Given the elevated risk of cardiovascular events and the higher prevalence of silent coronary artery disease (CAD) in diabetic versus non-diabetic patients, the need to screen asymptomatic diabetic patients for CAD assumes increasing importante. The aims of the study were to assess prospectively the prevalence and risk factor predictors of true silent myocardial ischemia  in asymptomatic type 2 diabetic patients.Stress myocardial perfusion gated SPECT (Single Photon Emission Computed Tomography) was carried out in 41 type 2 diabetic patients without history of cardiovascular disease (CVD) and 41 nondiabetic patients matched by age and gender.There were no significant differences between the two groups regarding either the classic CVD risk factors or left ventricular function. True silent ischemia was detected in 21.9% of diabetic patients but only in 2.4% of controls (p < 0.01). The presence of myocardial perfusion defects was independently associated with male gender and the presence of diabetic retinopathy (DR). The probability of having myocardial perfusion defects in an asymptomatic diabetic patient with DR in comparison with diabetic patients without DR was 11.7 [IC95%: 3.7-37].True silent myocardial ischemia is a high prevalent condition in asymptomatic type 2 diabetic patients. Male gender and the presence of DR are the risk factors related to its development. Patients with diabetes, in particular with type 2 diabetes, are at a 2- to 4- fold higher risk of cardiovascular mortality compared with nondiabetic subjects. In addition, diabetic patients are less likely to survive a first myocardial infarction than their nondiabetic peers. Therefore, early identification of coronary artery disease (CAD) in the diabetic population is needed. However, the fact that CAD is often asymptomatic in diabetic patients makes such identification a challenge. A number of studies have shown that silent myocardial ischemia - as evidenced by non-invasive tests such as the electrocardiogram stress test, myocardial scintigraphy or stress echocardiography- affects 20-50% of diabetic patients with additional risk factors . The terIn the largest study performed until now to evaluate the prevalence of silent myocardial ischemia in diabetic patients, the DIAD (Detection of Ischaemia in Assymptomatic Diabetics) study  patientsThe present case-control study was designed to assess prospectively the prevalence and risk factor predictors of true silent myocardial ischemia in asymptomatic type 2 diabetic patients without a history of cardiovascular disease (CVD).This study has been performed following the "Strengthening the Reporting of Observational Studies in Epidemiology" (STROBE) guidelines for reporting case-control studies .We used the following formula for the sample size calculation:n= estimated necessary sample sizeZα = Z-coefficient for Type I errorZβ = Z-coefficient for Type II error1 = estimated proportion of myocardial perfussion defects in controls (non diabetic population)p2 = estimated proportion of myocardial perfussion defects in cases (diabetic patients)p1 and p2p = mean value of pa priori that the prevalence of true silent ischemia would be of 2% (p1 = 0.02) in nondiabetic subjects and 20% (p2 = 0.20) in diabetic patients. We used a 5% of significance level and a power of 80%. The specific figures are displayed below:For this purpose we estimated Therefore, the minimum sample size was 36 subjects in each group.Exclusion criteria were: 1) history of CVD; 2) electrocardiographic evidence of Q-wave myocardial infarction, ischemic ST depression, T-wave changes, or complete left bundle branch block; 3) flat or downsloping ST segment depression > 1 mm at 80 ms after the J-point during an exercise test on a bicycle ergometer. On this basis 44 consecutive asymptomatic type 2 diabetic patients were propectively recruited from our outpatient diabetes clinic  [All patients were systematically screened with an exercise test on a bicycle ergometer and stress myocardial perfusion imaging using technetium-99m-methoxy-isobutyl-isonitrile single-photon emission computed tomography (SPECT). All patients did symptom-limited exercise on a stationary bicycle. One-day protocol gated SPECT was used: a first dose of 8 mCi, given 30-60 seconds before finishing exercise, and a second dose of 24 mCi, administered at rest, were separated by an interval of more than 45 minutes. The equipment used was a Siemens E-CAM dual head 90° gamma camera with a low energy high-resolution collimator and 180° semicircular orbit set in "step-and-shoot" mode, initiated at 45° right anterior oblique, with images every 3 degrees (25 second time frame). Acquisition was synchronized with the electrocardiogram R wave, with an 8 frame/cardiac cycle. Frames were reconstructed with this gamma camera using filtered backprojection . To quantify perfusion the left ventricle was divided into 17 segments, each of which was assigned a score from 0 to 4 . The summed stress score and summed rest score were obtained, with the summed difference score being the difference between the two. An ischemic patient was defined as showing the presence of a summed difference score ≥ 2. A moderate perfusion defect was defined as a segmental score ≥ 2 in > 1 segment, and severe perfusion defect was defined as a segmental score ≥ 3 in > 1 segment in stress images [2), smoking habit (past or current), hypertension (defined as blood pressure > 140/90 mmHg or anti-hypertensive drug use), dyslipemia , and diagnosis of CAD in parents or sibling before age 50.The cardiovascular risk factors evaluated were: obesity . The elapsed time between fundoscopic examination and gated SPECT was less than four weeks in all cases. For DR classification the International Clinical Diabetic Retinopathy Severity Scale was used . Blood at test and Fisher's exact test. To identify the factors independently related with clandestine ischemia a backward stepwise logistic regression analysis was performed.Bivariate associations were tested using Apart from diabetes there were no significant differences between the two groups regarding either the classic CVD risk factors  or left ventricular function Table . DiabetiThree out of 44 recruited patients were excluded from the study because they presented ST-segment depression > 1 mm during exercise test . In addition, the prevalence of true silent ischemia in diabetic patients with DR was significantly higher than in diabetic patients without DR . On the other hand, DR was higher in patients with true silent myocardial ischemia than in patients without it .In the logistic regression analysis, both male gender and DR were independently associated with the presence of true silent myocardial ischemia Table .Finally, we did not find any differences either in left ventricular volumes (end-systolic and end-diastolic) or in left ejection fraction between diabetic patients with and without myocardial perfusion defects measured during rest gated SPECT.In this case-control study we evaluated for the first time the prevalence of true silent myocardial ischemia in asymptomatic type 2 diabetic patients in comparison with a non-diabetic control group. Risk factors of CVD were similar between both groups. However, diabetic patients presented lower levels of LDL-cholesterol, this being accounted by the use of statins. We found that the prevalence of true silent myocardial ischemia is strikingly higher than in their nondiabetic peers. As previously reported in silent myocardial ischemia ,9.One of the most important findings of the present study is that DR is independently associated with the presence of true silent myocardial ischemia. In this regard, DR has been recently recognized as an indicator of risk for CAD in diabetic patients -12 and iper se used in the DIAD study did not improve the clinical outcomes of diabetic patients. By contrast, in another randomised study also including asymptomatic type 2 diabetic patients, it has been observed that the rate of cardiac events was significantly lower in patients screened for silent myocardial ischemia and revascularized when necessary than in patients who were not screened [One might wonder which diabetic patients would benefit from a SPECT for ruling out a silent ischemia and whether the information drawn from this test might be beneficial in terms of CAD prognosis. This is important not only for the management of diabetic patients but also in economic terms. In this regard, the main aim of the DIAD study was to test the hypothesis that systematic screening with stress myocardial perfusion imaging would identify higher-risk individuals and beneficially affect their risk of myocardial infarction or cardiac death. The conclusion of the study was that the identification of patients with myocardial perfusion defects did not serve to lower their risk over 5 years of follow-up . Howeverscreened . In thisscreened Although cost-effectiveness studies are still needed it is undeniable that type 2 diabetes could benefit from the identification of silent myocardial ischemia. In fact, a normal SPECT has a very high negative predictive value for myocardial infarction or cardiac death . In addiThe repercussion of true silent ischemia (in particular mild perfusion defects) on cardiac function in diabetic patients is a topic that remains to be elucidated. We did not find any differences either in left ventricular volumes (end-systolic and end-diastolic) or in left ejection fraction between diabetic patients with and without myocardial perfusion defects. However, it should be noted that diabetes can be associated with impaired myocardial performance assessed by echocardiographic tissue Doppler imaging even in the absence of significant CAD . In addiGiven that a generalized screening of asymptomatic type 2 diabetic patients with stress myocardial perfusion imaging is not a realistic approach , we wantOur results could also have therapeutic implications. There is now emerging evidence that intravitreal anti-VEGF  agents  are useful in the management of advanced DR . HoweverWe conclude that true silent myocardial ischemia is frequent in asymptomatic type 2 diabetic patients. In addition, DR is a high risk condition for true silent myocardial ischemia in asymptomatic type 2 diabetic patients, and point to these patients as a target to be screened for CAD. Further prospective studies evaluating the prognostic value of these findings and their impact in economical terms are warranted.bpm: beats per minute; CAD: coronary artery disease; CVD: cardiovascular disease; DIAD: Detection of Ischaemia in Assymptomatic Diabetics; LV: left ventricular; METs: Estimated Metabolic Equivalent; DR: diabetic retinopathy; SPECT: Single Photon Emission Computed Tomography; VEGF: vascular endothelial growth factor.The authors declare that they have no competing interests.CH, JC and RS: conceived the study; CH, JC, AC, GF and SA: collecting and analyzing data; CH: statistical analysis and writing the draft; JC and RS: obtaining funding and revising the manuscript. All authors read and approved the final manuscript.


