Dear Editor,1 Despite the recent success of BH3-mimetics targeting Bcl-2, Bcl-xL and Bcl-w, mechanism-based cell killing in vivo by targeting other important prosurvival proteins such as Mcl-1 or Bfl-1 has yet to be demonstrated. In the absence of small molecules targeting these prosurvival proteins, BH3-domain peptides, the prototypes for this class of drug, are useful because their binding specificity profile can be manipulated.3 Such peptides have been applied in in vitro studies to validate the targeting of particular prosurvival proteins in certain tumor types.5 However, in vivo applications require technically challenging chemical modifications of peptides, and while mouse xenograft models of virally-infected BH3 domain-expressing tumor cells can be used, this does not allow evaluation of effects of the ligand on normal tissues.BH3-mimetic drugs that antagonize Bcl-2 family prosurvival proteins are effective against some cancers, particularly those with abnormally high expression of the prosurvival protein target.in vivo and provide proof-of-principle for similar-acting drugs. To achieve this we adapted a previously described strategy allowing FLP-recombinase-mediated insertion of an expression cassette at an frt ‘landing pad' at the type I collagen (Col1a1) locus in mouse embryonic stem cells.6 The cassette comprises sequences encoding a BH3-domain protein under control of a tetracycline (tet)-regulated element (TRE) promoter. To develop a system that is broadly applicable to BH3 domains with different specificities, we employed the BimS BH3-only protein as a scaffold in which BH3 domains with different specificities could replace the native BH3 sequence  transactivator under the control of the cytomegalovirus (CMV) promoter, which provides high-level expression in many tissues including blood.8 Bitransgenic mice were then treated with doxycycline (Dox) to induce BimSBad expression  in platelet counts . Induction of BimSBadmut expression  seen in mice and humans treated with BH3-mimetic drugs of similar specificity. We envisage multiple applications for similarly engineered mice. For example, tet-regulated expression of different BimS variants in mice could reveal toxicities associated with neutralization of their prosurvival protein targets , and tissue-specific effects could be addressed by crossing onto mice where rtTA expression is driven by different promoters. Moreover, mice crossed to different tumor-prone models could provide in vivo evidence for the tumor-killing efficacy associated with different BH3 specificities not yet available through small molecule drugs, and extended to studies on combination therapies with existing drugs.We generated TRE-Bimpression . Westernnic mice . As ABT-t counts , a degrer 7 days , illustrpression  did not pression , indicat


A 68-day-old former 30-week infant presented with listlessness, apnea and bradycardia. The patient was intubated for airway protection. After intubation, breath sounds were auscultated bilaterally and a Pedi-Cap carbon dioxide detector had color change from purple to yellow. A nasogastric tube (NGT) was placed and a post-procedural chest radiograph was obtained .There are several features of esophageal intubation: low lung volumes, esophageal and gastric distention despite NGT placement and juxtaposition of the endotracheal tube (ETT) relative to the NGT.


Surprisingly, we detected a paucity of natural retinoids capable of transactivating RARA-dependent transcription in adult mouse bone marrow cells, and the trace activity we observed tended to be in erythroid-lineage cells, rather than in myeloid-progenitors. This suggests that the resistance to retinoid-mediated transactivation likely has a limited role in X-RARA-dependent leukemogenesis because natural retinoids are largely absent during normal myeloid maturation.The X-RARA fusion proteins have reduced sensitivity to all-RARB promoter, which may respond nonspecifically to Rara/Rxra and Rarg/Rxra heterodimers, and Rxra/Rxra homodimers.The presence and distribution of natural retinoids have not been studied in adult hematopoiesis. To detect retinoids capable of transactivating RARA, we developed a UAS-GFP reporter mouse. UAS promoter sequences are recognized by the yeast Gal4 transcription factor, and are not activated by mammalian proteins  or Gal4-RARG-IRES-mCherry , we observed a dose-dependent response to ex vivo ATRA, with subnanomolar sensitivity . This corresponds with the in vitro-measured Kd using radiolabeled ATRA .4 Synthetic, receptor-specific ligands induced receptor-specific GFP expression . The recipient mice were then maintained on standard chow  and then analyzed after complete engraftment (~6 weeks post-transplant). Surprisingly, we observed only trace GFPdim bone marrow cells in mice transplanted with Gal4-RARA-IC, suggesting that few bone marrow cells have active intracellular retinoids, and these exist at or below the threshold of RARA transcriptional activation (2 per day), before analysis on day 4. We observed a dose-dependent induction in GFP expression, suggesting that most bone marrow cells can respond to active retinoids when they are present  correlated with erythroid-lineage cells (Ter119+CD71+), not with myeloid progenitors (+Gr1+ and Kit+CD11b+) and erythroid progenitor cells  vs Kit+Lin−Sca+ (KLS)-enriched stem cells . In mice treated with 200 μg ATRA, the differences between specific progenitor compartments were blunted ,6 and RARA expression increases dramatically during myeloid maturation.7 Many of the X-RARA fusions have been proposed to act by reduced sensitivity to retinoid-dependent differentiation programs (a dominant negative effect).3 Therefore, we were surprised that the natural retinoids were largely absent during myeloid differentiation in vivo  expression and activity have been correlated with hematopoietic stem cells, and ALDH is the rate limiting step in ATRA synthesis.ous ATRA . In vivo+GFP− .We observed that even 200 μg of ATRA (given for short courses) was inadequate to activate retinoid-dependent transcription in all bone marrow cells in any hematopoietic compartment that we measured, and many cells remained mCherry+GFP− . SuccessThe UAS-GFP reporter system has the advantage of being highly modular , but this requires retroviral expression of the Gal4-fusion protein and subsequent transplantation/engraftment. We are currently generating a Gal4-RARA transgenic mouse to circumvent this issue. In addition, because GFP induction by the Gal4-RARA fusion protein requires functional co-activators, the GFP read-out integrates both the intracellular concentration of active ligands, and the intracellular co-activator/co-repressor environment. Cell populations that lack appropriate co-activators to induce response to ligand, or that contain trans-repressive effects from other transcription factors, will exhibit diminished or absent response to ligands. Thus, the read-out integrates both local retinoid concentrations and the transcriptional sensitivity to retinoids of the intracellular environment.in vivo. It is unknown whether the expression of PML-RARA or other hematopoietic stimuli might alter intracellular retinoid production, and whether this might lead to a pan-bone marrow endocrine effect or to a lineage-restricted retinoid production. The erythroid progenitor bias observed (in vivo retinoid availability in hematopoietic cells.This study has focused on intracellular retinoids that are present in bone marrow cells under steady-state conditions observed  at low din vivo. This suggests that these leukemic fusion proteins act predominantly through alternative mechanisms, and that retinoid-dependent transactivation likely has a limited role in normal, adult myeloid maturation.In conclusion, although X-RARA fusion proteins have been suggested to act by blocking retinoid-dependent transcriptional programs required for myeloid maturation, we observed a surprising paucity of natural retinoids capable of transactivating Gal4-RARA in primary mouse bone marrow cells


