sensory mismatch due to the substantial prediction error. Among those heterogeneous results, it was interesting to find significant difference of ALFF in the insula between the two groups in each frequency band. Moreover, the ALFF values of the left insula in facial synkinesis patients in the slow-6 band were found to correlate with the improvement of facial nerve function significantly. The insula was demonstrated to contribute to hand and eye motor movement in motor control , 23. TheMany studies paid attention to physiological signals arising from cardiac- and respiratory-related processes, for example, heart rate, respiration sources, and blood pressure, which could impact regional measures of ALFF –34. ThesThis study was an explorative neuroimaging study, and several limitations should be noted. Firstly, the sample size was relatively small, which might have limited the power of persuasion. Secondly, it might be better to compare patients who have recovered from facial paralysis and those who suffered from facial synkinesis to investigate alterations of spontaneous activity caused by facial synkinesis only, although the functional activities in the related brain regions gradually recovered to the normal level with the gradual recovery of facial nerve function –39. ConsWe reported multiple changes of band-specific ALFF in facial synkinesis patients. The increased ALFF value in the slow-6 band in the ipsilateral insula was positively related with facial nerve function (Sunnybrook scores). This study revealed frequency-specific changes in local spontaneous neuronal activity for severity correlation in facial synkinesis, which could be a potential indicator of disease progression. It also provided valuable spatial information about the functionally aberrant regions, which implied the possible involvement of motor control system in facial synkinesis. Although it is with limitation, this study may broaden our vision of the neural mechanism of facial synkinesis.


Exposure to ultrafine particles (UFPs) leads to adverse effects on health caused by an unbalanced ratio between UFPs deposition and clearance efficacy. Since air pollution toxicity is first direct to cardiorespiratory system, we compared the acute and sub-acute effects of diesel exhaust particles (DEP) and biomass burning-derived particles (BB) on bronchoalveolar Lavage Fluid (BALf), lung and heart parenchyma. Markers of cytotoxicity, oxidative stress and inflammation were analysed in male BALB/c mice submitted to single and repeated intra-tracheal instillations of 50 μg UFPs. This in-vivo study showed the activation of inflammatory response (COX-2 and MPO) after exposure to UFPs, both in respiratory and cardiovascular systems. Exposure to DEP results also in pro- and anti-oxidant  protein levels increase, although, stress persist only in cardiac tissue under repeated instillations. Statistical correlations suggest that stress marker variation was probably due to soluble components and/or mediators translocation of from first deposition site. This mechanism, appears more important after repeated instillations, since inflammation and oxidative stress endure only in heart. In summary, chemical composition of UFPs influenced the activation of different responses mediated by their components or pro-inflammatory and pro-oxidative molecules, indicating DEP as the most damaging pollutant in the comparison. Air pollution is a global public health emergency that affects people of all ages in every part of the world. Nowadays, addressing ambient air pollution is the government priority, and the World Health Organization  issues aNumerous epidemiological studies have showed the effects of air pollution on the respiratory and cardiovascular systems. Short-term exposure to air pollution at higher levels reduces life expectancy by aggravating pre-existing respiratory and cardiovascular diseases . CardiovIn Lombardy  diesel combustion and solid biomass burning are responsible for the 15% and the 50% of the primary fine particles production, respectively ,17. DiesIn-vivo and in-vitro toxicological studies have evidenced diverse cardiovascular effects due to the particles’ origins and composition ,24,25,26Cell-mediated immunity represents the first response triggered that indicates particles’ damage in pulmonary tissue. Under physiological conditions, healthy mice show a prevalence of alveolar macrophages (AMs) as resident immune cells in the lungs . In respIn parallel, in the BALf of mice subjected to acute UFPs treatment, we observed a significant increase of PMN percentage in parallel to a decrease of AM percentage. Although the trend of cell count was similar after both UFPs treatment, DEP-exposure led to a significant increase of PMNs  and a significant decrease of AMs  .The high levels of lactate dehydrogenase (LDH), increasing about 1.5-fold after UFP treatment, led us to hypothesize the occurrence of necrotic damage . The unvThe higher toxic potential of DEP is also revealed by the increased ET-1 concentration in the BALf, probably secreted from lung endothelial cells, as well as by AMs. ET-1 could promote acute lung injury, inducing cytokine production and activating neutrophils to release oxygen radicals . ActuallIndeed, IL-6 could dictate the profile of leukocyte recruitment during the inflammatory response  while TNWith the sub-acute treatment, we did not observe a significant change of BALf parameters among the three groups, suggesting some kind of adaptation to the inflammatory status caused by desensitisation after repeated exposures. After DEP-treatment, MPO protein level resulted the only parameter significantly higher than sham, although it appeared lower when compared with the acute exposure . We suppThe heart is the first organ directly reached by the bloodstream coming from the lungs, through the pulmonary circulation. Thus, although histologic analyses did not show infiltration of phagocytic cells overloaded with BB and DEP particles in the hearts of treated-mice (data not shown), UFP components or pro-inflammatory molecules produced in lung might immediately translocate and reach the cardiac tissue.Airway diseases have been already associated with cyclooxygenase 2 (COX-2) regulation at both gene and protein levels in-vitro and in-vivo following DEP treatment ,48,49, bTM 750 FAST. This probe is a matrix metalloproteinase (MMP) agent that is optically silent upon injection and produces a fluorescent signal after cleavage by inflammation related MMPs . Since the recommended optimal imaging time point is 6–24 h post-injection of the probe, the MMP activity evaluation after 3 h from single instillation was not possible. For this reason, in order to compare the effects of the single and the repeated exposures, in-vivo imaging was performed 24 h post-injection from the first (day 1) and the last (day 6) instillation in the sub-acute treatment.Thus, elevated MPO levels after DEP treatment might result in its higher passage in heart parenchyma with an increasing cardiovascular risk  althoughTM 750 FAST showed a 6-fold increase for BB , a pro-oxidant protein involved in NO production , showed In parallel to the activation of pro-oxidant proteins, in DEP-treated mice we observed the up-regulation of antioxidant and protective enzymes, such as heme oxygenase 1 (HO-1) and heat shock protein 70 (Hsp70) C,D. HO-1Moreover, MPO high levels in heart of DEP-treated mice could be another potential source of oxidative stress, since ROS produced by its enzymatic activity may affect the PAHs transformation to highly reactive intermediates ,69.The decrease of pro- and anti-oxidant protein levels in lung under sub-acute DEP treatment, compared to acute event, suggests that the antioxidant systems were active only until the pro-oxidant stimuli were high in the first deposition site. Then, the antioxidant machinery tended to burned out, although it might rest sensitive to successive acute events. On the other hand, frequent exposure to DEP might sustain prolonged oxidant stress environment in close or distant organs from pollution instillation locus.Oxidative stress and inflammation are pathophysiological events strictly connected, cause and consequence of each other in several diseases ,71, inclMoreover, during the inflammatory processes activated neutrophils and macrophages produces large amounts of ROS that might diffuse out of the phagocytic cells and induce localized oxidative stress and tissue injury . Indeed,Interestingly, after sub-acute treatment we observed a correlation among all the markers analyzed in heart parenchyma A. In parDEP and BB batches were provided by ENEA  in the framework of the project “Biological effects and human health impacts of ultrafine particles sources” lead by Prof Camatini of the POLARIS research centre. Particle sampling procedures and their characterization are extensively reported elsewhere . BrieflyMale BALB/cOlaHsd mice  were purchased from Envigo  with a health monitoring report based on FELASA recommendations. Mice were housed in plastic cages in groups of three for five days to acclimate to the housing facility. They were housed under controlled environmental conditions  with food and water administered ad libitum. Animal use and care procedures were approved by the Institutional Animal Care and Use Committee of the University of Milano-Bicocca (protocol 02-2014) and complied with guidelines set by the Italian Ministry of Health .3, it can increase severalfold at locations with high volumes of traffic or during high-pollution episodes [Animal testing has been carried out in the morning in the housing facility. Animals were randomly divided into three experimental groups (three mice for each group) for exposition to the different UFPs both in acute and sub-acute treatment: sham (isotonic solution); BB-treated mice and DEP-treated mice. The experiments were replicated twice, for a total of 6 shams, 6 BB- and 6 DEP-treated mice for each type of treatment. The sample size is in line with previously published data on air pollutant adverse effects ,32 with episodes . UFP dosAccording to our published data with PM , 3 h aftInstead, mice subjected to three repeated instillations of 50 μg UFPs/instillation every three days are consider representative of a sub-acute exposure B 30,31,,31,32,78g for 15 min at 4 °C [The BALf procedure, pellets, and supernatant recovery were performed as described previously ,73. Brie at 4 °C  and pellg for 7 min at room temperature. Subsequently, the smears were stained with Diff Quik  for cell differential count, according to the manufacturer’s instructions. Macrophages, polymorphonuclear leukocytes (PMNs) and lymphocytes were identified by their characteristic shapes.Total and differential