Neural circuits in neonatal animals contain numerous redundant synapses that are functionally immature. During the postnatal period, unnecessary synapses are eliminated while functionally important synapses become stronger and mature. The climbing fiber (CF) to the Purkinje cell (PC) synapse is a representative model for the analysis of postnatal refinement of neuronal circuits in the central nervous system. PCs are initially innervated by multiple CFs with similar strengths around postnatal day 3 (P3). Only a single CF is selectively strengthened during P3–P7 , and the strengthened CF undergoes translocation from soma to dendrites of PCs from P9 on (dendritic translocation). Following the functional differentiation, supernumerary CF synapses on the soma are eliminated, which proceeds in two distinct phases: the early phase from P7 to around P11 and the late phase from around P12 to P17. Here, we review our current understanding of cellular and molecular mechanisms of CF synapse elimination in the developing cerebellum. Formation of precise neuronal connections during development is a prerequisite for proper functions of the nervous system. At birth, neuronal connections are redundant, but they are refined and become functionally mature through activity-dependent competition among redundant synaptic inputs to each postsynaptic neuron. During the postnatal period, functionally important synapses are selectively strengthened and stabilized, whereas unnecessary surplus connections are weakened and eventually eliminated. Many studies indicate that these processes are dependent on neural activity particularly during the limited postnatal period known as the ‘critical period’ or ‘sensitive period’ –5.2+ transient due to activation of voltage-dependent Ca2+ channels (VDCCs) in PC dendrites [The climbing fiber (CF) to the Purkinje cell (PC) synapse in the cerebellar cortex is regarded as a representative model system for analyzing the mechanisms for developmental establishment of the functional connections in the central nervous system. In the adult cerebellum, each PC is innervated by a single CF (mono innervation) originating from the inferior olive of the contralateral medulla oblongata. Each CF forms hundreds of synaptic contacts on the proximal part of PC dendrites , 7. Therendrites . In earlendrites –11. Surpendrites –16. In t2+ transients [The cerebellar cortex is known to be subdivided into longitudinally elongated parasagittal bands –19. PCs ansients , 26. In ansients . These Cansients .Olivo-cerebellar axons reach the primitive cerebellum around E18 , 30. TheWhile molecules related to cell identity are differentially expressed in parasagittal bands in developing cerebellum and inferior olivery neurons , it remaWhile the microzonal projection is largely established at early postnatal days, each PC is innervated by multiple CFs. PCs are devoid of large primary dendrites and CFs mainly form terminals on the fine processes emerging from the PC somata. Adult-like, mono innervation is gradually established during postnatal development. Studies on the mechanisms underlying postnatal development of the CF to PC synapse were initiated from the analyses of spontaneously occurring mutant animals. Several mutant mice that have malformation of cerebellar cortex (reeler ), degeneAround P2–P3, most PCs are multiply-innervated by four or more CFs. At this postnatal age, individual multiply-innervating CFs elicit excitatory postsynaptic currents (EPSCs) with similar amplitudes. Then, a single CF in each PC undergoes selective strengthening such that it elicits larger EPSCs than do other CFs –51. QuanThese electrophysiological data in mice are supported by the morphological analyses in rats , 52. ImmIn addition to the increase in the synaptic terminal number, each CF terminal is also functionally strengthened. The EPSCs arising from weaker CFs have been shown to be suppressed by a low-affinity AMPA receptor antagonist, PDA, more potentially than those arising from the strengthened CF . This re2+-dependent long-term potentiation could be induced only in the CF inputs that elicited large excitatory postsynaptic potentials in early postnatal stage. Such activity-dependent competition may eventually result in selective strengthening of a single “winner” CF and weakening of the rest of CFs in each PC.Hashimoto et al.  have recThe territory of CF synapses on PCs expands from soma to dendrites during postnatal development , 56, 57,The CFs start to extend from soma to main dendrites from P9 (“capuchon” stage) . Simulta2+ containing extracellular solution which causes asynchronous release of transmitter quanta following CF stimulation [When the CF translocation is in progress, most of the PCs are still innervated by multiple CFs. Hashimoto et al.  have revmulation , 63  knockout mice, the CF innervation was largely confined around the PC soma even at P13 . HoweverIn the mouse cerebellar slices, the average number of CFs that innervate individual PCs does not decrease but slightly increases from P3 to P6 when the functional differentiation of multiple CFs occurs . The val2+ influx into the postsynaptic PCs are critical for the early-phase CF elimination.As we mentioned in the previous section, surplus CF elimination proceeds in at least two distinct phases: the early and the late phase of CF elimination. The early phase of CF elimination occurs from P7 to around P11 . MechaniIn addition, insulin-like growth factor I (IGF-1) is reported to be involved in the CF synapse elimination from P8 to P12 . Local aThe late-phase of CF elimination starts at around P12 in mice . While tAs mentioned in the “In mice deficient in the type 1 metabotropic glutamate receptor (mGluR1) or any of its downstream signaling molecules , the late-phase of CF elimination has been shown to be severely impaired –93. The Although mGluR1 is richly expressed at perisynaptic sites of PC dendritic spines facing PFs and CFs, mGluR1 can be activated readily by PF inputs –99, whilA receptor sensitizing agent, diazepam, to the cerebellar cortex. These results indicate that impaired CF synapse elimination in GAD67 heterozygote mice is the result of reduced activation of GABAA receptors within the cerebellum. Miniature inhibitory postsynaptic currents (mIPSCs) with large amplitudes and fast rise times, which presumably derived from BC terminals on the PC soma, were greatly reduced in GAD67 heterozygote mice. When paired whole-cell recordings were made from a putative BC and a PC, the amplitude of unitary IPSCs of GAD67 heterozygote mice was reduced to about half of that of wild-type mice. Because of the reduced inhibition to the PC soma, stimulation of weak CF in GAD67 heterozygote mice induced significantly larger Ca2+ transients in the PC soma than in wild-type mice. While the size of somatic Ca2+ transients by activation of weak CF was much smaller than that by the strongest CF in wild-type PCs, weak CF could induce somatic Ca2+ transients that were comparable in amplitude to those elicited by the strongest CF activation in GAD67 heterozygote PCs. Consequently, the Ca2+ influx around somatic CF synapses might be large enough to overcome synapse elimination signals that otherwise prune most of the somatic CF synapses during the late phase CF elimination [Recent analyses have revealed that GABAergic inhibition is also crucial for CF synapse elimination. Nakayama et al.  has recemination , 105.A neurotrophin receptor, TrkB, is also suggested to be involved in CF synapse elimination that starts from around P10–P12 , 107. ItThe current hypothesis for the mechanisms underlying the late-phase CF elimination is illustrated in Fig. 2+ influx are potential candidates. While several reports suggest the importance of neuronal activity in these processes, activity patterns in immature cerebellar circuits have not been well studied. What patterns of neural activity are important? How are such activity patterns generated? How much activity is required for proper maturation of cerebellar circuits? These issues should be addressed in intact cerebella from living immature animals in vivo.The scheme in Fig. The late-phase of CF elimination is critically dependent on the mGluR1 signaling cascade in PCs and GABAergic inhibition to PC somata. It is currently unknown whether these two pathways work in parallel or if they ultimately converge on the common process for the late phase of CF elimination. In addition, the final mechanisms that execute morphological pruning of surplus CF synapses are also unknown. On this point, a previous report suggests that surplus CF synapses might be engulfed by other cells . The CF 2+/calmodulin-dependent kinase IV (CaMKIV) are reported to have persistent multiple innervations, but it is unclear what stage of the postnatal process is impaired [2+/calmodulin-dependent protein kinase II (CaMKIIα) display multiple innervations at P21–P28, but this phenotype disappears in adulthood, suggesting delayed CF synapse elimination [As for other molecules potentially involved in CF elimination, null mutant mice lacking Caimpaired . It is amination . Such demination  and the mination . The rol Arc/Arg3.1 mediates the late phase of CF elimination [The candidate genes are picked up by their spatial and temporal expression profiles during postnatal development, or differential expression between wild-type mice and mutant mice with impaired CF synapse elimination. The functions of these candidate genes in cerebellar development are normally checked by gene knockout or pharmacological manipulations. However, gene targeting generally requires significant efforts and time. Pharmacological manipulation of molecules in the brain in vivo accompanies the problem of drug specificity and the difficulty in persistent, local, and stable application of drugs to the developing brain whose size and structure change continuously. For high-throughput screening of candidate molecules, establishment of culture system for analyzing synapse elimination have been attempted –122. In mination .