Background: Brain architecture can be divided into a cortico-thalamic system and modulatory “subcortical-cerebellar” systems containing key structures such as striatum, medial temporal lobes (MTLs), amygdala, and cerebellum. Subcortical-cerebellar systems are known to be altered in schizophrenia. In particular, intrinsic functional brain connectivity (iFC) between these systems has been consistently demonstrated in patients. While altered connectivity is known for each subcortical-cerebellar system separately, it is unknown whether subcortical-cerebellar systems’ connectivity patterns with the cortico-thalamic system are comparably altered across systems, i.e., if separate subcortical-cerebellar systems’ connectivity patterns are consistent across patients.Methods: To investigate this question, 18 patients with schizophrenia  and 18 healthy controls were assessed by resting-state functional magnetic resonance imaging (fMRI). Independent component analysis of fMRI data revealed cortical intrinsic brain networks (NWs) with time courses representing proxies for cortico-thalamic system activity. Subcortical-cerebellar systems’ activity was represented by fMRI-based time courses of selected regions-of-interest . Correlation analysis among ROI- and NWs-time courses yielded individual connectivity matrices  as main outcome measures, which were classified by support-vector-machine-based (SVM) leave-one-out cross-validation. Differences in classification accuracy were statistically evaluated for consistency across subjects and systems.Results: Correlation matrices based on allROIs-NWs yielded 91% classification accuracy, which was significantly superior to allROIs-allROIs and NWs-NWs . Considering separate subcortical-cerebellar systems, cerebellum-NWs and MTL-NWs reached highest accuracy values with 91 and 85%, respectively, while those of striatum-NW and amygdala-NW were significantly lower with about 65% classification accuracy.Conclusion: Results provide initial evidence for differential consistency of altered intrinsic connectivity patterns between subcortical-cerebellar systems and the cortico-thalamic system. Data suggest that differential dysconnectivity patterns between subcortical-cerebellar and cortical systems might reflect different disease states or patient subgroups. Widespread brain dysconnectivity is an essential element in the pathophysiology of schizophrenia : (i) We assessed slowly fluctuating ongoing brain activity , Olanzapine (n = 11), Clozapine (n = 4), Quetiapine (n = 2), Ziprasidone (n = 1), Risperidone (n = 5), Aripiprazole (n = 2), Paliperidone . All healthy subjects were free of psychotropic medication and any neurological and psychiatric disorder, current and in history.All patients included were diagnosed with paranoid schizophrenia during acute psychosis as indicated by clinical exacerbation and increased positive symptom scores on the PANSS Table . Seven oAll participants underwent 10 min of rs-fMRI with the instruction to keep their eyes closed and not to fall asleep. We verified that subjects stayed awake by interrogating via intercom immediately after the rs-fMRI scan. Before and after scanning, a medical examination of patients validated their stable condition and investigated whether they had feelings of odd situations during the scanning. No patient dropped out during the scanning session.2, matrix = 80 × 80, 32 slices, slice thickness = 4 mm, and 0 mm interslice gap; 300 volumes). T1-weighted anatomical MRI was based on magnetization-prepared rapid acquisition gradient echo sequence .MRI was carried out on a 3 T whole body scanner . FMRI was based on gradient echo EPI sequence , RMS (p > 0.25), or tSNR (p > 0.35).For each participant, the first three rs-fMRI scans were discarded due to magnetization effects. SPM8  was used for motion correction, spatial normalization into the stereotactic space of the Montreal Neurological Institute (MNI) and spatial smoothing with an 8 mm × 8 mm × 8 mm Gaussian kernel. To ensure data quality, particularly concerning motion-induced artifacts, temporal signal-to-noise ratio (tSNR) and point-to-point head motion were estimated for each subject  and , left and right Crus 1 and 2 following Krienen and Buckner  and , left and right hippocampus and parahippocampus ROIs following Kahn et al.  and .For each subcortical-cerebellar system, representative ROIs were defined based on previous studies, which investigated systems’ cortical iFC  and represented as typical correlation matrices. Correlation matrices for NWs-NWs and allROIs-allROIs connectivity were analogously created. Besides a global analysis including all ROIs and NWs as input for classification, also separateROIs-NWs correlation matrices were analyzed, including only one of the four subcortical-cerebellar system-ROIs at a time , thus facilitating comparison of classification accuracy obtained by each of the subcortical-cerebellar systems’ component.1,Y1), , …,  indicate the individual classification results, where Xi = 0 or Yi = 0 (misclassified), Xi = 1 or Yi = 1 (correct). We counted the number of pairs Xi > Yi. Let W be the number of pairs (Xi > Yi), N is the number of pairs that Xi and Yi are different, then W follows a binomial distribution W ~ B. The exact p-value was obtained by checking binomial distribution.The resulting correlation matrices of the two groups (feature vectors) were used for pattern recognition based classification via the WEKA toolboxICA analysis of rs-fMRI data from patients and controls identified 22 intrinsic networks, which cover mainly the cerebral cortex Figure .When separating individual patients from healthy controls via SVM classification accuracy for iFC patterns between all subcortical-cerebellar systems’ ROIs and cortical NWs  was 91% with high sensitivity and specificity levels of about 90% Table . ClassifWhen separating individual patients from healthy controls via SVM for different subcortical-cerebellar systems separately, classification accuracies were significantly higher for iFC patterns of cerebellum-NWs  and MTL-NWs  than for striatum-NWs  and amygdala-NW  and cortical intrinsic networks (NWs) in patients with schizophrenia. (i) Taking all subcortical-cerebellar systems of interest together, i.e., MTL, cerebellum, striatum, and amygdala , iFC separated patients from controls with an accuracy of about 90%, which was significantly superior in comparison to allROIs-allROIs and NWs-NWs connectivity. This result highlights the role of iFC between subcortical-cerebellar systems and cortical networks in contrast to both cortico-cortical and subcortical-cerebellar-subcortical-cerebellar systems’ connectivity in schizophrenia. (ii) Across subcortical-cerebellar systems, MTL-NWs and cerebellum-NWs connectivity showed highest classification accuracies of about 90%, which were significantly higher in comparison to iFC patterns of striatum-NWs and amygdala-NWs with accuracies of about 65%. This finding provides first evidence for differential across-system consistency of changes in intrinsic connectivity between different subcortical-cerebellar systems and the cortico-thalamic system. Data suggest that the whole pattern of cortical-subcortical-cerebellar system dysconnectivity might be differential for patients, maybe indicating different patient subgroups or disease states.Changes in iFC patterns between all subcortical-cerebellar and cortical networks are highly consistent in psychotic patients with schizophrenia indicated by classification accuracy, sensitivity, and specificity of about 90% Table . InteresIntrinsic connectivity changes between cerebellum and MTL respectively, and cortical networks were highly consistent in patients with accuracies of about 90% Table . This reIn contrast, iFC between striatum and amygdala, respectively, and cortical networks separated patients from controls with significantly lower accuracy levels of about 65% Table . AberranWidespread brain dysconnectivity is central to schizophrenia  Why did we select intrinsic networks as proxies for cortico-thalamic sub-systems instead of brain-atlas-based anatomically defined regions? Concerning brain activity, we focused on slowly fluctuating activity as measured by rs-fMRI and correspondingly on iFC of such ongoing activity. Overwhelming evidence indicates that iFC is organized by intrinsic networks particularly in the human cortex and exceeds anatomically defined cortex parcellations  Most patients of our study were treated with antipsychotic drugs (Table S1). Antipsychotic medication has been demonstrated to alter functional connectivity in patients with schizophrenia (Sambataro et al., Results provide preliminary evidence for more consistent iFC changes with cortical networks for MTL and cerebellum than for striatum and amygdala in schizophrenia. Differential iFC changes might reflect distinct disease states or patient subgroups.CS and HP designed the study; JB, MS, DS and HF recruited participants and acquired data; VR, CZ and AW acquired data; JS and HP analyzed data; CS, KK, VR, AW and HP interpreted data; CS, KK and HP drafted the article; all authors critically revised and approved the final version of the article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Increasing evidence suggests that after the first pro-inflammatory hours, sepsis is characterized by the occurrence of severe immunosuppression. Several mechanisms have been reported to participate in sepsis-induced immune alterations affecting both innate and adaptive immunity. Of these, the concept of ‘cell exhaustion’ has gained a lot of interest because some parallels can be drawn with the cancer field in which immunostimulation approaches through blocking immune checkpoints currently obtain remarkable success. Herein, perspectives regarding co-inhibitory receptors’ contribution to lymphocyte exhaustion in sepsis will be discussed in the context of a recently published study investigating the potential of PD-1 molecule expression  to predict mortality in septic shock patients. The work by Shao et al.  providesIn septic mice, administration of anti-PD1/anti-PD-L1 antibodies enhanced bacterial clearance by preventing lymphocyte depletion and alterations , 4. In aShao et al.’s study  agrees wImportantly, sepsis-induced impaired immune function is reversible and it remains possible to rejuvenate exhausted lymphocytes . Among mPD-1, Programmed Death-1; PD-L1, Programmed Death-ligand 1