cell counts have been performed according to the literature ,73. BriePro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), as well as the metalloproteinase MMP9, were analyzed by means of Luminex Screening Assay by LABOSPACE . The commercially available kits for alkaline phosphatase (ALP) activity , lactate dehydrogenase (LDH) activity  were employed according to the manufacturers’ instructions.Endothelin-1 (ET-1) and heat shock protein 70 (Hsp70) concentration were assayed by means of commercially available ELISA kits , according to manufacturer’s instructions.2+ ion is converted to reduced Cu+ ion by both antioxidant small molecule and protein. The reduced Cu+ is chelated with a colorimetric probe giving a broad absorbance peak around 570 nm, proportional to the total antioxidant capacity. All measurements performed were normalized to the total protein content evaluated by means of the bicinchoninic acid assay (BCA). Myeloperoxidase (MPO) analysis was performed by western blotting, as described in Total Antioxidant Capacity (TAC) was evaluated by means of TAC Colorimetric Assay Kit  according to manufacturer’s instructions, in order to analyse the overall cell capability to counteract reactive oxygen species (ROS). Briefly, CuLung and heart homogenates of sham, BB- and DEP-treated mice have been prepared and proteins were processed as previously described . BrieflyOnce excised, lungs and hearts from sham BB- and DEP-treated mice were washed with 0.1 M phosphate buffered saline , fixed by immersion in 10% buffered formalin o/n at RT, embedded in paraffin , cross-sectioned at 3 µm thickness by a rotary microtome, mounted on slides, and stained by Haematoxylin and Eosin (HE). Finally, they were observed under a light microscope . Representative images were captured with a digital camera . Data were obtained from two sham, two BB- treated and two DEP-treated mice.®, Danvers, MA, USA), and rabbit polyclonal myeloperoxidase (MPO) (1:200) . The secondary antibodies were appropriate horseradish peroxidase (HRP)-conjugated goat anti-rabbit (1:5000)  or donkey anti-goat (1:2000) . Immunoblot bands have been analyzed and the optical density quantified by LAS4000 ; all the data have been normalized to Ponceau staining  [Protein analysis was performed by SDS-PAGE electrophoresis/western blot, loading 30 μg each sample. Proteins were transferred to a nitrocellulose membrane , and revealed by immunoblotting with specific antibodies: rabbit polyclonal heme oxygenase-1 (HO-1) (1:200), rabbit polyclonal inducible nitric oxide synthase (iNOS) (1:200) , rabbit polyclonal cytochrome 1b1 (Cyp1b1) (1:200) , goat polyclonal heat shock protein 70 (Hsp70) (1:200), rabbit polyclonal cyclooxygenase 2 (COX2) (1:1000) is an imaging technique for improved fluorescence signals localization and quantification in deep tissue. After hair removal, male BALB/c mice were injected intravenously with MMPSenseTM 750 FAST probe, immediately before the first and the last instillation of the sub-acute treatment. MMPSenseTM 750 FAST probe is a matrix metalloproteinase (MMP) activatable agent that is optically silent upon injection but after cleavage by MMPs becomes fluorescent. The recommended optimal imaging time point is 6–24 h post-injection of the probe, to allow its distribution and the decrease of background signal; thus the evaluation after 3 h from single instillation was not possible. For this reason in-vivo imaging was performed during sub-acute treatment after 24 h from the first (day 1) and the last (day 6) instillation, in order to compare the effects of the single and the repeated exposures., Analyses was performed using the FMT 1500TM In Vivo Imaging System .Fluorescence molecular tomography  was calculated by the TrueQuant software. ROI was drawn with the same size each sample and so as to include the whole cardiopulmonary area. ROIs drawing was executed in a blind manner by an operator unaware of the experimental origin of the specimens in order to eliminate any operator bias.p ≤ 0.05 was used.Data were assembled with Galaxy (doi:10.1093/nar/gky379)  and analIn summary, DEP exposure was more harmful than BB. Indeed, although both of them induce inflammatory pathways, only DEP leads to strong oxidative stress activation in lung and heart parenchyma. Data obtained indicated that the chemical composition of UFPs is the key to the differential stress response. In fact, the main contribution in oxidative stress induction after DEP instillation is associated to higher concentrations of PAHs on diesel exhausted particles as compared to BB. On the contrary, the necrotic damage caused by both kind of particles could be specifically related to their physical nature.Here we showed that acute instillation elicited a stress response in both lung and heart characterizing a systemic damaging status. After repeated UFPs treatments, instead, BALf and lung parenchyma analysis did not disclose the presence of ongoing oxidative stress but at least a moderate cell-mediated immunity and inflammatory status. Therefore, we could hypothesize that lungs established some compensatory mechanisms in order to limit the toxic reaction and that the protective proteins, generated after a single treatment, achieved their function. Nevertheless, the inflammatory and oxidative stress pathways were activated in the cardiac tissue after repeated instillation.Since heart histologic analysis did not show any evident deposition of UFPs, that were instead persistent in the lung, alterations in heart parenchyma could be probably due to the translocation of harmful mediators produced in the lung. Therefore, a multiorgan analysis should be interesting to investigate the hypothesis of systemic damaging effects of UFP exposure.Our compelling evidence adds to a growing literature on the association between UFP exposure and adverse health outcomes. It is in line with studies performed on human subjects that highlighted the association of traffic-related air pollution with an acute decrement of lung function, elevated systemic inflammation , and the


In multivariable analysis, lung cancer patients with metastasis had significantly higher odds ratio for 28-day mortality than non-lung cancer patients without metastasis . Sepsis-related respiratory failure  and cardiopulmonary resuscitation  over respiratory failure without sepsis and acute organ dysfunction process measured by sequential organ failure assessment (SOFA) score  were independently associated with an increase in mortality rate. In conclusion, the treatment outcomes in lung cancer patients with metastasis who initiated mechanical ventilation in the ED were poor. Aggressive resuscitation versus end-of-life care in advance of an unexpected medical crisis should be considered in lung cancer patients with metastasis via a multidisciplinary approach with a consideration of underlying comorbid illnesses in the acute organ dysfunction processes.Few data are available regarding treatment outcomes in lung cancer patients with metastasis who initiated mechanical ventilation in the emergency department (ED). We aimed to evaluate 28-day mortality in lung cancer patients with metastasis who initiated mechanical ventilation in the ED. Patients with solid malignancy who initiated mechanical ventilation in the ED of a tertiary hospital were retrospectively identified and stratified into four groups according to the presence of lung cancer and metastasis. Among 212 included patients, the mortality rates by the 28 A previous study revealed that approximately 16% of all cancer-related ICU admissions are associated with lung cancer3. In addition, stage IIIB and stage IV lung cancer have shown annual increase of 6.6% in ICU use in the United States during the 1990s4. The prognosis of lung cancer is one of the worst among all cancers, and there is controversy regarding the usefulness of ICU care for this patient group.Lung cancer is one of the most common solid cancers, and often requires medical intensive care unit (ICU) admission due to acute illness6. In addition, among lung cancer patients admitted to the ICU, the need for mechanical ventilation and the advanced refractory cancer status were clinical factors predicting hospital mortality11. Considering the results of previous studies, the treatment outcomes in patients with far-advanced lung cancer  who initiated mechanical ventilation in the emergency department (ED) prior to ICU admission are expected to be even worse. Thus, the decision to triage these patients to receive mechanical ventilation in the ED should be made carefully. However, there have been few studies evaluating the treatment outcomes in patients with far-advanced lung cancer who initiated mechanical ventilation in the ED. Thus, we aimed to investigate the 28-day mortality in lung cancer patients with metastatic disease who initiated mechanical ventilation in the ED.Recently, it has been reported that the presence of lung cancer and emergency hospital admissions are particularly associated with higher mortality in patients with solid cancers who are admitted to the ICUBaseline characteristics of 212 cancer patients who were intubated for mechanical ventilation in the ED are summarized in Table th hospital day. Patients who died were more likely to have metastasis (p = 0.004) with higher frequency of abdominal metastasis (p = 0.005) and more progressive disease status (p = 0.020). In addition, patients who died were more likely to undergo cardiopulmonary resuscitation (CPR)  and less likely to have respiratory failure without sepsis  as an indication for mechanical ventilation, compared with those who survived. Patients who died also had a higher level of CRP , lactate , procalcitonin . Whereas the ratio of partial pressure to fractional inspired oxygen was lower in patients who died than those who survived , SOFA score was higher in patients who survived than those who died .Of the 212 patients with malignancy who were intubated for mechanical ventilation in the ED, 58.0% of the patients (n = 123) died within 28 hospital days. As shown in Table As shown in Table Regarding other clinical factors associated with 28-day mortality, compared to respiratory failure without sepsis, sepsis-related respiratory failure , CPR  and SOFA score  were significantly associated with 28-day mortality in both univariable and multivariable analyses. Although progressive disease was associated with increased 28-day mortality in univariable analysis , this was not significant in adjusted model (p = 0.299) , between January 2014 and December 2016, were identified. Demographic and clinical data including age, sex, BMI, comorbidities, site of primary malignancy and the presence of distant metastasis, indication for mechanical