We investigated substrate dependent paracrine signaling between subpopulations of bone marrow stromal cells (BMSCs) that may affect the formation, or perhaps malformation, of the regenerating tendon to bone enthesis. Polyacrylamide substrates approximating the elastic modulus of tendon granulation tissue and the osteoid of healing bone (10–90 kPa) were functionalized with whole length fibronectin (Fn), type-I collagen (Col), or a mixed ligand solution (Fn/Col), and BMSCs were cultured in growth media alone or media supplemented with soluble Col or Fn. More rigid substrates with a narrow mechanical gradient (70–90 kPa) robustly induced osteogenic cell differentiation when functionalized with either Col or Fn. On broader mechanical gradient substrates (with a linear elastic modulus gradient from 10–90 kPa), cell differentiation was markedly osteogenic on subregions of Fn functionalized substrates above 20 kPa, but osteogenic activity was inhibited on all subregions of Col substrates. Osteogenic behavior was not observed when cells were cultured on Fn substrates if Col was present either in the media or on the substrate (Fn/Col). Tenogenic differentiation markers were observed only on Col substrates with moderate rigidity (∼30–50 kPa). Tenogenic differentiation was unaltered by soluble or substrate bound Fn. Co-culture of narrow gradient subsections revealed that any inclusion of tenogenic substrates , caused otherwise osteogenic substrates to not develop markers of osteogenic differentiation, while increasing cell proliferation. These apparently paracrine effects could be mediated by bone morphogenetic protein-2 (BMP-2), as first confirmed by gene-level expression of BMP-2 and the transcription factor Smad8, and verified by BMP-2 media supplementation at levels similar to observed cell-secreted concentrations, which arrested osteogenic differentiation in 14 day cultures. Thus, cell instructive biomaterials with engineered mechanical and biochemical properties represent potentially powerful tools for directing BMSC differentiation to tendon and bone, however paracrine signals from tenogenic cells may delay osteogenesis at the healing enthesis. The native tendon to bone junction is an exquisitely designed tissue interface comprising a cellular transition from the tendon itself, to a non-mineralized fibrocartilage region, to a mineralized fibrocartilage region, and ultimately to the bone We previously demonstrated that BMSCs could be differentially induced to commit toward bone and tendon cell lineages using engineered substrates of given ligand chemistry and mechanical compliance In the present work we focused on the hypothesis that cell secreted cues can affect bone marrow stromal cell (BMSC) differentiation across remote sites of a MG substrate, and that these cues depend on both ligand chemistry and substrate stiffness. As before, we examined BMSC differentiation toward tenogenic and osteogenic lineages on gradient stiffness substrates functionalized with type-I collagen or fibronectin, but also explored mixed ligand functionalization, and the effects of stimulation by soluble fibronectin or collagen. To exclude the influence of cell-cell contacts across stiffness regions, we performed similar experiments using subsections of the broader mechanical gradient in the same culture well. We confirmed our hypothesis that cell-secreted (paracrine) cues from the tenogenic substrates could affect osteogenic differentiation within the system. We identified elevated signaling related to bone morphogenetic protein-2 (BMP-2) in tenogenic cultures that apparently interfered with (or delayed) the onset of osteogenic markers at the day 14 time point, on otherwise osteogenic substrates. Specifically, we found higher gene level expression of BMP-2 on MG30-50  collagen substrates, as well as on mixed and collagen substrates that were stimulated with soluble fibronectin. Direct measurements of BMP-2 secretion using immunosorbance techniques were substantially higher on collagen-presenting intermediate stiffness (i.e. tenogenic) substrates compared to fibronectin. Exogenous BMP-2 stimulation at levels similar to those observed in tenogenic cultures also interfered with differentiation on otherwise osteogenic substrates, while increasing cell proliferation. We thus conclude that BMP-2 secretion involved in BMSC differentiation can be modulated by strategic choice of cell substrate. BMP-2 signaling has been implicated in both osteogenic and tenogenic differentiation in vivo The mechanical compliance was determined to range from 10 to 150 kPa as a function of longitudinal position on the hydrogel substrate . As repoat 10 µg/ml (resulting in a bulk protein solution concentration of 20 µg/ml) was used, nodules were not observed although a comparable amount of fibronectin was present on the MG10-90 substrate (We first probed BMSC differentiation on MG10-90 substrates (moduli ranging from 10 to 90 kPa) that were functionalized with fibronectin or type-I collagen 10 µg/ml . Cells w10 µg/ml  that wer10 µg/ml . When a ubstrate . Next, cubstrate ). Consisubstrate .We confirmed osteogenic differentiation by using quantitative polymerase chain reaction (qPCR) to assess gene expression fold change of Runx2 and alkaline phosphatase (Alpp). Higher levels were seen on fibronectin MG10-90 substrates when cells were cultured with growth media . This waIn contrast to fibronectin functionalized MG10-90 substrates, the stiffest subregions of collagen functionalized MG10-90 substrates did not induce mineral plaques or gene level signaling related to osteogenic differentiation. We did confirm that collagen-functionalized subsections cut to isolate only the stiffest range of gradient (MG70-90) did induce osteogenic differentiation when cultured alone ,5. On fiBased on these results, we investigated the hypothesis that tenogenic regions of the intact MG10–90 substrates  were possibly interfering with osteogenic differentiation on stiffer subregions through paracrine signaling of growth factors and cytokines. To test this, we cultured subsections of the MG substrate in the same well spanning the same stiffness range to determine whether mineral plaques (osteogenic cells) could be obtained on stiff collagen subsections (MG70–90) when cultured in the same well as an intermediate compliance range (MG30–50) fibronectin functionalized subsections. Similarly, we cultured the stiff fibronectin subsection in the same well as the intermediate compliance collagen substrate subsections. In these studies, we found osteogenic plaques on the stiff collagen subsections (MG70–90) when cultured with the intermediate stiffness fibronectin subsection (MG30–50) . In contGiven its established primacy in tenogenic and osteogenic differentiation Cells sense the biochemical and mechanical properties of their environment, and use these powerful cues to regulate their function Soluble and insoluble cues from the microenvironment regulate BMSC-mediated tissue modeling and remodeling in wound healing processes. The established roles of BMSCs in healing are twofold, whereby they first proliferate and differentiate to provide cells that can regenerate damaged tissue We tested and confirmed our hypothesis that broad mechanical gradient (MG) substrates (MG10–90) can elicit dramatic differences in bone marrow stromal cell (BMSC) differentiation compared to substrates with a narrower range (more uniform) of mechanical properties. These differences further depended on ligand chemistry and presentation. As in our previous work, we found that tenogenic BMSC differentiation would only robustly occur on mechanical gradient cell culture substrates within a narrow range of elastic moduli (30–50 kPa) Based on work by others demonstrating the sufficiency of induced Smad-8/BMP-2 signaling to provoke tenogenic BMSC differentiation 5β1 integrin) number or engagement also represents grounds for future study. However, with soluble collagen in a substrate-bound fibronectin system, tenogenic differentiation did not occur. These findings align with investigations by Fan and co-workers that have shown that mesenchymal stem cells can respond differently to substrate presented ligand compared to soluble ligand, with differentially activated signaling pathways In tenogenic differentiation, it is apparently necessary to activate scleraxis from Smad8; recent findings illustrate that BMP-2 is a critical factor in Smad8 expression, both from cell expressed BMP-2 as well as exogenous application in soluble form The culture of subsections within the same well performed in this study were designed to address paracrine signaling and rule out cell-cell contact involvement in the altered osteogenic differentiation patterns observed using subsections of the broader range MG substrates (MG10–90). Central to these studies were the tenogenic subsections  and their influence on other substrate sections (or subregions of the MG10–90) by secretion of soluble cues. While we did include the MG10–30 substrate sections in the culture to more fully replicate the intact MG10–90 cultures, our reporting here focuses on the influence of the MG30–50 collagen functionalized substrate. As indicated by baseline levels of Runx2 and scleraxis expression in quantitative PCR analysis of the ∼20 kPa region of both intact and sectioned substrates, behavior of cells on the ∼20 kPa region of intact MG10–90 substrates differed somewhat from those on the isolated MG10–30 subsections – an effect we attribute to paracrine effects from other subregions of the intact substrates. However, we presume that these effects had little bearing on the interpretation of results from the culture of multiple subsections representing the overall mechanical gradient, since cell behaviors on stiffer substrates (40 kPa and above) were otherwise fully consistent between subregions of the intact MG10–90 substrates and analogous subsections with a narrower range of moduli.While two dimensional in vitro cell and tissue culture systems remain an indispensable tool for shedding first light on the cell mechanics modulation of cell function In summary, our data show that BMSC differentiation can dramatically differ for cells cultured on substrates with broad regional differences in mechanical properties, compared to more narrow gradient substrate cultures. More specifically, tenogenic differentiation of bone mesenchymal stromal cells seems to interfere with osteogenic differentiation at a 14 day time point on otherwise osteogenic substrates, inducing a more proliferative state. This is apparently related to cell secreted cues, such as BMP-2, that may regulate intracellular coordination of stromal cell populations at the healing tendon to bone interface.NuPage 4–12% electrophoresis gels (Invitrogen) were used as the substrate, with reproducible mechanical properties previously characterized and described by us Fibronectin, collagen I (Sigma), or a mixed solution of collagen and fibronectin at 10 µg/ml each was overlaid, incubated overnight, washed, and used afterwards for cell culture experiments. Different regions of the MG substrates were previously found to have equivalent ligand distributions as determined from immunofluorescence 2. Cells were used just before confluence. A cell suspension was created from near-confluent flasks by washing cells with PBS, overlaying with trypsin and neutralizing with growth media.Bone marrow stromal cells (BMSCs) were a kind gift from Dr. Simon Hoerstrup's lab (University of Zurich) and had been first verified in that laboratory for multipotency in osteogenic, adipogenic, and chondrogenic induction assays 2 were seeded and cultured for 14 days at 37°C with media changes every three days. In some cases, media was