Major depressive disorder (MDD) is characterized by mood, vegetative, cognitive, and even psychotic symptoms and signs that can cause substantial impairments in quality of life and functioning. Up to now, the exact pathogenesis of MDD remains poorly understood. Recent research has begun to reveal that the pro-inflammatory cytokines, particularly, tumor necrosis factor-α (TNF-α), play an integral role in the pathophysiology of depressive disorders and the mechanism of antidepressant treatment. On the base of several observations: it is found that subsets of MDD patients have enhanced plasma levels TNF-α; antidepressant treatments had linked with the decline of TNF-α; central administration of TNF-α gives rise to sickness behavior which shares features with depression; and a blockade of it can ameliorate depressive symptomatology in animal models and clinical trials. In this review article, we focus on recent evidence linking TNF-α and MDD looking at data from animal and clinical studies, illustrating the pathophysiological role, susceptibility and its therapeutic application in depression. We conclude by discussing future directions for research, in particular the opportunities for the development of novel therapeutics that target TNF-α. This will be very important for designing preventative strategies and for the identification of new drug targets and preventative strategies. Major depressive disorder (MDD) is a widespread, diverse and intermittent neuropsychiatric disorder characterized by a wide range of symptoms, such as cognitive functions and altered mood ,2,3. AlmIt has been widely accepted that depression may be a collection of partly distinct diseases, with overlapping causal pathways, initiating from the interaction among environmental and genetic factors ,8. AlterAmong the pro-inflammatory cytokines, multiple studies have focused on TNF-α being an endogenous pyrogen, which can cause inflammation, apoptotic cell death, and mediate the release of variety cytokines like IL-6, IL-8 and IL-1β by stimulated macrophages ,17. DysrTaken together, this evidence suggested that, compared to other inflammatory cytokines, TNF-α may play an integral part in the progress of MDD and the mechanism of antidepressant treatments. Previous studies have simply reviewed the experimental and cross-sectional studies that have supported the association of elevated serum concentrations of pro-inflammatory cytokines in depression. However, articles rarely aim to comprehensively review the leading role of specific inflammatory cytokines in depression from aspects of pathophysiological, genetic susceptibility, therapeutic applications and so on. Therefore, we integrate recent progress of the relationship between TNF-α and depressive disorders. In this paper, we begin review by providing a brief overview of TNF-α and the central nervous system (CNS), and then summarize evidence from clinical and animal studies, which suggest that TNF-α may be capable of causing mood swings and depression. Then, we focus on the more extensive rodent and human primate literature to illustrate the pathogenesis, susceptibility and therapeutic role of TNF-α in depression. We further elaborate on the role of TNF-α in depression with autoimmune diseases. We conclude by discussing future directions for research, in particular, the opportunities for the development of novel therapeutics that target TNF-α.TNF-α is produced from various kinds of cells such as lymphoid cells, cardiac myocytes, activated macrophages, endothelial cells, mast cells, fibroblasts, neurons and adipose tissue . It is pTNF-α has multi-biological effects primarily by binding to tumor necrosis factor receptors 1 (p55 receptor) and tumor necrosis factor receptors 2 (p75 receptor), giving rise to stimulation of complex signaling cascades that control various intracellular functions . TNFR1 hIt is well established that CNS is an immune-privileged organ. However, immune status is not clear and fluctuates with age and brain region . Brain iIn the brain, TNFR1 seems to show a constitutive pattern of expression, whereas TNFR2 is generally expressed under stimulatory conditions . The maxTissue injury and infection can stimulate the production of TNF-α in periphery. TNF-α is a large molecule and circulatory cytokines naturally cannot cross the blood-brain barrier (BBB) in normal physiological situations. However, it reaches the brain by using several immune-mediated pathways and transfers the signals from the periphery to CNS ,46. The et al. proved that the removal of TNFR1 and TNFR2 exhibited an antidepressant-like behavior in the tail suspension test (TST) and forced swimming test (FST) as compared with the wild type mice [Much research has collected substantial evidence from animal studies that support the role of TNF-α in depression ,49,50,51ype mice .in vitro studies with human whole blood, cultured lymphocytes and monocytes and studies with rat brain slices have reported that several classes of antidepressants are able to inhibit the production of pro-inflammatory cytokines including TNF-α [A recent meta-analysis calculating cytokine concentrations in MDD patients has found significantly higher concentrations of TNF-α in depressed subjects as compared with control subjects. Another study conducted in Europe recruited a psychiatric patient population, which had shown high levels of TNF-α and soluble TNF-receptors p55 and p75) in past history patients or those currently facing depression  and p75  and in tng TNF-α ,60.Altogether, these studies indicated that TNF-α may be capable of causing mood swings and depression, and central administration of it would be an innovative method to investigate the inflammatory component of depressive disorder.Many observation have shown that effects of the cytokine system, in which TNF-α is a part, on serotonin metabolism as well as on the hypothalamic-pituitary-adrenal (HPA)-axis, may induce changes in the structure and function of the brain, possibly leading towards the development of depression . There aThe HPA-axis is the main neuroendocrine system that controls stress related physiological response, and, as a result, drives how an organism might adapt its own behavior or environment in order to accommodate that stress . In a shThe stimulation of the cytokine system might be a possible cause of depression-related activation of the HPA-axis ,66. The Cytokine signaling molecules such as c-Jun N-terminal kinases (JNK), NF-κB, and signal transducers and activators of transcription-5 (STAT5) have been shown to control GR. Considerable evidence demonstrates that TNF-α encourages glucocorticoid resistance ,74,75,76et al. have suggested that the hyperactivity of chronic HPA system in depressed patients inhibited the production of inflammatory cytokines [In contrast, findings from Schuld ytokines . Anotherytokines .et al. concluded that TNF-α motivated the 5-HT uptake in mouse midbrain and striatal synaptosomes and rat embryonic raphe cell line [The pro-inflammatory cytokines depressogenic effects are mostly due to their action on the neurotransmitter serotonin (5-HT) homeostasis, and its deficiency is recognized as a key pathophysiologic feature of depressive disorders and the target of most antidepressants . Severalell line . Howeverell line . Moreoveell line ,50,51,85Recently, it has been demonstrated that TNF-α raised the brain monoamine metabolism, which caused anhedonia in mice. TNF-α has elevated the dopamine metabolite homovanillic acid without disturbing dopamine levels itself . On the Emerging research suggests that disturbances in tryptophan metabolism may be associated with pro-inflammatory cytokines and depression ,88,89. TN-methyl-d-aspartate (NMDA) receptors as well as producing neurotoxicity, resulting in glutamate excitotoxic cell death [It has been reported that TNF-α directly or indirectly affects the tryptophan metabolism by stimulating IDO ,95,96,97ll death . In contll death . Anotherll death . The rolll death .Therefore, we can hypothesize three mechanisms of how TNF-α may lead to depression or depressive symptoms: (1) modification of the HPA-axis; (2) activation of neurotransmitter transporter; (3) stimulation of IDO, which leads to tryptophan depletion and initiates the KYN pathway.Functional polymorphisms in the promoter region of regulatory genes predict phenotypes of interest in interaction with predisposing biological factors or behavioral ,105. SinTNF-α gene is positioned in the class III region of the major histocompatibility complex (MHC), on the small arm of chromosome 6 (6p21.1–21.3) [TNF-α gene region. According to the National Center for Biotechnology Information (NCBI), to date, more than 391 SNPs have been identified , with G to A substitution polymorphism, was the most extensively studied. There are two alleles at the polymorphic site, TNF-α -308G and TNF-α -308A. In normal populations, TNF-α-308G homozygosity is the predominant genotype [The human .1–21.3) . It spanentified . Among tgenotype . The A agenotype ,112,113.TNF-α gene polymorphisms in susceptibility to MDD has been extensively investigated in different ethnicities, especially rs1800629, but conflicting results were obtained due to the heterogeneity of the genetic background among populations, the number of cases in these studies, and the complexity of the pathogenesis of depression  and -857(C/T). This finding is in line with reports from Misener and colleagues , who fouet al. [Bosker et al.  used datExcess levels of TNF-α may play a pivotal role in the