Oxidative stress seems to be a role in the atherosclerosis process, butresearch in human beings is scarce.To evaluate the role of oxidative stress on human aortas of patientssubmitted to surgical treatment for advanced aortoiliac occlusivedisease.Twenty-six patients were divided into three groups: control group (n=10)formed by cadaveric organ donors; severe aortoiliac stenosis group ; and total aortoiliac occlusion group. We evaluated thereactive oxygen species concentration, nicotinamide adenine dinucleotidephosphate-oxidase, superoxide dismutase and catalase activities as well asnitrite levels in samples of aortas harvested during aortofemoral bypass fortreatment of advanced aortoiliac occlusive disease.P<0.05).Nicotinamide adenine dinucleotide phosphate oxidase activity was also higherin total aortoiliac occlusion group when compared to the control group. Furthermore, superoxide dismutase andcatalase activities were significantly higher in the severe aortoiliacstenosis and total aortoiliac occlusion groups when compared to the controlcases (P<0.05). Nitrite concentration was smaller in thesevere aortoiliac stenosis group in comparing to the other groups.We observed a higher level of reactive oxygen species in total aortoiliacocclusion group (48.3±9.56 pmol/mg protein) when compared to severeaortoiliac stenosis (33.5±7.4 pmol/mg protein) and control(4.91±0.8 pmol/mg protein) groups (Our results indicated an increase of reactive oxygen species levels andnicotinamide adenine dinucleotide phosphate-oxidase activity in human aorticsamples of patients with advanced aortoiliac occlusive disease. The increaseof antioxidant enzymes activities may be due to a compensative phenomenon toreactive oxygen species production mediated by nicotinamide adeninedinucleotide phosphate oxidase. This preliminary study offers us a morecomprehensive knowledge about the role of oxidative stress in advancedaortoiliac occlusive disease in human beings. It is widely accepted that oxidative stress playsimportant role in the pathogenesis of atherosclerosis[4]. Oxidative stress iscaused by an imbalance between the production of reactive oxygen species (ROS) andthe antioxidant capacity of the biological system[5]. ROS such as superoxideanion (O2-), hydroxyl radical (OH-), and hydrogenperoxide (H2O2), are produced through different pathways, butthe major source of ROS in the vasculature isnicotinamide-adenine-dinucleotide-phosphate (NADPH) oxidase. Superoxide dismutase(SOD) is the major cellular defense system to remove O2- invascular cells by converting O2- intoH2O2, while catalase (CAT) converts two molecules ofH2O2 into water and oxygen[7]. Nitric oxide (NO) -synthetized by nitric oxide synthase (NOS) - is a multifactorial free radical thatplays a key role in the physiological regulation of the cardiovascular system, andchanges in its production and/or bioavailability follow or even precede diseasessuch as atherosclerosis[8]. Superoxide anion may react with NO to formperoxynitrite (ONOO-), resulting in an increased cellulardamage7. Moreover,inactivation of NO by O2- limits the NO bioavailabilityleading to increased vasoconstriction, as commonly observed in the progression ofatherosclerosis[7].The cellular and molecular basis for atherosclerosis is complex and is not completelyunderstood, but research in human beings arerare[11]. In this study, we evaluated the role of oxidativestress in patients's aortas whith SAS and TAO submitted to aortobifemoral bypass.Thus, we proposed to compare the oxidative damage in different degrees of AOD inhuman beings.Some experimental studies have investigated the role of oxidative stress in theatherosclerosis process[2].The project was approved by the Research Ethics Committee of Santa Casa de PortoAlegre and all patients signed free and informed consent forms. We reviewed medicalrecords for patients with SAS and TAO electively submitted to aortobifemoral bypassby the first author as previously describedAll the patients were submitted to surgical procedure due to a limiting claudicationor critical limb ischemia . Patients with acuteaortoiliac occlusion, abdominal aortic aneurysm (AAA) thrombosis, and submitted toprevious aortoiliac intervention (endovascular or open repair) were excluded. Datawere collected on age, gender, comorbidities and clinical presentation for patientswith SAS or TAO. Laboratory profiles and surgical data were also routinelycollected. All the patients were submitted to computed tomography angiography toplan the surgical procedures.[12].Human specimens of infrarenal abdominal aorta were removed in the operating roomduring the proximal anastomosis of aortobifemoral bypasses. After removing thecalcifications, the aortic samples were immediately stored at -70°C for furtheranalysis of oxidative stress parameters. They were macerate in liquid nitrogen.After that, it was homogenized  in the Ultra-Turrax homogenizer. Posteriorly, it was performed asonification with the Hielscher Ultrasound Technology device[13].ROS concentration was measured by DCFH-DA fluorescence emission .Dichlorofluorescein diacetate is membrane permeable and is rapidly oxidized to thehighly fluorescent 2,7-dichlorofluorescein (DCF) in the presence of intracellularROS. The samples were excited at 488 nm and emission was collected with a 525 nmlong pass filter. It was expressed as nmols per milligram ofprotein[14]. SOD activity, expressed as U/mg protein, was basedon the inhibition of superoxide radical reaction withpyrogallol[15]. CAT activity was determined by following thedecrease in 240 nm absorption of hydrogen peroxide (H2O2). Itwas expressed as nmoles/mg protein[16]. NO in aortic samples was examined by measuringthe level of nitrite, an oxidative metabolite of endogenous NO, by using the Griessreagent, in which a chromophore with a strong absorbance at 542 nm is formed byreaction of nitrite with a mixture of naphthyletilenediamine (0.1%) andsulphanilamide (1%). The absorbance was measured in a spectrophotometer to give thenitrite concentration[17]. Protein was measured by the method of Lowry etal.[18],using bovine serum albumin as standard.Measurement of NADPH oxidase activity was assayed with spectrophotometricmethodAll data are expressed as the mean ± standard deviation. Comparisonsbetween two groups were performed by Fisher's exact test and statisticalanalysis for three groups included the ANOVA method followed by t test. Astatistical significance was assumed to be α=5%.Twenty-six patients were divided into three groups: control group (n=10) formed bycadaveric organ donors (aged 22 to 51 years old); SAS group (n=9) formed by patientswith SAS; and TAO group (n=7) formed by patients with chronic total aortoiliacocclusion. The clinical and surgical data are summarized in P<0.05). Patients with TAOdemonstrated higher levels of ROS (48.3±10.22 pmol/mg protein) when comparedto the SAS group (The aortic ROS levels were significantly higher in the SAS and TAO groups whencompared to the control group ( P<0.05).P>0.05).Measurement of NADPH oxidase activity demonstrated that TAO group had an incrementwhen compared to control group .MoreoveP<0.05) (P<0.05) (It was observed an increase of SOD activity in the SAS and TAO groups when comparedto the control group (P<0.05) . However(P<0.05) .P<0.05). Furthermore, therewas no difference in the nitrite levels between TAO and the control group(P>0.05) (Nitrite levels were significantly lower in the aortic samples of patients with SASwhen compared to the other groups ((P>0.05) ..Advanced AOD tends to occur in relatively young patients who have a history oftobacco abuse. Basically, patients with SAS and TAO are treated by theaortobifemoral bypass and the surgical results in these patients seems to besimilar. In atherosclerosis, ROSproduction can increment endothelial dysfunction, vascular smooth muscle cellsproliferation and apoptosis, and inflammatory response[20]. Another markers ofoxidative stress, such as levels of O2-, thiobarbituric acidand conjugated dienes were also evaluated in aortic samples of patients withatherosclerosis[21].The major finding of our study was to demonstrate the role of oxidative stress in theatherosclerosis in patients with different degrees of AOD. Our results demonstratedthat ROS levels were progressively higher as more severe as the AOD was. This way,patients with SAS or TAO had a higher ROS levels on their aorta samples; and the TAOgroup demonstrated a more elevated of ROS levels than SAS group. The role of ROS inthe onset and progression of atherosclerotic damage in aortas has been described inanimal models of different illnesses, being the ROS important mediators in thesignaling pathways of inflammation and atherogenesis[22] demonstrated that the earliest changes in anexperimental model of AAA were associated with the local production of ROS. In thestudy of Miller et al.[21], it was measured levels ofO2- and lipid peroxidation products in segments of AAA andin adjacent nonaneurysmal aortic tissue removed from patients undergoing electiveAAA repair. The results indicated that levels of ROS are locally increased in AAA,partially because of NADPH oxidase activity, and lead to marked increases inoxidative stress. There are evidences that NADPH oxidase expression and activity areupregulated in atherosclerosis[23]. Analysis of non-atheroscleroticversus atherosclerotic human carotid arteries demonstrated ahigher level of NADPH oxidase expression in atheroscleroticarteries[23]. We also observed an increase of NADPH oxidaseactivity in patients with TAO when compared to the control group. Haidari etal.[3]observed an increase of oxidative stress in atherosclerosis-predisposed regions ofthe normal C57BL/6 mouse aorta, implicating NADPH oxidase as a possible contributingenzyme.Xiong et al.[6]. Thus, knowledge of the activity of antioxidantsenzymes is very important. Under normal conditions, SOD is the first line of defenseagainst oxidative stress[5]. Dubick et al.[24] demonstrated a lower activity of SODin human aortic samples of patients with AAA and AOD in comparing tonon-atherosclerotic organ donors. In another work, the same group of authors alsocompared the SOD activity between aortic specimens of patients with aneurysmal andocclusive disease. These authors evidenced a reduced SOD activity in occlusive(n=14) compared to aneurysmal (n=29) group[25]. In contrast, our study showed asignificant increase of SOD activity in the SAS and TAO samples when compared to thecontrol group, maybe because a compensative phenomenon to minimize oxidative stress.Furthermore, the role of oxidative stress in different aortopathies was evaluated bySoto et al.[5]. In this study, there was an increase in SODactivity in aorta specimens of patients with hypertension, Marfan and Turner'ssyndromes[5].The imbalance between oxidants and antioxidants in favor of the former ones plays asignificant role in the pathogenesis of atherosclerotic vasculardisease2O2 molecules to breakthem into O2-; one acts as a reducing agent and the other asan oxidant agent. Overexpression of CAT prevents the stimulation of ROS and canprevent AAA formation in experimental setting[26]. Moreover, a diminished CAT expressionand activity were observed in polymorphonuclear neutrophils in patients withAAA[27].Soto et al.[5] also showed that CAT activity increased in thepatients with different aortopathies. Our results corroborate these findings thatthe increase of CAT activity in SAS and TAO groups may be due to overproduction ofH2O2 in the aorta from these patients.Catalase is another important antioxidant enzyme, which is found in the liver,kidney, and aorta. Catalase uses two H[11]. Endothelialdysfunction and downregulated NO would contribute to the stiffness, reduceddistensibility, and aortic complications such as AOD[5]. Our results showed adecrease of nitrite levels in SAS group may be due to endothelium dysfunction.Moreover, we did not observe an increase in nitrite levels in patients with TAO andthis can be explained at least in part because of the higher levels of ROS whichcould increase the nitrite levels by inducible NOS activation, an enzyme whichproduces high quantities of NO, resulting in more oxidativestress. Taking together, the higher levels of ROS andnitrite could facilitate intravascular thrombus formation by reducing theantiplatelets effects of NO[28]. Then, this aortic thrombus can provoke aninflammatory response in the aortic wall and can also limit the oxygen supply toendothelium resulting in increasing of NO production[29]. Liao etal.