ventilation, initial laboratory findings , and 28-day mortality were collected from the review of the medical records. The Institutional Review Board of Samsung Medical Center approved this study and the need for written patient consent was waived due to the retrospective nature of the study (IRB no. 2017-09-066). This study was performed in accordance with relevant guidelines/regulations of our institutions.Distant metastasis was defined as cancer involvement of distant organs, as seen on radiologic studies  with or without histologic confirmation. Indications for mechanical ventilation were categorized into respiratory failure without sepsis, sepsis-related respiratory failure, and CPR. Primary outcome was 28-day mortality, which was defined as all-cause mortality within 28 days from intubation. Malignancy of gastrointestinal system included malignancy of esophagus, stomach, small intestines, and large intestines. Malignancy of hepatobiliary-pancreas system included malignancy of liver, biliary system, gall bladder, and pancreas. Malignancy of genitourinary system included malignancy of urinary tract  and genital system .24. A two-sided p-value < 0.05 was considered to reflect statistical significance. All statistical analyses were performed using R Statistical Software (version 3.2.3) of the R Foundation for Statistical Computing, Vienna, Austria and Stata version 15.0 .Continuous variables are reported as medians with IQR and categorical variables as numbers with percentages. Continuous variables were compared using the Mann–Whitney U-test, and categorical variables were compared using the chi-squared test with Yates’ continuity correction. Clinical characteristics and 28-day mortality were evaluated with stratification based on cancer type and the presence of metastasis as follows: non-lung cancer patients without metastasis, non-lung cancer patients with metastasis, lung cancer patients without metastasis, and lung cancer patients with metastasis. A multivariable logistic regression model was used to evaluate the OR of 28-day mortality in lung cancer patients with metastasis over non-lung cancer patients without metastasis. The initial clinical variables entered into the model were age, sex, BMI, chronic liver disease, chronic pulmonary disease, indication of intubation (respiratory failure without sepsis [reference] vs. sepsis-related respiratory failure or CPR), performance status, disease status, ongoing treatment plan, groups stratified by cancer type and the presence of metastasis, multiple metastases defined as two or more metastatic lesions, and SOFA score. To handle missing data in the regression model, the missing-indicator method, which is a popular and simple method to handle missing data in clinical research was usedThe Institutional Review Board of Samsung Medical Center approved this study and the need for written patient consent was waived due to the retrospective nature of the study (IRB no. 2017-09-066). This study was performed in accordance with relevant guidelines/regulations of our institutions.Supplemental Table 1


Three midline regions , within the DMN and consistently implicated in mood disorders, were selected as seeds. Within- and between-network clusters of the DMN iFC changed with pubertal maturation differently in boys and girls (puberty-X-sex). Specifically, pubertal maturation predicted weaker iFC in girls and stronger iFC in boys. Finally, iFC was stronger in boys than girls independently of puberty. Brain–behavior associations indicated that lower connectivity of the anterior cingulate seed predicted higher internalizing symptoms at 2-year follow-up. In conclusion, weaker iFC of the anterior DMN may signal disconnections among circuits supporting mood regulation, conferring risk for internalizing disorders.This study examines the effects of puberty and sex on the intrinsic functional connectivity (iFC) of brain networks, with a focus on the default-mode network (DMN). Consistently implicated in depressive disorders, the DMN’s function may interact with puberty and sex in the development of these disorders, whose onsets peak in adolescence, and which show strong sex disproportionality . The main question concerns how the DMN evolves with puberty as a function of sex. These effects are expected to involve within- and between-network iFC, particularly, the salience and the central-executive networks, consistent with the Triple-Network Model. Resting-state scans of an adolescent community sample ( The pubertal rise in sex steroids is thought to further refine the organizational sex differences that are established early in life .Puberty and sex critically influence brain maturation in adolescence , neuroimaging studies that query the effects of puberty on neural development, are relatively sparse26 . A primary focus of these neurodevelopmental studies have targeted structural measures of the brain 24). Relatively few resting-state fMRI studies have investigated the effects of age and sex in adolescence. Across the relative sparsity of these studies, many different methods and targets have been used, which make the identification of consistent patterns too soon to draw. Some studies focus on specific structures. For example, Alarcon et al.23 examined iFC of subregions of the amygdala (seeds) in 122 healthy youths (10–16 years). Their findings show opposite age-related changes in connectivity in girls vs. boys, but these changes are not always in the same direction depending on the amygdala subregions. Others have conducted studies of whole-brain organization, using methods such as graph theory (for review ref. 32). For example, Satterthwaite et al. examined sex differences in 9–22-year-old youths33. They reported sex differences in the organization of whole-brain connectivity . This type of data underscore the presence of sex differences in brain connectivity in youths, but does not speak specifically to the direction of sex differences in specific networks. Unfortunately, an analogous situation characterizes this type of research in adults . Of note, a recent study using a large adult sample from the human connectome  identified the default-mode network (DMN) as being the best predictor of sex status, particularly for couplings involving the fusiform gyrus and ventromedial prefrontal cortex37. The direction of effects was not detailed. Taken together, this brief survey of the literature does not permit to integrate existing findings into specific hypotheses that could guide the present work. Finally, to our knowledge, no studies have yet investigated puberty-related changes in resting-state functional connectivity , particularly with respect to the DMN, which is shown to have a central role in the development of psychopathology38, and seems to be highly sensitive to sex status37. Of note, one reason for the sparsity of research on puberty stems from the difficulty of dissociating the effects of age from puberty.In contrast to the abundant neurodevelopmental research probing neural changes with age and sex . Therefore, understanding the effects of puberty and sex on the development of the DMN function might shed light on the neural mechanisms conferring vulnerability to mood dysregulation in adolescence.The present study takes advantage of a large community cohort of adolescents, all ~14-year old55. This model proposes that dysfunction or imbalance among three core canonical networks of resting-state fMRI might contribute to a number of psychiatric disorders. These networks consist of the DMN, the central-executive network and the salience network (SN). The DMN serves self-referential-related functions and comprises regions in the anterior medial prefrontal cortex (PFC), posterior cingulate cortex, middle temporal cortex, and hippocampus48. The central-executive network supports working memory, decision-making, and cognitive control. This latter network is particularly important for the regulation of emotion processing, which itself depends largely on subcortical regions . The central-executive network encompasses the dorsolateral PFC and dorsomedial PFC58. Finally, the SN supports the integration of internal and external stimuli into emotional and behavioral responses. The core nodes of the SN include the insula and dorsal anterior cingulate cortex59. The framework of the Triple-Network Model is used as a heuristic tool in the present work. This approach emulates the widely use of neural systems models to explain typical adolescent behaviors, such as increased risk-taking, emotional lability, or social transformation64.Highly relevant to this question is the Triple-Network Model1. However, we do not predict pubertal maturation to influence brain connectivity similarly in males and females, because puberty is by essence sexually dimorphic. Specific directional hypotheses are difficult to predict, based on the lack of prior work of typical changes of resting-state networks with puberty. Finally, exploiting the 2-year behavioral follow-up (16 yo) of this cohort, exploratory brain–behavior analyses are expected to reveal associations between the DMN iFC of the 14-year old and behavioral dimensions of internalizing, externalizing, or social problems when these adolescents reach 16 years of age.The present work focuses on how puberty and sex affect the DMN iFC, including couplings within and between networks, especially the salience and central-executive networks of the Triple-Network Model. We hypothesize sex differences in the pubertal maturation effects on the brain’s iFC. We also anticipate sex differences that are independent of pubertal maturationhttps://imagen2.cea.fr. Adolescents (n = 381) from the IMAGEN sample39 underwent fMRI scanning in a resting state. A sample size of n = 381 subjects was expected to provide sufficient power to detect reliable interaction effects of puberty by sex on iFC measures. Indeed, previous studies have reported significant clinical group effects on measures of intrinsic connectivity in pediatric samples smaller than n = 60 . Recruitment and assessment procedures, and exclusion and inclusion criteria are described elsewhere in detail39. In brief, participants and their parents were recruited via middle-schools in five European sites. Inclusion criterion was age between 13 and 15 years. Exclusion criteria were birth weight < 800 g, severe medical conditions, bipolar disorder, treatment for schizophrenia, and major neuro-developmental disorders. All participants were assessed for intelligence quotient (IQ) using the Wechsler Abbreviated Scale of Intelligence68.The IMAGEN consortium recruited over 2000 youths. Only five sites opted to collect resting-state scans. IMAGEN data are available from a dedicated database: 39 and the