Traumatic brain injury (TBI) results in significant disability due to cognitive deficits particularly in attention, learning and memory, and higher-order executive functions. The role of TBI in chronic neurodegeneration and the development of neurodegenerative diseases including Alzheimer's disease (AD), Parkinson's disease (PD), Amyotrophic Lateral Sclerosis (ALS) and most recently chronic traumatic encephalopathy (CTE) is of particular importance. However, despite significant effort very few therapeutic options exist to prevent or reverse cognitive impairment following TBI. In this review, we present experimental evidence of the known secondary injury mechanisms which contribute to neuronal cell loss, axonal injury, and synaptic dysfunction and hence cognitive impairment both acutely and chronically following TBI. In particular we focus on the mechanisms linking TBI to the development of two forms of dementia: AD and CTE. We provide evidence of potential molecular mechanisms involved in modulating Aβ and Tau following TBI and provide evidence of the role of these mechanisms in AD pathology. Additionally we propose a mechanism by which Aβ generated as a direct result of TBI is capable of exacerbating secondary injury mechanisms thereby establishing a neurotoxic cascade that leads to chronic neurodegeneration. Traumatic brain injury (TBI) is a leading cause of death and disability in industrialized countries. In the US alone, more than 1.7 million TBI's occur annually and currently, at least 5.3 million Americans are living with a long-term or life-long disability due to a TBI  from the primary injury causes direct mechanical damage to neuronal and glial cells, the vasculature and strains axons. The damage from the primary injury is immediate and irreversible and as a result is only amenable to preventative protective measures . The primary insult initiates the activation of a vicious cycle of secondary injury effects that include both systemic complications and cellular injury mechanisms that arise over the course of hours to several weeks following the primary injury. Systemic impairments including edema, increased intracranial pressure (ICP) and hemorrhage, all contribute to decreased cerebral blood flow (CBF) and impaired metabolism resulting in ischemia. The ischemia induced by these systemic impairments contributes to the initiation of biochemical and cellular cascades including glutamate excitotoxicity, calcium overload, free radical generation, mitochondrial dysfunction, inflammatory events, and pro-apoptotic gene activation. These cellular injury processes result in neuronal cell loss through necrotic  and programmed (delayed) cell death. Programmed cell death can occur by a number of mechanisms including apoptosis , which play important roles in cognition  an efflux of K+ ions, which is then followed by delayed calcium dependent neuronal degeneration. The influx of Na+ ions leads to membrane depolarization, opening voltage-dependent calcium channels, and removing the magnesium block on NMDA receptors causing a greater influx of calcium into the cytosol , proteases including calpains, and caspases, transcription factors including c-Fos, c-Jun, and c-myc, nitric oxide synthase (NOS) as well as DNA degrading endonucleases. The over-activation or overproduction of any of these molecules can lead to the degradation of cytoskeletal components , mitochondrial dysfunction, oxidative stress, and pro-apoptotic gene activation , controlled cortical impact (CCI) and more recently penetrating ballistic and blast models of TBI  peptidase inhibitor (ZJ-43) greatly improved cognitive performance following experimental TBI in rats by preventing the hydrolysis of the NAAG peptide neurotransmitter, which regulates synaptic release of glutamate  and can persist for up to 14 days  generating free radicals and causing oxidative damage to cells. Oxidative damage plays an important role in cell death pathway activation, axonal injury and impaired synaptic plasticity leading to cognitive deficits post-TBI  becoming activated. Activated glia secretes a range of proinflammatory cytokines and chemokines that are neurotoxic. Levels of the proinflammatory cytokines Tumor necrosis factor (TNF), Interleukin 1β (IL-1β) and Interleukin-6 (IL-6) have been shown to increase dramatically within hours of a TBI , Alzheimer's disease (AD), Amyotrophic lateral sclerosis (ALS), and the newly classified entity chronic traumatic encephalopathy (CTE)  or “punch–drunk” syndrome was first described in boxers in 1928  players demonstrated that football players have neurodegenerative mortality rates three times higher than the general US population and four times the national rate for the neurodegenerative sub-categories of AD and ALS  composed of hyperphosphorylated tau. Aβ is a 38–43 amino acid peptide that is generated by the sequential cleavage of the amyloid precursor protein (APP) by β-secretase (BACE1) and γ-secretase enzymes. Tau is a microtubule-associated protein that under normal conditions plays an important role in stabilizing axonal microtubules. While it is clear that both Aβ and tau play important roles in AD pathology, under the amyloid cascade hypothesis it is generally accepted that Aβ generation and amyloid plaque deposition are the initiating events in the pathology of AD and precede NFT formation. AD is a highly complex disease, the vast majority of cases are sporadic and most likely arise from the complex interactions of multiple genes with each other as well as the environment  family, in particular MMP-9 a potent inflammatory mediator, via HIF1α has been implicated in the loss of synapses witnessed post-TBI. TBI-induced expression of HIF-1α leads to synapse loss that closely correlates with the upregulation of MMP-2 and MMP-9. Synaptic loss was prevented by inhibiting HIF1α expression and MMP-9 upregulation by the administration of the anti-inflammatory drug minocycline  promoting LTP as well as the upregulation of the prosurvival gene BDNF. Conversely, activation of extrasynaptic NMDA receptors promotes the amyloidogenic pathway leading to Aβ production and has been shown to downregulate phosphorylation of CREB and enhance LTD, while also inducing a loss of mitochondrial membrane potential signaling activation of cell death pathways  and synaptic degeneration , an endogenous inhibitor of calpains, they can contribute to synaptic dysfunction and neuronal death following TBI and in AD. Calpain activation is a common pathological event in experimental models of TBI. It has been robustly demonstrated in rodents using a variety of injury techniques which give rise to both focal and diffuse injuries including: FPI, controlled cortical injury, weight drop, etc. Depending upon the type of injury distinct temporal patterns of calpain activation occur. In focal injuries activation occurs within minutes to hours, whereas it is delayed under conditions of diffuse injury. Additionally, calpain activation has been observed in human TBI subjects , and glial tangles (GT) are found in the frontal and temporal cortices. While the Tau detected in CTE biopsies is hyperphosphorylated as occurs in AD and other tauopathies its anatomical distribution is unique to CTE (Stern et al., While the pathology of CTE has been well described, little is known about the mechanisms by which, repetitive head trauma can lead to chronic neurodegeneration. This has been in part to due to the focus of most experimental TBI modeling on single events of mild, moderate or severe TBI rather than repetitive mild TBI events, and also due to the lack of appropriate models of repetitive mild TBI that encompass the rotational as well as linear acceleration/deceleration forces that appear to be important in repetitive TBI pathogenesis (see Viano et al., It is clear there are significant overlaps between the molecular pathways including known genetic risk factors such as APOε4 that lead to cognitive dysfunction following TBI and in neurodegenerative diseases such as AD Tables , 2. Howein vitro studies an in vivo animal models suggests that the activation of these biochemical pathways could potentially modulate Aβ and tau pathology. TBI has been demonstrated to lead to increased Aβ production and there is evidence that the injury induced modulation of BACE1 may play a key role in this increase. As experimental evidence indicates that Aβ is able to directly modulate many of the neurotoxic biochemical pathways induced by TBI, we postulate that Aβ may be the important link in initiating and sustaining a vicious neurotoxic cycle that leads to chronic neurodegeneration.It is clear that there is significant overlap between the secondary injury mechanisms and pathology observed following TBI and in the brains of AD patients. These common mechanisms include altered glutamatergic signaling, calcium dysregulation, inflammation, mitochondrial oxidative stress, and apoptosis. Importantly, it has been demonstrated that many of these processes remain activated for prolonged periods of time following injury, providing an important link between the acute injury and the persistent cognitive impairments that have been observed following TBI. Significant experimental evidence from However, in order to develop therapeutic interventions to prevent or reverse the cognitive dysfunction observed following TBI and in order to prevent the potential for development of dementia later in life, additional research must be performed to identify the molecular mechanisms that mediate the activation of these neurotoxic biochemical pathways. This will involve utilizing transgenic and knockout mice or the development of highly specific inhibitors that target molecular pathways of interest such as those implicated in neuro-inflammation following TBI. The need for a more thorough understanding of the molecular mechanisms linking TBI and chronic neurodegeneration is exemplified by the case of CTE. While there is evidence supporting a solid link between a history of repetitive mild TBI and the development of CTE and the pathology has been well characterized very little is known about the mechanisms that lead to this altered tau pathology. The development of new experimental animal models of repetitive TBI that more closely replicate CTE pathology along with detailed biochemical analysis of the brains of CTE patients will facilitate this.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.