supplemented with collagen or fibronectin at a final concentration of 10 µg/ml to examine the effect of soluble stimulation on fibronectin substrates or collagen substrates, respectively, as well as bone morphogenic protein-2 (BMP-2). At the 14 day time point, substrates were gently washed with PBS three times, and cells were fixed with 10% formaldehyde (Sigma) for 20 minutes at room temperature. The fixative was removed and substrates were carefully washed with distilled water three times. The substrates were then overlaid with 2% Alizarin Red (Sigma) (pH = 4.2) to stain for calcium present in the culture. The solution was incubated for 10 minutes at room temperature before being washed three times with distilled water. Images were acquired with a Nikon E600 upright microscope with a 10× objective. For each region of the MG10–90 substrate  or individual subsections of the MG substrates and ligand/culture condition, at least five non-overlapping fields were imaged. Experiments were performed three times.A cell suspension was created as described above, and 25,000 cells/cm2, and the total RNA was extracted using an RNeasy Kit (Qiagen) as per the manufacturer's protocols, and stored at −80°C until use. In the analysis of MG substrates (20–80 kPa), substrates were sectioned into three regions prior to isolating RNA. The cDNA was generated by using a cDNA Reverse Transcription Kit (Applied Biosystems). PCR was performed on resultant cDNA using SYBR green PCR Master Mix (SABiosciences) and upper and lower primers. Osteogenic markers probed included for alkaline phosphatase (Alpp), an enzyme expressed by osteoblasts TTwo markers of osteogenic differentiation and two markers for tenogenic differentiation were evaluated at day 14 of stromal cell culture by implementing reverse transcriptase polyermase chain reaction (RT-PCR). Substrates were prepared as described above, BMSCs were cultured at 25,000 cells/cm2 on MG70–90 collagen subsections, MG70–90 collagen substrates supplemented with BMP-2 in the media, and coculture of MG30–50 and MG70–90 substrates. Cells were cultured for 4 days, at which point they were trypsinized to release cells, neutralized, centrifuged for 6 minutes at 900 rpm, and resuspended in 150 µl of media. The cell suspension was plated in a 96 well plate, along with two freshly seeded wells at a seeding density of 25,000 cells/cm2. Cells were given 24 hours to settle before treating with media supplemented with 10% Alamar blue reagent (Invitrogen) for an additional 24 hours. As a control, prior to Alamar blue treatment one well with untreated cells was fixed for 15 minutes with formaldehyde. Readings were taken on a Spectra Max GeminiXS fluorescent plate reader with 570 nm excitation and emission readings at 600 nm, standardized to substrate culture area and normalized to the emission reading for untreated cells on tissue culture polystyrene.The Alamar blue assay is designed to measure cell proliferation from different culture conditions by conversion of nonfluorescent resazurin to fluorescent resorufin in living cells Bone morphogenic protein-2 (BMP-2) secretion was assessed using a colorimetric ELISA (R&D Systems). The ELISA uses well plates that are pre-coated with a monoclonal antibody specific for BMP-2. Briefly, a standard curve, ranging from 4 ng/ml to 0 ng/ml, was created from stock BMP-2 at 1 mg/ml and diluted with the provided dilution buffer. The standard curve and samples were overlaid in the pre-coated well plates with a provided assay diluent. The wells were incubated for two hours with agitation at room temperature, and washed four times with the provided wash buffer. Between each wash, the wells were shaken against paper towels and aspirated with a pipet to ensure complete removal of unreacted species. Next, a monoclonal antibody solution was prepared from the provided stock solution that binds to BMP-2 that is conjugated to an enzyme, overlaid on substrates, and incubated for two hours with agitation at room temperature. After washing substrates to remove any unbound antibody-enzyme reagent, a substrate solution was prepared from the provided kit and added to the wells. The color develops in proportion to the amount of BMP-2 bound in the initial step. The development is stopped with the stopping solution and read at 450 nm. Optical density readings were converted to concentrations from the best fit linear line to the standard curve.All experiments were performed three times. Statistics were performed with single-factor analysis of variance (ANOVA), and a confidence level of 95% (p≤0.05) was considered necessary for statistical significance. All error bars represent the standard error around the mean.


H17 CD4+ cell-mediated mechanism. Using preparative SDS gels, we separated the soluble proteins that compose the WCA in order to identify fractions that were immunogenic and protective. We screened these fractions for their ability to stimulate IL-17A secretion from splenocytes obtained from mice immunized with WCA and adjuvant. We identified 12 proteins within the stimulatory fractions by mass spectrometry; these proteins were then cloned, recombinantly expressed and purified using an Escherichia coli expression system. The ability of these proteins to induce IL-17A secretion was then evaluated by stimulation of mouse splenocytes. Of the four most stimulatory proteins, three were protective in a mouse pneumococcal serotype 6B colonization model. This work thus describes a method for identifying immunogenic proteins from the soluble fraction of pneumococcus and shows that several of the proteins identified protect mice from colonization when used as mucosal vaccines. We propose that, by providing protection against pneumococcal colonization, one or more of these proteins may serve as components of a multivalent pneumococcal vaccine.Mucosal or parenteral immunization with a killed unencapsulated pneumococcal whole cell antigen (WCA) with an adjuvant protects mice from colonization by a T Streptococcus pneumoniae remains an important public health issue, particularly in developing countries While pneumococcal conjugate vaccines (PCVs) have resulted in significant reductions in rates of invasive pneumococcal disease due to vaccine-serotypes, infection with Many strategies have been proposed and evaluated  to the remarkable impact of these vaccines on colonization: by CDC estimates, over 2/3 of cases prevented by pneumococcal conjugate vaccine is the result of indirect, or herd, protection 600 of 1.0 (corresponding to approximately 6.0×108 CFU/ml), washed in Lactated Ringer's solution supplemented with 10% sucrose, concentrated to an OD600 of 32, and inactivated by mixing with chloroform as previously described  g for 5 minutes and collecting the aqueous layer separate from the pellet. Protein estimation was performed using Total Protein Kit with bovine serum albumin as a standard (Pierce).The whole cell pneumococcal vaccine was prepared from strain RM200 that is derived from a parent Rx1 strain that is autolysin-negative, capsule-negative, and expresses a non-hemolytic pneumolysoid variant in the place of the hemolytic pneumolysin 7 CFU of a clinical type 6B pneumococcal strain  were used. All immunizations were performed by instilling 20 μl of a mixture of PBS, 1 μg of cholera toxin  and either 4 μg of the indicated purified pneumococcal protein or 100 μg of WCC as immunogens onto the nares of gently restrained unanesthetized mice. Control animals were immunized with CT mixed in PBS alone to control for any nonspecific effect of the adjuvant alone. Animals were immunized intranasally twice at one-week intervals. Spleens were harvested from immunized animals 2–4 weeks following last immunization  for stimulations with WCC supernatant fractions and purified pneumococcal proteins. Immunized animals that were subsequently challenged received an inoculum consisting of 10WCC supernatant (containing 800–900 μg of protein) was mixed with LDS 4x sample buffer and loaded into the 8 center lanes of a NuPAGE 4–12% Bis-Tris 12-lane gel  then resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for 30–40 minutes at 200V. The preparative gel was then equilibrated in elution buffer (2 mM phosphate buffer at pH 7.0) for three 20-minute equilibrations. Transverse elution of the proteins through the thickness of the preparative gel was performed at constant 300V for forty minutes (current ranged from 65–90 mAmp) in elution buffer in a mini whole gel transverse eluter system . Current was reversed for 15 seconds at the end of the elution and individual fractions were collected in fourteen chambers beneath the preparative gel and harvested into separate tubes. The protein content of each fraction was quantified by Total Protein Kit (Biorad).After determining which fractions were most immunogenic in the IL-17A splenocyte stimulation assays described below, MS identification of the proteins contained in the bands comprising the most immunogenic fractions was carried out at the Taplin Biological Mass Spectrometry Facility of Harvard Medical School, Boston, MA.his) tag. PCR products were ligated into the pQE30 expression vector and ligation products were transformed into competent Escherichia coli (XL/Blue) cells. Transformed cells were selected by growth on LB ampicillin and the identity of the insert confirmed by sequencing. Successfully transformed E. coli cells were grown in 1L LB ampicillin and protein expression was induced with 0.1 mM IPTG overnight at room temperature. Pelleted E. coli were lysed in lysis buffer  and sonicated. Protein was purified over a column of Ni-NTA-agarose beads that had been equilibrated with lysis buffer. The protein-bound column was washed with lysis buffer/40 mM imidazole, and proteins were eluted in 250 mM imidazole. Proteins were run on SDS-PAGE under reducing conditions and gels were Coomassie-stained to confirm protein expression and purity. Purified proteins were desalted on a PD-10 Sephadex column using PBS prior to use in cellular stimulation assays.Gene sequences for twelve of the proteins identified from immunogenic fractions were amplified by PCR using primers designed from the sequence published for serotype 4 strain TIGR4 . Two resSpleens were harvested from WCC-immunized C57BL/6 mice and were processed as previously described into a cellular suspension All statistical analyses were carried out using PRISM . Colonization densities and IL-17A values among immunization groups were compared by Mann-Whitney U test or by the Kruskal-Wallis test with Dunn's correction for multiple comparisons. P values <0.05 were considered to represent statistical significance.While the supernatant of WCC (WCCsup) contains approximately 15% of the total protein of freshly rehydrated noncentrifuged WCC, prior work has demonstrated that, with respect to protection against pneumococcal colonization, retention of the soluble portion of the vaccine yielded a 100-fold more potent whole cell preparation when compared with an ethanol-inactivated preparation in which the soluble fraction was removed by serial washings of the pellet Fractions were all brought to the same protein concentration and used to stimulate splenocyte suspensions obtained from WCC-immunized mice. The WCC and WCCsup elicited median IL-17A responses of 2100 and 3300 pg/ml respectively . The mosPeptides within the submitted bands were identified and matched to the proteomic sequence of pneumococcal strain TIGR4. Proteins with more than 2 identified peptide matches were considered positively identified. Using these methods, 37 proteins were identified in the stimulatory fractions but not in the non-stimulatory counterpart. The corresponding sequences of identified proteins were analyzed