The “hybrid” approach to multivessel coronary artery disease combines surgical left internal thoracic artery (LITA) to left anteriordescending coronary artery (LAD) bypass grafting and percutaneous coronary intervention of the remaining lesions. Ideally, the LITA to LAD bypass graft isperformed in a minimally invasive fashion. This review aims to clarify the place of hybrid coronary revascularization (HCR) in the current therapeutic armamentariumagainst multivessel coronary artery disease. Eighteen studies including 970 patients were included for analysis. The postoperative LITA patency varied between93.0% and 100.0%. The mean overall survival rate in hybrid treated patients was 98.1%. Hybrid treated patients showed statistically significant shorterhospital length of stay (LOS), intensive care unit (ICU) LOS, and intubation time, less packed red blood cell (PRBC)transfusion requirements, and lower in-hospital major adverse cardiac and cerebrovascular event (MACCE) rates compared with patientstreated by on-pump and off-pump coronary artery bypass grafting (CABG). This resulted in a significant reduction in costs for hybrid treatedpatients in the postoperative period. In studies completed to date, HCR appears to be a promising and cost-effective alternative for CABG in the treatment ofmultivessel coronary artery disease in a selected patient population. Coronary artery bypass grafting (CABG) is considered to be the “gold standard” in patients with multivessel disease and remains the treatment of choice for patients with severe coronary artery disease, including three-vessel or left main coronary artery disease . The use Angelini and colleagues reported the first hybrid coronary revascularization (HCR) procedure in 1996, and several patient series using hybrid coronary revascularization have been published since then . These s Nevertheless, this hybrid approach has not been widely adopted because practical and logistical concerns have been expressed. These concerns implicate the need for close cooperation between surgeon and interventional cardiologist, logistical issues regarding sequencing and timing of the procedures, and the use of aggressive anticoagulant therapy for percutaneous coronary intervention that may worsen bleeding in the surgical patient , 14, 16.This review aims to clarify the place of hybrid coronary revascularization in the current therapeutic armamentarium against multivessel coronary artery disease. First, the patient selection for the HCR procedure is clarified. Second, the results of previous patient series using the hybrid approach are summarized and interpreted. Finally, the cost effectiveness of the HCR procedure is analysed.The MEDLINE/PubMed database was searched in January 2012 using the medical subject headings (MESH) for “coronary artery disease” and “angioplasty, balloon, coronary” combined with the following free-text keywords: “multivessel coronary artery disease,” “minimally invasive coronary artery bypass,” “percutaneous coronary intervention,” and “hybrid coronary revascularization”. One hundred seventy-seven articles matching these search criteria were found, and the search for additional papers was continued by analysing the reference lists of relevant articles.n < 15) were also excluded. Studies examining the HCR procedure for multivessel coronary disease were included, while studies investigating the HCR procedure for left main coronary stenosis were excluded. Authors and medical centres with two or more published studies were carefully evaluated and were represented by their most recent publication to avoid multiple reporting of the same patients. A total of eighteen included studies remained eligible for analysis after applying these in- and exclusion criteria  studies were selected for inclusion. Publications in languages other than English were excluded beforehand. Letters, editorials, (multi)case reports, reviews, and small studies , packed red blood cells (PRBCs) transfusion rate, LITA patency, hospital length of stay (LOS), 30-day mortality, survival, and target vessel revascularization (TVR). Secondary outcome measures were intensive care unit (ICU) LOS and intubation time, as only a limited number of studies reported these outcome measures. In addition, the period of time between PCI and LITA to LAD bypass grafting and the cost effectiveness of HCR were examined. The long-term LITA patency was not included as an outcome measure, since only a limited number of studies report this outcome measure in a clear and concise manner.In-hospital major adverse cardiac and cerebrovascular events were defined as postoperative stroke, myocardial infarction (MI), or death during hospital stay. Only the Fitzgibbon patency class A (widely patent) was considered as a patent LITA to LAD bypass graft, while the Fitzgibbon patency class B (flow limiting) and C (occluded) were defined as a nonpatent LITA to LAD bypass graft. Hospital LOS was defined as the number of days spent in hospital from operation to discharge. If the need for repeated revascularization involved a coronary artery initially treated with either bypass grafting or PCI, this repeated revascularization was considered to be target vessel revascularization.  One observer extracted all available outcome measures of each article and a second observer checked and supervised the first observer thoroughly. When an article did not disclose one or more of these outcome measures or reported medians and ranges as central tendency instead of means and standard deviations, the study was excluded from the analysis of that particular variable.The results were analysed using IBM SPSS Statistics 19 software . Continuous data were presented as mean and standard deviation (SD), while categorical data were expressed as numbers and percentages. Nine hundred seventy patients undergoing HCR procedures were included for analysis Tables  and 2 6, 17–28. The classical indication for an HCR procedure is multivessel coronary artery disease involving LAD lesion judged suitable for minimally invasive LITA to LAD bypass grafting but unsuitable for PCI (type C), and (a) non-LAD lesion(s) (most of the time right coronary artery (RCA) and/or circumflex coronary artery (Cx) lesions) amenable to PCI (type A or B) , 26–28. 2), and previous left chest surgery are not well suited for minimally invasive LITA tot LAD bypass grafting [μmol/L), and allergy to radiographic contrast [Exclusion criteria for HCR consist of contraindications to minimally invasive LITA to LAD bypass grafting or PCI. LITA to LAD bypass grafting in a minimally invasive fashion requires single-lung ventilation and chest cavity insufflation. Therefore, HCR procedures are contraindicated in patients with a compromised pulmonary function  and a small intrathoracic cavity space , 27, 28.grafting , 27, 28.contrast , 27, 28.contrast , 27, 28.The best timing of the interventions remains a matter of debate. Three HCR strategies can be distinguished: (I) performing PCI first, followed by LITA to LAD bypass grafting or (II) vice versa; (III) combining LITA to LAD bypass grafting and PCI in the same setting in a hybrid operative suite. In the included studies, staged HCR procedures (I and II) were applied much more frequently than simultaneous procedures (III). In a “staged” procedure, in which PCI and LITA to LAD bypass grafting are carried out at separate locations and/or different days, both interventions can be performed under ideal circumstances  , 18, 29.When PCI is performed first, a staged procedure takes place with an unprotected anterior wall, which could pose serious health risks in case the LAD lesion is considered the culprit lesion . In addiNevertheless, the potential advantages of this strategy are threefold. First, revascularization of non-LAD vessels provides an optimized overall coronary flow reserve, thereby minimizing the potential risk of ischemia and myocardial infarction during the LAD occlusion for LITA to LAD bypass grafting , 12. SecWhen the LITA to LAD bypass graft is performed first, antiplatelet therapy is routinely started after surgery to prevent antiplatelet-related bleeding complications during surgery and is present at time of PCI , 13, 27.Nevertheless, combining the two procedures in one stage under general anaesthesia in a specific hybrid-operating room, which combines the potential of catheterization and cardiac surgery, has advantages compared with staged HCR procedures , 25, 28.This procedure also has its own drawbacks. Perioperative haemorrhage can become a problem because full antiplatelet therapy and incomplete heparin reversal are necessary instantly after MIDCAB to prevent a transient “rebound” increase in thrombin formation associated with stent thrombosis and ensure an optimal intraoperative DES placement , 14, 18.The latter is reflected in the small number of patients undergoing a simultaneous procedure in our sample of included studies , 25, 28.Tables As shown in The postoperative LITA patency seemed to be independent of the surgical technique of LITA to LAD bypass grafting, since LITA patency has shown to be approximately equal for all surgical techniques . The posBesides the technical improvements of LITA to LAD bypass grafting, innovations occurred in the field of PCI. This development was supported by the increased rate of DES implantation in later patient series compared to earlier patient series, which used percutaneous transluminal coronary angioplasty (PTCA) only or PTCA in combination with BMS implantation. Application of drug-eluting stents should lower the restenosis rate, but their potentially beneficial effect on the target vessel revascularization (TVR) is not supported by data from the included studies . The TVRA relatively small number of studies in our sample  comparedCurrently, only a few studies have explicitly explored the costs associated with hybrid coronary revascularization. De Cannière and colleagues were the first to quantify costs associated with HCR and to compare these costs with costs involved in conventional double CABG . Costs wP < 0.001). In conclusion, the difference in intraoperative costs was almost completely outweighed by the lower postoperative costs in the hybrid group. This resulted in slightly, but not significantly, higher overall costs in the hybrid group.  Kon et al. showed that shorter intubation times, shorter ICU and hospital length of stay, and less PRBC transfusions resulted in a significant reduction in costs for hybrid treated patients in the postoperative period . ConversThe nonhealthcare costs after HCR will presumably be lower than after CABG or OPCAB because both Kon et al. and de Cannière et al. showed that return to work was significantly faster in the hybrid group, leading to a marked reduction in absenteeism from work in hybrid treated patients , 12. ThiThis review is the largest and most comprehensive report to date comparing the clinical outcomes of patients who underwent either hybrid coronary revascularization or conventional on- or off-pump CABG for multivessel coronary artery disease. Three principal findings were revealed as follows: (1) hybrid treated patients showed a significantly faster recovery with lower PRBC transfusion requirements and less in-hospital major adverse cardiac and cerebrovascular events than patients treated by on- or off-pump CABG; (2) staged procedures were associated with considerable period of times between both procedures, leaving patients incompletely revascularized and in theory at risk for cardiovascular events for a considerable length of time; and (3) the invasiveness of surgical LITA to LAD bypass grafting appeared to influence the clinical outcome, with higher MACCE and 30-day mortality rates in patients treated by more invasive surgical techniques using CPB and/or median sternotomy.As with any review, this report shares the limitations of the original studies. First, the initial reports especially included a relatively small number of patients, which may have resulted in biased results due to outliers. Furthermore, almost all studies were performed retrospectively with inherent patient selection bias, since the decision to perform the HCR procedure was taken on an individual and highly selective basis according to cardiac surgeon and