The spread of non-communicable diseases (NCDs) presents a global crisis and is a major barrier to achievement of the Millennium Development Goals (MDGs). WHO identifies six important risk factors that are associated with NCDs: tobacco use, physical inactivity, overweight/obesity, high blood pressure, high cholesterol levels, and high blood glucose levels. Alert village is an Indonesian government policy implemented since the year 2006, aiming to empower the community to gain healthy living. One of the basic activities in Alert Village is the promotion of healthy behaviors in the community. This study aimed to explore the extent of Alert Village implementation and its role in the promotion of healthy behaviors.A qualitative study was conducted in Sakatiga Village, Ogan Ilir District, Indonesia using three methods: key informant in-depth interview, focus group discussion, and participant-observation. Two health office staffs, a midwife, a village head and two cadres were informants of in-depth interview. Focus group discussion was conducted to six selected community members. Data were analyzed descriptively.Results indicated that the extent of alert village implementation had run well. A village forum and a village health post were formed, moreover the midwife and the two cadres were active in dealing with some activities in health post. Some activities for healthy behaviors promotion had already been implemented in the village health post of Sakatiga. Health promotion efforts include: counseling and training of the cadres and the community members namely: promotion of community knowledge regarding NCDs risk factors, healthy food preparation for family and in particular pregnant and lactating mothers and healthy physical activity for elderly. But other facts, community had not actively participated in the implementation of village forum and it worked only when there was a health problem.In conclusion, Alert Village program is an acceptable way of health promotion of healthy behaviors in Sakatiga Village. It also has potential in producing beneficial health changes in the community.


Neurosteroids are synthesized in the brain and modulate brain excitability. There is increasing evidence of their sedative, anesthetic and antiseizure properties, as well as their influence on mood. Currently neurosteroids are classified as pregnane neurosteroids , androstane neurosteroids (androstanediol and etiocholanone) or sulfated neurosteroids (pregnenolone sulfate and dehydroepiandrosterone sulfate). Both preclinical and clinical findings indicate that progesterone derivative neurosteroids such as allopregnanolone and allotetrahydrodeoxycorticosterone play a role in mood disorders. Clozapine and olanzapine, which were shown to be effective in stabilizing bipolar disorder, elevate pregnenolone levels in rat hippocampus, cerebral cortex, and serum. In lithium-treated mice, the blood levels of allopregnanolone and pregnenolone were elevated compared to control levels. Women diagnosed with bipolar disorder typically show symptomatic exacerbation in relation to the menstrual cycle, and show vulnerability to the onset or recurrence of mood disorders immediately after giving birth, when the levels of neurosteroid derivatives of progesterone drop. Whereas in women who had recovered from bipolar disorder, the plasma concentration of allopregnanolone was elevated compared to either healthy controls or women with major depressive disorder during the premenstrual period. During depressive episodes, blood level of allopregnanolone is low. Treatment with fluoxetine tends to stabilize the levels of neurosteroids in depression. These findings converge to suggest that these steroids have significant mood-stabilizing effect. This hypothesis is consistent with the observation that a number of anticonvulsants are effective therapies for bipolar disorder, a finding also consistent with the antiseizure properties of neurosteroids. Further exploration of action of neuroactive steroids is likely to open new frontiers in the investigation of the etiology and treatment of mood disorders, particularly bipolar disorders. Pharmacotherapy of severe mental disorders has not changed significantly since the introduction of antipsychotic compounds in the 1950s. The current classes of drugs available to treat schizophrenia, bipolar disorder and major depression involve essentially the same mechanisms of action and the same neurobiological target ,3 and beThe development of new therapeutic targets in the treatment of severe mental disorders is hampered by the lack of external, biological markers of the nosographic phenotypes ,7, the sThere is robust epidemiological evidence that patients diagnosed with schizophrenia and mood disorders show gender differences in the onset, course and outcome of their disorders. Since the German ABC study  on schizophrenia, it is well known that females have two peaks in the onset of schizophrenia, contrary to males ,11. ReceMood disorders show a different picture, with women having an increased risk for developing major depression compared to males . The risPeripherally, steroids are produced mainly by the adrenal cortex and the gonads and are regulated by the hypothalamic-pituitary-adrenal axis and the hypothalamic pituitary-adrenal gonadal axis through negative feedback. The ovarian steroids regulate neuroendocrine, endocrine and behavioral functions through a number of cellular mechanisms. Typically, both estrogen and progesterone induce a relatively long-term action on neurons by activating a number of intracellular receptors that modulate the transcription and protein synthesis.The steroid hormones play an important role in both the central and peripheral nervous systems; they act during development, growth, maturation and cellular differentiation. The progesterone receptor and the nuclear hormone family of intracellular estrogen receptors, alpha and beta (encoded by two separate genes), are dimeric molecules that regulate transcription of target genes in the nuclei ,24. ThesBoth estradiol and progesterone receptors are found in the Central Nervous System (CNS) . ProgestBaulieu and co-workers were the first to observe that the brain concentrations of dehydroepiandrosterone sulfate were partially independent from adrenal and gonad secretion . SubsequCurrently neuroactive steroids are classified as pregnane neurosteroids , androstane neurosteroids (androstanediol and etiocholanone) or sulfated neurosteroids (pregnenolone sulfate or PS and dehydroepiandrosterone sulfate or DHEAS).Several enzymes are involved in the synthesis of neuroactive steroids: neuroactive steroids such as allopregnanolone, THDOC, and androstanediol are produced by 5α-reductase and 3α-hydroxysteroid oxidoreductase (3α-HSOR), which act by reducing the parent steroid in peripheral tissues, such as liver and skin . Both thIn the brain de novo synthesis of neurosteroids occurs in the cortex, the hippocampus and the amygdala, mainly in glutamatergic neurons . RegulatA receptor is the most important site where neuroactive steroids act as positive or negative regulators [There is evidence that neurosteroids do not produce most of their effects through an interaction with the steroid hormone receptors that regulate gene transcription. But, neurosteroids can regulate gene expression via the progesterone receptor. But this occurs only after conversion of the neurosteroids to typical steroids. The induction of DNA binding and transcriptional activation of the progesterone receptor requires intracellular oxidation of the neuroactive steroids into progesterone receptor active 5 alpha-pregnane steroids . Most efgulators , which iA receptor is the main target of action of neuroactive steroids. GABAA receptors are heteropentameric GABA-gated chloride channels: they are involved in fast inhibitory neurotransmission. The GABAA receptor, distributed in large quantity throughout the CNS, is a macromolecular complex consisting of five subunits, of which many homologs have been identified . In each receptor, these five subunits are assembled from among 19 different subunit isoforms [A receptors. The best-known and most widely distributed form in the CNS consists of two alphas, two betas, and a third subunit, which together constitute the chloride ion channel. Different assemblages of the five subunits determine differences in the functioning of the channel and differences in the response to the drugs acting on the receptor [The GABAisoforms . Composireceptor .A receptor, in subunit beta, determines the opening of the ion channel and chloride influx. The increase in negative charge leads to a hyperpolarization of the membrane, making it less susceptible to excitation [A receptors prevents a potential short circuit in the depolarization induced by excitatory neurotransmitters. The activity of these receptors is also modulated by a number of agents including benzodiazepines, barbiturates, some anesthetics and ethanol [Site-specific binding of the GABAcitation . The act ethanol .A receptors [A receptors, by increasing both the frequency and duration of the opening of the ion channel [A receptor, neuroactive steroids increase a massive influx of the ion and potentiate inhibitory GABAergic transmission [The neurosteroids allopregnanolone and THDOC are strong positive allosteric modulators of the GABAeceptors , but at eceptors . The neu channel . Due to smission .neuroactive steroids seems sufficient to significantly potentiate the function of the GABAA receptor [A receptor [A receptor: the action of these substances on the GABAA receptor depends on receptor assembly [In vivo, the basal plasma concentration of receptor . Neuroacreceptor . This reassembly .A receptors, acting on different sites from those bound by allopregnanolone and THDOC [A receptors.The steroid sulfates are non-competitive antagonists of the GABAnd THDOC . The negnd THDOC . The neund THDOC . Given tnd THDOC ; this inA receptors, is involved in the control of neurosteroidogenesis, with GABA inhibiting it [Studies carried out in amphibians showed that GABA, through its GABAiting it . TherefoThe role of allopregnanolone and THDOC has been studied in a number of pathologies including epilepsy , premensThese steroids show a protective action in epilepsy and elevate the threshold of convulsive crisis, particularly in so-called catamenial epilepsy, in which the crises worsen during the premenstrual phase. Indeed, clinical studies have demonstrated the potential therapeutic utility of synthetic analogs of allopregnanolone in the treatment of catamenial epilepsy . The redThe treatment with fluoxetine stabilizes the level of neurosteroids in depression and panic, and it has been hypothesized that at least part of the therapeutic effect of selective serotonin re-uptake inhibitors (SSRIs) could be through their influence on neurosteroids ,67. ReceIn women with bipolar disorders during euthymia, plasma concentration of the progesterone derivative allopregnanolone is elevated in the premenstrual period compared to healthy controls and women with major depressive disorder . This fiIrritable mood is another component of mood elevation in bipolar disorder. Anabolic/androgenic steroids increase sex drive and mental acuity. If abused, such steroids can cause irritability and impulsive aggression . Social More recently, the same group  found thIt can also be hypothesized that a dysregulated system in patients with bipolar disorder would cause low levels of neuroactive steroids during depression and mixed states. Thus, drugs able to correct the malfunctioning of systems based on neurosteroids could improve clinical status in patients with bipolar disorder spectrum conditions.Fluoxetine, which is effective in panic attack and major depression, also affect the levels of neurosteroids ,67. A roEven the two atypical antipsychotics clozapine and olanzapine, effective against the manic phase of bipolar disorder ,86, wereEffects of neurosteroids on mood fluctuation might extend across the bipolar disorder spectrum. Women of reproductive age with mental disorders may experience a fluctuating course of illness over the menstrual cycle. Some data suggest that for a subset of women there is a relationship between phases of the menstrual cycle and increased vulnerability for an exacerbation of ongoing mood disorders   inter-individual differences in neurosteroids synthesis, secretion and action at the target receptor; b) the conformation of the GABAA receptor as a function of alcohol and/or preceding treatment with drugs acting on it; the phase of the disorder, whether depressive or excitatory. Gender differences in neurosteroids functioning also would have an impact on the effectiveness of GABAergic compounds in bipolar disorder, and should be accounted for.Most antiepileptic compounds exert a direct or indirect GABA-mediated inhibitory action . Howeverazepines , which cynthesis -104. MorLiterature on the role of neuroactive steroids in mental disorders is sparse. Nonetheless we have made an attempt to present a narrative review of existing studies on neurosteroids acting on the GABAergic receptors. Current evidence suggests that the investigation of neuroactive steroids on mental disorders might open new frontiers in the investigation of the etiology and treatment of mood disorders, particularly bipolar disorder. Neurosteroids might be an endogenous mood stabilizer and the alteration of their functioning on a genetic or biochemical level might be responsible for the display of symptoms in individuals vulnerable to bipolar disorder.The authors’ declare that they have no competing interest.MGC conceived the idea of the paper and drafted the manuscript after discussion with KMB and AP. KMB and AP contributed to the molecular (particularly KMB) and clinical (AP) aspects of the paper. All authors read and approved the final manuscript.