bioinformatically; we prioritized antigens that were highly conserved (>85% amino acid homology) among the published pneumococcal sequences and with <10% homology to human proteins. This narrowed the panel of proteins to 12 proteins, which we examined in more detail; these are listed in E. coli and purified. Gels for each of the 12 proteins demonstrated predominant size-appropriate single bands suggesting successful purification. Each recombinant protein was used at 10 μg/ml to stimulate splenocytes from WCC-immunized mice. Several individual proteins elicited robust median IL-17A responses; in particular, SP2070, SP1534, SP0435 and SP0862 elicited the highest median IL-17A responses (range 260–2300 pg/ml) as shown in The 12 identified proteins were successfully expressed in H17 response and protect against colonization. The TH17 responses following immunization with the mixture of proteins or WCV were similar and in both cases significantly higher compared to that of mice immunized with CT alone , and the combination vaccine was as protective as the WCV in this experiment, with all but one nasal wash being free of detectable pneumococci . Additionally, SP1534 and SP0862 conferred statistically significant reduction in colonization compared with CT-immunized controls (over 1 log reduction in median density of colonization). Animals immunized with SP2092 were not protected, as predicted by the low IL-17A elicited by this protein in the splenocytes stimulation assay.Having shown that a combination of these proteins protected animals from colonization, we performed immunizations with individual proteins to determine which proteins contributed most to protection. Using each of the proteins contained within the combination plus an additional protein that was highly stimulatory in the splenocyte assay ; SP 0862We present a sequential method that we used to identify pneumococcal T cell antigens that, when administered as mucosal vaccines with CT, elicit IL-17A responses and provide protection against pneumococcal colonization. In particular, SP2070 was highly protective in our model and SP0862 and SP1534 both significantly reduced colonization.H17 dependent antigens in vitro 3) mass spectrometry of stimulatory fractions to identify comprising proteins and 4) identification of protective TH-17 antigens in vivo. A similar approach has been used to identify IFN-γ stimulatory antigens in mycobacterium tuberculosis In an effort to overcome the limitations of PCV-based approaches, many investigators have explored several serotype-independent approaches in vitro or in vivo for immunogenic and protective potential, respectively. A limitation of this approach as it was implemented here was that the starting material (WCCsup) represents only the soluble compartment of pneumococcal cellular growth, and does not address the potential of proteins in the nonsoluble compartment. Additionally, we did not evaluate different concentrations of the individual proteins used in splenocyte stimulations so it is conceivable that the optimal stimulatory potential of any individual protein was not evaluated. However, as all of the identified proteins were within a relatively close molecular weight range (approximately 20–60 kDa) the molar amount represented in each stimulation was not drastically different.There are several advantages to such a non-genomic approach to immunodominant antigen identification; namely, this method eliminates the need to generate a comprehensive antigenic expression library. The method used here can be completed relatively quickly and identifies a small number of immunodominant bands each comprised of several proteins which can be individually screened either We recognize that nasal mucosal cells such as nasal associated lymphoid tissue (NALT) or draining lymph nodes may represent more relevant immune cells than the splenocytes that were examined in the present study. Indeed, we have previously documented the presence of IL-17A response from nasal mucosal cells of WCV immunized mice H17 antigens that our group in collaboration with Genocea Biosciences and PATH identified following proteomic screening H17 antigens from the pneumococcal proteome that stimulated robust IL-17A from CD4+ T cells of mice immunized with a pneumococcal whole cell vaccine; in contrast, here we identified TH17 antigens from a single predominant protein band within an immunogenic fraction of the chloroform-soluble fraction of a whole cell preparation. There are many possible reasons why the results of the two screens differ, such as differences in the proteins present in the chloroform-soluble fraction vs. the most abundant proteins in the whole cell vaccine. Additionally, the comprehensive screen relied on complex statistical analyses for identification of a “hit," criteria that were not required for the screen presented here.Amongst the twelve antigens identified, one antigen SP1572 , has been shown to be protective against colonization and sepsis and was therefore not studied in this paper Since we are aiming to develop a pneumococcal vaccine to provide serotype-independent immunity, it will be important to test the efficacy of any of the protective proteins against strains of diverse serotypes and/or multilocus sequence types (MLST). From the identified panel of antigens,  we used several bioinformatic filters to further narrow the panel, including the requirement for high sequence homology across all sequenced pneumococcal strains. Of the 12 identified antigens, all but 2 had ≥98% homology at the amino acid level with the 22 available sequenced strains; SP 1572 and SP2092 had 89% and 95% homology, respectively. Plans to test the ability of these proteins to protect against strains of different serotypes and MLST are underway.Salmonella typhi Vi polysaccharide to provide protection against pneumococcal and/or S. typhi diseases are currently under investigation within our laboratory Intranasal immunization with these antigens provides protection against colonization, but such a route requires the use of an adjuvant such as CT, which is not appropriate for human use. In our experience, subcutaneous immunization of purified proteins with aluminum hydroxide did not induce IL-17A production unless proteins were introduced in a fusion conjugate construct In conclusion, using this sequential method of size separation of immunodominant fractions of the soluble compartment of a pneumococcal whole cell preparation and mass spectrometry to identify the comprising proteins, three out of the four top IL-17A stimulatory proteins tested in a colonization model were found to be protective while the lowest IL-17A stimulatory protein conferred no protection. The proteins identified with this method may represent promising candidates for inclusion in a protein-based pneumococcal vaccine.


We performed a network meta-analysis to investigate the optimal antithrombotic regime by indirectly comparing new antithrombotic regimes  in patients with acute coronary syndrome (ACS).12 inhibitors in patients with ACS. Major adverse cardiac events (MACE) were regarded as the efficacy endpoint, and thrombolysis in myocardial infarction (TIMI) major bleeding events were used as the safety endpoint. The net clinical benefit was calculated as the sum of MACE and TIMI major bleeding events.A systematic search of MEDLINE, EMBASE, and the Cochrane databases was performed to identify all phase 3 randomized controlled trials (RCTs) involving novel oral anticoagulants or oral P2YFive phase 3 RCTs with 64,476 ACS patients were included. Although there were no significant differences among new antithrombotic regimes, rivaroxaban 5 mg twice daily plus traditional DAPT might be the most effective in reducing the incidence of MACE, accompanying the highest risk of TIMI major bleeding. Ticagrelor plus aspirin presented slight advantage on the net clinical benefit over other new antithrombotic regimes, with the highest probability of being the best regimes for net clinical benefit (35.0%), followed by prasugrel plus aspirin (28.0%), and rivaroxaban 2.5 mg twice daily plus traditional DAPT (19.5%).Novel antithrombotic regime with ticagrelor plus aspirin brings a larger clinical benefit in comparison with other regimes, suggesting that it may be the optimal antithrombotic regime for patients with ACS. It is well known that the formation of thrombosis is the major pathophysiologic mechanism of acute coronary syndrome (ACS), and thus traditional dual antiplatelet therapy (DAPT)  has become the mainstay of treatment in patients with ACS. Nevertheless, there remains about 10% risk of recurrent thrombotic events within one year after percutaneous coronary intervention (PCI), even after the use of traditional DAPT 12 receptor inhibitors, e.g. Cangrelor [intravenous], Elinogrel [intravenous], prasugrel [oral] and ticagrelor [oral]) have been shown to have more potent therapeutic effect and have faster onset of action, as well as significantly decrease cardiovascular mortality after PCI as compared to clopidogrel Recently, more intensive antithrombotic regimes have been developed in order to overcome this issue, and the safety and efficacy of these therapies have been verified by a series of randomized clinical trials (RCTs). Newly developed antiplatelet agents , EMBASE (1966 to April 2013), and the Cochrane Central Register of Controlled Trials (until April 2013) to identify the eligible studies. An extensive manual search of the literature using the references of the original manuscripts, reviews, and meta-analyses was performed. No language restrictions were imposed. The search strategy was presented in The clinical trials were eligible for inclusion if 1) study design (phase 3 RCTs) involved patient randomization; 2) participants were diagnosed with ACS; and 3) comparisons were made between new oral P2Y12 receptor inhibitors with clopidogrel and novel anticoagulants with placebo in addition to DAPT. Trials would be excluded if the control group used single antiplatelet treatment, or if the sample size was less than 500.Two authors (Y. Y. and H. X.) independently determined the study eligibility and extracted the following data from the included studies: (1) study design; (2) participant and intervention characteristics; (3) treatment ; and (4) clinical outcomes . MACE was defined as a composite endpoint of cardiovascular death, myocardial infarction, or stroke. The net clinical benefit was calculated as the sum of MACE and TIMI major bleeding events. Any disagreements were resolved by consensus, and the principal investigators resolved any disagreements.The internal validity of the eligible studies was assessed according to the Cochrane Collaboration risk of bias tool 2 test and was quantified with the I2 test The κ statistic was used to assess the agreement between reviewers for study selection. In the pair-wise meta-analysis, the pooled odd ratio (OR) was calculated for each outcome using the Mantel-Haenszel method for random effects A network meta-analysis was conducted to compare the efficacy and safety among these new antithrombotic regimes. Due to indirect comparison among these antithrombotic regimes, it was not feasible to use an inconsistency model or a node-splitting model to statistically identify inconsistencies. We assumed the included studies were consistent based on the criteria of study selection and analysis using a consistency random effects model. This model was implemented in the Bayesian framework and estimated using Markov chain Monte Carlo (MCMC) methods p<0.10 indicates a significant publication bias among the included studies.Sensitivity analysis, including both phase 2 and phase 3 studies, was conducted to test how robust the final ranking