Clinical findings show that male infants with hypoxic-ischemic injury (HI) fare more poorly than matched females on cognitive outcomes. Rodent models of neonatal hypoxia-ischemia support this difference, with data showing that perinatal brain injury leads to long-term behavioral deficits primarily in male rodents and in female rodents treated with early androgens. Results support the idea that sex-specific gonadal hormones may modulate developmental response to injury and dovetail with overwhelming evidence of developmental androgen effects on typical brain morphology and behavior. However, mechanisms underlying sex differences in response to early brain injury may be more complicated. Specifically, activation of cell death pathways in response to HI may also differ by sex. In females, the preferential activation of the caspase-dependent apoptotic pathway may actually afford greater protection, potentially due to the actions of X-linked inhibitor of apoptosis (XIAP) within this pathway. This contrasts the pattern of preferential activation of the caspase-independent pathway in males. While an integrated model of sex-specific hormonal and genetic modulation of response to early injury remains to be fully elucidated, these findings suggest that infants might benefit from sex-specific neuroprotection following HI injury. Perinatal hypoxic-ischemic injury  represents a major cause of mortality and long-term neurologic morbidity in premature/very low-birth-weight (VLBW) infants <1500 g) and in term infants suffering birth trauma 500 g and, can oft In term infants, HI injury typically results from complications of birth  Not surprisingly, the long-term consequences of neonatal HI injury in both populations can be severe. Nearly 50% of term-born infants suffering severe HIE die within weeks of birth, while up to 25% of those surviving exhibit permanent neuropsychological dysfunction . SimilarDespite the overwhelming evidence of sex differences in outcome following neonatal HI injury, many researchers appear to remain naïve to the importance of sex in perinatal injury models and continue to utilize only male animals in research studies. However, recent work concerning hormones present during the perinatal period, as well as sex differences in mechanisms of cell death, have begun to illustrate dynamic and differing processes occurring in the neonatal brain following injury and emphasize the need for studies to include both sexes. This work suggests, first, that the substantially elevated level of testosterone present in human male fetuses during gestation through the first year of life –36 may eSex differences in androgen levels represent one principal difference in the male versus female neonatal brain and lead to substantial effects on brain morphology and subsequent behavior , 53–59. Human female fetuses are also exposed to androgens from the fetal adrenal glands, as well as the maternal adrenals, ovaries, and fat—though the amount is insufficient for masculinization. In humans, it is believed that a negative feedback loop between the fetal adrenal cortex and the anterior pituitary corticotrophins minimizes female adrenal androgen secretion, acting as a transient mechanism that safeguards early human female development from virilization . The fetAs noted above, the presence of testosterone during development represents one of the foremost differences between neonatal male and female brains. However, despite a large and dynamic literature concerning modulatory effects of gonadal hormones on pathologic and behavioral response to stroke injury in adults, research data concerning hormonal modulation of injury in neonates remains scant (but see ). A brie In neonatal animals, baseline sex differences have been seen with an early hypoxia model , 71 as wFrom these cumulative studies, it is evident that testosterone acts in some manner to exacerbate the response to early hypoxic-ischemic injury in rats, though the specific mechanism(s) of action remain to be defined. Moreover, since an aromatizable form of testosterone propionate was used in the above study , it is nIt must also be noted that evidence from animal models of adult stroke shows substantial beneficial and protective effects of estrogen modulation –77. In f These hormonal data, taken together, suggest that early androgen exposure in males may be a primary contributor to the modulation of sex differences in response to HI injury . However, another promising line of research aimed at exploring sex differences in response to brain injury has begun to examine possible sex differences in the mechanisms of cell death following injury. These findings have led researchers to believe that hormonal differences are not the only key factor modulating sex differences in response to injury, and that the apoptotic cascade may be differentially activated in the male and female brain following HI.independent-mediated cell death, a reduction of nicotinamide adenine dinucleotide  is caused by activation of poly(ADP-ribose) polymerase-1 , leading to release of apoptosis-inducing factor (AIF) and endonuclease G from the mitochondria to the nucleus of the cell, and ultimately cell death [dependent pathway, the increase in nNos ultimately leads to mitochondrial dysfunction and the translocation of cytochrome-c from the mitochondria to the nucleus, signaling apoptotic protease-activating factor-1 (APAF-1) and the formation of the apoptosome. The apoptosome binds with caspase-9 (the initiator caspase), which in turn cleaves downstream caspases 3, 6, and 7 (effector caspases) causing chromatin condensation, DNA fragmentation, and ultimately cell death [Apoptosis, or programmed cell death, can be triggered by various events including DNA damage, cytotoxic drugs, a lack of survival signals, and developmental death signals—among a variety of other mechanisms . With rell death –51. Throll death , 83–85, Interestingly, research has revealed the sexes to differentially favor one of these two pathways (though not exclusively), with females relying more heavily on the caspase-dependent pathway and males largely utilizing the caspase-independent pathway of cell death following HI insult. These data derive from injury models in both adult stroke (MCAO) and neonatal HI models , 86, 87.increased stroke damage in intact females and estrogen-replaced ovariectomized females, again suggesting a mediating role of estrogen [In adult stroke models, male and female Parp-1-deficient mice both display a reduction in Parp-1 and AIF production, suggesting the activation of the caspase-independent apoptotic pathway by both sexes. However, only male Parp-1-deficient animals exhibited a reduction in stroke-induced brain damage following MCAO . Likewisestrogen . Though estrogen ). These estrogen . Recent work exploring cell death mechanisms in neonatal models also indicates sex differences in the preferred apoptotic pathway following early HI , 42. WitFrom these studies, it is evident that male and female neurons undergoing apoptosis capitalize on pathways that show sex differences unlikely to be exclusively related to hormones (though these apoptotic pathways are not exclusive to sex). However, detailed assessment of the potential interaction between the early (or concurrent) presence of gonadal hormones, and the activation of sex-specific apoptotic cascades, remains to be defined. Nonetheless, apoptosis is a major contributor to neuronal cell death and tissue loss following neonatal HI, and the development of neuroprotectants aimed at targeting the mechanisms most utilized by each sex represents a valuable venue of investigation for therapeutic interventions.An alternative or additional explanation for sex differences seen in neonatal HI outcome involves endogenous inhibitors of apoptosis. During development, the apoptotic cascade is a highly regulated process critical for healthy development and maintenance of tissue. This process of programmed cell death is counterbalanced by antiapoptotic signals that promote the survival of cells. A family of proteins, known as inhibitors of apoptosis (IAPs), serve as endogenous inhibitors of cell death , 90 and in vitro studies have revealed XIAP to severely inhibit nuclear destruction and cytochrome-c-induced caspase activation [Of the known IAPs, X-linked IAP (XIAP) is recognized to be the most potent . XIAP eftivation . XIAP extivation . Moreovetivation . TherefoCurrently very little is known about the regulation of IAPs and XIAP in neonatal HI injury, though surprisingly, results from studies of XIAP knockout  and overClearly more research is needed on the role of IAPs in hypoxic-ischemic neuronal death, but the studies presented here emphasize the need for an improved understanding of innate mechanisms of protection in male and female neonates. Future studies will be needed to assess the potential interaction of hormonal exposure and genetic differences in sex chromosome gene expression within brain cells, since sex differences in response to early injury are almost certainly influenced by a combination of these factors.Neonatal HI is a major cause of infant mortality and long-term neurologic morbidity in both preterm and term-injured populations. It is evident that the consequences of neonatal HI injury are severe, yet the difference in outcome experienced between the sexes is surprising. Male infants not only exhibit increased risk for HI, but also display greater behavioral and cognitive disruption following HI injury as compared to matched female counterparts. Animal studies utilizing induced neonatal HI suggest that this sex discrepancy may be modulated by (1) the presence of sex-specific hormones , (2) sex differences in the preferred mechanisms of apoptosis, and/or (3) the protective effect of IAPs  on the caspase-dependent apoptotic pathway. Indeed, all three of these mechanisms may interact with each other, and sex differences in the effects of neonatal HI outcome likely reflect an interplay of both genetic and hormonal factors. One possible study to dissociate these interactive mechanisms could entail the use of a four-core genotype (FCG) mouse model (described in ), in whi In closing, further studies of the influence of both genetic and hormonal factors relevant to neonatal HI could have important clinical implications. For example, the modulation of hormonal mechanisms leading to increased damage in males, modulation of apoptotic cascades, or modulation of IAPs may all represent target candidates for therapeutic intervention in neonates suffering HI brain injury. Further, studies looking at neonatal HI while manipulating testosterone receptors, estrogen receptors, and/or aromatase blockers could potentially dissociate or clarify the mechanism of action promoting injury. Moreover, it seems plausible that a lack of exposure to placental hormones due to premature birth could also be detrimental to the neurological development of premature infants . Given the tremendous amount of research focusing on sex differences in adult stroke, we suggest that future research should similarly focus on sex differences in the consequences of neonatal HI. In fact, research in this area could yield beneficial sex-specific neuroprotectants, with far reaching implications for improved clinical practice and treatment.