Declaration of Helsinki. Seventy-seven participants were excluded from the analysis owing to excessive head motion  with a frame-to-frame Euclidean norm motion derivative > 0.25 mm; n = 72, 53 boys and 19 girls), poor spatial normalization by visual inspection (n = 2), or corrupted data (n = 1). Two participants lacked pubertal scores. The excluded group (n = 77), compared to included participants (n = 304), had more boys , but was similar in age, puberty status, and IQ . Characteristics of the sample are presented in Table n = 21 in common with40, n = 31 in common with Vulser et al.41, and n = 5 in common to all three samples).Written informed assent and consent were obtained, respectively, from all adolescents and their parents in accordance with the ethics committees of the participating institutionsBehavioral data were collected again in this sample 2 years later, at age 16 years. The attrition rate was 17% (53 subjects were not tested at follow-up), leaving a sample of 251 16-yo adolescents.www.dawba.com). The DAWBA is a self-administered questionnaire consisting of open- and close-ended questions completed by the participants and their parents that generates computerized probability levels of meeting DSM-IV and ICD-10 diagnoses, called “DAWBA bands” that are subsequently validated by experienced clinicians69. As part of the DAWBA, participants also completed a self-report inventory behavioral screening questionnaire, the Strengths and Difficulties questionnaire (SDQ70), which gives a measure of severity of problems within internalizing, externalizing, and social behavioral domains. Although the resting-state fMRI scanning only occurred at age 14 years, the behavioral assessments were repeated 2 years later, at age 16 years.Every participant completed a psychiatric assessment via the Development and Well-Being Assessment  and is highly correlated with physician ratings (Pearson’s R = 0.61)72.Pubertal status was assessed via self-report using the Pubertal Development Scale (PDS)MRI data were acquired at five sites using 3 T scanners: Phillips (Dublin), General Electrics (London), and Siemens . BOLD fMRI signal was acquired across 40 interleaved slices using the following parameters: TR = 2,200 ms; TE = 30 ms; flip angle = 75°; acquisition matrix = 64 × 64 × 40 with 2.4 mm slice thickness and 1 mm slice gap yielding an acquisition resolution 3.4 mm isotropic; 187 volumes collected over 6.5 minutes. High-resolution anatomical images were obtained using parameters based on the ADNI protocol, yielding a final voxel size of 1.1 × 1.1 × 1.1 mm.73. FreeSurfer version 5.374 was employed to segment the T1-weighted anatomical images. The ifirst four volumes of the functional run were discarded to allow for steady-state equilibrium. Functional volumes were slice-time corrected, aligned, and co-registered to the participants’ corresponding anatomical image. Functional volumes were then normalized to the Colin 27 Average Brain standardized template using 3dQwarp, which is a nonlinear transformation, and spatially smoothed with a 6 mm Full-Width Half-Maximum (FWHM) Gaussian kernel. All coordinates are reported in the Talairach and Tournoux system75.Preprocessing and analyses of BOLD fMRI data were conducted using AFNI76 who suggest that global signal regression can introduce spurious correlations into resting-state data sets, and advise against its use in preprocessing. In addition, we employed several strategies to minimize motion- and physiological-related variance, thus mitigating the need to apply additional measures, like global signal regression, to minimize variance related to motion and cardiac/respiratory processes .Acknowledging the debate as to whether global signal should be regressed out of resting-state data sets, we decided not to adopt this strategy. This decision was based on Saad et al.78, and (4) individual regressors corresponding with a frame-to-frame Euclidean norm motion derivative ≥ 0.25 mm, or volumes where ≥ 10% of voxels were determined to be outliers. This strategy followed the recommendations of Power et al.79.The following nuisance signals were regressed from the functional volumes: (1) six head motion parameters and their derivatives, (2) average time-series extracted from the ventricles, (3) time-series from local white matter within a 25-mm radius sphere surrounding each voxel using the ANATICOR approach80 were selected as seeds. These three seeds were retained because of their previously demonstrated association with subthreshold elevated40, as well as depressed41 mood symptoms in two independent subsets of the community cohort of 14 yo from the IMAGEN consortium (https://imagen.cea.fr39). MNI coordinates were all converted to Talairach coordinates using the Yale mni2tal GUI . Specifically, cubic ROIs (3 mm × 3 mm × 3 mm) were created within the left pregenual ACC , left medial PFC , and the left PCC . The left side was selected because the regions identified in previous studies were on the left side. Generically, these cortical regions have been implicated in the processing of salience and emotion encoding, particularly in the context of social and self-referential information (mPFC)82, visual stimuli (PCC)83, and autonomic visceral signals (pgACC)84.Three cortical regions of interest (ROIs) from the DMNSubsequently, average time-series from seed ROIs were extracted from the residualized functional images and were used to calculate Pearson correlations between the time-series from the seed ROIs and every voxel in the brain. Resulting statistical images were Fisher-transformed for group analyses.85. All group-level ANCOVA analyses statistically controlled for the effects of scanner site , as well as age. Group-level analyses were limited to all gray matter regions as determined by the parcellation of the Colin 27 Average Brain . Statistical thresholding was calculated using 3dClustSim’s Monte Carlo simulation via updated versions of 3dFWHMx and 3dClustSim to address the concerns of inflated false positive rates identified by Eklund86. These updates incorporate a mixed autocorrelation function that better models non-Gaussian noise structure87. The resulting maps were thresholded to p < 0.005 two-tailed, k = 37, which represents a global cluster correction at p < 0.05. Finally, to examine more stringently potential effects of group motion, we conducted an additional analysis including the additional covariate of individual average motion per TR. This analysis is presented in supplemental material (Table ANCOVA models (3dMVM) were used to determine the interaction of puberty-X-sex and the main effects of each factoral Table .Post hoc comparisons were conducted within SPSS v24 and interactions were probed using the PROCESS v21688 macro by calculating the beta value for the relationship between puberty and iFC for male and female participants, while controlling for age and scanner site. Levene’s test for equality of variances, and measures of skew and kurtosis are reported in Supplemental Table Average iFC values from clusters exhibiting a puberty-X-sex interaction, or main effects of sex or puberty, were extracted using 3dmaskave. To reduce the number of variables, two sets of principal components using SAS-9.4 program were extracted from the neural data at baseline (14 years old), and the behavioral data collected at 2-year follow-up (16 years old). All the iFC clusters significantly modulated by puberty and/or sex were entered into one factor analysis, and 15 items of the SDQ into a separate factor analysis. These factor analyses used ones as prior communality estimates. The principal axis method was employed to extract the components, and this was followed by a varimax  rotation.90. The SDQ has 33 items. A total of 16 items were removed a priori, because of no interest. These no-interest items included nine positive items , seven general items . Two additional items  were removed because they loaded on multiple factors. Accordingly, the final factor analysis was conducted on 15 items of the SDQ and the two DAWBA bands that probe generalized anxiety disorder and depression. These items and corresponding factor loadings are presented in Table Only two DAWBA bands were retained for this analysis, generalized anxiety disorder and depression, based on our main interest in these frequently comorbid diagnosesn = 5 clusters) and of the lMPFC seed (n = 3 cluster), all of which being modulated by Puberty-X-Sex. Therefore, factor-1 was labeled PCC/mPFC-pubXsex. Four clusters loaded on the second factor. All these clusters belonged to the lPCC-seed iFC, and were sensitive to Sex. Factor-2 was labeled PCC-sex Four clusters loaded on the third factor that was represented by clusters of the lACC seed. All these clusters were modulated by Sex. Factor-3 was thus labeled ACC-Sex.A similar approach was adopted to reduce the number of neural variables (iFC clusters). As reported below in the results, 17 clusters were of interest. Four clusters were removed because they loaded on more than one factor. The final 13 clusters and corresponding factor loadings are presented in Table Three multiple regression analyses were conducted to assess associations between the baseline iFC neural factors and each of the three follow-up (16 yo) behavioral factors.All significant puberty-X-sex interactions indicated that pubertal maturation was associated with increasingly weaker connectivity in females, but increasingly stronger connectivity in males. Slopes between puberty and iFC are reported separately for boys and girls in Table The anterior (lmPFC) and posterior (lPCC) midline cortical seeds revealed a similar pattern of clusters affected by the puberty-X-sex interaction Table . These cThe lpgACC-iFC showed no significant clusters in puberty-X-sex analyses.All sex effects followed the same pattern, i.e., stronger iFC in boys than girls.The iFCs of both the lPCC and lpgACC seeds were modulated by sex, but in quite distinct brain regions Table , Fig. 3.Finally, the third seed, the lmPFC-iFC revealed a main effect of sex in one cluster located within the right occipital cortex (BA 19) Table .x = −13.5, y = −79.5, z = −33.5; F = 20.32; 46 voxels) no clusters were found in cortical or subcortical regions.Other than a main effect of puberty on lmPFC-iFC with the cerebellum  models. Controlling for average motion per functional volume did not meaningfully impact the pattern of results reported above  relationship, where early pubertal maturation was accompanied by increased iFC (more representative of boys) and late pubertal maturation by decreased iFC (more representative of girls), analyses were repeated while controlling for quadratic effects. These analyses relied on power polynomial regression , whereas