interventional cardiologist discretion. Likewise, the inclusion and exclusion criteria used to select high-risk patients for the HCR procedure differed notably between the included studies, yielding a very heterogenic population. In addition, the used surgical techniques to perform the LITA to LAD bypass graft varied considerably, with learning curve issues and different levels of expertise and equipment. All these factors potentially contribute to heterogeneity, which may reduce the certainty of the evidence presented in this review. Moreover, the mean length of followup was generally short, almost never exceeding two years, which made it difficult to assess long-term clinical outcomes of hybrid treated patients. Therefore, this review relies mainly on in-hospital and short-term outcomes to assess the safety and feasibility of the HCR procedure. Another limitation was the lack of long-term systematic and routine angiographic followup of graft and stent patency in the majority of studies included in the present review, which precluded any conclusions about the graft and stent longevity of the HCR procedure. Furthermore, the comparative studies lacked randomization and nonblinded assessment of outcome, which might have led to selection bias and might have influenced outcome measures by preconceived notions about the superiority of the HCR procedure. Finally, postoperative pain, which might be higher in patients treated with conventional MIDCAB, was not included as outcome measure in the present review, because only a limited number of studies assessed this outcome measure. Notwithstanding these weaknesses and limitations, this review selected the best evidence currently available to give a broad and comprehensive overview of the preliminary results of the HCR procedure. Larger, multicenter, prospective, randomized trials with long-term clinical and angiographic followup and cost analysis comparing HCR with both conventional on-pump and off-pump CABG or multivessel PCI will be necessary to further evaluate whether this hybrid approach is associated with similar promising long-term results. In the meantime, the first prospective, randomized pilot trial to compare HCR with conventional CABG in patients with multivessel coronary artery disease has been started . These dThe large variability in HCR techniques makes it difficult to draw firm conclusions from the currently available evidence, but HCR appears to be a promising and cost-effective alternative for CABG in the treatment of multivessel coronary artery disease in a selected patient population. The HCR procedure was associated with short hospital stays (including ICU stay and intubation time), low MACCE and 30-day mortality rates, low PRBC transfusion requirements and TVR, high postoperative LITA patency rates, and high survival rates. These promising early outcomes warrant further research with larger sample size, multicenter RCTs to determine the definite place of HCR in the current therapeutic armamentarium against coronary artery disease. Until then, this review justifies the continued use of the hybrid approach, but careful patient selection and close cooperation between cardiac surgeons and interventional cardiologists will determine the clinical outcomes to a significant extent.


Recent advances in neuroscience have suggested that exercise-based behavioral treatments may improve function and possibly slow progression of motor symptoms in individuals with Parkinson disease (PD). The LSVT (Lee Silverman Voice Treatment) Programs for individuals with PD have been developed and researched over the past 20 years beginning with a focus on the speech motor system (LSVT LOUD) and more recently have been extended to address limb motor systems (LSVT BIG). The unique aspects of the LSVT Programs include the combination of (a) an exclusive target on increasing amplitude , (b) a focus on sensory recalibration to help patients recognize that movements with increased amplitude are within normal limits, even if they feel “too loud” or “too big,” and (c) training self-cueing and attention to action to facilitate long-term maintenance of treatment outcomes. In addition, the intensive mode of delivery is consistent with principles that drive activity-dependent neuroplasticity and motor learning. The purpose of this paper is to provide an integrative discussion of the LSVT Programs including the rationale for their fundamentals, a summary of efficacy data, and a discussion of limitations and future directions for research. Progressive neurological diseases, such as Parkinson disease (PD) impair speech, swallowing, limb function, gait, balance, and activities of daily living. Even with optimal medical management  these deficits cannot be controlled satisfactorily in the vast majority of individuals with PD and have a negative impact on quality of life –3. RecenNearly 90% of individuals with PD have speech and voice disorders that negatively impact communication abilities , 19. The Historically, speech treatment for individuals with PD was viewed as futile, in as much as treatment gains were minimal and short lived . Today, In contrast, traditional speech therapy typically involves multiple speech system targets , is low intensity , and does not systematically address the sensory processing deficits related to self-perception of loudness by individuals with PD  studies have been conducted , 28. DatIn addition, various physiologic changes such as increased movement amplitude of the rib cage (larger excursions) during speech breathing , increasTraining vocal loudness also has been studied for its distributed effects across the speech production system. In a series of smaller pilot studies (subsets of data from larger study) data have documented improvements in orofacial movements, as reflected in consonant articulation , tongue 15 PET in a small number of individuals with PD have documented changes in brain function immediately following LSVT LOUD [215O-positron emission tomography. Z-score images were generated by contrasting reading with rest conditions for pre- and post-LSVT LOUD sessions, and neural activity was correlated with the corresponding change in vocal SPL (loudness). Narayana et al. [Two brain imaging studies using OSVT LOUD –55. The Individuals with PD perform movements that are hesitant (akinesia), slow (bradykinesia), and with reduced amplitude (hypokinesia). Changing from one motor program to another (set-shifting) may be disturbed and sequencing of repetitive movements may occur with prolonged and/or irregular intervals and reduced and/or irregular amplitudes . Externa Most current therapies rely on compensatory behavior and external cueing in order to bypass deficient basal ganglia function , 63–70. Presently two trials on the effectiveness of LSVT BIG have been published.A noncontrolled study assessed effects of LSVT BIG in 18 individuals with PD . Data do In the recently published rater-blinded Berlin LSVT BIG Study improvement in motor performance was compared in 60 individuals with PD, randomly assigned to receive LSVT BIG, Nordic Walking (as group treatment) or domestic training without supervision  a change The Berlin LSVT BIG Study is one of the few studies comparing specific types of physiotherapy with both active comparators and inactive controls. Sage and Almeida  reportedWe hypothesize that training-induced increases in movement amplitude target the proposed pathophysiological mechanisms underlying bradykinesia/hypokinesia—inadequate muscle activation . The musexclusive focus. We hypothesize that a single, overlearned cue (louder voice/bigger movements) may minimize cognitive load and mental effort [The idea of targeting amplitude in rehabilitation for individuals with PD is not new. Training vocal loudness (amplitude) is consistent with approaches recommended for treating motor speech disorders that (a) create a single motor organizing theme, (b) have a maximum impact on other aspects of speech production, and (c) increase effort across the speech mechanism –83. Furtl effort  and possThe training mode of LSVT Programs is consistent with some principles that promote activity-dependent neuroplasticity , 87 incl“in my normal everyday life, I just exaggerate my movements. I keep things big when I reach for things, or when I bend or when I walk; and when I talk–I keep my voice loud.”Sensorimotor processing deficits during speech and movement have been well documented , 88–91. The hypothesized concepts underlying recalibration in LSVT Programs have yet to be systematically tested in pre/posttreatment experiments. However, there is evidence that cognitive training is possible in individuals with PD , includiThere are a number of limitations to the scope of research on LSVT Programs and we have highlighted some of the key areas below. First, there is a need to better define prognostic variables for who will respond best to LSVT Programs and what outcomes can be expected in individuals with a variety of factors, such as depression, dementia, apathy, orthopedic complications, and dyskinesias, as well as atypical PD and post-DBS surgery. While the majority of LSVT outcome data have been reported on individuals with idiopathic PD, single subject, case study and small group designs have documented post-LSVT-LOUD improvements in individuals after neurosurgery and with atypical parkinsonism –97. HoweOur ongoing work in LSVT LOUD is addressing questions related to the importance of the treatment target versus the mode of delivery. Specifically, we are comparing two treatment targets: vocal loudness training (LSVT LOUD) versus orofacial/articulation training (LSVT ARTIC) and the effects on measures of speech intelligibility, speech acoustics, facial expression, and swallowing. The two treatments are standardized and matched in terms of mode of delivery . LSVT LOUD focuses on training healthy vocal loudness across speech tasks , with focused attention on how it feels and sounds to talk LOUD, whereas, LSVT ARTIC focuses on high-force articulation or enunciation across speech tasks , with focus on how it feels to have high-effort enunciation. Preliminary data examining single-word intelligibility in noise conditions  and faci The impact of DBS on speech is an urgent area of research. Tripoliti and colleagues  are asseAdvances in computer and web-based technology offer potentially powerful solutions to the problems of treatment accessibility, efficacious dosage delivery, and long-term maintenance in rehabilitation –104. Pre Understanding neural mechanisms of both speech and movement disorders in PD as well as mechanism of treatment-related change are of great promise to help improve treatment outcomes. As part of our ongoing work we are examining neural changes (PET imaging) in individuals with PD across the LSVT LOUD, LSVT ARTIC, and Untreated groups. Hypothetically, intensive practice of speech enunciation by the LSVT ARTIC regimen should strengthen cortically mediated speech articulation in PD, beyond the improvement associated with LSVT LOUD. To our knowledge, this will be the first imaging study of comparison speech treatments in individuals with PD including long-term followup (3 months). Developing parallel imaging studies before/after LSVT BIG is of great interest to us both in terms of understanding reorganization of brain activation patterns following treatment but also to understand differences between using amplitude to treat speech versus limb motor systems. Finally, whereas studies of movement and limb/gait exercise in animal models of PD have contributed immensely to the literature, there have been no analogous models for studying vocalization deficits. Today, emerging models of vocal motor deficits following dopamine depletion in rodents (rats and mice) and songbirds offer promise for the feasibility and value of these models –114. Via Collectively these ongoing studies have the potential to improve our understanding of the underlying mechanisms of speech-treatment-related changes in individuals with PD and will help guide treatment improvements. Future research will address the underlying bases for treatment-related changes that have a beneficial impact on speech and movement and thus quality of life in individuals with Parkinson disease.