Stem cells are capable of renewing themselves through cell division and have the remarkable ability to differentiate into many different types of cells. They therefore have the potential to become a central tool in regenerative medicine. During the last decade, advances in tissue engineering and stem cell-based tooth regeneration have provided realistic and attractive means of replacing lost or damaged teeth. Investigation of embryonic and adult (tissue) stem cells as potential cell sources for tooth regeneration has led to many promising results. However, technical and ethical issues have hindered the availability of these cells for clinical application. The recent discovery of induced pluripotent stem (iPS) cells has provided the possibility to revolutionize the field of regenerative medicine (dentistry) by offering the option of autologous transplantation. In this article, we review the current progress in the field of stem cell-based tooth regeneration and discuss the possibility of using iPS cells for this purpose. Teeth consist of multiple hard tissues, including enamel, dentin, and cementum, and have an integrated attachment complex with alveolar bone through the periodontal ligament. After completion of tooth formation, the only vascularized tissue containing nerves is the dental pulp, which is encased in mineralized dentin. Because teeth have multiple functions, including feeding, articulation, and esthetics, their loss can cause not only physical but also psychological suffering that compromises an individual's self-esteem and quality of life -derived dental ectomesenchymal cells   are found in the papilla tissue in the apical part of the roots of developing teeth. The third molars and teeth with open apices are an important source of SCAPs. These cells have the potential to differentiate into osteoblasts, odontoblasts, and adipocytes and show higher rates of proliferation Future therapeutic approaches for the restoration of damaged dentin, pulp, cementum, and periodontal ligaments may make use of autologous stem cells such as DPSCs, SHEDs, SCAPs, and DFSCs that have been stored after removal from the patient.Dental epithelial stem cells were identified in the continuously growing rodent incisor  are quiescent epithelial remnants of Hertwig's root sheath (HERS) that remain in the adult tooth and play a role in cementum repair and regeneration  . This suggests that a specific signaling network for tooth organogenesis is missing.However, regardless of the presence of putative epithelial stem/progenitor cells, iPS cell-derived teratomas that formed in these conditions did not contain teeth, and no tooth germ-like structures could be found . Pax3, Snail, and Slug (NC-specific transcription factors), as well as human natural killer-1  showed higher expression in derived cells than in undifferentiated iPS cells , was significantly increased. Recombinant culture between NCLC and E14.5 dental epithelium in a collagen gel  stem cells and embryonic cells. The future establishment of this technique may considerably change therapeutic approaches to dental syndromes and diseases.in vitro that are relevant to the tissues and organs targeted for regeneration. To succeed in these challenges, further basic studies to elucidate the regulatory mechanisms of stem cells and tooth development are needed.However, some challenges remain to be addressed before successful tooth regeneration can be achieved. For example, natural tooth development generally takes several years to complete in humans, which is too long to wait for a patient who needs regenerated teeth. Therefore, we should address this issue carefully when considering clinical applications. In addition, because morphology and size differ depending on the tooth type, these aspects need to be addressed. We also need to further develop efficient protocols to induce stem cells to form cell types 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.


Adult neurogenesis represents a dynamic level of modulation upon the neuroplastic properties of the mature nervous system, that is essential to the homeostatic brain function. The adult neurogenic process comprises several sequential steps, all of which subjected to an assortment of cell-intrinsic and neurogenic-niche complex regulatory mechanisms. Among these, epigenetic regulation is now emerging as a crucial regulator of several neurogenesis steps. In particular, the active regulation of hippocampal neurogenesis and its repercussions in global hippocampal function are of special interest for the biomedical field, since imbalances at this level have been strongly related to the precipitation of several neuropsychyatric disorders, such as depression. Indeed, growing evidence supports that the detrimental effects on adult hippocampal neurogenesis, that have been associated with depression, might be epigenetically-mediated. Therefore, understanding the epigenetic regulation of the neurogenic process may provide a link between neurogenesis imbalances and the deterioration of the behavioural and cognitive domains frequently affected in depression, thus contributing to unravel the complex pathophysiology of this disorder.Here, we outline some of the major epigenetic mechanisms contributing to the regulation of hippocampal neurogenesis and discuss several lines of evidence supporting their involvement on the development of imbalances in the neurogenic process, often correlated to behavioural and cognitive deficits commonly observed in major depressive disorder. The beauty of research is that it ultimately defeats all established dogmas, even though some take very long to fall. Cajal's decree concerning the immutability of the central nervous system (CNS) has been reviewed and updated during the last decades, due to mounting evidence that substantiates the regenerative potential and plasticity of the CNS. Despite the initial reluctance manifested towards the first reports of post-natal neurogenesis, it is now well established that neurogenesis, a process that comprises the generation, differentiation and integration of new neurons in the preexisting brain neuronal networks, occurs in the adult brain, prevailing throughout life in specific brain areas, where neurons are persistently generated ,2. Such In the hippocampal formation, the precursor cell population resides throughout the SGZ, with specific gradients . After bAlthough it is now indisputably accepted that neurogenesis occurs in the adult brain, its functional relevance remains to be fully established. While it is clear that this phenomenon is confined to very discrete brain regions, the generation of new neurons in the post-natal period constitutes a new dimension of plasticity, with both direct and indirect impact on neuronal remodelling and repair, that is now regarded by the biomedical field as a promising therapeutical target in several neuropathological contexts. Notably, abnormal alterations in the hippocampal neurogenesis process have been implicated in an assortment of neuropsychiatric disorders -18. SeveIntegration of newly-born hippocampal neurons into pre-established neural networks seems to be achieved through highly regulated sequential steps: proliferation of neural stem cells, generation of amplifying progenitors, cell migration and, finally, maturation in the definitive destination with axon and dendrites formation and establishment of new synapses with preexisting surrounding cells -21  are believed to be the multipotent stem cells residing on the hippocampus ,22; theyImportantly, neurogenesis is a fine tuned process, rather than a mass phenomenon, during which most newborn cells are eliminated ,32. The Functional and structural chromatin properties are actively regulated in hippocampal NSCs. In fact, and despite being a relatively recent concept in the neuroscience field, the importance of epigenetics on the fine regulation of proliferation, fate specification and differentiation of NSCs is now becoming to be recognised as fundamental for the balanced production of new neuronal and glial cells, necessary for the homeostatic brain function. Therefore, it becomes gradually evident that both extracellular signalling and intracellular epigenetically regulated gene expression programs are dynamically involved in adult neurogenesis. Notably, the intracellular epigenetic program regulating adult neurogenesis is proposed to be quite similar to the epigenetic regulation occurring during development, but is also determined by a myriad of new extrinsic physiological and environmental stimuli , that alEpigenetic regulation is implicated in the first stages of adult neurogenesis, by promoting the maintenance of the self-renewal potential of adult NSCs. Molofsky and colleagues  highlighWhen exiting the mitotic phase, neural progenitor cells will eventually become committed to a specific neural cell lineage and start to differentiatiate. Neuronal or glial lineage commitment of NSCs involves a temporal-defined mutual regulation of several gene batteries. Commitment to a neuronal cell-fate, for instance, involves the repression of gliogenic genes; the alternative scenario of glial differentiation, requires the inhibition of genes responsible for neuronal specification. This is achieved through transcriptional and epigenetic regulation, that will integrate also the cell response to the extrinsic environment. In this context, HDACs and HATs are believed to exert an important role in the transduction of physiological signals to the stem cell genome, activating or repressing specific gene programs in NSCs. In general, HDACs catalyze the deacetylation of nucleossomes, that become highly condensed, obstructing the access of transcriptional activation factors to their binding sites and, therefore, resulting in transcriptional repression. In contrast, HATs catalyze the opposite reaction, resulting in global nucleossomal relaxation and, consequently, in increased transcriptional activity . A hint Repressor Element 1 Silencing Transcriprition Factor). REST was first described in 1995, as a repressor of neuronal genes containing a 23 bp conserved sequence, known as RE1  [Although several other epigenetic regulatory players in cell-fate specification have been identified, it becomes difficult to consider a unifying model through which all epigenetic regulatory actions are coordinated. Nevertheless, a promising candidate for orchestrating these epigenetic events is the DNA binding protein REST (or NRSF) ,48. Thisor NRSF) -51. Afteor NRSF) ,52. The or NRSF) ,54.Epigenetic regulation is now known to be also implicated in the final maturation of newborn neurons; at this stage it has an important role in promoting the integration of newly-born