Development of cardiac allograft vasculopathy represents the major determinant of long-term survival in patients after heart transplantation. Due to graft denervation, these patients seldom present with classic symptoms of angina pectoris, and the first clinical presentations are progressive heart failure or sudden cardiac death. Although coronary angiography remains the routine technique for coronary artery disease detection, it is not sensitive enough for screening purposes. This is especially the case in the first year after transplantation when diffuse and concentric vascular changes can be easily detected only by intravascular ultrasound. The treatment of the established vasculopathy is disappointing, so the primary effort should be directed toward early prevention and diagnosis. Due to diffuse vascular changes, revascularization procedures are restricted only to a relatively small proportion of patients with favorable coronary anatomy. Percutaneous coronary intervention is preferred over surgical revascularization since it leads to better acute results and patient survival. Although there is no proven long-term advantage of drug-eluting stents for the treatment of in-stent restenosis, they are preferred over bare-metal stents. Severe vasculopathy has a poor prognosis and the only definitive treatment is retransplantation. This article reviews the present knowledge on the pathogenesis, diagnosis, treatment, and prognosis of cardiac allograft vasculopathy. The leading causes of death during the first three years after heart transplantation are nonspecific graft failure and infections. Nonspecific graft failure may be caused by chronic graft rejection, while acute graft rejection accounts for no more than 11% of mortality. The major determinants of patient survival after three years are malignancy and cardiac allograft vasculopathy (CAV), also known as transplant coronary artery disease or cardiac transplant vasculopathy. It is detected by coronary angiography in 8% of patients by year 1, 30% by year 5, and 50% by year 10 after transplantation . The vas0 (no detectable angiographic lesions) = no vasculopathy; CAV1 (mild disease) = left main stenosis <50% or primary vessel stenosis <70% (including right coronary artery – RCA) or any branch stenosis <70%, without graft dysfunction; CAV2 (moderate disease) = left main stenosis <50% or single primary vessel stenosis >70% or isolated branch stenosis >70% in 2 systems, without graft dysfunction; CAV3 (severe disease) = left main stenosis ≥50% or stenosis >70% in two or more primary vessels or isolated branch stenosis >70% in three systems; or ISHLT CAV1 or ISHLT CAV2 with signs of graft dysfunction. Allograft dysfunction is defined as left ventricular ejection fraction ≤45% or evidence of significant restrictive pathology either on echocardiographic exam  or right heart catheterization . In this nomenclature, primary vessels stand for the proximal and middle thirds of the left anterior descending artery, the left circumflex, the ramus intermedius, and the dominant or co-dominant RCA. Branch vessels denote distal thirds of the primary vessels or large septal, diagonal, and obtuse marginal branch or any portion of a non-dominant RCA  proposed a grading system based on the combination of angiographic finding and graft function defined either by ultrasound or invasive hemodynamic measurement : CAV0  is a strong predictor of CAV development after heart transplantation . MultiplThe graft endothelium represents the borderline between the donor’s and recipient’s immune systems and plays a critical role in the pathophysiology of CAV. The increased expression of major histocompatibility complex (MHC) class I alloantigens on coronary endothelial cells of the transplant heart are directly recognized by CD8+ T-cells, resulting in the secretion of cytokines with further activation of endothelial cells. The activated endothelial cells express increased levels of MHC class II antigens, which later activate CD4+ T-cells. This can explain the predominance of CD8+ T-cells in the early vasculopathic lesions and the increased proportion of CD4+ T-cells in the advanced stage of the disease . EndotheMultiple cytokines with proliferative effects on coronary smooth muscle cells have been detected within vasculopathic lesions: interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), platelet derived growth factor, insulin-like growth factor-I, macrophage chemoattractant protein-1 (MCP-1), fibroblastic growth factor, vascular endothelial growth factor, transforming growth factor-alpha, and TGF-beta-1 -34. InteNon-immunologic factors associated with increased risk of CAV include age, sex, recipient body mass, diabetes mellitus, smoking, dyslipidemia, donor history of arterial hypertension, recipient history of previous coronary artery disease, cytomegalovirus (CMV) infections, defective fibrinolysis, polymorphism in angiotensin converting enzyme gene, early graft dysfunction due to preservation damage, ischemia-reperfusion injury, as well as the etiology of brain death of the donor . Non-immWhile older age and male sex of the donors are associated with higher risk, older age of the recipient is associated with lower risk for CAV ,38,39. CThe relation between CAV development and CMV infection can be explained by stimulation of immune response in the vessel wall via induced expression of endothelial MHC antigens and direct endothelial damage resulting in dysregulation of the NO pathway, activation of coagulation cascade, cytokine secretion, and vascular remodeling with vessel lumen loss ,44.Glucose intolerance and insulin resistance are associated with higher likelihood of CAV and increased mortality ,46. MetaThe paradoxical constriction of coronary arteries in response to acetylcholine is a sign of endothelial dysfunction and is predictive of vasculopathy ,54, whilThe complex interactions between the immunologic and non-immunologic factors cause endothelial damage with consequent endothelial activation and dysfunction. Endothelial activation stimulates expression of adhesion molecules and cytokine secretion, which further promote cell invasion, smooth muscle cells growth, and vascular matrix deposition, finally resulting in typical morphological vascular changes. Derangement of endothelial repair mechanism in transplant patients may also play a role. The level of circulating human endothelial progenitor cells (EPCs) was found to be significantly decreased in patients with CAV compared with matched patients without evidence of the disease. This may be explained by increased consumption of circulating pool of EPCs at the sites of graft endothelial injury. However, mobilization and survival of circulating EPCs was found to be suppressed by inflammation .Due to the graft denervation, CAV will unlikely be diagnosed based on typical symptoms of angina pectoris. However, it should be suspected during routine echocardiographic examination if signs of graft dysfunction, such as regional wall abnormalities and/or restrictive diastolic dysfunction, are found. When it becomes symptomatic, CAV is usually at an advanced stage and myocardial damage is irreversible. For this reason, it is necessary to perform screening measures to improve early diagnosis.Invasive screening methods include coronary angiography and intravascular ultrasound (IVUS). They cause some discomfort to the patient, involve the risk of renal damage from contrast agent application, and are resource consuming. Other methods include analysis of microvascular changes on routine endomyocardial biopsies and noninvasive methods like stress echocardiography, CT coronary angiography and measurement of serum biomarkers.Coronary angiography is used to establish the diagnosis of CAV in most transplant centers. Depending on the center, the baseline angiography is commonly performed several weeks after the transplantation, while the following ones are performed annually or biannually. In our center, it is routinely performed biannually. However, coronary angiography is performed annually or even more frequently after the diagnosis of CAV has been established or if there are signs of new-onset graft dysfunction on echocardiographic exam. Coronary angiography can identify luminal narrowing as well as slow contrast filling of the distal coronary vessels, suggesting the involvement of small coronary vessels and microcirculation. As coronary spasm may mimic concentric vessel narrowing in vasculopathy, intracoronary nitroglycerine should be applied before contrast injection in order to avoid false CAV diagnosis. Although angiography remains the routine technique for coronary artery disease detection, it is not sensitive enough for CAV detection due to diffuse, longitudinal, and concentric vascular changes. The positive predictive power of coronary angiography compared to IVUS as the gold standard is only 44% . ConsequUnlike angiography, IVUS can detect the abnormal concentric intimal thickening and is the most sensitive tool for CAV detection. The most rapid rate of intimal thickening occurs during the first posttransplant year . An incrNoninvasive tests include conventional electrocardiogram stress test, dobutamine stress echocardiography, coronary flow reserve (CFR) assessment by contrast-enhanced echocardiography, stress myocardial perfusion scintigraphy, positron emission tomography (PET) scanning, computed tomography (CT) angiography as well as serum biomarkers testing. These studies are less sensitive, but as they are not invasive and are generally less expensive, may serve as adjunctive methods of screening for myocardial ischemia . The besContrast enhanced CFR has been evaluated by clinical studies as a method of screening, but it requires high-quality imaging that is often impossible to perform due to unfavorable acoustic window in transplanted patients . AlthougBiomarkers like CRP or N-terminal pro-brain natriuretic peptide (NT-proBNP) were predictive of mortality but not of CAV development . Serum NImmunohistochemistry evidence of endothelial activation in the arteries and arterioles of endomyocardial specimens taken during the first three months after transplantation precede morphological changes in vasculopathy . StenotiCAV is a slowly progressive disease, though it may progress rapidly and result in graft failure, arrhythmias, or sudden cardiac death (SCD). Because of heart denervation, graft ischemia usually progresses without classic symptoms and SCD may be the first manifestation of vasculopathy. SCD may be prevented by cardioverter defibrillator implantation, though not entirely, as some of these deaths result from instantaneous and fatal heart failure . SignifiBecause CAV has substantially poor prognosis when it becomes symptomatic, the primary