Circadian and homeostatic sleep-wake regulatory processes interact in a fine tuned manner to modulate human cognitive performance. Dampening of the circadian alertness signal and attenuated deterioration of psychomotor vigilance in response to elevated sleep pressure with aging change this interaction pattern. As evidenced by neuroimaging studies, both homeostatic sleep pressure and circadian sleep-wake promotion impact on cognition-related cortical and arousal-promoting subcortical brain regions including the thalamus, the anterior hypothalamus, and the brainstem locus coeruleus (LC). However, how age-related changes in circadian and homeostatic processes impact on the cerebral activity subtending waking performance remains largely unexplored. Post-mortem studies point to neuronal degeneration in the SCN and age-related modifications in the arousal-promoting LC. Alongside, cortical frontal brain areas are particularly susceptible both to aging and misalignment between circadian and homeostatic processes. In this perspective, we summarize and discuss here the potential neuroanatomical networks underlying age-related changes in circadian and homeostatic modulation of waking performance, ranging from basic arousal to higher order cognitive behaviors. Aging can be defined in terms of life and time Martin,  and it iIn the 1970s and 1980s, coupled oscillator models were shown to reproduce the basic features of the timing of human sleep and wake episodes, with one oscillator representing sleep/wake and the other representing the circadian pacemaker driving the temperature cycle Wever, . AlternaThere is ample evidence that the interplay of circadian and homeostatic processes also determines the temporal modulation of sleepiness and alertness levels across the day, which in turn affects performance for different cognitive domains  studies in young volunteers yielded evidence that this interaction also influences cognition-related cortical  and subcortical  brain activity  was associated with a phase-advance in the circadian melatonin profile and an earlier occurrence of the melatonin peak within the sleep episode. Furthermore, older PER35/5 participants accumulated more nocturnal wakefulness, had increased EEG frontal delta activity (0.75–1.5 Hz), and decreased EEG frontal sigma activity (11–13 Hz) during (Non-REM) sleep compared with PER34/4 participants.Additionally, it is worth noting that the extent of age-related changes in circadian and sleep physiology substantially differs between individuals. For instance, the above mentioned polymorphism in the human clock gene PER3 may contribute to inter-individual differences in sleep and circadian physiology in older people  would help to elucidate their relationships.Healthy older individuals may experience twice as much time awake during a night sleep episode than young adults, suggesting that impaired sleep consolidation is associated with aging 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.


Equine infectious anemia (EIA), identified in 1843 [ DThe complex and lengthy maturation of immune responses to EIAV infection has also been observed in sera from HIV-infected patients as well as experimental infections by SIV, SHIV, and FIV, indicating a common theme in lentivirus immunology. However, the progressive changes in qualitative antibody properties may differ among the different viral infections. For example, EIAV immune maturation of the humoral immune response resembles that identified in HIV, whereas SIV, SHIV, and FIV antibody maturation differ principally in the evolution of conformation ratios or the predominant recognition of linear/conformational epitopes . ParalleExamination of the host cell-mediated immune response to EIAV reveals CD4 and CD8 MHC-restricted, EIAV-specific cytotoxic T-lymphocyte (CTL) activity apparent within three to four weeks post-infection, temporally correlating with the resolution of the primary viremia. After resolution of the initial acute episode, the breadth of EIAV-specific CTL activity differs greatly among experimentally infected ponies, ranging from readily detectable CTL to undetectable activity. The majority of the protective CTL responses appear to be directed towards Gag epitopes, although Env sequences have been loosely correlated with protective immunity ,48. NeveEIAV vaccine studies indicate a correlation between the extent of antibody maturation achieved by a particular immunization strategy and the potential for eliciting protective or enhancing vaccine immunity . AlthougLentiviral Env diversity has a tremendous effect on the host’s ability to control viral infection and disease. As the viral envelope protein is a primary source of antigenicity and interacts with the host immune system to a much larger extent than any other viral protein, it is often the main target in vaccine development. Thus, Env diversity has a remarkable impact on the development of an effective vaccine for lentiviruses in general as well as specifically for control of worldwide EIAV infections. Diverse experimental EIAV vaccine strategies have been evaluated for their ability to achieve mature protective immune responses. These exploratory vaccine trials include various live-attenuated (attenuated) virus strains, inactivated whole virus candidates, DNA, and particulate immunogens, viral or recombinant envelope protein subunit immunogens, and Gag subunit immunogens ,57,58,59The cumulative data on infection and candidate vaccine trials indicate that viral Env protein is indeed a primary determinant of vaccine efficacy and that the natural variation observed in Env quasispecies represents a major challenge to vaccine development. A significant, inverse, linear correlation between vaccine efficacy and increasing divergence of the challenge virus gp90 compared to the vaccine virus gp90 protein has been identified utilizing EIAV attenuated vaccine studies . The resStudies of EIAV pathogenesis, host immune response, and vaccine efficacy have provided many invaluable lessons for HIV/AIDS research. EIAV, like HIV, possesses an extremely variable genome with the majority of genetic mutations centralized within the Env protein. Env evolution and diversity have profound effects on host immune control of the infection and disease, as well as the development of an effective vaccine. The prominent demarcation of viremic episodes as well as the unique ability of the host to naturally achieve immunological control over disease made EIAV studies highly relevant to understanding basic principles of lentivirus pathogenesis. Furthermore, EIAV research has promoted fundamental understanding of the mechanisms by which Env variation circumvents vaccine immunity and informed the development of novel immunization strategies to maximize effective immune recognition of variant Env species.


Drought-induced tree mortality, including large-scale die-off events and increases in background rates of mortality, is a global phenomenon , where g is conductance of the surface boundary layer (Figure An increase in VPD affects both soil evaporation and plant physiology. Soil evaporation is affected by physical relationships with increased VPD under warmer temperature via Fick's law: E = r Figure . This car Figure ], transpr Figure . Such str Figure , 2011: ar Figure . If embor Figure . Carbon r Figure . IncreasEucalyptus (Eamus et al., Note that the VPD effects on physiology described above do not negate the direct negative impact rising temperature alone can have on plant survival. Although growth respiration is reduced during drought (Amthor and McCree, Pinus and Eucalyptus. Needed to compliment more mechanistic approaches are also experimentally determined climate-mortality envelopes that are specific to drought-induced tree mortality. Although much uncertainty remains about the specifics of the mechanisms underlying mortality, our principle point is that the risk posed by intensifying atmospheric moisture demands to future tree mortality and associated die-off events remains a critical but little-studied aspect in this domain. Additional study is required if we are to effectively predict and manage the consequences of future climate change and tree mortality. In summary, we need to shift focus to the critical amplifying role of VPD, not just of associated temperature, in driving tree mortality during drought because VPD changes impose fundamental curvilinear physical and physiological responses. Importantly climate models consistently predict VPD as well as temperature to increase in the future and these trends will almost certainly increase forest stress, tree mortality, and associated large-scale tree die-off events in many regions globally.The fundamental fine-scale relationships of increased temperature and associated increased VPD have profound global-scale implications for the future distributions of vegetation. VPD and temperature are core constituents of the three principle climatic determinants of the distributional patterns of vegetation, as exemplified in the Holdridge  life-zon


Environmental enrichment is a popular strategy to enhance motor and cognitive performance and to counteract the effects of various harmful stimuli. The protective effects of enriched environment have been shown in traumatic, ischemic and toxic nervous system lesions. Monosodium glutamate (MSG) is a commonly used taste enhancer causing excitotoxic effects when given in newborn animals. We have previously demonstrated that MSG leads to a delay in neurobehavioral development, as shown by the delayed appearance of neurological reflexes and maturation of motor coordination. In the present study we aimed at investigating whether environmental enrichment is able to decrease the neurobehavioral delay caused by neonatal MSG treatment. Newborn pups were treated with MSG subcutaneously on postnatal days 1, 5 and 9. For environmental enrichment, we placed rats in larger cages, supplemented with different toys that were altered daily. Normal control and enriched control rats received saline treatment only. Physical parameters such as weight, day of eye opening, incisor eruption and ear unfolding were recorded. Animals were observed for appearance of reflexes such as negative geotaxis, righting reflexes, fore- and hindlimb grasp, fore- and hindlimb placing, sensory reflexes and gait. In cases of negative geotaxis, surface righting and gait, the time to perform the reflex was also recorded daily. For examining motor coordination, we performed grid walking, footfault, rope suspension, rota-rod, inclined board and walk initiation tests. We found that enriched environment alone did not lead to marked alterations in the course of development. On the other hand, MSG treatment caused a slight delay in reflex development and a pronounced delay in weight gain and motor coordination maturation. This delay in most signs and tests could be reversed by enriched environment: MSG-treated pups kept under enriched conditions showed no weight retardation, no reflex delay in some signs and performed better in most coordination tests. These results show that environmental enrichment is able to decrease the neurobehavioral delay caused by neonatal excitotoxicity. The test was performed at a speed of 13 rpm. The pups were placed on the rotating drum and the time the animal could stay on the rota-rod was measured (maximum 2 min).Rat pups were tested for motor coordination twice a week between 2 and 5 weeks of age. (1) Grid-walking and footfault test: rats were placed on a stainless steel grid floor . Statistical analysis was performed using two-way analysis of variance (ANOVA) followed by Bonferroni-Dunn’s posthoc analysis. Results were considered significant when In summary, our present results show that enriched housing conditions can reduce the delaying effects of the excitotoxic glutamate treatment on neurobehavioral development in newborn rat pups.