neurons into the preexisting neural networks. HDACs and HATs appear to be once again decisive for neuronal maturation and early synaptogenesis ,56. UsinFinally, it is important to note that this epigenetic regulation is not only mediated through cell-intrinsic mechanisms, and that epigenetic mediators are likely stable transducers of extracellular signals  into the regulation of all phases of neurogenesis. The paracrine action of MeCP2 and Gadd45b are amongst the best known examples of this integration of intrinsic and extrinsic signals relevant for neurogenesis regulation -65. NotiAdult hippocampal neurogenesis represents an important, and formerly underestimated, form of neuroplasticity, namely in the hippocampal formation, a brain structure involved in several neuropsychyatric disorders ,18,66. IImpaired neuronal plasticity is increasingly viewed as central in the ethiopathogenesis of depression. In fact, during the last two decades a significant number of studies in this field revealed cell loss and neuronal atrophy, particularly in brain loci relevant for emotional behaviour control. Several mechanisms were proposed to be responsible for this neuronal atrophy, namely glucocorticoid and glutamate toxicity for both glia and neurons , decreasLastly, a third link between MDD and hippocampal neurogenesis, is reflected in the functional importance of the adult neurogenic process in some of the behavioural domains commonly affected in depressive patients, such as mood, anxiety and cognition -89. HoweDuring the last decade, compelling evidence has emerged for the participation of epigenetic regulatory mechanisms in adult hippocampal neurogenesis. Therefore, it appears reasonable to conceive the hypothesis that dysfunction in epigenetic regulatory mechanisms might mediate the neurogenic imbalances responsible for some neuropsychiatric conditions, such as depression. Although this idea is still controversial, several lines of research suggest that different epigenetic regulatory mechanisms of adult neurogenesis are affected in animal models of depression Figure .Mbd1-deficient mice, besides having decreased NSCs proliferation, as already described by Zhao et al., also exhibit significant deficits in several behavioural dimensions relevant for depression: increased anxious phenotype, detected in both elevated plus maze and light-dark preference tests; behavioural despair, observed in the forced swimming test; and cognitive deficits manifested during the execution of Morris water maze spatial learning tasks [A paradigmatic case supporting this hypothesis is the already mentioned methyl-CpG-binding protein MBD1. In fact, an elucidative work conducted by Allan and colleagues , showed ng tasks .Mrg15-deficient mice present significant deficits in proliferation of neural progenitors and in their subsequent differentiation [Another example derives from studies focused on MRG15, an active component of HDACs complexes, such as HDAC2 ,96. Indentiation . Interesntiation . These rde novo DNA methylation, has been reported to have an increased expression in depressive suicide completers. Interestingly, this increase was significantly more pronounced in women, a result that is in accordance with the gender preference of MDD  [Moreover, epigenetic regulators directly involved in post-mitotic neuronal maturation and differentiation, have also been associated with several behavioural and cognitive impairments present in several neuropsychiatric disorders. Work from Adachi et al. , for insn women) .In addition, pharmacological and non-pharmacological treatments of depression, such as ADs and electroconvulsive shock (ECS) therapy, respectively, provide additional endorsement of the hypothesis that the neurogenic precipitation of depression might be, at least partially, epigenetically mediated. In fact, imipramine, a tricyclic agent with a well described pro-neurogenic action ,79, has Finally, it is worth to mention that this epigenetic regulation is also implicated in the vulnerability to stress, enhancing the susceptibility to stress-related disorders, in which depression is included. The role of such epigenetic mechanisms has recently been highlighted. Indeed, REST4, a splicing variant of the epigenetic orchestrator REST, has been shown to have an increased expression in rats exposed to stress early in life; importantly, these animals display enhanced susceptibility to stress and increased susceptibility to depressive-like behaviour . In addiThe dynamic and environmentally driven modulation of neuroplasticity in the adult brain plays a crucial role in the ethiopathogenesis of depression. As discussed herein, the last two decades provided several lines of evidence supporting the implication of hippocampal neurogenesis in the maintenance of the homeostatic brain function and its importance in the pathophysiology of MDD Figure . HoweverOur comprehension of how adult neurogenesis is regulated begins now to be complemented with new insights into the complex epigenetic orchestration of this phenomenon, a regulatory dimension that is relatively new in neuroscience research. Yet, the scattered evidence gathered so far, opens a new dimension for unravelling the mechanistic explanation for the interplay of genes and environment that is central in several neuropsychiatric pathological scenarios. In fact, studies in this area endorse the role of epigenetic mechanisms as transducers of the environmental signals into transcriptional outcomes, strongly suggesting that they may participate in the precipitation of experience-dependent psychiatric disorders, like depression, and simultaneously mediate the pro-neurogenic action of commonly prescribed ADs. Therefore, epigenetic mechanisms begin to emerge as mediators through which environment modulates neurogenesis with long-lasting and stable repercussions in several behavioural and cognitive domains Figure . In thisThe authors declare that they have no competing interests.AP drafted the manuscript. LP and NS revised the manuscript and coordinated the work. All authors read and approved the final manuscript.


The regulated secretory pathway shared by excitable cells, including neurons and neuroendocrine cells is an intricate process that comprises multiple, tightly regulated steps. After their synthesis in the endoplasmic reticulum, hormones and neuropeptides have to be sorted and packed into large dense core vesicles  in the Golgi apparatus. Granules are transported toward the plasma membrane in a cytoskeleton-dependent manner and mature into competent organelles for secretagogue-induced exocytosis. Granules are then tethered to the plasma membrane, docked, and primed, before finally releasing their contents after fusing with the plasma membrane. To ensure that neurotransmission and neuroendocrine secretion operate correctly, all these steps must be tightly regulated and coordinated both spatially and temporally. Currently, when the field of intracellular trafficking has been honored by the 2013 Nobel Prize in Physiology or Medicine , this issue of Frontiers in Neuroendocrine Science is aimed to providing an up-to-date overview of the cellular and molecular mechanisms governing the regulated secretory pathway in neuroendocrine cells. Reviews presented here are widely covering this topic, from the architecture of the organelle involved in secretory cargo processing and sorting, the biogenesis of secretory granules, their specific transport toward the plasma membrane to the late steps of exocytosis, and the secretory granule membrane recapture.Early stages of the secretory pathway have been discussed by two groups. Emma Martinez-Alonso and colleagues discuss the Golgi complex architecture, as well as the regulatory proteins that govern extra- and intra-Golgi transport, and the still controversial, but not mutually exclusive, theoretical models proposed to explain cargo progression through the Golgi stack . The groCloser to the cell surface, several groups discuss the molecular mechanisms regulating the late stage of exocytosis. The group of Frédéric Meunier reviews recent insights of the role of the cortical acto-myosin network , whereasFinally, exocytosis cannot exist without a compensatory membrane intake process , which allows recycling of granule components and maintains organelle integrity. The groups of Stéphane Gasman and Alla Rynditch discuss the mechanisms that coordinate clathrin-mediated compensatory endocytosis with exocytosis, highlighting the specific role of the intersectin family of scaffold proteins in exocytosis and endocytosis , 9. The On a more physiological point of view, Maria-Luisa Durán-Pasten and Tatiana Fiordelisio present an example of how pituitary gonadotrophs receive and transduce extracellular signals to promote luteinizing (LH) and follicle-stimulating (FSH) secretion, highlighting the tremendous plasticity of the system for adapting to different physiological demands . Wei-JyeOn a more technical point of view, recent improvements in detection technologies, especially in optical microscopy, continually push the limits of sensitivity and resolution. The groups of Colin Rickman and Ute Becherer discuss how advances over the last decade in fluorescence microscopy provided spatial and temporal details on the subcellular organization of the molecular machinery governing the regulated secretory pathway , 16, wheCollectively, this compilation of reviews intends to illustrate the recent progress made to understand the complex regulation of the granule secretory pathways in neuroendocrine cells. We are grateful to all the authors who have contributed to this Research Topic and to the dedicated reviewers who helped us reaching the highest quality standards.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.


Acute generalized exanthematous pustulosis (AGEP) is a rare and severe subtype of drug eruption, characterized by acute, extensive, non-folicular, sterile pustules overlying an erythematous skin, associated with fever and leukocytosis.Case report of a 6-year-old female child with encephalopathy and chronic lung disease, who was treated with intravenous antibiotics, antipyretic and antiepiletics during hospitalization for sepsis. She evolved with diffuse erythemata and non follicular pustules on her trunk, flexural areas, palms and soles.The drug history, clinical presentation, cutaneous histopathological findings  and the prompt regression of the lesions after withdrawal of the suspected drugs were consistent to ratify the diagnosis of AGEP.AGEP is an uncommon reaction , usually seen after drug exposure and clinicians should keep in mind the possibility of this skin eruption.Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.