Early detection and adherence to infection prevention recommendations are necessary to avoid transmission.  Middle East respiratory syndrome coronavirus (MERS-CoV) infections sharply increased in the Arabian Peninsula during spring 2014. In Abu Dhabi, United Arab Emirates, these infections occurred primarily among healthcare workers and patients. To identify and describe epidemiologic and clinical characteristics of persons with healthcare-associated infection, we reviewed laboratory-confirmed MERS-CoV cases reported to the Health Authority of Abu Dhabi during January 1, 2013–May 9, 2014. Of 65 case-patients identified with MERS-CoV infection, 27 (42%) had healthcare-associated cases. Epidemiologic and genetic sequencing findings suggest that 3 healthcare clusters of MERS-CoV infection occurred, including 1 that resulted in 20 infected persons in 1 hospital. MERS-CoV in healthcare settings spread predominantly before MERS-CoV infection was diagnosed, underscoring the importance of increasing awareness and infection control measures at first points of entry to healthcare facilities. Epidemiologic aspects of this virus remain poorly defined, but human-to-human transmission of MERS-CoV in healthcare facilities is recognized as a means of spreading infection ; most were reported from Abu Dhabi during March–April 2014, when the Arabian Peninsula had a sharp increase in infections, primarily involving healthcare workers (HCWs) and patients with recent healthcare exposure . After laboratory confirmation, all case-patients were hospitalized with airborne precautions until they had 2 consecutive PCR-negative MERS-CoV tests on specimens collected at least 48 hours apart. Close contacts, which included anyone who lived with, visited, provided care for, or had other similarly close contact with case-patients while they were symptomatic, were identified from interviews and other sources . The contacts were interviewed and had nasopharyngeal, sputum, or tracheal aspirate samples collected for PCR testing, regardless of symptoms. ,Medical and public health records were used to categorize sources of exposure. Infections of case-patients who worked at, were admitted to, or visited a healthcare facility during the 14 days before symptom onset were considered to be healthcare-associated if exposure to a known MERS-CoV case-patient occurred exclusively in this setting. Healthcare exposure to a case-patient was characterized as either confirmed  . Healthcare-associated clusters consisted of >1 source case  and >1 secondary case . Healthcare-associated clusters could also include tertiary and quaternary cases . Clinical records were used to assess probable exposures for healthcare-associated case-patients with no confirmed exposure; cases with confirmed exposure were not assessed for probable exposure because confirmed exposure was assumed to confer the greatest risk.Case investigations were used to construct cluster diagrams depicting suspected healthcare-associated transmission pathways. Healthcare-associated clusters were defined as ,Nasopharyngeal swab, sputum, or tracheal aspirate samples were tested at a central laboratory  in Abu Dhabi by using real-time reverse transcription PCR (rRT-PCR) for the upstream E gene and open reading frame 1 (http://www.illumina.com/systems/miseq.html) . Full genome sequencing from original respiratory samples was determined by using the Sanger method  and next-generation sequencing approaches . Fisher exact test and independent Of 65 MERS-CoV case-patients identified during our investigation period  in Abu-Dhabi, 27 (42%) were healthcare associated; 19 (70%) of the 27 were HCWs; 6 (22%) were hospitalized patients, and 2 (7%) were hospital visitors . An addiAll 3 source case-patients in the healthcare-associated clusters were men with a median age of 59 years; 2 had a history of camel exposure in the 14 days before symptom onset . All werOf the 27 healthcare-associated case-patients, 17 (63%) were male; median age was 43 years. None had a history of camel exposure during the 14 days before symptom onset. Disease severity varied by type of case; source case-patients had the greatest disease severity . Fewer tFrom the epidemiologic and genetic investigation, we identified 3 healthcare-associated clusters at 3 hospitals during our investigation period. The clusters ranged in size from 3 to 21 case-patients .The source case-patient for cluster I  in Abu Dhabi on 3 occasions for fever, cough, shortness of breath, and pneumonia. He was examined in an ED room (with a curtain divider) under standard precautions and was given a surgical mask and oxygen, which staff reported he removed repeatedly because of difficulty breathing. On April 6, he was admitted to a general medical unit (unit A), where he received care for 5 hours before being transferred to the intensive care unit (ICU) and placed on airborne infection isolation precautions. A MERS-CoV diagnosis was confirmed on April 9, and he died the next day.PCR screening of respiratory specimens from 224 possible healthcare contacts from the ED, ICU, and medical wards identified 15 healthcare-associated case-patients . Ten werA second subcluster of illnesses was detected in unit A  when a 74-year-old hospital patient (patient III-Q) who had been admitted to the unit in February developed new onset of shortness of breath on April 12 and had a MERS-CoV–positive sample collected the same day. During the 14 days before symptom onset, the patient was continuously hospitalized, had no travel history, no animal contact, and no confirmed exposure to a case-patient; however, she had resided in a room adjacent to patient III-A (source case-patient of cluster III) in unit A for 5 hours on April 6–7. During this period, no close contact with patient III-A occurred, and no documented common healthcare contacts or common equipment was identified; consequently, this case constitutes a probable rather than confirmed exposure.Subsequent screening of 83 healthcare contacts of patient III-Q identified 2 healthcare-associated case-patients, including patient III-Q’s daughter , who had been staying in the patient’s hospital room, and a nurse who provided care to patient III-Q in unit A . Further screening of 12 patients who received care from the infected nurse while she was symptomatic identified 2 more case-patients , who were bedbound chronic care patients hospitalized for >4 weeks before their MERS-CoV–positive sample collection date. No cases were identified among the 50 additional healthcare contacts screened from Unit A; these contacts included the remaining 42 HCWs who worked on Unit A and the 8 patients who had shared a room with patient III-T. In total, 20 healthcare-associated cases  are attributable to a single introduction in the hospital.A final healthcare-associated case-patient who was also cared for in unit A was identified . All 23 PCR-confirmed case isolates included in the validation sample were verified by using the N2 assay at CDC.Genetic sequencing was performed on a subset of 8 isolates: 7 from case-patients in healthcare-associated clusters  and 1 from a non–healthcare-related case-patient from Abu Dhabi . Full geOf the 14 HCWs  who became infected with MERS-CoV after caring for a source case-patient, 13 (93%) were exposed before the patient’s diagnosis. PPE use during care was inconsistent among these HCWs . The 1 HMERS-CoV in healthcare settings accounts for >40% of all reported infections in Abu Dhabi. We found that healthcare-associated transmission occurred predominantly when HCWs, patients, and visitors were exposed to an infected person before recognition of MERS-CoV and implementation of appropriate infection prevention measures. These findings underscore the importance of early detection and intervention to limit spread of disease.,,–In the largest healthcare cluster in our investigation, 1 patient appears to have directly infected 12 persons in 1 hospital, resulting in a total of 20 healthcare-associated infections caused by secondary, tertiary, and quaternary transmission. Among ED HCWs, we estimate a 16% attack rate, ≈4 times higher than average household transmission estimates , other transmission pathways cannot be excluded. Genetic sequencing of a limited number of cases supports the epidemiologic characterization of cases and clusters, but we were unable to sequence and assess the molecular relatedness of all case isolates, a step that previous investigations have shown to be informative (In conclusion, large healthcare clusters of MERS-CoV illness contribute to substantial illness and also have potential for secondary consequences, including fear among HCWs and the public. MERS-CoV can clinically appear with mild or nonspecific respiratory symptoms, and patients may seek care without having known risk factors for infection. Maintaining a high index of suspicion in every patient encounter, especially at first points of patient entry such as EDs or primary healthcare settings, is imperative, particularly in regions reporting MERS-CoV cases. Early detection of cases, full adherence to infection prevention recommendations, and recognition of illness among HCWs are necessary factors to prevent further transmission of MERS-CoV in healthcare settings. Supporting healthcare facilities in these efforts remains a priority.


Ciliary dysfunctions may have deleterious consequences on mucociliary clearance. We propose a new approach based on coupling the isolated ciliary beat pattern and the global efficiency of ciliary beat on human ciliated cells.-1). We have performed an original quantitative analysis of ciliary beat dynamics (CBD) by following cilium tips. It allows to describe different parameters including ciliary beat frequency also measured by Fast-Fourier-Transform and Video-Kymography. We have also developed the micro-beads tracking method (MBT) to get an index of the global efficiency of ciliary beat. Here, micro-beads (4.5 µm) have been used as markers of the flow generated by beating cilia.Ciliated cells issued from nasal brushing (controls and primary ciliary dyskinesia patients) were recorded by high-speed video-microscopy (350 frames s-1 depending on the distance between micro-beads and beating ciliated edges (the fastest micro-beads being the closest). Interestingly, the addition of micro-beads created a stimulus that significantly increased ciliary beat frequency (~130%).In term of frequency measurement, Fast-Fourier-Transform, Video-Kymography and CBD gave similar results. CBD was very helpful to discriminate controls and primary ciliary dyskinesia patients especially when ciliary beat was partially maintained. For the moment, MBT, only applied in controls, allowed to observe a flow rate from 1 up to 150 µm/sCoupling Fast-Fourier-Transform, Video-Kymography, CBD and MBT is a promising approach to characterize ciliary beat under normal and pathological conditions, either congenital as primary ciliary dyskinesia or acquired as chronic rhino-sinusitis or bronchitis.


High-risk patients with multivessel disease (MVD) represent a surgical challenge carrying high mortality risk. These cases elicit discussion within heart teams regarding the actual benefit of undertaking major surgery on these patients and often lead to abandon the surgical option. Off-pump coronary artery bypass (OPCAB) provides good quality graft on left anterior descending (LAD) without exposing the patient to cardiopulmonary bypass, and, despite providing an incomplete revascularization, might be the ideal choice in patients with multiple comorbidities, not eligible to percutaneous or on-pump procedures.To compare survival in high-risk patients with MVD and no percutaneous option, treated with incomplete off-pump surgical myocardial revascularization or discharged on optimal medical therapy.83 high-risk patients with MVD were enrolled: 42 were treated with incomplete off-pump revascularization using left internal mammary artery (LIMA) to LAD; 41 were treated with optimal medical therapy (OMT), having refused surgery. Patients were followed-up by telephone interview. Primary endpoint was survival from all-cause mortality; secondary endpoints were survival from cardiac-related mortality and freedom from non-fatal major adverse cardiac events (MACE).During follow up, there were 11 deaths in OPCAB group and 27 deaths in OMT group. Death was due to cardiac factors in 6 and 15 patients, respectively. MACEs were observed in 6 patients in OPCAB group and 4 patients in OMT group. Both survival from all-cause mortality and cardiac-related events were in favor of the OPCAB group over the OMT which carried a propensity score-adjusted hazard ratio of 3.862 and 3.663, for all-cause and cardiac-related mortality respectively. There was no statistically significant difference concerning freedom from MACE.For high-risk patients with MVD, considered ineligible for on-pump complete revascularization surgery or percutaneous coronary intervention, incomplete revascularization with OPCAB LIMA-on-LAD offers benefits in survival when compared to OMT alone.


Veno-arterial extracoporeal membrane oxygenation (VA ECMO) is increasingly used for acute and refractory cardiogenic shock. Yet, in clinical practice, monitoring of cardiac loading conditions during VA ECMO can be cumbersome. To this end, we illustrate the validity and clinical applicability of a real-time cardiovascular computer simulation, which allows to integrate hemodynamics, cardiac dimensions and the corresponding degree of VA ECMO support and ventricular loading in individual patients over time.The online version of this article (doi:10.1186/s12967-015-0760-1) contains supplementary material, which is available to authorized users. With great interest we read the recent publication by Ostadal et al. Their experimental work underscores the clinical relevance to closely monitor left-ventricular (LV) loading conditions in cardiogenic shock supported by venoarterial extracorporeal membrane oxygenation (VA ECMO) . They reIn order to enhance bedside clinical decision support, we introduced a real-time, patient-specific simulation model of cardiovascular dynamics , 7. The The experimental data by Ostadal et al. are in agreement with our clinical translation enhanced by real-time cardiovascular simulation, both demonstrating unsatisfactory LV unloading during high-flow VA ECMO Table . ImportaReal-time cardiovascular computer modeling allows to simulate complex patient-specific clinical scenario’s as in extracorporeal support. Our findings support the notion that VA-ECMO flow should be maintained as low as possible in order to balance the simultaneous need of circulatory support and LV unloading.


EGFR-mutant lung cancer patients. In contrast to the lesions outside the central nervous system (CNS), molecular mechanisms of EGFR tyrosine kinase inhibitor (TKI) resistance in CNS lesions including LMC are largely unknown. In this study, we established an in vivo imaging model for LMC with EGFR mutant lung cancer cell lines harboring an exon 19 deletion in EGFR and evaluated the effect of first generation EGFR-TKIs, erlotinib, second generation afatinib, and third generation AZD9291. In PC-9/ffluc model, erlotinib treatment slowed the development of LMC. Importantly, treatment with afatinib or AZD9291 apparently delayed the development of LMC. Moreover, treatment with a higher dose of AZD9291, also associated with inhibited phosphorylation of EGFR downstream molecule S6, regressed LMC refractory to the aforementioned EGFR-TKI treatments. These observations suggest that the third generation EGFR-TKI AZD9291 may be an effective treatment for first or second generation EGFR-TKI resistant LMC caused by EGFR-mutant lung cancer.Leptomeningeal carcinomatosis (LMC) remarkably decreases the quality of life of  EGFR mutation more often compared with patients without the mutation , antibody diluent, and serum-free protein block were purchased from Dako. Formalin-fixed paraffin-embedded tissue skin sections were subjected to antigen retrieval and endogenous peroxidase blocking, and were incubated with primary antibody overnight at 4°C. Following overnight incubation, slides were rinsed and incubated with a peroxidase-labeled polymer. The tissue sections were then rinsed and stained with 3,3′-diaminobenzidine (DAB) substrate-chromogen and then counterstained with Hematoxylin Gill I (EMD Millipore) and bluing reagent (EMD Millipore).