controlling for other iFC principal components.Multiple regression analyses were conducted to determine whether the three iFC principal components uniquely predicted variance within the three behavioral principal components. The ACC-sex principal component was negatively associated with the principal component characterizing internalizing problems . Of note, these cortical regions have also been implicated in the processing of salience and emotion encoding, particularly in the context of social and self-referential information (mPFC)82, visual stimuli (PCC)83, and autonomic visceral signals (pgACC)84.Accordingly, the three seeds examined in the present study belong to the DMN55 and the focus on vulnerability to emotion dysregulation, three predictions regarding the iFC of the DMN were tested. First, pubertal maturation would impact the DMN iFC differently in girls and boys. Second, sex differences in the DMN iFC would be detected independently of puberty. Third, no sex-independent effects of puberty would be found, given that puberty is by essence sexually dimorphic. Finally, brain–behavior relationships would inform the potential contribution of the DMN to the onset of internalizing problems 2 years later. Findings were broadly in line with predictions.Based on the Triple-Network ModelAs expected, within- and between-network couplings of the DMN were affected by pubertal maturation differently in girls and boys. These effects were restricted to the lmPFC and lPCC seeds Table . The lmP92 reported lower DMN-iFC  in behavioral inhibited  vs. healthy children (8–17 yo), and showed that girls (not boys) exhibited a negative correlation of internalizing-symptom severity with DMN-iFC. Another study of 8–12-yo children also showed that patients with depression/anxiety  had similarly lower iFC, but this time in the ventral attention network, which was the exclusive focus of this work66. In contrast, a relatively large study in a community sample of 112 children 93 focused on the three networks of the triple-network model55 in relation to internalizing symptoms . Findings revealed no sex differences in the iFCs of the DMN, SN, or CEN. However, brain–behavior correlations identified a positive correlation between iFC within the SN and the levels of internalizing symptoms in girls, but not in boys. Specifically, higher SN-iFC predicted more severe symptoms in girls. In summary, this study93 reported a differential effect of sex on the association of SN-iFC with internalizing symptoms, affecting only girls. Compared with our current study, this brain–behavior relationship93 concerned a different network (SN) and was in the opposite direction to our findings . Furthermore, it is difficult to compare these results with the present findings, because of the differences in methodology , and of the significantly younger sample (11yo). Collectively, these pediatric studies concur on the association between dysfunction and internalizing symptoms, but the direction of this dysfunction is inconsistent, as is the specific couplings. In addition, the two studies probing the effect of sex revealed that brain–behavior associations concerned mainly girls. This observation supports our interpretation that the puberty-related reduction in the DMN iFC in girls might carry vulnerability for internalizing symptoms.In support of this thesis, the exploratory brain–behavior analysis across the whole sample revealed a negative association between the spgACC iFC component and internalizing symptoms 2 years later: The lower the connectivity at 14 yo, the more severe were internalizing problems at 16 yo. This relationship suggests that puberty, which is accompanied by decreased iFC in girls, might amplify risk for internalizing symptoms in girls. Findings from the literature using clinical samples or individuals at risk for internalizing disorders could inform this interpretation. Unfortunately, results are inconsistent, in terms of topology, direction of group differences, methodology, and sample characteristics. A number of studies in pediatric samples have reported that internalizing symptoms were associated with lower iFC in core networks. For example, Frost-Bellgowan et al. (2015)95, would be expected to contribute to the increased emotional intensity and lability in adolescence . These negative findings suggest that the iFC of the nodes/circuits underlying emotion/motivation processes do mature similarly in boys and girls. Although task-based fMRI studies have reported puberty-related changes of these structures in response to emotion/motivation probes , how sex influences these trajectories has not been reported.Negative findings regarding the distinct iFC modulation by puberty in girls and boys are notable. The lack of sex modulation of the effects of puberty on the lpgACC seed connectivity, and the lack of implication of the SN (particularly the insula) and the amygdala were surprising. Indeed, their established role in the coding of salience and emotional responses . In all cases, connectivity was higher in boys than girls. These sex effects concerned mainly two of the three seeds, the lPCC and lpgACC.97). Sex influenced the connectivity of the anterior DMN seed (spgACC) with the SN, and the connectivity of the posterior seed (lPCC) with the central-executive network. In line with this distinction, as discussed above, the anterior spgACC connectivity predicted internalizing symptoms 2 years later (age 16 years), whereas the posterior lPCC component was not associated with any of the behavioral components.From the perspective of the Triple-Network Model, sex impacted differently the between-network connectivity of the anterior (spgACC seed) and posterior (lPCC seed) DMN components Table  , without physical examination or hormonal assays. However, the PDS has been shown to have good psychometric properties and convergent validity from self- and physician-rated Tanner stages72. Second, as girls and boys were of similar age, the average pubertal level was higher in girls. Two different control analyses were conducted to validate the findings. First, the youngest boys were removed from analysis to equalize the puberty mean in girls and boys. Findings with these control analyses revealed that the pattern of results remained pretty much unchanged (supplemental material, Table These findings are not without limitations. First, puberty was measured by self-report (PDSThis study reveals that pubertal maturation influences iFC differently in boys and girls, and that sex can impact iFC independently of puberty. First, because mood dysregulation has its peak onset in adolescence, during pubertal maturation, it is reasonable to consider a role of puberty in the rise of incidence rate of mood symptoms. Second, because mood dysregulation, particularly depression and anxiety, occurs disproportionally in girls, sex is expected to uniquely modulate circuits of emotion regulation. For these reasons, analyses were focused on three regions previously identified as conferring risk for mood problems in adolescents. These three regions happened to belong to the DMN, which is recognized to be perturbed in pathological mood disturbances. Findings revealed that both puberty-X-sex interaction and sex main effects modulate within-network and between-network clusters of the DMN iFC. Notably, effects of puberty did not involve the amygdala or the SN, suggesting the notion that pubertal maturation might not significantly affect the iFC of these key centers of emotion processes. This is in contrast to the effect of sex, which did impact DMN–SN iFC. Tentatively, stronger iFC in boys might suggest tighter emotional control, also potentially serving a protective role against emotion dysregulation. Finally, the spgACC iFC significantly predicted internalizing symptoms 2 years later, supporting the association of this network with mood dysregulation.Suppl. Material


During a mean follow-up period of 41.9 months, 87 developed end-stage kidney disease (ESKD). The incidence rates of ESKD were comparable between the patients treated and untreated with urate-lowering agents. The Kaplan-Meier analysis indicated that kidney survival was also comparable between them. In the multivariate analysis, heart failure and low eGFR were the significant prognostic factors for kidney survival. However, pharmacologic urate-lowering therapy was not predictive of kidney survival. The overall GFR decline rate was also comparable between the groups (P = 0.13). The efficacy of pharmacologic urate-lowering therapy in delaying CKD progression remains controversial. Therefore, further randomized controlled trials are needed to confirm its efficacy in attenuating kidney function deterioration in patients with stage 3–4 CKD.Because the serum uric acid level increases as the glomerular filtration rate (GFR) decreases, hyperuricemia is associated with chronic kidney disease (CKD). Although hyperuricemia is a risk factor for CKD progression, the causal role of uric acid remains controversial in patients with CKD and asymptomatic hyperuricemia. This study included 588 patients with stage 3–4 CKD and asymptomatic hyperuricemia. Using propensity score matching, 165 pairs treated and untreated with pharmacologic urate-lowering therapy were matched. Kaplan-Meier curves were constructed to determine the effect of urate-lowering agents on kidney survival. The prognostic value for kidney survival was ascertained using Cox regression analysis. The GFR changes over time between the patients treated and untreated with urate-lowering agents were assessed using a linear mixed model analysis. The mean age of the matched patients was 63.2 ± 12.7 years, and 52 (15.8%) patients had diabetic nephropathy. The mean estimated GFR (eGFR) and serum uric acid level were 36.7 mL/min/1.73 m Uric acid is the poorly soluble circulating end-product of purine metabolism in humans owing to the loss of uricase activity . It has Although hyperuricemia is strongly associated with the development of CKD, whether it plays a role in the deterioration of kidney function or is only a marker reflecting such deterioration remains controversial in patients with CKD. In this context, some epidemiologic studies showed no relationship between hyperuricemia and deterioration of kidney function , 13. On The objective of this long-term observational study is to explore the effect of pharmacologic urate-lowering therapy on the kidney function of patients with CKD and asymptomatic hyperuricemia via propensity score matching.This study was performed in accordance with the Declaration of Helsinki principles and approved by the Institutional Review Board (IRB) of Kangdong Sacred Heart Hospital (Ref. no. 2019-01-005). This was a retrospective