In insulin-resistant states , hepatic production of glucose and lipid synthesis are heightened in concert, implying that insulin deficiency and insulin excess coexists in this setting. The fact that insulin may be inadequate or excessive at any one point in differing organs and tissues has many biologic ramifications. In this context the concept of metabolic compartmentalization in the liver is offered herein as one perspective of this paradox. In particular, we focus on the hypothesis that insulin resistance accentuates differences in periportal and perivenous hepatocytes, namely periportal glucose production and perivenous lipid synthesis. Subsequently, excessive production of glucose and accumulation of lipids could be expected in the livers of patients with obesity and insulin resistance. Overall, in this review, we provide our integrative perspective regarding how excessive production of glucose in periportal hepatocytes and accumulation of lipids in perivenous hepatocytes interact in insulin resistant states. Development of insulin resistance with obesity, pre-diabetes, and type 2 diabetes is a physiopathologic process where cells fail to respond normally to insulin.Hyperglycemia is then driven by excessive hepatic glucose productionIt is well recognized that lipolysis and weight loss are accelerated in the absence of insulin, underscoring the fact that insulin stimulates lipogenesis,The term insulin resistance oversimplifies a highly complex physiopathologic process for which a single overarching mechanism is not easily conceived.These questions and considerations have spawned a number of questions where answers may be found or conclusions drawn.Under physiologic conditions, the insulin sensitivity of various bodily tissues differs. Case in point, human skeletal muscle is more sensitive than subcutaneous fatty in terms of the effects of circulating insulin.Similarly, insulin resistance in insulin sensitive cells is not uniform but is tissue specific.Thus, insulin resistance is not a synchronous, all-or-nothing process but rather builds in select organs or tissues amidst normal insulin response.In parts of the body responding normally to insulin, the hyperinsulinism of insulin resistance likely is construed as a state of insulin excess. Hence, the catabolic activities of insulin resistance  may collide with complementary conditions that favor lipogenesis and obesity. So a vicious cycle can be set up with insulin resistance promoting weight gain, which promotes more insulin resistance. Subsequently, the multiple comorbidities of obesity, prediabetes, and type 2 diabetes that are attributed to insulin deficiency may actually stem in part from insulin excess. Likewise, the predisposition in patients with insulin resistance for aging,Development of hyperglycemia in overweight patients,Interestingly, with insulin resistance induced by a high-fat diet, a temporal sequence has been noted for each substrate during activation of hepatic gluconeogenesis, with progressive intensification for L-lactate, glycerol, and alanine on days 7, 14, and 56, respectively after dietary implementation.At this juncture, a new question surfaces: as with organs and tissues and the body as a whole, is the coexistence of insulin deficiency and insulin excess in insulin-resistant states applicable to isolated cells?To our knowledge, a number of metabolic pathways  are feasible in individual hepatocytes. Hence, gluconeogenesis (a process inhibited by insulin) and lipogenesis (which is stimulated by insulin) are achievable in the same liver cell. However, in the acini area, the hepatocytes are exposed to a spatial biochemical gradient that influences metabolism and gene expression, so cell specialization does exist to some degree, depending on locale.Parenchymal acini of the liver are divisible into 2 circulatory zones, based on proximity to afferent vessels.2 partial pressure of approximately 65 mmHg in periportal areas drops to 35 mmHg in perivenous zones.2 partial pressure regulates the expression of genes encoding glucose-metabolizing enzymes, , through O2-responsive transcription factors, such as hypoxia-inducible factor (HIF).Hepatocytes are also subject to differential regulatory control, due to gradients in oxygen, substrate, and hormone levels. Notably, OThe model of metabolic zonation assumes a functional specialization by each hepatic zone  first reach periportal zones, where glucagon receptors predominate for glycogenolysis and gluconeogenesis.Here again, a question can be raised: is the paradox of insulin resistance, namely the hyperproduction of glucose in liver alongside steatosis, adequately explained by the concept of metabolic compartmentalization?Liver metabolism comprises an immense spectrum of interrelated anabolic and catabolic functions which are performed simultaneously without futile cycles. Therefore, functional compartmentalization of the liver, as shown in Interestingly, decreasing periportal-perivenous gradients of oxygen tension and increasing periportal-perivenous gradients of insulin: glucagon ratio appear to be major factors in the zonation,A schematic of our theories is shown in Because insulin stimulates expansion of visceral fat, this pathologic process functions as a self-sustaining closed-loop system that will only be interrupted by weight loss (diet and/or exercise) and /or use of drugs to increase insulin sensitivity.The excess of free fatty acids (FFA) from splanchnic lipolysis is taken up by periportal hepatocytes . PKC induce insulin resistance by inhibiting insulin-stimulated phosphorylation of IRS proteins. Furthermore the excess of FFA results in activation of inflammatory toll-like receptors (TLR) signaling leading to increased As consequence of these metabolic changes,The enhanced FFA also increase the synthesis of triglyceride (TG) and very-low-density lipoprotein (VLDL) production in the perivenous zone,in vivo situation.All aspects of the human body must be considered to better understand the mechanisms of insulin resistance and to appreciate how insulin deficiency and insulin excess may coexist in this pathologic condition. The fact that insulin may be inadequate or excessive at any one point in differing organs and tissues has many biologic ramifications. Ultimately, attention must be paid to specific defects of insulin signaling in isolated cells and to subcellular compartments affected by the increased circulating insulin that accompanies insulin resistance. However, despite the enthusiasm for molecular aspects, we must never lose sight of the isolated organ and One key message is that insulin resistance accentuates differences in periportal and perivenous hepatocytes, namely periportal glucose production and perivenous lipogenesis. Subsequently, excessive production of glucose and accumulation of lipids are to be expected in the livers of patients with obesity and insulin resistance.However, there is still a great deal to be learned about the mechanisms linking insulin resistance and metabolic compartmentalization in the liver. Therefore, future studies on the regulation of zonal gene expression in parenchymal and nonparenchymal liver cells will provide advances in our understanding of the impact of insulin resistance on metabolic compartmentalization in the liver.


As high-resolution functional magnetic resonance imaging (fMRI) and fMRI of cortical layers become more widely used, the question how well high-resolution fMRI signals reflect the underlying neural processing, and how to interpret laminar fMRI data becomes more and more relevant. High-resolution fMRI has shown laminar differences in cerebral blood flow (CBF), volume (CBV), and neurovascular coupling. Features and processes that were previously lumped into a single voxel become spatially distinct at high resolution. These features can be vascular compartments such as veins, arteries, and capillaries, or cortical layers and columns, which can have differences in metabolism. Mesoscopic models of the blood oxygenation level dependent (BOLD) response therefore need to be expanded, for instance, to incorporate laminar differences in the coupling between neural activity, metabolism and the hemodynamic response. Here we discuss biological and methodological factors that affect the modeling and interpretation of high-resolution fMRI data. We also illustrate with examples from neuropharmacology and the negative BOLD response how combining BOLD with CBF- and CBV-based fMRI methods can provide additional information about neurovascular coupling, and can aid modeling and interpretation of high-resolution fMRI. To accurately predict high-resolution BOLD responses, some of these features need to be modeled explicitly  concentration, and is therefore sensitive to changes in cerebral blood flow (CBF), blood volume (CBV), and tissue oxygen consumption. In healthy brain, neural activity typically leads to a concurrent increase in oxygen consumption, blood flow, and volume. The inflow of oxygenated blood exceeds the ODifferent compartments can be used to model the different vascular elements , or vasculature vs. tissue, or different cortical layers. The general equation for the amount of deoxyhemoglobin (Q) in a compartment is a function of the dHb entering and leaving the compartment and its production:in and out are functions describing the amount of dHb entering and leaving the compartment, and thus these are CBF-dependent terms (CBF · [dHb]).leading to n coupled differential equations for n compartments. Note that the production term only arises for compartments containing cortical parenchyma, and that Some properties make fMRI modeling challenging, and additional terms or functions are needed to describe the system:Vasodilation in response to neural activity renders the volume of compartments time-dependent, leading to time-dependent volume terms in the equations.The MRI signal does not vary linearly with Q. The degree of signal attenuation following an increase in dHb differs for extravascular or intravascular water protons, or for water near capillaries or near veins, and depends on the size and orientation of the vessel . However, in some cases, for instance in disease, development, or after pharmacological intervention, CBV, CBF, and CMRO2 do not increase or decrease simultaneously -BOLD to the static gradients around large vessels, or that at higher magnetic field strength the sensitivity of gradient echo (GE)-BOLD shifts from sensitivity to predominantly veins at low field to an increased sensitivity to capillaries at higher field , but should be accounted for in laminar fMRI and high-resolution models. This can be done by including sensitivity to extravascular vs. intravascular water in the model, or by adding model parameters reflecting vessel size.The previous section described sources of heterogeneity at the mesoscopic level. Biological sources of heterogeneity within the cortex and heterogeneity resulting from the properties of the acquisition methods were described. Although all these can in principle be parametrized in multicompartment models, this would lead to highly complex models with many degrees of freedom. Judicious choice of which additional model parameters to include, and which assumptions or simplifications are reasonable, based on the study questions and acquisition method is therefore paramount.2), etc., and these techniques often provide complementary information. Optical imaging is used to answer questions about cortical function and the hemodynamic response, and the dynamics of BOLD and optical imaging responses are often compared. There are similarities but also differences between the methods and in the ideal case, one can inform the other.Many other techniques are used to study neurovascular coupling, such as optical imaging, functional photoacoustic imaging, functional ultrasound imaging, autoradiography, tracer techniques, histology, electrochemistry (e.g., probes to measure POThe optical field has contributed much to the elucidation of the BOLD mechanism. Intrinsic optical imaging, in which tissue is illuminated and reflected light is measured, has superior in-plane resolution and the ability to discriminate oxygenated and deoxygenated hemoglobin. Arteries and veins are easily resolved, while the more diffuse background signal originates from capillaries. These properties, and because combination with CBF measurement and electrophysiology is straightforward, make it a valuable tool for comparison with fMRI, and the availability of both the oxy- and deoxyhemoglobin signal aids in making inferences about metabolism. The drawback of intrinsic optical imaging is that it is dominated by signal from the cortical surface and superficial layers. Although some signal originates