of these new antithrombotic regimes was relative to eligibility criteria. Publication bias was assessed by the Begg's funnel plot and the Egger weighted regression statistic where a value of All analyses were performed using STATA (version 11.0) and ADDIS . The meta-analysis was prepared in accordance with the PRISMA  statement Four-hundred and sixty-three records were retrieved from the initial search. Seventeen studies were reviewed in full-text. Five phase 3 randomized control trials . However, an increased risk of TIMI major bleeding was identified in new antithrombotic treatment group  with significant heterogeneity . To take the benefit and the bleeding risk together, we found that the use of new antithrombotic agents was associated with a net benefit compared with traditional DAPT . No significant heterogeneity was identified across the included studies  with modest heterogeneity  12 receptor inhibitor, ticagrelor, showed a trend toward achieving the greatest beneficial effect compared to other regimes, which gives it the highest probability of being the optimal therapy.The finding of this analysis demonstrated that the use of new antithrombotic agents resulted in significant reduction in MACE with an increased risk of major bleeding. There was a significant net benefit towards new antithrombotic regimes compared with traditional DAPT. Although no statistical significance was identified, the new P2Y12 receptor inhibitors 12 receptor inhibitors (e.g. prasugrel or ticagrelor) with novel oral anticoagulants (rivaroxaban or apixaban). It was required that large-scale RCTs directly comparing clinical value of these new antithrombotic agents were performed in order to achieve the sufficient power.There were several small-scale studies directly comparing the antiplatelet effect directly of ticagrelor with prasugrel. In ACS patients with diabetes, ticagrelor presented a significantly higher platelet inhibition than prasugrel It has been reported that multiple medications may reduce patients' compliance and fixed-dose combination antihypertensive medication resulted in better compliance than the single agent It is no doubt that these new antithrombotic regimes are associated with reduced rate of recurrent ischemic events. However, more potent platelet/factor Xa inhibition increases the risk of bleeding. In the current study, we found that rivaroxaban in combination with DAPT seemed likely to be the most efficacious in reducing MACE in ACS patients. However, the clinical benefit may be significantly offset by the increase in major bleeding events. This conclusion was confirmed by the recent meta-analysis, in which the administration of novel oral anticoagulant agents did not provide the net clinical benefit compared toplacebo in ACS patients, due to dramatic increase in major bleeding events Several limitations of this meta-analysis deserve comment. Firstly, in order to reduce heterogeneity, we included only phase 3 studies in our meta-analysis. Nevertheless, we conducted a sensitivity analysis by combining phase 2 studies with phase 3 studies, and we found the similar results to our original findings. Secondly, there was slight difference in baseline characteristics of enrolled patients in the meta-analysis due to different eligible criteria in the enrolled studies. Thirdly, although combining the major bleeding with the ischemic endpoints into a composite endpoint  has been widely used in contemporary trials 12 receptor inhibitor, ticagrelor. The findings may provide a support for ticagrelor plus aspirin to be an optimal antithrombotic regimen for patients with ACS.New antithrombotic agents are associated with significantly reduced risk of MACE, as well as an increased risk of major bleeding, in comparison with traditional DAPT. Although no significant statistical differences were identified among these new antithrombotic regimes, there was a trend in net clinical benefit favoring the new P2YFigure S1Risk of bias assessment.(TIF)Click here for additional data file.Table S1Major inclusion and exclusion criteria of the included studies. UA =  unstable angina; NSTEMI =  non ST elevated myocardial infarction; ACS =  acute coronary syndrome; STEMI =  ST elevation myocardial infarction; PCI =  percutaneous coronary intervention; CABG =  coronary artery bypass; NYHA =  New York Heart Association.(DOCX)Click here for additional data file.Table S2Medical history and medication of included studies. MI =  myocardial infarction; CABG =  coronary artery bypass; ACEI =  angiotensin converting enzyme inhibitors; ARB =  angiotensin II receptor blocker.(DOCX)Click here for additional data file.Table S3The distribution of probabilities of each treatment being ranked at each of the possible 6 positions. DAPT  =  dual antiplatelet therapy; TIMI  =  thrombolysis in myocardial infarction.(DOCX)Click here for additional data file.Text S1Search strategy (via EMBASE.com).(DOC)Click here for additional data file.


As a leading cause of adult blindness, diabetic retinopathy is a prevalent and profound complication of diabetes. We have previously reported duration-dependent changes in retinal vascular permeability, apoptosis, and mRNA expression with diabetes in a rat model system. The aim of this study was to identify retinal proteomic alterations associated with functional dysregulation of the diabetic retina to better understand diabetic retinopathy pathogenesis and that could be used as surrogate endpoints in preclinical drug testing studies.A multi-modal proteomic approach of antibody (Luminex)-, electrophoresis (DIGE)-, and LC-MS (iTRAQ)-based quantitation methods was used to maximize coverage of the retinal proteome. Transcriptomic profiling through microarray analysis was included to identify additional targets and assess potential regulation of protein expression changes at the mRNA level. The proteomic approaches proved complementary, with limited overlap in proteomic coverage. Alterations in pro-inflammatory, signaling and crystallin family proteins were confirmed by orthogonal methods in multiple independent animal cohorts. In an independent experiment, insulin replacement therapy normalized the expression of some proteins  while other proteins  were only partially normalized and Fgf2 and Crybb2 expression remained elevated.These results expand the understanding of the changes in retinal protein expression occurring with diabetes and their responsiveness to normalization of blood glucose through insulin therapy. These proteins, especially those not normalized by insulin therapy, may also be useful in preclinical drug development studies. Approximately 97% of patients with Type 1 diabetes develop some degree of diabetic retinopathy (DR) within 25 years of diagnosis Research using rat models of diabetes have contributed to the concept of diabetic retinopathy as a progressive neurovascular complication that includes vascular, inflammatory, and neuronal components Reports detailing the retinal proteome in human subjects with DR or animals models of DR are limited. The difficulty in obtaining human retina samples has led to studies using vitreal protein collected from subjects. These studies have provided novel and important insights including the potential role of carbonic anhydrase in vascular leakage The present study used a multi-modal proteomic discovery approach . A numbeFive independent experiments were performed in this study. The rat biometric data are summarized in Of the 59 growth factors, cytokines, and signaling molecules examined in the directed Luminex analysis 46 were observed at detectable levels . Four prLabeled retinal proteins from control and diabetic rats were separated by isoelectric point and molecular weight, producing consistent spot patterns on analytical and preparative gels . 1231 pr368 protein spots, including all spots matched to those with significant differential expression and a selection of other spots, were picked for MS-MS/MS protein identification. 288 proteins spots were confidently identified, including 32 of the 48 protein spots with significant differential expression and a fold change ≥1.1 or ≤0.9 . SixteenIn the quantitative LC-MS/MS analysis with isobaric tags, 455 proteins were confidently detected with at least two peptides identified with greater than the 95% confidence required for accurate quantitation. A total of 27 proteins were significantly regulated  with a fold change filter of ≥1.1 or ≤0.9. Differential expression was distributed equally between down- (14 proteins) and up- (13 proteins) regulated species . Protein2 was >0.989 for all control comparisons and >0.982 for all diabetic comparisons. A total of 1274 changes  were observed, with 583 up-regulated and 691 down-regulated probes as illustrated by a heatmap schematic   . The fulComplementarity of the multi-modal proteomic approach used here was assessed by comparing identities and biophysical properties of unique species detected/identified by technology. Overlap in proteomic coverage was limited , with noPotential reasons for the differential coverage of the proteome between proteomic methods are inherent differences in the techniques that may select for proteins with certain biophysical properties . The general biophysical properties of proteins identified by each proteomic approach were similarly distributed across molecular weight and GRAVY score ranges . In eachTo confirm discovery findings, immunoblot confirmation analysis was performed using two sets of animals, independent from the discovery experiments .To test the effect of restoration of normoglycemia by exogenous insulin on retinal protein and mRNA expression, rats received continuous insulin administration by implantation of subcutaneous pellets for the last 1.5 months of the 3 month diabetic period (Experiment 5). Expression of the 10 proteins confirmed in the previous animal experiments was quantified by immunoblotting. A number of different patterns in protein expression were observed, with some changes not normalized by insulin, others partially normalized and some completely returned to control levels. Fgf2 protein and mRNA expression were not normalized by insulin treatment, with levels remaining significantly elevated compared to controls . InsulinThe results of the combined proteomic, transcriptomic, and confirmation experiments both validate previously reported changes in retinal protein expression with diabetes and identify novel changes. These findings have implications for both our understanding of the pathogenesis of DR and as preclinical drug development biomarkers. Additionally, the combination of discovery approaches used provides comparative insight into commonly used quantitative proteomic techniques.The proteomic profiling and confirmation experiments detailed in this report could be used as the first stages towards developing a panel of protein biomarkers for use in preclinical studies for disease monitoring and evaluation of novel treatment efficacy, similar to previously reported mRNA biomarkers In agreement with previous reports, diabetes induced retinal expression of multiple crystallin isoforms was observed. Crystallin isoforms Cryaa , Crybb2 , Cryab  and Cryba3  were confirmed to be induced after 3 months of diabetes. Interestingly, Cryba3 protein expression was only partially normalized by insulin treatment and Crybb2 protein expression was unaffected.Crystallin proteins are associated with a variety of functions ranging from lens structure to stress response, but little is known about their potential involvement in diabetic retinopathy. Alpha crystallins, which are similar to small heat shock proteins, have been reported to have neuroprotective effects Dbi (diazepam binding inhibitor) and Anxa5 (Annexin