DNA methyltransferases establish methylation patterns in cells and transmit these patterns over cell generations, thereby influencing each cell's epigenetic states. Three primary DNA methyltransferases have been identified in mammals: DNMT1, DNMT3A and DNMT3B. Extensive in vitro studies have investigated key properties of these enzymes, namely their substrate specificity and processivity. Here we study these properties in vivo, by applying novel statistical analysis methods to double-stranded DNA methylation patterns collected using hairpin-bisulfite PCR. Our analysis fits a novel Hidden Markov Model (HMM) to the observed data, allowing for potential bisulfite conversion errors, and yields statistical estimates of parameters that quantify enzyme processivity and substrate specificity. We apply this model to methylation patterns established in vivo at three loci in humans: two densely methylated inactive X (Xi)-linked loci ( DNA methyltransferases establish methylation patterns in cells and transmit these patterns over cell generations, thereby influencing each cell's epigenetic states. , thus maintaining methylation at these CpG sites over cell generations Regarding processivity, in vitro experiments suggest that mouse DNMT1 acts processively, binding to DNA and then remaining active over a stretch of consecutive nucleotides De novo methyltranferases DNMT3A and 3B Despite the availability of significant in vitro data, important questions remain to be addressed regarding the in vivo properties of the DNA methyltransferases. Here, we investigate in vivo substrate preferences and levels of processivity of human DNMT1 and DNMT3s by analyzing double-stranded DNA methylation patterns established in vivo, measured using hairpin-bisulfite PCR An important feature of our analyses is that they are based on a statistical model, which quantifies processivity as a probability, thus allowing for statistically testing whether processivity exisits, and which assesses the statistical uncertainty in the estimate of this probability, thus facilitating comparison of inferred levels of processivity among data sets. Our HMM also explicitly accounts for potential measurement errors in the observed data; these errors have been generally unaccounted for in other in vivo and in vitro studies. Additionally, this HMM can also be used to infer the set of enzymatic activities that most likely gave rise to each observed methylation pattern, and to infer, probabilistically, which strand in each double-stranded methylation pattern is the parent strand, and which is the daughter strand (this information is not directly measurable in the hairpin-bisulfite experiment), allowing for investigation of strand-specific behavior of these enzymes .We model the observed double-stranded methylation patterns as having arisen from a process where the methyltransferases (DNMT1 and the DNMT3s) were either associated or not associated with the DNA at a CpG site. We model these association/non-association states as a Markov process along the DNA. The model for the observed data based on these unobserved (“hidden”) states is then a Hidden Markov Model HMM . For in More specifically, the hidden Markov process in the HMM can be decomposed into three independent Markov processes: the first representing association of DNMT1, which we assume to act only on the daughter strand Under this parameterisation, two events contribute to the probability of a methyltransferase staying associated at two consecutive sites 1 bp apart  provides additional information. Whereas randomly-distributed hemis in the data suggest that the methyltransferases may have been associated with or dissociated from the dyads randomly, clustered hemis suggest nonrandom enzyme activities. In particular, runs of hemis of the same orientation , if observed more often than expected by chance, provide evidence for processivity. For example, the in vitro data on mouse DNMT1 from Goyal et al. The methylation patterns at the ed dyads . AnalyseWe find that the methylation patterns at both of these Xi-linked loci provide strong evidence for substantial processivity of DNMT1: the estimated dissociating probability for the DNMT1 process, s near 0 . To inveg narrow . This esg narrow . The higRecall that our HMM also allows that DNMT1, when associated with the DNA, adds a methyl group to the daughter strand with probability oth loci  and  to 0.06 at  , consistAt both Xi-linked loci, we estimated that the average frequency of association  . Furtherof  . Thus, tpG sites . The medSimilar to the ribution . This unBisulfite conversion used in the experiment can give rise to two types of measurement errors Another source of possible measurement errors is PCR crossover, which can occur during PCR amplification with probability less than 1% per molecule data set , one of Most previous studies of DNTM1 were conducted in vitro and investigated mouse DNMT1 Although both in vivo and in vitro data show strong levels of processivity, there are noticeable quantitative differences between the two sets of estimates: our estimates from these published in vitro data suggest a shorter mean association tract length  and 5A aIn this article, we have developed a novel hidden Markov model to infer complex methyltransferase activities from double-strand methylation patterns established in single molecules. This model complements our earlier model Applying our model to four in vivo human data sets collected at three loci, we find strong evidence for a high level of processivity of DNMT1 at two Xi-linked loci, with the mean association tract lengths being a few hundred to a few thousand bp, whereas the methylation patterns at the autosomal locus Our analysis of in vivo data suggests low contributions from the DNMT3s in these in vivo somatic cells. To study the properties of the DNMT3s, an alternative is to analyze double-stranded methylation patterns from in vitro experiments. Such data are indeed available at least for DNMT3A Consider the Each entry in We made two assumptions in the calculation of the emission probabilities: (i) measurement errors did not occur in the collection of our data; relaxation of this assumption to incorporate error is described below; and (ii) there is no active removal of methyl groups on the parent strand when DNA is replicated To distinguish between the DNMT1 process and the DNMT3 process on the daughter strand, in the simplest version of the HMM we draw on the existing evidence that the two classes of methyltransferases exhibit different substrate preferences ly  and  .We consider two types of measurement errors due to bisulfite conversion es  and  , whereasnt error .http://stephenslab.uchicago.edu/software.html. This program includes also all the data sets analyzed here.The models and analyses presented here are implemented in the computer program MethylHMM, which can be downloaded from We used hairpin-bisulfite PCR Materials S1(PDF)Click here for additional data file.


Coronary artery disease remains the leading cause of death in developed countries. Major recent studies such as SYNTAX and FREEDOM have confirmed that coronary artery bypass grafting (CABG) remains the gold standard treatment in terms of survival and freedom from myocardial infarction and the need for repeat revascularization. The current review explores the use of new technologies and future directions in coronary artery surgery, through 1) stressing the importance of multiple arterial conduits and especially the use of bilateral mammary artery; 2) discussing the advantages and disadvantages of off-pump coronary artery bypass; 3) presenting additional techniques, e.g. minimally invasive direct coronary artery bypass grafting, hybrid, and robotic-assisted CABG; and, finally, 4) debating a novel external stenting technique for saphenous vein grafts. Late survival after coronary artery bypass grafting (CABG) is improved when the left internal mammary artery (LIMA) is grafted to the left anterior descending artery (LAD).13n = 7,435) or the MultArt group . Propensity score analysis matched 1,153 patients. Operative mortality was 0.8% (n = 10) in the MultArt and 2.1% (n = 154) in the LIMA–SV . Late survival was greater for MultArt versus LIMA–SV , in unmatched groups and 83% and 70% versus 80% and 60%, respectively (P = 0.0025), in matched groups). MultArt subgroups with bilateral internal mammary artery (BIMA)–SV (n = 589) and BIMA only (n = 271) had improved 15-year survival , and patients with BIMA–RA (n = 147) and LIMA–RA (n = 169) had greater 10-year survival  versus LIMA–SV. In multivariate analysis, MultArt grafts remained a strong independent predictor of survival . These findings suggest that in patients undergoing isolated coronary artery bypass graft surgery with LIMA to left anterior descending artery, arterial grafting of the non-left anterior descending vessels conferred a survival advantage at 15 years compared with SV grafting. It is still unproven whether these results apply to higher-risk subgroups of patients.A recent observational, retrospective studyDespite previous reports of greater benefit from left than right coronary system grafting with the second arterial graft,14Almost three decades ago, in a seminal study, the Cleveland Clinic Group reported that a single internal mammary artery (SIMA) resulted in superior survival benefit as well as a reduced subsequent incidence of myocardial infarction, recurrent angina, and the need for repeat revascularization.The most likely explanation for the survival benefit of IMA grafting is its greatly superior rates of patency in comparison to