Aging is associated with a tissue degeneration phenotype marked by a loss of tissue regenerative capacity. Regenerative capacity is dictated by environmental and genetic factors that govern the balance between damage and repair. The age-associated changes in the ability of tissues to replace lost or damaged cells is partly the cause of many age-related diseases such as Alzheimer's disease, cardiovascular disease, type II diabetes, and sarcopenia. A well-established marker of the aging process is the length of the protective cap at the ends of chromosomes, called telomeres. Telomeres shorten with each cell division and with increasing chronological age and short telomeres have been associated with a range of age-related diseases. Several studies have shown that chronic exposure to exercise  is associated with telomere length maintenance; however, recent evidence points out several controversial issues concerning tissue-specific telomere length responses. The goals of the review are to familiarize the reader with the current telomere dogma, review the literature exploring the interactions of exercise with telomere phenotypes, discuss the mechanistic research relating telomere dynamics to exercise stimuli, and finally propose future directions for work related to telomeres and physiological stress. Broadly, aging is defined as the accumulation of cellular damage that results in a loss of cellular and organismal fitness. Aging is marked by a substantial decrease in the regenerative potential of several cell types, including immune cells and skeletal muscle cells , 2. BothInterestingly, all of the aforementioned age-related diseases are modified by physical activity. Further, recent evidence indicates that telomere length is also associated with several age-related diseases, and that telomere length and the suite of proteins that maintain telomere length are altered by changes in physical activity level . Thus, tn-3′) at the ends of linear chromosomes [Recently, short telomeres have become a widely accepted molecular/cellular hallmark of aging . Telomeromosomes . With eaomosomes , 11). Inomosomes –15. Theromosomes , 14. Thuomosomes –21). An omosomes –24). WhiRepeated cell division is one mechanism for reduced telomere length; however, other mechanisms also contribute to the total rate of telomere shortening in cells . For exaCertain cell types, even with continuous cell division, are able to maintain their telomeres despite the end-replication problem. In highly mitotic cells , the gradual telomere shortening due to the end-replication problem is overcome by the enzyme telomerase . TherefoTelomerase is a ribonucleoprotein that consists of two central components: a protein reverse transcriptase component (TERT) and an RNA template (TERC) , 27, 28.Shelterin acts as both a positive and negative regulator of telomere length , 35 and Shelterin consists of six proteins: telomere-repeat binding factors (TRF) 1 and 2, protection of telomeres (POT1), RAP1, TPPI, and TIN2  that are related to telomere maintenance. Physiological stressors such as lifestyle choices and psychological stress have been shown to influence telomere length and telomerase enzyme activity , 41–45. Regular physical activity and exercise training (both resistance exercise and endurance exercise) are known to reduce the risk of developing many age-related chronic diseases such as cardiovascular disease, certain cancers, type II diabetes, and sarcopenia . ExercisWe performed a systematic review of the relevant literature using the NCBI PubMed database. The following search terms were used: telomere length and exercise (45 results), telomere length and physical activity (74 results), telomerase and exercise (21 results), and telomerase and physical activity . Studies were included in the review if they met the following criteria: (1) measured telomere length (by any method); (2) determined physical activity status either by physiological measure  or survey . The final number of studies included in the review concerning telomere length and physical activity/exercise was 23  and for this reason very few well-controlled exercise training intervention studies have been performed in humans. The majority of the research on human telomere length and exercise has been done retrospectively on banked DNA samples from immune cells , 56. SinMultiple cross-sectional studies have described the associations between physical activity and/or exercise training and telomere length in immune cells, with three different relationships reported: a positive association, no association, and an inverted U relationship. In the inverted U relationship, sedentary individuals and extremely active individuals have shorter telomeres than moderately active individuals. The cross-sectional nature of these studies, their small sample size, variation in collection of exercise and physical activity data, methods of telomere length determination, cell types used for DNA extraction, and the various ages of the individuals in the study cohorts likely explain the discrepant results.Several studies have reported a positive association between physical activity and telomere length, in that active individuals have longer telomeres in immune cells compared to sedentary individuals , 57–60. Studying a group of marathon runners compared to sedentary age- and sex-matched individuals, Mathur et al.  found noA few studies have described an inverted U relationship between physical activity and telomere length where moderately active individuals exhibit longer telomeres compared to both sedentary and extremely active individuals. Ludlow et al.  showed tSeveral factors must be considered when interpreting the mixed associations between telomere length and exercise/physical activity levels. Specifically, sampling bias, cell type, age of individuals when measures were made, and the timing of sample collection may all influence study outcomes. For instance, because circulating stem cells are released following exercise –81, if aAnother important factor that should be considered is the method of telomere length determination . TelomerWhile the current research findings are divergent, the overall consensus from these studies is that moderate levels of physical activity are associated with longer telomere length in immune cells. To expand upon these findings, future studies should focus on how moderate exercise can maintain the shortest telomeres and not just mean telomere length in leukocytes. Cross-sectional studies have provided several key insights, but the limitations of these types of studies have produced mixed results that may be biased by sample collection, cell type utilized, and telomere length measurement methodology.Randomized control clinical exercise trials would eliminate some of the bias associated with cross-sectional studies and help to clearly define the effects of exercise (both acute and long-term training) on telomere length and telomere biology. However, to date few longitudinal studies investigating the relationship between telomere length and exercise have been performed in humans. Shin et al.  exerciseRecently, Ornish et al. , 50 compVery few studies have investigated the effects of acute exercise  on telomere length. Endurance exercise, specifically marathon running, is a potent immune cell proliferative stress  and causThe consensus from the above studies in immune cells is that telomere length decreases with age in sedentary individuals, longer telomeres are observed in individuals who are moderately active , and extreme long-duration endurance training for an extended portion of one's lifetime may result in telomere shortening. These divergent responses are likely directly related to the antioxidant capacity of the immune cells and the proliferative demand placed on the progenitor cells by the exercise stimulus.Skeletal muscle has unique telomere biology when compared to other tissues. Skeletal muscle consists of a syncytium of multinucleated muscle fibers that are postmitotic; thus, telomere length should remain stable in this population of nuclei, with the rare exception of DNA damaging stimuli . In addiMuscle contraction, such as heavy-resistance-type exercise, is known to cause injury to skeletal muscle and thus is a stimulus for satellite cell proliferation. Recently, long-duration and high-intensity endurance exercise was shown to cause satellite cell replication in skeletal muscle . Since eTwo studies by the same group have investigated the effects of long-term endurance training and found that exercise may cause telomere shortening in skeletal muscle of chronically trained individuals. Though the sample size was small, Collins et al.  investigSince heavy-resistance exercise is a well-known muscle damaging and satellite cell proliferative stimulus, researchers have investigated the effect of chronic resistance training on telomere length in skeletal muscle. Kadi et al.  comparedNot all data in skeletal muscle indicate that telomere shortening occurs with physical activity. Ponsot et al.  investigIn summary, the data for how exercise and physical activity influence telomere biology in human skeletal muscle are mixed and need clarification with longitudinal experiments. The effect of exercise on skeletal muscle telomere length is likely directly linked to the proliferative demand the exercise places upon the skeletal muscle. Thus, the more damaging the exercise stimulus (either endurance- or resistance-type exercise) to the muscle fibers and the greater the proliferative demand upon the muscle stem cells, the faster the rate of skeletal muscle telomere shortening. Furthermore, the longer the exercise duration, both in terms of length of individual exercise bouts and years of training an individual performs, the greater the observed decrease in telomere length. Another potential mechanism to consider is the antioxidant capacity of the skeletal muscle, which could be overwhelmed by contraction-induced reactive oxygen species (ROS) and result in damage to telomere DNA and the stochastic loss of telomere length, as has been shown in other tissues exposed to ROS –100. FurAre Telomere Lengths Synchronized across Tissues? Telomere length in mammalian species is tissue-specific [specific , 103. Thspecific , while ispecific , telomerspecific . Danialispecific  collectespecific  observedThe data from the skeletal muscle telomere and exercise literature seem to directly conflict with Daniali et al.'s  data. AcFew reports have described the effects of physical activity, exercise training, or acute exercise on telomerase, shelterin, and other telomere-associated proteins , 74. Of Strenuous exercise is a known proliferative stress for immune cells . MitogenIn support of this hypothesis, Werner et al.  showed tSince human telomeres are slow to shorten , groups have turned to model organisms, such as rodents, to explore the relationship between exercise