Mass spectrometry-based analyses of the low-molecular-weight fraction of serum proteome allow identifying proteome profiles (signatures) that are potentially useful in detection and classification of cancer. Several published studies have shown that multipeptide signatures selected in numerical tests have potential values for diagnostics of different types of cancer. However due to apparent problems with standardization of methodological details, both experimental and computational, none of the proposed peptide signatures analyzed directly by MALDI/SELDI-ToF spectrometry has been approved for routine diagnostics. Noteworthy, several components of proposed cancer signatures, especially those characteristic for advanced cancer, were identified as fragments of blood proteins involved in the acute phase and inflammatory response. This indicated that among cancer biomarker candidates to be possibly identified by serum proteome profiling were rather those reflecting overall influence of a disease (and the therapy) upon the human organism, than products of cancer-specific genes. Current paper focuses on changes in serum proteome that are related to response of patient's organism to progressing malignancy and toxicity of anticancer treatment. In addition, several methodological issues that affect robustness and interlaboratory reproducibility of MS-based serum proteome profiling are discussed. Biological factors , whose status and/or quantity reflect the risk of a disease, severity of an illness, or the effects of therapy are called markers or biomarkers. In oncology, appropriately selected sets of markers can provide information about carcinogenic triggers to which the organism was exposed, detect early changes  that appear prior to the occurrence of overt forms of cancer, as well as monitor efficacy and toxicity of the treatment. Such factors  are present in tumor tissues or body fluids, and encompass a wide variety of molecules, including transcription factors, cell-surface receptors, and secreted proteins. Several protein tumor markers have been used for decades in the traditional oncology for detection of cancer, for example, prostate cancer antigen (PSA) or cancer antigen 125 kD (CA125). In fact, effective tumor markers are in great demand since they have the potential to reduce cancer mortality rates by facilitating diagnosis of cancers at early stages and helping to plan tailored treatment. Cancer biomarkers can be divided into prognostic and predictive. Prognostic factors, which include tumor size, histological type of cancer, grade, and nodal status, allow the determination of prognosis regardless of a treatment. Currently list of prognostic molecular factors has been extended considerably including hormone receptors, markers of angiogenesis, and proliferation . Howeverinter alia, by Liotta and Petricoin + molecular ions); the analyte molecules are not fragmented in normal conditions. MALDI spectrometers are used for the analysis of proteins and peptides with molecular weights ranging from 500 Da to as much as several hundred thousand Da [m/z value and can be converted into mass of the analyte particles [m/z values) of fragmentary ions are determined in the so called tandem mass spectrometry (MS/MS). On the basis of fragmentary ion sizes, the appropriate computational analysis allows establishing of amino acids sequence in the peptide chain and identification of corresponding protein [Constantly improved mass spectrometric methods are widely used in proteomic studies. By using different techniques of MS it is possible to read the molecular weights of specific peptides and proteins in complex mixtures containing only pM of particular molecules. Depending on a type of analyte various techniques of ionization and separation of ions in the analyzers are used in the field. Two ionization methods: electrospray ionization (ESI) and matrix-assisted-laser(induced)desorption ionization (MALDI) are the most commonly used for protein analyses in current clinics-oriented studies. In MALDI, the analyte molecules  are cocrystallized with matrix molecules that absorb UV light; cinnamic acid derivatives  can be used for further sample identification and classification . The. Them/z on (rev. . These sAuthors of those works (as well as other works not quoted in the table) proposed multicomponent classifiers that could be used for detection and/or classification of cancer. This approach seems to be accepted as the first stage of the biomarker's discovery pipeline—identification of marker candidates. However due to apparent problems with standardization of methodological details, both experimental and computational, none of proposed serum peptide signatures analyzed directly by mass spectrometry has been approved for routine diagnostics yet. Noteworthy, several components of proposed cancer signatures, especially those for advanced cancer, were identified as fragments of blood proteins involved in the acute phase and inflammatory response , 48, 54.One of the major challenges of MS-based serum/plasma proteomics is the high dynamic range in the abundance of particular blood proteins, exceeding 10 log scale . The repThe presence of albumin  is the major factor changing the yield of ionization and affecting the ability to register low abundance serum components . Such inm/z axis among all spectra. Normalization is used for scaling the spectra—the most frequently used algorithms base on total ion current (TIC) value or constant noise. All procedures reduce dimensionality of data and improve quality of further analyses. There are several algorithms accepted for procedures and most of proteomics studies used comparable methods for spectra preprocessing. However, there is no single protocol generally accepted as the standard. In fact some of the key parameters are tuned and adjusted for particular analyses, and type and order of preprocessing steps may significantly differ among different analyses [Proteomics studies generate high-dimensional data sets where traditional statistical and computational methods are not sufficient for analyses, hence novel “nonstandard” mathematical approaches are frequently required. A typical proteomic analysis of spectral data consists of three steps: (i) preprocessing of data, (ii) identification of spectral components, and (iii) statistical analyses and classification based on identified components. The first step includes: “smoothing” of the spectrum, removing the baseline, normalization, and averaging of technical replicates. The simplest method of spectra “smoothing” is averaging among several neighboring points. The baseline correction flattens specific noise named baseline. Interpolation is performed to standardize points on the analyses –78. Theranalyses . Alternaanalyses . The objMany different experimental and computational procedures are used in the field for sample preprocessing and data analysis. All these procedures are modified and adjusted for specific objectives and sample or data sets to deliver optimal results. However, the major drawback of the situation is the lack of “golden standard” in sample and data processing, which makes impossible direct comparison of peptide signatures delivered in different studies of blood proteome of cancer patients. Hence, comparative analysis and interpretation of such data requires caution and should be preferably performed based on sequence-identified components of peptide profiles.α1-proteinase inhibitor, α1-acid glycoprotein, serum amyloid A (SAA), haptoglobin, ceruloplasmin, α2-macroglobulin, and C-reactive protein (CRP). Interestingly, many acute-phase proteins were identified as potential markers of the process of carcinogenesis [Precise and adequate assessment of degree of tumor progression is a key factor for proper selection of the optimal treatment. However, the traditional anatomy-based classification of cancer stages, the so-called TNM classification –83, turnogenesis –96. An iogenesis , 98. Othogenesis . Thus SAogenesis . Several components of cancer signatures proposed in MS-based serum proteome studies, especially those characteristic for advanced cancer, were identified as fragments of blood proteins involved in the acute phase and inflammatory response . Tm/z  =  2790 Da) that specifically increased in its abundance, yet the presence of this peptide did not correlate with the outcome of therapy [m/z  =  7790 and 9285 Da), which changed their abundances in response to the treatment. These peptides were identified as kininogen and apolipoprotein A2, respectively [m/z  =  2276, 4892, and 6194 Da) increased their abundance in serum collected posttreatment. Noteworthy, both pretreatment and posttreatment samples retained specific features of mass profiles that differentiated them from serum samples collected from healthy donors [m/z values 2184 and 5403 Da) whose changes correlated significantly with the type of treatment employed  [A few studies have focused on application of MS-based serum proteome profiling to identify possible therapy-related features that could be used for monitoring of progression, efficacy, and toxicity of the treatment. Some of these works were related to analysis of serum/plasma of patients undergoing chemotherapy because of breast cancer. SELDI-ToF analysis of the plasma proteome of breast cancer patients who underwent paclitaxel-based neoadjuvant treatment revealed one peptide ( therapy . Similarectively . Such tay donors . We havesurgery) . There isurgery) . There are also a few works focusing on changes in serum proteome composition induced specifically by exposure of cancer patients to ionizing radiation. SELDI-based proteome profiling was applied in comparative analysis of serum samples collected before and during or directly after radiotherapy of cancer patients. Analyzed group consisted of 68 patients treated with radiation due to 18 different malignancies, who received total doses in a range of 1.5 to 86.4 Gy (median 48.6 Gy), with the time interval between consecutive blood samples in a range of 1 to 55 days . In spite of a very heterogenous material the authors identified differences between preexposure and postexposure samples. Registered serum proteome profiles were used to build multicomponent classifiers that differentiated unexposed and exposed samples, as well as samples collected from patients exposed to higher and lower doses of radiation . We perfAll these data indicate collectively high potential applicability of MS-based serum proteome profiling in monitoring the response of cancer patients to chemo- and radiotherapy. Changes in abundance of particular low-molecular-weight components of blood proteome observed in the course of anticancer therapy could reflect several different processes, including direct toxic effects of the treatment, healing of toxic reactions, and effects related to removal of a tumor. Not surprisingly among serum proteins that changed their abundance during the treatment several were identified as acute-phase proteins. This confirmed that processes reflecting the general reaction of patient's organism to the malignancy and its treatment were major targets of MS-based profiling of the low-molecular-weight fraction of serum proteome.In spite of tens of published studies the real potential of MALDI/SELDI-based serum proteome profiling in clinical diagnostics is not clearly defined so far. Even though these studies have showed that multipeptide signatures selected in numerical tests have some potential value for classification and differentiation of cancer, none of proposed serum peptide signatures has been approved for routine diagnostics. Lack of standardization of methodological details, both experimental and computational, is an important problem in MS-based blood proteome profiling. It appears that guidelines for standardization of these multiparametric analyses should be generally accepted in the field and that identified marker candidates need to be confirmed in multicenter bias-free prospective validation studies.