[11]founded an overexpression of NOS which was positively correlated with the degree ofinflammation in the aortic wall in patients with AAA.NO is usually quoted for its vasodilating properties and protective role in manyvascular diseases and previous reports have indicated that decreased activity andproduction of NO play an important role in vascular disease. To our knowledge, this is the first study in whichhuman aortic segments of patients with different degrees of AOD were directlycompared.The major limitation of this study is the small size of patients and aortic samples,preventing some correlation with clinical and angiographic findings. However,previous studies performed with human aortic samples have also been described withsmall number of samples and have obtained aortas from autopsy or organ donorpatients as control group[30], this preliminary study offer us amore comprehensive knowledge about the role of oxidative stress in atheroscleroticaortas in human beings.Our study on human aortic samples showed that oxidative stress seems to be relatedwith the degree of AOD and the increase of antioxidant enzymes (SOD and CAT)activities may be due to a compensative phenomenon. Moreover, nitrite overproductionoccurred in patients with chronic total aortic occlusion (TAO group) while itsreduction was observed in severe aortoiliac stenosis (SAS group). Althoughantioxidant therapies did not have a consistent effect in some clinical trials forprevention atherosclerosis complications


INV signaling pathway in the tumor cell upon macrophage contact. This heterotypic tumor cell – macrophage interaction results in the upregulation of MenaINV through the activation of MENA transcription. Notch1 and MenaINV expression are required for tumor cell transendothelial migration, a necessary step during intravasation. Inhibition of the Notch signaling pathway blocked macrophage-induced invadopodium formation in vitro and the dissemination of tumor cells from the primary tumor in vivo. Our findings indicate a novel role for Notch1 signaling in the regulation of MenaINV expression and transendothelial migration and provide mechanistic information essential to the use of therapeutic inhibitors of metastasis.The process of intravasation involving transendothelial migration is a key step in metastatic spread. How the triple cell complex composed of a macrophage, Mena over-expressing tumor cell and endothelial cell, called the tumor microenvironment of metastasis (TMEM), facilitates tumor cell transendothelial migration is not completely understood. Previous work has shown that the physical contact between a macrophage and tumor cell results in the formation of invadopodia, actin-rich matrix degrading protrusions, important for tumor cell invasion and transendothelial migration and tumor cell dissemination. Herein, we show that the macrophage-induced invadopodium is formed through a Notch1/Mena Metastasis is the primary cause of morbidity and mortality of breast cancer patients. The process of breast cancer metastasis requires tumor cells to migrate towards blood vessels where they intravasate. Intravasation of tumor cells during metastasis remains poorly understood, although it has become increasingly apparent that components of the tumor microenvironment such as macrophages, contribute to the efficacy of this process. Within invasive breast carcinoma we have identified multicellular microanatomical structures called the tumor microenvironment of metastasis (TMEM) that serve as the functional sites of tumor cell intravasation, and have been validated as a prognostic marker of metastatic outcome in patients1234INV results in a highly motile, invasive phenotype89Migratory and disseminating breast tumor cells at TMEM over-express the ENA/Vasp family member Mena  when compared to normal breast epithelium4INV mRNA, whereas Mena11a demonstrates a negative correlation in the same patient cohortINV in invasive-intravasation-competent tumor cells in vivo, the signals regulating the expression of the MenaINV isoform have remained unknown.The number of TMEM sites in patient samples positively correlates with the abundance of Menain vivo models such as chicken chorioallantoic membrane (CAM) models, tumor xenographs in mice and zebrafish and transgenic mouse models that invadopodia can be observed in mammalian cells in vivo and that these structures play a critical role in the cells ability to breech the basement membrane for invasion171819in vivo reduces the numbers of circulating tumor cells and metastasisIn addition to the macrophage-regulated vascular permeability events that contribute to tumor cell intravasation and metastasis, tumor cells form an invasive actin-rich invadopodium that enables the tumor cell to degrade extra-cellular matrix proteins and migrate, invading across barriers like blood vessel walls and intravasate121315A major signaling pathway that is involved in cell contact-mediated communication is the Notch signaling pathway. In addition to critical roles in development, Notch signaling has been implicated in cancers such as breast, lung and pancreatic cancers and leukemia, where activation of Notch pathways can promote proliferation, prevent differentiation, and promote metastasis212223Herein, we explore the contribution of the Notch signaling pathway to TMEM function; in particular macrophage-dependent tumor cell invadopodium formation and its relationship to Mena expression during transendothelial migration and tumor cell dissemination.To evaluate if Notch signaling is required for macrophage – induced invadopodium formation , cells wIncreased Notch1 signaling is associated with a greater chance of metastasis and poor prognosisINV (INV isoform was highly upregulated (~45x) while Mena11a isoform levels remain unchanged. This finding is of great interest because high expression of the MenaINV isoform has been implicated in the increased motility, invadopodium assembly and invasion by tumor cells1834Notch1 has well defined roles in the regulation of gene and protein expression at the transcriptional level, although a role for Notch1 in the regulation of the macrophage-induced invadopodium pathway is unknown. We sought to determine if macrophage – induced Notch1 signaling in tumor cells results in expression changes in genes with known roles in invadopodium formation and function. We performed a qRT-PCR analysis to determine changes in mRNA levels of the known invadopodium pathway regulatory proteins . The co-122829303132INV , but theINV upon co-culture with macrophages was a result of cell-cell contact and not soluble signaling factors. We performed co-culture experiments utilizing 3 μm pore transwells, which allow for the passage of soluble factors but not the tumor cells. Tumor cells were either plated in the transwell alone, with the two cell types mixed in the top of the well, or with macrophages added to the bottom of the well to prevent tumor cell – macrophage contact , which yields ~97% pure tumor cell samples1in vivo express elevated MenaINV, when cells were cultured with macrophages there is a significant increase in MenaINV mRNA  probes to MENA. The different Mena isoforms are splice variants transcribed by the same gene6To test the hypothesis that the increase in MenaIn addition, we measured the probability of active MENA transcription as a function of the distance of a tumor cell to the nearest macrophage in control conditions or in the presence of DAPT . When tuThese FISH data and the qPCR data above indicate that when tumor cells come into direct contact with a macrophage there is a rapid Notch-dependent induction of MENA transcription.INV mRNA results in increased MenaINV protein in the tumor cells, we developed a polyclonal anti- MenaINV specific antibody  activity, we either depleted Notch1 in tumor cells with siRNA or inhibited Notch signaling with DAPT and quantified the tumor cell iTEM activity in the presence and absence of macrophages. Notch1 depletion had no effect on the iTEM activity of tumor cells alone, but either depletion of Notch1 with siRNA or its inhibition with DAPT inhibited macrophage-induced tumor cell iTEM activity .in vitro as well as invasion in vivo has been well characterized using the rat mammary adencocarcoma MTLn3 cells. Like MDA-MB-231 cells, MTLn3 cells can form invadopodia and undergo migration in vivo when they upregulate MenaINV expression6Formation of invadopodia in vivo as described previously1210141014We utilized MTLn3 cells expressing photo-convertible Dendra2 to measure the effects of Notch1 inhibition on loss of tumor cells from the primary tumor To find out if cells leaving the tumor site were correlated with circulating tumor cells, mice with MTLn3 xenographs were treated with control or Notch1 blocking IgG for 6 hours and the circulating tumor cells were measured after treatment. There was a significant decrease in the number of circulating tumor cells in mice that were treated with the Notch1 blocking antibody compared to control treated mice , consistINV expression via activation of transcription. We found that Notch1 is required for transendothelial migration of tumor cells and dissemination of tumor cells from the primary tumor. These results are consistent with previous work showing that MenaINV expression is required for transendothelial migration by tumor cellsINV inhibits macrophage-mediated transendothelial migration of tumor cellsINV over-expression drives invadopodium assembly and function, and transendothelial migrationTumor cell dissemination via blood vessels in breast tumors requires tumor cells to undergo transendothelial migration. This occurs uniquely at TMEM sites where macrophages are in direct contact with tumor cells and endothelial cellsINV to that of Mena11a is associated with TMEM assembly, metastatic recurrence and death of breast cancer patients1393INV were not known. Here we show that the expression of the MenaINV isoform is induced by activation of Notch1 signaling in tumor cells. In this regard our findings indicate a novel role for Notch1 signaling in the regulation of MenaINV expression and transendothelial migration at TMEM sites.The relative expression of MenaThe induction of invadopodia in tumor cells by macrophages identifies an important step in tumor cell dissemination. Invadopodia are F-actin-rich protrusions on tumor cells capable of degrading extracellular matrix and assisting in tumor cell chemotaxis and migration2942INV is upregulated, while the invasion-suppressing Mena11a isoform is downregulated in migratory/disseminating tumor cells689103537INV-high/Mena11alow) is associated with directional cell migration towards chemotactic factors such as EGF and HGF, matrix degradation, TMEM assembly and transendothelial migration810343537539INV dramatically increases the sensitivity of receptor tyrosine kinases to their ligands EGF, IGF1 and HGF to increase cell protrusion and locomotion of tumor cells toward blood vessels910The analysis of the gene expression pattern of migratory and disseminating tumor cells in breast tumors revealed an “invasion signature” that is associated with distant recurrence in breast cancer patients5359343940in vivo invasiveness1213INV expression and invadopodium formation110INV upregulation is achieved in tumor cells and opens the future exploration of how different