medical record-based study, and the study subjects were de-identified. The IRB waived the need for written consent from the participants.2  age of ≥ 18 years; 2) estimated glomerular filtration rate (eGFR) of 15 to 60 mL/min/1.73 mequation ) for > 3The date of the first occurrence of a serum uric acid level of > 7 mg/dL was defined as the index date. The inclusion and exclusion criteria were applied on the basis of the index date. To evaluate the effect of long-term hyperuricemia on kidney function, the serum uric acid levels were also investigated during the follow-up period. To capture longitudinal changes in serum uric acid, the area under the serum uric acid level-time curve was calculated and adjusted on the basis of the total follow-up period . In otheAmong eligible patients followed-up in nephrology clinic at Kangdong Sacred Heart Hospital, the decision to treat with urate-lowering agents was made by nephrologists. Patients in treated group received urate-lowering agents from the index date. Urate-lowering agents were titrated or switched to other alternative drugs for reaching the target uric acid level (< 6 mg/dL).The initial study endpoint was the development of end-stage kidney disease (ESKD), defined as a condition needing maintenance dialysis or kidney transplantation. Meanwhile, the changes in the eGFR over time were evaluated according to whether the patients were treated with urate-lowering agents. In addition, subgroup analyses of eGFR decline slopes were conducted for sex, age, diabetes, CKD stage, heart failure, proteinuria, and serum uric acid level.http://cran.r-progect.org/). The Kolmogorov-Smirnov test was used to analyze the normality of parameter distribution for continuous variables. Continuous variables were expressed as means ± standard deviations or medians (interquartile ranges) and categorical variables as numbers (percentages). Propensity score matching was performed using the MatchIt package in R and variables collected at the index date. Patients with the nearest propensity scores in the two groups (patients treated and untreated with urate-lowering agents) were matched using a 1:1 scheme without replacement using a greedy algorithm. Greedy algorithm is a simple, intuitive algorithm that is used in optimization problems. The algorithm makes the optimal choice at each step as it attempts to find the overall optimal way to solve the entire problem. Propensity score matching allowed the comparison of outcomes between individuals with a similar likelihood. Differences between the two groups were assessed using the t-test, Wilcoxon’s rank sum test, and chi-squared test. Standardized mean differences were also estimated to evaluate the pre-match and post-match imbalance. Kidney survival curves were generated using the Kaplan-Meier method, and between-group survival was compared using the log-rank test. The prognostic values for kidney survival were analyzed by performing Cox regression analysis. The slope of the decline in kidney function over time was calculated using a linear regression analysis of the serial eGFR for each patient. The changes in the eGFR over time were compared between the two groups using a linear mixed model analysis. In this analysis, the patients treated and untreated with urate-lowering agents and follow-up period were treated as fixed effects, and each patient was treated as a random effect such that each subject had a unique intercept and slope. All probabilities were two-tailed, and the level of significance was set at 0.05.Statistical analyses were performed using the SPSS 19.0  and R version 3.5.1  patients.The baseline characteristics of the patients treated and untreated with urate-lowering agents are shown in Ninety patients were prescribed with allopurinol as the urate-lowering agent. The starting dose of allopurinol ranged from 50 mg/day to 100 mg/day, with 100 mg/day being the most common . The maintenance dose of allopurinol was 100 mg in 65 patients, 200 mg in 23 patients, and 300 mg in two patients. Thirty-four patients switched to febuxostat with a maintenance dose of 40 mg/day. The reason for discontinuation of allopurinol was inefficacy or presence of side effects. Meanwhile, 75 patients were prescribed with febuxostat. The starting dose of febuxostat ranged from 20 mg/day to 40 mg/day, with 20 mg/day being the most common . The maintenance dose of febuxostat was 40 mg in 71 patients and 80 mg in four patients.P < 0.001) and actual serum uric acid level  were significantly lower in the patients treated with urate-lowering agents than in those untreated with such. Among the patients treated with urate-lowering agents, the percentage of those whose serum uric acid levels decreased to 6.0 mg/dL during the first 3 months was 98.2%.The baseline serum uric acid levels were comparable between the patients treated and untreated with urate-lowering agents. However, the final serum uric acid level . The Kaplan-Meier analysis indicated that kidney survival was also comparable between them .The eGFR during the follow-up period is shown in The subgroup analysis of the eGFR decline slope was conducted according to sex, age, heart failure, diabetes, proteinuria, CKD stage, and serum uric acid level. It revealed no significant difference between the patients treated and untreated with urate-lowering agents .We evaluated the efficacy of urate-lowering agents in attenuating the deterioration of kidney function and effects on the associated clinical factors in patients with stage 3–4 CKD and asymptomatic hyperuricemia. Although clinical factors, such as diabetes, proteinuria, heart failure, and baseline eGFR, affect the deterioration of kidney function, there was no significant difference in kidney survival between the patients treated and untreated with urate-lowering agents. In addition, the subgroup analysis of the eGFR decline slope according to sex, age, heart failure, diabetes, proteinuria, CKD stage, and serum uric acid level revealed no significant difference between them.There is accumulating evidence that hyperuricemia has a causal relationship with the deterioration of kidney function –12; howeIt is well known that certain clinical factors, such as diabetes, proteinuria, heart failure, and baseline eGFR, could exacerbate the eGFR decline rate in patients with CKD –31. In oXanthine oxidase inhibitors with either allopurinol or febuxostat are recommended as the first-line pharmacologic urate-lowering agents . AllopurOur study has certain limitations. First, the study was an uncontrolled retrospective study. However, propensity score matching was used to reduce any bias owing to confounding variables commonly found in uncontrolled data. In addition, in most patients treated with urate-lowering agents, the uric acid levels were consistently maintained at < 6.0 mg/dL for a long period. Second, only a small number of patients were enrolled in this study. Therefore, the interpretation of the subgroup analysis findings may be limited. Third, different agents  were prescribed as the urate-lowering agents in our study.In conclusion, considering the side effects of urate-lowering agents in patients with CKD, there is a need to establish their efficacy according to certain pathologic conditions in patients with stage 2–3 CKD. Therefore, a large-scale prospective trial is needed to confirm such.S1 FigULT, urate-lowering therapy.(TIF)Click here for additional data file.


Similarly, in non-PNV, change of luminal area , baseline CNV area , and aflibercept vs. ranibizumab  were chosen as predictors for greater VA improvement. In PNV, however, none of these factors was chosen as predictors for dry macula or VA improvement by the model selection. The result of the present study implied that structural response after anti-VEGF might be different between non-PNV and PNV in the treatment with anti-VEGF agents.Pachychoroid neovasculopathy (PNV) shares some anatomical features with other pachychoroid spectrum diseases, but little is known about the characteristics on the treatment with anti-vascular endothelial growth factor (VEGF). We investigated the effect of choroidal structure and responses to anti-VEGF on the prognosis of pachychoroid neovasculopathy (PNV) and other types of neovascular age-related macular degeneration (non-PNV). Twenty-one eyes with PNV and 34 eyes with non-PNV who had anti-VEGF treatment were retrospectively reviewed. Choroidal neovascularization (CNV) area at baseline was measured with fluorescein angiography (FAG). The luminal and stromal area in the choroid was measured by enhanced-depth-imaging (EDI) OCT at baseline and 1 month. The association between dry macula or LogMAR VA  at 1 month and baseline values or changes in the luminal or stromal area at 1 month, baseline CNV area, or anti-VEGF drugs were analyzed in patients with or without PNV. In non-PNV, change of luminal area (coefficient = 7.0×10 Age-related macular degeneration (AMD) is a major cause of progressive visual impairment in the developed world , 2. EspeRecent studies have revealed that there is a subtype of nAMD with distinct characteristics, described as pachychoroid neovasculopathy (PNV) , 4. PNV Previous studies have shown different clinical findings between PNV and non-PNV. Compared to patients with nAMD without PNV (non-PNV), PNV occurs in younger patients . PolypoiThe response of PNV [or eyes with choroidal vascular hyperpermeability (CVH)] to anti-vascular endothelial growth factor (anti-VEGF) treatment is thought to be different from that of non-PNV. Patients with PNV are more likely to show persistent fluid after three loading doses of ranibizumab than non-PNV patients . In contTo the best of our knowledge, this is the first report to study anatomical changes of the choroidal structure and its association with dry macula or visual function in PNV or non-PNV eyes treated with anti-VEGF agents.The purposes of the present study were (1) to assess the short-term response of choroidal structure to anti-VEGF treatment using enhance-depth imaging (EDI) optical coherence tomography (OCT) and (2) to characterize the differences between PNV and non-PNV.Fig 1.This was a retrospective study that was approved by the Institutional Review Board (IRB) of the University of Tokyo. All data were fully anonymized before we accessed them. Written informed consent was not required by the IRB but participants who did not grant authorization to use their medical records for the research were excluded from analyses. We retrospectively reviewed the medical history of patients with typical AMD and PCV undergoing intravitreal injections of anti-VEGF from August 2016 to December 2017 at the outpatient clinic of Tokyo University Hospital. All patients underwent single intravitreal