Prostate biopsy for prostate cancer (PCa) is invasive with associated morbidity and several diagnostic limitations, suggesting the need for a new approach. Recently, several nucleic acid cancer biomarkers  have been identified and shown promise for improving cancer diagnostics. However, the abundance of these biomarker classes in the circulation is low, impeding reliable detection and adoption into clinical practice. In order to stimulate the release of these intracellular biomarkers, the exposures optimized for mechanical disruption of cells may be desirable.Here, two approaches based on HIFU-induced bubble activity were tested for their ability to stimulate release of cancer-associated microRNAs in a heterotopic syngeneic rat prostate cancer model. In the first approach, tumor tissue was locally liquefied with boiling histotripsy (BH) - a HIFU technique utilizing millisecond-long pulses to create boiling bubbles via rapid shockwave heating. The interaction of shocks with the ensuing vapor cavity fractionates tissue with negligible thermal effect. In the second approach HIFU-induced inertial cavitation was used for permeabilization of tumor tissue and vasculature.Putative miRNA biomarkers were identified using RT-PCR array profiling of the syngeneic MatLyLu rat PCa cell line. Adult intact male Copenhagen rats were then subcutaneously grafted with the MatLyLu cells. When the tumors were >1cm, the rats were assigned to one of two HIFU treatment groups: HIFU optimized for inertial cavitation activity , BH  or a to a control group (N=6) that received sham treatment. Treatments were performed in a heated water tank using a single-element 1.5 MHz HIFU transducer  under ultrasound image guidance. Blood samples were collected immediately prior to treatment and serially over a 24-hour time course. Specimens were immediately processed into plasma and miRNAs extracted. Plasma concentrations of candidate tumor-derived miRNAs were measured via quantitative RT-PCR and compared with ANOVA and the Mann-Whitney test.Following sham procedure, no significant changes were observed in the relative plasma concentrations of any evaluated miRNA. Conversely, following both cavitation-based and BH treatments, the relative plasma concentrations of the putative PCa-associated miRNAs miR-34c and miR-196a increased significantly while the relative concentration of the broadly expressed, non-PCa specific miR-16 was not significantly altered. PCa-associated miRNA concentrations peaked at 0.25 hr (10-23-fold) from initiation of HIFU treatment, remained significantly elevated for 3 hours, and then returned to baseline within 24 hours. Histologic examination of excised tumor confirmed complete fractionation of targeted tumor by BH and localized areas of intraparenchymal hemorrhage and tissue disruption by cavitation-based treatment. These data suggest a clinically useful application of HIFU-induced bubbles for non-invasive molecular biopsy.


Escherichia coli. We created Deconvoluter—ordered libraries with selectable insertions every 50 kb in the E. coli genome. The Deconvoluter method uses these for replacement of untagged mutations in the genome using a phage-P1-based gene-replacement strategy. We validate the Deconvoluter resource by deconvolution of 17 of 17 phenotype-altering mutations from a screen of N-ethyl-N-nitrosourea-induced mutants. The Deconvoluter resource permits rapid unbiased screens and gene/function identification and will enable exploration of functions of essential genes and undiscovered genes/sites/alleles not represented in existing deletion collections. This resource for unbiased forward-genetic screens with mapping-by-sequencing (‘forward genomics’) demonstrates a strategy that could similarly enable rapid screens in many other microbes.With the wide availability of whole-genome sequencing (WGS), genetic mapping has become the rate-limiting step, inhibiting unbiased forward genetics in even the most tractable model organisms. We introduce a rapid deconvolution resource and method for untagged causative mutations after mutagenesis, screens, and WGS in  Escherichia coli, which has been favored historically by geneticists, physicists turning to biology, and others because of its relative simplicity and the ease of propagation in the laboratory , rig