macrophage ligands might activate Notch signaling on tumor cells to lead to phenotypes associated with tumor metastasis.Previous studies have shown that heterotypic interactions among cells surrounding intratumoral vasculature can promote cancer cell dissemination. For example, fibroblast-derived lysly oxidase, a matrix cross-linking enzyme that stiffens collage fibers, driven by myeloid cell-derived TGFβ, promotes cancer metastasisINV expression shown here opens the possibility of developing additional markers that might be used in combination with TMEM sites to better predict the risk of distant metastatic recurrence of breast cancer patients and their response to treatment. We think that combining measures of MenaINV expression with the presence of TMEM sites could be useful in determining the relative activity of TMEM sites in transendothelial migration and the response to inhibitors designed to suppress either MenaINV expression and/or TMEM activity. Further work will be required to explore this possibility in patient cohorts of known outcome.Our results are directly relevant to how Mena isoform expression and TMEM number can predict distant recurrence in breast cancer patients34539INV in primary mammary tumor cells and its consequences has been well described in previous studies81034INV have remained unknown until now. Our finding of the increased expression of MenaINV in response to Notch1 signaling between tumor cells and macrophages, but not between tumor cells and endothelial cells, restricts the origin of MenaINV expression in the TMEM tumor cell, and therefore invadopodium assemblyINV expression and invadopodia in tumor cells. In this regard neutrophils have been tested for their ability to induce invadopodia and transendothelial migration in a previous study and fail to do eitherin vivoThe spatial heterogeneity of expression of MenaThere is significant interest in targeting Notch1 signaling for the treatment of a number of cancers but previous studies have shown chronic Notch inhibition can lead to detrimental secondary effects and in some cases increased vascular tumor development21in vitro at 5 μg/mL and in vivo via intraperitoneal injection at 1 mg/kg.MDA-MB-231 cells were cultured in DMEM supplemented with 10% FBS and antibiotics. MDA-MB-231 cells were serum-starved in DMEM supplemented with 0.5% FBS/0.8% BSA 16 h before macrophage induction studies. MTLn3 cells, derived from the 13762NF rat mammary adenocarcinoma, were cultured in α-MEM supplemented with 5% FBS and antibiotics. MTLn3 cells were serum-starved in α-MEM supplemented with 0.5% FBS/0.8% BSA 4 h before macrophage induction studies. BAC1.2F5 cells were cultured in MEM supplemented with 10% FBS, 2 mm l-glutamine, 22 μg/ml l-asparagine and 3000 U/ml of purified human recombinant CSF-1 . Human umbilical vein endothelial cells  were cultured in EGM-2  and only used between passage 1–4. DAPT (Sigma 10 μM) was used for gamma-secretase inhibition experiments as indicated in the results. When used, the DAPT or vehicle was added at the beginning of the experiments. Notch1 function blocking antibody (R&D Systems) was used INV siRNA from AmbionControl non-silencing siRNA was from Qiagen. Human-specific Notch1 siRNA pool was from Dharmacon and panMena and MenaThe 405 gelatin-labeled Mattek dishes were prepared as previously described43To detect macrophage-induced invadopodia, MDA-MB-231 cells were serum-starved for 16 h. BAC1.2F5 cells were cell tracker-labeled . A total of 25 K MDA-MB-231 cells were incubated with 125 K BAC1.2F5 cells in serum-starvation media on 405-labeled gelatin-coated dishes for 6 h, fixed and immunostained for invadopodium markers as described above. For MTLn3 experiments, tumor cells were serum starved for 4 hrs before being plated with BAC1.2F5 cells for 6 hrs as described above.MENA Ct values in the carcinoma samples were first normalized to GAPDH. qRT-PCR analyses were performed using a SyBR Green kit (Qiagen) and analyzed with a Qiagene Rotor Gene-Q detector and associated software.qRT-PCR for Mena splice variants was performed as described previouslyFor primary cancer cells from two human invasive ductal carcinomas fine needle aspiration (FNA) was completed as previously describedThe TN human tissue transplant HT17 was previously described148 oligodeoxynucleotide probes for MENA were designed with online Stellaris RNA FISH probe designer (Biosearch Tehchnologies). Each probe was 20 nt long and contained a 5′ amino-modified nucleotide that was chemically coupled to CAL Fluor 610 fluorescent dye. The sequences for probes used to detect MENA mRNA are provided in E. Coli tRNA (1 mg/ml), vanadyl ribonucleoside complex (0.2 mg/ml), and bovine serum albumin (0.2 mg/ml). The cells were hybridized for 3 hours at 37 °C and washed with 10% formamide/2xSSC. The nuclei were stained with DAPI and the coverslips were then mounted with ProLong Gold antifade reagent (Invitrogen).For FISH experiments, MDA-MB-231 cells we starved overnight as described above. Tumor cells were plated in the presence or absence of BAC1.2F5 macrophages that were labeled with cell-tracker red on glass coverslips for 1 h then fixed with 4% paraformaldehyde for 20 minutes at room temperature. After washing away the fixative, the cells were stored in 70% (v/v) ethanol at 4 °C. Prior to hybridiazation, stored coverslips were washed with 1 × PBS and pretreated with 10% formamide/2 × SSC at room temperature for 5 minutes. The converslips were then inverted onto 20 μl of hybridization solution containing MENA FISH probes (0.125 μM), dextran sulfate (10%), 2xSSC, 10% formamide, Images were acquired on an Olympus BX61 epi-fluorescence microscope with an UPlanApo 60x, 1.35 numerical aperture oil immersion objective (Olympus). X-Cite 120 PC (EXFO) light source was used for illumination with filter sets 31000 (DAPI), 41001 (Cell-tracker Green), and SP-103v1 (CAL Fluor 610) (Chroma Technology). Vertical stacks of 30 images with a Z step size of 0.2 μm were acquired using a CoolSNAP HQ camera (Photometrics) with 6.4 μm pixel size CCD. IPLab (BD Biosciences) software platform was used for instrument control as well as image acquisition. Automated detection and counting of mRNAs was performed by fitting Gaussians to fluorescent spots with FISH-quant as described previously60INV INV exon sequence.Chicken poly-clonal antibodies were generated by Covance. Animals were immunized with a peptide containing the unique MenaINV, when stained with the affinity purified anti-MenaINV IgY, contain a single faint endogenous MenaINV band in both cell types as described previouslyINV in the over-expressing cells consistent with the specificity of this antibody  were injected into the mammary glands of 5 to 7 well old SCID mice and allowed to grow tumors for 3 weeks, until the tumors reached approximately 0.8–1.0 cm. Circulating tumor cell count was determined as previously describedin vivo dissemination assay, a mammary imaging window was implanted and dissemination was measured as previously described3536For the How to cite this article: Pignatelli, J. et al. Macrophage-dependent tumor cell transendothelial migration is mediated by Notch1/MenaINV-initiated invadopodium formation. Sci. Rep.6, 37874; doi: 10.1038/srep37874 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Recent discovery of mutations in CSF3R and activation of JAK-STAT signaling in chronic neutrophilic leukemia (CNL) and atypical CML (aCML) provided the rationale for tyrosine kinase inhibitor (TKI) therapy targeting JAK2 . Mutatioin vivo mouse models show that the truncation mutations are not leukemogenic and are insensitive to dasatinib. Surprisingly, compound mutations induced aggressive leukemia and are resistant to both ruxolitinib and dasatinib treatments [in vivo data that supports the notion that secondary mutations  will be resistant to ruxolitinib. Besides, the timing of acquisition of CSF3R mutations is critical for TKI response [Patients harboring compound-mutations were presumed to be non-leukemic or have weakened leukemia, and most-likely to be sensitive to both inhibitors. In contrast to the previous finding, we observed that both proximal and truncation mutations are sensitive to ruxolitinib, but resistant to dasatinib. Moreover, eatments . This obeatments . Ongoingresponse . For insresponse . NonetheClinical efficacy of targeted therapy has been attributed to oncogene dependence . To idenGiven both JAK2 and MEK inhibitors are predominantly cytostatic (do not eradicate the leukemic clone), identifying drug combinations that harness ‘MEK dependence’ to induce cell death in leukemic cells might be more effective in targeting resistance and perhaps effect curative response. Because CSF3R leukemic mutations activate both Jak2 and Mek, a combination of both inhibitors might exert better therapeutic response. But, a combination of ruxolitinib and trametinib failed to show a better response than each inhibitor alone [In addition to KSR1, we identified overexpression of Pak6 and Bcl2l1 genes in cells expressing leukemic CSF3R. We anticipate that a combination comprising trametinib and Bh3 mimetics (antagonizes Bcl2) or Pak6 inhibitors might induce clonal selectivity. Alternatively, targeting KSR1 by a recently developed small molecule inhibitor alone or in combination with MEK or JAK inhibitors might be more effective in suppressing the resistance . Perhaps


Amyotrophic lateral sclerosis (ALS) and Parkinson’s disease, both progressive neurodegenerative diseases, affect >1 million Americans ,International Classification of Diseases,9th Revision (ICD-9) codes until 1998 and 10th Revision (ICD-10) codes thereafter. ALS deaths were defined as decedents with underlying or contributing cause of death codes 335.2 (ICD-9) or G12.2 (ICD-10) and Parkinson’s disease deaths as those with underlying or contributing cause of death codes 332 (ICD-9) or G20 (ICD-10). Usual occupation,ALS and Parkinson’s disease deaths were identified using Because cause-specific PMRs are mutually dependent, a higher mortality proportion for one cause results in a lower mortality proportion for another cause  were ≥1.50 . In contMost previous studies of occupation and ALS and Parkinson’s disease have focused on exposures to toxicants  that occur more frequently in lower SES occupations  , individual exposure data for specific agents or experiences, and occupation categories formed on the basis of exposure to specific agents or experiences and linked to job exposure matrices for exposures of interest. Adjusting for potential confounding by cigarette smoking and socioeconomic status, using incidence rather than mortality to ascertain outcomes, and incorporating information regarding the timing of exposures relative to the timing of outcomes might help further elucidate the reasons for these findings, so that strategies for prevention could be developed.Amyotrophic lateral sclerosis (ALS) and Parkinson’s disease are progressive neurodegenerative diseases that affect >1 million Americans. Factors consistently reported to be either positively or inversely associated with ALS and Parkinson’s disease are primarily demographic or behavioral. The role of occupation in these diseases is relatively understudied and poorly understood.This study described the burden of ALS and Parkinson’s disease mortality by usual occupation in a large, complete, representative, and population-based sample in the United States and found higher ALS and Parkinson’s disease mortality among workers in occupations associated with higher socioeconomic status (SES).Although the reasons for the findings of this study are not understood, it provides information for future targeted studies among workers in higher SES occupations to identify risk factors for ALS and Parkinson’s disease. These studies should use designs that provide evidence for causality, detailed exposure assessment, and adjustment for additional potential confounders.