injection of an anti-VEGF drug, ranibizumab or aflibercept and were examined at their visit after one month. They underwent the ancillary multimodal imaging, i.e., OCT  using the EDI technique. Inclusion criteria were patients > 50 years of age with treatment and treatment-naïve CNV confirmed by fluorescein dye leakage on angiogram and the presence of one of the following on OCT: subretinal fluid, intraretinal fluid, or sub-RPE fluid. Patients who had already been treated with anti-VEGF before the data was acquired for analysis were considered as “having a treatment history”. The exclusion criteria were diagnosis of other pathologies  and history of other treatments than intravitreal anti-VEGF drug administration such as photodynamic therapy, laser photocoagulation for AMD. Also, the exclusion criteria included inadequate cooperation to obtain satisfactory images. The flowcharts of patient selection were shown in Areas of CNV were measured with fluorescein angiography before intravitreal anti-VEGF injection. The choroidal area on the fovea was assessed using EDI-OCT . Binarization was performed separately using the methods of Sonoda et al , and we In this study, PNV was diagnosed if all of the following criteria were satisfied: (1) CNV in either eye; (2) subfoveal choroidal thickness ≥ 200 μm in both eyes; (3) no drusen or only non-extensive  hard drusen ≤ 63 μm) in both eyes ; (4) CSC or PPE characteristics; namely, choroidal vascular hyperpermeability, retinal pigment epithelium (RPE) abnormality independent of CNV lesions, the presence of dilated choroidal vessels or thickening below the Type 1 CNV, or a history of CSC 3 μm in b. Other et-test for continuous variables. The proportion of patients with “Dry macula,” which was defined as absence of intraretinal or subretinal fluid on OCT and absence of hemorrhage by funduscopy based on the previous report [9 patterns, based on the second order bias corrected Akaike Information Criterion (AICc) index . We used correlation and multiple regression cross-sectional analyses to determine associations of baseline parameters or changes in 1-month parameters.The baseline characteristics or their change before and after the treatment in patients with PNV and non-PNV was analyzed using the chi-squared test for categorical variables and s report  was compThe AIC is a well-known statistical measure used in model selection, and the AICc is a corrected version of the AIC, which provides an accurate estimation when the sample size is finite . The degAll statistical analyses were carried out using the statistical programing language “R” .Table 1.We retrospectively reviewed 55 eyes of 55 patients with PNV and non-PNV. There were 21 eyes with PNV and 34 eyes with non-PNV. Among 55 patients in total, 89% of cases had a history of anti-VEGF treatment, and 11% was treatment-naïve patients. The baseline characteristics of the patients before treatment are shown in 2  in the PNV group and 50,595 μm2  in the non-PNV group. Patients with PNV had significantly larger luminal areas than those with non-PNV . The average central choroidal thickness (CCT) was 306 μm  in the PNV group and 193 μm  in the non-PNV patients. The CCT in PNV was significantly larger than those in non-PNV. At baseline, all patients showed intraretinal/subretinal fluid or hemorrhage. Their visual and anatomical outcomes are shown in Table 2.The average luminal area was 69,413 μmThe best-corrected visual acuity (BCVA) improvements at 1 month did not significantly differ between the two groups. Dry macula rate was 67% in total, 81% in PNV, and 59% in non-PNV. There was no significant difference between the PNV and non-PNV patients in dry macula rate. The visual change in PNV and non-PNV after one year were -0.07±0.22 and -0.07±0.27, respectively. There was no difference between the two groups.Table 3, in the optical model for dry macula in non-PNV, anti-VEGF , CNV area at baseline  and greater luminal area change  were included in the optimal model for dry macula. Additionally, anti-VEGF , CNV area at baseline , and greater luminal area change  were included in the optimal model for LogMAR VA change in non-PNV. However, none of these variables were included in the optimal model for dry macula or LogMAR VA change in PNV.As shown in In the present study, we analyzed the short-term changes of choroidal structure after anti-VEGF treatment using EDI OCT, and characterized the distinct response of PNV compared to non-PNV. In patients with non-PNV, we found that greater decrease of luminal area at 1 month, smaller baseline CNV area, and aflibercept compared to ranibizumab treatments were significantly associated with dry macula or better LogMAR VA changes. Considering luminal area decreases after anti-VEGF treatment in non-PNV , 21, greIn patients with PNV, however, neither the luminal area at baseline nor decrease in the luminal area was significantly associated with dry macula or LogMAR VA change at 1 month. This might be because, compared to non-PNV, dilation of the luminal area in PNV is not associated with upregulated VEGF induced by active CNV, and the choroidal vessel dilation is not reversed by the inactivation of CNV .The luminal area change after anti-VEGF administration were significantly associated with dry macula rate or greater LogMAR VA improvement in eyes with non-PNV; however, these associations were not observed in PNV. A recent study reported that eyes with active PNV presented with exudative changes in the macula accompanied with visual loss . HoweverThere are several limitations of this study. This was a retrospective non-randomized study, and the numbers of cases were relatively small. A further study would be needed to validate the current results preparing much larger number of samples. The measurements were performed by independent investigators completely masked to the patient information, but they were mostly carried out manually. In addition, the cases had short-term follow-up periods. Additionally, both treatment-naïve patients and the patients with a treatment history were included in the analysis. Although all patients exhibited exudative changes before treatment and multivariate analyses in the present study showed treatment history was not associated with treatment outcomes, possible bias associating with previous treatments could not be fully eliminated. Further analysis including only treatment-naïve patients might be needed to characterize PNV or non-PNV in more detail.The change of the luminal area after anti-VEGF administration were significantly associated with dry macula rate or VA improvement in eyes with non-PNV; however, these factors were not chosen as predictors in eyes with PNV. The results of the present study suggested that structural response after anti-VEGF might be different between non-PNV and PNV.S1 Dataset(XLSX)Click here for additional data file.


Thus, an important challenge is to find measures to eliminate these cancer stem cells, which will stop tumor growth and prevent disease-recurrence. However, knowledge about molecular features critical for the survival of prostate cancer stem cells (PCSC) is meager. Here we report that inhibition of 5-lipoxygenase (5-Lox) by shRNA or MK591 dramatically kills PCSC by inducing apoptosis, suggesting that 5-Lox plays an essential role in the survival of PCSC. Interestingly, MK591 treatment decreases protein levels and inhibits transcriptional activities of Nanog and c-Myc. Since Nanog and c-Myc play important roles as stemness factors, our findings indicate that the 5-Lox activity plays a causal role in maintaining prostate cancer stemness via regulation of Nanog and c-Myc, and suggest that further exploration of 5-Lox-mediated signaling in PCSC may lead to development of novel, target-based, durable strategies to effectively block development and growth of prostate tumors, and prevent prostate cancer recurrence.The cancer stem cell (CSC) concept suggests that neoplastic clones are maintained exclusively by a rare group of cells possessed with stem cell properties. CSCs are characterized by features that include self-renewal, pluripotency and tumorigenicity, and are thought to be solely responsible for tumor recurrence and metastasis. A hierarchically organized CSC model is becoming increasingly evident for various types of cancer, including prostate cancer. The CD44  The concept of cancer stem cell propounds that a small fraction of cells in tumors are capable of self-renewal and to differentiate into various types of tumor cells, and are responsible for tumor formation, growth, progression and recurrence, while the majority of cells that make up bulk of the tumors are non-tumorigenic end cells –3. The dCancer cells with stem-like properties have been identified in prostate tumors and characterized to be the population responsible for generating the diverse cell types within prostate tumors –12. CSCsOur strategy was to utilize an established, clinically-relevant model of human PCSCs to interrogate the mechanisms of stem cell vulnerabilities and to find effective measures for controlling the population of PCSC which express both pluripotency markers  as well as the tumor-progenitor markers  –29. EspeA flow chart depicting the process of isolation of prostate cancer stem cells from patient prostate tumors is shown in Figure Earlier we reported that under standard culture condition prostate cancer cells express 5-Lox but non-cancer cells do not, and that the 5-Lox activity plays a critical role in the survival of prostate cancer cells –26. HoweStemness in normal as well as cancer stem cells is maintained by a list of transcription factors such as, Nanog, c-Myc, Oct-4 and Sox-2 –17. ThesBased on previous observations of a critical role of 5-Lox activity in prostate cancer stem cell survival, we wanted to explore downstream signaling mechanism(s) regulated by 5-Lox using MK591, a specific inhibitor of 5-Lox activity. We found that MK591 dramatically induces mitochondrial-membrane depolarization, which is indicated by the loss of permeability barrier of mitotracker-red Figure . ExternaPreviously we have addressed the mechanism behind 5-Lox inhibition-induced apoptosis-triggering in prostate cancer cells, which revealed the involvement of c-Jun N-terminal Kinase and caspase-3 . To explHow inhibition of 5-Lox triggers apoptosis in PCSC is an intriguing question. We addressed this by examining the effect of 5-Lox inhibition on well-characterized survival-regulating kinases. Previously, we reported that inhibition of 5-Lox induces apoptosis in prostate cancer cells without inhibition of the Akt, or ERK, but via a rapid decrease in the membrane-localization as well as enzymatic activity of PKCε , 37. Invin vitro invasion