Here, preformed donor-specific alloreactive T-cell responses were evaluated using the IFN-γ ELISPOT assay in a large consecutive cohort of kidney transplant patients (n = 90), to assess the main clinical variables associated with cellular sensitization and its predominant time-frame impact on allograft outcome, and was further validated in an independent new set of kidney transplant recipients (n = 67). We found that most highly T-cell sensitized patients were elderly patients with particularly poor HLA class-I matching, without any clinically recognizable sensitizing events. While one-year incidence of all types of biopsy-proven acute rejection did not differ between T-cell alloreactive and non-alloreactive patients, Receiver Operating Characteristic curve analysis indicated the first two months after transplantation as the highest risk time period for acute cellular rejection associated with baseline T-cell sensitization. This effect was particularly evident in young and highly alloreactive individuals that did not receive T-cell depletion immunosuppression. Multivariate analysis confirmed preformed T-cell sensitization as an independent predictor of early acute cellular rejection. In summary, monitoring anti-donor T-cell sensitization before transplantation may help to identify patients at increased risk of acute cellular rejection, particularly in the early phases after kidney transplantation, and thus guide decision-making regarding the use of induction therapy.Preformed T-cell immune-sensitization should most likely impact allograft outcome during the initial period after kidney transplantation, since donor-specific memory T-cells may rapidly recognize alloantigens and activate the effector immune response, which leads to allograft rejection. However, the precise time-frame in which acute rejection is fundamentally triggered by preformed donor-specific memory T cells rather than by  Outstanding progress has been made in recent decades in assessing the humoral alloimmune sensitization against donor HLA antigens in kidney transplant patients, and has led to a major reduction in acute antibody-mediated rejection (ABMR) rates immediately after transplantation. However, no comparable success has been achieved in the monitoring of the anti-donor T-cell immune response. As a consequence, acute T-cell mediated rejection (TCMR) is still an unpredictable event, and this uncertainty negatively affects decision-making in daily clinical practice.In fact, there is a considerable inconsistency between what we know from basic immune biology and what we have learnt from clinical transplantation. It is well accepted that T cells are key initiators, mediators and effectors of the alloimmune response, thus playing a key role in allograft rejection –3. In fade novo donor-specific T-cell alloreactivity and subclinical TCMR in protocol biopsies, suggesting a specific time-frame relationship between preformed donor-specific memory T cells or de novo alloreactive naïve T cells and their impact on kidney allograft outcome.Importantly, monitoring T-cell sensitization against donor or even a panel of reactive antigens has been shown to be feasible and reliable using the highly sensitive IFN-γ ELISPOT assay, and has also been shown to correlate with worse allograft function after kidney transplantation –17. In tHere, we analyzed the presence of pre-transplant donor-specific T-cell sensitization in a large consecutive cohort of 90 kidney transplant recipients in whom the type of immunosuppression was given without knowing their baseline anti-donor T-cell sensitization status and the data obtained was further validated in a new independent group of kidney transplant recipients (n = 67). We aimed to investigate the main clinical variables associated with cellular sensitization and the specific post-transplant time-frame in which preformed donor-specific (d-s) memory T cells may negatively challenge allograft outcome.Ninety adult kidney transplant recipients from our Renal Transplant Unit at Bellvitge University Hospital (n = 90) attended between 2011 and 2012 were retrospectively analyzed on the basis of the availability of donor stimulator cells consisting of donor splenocytes in deceased-donor transplants or of donor peripheral blood mononuclear cells (PBMC) in living-donor transplants. The Bellvitge University Hospital ethics committee specifically approved the study, and all patients gave written informed consent. None of the transplant recipients had received immunosuppression before providing PBMCs to perform the ELISPOT assay. The pre-transplant ELISPOT result was not available to the clinicians before or after transplantation, and so had no influence on the choice of immunosuppression and clinical management after the transplantation. The mean time of follow-up of this cohort was 21 months (range 6–48 months).Main baseline demographic variables in all patients were collected at the time of enrollment and included donor source and age, time on dialysis, number of human antigen leukocyte (HLA) mismatches, number of previous transplants, presence of donor-specific circulating antibodies by solid phase assays (Luminex®), cause of end-stage renal disease (ESRD) and recipient ethnicity (caucasian or otherwise). Furthermore, most relevant clinical variables associated with clinical transplant outcome such as type of maintenance IS (CNI-based or not), steroid withdrawal, use and type of induction therapy (T-cell depletion or not), delayed graft function (DGF), six and twelve-month allograft function , CMV infection, biopsy-proven acute rejection (BPAR), type  and time of rejection  were also pooled together for the analysis. DGF was defined as the need for dialysis during the first week after transplantation. The use of either T-cell depleting or monoclonal antibody induction therapy was based on the observed presumable risk derived from the percentage of PRA and HLA mismatch data. CNI were used as maintenance IS treatment in all but two patients who received mTor-inhibitors (Everolimus) as main immunosuppression.Estimated glomerular filtration rate (eGFR) was calculated using the simplified Modification of Diet in Renal Disease equation (MDRD). All patients in the study showed a negative pre-transplant Complement-dependent cytotoxic (CDC) cross-match test.A second independent validation cohort of kidney transplant patients (n = 67) was used to evaluate the predictive value of pre-transplant donor-specific T-cell alloreactivity on the advent of early T-cell mediated allograft rejection. These set of patients were consecutively transplanted from November 2011 to October 2013. The clinical management of all these patients was done irrespective of the pre-transplant donor-specific T-cell Elispot result, as the test was not done.Automated nucleotide sequencing was performed from genomic DNA by selective amplification (PCR) of target exons from each locus for a particular allele. Loci sequenced included HLA class I  and II (DRB1/3/4/5 and DQA1/DQB1). Nucleotide sequencing was done as previously described .Screening for circulating anti-HLA class I and II alloantibodies in peripheral blood was done by FlowPRA screening beads (One-lambda Inc.). Antibody specificities of positive samples were determined as previously described  using th5 PBMC.Donor-specific IFN-γ ELISPOT assays were done following recently described techniques ,21. PeriRenal allograft biopsies were performed in patients undergoing acute clinical graft dysfunction. All renal histologies were analyzed following the Banff ‘09 score, and histological analyses were blindly evaluated.2-test for categorical variables and the one-way analysis of variance or Student’s t-test for normally distributed data for quantitative variables, and the nonparametric Kruskal-Wallis or Mann-Whitney U-test for non-normally distributed variables.All data are presented as mean ± sd. Groups were compared using the XA sensitivity/specificity ROC curve analysis was done to determine the ELISPOT test value for the prediction of the advent of early T-cell mediated acute rejection. Stepwise linear regression and binary logistic regression analysis were performed to determine the independent correlation of several independent variables with the presence of early cellular acute rejection. The statistical significance level was defined as p<0.05.The main baseline clinical and demographic variables were assessed among all patients for their association with pre-transplant T-cell sensitization. As shown in pneumocystis juvencii. Three allografts were lost within the first year, one due to a vein thrombosis, another due to unresolved urine leakage , and a third in a highly T and B-cell sensitized patient due to a severe BPAR (mixed ABMR and TCMR) who did not respond to intensive rescue immunosuppressive treatment.Two patients died during the first year; neither presented pre-formed donor-specific T-cell sensitization. One died of a cardiac arrest nine months after transplantation and the other of an opportunistic infection caused by T-cell alloreactive and non-alloreactive patients were comparable in terms of main baseline clinical variables such as the use and type of induction therapy, the choice of maintenance immunosuppression  as well as regarding steroid withdrawal . High prIn order to establish whether any specific time frame could most likely distinguish all TCMR events significantly associated with pre-transplant T-cell sensitization, a receiver-operating characteristic (ROC) curve analysis was carried out taking into account all time-points of BPAR episodes and the pre-transplant ELISPOT data. Interestingly, the first eight weeks after transplant proved to be the most accurate time frame for identifying TCMR episodes that were related to the pre-transplant donor-specific T-cell immune response, with a notably high sensitivity and specificity   . Now, usEven though pre-transplant anti-donor alloreactive patients showed worse 6 and 12-month allograft function, these values were probably influenced by the significant higher donor age of alloreactive transplant recipients as compared to non-alloreactive recipients (data not shown). Nonetheless, a numerically worse allograft function was consistently observed among pre-transplant alloreactive patients when stratified both by type of kidney transplant (living or deceased donors) as well as by donor age (older or younger than 50 years old) (data not shown).Since pre-transplant T-cell sensitization correlated positively with both recipient and donor ages and are matched for transplant selection, we were particularly interested in determining whether recipient age and pre-transplant cellular sensitization could influence the risk of early TCMR. Therefore, we stratified recipient age as a binary variable (below or above 50 years), and evaluated the risk of early TCMR depending on the pre-transplant ELISPOT test (R<50/ELSPOT- (n = 30); R<50/ELSPOT+ (n = 14); R>50/ELSPOT- (n = 23) and R>50/ELSPOT+ (n = 23). As shown in Whether the use and type of induction therapy could impact on preformed highly alloreactive anti-donor T-cell frequency was further evaluated. While the use of any type of induction therapy did not discriminate those patients developing any kind of BPAR , patients receiving T-cell depletion with rATG showed a significantly lower incidence of early TCMR as compared to those that did not  . FurtherThe most relevant clinical variables influencing the outcome of early TCMR  were assessed with univariate and multivariate Cox regression analysis. Although non-caucasian ethnicity, longer ischemia time, non-use of T-cell depletion and a positive pre-transplant ELISPOT were associated with an increased risk of early TCMR, only the presence of baseline anti-donor T-cell sensitization and non-use of T-cell depletion as induction therapy were significant independent correlates of risk for early TCMR .The impact of preformed donor-specific T-cell alloreactivity was validated in a second independent cohort of kidney transplant patients with similar baseline clinical and immunological characteristics. As shown in In this study, we show that high frequencies of donor-specific alloreactive memory/effector T cell responses before kidney transplantation is frequent among patients waiting for a kidney allograft. Furthermore, we report for the first time that this preformed anti-donor T-cell sensitization seems to have a direct negative impact on kidney allograft outcome by favoring TCMR during the early period of time after transplantation, especially among highly T-cell sensitized individuals against donor antigens and patients not receiving T-cell induction therapy. Of note, this observation was further validated in a subsequent independent cohort of kidney transplant patients with high sensitivity and negative predictive value.de novo naïve T-cell activation, potentially reflecting insufficient immunosuppressive exposure. In fact, in the multivariate analysis performed, the presence of high frequencies of circulating donor-specific memory/effector T cells prior to transplant surgery was shown to be an independent predictor of early TCMR, but not of all the immune-mediated clinical events occurring later on. A plausible explanation of the relatively frequent detection of anti-HLA cellular alloreactivity before kidney transplantation may be heterologous immunity, in which T cells, initially primed against infectious agents and environmental antigens, cross-react with allogeneic MHC molecules [Unlike acute antibody-mediated rejection (ABMR), TCMR remains an unpredictable process in clinical practice since no accurate immune monitoring is currently done. However, in recent years, a strong deleterious association has been observed between the presence of alloreactive memory T cells and clinical and also subclinical immune-mediated allograft injury or allograft dysfunction in humans ,13,22,23olecules ,25. NeveA conclusion that can be drawn from our study is the importance of an optimal HLA matching, especially within class I molecules between donor/recipient pairs in order to reduce the chance that pre-formed memory T cells recognize donor alloantigens. Likewise, we recently showed that the higher the number of HLA mismatches, the greater the frequency of donor-specific memory T-cell responses, particularly among T-cell subsets primed by the direct pathway of alloantigen presentation . Even threpertoire, these cells have a significantly poorer capacity to mount effective recall effector responses compared with young memory/effector T cells [milieu triggered in these grafts could facilitate a much more effective effector T-cell response among T-cell sensitized individuals due to the higher immunogenicity. In our study, although a similar higher T-cell sensitization was found among individuals receiving older allografts, the most relevant variable influencing the outcome was recipient’s age. This observation stresses the fact that transplant rejection is much more likely to happen in highly T-cell sensitized younger individuals, whereas in older sensitized transplant patients it would be facilitated if receiving highly immunogenic tissues from older donors.Even though older individuals showed increased frequencies of alloantigen-specific memory T cells as compared to younger patients, younger T-cell alloreactive individuals seem to have a more effective anti-donor effector immune response, as shown by the significantly higher incidence of early TCMR particularly among younger alloreactive T-cell sensitized patients. A plausible explanation is that although aged recipients exhibit higher numbers of memory T cells with a broader antigen  T cells –30. Inte T cells  recentlyAn important issue still under debate in clinical kidney transplantation is the choice and type of induction therapy. In our study we found that the use of T-cell depletion as induction therapy with rATG, in combination with CNI drugs, seemed to provide significantly better protection for T-cell sensitized patients against developing TCMR than the use of anti-IL2 receptor monoclonal antibodies or no induction therapy. Indeed, we found a significant reduction of early TCMR among highly T-cell sensitized patients receiving rATG as compared to transplant recipients using other type of induction treatment or no induction, whereas the use of rATG did not provide any benefit as compared to other induction treatments when given in non T-cell alloreactive individuals thus, suggesting the usefulness of this assay to individualize the use of such aggressive induction therapy. This finding is in fact in line with previous reports from others and us ,32,33 adThe new observations reported in this work underline the high interest of this assay for being implemented in clinical practice as it provides crucial information to transplant clinicians not currently available; on the one hand, the likelihood of T-cell mediated rejection in the very early phases after transplantation that may not be inferred with patients’ baseline clinical background on the other, help further refine decision-making regarding the use of T-cell depletion as induction therapy, particularly among the most fragile transplant population, that is in the older range of age, in whom in absence of humoral sensitization, this potent immunosuppression could be safely avoided.A main limitation of this study is its retrospective nature. However, the multivariate analysis performed, together with the results obtained, which corroborate those of some previous reports, strengthen our observations and should alert the transplant community to the urgent need to perform prospective, observational studies as well as interventional trials to test our results.The high sensitivitiy and particularly high negative predictive value of the ELISPOT test for early TCMR is of great relevance due to its capacity to rule out the disease. Therefore, patients with no evidence of T-cell sensitization would be suitable candidates to enroll in such clinical trials, which should ideally be conducted under the auspices of international collaborative networks.High levels of donor-specific alloreactive memory T cells may be relatively frequent prior to transplantation, despite the absence of any clinically recognizable sensitizing events. They may directly facilitate the advent of TCMR early after transplantation, particularly in patients not receiving T-cell depleting agents as induction therapy. Therefore, screening for anti-donor T-cell sensitization should be seriously considered in kidney transplant patients. At this point, prospective randomized trials are clearly warranted.