pathophysiology of depression. Consequently, several TNF-α antagonists have been developed to block the action of TNF-α in depressive patients . Indeed,et al. reported that chronic administration of infliximab decreased chronic unpredictable mild stress (CUMS)-induced depression-like behaviors back to or lower than presumably the normal levels observed in saline-control rats [et al. explored whether long-term etanercept treatment could affect the anxiety- and depression-like neurobehaviors in rats without a chronic inflammatory or stressful condition by various behavioral methods. The anxiety- and depressive- like neurobehaviors of the animals as assessed with an elevated plus maze and forced swimming tests, respectively, were found significantly decreased after the etanercept treatment. However, etanercept did not alter total locomotors activity levels, which indicated that differences observed in emotional tests were not mediated by the psychomotor activation of etanercept. These findings suggested that TNF-α has a role in the modulation of emotional processes, and its inhibition may represent a novel strategy for the treatment of affective disorders [et al. demonstrated treatment with etanercept significantly that reduced the restraint-induced depression-like effects, resulting in reduced immobile time in the FST and intensified climbing behavior, both similar to the antidepressive-like effect of imipramine. They speculated that antidepressant effects of etanercept may be caused by enhancement of serotonergic or noradrenergic neurotransmission or normalization of stress hormone secretion [et al. has showed that chronic infliximab treatment prevented the CUMS-induced cognitive impairments as well as the reduction in the levels of hippocampal brain-derived neurotrophic factor (BDNF). These results suggest that infliximab improves the spatial and emotional memory impairments induced by chronic stress in rats, likely through its effects on hippocampal function by modulating inflammation [Using chronic unpredictable mild stress combined with separation, Karson rol rats . In anotisorders . Meanwhiecretion . Very reammation .et al. showed that adalimumab treatment reduced symptoms of depression and improved health-related quality of life (QOL) in addition to improving psoriasis [et al. [et al. [et al. also reported the beneficial effects of infliximab on depression and psychological well-being in active CD [et al. have demonstrated that the administration of infliximab in CD significantly reduced depression scores and improved the QOL as well as fatigue in a four-week follow-up study, although a clear role of cytokines could not be substantiated [It is well known that TNF-α blockers are very effective in the treatment of chronic inflammatory disorders like psoriasis, Crohn’s disease (CD), and ankylosing spondylitis (AS), which can co-exist with depression. However, recent clinical trials have shown that TNF-α blockers were also effective in decreasing depressive symptoms associated with these disorders. In one of these trials, Tyring and co-workers firstly demonstrated that, compared to placebo administration, etanercept treatment led to an improvement of at least 50% in depression rating scales at week 12. However, the improvement in symptoms of depression was not strongly correlated with the improvement in psoriasis area and severity index (PASI) in this study . Subsequsoriasis . In cont [et al.  they rep [et al.  have fou [et al. . Then, Pctive CD . Likewisantiated . Up to nantiated ,136. Theet al. for the first time performed a randomized double-blind, placebo-controlled clinical trial of infliximab versus placebo for antidepressant non-responders with major depression. Surprisingly, no differences in clinical response were found between infliximab and placebo in the group as a whole. However, there was a significant interaction between baseline inflammation and treatment response, with patients exhibiting high initial markers of inflammation performing clinically better [et al. further examined additional predictors and targets of response to infliximab in patients with TRD, they found baseline transcriptional signatures reflective of alterations in glucose and lipid metabolism predicted antidepressant response to infliximab, and infliximab response involved regulation of metabolic genes and inhibition of genes related to innate immune activation [et al. [et al. [In view of the association between inflammatory cytokines and treatment resistance ,138, it y better . Mehta etivation . Studiestivation ,142. TNF [et al.  Moreover [et al.  suggesteet al. also reported that MDD patients had higher plasma TNF-α levels compared to healthy subjects, and this value further increased after eight-week venlafaxine treatment [The research on biomarkers that predict antidepressant response has been recognized as remarkably difficult, and the literature is spoiled with conflicting observations regarding an array of putative genetic and physiological predictor variables. Given that TNF-α has been recognized to be involved in the pathogenesis of MDD, it is worthy to examine whether TNF-α is associated with antidepressant medication treatments and could serve as putative biomarkers for clinical response. Multiple studies have evaluated the effects of antidepressants on TNF-α, and the results are not consistent. Owing to heterogeneity and the complex nature of MDD, TNF-α level was increased , decreasreatment ; In a foreatment .et al. recently conducted a meta-analyses examining data from 35 studies (TNF-α in 11 studies) that investigated pro-inflammatory cytokines before and after treatment in depressed patients together with a measure of clinical response [Faced with controversial clinical findings, Strawbridge response . TNF-α lThere are several ways that TNF-α could contribute to the action of antidepressants . First, et al. observed increased levels of TNF-α in childhood-onset SLE patients with moderate/severe depression as compared to patients with no/mild depression [Patients with autoimmune disease more frequently suffer from major depressive episodes. This fact might be associated with the physical disability and multiple stresses existing with chronic disease conditions. Currently, studies designed with the objective to explore the association between depression and pro-inflammatory cytokines in patients suffering with depression and autoimmune diseases, such as systemic lupus erythematous (SLE) and multiple sclerosis (MS), are not common. A recent data found that serum TNF-α levels were significantly higher in SLE patients with depression. Additionally, it was also independently associated with more severe depression symptoms in these patients. These finding propose a potential impact of TNF-α on depression symptoms in SLE patients . Similarpression . There ipression . The relpression . Anotherpression . Pro-infpression . ConsequAs mentioned above, both direct and indirect evidence recommends that TNF-α might contribute to the etio-pathogenesis of MDD and the mechanisms of antidepressant treatment. Peripheral TNF-α motivated via infection and tissue damage can cross the BBB through fast transmission pathway involving primary afferent nerves, a saturable transport system, or a slow transmission pathway. The secretion of TNF-α has been revealed to elevate the levels of ACTH, cortisol and CRH, which have a direct effect on the HPA-axis. The upregulation of the HPA-axis can cause depression. Furthermore, TNF-α also activates neurotransmitter transporters and indirectly consume serotonin and its precursor tryptophan, which leads to decreased monoamine neurotransmitters in synaptic cleft.Interestingly, anti-TNF-α antagonists have already been revealed to have antidepressant effects or improve antidepressant response. Moreover, high levels of TNF-α and other inflammatory markers forecast non-responsiveness of patients to standard antidepressants. Therefore, normalization of peripheral TNF-α may be regarded as alternative treatment or preventative strategies, and potential biomarkers for treatment response. Upcoming research needs to advance the existing discoveries and initiate the teasing out of some of the unknown issues highlighted such as—can TNF-α inhibitor therapy recover certain subgroups of populations with depression who present with higher inflammatory biomarkers, and may existing levels of TNF-α or other inflammatory biomarkers in individuals with depression indicate who is likely to respond to therapy? Moreover, maybe there are some other characteristics  that recognize who are most likely to respond to TNF-α inhibitor therapy? Some antidepressant effects are observed by presumably blocking peripheral TNF-α, for example with TNF-α antibodies and neutralizing agents that are administered peripherally and do not cross the blood brain barrier. What are the proposed mechanisms underlying the actions of these agents on brain behavior and function? Whether TNF-α gene polymorphism’s association of its blood levels and treatment in MDD patients? In the experiment to figure out the mechanism, there is still uncertainty about whether TNF-α inhibitors have any direct effect on depression or whether they are indirectly improving depression by recovering the underlying physical condition.In conclusion, recognizing the influence of TNF-α on neurotransmitter systems and obtaining a better understanding of the consequences of altered inflammatory responses may support not only MDD but also other neuropsychiatric illnesses.