from deeper layers, resolving power in the depth-dimension is absent Hillman, .in vivo, pinpointing the exact location of vasoconstriction and dilation and the signaling pathways involved, as well as flow in microvessels (Kleinfeld et al., Two-photon imaging can reach cellular resolution and has greatly increased our understanding of neurovascular coupling. Single microvessels can be imaged Neurovascular coupling research benefits from the comparison of optical imaging and high-resolution fMRI. However, optical methods' lack of resolving power in the depth dimension also complicates the comparison. fMRI voxels typically cover the entire cortical thickness, but their signal is compared with hemodynamic responses measured with optical imaging derived from the cortical surface and superficial layers. This comparison is only valid when the responses of the deeper layers are the same as those of the surface layers. Laminar fMRI can provide additional information by virtue of its ability to separate surface- and deep voxels and can thereby improve the comparison with optical imaging. The ability to resolve single arteries and venules with fMRI (Yu et al., in vivo.The interpretation of neurovascular coupling at the mesoscopic scale is also expected to improve by comparison with new methods such as functional ultrasound and functional photoacoustic imaging that can measure blood flow, volume and oxygenation at high spatial and temporal resolution (Macé et al., Further exciting developments are the imaging and modeling of entire vascular and neural networks, which allows building increasingly sophisticated models of the cortical anatomy and blood flow in the cortical network (Guibert et al., in vivo.In this paper we discussed the properties of high-resolution fMRI and factors that need to be taken into account when extending current macroscopic fMRI models to the mesoscopic scale that becomes accessible with high-resolution fMRI. Those factors are biological, for instance laminar differences in neurovascular coupling that become visible at high-resolution, and methodological, like the differences between GE- and SE-BOLD acquisitions. For modeling of BOLD-responses at the mesoscopic scale this means that additional compartments and variables need to be included, needing more elaborate models and judicious choice of the models' assumptions. Aside from this complication, high-resolution fMRI provides an opportunity to improve BOLD-models, since more information is available. An obvious example is the information about different cortical layers that becomes available, but high-resolution fMRI can also separate the responses in vessels from the responses in parenchyma and reduce partial volume effects. The extension of fMRI models to the mesoscopic scale holds the promise of better understanding of the BOLD signal, neurovascular coupling, and the possibility of elucidating neural circuitry Acquired data: JG, YB, provided resources and supervision: NL, wrote first draft: JG. All authors edited and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Abnormalities in the integral components of bone, including bone matrix, bone mineral and bone cells, give rise to complex disturbances of skeletal development, growth and homeostasis. Non-specific drug delivery using high-dose systemic administration may decrease therapeutic efficacy of drugs and increase the risk of toxic effects in non-skeletal tissues, which remain clinical challenges in the treatment of skeletal disorders. Thus, targeted delivery systems are urgently needed to achieve higher drug delivery efficiency, improve therapeutic efficacy in the targeted cells/tissues, and minimize toxicities in non-targeted cells/tissues. In this review, we summarize recent progress in the application of different targeting moieties and nanoparticles for targeted drug delivery in skeletal disorders, and also discuss the advantages, challenges and perspectives in their clinical translation. The skeletal system provides a strong support and protection for the soft tissues and organs in the human body. The skeletal system includes bone matrix, bone mineral and bone cells. Any defects in these components may hinder skeletal development, growth and homeostasis, which results in skeletal disorders. The most common skeletal disorders are osteoarthritis, bone cancer, and osteoporosis. These disorders result from a wide variety of causes, some of which, such as vitamin D deficiency, are easily treatable, while the pathogenesis of some others are more complicated . The druco-glycolic acid) (PLGA) nanoparticles. Some of nanoparticles have been also approved by the FDA (Food and Drug Administration)  (PEG-DSPE) and l-glutamic acid, N-(3-carboxy-1-oxopropyl)-1,5-dihexadecyl ester (SA). Although the utility of PEG would reduce the uptake of MPS, the liposome size could modulate the stealth property of PEG [99mTC), a metastable nuclear isomer as medical radioisotope, to the bone marrow after intravenous administration in rabbits. This design would open up a wide variety of new therapeutic applications [Because the MPS will take up the delivery system, researchers took advantage of this mechanism to develop a bone marrow-targeted delivery system for diagnostic and therapeutic reasons. Mononuclear phagocyte system (MPS) organs have sinusoids with a fenestrated endothelium in association with macrophages, which express several types of receptors on their surface for the uptake of specific nanoparticles. The bone marrow is a part of the MPS. Thus, the nanoparticles in the blood circulation would be taken up by the MPS and gradually go to bone marrow . The lipy of PEG . In addiy of PEG . Due to ications .Lipid nanoparticles (LNPs) have been widely used for medicines, due to their lipid biocompatibility and versatility. Unlike liposomes in the same category, LNPs show considerable kinetic stability and rigid morphology. Their low cytotoxicity, production scalability, the modulation of drug release, the avoidance of organic solvents, and wide potential application spectrum is greater than liposomes . In addiin vivo are still obstacles in their clinical translation. siRNA and microRNA are hydrophilic negatively charged macromolecules, very labile in biological fluids: the systemic administration of naked siRNAs or microRNA does not result in effective therapeutic responses. Then, the interaction between active molecules and biological membranes is necessary to initiate the entrance into cells, but this process is not spontaneous because the negatively charged surface of siRNA and microRNA and cell membranes hampers the interaction, and their hydrophilic character prevents the passing through lipophilic cell membranes. But the LNPs can carry large amount of siRNAs and microRNA to enter into cells by endocytosis [In addition, the emergence of therapeutic siRNA and microRNA provides a novel avenue in molecular therapy of skeletal disorders. However, the safety, efficiency and targeted delivery of therapeutic siRNA and microRNA ocytosis . The LNPocytosis . The staocytosis . This apco-glycolic acid) (PLGA) is one of the most successfully developed biodegradable polymers. The PLGA has attracted considerable attention due to its attractive properties. It has been approved by the FDA and the European Medicine Agency as a drug delivery system with characters of biodegradability, biocompatibility, well-described formulations, various types of drug encapsulation and sustained release. PLGA is made up of lactic acid and glycolic acid, which would be easily metabolized by the body via the Krebs cycle. Thus, there is a minimal systemic toxicity associated with the use of PLGA for drug delivery or biomaterial applications [Poly and by disruption of epithelium in these inflamed sites, resulting in a preferential accumulation of these nanoparticles. In addition, the use of PLGA could not induce “crystal-induced pain” because of its biodegradability, biocompatibility and small size. The PLGA nanoparticle encapsulated glucocorticoids were directly injected inside the rat joint cavity. The results showed that the PLGA nanoparticles were preferentially phagocytozed by macrophages, which would specifically deliver glucocorticoids to the inflamed cells. It could also facilitate the medicines remaining in synovium, which is the pathological site of rheumatoid arthritis .The PLGA nanoparticles modified with poly aspartic acid peptide (poly-Asp) were designed as a bone-tissue targeting delivery system. This bone-targeting delivery system has strong affinity to high hydroxyapatite (HA), which is characterized by the bone resorption surfaces . It indi. The bone tissue-targeting delivery systems deliver drugs to the whole skeletal tissues, not the specific functional cells in bone, which reduce the efficiency and increase toxicity on neighboring non-targeted cells during treatment. In addition, some targeting moieties, such as pamidronate, alendronate and tiludronate, have dual effects on osteoblasts and osteoclasts and potential induction of jaw osteonecrosis. Moreover, one of alternative approaches in the clinic for bone regeneration and bone repair is calcium phosphate biomaterials [Since the concept of targeted drug delivery systems appeared in 1906, this concept has been gaining much attention and introduced as therapeutic approaches in skeletal disorders gradually. In recent years, the bone-seeking moieties were discovered and applied in targeted delivery system of molecular therapy to enhance and prolong pharmacological effects in bone, reduce side effects in non-targeted issues, and improve compliance because of less frequent need for medication. Previously, we gave more attention on the targeting moieties for bone tissues, which is an important part in various bone-targeting delivery systems. However, bone tissues are made up of different type of cells, such as osteoclasts, osteoblasts, bone marrow mesenchymal stem cells, and osteocytesaterials . This meThe new generation of targeting moieties, such as peptides and aptamers, appeared to improve the accuracy of targeting in order to increase the therapeutic efficiency and reduce adverse effects to the untargeted cells. The most important one is the aptamer, which could exhibit cell-type specific seeking and exert the modulation of specific functional cells in bone. The emergence of cell-SELEX technique will allow a big stride forward to screen specific cell-type targeting aptamers to bone. The osteoblast-targeting aptamer has been screened as targeting moieties in the osteoblast-targeting delivery systems. Although BMSCs-specific aptamers have been used as targeting ligands in the conjugate, the administration of conjugate by intra-bone marrow will limit its utility. In addition, it is still lacks targeting moieties for cartilage cells and osteoclasts. With the development of this technique, these drawbacks of targeted delivery systems in of skeletal therapy could fill up in the near future.The conventional nanoparticles have their own limitation, which drove the nanoparticles for molecular therapy in skeletal diseases to upgrade, to become biostable, biocompatible and biodegradable. The nanoparticles become an important and significant partner to bone-seeking agents during the development of targeted delivery system in skeletal diseases. The components and the size of nanoparticles are two important factors during the development of targeted delivery systems in skeletal therapy. The use of polyethylene glycol (PEG) in nanoparticles could avoid the clearance of the MPS to some extent, which protects drugs from degradation before arrival to the specific pathological sites. However, limited therapeutic efficacy and loss of their long circulating property of PEG-conjugated nanoparticles have been recently reported in animals and humans following repeated injection due to the induction of anti-PEG antibody . Thus, tAlthough many challenges for targeted delivery systems of molecular therapy in skeletal disorders have come to the forefront, such as manufacture cost, biostability, pharmacokinetic and therapeutic efficiency, the therapeutic strategy of targeted delivery systems used in skeletal diseases still have attractive advantages. The evaluation of targeted delivery systems in skeletal diseases requires complicated and costly safety and efficacy experiments, but the engineering of this targeted delivery system is fairly simple owing to cost-effective, and easy manufacture. Thus, the targeted delivery systems for molecular therapy in skeletal disorders have an immense clinical potential leading to a new generation of disease treatment. The recent exploration of various targeted delivery systems for molecular therapy in skeletal disorders is worthy of note.