of prostate cancer stem cells through extracellular matrix  dramatically decrease the x Figure . Moreovex Figure . Note: Is Figure .Our findings, for the first time, document that 5-Lox plays an essential role in the survival of prostate cancer stem cells, and that inhibition of 5-Lox kills these cells via induction of c-JNK-mediated apoptosis. Our observation of the massive induction of apoptosis in prostate cancer stem cells by specific inhibition of 5-Lox, opened up a unique possibility that both the progression and recurrence of prostate tumors can be vertically checked by eliminating these self-perpetuating and pluripotent cells by specific inhibitors of 5-Lox, such as MK591 Figures . We founin vitro invasion and completely blocks the anchorage-independent colony forming abilities of PCSCs on soft-agar at sub-lethal doses . These findings suggest that downstream mechanisms which mediate the survival-promoting effects of 5-Lox metabolites in PCSC involve the activity of PKCε, but not the more prevalent Akt or ERKs Figure . Cancer s Figure . Previouviz tumor growth, recurrence and metastatic spread. The important question now is how to eliminate the cancer stem cells without killing normal stem cells because they are vital for maintaining tissue homeostasis? Since the inexorable growth of tumor is maintained by this rare group of stem/progenitor cells within it, we can expect that the cancer stem cells, because of their unique biological features, may be more dependent on certain cellular processes than normal cells and thus, will be more vulnerable to some agents that block those processes. However, sorting out unique pathways that are critical for cancer stem cells, but not for non-tumor cells, though of utmost importance, is incredibly complex.CSCs are a rare sub-population of cells present within the tumor, and are responsible for tumor-initiation, maintenance and tumor-recurrence. They possess unique biological properties, such as self-renewal and pluripotency. Moreover, they are thought to be more resistant to conventional chemotherapy and, as a result, are responsible for disease relapse. The core principle of cancer stem cells suggests that somewhere at the heart of a tumor there lies a handful of aberrant, genetically altered cells that are critical to maintain the malignant characteristics. This idea bears the potential to explain why tumors often regenerate long-time after even being destroyed by standard anticancer regimen. Moreover, it points to an alternative strategy for developing new generation of anticancer drugs, suggesting that these agents should be more selective towards the lethality of cancer stem cells and not to kill just any cells to shrink the tumor mass. Interestingly, cancer stem cells share many characteristics with normal stem cells, which include self-perpetuation and differentiation to other cells, and with growing evidence that cancer stem cells indeed exist in a wide variety of tumor types, it is changing the landscape of tumor biology research. It is also becoming increasingly relevant to understand the basic molecular mechanisms that regulate their self-renewal and differentiation because deregulation of genes involved in these pathways likely participate in basic characteristics of cancer The stem cell concept is enticing because of the potential it can provide to more durable and widespread cancer cures by identifying and attacking the tumor's most notorious cells. Evidence is slowly accumulating to document that prostate cancer stem cells/tumor-initiating cells are key drivers in initiation and progression of prostate tumor. Therefore, successful therapy must eliminate these cells, which is hampered by their high inherent resistance to common therapeutic modalities. The discovery of cancer stem cells has led to formulation of new strategies in future therapy of prostate cancer. The so called “cancer normal cells” can be eliminated by conventional therapies  relatively easily, though these are rarely successful in attacking “cancer stem cells.” To be successful, the new approach should therefore be to develop therapeutic approaches to destroy these cancer stem cells. Thus far, only a handful of studies have addressed the cancer stem cell killing potential of standard apoptosis-inducing therapies and the underlying molecular mechanisms of apoptosis-resistance in these cells. Though our knowledge of PCSCs and their microenvironment remains a relatively new field, our current observations of PCSC's critical dependence upon 5-Lox activity for survival, as well as for the maintenance of self-renewal markers, make this regulatory mechanism ripe for developing experimental agents for effective management of prostate cancer. However, though it may be of high significance for development of durable prostate cancer therapy, whether 5-Lox activity plays any role in the survival of prostate cancer stem cells was never addressed before. This report documents that inhibition of 5-Lox, triggers rapid and wide-spread apoptosis in prostate cancer stem cells via activation of c-Jun N-terminal Kinase, and that this process is associated with down-regulation of PKCε, but not Akt or ERKs as we observed in regular prostate cancer cells –26, 37. +, CD44+, ALDH1+, ABCG2+, Nanog+, c-Myc+) were purchased from Celprogen, Torrance, CA, USA [+ human cord blood stem cells were isolated by Dr. N. Varma from fresh cord blood  using antibody-coated magnetic beads . Human foreskin fibroblasts (HFF) and DU-145 prostate cancer cells were purchased from American Type Culture Collection  and were grown in RPMI or DMEM medium 1640 . All the media were supplemented with 10% FBS and antibiotics. Antibodies against Nanog, c-Myc and survivin were purchased from R and D Systems , and antibodies against, Sox2, CD44, CD133, ABCG-2, cyclin D1, CDK4, and Bcl-xl were from Santa Cruz Biotechnology . Polyclonal antibodies against Akt, phospho-Akt (Ser473), phospho-ERK1/2 (Thr202/Y204), phospho-JNK (Thr183/Y185), and phospho-Stat3 (Ser727), were purchased from Cell Signaling Technology . Monoclonal anti-5-Lox antibody was purchased from BD Biosciences . Anti-beta-actin antibody, ibuprofen and cisplatin were purchased from Sigma Chemical CO . MK591 was obtained as a generous gift from Dr. Robert N. Young .Well-characterized human tumor-derived prostate cancer stem cells  as described before –26.5) were plated in complete growth medium supplemented with 10% FBS overnight onto 60mm diameter tissue culture plates  and allowed to grow for 48 hours. On the day of experiment, the spent culture medium was replaced with 2ml fresh RPMI medium and the cells were treated with inhibitors. Control cells were treated with solvent only (0.2% DMSO). Photographs were taken with a Nikon digital camera attached to a LEICA fluorescence microscope at ×400. Image acquisition and data processing were done with a Dell computer attached to the microscope using Q-Capture 7 software.Cells (~3 × 105) were plated in complete medium and allowed to grow for 48 hours. On the day of experiment, the spent culture medium was replaced with fresh 2ml medium and the cells were treated with MK591 or ibuprofen for 24 hours at 37°C. Then the cells were treated with FITC-labeled annexin-V and propidium-iodide for 15 minutes in the dark using an Annexin V-Binding Detection Kit following a protocol supplied by the manufacturer (BD Biosciences). After washing, cells were photographed using a Nikon digital camera attached to a LEICA fluorescence-microscope at 20×. Image acquisition and data processing were done with a Dell computer attached to the microscope using Q-Capture 7 software.Cells (~3 × 105) were plated in 60mm diameter tissue culture plates and allowed to grow for 48 hours. The old medium was then replaced with 2ml fresh medium and the cells were treated with inhibitors. After treatment, cells were harvested, washed, and lysed in lysis buffer . Proteins were separated by 12% SDS–PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 5% nonfat-milk solution and then blotted with appropriate primary antibodies followed by peroxidase-labeled secondary antibody. Bands were visualized by enhanced chemiluminescence (ECL) detection kit from Pierce Biotech  and analyzed with a densitometer using Kodak imaging software. Unless otherwise mentioned, protein blots were analyzed in three independent experiments.Cells , expanded, and re-plated in 96 well culture plates in triplicates. Cells were then treated with MK591 or solvent only, and the luciferase activity was measured by a Luciferase Assay Kit from Promega Corporation  as we reported recently , 26. IbuInvasion chambers (BD Biosciences) were soaked with 50 μl DMEM medium for 30 minutes at room temperature in cell culture hood. Then, a 50 μl single cell suspension of ~40,000 PCSCs in DMEM was gently placed on top of the matrigel with or without drugs. These chambers were then placed in twenty four well cell culture plates (one in each well) containing 500 μl of DMEM medium plus 2% serum as attractant. The chambers were incubated overnight in the incubator at 37°C. On the next day, matrigels inside the chambers were carefully scraped out and the membranes were fixed in methanol, stained with crystal violet, mounted on glass slides and counted under microscope.2 incubator for a maximum period of three weeks. Cell growth medium and inhibitors were exchanged every fourth day. At the end of incubation, cells were stained with 0.25% crystal violet and colonies were counted under a Leica microscope at ×150.Colony formation assays were performed in six well plates by placing ~5,000 Cells in 0.5 ml of 0.3% soft-agar on top of a 2 ml base layer of 0.6% agar. Plates were allowed to settle and then the wells were covered with 2 ml fresh medium containing 10% FBS with or without inhibitors. Plates were incubated at 37°C in the CO5) were plated in 60 mm dishes and allowed to grow for 48 hours. Cells were then treated either with the experimental agents or the solvent vehicle for varying periods of time up to 24 hours. At the end of incubation periods, cells were lysed and the degradation of chromatin DNA to nucleosomal fragments was measured by Cell Death Detection ELISAplus as described before [Apoptosis was quantitatively measured by detecting degradation of nuclear DNA to nucleosomal fragments by sandwich-ELISA. Cells (~3 × 10d before , 26, folOur findings revealed a new mechanism in the regulation of stemness and survival of prostate cancer stem cells, which suggests that by blocking 5-lipoxygenase activity with suitable agents we can effectively eliminate prostate cancer's most-resistant cells for development of a curative therapy of clinical prostate cancer and to prevent prostate cancer recurrence.