Heart failure with preserved ejection fraction, i.e. HFpEF, is highly prevalent in ageing populations, accounting for more than 50 % of all cases of heart failure in Western societies, and is closely associated with comorbidities such as obesity, diabetes and arterial hypertension. However, all large multicentre trials of potential HFpEF treatments conducted to date have failed to produce positive outcomes. These disappointing results suggest that a ‘one size fits all’ strategy may be ill-suited to HFpEF and support the use of tailored, personalised therapeutic approaches with specific treatments designed for specific comorbidity-related HFpEF phenotypes. The accumulation of a multitude of cardiovascular comorbidities over time leads to increased systemic inflammation, oxidative stress and coronary microvascular endothelial inflammation, eventually resulting in degradation of cyclic guanosine monophosphate (cGMP) via multiple pathways, thereby reducing protein kinase G (PKG) activity. The importance of cGMP-PKG pathway modulation is supported by growing evidence that suggests that this pathway may be a promising therapeutic target, evidence that is mainly based on its role in the phosphorylation of the giant cytoskeletal protein titin. This review will focus on the preclinical and early clinical evidence in the field of cGMP-enhancing therapies and PKG activation. Greater molecular understanding of cardiac mechanotransduction in normal and failing hearts has provided novel perspectives on the role of cyclic guanosine monophosphate (cGMP)-protein kinase G (PKG) signalling in heart failure with preserved ejection fraction, i.e. HFpEF –5; Fig. .While initial descriptions of HFpEF focused on left ventricular (LV) diastolic dysfunction, it is now clear that HFpEF actually involves a complex interplay of factors, including LV and vascular stiffness, left atrial impairment, chronotropic incompetence and decreased pulmonary vascular capacitance . Recent In diastole, the ECM contributes to passive stiffness, helping to prevent overstretch, myocyte slippage and tissue deformation during ventricular filling. ECM components also serve as modulators of growth and tissue differentiation. Certain forms of excessive collagen deposition are associated with the chronic pressure overload that occurs in hypertensive heart disease and aortic stenosis, leading to an increase in myocardial stiffness. Collagen is a major determinant of ECM-based stiffness, and factors such as total collagen levels, the expression of collagen type I relative to type 3 , and thePatients with HFpEF show characteristic features, being generally elderly, more often female, and display a high prevalence of non-cardiac comorbidities such as overweight/obesity, hypertension, diabetes, chronic obstructive pulmonary disease, anaemia and chronic kidney disease. Interestingly, systemic inflammation and endothelial dysfunction are important hallmarks of these comorbidities, and may also drive myocardial dysfunction and remodelling in HFpEF through coronary microvascular endothelial inflammation , 12. ThiConsiderable evidence now supports a role for oxidative stress and inflammation in HFpEF progression, in addition to the endothelial dysfunction that is common to many forms of vascular disease , 14, 15.cGMP is synthesised by two distinct pathways: the first requires activation of soluble guanylate cyclase (sGC) by NO, while the second requires particulate guanylate cyclase (pGC) coupled to natriuretic peptide (NP) , 19; Fig9; Fig1. in vivo administration of the PDE5A inhibitor sildenafil, which facilitates increased myocardial PKG activity by inhibiting the breakdown of cGMP by PDE5A [One finding of potential clinical importance was the favourable reaction of slow LV relaxation and high diastolic LV stiffness in HFpEF to increased PKG activity following by PDE5A –5. This by PDE5A . All of by PDE5A , 19, 22.The relevance of the NO-cGMP-PKG signalling network is further emphasised by the actions of sildenafil in many studies. Inhibition of cGMP degradation enhances NO-mediated vasodilation by sildenafil, restores LV relaxation kinetics in mice exposed to transverse aortic constriction , and redbona fide therapeutic target in heart failure. Conversely, the failure of the RELAX trial might also be related to the subcellular microdomains of cGMP recruited by inhibition of PDE5A, which are NO dependent and therefore ineffective in recruiting a preload reserve under conditions of reduced NO bioavailability.Given the known properties of sildenafil, a long-term (24-week) trial of sildenafil  in patients with HFpEF reported an unexpected outcome , produciIn light of the many caveats surrounding the RELAX trial it is worth recalling that other small studies have demonstrated benefits of sildenafil in patients with high blood pressure, right ventricular dysfunction and pulmonary arterial hypertension. In some studies sildenafil was reported to be beneficial for right ventricular function and pulmonary pressures in HFpEF patients with pulmonary hypertension . This isin vivo.One of the abovementioned studies showed that 30 % of HFpEF patients have diastolic dysfunction with no increase in LV collagen , but wit2+-ATPase (SERCA2a) and ryanodine receptor 2 (RyR2) [in vitro. In line with this, in patients with chest pain but no evidence of coronary lesions, intracoronary infusion of sodium nitroprusside resulted in a slightly decreased LV peak systolic pressure and an acute increase of LV end-diastolic capacitance [Previous clinical trials of PDE5A inhibitors, NO donors, sGC activators and neprilysin inhibitors have focused on acute vasodilatory effects. This focus is now shifting towards their potential direct myocardial effects, independent of afterload reduction. Nitroxyl donors, such as 1-nitrosocyclohexyl acetate, may have therapeutic benefits in heart failure due to the direct action of nitroxyl on myofilaments, sarco/endoplasmic reticulum Ca2 (RyR2) , which a2 (RyR2) , inducinacitance , 35. In acitance , 12, 13.acitance . Finallyacitance , 12, 13.acitance –39. Furtacitance –39.The responsiveness of sGC to NO and its subsequent ability to generate cGMP is impaired by oxidation Fig. . NOS cansGC activators are ideal substitutes for NO under conditions of endothelial dysfunction, such as increased oxidative stress , 41. So Stimulators of sGC reduce renal and cardiac organ damage in experimental high and low renin models . RecentlANP and BNP are released by atrial and ventricular cardiomyocytes, respectively Fig. , in respAs previously discussed, inhibitors of cGMP degrading enzymes, in particular of PDE5A and PDE9A , enhance cGMP-driven effects and consequently increase cardiac muscle relaxation via PKG-mediated phosphorylation of TnI and titin , 28, 31;;1. A rolHFpEF is a major public health problem that lacks effective evidence-based therapies. The cGMP pathway plays a central role in the derangements integral to HFpEF pathophysiology. The capacity of PKG to phosphorylate titin and lower titin-based stiffness has formed the basis for several therapeutic interventions that activate this pathway. The body of evidence surrounding cGMP enhancement thus provides a compelling rationale for further investigation of this pathway in HFpEF. Current evidence also suggests that the cGMP-PKG deficiency seen in HFpEF is a result of a wider pro-inflammatory state that is accompanied by widespread endothelial dysfunction and oxidative stress. This then leads to a reduction in or inactivation of several key signalling pathways, which subsequently affects the pool of cGMP. These changes are not only attributable to an increased rate of cGMP degradation via PDE5A, but may also be partly dependent on the interplay of several signalling pathways. Research priorities therefore include the elucidation of the key signalling pathways that lead to elevated cGMP, increased titin phosphorylation and reduced passive stiffness in cardiomyocytes. A better understanding of these key issues will have direct consequences when considering their relative therapeutic potentials for improving diastolic function.DFG (HA 7512/2 − 1), MERCUR (GZ An-2015-0031), FoRUM (F808N-14), FoRUM (Az. F765-13).