vein grafts. Whereas 10 years after bypass grafting up to three-quarters of all vein grafts are occluded or severely diseased, in contrast the patency rates of IMA grafts remain in excess of 90% even into the second decade of follow-up.5The IMA graft is a unique artery both in anatomical structural terms (with a high proportion of elastic rather than muscle or adventitia composition) and in physiological function (it produces much greater levels of nitric oxide and decreased release of vasoconstrictors in comparison to other vessels), resulting in potent anti-atherosclerotic effects.Despite strong clinical evidence in favor of the use of BIMA grafts, their use in current practice remains disappointingly low, being around 5% of patients in the USA and fewer than 10% in Europe.In an effort to add more scientific data to the debate of SIMA or BIMA grafting, the Arterial Revascularization Trial (ART) randomized 3,102 patients in 28 centers in seven countries.The one note of caution was that there was indeed an increase in sternal wound reconstruction from 0.6% in the SIMA group to 1.9% in the BIMA group, i.e. an absolute difference of 1.3% or a number needed to harm of 78 patients. However, it is noteworthy that while one-quarter of all patients in the ART Trial had diabetes almost half the patients requiring sternal wound reconstruction had diabetes. It is highly likely that with more judicious patient selection (avoiding BIMA grafts in obese diabetics or those with impaired lung function) and more precise harvesting techniques While the results of recent trials of CABG versus stents in general populations  and in diabetics  confirm the significant superiority of CABG over stents in terms of superior survival and freedom from subsequent myocardial infarction or repeat revascularization, the low use of BIMA grafts in current practice is a poor reflection of optimal surgical therapy. The recommendations in guidelines support the use of more arterial grafts during CABG,For almost three decades there has been controversy as to the potential benefits of off-pump CABG in relation to on-pump CABG. The initial rationale for off-pump CABG was mainly driven by economic considerations in developing countries where the economic cost of cardiopulmonary bypass made CABG an unrealistic proposition in many patients. Despite much skepticism by its opponents, off-pump CABG was gradually introduced into developed countries where its proponents recognized its potential to mitigate the adverse effects of extracorporeal circulation in an increasingly elderly population undergoing CABG. The views of supporters and opponents of off-pump CABG have remained essentially unchanged in the intervening period.A meta-analysis by Afilalo and colleagues24P = 0.24). In particular, there was no difference in the incidence of individual components of the primary outcome in terms of death, myocardial infarction, stroke, or new renal failure. Furthermore, and in contrast to previous studies, there was no significant increase in the incidence of repeat revascularization for off-pump CABG at 1 year. Additionally, there was no difference in neurocognitive outcomes at 1 year between the two groups. The most likely explanation of the differences between the findings of the CORONARY Trial and two of the largest previous trials reporting inferior outcomes for off-pump CABG is that the CORONARY Trial not only enrolled a far greater number of patients but, crucially, recruited surgeons with a far higher level of surgical expertise in off-pump surgery.Two recently published trials provide far more definitive answers. First, the CORONARY Trial, which enrolled 4,752 patients in 79 centers in 19 countries, had previously reported no significant difference at 30 days in the primary composite outcome of death, myocardial infarction, stroke, or new renal failure between the two techniques.P = 0.74) or at 12 months . Of particular note in this trial is the fact that the surgeons were highly experienced; for off-pump surgeons the average number of off-pump surgeries was 514 and for on-pump surgeons 1,378. Although the average number of coronary anastomoses was 2.7 in the off-pump group and 2.8 in the on-pump group (P < 0.001), this is highly unlikely to be of any clinical significance. The only remaining question now would appear to be whether off-pump surgery in association with a no-touch aortic technique significantly reduces the risk of perioperative stroke. It is noteworthy that in the GOPCABE Trial the most common reason for conversion from on-pump to off-pump CABG after the skin incision was a calcified ascending aorta.A second trial (GOPCABE), which randomized 2,539 patients aged 75 years or older to on-pump and off-pump CABG, has been published very recently.In summary, the postulated benefits of off-pump surgery have not materialized in clinical practice for most patients, possibly due to the fact that advances in extracorporeal perfusion have made cardiopulmonary bypass much safer. For most patients undergoing CABG today the use of bilateral internal mammary arteries is far more important than whether surgery is performed on or off pump.Minimally invasive direct coronary artery bypass grafting (MIDCAB) utilizes a small anterior left thoracotomy incision and harvesting of the left internal mammary artery with an anastomosis performed to the left anterior descending artery without cardiopulmonary bypass. MIDCAB was initially described for single-vessel bypass to the left anterior descending (LAD) artery.29Patients for the MIDCAB approach are to be selected carefully; the ideal candidate would have severe stenosis or complete occlusion of the proximal LAD. It is imperative that the distal LAD is visualized either by collateral filling or by computed tomographic angiography in cases in which the patient has complete occlusion. Importantly, obesity is a relative contraindication; although the LIMA takedown is technically possible in obese patients, the pressure placed on the wound edges by the retractor can lead to tissue necrosis and wound infections. Similarly, female patients with large breasts are at increased risk of wound necrosis.30The most pivotal factors in the postoperative management of MIDCAB patients are analgesia and early mobilizationThe overall reported results of MIDCAB have been excellent,Recently, several studies reported a fruitful use of a hybrid approach combining minimally invasive LIMA–LAD bypass procedures with catheter-based interventions on the circumflex or right coronary arteries for the treatment of multivessel disease. In most series, the catheter-based interventions, which generally necessitate the placement of a drug-eluting stent, were performed several days before or several days after the surgical revascularization,A very recent studyTheoretically, hybrid procedures provide a complete revascularization while keeping the survival benefit and angina relief of a LIMA–LAD graft and avoiding the morbidity of sternotomy.The surgical robot is an elegant microprocessor-controlled, electromechanical instrument that allows the surgeon to remotely manipulate fully articulating videoscopic instruments by way of master–slave servos and microprocessor control. These long, thin instruments, which can be inserted into the closed chest through half-inch incisions, are designed to allow multiple degrees of freedom and can precisely emulate the surgeon’s movements at the control console.Since the introduction of surgical robotics in the 1990s, there has been a progressive increase in utilization for thoracic surgical procedures. Although mitral valve and non-cardiac thoracic procedures account for the majority of cases, there are increasing reports of robotic-assisted coronary revascularization procedures. These reports include robotic LIMA harvest followed by a traditional MIDCAB43Because of the added expense and difficulty with learning the technique, the routine use of surgical robotics in CABG surgery does not seem likely in the near future. The robot has and will continue to evolve. Improved video resolution, lower-mass arms, the addition of a fourth tele-manipulator, and the availability of an elegant robotic coronary stabilizer will likely increase its effectiveness and extend its application. Refinement of automated distal anastomotic devices may further increase the growth of robotic coronary revascularization surgery.Coronary artery disease (CAD) remains the leading cause of death in developed countries, and recent studies such as SYNTAX and FREEDOM have confirmed that CABG remains the gold standard treatment in terms of survival and freedom from myocardial infarction and the need for repeat revascularization. Despite strong evidence of an additional survival benefit of BIMA over a SIMA, only around 5%–10% of patients receive BIMA or additional arterial grafts. The saphenous vein graft (SVG) is still the most commonly used conduit because of its abundance, ease of harvest, and “user friendliness.” However, its main disadvantage is its relatively poor long-term patency compared to IMA grafts, with graft failure in as many as 20% of veins within the first year and in as many as 50% at 10 years and with further significant disease in half of the remaining patent grafts (in comparison to perfect patencies of 90%–95% of IMA grafts). SVG failure can result in major adverse clinical sequelae .Vein graft failure appears to result from both medial and neo-intimal thickening, caused by migration and proliferation of smooth muscle cells and the late appearance of mature lipid-laden atherosclerotic plaques. These