and telomere dynamics. However, rodents require special considerations when studying telomere biology. In this section, we discuss the positives, negatives, and caveats to using rodents for telomere-related studies and also describe the work that has been completed using mice as model organisms in this area of investigation.mus musculus cataneous, CAST/Ei) or models where gene disruption of telomerase components, mTERT and mTERC, has been performed [Though the biology of telomere mechanics is similar between humans and rodents, human telomeres are typically only 7–15 kilobases in length , while rerformed –115.Naturally occurring shorter telomere rodents, such as CAST/Ei mice, are typically wild derived, more recently inbred, and have telomere lengths (15–20 kb) much closer to those of humans (7–15 kb) , 116. SeAnother way to induce shorter telomere lengths in rodents is by knocking out either the protein catalytic subunit (mTERT) or the RNA component (mTERC) of telomerase. These models are unique in that the first generation of knockout animals retain wild-type telomere lengths while offspring generations three through six display shorter telomere lengths and aging phenotypes compared to age-matched wild-type controls . In addiTo date only two groups have investigated the effect of long-term exercise training on telomere length in rodent models , 63, 117Ludlow et al.  performeSeveral groups have investigated how exercise may alter the expression of telomere-related proteins in rodent tissue. Werner et al. , 117 perIn a different strain of mice (CAST/Ei), Ludlow et al.  observedOverall these data indicate that telomere-binding proteins and telomerase adapt to exercise training in a tissue-specific fashion. Some tissues upregulate telomerase enzyme activity, while other tissues seem to alter the expression of shelterin components and DNA damage response and repair genes. These data indicate that exercise likely results in tissue-specific adaptation of the telomere maintenance pathways; however, regardless of tissue-specific effects, it is important to note that exercise results in a phenotype that is teloprotective in most tissues studied to date.Trf1 following acute exercise in rodent skeletal muscle [Elucidating the mechanisms of how telomere length is preserved or lost following exercise is important for understanding how telomeres respond to physiological stressors. To date most mechanistic studies on exercise have focused on the stress response and growth/cellular proliferation pathways. Only three studies have investigated exercise-specific signaling mechanisms associated with altered telomere biology, all of which have been performed in rodent cells and tissues , 63, 117l muscle . These dExploration of the effect that physiological stressors such as exercise and exercise training have on the structure-function relationship of the genome is fertile ground. Researchers should consider the following five directions of importance for future research: (1) determining whether or not telomere length shortens at equal rates across somatic tissues in response to exercise training; (2) if an inverted U relationship exists between physical activity and telomere length; (3) the tissue-specific functional consequences of short telomeres in trained versus untrained individuals; (4) the role of oxidative stress and inflammation during and following exercise and the effects on telomere biology; and (5) the specific pathways  that cause the adaptation and response of telomerase and shelterin to exercise and how these adaptations result in altered telomere length.Numerous studies have implicated a telomere-protective phenotype induced by moderate levels of physical activity, indicating an important cellular adaptation that may slow the onset of symptoms or prevent certain age-related diseases. In contrast, several lines of evidence in both immune cells and skeletal muscle indicate that telomeres may actually shorten in response to long-term high-intensity endurance training. As such, the tissue-specific response of telomeres should be investigated, with specific consideration given to the proliferative demands placed on the tissue by the exercise stimulus and the antioxidant capacity of the individual tissues. Understanding how telomeres adapt on a tissue-specific basis and if immune cells are predictive of the adaptive response of other tissues is a necessary next step in this field. Additionally, determination of the type, time, intensity, and frequency of exercise that results in an excess proliferative demand on immune and skeletal muscle tissues and results in loss of telomere length is important. A multidisciplinary approach must be taken to tackle these important questions and to further solidify telomere length as a useful biomarker in monitoring the long-term effects of environmental and physiological stressors, such as exercise training.


Anopheles maculipennis s.l. mosquitoes the apparently nearly complete genome of one previously undescribed virus related to chronic bee paralysis virus, and, from a pool of Ochlerotatus caspius and Oc. detritus mosquitoes, a nearly complete entomobirnavirus genome. We also reconstructed long sequences (1503-6557 nt) related to at least nine other viruses. Crucially, several of the sequences detected were reconstructed from host organisms highly divergent from those in which related viruses have been previously isolated or discovered. It is clear that viral transmission and maintenance cycles in nature are likely to be significantly more complex and taxonomically diverse than previously expected. We investigated whether small RNA (sRNA) sequenced from field-collected mosquitoes and chironomids (Diptera) can be used as a proxy signature of viral prevalence within a range of species and viral groups, using sRNAs sequenced from wild-caught specimens, to inform total RNA deep sequencing of samples of particular interest. Using this strategy, we sequenced from adult  The emergence of new infectious diseases, many of which are caused by RNA viruses, is a major threat to human, animal and plant health and agriculture. RNA viruses demonstrate remarkable capacity to evolve due to large population size, short generation times and high mutation and recombination rates. Many are also vector-borne, potentially increasing the prevalence and range of a given virus in natural ecosystems. Understanding the mechanisms underlying viral emergence is key for the rational design of antiviral therapies and control strategies. A first step in this process is the characterisation of the true distribution of virus genetic variability, both spatially and taxonomically across different host species. Recent research has demonstrated that there exists a vast diversity of previously undiscovered viruses in the natural environment . For exaStegomyia aegypti [=Aedes aegypti] mosquitoes infected with dengue virus (DENV) or Sindbis virus (SINV), Culex quinquefasciatus mosquitoes orally exposed to West Nile virus (WNV) and Drosophila infected with flock house virus (FHV) . The first isolations of ISFs from the natural environment include CFAV from Puerto Rico and Kamiti River virus (KRV) from Kenya [Culex flavivirus (CxFV) has been isolated and characterised from Culex mosquitoes from Asia, the Americas and Africa [Aedes flavivirus (AeFV) from Stegomyia species in Japan [Culex tritaeniorhynchus in Vietnam [Mansonia africana nigerrima in Uganda [Culex, Aedimorphus, Ochlerotatus and/or Stegomyia that may represent DNA integrations into genomes of mosquitoes [Culex mosquitoes [Culex genome will eventually be found to contain some integrated flaviviral-like sequence in common with Aedimorphus, Ochlerotatus and/or Stegomyia species [We identified a partial flavivirus-related sequence in adult chironomids. The sequence was most closely related to Culex flavivirus and other ISFs. Cell fusing agent virus (CFAV) was the first ISF to be discovered over 30 years ago during laboratory studies, in the supernatant medium from a om Kenya ,2. More d Africa ,62,62,64in Japan , Quang B Vietnam  and Nakin Uganda , which an Uganda . Our stusquitoes ,72,73,74squitoes . They sc species . The ISFCulex pipiens from a laboratory colony [bona fide viruses. Such controls must be incorporated in future studies, namely testing via (i) lack of reverse transcriptase and (ii) DNase-treatment of samples.Regardless of whether the ISF-like sequences detected in the current study originate from virus infection or genome integrations, either would likely result from a long history of contact and interaction between ISF-like viruses and a range of insect taxa. We may hypothesise a common source of virus association or infection for all three dipteran groups , potentially related to a shared environment and/or transmission and maintenance cycles. Previous studies have presented evidence for vertical transmission of ISFs: CFAV was isolated from both sexes of a number of mosquito species  and the y colony . Chironoy colony  and sharBunyaviridae in adult culicine mosquitoes. The contig contained a single long ORF that showed greatest similarity to members of the genus Phlebovirus, and the currently unclassified GOUV which was originally detected in specimens of the mosquito genera Anopheles (anophelines), Culex and Uranotaenia (culicines) in West Africa. Here, we provide evidence for a related novel virus from mosquitoes collected in France. Further field studies are necessary to allow the potential full isolation of this strain, which considering its divergence from GOUV, may constitute a novel genus within the family Bunyaviridae. In addition, we detected sequences in adult anopheline mosquitoes and adult culicine mosquitoes with similarity to the orbivirus VP1/RdRp-encoding segment (segment 1). Orbiviruses infect a range of hosts, including cattle, goats and sheep. The three economically most important orbiviruses are bluetongue virus, African horse sickness virus and epizootic hemorrhagic disease virus, all of which are transmitted by Culicoides species (midges of the dipteran family Ceratopogonidae). The detection of novel orbivirus sequences in both culicine and anopheline mosquitoes may warrant further study, with the aim of viral isolation and full characterisation. From adult chironomids and adult culicine mosquitoes, we also identified sequences related to Spissistilus festinus virus 1 (SpFV1) and Circulifer tenellus virus 1 (CiTV1). SpFV1 and CiTV1 were originally isolated from hemipteran insects  whereas the current study provides evidence for related sequences present in dipteran insects (both chironomids and culicine mosquitoes). We also detected a sequence related to the large segment of members of the family Culex flavivirus CxFV, Nakiwogo virus NAKV and Kamiti River virus KRV (flaviviruses), related to isolates first detected in the laboratory, e.g. DXV and CFAV. First, these novel naturally occurring