The TNF-α−308 (G/A) and TNF-α−238 (G/A) single-nucleotide polymorphisms SNPs are the most extensively studied. However, all these studies are conducted in Caucasian and Asian populations. Thus, for the first time in Africa, we sought to investigate whether polymorphisms in TNF-α gene were associated with the development of gastric pathology in Morocco. Two SNPs located in the promoter region (positions −308 and −238) in TNF-α gene were genotyped in 244 individuals . Odds ratios (ORs) and 95% confidence intervals (CI) were estimated using logistic regression analysis. The TNF-α−238 (G/A) genotype was significantly associated with a high risk of gastritis and gastric cancer (GC) . Furthermore, a new polymorphism located in the promoter region at position −193 in TNF-α gene was identified. The distribution of this SNP was markedly different in patients suffering from ulcers. The association between TNF-α−193 (G/A) genotype and high risk of ulcer was significant (P = 0.03). These results suggest that the TNF-α−193 (G/A) allele has a protective function against gastric cancer by developing ulcer.Polymorphisms in tumor necrosis factor alpha (TNF- Helicobacter pylori (H. pylori) are at increased risk for developing GC [ H. pylori plays a major role in gastric carcinogenesis [Gastric cancer (GC) is the fourth most common malignancy worldwide , with a oping GC . It is nogenesis  through ogenesis , 6. Howeogenesis , 6, 7.α) and its receptor, are associated with an increased risk of GC [α play a crucial role on gene transcription levels [α gene, mainly in the 5′-promoter region [α−308 (G/A) is associated with an increased production of TNF-α [α−238 (G/A) are less clear, but, because a putative repressor site is located in a 25-base stretch that includes position −238, this polymorphism may be functional [α−308 (G/A) and TNF-α−238 (G/A) single-nucleotide polymorphisms SNPs are the most extensively studied. However, all these studies are conducted in Caucasian and Asian populations. In Africa, up to today, no particular study was interested in the association between polymorphism in the promoter of the TNF-α gene and high risk of GC. Thus, the aim of the present work was to study the frequency of SNPs in TNF-α gene in Moroccan patients with gastritis, ulcer, and gastric cancer.Host genetic factors are emerging as key determinant of diseases for many cancers . Geneticsk of GC . Indeed,n levels . Severalr region . Two SNPnctional , 13. TheIn this case control study, a total of 244 GC patients were enrolled . A comprehensive set of sociodemographic, clinical, and laboratory data have been collected. Written informed consent was obtained for all subjects.α−308 (G/A) and TNF-α−238 (G/A) SNPs, a 266-bp fragment of the 5′ flanking region of the TNF alpha gene was amplified from genomic DNA extracted from blood samples, by polymerase chain reaction (PCR). A total of 0.2 μg of genomic DNA was used, along with 200 μM of dNTP, 1.5 m M of MgCl2, 0.5 μM of primers  PCR, except, for the annealing temperature, it was 55°C.To allow simultaneous determination of genotypes at the TNF- primers , and 1 uμL of exonuclease, 1 μL alkaline phosphatase, 0.5 μL of buffer in a final volume of 7 μL, and 10–50 ng of PCR product and incubated at 37°C for 30 min and 80°C for 15 min. DNA cycle sequencing was carried out in 20 μL reactions using 5 μL of purified PCR product, 1 μL of 3.2 pmol primer, and 1 μL and ABI Prism BigDye 3.1. The forward and reverse sequencing primers were the same as those used in the respective PCR amplifications, in separate sequencing reactions. Conditions for the sequencing of PCR products were 25 cycles with 10 seconds at 96°C, 5 seconds at 50°C, and 4 min at 60°C. Residual dideoxy terminators were removed by ethanol precipitation and sequences were analyzed on an Applied Biosystems 377 DNA sequencer.The PCR products were analyzed by electrophoresis on 2% agarose gel and purified using 0.5 α−308 (G/A) and TNF-α−238 (G/A) genotype frequencies between cases and controls were assessed by test khi2 statistics. Crude odds ratios (ORs), adjusted ORs, and the simultaneous effect of the genetic polymorphisms with 95% confidence intervals (CIs) were estimated by logistic regression analysis. ORs and unconditional logistic regression models were computed using the program Epi Info 2002 (available: ftp://ftp.cdc.gov/pub/Software/epi_info/). Differences were considered to be significant at a P value < 0.05. All statistical tests were 2-sided.Evidence for deviation from Hardy-Weinberg equilibrium of alleles at individual loci was assessed by exact tests. Differences in the proportion of the TNF-α polymorphisms at positions −308 and −238. Despite the disparity of −308 SNPs in the world, it is absent in our population. However, the analysis of the TNF-α promoter from position −106 to position −372 revealed the presence of a new mutation in position −193. This is a transition from guanine to adenine G/A. The frequency of occurrence of the TNF-α−193 A allele in our population is 17.35% in patients suffering from gastric diseases and 13.51% in controls. The distribution of the three genotypes GG, GA, and AA is irregular between controls and patients. The frequencies of the TNF-α−193 A allele and the GA + AA genotypes were slightly higher in patients suffering from ulcers. In addition, ulcer presence was significantly more frequent in A carrier allele group TNF-α−193 (G/A)  .α gene with gastritis and adenocarcinoma. Taken together, these results strongly support our hypothesis suggesting that TNF-α−238 (G/A) polymorphism may be involved in gastritis susceptibility, which progresses in adenocarcinoma  and TNF-α−238 G/A, which were extensively studied [α polymorphisms in gastric cancer in African populations. In case of our study, we identified a new SNP located in the position −193 never shown before, using sequencing. This is a transition from guanine to adenine (G/A). The TNF-α−193 (G/A) has been significantly associated with high risk of ulcer. This result suggests that the TNF-α−193 (G/A) allele has a protective function against gastric cancer by developing ulcer. Indeed, Correa has established a cascade of events leading to GC more than 20 years ago. Chronic inflammation induced by Hp may progress further through the premalignant stages of gastric atrophy, intestinal metaplasia, dysplasia, and finally adenocarcinoma [α are based on the research of known SNPs using PCR-RFLP or sometimes the Taq Man technics. Therefore, we limited ourselves to look for new SNPs that may be specific to certain populations. Proof of this study's originality identifies a novel SNP TNF-α−193 (G/A) by using sequencing technics.Currently, several molecular epidemiological studies have been conducted to investigate the association between the SNPs in TNF-cer risk . Some co studied , 20. Howarcinoma . The relα−308 (G/A), TNF-α−238 (G/A) polymorphism, and gastric cancer was a paradox [α−308 G/A polymorphism and gastric cancer. The results of these studies remain inconclusive or inconsistent. Example of TNF-α−308 (G/A), while our study has not found association between this SNP and gastric pathology like Japan [α polymorphisms as a risk factor for gastric cancer in Caucasian populations, but not in Asian populations [α−308 (G/A) polymorphism was correlated with gastric cancer risk in Caucasians, but not in East Asians and other ethnic groups. Likewise, Gorouhi et al. showed that the statistically significant association between TNF-α−308 (G/A) and gastric cancer was limited to western populations [To date, the association between TNF- paradox . There ake Japan , China [ke Japan , other ske Japan , 26. Morulations , 27, 28.ulations . They deulations . This asα−238 (G/A) is the second commonly studied SNP site following −308 among all polymorphisms located in the promoter region of TNF-α gene [α−238 was significantly associated with high risk of gastritis and GC. These results are similar to those found by others [α−238 and high risk of GC. In contrary, another meta-analysis showed no significant association concerning TNF-α−238 frequency for genotypes G/G, A/A, and G/A [α−238 (G/A) is associated with GC [α−238 polymorphism and risk of gastric diseases yielded contradictory results.Nonetheless, TNF-F-α gene , 29. Itsy others . Yu et ay others . That st and G/A , 15, 20. with GC . To our