Parkinson disease (PD) is a chronic and progressive movement disorder classically characterized by slowed voluntary movements, resting tremor, muscle rigidity, and impaired gait and balance. Medical treatment is highly successful early on, though the majority of people experience significant complications in later stages. In advanced PD, when medications no longer adequately control motor symptoms, deep brain stimulation (DBS) offers a powerful therapeutic alternative. DBS involves the surgical implantation of one or more electrodes into specific areas of the brain, which modulate or disrupt abnormal patterns of neural signaling within the targeted region. Outcomes are often dramatic following DBS, with improvements in motor function and reductions motor complications having been repeatedly demonstrated. Given such robust responses, emerging indications for DBS are being investigated. In parallel with expansions of therapeutic scope, advancements within the areas of neurosurgical technique and the precision of stimulation delivery have recently broadened as well. This review focuses on the revolutionary addition of DBS to the therapeutic armamentarium for PD, and summarizes the technological advancements in the areas of neuroimaging and biomedical engineering intended to improve targeting, programming, and overall management. Parkinson disease (PD) is a chronic neurodegenerative disorder classically characterized by motor manifestations including slowed voluntary movements, resting tremor, muscle rigidity, and impaired gait and balance Stacy, . With a In advanced stages of PD, when medications no longer adequately control motor symptoms, deep brain stimulation (DBS) offers a powerful therapeutic alternative. DBS is a surgical therapy involving the implantation of one or more electrodes into specific regions of the brain, which deliver electrical stimuli to modulate or disrupt abnormal patterns of neural signaling within the targeted region. The results are often dramatic, with improvements in motor function and motor complications having been repeatedly demonstrated. Given such robust responses, emerging indications for DBS are being investigated. In parallel with expansions of therapeutic scope, advancements within the areas of neurosurgical technique and the precision of stimulation delivery have recently broadened as well. This review summarizes the revolutionary addition of DBS to the therapeutic armamentarium for PD as well as recent advances in treatment delivery.Prior to the discovery of levodopa, surgical interventions were the most efficacious treatment for PD symptoms, but primarily focused on the reduction of bothersome tremor. Early approaches targeted the pyramidal tracts, with lesioning either at the point of origin in the cortex or the descending pathways through the brainstem and cervical spinal cord Cooper, . AlthougSurgical intervention for PD faded in popularity in the early 1970s after the arrival of levodopa, which radically transformed the pharmacological treatment of PD. Levodopa was soon accepted as the preferred treatment for PD while demonstrating a positive impact on disability, quality of life, and longevity. However, it also became clear that patients on levodopa and other antiparkinsonian drugs often develop significant drug-induced complications, such as involuntary movements (dyskinesias), motor fluctuations , hallucinations, and psychosis. Such complications, combined with a growing understanding of basal ganglia and cerebellar circuitry, rekindled interest in earlier surgical procedures.In 1987, Dr. Alim-Louis Benabid explored high frequency stimulation of the thalamus while operating on an elderly man who had previously undergone contralateral ablation for tremor. Given his concerns for creating bilateral lesions, Benabid implanted a wire into the untreated thalamus with four metal contacts at its tip, which was then connected to an external battery source. By passing high-frequency electrical stimulation through the wire, Benabid and colleagues demonstrated that chronic electrical stimulation of the subcortical brain (DBS) was as effective as thalamotomy for tremor suppression , a cholinergic nucleus located within the mesencephalic locomotor region (MLR), functionally defined as the area of the brainstem responsible for locomotion Figure . InitialOther locations of interest include the centromedian thalamus, zona incerta, and the substantia nigra (SNr). The SNr is also part of the MLR, with dense reciprocal interconnections to the PPN and brainstem circuitries. Early investigations have shown promise, demonstrating improvement in gait freezing with combined SNr/STN-DBS . The electrode is implanted into a brain target and attached to an implanted pulse generator (IPG), which provides stimulation as a continuous spherical electric field around the active contact(s). The electrical parameters for chronic stimulation are selected during DBS programming, the goal of which is to screen for the optimal electrode polarity, amplitude, pulse width, and frequency which avoids adverse effects of stimulation, while optimizing benefits and minimizing current consumption. At present, this is most commonly achieved by choosing either monopolar or bipolar stimulation and varying combinations of active contacts, thus adjusting current flow through the desired target area  is capable of delivering multiple source constant current, enabling the allocation of completely different stimulation parameters independently to each of eight contacts of the electrode Figure . A recenGiven the close proximity of subcortical DBS targets to surrounding neuronal structures and white matter pathways, there remains a need for true current steering and true electric field shaping around all three directional axes. Two approaches have been investigated to address this need, including a novel electrode with 32 contacts distributed evenly around the circumference of the lead Figure  and a leFurther, advancements in DBS lead design continue with the goal of enhancing the spatial precision of stimulation  recorded from the STN show an exaggerated oscillatory activity in the beta range (13–30 Hz) while at rest. Resting state beta band power has the potential to be a useful metric to guide an adaptive DBS system, and has a number of potential advantages. Parkinsonian motor symptoms, including bradykinesia and rigidity, are related to strong and sustained activity in beta-frequencies , which enables direct visualization of brain anatomy and the intended targets utilized for PD. Stereotaxy determines accurate localization of intracranial targets by ascertaining triplanar coordinates with reference to a fixed point, thus providing access to surgical targets deep within the brain in a minimally invasive fashion. The target is then constructed by use of graphic tools embedded within available stereotactic navigation software and registered in a stereotactic coordinate system. This planning stage allows pre-operative target adjustments based on individual patient anatomy and the selection of a skull entry point that avoids interrupting vessels of the cortical surface and sulci, ventricles, or other eloquent brain structures.Functional neurosurgery involves the precise surgical targeting of anatomic structures to modulate neurologic function, such as abnormal movements. As such, accuracy and precision of electrode placement is of great importance. Most surgical teams utilize some form of electrophysiological mapping or stimulation testing to ensure that the optimal location within the target has been implanted. This exploration may consist of microelectrode recording of single neurons, local field potential recording of populations of neurons, or macrostimulation of the intended target with direct observation of the patient for improvement in symptoms and/or stimulation-induced side effects. Microelectrode recording provides adjustment for interpatient variability in physiology and symptomatology, and recording during passive movement of a limb can confirm an association with motor function and help ensure implantation within the motor subterritories of the intended brain target. However, these techniques require that the patient be awake for much of the procedure, which is difficult for some to tolerate. In addition it is time consuming, technically demanding, and often necessitates multiple brain penetrations.Electrophysiological mapping helps ensure proper anatomic position of the DBS electrode in the brain, which is essential for safety, selection of stimulation parameters, and the ultimate success of the therapy. Various types of imaging modalities may also aid surgical targeting and both computed tomography (CT) and MRI have been successfully integrated into the intraoperative workflow of DBS surgery. One approach involves imaging the implanted microelectrode with registration back to the pre-operative planned trajectory. The physiologic data are then interpreted in light of the position of the electrodes on the scan. A subsequent track can be created, or the DBS lead can replace one of the microelectrodes. Imaging of the DBS lead after placement assures the surgeon that its position is acceptable. Evidence supports that this approach allows for correction of the average 2-mm targeting error seen with traditional stereotactic approaches  guidance without the use of microelectrode recordings or intraoperative test stimulation . Such advanced neuroimaging techniques have helped validate many components of these circuits, while also refining some overlap between pathways and enhancing information about smaller subregions , as well as image reconstruction methods such as susceptibility-weighted phase imaging (SWPI) and quantitative susceptibility mapping (QSM), have shown promise in improving target contrast and better defining target borders  and T2-weighted magnitude imaging , Saraepta Therapeutics, and Sunovion Pharmaceutics, Inc. Dr. MS has also received grants from the Michael J. Fox Foundation, the NIH, the Parkinson Study Group, and Pharma 2B, royalties from Informa Press for the Handbook of Dystonia and Duke University for the Wearing Off Questionnaire. He has also received payment for development of educational presentations from the University of Kansas, the University of Miami, and the University of Rochester. Dr. MS also received paid travel accommodations from the Cleveland Clinic Neurological Institute, the Movement Disorder Society, and the National Parkinson Foundation.