strains provide useful comparisons for research on cell culture adapted strains such as DXV, and tools to examine the response of multi-passaged insect cell lines such as C6/36. In the wider picture, it is becoming clear in an increasingly large number of viral families that there is a diverse and highly prevalent background of transmission and maintenance of numerous viral strains across a range of host taxa against which known pathogens are evolving. The relevance of this genetic diversity in terms of potential competitive exclusion, superinfection or potential recombination events requires extensive study.We see parallels among many of the viral families encountered in this study, particularly the entomobirnaviruses and flaviviruses, which include recently discovered potential strains of highly divergent viruses in a range of species, e.g. ESV, CYV and MXV (entomobirnaviruses) and bona fide viruses from RNA transcripts of genome-integrated virus sequences. In the cases of the CBPV-like virus AACV and the entomobirnavirus CAZV, the presence of both segments, the high coverage, the presence of multiple well-supported (and often synonymous) polymorphisms and the presence of terminal motifs strongly suggest that these sequences derive from bona fide replicating viruses. The origin of the other sequences identified is less certain. Further investigations aimed at the isolation and full characterisation of viruses from a wide range of insects is a priority. The narnavirus-, mitovirus- and partitivirus-like sequences obtained in the current study may derive from fungal (or other) contaminants of the insect preparations, either external or internal (e.g. insect gut contents). Similarly, a number of plant-virus-like sequences (not reported) were detected in samples 7 and 9. During the analysis, we found a number of other virus-related sequences (not reported) that appeared to have fragmented ORFs , and some of these likely represent transcribed genome-integrated sequences. A large number of rhabdovirus-like sequence fragments were identified but were not further analysed as genome integration of rhabdovirus-like sequences has been shown to be particularly common in arthropod genomes [Although the sequences identified in the present study are certainly of viral origin, our methods do not allow us to definitively distinguish sequences of  genomes ,45. bona fide (non-integrated) virus sequence, e.g. if the host species sample preparation is not genetically uniform or if related sequences have been integrated at multiple sites within the same genome. Once integrated, such sequences evolve at a rate many orders of magnitude more slowly than replicating RNA viruses, and thus such sequences may provide a window on the time-scale of evolution of RNA viruses, which appears to occur at a much slower rate than what one would infer from extrapolating short-term evolutionary rates [In some cases, genome-integrated sequences appear to provide a functional benefit to the host, and are retained (exaptation) and may be subjected to purifying selection ,76,77. TMost of the sequences we identified contained the viral RdRp. This may be because the RdRp tends to evolve more slowly than other regions of an RNA virus genome; hence, it is easier to identify the RdRp in highly divergent viruses based on blastp analysis. This is also true for integrated sequences: whereas the RdRp of ancient RNA viruses, e.g. 10-100 mya, may resemble the RdRp of extant RNA viruses, other proteins of integrated ancient RNA viruses may not be detectable by blastp comparison to extant viruses. It is possible that applying structure-based homology search tools, such as hhpred , to our a priori knowledge. Except for the entomorbirnavirus-related sequences, all exhibited low amino acid sequence identities (24-42%) to known viruses.In this study, we report the discovery of viruses in wild-caught dipteran insects using deep sequencing of RNA. In our approach, total small RNAs were isolated from a field collection, sequenced via Illumina and assembled into contigs via Velvet and analysed using a custom-built bioinformatics workflow process. Results were used to inform total RNA sequencing of samples of particular interest . This strategy enabled the construction of two complete or nearly complete virus genomes and numerous other novel virus sequences with minimal


Steatornis caripensis: Caprimulgiformes), is nocturnal and eats fruit. The other is a selection of diurnal, insect-eating swiftlets  from across the Indo-Pacific. Bird echolocation is restricted to lower frequencies audible to humans, implying a system of poorer resolution than the ultrasonic (>20 kHz) biosonar of most bats and toothed whales. As such, bird echolocation has been labeled crude or rudimentary. Yet, echolocation is found in at least 16 extant bird species and has evolved several times in avian lineages. Birds use their syringes to produce broadband click-type biosonar signals that allow them to nest in dark caves and tunnels, probably with less predation pressure. There are ongoing discrepancies about several details of bird echolocation, from signal design to the question about whether echolocation is used during foraging. It remains to be seen if bird echolocation is as sophisticated as that of tongue-clicking rousette bats. Bird echolocation performance appears to be superior to that of blind humans using signals of notable similarity. However, no apparent specializations have been found so far in the birds' auditory system (from middle ear to higher processing centers). The advent of light-weight recording equipment and custom software for examining signals and reconstructing flight paths now provides the potential to study the echolocation behavior of birds in more detail and resolve such issues.The discovery of ultrasonic bat echolocation prompted a wide search for other animal biosonar systems, which yielded, among few others, two avian groups. One, the South American Oilbird ( Steatornis caripensis, von Humboldt, In 1794, Lazzaro Spallanzani reported that blinded bats oriented in complete darkness, and, except for the fluttering of their wings, did so silently. Almost 20 years later, Alexander von Humboldt entered a cave in Venezuela and heard resident Oilbirds  within the otherwise non-echolocating family Pteropodidae . Given the benefits of biosonar under conditions of poor visibility, seals and owls had been proposed as possible echolocators  than any other vertebrate  and capable of slow, maneuverable flight, with estimated flight speeds of 0.5–7 m/s, and of hovering in narrow spaces Snow, . Like otAerodramus) and non-echolocating  genera  also echolocate  than Oilbirds and all species have long, narrow wings  are assumed, but remain unconfirmed  folds the external tympaniform membranes into the syrinx  lumen toward the internal tympaniform membranes. The membranes are then set into vibration by the expiratory airflow. In Oilbirds, clicks are actively terminated by contraction of the single pair of intrinsic syringeal muscles . In contrast, the social vocalizations of Oilbirds are terminated passively by relaxation of the sternotrachealis muscles  in both groups is driven by subsyringeal pressure, initiated during expiration, and controlled by two antagonistic muscle pairs. Contraction of an extrinsic muscle pair  at Caripe, Venezuela Griffin, . SignalsKonishi and Knudsen  reported−1 . Fullard et al. , a simple cochlea Martin, , and thuExisting descriptions of echolocation signal parameters from Oilbirds also reveal discrepancies concerning signal duration Table  and raisA. hirundinacea detected vertical rods down to 10 mm diameter and potentially even smaller. These data suggest that swiftlets receive useful echo information via the higher frequency portions of their clicks, even though these components contain less energy. However, for this to be plausible the birds must hear, at least to some extent, higher frequencies. This is not supported by data from single neuron recordings from the midbrain auditory nucleus of Collocalia spodiopygia, which indicate best frequency thresholds from 0.8 to 4.7 kHz  at levels above chance  use double clicks to point their sound beam to the right and left of a target to trade localization over detection  and in Australia (A. spodiopygia) and never heard clicks from night-flying birds except as they returned to their roosts. Notably, however, Atiu Swiftlets (A. sawtelli) and Papuan Swiftlets (A. papuensis) click not only in their caves but also outside at night, apparently while hunting insect prey in low light  has spent considerable time listening for echolocation clicks from swiftlets on Papua New Guinea  are acoustically conspicuous to human observers. Two examples are the “screaming” parties of Common Swifts on the wing . At the same time click-type signals do not require the laryngeal specializations observed in bats necessary to produce a sufficiently short frequency-modulated signal. In the non-echolocating Chimney Swifts, none of the frequency-modulated and/or harmonic signals reported by Bouchard  would beA recent phylogenomic study of the birds embeds swiftlets within what appears to be the paraphyletic Caprimulgiformes, the avian order that includes Oilbirds  and custom-designed computational software should provide better quality recordings of biosonar signals from Oilbirds and swiftlets. Experiments could be designed to compare signals of birds flying in different contexts, for example, field versus captivity, open space versus cave interior and multiple versus single birds, to help resolve current uncertainties about signal design. Further, such recordings should help identify The highly specialized echolocation systems of toothed whales and laryngeal echolocating bats have provided and continue to provide fascinating insights into the mammalian auditory system and active sensory processes in animals across taxa. By comparison, echolocation in birds has received almost no attention. This is perhaps because we have implicitly regarded bird biosonar as unsophisticated and, thus, less interesting. Perhaps, less cynically, it is simply because bats are found everywhere, save past the tree-line and on a few isolated Oceanic islands, while echolocating birds are far less wide-spread and in general more difficult to gain access to than are bats.in vitro neuromuscular preparations to study the biomechanic mechanisms involved in avian and mammalian sound production (Elemans et al., Deployment of portable tags with hydrophones and accelerometers has contributed greatly to the understanding of toothed whale acoustic behavior in deep waters where the animals roam beyond visual inspection (Madsen et al., R. aegyptiacus uses echolocation to detect and discriminate objects better than previously suspected (Yovel et al., The tongue-clicking pteropodid bat 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.