effort is directed to early prevention, detection, and initiation of treatment. Only two types of drugs were found to provide significant protection from CAV: statins and mammalian target of rapamycin (mTOR) inhibitors.Statins are considered mandatory in transplant patients because they improve patient survival, reduce both the incidence and severity of vasculopathy, as well as the incidence of graft rejection . They arSirolimus (SRL) and everolimus (EVL), also known as mTOR or proliferation signal inhibitors (PSI), inhibit T-cell and B-cell proliferation driven mainly by interleukins. Eisen et al showed superiority of EVL over azathioprine for the prevention of CAV in de novo heart transplant patients already on cyclosporine and corticosteroid therapy . After 1P = 0.056). It also showed significantly lower incidence of retransplantation or death , while only few patients are candidates for coronary artery bypass grafting (CABG). Revascularization procedures are associated with poor long-term results and are considered palliative due to the diffuse and progressive nature of vascular changes. Heart failure medications should be introduced with the signs of graft failure, while in patients with severe disease, the only option remains to be retransplantation.Short-term enhancement of immunosuppressive therapy may slow down the progression of CAV (three days of methylprednisolone plus antithymocite globulin). This effect is more pronounced if applied early, ie, within the first posttransplant year. However, due to significant risk of infections and malignant diseases, this approach is not adopted in routine practice .There is evidence that both EVL and SRL prevent CAV development, but established vasculopathy is treated only with SRL . A studyRaichlin et al replaced CNI with SRL as a primary immunosuppressive drug in patients with vasculopathy and/or renal impairment . The secPCI is considered palliative because it does not influence the progressive nature of CAV. No studies tested the effect of PCI on the graft and patient survival. Optimal candidates for PCI are patients with focal and one vessel disease , who repBecause of the diffuse nature of transplant vasculopathy and high perioperative mortality, surgical revascularization is an option in a very small number of patients with CAV. Coronary bypass grafting is associated with high perioperative mortality (36%) and only few patients with CAV are candidates for this type of revascularization .According to the present guidelines for SCD prevention in heart failure patients, implantable cardioverter defibrillators are used for the prevention of SCD in patients with severe CAV and reduced systolic function . HoweverAs present CAV prevention and treatment modalities are very limited, there is a need for novel strategies. Alloimmune response may be prevented by induction of allograft tolerance by targeting local or circulating dendritic cells or by modulating T-cells co-stimulation pathways . Graft s


Syphilis co-infection risk has increased substantially among HIV-infected men who have sex with men (MSM). Frequent screening for syphilis and treatment of men who test positive might be a practical means of controlling the risk of infection and disease sequelae in this population.We evaluated the cost-effectiveness of strategies that increased the frequency and population coverage of syphilis screening in HIV-infected MSM receiving HIV care, relative to current standard of care.We developed a state-transition microsimulation model of syphilis natural history and medical care in HIV-infected MSM receiving care for HIV. We performed Monte Carlo simulations using input data derived from a large observational cohort in Ontario, Canada, and from published biomedical literature. Simulations compared usual care  to different combinations of more frequent (3- or 6-monthly) screening and higher coverage (100% screened). We estimated expected disease-specific outcomes, quality-adjusted survival, costs, and cost-effectiveness associated with each strategy from the perspective of a public health care payer.Usual care was more costly and less effective than strategies with more frequent or higher coverage screening. Higher coverage strategies (with screening frequency of 3 or 6 months) were expected to be cost-effective based on usually cited willingness-to-pay thresholds. These findings were robust in the face of probabilistic sensitivity analyses, alternate cost-effectiveness thresholds, and alternate assumptions about duration of risk, program characteristics, and management of underlying HIV.We project that higher coverage and more frequent syphilis screening of HIV-infected MSM would be a highly cost-effective health intervention, with many potentially viable screening strategies projected to both save costs and improve health when compared to usual care. The baseline requirement for regular blood testing in this group  makes intensification of syphilis screening appear readily practicable. Major urban centers in the developed world have witnessed a dramatic re-emergence of syphilis in recent years, with the epidemic concentrated among men who have sex with men (MSM) and HIV-infected individuals The disproportionate and increasing burden of syphilis in MSM and HIV-infected individuals has led to calls for novel syphilis control strategies targeting these populations The current syphilis testing algorithm in Ontario, Canada includes a treponemal screening test followed by a confirmatory non-treponemal test . Although the treponemal test is more sensitive than the non-treponemal test for detecting early infection, once the test is positive, it often remains so for life, resulting in low specificity in individuals with previously treated syphilis Given the current burden of syphilis in HIV-positive MSM, it is unclear if the benefits associated with preventing new cases of neurosyphilis and tertiary syphilis via more frequent screening would outweigh the costs and health consequences associated with unnecessary treatment. Our objective was to evaluate whether strategies of enhanced (more frequent and/or higher coverage) routine syphilis screening in HIV-positive MSM receiving HIV medical care would be effective and economically attractive, relative to the current standard of care.We developed an individual-level state-transition (“microsimulation”) model In our base case, we compared usual care to more frequent screening, and screening with higher population coverage. For the usual care strategy, 57% of the population received syphilis screening annually Our Markov model consisted of mutually exclusive and collectively exhaustive health states: uninfected; primary syphilis, secondary syphilis and early latent syphilis (“early syphilis”); late latent syphilis; neurosyphilis; tertiary syphilis; treated late syphilis; and death Figure 2In the base case, incidence of syphilis infection was based on estimated rates observed in the OCS for MSM, with infection rates higher in men with prior syphilis In all strategies, syphilis infection was managed according to the current Canadian guidelines with early syphilis treated with a single dose of benzathine penicillin, late syphilis treated with three once-weekly doses, and neurosyphilis treated with high dose intravenous penicillin Costs are presented in We performed cost-effectiveness analyses from a public Canadian health care payer perspective, which approximates a societal perspective in a country like Canada with publicly funded health services, but ignores patient time and travel costs. Cost effectiveness of each strategy was estimated as the incremental cost per QALY gained relative to the next most expensive strategy. Since the effects of untreated syphilis often takes decades to manifest, we used a lifetime time horizon. A base case discount rate of 5% was applied to future costs and outcomes To ensure that our model was reproducing observed trends in reported syphilis infection, we compared the expected incidence of diagnosed early neurosyphilis in our modeled cohort under the usual care scenario to estimated rates of neurosyphilis between 2008 and 2012 In the absence of enhanced (more frequent and/or higher coverage) screening, approximately 2.9 per 10,000 men were projected to be diagnosed with neurosyphilis annually, which is within the range of rates of 0.7–9.1 (mean 4.5) per 10,000 HIV-infected males in Toronto between 2008–2012. Compared to usual care, higher (100%) coverage 3-monthly screening was projected to reduce incidence of diagnosed early neurosyphilis , while increasing the diagnosis of infectious syphilis (from 3.7 to 4.7 per 100 person-years). Unnecessary treatment of uninfected men due to false positive test results also increased (from 0.5 to 3.5 per 100 person-years) Figure 3Compared to the usual care  strategy, higher (100%) coverage 3-monthly screening was projected to cost more ($98.69) and be more effective (0.015 QALY) Table 2.To capture second-order uncertainty, we performed 1000 simulated trials, each with 1000 participants randomly assigned to each screening strategy. The preferred strategy varied depended on the willingness-to-pay threshold Figure 4As uncertainty in model parameters was incorporated into second order Monte Carlo simulations, we performed additional sensitivity analyses using alternate assumptions about disease epidemiology, natural history and programmatic features Table 3.In other sensitivity analyses, higher coverage strategies dominated usual coverage strategies  but the optimal screening interval was sensitive to input parameters. In our base case we assumed that treatment with combination ART would begin when CD4 counts reached 500 cells/mL; when we assumed ART at a CD4 count of 350 cells/mL, higher coverage every 3 months became costly relative to every 6 months  but might still be considered cost-effective in high income countries We project that a universal syphilis screening program in HIV-infected MSM under medical care has the potential to improve health and save costs, relative to usual care. However, increased screening may result in unnecessary treatment of men with false positive tests and lead to excess adverse events associated with testing and treatment . Although previous studies have examined the disease dynamic impacts of screening frequency, Perhaps most importantly, we project that both increases in test frequency and coverage for MSM with HIV infection would increase effectiveness relative to current standard of care, and that all more-intense screening regimens  would decrease, rather than increase, net healthcare costs due to aversion of downstream sequelae of untreated syphilis infections. Although the projected increase in effectiveness observed with enhanced (higher coverage and/or increased frequency) screening was small compared to usual care, this reflects the fact that the major health consequences of syphilis occur years, and often decades, after initial infection We restricted our analysis to HIV-positive MSM because surveillance data show this to be the population at greatest risk of syphilis in much of North America Our study is subject to several limitations. Our estimates of syphilis acquisition risk are based on HIV-positive MSM in Ontario Lastly, we used a traditional static risk Markov model to evaluate the health-economic attractiveness of syphilis screening in MSM. Such models are limited in their ability to capture the indirect effects of disease treatment and prevention that are characteristic of communicable disease control programs In summary, we have shown that when rates of syphilis acquisition are high, implementing routine syphilis screening in MSM currently under medical care for HIV is expected to be a highly cost-effective intervention. This strategy has been implemented in Australia and has been demonstrated to increase the detection of early asymptomatic syphilis