changes can compromise flow directly or promote thrombotic occlusion. Diffuse neo-intimal tissue proliferation, the origin of vein graft disease, develops in 75% of grafts within 1 year of implantation. This occurs because the vein graft is exposed to a “new” mechanical environment in the arterial circulation, with relatively high pressures and shear stress. In the first few weeks, shear-induced remodeling leads to luminal enlargement followed by a later phase typified by wall tension-induced remodeling leading to wall thickening  and stiffening. It is also believed that luminal irregularities of the native vein and its valves are additional triggers for aggressive focal intimal hyperplasia, further increasing the risk of vein graft failure. Neither antiplatelet therapy nor avoidance of surgical preparative injury has been shown conclusively to eliminate medial and neo-intimal thickening in either experimental models or human vein grafts.in vitro and in vivo experimental attempts have been made to reach the same pivotal goal. The employed experimental strategies include the use of 1) pharmacological agents, such as lidocaine, which was studied in vitro using standard tissue bath techniquesIn addition to the clinically well-established ways of improving vein graft patency, such as a low-cholesterol diet and smoking cessation,51; notably, the “no-touch” technique seems to produce a superior graft with long-term patency comparable to the internal thoracic artery. Moreover, reducing the distending pressure while harvesting the SVG was suggested to increase the SVG patency.47Various vein harvesting techniques were explored in order to extend the SVG patency, including the “no-touch” saphenous vein harvesting, which refers to the saphenous vein removal with minimal trauma and preservation of the normal architectureUsing an external stent to prevent vein graft dilation and mitigate luminal irregularities and wall tension has been hypothesized to reduce intimal hyperplasia and consequently vein graft failure. However, previous attempts at external stenting of vein grafts have failed for a variety of reasons. VGS FLUENT , a novel external support device for SVGs, is a cobalt chrome braid, with a unique combination of different types of wires which provide it with axial plasticity (i.e. can stretch to cover the entire length of a vein graft) and radial elasticity . The stent maintains its position without any additional fixation such as using glue and can be applied in less than a minute without affecting current grafting technique.A CABG study in sheep demonstrated the FLUENT’s safety along with excellent efficacy in reducing intimal hyperplasia, preventing vein graft dilation/deformation, and eliminating thrombus formation. Following these successful animal studies the FLUENT has been evaluated in a randomized controlled study  in the UK, which recruited 30 patients in Oxford and Brompton/Harefield who, in addition to an IMA graft to the LAD, required vein grafts to the right coronary artery and the circumflex artery. Patients were randomized for one vein graft to receive the stent and the other to act as a control. Patients are now undergoing 12-month-postprocedure angiography , intrava


Cholangiocarcinoma (CC) encompasses a group of related but distinct malignancies whose lack of a stereotyped genetic signature makes challenging the identification of genomic landscape and the development of effective targeted therapies.Accumulated evidences strongly suggest that the remarkable genetic heterogeneity of CC may be the result of a complex interplay among different causative factors, some shared by most human cancers while others typical of this malignancy.Currently, considerable efforts are ongoing worldwide for the genetic characterization of CC, also using advanced technologies such as next-generation sequencing (NGS). Undoubtedly this technology could offer an unique opportunity to broaden our understanding on CC molecular pathogenesis. Despite this great potential, however, the high complexity in terms of factors potentially contributing to genetic variability in CC calls for a more cautionary application of NGS to this malignancy, in order to avoid possible biases and criticisms in the identification of candidate actionable targets. This approach is further justified by the urgent need to develop effective targeted therapies in this disease.A multidisciplinary approach integrating genomic, functional and clinical studies is therefore mandatory to translate the results obtained by NGS into effective targeted therapies for this orphan disease. Cholangiocarcinoma (CC) is a complex disease encompassing a group of related but distinct malignancies that may emerge at different anatomic sites along the intrahepatic and extrahepatic biliary tree .CC treatment represents a major challenge for oncologists. To date radical surgery, suitable only in a limited number of patients, has offered the only chance of cure . SystemiThe addition of targeted therapies in CC management was expected to broaden the therapy options and thereby improve the survival rate of these patients. However, targeted therapies have failed or shown only marginal benefits in several clinical trials with different drugs alone or combination with chemotherapy Table  and 2 5. The devCurrently, considerable efforts are ongoing worldwide for the genetic characterization of CC, also using advanced technologies such as next-generation sequencing (NGS). NGS has opened the possibility to identify the full repertoire of tumor-borne genetic alterations , 22 and,The genetic characterization of adult solid tumors is more complex. The many mutational events accumulated during the disease course can indeed hamper the discrimination between driver and passenger mutations and hence the identification of the mutational landscape in these cancers.An even more complex scenario probably occurs in CC. Although some studies have focused on the genetic characterization of CC, the overall molecular pathogenesis of this malignancy still remains poorly defined. Recent findings suggest that this process may involve a multitude of mutational events, including mutations in KRAS, TP53, BRAF, PTEN, SMAD4, IDH1, IDH2, BAP1, ARID1A and PBRMI genes, as well as FGFR translocations -37. SomeAccumulating evidence strongly supports the notion that the remarkable genetic heterogeneity of CC may be the result of a complex interplay among different factors, some shared by most human cancers, while others typical of this malignancy.O. viverrini infection and CCs not related to such exposure has identified two distinct mutational patterns in the two subgroups, with a major frequency of BAP1, IDH1 and IDH2 mutation in non-O. viverrini-related CCs and a major mutation rate in TP53, KRAS, SMAD4, MLL3, ROBO2, RNF43 and PEG3 genes in O. viverrini-related CCs, respectively [Recent findings suggest that the large number  of established or putative risk factors associated with CC Table  40-47,  may playectively . Mutatioectively , 50. Actectively , 52. TheThe induction of distinct genetic profiles in CC by different risk factors could rely not only on the different carcinogenetic mechanisms driven by such risk factors, but also on the existence along the biliary tree of two distinct stem cell niches, the canals of Hering harboring hepatic stem cells (HpSCs) and the peribiliary glands harboring biliary tree stem/progenitor cells (BTSCs) . Cell poThe genetic characterization of CC may be further complicated by the presence of tumor clonal heterogeneity , a hallmark of almost all human cancers -65. IndeThe dynamic changes in time and in space of tumor subclonal composition may lead to an underestimated or biased identification of the mutational landscape in heterogeneous tumors if genetic characterization is carried out only on a single tumor biopsy . This apOverall, the highly complex array of causative factors (summarized in Figure Given the marked genetic variability of CC, it seems likely that the best chance of response could be achieved by a combination of drugs targeting different driver mutations in this disease. Obviously, the ideal therapeutic regimen would include drugs specifically tailored to the unique genetic make-up of the individual patient, which is the cornerstone of “true” personalized therapy in cancer treatment . This apV600E mutation, which is associated with a clinical response to BRAF inhibitors in melanoma but not in colon cancer [An excellent clinical response to doublet-targeted therapy with the BRAF and MEK inhibitors dabrafenib and trametinib has also been reported in an ICC patient harboring this mutation [However, whether BRAFV600E represents a driver mutation in ICC and whether other ICC patients with this mutation could benefit from treatment with BRAF inhibitors await clarification.More recently a new strategy, the so-called basket trial, suggests the use of targeted therapies exclusively on the basis of identified actionable targets, irrespective of tumor histology and other clinical/biological variables . Despiten cancer , 75. An mutation . HoweverIn summary, the identification of candidate actionable mutations in CC is challenging because of the many factors potentially contributing to genetic heterogeneity in this malignancy. Undoubtedly, NGS may offer an unique opportunity to broaden our understanding of CC molecular pathogenesis; however, a multidisciplinary approach integrating genomic, functional and clinical studies is mandatory to translate the results obtained into effective targeted therapies for this orphan disease.


Arabidopsis thaliana ecotypes  were characterized in the context of habitat of origin. Temperatures of the habitat of origin decreased linearly with increasing habitat latitude, but habitat precipitation was greatest in Italy, lowest in Poland, and intermediate in Sweden. Plants of the three ecotypes raised under three different growth temperature regimes  exhibited highest photosynthetic capacities, greatest leaf thickness, highest chlorophyll a/b ratio and levels of β-carotene, and greatest levels of wall ingrowths in phloem transfer cells, and, in the Col-0 and Swedish ecotypes, of phloem per minor vein in plants grown at the low temperature. In contrast, vein density and minor vein tracheary to sieve element ratio increased with increasing growth temperature – most strongly in Col-0 and least strongly in the Italian ecotype – and transpirational water loss correlated with vein density and number of tracheary elements per minor vein. Plotting of these vascular features as functions of climatic conditions in the habitat of origin suggested that temperatures during the evolutionary history of the ecotypes determined acclimatory responses of the foliar phloem and photosynthesis to temperature in this winter annual that upregulates photosynthesis in response to lower temperature, whereas the precipitation experienced during the evolutionary history of the ecotypes determined adjustment of foliar vein density, xylem, and transpiration to temperature. In particular, whereas photosynthetic capacity, leaf thickness, and foliar minor vein phloem features increased linearly with increasing latitude and decreasing temperature of the habitats of origin in response to experimental growth at low temperature, transpiration rate, foliar vein density, and minor vein tracheary element numbers and cross-sectional areas increased linearly with decreasing precipitation level in the habitats of origin in response to experimental growth at high temperature. This represents a situation where temperature acclimation of the apparent capacity for water flux through the xylem and transpiration rate in a winter annual responded differently from that of photosynthetic capacity, in contrast to previous reports of strong relationships between hydraulic conductance and photosynthesis in other studies.Acclimatory adjustments of foliar vascular architecture, photosynthetic capacity, and transpiration rate in  Arabidopsis thaliana . In an initial 5-year characterization in the field ; there are 1176 km of latitude between the Italian and Polish sites and 1122 km of latitude between the Polish and Swedish sites. All three habitats of origin are low altitude sites near major bodies of water: the Swedish site is just over 80 m above sea level and less than a kilometer from the Gulf of Bothnia , the Polish site is just short of 60 m above sea level and approximately 160 km from the Baltic Sea, and the Italian site is approximately 225 m above sea level and 35 km from the Tyrrhenian Sea (Mediterranean Sea). The symbols pinpointing the location of the sites of origin  are used to designate the ecotypes originating from these three sites in all subsequent figures. In addition, the colors used to identify the countries of origin  are also used to identify the three sites/populations in Figures 2, 3, 5, and 10.From a functional standpoint, both ecotypes were shown to exhibit acclimation to experimental growth temperature and light environment, with more pronounced acclimatory adjustments in chloroplast pigments and tocopherols, photosynthesis, plant and leaf structure, and foliar vasculature (particularly the phloem) in the Swedish compared to the Italian ecotype , 2016. TA. thaliana thus allow for characterization of phenotypic plasticity that has presumably evolved in response to conditions experienced by these distinct populations from the three sites – with otherwise remarkably similar features – along an extensive latitudinal gradient. In order to evaluate the acclimation potential within each ecotype, plants were grown in growth chambers at different temperatures spanning more than 20°C. Given the many generations over which the Col-0 line has been maintained under controlled laboratory conditions since its original collection in 1955  Heynhold were investigated. Seed of Col-0 was obtained from The Arabidopsis Information Resource1 for comparison with the Swedish and Italian ecotypes  containing Canadian Growing Mix 2  under controlled conditions in growth chambers , receiving water daily and nutrients every other day. Multiple plants of each ecotype were grown under three independent day/night air temperature regimes of 8°C/12.5°C (low or cool), 25°C/20°C (moderate), and 35°C/25°C (high or hot) under a common photon flux density of 400 μmol m-2 s-1 (provided by a mixture of cool white fluorescent bulbs and incandescent bulbs) during a 9 h photoperiod as described in Figure 7. Fully expanded leaves from three to five non-flowering plants were evaluated for each ecotype and growth temperature.Three ecotypes of lim.org/  and the 2 leaf area), vein architectural features , leaf thickness, pigment levels , photosynthetic capacity , and transpiration rate  during exposure to the growth photon flux density of 400 μmol m-2 s-1 at a leaf temperature of 28.7 ± 1.6°C  and a vapor pressure deficit of 2.74 ± 0.07 kPa  were determined as described in 2 exchange because the former is unaffected by multiple resistances to CO2 diffusion  into the sites of carboxylation (n = 3 to 4 plants), photosynthetic capacities (n = 3 to 4 plants), transpiration rates (n = 4 to 5 plants), and pigment data (n = 3 to 4 plants) are presented as mean values ± SD , whereas leaf thickness (three cross-sections from each of 3 to 4 plants) and all of the vein architectural features (n = 7 to 10 minor veins per plant from 3 to 4 plants) are presented as mean values ± SE. With the exception of Figures 5 and 10 that depict the calculated difference between metrics from plants grown under the low compared to the high temperature regimes and utilize the country of origin color coding , the symbols in Figures 12 are color coded according to the foliar chlorophyll a/b ratio and resulting subtle differences in visual appearance of the plants grown under the different temperature regimes : blue-green (highest chlorophyll a/b ratio) for the low growth temperature regime, green (intermediate chlorophyll a/b ratio) for the intermediate growth temperature regime, and olive-green (lowest chlorophyll a/b ratio) for the high growth temperature regime.Vein density .Figures 2A–C). We estimate that the Col-0 ecotype germinates from mid-September through October, overwinters as a rosette, and flowers and sets seed in April and May, and thus experiences photoperiods intermediate to those of the Italian and Swedish ecotypes ranging from about 7.6 h at winter solstice to close to 17 h at the end of its life cycle. The range of temperatures experienced by each ecotype during the growing season in its native habitat increases with increasing latitude, from a differential between mean monthly minima and maxima of 18°C (2.7°C to 20.6°C) at the Italian site, to 24.7°C (-5.0°C to 19.7°C over the estimated growing period) at the Polish site, and 31°C (-12.6°C to 18.4°C) at the Swedish site . Average annual temperature maxima and minima in the three sites vary linearly with latitude . Precipitation patterns, on the other hand, do not follow a latitudinal gradient , but nonetheless present a range of variation among the habitats of origin: during the respective growing seasons, the habitat of the Italian ecotype receives the most precipitation (577 mm over seven months), followed by the Swedish site (529 mm over 10 months), with the Polish site receiving the least precipitation (362 mm over the estimated growing period of nine months).In their respective original habitats, the Italian ecotype experiences the smallest annual variation in photoperiod (from just over 9 h of daylight on the winter solstice to about 14 h at the end of seed set in April) during an annual life cycle, whereas the Swedish ecotype experiences photoperiods ranging from just under 5 h in December to 20 h at the end of June  and leaf thickness  of the three ecotypes grown in climate-controlled growth chambers under two different experimental temperature regimes increased linearly with either increasing latitude of origin  or decreasing average annual temperature in each respective habitat of origin , particularly for cool-grown leaves. These linear increases in photosynthetic capacity and leaf thickness for the cool-grown leaves were paralleled by linear increases in several features of the foliar phloem, including the cross-sectional area per minor vein of the sugar-loading and -exporting sieve elements , the number of phloem cells per minor vein that facilitate sucrose loading into sieve elements , and increases in cell membrane length (and presumably membrane area) resulting from invagination of phloem parenchyma cell walls . In contrast to the responses of cool-grown leaves, hot-grown leaves did not display such increases in phloem features . There were also significant ecotypic differences in photosynthetic capacity, leaf thickness, and sieve element cross-sectional area per vein, significant ecotype × temperature interactions for the latter two parameters as well as for the number of loading cells per minor vein, and all of these features varied significantly with growth temperature in controlled growth chambers (Table 1).Photosynthetic capacity . While linear regressions for photosynthetic capacity  and cross-sectional area of sieve elements comprising the minor veins  with latitude or average annual temperature at ecotype origin were significant, the phloem features of phloem cell numbers and cell wall ingrowth levels, together serving as proxies for total phloem cell membrane area, were not. In particular, leaves of Col-0 exhibited levels of phloem transfer cell wall ingrowths that fell slightly below the line  and numbers of phloem loading cells  that fell slightly above the line; it is thus attractive to speculate that these two features together do effect a level of total phloem cell membrane area adjustment in the Col-0 ecotype in line with that of the other two ecotypes.When expressed as the difference between hot-grown and cool-grown leaves , photosynthetic capacity, leaf thickness, and minor vein phloem metrics exhibited linear increases with increasing latitude and also with decreasing temperature in the habitat of origin  and decreasing annual temperature in the respective habitats of origin , and this was due to increases in the cross-sectional areas of both the phloem  and xylem . Both of the latter metrics varied significantly as a result of growth temperature, while minor vein cross-sectional area also varied significantly among ecotypes and the phloem cross-sectional area showed significant ecotype × temperature interactions (Table 1). However, for  hot-grown leaves, only the cross-sectional area of the xylem increased with increasing latitude  and decreasing annual temperature  in the respective habitats of origin; these latter trends did not differ between  cool- and hot-grown leaves, but there was a significant difference in xylem cross-sectional area among the ecotypes (Table 1).Foliar minor vein cross-sectional area in  cool-grown leaves increased linearly with increasing latitude , with some features exhibiting positive and others negative correlations. While cross-sectional area of the minor veins decreased with increasing growth temperature under controlled experimental conditions , due in large part to decreases in phloem tissue , vein density (minor vein length per leaf area) increased with increasing growth temperature , as did the ratio of tracheary to sieve elements in minor veins  and the fraction of vascular cells per minor vein represented by tracheary elements . Three additional parameters decreased with increasing growth temperature: photosynthetic capacity , β-carotene levels , and chlorophyll a/b ratio . In addition to  growth temperature having a highly significant impact on all of these features, the fraction of vascular cells comprised of tracheary elements was significantly different among ecotypes, and vein density and the minor vein ratio of tracheary to sieve elements exhibited significant ecotype × temperature interactions (Table 1).Furthermore, a number of foliar features also exhibited significant, linear relationships with temperature during  plant growth across ecotypes (Table 1). When combining data for leaves of the three ecotypes grown under either low or high temperature in controlled conditions, transpiration rate was found to increase with increasing vein density , as did several minor vein xylem features, including ratios of either the numbers  or the cross-sectional areas of tracheary to sieve elements , and the number of tracheary elements per minor vein , all of which were significantly influenced by  growth temperature (Table 1). On the other hand, photosynthetic capacity , the fraction of minor veins occupied by phloem , and the cross-sectional area of minor vein phloem  decreased linearly with increasing vein density. One vascular feature that may contribute to the variation in foliar vein density is the level of free-ending veinlets, of which the greatest range of phenotypic plasticity (greatest difference between cool- and hot-grown leaves) was seen in the Col-0 ecotype  and that also varied significantly among ecotypes, with  growth temperature, and with ecotype × temperature interactions (Table 1). One intriguing feature of these relationships is that, for vein density as well as the ratios of tracheary to sieve element number and area , the Italian ecotype (circles) exhibited the smallest difference between  cool- and hot-grown leaves, the Swedish ecotype (squares) exhibited an intermediate difference, and the Col-0 ecotype (triangles) exhibited the greatest difference.When evaluated under a common set of experimental conditions, transpiration rate was significantly influenced by the temperature of the plants during growth, and also displayed significant ecotype × temperature interactions , transpiration rate, foliar minor vein density, and the minor vein ratio of tracheary to sieve element numbers and cross-sectional areas of plants grown under controlled conditions did not vary regularly with latitude or average temperatures for the respective sites from which each ecotype originated (relationships not shown). Therefore, these latter parameters were instead evaluated for a possible association with variation in precipitation levels among the three sites of origin. For  hot-grown leaves of the three ecotypes, transpiration rate , vein density , and tracheary to sieve element ratios  exhibited trends for decreases with increasing levels of habitat-of-origin precipitation, whereas for  cool-grown leaves all three vascular features exhibited trends for increases with increasing levels of habitat-of-origin precipitation level . Moreover, the extent of phenotypic plasticity (difference between cool- and hot-grown leaves) of the latter three vascular metrics was negatively correlated with average precipitation  in the respective habitats of origin . Intriguingly, when normalized for differences in vein density – by multiplying the difference in vascular cell numbers or cross-sectional area by the difference in vein density (VD) – the correlations between average precipitation in the respective habitats of origin and the extent of the differences in tracheary to sieve element ratios between  cool- and hot-grown leaves became stronger yet  and significant for average monthly precipitation during the growing season in the habitat of origin .In contrast to photosynthetic capacity and foliar minor vein phloem features of plants grown under controlled conditions, which varied with latitudinal and temperature gradients between the habitats of origin  and cross-sectional areas  as well as with the product of vein density and  tracheary element number or  tracheary element cross-sectional area of minor veins, transpiration rate was significantly and positively correlated with all of these tracheary element features .While the capacity for photosynthetic oxygen evolution exhibited A. thaliana , and for the number of tracheary elements per vein to increase with increasing vein density, may contribute to an increased capacity to deliver water to the leaves in support of higher levels of transpirational cooling in hot-grown leaves. The absence of a positive association between these xylem features and photosynthesis contrasts with associations observed between foliar hydraulic conductance, vein density, and photosynthesis in studies of other species . While we common .A. thaliana may also be associated with possible differences in response between summer and winter annuals. While foliar vein density and photosynthetic capacity exhibited concomitant upregulation in a summer annual and two biennial species  exhibited upregulation of photosynthesis with no difference in vein density in certain controlled environments , with vein density exhibiting the greatest differential response in the Col-0 ecotype (bracketing that of the Swedish ecotype), a pattern that did not follow the latitudinal and temperature gradients of the ecotypes’ respective habitats.The different response of vein density and photosynthetic capacity to leaf growth environment in  species , winter ronments  or an upronments . MoreoveRather than with temperatures at the respective habitats of origin, the extent of vein density adjustments to experimental growth temperature was inversely correlated with average precipitation in the habitat from which each ecotype originated, as was the extent of the adjustment in the ratio of tracheary to sieve elements to experimental growth temperature . Moreover, the number of tracheary elements per minor vein and transpiration rate were both positively correlated with vein density. These findings suggest that (i) water delivery to the leaves is enhanced not only by a greater foliar vein density but also by a greater number of tracheary elements per vein and (ii) the ability to adjust vein density and the number of tracheary elements per vein to growth at hot temperature is particularly important in natural habitats with lower precipitation levels. Under low temperature conditions, the gradient in water potential from leaf to the atmosphere is relatively low, and the needs of the phloem could presumably be met by a lower tracheary to sieve element ratio, whereas a greater gradient in water potential from the leaf to the atmosphere at high growth temperature presumably necessitates a higher tracheary to sieve element ratio. While water delivery to mesophyll and epidermal cells of the leaf is one important function of xylem conduits, another function of tracheary elements is delivery of water to the phloem to facilitate the pressure-driven flow of viscous, sugar-laden sap in sieve elements from leaves to the plant’s sinks . For adeA. thaliana ecotypes in the present study. Such upregulation presumably counteracts the impact of low temperature on the activity of Calvin cycle enzymes, the activity of the membrane-spanning transport proteins, and the viscosity of the phloem sap  growth under low temperature, while the Polish ecotype that experiences intermediate temperatures and the Italian ecotype that experiences the relatively highest temperatures during their respective growing seasons exhibit intermediate and lower levels of such upregulation, respectively, are consistent with this interpretation.A growing body of evidence supporting a relationship between foliar phloem features that facilitate the export of reduced carbon compounds from the leaf and photosynthesis has been generated over the past decade . Given tloem sap ,b, 2014bPlantago under common conditions (plus a seventh characterized in the field) revealed that species from drier habitats had higher foliar vein densities  in a common garden and found that plant hydraulic conductivity was not correlated with precipitation level but instead with increasing temperature of the habitat from which the species or populations originated  to meet their greater sink activity compared to woody species with lower sink activity and rates of photosynthesis.Perhaps these seemingly divergent findings can be reconciled by consideration of the habitats in which the different species are found and their diverging strategies for acclimating to the range of conditions to which each species is adapted. Firstly, the habitats to which woody species such as conifers and oaks are adapted may simply extend over a much broader range of aridity  than the habitats in which herbaceous species grow and/or the long-lived nature of woody species may require the latter species to persist through seasonal periods of even lower water availability and greater evaporative demand. Consequently, such woody species may place an emphasis on the prevention of cavitation  with decreasing precipitation level. For herbaceous species, an emphasis may instead be on greater hydraulic conductivity to meet the evapotranspirational demand of the leaves at elevated temperatures. The current findings thus contribute to the debate concerning the trade-off (or lack thereof) between hydraulic efficiency of the xylem versus its susceptibility to cavitation . It is aTo briefly summarize, photosynthetic capacity and foliar phloem features presumably indicative of foliar sugar-loading and sugar-export capacity varied in a consistent manner with latitude and temperature of the habitat of origin (greatest in the Swedish ecotype from high latitude and lowest prevailing temperature), whereas vein density and water-transporting tracheary elements varied with precipitation level of the habitat of origin (greatest in the Col-0 ecotype from Poland with the lowest level of precipitation). The nature of these relationships depended on conditions experienced by the plants during growth. The phloem and photosynthesis relationships could be observed in the plants grown under low temperature, whereas the xylem and transpiration relationships could be observed in the plants grown under hot temperature. Moreover, the level of responsiveness in the characterized features was most strongly clarified by comparing the range over which the features acclimated to the two extreme experimental growth temperatures utilized in the present study. Growing all three ecotypes under common conditions at an intermediate temperature revealed very little of the ecotypic differentiation that this species harbors. Similarly, growth of the plants at a higher light intensity masked the extent to which the ecotypes respond to temperature see .Future studies exploring ecotypic differences within a species should thus utilize more than one common experimental growth condition. The straightforwardness of the relationships illuminated in the current study may have been a result of the consistent nature of the three habitats from which the ecotypes were obtained . Were additional ecotypes from more diverse climatic conditions such as higher altitudes, from inland sites further from major bodies of water, or from sites further to the east in latitude  or west in latitude  to be characterized in the same manner, it is possible that their responses may not align closely with those observed in the present study. Moreover, to further understand the relationships evaluated in the current study, additional species from a range of environments should also be characterized. For instance, it is possible that some species responding to declining winter temperatures through shedding of leaves (winter deciduous) or downregulation of photosynthesis (characteristic of many sclerophyllous evergreen species) may exhibit different relationships between xylem features associated with hydraulic conductivity and habitat temperatures and precipitation levels than herbaceous annual and biennial species that respond to low winter temperatures through upregulation of photosynthesis ,b. RelatBD-A and WA designed the experiments, CC, OM, and JS carried out the experiments and collected the data, JS assembled the climatological data, executed the statistical analyses, and rendered the figures, and WA and BD-A analyzed the data and wrote the manuscript, with input from JS.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.