Archaeopteryx is an iconic fossil taxon with feathered wings from the Late Jurassic of Germany that occupies a crucial position for understanding the early evolution of avian flight. After over 150 years of study, its mosaic anatomy unifying characters of both non-flying dinosaurs and flying birds has remained challenging to interpret in a locomotory context. Here, we compare new data from three Archaeopteryx specimens obtained through phase-contrast synchrotron microtomography to a representative sample of archosaurs employing a diverse array of locomotory strategies. Our analyses reveal that the architecture of Archaeopteryx’s wing bones consistently exhibits a combination of cross-sectional geometric properties uniquely shared with volant birds, particularly those occasionally utilising short-distance flapping. We therefore interpret that Archaeopteryx actively employed wing flapping to take to the air through a more anterodorsally posteroventrally oriented flight stroke than used by modern birds. This unexpected outcome implies that avian powered flight must have originated before the latest Jurassic. Archaeopteryx had a mix of traits seen in non-flying dinosaurs and flying birds, leading to debate on whether it had powered flight. Here, Voeten et al. compare wing bone architecture from Archaeopteryx and both flying and non-flying archosaurs, supporting that Archaeopteryx had powered flight but with a different stroke than that of modern birds. As the oldest potentially free-flying avialian known3, Archaeopteryx represents the prime candidate to consider in resolving the initial chapter of bird flight. Although the traditional dichotomy between an arboreal and a cursorial origin of avian flight4 has relaxed towards the consideration of intermediate perspectives5, the question whether the first flying bird-line dinosaurs took flight under their own power remains unanswered.The earliest phases of avian evolution and development of avian flight remain obscured by the rarity of representative fossil material and consequential limited phylogenetic resolution6 and continuous morphological and structural adaptation to the biomechanical loading regimes experienced during life7. Therefore, the avian wing skeleton informs on this stress regime through the application of beam theory mechanics10.Skeletal adaptations that structurally accompany known locomotor modes provide reliable proxies for inferring the habits of extinct tetrapods. The cross-sectional geometry of limb bones is largely determined by evolutionary selection on the interplay between strength and weight11. Using PPC-SRµCT at the European Synchrotron Radiation Facility with a novel data acquisition protocol  now offers non-destructive alternativesens Fig.  because ted Fig.  and compArchaeopteryx exhibits a vascular density in the range of modern birds, which proposes substantial metabolic performance. Cortical vascular density strongly varies between two specimens of Archaeopteryx studied, which we interpret, based on body size differences, to reflect ontogenetic disparity. Relative cortical thickness of archosaurian anterior limb bones successfully discriminates between known non-volant and volant forms, and confidently indicates that Archaeopteryx was volant. Mass-normalised torsional resistance in the same set of limb bones describes a gradient within modern volant birds that ranges from flight strategies relying on occasional or intermittent flapping to gain altitude to hyperaerial specialists employing prolonged gliding or soaring in their flight. The three specimens of Archaeopteryx were found to unanimously ally with birds that incidentally employ flapping flight to evade predators or cross physical barriers, through which we interpret that Archaeopteryx actively used its wings to take to the air. Since the morphology of the flight apparatus in Archaeopteryx is known to be incompatible with the flight stroke executed by modern volant birds, we furthermore conclude that Archaeopteryx adopted a different flight stroke than used by birds today. Finally, we found that the evolution from primitive long-tailed pterosaurs to more derived short-tailed pterosaurs was accompanied by qualitatively comparable modifications to wing bone geometry as those that distinguish Archaeopteryx and principally flapping birds from hyperaerial birds, respectively.Raw virtual slice data revealed that the long-bone cortex of 13, the bone cortex of Archaeopteryx is well vascularised , but higher than the hoatzin , whereas the average cortical vascular density of the smaller fifth specimen is higher than in most neognaths assessed  exhibit humeral and ulnar relative torsional resistance values overlapping with those of intermittent bounding20 flyers . However, burst-flying20 birds attain body mass values that are, on average, two orders of magnitude higher than those of intermittent bounding20 flyers, which is informative when discriminating these two groups. Conversely, flap-gliding20 birds have a similar to higher humeral and ulnar relative torsional resistance compared to burst20-adapted and most continuously flapping20 flyers at body masses that are, on average, one order of magnitude lower. Notably, the two large non-domesticated anatids in our data set share elevated relative torsional resistance values compared to other continuously flapping birds. Soaring birds20 may attain comparatively high body mass values, yet exhibit distinctly elevated normalised torsional resistance values relative to their body mass throughout  of the referred parameters places Archaeopteryx. Linear discriminant analysis (LDA) principally separates volant and non-volant archosaurs along discriminant axis (DA) 1  1 Fig.  through Archaeopteryx with short21 or burst20 flyers in our set , and this uncertainty undoubtedly carries over to the characterisation of extinct taxa with unknown locomotory strategies. Nevertheless, the position of Archaeopteryx is chiefly shared with short21/burst20 flyers  amniotes, such as the extant flying lizard Archaeopteryx was capable of active flight26 have not received universal support, largely because three skeletomorphological conditions considered essential for a functional avian flight stroke  were not yet present in Archaeopteryx30. Such challenges in reconciling Archaeopteryx’s dromaeosaurid-like pectoral morphology30 with the modern avian dorsoventral flight stroke exemplify that avian powered flight may have worked through alternative configurations in the past. A putative aerodynamic control function for the long, stiff, frond-feathered tail2 and hindlimb plumage31 argue for an alternative aerial posture compared to modern birds. Archaeopteryx’s large coracoids30 and robust, flattened and more dorsally positioned furcula lacking hypocleidial communication with the sternum2 could have provided support for an anterodorsally-posteroventrally oriented flight stroke cycle that was morphologically closer to the “grabbing” motion of maniraptorans25 and did not or hardly extend over the dorsum.Earlier conclusions that Archaeopteryx’s humeral and ulnar cross-sectional geometry shares more flight-related biomechanical and physiological adaptations with modern volant birds than previously known, which we argue to reflect the shared capability of powered flight. Confuciusornis from the Early Cretaceous of China also lacked the supracoracoideal pully30 and sufficient dorsal humeral excursion5 to permit a modern avian flight stroke. However, a variety of Early Cretaceous enantiornitine and euornithine birds  is a nearly complete and largely articulated skeleton of the smallest Archaeopteryx specimen known to date. It is also known as the Eichstätt Specimen and housed at the Jura Museum in Eichstätt, Germany (JM). The seventh specimen of Archaeopteryx2 (BSP 1999 I 50) is represented by a comparably complete skeleton exhibiting a substantial degree of articulation. It is formally named Solnhofen-Aktien-Verein Specimen but generally referred to as Munich Specimen, and is kept at the Paläontologisches Museum München in Munich, Germany (PMM). Skeletal elements of both the fifth and seventh specimen of Archaeopteryx have experienced brittle deformation during post-depositional compaction that resulted in splintering of the bone cortex. The ninth specimen of Archaeopteryx2 (BMMS-BK1a) preserves a partially disarticulated right wing skeleton of comparably large size that is presently housed at the Bürgermeister–Müller–Museum in Solnhofen, Germany (BMM). It is officially named “Exemplar der Familien Ottman & Steil”, also known as Bürgermeister–Müller Specimen, and colloquially referred to as “Chicken Wing”. Although a certain degree of post-depositional compaction is evidenced by the presence of several fractures that propagate through the long bone cortex, cortical splintering has not occurred. Its elements have therefore largely preserved their original three-dimensional geometry2.Specimens of Comparative material  at beamlines BM05 and ID19 of the European Synchrotron Radiation Facility. An ulnar cross section of aff. Deinonychus antirrhopus was imaged and subsequently paired with a humeral section of D. antirrhopus from literature32 through morphological and dimensional comparison.The humeral and ulnar cross-sectional geometry of the three specimens of 33. The fifth and seventh specimen of Archaeopteryx were imaged in accumulation mode, a novel acquisition protocol developed for imaging fossils encased in lithic slabs. The motivation for and implementation of the accumulation mode are explained in Supplementary Note Synchrotron X-ray tomography was conducted by utilising an optimised polychromatic beam with sufficient coherence to permit the application of PPC-SRµCT. Propagation phase-contrast imaging relies on a certain propagation distance between the sample and the detector that allows for the exploitation of the phase-contrast effect towards emphasising low-contrast features33 version of the algorithm developed by Paganin et al.34. Virtual two-dimensional cross-sectional slides were extracted directly from the reconstructed volumes at the developmental mid-diaphyseal plane oriented perpendicular to the local bone long axis in VGStudio MAX 2.2 . Avian ulnae that carry a quill knob (ulnar papilla) at mid-diaphysis were virtually sampled at the level nearest to mid-diaphysis where no quill knob was present. The data set was supplemented with avian samples used in earlier studies35. Furthermore, scaled figures depicting complete perpendicular humeral and ulnar cross sections of avian and non-avian archosaurs were sourced from literature39 and processed in tandem with data obtained through PPC-SRµCT.Three-dimensional volume reconstruction was conducted through filtered back projection following a phase retrieval protocol that relies on a homogeneity assumption by using a modified40, the geometry of Archaeopteryx wing elements was assumed to have experienced only brittle deformation during unidirectional compaction with insignificant movement of bone fragments perpendicular to the visualised cross sections. Two-dimensional restoration was conducted with image editing software by virtual extraction of the bone fragments and visually applying optimal fit of local fracture geometry, periosteal and endosteal curvature across adjacent fragments, and internal structures . For the ninth specimen of Archaeopteryx, humeral and ulnar parameters were obtained by averaging the values found for two reconstructed circa mid-diaphyseal cross sections each. As the humeral and ulnar geometry of the fifth and seventh specimen are distorted to a markedly larger degree than those of the ninth specimen, they are represented by the