Arquivos Brasileiros de Cardiologia (ABC) has been the officialpublication of the Brazilian Society of Cardiology for more than 60 years, and has provedto be one of the most prestigious scientific journals in Latin America, approaching allaspects of cardiology, including the surgical ones. In August 1986, the RevistaBrasileira de Cirurgia Cardiovascular (RBCCV), specifically directed to thesurgical aspects of heart diseases, began to be published. Thus, the editorial policy ofthe ABC has progressively privileged publications focused on the clinical treatment ofcardiovascular diseases over surgical publications of common interest to cardiologists andcardiac surgeons1. Despite thesubstantial changes in the treatment of heart diseases, deriving from the appearance of newdrugs and percutaneous devices, cardiac surgeries are still the treatment of choice to asubstantial number of heart diseases, mainly the congenital, valvular and even coronaryones, whose onus of surgery indication is invariably on cardiologists. Thus, thosespecialists should be aware of several aspects of cardiac surgery, mainly those concerningtheir risks and results, so that they can base their indications on evidence.The 2. Thus, the optimization of arterial grafts has been widespread withthe use of the radial artery (RA) and composite grafts. However, only few professionals areaware of the controversies involving risky situations, possible limitations to the use ofBITA3, the patency index of RAgrafts and their influencing factors. The RA was the second arterial graft introduced inthe clinical practice for coronary artery bypass grafting, and has drawn the interest ofBrazilian surgeons since the 1990s4.Skeletonization of the left ITA (LITA) can favorably change the flow capacity of the graft,leading to the assumption that the behavior of the RA, as a coronary graft, is similar tothat of the skeletonized LITA. Considering the potential role of the RA as a second optionof coronary grafting and the concept of complete coronary artery bypass grafting with theexclusive use of arterial grafts, Bonini et al5 have carried out a prospective randomized study comparing 40 patientsdistributed into two groups. In group I, skeletonized RA was used in 20 patients, and, ingroup II, RA with adjacent tissues was used in 20 patients. After the surgical procedure,flow velocity was measured. The results showed that the morphological and pathologicalcharacteristics, as well as the hemodynamic performance of the free RA grafts, regardlessof being skeletonized or having adjacent tissues, were similar. However, a larger number ofnon-obstructive lesions was observed in the group with the RA graft preserving the adjacenttissues5.The great majority of cardiologists might know that the use of bilateral internal thoracicarteries (BITA) for coronary artery bypass grafting improves survival as compared to theuse of only one single internal thoracic artery (ITA)6, as well as of theirlimitations, mainly regarding the most recent options of percutaneous therapies7.The applicability of risk scores to cardiac surgery is another relevant internationalmatter, but not well defined in centers outside North America and Europe. Garoffalo etal8 have assessed the ability ofthe Parsonnet-Bernstein 2000 score and EuroSCORE to predict the in-hospital mortality ofpatients undergoing cardiac surgery at a reference hospital in Brazil, and have identifiedrisk predictors. The use of those scores has underestimated in-hospital mortality,suggesting inadequate preoperative assessment of the patients undergoing cardiac surgery.That study has stressed the need to develop local scores based on the reality of thepopulations to better assess the risk of cardiac surgery3.In addition, small is the number of cardiologists aware of the models to calculate theperioperative risk for morbidity and mortality available and popular among cardiacsurgeons9, inthe United States, have compared long-term survival of patients on dialysis afterangioplasty, coronary stenting and coronary artery bypass grafting. That retrospectivestudy has reported better long-term survival of patients on dialysis after coronary arterybypass grafting than after percutaneous coronary intervention and coronary stenting,emphasizing the relatively poorer results of patients with diabetes. That study hassupported the need to develop large clinical registries and prospective studies on coronaryrevascularization procedures for patients on dialysis9. Miranda et al10have approached that important question, analyzing retrospectively 50 consecutive andnon-selected patients on dialysis, undergoing coronary artery bypass grafting at a tertiaryuniversity-affiliated hospital between 2007 and 2012. Those authors have shown thatcoronary artery bypass grafting is feasible in patients on dialysis, although followed byhigh morbidity and in-hospital mortality. In addition, they have emphasized the frequentexclusion of that group of patients from large cardiac studies. That detail might evencontribute to the difficulty in selecting a better approach and to the still modestsurgical results as compared to those of patients with preserved kidney function10.Considering the controversies about the ideal method of coronary artery bypass grafting forpatients on dialysis, Herzog et alIt is therefore evident that, although the ABC prioritizes articles on clinical therapiesand their guidelines, the restriction upon articles with a surgical bias can deprivecardiologists from essential information to support their clinical decision-making,especially regarding the case-to-case analysis in daily practice. It is worth noting thepublication in the ABC of several articles of common interest to both cardiac surgeons andcardiologists in recent years, therefore providing a relevant service to cardiology andcardiovascular surgery.


Cannabis sativa developed lower intraocular pressure (IOP). This led to the isolation of phytocannabinoids from this plant and the study of their therapeutic effects in glaucoma. The main treatment of this disease to date involves the administration of drugs mediating either the decrease of aqueous humour synthesis or the increase of its outflow and thus reduces IOP. However, the reduction of IOP is not sufficient to prevent visual field loss. Retinal diseases, such as glaucoma and diabetic retinopathy, have been defined as neurodegenerative diseases and characterized by ischemia-induced excitotoxicity and loss of retinal neurons. Therefore, new therapeutic strategies must be applied in order to target retinal cell death, reduction of visual acuity, and blindness. The aim of the present review is to address the neuroprotective and therapeutic potential of cannabinoids in retinal disease.The functional significance of cannabinoids in ocular physiology and disease has been reported some decades ago. In the early 1970s, subjects who smoked The study of cannabinoids in the central nervous system (CNS) has been the primary focus of many investigations, not only due to their psychotropic effects, but also due to their involvement in neuroplasticity and their therapeutic use in conditions such as obesity, pain, and neurodegenerative diseases. Fewer studies have focused on the role of cannabinoids in the retina, as well as other critical players of the visual system , 2.Cannabis sativa) developed lower intraocular pressure (IOP). This finding was subsequently reproduced and the results suggested that cannabinoids isolated from cannabis may be useful in the treatment of glaucoma , have been found in the retina of rodents and other mammals , 9, 22. 2+ and/or K+ channels in bipolar cells and photoreceptors -D-aspartate release from bovine retina, and Ca2+ evoked [3H]-noradrenaline and [3H]-dopamine release in guinea pig retina -D-aspartate release from isolated bovine retinas, as mentioned above  dopamine [DA also regulates the release of other retinal neuromodulators, such as NO (nitric oxide) and the neuropeptide somatostatin. It enhances NO release, while a reciprocal effect was also observed, namely, NO attenuating DA release –97. NO, ircuitry . CB1 agodopamine . HU-210 dopamine . TherefoHowever, other mechanisms may also be involved. HU-210 was shown to provide neuroprotection and counteract the oxidative stress and cellular injuries observed in diabetic encephalopathy, a CNS neuropathy and one of the most common complications of diabetes, through a cannabinoid receptor-independent mechanism .The aim of this review was to summarize the knowledge acquired to date on the function of endogenous and synthetic cannabinoids in the retina, the receptors they activate to mediate their actions, and the neuroprotective role of cannabinoids in retinal models of disease. The studies presented in this review support the involvement of the endocannabinoids, AEA and 2-AG, and synthetic cannabinoids in the neuroprotection of the early and final events underlying the pathophysiology of retinal ischemia. It appears that these agents have the pharmacological profile to target the neurodegenerative/proapoptotic components of retinal disease.The available treatments for neurodegenerative retinal diseases have limitations and further research is needed in order to provide efficacious therapies. Cannabinergic agents, CB1 or TRPV1 agonists and inhibitors of endocannabinoid metabolic enzymes, may prove to be efficacious therapeutics in the treatment of glaucoma and diabetic retinopathy.In order to provide “LEAD” cannabinergic agents, further studies are essential (a) to study the pharmacokinetic properties of cannabinoids that would suggest their use as effective topical retinal therapies and (b) to ascertain their pharmacological efficacy in chronic use. These studies will assess whether cannabinergic agents can play an important role alone or in a multidrug treatment to provide efficacious therapy and improve the eye sight of millions of people worldwide who are afflicted with neurodegenerative retinal disease.