An analysis of the intrinsic and regulator-activated Dbp5 ATPase cycle is necessary to define how these factors control Dbp5 and mRNA export. Here, we report a kinetic and equilibrium analysis of the Saccharomyces cerevisiae Dbp5 ATPase cycle, including the influence of RNA on Dbp5 activity. These data show that ATP binds Dbp5 weakly in rapid equilibrium with a binding affinity (KT ~ 4 mM) comparable to the KM for steady-state cycling, while ADP binds an order of magnitude more tightly (KD ~ 0.4 mM). The overall intrinsic steady-state cycling rate constant (kcat) is limited by slow, near-irreversible ATP hydrolysis and even slower subsequent phosphate release. RNA increases kcat and rate-limiting Pi release 20-fold, although Pi release continues to limit steady-state cycling in the presence of RNA, in conjunction with RNA binding. Together, this work identifies RNA binding and Pi release as important biochemical transitions within the Dbp5 ATPase cycle and provides a framework for investigating the means by which Dbp5 and mRNA export is modulated by regulatory factors.mRNA export from the nucleus depends on the ATPase activity of the DEAD-box protein Dbp5/DDX19. Although Dbp5 has measurable ATPase activity alone, several regulatory factors (e.g., RNA, nucleoporin proteins, and the endogenous small molecule InsP  •mRNA export from the nucleus requires DEAD-box protein Dbp5/DDX19 ATPase activity.•Kinetics and thermodynamics of intrinsic Dbp5 ATPase reveal RNA's effect on Dbp5.•i release.Intrinsic Dbp5 ATPase is limited by slow ATP hydrolysis and slower P•i release, but it and RNA binding still limit RNA-stimulated ATPase.RNA activates P•i release define RNA-stimulated Dbp5 ATPase for further regulation.RNA binding and P Critical to deciphering the effect of regulators on the ATPase cycle of Dbp5, or any DBP, is a quantitative characterization of the intrinsic ATPase cycle, as it is only with this base knowledge that we can undertake future in-depth studies on the mechanism of regulation by trans-activating factors and apply this to understand the in vivo cellular function of DBPs. Deducing how trans-activating factors modulate the ATPase cycle of individual DBPs and the functions that emerge from this regulation will also further our understanding of this important class of enzymes as a whole, which functions in almost all aspects of RNA biology.The inherent ATPase cycles of some DBPs are additionally regulated through ancillary domains that mediate interactions with regulatory factors that can include RNAs and/or proteins. For example, Dbp5 is a DBP required for nuclear mRNA export that is stimulated by RNA and further regulated by two nucleoporins (Gle1 and Nup159) and an endogenous small molecule , common tools for kinetic studies of ATPases Here, we have determined the minimal ATPase cycle and calculated the rate and equilibrium constants of individual biochemical transitions for Dbp5. This work has identified slow ATP hydrolysis and even slower subsequent phosphate release as key bottlenecks in the ATPase cycle of Dbp5 that function as targets for modulation by RNA and other regulatory cofactors. We also show that the fluorescent nucleotides versus free), we are building a framework for understanding Dbp5 functions in vivo, which may vary and be dependent upon different regulators given the assorted functions Dbp5 has been proposed to perform Having defined the minimal catalytic ATPase cycle of Dbp5, we show that, in the presence of RNA, phosphate release remains a limiting step in the hydrolysis cycle of Dbp5. However, we also observe evidence of a new RNA-binding-associated rate-limiting step in the kinetic scheme of Dbp5, which identifies an additional feature in the minimal kinetic scheme of RNA-stimulated ATPase activity. Overall, by understanding the steps that limit Dbp5 function in various biochemical states . Under our experimental conditions using both the NADH-coupled assay to detect ADP product formation and PiBiP to detect phosphate product accumulation kcat is 0.04–0.08 s− 1 in the presence of saturating ATP  is 1.3–1.9 mM  were measured by competition with mant-labeled nucleotides, and constants related to ATP hydrolysis and product release were measured with Pki+ mT and ki− mT, i = 1, 2, 3; KmT; the mantATP equivalent of KT in k− mT1/k+ mT1 is 7 μM (The Förster resonance energy transfer (FRET)-based fluorescence enhancement associated with mant-labeled nucleotide binding has been successfully used as a spectroscopic probe to characterize the ATPase cycles of DNA  is 7 μM .overall mantATP binding affinity  is in the range 1 μM  has been reported 3 and RNA to assess the influence of the mant moiety and to identify any structural perturbations in the complex.The structure of Dbp5 with bound nucleotide has not been determined by X-ray crystallography, but the structure of Dbp5 bound to RNA and the ATP analog  to 20 nM  and the ADP affinity from 360 μM  to 1 nM . Although these mant-nucleotide binding constants are considerably tighter than observed  reveals a similar overall structure of the ATPase and bound ligands  3-bound structure by the mant moiety  and competing with mant nucleotides for Dbp5 binding kmT1,obs with increasing [ATP] to estimate an unlabeled ATP binding affinity (KT) of 6 mM  dependence of the turnover rate  to Eq. Fluorescently labeled phosphate binding protein (Par phase . The linKT) of 4 mM   of Dbp5 (with bound nucleotide) at sub-saturating [ATP], and its value of 3 (± 3) mM agrees well with the Michaelis constant KM,RNAi burst from the pre-formed Dbp5-ADP-Pi state following RNA binding , which equates to a Pi release rate constant that is ~ 25-fold faster than ATP resynthesis  and a weak KM,ATP (1.3–1.9 mM). The slow kcat results from slow ATP hydrolysis coupled to even slower Pi release, both of which limit overall cycling, though Pi release is the major determinant. It is experimentally difficult to measure hydrolysis directly by chemical quench flow iBiP fluorescence time courses provide sufficient evidence to implicate both hydrolysis and Pi release as slow and relatively irreversible transitions that limit Dbp5 ATPase cycling in the absence of RNA and other regulatory factors. Current structural and biochemical data support a role for protein regulators  in facilitating enzyme recycling post-hydrolysis Dbp5 has a slow maximum turnover rate . This is the first example to our knowledge of such dramatic differences in behaviors between native and mant-labeled nucleotides with the DBP family of enzymes. While the origin of this fluorophore effect is still unclear, additional hydrophobic interactions with the mant fluorophore  becomes partially rate limiting and, together with Pi release (k− RPi = 1–5 s− 1), limits steady-state Dbp5 ATPase cycling in the presence of RNA. This suggests that the interaction time between Dbp5 and RNA is ≥ 0.4 s Dbp5 steady-state ATPase cycling is accelerated 20-fold by RNA . Consequi release in the absence of RNA KH) and Pi release (KPi) of intrinsic Dbp5 ATPase activity actually favor progression through the ATPase cycle, the slow rate constants associated with both transitions bottleneck the intrinsic cycle at similar positions  as with DbpA and Mss116 cycling. In this sense, Dbp5 appears to be typical of the DBP family.The intrinsic Dbp5 ATPase cycle appears very similar to that of other RNA helicases. Like Dbp5, both DbpA and Mss116 display unfavorable hydrolysis and fully or partially rate-limiting Pi, and Dbp5-ADP are favored  as compared to sub-millimolar binding affinities for most other studied DBPs  favored .i state when coupled with even slower Pi release  rate constant both in the presence and in the absence of RNA, which maintains Dbp5 in an ADP-P release . AlthougAlternatively, it has been demonstrated that Dbp5 shuttles through the nucleus 259 of 15,400 M− 1 cm− 1. Polyuridylic acid (polyU RNA)  was dialyzed extensively against distilled deionized water and ethanol precipitated prior to use. RNA concentrations were determined by absorbance using ε260 of 9660 M− 1 cm− 1 and are in units of nucleotides. Mant-labeled nucleotides  concentrations were determined by absorbance . One molar equivalent of MgCl2 was added to all nucleotides immediately before use.All chemical reagents were of the highest purity commercially available. Millipore MiliQ® water (filtered through a 0.2-μm filter) or Sigma RNase-free water was used in all buffers. ATP  and ADP  concentrations were determined by absorbance using ε2, and 2 mM DTT.All assays were performed at 25 ± 0.1 °C in 30 mM Hepes(pH 7.5), 100 mM KCl, 2 mM MgClEscherichia coli as previously described E. coli phosphate binding protein (PiBiP) was purified and labeled with MDCC ethyl)amino)carbonyl coumarin (Thermo Fisher Scientific Inc.) as previously described N-terminal 6 × His-tagged Dbp5 was expressed and purified from 6 in 10% glycerol as previously described 2 +-NTA agarose (Qiagen) followed by size-exclusion chromatography over a Superdex 200 (10/300) GL column  in 10 mM Hepes(pH 7.5), 100 mM NaCl, 1 mM DTT, and 0.25 mM InsP6 in 10% glycerol. Purified ∆90Dbp5 was concentrated after the addition of 5 mM MgCl2. The ∆90Dbp5-mantADP·BeF3 complex was formed by mixing concentrated ∆90Dbp5 in solution with a 1.2:1 molar ratio of rU10 RNA (IDT), incubating 15 min, and then mixing mantADP·BeF3 (prepared in a 1:3:15 ratio of ADP:Be:F) to a final concentration of 1 mM, followed by another 15 min of incubation. Crystallization of ∆90Dbp5-mantADP·BeF3 was performed by hanging-drop vapor diffusion at 18 °C by mixing 100 nL of the complex at a protein concentration of 16 mg mL− 1 with 100 nL of reservoir solution containing 200 mM Mg(NO3)2 and 18% polyethylene glycol 3350 and incubating 4 days over 