Leu-Gly). However, in this study, we sequenced the coding region for OXT in 22 species covering all New World monkeys (NWM) genera and clades, and characterize five OXT variants, including consensus mammalian Leu8-OXT, major variant Pro8-OXT, and three previously unreported variants: Ala8-OXT, Thr8-OXT, and Phe2-OXT. Pro8-OXT shows clear structural and physicochemical differences from Leu8-OXT. We report multiple predicted amino acid substitutions in the G protein-coupled OXT receptor (OXTR), especially in the critical N-terminus, which is crucial for OXT recognition and binding. Genera with same Pro8-OXT tend to cluster together on a phylogenetic tree based on OXTR sequence, and we demonstrate significant coevolution between OXT and OXTR. NWM species are characterized by high incidence of social monogamy, and we document an association between OXTR phylogeny and social monogamy. Our results demonstrate remarkable genetic diversity in the NWM OXT/OXTR system, which can provide a foundation for molecular, pharmacological, and behavioral studies of the role of OXT signaling in regulating complex social phenotypes.Oxytocin (OXT) is an important neurohypophyseal hormone that influences wide spectrum of reproductive and social processes. Eutherian mammals possess a highly conserved sequence of OXT (Cys-Tyr-Ile-Gln-Asn-Cys-Pro- Our two findings showing ligand-receptor coevolution and phylogeny-trait association suggest that the OXTR/OXT system may be a factor contributing to social monogamy, a conclusion that is supported by pharmacological and neuroanatomical evidence that OXT is an important modulator of sociality in NWM [Callithrix): pair-bonded marmosets treated with Pro8-OXT (but not Leu8-OXT) showed reduced social and sexual interactions with opposite-sex strangers [The diversity in OXTR/OXT structures among primates may have important implications for understanding diversity in social systems. In prosimian and Old World primates, the regular expression of social monogamy is rare or absent; in hominoid primates (apes and humans), social monogamy is noted only in gibbons, siamangs and humans . By conty in NWM , 33. We trangers . These bIn addition, we recently characterized coding region for arginine vasopressin (AVP), a nonapeptide neurohormone that is closely related to OXT that also plays an important role in modulating social behavior in mammals . In starSaimiri) that coding sequence variation in ligand produces corresponding differences in OXT and OXTR mRNA and protein structure [in vivo pharmacological and behavioral studies with altered OXT and OXTR will further characterize the functional role of OXT/OXTR system diversity in social monogamy in primates.As described in detail previously , social tructure . This vaS1 Table* DNA = extracted and purified DNA sample provided by institution; otherwise, we extracted DNA from the source tissue indicated.(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 TableN-terminus; TM = transmembrane region; IC = intracellular region; EC = extracellular region; C-term, C-terminus.N-term = (DOCX)Click here for additional data file.


Intestinal monocytes/macrophages sustain the intestinal immune homeostasis and might be an attractive therapeutic target for the management of inflammatory bowel disease (IBD). Granulocyte macrophage colony-stimulating factor (GM-CSF) exerts beneficial effects on intestinal inflammation and promotes signal transducer and activator of transcription 3 (STAT3)-mediated expansion of myeloid-derived suppressor cells (MDSCs). However, the full action mechanism of GM-CSF, and especially whether monocytes mediate its therapeutic effects in vivo, had not been previously elucidated.  This review article summarizes recent developments in the immunology of mucosal diseases and describes new aspects of the role of myeloid regulatory cells in IBD and the function of GM-CSF in maintaining the intestinal immune homeostasis in Crohn’s disease (CD). This review article highlights the exploration of stimulating in addition to suppressive therapies for patients with IBD and underpins that myeloid regulatory cells might become a promising novel cell-based therapeutic option. Crohn’s disease (CD) is a chronic relapsing disease and is a manifestation of a dysregulated immune response against the microorganisms of the intestinal flora in genetically susceptible individuals . VariousMonocytes and their derivative cells play an important role in the pathophysiology of IBD. Intestinal macrophages derived from blood monocytes play a key role in sustaining the innate immune homeostasis in the intestine . IntestiGM-CSF also triggers several different signaling pathways in myeloid-derived suppressor cells (MDSCs) that mainly involve the signal transducer and activator of transcription (STAT) family of transcription factors. MDSCs are a heterogeneous group of immature cells that includes precursors of macrophages, granulocytes, DCs, and myeloid cells at earlier stages of differentiation. MDSCs expand rapidly during inflammation, infection, and cancer and suppress T cell responses . Althougin vivo, had not been previously elucidated.The potential for future cell-based therapies for IBD is enhanced by the advances being made in the understanding of the innate immune system in the intestine. Based on this, any therapeutic approach aimed at modulating monocytes/macrophages as a key player in innate immunity could be a potent way to modulate/manage intestinal inflammation in patients with chronic intestinal inflammation. However, the full action mechanism of GM-CSF, and especially whether monocytes mediate its therapeutic effects in vitro using microarray technology and functional assays taking all potential functions of human GM-CSF-activated monocytes into account . Our findings suggest that the early imprinting of monocytes after activation with GM-CSF is of crucial importance, because monocytes play an important role during the recruitment phase of the innate immune response and have the potential to regulate adaptive immune mechanisms.We hypothesized that GM-CSF might activate monocytes in a way that modulates their function during intestinal inflammation. We analyzed the immunoregulatory potential of GM-CSF on human monocytes + T cells, we observed that GM-CSF-activated monocytes attract T cells and shape their differentiation toward Th2 cells by upregulating T cell-derived IL-4, IL-10, and IL-13 and downregulating T cell-derived IFNγ in vitro. In addition, cocultures of GM-CSF-activated monocytes and naïve T cells led to an induction of Treg, which produce anti-inflammatory IL-10 and TGFβ and have important anti-inflammatory functions [We showed that GM-CSF-activated monocytes combine (i) an anti-inflammatory phenotype, (ii) features of augmented innate immune functions , (iii) the ability to facilitate epithelial healing, and (iv) the regulatory potential on adaptive immunity. GM-CSF-activated monocytes represent an intermediate cell type, combining cell-surface expression characteristics and functional features of different M2-type macrophage subsets . In contunctions .Finally, we showed that GM-CSF may regulate the homing molecules CCR2 and CCR6 on human monocytes , which ain vitro, we next aimed to address the functionality of these cells in vivo in the context of CD. Because systematic analyses in the human system are not feasible, we went on analyzing the effects of GM-CSF-activated monocytes on the murine system. In the first step, we activated bone-marrow-derived murine monocytes with GM-CSF in vitro and analyzed the phenotype and functional properties of murine GM-CSF-activated monocytes in vitro.After having analyzed the specific activation pattern of human monocytes in response to GM-CSF in vitro that this population has a lower capacity for phagocytosis and adhesion but triggers increased generation of reactive oxygen species, which are required for pathogen clearance. Moreover, after lipopolysaccharide treatment, proinflammatory cytokine production was found to be higher in GM-CSF-activated monocytes compared with untreated monocytes. However, despite their phenotypical and functional diversity, murine GM-CSF-activated monocytes mainly seem to represent monocytes in a developmental stage passing toward M2-polarized macrophages with an upregulation of M2 markers and important anti-inflammatory functions [Our gene expression analyses showed that murine GM-CSF-activated monocytes have a similar gene regulation profile when compared with the GM-CSF-dependent gene expression in human monocytes . Like huunctions .In addition, we performed flow cytometry-based staining of various gut homing molecules on monocytes and found, in agreement with our studies on human GM-CSF-activated monocytes, CCR2, necessary for migration toward inflammatory sites, and CCR6, used for the migration toward lymph follicles such as Peyer patches, to be upregulated on murine GM-CSF-activated monocytes .+CD25− T cell-induced experimental colitis when they received GM-CSF-activated monocytes but not untreated monocytes. In agreement with earlier work, we found that Rag1-deficient mice that did not receive GM-CSF-activated monocytes but GM-CSF intraperitoneally after the transfer of T cells were not completely protected from colitis but showed reduced disease severity. Our data demonstrate that the protective effect of monocytes depends on their GM-CSF pre-activation in a T cell-dependent model of colitis [In the next step, we tested whether GM-CSF-activated monocytes have a protective and therapeutic effect against intestinal inflammation in vivo. Since none of the currently available animal models of IBD reflects all aspects of IBD in humans, we used various mouse models of chronic colitis. Firstly, we showed that Rag1-deficient mice  were protected from disease progression during CD4 colitis . Secondl colitis . Thirdly colitis .+ Tregs in the lymph follicles of mice with experimental colitis when compared with untreated mice or those treated with nonactivated monocytes. In an in vitro model in which GM-CSF-activated monocytes were cocultured with naïve T cells, we observed increased proliferation and differentiation of Foxp3+ Treg cells and found that this occurred through a mechanism that involves the CD39-mediated conversion of ATP into adenosine—a mechanism that was previously reported to induce Treg cell differentiation [We next sought to decipher the mechanism by which GM-CSF-activated monocytes confer their therapeutic effects on intestinal inflammation. We found that, compared with control monocytes, the GM-CSF-activated monocytes were taken up faster by the inflamed intestine and stayed there longer, especially in Peyer patches , 16. In + Treg cells. This is in agreement with the work of others, who showed that GM-CSF can influence the interactions between DCs and T cells and in this manner determine a pro- versus anti-inflammatory adaptive immune response via Th1 and Th17 cell responses or the induction of Treg cells, respectively [Taken together, these important data show that GM-CSF-activated monocytes are a specific population of monocytes that has a protective effect against intestinal inflammation Fig. . The benectively .Fig. 2MeGM-CSF bioactivity varies inversely with increasing serum levels of endogenous neutralizing GM-CSF autoantibodies (Ab). GM-CSF Ab as well as defective GM-CSF receptor expression have been described in IBD –25. IncrOur phenotypic and functional studies showed that peripheral blood monocytes from CD patients in clinical remission are not impaired compared to healthy controls . We next757F/F mice is associated with protection from DSS-induced colitis. We hypothesized that the protective role of gp130-dependent STAT3 activation in experimental IBD involves the expansion and activation of MDSCs, in addition to the previously reported proliferative, regenerative, and survival effects on intestinal epithelial cells. We therefore induced acute and also chronic colitis with DSS and analyzed the effects in mice with systemic hyperactivation of STAT3 (gp130757F/F) ± myeloid-specific STAT3-deficiency (LysMcre/STAT3flox). We showed that the resistance to DSS-induced acute colitis in gp130757F/F mice is via myeloid cell-specific STAT3 activation, expansion of granulocytic MDSCs in the colon, and increased production of suppressive and protective mediators. Interestingly, gp130757F/F mice were not resistant to chronic DSS-induced colitis [Several studies have investigated the mechanisms of MDSC T cell suppressive activity in health and disease including experimental colitis models and human IBD , 9. MDSC colitis .Thus, our study identified new immunoregulatory mechanisms of STAT3 during intestinal inflammation. Importantly, the adoptive transfer of MDSCs in different animal models of IBD ameliorates colitis and suggests that MDSCs may be used as the basis for a novel adoptive cellular cell therapy in IBD (–36. In aThe work of others and our in vitro and in vivo data show that GM-CSF-activated monocytes are a specific population of monocytes that has a protective effect against intestinal inflammation as shown in various independent mouse models of experimental colitis. The beneficial effects of GM-CSF-activated monocytes are T cell dependent and involve the proliferation and differentiation of Th2 and Treg cells in the intestine (Fig.