single best-preserved cross section present in the circa mid-diaphyseal domain.Based on the characteristically unfolded nature of the Solnhofen PlattenkalkCompsognathus and Rhamphorhynchus used in this study were recovered from the Solnhofen Plattenkalk as well, and were reconstructed following the same protocol as the Archaeopteryx material  in the public-domain image analysis software ImageJ (https://imagej.nih.gov/ij/).All transverse cross sections were converted to binary cortical bone profiles by tracing the periosteal and endosteal surfaces and subsequently filling the area of the original cortical bone whiteArchaeopteryx specimens are often considered to be juveniles, which has been specifically concluded for the specimens of Archaeopteryx included in this study through relative size and bone surface texture13. The sampled Compsognathus specimen was also reported to represent a juvenile individual42. The studied Rhamphorhynchus individual is of comparably small size, suggesting juvenility as well. Gender composition across the data set is generally unknown and was therefore not considered.Most species in our data set are represented by the humerus and ulna of a single adult individual, although some were included as a composite of elements sourced from two individuals, or as average values derived from elements from two, three or four individuals  short flight, (2) forward flapping/bounding flight, (3) high-frequency flapping flight, (4) undulating flight and (5) gliding–soaring flight, which were assigned following the proposed taxonomical designations21. Taxa not included in their work were assigned flight modes according to the provided description21. Geococcyx californianus was classified as ‘short flight’ rather than as ‘forward flapping/bounding flight’ proposed for Cuculidae. A second, more recent avian flight mode division by Close et al.20 separates (1) burst flight, (2) intermittent bounding flight, (3) continuous flapping flight, (4) flap-gliding flight and (5) soaring flight, and was applied through description. We chose to score volant wing-propelled divers separately in both subdivisions as their aquatic locomotory strategy is known to profoundly influence wing bone morphology43 and, consequently, the expression of flight-related adaptations recorded therein43. Both referred avian flight classifications were complemented with the following locomotor categories: (6) long-tailed pterosaurian flight, (7) short-tailed pterosaurian flight, (8) (avian) non-volant wing-propelled diving, (9) ratite bipedal, (10) (non-avian) dinosaurian bipedal, (11) (non-avian) dinosaurian omnipedal and (12) crocodilian quadrupedal.Avian flight mode categorisation notoriously suffers from the qualitative, non-discrete nature of faunal flight strategies45 and literature47 as species averages , was assigned a reconstructed body mass of 2000 g. Body mass for the Rhamphorhynchus sp. MdC 20269891 was reconstructed through the relation between body mass and wing span for basal pterosaurs proposed by Witton52. Total wing length for MdC 20269891 was measured as the cumulative length of the humerus (19 mm), radius (34 mm), wing metacarpal (14 mm), phalange I (47 mm), phalange II (40 mm), phalange III (35 mm) and phalange IV (44 mm) taken from photographic and scan data, and amounts to 233 mm. In the Dark Wing specimen of Rhamphorhynchus muensteri , the distance between left and right glenoid measures 1.56 × humeral length, which proposes an original interglenoid distance of 30 mm for for McD 20269891. Its corresponding wingspan, calculated as twice the wing length plus the interglenoid distance, amounts to 0.496 m. From the relation of Witton52 follows a reconstructed body mass of 95 g. The body mass for the Brasileodactylus araripensis individual in our study was inferred through close morphological and dimensional agreement between its humerus  and the humerus of AMNH 22552 54, for which a reconstructed wingspan of 3270 mm was reported55. From the described relation between wingspan and body mass in pterodactyloids56 follows a reconstructed body mass of 6540 g.Body mass values for extant taxa were either directly available for the referred individuals or sourced from online databasesAlligator mississippiensis (141 cm38) and the domestically bred Crocodylus niloticus  were reconstructed through specific allometric scaling relations between body length and body mass offered in literature58.Body mass values for the studied specimens of 12 (CA/TA) and mass-normalised resistance against torsional forces12 (J/M) were quantified for archosaurian humeri and ulnae  and minimum second moment of area (Imin) that quantify resistance against deflection along the respective orthogonal major and minor principal axes . J was normalised over body mass to permit comparison in a highly body mass-diversified comparative framework that spans well over five orders of magnitude  by thresholding the cortical domain and subsequently counting the amount of elements within canal size range using the Analyse Particles function of ImageJ 1.49.Cortical vascular density, expressed as the amount of canals per mm67. These approaches traditionally assume that the degree and orientation of ellipticity reflect an adaptation that offers optimised resistance against bending, with the direction of Imax corresponding to the orientation of the maximum bending moment. However, an opposite functional interpretation of cross-sectional element ellipticity in which a preferred bending direction is achieved through orientation of Imin has also been proposed specifically for avian wing bones68. Such conflicting explanations of the same parameter illustrate the complexity of interpreting cross-sectional bone ellipticity in a functional context and thereby obscure the information offered by other characters when assessed in a multivariate context. We therefore chose not to include quantified bone ellipticity measures in our comparative study.The ratio of Imax over Imin provides a reliable measure for the ellipticity of the transverse bone shaft and has been considered as such in biomechanical explorationsMancalla cedrosensis were placed at the younger bound date of their occurrence. Alopochen sirabensis is placed at 656 AD, which represents the median calibrated radiocarbon age for the last-occurrence date for the species69. The 19th century terminal node for Pinguinus impennis was dated through its well-documented last observation in 1844 AD70. Topology and timing within the extant avian subset were largely adopted from the well-resolved phylogeny by Jarvis et al.71 (Supplementary Data 72 was found to conflict PaleoDB on numerous crucial accounts. Several higher-order divergence times in Aves were obtained from PaleoDB following the procedure described above. Two specific inconsistencies in PaleoDB were negotiated through literature (see Supplementary Note Archaeopteryx specimens were included as a polytomy at + 4 MY with respect to the older bound date for the genus in recognition of taxonomic and ontogenetic uncertainty2. The phylogenetic tree was constructed in Mesquite 3.0473.Mesozoic topology and timing used in this study (Supplementary Data 74. For each parameter, 10000 unbound simulations were performed along the constructed tree (Supplementary Fig. The relations between individual geometric parameters and locomotor divisions in the training taxa set were statistically assessed through phylogenetic analysis of covariance using the PDAP module of Garland et al.75 scores for the studied taxa, founded on humeral and ulnar CA/TA and J/M (Supplementary Data 76 in the R-environment77 through RStudio 0.99.48478. The phylogenetic PCA scores were subsequently subjected to Partitioning Around Medoids specified to two clusters with the pam function of the cluster package79 in RStudio 0.99.484.Phylogenetic PCAArchaeopteryx serve as training taxa that represent known locomotor modes and thus form a morphological reference environment for discriminant analysis. The optimum value of Pagel’s λ, the scaling factor of autocorrelation for a certain parameter on a given phylogenetic tree80 to be applied in phylogenetically informed discriminant analysis, was found using the approach described by Schmitz et al.81 in RStudio 0.99.484 (Supplementary Fig. 82, as was one-way MANOVA (Supplementary Tables The archosaurian  groups outside All data underlying the study are available in Supplementary Data Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5


NIPBL are responsible for the majority of cases of CdLS. Through RNA-sequencing of human induced pluripotent stem cells and in vitro-derived cardiomyocytes, we identified hundreds of mRNAs, pseudogenes, and non-coding RNAs with altered expression in NIPBL+/− patient-derived cells. We demonstrate that NIPBL haploinsufficiency leads to upregulation of gene sets identified in functions related to nucleosome, chromatin assembly, RNA modification and downregulation of Wnt signaling, cholesterol biosynthesis and vesicular transport in iPSC and cardiomyocytes. Mutations in NIPBL result in the dysregulation of many genes responsible for normal heart development likely resulting in the variety of structural cardiac defects observed in the CdLS population.Cornelia de Lange syndrome (CdLS) is a complex disorder with multiple structural and developmental defects caused by mutations in structural and regulatory proteins involved in the cohesin complex. NIPBL, a cohesin regulatory protein, has been identified as a critical protein responsible for the orchestration of transcriptomic regulatory networks necessary for embryonic development. Mutations in  It is a rare, dominant, genetic multisystemic disorder caused by a disruption of the normal function and regulation of the cohesin complex; a key regulator of cell division and gene expression during development2. CdLS arises from heterozygous mutations in cohesin structural and regulatory proteins. There have been five causative genes identified in CdLS - NIPBL, SMC1A, SMC3, RAD21, and HDAC88. About 60% of all diagnosed cases result from mutations in NIPBL, which is required for the loading and unloading of cohesin onto DNA10. It has been shown that as little as a 15% decrease in NIPBL expression can lead to multiorgan defects, including disruption of gut and heart development during embryogenesis14. Although cohesin’s canonical role in sister chromatid segregation is not disrupted in CdLS, the precise cellular and molecular mechanisms underlying morbidity or mortality still remain enigmatic. Several key biological and cellular features of this diagnosis have been documented, including altered transcriptional regulation, DNA replication, homologous recombination-mediated repair, genome compartmentalization, and RNA biogenesis17. Typical characteristics of children with CdLS may include clinical features of intellectual disability, growth retardation, facial dysmorphism, microcephaly, limb anomalies, hypertrichosis, and congenital heart defects.Cornelia de Lange syndrome (CdLS) is one of a family of disorders known as 18. CHD is a significant cause of morbidity and mortality in children with CdLS, affecting 30–40% of patients19. Cardiac abnormalities vary across patients with most involving structural anomalies; atrial and ventricular septal defects and pulmonic stenosis tend to be most frequently diagnosed, with more severe, yet occasional, anomalies including Tetralogy of Fallot (TOF) and single ventricle malformations. To better investigate these congenital anomalies, several NIPBL deficient animal models have been created. NIPBL haploinsufficient mice manifest several features of CdLS and are significantly smaller than wildtype siblings