Cre activity, which is induced in response to tamoxifen treatment, has been frequently exploited in mouse models of heart disease to investigate the effects of knocking out disease-associated genes in specific cardiac cells. Two articles published in this issue report results of investigations into Cre-recombinase-associated toxicity in mouse cardiac tissue. Nadia Rosenthal’s group systematically characterised the effects of Cre activity on cardiac morphology, physiology and function (1470page ). Their analysis revealed that a significant proportion of treated mice develop cardiac fibrosis in response to Cre activity. Interestingly, the severity of the phenotype varied in different genetic backgrounds, and toxicity was dependent on the frequency of tamoxifen injections. In the second study, Bernhard Kühn and colleagues substantiate these findings by demonstrating that Cre activity leads to cardiac toxicity and cardiomyocyte apoptosis in a dose-dependent manner (1459page ). Importantly, both studies ruled out the possibility that toxicity is caused by tamoxifen rather than Cre activity. Collectively, these analyses confirm that the Cre-loxP system can have unwanted, toxic effects in target tissue, highlighting the need for careful experimental design in studies utilising this tool and related genome-editing technologies.Bacterial endonucleases have become powerful tools for mammalian genome editing, and new technologies such as TALENs and CRISP/Cas9 are increasingly used in biomedical research. However, unwanted ‘off-target’ effects of these nucleases could limit their usefulness. The Cre-


In vitro and in vivo studies have demonstrated enhanced hypoxia and formation of reactive oxygen species (ROS) in the kidney following the administration of iodinated contrast media, which play a relevant role in the development of contrast media-induced nephropathy. Many studies indeed support this possibility, suggesting a protective effect of ROS scavenging or reduced ROS formation with the administration of N-acetylcysteine and bicarbonate infusion, respectively. Furthermore, most risk factors, predisposing to contrast-induced nephropathy, are prone to enhanced renal parenchymal hypoxia and ROS formation. In this review, the association of renal hypoxia and ROS-mediated injury is outlined. Generated during contrast-induced renal parenchymal hypoxia, ROS may exert direct tubular and vascular endothelial injury and might further intensify renal parenchymal hypoxia by virtue of endothelial dysfunction and dysregulation of tubular transport. Preventive strategies conceivably should include inhibition of ROS generation or ROS scavenging. Contrast media- (CM-) induced nephropathy (CIN) is an acute deterioration of renal function following administration of CM in the absence of any other known reason. CIN remains a leading cause of iatrogenic acute kidney injury, accounting for some 10% of in-hospital acute renal failure, despite adherence to protocols of risk assessment and prevention strategies. This reflects the unremitting increase in radiocontrast procedures for computerized tomography and vascular interventions, especially in high risk and elder patients with major comorbidities .CIN is commonly defined as an increase in serum creatinine concentration >0.5 mg/dL or greater than 25% of its previous value within 3 days after contrast medium administration, in the absence of other different causes .There are several predisposing factors to the development of CIN . PreexisWhile there is full agreement about the greater nephrotoxicity of high-osmolarity CM compared to others, it is not clear whether isoosmolar CM are less nephrotoxic than low-osmolarity CM, since clinical studies offer controversial results; all CM, however, may determine acute renal damage. Among procedure-related factors, the amount of injected CM is a crucial factor in determining CIN, since ROS generation appears to be strictly proportional to it .CIN is the result of combined hypoxic and toxic renal parenchymal injury; the latter is presumably mediated by reactive oxygen species (ROS). Experimental findings in vitro and in vivo illustrate that renal hypoxia induced by administration of iodinated CM enhances ROS formation within the kidney. Moreover, the protective effect on renal function of ROS scavengers or reduced ROS formation with specific drugs provides indirect evidence that ROS might be involved in the pathogenesis of CIN.2−) and hydroxyl radicals (OH−) by NAD(P)H-oxidase [Under physiological conditions, tubular transport is associated with ROS formation, mostly in the renal medullary thick ascending limb, where the extremely dense mitochondrial population represents a major source for generation of superoxide anions O− and hyAdministration of CM markedly decreases renal oxygenation in medullary structures, without reducing tubular reabsorption, as the result of neurohumoral vasoconstrictive stimuli, following the release of prostaglandins and endothelin from endothelial cells exposed to CM. This latter agent is markedly enhanced following radiocontrast studies, due to the activation of endothelin-converting enzyme-1.2O2), via xanthine oxidase. The same enzyme also helps xanthine generate uric acid and additional amounts of H2O2 that scavenge NO, further impairing renal medullary microcirculation. ROS and NO interactions play a crucial role in renal oxygenation and in the generation of CIN. Superoxide radicals, in fact, reduce NO bioavailability through the formation of peroxynitrite, further worsening vasoconstriction but also endothelial dysfunction, since in physiologic conditions NO prevents ROS-mediated endothelial cell injury and reduces transport-dependent ROS formation in the medullary thick ascending limbs (mTALs).This results in altered oxygen balance, leading to depressed activity of mitochondrial scavengers and enhanced formation of ROS. During hypoxia, greater amounts of ATP are hydrolyzed to ADP and AMP, further metabolized to adenosine and inosine by 5′-nucleotidase, and subsequently to hypoxanthine that generates xanthine and hydrogen peroxide  could not clearly demonstrate a direct role of CM in generating ROS, since in most of these experiments hydrogen peroxide, superoxide anion, or MDA levels were not increased after CM, despite the fact that cellular necrosis and apoptosis were observed, nor could addition of scavengers attenuate the extent of tubular cell damage , with thThe discrepancy with “in vivo” data mostly relies on the different environment in which tubular cells are cultured, far away from that observed in patients.Nonetheless, cellular studies offer the unique opportunity to evaluate the activation of intracellular signaling pathways involved in cellular apoptosis or necrosis, in the attempt to develop specific therapies to be used in vivo . Recent In these last years, in the attempt to reduce the incidence of CIN, greater attention has been devoted to the use of low- or isoosmolar agents at reduced doses, to the use of specific infusion protocols to hydrate patients, to the elimination of coexisting nephrotoxic agents, and to careful selection of patients. Nevertheless, CIN remains a major adverse iatrogenic complication through the use of CM . Clinicade novo cytosolic glutathione synthesis; in vitro studies have shown the ability of NAC to protect in a dose-dependent fashion cultured renal tubular cells incubated with high concentrations of low- and isoosmolar CM [NAC is a thiol-containing antioxidant able to permeate cell membranes that acts as a cysteine donor for molar CM .Tepel et al. first reported that administration of NAC was able to drastically reduce the risk of CIN: in a randomized, prospective study 83 patients with chronic stable renal failure undergoing CT scans were enrolled and pretreated with NAC or placebo. The incidence of CIN, defined as a rise in plasma creatinine >0.5 mg/dL within 48 hours, resulted only in 2% of NAC-treated patients, compared to 21% of the placebo group .The efficacy of NAC in CIN prevention was questioned by a number of subsequent clinical trials and meta-analyses showing opposite results , 30, 31,NAC pretreatment is also able to improve renal blood flow, as shown in rats, through a direct renal vasodilation  and to aThis could suggest that NAC enhances eNOS activity and NO generation, but its renal protection could be dissociated from its antioxidant effect.Bicarbonate infusion has also been proposed as a renoprotective factor in patients undergoing contrast studies; the rationale is that the alkalization might reduce the formation of hydroxyl radicals from hydrogen peroxide and of peroxynitrite. Although the increase of extracellular pH may ameliorate ROS-mediated proximal tubular injury in vitro , the impIn 119 patients with chronic renal failure, randomized to bicarbonate or saline, Merten et al. have reported a protective effect of sodium bicarbonate infusion on CIN: its solution (1.4%), given as a bolus either 1 hour before CM and for 6 hours after the procedure, was able to attenuate the incidence of CIN compared with the control saline treated patients .In a large prospective study in patients with chronic renal impairment undergoing coronary interventions, the combined administration of NAC and bicarbonate was superior to NAC alone, independent of whether ascorbic acid was present : the incThe results of repeated meta-analyses, however, consistently demonstrate a better outcome with bicarbonate infusion compared to saline-hydration, with a 50% reduction in CIN incidence –54, despP = NS) [Most recently the results of the BOSS trial, which compared sodium bicarbonate and saline for the prevention of a composite of death, renal replacement therapy, or progressive kidney failure over 6 months in patients undergoing any type of angiography, showed no difference on the primary composite endpoint in two group  .A report in patients with chronic renal failure has suggested that the antioxidant probucol may also prevent CIN (8% incidence in probucol-treated patients versus 15%) , and fewA further approach to CIN prevention is offered by inhibition of tubular carbonic anhydrase, that results in bicarbonaturia and alkaline urine that might attenuate ROS attack at the apical membrane of tubular epithelium; interestingly, acetazolamide administration ameliorated renal dysfunction in a CIN rat model , a resulTo date, however, the real impact of bicarbonate and acetazolamide on tubular intracellular pH and ROS formation during CM administration has not been evaluated.Data on other drugs effective in reducing CIN potentially by reducing ROS, like statin and ascorbic acid, remain controversial, but recent data suggest that a single high loading dose of atorvastatin administered within 24 hours before CM exposure is effective in reducing the rate of CIN patients at low to medium risk .More recently, our research group evaluated whether a novel isoform of a recombinant Manganese SOD (rMnSOD) could provide an effective protection against CIN: this molecule shares the same ability of physiological SODs in scavenging reactive oxygen species (ROS) but differs from extracellular SODs in that it enters inside the cells after its administration thanks to the presence of a peculiar leader peptide that allows the internalization of the molecule.P < 0.001). In rats treated with high-osmolarity CM, ROS production was almost doubled compared to normal placebo-infused rats (P < 0.01) but returned to normal values in rats pretreated with rMnSOD, where a significant increase of SOD activity was detected . Renal hemodynamics confirmed that the administration of CM determined a striking fall of GFR in CM-treated rats , greatly blunted by rMnSOD administration ; administration of CM was associated with presence of both tubular necrosis and intratubular casts, that were both greatly reduced in SOD-treated rats (both P < 0.01). Our conclusions indicate that the scavenging activity of rMnSOD was able to reduce renal oxidative stress and to prevent the reduction of GFR and the renal histologic damage that follows CM administration [We studied the effects of rMnSOD on oxidative damage in a rat model of CIN in uninephrectomized rats; in normal rats, pretreatment with rMnSOD reduced renal superoxide anion production, induced by the activation of NAPDH oxidase, by 84% (stration .In summary, experimental and clinical trials, using NAC, bicarbonate infusion, Probucol, acetazolamide, and rMnSOD seem clearly to suggest that ROS are involved in the pathogenesis of CIN. Nevertheless, whether the efficacy of these interventions are related to ROS antagonism or to the modification of oxygen availability is yet to be defined.The pathogenesis of CIN is a paradigm of hypoxic-toxic injury, involving altered renal microcirculation, hypoxia, and ROS-mediated cellular injury. Hypoxic damage develops especially in high-risk patients, in whom renal protective mechanisms, which maintain renal medullary oxygen balance and prevent ROS generation and action, are hampered.Formation of ROS likely results from the evolving hypoxia and reoxygenation, activating a feed-forward loop of endothelial/vascular dysfunction, upregulation of tubular transport, and the induction of oxygen-consuming reparative mechanisms, with consequently intensified hypoxia. By interfering with hypoxia-adaptive cell responses, ROS might further intensify renal parenchymal injury and dysfunction. Improvement of renal medullary oxygenation and inhibition of ROS formation or ROS scavenging are, therefore, reasonable therapeutic interventions in the prevention of CIN. However, the protective properties attributed to NAC and to bicarbonate infusion or ROS scavenger agents and their putative action through defusing oxidative stress have yet to be established.