MicroRNAs (miRs) regulate hepatic steatosis, inflammation and fibrosis. Fibrosis is the consequence of chronic tissue damage and inflammation. We hypothesized that deficiency of miR-155, a master regulator of inflammation, attenuates steatohepatitis and fibrosis.Wild type (WT) and miR-155-deficient (KO) mice were fed methionine-choline-deficient (MCD) or -supplemented (MCS) control diet for 5 weeks. Liver injury, inflammation, steatosis and fibrosis were assessed.MCD diet resulted in steatohepatitis and increased miR-155 expression in total liver, hepatocytes and Kupffer cells. Steatosis and expression of genes involved in fatty acid metabolism were attenuated in miR-155 KO mice after MCD feeding. In contrast, miR-155 deficiency failed to attenuate inflammatory cell infiltration, nuclear factor κ beta (NF-κB) activation and enhanced the expression of the pro-inflammatory cytokines tumor necrosis factor alpha (TNFα) and monocyte chemoattractant protein-1 (MCP1) in MCD diet-fed mice. We found a significant attenuation of apoptosis (cleaved caspase-3) and reduction in collagen and α smooth muscle actin (αSMA) levels in miR-155 KO mice compared to WTs on MCD diet. In addition, we found attenuation of platelet derived growth factor (PDGF), a pro-fibrotic cytokine; SMAD family member 3 (Smad3), a protein involved in transforming growth factor-β (TGFβ) signal transduction and vimentin, a mesenchymal marker and indirect indicator of epithelial-to-mesenchymal transition (EMT) in miR-155 KO mice. Nuclear binding of CCAAT enhancer binding protein β (C/EBPβ) a miR-155 target involved in EMT was significantly increased in miR-155 KO compared to WT mice.Our novel data demonstrate that miR-155 deficiency can reduce steatosis and fibrosis without decreasing inflammation in steatohepatitis. MicroRNAs (miRNAs) are small non-coding RNAs that regulate the expression of numerous target genes at the transcriptional or translational level and play important roles in liver diseases . AlteredA clinically relevant challenge in NASH research is to define factors that lead to progression of steatosis to steatohepatitis and fibrosis. Increasing evidence suggests the role of innate immunity, pattern recognition receptors, including TLR4 and TLR9, stimulated by various microbial and endogenous danger molecules in the development of steatohepatitis and fibrosis ,7. miRNAPersistent and excessive liver damage leads to chronic inflammation and fibrosis . ImpairmHere we hypothesized that miR-155 has a role in the development and progression of nonalcoholic steatohepatitis and fibrosis. Our novel data show that miR-155 deficiency promotes inflammation, and increases some inflammatory cytokines/chemokines such as TNFα and MCP1 in MCD-steatohepatitis. This shows the complex role of miR-155 in the inflammatory pathways and also emphasizes the importance of its negative regulatory role in inflammation. Our findings also revealed that despite of the significant inflammation, miR-155 deficiency attenuates steatosis and fibrosis in NASH suggesting that miR-155 regulates fibrosis, at least partially, independent of inflammation in the liver.; n = 5–10. miR-155 deficient (knock out/KO) mice with the appropriate control groups were fed with MCD or MCS diet for 5 weeks (n = 6–10). miR-155 KO mice were purchased from Jackson laboratory  and breeding colony was maintained in the animal facility of UMMS.This study was approved by the University of Massachusetts Medical School Institutional Animal Use and Care Committee. Six-to-eight week-old female C57Bl/6 wild type (WT) mice were fed with methionine-choline-deficient (MCD) diet for 3, 6 or 8 weeks; controls received a DL-methionine (3 g/kg) and choline bitartrate (2 g/kg) supplemented (MCS) diet , while cellular miR-155 expression was evaluated by qPCR as described  is one of the most common liver diseases worldwide . NASH isHere we report novel findings on the dual role of miR-155 on regulating steatosis and fibrosis in the methionine-choline deficient (MCD) model of steatohepatitis. We show that there is an increased miR-155 expression in both parenchymal and non-parenchymal cells in MCD diet-fed livers and we demonstrate that miR-155 deficiency attenuates steatosis in the MCD model. Our findings reveal that miR-155 regulates fibrosis at least partially independent of inflammation in NASH, since miR-155 deficiency attenuated MCD diet-induced fibrosis despite of sustained significant inflammation and liver injury. Finally, we propose that miR-155 affects fibrosis at multiple levels, via direct and indirect targets, including PDGF, Smad3 and C/EBPβ in NASH.In the HFD model, hepatic steatosis was significantly enhanced by miR-155 deficiency .Increased fatty acid uptake, increased fatty acid synthesis, impaired fatty acid oxidation or impaired VLDL secretion might all contribute to hepatic steatosis . While iBeyond fatty acid metabolism, dietary cholesterol has also been shown to contribute to the development of fatty liver and steatohepatitis ,44,45. NOverall these results suggest that miR-155 targets lipid metabolism via multiple mechanisms and it might vary depending on the model of steatohepatitis. Further studies are needed to clarify the exact pathomechanism via which miR-155 targets genes in the lipid metabolism.Hepatic steatosis is a risk factor; a preceding step for nonalcoholic steatohepatitis and its progression to fibrosis according to the two-hit hypothesis model . HoweverSteatohepatitis, and in general any type of chronic inflammation and cell death, tissue damage of the liver, potentially leads to liver fibrosis. One of the proposed mechanisms of the fibrosis development is the monocyte/macrophage recruitment/activation and inflammatory and fibrogenic cytokine production ,50. The According to previous studies IL-1β and TNFα influences fibrosis via hepatic stellate cell survival and not activation , while oStellate cells and their transdifferentiation into extracellular matrix producing myofibroblasts is the central event in fibrosis, however, other contributors of fibrogenic cells exist too, including portal fibroblasts, bone marrow-derived mesenchymal cells and EMT . During In conclusion, here we show novel data that miR-155 deficiency attenuates liver steatosis and fibrosis, but not liver injury and inflammation in the MCD model of steatohepatitis . The reg


Anti-cancer drugs might exert their action during physiological pathways of apoptosis, leading to tumour cell destruction. One of the most effective anti-cancer agents used in the treatment of colorectal cancers is 5-fluorouracyl (5-FU), but tumour chemoresistance is a major limiting factor of its use. The purpose of this study was to assess whether different doses of plant bioactive compounds as genistein (GST) or resveratrol (RSV) might increase the anti-carcinogenic and anti-proliferative effects of 5-FU treatment and modulate gene The compound-mediated cytotoxity was measured by real-time cell analysis (RTCA) using xCELLigence System, a cell-based label-free platform technology that utilises the inherent morphological and adhesive characteristics of the cell. DNA progression through cell cycle phases was estimated by using PI staining (BD Cycletest Plus/DNA Reagent kit), while apoptosis was assessed by Annexin-V/FITC and PI double staining. In addition, antigen expression was evaluated by indirect immunofluorescence followed by flow-cytometry acquisition and analysis by FACS Canto II flow-cytometer. Gene expression was measured by qRT-PCR using Applied Biosystems® 7300 System.Both GST and RSV single treatments, or combined with 5-FU induced a decrease of S% cell cycle phase, and an increse of apoptotic events. Different concentrations of GST or RSV blocked the treated cells in G2M; cells treated with RSV and 5-FU were blocked in G0G1 phase, while combined treatments with GST and 5-FU blocked the cells in G2M phase. The gene and antigen expression of molecules associated to cell proliferation and apoptosis were modulated by single and combined treatments, and additive effects of RSV or GST to 5-FU treatment were observed.The chemo-preventive efficacy has been associated to enhanced apoptosis, therefore any therapeutic strategy that specifically triggers apoptosis in cancer cells might have potential immunotherapeutic value. By combining flavonoids with anti-cancer drugs, an increase of the antitumoral effects might be achieved, specifically in highly invasive cancer cells, while in non-tumoral cells the cytotoxic side effects could be reduced.