Introduction. Although residents frequently lead end-of-life (EOL) discussions in the intensive care unit (ICU), training in EOL care during residency has been required only recently, and few educational interventions target EOL communication in the ICU. This study evaluated a simulation-based intervention designed to improve resident EOL communication skills with families in the ICU. Methods. Thirty-four second-year internal medicine residents at a large urban teaching hospital participated in small group sessions with faculty trained in the “VitalTalk” method. A Likert-type scale questionnaire measured self-assessed preparedness before, immediately following, and approximately 9 months after intervention. Data were analyzed using Wilcoxon rank-sum analysis. Results. Self-assessed preparedness significantly improved for all categories surveyed , including discussing bad news , conducting a family conference , discussing treatment options , discussing discontinuing ICU treatments , and expressing empathy . Improvement persisted at follow-up for all items except “expressing empathy.” Residents rated the educational quality highly. Conclusion. This study provides evidence that brief simulation-based interventions can produce lasting improvements in residents' confidence to discuss EOL care with family members of patients in the ICU. Conducting end-of-life (EOL) discussions with patients' families is an essential skill for residents in the intensive care unit (ICU). Approximately 22% of all US deaths occur in the ICU , and 75%Although the Accreditation Council for Graduate Medical Education has mandated training in EOL care for internal medicine residents , a natioThis study used a pretest-posttest design to evaluate the effectiveness of a brief simulation-based training program intended to improve residents' EOL discussions with families in the ICU. The course was modeled on prior simulation-based interventions , 18, 19.All of the 35 residents in internal medicine at our academic medical center preparing to start postgraduate year 2 in June 2013 were eligible to participate in the study. The EOL educational intervention was a required educational activity for eligible participants and implemented along with standard clinical rotations. This study was deemed exempt from informed consent per the health system's institutional review board.http://www.VitalTalk.org) to learn to facilitate small-group ICU communication skills sessions using simulated patient family members. Residents participated in three 90-minute sessions in groups of 5 to 7 under the guidance of a primary faculty facilitator and a cofacilitator. Simulations were repeated until all eligible residents participated.Two faculty members attended two 3-day workshops , and transitioning goals of care . The eduA survey on attitudes toward EOL care was administered prior to the initial small group session for baseline measurement, immediately following the educational intervention, and approximately 9 months later. The survey contained 6 questions measuring self-assessed preparedness to deliver various aspects of EOL care using a 5-point Likert scale. Data on demographic information, future career plans, prior structured educational experiences, and prior bedside teaching relevant to EOL communication skills were also collected. A question regarding previous formal training in discussing a clinical trial with critically ill patients in the ICU, to which few residents were expected to have been exposed, served as a measure of internal validity.The primary outcome of the study was change in resident self-assessed preparedness to discuss EOL care with patients' families. Secondary outcomes included assessment of the structured and bedside EOL education received by residents during medical school and the first year of residency, as well as satisfaction with the course.Descriptive statistics were used to analyze demographic information. The Friedman test and Wilcoxon rank-sum tests were used to compare results of the assessments. All analyses were performed using SAS version 9.4 . Data were deidentified after completion of all analyses; data could not be immediately deidentified because subject identifiers were required for pairing pretest and follow-up data points. No identifiable participant data were shared with residents' supervisors.Of the 35 internal medicine residents eligible for the study, 34 attended the workshop. One was not able to attend due to scheduling conflict. A total of 33 completed both the presurvey and immediate postintervention survey and 15 residents completed the 9-month follow-up survey. Most residents reported previous structured teaching on discussing code status (88%), expressing empathy (88%), and giving bad news to a family (82%). Less structured teaching was reported for discussing discontinuing intensive care treatments (36%) or discussing religion or spiritual issues with patients and families (24%). Few reported discussing a clinical trial for a patient in the ICU (3%). The same pattern prevailed for previous bedside teaching . Most rep < 0.01), conducting a family conference , expressing empathy , discussing treatment options and palliative care with families of critically ill patients , responding to families who deny the seriousness of their loved one's illness , and discussing discontinuing intensive care treatments . The Friedman test revealed significant differences between the 3 assessments. Wilcoxon signed-rank tests demonstrated that scores improved to initial baseline for all items at the 9-month follow-up (p = 0.12).Self-assessed preparedness significantly increased immediately following the intervention for all items surveyed . Mean scollow-up  except fThe intervention was favorably evaluated by the residents, including the utility of the interactive didactics, use of actors for skill practice, and readability and utility of the presession modules . AdditioThis study showed that resident self-assessed preparedness for EOL communication with families in the ICU significantly improved after a brief didactic and simulation-based intervention. Scores remained significantly higher at 9-month follow-up compared to the preintervention baseline, and the course was evaluated favorably by residents. These results were observed in a sample of residents who reported considerable previous training in EOL communication during medical school and their intern year.Our findings are concordant with previous work using a similar curriculum with critical care fellows in the ICU  and non-Lorin et al. reported improvement in objective evaluations of EOL discussion skills among medical students on their ICU clerkship following a brief intervention consisting of a lecture, discussions, and interaction with a simulated patient . Han et We utilized the VitalTalk curriculum for its distinction from other interventions in the use of simulated family members who were not limited to scripted responses, the group setting with real-time peer feedback, and the ability to “rewind” and “replay” segments of the interaction based on feedback. These unique features may have contributed to its observed efficacy in our study sample, although the relative value of each of these attributes merits further investigation.Additional strengths of the VitalTalk program include its “train-the-trainer” model that leverages the skills of national experts to improve the care provided by a large number of trainees, its standardization, and its availability to faculty across the country. Of note, the intervention required only 90 minutes of residents' time. Previous work has shown that time away from clinical responsibilities is a significant impediment to education focused on communication skills .While these results are encouraging, they must be interpreted in light of several limitations. No objective measures of communication quality were available for analysis, and self-reported outcomes have been shown to correlate poorly with patient- or family-reported outcomes . Since aThe present findings have implications for current practice. Though training in EOL care is currently mandated by the Accreditation Council for Graduate Medical Education, the methods currently employed to satisfy this requirement remain inadequate and heterogeneous. Standardization of these curricula might more reliably improve patient care than the current system. That empathy alone did not significantly increase from baseline to follow-up also has implications for graduate medical education. It is possible that immediate improvements in self-assessed empathy were offset over time by the well-documented “empathy decline” among residents during their training .Moving forward, several unanswered questions remain regarding the optimal strategy to improve EOL care provided by residents. Future work may involve comparison of VitalTalk with other published interventions as well as assessing patient and family satisfaction along with objective measures of quality care, such as ICU length of stay and timing of enrollment in hospice. Additionally, strategies specific to the ICU should be integrated into broader systems designed to improve EOL care across the continuum of medical acuity, including outpatient clinics, the emergency department, and non-ICU inpatient units.Implementation of the VitalTalk program at a large urban teaching hospital significantly improved resident self-assessed preparedness for EOL communication with family members in an ICU setting. Interventions that improve EOL care are possible without unduly burdensome time commitments and can be perceived as valuable by residents.