While small bone defects heal spontaneously, large bone defects need surgical intervention for bone transplantation. Autologous bone grafts are the best and safest strategy for bone repair. An alternative method is to use allogenic bone graft. Both methods have limitations, particularly when bone defects are of a critical size. In these cases, bone constructs created by tissue engineering technologies are of utmost importance. Cells are one main component in the manufacture of bone construct. A few cell types, including embryonic stem cells (ESCs), adult osteoblast, and adult stem cells, can be used for this purpose. Mesenchymal stem cells (MSCs), as adult stem cells, possess characteristics that make them good candidate for bone repair. This paper discusses different aspects of MSCs that render them an appropriate cell type for clinical use to promote bone regeneration. Bone is a highly specific, dynamic tissue capable of maintaining viability under mechanical stress and external continuous compression. This capability of bone tissue diminishes with increasing age . FurtherHistorically, the definitive presence of MSCs was discovered about 40 years ago by Friedenstein et al. in bone marrow tissue. They described these cells as mononuclear nonphagocytic cells with fibroblast-like phenotype and colongenic potential capable of adhering to the culture surface in a monolayer culture . Later, It should be mentioned that stem cells are defined by two key characteristics: the ability of multilineage differentiation and the capacity of self-renewal . Of thesAmong candidate cells for bone regeneration, embryonic stem cells (ESCs) possess ethical issues limiting their application in bone regeneration. ESCs are derived from the blastocyst inner cell mass and can be directed toward differentiation into varying cell lineages, including osteoblastic cell lineages under suitable culture conditions –32. To dMSCs have been reported to constitute about 0.01%–0.001% of the marrow mononuclear population . These cThe presence of cells with multipotent differentiation capacity in adipose tissue is promising due to the ease of accessibility of adipose tissue and its abundance in the body. Adipose tissue can be an appropriate substitute for marrow in regenerative medicine and tissue engineering , 47. AdiThe umbilical cord from a newborn baby contains two arteries and a vein covered with mucus connective tissue rich in hyaluronic acid, referred to as Wharton's jelly. According to studies, MSC-like cells can be derived from various components of this cord . For exaSeveral stem cell types in dental tissue have been reported including dental pulp stem cells (DPSCs), stem cells from human exfoliated deciduous teeth (SHED), stem cells of the apical papilla (SCAP), periodontal ligament stem cells (PDLSCs), and dental follicle progenitor cells (DFPCs) , 62. SinMSCs osteogenic property was the first reported differentiation capacity when they were discovered. Indeed, even prior to definitive isolation of MSCs from bone marrow, some transplantation experiments clearly showed the osteogenic capacity of marrow tissue. Friedenstein et al. were the first to isolate and describe the cellular equivalent of osteogenic features of marrow tissue .μM concentrations [Osteogenic differentiation is a highly programmed process that consists of many stages including proliferation, differentiation, matrix deposition, mineralization, and matrix maturation. The general protocol for in vitro bone differentiation of MSCs involves incubation of cell monolayer in a culture medium containing dexamethasone, beta glycerol phosphate, and ascorbic acid for a period of two to three weeks . Dexamettrations . In additrations .MSC in vitro bone differentiation results from the activation of some well-known molecular signaling pathways. Each osteogenic reagent activates a molecular pathway that leads to a differentiated phenotype. Although the osteogenic effects of a number of these reagents have long been known, specific pathways by which the effects are mediated remain to be clarified. The activation of wingless-type MMTV integration site family of the protein (Wnt) signaling pathway , mitogen Activation of signaling pathways by osteogenic supplements eventually leads to activation of osteoblast-specific signal proteins and specific osteoblastic transcription factors. Cbfa1  also called as Runx2 (runt-related gene 2) is one of the most studied transcription factors expressed in MSCs upon their commitment toward osteogenic differentiation , 79. RunOptimal conditions for MSC in vitro bone differentiation are well been established. For example, the addition of rhBMP-2 to osteogenic medium can facilitate proliferation and osteogenic differentiation of BMSCs both in vitro and in vivo. The use of alpha MEM versus DMEM and application of low-passaged versus high-passaged cells can end with higher expression of osteogenic genes and more culture mineralization –85. StudMSCs possess immunologically specific characteristics; therefore they would be general donors for therapeutic applications. Immunologic phenotypes of MSCs are MHC I+, MHC II−, CD 40−, CD80−, and CD 86− . Graft rAccording to research, MSCs secrete soluble factors that inhibit CD4+ and CD8+ T-cell activation as well as proliferation. Among these factors are indoleamine 2,3-dioxygenase (IDO), nitric oxide, TGF-beta, and prostaglandin E 2 –95. It hImmunomodulatory features of MSCs make them an important cellular candidate for cell-based treatment of tissue defects in an allogenic setting. For this reason, there is hope that MSCs could replace autologous and allogenic bone grafts which have known exhibited limitations in terms of availability and risk of pathogen transmission, respectively. At the allogenic approach, it will be possible to develop a cell bank to maintain MSCs from every donor for use in cell therapy. Prior to routine application of the cells in the clinic, an exact understanding of the immunologic features of MSCs and the underlying mechanism of action is needed , 101.One of the most important capabilities of MSCs is their migration capacity in response to signals produced by an injured bone , 103. AtThe precise mechanisms of cell trafficking in blood, transmigration through endothelial cell, and homing to the injured site are not thoroughly understood, but it has been speculated that chemokines and their receptors regulate this process . ChemokiKitaori et al. have reported that SDF1 expressed by periosteum mediates bone repair in the murine femoral model by recruiting MSCs to the fracture site . The SDFConsidering the relationships of cell migration with the chemokine concentration gradient, it can be concluded that the application of MSCs must be performed at the time when the chemokine concentration gradient is established at an adjacent area to the injured site.MSCs could be ideal carriers for therapeutic genes at a cell-mediated gene delivery strategy owing to their unique characteristics that include ease of isolation, culture, and expansion as well as their immunomodulatory property .In the normal process of bone development and repair, cytokines and osteoinductive growth factors play a major role by recruiting osteogenic progenitors at the bone formation site and promoting their differentiation into bone cell lineages . TherefoMany investigators have tried to regenerate bone by transfecting MSCs with the BMP gene. For example, Lieberman et al. have indicated that autologous BMSCs expressing Ad-BMP2 can considerably promote segmental femoral defects in rat models when compared with BMSCs expressing Ad-LacZ . TransplIt has been shown that Ad-Runx2-MSCs transplanted in murine calvarial defects produce more bone tissue compared to MSCs . Recent http://clinicaltrials.gov/.MSCs as adult stem cells are free from ethical concerns, residents of multiple tissues, able to efficiently differentiate along an osteogenic lineage, possess non-immunogenic properties, have injury-seeking capabilities, and can be used as vehicles for bone gene therapy. These characteristics make MSCs safe and promising candidates for use in bone engineering and regeneration. Currently, several clinical trials are being performed on problematic human bone lesions, including nonunion fractures, delayed union, bone cysts, and bone neoplasms, among others. These ongoing registered trials are available at the following clinical trial website: