The development of the mammalian cerebellum is orchestrated by both cell-autonomous programs and inductive environmental influences. Here, we describe the main processes of cerebellar ontogenesis, highlighting the neurogenic strategies used by developing progenitors, the genetic programs involved in cell fate specification, the progressive changes of structural organization, and some of the better-known abnormalities associated with developmental disorders of the cerebellum. The work done on cerebellar development from the late nineteenth century until the 1970s provided substantial and significant information; however, it was only descriptive and barely addressed the mechanisms involved. Over the last two decades, thanks to the technological revolution in molecular biology, our understanding of cerebellar development has drastically changed. We are now going through an exceptional period in our understanding of the mechanisms that underlie the complex development of the cerebellum. An understanding of cell specification regulated by the expression of region-specific combinations of transcription factors or proneural genes, and the formation of synaptic circuits, seems within reach.Ferdinando Rossi, a few months before his death, undertook the monumental task of writing a monograph on the spectacular advances in our understanding of cerebellar development achieved in the last 20 years. Sadly, Ferdinando died a few months after beginning his monograph. This consensus paper, based on Ferdinando’s initial design, summarizes many of these advances and is dedicated to his memory.The review comprises 18 brief sections, ranging from the early molecular specification of the cerebellar anlage to its mature architecture and pathology. It also includes information on neurogenesis, mainly the specification and origins of neuronal and glial progenitors. An important part of the paper is devoted to Purkinje cells (PCs) as key neurons of the cerebellar cortex responsible for the proliferation of granule cells (GCs) and the establishment of “crude” projection maps with extracerebellar afferent fibers. Finally, the biochemical heterogeneity of PCs allows for a cortical subdivision into distinct functional bands, a presumptive protomap for the development of circuit topography  8.5 in mice) at the interface of the OTX2- and GBX2-positive neuroepithelial domains. The co-expression of Otx2 and Gbx2 in the IsO territory essentially disappears by HH11–12 (chick) and E10 (mouse), and both domains become thereafter mutually excluded and complementary. The limit determined by Otx2 and Gbx2 marks the mid-hindbrain molecular boundary (MHB)  as well as knock-in mice (SCA1154Q/2Q) exhibit motor impairments and PC degeneration [ATXN1[82Q] model [staggerer mouse [Rora gene encoding RORα, and is viewed by some as an extreme model of SCA1 [Spinocerebellar ataxia type 1 (SCA1) is caused by a CAG repeat expansion in the Ataxin1  mouse harbors a dominant gain-of-function mutation in the TRPC3 ion channel, resulting in adult-onset PC loss but also impairments in PC dendritic arborization during cerebellar development [Besides models of the human SCAs, several other genetic mouse mutants have highlighted the relationship between abnormal PC development and ataxia. For example, the ataxic elopment . TRPC3 ielopment , 438. Inelopment , 440, SCelopment , and SCAelopment  models. In summary, PC developmental abnormalities are clearly evident in a wide range of ataxic mouse mutants including models of the degenerative SCAs. The observed PC developmental defects commonly include impaired dendritic arborization, resulting in synaptic deficits affecting CF and PF connections and ultimately altering PC physiology. It will be important to better understand the underlying—likely common—molecular mechanisms by which mutations in distinct genes cause abnormal PC development and function. These could offer attractive future therapeutic targets to alleviate motor dysfunction in cerebellar ataxia.MB is the most common pediatric brain tumor and is the most common cause of pediatric death from cancer. Histologically, cases are classified into classic, nodular/desmoplastic, and large cell/anaplastic subtype , and proThe current consensus is that MB can be sub-classified based on genetic, epigenetic, and transcriptomic characteristics , 445 intPtch1 is conditionally inactivated in GCPs. Importantly, constitutive activation of SHH signaling induces neoplastic transformation of more undifferentiated progenitor cells only upon commitment toward a GC lineage [Atho1, a marker of the GC lineage, and showed activation of Gli1, Cyclin D1, and MycN, which are SHH target genes. Their gene expression profiles were more similar (differing by 34 genes) to tumor cells than GCPs (differing by 75 genes) [GCPs are the main cell of origin of SHH-MB, as shown in mouse models in which  lineage . Pre-neo5 genes) . SHH-MBs5 genes) .MRI studies have shown differences in the location of MB subgroups, and MB location (as well as enhancement pattern) can predict the molecular subgroup of pediatric MBs. SHH tumors are mainly detected within the cerebellar hemispheres –451 consMouse models have shown that WNT-MBs, characterized by activating mutations in the Wnt pathway effector CTNNB1, arise from cells outside the cerebellum, in the embryonic dorsal brainstem . These sMRI studies in patients have shown that WNT tumors are often found within the fourth ventricle (cerebellar peduncle/cerebellopontine angle cistern) and infiltrated the dorsal brainstem , 450, wiRb+Tp53 mutant mice were generated where MBs developed from neural stem cells in the cerebellar white matter [Myc+Tp53 mutant mice developed MBs from cerebellar neural stem cells and from GCPs, although the tumors that formed from GCPs lost their lineage specific markers first [Cerebellar stem cells are a third likely cell of origin for human MBs. Models using e matter , 454. Thrs first . This moIn the MRI studies to date, Group 3 and Group 4 MBs are characterized by brainstem contact with most of the tumors growing within the vermis. Most also contact both the cochlear and cuneate nuclei and always infiltrate the fourth ventricle , 451. GrGroup 4 comprises 30 % of MBs and it is the least well characterized molecularly. At present, it is also unclear from which cells these tumors originate. This subgroup has a male-to-female ratio of 2:1 and has an intermediate prognosis, similar to the SHH subgroup . Group 3In summary, “-omics” datasets on large series of MB combined with the results of ontogeny studies performed in mouse models aiming at characterizing the cell of origin of the various subgroups together with magnetic resonance imaging (MRI) studies in patients lend additional support to the notion that MB is in fact a disease arising from deregulated cellular and molecular mechanisms involved in the development and homeostasis of the cerebellum.The goal of our consensus paper is to provide an updated view of our current knowledge on cerebellar development. The broad spectrum of the work reviewed in such a short format leaves little room for specific debate. Nevertheless, many of the concepts reported here have already reached consensus.The seminal discovery of the “isthmic organizer” and its inductive role has led to an understanding of the molecular regionalization of the neural tube with its precise frontiers between domains expressing homeobox-containing genes. The interface between caudal GBX2 and rostral OTX2 expressing domains marks the location of the “isthmic organizer” which, through Fgf8 secretion, initiates the molecular cascade required for the specification of cerebellum.Next come the germinal epithelia of the cerebellum. For over a century, the cerebellum was known for the dual origins of its neuronal populations: the VZ and the RL. However, the categories of neurons generated in each of these epithelia have only recently been established. Genetic fate mapping proved that glutamatergic neurons , specified by the transcription factor ATOH1, originate from the RL and migrate via the EGL. In contrast, GABAergic neurons , specified by PTF1a, originate from the VZ. Moreover, both neuroepithelia are divided into subdomains, each one specified by distinct transcription factors to generate the corresponding population of neurons.+ and those born later will be ZII−, indicating that specification might exist among progenitors. Related to PC compartmentalization, the formation of circuit topography and the elimination of CF multiple innervation opens the vast domain of synaptogenesis and the role of functional activity in the fine-tuning of the cortical circuit’s specificity—a chapter that, at this moment, remains little explored and is an anticipated topic for future consensus papers. Similarly, the ways in which cerebellar INs and glia become integrated into the mature cerebellum are also beginning to be understood. Finally, in parallel with these events, we are now seeing how the development of the characteristic cerebellar foliation fits into the picture.Following specification comes neuronal migration and differentiation. Despite recent progress, the data are less complete. For example, broad areas of the early development of PCs remain obscure, even while later events such as dendritogenesis are well understood. Our knowledge on the mechanisms leading to the biochemical heterogeneity of PCs and their aggregation into longitudinal and transverse compartments has improved: there is a dichotomy among PCs, those born between E10 and E11.5 will become ZIIWe have also updated on the role of the TH (a cerebellists’ old friend because of its multiple effects on cerebellar development) describing recent advances using transgenic animals with conditional nuclear TH receptor knock-outs targeted to PCs or/and IN that allow for the suppression of secondary effects on other neurons or peripheral organs.Finally, our review has briefly touched upon cerebellar pathology. For example, the analysis of the role of SHH, secreted by PCs as mitogen for GC proliferation, provides a basis for better understanding of the origins of MBs, and inherited disorders of cerebellar development can now be placed in a stronger developmental context.In conclusion, following the outline proposed by Ferdinando Rossi in his planned monograph, the data presented here provide our brief consensus of the current knowledge on cerebellar development.


Enduring diabetes increases the probability of developing secondary damage to numerous systems, and these complications represent a cause of morbidity and mortality. Establishing the causes of diabetes remains the key step to eradicate the disease, but prevention as well as finding therapies to ameliorate some of the major diabetic complications is an equally important step to increase life expectancy and quality for the millions of individuals already affected by the disease or who are likely to develop it before cures become routinely available. In this review, we will firstly summarize some of the major complications of diabetes, including endothelial and pancreatic islets dysfunction, retinopathy, and nephropathy, and then discuss the emerging roles exerted by the neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) to counteract these ranges of pathologies that are precipitated by the prolonged hyperglycemic state. Finally, we will describe the main signalling routes activated by the peptide and propose possible future directions to focus on developing more effective peptide-based therapies to treat the major complications associated with longstanding diabetes. The prevalence of diabetes is increasing worldwide. According to the World Health Organization (WHO), the total number of people affected by diabetes is expected to rise to an estimated 300 million cases by the year 2025 . The onsβ-cell insulin secretion, and discuss the major intracellular events implicated and how these are hampered by peptide treatment. The final purpose will be to shed more light into PACAP function for future exploitation in therapies aimed at arresting or preventing the development of complications associated with disease progression.In this review we will outline some of the most recent advances from our research group and others in relationship to the role of a promising neuropeptide candidate endowed with potential beneficial effects to treat or ameliorate certain aspects of diabetes, namely, the pituitary adenylate cyclase-activating polypeptide (PACAP). We will emphasize how the neuropeptide interferes and in some cases prevents the development of specific pathological consequences of the disease, such as micro- and macroangiopathy, retinal dysfunction, and defective pancreatic β-pancreatic cells destruction that leads to absolute insulin deficiency. This failure to produce insulin can be autoimmune-mediated or idiopathic. Type 2 diabetes constitutes 90–95% of all diabetes cases and results from insulin resistance, inadequate compensatory insulin secretory response, or both. It is characterized by reduced responsiveness of peripheral cells in the body to insulin and, consequently, reduced ability to transfer glucose out of the circulation [The term diabetes mellitus indicates a group of metabolic disorders characterized by high blood sugar and impaired insulin signaling. The disease is usually classified into type 1 diabetes, type 2 diabetes, and other specific types including gestational diabetes. Type 1 diabetes, also known as insulin-dependent diabetes, represents about 5–10% of all cases of diabetes. It is characterized by Elevated glucose levels result in an increased production of reactive oxygen species (ROS) and inflammatory mediators strictly involved in vasculature dysfunction. The altered system not only causes hypertension, ischemia, and/or altered vascular permeability but also contributes to other complications of diabetes , 7.In the last decades, a big effort was put into finding new therapeutic approaches to target either the full range of diabetic complications or the damaged tissues/organs. Although several classes of antidiabetic drugs are currently available, achieving and maintaining long-term glycemic control are often challenging and not sufficient alone, so a significant need for novel antidiabetic drugs remains. It has been discovered that neuropeptides and their analogues, activating different signalling pathways through their receptors, are emerging as new therapeutic options. Among these, one of the most promising human peptides is the pituitary adenylate cyclase-activating polypeptide (PACAP). This review outlines the knowledge accumulated over the past years on the role of this neuropeptide in the major complications of diabetes, particularly emphasizing its potential for new therapeutic approaches.203H331N63O53S; for details on PACAP38 chemical structure please refer to  In vitro and in vivo studies have shown that the peptide acts through the PAC1 receptors to stimulate various downstream executors of the protein kinase A and C (PKA and PKC) pathways [β-arrestin signalling, and mitogen-activated protein (MAP) kinase in some tissues [PACAP  sites are also detectable in the retina and the nervous tunica of the eye, both known to be highly sensitive to long-lasting glucose insult. Indeed, immunohistochemistry studies have shown that PACAP-like IR is evident in a population of sensory neurons in the rat uvea , 30 and + amacrine cell processes make synaptic contacts with retinal ganglion cell (RGC) terminals, as well as amacrine and bipolar cell processes. PACAP+ amacrine cells also establish synaptic contacts with each other. PACAP+ axon terminals establish synapses with ganglion cells, bipolar cells, amacrine cells, and horizontal cells in the GCL, NFL, and IPL.In the IPL, PACAP in vivo, PAC1+ has been shown in rat primary cultures of Müller cells [Studies concerning PAC1 receptor distribution have indicated that binding sites for PACAP and VIP, positively coupled to adenylate cyclase, are expressed in the retina of different mammalian species . PAC1-IRer cells . For a cDiabetes is characterized by a high risk of developing chronic complications, with micro- and macrovascular impairments in multiple organs. Today it is well established that increased oxidative stress impairs endothelial function and it is thought to mediate vascular disease.Vascular function depends upon the balance of oxidant and antioxidant mechanisms. During diabetes, an unbalance between anti-inflammatory and antithrombotic homeostasis in favor of proinflammatory and thrombogenic elements is evident. Heightened oxidative stress and altered expression of a variety of genes related to atherogenesis and angiogenesis have also been described in the vasculature exposed to elevated levels of glucose , 36–39. in vitro, such as longstanding high glucose exposure in murine microvascular endothelial cells [In addition, trophic factor alterations associated with early and late stages of diabetes seem to aggravate hyperglycemia-induced proliferative response, resulting in increased basement membrane thickening and aberrantly regulated vasculogenesis. On the other hand, molecules like PACAP, though being commonly recognized as trophic factors, have demonstrated to also possess opposite biological roles in the vasculature system, most likely depending on the surrounding microenvironment or other coexisting factors, and have been therefore defined as “nonclassic endogenous regulators of angiogenesis” . To suppal cells  or in aoal cells , acting al cells . In thatal cells . It is tThe importance of PACAP in relationship to circulatory dysfunction caused by long-standing diabetes does not seem to be solely limited to restoring microvasculature, as the combined vasodilatory effects together with the growth-inhbitory functions of the peptide can be extended to the circulatory system in general. In particular, the PACAP homolog VIP has captured scientific interest because of its growth inhibitory properties on smooth muscle cells , inferri in vitro and in vivo evidences discussed here, it appears that PACAP, by recruiting different receptors in a cell type-dependent fashion, activates a series of physiological/compensatory responses finalized to reestablish vascular homeostasis. However, further investigations might still be needed to better define the specific actions mediated by each binding receptor, as this will set the stage to develop new highly selective PACAP analogues or receptor agonists encompassing therapeutical activity for the treatment of vascular complications associated with diabetes.Based on theβ-cell insufficiency in the pancreas, which results in defects in insulin secretion and hyperglycemia [β-cell mass and function in diabetic patients and causes diabetic complications, including diabetic nephropathy, neuropathy, and retinopathy [β-cells. PACAP protein was colocalized to secretory vesicles of insulin and glucagon-containing cells in human and rodent pancreases [ In situ hybridization studies have also shown that both PAC1 and VPAC2 receptors are expressed in the β cells, being the predominant PACAP binding receptors in these cells, while VPAC1 receptors were detected only in vessels surrounding the islets [ in vivo and in vitro studies have shown that PACAP, in an autocrine and/or paracrine manner, produces trophic effects on β-cells. It stimulates the main endocrine pancreatic function (insulin production and release), preserves β-cell responses to glucose, and regulates both proliferation and cell viability [β-cell population have taken advantage on genetically engineered mice with either a targeted deletion of the PAC1 receptor gene (PAC1−/−) or of the VPAC2 receptor gene (VPAC2−/−). Either knockout animal model displays reduced insulin response to both gastric and, to a lesser extent, intravenous glucose administration. These mice also display impaired glucose elimination. PAC1 deletion is also associated with a robust reduction of glucose-stimulated insulin secretion, suggesting that PACAP through PAC1 receptors might also play a role in modulating the insulin secretory response to glucose itself. Regarding VPAC2−/− models, although these mice develop normally, they exhibit increased metabolic rate and increased insulin sensitivity, accompanied by reduced body weight due to reduced fat mass. As for PAC1 receptors, these observations suggest that VPAC2 receptors might as well be necessary for physiological insulin secretion [A hallmark of both types I and II diabetes is the progressive glycemia , 48. Paninopathy , 50. Aboncreases . In situe islets , 52. Acciability –57. Studecretion . However in vitro of DMO, hence showing that PACAP is able to also prevent the disruption of the BRB, mainly by modulating the expression of important tight junctions such as zona occludens-1 (ZO-1) and claudin-1, both essential for the proper functionality of the retinal barrier during diabetes [Diabetic retinopathy (DR) is one of the most significant and disabling chronic complications of diabetes mellitus . DR can diabetes .β1 in the kidney, or hampering the proapoptotic p38 MAPK pathway [ in vitro models, such as vasodilation and renin secretion [ In vivo models of diabetic nephropathy have also proven the protective role of PACAP in this pathological condition. Its ameliorative effects against high glucose-driven kidney impairment seem to be in part due to inhibition of apoptotic, fibrotic, and oxidative pathways, paralleled by anti-inflammatory properties of the peptide [Alterations in renal function and structure are found even at the onset of diabetes mellitus. Diabetic nephropathy is characterized by initial proteinuria followed by a decline in glomerular filtration rate and ultimate progression to uraemia. Diabetic nephropathy is the leading cause of end-stage renal failure and about 30–40% of patients need renal transplantation . The mai pathway . Indeed,ecretion  and protecretion . In vivo peptide , 77. AltA further severe complication in most cases secondary to angiopathy is diabetic neuropathy, which affects the peripheral nervous system. Over half of all patients affected by diabetes develop some form of neuropathy, resulting in sensory loss, pain, and autonomic dysfunction. These manifestations of neuropathy can severely reduce a patient's quality of life. As for the previous complications discussed above, duration of diabetes and glycemic control are important risk factors for neuropathy in both patients with type 1 and type 2 diabetes . ActuallThe present review summarizes the findings on the neuroprotective potential of PACAP in major diabetes complications. Based on these data, PACAP emerges as a powerful and promising candidate in the treatment of those pathological conditions associated with long-lasting diabetes. From the present review it arises that diabetic complications are the main consequence of significant changes in vascular structure and function, along with other metabolic alterations. PACAP appears to be able to effectively counteract and/or prevent the majority of these changes, making it a potentially valuable candidate to treat diabetes complication. Unfortunately, a major hurdle that has significantly limited PACAP therapeutic use in T2D-related injuries depends on the rapid hydrolysis of the peptide occurring when injected systemically. Future efforts are therefore necessary to develop a peptide analogue which is more stable, can easily cross human biological barriers  to bind its receptors in affected organs/systems, and possesses adequate pharmacokinetics. Alternatively, other routes could be to take advantage of new generation viruses for PACAP gene delivery to treat this still unmet medical need, but further studies in this direction are yet to be carried out to exclude any possible associated risk.