Metabolic syndrome, a network of medical disorders that greatly increase the risk for developing metabolic and cardiovascular diseases, has reached epidemic levels in many areas of today's world. Despite this alarming medicare situation, scientific understandings on the root mechanisms of metabolic syndrome are still limited, and such insufficient knowledge contributes to the relative lack of effective treatments or preventions for related diseases. Recent interdisciplinary studies from neuroendocrinology and neuroimmunology fields have revealed that overnutrition can trigger intracellular stresses to cause inflammatory changes mediated by molecules that control innate immunity. This type of nutrition-related molecular inflammation in the central nervous system, particularly in the hypothalamus, can form a common pathogenic basis for the induction of various metabolic syndrome components such as obesity, insulin resistance, and hypertension. Proinflammatory NF-κB pathway has been revealed as a key molecular system for pathologic induction of brain inflammation, which translates overnutrition and resulting intracellular stresses into central neuroendocrine and neural dysregulations of energy, glucose, and cardiovascular homeostasis, collectively leading to metabolic syndrome. This article reviews recent research advances in the neural mechanisms of metabolic syndrome and related diseases from the perspective of pathogenic induction by intracellular stresses and NF-κB pathway of the brain. Looking back upon the time when the human species had to spent tremendous efforts to gather enough food for survival, there is no doubt that modern industrialization has succeeded at making many choices of calorie-abundant food easily available with little physical efforts. However, this over-correction of food crisis has led to an opposite pattern of medical problems by introducing a worldwide outbreak of overnutrition-related diseases such as obesity, type 2 diabetes (T2D), and cardiovascular diseases (CVDs) -7. WhileReactive oxygen species (ROS) refer to a class of radical or non-radical oxygen-containing molecules that have high oxidative reactivity with lipids, proteins, and nucleic acids. There are many potential sources of ROS in cells . MitochoThe brain utilizes a large amount of oxygen and ATP to support its normal functions, resulting in a high susceptibility to oxidative stress ;70;71;82Endoplasmic reticulum (ER) is the cellular organelle responsible for protein synthesis, maturation, and trafficking to secretory pathways. Since cellular metabolic demands undergo fluctuations depending on systemic physiological conditions, ER uses its unfolded protein response (UPR) machinery to fine tune its protein synthesis, folding, and secretion accordingly . Three EHowever, when cellular metabolic challenges that trigger ER stress is severe or persistent as in pathological settings, UPR may not be sufficient to neutralize ER stress, which leads to ER stress-related pathological changes at molecular, cellular, and systemic levels. Such unresolved ER stress can induce cell apoptosis ;94, whicAutophagy is an evolutionarily conserved lysosomal degradation pathway that plays essential roles in maintaining cellular homeostasis and promoting cell survival, growth, and differentiation against adverse conditions ;107. To Tissue-specific impairment of autophagy, though not lethal, leads to the development of diverse diseases such as infection -120, canOne lingering question is: what are the inducers of hypothalamic autophagy defect in metabolic syndrome? A plausible answer is brain autophagy defect may occur secondarily to oxidative stress and ER stress in the development of central metabolic dysregulations, presumably when intracellular accumulation of damaged mitochondria, ER, and misfolded proteins exceeds the degradative capacity of autophagy machinery. This hypothesis can be inferred from the observation that metabolic disorders related to central autophagy defect are late onset , and indIn addition to the primary roles of receptor-independent intracellular stress pathways in overnutrition-induced central metabolic dysregulations, immune receptor-mediated pathways can also function in the hypothalamic inflammatory mechanisms of metabolic syndrome. In this regard, toll-like receptor (TLR) pathway has received substantial research attentions. TLRs are an important class of membrane-bound pattern recognition receptors in classical innate immune defense, primarily functioning to promote synthesis and secretion of immune response molecules upon binding by “non-self” molecules  ;142. MosIn addition to TLRs, cytokine receptors can also participate in the central induction of metabolic syndrome and related diseases, given that a prominent pathologic feature of these diseases, especially in the late stage, is the prevalent presence of cytokines in the circulation and various tissues of the body -30. ThesEvidences supporting the involvement of cytokine receptors in central metabolic dysregulations mainly come from studies using central administration of cytokines or genetic deletions of cytokines or receptors. However, both approaches have pros and cons. The pharmacological studies, while having the advantage of directly targeting the brain, have major issues with dose usage. Most of the time, the doses applied did not reflect the pathophysiological levels in metabolic syndrome and related diseases, which unintentionally skewed the interpretations of their biological effects. The genetic knockout approach, while providing clean loss-of-function models to infer gene functions, has major limitations with site-specificity, i.e., most cytokine or cytokine receptor knockout models available now target the whole body instead of a specific tissue or cell type. Nonetheless, significant evidences have been recently documented demonstrating the role of cytokine receptor pathways in the development of metabolic syndrome components. For example, central administration of TNF-α at low doses faithfully replicated the effects of central metabolic inflammation in enhancing eating, decreasing energy expenditure ;159, andMammalian IκB kinase β (IKKβ) and nuclear factor-κB (NF-κB) comprise a master proinflammatory pathway that has pivotal roles in classical innate immune response . In quieNF-κB is a redox-sensitive transcription factor whose activity is affected by cellular oxidative state ;167. A mUPR signaling of ER can actively respond to changes of cellular nutrient state such as increased protein synthesis, glucose or energy deprivation, hypoxia, or even elevated intracellular lipid storage . The undFurther interests were diverted to how ER stress transduces overnutrition signals into metabolic derangements, and metabolic inflammation was brought upon as a mediator, as all three branches of UPR pathways are linked to proinflammatory IKKβ/NF-κB or JNK signaling -199. HowAs a stress-response mechanism against adverse growth conditions such as nutrient depletion, and as a machinery to maintain normal intracellular environment, autophagy is key to cell/organism survival and proper functioning ;107. UnsThe connection between autophagy defect and proinflammatory activation of NF-κB pathway can also be inferred in metabolic syndrome, since both autophagy defect -133;200 Both TLR pathway and cytokine receptor pathways are closely related to IKKβ/NF-κB signaling in the central pathogenesis of metabolic syndrome. Overnutrition, especially in the form of HFD feeding, was shown to activate TLR4 signaling and downstream IKKβ/NF-κB pathway ;37, leadThe close link between IKKβ/NF-κB and cytokine receptor signaling in metabolic inflammation is beyond doubt, given that many cytokines and their receptors are both upstream activating components and downstream transcriptional targets of NF-κB activation . For exaThe CNS, particularly the hypothalamus, is the central regulator of energy and body weight balance -62. ThisThe hypothalamus plays a central role in controlling glucose homeostasis through coordinating the regulatory networks formed between multiple organs including brain, liver, pancreas, adipocytes, and skeletal muscles ;235-239.Many clinical and epidemiologic studies have demonstrated the therapeutic/preventive effects of anti-cellular stress agents against CVDs. For example, dietary supplementation of antioxidant vitamin E can reduce the development of atherosclerosis through increased production of oxidation-resistant low-density lipoprotein . AntioxiResearch in the past decade has established that metabolic syndrome can result from innate immune activation in response to overnutrition. While this type of inflammation exists broadly across different tissues, the CNS is in a primary and wide-impact position for the induction of metabolic syndrome by nutritional inflammation. An inflammatory state in brain regulatory centers such as the hypothalamus disrupts its metabolic sensing function, which in turn affects downstream neural and neuroendocrine regulation of a wide range of physiological processes such as energy balance, glucose metabolism, and cardiovascular homeostasis. Dysregulations of these processes often happen concurrently and manifest as a cluster of highly associated metabolic disorders such as obesity, insulin resistance, and hypertension. Research in the past few years has significantly differentiated the hypothalamic inflammatory pathways underlying these metabolic disorders, and overnutrition-induced intracellular stresses have been recognized as key activators of metabolic inflammation in the hypothalamus. This new knowledge not only provides a conceptual framework for further dissecting the pathogenesis of metabolic syndrome related diseases, but also indicates potential interventional strategies of counteracting neuroinflammation against metabolic diseases. Regardless of this exciting status quo, many important questions still remain to be addressed experimentally. It is fair to say that current understandings on the central inflammatory mechanisms of metabolic syndrome and related diseases are still in a primitive stage. However, in light of its great significance from both biomedical research and therapeutic application perspectives, we expect major research endeavors being drawn to this field and more advances being made in the near future. We also anticipate that eventually these findings will be translated into novel and effective treatments/preventions against miscellaneous overnutrition-induced metabolic diseases.