100 μL of reservoir solution in a sealed chamber. Crystals were transferred to a cryoprotectant solution containing 18% polyethylene glycol 3350, 9 mM Hepes(pH 7.5), 180 mM Mg(NO3)2, 4.5% glycerol, 1 mM DTT, 90 mM NaCl, 0.25 mM InsP6, 4.5 mM MgCl2, and 1 mM mantADP·BeF3 (prepared in a 1:3:15 ration of ADP:Be:F) and were flash frozen in liquid nitrogen.Dbp5 crystal structures used the construct ∆90Dbp5 (Dbp5 residues 91–482) instead of full-length Dbp5 P212121. The structure of ∆90Dbp5-mantADP·BeF3 was determined by molecular replacement with Phaser using the entire Dbp5 subunit (PDB ID 3PEY) as a search model Data were collected at 100 K on the NECAT 24-ID-E beam line of the Advanced Photon Source at the Argonne National Laboratory and processed with HKL v0) was fitted to the Michaelis–Menten equation:kcat is the maximal turnover rate of Dbp5 at saturating ATP in units of s− 1, [H]T is the total Dbp5 concentration, KM,ATP is the Michaelis constant for ATP, and [T] is the total [ATP]. The [ADP] under our conditions of 2 mM ATP is ∼ 7 μM Time courses of Dbp5 (0.6–1 μM) steady-state ATPase assayed by absorbance change at 340 nm using the NADH-coupled assay were acquired on a Perkin-Elmer Lambda 20 UV–Vis spectrophotometer thermostatted at 25 °C T is the total RNA concentration in nucleotides RNA-stimulated steady-state ATPase experiments were performed as detailed above, using 100 nM Dbp5 and 20 mM ATP. Data were fitted to the quadratic form of the Briggs–Haldane equationk) and equilibrium constants  was used for analysis and modelingi is inorganic phosphate. We assume that product release is sequential with Pi preceding ADP. Several processes  are likely to be associated with multiple transitions  but are omitted for simplicity Transient kinetic measurements were performed with an Applied Photophysics SX2.0 stopped-flow apparatus thermostatted at 25 ± 0.1 °C ex = 280 nm) was monitored through a 400-nm-long pass colored filter. Inner filter effects from mant-nucleotide absorption are minimal at the mant-nucleotide concentrations used (< 120 μM). Time courses of association were acquired under both pseudo-first-order and nonpseudo-first-order conditions. Data acquired under pseudo-first-order conditions were fitted to a linear sum of exponentials. Time courses acquired under nonpseudo-first-order conditions were fitted with the following expressionF0 is the signal at time (t) = 0, A0 and k0 are the signal amplitude and observed rate constant of the first phase, and r is an indication of the deviation of the bimolecular binding term  dependence of the observed mant-nucleotide binding rate constant, as defined by k0 is the observed mant-nucleotide rate constant in the absence of competing unlabeled nucleotide ([N] = 0), and k∞ at saturating [N].Unlabeled nucleotide binding was measured by kinetic competition with mant-labeled nucleotides ex = 436 nm, 463-nm-long pass colored glass emission filter) of the iMDCC-labeled PiBiP upon binding phosphate. Contaminating Pi was removed from all equipment and buffers with a Pi “mop” consisting of 0.5 mM 7-methylguanosine and 0.01 U/mL purine nucleoside phosphorylase (Sigma). Since the process of mop's scavenging free Pi in solution is very slow on the seconds timescale and PiBiP binds Pi much faster (< 100 ms) and extremely tightly, the presence of mop has minimal effect on transient and steady-state Pi release measurements, especially at short timescales iBiP fluorescence was converted into [Pi] following calibration with phosphate standard solutions. Time courses of Pi liberation ([Pi]) measured after rapidly mixing 0.5 μM Dbp5 with an equilibrated mixture of 5 μM PiBiP and varying ATP concentrations were fitted to the following expression defining a lag phase followed by a linear phasePhosphate release from Dbp5 was assayed from the eightfold increase in fluorescence  is the turnover rate. The lag phase is defined by an exponential with observed rate constant λ. The [ATP] dependence of the observed lag phase rate constant was fitted to a hyperbola in the following formiBiP. Pi mop was present in all solutions. Data were analyzed as detailed above.Single-mix experiments were conducted above with the following modifications: 0.4 μM Dbp5 was pre-equilibrated with varying concentrations of RNA and was rapidly mixed with 2.5 mM ATP pre-equilibrated with 5 μM PiBiP and Pi mop. Using the dual-mix mode of the SX20 stopped flow, we mixed 5 μM Dbp5 with 4 mM ATP and aged it for 0.05, 6, or 25 s. After aging, we rapidly mixed the Dbp5/ATP solution with 0 or 10 mM RNA (nucleotide). Final concentrations were 2.5 μM Dbp5, 2 mM ATP, 0 or 10 mM RNA, and 5 μM PiBiP. Time courses of [Pi] release in the absence of RNA were fit to a sum of bimolecular binding and a linear function. Time courses of [Pi] in the presence of RNA were fit to the following sum of bimolecular binding and combined lag/linear phase:Dual-mix experiments were conducted as above, with the modification that all solutions contained both 5 μM P− 1 phosphoenolpyruvate kinase, and 48.5% 18O-labeled water. Reactions were run at room temperature for several hours to generate 2–3 mM Pi, then quenched with an equivalent volume of 1 N HCl. Pi was isolated and analyzed by coupled gas chromatography/mass spectrometry (GCMS) i samples from hydrolysis of unlabeled ATP in 18O-enriched water yields the distribution of species with zero to four 18O oxygens per Pi. The Pc value was not determined by global fitting of this complete distribution because the starting reactions contained variable amounts of unenriched Pi that result in an excess of the 18O0 species. At the low extents of reversal observed with Dbp5, most of the Pi contains only one water-derived oxygen with only a small amount of Pi that contains two water-derived oxygens. The amount of the species with two water-derived oxygens increases with increasing Pc value. The ratio of the 18O2:18O1 species with 2 versus 1 labeled oxygens was therefore used to determine Pc because this ratio only depends on the enrichment in the water and the Pc value and is uninfluenced by the amount of contaminating unenriched Pi.ATPase reactions (5–10 μM Dbp5 and 5–10 mM ATP) were conducted in 5–6 mM phosphoenolpyruvate, 50 U mLOxygen isotope exchange experiments were also performed with RNA-stimulated Dbp5 ATPase activity. Steady-state reactions in the presence of RNA used 2 μM Dbp5, 2.5 mM ATP, and 8 mM polyU RNA. Data were analyzed as above.i release acquired by PiBiP fluorescence were performed using Scheme 1 and KinTek Global Kinetic Explorer Software (KinTek Corp.) kcat; e.g., ≥ 10 s− 1) transition accounting for ATP regeneration from liberated ADP was also included to account for the activity of lactate dehydrogenase and phosphoenolpyruvate kinase in the NADH assay.Kinetic simulations by global fitting of both steady-state ATPase time courses acquired with the NADH assay and transient/steady-state P3 have been deposited in the Protein Data Bank under the accession number 5ELX.Coordinates of the crystal structure of Dbp5 bound to RNA and mantADP·BeF


Peripheral blood monocytes were isolated from 10 acute Charcot patients, 8 diabetic patients, and 9 healthy control subjects and cultured in vitro on plastic and bone discs. Osteoclast formation and resorption were assessed after treatment with (1) macrophage-colony stimulating factor (M-CSF) and RANKL and (2) M-CSF, RANKL, and neutralising antibody to TNF-α (anti-TNF-α). Resorption was measured on the surface of bone discs by image analysis and under the surface using surface profilometry. Although osteoclast formation was similar in M-CSF + RANKL-treated cultures between the groups (p > 0.05), there was a significant increase in the area of resorption on the surface (p < 0.01) and under the surface (p < 0.01) in Charcot patients compared with diabetic patients and control subjects. The addition of anti-TNF-α resulted in a significant reduction in the area of resorption on the surface (p < 0.05) and under the surface (p < 0.05) only in Charcot patients as well as a normalisation of the aberrant erosion profile. We conclude that TNF-α modulates RANKL-mediated osteoclastic resorption in vitro in patients with acute Charcot osteoarthropathy.We hypothesised that tumour necrosis factor- Charcot osteoarthropathy is a severe complication of diabetes, which is associated with significant morbidity and mortality –5. Inflaκβ ligand (RANKL) [We have recently demonstrated that, in acute Charcot osteoarthropathy, there is increased osteoclastic activity in response to the osteoclastogenic cytokine receptor activator of nuclear factor- (RANKL) . Osteocl (RANKL) . Resorpt (RANKL) .α (TNF-α) [α induces expression of RANKL in osteoblastic cells, but it can also act directly on osteoclastic precursors (monocytes) to potentiate RANKL-induced osteoclastogenesis and thereby activity [α may also modulate osteoclastic activity in acute Charcot osteoarthropathy. Thus the aim of this study was to determine the role of this cytokine by comparing the extent of osteoclast formation and resorption in M-CSF + RANKL-treated cultures with and without the addition of neutralising antibody to TNF-α (anti-TNF-α).The reason for this increased resorbing activity is unknown, but it is possible that it is driven by uncontrolled inflammation due to upregulation of proinflammatory cytokines and in particular tumour necrosis factor- (TNF-α) . As an oactivity . This cyactivity , 11 and activity  and alsoactivity  and we hSamples from peripheral blood were obtained from 10 consecutive patients with recent onset of acute Charcot osteoarthropathy, 8 diabetic patients with no history of Charcot osteoarthropathy, and 9 healthy control subjects. All patients with Charcot osteoarthropathy presented with a unilateral red hot swollen foot and radiological evidence of acute Charcot fractures, demonstrated on plain foot and ankle radiographs . All par6 cells were cultured on 24-well plates and 5 × 105 cells were cultured on bovine bone discs  in duplicate to assess osteoclast formation and resorption, respectively. Cultures were maintained in α-minimal essential medium  supplemented with penicillin (50 U/mL)/streptomycin (50 μg/mL) , L-glutamine (2 mM) , and 10% heat-inactivated FBS  under the following conditions:Cultures with M-CSF 25 ng/mL (added at day 0)  and soluble RANKL 100 ng/mL (added at day 7) : M-CSF + RANKL-treated cultures served as a positive control.α 10 μg/mL (added at day 0), , and soluble RANKL 100 ng/mL (added at day 7): M-CSF + RANKL + anti-TNF-α-treated cultures were used to assess the role of TNF-α on osteoclastogenesis. The rationale for this study was to inhibit TNF-α modulation on peripheral blood monocytes by using excess concentration of anti-TNF-α, added from the beginning until the end of the cell culture treatment [Cultures with M-CSF (added at day 0), anti-TNF-reatment .Culture medium was refreshed every 3-4 days supplemented with the appropriate agents as described above. After 17 days in culture, 24-well plates were stained for tartrate-resistant acid phosphatase (TRAP). Plates were viewed by light microscopy and TRAP-positive cells with three or more nuclei were counted as osteoclasts. The ability of these cells to resorb bone was demonstrated by culturing PBMCs on bovine bone discs for 21 days. The bone discs were stained with toluidine blue and mounted on glass slides. Resorption was quantitated by two methods: (1) area of resorption on the surface (%) assessed by image analysis after light microscopy and (2) area of resorption under the surface (μm2) assessed by surface profilometry, as previously described [μm), as previously described [μm, scan duration: 60 seconds, vertical measurement range: 65.5 μm, scan resolution: 0.056 μm/scan. On average, 75 pits per condition/per subject were analysed and the median area of disc erosion was calculated in μm2 using Origin Pro 8.6 software.Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood as previously described . The PBMescribed . The eroescribed . The styμm), maximum depth (μm), and full-width-half-maximum (FWHM) (μm), where the width was measured at the half of the maximum depth. The median width, depth, and FWHM for the unidented, bidented, and multidented pits were calculated for each subject [According to their shape, pits were defined as unidented (erosion with one dent starting from and finishing at the level of the unresorbed surface), bidented (erosion with two clearly defined dents starting from and finishing at the level of the unresorbed surface), and multidented (erosion with three or more dents starting from and finishing at the level of the unresorbed surface). Each pit was characterised by the following parameters: width at the surface ( subject .U test (two groups) or Kruskal-Wallis test (three groups), as appropriate. Chi-square test was used for categorical variables. Differences were considered significant at p < 0.05.Data were analysed with Predictive Analytics Software 18 statistical package and expressed as median (25th–75th percentile). Differences between study groups and culture treatments were analysed using the nonparametric Mann-Whitney Patients with acute Charcot osteoarthropathy were matched for age, gender, and type and duration of diabetes with the diabetic patients and for age and gender with the healthy control subjects. The age, gender distribution, and type and duration of diabetes were not significantly different between the Charcot patients and diabetic patients nor were the age and gender distribution between the Charcot patients and healthy control subjects .Observation of the cell culture plates with light microscopy showed no difference in osteoclast formation in M-CSF + RANKL-treated cultures between the three groups . The medα to M-CSF + RANKL treatment did not lead to a significant difference in the median number of TRAP-positive multinucleated cells in Charcot patients, diabetic patients, and healthy control subjects  were greater in Charcot patients compared with diabetic patients and healthy control subjects Figures . The addpatients . Althougpatients  and multpatients  was not  Figures .α to M-CSF + RANKL resulted in a significant increase in the percentage of unidented pits  and a significant decrease in the percentage of multidented pits , whereas the percentage of bidented pits remained unchanged   . There w > 0.05) . in vitro study has shown that there was a significant reduction in the resorbing activity of M-CSF + RANKL-treated osteoclasts derived from Charcot patients in response to anti-TNF-α treatment. The addition of anti-TNF-α resulted in significant reduction in the area of resorption on bovine bone discs both on the surface, as assessed by image analysis, and also under the surface, as assessed by surface profilometry. The aberrant erosion profile, pit morphology, and pit distribution in M-CSF + RANKL-treated cultures in Charcot patients were reversed after the addition of anti-TNF-α. These findings confirm the hypothesis that the proinflammatory cytokine TNF-α modulates increased osteoclastic activity in acute Charcot osteoarthropathy.ThisIn the present study, we have shown that newly derived osteoclasts from monocytes isolated from Charcot patients exhibit an enhanced response to RANKL. Osteoclastogenesis is pivotally dependant on M-CSF  and RANKL (key factor for osteoclast differentiation and regulation) , 16. In α as a promoter of the observed enhanced osteoclast function. This cytokine has been linked with inflammatory bone loss [α [α are raised [α has been noted [α to M-CSF + RANKL-treated cultures significantly decreased osteoclast function. This is in agreement with previous data showing that TNF-α is more potent for osteoclast activation than for osteoclast formation [α led to a significant reduction in the area of resorption on the surface and under the surface in cultures from Charcot patients.In Charcot osteoarthropathy bone loss is limited to the inflamed affected foot , 20 and one loss  and immu loss [α . In the e raised . Moreoveen noted . In thisormation . The inhα reversed the observed differences in pit parameters and erosion profile between the study groups and in Charcot patients there was a notable “normalisation” of the erosion profile and pit morphology. This suggests that osteoclasts generated in M-CSF + RANKL-treated cultures, prior to inhibition of TNF-α, exhibit a highly aggressive resorptive profile. This exuberant resorptive activity was reduced after the addition of anti-TNF-α, providing further evidence to support the role of this cytokine in the osteoclastogenesis of acute Charcot osteoarthropathy.As well as increased resorption, in our study, there were a pathological erosion profile and aberrant morphological appearance of resorption pits on bone slices in M-CSF + RANKL-treated cultures in Charcot patients compared with healthy control subjects  and alsoAs well as changes in the morphological appearance of pits, there was a difference in the distribution of the shape of the pits in Charcot patients between the two culture treatments. The resorption pits in M-CSF + RANKL-treated cultures of the Charcot patients were predominantly multidented and bidented whilst the unidented pits were less frequently seen although in diabetic patients and in healthy control subjects the pit distribution remained unchanged between the two culture treatments.