Z and energy (HZE) particle radiation is a function of the relative contribution or extent of NTE or due to unique NTE. We addressed this problem using a radiation/genetic mammary chimera mouse model of breast cancer. Our experiments suggest that NTE promote more aggressive cancers, as evidenced by increased growth rate, transcriptomic signatures, and metastasis, and that HZE particle NTE are more effective than reference γ-radiation. Emerging evidence suggest that HZE irradiation dampens antitumor immunity. These studies raise concern that HZE radiation exposure not only increases the likelihood of developing cancer but also could promote progression to more aggressive cancer with a greater risk of mortality.Clear mechanistic understanding of the biological processes elicited by radiation that increase cancer risk can be used to inform prediction of health consequences of medical uses, such as radiotherapy, or occupational exposures, such as those of astronauts during deep space travel. Here, we review the current concepts of carcinogenesis as a multicellular process during which transformed cells escape normal tissue controls, including the immune system, and establish a tumor microenvironment. We discuss the contribution of two broad classes of radiation effects that may increase cancer: radiation targeted effects that occur as a result of direct energy deposition, e.g., DNA damage, and non-targeted effects (NTE) that result from changes in cell signaling, e.g., genomic instability. It is unknown whether the potentially greater carcinogenic effect of high  Z) and energy (HZE) charged particles that are characterized by high linear energy transfer (LET) along the particle track, i.e., densely ionizing, in contrast to most terrestrial low LET radiations that are sparsely ionizing. The unique pattern of energy deposition incurred by HZE particle traversal is of often the primary focus in evaluating the biological effects of the galactic cosmic radiation on astronauts  for densely ionizing HZE particles is several-to-many fold greater than sparsely ionizing radiation. HZE particles have a high RBE for many biological end points  or altered phenotype; second, the designation of so-called “bystander” radiation effects, in which non-irradiated cells respond to signaling by irradiated cells . NTE canThe crucial question is to determine under what conditions and to what extent NTE contribute to human health risks. Recent experimental studies of radiation carcinogenesis following low- and high LET radiation exposures are concerned with how complex organismal responses to radiation interact across levels of organization and time scales to impede or promote malignant processes . MechaniThe understanding of cancer as a result of systemic failure, in which many cells other than those with oncogenic mutations/alterations determine the frequency and characteristics of clinical cancer, underscores tissue dysfunction, in which cancer cells are highly intertwined with the microenvironment , 12. BotRecent studies demonstrate that host biology is altered even before cancer is evident. A systems biology approach by Hanash and colleagues characterized the plasma proteome response in the inducible HER2/neu mouse model of breast cancer during tumor induction, progression, and regression. Mass spectrometry data derived from approximately 1.6 million spectra identified protein networks associated with tumor development. Some networks were derived from the tumor microenvironment and some from tumor cell secreted or shed proteins. The observed alterations developed prior to cancer detection, increased progressively with tumor growth, and reverted toward baseline with tumor regression. Importantly, these findings were mirrored with findings resulting from in-depth profiling of circulating proteins using prediagnostic plasma samples from women who participated in the Women’s Health Initiative study and who subsequently developed breast cancer –27.in vivo and neoplastic transformation in culture of irradiated tracheal epithelial cells is inversely correlated to the number of cells seeded  that support tumor growth. Indeed, the pre-metastatic niche, first described by Lyden and colleagues, pre-dates and facilitates metastatic disease . Other oncogenes, such as Bcl2, inhibit apoptosis leading to necrotic tumor cell death and release of damage-associated molecular pattern molecules that activate innate immune cells via toll-like receptors  of 350 MeV/amu 26Si particles and compared to contemporaneous γ-irradiated (100 cGy) and sham-irradiated mice  and non-cell-autonomous (inflammation and innate immunity) components that were differentially expressed in the susceptible animals. Interestingly, the highly susceptible mice exhibited increased levels of anti-inflammatory genes within the inflammation associated network, leading to the conclusion that chronic and acute inflammation are, respectively, tumor-promoting versus suppressive . Engineered loss or misregulation of p53 increases the detection sensitivity. Radiation induces the same spectrum of tumors in p53-deficient mice that lack one or both p53 alleles; however, the survival time is dramatically reduced after a single exposure to ionizing radiation . Likewistibility –110. Thetibility , 111.Trp53 null tumor latency, increased frequency of tumor formation at a year posttransplantation, and that tumors arising in irradiated hosts grew more rapidly  at 18 months posttransplantation compared to sham-irradiated F1Bx hosts. The revelation that NTE delay rather than accelerate mammary cancers in genetically diverse hosts underscores the outcome of radiation exposure in terms of carcinogenesis depends of genetic background.Recent experiments focus on the genetic contribution to NTE using the mammary chimera . Radiore rapidly . ConsistTrp53 null mammary tumor latency of the radioresistant SPRET/EiJ genome using genome-wide genotyping. Only two loci were associated with tumor latency in sham-irradiated mice. Tumors in mice homozygous for the BALB/c allele at loci on chromosomes 2 and 14 appeared with a significantly shorter latency than those mice, in which one allele was from BALB/c and the other from SPRET/EiJ at these loci. Interestingly, neither of the loci affected latency in irradiated hosts. In contrast, 15 genetic loci were associated with tumor latency in irradiated mice, 11 alleles confer resistance to tumor development, and 4 alleles conferred susceptibility.Introgression was used to determine the genetic loci that affected Together, the use of systems genetics with the radiation-chimera model provides new insight into the processes that mediate carcinogenic susceptibility to radiation. To further explore stromal genetic associations with cancer risk after exposure to low LET radiation, we used ingenuity pathway analysis (IPA) to identify 696 candidate genes located within the identified loci. Of these, 185 genes were within 4 loci on chromosomes 2, 11, 14, and 16 where homozygous BALB/C alleles associate with increased latency for cancer arising in irradiated mice. These genes were enriched in four pathways, γ-glutamyl cycle, leukotriene biosynthesis, alanine biosynthesis III, and glutathione biosynthesis. In contrast, 511 genes enriched for 24 pathways were within 11 regions where heterozygous SPRET/EiJ alleles associate with increased latency. Importantly, these 11 loci were enriched for genes involved in regulating the immune response including signaling pathways of natural killer cells and cytokines. Radiation-induced activation of pathways that control release of inflammatory cytokines varies among mouse strains , 114 andThe observation that many more genetic loci are linked with tumor latency in the radiation-treated cohort than in the sham-irradiated cohort suggests the interesting idea that genetic contribution is actually specific to NTE, in contrast to the widely held belief that radiation exaggerates inherent susceptibility. This is exemplified by the work of Onel and colleagues who identified PRDM1 (Blimp-1), a transcriptional regulator of cell specification, with the risk of second malignancies only in those treated with radiation for childhood malignancy . In indiIdentifying mechanisms of NTE is essential to understand the biology of irradiated tissues. Two fundamental aspects of NTE in carcinogenesis warrant careful consideration for further understanding of cancer risk in irradiated populations. First is that radiation NTE may alter the shape of the dose response. Recent modeling by Cucinotta and colleagues suggest that NTE may be particularly important in the low-dose region of concern for occupational exposures. Second, NTE are targetable; the biology that ensues after exposure is persistent and may be “reset” after the fact to limit carcinogenic potential. This offers the possibility of protecting those at greatest risk, for example, children who are treated with charged particles for childhood malignancy, in which the clear benefit of dose distribution may come at the price of long-term cancer risk. Moreover, NTE will likely provide insight into the use of particles for cancer therapy as there are common microenvironment components, such as the immunoregulatory axis and the vasculature, that are likely critical to treatment outcome.Dr. MHB-H outlined and wrote the manuscript. Dr. J-HM edited and contributed to this manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Objective: Although the mixed orbicularis oculi muscle lacks the muscle spindles required to induce reflex contraction of its slow-twitch fibers, the mechanoreceptors in Müller's muscle function as extrinsic mechanoreceptors to induce reflex contraction. We hypothesize that strong stretching of these mechanoreceptors increases reflex contraction of the orbicularis oculi slow-twitch muscle fibers, resulting in blepharospasm. Methods: We examined a 71-year-old man with right blepharospasm and bilateral aponeurosis-disinserted blepharoptosis to determine whether the patient's blepharospasm was worsened by increased trigeminal proprioceptive evocation via stretching of the mechanoreceptors in Müller's muscle owing to a 60° upward gaze and serrated eyelid closure, and whether local anesthesia of the mechanoreceptors via lidocaine administration to the upper fornix as well as surgical disinsertion of Müller's muscle from the tarsus and fixation of the disinserted aponeurosis to the tarsus decreased trigeminal proprioceptive evocation and improved patient's blepharospasm. Results: Before pharmacological desensitization, 60° upward gaze and serrated eyelid closure exacerbated the patient's blepharospasm. In contrast, these maneuvers did not worsen his blepharospasm following lidocaine administration. One year after surgical desensitization, the blepharospasm had disappeared and a 60° upward gaze did not induce blepharospasm. Conclusions: Strong stretching of the mechanoreceptors in Müller's muscle appeared to increase reflex contraction of the orbicularis oculi slow-twitch muscle fibers, resulting in blepharospasm. In addition to botulinum neurotoxin injections into the involuntarily contracted orbicularis oculi muscle and myectomy, surgical desensitization of the mechanoreceptors in Müller's muscle may represent an additional procedure to reduce blepharospasm. In this investigation, we clinically evaluated the possibility that reduced trigeminal proprioceptive achieved by desensitizing the mechanorceptors in Müller's muscle decreases reflex contraction of the orbicularis oculi slow-twitch muscle fibers in a patient with unilateral blepharospasm.The patient was a 71-year-old man who sustained right blepharospasm and bilateral aponeurosis-disinserted blepharoptosis . No blooBefore pharmacological desensitization of the mechanoreceptors in Müller's muscle, 60° upward gaze, right lateral gaze, and serrated eyelid closure exacerbated the patient's blepharospasm . After p-19In our previous reports regarding reflex contraction of the levator, frontalis, and orbicularis oculi slow-twitch muscle fibers,7-29,31-Cortical control of eyelid closure is not well understood.25Both upward gaze and right lateral gaze increased reflex contraction of the orbicularis oculi slow-twitch fibers in our patient. The levator and superior rectus muscles form an angle of 23° with the visual axis in primary gaze. Because the length of the levator muscle on the side of the lateral gaze is longer than that on the side of the medial gaze, the mechanoreceptors in Müller's muscle on the lateral gaze side appears to be have been stretched more. Accordingly, the stronger trigeminal proprioceptive evocation on the lateral gaze side may have enhanced reflex contraction of the orbicularis oculi slow-twitch muscle fibers more visibly than the weaker trigeminal proprioceptive evocation on the medial gaze side. Voluntary serrated eyelid closure appears to consist of increased voluntary contraction of the extraocular muscles, including the levator nonskeletal fast-twitch muscle fibers, strong trigeminal proprioception evocation by strong stretching of the mechanoreceptors in Müller's muscle, and subsequently increased involuntary reflex contraction of the orbicularis oculi slow-twitch muscle fibers. Predictably, voluntary serrated eyelid closure caused involuntary serrated eyelid closure as blepharospasm . As far In conclusion, in addition to botulinum neurotoxin injections into the involuntarily contracted orbicularis oculi muscle and myectomy,


We have read with great interest the article entitled 'Left and Right Ventricle Leads Switch as a Solution for T Wave Oversensing - How a Good Idea Turned out Bad' by Alzand et al. in the last issue of the journal . They haAtrial tachycardia/atrial fibrillation accounted for the majority of cardiac resynchronization therapy loss and inappropriate therapies. However, many programmable causes of cardiac resynchronization therapy loss and inappropriate therapies such as TWOS might also be detected. We should mention about potential solutions to overcome TWOS problem. Initially, programmable features of the device should be performed before the opening of the pace pocket due to various complications including device-related infection. As the authors stated that the right ventricular lead sensitivity can be decreased to acceptable values. Extremely decreasing sensitivity (>0.6 mV) is, however, already not a solution because of undersensing of ventricular tachyarrhythmia. Some devices have special programs including programmable threshold start, decay delay, upper threshold, T-wave blank period, high-pass filtering, differentiating waveform characteristics and frequencies of R and T waves, and ventricular tachycardia discriminators including morphology to suppress TWOS. Changing ventricular sensing configuration can result in higher R-wave amplitude (>3 mV) and loss of T-wave sensing. Shortening of the post-ventricular atrial refractory period is another option to overcome TWOS. However, this carries the risk of inappropriate therapies. Changing timing and sequence of ventricular pacing with various V-V pace delay settings can also solve the problem of TWOS. Because with changing sequence and timing of V-V pace ventricular repolarization preceding depolarization and T-wave morphology change. The last resort can be increasing therapy zones to high ranges and detection intervals to prevent delivery of inappropriate therapies although it seems inappropriate due to not delivery of programmed therapies to real tachyarrhythmia. If programmed parameters do not work invasive options including repositioning of the lead or implantation of a new lead with good sensing and pacing values should be performed. Therefore, the authors should have tried to perform some of above mentioned programmable parameters to overcome TWOS before implanting a new lead although the R-wave amplitude of the defibrillator lead was 1.5 mV. Second, the authors should have tried to perform repositioning of the defibrillator lead before implanting a new lead although it seems as a more complex procedure. Lastly, the locations of figures 1 and 3 seem wrong.