Lyssavirus species and this novel lyssavirus provided strong empirical evidence that this is a new lyssavirus species, designated Ikoma lyssavirus.Evidence in support of a novel lyssavirus was obtained from brain samples of an African civet in Tanzania. Results of phylogenetic analysis of nucleoprotein gene sequences from representative  Lyssavirus species have been classified: Rabies virus (RABV), Lagos bat virus (LBV), Mokola virus (MOKV), Duvenhage virus (DUVV), European bat lyssavirus types -1 and -2, Australian bat lyssavirus, Aravan virus, Khujand virus, Irkut virus, and West Caucasian bat virus (WCBV) . LBV, MOKV, DUVV, and SHIBV are detected exclusively in Africa, whereas RABV is detected worldwide. The predominant RABV variants circulating in Africa are the mongoose and canine biotypes. In South Africa, canine RABV is considered to have been introduced in the eastern Cape Province after importation of an infected dog from England in 1892 and subsequently spread, infecting domestic and wild carnivores  displaying clinical signs consistent with rabies was killed by rangers in Ikoma Ward within Serengeti National Park , including 12 canine RABV sequences from domestic and wild animals in the Serengeti ecosystem (64.1%–65.1% identity). The posterior probabilities indicated that the IKOV and WCBV grouping was strongly supported, despite low sequence identity. Further phylogenetic analysis of representatives from other rhabdoviruses demonstrated that IKOV is a member of the Lyssavirus genus (41.6%–50.9% identity to representative rhabdovirus sequences that are available for this region of the genome) (data not shown).RNA was extracted by using TRIzol Reagent , and a pan-lyssavirus reverse transcription PCR yielded a specific 606-bp amplicon . A caninWe describe evidence, based on genomic sequences obtained from the brain sample of an African civet with clinical signs consistent with rabies, for the existence of a novel lyssavirus designated IKOV (Ikoma lyssavirus). The sample was frozen and thawed several times before being sent to AHVLA, which had a detrimental effect on the sample quality and resulted in viral RNA degradation and loss in viral viability. The results of confirmatory FATs performed at AHVLA were inconclusive, and attempts to isolate virus by using the rabies tissue culture inoculation test and the mouse inoculation test were unsuccessful. Despite the lack of isolated virus, the pan-lyssavirus sensitivity and specificity of the fluorescent antibody test  and direct rapid immunohistochemistry test support the assertion that a novel lyssavirus exists in the region. In addition, despite the poor quality of the sample, molecular techniques identified a lyssavirus-specific amplicon that was confirmed to be unique by phylogenetic analysis and to be highly divergent from known circulating RABV strains. A real-time PCR also detected this unique lyssavirus sequence, confirming that both molecular tests are pan-lyssavirus specific and are sufficient for the detection of highly divergent novel lyssaviruses  was restricted to the RABV mongoose lineage . Ad


Patent ductus arteriosus (PDA) is a common clinical condition in preterm infants. Preterm newborns with PDA are at greater risk for several morbidities, including higher rates of bronchopulmonary dysplasia (BPD), decreased perfusion of vital organs, and mortality. Therefore, cyclooxygenase (COX) inhibitors and surgical interventions for ligation of PDA are widely used. However, these interventions were reported to be associated with side effects. In the absence of clear restricted rules for application of these interventions, different strategies are adopted by neonatologists. Three different approaches have been investigated including prophylactic treatment shortly after birth irrespective of the state of PDA, presymptomatic treatment using echocardiography at variable postnatal ages to select infants for treatment prior to the duct becoming clinically significant, and symptomatic treatment once PDA becomes clinically apparent or hemodynamically significant. Future appropriately designed randomized controlled trials (RCTs) to refine selection of patients for medical and surgical treatments should be conducted. Waiting for new evidence, it seems wise to employ available clinical and echocardiographic parameters of a hemodynamically significant (HS) PDA to select patients who are candidates for medical treatment. Surgical ligation of PDA could be used as a back-up tool for those patients who failed medical treatment and continued to have hemodynamic compromise. The incidence of patent ductus arteriosus (PDA) in preterm infants varies between 40% and 60% on the third day of life, depending on the estimated gestational age –4. ThereIn recent years there has been a growing debate about whether or not to treat a persistent PDA in neonates. The preterm PDA has shifted from being viewed as a pathologic condition causing morbidities and mortality in the preterm infant to being proposed as an innocent physiological bystander –8.The association between PDA and common neonatal morbidities and mortality is the main reason why neonatologists tried to close the PDAs for decades. A ductal left-to-right shunt will cause increased pulmonary blood flow. In preterm infants with respiratory distress syndrome, who exhibit low plasma oncotic pressure and increased capillary permeability, a PDA can result in an interstitial and alveolar pulmonary edema and decreased lung compliance. This, in turn, will lead to higher ventilator settings prolonged ventilation with potentially high oxygen load  and incrDoppler ultrasonographic studies have demonstrated impaired cerebral blood flow (CBF) in preterm infants with a PDA suggesting a role in the pathogenesis of IVH . MoreoveIn preterm infants, the ductal tissue matures and becomes less regulated by prostaglandins with advancing postnatal age , 25. AccA recent study enrolling 129 preterm infants (501–1500 g birth weight) in 4 different NICUs in the Netherlands has demonstrated that the BPD rate and the combined outcome of death after day 7 and/or BPD was higher in the period where permissive tolerance of PDA was applied  compared to the period where traditional management with indomethacin and/or surgical ligation used early to close PDAs. However this was not associated with significant changes in other neonatal morbidities .Functional closure of the ductus arteriosus occurs in almost 50% of full-term infants by 24 hours, in 90% by 48 hours, and in all by 72 hours after birth. In healthy preterm infants of ≥30 weeks' gestation, duct closure occurs by the fourth day after birth, while preterm infants of <30 weeks' gestation, with severe respiratory distress, have a 65% incidence of PDA beyond the fourth day of life –31.A prospective study demonstrated a spontaneous closure of the PDA in the first 10 days of life in at least 35% of extremely low-birth-weight (ELBW) infants and up to 70% in neonates of >28 weeks' gestation. There was a direct relationship between gestational age and spontaneous closure and for each additional week above 23 weeks, the odds of spontaneous closure increased by a ratio of 1.5 .Clinical trials have failed to demonstrate a meaningful long-term advantage of therapeutic intervention for the ductus arteriosus. Meta-analyses of RCTs have not demonstrated any beneficial effect for early PDA closure on neonatal morbidities including BPD, NEC, neurosensory impairment, death, or the combined outcomes of death or BPD and death or neurosensory impairment , 33–38. COX inhibitors have many systemic side effects, as they constrict not only the ductus arteriosus but also the arteries that supply blood to the heart, brain, kidneys, and gut. Indomethacin produces significant reductions in renal , mesenteNot only are surgical interventions to close the ductus arteriosus associated with additional short-term complications –61, but Most neonatologists agree that not all PDAs require treatment, but definitely there is a subgroup of PDA that should be closed. There is individual variability among preterm infants, affecting who will respond to which drug and which dose. It is of paramount importance to predict the responders versus the nonresponders, those who will close after repeated courses and those who will close only if given a higher dose. Several studies have tried to define the target population that should be treated using different clinical, echocardiographic, pharmacokinetic, and biochemical parameters.Lower birth weights and gestational ages have been associated with pharmacologic treatment failures and eventual need for surgical ligation . MoreoveEchocardiography is widely used to define HS-PDA requiring treatment and to exclude duct-dependent congenital heart disease. Although there are no stringent echocardiographic criteria to define the need for therapeutic intervention, several echocardiographic parameters have been correlated with PDA hemodynamic significance and with therapeutic responsiveness. These parameters include: PDA diameter >1.4 mm, the internal diameter of the ductus/body surface area ratio, a low-velocity pulsatile flow pattern, left atrium to aorta ratio >1.4, and diastolic reverse flow in the aorta, mesenteric, cerebral, and renal arteries –71. LongFew studies have evaluated individualizing COX inhibitors dosing based on targeting “therapeutic” plasma concentrations. Al Za'abi et al.  failed tBiomarkers such as B-type natriuretic peptide (BNP), aminoterminal B-type natriuretic peptide (NT-proBNP), and cardiac troponin T (cTnT) may be used to identify significant PDA and determine indication, timing, and treatment options . They arPlasma BNP correlated with magnitudes of the ductal shunt –81. Hsu Both NT-pBNP  and cTnTIn an interventional study in preterm infants <33 weeks' gestation, indomethacin therapy was given if plasma NT-proBNP concentration was ≥10,180 pg/mL on the 2nd day of life, the cutoff for predicting HS-PDA. On day 2, 19 (38%) infants had plasma NT-proBNP above the cutoff and received indomethacin therapy; none of them developed later HS-PDA, while 1 of 31 infants with NT-proBNP below the cut-off level developed clinical HS-PDA. Overall, no enrolled infants had either reopening of ductus or PDA ligation .Recently, urinary NT-proBNP was found to be a simple and noninvasive alternative. Urinary NT-proBNP/creatinine ratios on day 14 were higher in 14 ventilated infants who did not respond to pharmacological treatment and subsequently required surgical PDA closure than in ventilated infants with successful pharmacological PDA closure .Timing of PDA treatment has gained the interest of scientists over the past years. Early intervention for asymptomatic duct carries the benefit of higher success rate but increases