While the capacity of the olfactory epithelium (OE) to generate sensory neurons continues into middle age in mice, it is presumed that this regenerative potential is present throughout all developmental stages. However, little experimental evidence exists to support the idea that this regenerative capacity remains in late adulthood, and questions about the functionality of neurons born at these late stages remain unanswered. Here, we extend our previous work in the VNO to investigate basal rates of proliferation in the OE, as well as after olfactory bulbectomy (OBX), a commonly used surgical lesion. In addition, we show that the neural stem cell retains its capacity to generate mature olfactory sensory neurons in aged animals. Finally, we demonstrate that regardless of age, a stem cell in the OE, the horizontal basal cell (HBC), exhibits a morphological switch from a flattened, quiescent phenotype to a pyramidal, proliferative phenotype following chemical lesion in aged animals. These findings provide new insights into determining whether an HBC is active or quiescent based on a structural feature as opposed to a biochemical one. More importantly, it suggests that neural stem cells in aged mice are responsive to the same signals triggering proliferation as those observed in young mice. The adult nervous system is limited in its capacity to repair itself. Repositories of neural stem cells are found in the subgranular zone, subventricular zone, and in the neuroepithelium lining the nasal cavity. However, the mechanisms responsible for widespread aging-induced diminished neurogenesis are unclear. It is possible the function of the stem cell niche surrounding neural stem cells declines with age, or perhaps the regenerative capacity of the stem cell itself is exhausted. In addition, the fundamental question of whether regeneration in the olfactory system is simply a recapitulation of development or is in fact its own process also remains unanswered  supplies inhibitory interneurons to the olfactory bulb, while the other  generates excitatory projection neurons resident in the olfactory epithelium (OE). The basal stem cells that give rise to this latter group in the OE are accessible, easily studied, and are a rich source of adult neural stem cells that may have potential for clinical research , mature olfactory sensory neurons (OSNm), and sustentacular cells (Sus). Both types of basal cells, the HBCs and GBCs, are multipotent and give rise to sensory neurons , is intact even in very old animals, particularly in response to injury late in life , sensory deprivation (via naris occlusion), axotomy, and chemical ablation of the OE. Target ablation and axotomy result in the initial degeneration of sensory neurons in the OE, followed by an upregulation of proliferation of mitotic progenitor cells and subsequent robust regrowth of millions of new sensory neurons in a period of several weeks. The OE is largely reconstituted by approximately thirty days , cytokeratin 5 , olfactory marker protein  and PCNA ; Abcam, Cambridge, MA; catalog number Ab29; dilution 1:200).Mice were anesthetized by intraperitoneal injection with a mixture of ketamine/xylazine  and perfused transcardially with heparinized (4 unit/mL) phosphate buffer saline (PBS) (pH 7.4) followed by 4% paraformaldehyde  in 0.1 M phosphate buffer . Olfactory epithelia were dissected, decalcified, cryoprotected (30% sucrose) and frozen in OCT. Cryosections (12 μm) were incubated in blocking solution containing 0.5% Triton X-100 and 5% normal donkey serum in 0.1 M PB for 1 h at room temperature. Antigen retrieval was performed with Retrievagen A  or Retrievagen B pH 9.5, 550527; BD Biosciences) if necessary. Primary antibody incubation was performed overnight at 4°C. Sections were then incubated with Alexa Fluor 488 and 594 conjugated secondary antibodies  for 2 h at room temperature. TOTO-3  was added in a wash step after secondary incubation for visualization of nuclei. Sections were mounted in Vectashield  to prevent photobleaching. Optical sections (1 μm) were taken through the depth of the section with a Zeiss LSM700  confocal microscope and analyzed with ImageJ software. Figures were assembled with Adobe Photoshop and Illustrator software.To chemically ablate the olfactory epithelium, methimazole  was administered via single intraperitoneal injection (50 mg/kg). To surgically ablate the OE by severing the axons of mature sensory neurons, OBX was performed according to standard methods  injection and was detected with an antibody recognizing BrdU (described above). For acute labeling experiments, 100 mg/kg BrdU was injected 2 h prior to sacrifice. For maturation studies (see below), two injections 2 h apart of 50 mg/kg BrdU were given; the animals were then sacrificed 30 days following injection. For OBX studies, 100 mg/kg was injected 2 h prior to death on the fifth or thirtieth day after OBX was performed.Immunohistochemistry). In all animals, at least 10 coronal sections throughout the OE were examined to yield a minimum of 25 BrdU positive cells examined per animal. Optical sections (1 μm) were taken with a 40x objective through the depth of the section with a Zeiss LSM700 confocal microscope and analyzed with ImageJ software. Complete overlap in all visual planes with a nuclear marker (TOTO-3) was required in order to verify that examination was of a single BrdU-labeled cell. Z-projections through the middle of BrdU/TOTO-3 positive nuclei were then made to verify that OMP labeling surrounded the entire nucleus. Cells that exhibited partial labeling with OMP or those in which the plane of cryosection bisected the nucleus were not included in the analysis.Cryosections were prepared as described above (see Immunohistochemistry). In all animals, every fifth section (60 μm increments) of olfactory epithelia were processed for BrdU and OMP immunohistochemistry, counted immediately with a Leica DMR microscope  to minimize effects of photobleaching, and photographed with a SPOT digital camera . The area of OMP immunoreactivity was used to define the reference space and was measured with ImageJ (NIH) software to allow for normalization of counts. Counts were performed by a trained experimenter blind to condition in an unbiased, random manner with systematic sampling. The total number of BrdU-positive cells was not calculated but rather the number of BrdU-positive cells per unit area, as BrdU labeling observed at the dosages utilized here is a rare event. For quantification of HBC number, at least two positions along the dorsal septum in each animal were imaged with a Zeiss LSM 700 confocal microscope. The number of Krt5+ cells was counted with NIH ImageJ Cell Counter, the length of the counted OE was recorded, and the number of cells per mm was calculated for each animal. Three to five animals were analyzed per group.Thin cryosections (12 μm) were prepared as described above . For analysis of basal proliferation levels, all data were analyzed for statistical significance by One-Way ANOVA and Student Newman-Keuls (SNK) pairwise multiple comparison between age groups. For maturation studies, data were expressed as a ratio defined by the number of cells that were both OMP-positive and BrdU-positive divided by the number of BrdU-positive cells, regardless of OMP status. Ratio data were analyzed with a One-Way ANOVA and SNK multiple comparison between age groups. For OBX studies, olfactory epithelium measurements were performed in the same tissue section to control for effects of position, permitting the analysis of control epithelium (olfactory bulb intact) and contralateral lesioned epithelium (olfactory bulb removed) in a single section. Data were analyzed with Two-Way repeated measures ANOVA for the effects of age and treatment (OBX), followed by Bonferroni multiple comparisons test between lesion and non-lesion control for each age group. For HBC cell number and morphology studies, data were analyzed by Two-Way ANOVA for the effects of age and treatment (methimazole lesion).We first examined whether proliferation in the adult olfactory epithelium continues to decline with age. Although previous work has shown that proliferation does decline, these studies examined animals only to middle ages. Kondo et al. recently found that the rates of proliferation and cell death decline in animals up to 16 months of age  labeling paradigm to label proliferative cells, where mice were injected with 100 mg/kg BrdU 2 h before sacrifice. As we observed BrdU incorporation in both basal Figures ; arrows While we observed an inverse relationship between age and the level of proliferation in an intact epithelium, it was not clear from these data whether the proliferative capacity of neural stem cells also declined in aged animals. We therefore asked whether a lesion challenge, namely unilateral OBX, would result in an increase in proliferation in the OE. Lesion of the axons extending from the olfactory epithelium to the olfactory bulb by OBX results in a wave of apoptotic cell death that selectively targets all mature neurons in the epithelium within 5 days of the lesion. In young animals the robust regenerative capacity of the basal cells repopulates the epithelium with > 8–10 million new neurons within 3–4 weeks  were administered 2 h apart, followed by sacrifice either 10 or 30 days after BrdU injection. We then examined cells for coincident labeling of both BrdU and olfactory marker protein (OMP), a marker of neuronal maturity Figure . AlthougThe identity of the neural stem cell in the adult olfactory epithelium is somewhat controversial as both types of basal cells are neurogenic progenitor cells , we found that, as expected, in intact epithelium the HBC population is found restricted to the base of the epithelium with a flattened morphology Figure . Krt5 haIn aged mice, as the olfactory epithelium recovered from the lesion, HBC number declined; by 30 days post-lesion, HBC number in methimazole-treated animals was no different from saline-injected control animals (data not shown). We did not observe a decline in HBC number by 30 days post-lesion in young animals, however; in 2 month old methimazole-treated animals, HBC number remained elevated above that observed in saline-treated animals (data not shown). Hence it is possible that while the olfactory epithelium is recovering in aged animals, the percentage of newborn cells surviving is higher than that in young animals, as previous data indicate the aged OE is in fact capable of a robust response to a prolonged surgical lesion Figure .+ immunoreactive cells as having either a flat or pyramidal morphology. Cells exhibiting pyramidal morphology more frequently expressed a marker of proliferation, PCNA (proliferating cell nuclear antigen), than cells with a flattened morphology  and aged adult (18 month) mice. When we analyzed cross-sectional area according to morphology, we found a significant increase in cell area from a flat to a pyramidal morphology (data not shown). We also observed that in saline-injected mice, regardless of age, a significant majority of HBCs were flat in morphology while in methimazole-injected animals, a significant majority of HBCs were pyramidal in morphology Figures . We contNeural regeneration is rare in the nervous system, but could have an important impact on recovery from traumatic or pathologically induced brain injuries and on degenerative processes of normal aging. The potential of the basal stem cells to generate sensory neurons in a young adult olfactory epithelium (OE) has been known for more than 40 years, as they continually send new fledgling axons to stereotyped positions in the OB with each cell birth and growth cycle , axotomy, and chemical ablation of the OE. Our next question was whether the aged OE could robustly respond to injury paradigms. We chose to use two separate lesion techniques, namely a surgical (OBX) and a chemical (Methimazole) ablation.When we lesion the olfactory epithelium acutely by OBX, the basal cells return to action and initiate a significant increase in the basal rate of proliferation. Thus, although the basal stem cells in aged animals have become quiescent, they retain the capacity to meet the challenge of traumatic cell loss—even in 24 month old mice. When challenged with a sustained OBX injury, increased proliferation persists in aged mice, as in younger animals. These data demonstrate that certainly the proliferative capacity and perhaps the regenerative capacity of the epithelium remains intact with aging and that there is a viable population of neural stem cells even in very old animals. Suzukawa et al.  suggest We are particularly interested in the behavior of the HBC population of stem cells. While these cells are particularly active during the first third of the life of the animal, apparently for the purpose of continued post-natal growth of the tissue, they enter a quiescent phase at early post-natal stages, where previously pyramidal shaped basal cells change to a flattened morphology and complete lack of incorporation of the proliferative marker BrdU (Suzuki and Takeda, The results presented here, in concert with data from other laboratories, present numerous interesting questions. First, what is the biochemical switch that regulates the proliferative vs. quiescent state of HBCs? Recently it has been demonstrated that p63 down-regulation acts as a switch to activate HBCs following injury with the olfactotoxic gas methyl bromide (Schnittke et al., We have provided here a perspective of neuronal regeneration in the olfactory epithelium that is different from that seen anywhere else in the nervous system. Particularly the dual profile of the HBC documented here may lead to new and intriguing mechanisms for maintaining stem cell growth potential in aging neuronal tissue.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.


Studies using inhibitors of ethylene biosynthesis or of ethylene action and analysis of mutant lines altered in genes involved in the ethylene signaling pathway  demonstrate the involvement of ethylene in the regulation of germination and dormancy. Ethylene counteracts ABA effects through a regulation of ABA metabolism and signaling pathways. Moreover, ethylene insensitive mutants in Arabidopsis are more sensitive to ABA and the seeds are more dormant. Numerous data also show an interaction between ABA, GAs and ethylene metabolism and signaling pathways. It has been increasingly demonstrated that reactive oxygen species (ROS) may play a significant role in the regulation of seed germination interacting with hormonal signaling pathways. In the present review the responsiveness of seeds to ethylene will be described, and the key role of ethylene in the regulation of seed dormancy via a crosstalk between hormones and other signals will be discussed.Ethylene is an important component of the gaseous environment, and regulates numerous plant developmental processes including seed germination and seedling establishment. Dormancy, the inability to germinate in apparently favorable conditions, has been demonstrated to be regulated by the hormonal balance between abscisic acid (ABA) and gibberellins (GAs). Ethylene plays a key role in dormancy release in numerous species, the effective concentrations allowing the germination of dormant seeds ranging between 0.1 and 200 μL L Phase III is characterized by radicle protrusion  in particular regulates germination and dormancy of numerous species via a complex hormonal signaling network  and gibberellins (GAs) in the regulation of seed germination and dormancy in response to environmental signals is well documented and discussed in recent reviews . ABA is  network .The role of reactive oxygen species (ROS) in seed biology has progressively emerged and evolved this last decade. Originally considered as harmful compounds, causing deleterious reactions toward a wide range of biomolecules and thus to seeds, ROS are now widely acknowledged as signaling compounds regulating the germination process through an hormonal network .In this review, we describe how ethylene interacts with other plant hormones in regulation of germination and dormancy, concentrating on its interactions with ABA, GAs, and ROS.Helianthus annuus) seeds. Interestingly, a close relationship between the ability to produce ethylene and seed vigor has been reported in various species including rape  and ACC are the main intermediates . S-AdoMet synthesized from methionine by the S-AdoMet synthetase (or SAM synthetase), is converted to ACC, the direct precursor of ethylene, by ACC synthase . The by-product 5′-methylthioadenosine (MTA) is recycled back to methionine through the Yang Cycle . Autocatalytic synthesis of ethylene via induction of ACC and ACO transcription is well known in fruit ripening , ACO1 and ACO2 have been demonstrated to be the major ACOs involved in ethylene synthesis . Recently, it was demonstrated that EIN2 is phosphorylated by CTR1 kinase in the absence of ethylene, and that EIN2 protein level is regulated through its degradation by the proteasome . It breaks the embryo dormancy in apple  released by wet ash stimulates germination , inhibitors of ACO activity, or 2,5-norbornadiene  and silver thiosulfate (STS), inhibitors of ethylene action, allowed demonstration of the involvement of endogenous ethylene in germination and dormancy breakage  and aminooxyacetic acid (AOA), inhibitors of ACS activity, CoClbreakage . In contbreakage , sunflowbreakage , cocklebbreakage , Amarantthus sp. , chickpethus sp. , and sugthus sp. . This efAmaranthus retroflexus . Seeds of ethylene insensitive etr1 (ethylene resistant) as well as ein2 (ethylene insensitive 2) mutants display enhanced primary dormancy relative to wild type, probably due to high ABA sensitivity, whereas ctr1 (constitutive triple responses) mutant have a slightly enhanced rate of germination , SlERF2 transcript abundance is higher in germinating seeds than in non-germinating ones, and its overexpression in transgenic lines results in premature seed germination , prohibitin 3 or ERF have been shown to be up-regulated in after-ripened seeds at 12 or 24 h of imbibition. The number of genes related to ethylene involved in germination is underestimated since only direct known ethylene signaling components are targeted for analysis in omic studies. It has been shown that treatment with ACC of 7 days germinated seedlings triggers change in expression of 544 genes, among them 244 were common to seeds given an ABA treatment , and Arabidopsis  are hypersensitive to ABA, whereas seeds of ein3, ein4, ein5, and ein7 germinate normally. Conversely, eto1, eto3, and ctr1 mutants (characterized by an increase in C2H4 production) exhibit a reduced sensitivity to ABA  with aba2 mutant, demonstrate that ABA and C2H4 may also act in parallel, since they exibit phenotypes resulting from both ABA deficiency and altered ethylene sensitivity . Both hormones promote the germination of primary dormant seeds of Arabidopsis  in Arabidopsis in the presence of GA4  and seeds treated with GA3 or ethephon, but inhibition of ethylene biosynthesis by AOA (2-aminoxyacetic acid) results in an increase in this transcript indicating the involvement of ethylene in the regulation of GA biosynthesis (SoGA3ox2 and SoGA20ox2) and degradation (SoGA2ox6) during imbibition of Sisymbrium officinale seeds in the presence of GA4+7, ethylene, and inhibitors of GA synthesis or ethylene synthesis and signaling, indicate that GA biosynthesis is strongly regulated by GA and ethylene , RGL1 (RGA LIKE1), RGL2 and RGL3 , RGA  embryonic axes during imbibition induces ethylene production, which promotes cell elongation in the radicle. However, in that case, ethylene was measured after the onset of radicle protrusion, and this production was probably more related to the kinetics of seedling elongation than on a direct effect of H2O2. In contrast, in pea, which germination is not strongly regulated by ethylene, 2O2 treatment results in a reduction in PsACS2 transcript abundance consistent with a decrease in ACC content. These results suggest that ROS and ethylene probably do not interact directly, but rather through a complex hormonal network  involved in GAs synthesis. Ethylene also probably modifies the GAs signaling pathway via a regulation of DELLA proteins, as demonstrated in growth processes (2H4 network, there are antagonistic interactions between ABA and GAs, C2H4 and brassinosteroids, jasmonates and auxins (Seed germination is regulated by ethylene in a complex signaling network, which is also operational in numerous developmental processes, including vegetative growth, flowering timing, fruit ripening and organ senescence and abscission . As mentrocesses , 2007. Td auxins . ROS alsd auxins . It woulOmics studies are now available in the field of seed germination but efforts to develop transcriptomic analysis of ethylene action are required to understand ethylene involvement in seed germination. Analysis of the effects of ethylene on specific cellular processes highlighted by dormancy and germination studies such as transcription regulation, cell cycle activity and endosperm weakening should help to understand the regulatory network of germination process in seeds. Moreover, although hormonal signaling network share common components, they may work in specific territories in seeds.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.


The respiratory diseases caused by rhinovirus, respiratory syncytial virus, and influenza virus represent a large social and financial burden on healthcare worldwide. Although all three viruses have distinctly unique properties in terms of infection and replication, they share the ability to exploit/manipulate the host-cell nucleocytoplasmic transport system in order to replicate effectively and efficiently. This review outlines the various ways in which infection by these viruses impacts on the host nucleocytoplasmic transport system, and examples where inhibition thereof in turn decreases viral replication. The highly conserved nature of the nucleocytoplasmic transport system and the viral proteins that interact with it make this virus–host interface a prime candidate for the development of specific antiviral therapeutics in the future. Viral respiratory disease (VRD) results in the hospitalization and deaths each year of millions of people worldwide, representing a large social and financial burden on healthcare globally. Although 100s of viruses can potentially cause VRD, the main causative agents are, Influenza virus, RSV, and HRV. Influenza virus, an orthomyxovirus, is responsible for an estimated 3–5 million cases of severe illness and 250–500 thousand deaths worldwide per year, with an economic impact of $87.1 billion in the US alone . During The pneumovirus RSV is the single greatest cause of lower respiratory tract illness (LRTI) and bronchiolitis in infants and the elderly, with an estimated 64 million infectious cases and 160,000–600,000 deaths recorded worldwide each winter . RSV relInfluenza virus, RSV, and HRV alone represent a huge burden of disease and economic strain worldwide. Unlike influenza virus, where a seasonal vaccine is available, there are currently no efficacious vaccines or treatments for either RSV  or HRV . ResearcThe nucleus is a specialized compartment within eukaryotic cells where the genetic information is contained, surrounded by the lipid double membrane structure of the NE representing the boundary between the genome and the cytoplasm . SpecifiFigure 1) is mediated by members of the IMP superfamily of transporters, of which various α and β subtypes exist that recognize and bind to specific and highly conserved NLSs on their respective cargo proteins  or one of the IMPβs alone , before translocating through the pore via transient and sequential interactions between IMPβ and the nups  resulting in NLS-cargo release  is required to allow potential cargo proteins to bind. The RanGTP/XPO/cargo trimeric complex then passes through the NPC  to the cytoplasm via sequential interactions between the XPO and nups of the NPC. Once in the cytoplasm, hydrolysis of RanGTP to RanGDP  effects release of the NES containing cargo protein into the cytoplasm.Signal-dependent protein nuclear import  encountered, so it is imperative that the host innate immune response is able to identify and eliminate these threats efficiently. During viral infection, the family of TLRs  in concepicornaviridae family, is a non-enveloped icosahedral virus that at its core possesses a positive sense single-stranded RNA (+ssRNA) genome that encodes 11 proteins initially expressed as a single polyprotein. To date there have been 156 different HRV serotypes identified , contributing to reduced cellular transcription and translation, although viral transcription/translation continues unabated. The disruption of host-cell nuclear transport has been attributed to the specific proteolysis and degradation of the FG-containing nups 62, 98, and 153 within the NPC by the viral proteases 2A and 3C , leading to disruption of classical nucleocytoplasmic shuttling (Table 1). The general disruption of nuclear transport can be observed early in HRV infection whereby endogenous nuclear proteins such as the RNA associated La and Sam68 proteins  and passive (size exclusion) nuclear transport through degradation of nups 358, 214, and 153  further contributing to halting host-cell translation . Although the precise role(s) and kinetics of 2A and 3C protease-mediated nup degradation remain to be determined in vivo, it is clear that disruption and degradation of the NPC and cleavage of essential transcription factors by 3C is central to host cell shutdown, through deregulation of host cell nucleocytoplasmic transport to prevent the infected cell mounting an antiviral response.During HRV infection, the production of INF-β mRNA through the Type 1 IFN response is attenuated, leading to dampening of the antiviral response . AlthougIn a similar fashion to HRV, the paramyxovirus RSV replicates entirely within the cytosol of infected cells. The outer surface of the RSV virion is comprised of a lipid-rich membrane that encompasses the nucleocapsid within which resides the ssRNA viral genome that encodes all 11 viral proteins . AlthougFigure 2xiv), dependent on interaction with IMPβ1 (Table 1). Nuclear M inhibits host cell transcription  protein, which associates with the nucleocapsid and envelope glycoprotein complexes within the virion, and is believed to be a key driver of virus assembly in the infected cell , 2009a. Figure 2xvi; Later in infection, M is ferried to the cytoplasm by XPO1 .Unlike RSV and HRV, which replicate their viral genomes within the host-cell cytoplasm, Influenza virus must transport its genome, in the form of a RNP complex, into the host-cell nucleus in order for replication to occur. The key components of the RNP are the vRNA binding nucleoprotein NP;  and the Figure 2ix; Table 1; Figure 2xi) via 2 NESs on NS2, which helps effect transport of the complex out of the nucleus via the NPC. M1 also appears to contribute to vRNP nuclear export through a NES that would appear to be recognized by an export protein other than XPO1 ; mutation of the NES results in vRNPs nuclear accumulation  for vRNP-M1-NS2-XPO1 export (Table 1); the heat shock protein Hsc70  viral mRNA which is exported to the cytosol utilizing the host-cells mRNA transport machinery to undergo translation to produce new viral proteins. A second round of replication then proceeds, whereby positive sense RNAs are produced to serve as templates for the production of negative sense vRNA genomes, which in turn combine with newly synthesized and nuclear localized NP, PB1, PB2, and PA proteins to form new vRNPs . These vmulation . Additioin Hsc70 , which iin Hsc70 , and NXFin Hsc70 , integrain Hsc70 .Although there are currently a number of therapeutic and prophylactic approaches to manage HRV, RSV, and Flu, there is an ever-present need for new specific and low toxicity treatments for all three.in vitro and in early challenge trials, a significant reduction in viral load was not achieved during trials with natural infection due to the requirement for the drug to be taken immediately prior or just after infection  is performed on a compound library to identify molecules that disrupt the interaction between the viral protein-IMP/XPO of interest, followed by specificity counter-screening  to identify compounds that directly inhibit IMP/XPO function. Only compounds shown to specifically inhibit viral protein-IMP/XPO are selected for further cell based antiviral activity analysis  and structural refinement . Refined molecules are re-screened  to confirm activity and specificity before evaluation in animal models . This strategy has been used successfully to identify antivirals targeting nuclear import for HIV and DENV  rather than the IMP or XPO directly, which would potentially block transport of all host cell proteins that use the IMP/XPO for normal trafficking. High-throughput screening where a counterscreening strategy has been incorporated has proved efficacious in identifying compounds that have proved to be specific inhibitors , with loand DENV  – see neTable 1 thus at least in theory representing potential targets for drug development. Targeting the host cell nuclear import/export machinery itself can clearly have an effect on virus production  retinamide] of DENV non-structural protein five nuclear import through IMPα/β1 can protect against infection by all four serotypes of DENV, including severe, antibody-dependent enhanced disease in a lethal mouse model  to identify all inhibitors that were likely to be directed against IMPs rather than the host-virus interface; only compounds specifically disrupting viral:host protein interaction were pursued, resulting in successful and rapid identification of the inhibitors that ultimately proved to be highly specific for the host-virus interface. This represents an exciting strategy in the future, potentially for HRV 2A/3C nuclear import, for RSV M nuclear import and export, and nuclear import/export of the various influenza virus proteins. Targeting the viral protein-IMP  interface in the case of these various proteins would appear to be an exciting possibility, with great potential to generate novel, specific antivirals with low toxicity, and little risk of selecting for viral resistance.The approaches of 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.


In these last years, along with the description of new NK cell types playing key roles within different body compartments, it is becoming conceivable that NK cell receptors might have been shaped to fulfill a complex array of functions beyond the simple recognition of pathological cells , 2. SuchLi and Mariuzza  describe+ NK cells, which are, nevertheless, characterized by reduced NCR-mediated function. In this context, aKIRs have been recently shown to participate in the education of NK cells, rendering them hyporesponsive. Interestingly, education by aKIRs shares features with the hyporesponsiveness induced by chronic stimulation of other activating receptors expressed by NK cells  is still unclear. Nevertheless, aKIRs are likely to play a role in infectious diseases, cancer, and autoimmunity. For example, aKIRs-mediated recognition of viruses and consequent NK cell priming has been proposed to occur and to be directly involved in the pathogenesis of the chronic lymphoproliferative disorder of NK cells (CLPD-NK) . RemarkaNK cells .All the above mentioned articles can provide hints on the process of co-evolution that may have affected KIRs, their autologous ligands and exogenous molecules or peptides, as well as the mechanisms controlling the strength of NK cell response to stimuli. A possible receptor/ligand co-evolution may have also concerned several non-MHC-specific activating receptors. A living recapitulation of how this co-evolution may have occurred is represented by the struggle between NK cells and tumors. Cerboni et al.  and HuerRemarkably, the quantitative deficiency of NK cells or the altered expression/function of their receptors witness the negative influence exerted not only by tumor microenvironment but also by viruses. Lugli et al.  and Marr+ cell types: the conventional NK cells (belonging to group 1 ILCs) and the IL22-producing NCR+ILC3 cells (belonging to group 3 ILCs). Depending on the environmental mediators, engagement of NKp44 and PRRs can mediate release of the ILC3 signature cytokine IL-22 or pro-inflammatory soluble factors thus modulating both epithelial and immune cell functions at mucosal interfaces.As above mentioned, because of their polyvalent role, certain receptors may have been exposed to multiple shaping pressures. For example, NK cells that populate decidua (dNK) in the first trimester of pregnancy can use NKp44, NKp30, and KIR2DL4 both to control infections and to promote regulatory interactions [with trophoblasts or dendritic cell (DC)] important for placental development . The abuAlso, NKp30 can fulfill different tasks, as it can serve as a tool for eliminating altered cells and promoting regulatory interaction with autologous cells. Reiners et al.  shows thDifferent studies have highlighted the important immunomodulatory role of the crosstalk between NK cells and DCs. Ferlazzo and Morandi  describeThe functional interaction with normal cells (such as DCs or trophoblasts) implies that several NK receptors can recognize autologous ligands. These considerations indicate that a potential role for NK cells also in the development of autoimmune diseases. Poggi and Zocchi  and Enk In conclusion, the analysis of the NK cell functions and receptors as discussed in this Research Topic indicates that, in spite of the continuous flow of discoveries that have significantly changed key concepts on the biology of NK cells, “the recognition of self,” originally postulated by Kärre more than 20 years ago as strategy to drive NK cell cytotoxicity , still rThe 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.


This article will review recent impact of massively parallel next-generation sequencing (NGS) in our understanding and treatment of cancer. While whole exome sequencing (WES) remains popular and effective as a method of genetically profiling different cancers, advances in sequencing technology has enabled an increasing number of whole-genome based studies. Clinically, NGS has been used or is being developed for genetic screening, diagnostics, and clinical assessment. Though challenges remain, clinicians are in the early stages of using genetic data to make treatment decisions for cancer patients. As the integration of NGS in the study and treatment of cancer continues to mature, we believe that the field of cancer genomics will need to move toward more complete 100% genome sequencing. Current technologies and methods are largely limited to coding regions of the genome. A number of recent studies have demonstrated that mutations in non-coding regions may have direct tumorigenic effects or lead to genetic instability. Non-coding regions represent an important frontier in cancer genomics. Cancer, in its many forms, accounted for 8.2 million deaths in 2012 , exome sequencing covers only the 1% of the genome that is translated into protein, greatly reducing the technical burden of data collection and analysis. Ley et al. piloted the use of NGS to study the exomes of 140 samples of human acute myeloid leukemia (AML) cells in 2003, identifying 6 previously described and 7 undescribed mutations relevant for AMP pathogenesis. The investigation searched for mutations capable of altering gene function and identified the FLT3 gene as a distinguishing mutant in AML patients , luminal B , HER2-enriched , and basal-like . 40% of luminal A tumors possessed a mutated BRCA1, BRCA2, and other biomarkers introduced genetics as an important consideration  technologies enables clinicians to make improved diagnostic and treatment decisions. For example, breast cancers have traditionally been diagnosed by mammogram, physical exam, and histology. The discovery of BRCA1, BRCA2, APC, and TP53  and trastuzumab (HER2). Targeted therapies have the potential to be more effective and less toxic than traditional chemotherapies for patients suffering from cancer , sequencing ctDNA is another attractive method for analyzing tumor load and treatment effectiveness  and nanopore sequencing (Oxford Nanopore) are perhaps the most well-known. Conceptually, both of these systems detect nucleotide incorporation onto a single DNA strand in real time. SMRT uses a system of fluorescent signals to monitor nucleotide incorporation while nanopore detects voltage across a lipid bilayer as as DNA is ratcheted across an α-hemolysin nanopore  associated with poor prognosis  was only able to sequence 51 out of 56 genes with adequate coverage (Dewey et al., Brca1 in mice may be due to de-repression of satellite DNA transcription. The authors propose that tumor suppressor functions of BRCA1 primarily stem from its role as a regulator of heterochromatin (Zhu et al., Current findings suggest that a variety of defects in non-coding regions can contribute to neoplastic pathogenesis (Chmielecki and Meyerson, Complete genomic sequencing may also reveal epigenetic changes in cancer that were previously inaccessible due to limitations in the coverage and resolution of last-generation technologies The epigenetics of nucleosomes for example, have eluded investigation in the past, but new protocols utilizing NGS which enable genome-wide studies of nucleosome positioning at high sensitivity might allow networks of chromatin modulation to finally be probed (Wei et al., In this article, we discussed the past, present, and future of DNA sequencing in cancer genomics. As advancements in sequencing technology enabled investigators to study cancer genomes in greater breadth and depth, the field has produced novel insights into tumor pathogenesis, identified clinically useful biomarkers, and developed increasingly precise diagnostics and targeted therapeutics. However, it is important to note that our understanding of the genome is limited, with <5000 of 23,000 known (Park et al., 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.


Survival Motor Neuron 1 (SMN1) gene, leading to a pathological decreased expression of functional full-length SMN protein. Emerging studies suggest that the small GTPase RhoA and its major downstream effector Rho kinase (ROCK), which both play an instrumental role in cytoskeleton organization, contribute to the pathology of motoneuron diseases. Indeed, an enhanced activation of RhoA and ROCK has been reported in the spinal cord of an SMA mouse model. Moreover, the treatment of SMA mice with ROCK inhibitors leads to an increased lifespan as well as improved skeletal muscle and neuromuscular junction pathology, without preventing motoneuron degeneration. Although motoneurons are the primary target in SMA, an increasing number of reports show that other cell types inside and outside the central nervous system contribute to SMA pathogenesis. As administration of ROCK inhibitors to SMA mice was systemic, the improvement in survival and phenotype could therefore be attributed to specific effects on motoneurons and/or on other non-neuronal cell types. In the present review, we will present the various roles of the RhoA/ROCK pathway in several SMA cellular targets including neurons, myoblasts, glial cells, cardiomyocytes and pancreatic cells as well as discuss how ROCK inhibition may ameliorate their health and function. It is most likely a concerted influence of ROCK modulation on all these cell types that ultimately lead to the observed benefits of pharmacological ROCK inhibition in SMA mice.Spinal muscular atrophy (SMA) is the most common genetic disease causing infant death, due to an extended loss of motoneurons. This neuromuscular disorder results from deletions and/or mutations within the  Spinal muscular atrophy (SMA) is a devastating neurodegenerative disease, affecting approximately 1:6,000–10,000 live births per year Pearn, . This auSurvival Motor Neuron 1 (SMN1) gene and subsequent reduction of the SMN protein  and a duplicated centromeric copy (SMN2), both located on chromosome 5q13   lead to a reduced differentiation potential, shorter neurites and more importantly, perturbation of the actin cytoskeleton and key modulators of actin dynamics. Indeed, in Smn-depleted PC12 cells, there is an increase in RhoA-GTP, the active form of the kinase, which is known to inhibit neuronal outgrowth and differentiation , the lifespan of an intermediate SMA mouse model is dramatically increased  of SMA mice  promotes astrocytic stellation  and insulin receptor substrate 1 (IRS1)] through phosphorylation of IRS1. All together, these observations point to a key regulatory role for the RhoA/ROCK pathway in specific pancreatic functions as well as in the general maintenance of glucose homeostasis.RhoA/ROCK signaling modulates various pathways responsible for pancreatic function and glucose homeostasis Figure . For exain vivo still needs to be evaluated. In the same manner, the observation that ROCK enhances insulin signaling and glucose storage in muscle cell lines should be verified in animal models. Lastly, it has been reported that β cell proliferation is modulated by an interaction between CREB, a transcription factor that mediates hepatic glucagon signaling  administration, neuronal depletion of phosphatase and tensin homolog (PTEN) and systemic trichostatin A administration have previously been shown to also influence glucose metabolism , a motoneuron pathology closely related to SMA (reviewed in Achsel et al., 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.


Social environment is a well-recognized determinant in health and wellbeing. Among breast cancer patients, inadequate social support is associated with a substantial increase in cancer-related mortality. A common explanation is that socially isolated individuals fare worse due to reduced instrumental support . However, the ability to replicate the detrimental effects of social isolation on mammary tumor growth in rodents strongly suggests an alternative explanation; i.e., socially isolated individuals have a physiological milieu that promotes tumor growth. This review summarizes the clinical and basic science literature supporting social influences on breast cancer, and provides a conceptual physiological framework for these effects. We propose that social environment contributes to the vast individual differences in prognosis among breast cancer survivors because social environment is capable of altering basic physiological processes, which in turn can modulate tumor growth. Appreciation of the role of social environment in breast cancer progression could promote the identification of patients at increased risk for poor outcomes. In addition, characterization of the underlying physiological mechanisms could lead to targeted disruption of detrimental pathways that promote tumor progression in socially isolated individuals, or exploitation of protective pathways activated through social engagement as novel therapeutic complements to contemporary treatments. Social milieu dramatically affects the pathophysiology of a wide range of diseases. Nearly 25 years ago social isolation was identified as one of the most important risk factors for all-cause mortality , and theSocial isolation can be defined and quantified in terms of either objective criteria, such as social network size or number/frequency of interactions with others, or subjective criteria, such as an individual’s level of perceived social isolation ; but generally isolation refers to a complete or near-complete lack of interaction. Social environments are also assessed in terms of the level of social support provided, including both emotional support and instrumental support (help with daily tasks/transportation/treatment). The various social measures are often correlated and may share similar underlying biologic and neurologic effectors, yet concurrence on health outcomes is not 100 % among the various measures ; part ofThe “social control theory” is often evoked to explain the effects of social isolation on human health; it posits that people with strong social networks are healthier because network members discourage detrimental health behaviors and encourage beneficial health behaviors . HoweverThere are currently two leading physiological frameworks that have been developed to explain how social relationships may influence health outcomes. The “stress buffering model” posits that affiliative social interactions provide a buffer against the physiological and psychological effects of acute and chronic stressors, which in turn negatively influence health ; this hyAlthough relatively few BC studies have directly assessed the effects of social isolation on outcomes, converging evidence from more than a dozen generally small to medium size studies conducted over the past 20 years indicate that various forms of social engagement extend the lives of patients with BC and result in a higher quality of life (QOL) after the diagnosis –15. The In summary, the clinical data demonstrate a role for social environment in modifying symptomology, QOL, and mortality among BC survivors. Whether the effects of social environment are direct or indirect remains to be demonstrated conclusively; there is one early study that reported that both social involvement and stress are independently related to BC survival, although social factors did not appear to moderate the effects of stress on survival . These dClinical studies can provide a wealth of evidence on correlation between biological markers and measures of social factors. However, due to the crucial role of the brain in processing social cues and coordinating complex behaviors, and the nature of the physiological changes induced by social interaction, mechanistic studies need to rely heavily on animal models for establishing causal relationships between putative biological mediators and BC outcomes. Fortunately, the effects of social isolation on wellbeing and longevity have been demonstrated in a wide range of species. In particular, rodent models of social isolation have proven highly effective at recapitulating human health outcomes that are strongly influenced by social milieu, including cardiovascular disease, cerebrovascular disease, wound healing, depression, and neuropathic pain . LikewisAll living creatures experience stress; in the short term, increased activation of the sympathetic nervous system (SNS) and hypothalamic-pituitary-adrenal (HPA) axis is typically an adaptive response to a stressor. However, as activation of these two systems becomes chronic and grows resistant to regulation, the response becomes maladaptive, increasing the individual’s susceptibility to a wide range of diseases . Social 1) and beta-2 (β2) adrenergic receptors. Retrospective studies indicate that the use of β1/β2 antagonists (beta blockers) is associated with the presentation of less advanced BC, reduced metastasis, increased relapse-free survival, and reduced BC-related mortality [1/β2 antagonist) co-administration increases the efficacy of chemotherapy in patients with BC (NLM Identifier NCT01847001). There are several direct and indirect mechanisms through which β-adrenergic signaling may be influencing BC development, including tumor cell invasion [Converging evidence from several studies indicates that sympathetic nervous system (SNS) activation can facilitate BC progression. The SNS primarily exerts its effects through the production of adrenaline (epinephrine) from the adrenal medulla and noradrenaline (norepinephrine) from post-ganglionic neurons. In the context of cancer, most of the research to date has focused on signaling via beta-1  is associated with reduced survival duration and the suppression of natural killer cell (NKC) counts . NKCs haInterestingly, however, chronic synthetic glucocorticoid use for other health reasons was not associated with increased risk of BC or its recurrence in a large cohort of Scandinavian women , 51. FurAs described above, social influences on a wide array of health conditions, including mammary cancer can be reproduced in rodents by comparing individually housed versus pair-housed or group-housed rodents. Indeed, rodent studies have been remarkably consistent in reporting a detrimental effect of social isolation on mammary tumor progression. Specifically, social isolation in both a Sprague-Dawley rat model of spontaneous BC and a transgenic mouse model of triple-negative BC (C3(1)/SV40 Tag) results in increased tumor burden and invasiveness or malignancy , 25. IntTo date, none of the rodent studies demonstrating social modulation of mammary tumors have conclusively established a causal mechanism, although there are several promising leads Fig. . One likSurprisingly, only one rodent study has specifically examined the role of the SNS in social modulation of mammary cancer . As descOne of the greatest advantages of using mouse models to study BC is the ability to isolate specific cellular components within the tumor microenvironment. For example, several genes involved in cancer-associated glycolysis and lipogenesis , ATP citrate lyase (Acly), and Hexokinase 2 (Hk2)) are upregulated in the mammary tissue of socially isolated mice . FurtherThere are substantial biological differences among cancers originating in different tissues; however, through the analysis of shared systems and pathways in a variety of cancers, insight can be gained into possible mediators that may play a role in BC. For example, social factors have been demonstrated to influence ovarian cancer outcomes. Increased social attachment, a subtype of emotional support, has been associated with a lower likelihood of death and increased survival time in follow up of patients with diagnosed epithelial ovarian cancer . HoweverOxytocin (OT) has remained understudied as a possible mediator of social influences on mammary cancer, though it is released during social and physical contact, serves important biological functions in the mammary gland, and modulates both the HPA axis and SNS. OT has a demonstrated role in the mechanisms through which social isolation affects a wide range of disease outcomes, including wound healing, pain responses, atherosclerosis, cerebral ischemia, and depressive-like behavior . AdditioThe synthesis of OT within tumors has also been demonstrated , and itsIn summary, social isolation is detrimental to overall health and has been identified as an exacerbating factor in many disease states. The clinical literature on BC convincingly demonstrates an association between social isolation and decreased survival following BC; several potential hormonal, angiogenic, and inflammatory markers and mediators have been identified. The critical next step will be full characterization of the physiological mechanisms underlying social influences on BC progression, including the establishment of causation for key factors. An improved understanding of the biological pathways will lead to identification of the most meaningful biomarkers and will provide context for correlations reported in clinical studies. Improved understanding of the biology also could reveal novel therapeutic targets for pharmaceutical development, identify individuals whose biology puts them at increased risk of BC recurrence or mortality, and clarify the pathways through which psychosocial interventions improve BC outcomes.


The use of animal host-targeted pesticide application to control blacklegged ticks, which transmit the Lyme disease bacterium between wildlife hosts and humans, is receiving increased attention as an approach to Lyme disease risk management. Included among the attractive features of host-targeted approaches is the reduced need for broad-scale pesticide usage. In the eastern USA, one of the best-known of these approaches is the corn-baited “4-poster” deer feeding station, so named because of the four pesticide-treated rollers that surround the bait troughs. Wildlife visitors to these devices receive an automatic topical application of acaricide, which kills attached ticks before they can reproduce. We conducted a 5-year controlled experiment to estimate the effects of 4-poster stations on tick populations in southeastern Massachusetts, where the incidence of Lyme disease is among the highest in the USA.We deployed a total of forty-two 4-posters among seven treatment sites and sampled for nymph and adult ticks at these sites and at seven untreated control sites during each year of the study. Study sites were distributed among Cape Cod, Martha’s Vineyard, and Nantucket. The density of 4-poster deployment was lower than in previous 4-poster studies and resembled or possibly exceeded the levels of effort considered by county experts to be feasible for Lyme disease risk managers.Relative to controls, blacklegged tick abundance at treated sites was reduced by approximately 8.4%, which is considerably less than in previous 4-poster studies.In addition to the longer duration and greater replication in our study compared to others, possible but still incomplete explanations for the smaller impact we observed include the lower density of 4-poster deployment as well as landscape and mammalian community characteristics that may complicate the ecological relationship between white-tailed deer and blacklegged tick populations. Ixodes scapularis) are the primary vector of Lyme disease between wildlife and human populations in eastern North America, so their abundance during periods of outdoor human activity is a key determinant of Lyme disease risk [Borrelia burgdorferi. The biology of this spirochete and the multi-host two year life cycle of blacklegged ticks have produced a highly complex ecological system that continues to challenge ecologists, public health experts, natural resource managers, integrated pest management (IPM) practitioners, and land use planners. Risk management solutions are in various stages of development, some of which require changes in land use practices or the use of biocontrol agents or pesticides that may be harmful to non-target organisms. However, because of the complexity of the Lyme disease ecological system [Blacklegged ticks (ase risk . Methodsl system  and the Odocoileus virginianus) are important hosts for adult blacklegged ticks seeking bloodmeals, so their overabundance in the eastern US was historically assumed to be a significant determinant of Lyme disease risk [-2) [White-tailed deer (ase risk . Massachisk [-2) . Extirpaisk [-2) ). Howeveisk [-2) . Moreoveisk [-2) . These cAs an alternative to direct population control of white-tailed deer, the use of deer-targeted pesticide application via “4-poster” feeding stations to control tick populations is now included among the risk management techniques being tested and in some cases implemented in areas of high Lyme disease incidence . BecauseBorrelia burgdorferi, and if transmission occurs, then the larva becomes infected. After feeding to repletion, larvae moult into nymphs. After overwintering, each nymph seeks a new host for what is typically the second blood meal in its life cycle. This second host is an additional opportunity for the tick to acquire the Lyme disease spirochete. In late summer, these nymphs moult into the adult stage and seek their final blood meal. All stages of feeding ticks are potentially affected by exposure of their hosts to 4-poster treatments, but this exposure is expected to be highest for adult ticks because of their relatively high abundance on large vertebrates. The impacts of 4-posters include direct mortality to larvae or nymphs attached to 4-poster visitors and reduced numbers of eggs due to reductions in adult populations. Our study was designed to estimate the magnitude of these effects by repeated sampling of nymph and adult ticks at 4-poster sites and untreated control sites.White-tailed deer frequently carry heavy burdens of adult stage blacklegged ticks seeking their final blood meal. However, blacklegged ticks have a complex life cycle involving multiple hosts (reviewed in ). After et al. [Several previous studies have reported large reductions in tick abundance in areas treated with 4-posters relative to untreated control areas. Most notably, a coordinated six-year study in the northeastern US reported approximately 70% reduction in nymphs at the end of the study -13. Onlyet al.  appears -2 (0.016 stations acre-1). We were interested in estimating 4-poster efficacy for coastal southeastern Massachusetts and used a single controlled experiment with site replication. We expected our study to produce a geographically narrower but more statistically robust confirmation of the broader regional findings reported by Pound et al.[-2 (< 0.007 stations acre-1), or approximately 40% of the density used in the northeast regional study, would produce measurable effects at a more feasible deployment density for area resource managers.The northeast regional study deployed > 100 4-posters across its five study areas at a density of 4 to 5 stations kmnd et al.. In addiWe conducted our 4-poster study in southeastern Massachusetts, where Lyme disease poses a serious health risk. Massachusetts ranks among the top 10 states in Lyme disease annual reporting to the US Centers for Disease Control . In rece-2), based in part on results from previous studies [Deer 4-poster stations were activated in the fall of 2007 (mid August to mid November) and in spring (mid March to mid June) and fall of all subsequent years (2008–2011) at precisely the same locations each year . Closure of stations during winter was partly the result of regulations prohibiting wildlife provisioning during the hunting season. At each site, multiple stations were distributed at approximately one station per 150 acres (1.65 stations km-2) was considered indicative of what tick control programs can realistically be expected to maintain in the study area.Selection of sites for this study was based on: 1) history of an active blacklegged tick population; 2) evidence of white tailed deer; 3) accessibility for maintenance and input of corn bait and permethrin; and 4) distance from residences (> 91 m). This resulted in seven treatment sites on Nantucket, Martha’s Vineyard and Cape Cod  of corn consumed. Inputs to each station, including the amount of corn consumed monthly, the amount of permethrin added, the number of station visits, as well as any necessary replacements or repairs were recorded. Beginning in spring 2007 (before station deployment), nymph ticks were sampled at all treatment and control sites in May, June and July of each year using a cloth dragging procedure [2 (50.8 × 90.4 cm) double-sided white flannel cloth was dragged along the ground at the edge of a trail or wooded road for 30 seconds at approximately one yard per second. This procedure was repeated along fixed transects in October and November of each year for collection of adult ticks. This resulted in a total of 9890 drags approximately evenly distributed across sites . Life stage, treatment, and time were treated as fixed effects. Each statistical formulation was fitted using either days or years elapsed since the beginning of the study. Each of the 42 transects in the study was assigned a unique ID and treated as a random effect. The random effects were modeled as effects on intercepts only and were included because of the expected correlation between repeated samples taken from each transect over the course of the study. This is intended to address microclimate or other unknown but persistent differences between sites. The negative binomial distribution was used because of the high variance to mean ratio in the data, as is common in tick sampling data due to patchy spatial distribution . This sWe used Abbott’s formula  to comput = 0 and t = t, where E denotes mean abundance at control (cntrl) and treated (trt) sites predicted from the statistical models.represents the effect of treatments between time Pelage swab samples from white-tailed deer carcasses were collected prior to meat processing at hunter check-in stations on Chappaquiddick Island, Edgartown, MA, which we assume supports a closed deer population (no immigration or emigration). These samples were collected by wiping a cotton gauze pad on the neck, throat and chin area of each deer for thirty seconds. The samples were placed in amber glass vials and stored frozen. Samples were shipped on ice by overnight delivery to the Massachusetts Pesticide Analysis Laboratory for permethrin residue analysis using hexane extraction followed by gas chromatography with electron capture detection and mass spectrometry. Data from island hunters were used with these residue analyses to estimate the proportion of deer treated topically within the treatment zone.c) of the models we used to analyze tick sampling data  and most parsimonious  collected from harvested deer on Chappaquiddick Island were 0.12, 0.69, 0.47, and 0.7 detections per deer for 2007, 2008, 2010, and 2011, respectively. The low number for 2007 and low rate of corn replenishment during station maintenance in that year suggest the possibility of a period of low deer usage during initial habitation to the station locations.The rates of pesticide residue detections on pelage swabs . Other differences between studies may include deer densities and the operational periods for which the stations were maintained. Also, our study used permethrin as the acaricidal ingredient whereas the USDA study used amitraz. We are unaware of any known differences in effectiveness of these ingredients when used in 4-posters, but permethrin has been shown to be considerably more toxic than amitraz to several species of Amblyomma ticks [We detected a relatively modest effect of 4-posters on blacklegged tick abundances in our coastal Massachusetts study area. Thus, our experiment supports previous findings that 4-posters reduce tick abundance, but the effect size we observed was smaller Figure . Our stuet al. . Althoug. [et al. and othe. [et al., are prol.[et al., perhapsl.[et al.; also sel.[et al.). Distanma ticks ,25.-2 (2.8 – 3.1 deer km-2), primarily through recreational hunting allowances [-1 yr-1 for Cape Cod, Martha’s Vineyard and Nantucket, respectively). Since station density was similar across sites, these consumption rates should be roughly indicative of deer density if relative corn consumption by non-target species is also similar across sites. Indeed, the State of Massachusetts estimates deer densities on Cape Cod to be much closer to its management goal than on the islands, where densities may be more than 15 deer km-2[In the region of our study, the Commonwealth of Massachusetts seeks to manage white-tailed deer abundances at a density of 6–8 deer milowances . Howeverdeer km-2.et al.