Dementia is defined as chronic deterioration of intellectual function and cognitive skills significant enough to interfere with the ability to perform daily activities. Recent advances in the treatment of dementia have renewed interest in the use of various neuroimaging techniques that can assist in the diagnosis and differentiation of various subtypes. Neuroimaging and computational techniques have helped the radiological community to monitor disease progression of various neurodegenerative conditions presenting with dementia, such as Alzheimer disease, frontotemporal lobe dementia (FTLD), progressive supranuclear palsy (PSP) and multisystem atrophy-cerebellar variant (MSA-C), and their response to newer therapies. Prompt identification of treatable or reversible forms of dementia, such as tumours, subdural haemorrhage and intracranial dAVF, is crucial for the effective management of these conditions. It is also prudent to recognize the imaging spectrum of metabolic, infective and autoimmune diseases with rapidly progressing dementia, such as methanol toxicity, central pontine myelinolysis (CPM), delayed post hypoxic leukoencephalopathy (DPHL), HIV, Creutzfeldt-Jakob Disease (CJD), Sjogren's syndrome, multiple sclerosis (MS), radiation necrosis and Fragile X-Associated Tremor/Ataxia Syndrome (FXTAS), which are difficult to treat and often require palliative care. This pictorial review emphasizes various non-Alzheimer’s dementia entities and discusses their imaging highlights.Teaching PointsNon Alzheimer’s dementia constitutes a broad spectrum of conditions.• Neuroimaging plays an important role in differentiating treatable from irreversible dementia.• Neuroimaging is often non-specific in early stages of neurodegenerative conditions with dementia.• Neuroimaging plays an important role in the multimodal approach towards management of dementia.•  Dementia is a disorder characterized by global impairment in cognition, social and occupational functioning severe enough to interfere with daily functioning and quality of life. Various studies have confirmed an exponential increase in dementia with age and a higher prevalence in older females . OverallAlzheimer disease (AD) is the most common cause of primary dementia, constituting 60 % of all cases. Vascular dementia is the second most common form of dementia, which often coexists with AD. There are various other non-Alzheimer conditions, which may present with cognitive impairments and can be divided into the broad categories of neurodegenerative, inflammatory/infective, metabolic/genetic and miscellaneous. This pictorial review emphasizes the role of neuroimaging in evaluating various causes of non-Alzheimer’s dementia. We also highlight conditions with reversible dementia, where neuroimaging plays a significant role in management.Binswanger and Alzheimer first described VaD and recognized the role of multiple infarctions and chronic ischemia in its etiopathogenesis. VaD is the most common cause of non-Alzheimer dementia in the aging population and often contributes to cognitive impairment in AD and other forms of so-called “mixed dementias” . Unlike The diagnostic approach to patients with suspected VaD is similar to that of any other form of cognitive decline. Neuroimaging should be performed where there is history of stroke, vascular risk factors and clinical features atypical of AD. Magnetic resonance imaging (MRI) is considerably superior in sensitivity and specificity in detecting ischemic lesions compared to computed tomography (CT); however, radiological criteria are not adequate enough in differentiating between post-stroke patients with and without dementia . MicroanCADASIL is an inherited vasculopathy with subcortical dementia of Binswanger type. It affects the younger population, beginning in the fourth to fifth decade, and commonly presents with recurrent transient ischemic attacks, strokes, migraine and cognitive decline . The etiChronic SDH is the most frequent cause of traumatic intracranial bleeding in the elderly population, especially in the background of cerebral atrophy. The mechanism of dementia can be explained by direct brain compression, reduction in whole brain cerebral blood flow (CBF) and generalized astrocyte reactivity. Approximately 45 % of patients with chronic SDH can have amnesia and cognitive deficits. This is considered to be one of the treatable forms of dementia and reversible upon surgical evacuation. On CT, chronic SDH often appears as a hypodense concavo-convex collection. On T1W and FLAIR MRI, it appears minimally hyperintense in comparison to CSF due to low paramagnetic methemoglobin and high protein content Fig. . MembranDementia with Lewy bodies (DLB) is considered as the second most common type of degenerative dementia after Alzheimer disease (AD). Cognitive dysfunction is the frequent initial presentation of DLB, with dementia eventually occurring in all cases. Unlike Alzheimer disease (AD), the patients with DLB often present with early impairments in attention, executive and visuospatial function, with memory affected later in the course of the disease . Other cDementia is common and typically appears late in Parkinson disease (PD). Milder cognitive impairment represented by executive dysfunction and impaired visuospatial function is usually noted early in the disease . Other aMSA-C was formerly called olivopontocerebellar degeneration, with onset at a mean age of 56 to 60 years. It is a sporadic disease with a combination of Parkinsonism symptoms, autonomic failure and cerebellar ataxia. Dementia is uncommon and of the frontal type, but is found in a minority of patients . There i2) compared to idiopathic Parkinson disease (103 mm2) or multiple system atrophy-parkinsonism (MSA-P) (97 mm2) . La. La38]. Sjogren’s syndrome is one of the most common autoimmune diseases, affecting the exocrine glands and manifesting as sicca syndrome. CNS involvement is noted in approximately 20 % of patients, presenting with transverse myelitis and aseptic meningitis. It can manifest as steroid-responsive treatable dementia, which is caused by angiitis or nonvasculitic autoimmune inflammatory meningoencephalitis (NAIM). Cognitive and behavioural disorders like forgetfulness and bradyphrenia are some known presentations. MR imaging can often be normal or show non-specific perivascular and subcortical small punctate white matter lesions Fig.  40, 41], 4140, 4MS is a demyelinating disease involving both white and gray matter. Focal neurological deficits caused by lesions in an eloquent long tract, like optic neuritis or a spinal cord syndrome, are common presentations. Nearly one in 20 patients can manifest with dementia and the common imaging finding is nonspecific generalized atrophy Fig.  42]. Th. Th42]. There are three basic mechanisms of developing hypoxic-ischemic brain injury- hypovolemia (cardiac failure), hypo-oxygenation (respiratory failure) and impaired oxygenation (CO poisoning) . InitialMethanol is a highly toxic, colourless liquid that resembles ethanol in taste and smell. Intoxication is common after suicidal or accidental ingestion and fraudulent adulteration of alcoholic beverages. It results in severe metabolic acidosis and optic necrosis or demyelination leading to vision loss. Severe toxicity may progress to Parkinson-like extrapyramidal syndrome, cognitive deficits/dementia, permanent neurological deficits and death . Both CTCentral pontine myelinolysis is an acute demyelinating process affecting the central pontine fibres and frequently associated with chronic alcoholism, nutritional deficiency and rapid correction of hyponatremia (osmotic demyelination). Spastic quadriparesis, pseudobulbar palsy, altered level of consciousness and coma are usual presentations. Dementia component and behavioural changes are likely from callosal involvement . SymmetrFXTAS is a late onset X linked genetic cause of mental retardation and autism due to trinucleotide repeat mutation of the Fragile X Mental Retardation 1 (FMR1) gene, and is commonly seen in males between the ages of 50 and80 years. The clinical presentation includes intentional tremor, ataxia, neuropathy, and personality changes in association with memory loss. Atrophy of the cerebral and cerebellar hemispheres, middle cerebellar peduncles, pons, medulla and corpus callosum are the important imaging features. Symmetric T2W/FLAIR hyperintense lesions in the cerebellar hemispheres close to the dentate nucleus, pons and bilateral middle cerebellar peduncles (MCP sign) are commonly noted Fig.  & b. In dAVF is an acquired pathological shunt between intracranial arteries and dural venous sinuses, meningeal or cortical veins. It mostly presents in the fifth or sixth decades and the common locations are in transverse, sigmoid and cavernous sinuses . Many dANormal pressure hydrocephalus represents ventricular dilatation and clinical triad of cognitive decline, gait disturbance and urinary incontinence with normal CSF opening pressure. Management of this condition is a dilemma because of diagnostic uncertainty, and requires careful patient selection for CSF shunting. The aetiology is still debatable and explained by two existing theories: 1) Impaired CSF absorption due to prior meningitis or subarachnoid haemorrhage and 2) Decreased white matter tensile strength due to ischemic changes . ImagingPatients irradiated for nasopharyngeal and oropharyngeal tumours can also present with cognitive and memory impairment due to bilateral temporal lobe involvement . RadiatiBoth extra and intra axial tumours, when situated near eloquent areas, may present with slowly progressing neuropsychological, behavioural disturbance and dementia. It is potentially a reversible cause of dementia if amenable to surgical resection. Common locations are being middle cranial fossa, basal frontal regions, thalamus, corpus callosum, cingulate gyrus and limbic system Fig. . NeuroimNon-Alzheimer’s dementia comprises several pathologies varying from rapid onset to gradually progressive disease spectrum. Often there is considerable clinical overlap, and neuroimaging features tend to be non-specific during the initial stage of disease. The rationale of conventional neuroimaging is not to offer definitive diagnoses, but to suggest possible differential diagnoses and track evolving changes on follow-up studies. Newer molecular imaging bio-markers combined with conventional neuroimaging techniques hold promise in assessing applications of novel disease-modifying drugs for treatment and response evaluation. An important aspect of neuroimaging is also to recognize reversible or treatable causes of dementia that can potentially be treated or halt further disease progression.