the risk of over exposure, while late intervention for symptomatic duct minimizes overexposure but increases the risk of treatment failure and surgical ligation. Three different strategies have been investigated including prophylactic treatment shortly after birth without reference to the state of PDA, presymptomatic treatment using echocardiography at variable postnatal ages to select infants for treatment prior to the duct becoming clinically significant, and symptomatic treatment once PDA becomes clinically apparent or hemodynamically significant.The decline of the pulmonary vascular resistance and pulmonary artery pressure occurs earlier in preterm infants, usually after the first 24 hours of life ; thus moWhen first introduced, predefined gestation or weight-based criteria for prophylactic therapy were needed to minimize unnecessary exposure. Most of the conducted researches on prophylactic therapy included preterm infants <32 weeks' gestation and VLBW infants <1500 grams –98.Mahony et al.  studied Prophylactic ibuprofen therapy at a dose of 10 mg/kg in the first 24 hours of life followed by 5 mg/kg after 24 hours and 48 hours did not show any superiority over prophylactic indomethacin therapy. In a meta-analysis of seven studies comparing prophylactic ibuprofen with placebo, prophylactic ibuprofen was found to decrease the incidence of PDA on day three, decreased the need for rescue treatment with indomethacin, and decreased the need for surgical ligation . HoweverBased on the above evidence of nonbeneficial short-term effect and the absence of long-term benefits, the use of prophylactic indomethacin or ibuprofen practice has been abandoned by most neonatologists.The concept of presymptomatic treatment of PDA was to restrict the use of therapy to a group of infants with an asymptomatic duct, rather than treating all preterm infants prophylactically, getting a greater chance of benefit together with limiting the possibility of significant side effects.In a meta-analysis of 3 trials, it was reported that presymptomatic treatment of PDA reduced the incidence of symptomatic PDA and duration of supplemental oxygen with no effect on the rate of mortality, BPD, IVH, ROP, or length of ventilation . The thrPresymptomatic treatment, within 72 hours of life, of PDA with intravenous (IV) ibuprofen was found to be effective in the early closure of PDA in preterm neonates with a trend toward decreased PVL. However, other outcomes including death, IVH, NEC, daily fluid intake/output, liver function, BPD, and ROP did not differ .According to the above evidence, presymptomatic indomethacin or ibuprofen therapy for PDA in preterm infants is not recommended.Expectant approach of treating PDA at a later time, only when signs indicate hemodynamic significance, allows for possible spontaneous closure. It carries the advantage of minimizing the risk of exposure of preterm infants to the hazards of therapy but the disadvantage of late intervention such as lower success rate and increased exposure of preterm infants to the hazards of HS-PDA. One of the challenges of expectant symptomatic approach is to clearly define a HS-PDA as discussed earlier. Gersony et al.  randomizOnly one clinical trial has compared late indomethacin therapy with continued medical management without indomethacin and found a significant decrease in the rate of mechanical ventilation with a trend toward decreasing NEC and ROP in the indomethacin-treated group. However, the sample size of this trial was too small to make any firm conclusions .As the available evidence does not support prophylactic or presymptomatic approach for PDA, expectant symptomatic intervention for HS-PDA seems to be the most reasonable approach of care. Further studies are needed to evaluate the validity of expectant symptomatic therapy compared to conservative treatment.Although fluid restriction has been widely recommended in management of PDA –116, itsThere is no enough evidence to support the routine use of diuretics for prevention or treatment of congestive heart failure in HS-PDA, and a systematic review of the coadministration of furosemide in indomethacin-treated neonates showed a trend toward failure of ductal closure in furosemide-treated patients because furosemide increases prostaglandins production and could potentially decrease the ductal response to indomethacin . In a stOxygen therapy has been proposed in the pathogenesis of duct closure in preterm infants. In a retrospective study including 263 ELBW infants, infants treated with lower oxygen saturation target range policy (83–89% versus 89–94%) had more incidence of HS-PDA; however, none of these infants required surgical ligation later on .Indomethacin is the most widely used nonselective COX inhibitor for PDA closure. In a large national collaborative trial involving 421 preterm infants (<1750 grams) with HS-PDA, duct closure was observed in 79% in indomethacin-treated infants versus 35% with placebo . Van OveSeveral dosing regimens of indomethacin have been used for prophylaxis and treatment of PDA . The mosFive RCTs were included in a meta-analysis comparing prolonged (>4 doses) and short course (<4 doses), there was no significant difference between PDA closure, need for ligation, reopening or retreatment, mortality, BPD, or IVH. There was an increased incidence of NEC [number needed to harm (NNH) = 13] and renal impairment (NNH = 6)  in the pSperandio et al.  reportedIntravenous indomethacin is usually given as bolus over 30 min, only two small trials compared continuous with intermittent bolus administration and reported no statistically significant differences in PDA closure at day 2 or day 5, rates of reopening of PDA, neonatal mortality, IVH, and NEC .Adverse effects might occur frequently during indomethacin treatment including hyponatremia, oliguria, active bleeding, and impaired renal function, which are transient and seem to have no long-term sequelae. NEC (stage II and III), IVH, and focal gastrointestinal perforation are rarely found during therapy. These side effects of indomethacin are due to the nonselective vasoconstrictive effect of the drug and the reduction of blood flow through various organs , 132.Indomethacin is contraindicated in preterm infants with the following: proven or suspected life-threatening infection that is untreated, active bleeding especially gastrointestinal or intracranial, coagulation defects and/or thrombocytopenia, significant impairment of renal function, suspected or proved NEC, and PDA-dependent congenital heart diseases .Ibuprofen, another COX inhibitor, is effective in closing PDA without reducing cerebral, intestinal, or renal blood flow , 135. InThe initial dosing guidelines consist of 10 mg/kg loading dose followed by 5 mg/kg/d every 24 hours twice, total of 3 doses in 3 days , 142. A n = 166) comparing oral ibuprofen with IV ibuprofen showed higher PDA closure rate with oral ibuprofen in comparison with IV ibuprofen and did not show a significant difference in adverse effects [Oral ibuprofen is an alternative for the treatment of PDA. Oral ibuprofen was more effective than IV ibuprofen (84.6% versus 62%) for ductal closure in VLBW infants. Oral, but not IV, ibuprofen was associated with rise in cystatin-C levels, a marker of impaired renal function, in preterm infants indicating that infants with borderline renal function may need careful monitoring . Erdeve  effects .Ibuprofen therapy, particularly the oral form, was found to be associated with adverse events mainly on gastrointestinal tract . SpontanAccordingly, ibuprofen is contraindicated in treatment of PDA in preterm infants with renal failure, hyperbilirubinaemia, gastrointestinal perforation, severe thrombocytopenia , life-thStudies comparing ibuprofen to indomethacin therapy showed similar efficacy of both drugs for PDA closure, less nephrotoxic effects of ibuprofen, less adverse peripheral vasoconstrictive effects for ibuprofen, with no difference in mortality, IVH, and BPD . Two recRecently, paracetamol has been shown to be an alternative treatment for closure of PDA because of its safety profile and low cost. The effect of paracetamol is through prostaglandin synthetase inhibition; this action is at the peroxidase segment of the enzyme. Peroxidase is activated at tenfold lower peroxide concentrations than COX. Therefore, paracetamol-mediated inhibition is facilitated at reduced local peroxide concentrations. This would permit peroxidase inhibition to be optimally effective under conditions in which COX inhibition is less active , 161.Hammerman et al.  reportedIn 1963 surgical PDA ligation was first performed in a preterm infant . Since tAlthough surgical closure of PDA in preterm infants is considered a well-tolerated procedure, many short-term complications were identified including unilateral vocal cord paralysis –55, diapSeveral retrospective studies demonstrated lack of pulmonary benefit from surgical ligation of PDA and even increased risk for BPD , 177. A Several observational retrospective studies analyzed the neurodevelopmental outcome of preterm infants with surgically-ligated PDA, in comparison with those who were not subjected to surgical ligation. A secondary analysis of data from TIPP trial revealed increase risk of neurosensory impairment and cognitive delay at 18 months in infants who were subjected to surgical ligation . FurtherP = 0.046) [There are two approaches for surgical ligation of PDA; the early surgical ligation approach involves surgical ligation as soon as possible for all PDAs that failed to close after indomethacin therapy irrespective of ventilatory requirements or the degree of left-to-right shunt , whereas= 0.046) . Given tThe decision of treatment of PDA should be individualized, according to clinical, echocardiographic, and biochemical parameters that validate hemodynamic significance of PDA. As the available evidence does not support prophylactic or presymptomatic approach, expectant symptomatic treatment for HS-PDA seems to be the most reasonable approach. A suggested timeline algorithm for timeline management of PDA in preterm infant according to the best available evidence is shown in Fluid restriction has no beneficial effects on pulmonary or systemic hemodynamics in infants with PDA. Medical management involves the use of COX inhibitors: indomethacin and ibuprofen lysine. They are equally effective in closing the PDA. Infants whose ductus arteriosus is surgically ligated may be at a greater risk for poor neurodevelopmental outcome, BPD, and severe ROP. Until new evidence becomes available, it seems wise to reserve surgical closure for those neonates who have failed medical therapy and have echocardiographic evidence of a large duct or ongoing significant oxygen and ventilatory requirements.