and display structural heart defects20. Studies have shown that these animal models correlate with patient clinical phenotypes, one study found 77% of NIPBL+/− mice had incomplete ventricular septation, compared to 14% of the wildtype mice12. New allelic series (NIPBLFLEX) models that allow for creation and efficient rescue of NIPBL-expression by targeting multiple lineages at early developmental stages suggest that multiple germ layers and cell lineages contribute to the CHD risk in CdLS patients12. Although this system has many advantages, the mouse models have obvious limitations. For example, the transgenic mice do not carry the same mutations as human patients. Although the conditional mutant phenotypes are observed in cardiac morphogenesis, these models are unable to validate if these defects occur in very early embryos. Recent advancements in pluripotent stem cell technology have overcome these limitations, and have allowed us to generate patient-specific strategies for studying the pathogenic mechanism of CdLS in multiple cell types and at the earliest stages of embryogenesis.Congenital heart disease (CHD) is the most common life-threatening birth defect in humans affecting approximately 1% of all newborns+/−patient generated samples and overlapping biological and cellular processes involve increased chromatin modifications, nucleosome assembly and transcriptional regulation, and reduced immunological functions, regulation of apoptosis and proteasome dynamics. Alteration in expression of many known CHD associated genes suggest that variability in CdLS birth defects results from a constellation of cardiac signaling events.It has been hypothesized that the multi-organ defects commonly observed in CdLS patients are due to a global disruption of transcriptional regulation; which, due to an overall NIPBL deficiency, leads to altered epigenetic, biological and cellular responses. To access the impact of NIPBL on gene expression in human pluripotency and early cardiac development, we performed genome wide transcriptome analysis from pluripotent stem cells and cardiomyocytes from male and female NIPBL patient samples. Here, we report that hundreds of genes are differentially expressed genes at the iPSC and cardiomyocyte stage between control and NIPBLDrosophila24, murine26, zebrafish20 and human skin, blood and lymphoblastoid cell lines27. The cohesin-accessory protein NIPBL, has been previously implicated in regulating the pluripotent cell state and has been identified as a key component in defects of limb and cardiac development in NIPBL+/− patients presenting with structural heart defects29; however, the mechanism by which it affects cardiac development has remained unclear.To investigate the impact of NIPBL mutations in CdLS patient samples, we focused our work on the role they play in cultured human cells, both in the undifferentiated and cardiomyocyte states. The role of NIPBL has been extensively studied in 16. To generate a model to study transcriptional disturbances underlying the cardiac phenotypes seen in CdLS, we established iPSCs using episomal reprogramming methods31 , and stained positively for intracellular pluripotency markers (OCT4 and Nanog); displaying similar levels of core pluripotency RNA gene expression by RT-PCR in comparison to human embryonic cells (H9)  were present , and RNA polymerase modification   and compared the gene expression profiles to four unaffected control subjects. All samples were quality controlled for pluripotency markers and genome stability prior to RNA-analysis, and NIPBLSCs Fig. . Additioon) Fig. , and dowon) Fig. . Overallin vitro. CdLS patients exhibit multiple forms of congenital heart defects. In order to develop a model amenable for studying early stages of heart development, we investigated the capacity of NIPBL+/−-iPSCs to generate uniform populations of contracting cardiomyocytes. This was performed using a cardiac-directed monolayer differentiation through modulation of the WNT and insulin pathway at specific time intervals  and VCAM1 + (CD106)res Fig. . Upon cores Fig. . Positivity Fig. . Althougnes Fig. .Figure 3+/− cardiomyocytes when compared to unaffected controls (*p < 0.05). The complexity of gene regulation defects in NIPBL+/− cardiomyocytes extends to many biological and cellular phenotypes. We identified a number of Gene Ontology (GO) terms enriched in cardiomyocytes . In addition, we identified a set of coronary heart disease (CHD)-associated genes consisting of several transcription factors, structural proteins, several receptors, and signaling molecules36. We found that twelve were significantly dysregulated at a p-value < 0.05, but extending the confidence level to p-value < 0.15 an additional 9 genes were identified to be altered in NIPBL+/− cardiomyocytes. This showed that 21/53 CHD-associated genes were differentially expressed in our NIPBL+/− patient-derived cardiomyocytes  to be significantly altered in our NIPBL+/− cardiomyocytes.When specifically focused on the gene ontology associated with heart development (G0: 00075075), we identified 10 genes that were significantly altered in NIPBL+/−-iPSC and -CMs correlated with previously identified gene set analysis in the literature. It was found that a set of transcriptome data generated from NIPBL patient-derived iPSCs and CMs correlated with murine data described by Remeseiro et al. for the protocadherin cluster  gene cluster also showed significant dysregulation in either iPSC (5 of 64 genes) or CM (10 of 64 of genes) when compared to control samples but no genes overlapping in both cell types , respectively in both NIPBL+/− cell types. We used String database to determine the overlapping pathways enriched in our NIPBL samples and showed that the nucleosome is primarily affected, followed by ECM-receptor interaction, focal adhesion, Wnt signaling, purine metabolism, cGMP-PKF, and PI3K-Akt signaling pathways were affected to the greatest degree  resulted in no difference in reprogramming efficiency, loss of genomic stability, or alterations in cell morphology, pluripotency markers or proliferation. This is in contrast to Kagey et al. (2010), which showed that reduction of NIPBL using shRNA resulted in loss of pluripotent stem cells morphology, and reduction of key pluripotency mRNAs . Patient samples carrying mutations in NIPBL do not have a loss of mRNA expression greater than 40% of normal suggesting a threshold of expression necessary for maintaining cellular embryonic and somatic survival. Thus, using shRNA to knockdown NIPBL expression could have resulted in levels (<60%) which was not compatible with maintaining normal pluripotent stem cells characteristics. Although there were similarities in pluripotency characteristics in NIPBL+/− compared to controls, iPSC transcriptome profiles show an array of gene disturbances similar to that observed in NIPBL shRNA knockdown studies. Our group and others have previously shown global transcriptional differences in CdLS samples; human fibroblast and lymphoblastoid cell lines, drosophila brains, murine and zebrafish tissues highlighting why it has been termed a “transcriptomopathy” and a member of a growing list of disorders of transcriptional regulation (DTRs)38. Given the strong involvement of transcriptional regulation and epigenetic factors in CdLS it was not surprising that we identified gene set alterations involving gene expression regulation, RNA polymerase, RNA modification, chromatin and that nucleosome-related genes were amongst the top hits in our bioinformatic analysis of NIPBL+/−-iPSCs.CdLS is a genetically heterogeneous disorder with multiple structural and developmental deficits. Elucidating the mechanisms by which disruption of cohesin function results in these pleiotropic manifestations is challenging. A number of non-human models have been developed for 36. It is estimated that 25% of CdLS patients have some form of CHD but the etiology remains unclear; however, it has been suggested that small changes in many genes results in the wide variation of structural birth defects39. When we performed gene set enrichment analysis for heart development (GO: 00007507), 10 genes were identified to be dysregulated at >2-fold change, and FDR <0.05, but more than 80 genes with a p-value of <0.05 did not make the cut off for 2-fold change. The wide-range of phenotypic variability in NIPBL haploinsufficient patients is documented and can have CHDs ranging from significant structural heart defects to minor findings including innocent murmurs and persistent fetal circulatory issue19. We investigated the expression of known CHD-related genes to identify if one or a series of genes or pathways was significantly perturbed in NIPBL+/− cardiomyocytes. Overall, genes involved in transcriptional regulation , receptor signaling , and structural proteins  were found to be consistently dysregulated in our cellular assays. Our results suggest that the CHDs seen in individuals with CdLS caused by NIPBL mutations are likely not a result of disruption in any one cardiac gene or developmental pathway, but rather result from a constellation of multiple gene/pathway dysregulation that in turn manifests in the broad spectrum of structural CHDs observed in CdLS patients. Although not studied here, these transcritptional dysregulations are likely additionally influenced by individual patient genetic background and environmental modifiers. Various CdLS models have been studied to define patterns of transcriptional dysregulation, which correlated with our findings in this study. Some of the greatest changes in expression occur within large gene clusters responsible for developmental gene regulation, such as the HOX, Pcdh, and FOX gene clusters43. While many promoters are unaffected by cohesin disruption, certain promoter regions such as those for the protocadherins and HOX gene clusters have been shown to be positively regulated by cohesin loading44. Consistent with these findings, more than 30% of Pcdh genes are significantly downregulated in both iPSCs and CMs. Regulation of Pcdh and HOX expression is hampered in NIPBL mutant murine and zebrafish models, and the underlying mechanism has been suggested to be dependent of the positional effects of the given Pchd or HOX gene within the clusters. The transcriptional activity of the clustered Pcdhs has been directly correlated with binding of CTCF/cohesin complex to active promoters and enhancers45, and regulation of expression is mediated through DNA-looping interactions within the Pcdh locus41. Many of the differences observed between Pcdh, HOX, and FOX genes could be associated with differences in embryonic developmental states and chromatin condensation effects during cell differentiation processes which are affected in both the iPSCs and CMs of NIPBL+/− patient derived samples46. Our data shows significant disturbances in chromatin associated complexes (nucleosome and DNA bending), and modifiers of gene expression through epigenetic modification . Interestingly, we find that 17% of the genes identified by Yuen et al. (2016) for the PKR stress response pathway in lymphoblastoid cell lines (LCLs) to be correlated with NIPBL+/−-iPSC and CMs.The development of congenital heart defects involve perturbations of pathways responsible for cardiac morphogenesis, nutrient metabolism, myocyte specification, and neural crest and germ layer differentiation (CHDs)+/−-iPSCs and cardiomyocytes. The most significant GO enrichment was related to genes of the nucleosome. These data show significant increased expression of histone coding genes and inhibition of methylation specific modifiers, which support the role of NIPBL in maintaining DNA methylation and gene silencing