MicroRNAs (miRNAs) are small RNA molecules (approximately 22 nucleotides long) that take part in the post-transcriptional regulation of gene expression  reverse transcriptase may contain miRNAs.T values of these miRNAs were found in almost the same Log2 proportions as the volumes of reverse transcriptase. These results implied that the miRNAs found in AMV reverse transcriptase might explain the high background signals.In practice, DEPC-treated water or ultrapure water (Invitrogen) served as no-template-controls for miRNA quantitation. However, certain miRNAs  exhibited high background signals in these no-template-controls when AMV reverse transcriptase was used  and sequenced. Importantly, only the sequences containing the adaptor sequence were counted as the correct sequences. This procedure guaranteed that the small RNAs within the reverse transcriptase could not be counted in the RNA samples during the ensuing sequence determinations. As shown in the supplementary information, after comparing the sequencing results with an miRNA database, Solexa sequencing revealed hundreds of copy numbers (readings) of many miRNAs were existed in commercial AMV transcriptase . Most commercial AMV reverse transcriptases are produced using the first method. The AMV is used to infect chicks or other birds, after 7 days, AMV particles are purified from the host blood plasma. We checked the miRNA profile of chick plasma by RT-qPCR  particles isolating from the host plasma or cultured cells infected with AMV  and performed Solexa sequencing. We found a large quantity of small RNAs (under 50 nucleotides) in the M-MLV reverse transcriptase. The most abundant small RNAs in M-MLV reverse transcriptase (copy numbers > 10000) are listed in Table E. coli genome. The above results indicated that commercial M-MLV reverse transcriptase also contains small RNAs derived from their artificial E. coli host. Although these small RNAs may not interfere with miRNA quantification, they could interfere with E. coli small RNA identification and characterization.Commercial M-MLV reverse transcriptase was produced by recombinant protein expression in -MLV Co.  and perfT values of four miRNAs were decreased about 1–2 cycles, which indicates that the miRNAs in AMV solution might be separated from the reverse transcriptase in this condition. Consistently, the function test showed that filtration treatment did not impair the capability of the AMV reverse transcriptase to perform their function in different concentration . After centrifuge, the remaining solutions above the filters were collected. To examine the function of filtration-treated AMV reverse transcriptase, undiluted or diluted transcriptase in different concentrations (100 and 10000 dilution) were used in RT reaction with synthetic miR-16 molecules served as RNA template. To evaluate the miRNAs remaining in the AMV solution, the rest of the solutions were extracted with Trizol to obtain the RNA contents. The extracted RNAs were quantified by RT-qPCR. As shown in Fig. T value for each miRNA can be figured out. If CT value in experimental group is far below the base line CT value, miRNA contamination from reverse transcriptase may not affect the results; otherwise the possibility of false negative data should be taken into consideration because the high background might conceal the changes of miRNA levels. Although no-template control has not solved the problem of miRNA contamination in reverse transcription, it does provide a reference to get rid of bad data. A reverse transcription procedure is needed when engaging in miRNA sequencing or RT-PCR quantitation. In Solexa, a pair of Solexa adaptors was added to the 5′ and 3′ ends of RNAs to form a 90-bp fragment  with the help of ligase. The ligase was then inactivated, and reverse transcriptase was added. Afterwards, the 90-bp fragment was amplified through adaptor primers. This procedure guaranteed that the small RNAs present in reverse transcriptase could not be detected during the ensuing sequence determination. We suggest that the strategy of adding adaptors first might be applied to the RT-PCR technique to avoid interference from unwanted miRNAs in future. Choosing M-MLV reverse transcriptase or cloned AMV reverse transcriptase in miRNA studies could eliminate interference from miRNA contamination in reverse transcriptases.From the above study, we think that a no-template control is necessary for miRNA quantification using RT-qPCR assay, because of the uncertain miRNA contents of AMV from different sources. With no-template control, an accurate base line of CT value (data not shown), which provided a possibility that small RNA in reverse transcriptase might be a functional co-factor rather than contamination. Although our preliminary studies revealed that partially depleting part of miRNAs in AMV by mild physical methods did not interrupt the function of AMV, we could not exclude the possibility that miRNAs in AMV or small RNAs in M-MLV is a potential co-factor for reverse transcriptase, because filtration could not eliminate all small RNAs in reverse transcriptase. We propose that the small RNAs existing in reverse transcriptase might be functional. Based on our discovery and hypothesis, further studies should focus on the analysis of crystal structure of reverse transcriptase and explore the possible binding site for small RNA.RNase is usually strictly limited during reverse transcriptase production to avoid RNase contamination; otherwise the contaminated RNase will degrade the RNA template in following reverse transcription. Therefore, small RNAs might be co-purified during reverse transcriptase production. According to our results, both AMV and M-MLV reverse transcriptases contain small RNA fragments coming from their host, respectively. During previous studies, we were surprised to find adding synthetic miRNA molecule into reverse transcription system could slightly change the CE. coli strains or in insect cells, which excluded the possibility of mammalian miRNA contamination. Interestingly, Solexa sequencing detected a large number of small RNAs (approximately 20–25 nucleotides) that matched the E. coli genome. A special purification step aimed at small RNAs removal might not be included in the production process. All of these problems contribute to high background signal in small RNAs quantification. The uncertain miRNA contamination status of reverse transcriptase would affect data analysis, and it would even prevent certain changes from being detected . Therefore, the small RNAs in contaminated transcriptase might interfere with quantification in certain cases. This study first identified the interference caused during miRNA RT-qPCR and then identified the source of these miRNAs, providing an optimization protocol for the detection of miRNAs to avoid unnecessary interference during data analysis.In summary, small RNAs exist in commercial reverse transcriptase. According to the product description, some commercial AMV reverse transcriptases were derived from avian myeloblastosis virus, which suggested that miRNAs might be derived from the host of the virus particles. Our miRNA profiling study compared the miRNA profile of AMV with the miRNA profile in bird serum. Commercial M-MLV reverse transcriptase and cloned AMV reverse transcriptase are usually produced by Supplementary material 1 (DOC 33 kb)Supplementary material 2 (JPEG 195 kb)Supplementary material 3 (XLS 40 kb)Supplementary material 4 (XLS 4616 kb)Below is the link to the electronic supplementary material.