A5) were identified as targets of interest in our iTRAQ analysis. Dbi is an insulin-regulated transport molecule with numerous functions and protein interactions ranging from regulation of lipid metabolism Although well-characterized for its utility as a marker of early apoptosis due to its high binding affinity for phosphatidylserine, Anxa5 mediates a variety of cellular functions including adhesion and calcium flux in addition to phosphatidylserine-catalyzed inflammation and apoptosis. This glycoprotein is expressed in vascular cells Our data agree with a previous report of upregulated Fgf2 protein levels after 3 months of diabetes in the Sprague Dawley rat Stat3 activation by diabetes and subsequent blood retinal barrier damage can be prevented by simvastatin treatment Lgals3  is an abundant protein expressed in the nucleus, cytoplasm, mitochondria, and plasma membrane of multiple cell types including inflammatory macrophages Likewise, ceruloplasmin (Cp), an iron binding and metabolizing α2 globulin, was upregulated with diabetes in this work. This protein is a component of the acute phase response, and is induced by inflammation Previously, in focused studies examining specific proteins changes in expression of para-inflammatory and microvascular-related proteins  have been reported in rodent models of DR. Some of these proteins, such as Icam1, were not observed with any of the three proteomic methods used in this study. For other proteins, no changes in expression were observed . Increased Vegf protein expression has been reported in the Sprague Dawley STZ model at after 1 and 6 months of diabetes The overarching finding from the comparison of the methods used in this study is that the different techniques implemented are complementary rather than redundant. Comparison of the biophysical properties of proteins identified by the different methods demonstrated no preferential discrimination of protein molecular weights between iTRAQ and DIGE. However, the distribution of identified iTRAQ proteins was skewed toward hydrophilic proteins while those identified by the DIGE analysis were more hydrophobic. Proteins detected by the Luminex analysis were evenly distributed across molecular weights and hydropathicity. Since Luminex assays are predesigned to select specific proteins, the technical capabilities of this assay are highly dependent on the specific antibodies used. The inclusion of transcriptomic analysis provided novel targets not observed in any of the proteomic analyses. Interestingly, all targets chosen from the transcriptomic analysis were confirmed at both the mRNA and protein levels. In total, the comparison of mRNA and protein expression levels demonstrates that while mRNA changes are not determinative of protein expression changes in many cases they are highly correlated.This study identified a number of retinal protein expression changes with diabetes. Of particular interest for future studies are those proteins that are not normalized (Fgf2 and Crybb2) or only partially normalized  by insulin treatment, as these persistent changes reflect the limitations of insulin to restore the retina to a nondiabetic state. The lack of complete normalization by insulin treatment offers the potential that these proteins may play a role in the ‘metabolic memory’ All animal experimental procedures were approved by and carried out in accordance with the Penn State College of Medicine Institutional Animal Care and Use Committee . Animal surgery and sacrifice was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.All rats were maintained by the Penn State JDRF Animal Models Core in accordance with the Penn State College of Medicine Institutional Animal Care and Use Committee guidelines under specific pathogen-free conditions and monitored by quarterly sentinel testing. Diabetes was induced in male Sprague-Dawley rats   by intraperitoneal streptozotocin as previously described Quantitative two-dimensional in-gel electrophoresis (DIGE) was performed as detailed previously For each protein spot, the ratio of background-subtracted sample-specific signal (Cy3 or Cy5 density) to normalization pool signal (Cy2) was calculated. Only protein spots detected on ≥6/8 analytical gels were included in quantitation analysis. One-way ANOVA (p<0.05) and a 1.1-fold change cutoff were used to determine protein spots significantly regulated with diabetes.Rattus taxonomy of the NCBI database downloaded on Feb 16 2008 . In the final combined MS and MS/MS database searches, identifications required a MASCOT confidence interval ≥95%.MALDI-ToF/ToF mass spectrometry was performed as described previously 2PO4 for LC-SCX separation. SCX separations were performed on a passivated Waters 600E HPLC system, using a PolySULFOETHYL Aspartamide column  prior to reverse phase C18 nanoflow-LC separation performed using a Tempo LC MALDI Spotting System (ABI-MDS/Sciex) and a Chromolith CapRod column (Merck).8-Plex iTRAQ (Applied Biosystems) sample preparation and labeling were performed according to manufacturer's instructions as previously described MALDI target plates were analyzed in a data-dependent manner on an ABI 4800 MALDI TOF-TOF. Both MS and MS/MS default calibrations were performed using the Applied Biosystems Calibration Mixture 1. MS Spectra were acquired from each sample spot and selected peaks were analyzed by MS/MS with collision-induced decay.Rattus subset of the NCBI database downloaded on July 22 2008  with a concatenated with a reversed “decoy” version. Confident protein identifications required a ProteinPilot Unused Score of at least 1.3  and a Local False Discovery Rate estimation with a decoy database of no higher than 5% using PSPEP software Protein identification and quantitation were performed using the Paragon algorithm as implemented in Protein Pilot 2.0 software (ABI/MDS-Sciex). Database searches were carried out against the Retina protein profiling was performed at Rules-Based Medicine  using standard Luminex technology Retina homogenization and RNA isolation was performed by standard Tri Reagent/BCP disruption and phase separation followed by over-night isopropanol precipitation at -20°C, as previously described in vitro transcribed to synthesize cRNA by incubation with T7 10× Reaction buffer, T7 Enzyme mix and Biotin-NTP mix. cRNA was purified according to manufacturer's instructions and quantitated using a NanoDrop ND1000 spectrometer. 750 ng of purified cRNA was prepared for hybridization and incubated with RatRef-12 BeadChips prior to washing and staining with streptavadin-Cy3 stained. BeadChips were then dried and subsequently scanned using a BeadStation BeadArray Reader.Microarray analysis was performed with Illumina RatRef12 microarrays  according to standard procedures After initial quality control of arrays, one array from the diabetic group was removed. GenomeStudio (Illumina) flat files were imported into GeneSpring GX11 (Agilent) software for data analysis. Using the detection p-values initially generated in GenomeStudio, probes were required to have detectable signal  in all arrays for at least one of the two groups in the experiment to be included in the subsequent statistical analyses. This filtering eliminates from further analysis transcripts not reliably detected, while retaining genes potentially expressed in only one group . The full complement of microarray data is available in the Gene Expression Omnibus To determine differential expression, a combination of statistical p value  and fold-change (≥1.2 fold) cutoffs were used in accordance with standards in data analysis −ΔΔCt analysis method were used to quantitate relative amounts of product using β-actin as an endogenous control Quantitative PCR confirmations were performed as described previously 3VO4, 2 protease inhibitor tablets/15 mL). Insoluble material was removed by centrifugation at 10,000×g for 10 minutes at 4°C. Protein concentrations were determined by Pierce BCA reagent. Samples were separated on Criterion 4–20% gradient gels (Bio-Rad) and transferred to either PVDF or LF-PVDF membranes  depending on the manner of subsequent visualization (chemiluminescence or fluorescence). Membranes were incubated with primary and secondary antibodies as described in Soluble retinal protein was isolated from independent sets of animals  and one-way ANOVA with pair-wise Student Newman Keuls post-hoc tests for multiple group comparisons.Coverage and differential expression between proteomic and transcriptomic approaches were evaluated. Protein accession numbers and gene IDs were converted to gene symbols using Ingenuity Pathway Analysis (IPA) software to eliminate potential redundancy and incomplete mapping of identifiers. Proteins with multiple identifications in a method  were compressed to one unique gene symbol. Overlap between Luminex, DIGE, and iTRAQ coverage  was visualized by Venn diagram. Total proteomic coverage  was then compared to transcriptomic coverage. Similarly, Venn diagrams were created to illustrate commonalities and differences in differentially regulated species detected by proteomic and transcriptomic methods.http://www.expasy.org). Molecular weights and grand average of hydropathicity (GRAVY) scores were retrieved for each unique protein, and binned according to size (0 kDa to >150 kDa) and score (<−1.0 to >0.4) for comparison of frequency distributions.The biophysical properties of detected/identified proteins for each proteomic method  were determined. NCBI gi| accession numbers were translated to UniProt/Swiss-Prot accession numbers and imported into the Swiss Institute of Bioinformatics Expert Protein Analysis System (ExPASy) ProtParam tool  and diabetic (n = 4) rats collected after 3 months of hyperglycemia was analyzed by Illumina Rat Ref12 microarray. 11,874 of 22,517 probes on the microarray had detectable signals. The 1,274 probes with significantly different expression between control and diabetic groups , including 583 down-regulated and 691 up-regulated genes, are shown.(TIF)Click here for additional data file.Figure S2Comparison of biophysical properties of identified proteins. The biochemical characteristics of proteins identified by the three complementary proteomic methods are illustrated by frequency plots. (A) Molecular weight distributions of unique species identified in iTRAQ, DIGE and Luminex approaches are depicted as the percent of the total protein coverage for each method. Proteins identified in iTRAQ and DIGE were similarly distributed across a broad molecular weight range, while the directed Luminex approach provided better coverage of smaller (10–30 kDa) species. (B) The relative hydrophobicity/hydrophilicity of proteins identified by the three approaches were compared using grand average of hydropathicity (GRAVY) scores for each protein. All three methods provided broad coverage ranging from hydrophilic (negative score) to hydrophobic (positive score) proteins. A greater percentage of the proteins observed in the iTRAQ analysis were hydrophilic in nature while DIGE or Luminex approaches had a greater percentage of hydrophobic proteins, further demonstrating the complementarity of the three approaches.(TIF)Click here for additional data file.Table S1Primary data from Luminex analysis(XLS)Click here for additional data file.Table S2Primary data from DIGE analysis(XLS)Click here for additional data file.Table S3Primary data from iTRAQ analysis(XLS)Click here for additional data file.Table S4Differentially expressed genes from transcriptomic analysis(XLS)Click here for additional data file.Table S5Primer/probe sets used in qPCR confirmations(DOC)Click here for additional data file.Table S6Antibodies used in immunoblot confirmations(DOC)Click here for additional data file.