Paramyxoviruses are a family of negative sense RNA viruses whose members cause serious diseases in humans, such as measles virus, mumps virus and respiratory syncytial virus; and in animals, such as Newcastle disease virus and rinderpest virus. Paramyxovirus particles form by assembly of the viral matrix protein, the ribonucleoprotein complex and the surface glycoproteins at the plasma membrane of infected cells and subsequent viral budding. Two major glycoproteins expressed on the viral envelope, the attachment protein and the fusion protein, promote attachment of the virus to host cells and subsequent virus-cell membrane fusion. Incorporation of the surface glycoproteins into infectious progeny particles requires coordinated interplay between the three viral structural components, driven primarily by the matrix protein. In this review, we discuss recent progress in understanding the contributions of the matrix protein and glycoproteins in driving paramyxovirus assembly and budding while focusing on the viral protein interactions underlying this process and the intracellular trafficking pathways for targeting viral components to assembly sites. Differences in the mechanisms of particle production among the different family members will be highlighted throughout. Paramyxoviridae, a family of enveloped viruses with negative strand, non-segmented RNA genomes, cause significant disease in humans and animals. Important human pathogens within this family include measles virus (MeV), mumps virus (MuV) and human respiratory syncytial virus (HRSV), which is the single largest cause of respiratory tract infections in the pediatric population [in vitro [The pulation . In addipulation ,3,4. Parpulation ,6, cattlpulation ,10,11,12pulation ,14. A suin vitro ,17,18,19in vitro ,21,22.The three key components in production of infectious paramyxovirus particles, the surface glycoproteins, the matrix proteins and the RNPs, must coalesce at the plasma membrane to initiate budding. Interactions among these three components are critical for glycoprotein incorporation and particle assembly. The matrix protein is generally considered the main driver of paramyxovirus assembly and can interact with both the glycoproteins and the core RNPs in an orderly manner. However, paramyxovirus surface glycoproteins are not simply bit players in this process, but instead can play important roles in directing the process of particle formation.pneumovirinae subfamily. Interestingly, the attachment proteins of pneumoviruses differ significantly from attachment proteins within the paramyxovirinae subfamily, as they are much smaller in size and are not strictly required for membrane fusion and entry [pneumovirinae subfamily, and cellular factors which interact with both RSV F and HMPV F have been identified [Entry of paramyxoviruses into target cells requires the concerted effort of two glycoproteins on the viral membrane: the attachment protein and the F protein. The attachment protein is generally responsible for primary adsorption of the virus to the cell surface by binding proteinaceous or sialic acid receptors ,24,25. Fnd entry ,31,32,33entified ,35,36,37C-terminal globular head domain ; and the G protein of HeV and NiV, suggests that the attachment proteins exist as a dimer-of-dimers, with each monomer comprising a short cytoplasmic tail, a single membrane-spanning domain and a large ectodomain made up of a membrane-proximal stalk region and a d domain A [38,39,C-terminal cytoplasmic tail, and a single-pass transmembrane domain which anchors the protein to plasma membrane . Par of VLPs . Specifi of VLPs . Similar of VLPs ,96,139.In addition to their contribution to the budding process, paramyxovirus glycoproteins are also implicated in assembly of other viral components. The fusion protein of RSV was shown to be responsible for incorporation of G and SH proteins into budded virions and for their co-localization with N at plasma membrane assembly sites; however, F deletion had no effect on M assembly into virions . A key fin vitro [Paramyxovirus surface glycoproteins contain short cytoplasmic tails which extend on the inner side of the plasma membrane. The length and amino acid sequences of these domains in the fusion and attachment proteins vary significantly among different paramyxovirus members . Severalin vitro ,31. Thisin vitro  and a roin vitro . The impin vitro ,138. Forin vitro ,142. Forin vitro ,143. In in vitro ,142. Forin vitro . Deletioin vitro ,113,145.in vitro  (HA) andin vitro ,148. Figin vitro ,153,154.in vitro ,131. A fin vitro . Specifiin vitro ,153. TheDuring paramyxovirus replication, the glycoproteins, matrix proteins and RNPs are synthesized at distinct sites in the cytoplasm and must be transported to the plasma membrane for coordinated assembly. The different viral components reach the plasma membrane by different mechanisms and interact with each other in an orderly manner either during trafficking or at the cell surface prior to packaging into virions. Paramyxovirus proteins are carried to the cellular plasma membranes by utilizing various cellular machineries including endocytic and exocytic pathways, in addition to vesicular trafficking and the cytoskeleton.N-glycosylation can also be essential for the proper folding, stability, intracellular transport, and surface expression of the paramyxovirus glycoproteins. Removal of N-glycans from the glycoproteins of NDV, CDV, PIV5, SeV, HeV and NiV had a significant effect on their exocytic transport and surface expression [N-glycans did not affect transport of the RSV F protein to the cell surface indicating that the degree to which N-glycosylation influences proper folding and transport varies among paramyxovirus glycoproteins [Paramyxovirus glycoproteins are synthesized in the endoplasmic reticulum (ER) and traffic through the secretory pathway, and in some cases through endocytic pathways, to the plasma membrane. Proper trafficking is needed for incorporation into budding virions or induction of cell-cell fusion for direct cell-to-cell transmission of virus particles. For some paramyxoviruses, data indicate that the fusion and attachment proteins can interact following their synthesis in the ER, and thus are transported to the cell surface as a metastable protein complex. This has been suggested to occur for NDV, MeV and human parainfluenza viruses 2 and 3 ,156,157.pression ,171,172.proteins ,174.Trafficking of viral glycoproteins has been demonstrated to involve tyrosine-based and di-leucine motifs which are involved in protein trafficking in both secretory and endocytic pathways. Several paramyxovirus glycoproteins have endocytic signals and can undergo internalization following trafficking to the plasma membrane ,177,178.Paramyxoviruses can infect cells that are polarized in nature, such as neurons, epithelial cells, endothelial cells and lymphocytes. The plasma membrane in polarized cells is divided into two discrete domains, the apical domain and the basolateral domain, and this polarity is maintained by sorting of proteins and lipids in the TGN or secretory pathways and recycling endosomes . SeveralThough originally synthesized in the cytoplasm, the matrix protein and the RNPs must subsequently translocate to viral budding sites at the plasma membrane; however, very little is currently known on the mechanisms underlying transport of these critical viral structural components. In the classical model for paramyxovirus assembly, the matrix protein is thought to interact with the RNP at the cell membrane to mediate its insertion into budding sites for the production of infectious virus particles. Substantial data, however, support an alternative model in which the matrix protein associates with the RNP complex in the cytoplasm prior to translocation to the plasma membrane. Data on both MeV and SeV suggest that the M protein binds to the RNP in the cytoplasm, and the two components are then co-transported to the plasma membrane ,108. FurData from live cell imaging revealed an important role of the host cytoskeleton in the trafficking of paramyxovirus RNP complexes. Filamentous RNPs of RSV show myosin-motor driven directional movement on the actin cytoskeleton . In the Although the entire replication cycle of paramyxoviruses occurs in the cytoplasm, the matrix proteins of HRSV, SeV, NDV and NiV have been shown to traffic through the nucleus early during virus infection. In the case of RSV, localization of M in the nucleus occurs through interaction of a nuclear localization signal (NLS) with the nuclear import receptor, importin β1, and its exit to the cytoplasm at later stages of infection is mediated by a nuclear export signal (NES) that directs Crm-1dependent nuclear export . NuclearParamyxoviruses have a host-derived, lipid bilayer envelope containing the membrane spanning envelope glycoproteins. The assembly of viral membranes is a sophisticated and specific process that involves coalescence of viral components at discrete sites on cellular membranes and exclusion of the majority of host cell membrane proteins. Recent data support the idea that viral glycoproteins of enveloped RNA viruses are not randomly distributed on the cell surface but instead are clustered within lipid raft membrane microdomains to form the nucleation points for budding . Lipid rBudding of enveloped viruses is a complex process that requires induction of membrane curvature followed by membrane scission and release of virus particles. Induction of membrane curvature and the final membrane fission event requires manipulation of the lipid-lipid interactions within cellular membranes, and is driven by interactions of viral proteins with membrane lipids in addition to viral-viral and viral-host protein interactions . The mecAlthough M proteins are considered the driving force for budding of paramyxoviruses, increasing evidence demonstrates that the glycoproteins can also play a role. Several paramyxovirus glycoproteins can induce VLP formation by themselves or must be present with the M protein for efficient VLP formation, indicating that surface glycoproteins in these cases are needed either to recruit M to assembly sites or to initiate budding. A major role of glycoproteins in paramyxovirus budding is well characterized for the SeV fusion protein. Sendai F induces VLP release when expressed alone in cells, and silencing of the F gene reduces virus production by 70% . The abiN-terminal domain. The viral protein-protein interactions involved in particle assembly have been demonstrated for a variety of paramyxoviruses. While there are differences between members of the family in terms of specific host cell protein interactions or points of viral protein-protein interactions, the general concepts of coordinated assembly are consistent. However, the mechanisms by which different viral components reach the plasma membrane and coordinate their localization at assembly sites still require further investigation. It is evident from the studies summarized in this review that fundamental differences exist among the different family members in the mechanisms that underlie coordinated targeting to the assembly site, and three different models for paramyxovirus assembly can at present be deduced , the fusion protein can bind the matrix protein in the ER, and the two are transported as a complex to the plasma membrane where they create a nucleation site for assembly. Incorporation of the attachment protein likely occurs though interactions with M or with F. The RNP can traffic by itself to the assembly site and is packaged within particles upon binding to M or one of the glycoproteins. Studies of RSV suggest a third model of paramyxovirus assembly, where the formation of an assembly complex containing F, M and the RNP core occurs in inclusion bodies in the cytoplasm, with a role of the cytoplasmic tail of F in targeting M-RNP to assembly sites. While these models, and potentially others to be generated after future research, provide insight into glycoprotein incorporation and paramyxovirus assembly, several critical questions remain to be answered to obtain a complete understanding of paramyxovirus particle formation.One of the key unanswered questions is how are assembly sites initiated? Is the clustering of surface glycoproteins in membrane raft domains sufficient to create an outward bud in the plasma membrane to which other viral components are recruited, or is the interaction of M with the cytoplasmic tail of glycoproteins and its self-oligomerization the main driver for the formation of assembly nucleation sites? The requirements for the formation of budding precursor sites may vary among different paramyxoviruses. For RSV, the fusion protein seems to be the significant contributor for the formation of short viral filaments without the need for M or the host cytoskeleton. On the other hand, the actin cytoskeleton and the interaction between M and the cytoplasmic tail of F appear to drive SeV particle formation at the plasma membrane. Another significant area of study is to determine the cellular pathways that are utilized by the matrix proteins and the RNP core that allows their delivery to assembly sites and subsequent packaging of the RNA genome into virions. Important questions also remain regarding how membrane budding and the final scission process are established for paramyxoviruses, particularly for those viruses that do not utilize the well-characterized ESCRT proteins. What are the cellular factors that play a role in budding of paramyxoviruses, and how are they recruited by viral proteins? Thus, further studies are still required to clarify multiple aspects of paramyxovirus particle production and to uncover the differences that exist in the molecular mechanisms utilized by different paramyxoviruses to form new infectious particles. Future discoveries in this field may contribute to the development of antivirals against paramyxovirus infections.