Victoriapithecus, reveals an unexpectedly small endocranial volume (ECV) relative to body size and a large olfactory bulb volume relative to ECV, similar to extant lemurs and Oligocene anthropoids. However, the Victoriapithecus brain has principal and arcuate sulci of the frontal lobe not seen in the stem catarrhine Aegyptopithecus, as well as a distinctive cercopithecoid pattern of gyrification, indicating that cerebral complexity preceded encephalization in cercopithecoids. Since larger ECVs, expanded frontal lobes, and reduced olfactory bulbs are already present in the 17- to 18-Myr-old ape Proconsul these features evolved independently in hominoids (apes) and cercopithecoids and much earlier in the former. Moreover, the order of encephalization and brain reorganization was apparently different in hominoids and cercopithecoids, showing that brain size and cerebral organization evolve independently.Analysis of the only complete early cercopithecoid (Old World monkey) endocast currently known, that of 15-million-year (Myr)-old  The evolution of the brain in Old World monkeys (cercopithecoids) is poorly understood. Here the authors describe a complete endocast of Victoriapithecus, a 15 Myr old cercopithecoid, which shows that the brain size was much smaller and the olfactory bulbs much larger than in any extant catarrhine primate. The relationship between the external morphology, cytoarchitecture and function of the brain is better understood for macaques than for other non-human primates because of their extensive use in neuroscience research12357810111213Victoriapithecus from Maboko Island, Kenya currently includes the only intact neurocranium of a Miocene catarrhine before 6 MyrVictoriapithecus represents a cercopithecoid clade that postdates the earliest fossil hominoid (Rukwapithecus) and cercopithecoid (Nsungwepithecus) by 10 million years (Myr); however, retention of a crista obliqua on the upper molars indicates that it is more primitive than the last common ancestor of extant Colobinae and Cercopithecinae16The well-preserved 15-Myr-old adult male cranium KNM-MB 29100 of 3 was obtained  when using a body mass of 6–7 kg for KNM-MB 29100 (ref. Aegyptopithecus and stem anthropoid Simonsius (=Parapithecus) fall somewhat further below the strepsirrhine regression than Victoriapithecus, indicating that 15 Myr ago the latter had only a slightly larger relative brain size than 32-Myr-old Oligocene stem catarrhines22The endocast of KNM-MB 29100 is well preserved and shows remarkably clear impressions of the cerebral sulci and gyri . After cobtained , substan kg refs , 20. In arrhines , Fig. 2a of 5 kg , and furMiopithecus talapoin has an ECV similar to Victoriapithecus, but an average body mass of 1.5–1.9 kg  if female and 53–71 cm3  if male21Victoriapithecus was clearly frugivorous, diet did not contribute to its extremely small brain sizeAmong cercopithecoids  kg ref. . CercopiVictoriapithecus, the estimated ECV for the 8- to 9-Myr-old colobine Mesopithecus is substantially larger, falling within the extant catarrhine cluster but just below the modern colobine regression line relative to its inferred body mass5Theropithecus are similarly larger relative to body mass than Victoriapithecus but are all smaller than living T. gelada, and plot towards the edge of the catarrhine cluster where species with the smallest ECVs per body mass are found35Victoriapithecus. While ECV increases evolved independently in colobine and cercopithecine subfamilies, neither lineage had reached modern ECV level until the Holocene.Compared with Afropithecus) were substantially more encephalized than contemporary cercopithecoids, although assessing exactly how different their ECVs were will require an improved fossil record. ECV estimates for incomplete skulls of 17- to 18-Myr-old Proconsul242526Dryopithecus2526Oreopithecus25262930313252223Victoriapithecus by 34% compared with the direct measurements obtained here, these apparently large hominoid ECV estimates could be an artefact of methodology. The earliest conclusive evidence that hominoids reached ECV levels of extant apes comes from the late-Miocene Sahelanthropus cranium, for which ECV falls within the range of chimpanzees relative to its estimated body mass3940Existing evidence indicates that Miocene apes (excluding Victoriapithecus differs from modern anthropoids in having much larger olfactory bulbs that project anteriorly as in strepsirrhines and Aegyptopithecus233), falling within the lower range of strepsirrhines and upper-most range of anthropoids, similar to Aegyptopithecus and Simonsius  used to detect odorant molecules of high molecular weight such as water-soluble pheromonesAegyptopithecus but absent in Victoriapithecus and living crown catarrhines47484950Afropithecus and Proconsul similarly lack VNO-related structures; however, the presence of an atrioturbinal ridge in two small-bodied early Miocene non-cercopithecoid catarrhines Limnopithecus and Kalepithecus indicates that some catarrhine lineages retained VNO function during the MioceneAegyptopithecus and these small-bodied catarrhines Victoriapithecus, Afropithecus and Proconsul would have relied only on their main olfactory bulbs rather than VNO for the detection of socially relevant olfactory stimuli, possibly including pheromones  and arcuate sulci523Cebus. Aegyptopithecus also has smooth occipital and temporal lobes lacking inferior occipital sulci, dimpling of the temporal lobes and anterior and posterior middle temporal sulci, all of which occur in Victoriapithecus. Principal sulci are shared by all crown catarrhines and must have been retained from an ancestral condition more recent than Aegyptopithecus. The presence of arcuate sulci in Victoriapithecus, all other cercopithecoids, the 10-Myr-old hominoid DryopithecusProconsul552556Cebus. In macaques, areas around the arcuate sulcus are involved in visual working memory, hand–eye coordination and mirror neurons activated by observing the movements of others59Victoriapithecus but not in Aegyptopithecus, concerns an area where sharply tuned colour-selective neurons are concentrated in macaque brainsVictoriapithecus compared with Oligocene anthropoids suggests that it had already evolved a more complex visual system than Aegyptopithecus in spite of their similarly small ECVs.In contrast to Victoriapithecus and extant cercopithecoid cerebral cortices are indicated by the more anterior position of various sulcal landmarks relative to endocast length and height in the former , Turkanapithecus (17 Myr) and Dryopithecus (10 Myr) are substantially broader and less constricted anteriorly than both Aegyptopithecus and Victoriapithecus. Only Afropithecus (17 Myr) has a small V-shaped frontal cortex among Miocene hominoids.Differences between e former . VictoriVictoriapithecus motor cortex (the frog's thigh) appears to have been narrower and the superior temporal gyrus shorter than in extant cercopithecoids as indicated by the more anterior position of the confluence of lateral and superior temporal sulci. In addition, the inferior temporal lobe is large and uniquely has a large posterior inferior temporal region that is continuous with the occipital lobe, creating a distinct thickening in the occipital lobe between the short lunate and upwardly curving inferior occipital sulcus that is much less anteriorly positioned than in any extant cercopithecoid we observed.The Victoriapithecus cerebral cortex is more similar to those of cercopithecines than colobines. Only extant colobines and the late-Miocene colobine Mesopithecus have intraparietal sulci that diverge laterally at their posterior ends as the superior parietal lobule (SPL) expands and impinges on a shorter but more gyrified occipital lobe56Victoriapithecus shares with extant cercopithecines and the late-Miocene colobine Libypithecus intraparietal sulci that are straight and converge posteriorly as they approach the lunate sulcus, posterior ends of the superior parietal gyrus that are V-shaped and a more anteriorly positioned lunate sulcusAegyptopithecus and Victoriapithecus share relatively large occipital lobes and unexpanded SPLs with cercopithecines . With cercopithecoids showing cerebral and visual system complexity preceding encephalization and olfactory bulb reduction, and hominoids exhibiting frontal lobe expansion and encephalization before gyrification, it appears that some morphological and functional similarities between extant macaque and hominoid brains may have evolved convergently. The combination of a modern cercopithecoid sulcal pattern with strepsirrhine ECV and olfactory bulb size in KNM-MB 29100 was CT scanned with the BIR ACTIS 225/300 of the Max Planck Institute for Evolutionary Anthropology, Leipzig, at the time installed at the National Museums of Kenya in Nairobi. The isotropic voxel size is 0.044 mm. Avizo 7.1 and 8.0  and Geomagic Studio 2013 (Geomagic Inc.) were used for visualization, segmentation, reconstruction and quantification.3. Since parts of the right frontoparietal area are not as well preserved as the left side, we also calculated ECV values for two endocast reconstructions based on the left half combined with its mirror image. One version uses an overall best-fit midsagittal plane, whereas the other applies the additional constraint that the original foramen magnum size is maintained. The associated ECVs, 35.5 and 36.2 cm3, respectively, bracket the value obtained for the full endocast.To calculate the ECV of KNM-MB 29100, the preserved endocast was corrected for distortion in three areas . The endHow to cite this article: Gonzales, L.A. et al. Cerebral complexity preceded enlarged brain size and reduced olfactory bulbs in Old World monkeys. Nat. Commun. 6:7580 doi: 10.1038/ncomms8580 (2015).Supplementary Figures 1-2, Supplementary Tables 1-2 and Supplementary ReferencesThe KNM-MB 29100 endocast rotating around a supero-inferior axis. The surface reconstruction, based on high-resolution CT images, is of the endocast as preserved in the fossil, without corrections for distortion.