Troponin plays a central role in regulating the contraction and relaxation of vertebrate striated muscles. This review focuses on the isoform gene regulation, alternative RNA splicing, and posttranslational modifications of troponin subunits in cardiac development and adaptation. Transcriptional and posttranscriptional regulations such as phosphorylation and proteolysis modifications, and structure-function relationships of troponin subunit proteins are summarized. The physiological and pathophysiological significances are discussed for impacts on cardiac muscle contractility, heart function, and adaptations in health and diseases. These TnI and TnT isoform genes are closely linked in three tandem pairs in the vertebrate genomes: Fast TnI-fast TnT (TNNI2-TNNT3), cardiac TnI-slow TnT (TNNI3-TNNT1) and slow TnI-cardiac TnT (TNNI1-TNNT2) , which are primarily occupied by Mg2+ in resting muscle cells and can become partially bound with Ca2+ during the activation of contraction , whereas Site I had lost the ability of binding Ca2+  in embryonic cardiomyocytes  under the regulation of adrenergic signaling cascades  in the region binding the C domain of TnC and Thr144 in the inhibitory region, slowing cardiac relaxation and increasing the duration of calcium transient and twitch contraction  to increase the Ca2+ sensitivity of cardiac myofibrils  at Thr31, Thr51, Thr129, and Thr143, among which Thr31 is a preferential site  have also been identified with decreased phosphorylation in heart failure, whereas phosphorylation of Ser166/Thr181/Ser199 in the C-terminal region and Ser77/Thr78 at the TnI-TnT interface (I-T arm) was increased  are summarized in Figure The half-life of cardiac TnI in adult cardiomyocytes is estimated to be ~3.2 days and there is a pool of unassembled cardiac TnI in the cytoplasm Martin, , indicatCardiac TnI is also a substrate of intracellular modifying proteases, with a demonstrated sensitivity to μ-calpain  in transgenic mouse heart resulted in a phenotype of myocardial stunning, and systolic and diastolic dysfunctions  increased myocardial relaxation and improved ventricular filling, similar to the effect of PKA phosphorylation   exclusively expresses slow skeletal muscle TnT instead of cardiac TnT while all other myofilament proteins, including cardiac TnI and cardiac myosin, remain to be the normal cardiac isoform  , TnT2 (splice out exon 4), TnT3 (splice out exon 5), and TnT4 (splice out exon 4 and 5), numbered in the order of decreasing molecular weight , which are located in the C-terminal region containing binding sites for TnI, TnC, and tropomyosin , which inhibited tension development and ATPase activity in skinned fibers  has been found to dephosphorylate cardiac TnT  decreased the phosphorylations of cardiac TnT by 30% and cardiac TnI by 46%, mainly at Ser23/24, in vivo as compared with wild-type controls  increased in primary cultures of adult mouse cardiomyocytes upon ouabain-produced Ca2+ activation of actomyosin ATPase  in cardiac TnI are located in the C-terminal half of the polypeptide chain (residues 128–210)  of cardiac TnI (Liu et al., Another example is that the S69D and D73N mutations of cardiac TnC corrected the abnormal CaFigure The TnC, TnI, and TnT subunits of cardiac troponin function interactively as regulators of myofilament activation and force generation. Based on biochemical, biophysical, physiological and pathophysiological studies, mounting evidence for the molecular evolution, gene regulation, alternative splicing, and posttranslational modifications of cardiac troponin subunits has laid a solid foundation for understanding their structural diversity, structure-function relationships, adaptive regulations, and pathogenic mutations. For troponin's central role in muscle thin filament regulation and contractility, further elucidation of troponin structure and function will powerfully forward the prevention, diagnosis, and treatment of heart diseases.In order to advance troponin research and translate the knowledge into clinical applications, there are several important questions remain to be answered. With modern molecular engineering methodology, it is important and feasible to fine map the interaction sites between troponin subunits and the allosteric and conformational relationships that are essential in regulation of cardiac muscle contraction. Alternative splicing is an important regulatory pathway of cardiac TnT and aberrant splicing of it has close relation with cardiomyopathy. However, the mechanism of cardiac TnT expression via alternative splicing is not well understood. The cell signaling pathway that controls RNA splicing and the production of cardiac TnT variants remains to be investigated. Restrictive N-terminal truncations of cardiac TnI and cardiac TnT are novel posttranslational regulatory mechanisms that have potent roles in cardiac adaptation to stress conditions. However, the cellular mechanisms that induce restrictive N-terminal truncations of cardiac TnI and cardiac TnT have not been established. It is worth noting that no single stress condition has been found to be able to produce restrictive N-terminal truncations of both cardiac TnI and cardiac TnT, indicating distinct mechanisms in the posttranslational regulation of the two troponin subunits that are structurally and functionally closely related. It is also an intriguing observation that the N-terminal truncation of cardiac TnI selectively enhances diastolic function whereas the N-terminal truncation of cardiac TnT selectively reduces systolic velocity of the hearts. To understand how structural modifications of the two subunits of troponin regulate muscle contraction and relaxation in a highly selective manner would lead to development of new therapeutic approaches for the treatments systolic and diastolic heart failures.Continued in depth research is required to answer these and new emerging questions toward the goal of fully understanding the function of troponin in cardiac muscle contraction in order to improve the treatment and prevention of myocardial diseases and heart failure.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.


Septic shock remains a major problem in Intensive Care Unit, with high lethality and high-risk second lines treatments. In this preliminary retrospective investigation we examined plasma metabolome and clinical features in a subset of 20 patients with severe septic shock (SOFA score >8), enrolled in the multicenter Albumin Italian Outcome Sepsis study . Our purpose was to evaluate the changes of circulating metabolites in relation to mortality as a pilot study to be extended in a larger cohort. Patients were analyzed according to their 28-days and 90-days mortality. Metabolites were measured using a targeted mass spectrometry-based quantitative metabolomic approach that included acylcarnitines, aminoacids, biogenic amines, glycerophospholipids, sphingolipids, and sugars. Data-mining techniques were applied to evaluate the association of metabolites with mortality. Low unsaturated long-chain phosphatidylcholines and lysophosphatidylcholines species were associated with long-term survival (90-days) together with circulating kynurenine. Moreover, a decrease of these glycerophospholipids was associated to the event at 28-days and 90-days in combination with clinical variables such as cardiovascular SOFA score  or renal replacement therapy . Early changes in the plasma levels of both lipid species and kynurenine associated with mortality have potential implications for early intervention and discovering new target therapy. Septic shock is the most severe complication of sepsis and is one of the main causes of death in intensive care units (ICUs)In the recent, large, multicenter, randomized clinical trial ALBIOS  that enrolled 1818 patients with severe sepsis or septic shock, 90-day mortality of the latter patients was 46.7%5Indeed, several recent studies have focused on investigating plasma metabolomic profiles as predictive signatures of ICU mortality in adult patients789In the present study, we used a targeted mass spectrometry-based quantitative metabolomics approach focusing our attention on several series of metabolites, such as glycerophospholipids, aminoacids, biogenic amines and acylcarnitines, some of which have already been identified as part of key biochemical pathways in septic shock. Metabolic signatures showing alteration in circulating kynurenine, fatty acids, lysophosphatidylcholines species and /or carnitine esters have already been reported in different settings of septic shock patients789101112131415We applied our metabolomic strategy on a selected subset of patients with severe septic shock (serum lactate concentration >4 mmol/L) with multiple organ failures (SOFA score >8) enrolled in the ALBIOS study. The severe condition under study is, fortunately, not frequent in septic shock patients, but it represents a pathological state associated with a mortality of 20% in the next 24 hoursOur explorative study was designed to provide absolute quantitative information on changes in plasma metabolite levels measured one day  and one week after development of severe septic shock in ICU, and to relate these changes with mortality. The two time points were chosen to verify the hypothesis that the metabolic changes over the time period reflect not only initial clinical characteristics but also the progression of the disease and long-term survival. Association between metabolic patterns and mortality was assessed with univariate and multivariate analyses adjusted for clinical relevant variables.The primary goal of this pilot investigation was to verify the feasibility of our metabolomic approach (e.g. metabolic profiles characterizing non-survivors) that is intended to be used in our ongoing larger scale study ShockOmics (NCT02141607), aimed at elucidating early multilevel markers signatures which could reveal the metabolic pathways involved, a necessary step for a actual target therapy.All the patients with severe septic shock enrolled in