The poor or lack of injured adult central nervous system (CNS) axon regeneration results in devastating consequences and poor functional recovery. The interplay between the intrinsic and extrinsic factors contributes to robust inhibition of axon regeneration of injured CNS neurons. The insufficient or lack of trophic support for injured neurons is considered as one of the major obstacles contributing to their failure to survive and regrow their axons after injury. In the CNS, many of the signalling pathways associated with neuronal survival and axon regeneration are regulated by several classes of receptor tyrosine kinases (RTK) that respond to a variety of ligands. This paper highlights and summarises the most relevant recent findings pertinent to different classes of the RTK family of molecules, with a particular focus on elucidating their role in CNS axon regeneration. In the mammalian central nervous system (CNS), the failure of spontaneous regeneration of injured axons leads to devastating consequences and poor functional recovery. Severe injuries to CNS axons not only damage plasticity of synapses but also provoke complex degenerative cascades, leading to glial and neuronal apoptosis. The vast majority of injured CNS neurons progressively fails to regenerate beyond the lesion site to reestablish functional synaptic transmission and only a small number of axons show compensatory sprouting, resulting in poor functional recovery –5. Lack In general, functional axon regeneration is a multifactorial process; a myriad of molecules and a combination of signalling pathways are often involved. Two important prerequisites are essential for successful regeneration. Firstly, the injured neurons must be competent to survive after injury, since the replacement of compromised cells is a critical step in the healing process, and having the intrinsic ability to reexpress growth-promoting genes is necessary to stimulate axon regeneration. Secondly, there should be a permissive environment to support spontaneous axon growth and facilitate reinnervation of their target tissues , 12. In It is widely believed that most injured adult CNS neurons are intrinsically incapable of axon regeneration , 15, 16.The insufficient growth potential of CNS neurons results from the failure of the transition from the normally “transmitting” to the “growth mode” after injury, mainly due to their multiple collateral axons which remain connected to their various target tissues . By conOn the basis of several studies, the lack of axon regeneration of mature CNS neurons is ultimately due to a paucity of growth promoting cues, and especially the availability of growth promoting factors and their heightened susceptibility to a plethora of axon-growth inhibitory ligands. These include central myelin/oligodendrocyte-derived Nogo, myelin associated glycoprotein (MAG), and oligodendrocyte myelin glycoprotein (OMgp), the astrocyte-rich glial scar-derived inhibitory molecules such as chondroitin sulphate proteoglycans (CSPG) and tenascin that promote growth cone collapse and slow Wallerian degeneration with poor remyelination after injury , 29, 30.Indeed, axonal growth determinants in the CNS are complicated and the existing challenges to overcome these inhibitory cues and obtain complete functional recovery are substantial. To date, there remains a controversy regarding how injured neurons respond to these intrinsic and extrinsic cues to provoke the cascade of signalling pathways that disrupt or abort axon regeneration after injury. Several studies indicate that optimization of axon regeneration is a counterbalance between the intrinsic growth promoting ability of severed neurons and the growth inhibitory signals from the nonpermissive CNS environment , 29, 31.RTK are high affinity, cell surface receptors for many polypeptide growth factors, cytokines, and hormones. RTK-mediated signals play pivotal and diverse roles in the regulation of various physiological functions, ranging from cell proliferation, differentiation, cell adhesion, cell migration, survival, and apoptosis , 35. In via the lateral dimerization of their two cytoplasmic catalytic domains as a starting point and the subsequent intermolecular autophosphorylation upon binding to extracellular ligands. The autophosphorylation of particular dimers of RTK in turn activates the signalling cascades to phosphorylate their corresponding cytoplasmic substrates , transforming growth factor-α (TGF-α), and amphiregulin, to regulate a wide range of biological functions. These include cell differentiation, development, proliferation, angiogenesis, and survival of fibroblasts and epithelial cells [EGFR was the first family member of the RTK family to be discovered and comprises of four homologous receptors such as ErbB-1 (EGFR itself), ErbB-2 (HER2), ERbB-3 (HER3), and ErbB-4 (HER4). They all elicit intracellular signalling pathways al cells –46. Receal cells –49.On the basis of several studies, the upregulation and transactivation of EGFR elicits both disinhibited axon regeneration of transected ON axons and survival of RGC in chronic glaucoma models , 51. Alt Koprivica et al.  proposedin vivo and in vitro observations showed that AG1478 and PD168393 promoted neurite outgrowth of RGC by off-target effects on glia, independently of EGFR [in vivo paradigm, pEGFR was absent in axons and their corresponding RGC somata while the majority of pEGFR was expressed in ON glia (astrocytes and oligodendrocytes) [in vitro studies, these EGFR antagonists caused the release of RGC/glia-derived neurotrophins together with elevated cyclic adenosine monophosphate levels (cAMP). This in turn reduced the levels of pEGFR, enhanced the production of mobilizing axogenic proteins, and promoted regulated intramembranous proteolysis of p75NTR, thereby inactivating the Rho/ROCK pathway to disinhibit neurite outgrowth in axotomized RGC [Contrary to this paper, the growth-promoting effects of EGFR antagonists in a similar ON crush model were shown to be mediated by off-target actions of these compounds on resident glial cells in the inhibitory environment. For example, both  of EGFR , 53, 54.Interestingly, EGFR-mediated signalling pathways are implicated in cell growth and proliferation in certain nonneuronal cells such as reactive microglia/macrophages and astrocytes in the CNS . While tAll these studies have postulated contradictory reasons for the disinhibited axon regeneration upon suppression of EGFR activity and the resulting astrocyte activation in the ON injury paradigm. Indeed, the understanding of the molecular mechanisms behind pharmacological inhibition of EGFR activity and its effects on the inhibitory signalling cascade in axon regeneration is incomplete. However, these observations argue strongly that injury-induced inhibition of ON regeneration is not going to be explained solely by the inhibition of EGFR but rather other combinatorial factors and signalling pathways need to be considered in the context of future therapeutic strategies in injured ON axon regeneration.In most cases, injury to the brain and spinal cord causes major motor and sensory deficits resulting from Wallerian degeneration followed by abortive axonal sprouting and eventual neuronal loss. A convergence of evidence has indicated that inhibition of EGFR can be both neuroprotective and axon growth stimulatory by the modification of astrocyte-mediated inhibitory responses after spinal cord injuries , 58. AltInterestingly, intrathecal administration of the EGFR inhibitor PD168393 enhanced robust regeneration of 5-HT-immunoreactive and Th-immunoreactive axons in the spinal cord accompanied by recovery of hind limb and bladder function in addition to improved sensory function after contusion spinal cord injury in rats. . This stα (TGF-α), a known ligand of EGFR, at the lesion site, enhanced axon outgrowth at the rostral lesion border after spinal cord injury. The presence of an astrocyte-mediated growth permissive environment following upregulation of TGFα raises the interesting issue that EGFR-TNFα mediated signalling triggers proliferation and migration of astrocytes but intrinsically modulates its state to a functional growth-supportive phenotype that facilitates robust axonal outgrowth after spinal cord injury [A contradictory finding has pointed out that activation of EGFR by overexpression of transforming growth factor-d injury . Althougd injury . Howeverd injury –66. BaseNTR, which belong to the tumour necrosis factor (TNF) receptor family [in vivo expression and the level and localization of their corresponding receptors. Although their higher order structural features enable them to bind the same receptors, they share several features in their signalling [NTR is relatively higher than that of Trk receptors and p75NTR is capable of interacting with all neurotrophins to trigger or inhibit Trk signalling, regulating cell survival and in some instances death of both neurons and glia through co-operation with other Trk receptors [Neurotrophins, members of the nerve growth factor (NGF) family mediate physiological activities through their specific receptors expressed on target tissues or cells. Depending on the binding affinity of neurotrophins, their corresponding receptors are classified into high affinity RTK and the low affinity pan-neurotrophin glycoprotein receptor p75r family –69. The r family , 70. Trkgnalling . Surprisgnalling , 72. Theeceptors , 74.During development, activation of Trk-NGF signalling regulates nonmitogenic effects including differentiation, proliferation, and survival of certain neuronal populations in the nervous system . There iβ RGCs in cat retina [Emerging evidence indicate that neurotrophins and their corresponding receptors play pivotal roles in overcoming the problems of neuronal survival, axon elongation, expression of key enzymes for neurotransmitter synthesis, and remyelination. However, the presence of a permissive environment together with growth promoting cells and/or the use of axonal bridging matrices in the lesion site is a prerequisite for neurotrophins to mediate successful axonal growth in the CNS , 9, 78. t retina . via p75NTR mediated cosignalling in vivo. TrkB and TrkC are expressed in unlesioned and lesioned corticospinal neurons while relatively low levels of p75NTR expression is evident after injury, suggesting that the potency of each receptor to elicit their function is determined at the level of their expression in relevant cells [The major obstacles for systemic application of neurotrophins are the blood brain barrier (BBB) and the truncated TrkB receptor on astrocytes in devising treatments for CNS neuropathies and injuries. Exogenous application of BDNF and preconditioning lesions  enhance regeneration of ascending sensory axons but also enhanced functional recovery together with expression of p-CREB, pERK, and GAP-43 after spinal cord injury, indicating that BDNF is anterogradely transported by axons . Howevernt cells .