status and chromatin compaction and architecture48. We identified ECM-receptor interaction and focal adhesion defects which are important in intracellular signaling and cell-adhesion kinases responsible for cell survival and cell specification49. We also identified Wnt signaling alterations, which has broad implication for embryonic development and also substantial impact on cardiovascular development including cardiomyocyte polarity and left-right patterning50. Pistocchi et al.51 showed a reduction in canonical Wnt pathway with a subsequent decrease in Ccnd1 expression in a nipblb-loss-of-function zebrafish model, which correlates with our iPSC and CM data which show altered Wnt signaling and significant reduction in Ccnd2 expression and slight reduction in Ccnd1 levels (not significant) in NIPBL patient samples. Dysregulation in Wnt signaling caused cardiac neural crest development defects in Rad21-depleted animals52. Interestingly, NIPBL mutations decreased the expression of cholesterol biosynthesis and glycolipid metabolism genes. Cholesterol and lipid metabolism is essential for embryonic development, abundant in the cardiovascular and central nervous system, and critical for plasma membrane components and signal pathways55.Using an unbiased evaluation of RNA sequencing data, we identified unique disease-specific protein interaction networks overlapping in NIPBL+/−-iPSC and cardiomyocytes reveal considerable global transcriptional variations as compared to unaffected control cell lines. Perturbation of multiple biological, molecular and cellular processes highlight the complexity of primary and downstream effects mediated by dysregulation of the cohesin complex. This suggests that global alterations in gene expression during early cardiac development in CdLS is not directly related to a “monogenic” associated alteration in cardiac signaling, but rather consists of a cascade of effector genes which are regulated by epigenetic and DNA looping related alterations responsible for the phenotypic variations seen in CdLS-related structural heart defects.In summary, our work has demonstrated that NIPBLNIPBL gene  and all human protocols for this work were approved by the human subject Research Internal Review Board (IRB 98-001439) at The Children’s Hospital of Philadelphia. The methods performed in these studies followed all relavent guidelines and regulations set forth by institutional guidelines. Informed consent was obtained from all subjects involved in this work. Unaffected control induced pluripotent stem cells were generated as described previously56. iPSC-like colonies were expanded on MEFs in HES media containing 5 ng/mL bFGF, further characterization included quantitative polymerase chain reaction (PCR), immunofluorescence, flow cytometry for pluripotency marker expression and teratoma analysis. Additional details are provided in supplementary methods.Primary skin cells were collected from four Cornelia de Lange (CdLS) probands carrying heterozygous mutations in the 2 and 5% CO2. Cell lines were feeder depleted twice and seeded onto matrigel-coated dishes. Cells were maintained until approximately 60% confluence in HES media (50%) and iMEF-conditioned media that was collected 36 h after plating iMEFs. The second feeder depletion occurred between 2-3 days prior to differentiation to completely eliminate residual iMEFs. iPSCs were dissociated into single cells using Accutase (Life Technologies) and plated onto matrigel-coated dishes  in HES:MEF-conditioned (50:50) medium +20 ng/mL of bFGF supplemented with Rho-kinase inhibitor (10 μM). Next day, iPSCs were fed with mTesr media (Stem Cell Technologies) and maintained to reach 100% confluency. On the first day, IPS cells were changed to cardiac induction media  containing 12 μM CHIR (Selleck). Cells were maintained in cardiac media for 24 hours in a 37 C 5%O2 and 5%CO2 incubator, then switched to cardiac base media for 48 h. On day 4, media was changed to CM1 containing WNT inhibitors . Cells were placed in a 37 C 5% CO2 incubator for the remainder of cardiac differentiation. On day 6, media was replenished with CM1 without WNT inhibitors until day 8. On day 8, culture media was replenished with cardiac media . Contracting culture generally occurred between day 8-10 of differentiation. On day 12, cardiomyocytes were expanded using 0.2% collagenase II  plus DnaseI for 20 minutes in 37 C 5% CO2 incubator, cultures were washed with 1XPBS to removed floating cells and then treated for an additional 20-min with 0.2% collagenase to detach cardiomyocytes. Cardiomyocytes were less resistant to early detachment then other non-cardiomyocyte cells. This step was used to enrich cardiomyocyte cells at early passaging time points. Cells were replated at high density (1 × 106 cells/well of 6-well) in CM2 media containing 10 uM Rock inhibitor  and culture media changed 16 h post-plating with CM2 media only. This high density ensured equal cell number and contracting cultures across cell line for future analysis. Cells were maintained until day 18 then harvested for transcriptome analysis.iPSCs were cultured on 0.1% gelatinized plates containing iMEFs in 37 C 5%O35. For intracellular staining, cells were fixed using BD Perm/Fix for 20-min at 4 C, washed 2 × with FACS buffer. Cells were incubated in either anti-mouse IGG1 isotype control or anti-cTNT2  for 30-min at RT. Goat anti-mouse-488  antibodies were added per 5E5 cells and incubated at RT for 30-min before washing with FACS buffer. We analyzed stained cells on a BD FACS Aria and data analyzed using FlowJo Version 10.0.8 software (TreeStar).Culture of cardiomyocytes were collected using accutase to ensure single cell suspension. A portion of cells were incubated with antibodies anti-CD107a  and anti-CD172a  in PBS supplemented with 0.5%BSA + 0.1% Na Azide (FACS buffer) and incubated at RT for 15 min as previously describedhttps://support.illumina.com). STAR was run in its 2-pass mode: the first pass aligned reads to the reference genome and transcriptome using known splice junctions while allowing for detection of novel junction sites. Junction sites detected from all libraries by the alignment were collected, combined and filtered. The second pass re-aligned reads to the references using both known and novel junction sites. Aligned reads were mapped to known genes and reads with both ends uniquely mapped to the sense strand of the same gene were assigned to that genes. The mapping procedure generated a data matrix of read counts. Sample analysis using the matrix did not identify any batch effect or outlier sample. DESeq. 2 method was used to test for differential gene expression between WT and CdLS groups. GSEA (http://software.broadinstitute.org/gsea) method was used to identify gene sets enriched with differential expression. Top differentially expressed coding genes were clusterd by the iGraph (http://igraph.org) algorithm using the protein-protein interaction information downloaded from the STRING (https://string-db.org) database. The RNA- seq data is available through the GEO database library with the access ID: GSE102873. RNA was collected from two separate clones and a pool of these clones for a total of three technical replicates per individual for all transcriptome analysesRNA-seq reads saved in.fastq files were aligned to human reference genome and transcriptome using the Spliced Transcripts Alignment to a Reference, or STAR, program (version 2.5.1b). The NCBI GRCh38 reference genome and transcriptome were downloaded from the iGenomes . Statistical analysis was performed with Student T-test in Excel. Supplementary Experimental Procedure and FiguresSupplementary Dataset


The majority of transplanted kidneys are procured from deceased donors which all require exposure to cold storage (CS) for successful transplantation. Unfortunately, this CS leads to renal and mitochondrial damage but, specific mitochondrial targets affected by CS remain largely unknown. The goal of this study is to determine whether pathways involved with mitochondrial fusion or fission, are disrupted during renal CS.Male Lewis rat kidneys were exposed to cold storage (CS) alone or cold storage combined with transplantation (CS/Tx). To compare effects induced by CS, kidney transplantation without CS exposure  was also used. Mitochondrial function was assessed using high resolution respirometry. Expression of mitochondrial fusion and fission proteins were monitored using Western blot analysis.Optic Atrophy Protein (OPA1), and altered the mitochondrial protease, OMA1, which regulates OPA1 processing. CS followed by Tx (CS/Tx) reduced complex I, II, and III activities, and induced a profound loss of the long and short forms of OPA1, mitofusin 1 (MFN1), and mitofusin 2 (MFN2) which all control mitochondrial fusion. In addition, expression of DRP1, along with its primary receptor protein, mitochondrial fission factor (MFF), were also reduced after CS/Tx. Interestingly, CS/Tx lead to aberrant higher molecular weight OMA1 aggregate expression.CS alone (no Tx) reduced respiratory complex I and II activities along with reduced expression of the primary mitochondrial fission protein, dynamin related protein (DRP1), induced loss of the long form of Our results suggest that CS appears to involve activation of the OMA1, which could be a key player in proteolysis of the fusion and fission protein machinery following transplantation. These findings raise the possibility that impaired mitochondrial fission and fusion may be unrecognized contributors to CS induced mitochondrial injury and compromised renal graft function after transplantation. Renal transplantation has been a clinical reality for almost 50 years, and improved success in recent years is attributed to the advent of tissue-typing protocols and superior immunosuppressive regimens. While immune mechanisms are clearly important to allograft survival, the existence of alloantigen independent mechanisms is undeniable. Kidneys from living donors have better long-term graft outcome –3 than tThe purpose of CS is to lower the kidney’s metabolic activity and oxygen demand during the wait for transplantation, but nevertheless, injury occurs . Severaoverlapping with the m-AAA protease) causing proteolysis of the long to short form of OPA1 resulting in impaired fusion and mitochondrial fragmentation [Several studies, including our own, have revealed that renal CS injury induces reactive oxygen species (ROS) and this contributes to impaired mitochondrial respiration, loss of ATP, and cell death ;14;18–23entation –32. YME1entation ;33.Abnormalities of mitochondrial fission or fusion during renal CS/Tx have not been reported previously. We have data suggesting, for the first time, the relationship between extensive mitochondrial dysfunction and decreased OPA1, MFN1/2, DRP1, MFF, YME1L and an altered conformation of OMA1 in kidneys exposed to CS/Tx compared to transplantation without CS. These findings raise the possibility that impaired mitochondrial fission/fusion may be an unrecognized contributor to CS-induced injury and compromised renal graft function after transplantation.Male Lewis rats weighing 200–250g were used as transplant donors and recipients in this study. All animal protocols were approved by the Institutional Animal Care and Use Committee , at the University of Arkansas for Medical Sciences (UAMS), and all animal experiments described below were performed in compliance with the IACUC at UAMS using NIH guidelines  solution at 4o C for 18 hr. The right kidneys of donor rats are referred to as CS group (n = 10). For the recipient rat surgery, rats were anesthetized using isoflurane, the native left kidney was removed, and the donor