the multicenter ALBIOS clinical trialWe applied a mass spectrometry-based quantitative metabolomic profiling in order to unambiguously identify and quantify lipids, aminoacids, biogenic amines and acylcarnitines in the plasma of the study subjects. To ensure data quality and robust statistical analysis the following filtering criteria were applied: (i) metabolites measured with more than 20% missing data (no detectable peak) were excluded for any further data elaboration; (ii) metabolites for which the plasma concentration was below the limit of detection (<LOD) in at least ≥50% of analyzed samples were excluded.In total, 137 metabolites were selected after quality control: 1 hexose, 21 amino acids, 14 biogenic amines, 3 acylcarnitines, 14 sphingomyelins (SM), 84 glycerophospholipids. Concentrations of all analyzed metabolites for each patient are expressed as μM and shown in We first assessed by univariate analysis whether metabolite levels significantly changed from D1 to D7, after stratification by survival status. Profiles of specific metabolites differed significantly between NS and S . The majThe changes of metabolites from D1 to D7, reported in Multivariate analysis was performed using Elastic Net in order to predict the outcome of the patients at 28 or 90 days. Such technique permits to weigh the contribution added by each metabolite to the prediction model and to set to zero the coefficients of those that are not predictive . The coeWe next checked for a possible redundancy of prognostic information among circulating metabolites and clinical variables.This study is a preliminary investigation to characterize the metabolomic profiles of patients with severe septic shock and multiple organ failures at the time of its development, and to integrate them with the clinical manifestation characterizing this syndrome. It was meant as a preparatory study to the ongoing clinical trial  aimed at elucidating early multilevel markers signatures that might help clinicians in understanding the particular pathophysiology characterizing a specific clinical phenotype of septic shock, in prioritizing individual patient treatment, and in developing therapy that acts on specific target of the syndrome.The present study identifies several metabolomic alterations, previously reported in patients with severe sepsis and septic shock78910111215Non-survivors were characterized by a significant elevation of the polyamine pool , from D1 to D7, especially in those who died at 90 days . Since p1921+ and CD8+ effector T cell suppression as well as in generation and activation of regulatory T cells23A peculiarity of NS at days 28 and 90 was the significant increase from D1 to D7 of plasma KYN . Indeed,Decreased plasma level of PC and LPC species was a prominent component of the metabolic phenotype in NS , in acco7131415A general explanation for these findings is that the lowered PC circulating level found in non-survivors might be due to reduced or unbalanced fatty acid substrates for their biosynthesis, consistent with a deregulated mitochondrial and/or peroxisomal beta-oxidation occurring early in sepsiset al.313211A further bio-signature characterizing non-survivors was their reduction over time in circulating mono-saturated and saturated LPCs. Such changes in LPC concentration is in concordance with Park In the multiparameter model, low unsaturated long-chain PC species and LPC a C24:0 were associated with long-term mortality (90 days) together with circulating KYN. Moreover, LPC a C24:0 and PC aa C32:3 were negatively correlated to the event at 28 days and 90 days in combination with clinical variables, in particular, the cardiovascular SOFA score for the 28-day mortality model and the necessity of renal replacement therapy  during the 7 day ICU staying for the 90-day mortality model. The recurrent decrease in LPC a C24:0 may denote an alteration in very long chain fatty acids, such as lignoceric acid as preferred substrates for the peroxisomal beta-oxidation. Consequently, a down-regulation of the peroxisomal lignoceryl-CoA ligase activity might be hypothesized. Indeed, reduced peroxisome proliferator-activated receptor α (PPARα) expression has been associated with decreased survival in pediatric patients with septic shockOur study has several limitations. First, a targeted approach restricts, by its nature, the panel of candidate markers and focuses only on few metabolic pathways. Second, the sample size is limited, and confirmatory studies are necessary. Since many patients with such severity of septic shock do not reach day 7 of ICU staying, we end up with a limited number of patients fulfilling our study criteria. To note that the primary purpose of this investigation was to examine the feasibility of our metabolomics approach in determining changes in circulating metabolites able to characterize the progress of septic shock condition and to reveal the involved pathways.Third, we measured metabolites at two time points only within one week from the diagnosis of septic shock, and metabolites with temporal changes out of this time window might provide a more precise insight for the clinical progression of the disease. Nevertheless, we identified a combination of circulating metabolites altered during the early course of severe septic shock and associated with mortality.This preliminary investigation was therefore very informative in capturing possible evolution and variations of metabolic signatures during a full blown, durable and well-established pathophysiologic manifestation of severe septic shock. Focusing on a homogeneous group of patients rather than on a larger number of scattered phenotypes allowed for a better control of potentially confounding factors. Therefore, the metabolic changes observed in our samples pertains more closely to the selected pathophysiological condition and it should be proved in a larger cohort by including different phenotypes such as not only the severe patients and multimarkers information such as multi-omics data.In conclusion, the data presented here confirm the feasibility of our approach in determining changes circulating metabolites able to characterize the progress of septic shock condition. Our results are in line with recent findings indicating that lipid homeostasis and tryptophan catabolism might influence mortality in septic shock. The association of early changes in the plasma levels of both lipid species and KYN with mortality, with possible implications for early intervention is the most important result of our study Although our analyses cannot determine causality, it suggests that alterations in KYN and lipid species might represent not only risk factors for patients with severe septic shock but important pathophysiologic mechanisms deserving further investigations.This pilot retrospective metabolomic investigation was a substudy of the multicenter, randomized Albumin Italian Outcome Sepsis (ALBIOS) clinical trial, which enrolled patients with severe septic or septic shock from 100 ICU in Italy (NCT00707122). Details of the ALBIOS study and protocol are fully described in the original articleInclusion criteria for the present study were the presence of septic shock20), FiO2 (%); (iv) blood gas analysis: central venous O2 saturation, venous partial pressure of CO2, arterial partial pressure of CO2, arterial partial pressure of O2, central venous partial pressure of O2, arterial and venous pH; (v) laboratory and clinical parameters: serum concentrations of creatinine (mg/dL), biliuribin (mg/dL), and lactate (mmol/L), platelet count (x103 cells/mm3); (vi) Sequential Organ Failure Assessment Score (SOFA)Only 20 among the 997 patients enrolled in ALBIOS trial and with plasma samples stored in the biobank fulfilled the inclusion criteria see . The folOutcome was defined as mortality at 28 and 90 days. For each patient, plasma samples were available at day 1  and at day 7  after diagnosis of septic shock for the present metabolomics analysis.A targeted quantitative approach using a combined direct flow injection and liquid chromatography (LC) tandem mass spectrometry (MS/MS) assay  was applied for the metabolomics analysis to EDTA-plasma samples stored at −80 °C at the ALBIOS-biobank. The method combines derivatization and extraction of analytes with the selective mass-spectrometric detection using multiple reaction monitoring (MRM) pairs. Isotope-labeled internal standards are integrated into the platform for metabolite absolute quantification. This strategy allows simultaneous quantification of 186 metabolites . A summary of the metabolomic analysis and the list of all the measurable metabolites are provided in In the present analysis, patients were analyzed according to their survival status at 28 and 90 days after study enrollment. All quantified metabolites were considered in the analysis, together with the clinical parameters available at study enrollment and those related to patient condition at day 1 and at day 7  and 2.The comparisons between survivors (S) and non-survivors (NS) were performed by unpaired Wilcoxon Test for clinical parameters and metabolite concentrations (μM). The changes of the parameters from day 1 to day 7 were evaluated by means of the paired Wilcoxon signed rank test separately for the S and NS groups. Finally, the variations of the metabolites concentrations were compared between S and NS group by unpaired Wilcoxon test. We calculated also the false discovery rate (FDR) to overcome the problem of the large number of statistical comparisons. To compute the FDR, we used a bootstrapping technique of oversampling with replacement to obtain a sample size of 20 patients for each group (S and NS).Multivariate analysis was performed using the Elastic Net technique, a shrinkage regression method effective in case of several highly correlated variablesHow to cite this article: Ferrario, M. et al. Mortality prediction in patients with severe septic shock: a pilot study using a target metabolomics approach. Sci. Rep.6, 20391; doi: 10.1038/srep20391 (2016).