Drosophila melanogaster protects against anoxia-reoxygenation (A-R) induced injury.Paraoxonase 1 (PON1) is a protein found associated with high density lipoprotein (HDL), thought to prevent oxidative modification of low-density lipoprotein (LDL). This enzyme has been implicated in lowering the risk of cardiovascular disease. Anoxia-reoxygenation and oxidative stress are important elements in cardiovascular and cerebrovascular disease. However, the role of PON1 in anoxia-reoxygenation or anoxic injury is unclear. We hypothesize that PON1 prevents anoxia-reoxygenation injury. We set out to determine whether PON1 expression in Wild type (WT) and transgenic PON1 flies were exposed to anoxia (100% Nitrogen) for different time intervals (from 1 to 24 hours). After the anoxic period, flies were placed in room air for reoxygenation. Activity and survival of flies was then recorded.Within 5 minutes of anoxia, all flies fell into a stupor state. After reoxygenation, survivor flies resumed activity with some delay. Interestingly, transgenic flies recovered from stupor later than WT. PON1 transgenic flies had a significant survival advantage after A-R stress compared with WT. The protection conferred by PON1 expression was present regardless of the age or dietary restriction. Furthermore, PON1 expression exclusively in CNS conferred protection.Our results support the hypothesis that PON1 has a protective role in anoxia-reoxygenation injury, and its expression in the CNS is sufficient and necessary to provide a 100% survival protection. Paraoxonases (PON) are a family of enzymes whose enzymatic activity is not well understood. There are three known members of the PON family in humans, named PON1, PON2 and PON3. PON1 is a 45 kDa glycoprotein expressed in different tissues including kidney, colon, and the liver and mainly found associated with HDL in the bloodstream which in turn can deliver the protein to various tissues. Epidemiological data shows an association between PON1 activity, systemic oxidative stress and cardiovascular risk in humans Reperfusion of hypoxic/anoxic tissues induces a massive production of reactive oxygen species (ROS) causing important oxidative damage Although most epidemiological studies suggest that PON1 protection against cardiovascular disease is due to its antioxidant effects, little is known about the role of this protein in anoxia-reoxygenation injury and other oxidative-mediated pathologies.Drosophila melanogaster, as flies do not contain the PON gene or any homologs Determining the role of PON1 in different injury models is challenging, as in the mammalian systems there are three PONs, therefore, to avoid the potential of compensatory mechanisms in a PON1 knockout, a triple knockout  is required. One strategy that other investigators have developed to overcome the above challenge is testing the role of PON in an invertebrate model, specifically, Drosophila melanogaster, as a model of AR injury as previously reported by Vigne and colleaguesWe decided to test our hypothesis in model of anoxia-reoxygenation (AR) in We hypothesized that PON1 expression will protect flies from AR injury. To test this hypothesis we conducted a series of experiments where transgenic flies expressing tubulin (WT) and flies expressing PON1 were exposed to AR injury. We demonstrated that PON1 expression confers a survival advantage against AR injury, and specifically PON1 expression in the CNS protects against AR induced mortality.tub promoter was used for the ubiquitous transgenic expression of PON1 (3). The neuron specific Elav driver and gut driver were used instead of tub promoter to achieve expression of PON1 exclusively in the CNS and gut, respectively.All flies were maintained and cultured on standard yeast-agar-sucrose-cornmeal medium. Flies were bred and tested at 25°C. The binary GAL4-UAS system and Drosophila stocks were reared on standard cornmeal-agar-molasses medium at room temperature (21–25°C). The tubulin-Gal4 transgenic line  was obtained from the Bloomington stock center .+/tub and PON1 transgenic flies were placed in vials  inside a plastic chamber see . 100% NiIn a subset of flaks exposed to 1h of anoxia, flasks were examined every 5 min after exposure to rom air and number of active flies were recorded. All flies that remained motionless at the bottom of the flask were considered inactive, and all flies that were moving or at the wall of the flask were considered active.Guts from flies were dissected and stained using Dihydroethidium (DHE), a superoxide indicator. Guts were then observed under fluorescence microscope.The entire gastrointestinal tract was removed intact from females for staining and further processing by grasping below the head and at the tip of the abdomen with fine forceps and gently pulling.Female adult fly guts  were dissected as previously described under 1× Schneider's Drosophila Media . The guts were then stained with DHE using a previously described method 2−) levels in whole flies, a lucigenin based ROS assay was performed on fly lysate as previously describedTo assess superoxide (O1118 strain was used for transgenic injections. P element-mediated transformation and subsequent fly crosses were performed following standard techniques The y wDrosophila melanogaster embryos (y w1118) using standard techniques . y w1118; ; tub-GAL4/TM3,Sb virgin females were crossed to y w1118; ; UAS-PON1 males to obtain the experimental lines y w1118; ; UAS-PON1/tub-GAL4, which is referred to as UAS-PON1/tub-GAL4. Control flies  were obtained by crossing w1118 males with y w1118; tub-GAL4/TM3, Sb virgin females and are denoted as tub/+. F1 progeny were tested for expression of PON1 by performing western blots using an antibody to PON1 . The neuron specific Elav driver and gut driver were used instead of tubulin driver to achieve expression of PON1 exclusively in the CNS and gut, respectively.The binary GAL4-UAS system and tubulin (tub) promoter were used for the ubiquitous transgenic expression of PON1. Human PON1 cDNA was cloned into the pUAST plasmid and subsequently injected into All experiments were performed at least 3 times separately with 20 flies per condition per experiment. Statistical analyses were performed in Graphpad Prism 5. Unpaired t-test, paired t-test and Mann-Whitney U test were performed as indicated. A p-value<0.05 was considered statistically significant Drosophila after AR exposure Drosophila melanogaster protected against AR induced mortality. Wild type +/tub flies (WT) and transgenic flies expressing Paraoxonase 1 (PON1) were exposed to different periods of anoxia . After anoxic period, flies were placed at room air for reoxygenation. Survival was recorded 48h after AR stress. Oxygen levels of <1% were achieved after 5 min of injecting 100% Nitrogen into the chamber. Within one minute of achieving this level of anoxia, flies stop flying and lay at the bottom of the vial, motionless. They remained stuporous throughout the anoxic time. After reoxygenation, flies became active from the anoxia-induced stupor and start flying.Vinge and colleagues have reported that mortality increases in As shown in One common polymorphism is at position 192, were a glutamine (192Q) is replaced by an arginine (192R). These alloenzymes have differences in their paraoxonase and arylesterase activity Since the mechanisms of AR injury are mediated by ROS, guts from flies of each variant were stained using Dihydroethidium, a superoxide indicator. Transgenic flies have a slight difference in food intake of about 10% (data not shown), and since dietary restriction increases survival after AR We also investigated whether aging will influence AR induced mortality. Flies were paired to ages ranging from one to four days and exposed to three hours of anoxia. 48h survival was registered. As shown in In order to determine whether regional expression of PON1 would confer survival advantage against AR, we developed two strains of flies with organ specific promoters that expressed PON1 protein only in the central nervous system (CNS) or in the Gut. In a similar manner of previous experiments, flies were exposed to three hours of anoxia and survival was registered 48 hours later. PON1 CNS transgenic flies have higher survival than +/tub CNS flies, similar to transgenic flies with a universal expression of PON1, while flies expressing PON1 in the gut survived similarly to WT flies .Oxidative stress is an essential element of ischemia reperfusion injury, where perfusion of anoxic tissues produces an increase of ROS production beyond the antioxidant capability of the organism causing oxidative damage of the cell membrane by lipid peroxidation. This is the hallmark of multiple pathological processes including myocardial infraction, stroke, and transplant. During the last decade, paraoxonase enzymes have been shown to be an important element in the physiopathology of oxidative stress. It is well known that PON1 prevents the oxidation of LDL, and it is necessary for the antioxidant properties of HDL on LDL PON1 activity has been implicated in having a protective effect in ischemia colitis Drosophila melanogaster confers a survival advantage against AR injury. Our results also show that flies expressing PON have lower levels of superoxide at baseline, suggesting that PON1 has antioxidant effects, protecting flies from oxidative stress after anoxia-reoxygenation. PON activity not only prevents oxidative stress that promotes atherosclerotic plaque formation but also may be relevant in more rapid burst of oxidative stress as in AR injury. More recently, it has been reported that PON1 forms a complex with HDL and myeloperoxidase, an important oxidative enzyme, partially inhibiting myeloperoxidase activity Anoxia-reoxygenation, as well as ischemia reperfusion injury, is mainly mediated by the overproduction of ROS. Our results show that transgenic expression of PON1 in Transgenic flies had a delayed recovery from AR-induced stupor. This effect can potentially explain some of the protection conferred by PON1 expression, as flies are extremely resistant to hypoxia and anoxia, and they respond to low oxygen and anoxia by decreasing their activity, and oxygen utilization Also, our results suggest that protection is due to PON1 expression in the brain; as expression of PON1 in flies was sufficient and necessary for protection against AR injury in the same proportion that universal expression of PON1. In humans, PON1 mRNA has been found mainly in kidney, colon and liver Epidemiological data shows a clear association of ischemic stroke incidence, survival and PON activity, where subjects with 192R have a higher risk for ischemic stroke than 192Q PON1 is only one of three enzymes in the family of PON in humans. PON are promiscuous enzymes and its native enzymatic function is not clear. PON1 is the only one with paraoxonase activity, while all members of the family have different degree of lactonases and acetylesterase activity, whose relevance for antioxidant effect remains to be determined. While PON1 is expressed mainly in the liver and found associated to HDL, PON2 is ubiquitously expressed and its role in oxidative damage by anoxia reoxygenation is potentially more relevant and it still remains to be tested. Giordano G and colleagues reported that PON2 is expressed in the brain and protects CNS cells against oxidative stress and confers gender-dependent susceptibility to such stress