α1-tubulin [ While endogenous NT-3 promotes death of axotomized spinal neurons, non-physiological upregulation of NT-3 suppresses the expression of TrkC receptors and promotes survival of these neurons, by mimicking BDNF function through TrkB receptors and/or upregulating the expression of BDNF –86. The -tubulin . In a la-tubulin  reported-tubulin . NTR appears to inhibit BDNF-mediated neurite outgrowth from CNS neurons after injury [+ neighbouring cells, removing excess BDNF and promoting axonal pruning via a dominant negative effect and inhibiting the activity of normal TrkB receptors in neurons [Truncated TrkB, a spliced form of TrkB lacking the catalytic tyrosine kinase domain, plays an important role in BDNF-mediated neurogenesis and synaptogenesis, exhibiting differential responsiveness for axon regeneration after axotomy. For example, upregulation of truncated TrkB and p75r injury , 89, 90.r injury  further  neurons . Paradox neurons . The loc neurons . Based oin vivo [NTR can be used as better therapeutic candidates in CNS axon regeneration since the BDNF/  p75NTR complex potentially compromises the ability of BDNF to activate TrkB to enhance growth of axotomized CNS axons [in vivo uses of neurotrophins as therapeutics in the treatment of CNS injuries. These issues will be briefly discussed later in this paper.The importance of paired immunoglobulin-like receptor B (PIR-B)-mediated inhibition of TrkB activity and its role in axon regeneration has recently been reported. PIR-B is considered as a high affinity receptor for myelin-derived axon growth inhibitory ligands such as Nogo, MAG, and OMgp  and inhiin vivo . This stNS axons . On the Even though both Trk receptors and EGFR have been extensively studied and subjected to therapeutic approaches in the CNS and particularly after spinal cord and ON injury, other growth factors such as insulin-like growth factor- receptor (IGF-1R), fibroblast growth factor receptor (FGFR), ciliary neurotrophic factor receptor (CNTFR), and glial cell-derived neurotrophic factor (GDNFR) remain of considerable potential targets in CNS injury. More recently EphA4 receptor, also a subfamily of RTK has been implicated in CNS injury. The inhibition of EphA4 promotes axon regeneration and functional recovery by blocking astrocyte gliosis in spinal cord injury models and this receptor type may be subjected to considerable attention in future studies for the treatment of spinal cord injuries .β) superfamily and preferentially binds glycosylphosphatidylinositol (GPI)-anchored protein receptor (GFR), which is dynamically located on the plasma membrane. The GDNF receptor family comprises of GFRα1, GFRα2, GFRα3, and GFRα4. Among them, GFRα2 is highly expressed in the cortex, basal forebrain, specific layers of the olfactory bulb, cerebellum and motor nuclei [α2, such as GFRα2a and GFRα2c, also promote cAMP-mediated axon growth [GDNF is a family member of the transforming growth factor-beta (TGF-r nuclei , 98. Then growth . Initialn growth , 101. Itn growth .Ex vivo gene delivery of GDNF has been shown to promote extensive growth of motor and dorsal column sensory axons and remyelination of regenerating axons by recruiting more Schwann cells to the lesion site after partial and complete spinal cord transections [GDNF is generally considered a potent neurotrophic factor for motor neurons and enhances their survival and axon regeneration after implantation of peripheral nerve grafts in spinal root avulsion models . Ex vivosections . GDNF-mesections . The sursections  and disrsections . GDNF-ensections . These sCNTF, a potent survival factor for neurons and oligodendrocytes, elicits its signals after binding its receptor, CNTFR, promoting neurotransmitter synthesis, neuronal survival, and neurite outgrowth in certain neuronal populations. Previous studies reported that CNTF stimulates neurite outgrowth from spinal cord neurons and the neurite growth-promoting effects of CNTF, however, does not appear to be a consequence of its survival-promoting effect . Unlike The axon growth promoting and neuroprotective effects of CNTF have been extensively studied in injured RGC and accumulating evidence suggests that CNTF profoundly enhances both survival and axon regeneration through activation of the JAK/STAT3 and PI3K/Akt pathways –113. Genin vitro [The effects of IGF-1 have also been studied in various neuronal populations. In one study, IGF-I gene delivery enhanced adult corticospinal neuron survival but failed to promote their axon regeneration after injury . In addiin vitro . IGF-1 ain vitro  IGF-1 mein vitro . In contin vitro . To suppin vitro . Based oIn order to obtain complete functional regeneration, surviving injured adult neurons must respond either by reexpressing growth associated molecules, respond to exogenously applied neurotrophins, or respond to other appropriate stimuli to trigger robust axon regrowth. In contrast to the PNS, there is a differential cell body response after injury in the CNS. Regardless of injury sites being either proximal or distal to the cell body, axotomized motor neurons regenerate their axons into PN grafts, while graft-induced regeneration of rubrospinal axons only occurs after axotomy proximal to their cell bodies, indicating that appropriate cell body responses to injury are a prerequisite for CNS axon regeneration . AnotherIt is well documented that both PNS and CNS neurons require trophic factor-derived signals for their survival and regeneration , 125–127via their axons [The ability of particular neurotrophic factors to promote survival and axon regeneration depend on the localization of their receptors in specified locations that are then retrogradely transported to the cell body ir axons . For exair axons . TherefoNumerous experimental strategies to promote axon regeneration have shown their feasibility in animal models of acute spinal cord injury, but their effectiveness often declines with a delay in administration of neurotrophic factors. Appropriate timing is therefore crucial for the repair of injuries and their potential for axon regeneration and restoration of lost function. Once sprouting injured axons have encountered growth-inhibiting cues in the external environment, they lose their ability to respond to growth promoting factors as well as other intracellular growth mediators, including signals derived from RTK , 139.Several experimental strategies have been employed to minimize tissue damage during the exogenous application of neurotrophic factors and to enhance axon growth and functional restoration after CNS injury. The mode of administration of growth factors has been under scrutiny since some methods downregulate the effectiveness as a result of damage around neuronal tissues, altering morphological as well as functional recovery. Currently, there are several fascinating techniques, which exist, including implantation of appropriate cells and tissues at the site of injury, gene therapy, and electrical stimulation. Among them, gene therapy is the most popular method to deliver neurotrophic factors and holds the most therapeutic promise , 140.In summary, there are now several experimental strategies that have been established to promote neuronal survival and regeneration of their axons to improve function after CNS injury. Among these approaches, upregulation of neurotrophic factors and their receptors, many of which belong to the RTK family, have a great impact on CNS axon regeneration. These strategies target many of the cellular processes behind axon regeneration such as demyelination, axon retraction, sprouting, and neuronal death. Thus far, combinatorial signalling pathways that mediate axon regeneration can be targeted for sufficient functional impact. However, simultaneous and spontaneous administration of cAMP and neurotrophins are more effective in promoting axon regeneration in CNS injury models. Faced with the significant barriers to axon regeneration in the CNS, it may be that only a modest level of axon regeneration is sufficient to promote substantial functional restoration—a feat, which is probably achievable in the not too distant future. Despite extensive studies and encouraging data on a variety of neurotrophins and their roles in promoting survival of injured adult neurons and regeneration of their axons, many challenges yet remain for RTK and their ligands prior to their therapeutic use in humans. The inherent complexity of the adult CNS, the plethora of RTK and their ligands as well as their different roles apart from neuronal survival and axonal regeneration make the therapeutic approaches more complex. Therefore, many issues need to be resolved before use in CNS regeneration, stroke, and other neurological disorders including Parkinson's and Alzheimer's diseases. Hence, a combinatorial approach is the most promising strategy to enhance the ability of injured axons to promote CNS axon regeneration.


Small-cell lung cancer (SCLC) is the most aggressive lung cancer subtype and lacks effective early detection methods and therapies. A number of rare paraneoplastic neurologic autoimmune diseases are strongly associated with SCLC. Most patients with such paraneoplastic syndromes harbor high titers of antibodies against neuronal proteins that are abnormally expressed in SCLC tumors. These autoantibodies may cross-react with the nervous system, possibly contributing to autoimmune disease development. Importantly, similar antibodies are present in many SCLC patients without autoimmune disease, albeit at lower titers. The timing of autoantibody development relative to cancer and the nature of the immune trigger remain to be elucidated. Here we review what is currently known about SCLC-associated autoantibodies, and describe a recently developed mouse model system of SCLC that appears to lend itself well to the study of the SCLC-associated immune response. We also discuss potential clinical applications for these autoantibodies, such as SCLC diagnosis, early detection, and therapy. Lung cancer is the leading cause of cancer-related death in the world, claiming the lives of 1.3 million individuals worldwide in 2007 , accountThere are as yet no effective early detection tools for SCLC. It is most often diagnosed due to symptoms associated with disseminated disease, such as bulky intrathoracic malignancy or distant metastases. Cough, shortness of breath, and chest pain are the most common local symptoms, and distant signs of the disease include weight loss and weakness. After presentation of symptoms, histological analysis of bronchoscopic biopsy samples and cytological study of fine-needle aspiration (FNA), transbronchoscopic needle aspiration (TBNA), or endoscopic ultrasound (EUS)-guided fine-needle aspiration (EUS-FNA) samples are common approaches to confirm SCLC diagnosis -12. The Paraneoplastic neurologic syndromes (PNS) are defined as cancer-associated neurological diseases that damage neuronal tissues in a site remote from the tumor (unrelated to metastasis) . PNS patThe mechanism by which autoimmunity develops in SCLC remains to be elucidated. For example, it is unclear whether the antigens that prompt an immune response are in any way different from those in normal tissue. If these antigens exhibited cancer-specific changes responsible for triggering an antibody response, they might not only be useful for the development of tools for (early) detection, but also for imaging and treatment. Thus, elucidating the basis of immune reactivity in SCLC is of great importance. The study of SCLC-associated PNS offers a potential window into the relationship between SCLC and the immune response.Many SCLC-associated autoantigens are normally expressed in the nervous system, but they become abnormally expressed in the tumor, classifying them as onconeural antigens. The biological characteristics of these autoantigens can be broadly divided into four categories: 1) neuromuscular junction proteins; 2) vesicle-associated nerve-terminal proteins; 3) neuron-specific DNA or RNA-binding proteins; and 4) neuronal signaling proteins . SeveralThe nomenclature of SCLC-associated antibodies and antigens has evolved over time, largely because the specific antigens were originally unknown, and the antibodies were classified based on reactivity with whole cells or whole cell lysates. For example, ANNA  reactivity was later reclassified as anti-Hu to reflect the fact that these antibodies