left kidney (exposed to CS) was transplanted using end-to-end anastomosis as described previously [18hr cold storage plus transplantation (CS/Tx) group (n = 10). The rat survival rate was greater than 95% for the CS/Tx groups. Animals were not randomized other than if a donor or recipient rat kidney displayed multiple renal arteries or veins making the transplant procedure more difficult, these rats were used for control or sham animals described below.Orthotopic renal transplant surgery was performed in male Lewis rats as illustrated in lication . For theeviously . The surFig 1). After 24 hr, the transplanted kidney was harvested under anesthesia, and the organs were referred to as Autotransplant (ATx) group (n = 8).The autotransplant (ATx) surgery was included in these studies so that the impact of CS could be isolated from the impact of transplant surgery alone with regard to alterations in mitochondrial fusion/fission protein expression. ATx is performed as described in the recipient surgery method, except that the left kidney was removed, flushed with saline and immediately transplanted in the same rat without CS exposure, followed by right nephrectomy . The right kidney from a healthy rat was removed and saved as Control kidney (n = 10). The left kidney remained functioning for 24 hr, and then the sham kidney and blood were harvested and saved as the Sham group (n = 10).Rats underwent identical surgery (right nephrectomy), but without the renal transplantation , or flash frozen for biochemistry/western blots. The ‘control’ kidney was compared to the CS alone kidney since both kidneys were harvested from healthy rats. The ‘sham’ kidney served as the control for both transplant models (ATx and CS/Tx) since all underwent a nephrectomy .TM, and cartridges (CHEM8+) as described by the manufacturer .Blood chemistry was determined in heparinized blood  using a hand-held clinical chemistry analyzer, iSTAT3) representing cortex and fixed with fixative solution ) for 2hr at room temperature followed by storing at 4°C. The fixed biopsies were then sent to the histology core laboratory at Georgia Health Sciences University for further processing and analysis.Fresh kidneys were cut into small pieces  by high resolution respirometry (HRR) , according to substrate-inhibitor-titration (SIT) protocol as described earlier ;34;35. B3VO4 , 2.5 mM NaF , 1mM DTT  and protease inhibitor cocktail . Protein concentrations were determined by Coomassie Plus Protein Assay Reagent . Renal extracts (30 μg) were resolved onto SDS-PAGE gel and then transferred to PVDF membrane. Western blot analysis was performed using antibodies against proteins: DRP1 ; MFF ; MFN1 ; MFN2 ; OPA1 ; YME1L ; OMA1 ; Core 2 ; MnSOD  and β-actin . Actin expression was used as a loading control for all western blot experiments. Probed membranes were washed three times and immune-reactive proteins were detected using horseradish peroxidase conjugated secondary antibodies  and enhanced chemiluminescence . Densitometry evaluation on scanned membrane was performed using AlphaEase FC software.Renal extracts from whole kidney homogenates were prepared by using radioimmuno-precipitation assay (RIPA) buffer  with 1 mM PMSF , 1.2 mM Nat-test was used when comparing differences between the mean of two groups  at a 95% level of confidence. Likewise, western densitometry analysis comparing Control versus CS alone was done using an unpaired Student’s t-test. Differences with a P value < 0.05 were considered statistically significant.Results are presented as mean ± standard error of the mean (S.E.M.) using Graph Pad Prism software. Mitochondrial respiration data and western densitometry analysis between all transplant groups  were analyzed by a one-way factorial analysis of multivariance (ANOVA) followed by Tukey’s post-hoc test using multiple group comparisons. For renal function analysis an unpaired Student's Fig 2). Rat kidneys exposed to CS (18 hr) followed by transplantation in a new recipient rat (CS/Tx) resulted in a greater inactivation of complex III when compared to sham kidneys  or CS alone kidneys. Compared to kidneys from sham rats, kidneys subjected to CS/Tx showed impaired renal function 24 hr after transplantation with increased serum creatinine and BUN levels . Our earlier study showed that ATx did not alter mitochondrial complex activity, but induced renal injury when compared to sham [Fig 3B). However, CS/Tx resulted in profound fragmentation and rounding of renal mitochondria .The activities of renal mitochondrial respiratory complexes I, II and III were assessed by high resolution respirometry (HRR), which has the advantage of using an intact tissue biopsy, rather than relying on mitochondrial isolation. Isolated rat kidneys were exposed to CS alone (18 hr) which resulted in significant inhibition of complexes I and II activity when compared to controls (rats with both native kidneys intact) . Interestingly, CS alone also significantly reduced DRP1 expression (~75 kDa) compared to control kidneys . MFF expression (~38 kDa) was only reduced with combined CS/Tx , not after CS alone or ATx . Actin expression was used as a loading control for all western blot experiments.In an attempt to dissect pathways responsible for the profound mitochondrial fragmentation during CS/Tx, rat tissues from the experiment described in Fig 5A & 5B). Conversion of the long form of inner mitochondrial fusion protein OPA1  to the short form  is indicative of OPA1 proteolysis and impaired mitochondrial fusion. Interestingly, CS alone induced conversion of the long form of OPA1  to the short form   suggesting reduced inner membrane fusion during CS. However, CS/Tx lead to extensive loss of both the long and short form of OPA1  suggesting severe impairment of inner mitochondrial membrane fusion.We next evaluated key proteins involved with mitochondrial fusion including mitofusins (MFN1/2) and optic atrophy protein type 1 (OPA1), which are GTPase’s that regulate the fusion process of the outer and inner membranes, respectively. Neither ATx nor CS alone (18 hr) altered expression of outer membrane fusion proteins, MFN1 or MFN2 (both ~75 kDa); however, CS combined with Tx (CS/Tx) significantly lowered expression of both MFNs . We show decreased OMA1 expression at its predicted molecular weight of 40 kDa after ATx and CS alone, and a more robust decline after CS/Tx . Decreased OMA1 protein expression is sometimes used to assess OMA1 activity since it is known that OMA1 can cleave itself [ The reason or function of these OMA1 aggregates remains unknown but might be involved with an aberrant activity of OMA1 during CS/Tx.As mentioned earlier, OMA1 and YME1L are mitochondrial proteases known to cleave OPA1 to the short form. OPA1 cleavage is commonly used as an indirect assay for activation of these proteases, since there are no sensitive or specific activity assays available. Interestingly, CS/Tx lead to a reduction of YME1L, while CS alone or ATx did not alter YME1L protein levels  as well as Core2, a complex III subunit protein (found in inner membrane), were not reduced after CS alone or after CS/Tx , suggesting exacerbation of CS-induced mitochondrial dysfunction. The profound loss of ALL mitochondrial dynamic proteins examined  after CS/Tx may indicate altered mitochondrial biogenesis (synthesis), but the data showing no change in MnSOD or Core 2 indicate that this is unlikely. The reduced expression in the short form of OPA1 was unexpected and may indicate involvement of other protease systems (e.g. ubiquitin proteasome pathway) during CS/Tx, which deserves further study. Our recent study [The current study clearly demonstrates for the first time that renal CS alone (18 hr) leads to damaged mitochondria and altered mitochondrial fission and fusion pathways. One important aspect of this study is the value of evaluating CS alone and combined with transplantation, since our data revealed complex I and II were partially inactivated with CS alone-nt study  using thFig 6), suggesting possible binding to substrates or altered conformation of OMA1. To our knowledge very few studies have evaluated altered OMA1 protein levels in disease models, the majority of studies utilize labeled (e.g. myc tagged) overexpressed OMA1 in cell models exposed to different stressors. And since very little is understood about how OMA1 is regulated in disease, we postulate that our study will yield new information that will help unravel the complexities associated with this important mitochondrial protease.OMA1 is considered a stress-induced protease that can function independently of ATP, whereas YME1L is constitutively active, requires ATP, and is needed for normal mitochondrial function ;30;36;37in vitro models [In yeast it has been shown that OMA1 can exist as higher molecular weight oligomers in native gels, and that stress can alter OMA1 conformation and activity, but the mechanisms involved and whether this occurs in mammalian systems remains unknown ;39. Receo models . These so models , which mo models ;33; howeo models . Thus, iVery few disease models have demonstrated reduced expression of key mitochondrial fission and fusion proteins to the extent we have shown in the current study following CS/Tx. In most instances, enhanced fission (e.g. increased DRP1) and reduced fusion leads to the classic fragmented mitochondria along with a decline in mitochondrial function. For example, Funk and Schnellmann showed that expression of DRP1 increased and MFN2 declined after renal ischemia/reperfusion . Anotherin vivo revealed clear renal and mitochondrial damage [There are a few limitations of our current study focused on selection of the animal model. Since the primary focus of this study was to evaluate the impact of CS alone, we chose to use a syngeneic (Lewis to Lewis) transplant model, rather than a more clinically relevant allograft (Fisher to Lewis) transplant model, which would otherwise add confounding effect from the immune system. Given our results showing profound renal and mitochondrial damage, we are concerned that adding an immune insult to the CS effect may increase mortality. Many studies using an allograft renal transplant model elect to keep the native kidney intact for up to ten days after surgery, presumably to prevent mortality. This paradigm would limit our ability to determine the acute (within 1–3 days) impact of CS plus immune activation on mitochondrial fission/fusion in the transplanted kidney. Another drawback to our model, is that we used static CS as the method of preservation, rather than the clinically preferred machine preservation, which involves constant pumping of cold preservation solution through the kidney. We selected the static CS method due to the obvious technical difficulty in pumping a rat kidney, but also because there are still many transplant centers that rely on static CS due to cost restrictions or to transportation of the donor kidney to the center. However, our prior publication using a novel method of cold machine preservation in a rat kidney l damage .Fig 8). Pharmacological strategies designed to blunt the OMA1 mediated effect could prove to be translationally beneficial.Long-term renal function after transplantation is improved when kidneys are harvested from living rather than deceased donors. The biggest difference in pre-surgical organ preparation is that deceased-donor kidneys are subjected to cold preservation (static or pumping) while a suitable recipient is located, whereas kidneys from living donors do not undergo lengthy cold preservation. The early work of Belzer and Southard revolutionized the world of preservation solutions –44 and mS1 File(PDF)Click here for additional data file.