Salvia miltiorrhiza (Danshen in Chinese). Research fromour laboratories and others have revealed the synergy of a tanshinones-TRAILcombination in diverse types of cancer cells through up-regulation of DR5 and/ordown-regulation of antiapoptotic proteins such as survivin. Thus, in addition totheir anticancer mechanisms, tanshinones as TRAIL sensitizers hold great potentialto be translated to TRAIL-based therapeutic modalities for combatting cancer.An ideal cancer therapy specifically targets cancer cells while sparing normaltissues. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) elicitsapoptosis by engaging its cognate death receptors (DRs—namely, DR4 and DR5. Thecancer cell-selective proapoptotic action of TRAIL is highly attractive for cancertherapy, but clinical application of TRAIL is rather limited due to tumors’ inherentor acquired TRAIL resistance. Combining TRAIL with agents that reverse resistance toit has proved promising in the sensitization of TRAIL-induced apoptosis. Noteworthy,natural compounds have already been validated as potential resources for TRAILsensitizers. In this review, we focus on the recently identified TRAILsensitizingeffect of tanshinones, the anticancer ingredients of the medicinal plant  Salvia miltiorrhiza Bunge (Lamiaceae) (a.k.a. Danshen)Figure  that hasin vitro and in vivo studies have revealed the anticancer actions as well as theunderlying mechanisms of these main tanshinones  is aneffective chemotherapeutic for acute promyelocytic leukemia (APL) that works byinducing APL cell differentiation, but resistance to ATRA does eventuallydevelop. Notably, tanshinone IIA has effectively induced APL celldifferentiation in both ATRA-sensitive and –resistant cell lines, likely throughinducing CCAAT/enhancer-binding protein β (C/EBPβ)-mediated differentiation[All-ntiation.TRAIL is a type II membrane protein belonging to the TNF death ligandsuperfamily, which also includes TNFα and Fas ligand (FasL/CD95L) . TRAIL iThe ability to induce cancer cell-selective apoptosis makes TRAIL anattractive choice for cancer therapy. Indeed, preclinical trials using solubleforms of recombinant TRAIL have shown a promising tumoricidal effect and, unlikeTNFα and CD95L, barely caused systematic toxicity . ClinicaOur knowledge regarding the mechanisms of TRAIL resistance in tumor cells hasadvanced greatly in recent years. In general, the deregulation of the moleculesinvolved in TRAIL-initiated apoptotic pathway is closely linked to the developmentof TRAIL resistance , 42. BriGiven an acquired resistance to TRAIL commonly develops in most human tumors,current clinical trials for TRAIL-based therapies employ combination strategiesusing agents that overcome TRAIL resistance, thus restoring sensitivity toTRAIL-induced apoptosis -45. InteReversing the mechanisms of TRAIL resistance forms the functional basis ofTRAIL-sensitizing agents. Indeed, the majority of TRAIL sensitizers reported todate synergize with TRAIL by inducing up-regulation of DRs (particularly DR5),thus highlighting DRs’ expression levels as the primary point of control forTRAIL-induced apoptosis. In this context, DR5 is up-regulated through distinctmechanisms of action . Mostnaet al. identified crytotanshinone as a TRAIL sensitizerin the human melanoma cell line A375 and the lung adenocarcinoma cell line A549,both refractory to TRAIL [et al. recently reported thattanshinone I restores the sensitivity of the TRAIL-resistant human prostate cancercell lines PC-3 and DU145 to TRAIL-induced apoptosis [Using the TRAIL-resistant human ovarian cancer cell lines TOV- 21G and SKOV3as cellular models, in early 2013 we published the first report demonstrating theTRAIL-sensitizing effect of crytotanshinones, tanshinone I, and tanshinone IIA. Later oto TRAIL .Likewispoptosis . These sDR5 along with increasedcell-surface DR5 expression following tanshinone IIA stimulation [We made the pioneering discovery that crytotanshione, tanshinone I, andtanshinone IIA all exert TRAIL-enhancing action on the TRAIL-resistant humanepithelial ovarian cancer (EOC) cell lines TOV-21G and SKOV3, with tanshinone IIAshowing the best potency .Subsequmulation .DR5 promoter.The functional significance of the ROS-JNK-CHOP signaling axis in tanshinoneIIA-mediated DR5 up-regulation is clearly supported by the failure of tanshinoneIIA to induce DR5 when CHOP is depleted by CHOP shRNA, JNK-specific inhibitorSP600125, or ROS scavenger N-acetylcysteine (NAC) [The levels of DRs on the cell surface is essential for TRAIL to induceeffective apoptotic signaling, whereas low levels of cellsurface DRs confer TRAILresistance. Along this line, the functional blockade of DR5 by the DR5/Fc chimerprotein abolished tanshinone IIA’s action to sensitize TRAIL, indicating that DR5up-regulation primarily determines tanshinone IIA as a TRAIL sensitizer in thecontext of EOC cells. Mechanistic studies on how tanshinone IIA up-regulates DR5uncovered that tanshinone IIA triggers reactive oxygen species (ROS) production toinduce JNK activation, leading to the transcriptional up-regulation ofCAAT/enhancer-binding protein homologous protein (CHOP), a well-establishedtranscriptional activator of the Down-regulation of IAPs such as survivin or XIAP represents an alternativeapproach to overcome TRAIL resistance aside from DR5 up-regulation . Surviviet al. demonstrated that Tanshinone Isynergistically sensitizes TRAIL-induced apoptosis in these TRAIL-resistant cells[DR5 promoter, and this was accompanied byan increase in cell-surface DR5 levels. Not surprisingly, the depletion of DR5severely lowered the potency of tanshinone I to enhance TRAIL-induced apoptosis,confirming the essential role of DR5 up-regulation in tanshinone I-mediated TRAILsensitization. Furthermore, Shin et al. made aunique, noteworthy discovery that microRNA 135a-3p (miR135-3p) elicited by thetanshinone I-TRAIL combination accounts for the up-regulation of DR5, whereas themiR135-3p inhibitor attenuated the apoptosis induced by the tanshinone I-TRAILcombination. However, how miR135-3p up-regulates DR5 was not demonstrated in thereport. Likewise, although tanshinone I evoked ROS production in these cell lines,the roles of ROS in miR135-3p induction and TRAIL sensitization by tanshinone Iwere not addressed either.Using the human prostate cancer cell lines PC-3 and DU-145 as cellular models,Shin nt cells. Furtheret al. identified cryptotanshinone, amongtanshinone I, dihydrotanshione I, and tanshinone IIA, as the most potenttanshinones to induce DR5 expression in the TRAILresistant human melanoma A375cells [DR5 transcription for enhancing TRAIL’s proapoptoticaction. It is also noteworthy that p53 is not required for cryptotanshinone toup-regulate DR5, as evidenced by the comparable induction of DR5 in the HCT116cell lines with or without p53 expression. As a matter of fact, the p53-independent nature of DR5 up-regulation by cryptotanshinone is advantageous,considering that the majority of human cancer cells are deficient in p53-mediatedapoptosis.To employ DR5 up-regulation as the strategy of to overcome TRAIL resistance,Tse 75cells . Similarin vitro level so far. Therefore, preclinical validationof tanshinones- TRAIL synergy in xenograft or orthograft mouse cancer models isnecessary for subsequent clinical trial design, which will hopefully lead to thefuture translation of tanshinones to TRAIL-based cancer therapy.TRAIL, which benefits from its malignant cell-selective proapoptotic action, isan ideal cancer therapeutic agent, but its potential is ironically sabotaged byeither intrinsic or acquired resistance commonly developed by tumor cells. Acombination of TRAIL with agents that overcome TRAIL resistance mechanisms has beenvalidated in preclinical and clinical trials as a promising strategy to boostTRAIL’s efficacy. In this regard, tanshinones as effective TRAIL sensitizers holdgreat potential to be included in TRAIL-based cancer therapeutic regimens. As forthe TRAIL-sensitizing mechanisms of tanshinones, it appears that ROS-mediated DR5up-regulation is the primary modes of action . It would be informative to decipher additional targets of TRAILresistance mechanisms likely modulated by tanshinones in the context of varioustypes of tumor cells, as a detailed molecular understanding of tanshinones-elicitedTRAIL sensitization would allow for a rational design for more effectivetanshinones-TRAIL synergy. Furthermore, although only cryptotanshinone, tanshinoneI, and tanshinone IIA are validated as TRAIL sensitizers to date, additional membersof tanshinones that are more effective to circumvent TRAIL resistance are likelypresent and remain to be discovered. It is also worth noting that the action oftanshinones as TRAIL sensitizers have been demonstrated only at the Salvia miltiorrhiza. We are also grateful toour students for contributing to the tanshinones-TRAIL synergy studies, andapologize for the literature of tanshinones not being included in this article dueto limited space.This work was supported by grants from Central Taiwan University of Science andTechnology, Taichung, Taiwan (CTU104-P- 16); National Chung Hsing University andAgricultural Research Institute, Council of Agriculture, Executive of Yuan, R.O.C.(NCHU-TARI 9904 and NCHU-TARI 10104); Taichung Veterans General Hospital andNational Chung Hsing University, Taichung, Taiwan (TCVGH-NCHU997606); and TheMinistry of Education, Taiwan, R.O.C. under the ATU plan. We thank Dr. Jui-Sheng Lai for kindly providing the photographs ofThe authors declare no conflicts of interest for this work.


Atg7), concurrently with G12DKras activation with or without intact Trp53 in two mouse models for non-small-cell lung cancer (NSCLC). In both models, Atg7 deficiency caused tumor cells to accumulate dysfunctional mitochondria, and acquire metabolic, growth and survival defects associated with reduction in tumor burden. Importantly, Atg7 deficiency altered the fate of G12DKras-induced carcinomas to that of oncocytomas, rare, predominantly benign tumors characterized by the accumulation of defective mitochondria. Surprisingly, lipid accumulation was observed in Atg7-deficient tumors only when Trp53 was deleted. Atg7-deficient tumor-derived cell lines (TDCLs) had compromised starvation survival and formed lipidic cysts instead of tumors, suggesting defective utilization of lipid stores. Atg7 deficiency reduced fatty acid oxidation (FAO) and increased sensitivity to FAO inhibition, indicating that with Trp53 loss, RAS-driven tumors require autophagy for mitochondrial function and lipid catabolism. Thus, autophagy is required for carcinoma fate, and cancers require autophagy for distinct roles in metabolism that are oncogene- and tumor suppressor gene-specific. To test the role of autophagy in oncogenic signaling pathways downstream of RAS, Atg7 was deleted in a mouse model of V600EBRAF-induced lung cancer in the presence or absence of the tumor suppressor Trp53. Atg7 deletion initially induced oxidative stress and accelerated tumor cell proliferation in a manner indistinguishable from Nrf2 ablation.Autophagy degrades and recycles proteins and organelles to support metabolism and survival starvation. Oncogenic RAS upregulates autophagy required for mitochondrial function, stress survival, and engrafted tumor growth. We deleted an essential autophagy gene, autophagy-related-7 (Atg7 and Nrf2 had no additive effect suggesting that both genes modulate tumorigenesis by regulating oxidative stress, revealing a potential mechanism of autophagy-mediated tumor suppression. At later stages of tumorigenesis, Atg7 deficiency resulted in an accumulation of defective mitochondria, proliferative defects, reduced tumor burden, conversion of adenomas and adenocarcinomas to oncocytomas, and increased mouse lifespan. Autophagy-defective tumor-derived cell lines were defective in their ability to respire, survive starvation and were glutamine-dependent, suggesting that autophagy-supplied substrates from protein degradation sustains V600EBraf-tumor growth and metabolism.Compound deletion of