A case of atypical (“pagetoid”) compound Spitz nevus on the face of a 2-year-old girl is reported with a review of the literature. The nevus was composed of broad but laterally demarcated compound proliferation of enlarged fusiform and epithelioid melanocytes, with florid pagetoid scatter above the junction. Immunohistochemical analyses revealed the Ki-67 proliferation index to be relatively low. Given the histomorphological overlap with melanoma, an array-based comparative genomic hybridization approach revealed a subthreshold gain in chromosome 1q and gain in distal chromosome 17q, with no other associated chromosomal gains or losses. These molecular aberrations suggested a partially transformed tumor, without adequate evidence for a molecular diagnosis of melanoma. Because of the diagnostic dilemma posed by these lesions, recent research has focused on molecular alterations that may help differentiate Spitz tumors from malignant melanomas. Spitz nevus is an uncommon melanocytic lesion composed of epithelioid and spindled cells, with a remarkable histological resemblance to malignant melanoma. First described in 1948, the diagnosis and management of Spitz tumors are still heavily debated today. Spitz nevi display a morphological and biological spectrum ranging from benign to malignant, with 3 main classifications: Spitz tumor, atypical Spitz tumor, and malignant melanoma.We present a case of 2-year-old girl with an atypical Spitz tumor, with peculiar clinical, histopathological, and molecular based findings.An otherwise healthy 2-year-old Caucasian female patient presented with a pigmented lesion on her left cheek since birth. The mother reported that the lesion had demonstrated recent enlargement. Medical and surgical history and systematic review were unremarkable. She had no family history of melanoma. Skin examination was notable for a 1-cm diameter heavily pigmented lesion with regular borders located at the junction on the lower eyelid. The rest of the skin examination was unremarkable.Excisional biopsy with 1-mm margins was performed. Histopathological examination revealed a broad but laterally demarcated compound proliferation of enlarged fusiform and epithelioid melanocytes. Cells were distributed within a broad zone of acanthosis and relatively florid pagetoid scatter above the junction. Numerous well-formed pink globules were noted within the expanded epidermis, and eosinophilic hyalinization of dermal papillae were noted. Within the underlying dermis, there were maturing nests and cords of cytologically similar melanocytes coupled with an infiltrate of lymphocytes and melanophages. Rare mitotic figures were observed within the dermal component. Immunohistochemical analyses revealed the Ki-67 proliferation index to be relatively low.Given the histomorphological overlap with melanoma, an array-based comparative genomic hybridization (CGH) approach was used on tumoral DNA extracted from the tissue block following microdissection.Atypical Spitz nevi are described as conventional Spitz nevi with 1 or more atypical features with indeterminate biological potential. Features of atypical Spitz include larger size (>6 mm), irregular borders, irregular topography, or ulceration. On histology, asymmetry, ulceration, pagetoid melanocytosis, lack of zonation and maturation, deep mitoses, and atypia can be seen. There are no clear histomorphological criteria that differentiate atypical Spitz nevi from malignant melanoma.A recent study among a panel of 13 expert dermatopathologists assessing atypical Spitz tumors found low interobserver agreement in categorizing lesions as malignant versus nonmalignant.Ancillary studies including immunohistochemistry, CGH, and fluorescence in situ hybridization have proved promising to help distinguish melanocytic from nonmelanocytic lesions, but sensitivity and specificity of studies are not ideal. Immunohistochemical staining may be useful for detection of mitoses to determine the proliferation activity of a lesion. The rate of staining with proliferation markers is generally lower in atypical Spitz nevi than that in malignant melanomas. Increased expression of Ki-67, HMB-45, cyclin D1, and fatty acid synthase are more suggestive of melanoma.This case report underlines the ambiguity and difficulty in diagnosing atypical Spitz nevi, and the clinical management is still a matter of debate. In support of the literature, it may be beneficial to view atypical Spitz nevi as a heterogeneous group of biologically different lesions rather than as a continuum. For example, the lesions can represent a large proportion of morphologically atypical but biologically benign lesions as well as a small group of true melanomas.


Cells employ a complex network of molecular pathways to cope with endogenous and exogenous genotoxic stress. This multilayered response ensures that genomic lesions are efficiently detected and faithfully repaired in order to safeguard genome integrity. The molecular choreography at sites of DNA damage relies heavily on post-translational modifications (PTMs). Protein modifications with ubiquitin and the small ubiquitin-like modifier SUMO have recently emerged as important regulatory means to coordinate DNA damage signaling and repair. Both ubiquitylation and SUMOylation can lead to extensive chain-like protein modifications, a feature that is shared with yet another DNA damage-induced PTM, the modification of proteins with poly(ADP-ribose) (PAR). Chains of ubiquitin, SUMO, and PAR all contribute to the multi-protein assemblies found at sites of DNA damage and regulate their spatio-temporal dynamics. Here, we review recent advancements in our understanding of how ubiquitin, SUMO, and PAR coordinate the DNA damage response and highlight emerging examples of an intricate interplay between these chain-like modifications during the cellular response to genotoxic stress. Our genetic material is under constant cellular surveillance and care. Maintaining genome stability is indeed a vital task, not only under conditions when external toxins or physical strains challenge the integrity of the genome, but also in the course of normal cellular metabolism, when reactive metabolites and physiological DNA transactions can lead to a plethora of lesions. If these damages are not detected and faithfully repaired, cells run the risk of accumulating mutations that can erode genome function, vitiate cell fate, or compromise cell survival. Faced with such threats cells have developed sophisticated mechanisms to sense and repair damaged DNA. These mechanisms, which are collectively referred to as the DNA damage response (DDR), not only ensure that most lesions are efficiently repaired, but they also coordinate genome integrity maintenance with other cellular functions such as transcription, DNA replication, and cell cycle progression . The DDRTo fulfill its task, the DDR employs a multitude of tightly regulated posttranslational protein modifications (PTMs). In addition to modulating protein functions locally at the damage site, PTMs play important roles in spreading the DNA damage signal to the surrounding chromatin  and in aFigure 1A)  pathway is independent of intact template DNA sequences and can re-ligate broken DNA ends throughout the cell cycle. In contrast, faithful repair by homologous recombination (HR) depends on an undamaged template DNA and is thus restricted to the S/G2 phases of the cell cycle when sister chromatids are available. While NHEJ is generally considered error-prone due to the risk of nucleotide loss from DNA ends, HR is considered to be more accurate due to template-based repair. The choice between NHEJ and HR is tightly controlled, and imbalances in its regulation can lead to genome instability and accelerate cancer development . Interesgure 1A) . Of notegure 1A) , thus liFigure 1A).Importantly, ubiquitin conjugation is not only involved in generating the ubiquitin code that is recognized by 53BP1, but also fosters 53BP1 accumulation by RNF8/RNF168-dependent and VCP/p97-mediated removal of proteins from damaged chromatin . As was While these reactions build up an important anti-resection barrier that shields broken DNA ends from unscheduled nucleolytic digestion, ubiquitin conjugation also plays a role in promoting HR. For example, the ubiquitin E3 ligases TRIP12 and UBR5 cooperate to keep RNF168 levels in check and thereby prevent excessive 53BP1 function . More reFigure 1B)  . SUMOylagure 1B) . While mFigure 1C)  in response to genotoxic stress, DNA break-associated ADP-ribose polymers provide a landing platform for a plethora of PAR-binding proteins . This ingure 1C) . The relGiven that all three catenarian modifications, ubiquitylation, SUMOylation, and PARylation, orchestrate the protein accumulations around DNA break sites, significant crosstalk exists. In the following paragraphs, we highlight emerging examples of such interplay and how it affects the accrual of genome caretakers at damaged chromatin.Figure 2A)  . Interesgure 2A) , and SUMIn analogy to SUMO-targeted ubiquitylation by STUbLs it was recently discovered that also PAR serves as recognition signal for selected ubiquitin ligases . The besWhile these events likely evolved to prevent excessive interactions of repair factors with DNA break sites, PAR-dependent ubiquitylation also assists the early recruitment of genome caretakers. For instance, PAR formation was shown to be required for the recruitment of the BAL1/BBAP ubiquitin ligase complex, whose activity promotes the retention of the RAP80-BRCA1 complex . This meAnother RING-type ubiquitin E3 ligase, whose role in genome integrity maintenance is linked to PAR formation, is the mitotic regulator CHFR. A PAR-binding zinc finger motif (PBZ) mediates its interaction with genotoxic stress-induced PAR and was shown to be required for the CHFR-dependent antephase checkpoint . InteresFigure 2B).Taken together, PARylation assists the early recruitment of genome caretakers, including various ubiquitin ligases, which further promote chromatin modifications and lead to the formation of a dedicated repair compartment, but it also participates in temporarily restraining protein access to repair sites and in the timely and PTUbL-mediated removal of repair factors once they have fulfilled their duties    . Finallygure 2D) . Thus, iWhile distinct in their chemical nature and regulatory mechanism, ubiquitylation, SUMOylation, and PARylation share the feature of being chain-like protein modifications. The composition of modification chains, their length, linkage type and branching frequency contains information that can be used by complex regulatory circuits and signaling pathways such as the DDR. Recent work has elucidated how cells employ a sophisticated sequence of reactions with remarkable temporal and spatial resolution to shield genomic lesions and build up dynamic functional platforms that promote repair. The information content imbedded in this response is immense, and the use of modification chains may thus support the need for lesion-specific chromatin barcodes that dynamically change as repair reactions progress.The multivalent protein recruitment polymers formed by ubiquitylation, SUMOylation, and PARylation often cooperate to achieve robust responses. To this end, they act successively or in parallel, and frequently use positive feedback loops to amplify the signal and increase its specificity. They also employ time-delayed negative feedback to terminate reactions and disassemble complexes, which are no longer needed and constitute roadblocks for downstream events. While recent work has started to elucidate the crosstalk between these modifications, how their combinatorial use and dynamic interplay reshapes the chromatin environment surrounding different types of genomic lesions, dictates repair pathway decisions, and determines repair fidelity remains incompletely understood. Moreover, almost nothing is known about mixed chain modifications, e.g., PARylation of ubiquitin or SUMO chains, and how they might be employed by the DDR. Quantitative time-resolved proteomics and imaging approaches that provide spatial information about protein redistribution and can relate this to cell cycle information are powerful tools to address these issues. The insights gained will not only deepen our understanding of the DDR, but may also provide additional clues to the mechanisms that underlie the toxicity of inhibiting chain-like modifications in cancer treatments.All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.