target the Hu proteins -26. CurrThe SCLC-associated autoantigens/autoantibodies identified to date are summarized in Table Drosophila melanogaster protein Elav  which plays a role in eye and central nervous system development of the fly  fluorodeoxyglucose positron emission tomography (FDG-PET) scanning, a method that has been used to detect SCLC tumors in paraneoplastic patients positive for anti-Hu antibodies, has not been shown to decrease mortality [To date, imaging has shown limited promise for the early detection of SCLC. [ortality . This suortality -151. Howortality . In addiortality ,154, potortality ,155,156.ortality , might swithout PNS. In our studies of the immune response in SCLC-prone mice, we observed one mouse with high titer antibodies and in situ neuroendocrine lesions, but no invasive cancer [Autoantibodies against tumor-associated antigens are more stable and specific than other serum-derived proteins , making e cancer . This, aspecific for a malignant condition is one great challenge in developing a serological detection tool. In the case of autoantibodies, multiple environmental factors, pathogen invasion, and autoimmune disease can result in the production of a high-level of IgG and IgM autoantibodies that recognize various antigens and thus reduce the specificity of the antibody [Identifying biomarkers highly antibody -161. Theantibody ). In addantibody . Unfortuantibody . The latantibody . In otheantibody ,164. TheBackground reactivity does not appear to occur for all antigens. In a study examining anti-recoverin response, no healthy individuals showed positive reactivity . Howeversensitivity. It is clear that a detection test utilizing any single SCLC antigen will show insufficient sensitivity because any single antibody is present in only a fraction of SCLC patients  study  and the not precede cancer development, or does not precede it by sufficient time, these antibodies would still be useful tools to establish or confirm a SCLC diagnosis. In PNS patients, detected autoantibodies often trigger a tumor search. One could envision that an antigen panel to confirm suspected SCLC could be established. Retrospective studies of archival human serum and plasma aimed at exploring SCLC-associated antibodies for early detection will provide data that would also be very useful to determine utility for diagnosis. Ultimately, however, a prospective study in which serum and plasma from suspected SCLC are tested and correlated with the confirmed clinical diagnosis, would be required.While early detection of SCLC would be the holy grail, a much simpler application of SCLC-associated autoantibodies is the establishment of a diagnosis. If the timing of autoantibody generation does How could SCLC-associated autoantibodies affect survival? For one, in patients exhibiting neurological symptoms, a diagnosis of SCLC-related autoimmunity will prompt a tumor search, which would allow the cancer to be detected at an earlier time point when intervention might be more effective. The fact that autoantibodies can precede the diagnosis of the tumor ,173 suggwithout paraneoplastic disease, low titers of anti-Hu autoantibodies are correlated with comparatively indolent tumor growth relative to antibody-negative patients [Secondly, the immune response may itself be protective. A number of studies suggest that patients with SCLC-related autoimmune disease have a better prognosis than patients with histologically identical tumors without a paraneoplastic disorder ,174-176.patients ,88. The patients -142. Othpatients ,144. ThiAbnormal or ectopic expression of antigens by a non-neuronal cell type could activate immuno-competent cells that promote an inflammatory response. The inflammatory response can trigger apoptosis, which could then initiate an antitumor immune response ,108. ApoThe development of SCLC vaccines presents a formidable challenge because an immune response against antigens expressed both in the tumor and nervous system may cause neurotoxicity. The main objective in treating a patient with SCLC-related PNS is to cure the underlying cancer and to improve or stabilize the neurological dysfunction . Interesin vitro evolution in small scaffolds, like knottin peptides [A very different situation arises if the antigens triggering the immune responses are mutated or post-translationally modified in the tumor compared to the native nervous system. In that case, a strategy to specifically target the modification would be an option. However, when using immunizations aimed at inducing a cancer-specific response to a modified protein, a unique tumor-specific epitope may not preclude a destructive autoimmune response; reactivity might expand through epitope spreading. For any immunotherapy strategy, finding the right balance between attacking the tumor and avoiding neurotoxicity is imperative. A more promising avenue may be to develop molecules tailored to recognize the antigen using peptides . Such "mThe potential to use the immune system or targeted molecules to attack the tumor underlines the importance of obtaining a detailed mechanistic understanding of SCLC-associated autoimmunity. Until the nature of the antigen and the immune response are clarified, it will be very difficult to devise fruitful applications. The biggest challenge may be that different SCLC-associated immune responses may occur through distinct mechanisms.panel of antigens might be used to detect antibodies in the blood of those at most risk for SCLC. It is unclear whether such a panel could provide high enough sensitivity and specificity and whether the antibodies arise with enough lead time to allow intervention with an appreciable impact on survival. Effective intervention would only be possible if the tumor could be resected before it metastasizes or if improved treatments become available. Establishing the timing of an autoimmune response relative to cancer development is therefore of great importance. The identification of the autoimmune trigger (if it is distinct from the wildtype neuron-specific protein) in the different SCLC-associated PNS is crucial. Any identified epitopes might be of use as therapeutic targets, for example using tailored antibodies or synthetic molecules. A recently developed SCLC mouse model that has been shown to develop an anti-Hu response provides powerful new tool for investigation in this important field, and could provide insight into many of the questions raised above.Identifying SCLC-associated autoantigens that may elicit an antibody-based immune response may yield new methods for SCLC detection, screening, treatment, and imaging. Antibody responses against a wide variety of antigens are seen in SCLC patients. How common these antibodies are in patients and what the background response is in healthy individuals varies by antigen. The exact mechanism whereby these antibodies develop remains to be elucidated. While the examination of antibodies for a single autoantigen as a diagnostic or screening tool for SCLC would not be sufficiently sensitive given the fact that only a fraction of SCLC patients shows a response to any given antigen, in theory, a The authors declare that they have no competing interests.MK and IALO reviewed the literature, and drafted, edited, and finalized the manuscript.


Francisella novicida, and is essential for its virulence. Here we describe the Cas9 regulatory system in detail, as well as data on its role in controlling virulence traits of Neisseria meningitidis and Campylobacter jejuni. We also discuss potential roles of CRISPR-Cas systems in the response to envelope stress and other aspects of bacterial physiology. Since ~45% of bacteria and ~83% of Archaea encode these machineries, the newly appreciated regulatory functions of CRISPR-Cas systems are likely to play broad roles in controlling the pathogenesis and physiology of diverse prokaryotes.CRISPR-Cas systems are bacterial defenses against foreign nucleic acids derived from bacteriophages, plasmids or other sources. These systems are targeted in an RNA-dependent, sequence-specific manner, and are also adaptive, providing protection against previously encountered foreign elements. In addition to their canonical function in defense against foreign nucleic acid, their roles in various aspects of bacterial physiology are now being uncovered. We recently revealed a role for a Cas9-based Type II CRISPR-Cas system in the control of endogenous gene expression, a novel form of prokaryotic gene regulation. Cas9 functions in association with two small RNAs to target and alter the stability of an endogenous transcript encoding a bacterial lipoprotein (BLP). Since BLPs are recognized by the host innate immune protein Toll-like Receptor 2 (TLR2), CRISPR-Cas-mediated repression of BLP expression facilitates evasion of TLR2 by the intracellular bacterial pathogen  This alcas genes have degenerated, providing an explanation for why the chromosome itself is not targeted , as well as RNase III, for maturation of crRNAs  are each characterized by unique Cas proteins involved in maturation of the crRNAs, targeting of foreign nucleic acid, and nucleic acid cleavage , form a dual RNA:protein complex capable of targeting the BLP transcript , which lack endonuclease activity, but retain the ability to bind DNA  mutants had significant delays in aggregation, sporulation, and chemotaxis. This correlated with low levels of transcript for a necessary activator of fruiting body formation , it was observed that histidyl-tRNA transcript levels were reduced, and that the bacteria exhibited a growth defect  (Aklujkar and Lovley, cas genes are involved in this process is yet unknown. In addition to these examples of self-targeting spacers involved in gene regulation through unknown mechanisms, there are numerous examples of self-targeting crRNA spacers (Stern et al., Aggregatibacter actinomycetemcomitans encodes a spacer putatively targeting the important metabolic enzyme glgP (Jorth and Whiteley, glgP production (Jorth and Whiteley, Additionally, the Archaeal Type I system encoded by Listeria monocytogenes encodes an isolated crRNA locus, consisting of five identical repeats, and four unique spacer sequences (Mandin et al., cas genes, and is present even within L. monocytogenes strains that are devoid of any cas genes (Mandin et al., feoAB iron transport system (Mandin et al., feoAB is an important virulence factor in numerous organisms, it is likely that rliB plays an important role in the virulence of L. monocytogenes. Interestingly, this orphaned system is still capable of acting canonically against plasmid transformation, provided there are cas genes produced in an exogenous locus. However it is unknown if this occurs through targeting of DNA or RNA substrates (Sesto et al., There also exist examples in which crRNAs may have regulatory roles, even in the absence of Cas proteins. E. coli play a role in DNA repair (Babu et al., While the aforementioned examples of alternative CRISPR-Cas function focus on regulatory roles, there may be more indirect mechanisms by which CRISPR-Cas systems contribute to virulence. For example, it has been observed that both Cas1 and the crRNA array in the Type I CRISPR-Cas system of While now very well established to play roles in bacteriophage and foreign genetic element defense, the alternative functions that CRISPR-Cas systems play in the ability of bacterial pathogens to evade and dampen host defenses, and ultimately survive and replicate within the host, have only recently begun to be appreciated. Furthermore, with the continued observations that some CRISPR-Cas systems can target RNA substrates (Hale et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.