[personal communication, M. Maquire, Cape Cod Cooperative Extension). These complexities, the existence of alternative tick hosts that might support tick abundance in the absence of deer or compensate for high mortality on treated deer, and the unknown degree to which these other hosts visit the 4-posters are all potentially important factors in the interpretation of tick abundance data such as ours.Experimental exclusion of deer has been shown to affect the density of blacklegged ticks ,28 . If theSince there is no currently available pharmacological solution to Lyme disease, risk management focuses on reducing the likelihood of tick bites. The suite of management techniques includes modification of landscapes to reduce habitat suitability for ticks and their hosts, hunting programs to control deer populations, application of pesticides to the landscape, application of pesticides targeted to potential hosts  and increase of human awareness to modify behavior and promote personal protection practices. Some of these methods have been shown to affect the Lyme disease ecology (and presumably risk), but to varying degrees that depend on the ecological context, scale, and other details of the application. Landscape-scale experimental and observation programs that incorporate ecological and epidemiological approaches would help to identify those critical contextual details that should inform the balance of techniques. At that point, holistic and sustainable risk management strategies would be within reach.The relatively modest effect of 4-posters on tick abundance in this five-year experiment, compared to larger effects seen in other studies, can possibly be explained by landscape characteristics, deer density and vertebrate host community composition in our study area, and the density of 4-poster stations we deployed. An important management implication is that the role of deer in the Lyme disease system may be more complicated than previously expected. It is important to weigh this possibility against concerns from the wildlife management community about the effects of wildlife provisioning and increased social contact between wildlife visitors at the 4-poster stations . This means that 4-posters deserve further study, experimental application, and refinement, but do not represent a low cost ‘silver bullet’ in the control of Lyme disease except perhaps under specific circumstances that remain to be identified. This is unsurprising given the complexity of the Lyme disease ecological system. 4-posters should be considered part of a broader suite of strategies, the most sustainable of which in the long term will embrace the strong linkages between ecological health and human disease risk and will support the differing mandates of environmental stewardship, wildlife management, and public health organizations.The authors declare that they have no competing interests.All authors made substantial contributions to conception and design of this study and the interpretation of results. In addition, RK, BH and RH contributed to the review and oversight of field research. LD carried out and/or supervised the 4-poster station maintenance and tick and deer residue sampling. JG designed and performed the statistical analyses and modeling, prepared the manuscript and coordinated its review and revision by the participating authors, all of whom read and approved the final version.


Mitochondrial oxidative stress is a common feature of skeletal myopathies across multiple conditions; however, the mechanism by which it contributes to skeletal muscle dysfunction remains controversial. Oxidative damage to proteins, lipids, and DNA has received the most attention, yet an important role for reversible redox post-translational modifications (PTMs) in pathophysiology is emerging. The possibility that these PTMs can exert dynamic control of muscle function implicates them as a mechanism contributing to skeletal muscle dysfunction in chronic disease. Herein, we discuss the significance of thiol-based redox dependent modifications to mitochondrial, myofibrillar, and excitation-contraction (EC) coupling proteins with an emphasis on how these changes could alter skeletal muscle performance under chronically stressed conditions. A major barrier to a better mechanistic understanding of the role of reversible redox PTMs in muscle function is the technical challenges associated with accurately measuring the changes of site-specific redox PTMs. Here we will critically review current approaches with an emphasis on sample preparation artifacts, quantitation, and specificity. Despite these challenges, the ability to accurately quantify reversible redox PTMs is critical to understanding the mechanisms by which mitochondrial oxidative stress contributes to skeletal muscle dysfunction in chronic diseases. Oxidative stress plays an important role in skeletal muscle health and disease. In the past, most attention has been focused on skeletal muscle dysfunction caused by oxidative damage to proteins, lipids, and DNA. This damage alters the structure of macromolecules in an irreversible manner and requires turnover and production of new macromolecules to reverse the effects. However, there is growing appreciation of the role of reactive oxygen and nitrogen species (ROS/RNS) in reversible redox post-translational modifications (PTMs) in both muscle adaptation and the effects of chronic disease and aging on skeletal muscle H oxidases, along with xanthine oxidase are the main contributors of contraction induced extracellular superoxide production as reviewed by Powers and Jackson  production is elevated in aging skeletal muscle  or CuZnSOD (inner membrane space). H2O2 then interacts with the GSH-thioredoxin redox buffering system or is rapidly decomposed to water and oxygen by the enzyme catalase in the cytosol.In contrast, elevation of superoxide production associated with chronic degenerative disease appears to be primarily mitochondrial in origin. Mitochondrial superoxide or hydrogen peroxide  redox couples that are present in both the cytosolic and mitochondrial matrix compartments. This redox system, for which NADPH/NADP+ provides reducing power, transduces information on the redox status to the cell by interacting with the proteome through the thiol group on cysteine (Cys) residues. Of the greater than 200,000 Cys residues present in the proteome over 20,000 are thought to be redox sensitive, meaning that they can undergo reversible redox modification as described above  and sulfonic acid (+4) which are considered irreversible . Increasing evidence suggests that redox regulation of cellular processes is the result of signaling rather than non-specific oxidation  and S-glutathionylation (SSG) are two of the most studied reversible redox PTMs Figure . These min vivo redox PTMs. To minimize oxidation artifacts during cell or tissue processing, two techniques are commonly applied. These are trichloroacetic acid (TCA) quenching and cell lysis directly with a relative high concentration of alkylation reagents, such as N-ethylmaleimide (NEM) and iodoacetamide (IAA), to block free thiols  thiol oxidation by air through sample processing; (2) reduction of PTMs by antioxidant enzymes during sample storage or processing  for S-nitrosylated proteins -based proteomics approaches have been developed for identifying and quantifying redox PTMs at the proteome level. For example, in BST the biotinylated peptides (formerly SNO-modified) were enriched by avidin chromatography and the biotin tags were removed to allow the detection of SNO modified Cys sites  in order to better identify functionally important Cys sites. To date, such measurements were only demonstrated by a few approaches such as OxICAT  associated with the PTM, and the specificity of target /GSH redox buffering system that would facilitate translation of oxidative stress to redox PTMs. Finally, many mitochondrial proteins, such as 2-oxoglutarate dehydrogenase, isocitrate dehydrogenase, Complex I, and glutaredoxin isoforms 1 and 2, have conserved cysteine residues associated with dehydrogenase or redox transfer reactions  compared to state 3 respiration (ADP and substrates), however, the GSH/GSSG ratio of freshly isolated mitochondria in the absence of respiratory substrates was more oxidized than either condition  or sulfonic (-SO3H) acid. In the case of complex I, irreversible oxidation results in loss of enzyme activity , which can be reversed by glutaredoxin, prevents the irreversible oxidation to sulfinic (-SOin vivo following depletion of GSH with buthionine sulphoximine (BSO). Two of these residues, cys-531 and 704 on the Ndufs1 subunit were also found to be oxidized in oxidatively stressed bovine hearts  also plays an important role in regulating mitochondrial ROS production through control of the membrane potential , P41-GM103493 (WQ), and T32AG000057 (PK).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.


Worldwide, ticks transmit more pathogens than other arthropods. Around 60 bacteria, 30 parasites and 100 viruses have been registered as tick-borne pathogens; a third of these pathogens are responsible for zoonoses. Usually, detection of tick-borne pathogens depends on the tick species collected: assays are performed for a restricted number of pathogens that are known to be transmitted by a particular tick species collected at a particular site. To better understand the epidemiology of tick-borne pathogens, it will be important to detect for each sample (one tick or one pool of ticks) most of the diseases they potentially transmit, regardless of the tick species. The aim is therefore to develop a new epidemiologic investigative tool which could detect high number of tick-borne pathogens by real time PCR.Francisella tularensis, Coxiella burnetii, Neoehrlichia mikurensis, 5 species of Anaplasma, 3 species of Ehrlichia, 8 species of Borrelia, 2 species of Bartonella, 4 species of Rickettsia, 10 species of Babesia and 2 species of Theileria, (ii) 5 species of ticks whose 3 species of Ixodes and 2 species of Dermacentor. Sensitivity of primers and probe has been tested on a dilution range of reference DNAs of the targeted pathogens on a Lightcycler 480. Specificity then has been tested on a Biomark™dynamic array. The chip was secondly evaluated on field samples corresponding to 47 pools of 25 nymphs collected in two sites in France, the Netherlands and Denmark . We succesfully detected and determined the prevalence of Anaplasma phagocytophilum, Neoehrlichia mikurensis, Rickettsia helvetica, Bartonella henselae, five different genospecies of Borrelia burgdorferi s.l., the recently identified pathogen Borrelia miyamotoi, and two parasite species Babesia divergens and Babesia venatorum. This fast and low-cost tool allows comprehensive testing of tick-borne pathogens and can be customized to fit regional demands or to accommodate new or emerging pathogens. The tool represents a major improvement for surveillance and future epidemiological studies.We developed a chip  targeting pathogens of worldwide distribution transmitted by ticks. The designed epidemiologic arrays may detect 48 pathogens in 48 samples corresponding to 2304 qPCR reactions on the same time. Specific primers and probe have been designed for each pathogen and their specificity have been tested in silico with Blast. To begin, we targeted: (i) 37 pathogens whose