α to M-CSF + RANKL-treated cultures resulted in a significant reduction in the percentage of the multidented pits as well as a significant increase the percentage of the unidented pits. Thus, the inhibition of TNF-α normalised the resorptive behaviour of Charcot osteoclasts in which resorption alternated with migration as indicated by a significant increase in the percentage of unidented pits. It is possible that observed aberrant pit morphology and distribution were due to a TNF-α modulation of the resorption cycle ( in vitro studies have shown that agents which can upregulate cathepsin K expression prolong the resorption cycle and resorption events more frequently present as trenches (continuous resorption) [α stimulate the osteoclasts to produce cathepsin K, which is the major protease responsible for the degradation of collagen [α via enhanced cathepsin K expression may lead to imbalance between the relative rate of collagenolysis and demineralisation. This mechanism may explain the continuous resorptions which we observed as multidented pits in the M-CSF + RANKL-treated cultures in contrast to the more frequently noted intermittent resorptions (unidented pits) after the addition of anti-TNF-α [The addition of anti-TNF-on cycle . During orption) . In contcollagen . In Charti-TNF-α  (Figure α in the RANKL-mediated osteoclastogenesis (α [α-primed osteoclastic precursors in the presence of increased local expression of RANKL differentiate into highly aggressive osteoclasts with extensive resorbing activity characterised by increased survival and reduced apoptosis and migration. This increased osteoclastic activity may be due to cathepsin K upregulation, which requires further studies, as this may provide scientific basis for novel intervention with cathepsin K inhibitors. Overall, these aberrantly activated osteoclasts play a key role in the pathological bone destruction of the acute Charcot foot.These findings have important implications for understanding the pathogenesis of this condition. Our data underscores the potent role of TNF-ogenesis . Trauma nesis (α . In the nesis (α . Furthernesis (α  and its α modulated RANKL-mediated osteoclastic resorption in vitro in patients with acute Charcot osteoarthropathy and these observations shed light into the pathogenesis of this devastating condition.In conclusion, using a traditional osteoclast resorption assay together with surface profilometry, this study has demonstrated for the first time that the proinflammatory cytokine TNF-


Functional Ecology from 2004 to 2013 and citation data for papers published in this journal from 1987 to 2011 to examine how title features changed and whether a manuscript's title structure was predictive of success during the manuscript review process and/or impact (citation) after publication. Titles of manuscripts submitted to Functional Ecology became marginally longer (after controlling for other variables), broader in focus (less frequent inclusion of genus and species names), and included more humor and subtitles over the period of the study. Papers with subtitles were less likely to be rejected by editors both pre- and post-peer review, although both effects were small and the presence of subtitles in published papers was not predictive of citations. Papers with specific names of study organisms in their titles fared poorly during editorial (but not peer) review and, if published, were less well cited than papers whose titles did not include specific names. Papers with intermediate length titles were more successful during editorial review, although the effect was small and title word count was not predictive of citations. No features of titles were predictive of reviewer willingness to review papers or the length of time a paper was in peer review. We conclude that titles have changed in structure over time, but features of title structure have only small or no relationship with success during editorial review and post-publication impact. The title feature that was most predictive of manuscript success: papers whose titles emphasize broader conceptual or comparative issues fare better both pre- and post-publication than do papers with organism-specific titles.A poorly chosen article title may make a paper difficult to discover or discourage readership when discovered, reducing an article's impact. Yet, it is unclear how the structure of a manuscript's title influences readership and impact. We used manuscript tracking data for all manuscripts submitted to the journal  Nearly all published scientific papers have a title, and this title is the first part of the paper that prospective readers encounter. The title gives readers a summary of the content of the manuscript, provides keywords and index terms in electronic databases . We then use publicly available citation data (Web of Science) to quantify the relationship between title structure and citations for papers that are published by this same journal, Functional Ecology, from 1987 (volume 1) to 2011 (volume 25).In this study, we examine (1) how the features of manuscript titles  have changed over time, and (2) whether the structure of a manuscript's title is predictive of success of a manuscript during the manuscript review process and impact of a manuscript (using citations counts as a proxy) after publication. Our study is unusual in that we make use of a comprehensive dataset including detailed peer-review data for all manuscripts submitted to one specific ecological journal, Functional Ecology uses ScholarOne Manuscripts  to manage manuscript submissions and peer review. We extracted data from ScholarOne (on 19 December 2014) for all “standard” papers submitted to Functional Ecology between 2004 and 2013 (inclusive). “Standard” papers include all typical research studies , but exclude review papers, commentaries, perspectives, editorials, and other types of papers not considered typical research manuscripts. Over this time period (2004–2013), the journal received 6795 total submissions, of which 6257 were standard papers included in our analyses . For our analyses, we examined these two steps separately, with all papers being categorized as “reviewed” or “not reviewed” after the first stage, and then “rejected” or “not rejected” after the second stage.The editorial review process is generally broken into two steps at We only examine the fate of papers during their initial submission to the journal; a small subset of papers, only 1–2% of invited revisions, are rejected after revision, but these papers are treated as “not rejected” in our analysis because revision was invited. Papers that are rejected but with resubmission invited  are counted twice in our dataset (if resubmitted) because each submission has a unique manuscript number and is treated editorially as a separate paper. Second submissions have a much higher success rate than normal submissions (because they were invited) and thus create a potential bias in our dataset. We considered deleting these papers from the dataset, but this was problematic because paper titles and (less often) author lists can change between the first and second submission, making identifying resubmitted papers difficult. However, only 2.7% of submissions in our dataset were “reject with resubmission” decisions, such that any potential error is at most small. Our analysis does account for papers whose initial decision was overturned on appeal  because this is updated in ScholarOne; our analysis thus uses the final decision postappeal rather than the pre-appeal decision, except for rare cases where such papers are resubmitted as new manuscripts and thus have a new manuscript number.Our dataset also includes how many reviewers were invited for each paper, how many agreed/declined to review (or failed to respond), and how long the peer-review process took for each individual paper.Web Of Science on 17 December 2014. For our analyses of how title structure has changed over time, we include data on all published standard papers (defined above) from 1987 (volume 1) to 2014 (volume 28) (inclusive). However, for analyses of citation counts we only include standard papers published between 1987 and 2011. We exclude papers published after 2011 (2012 to present) because citation counts were generally low with a lot of papers yet to be cited.Citation data were extracted via the Web interface available to library subscribers of Functional Ecology published a total of 2785 papers during the 25-year period included in the citation analysis, of which 2435 were standard papers and included in our analyses of citation counts. An additional 458 papers were published in 2012–2014, of which 415 were standard papers and included in our analysis of the change in title structure over time, but not the analysis of citations.Web of Science database are as published by the journal, but all titles in the ScholarOne database have been entered by authors. A small subset of author-entered titles are known to deviate slightly from the actual title of the submitted paper as printed on the document  submitted by the author. Unfortunately, we cannot access all of the original manuscripts to reconcile such discrepancies – older documents are archived by ScholarOne and only some are available without a substantial per-manuscript fee. We did, however, proof the entire dataset for typos in titles that would affect word count or other classification variables described below.Our focus in this study is on the structure of titles. Titles from the Titles were categorized by hand as being compound titles  or being a question versus statement. We also categorized titles as being amusing or not. Amusing titles generally had subtitles; in most cases, the amusing part of the title was before the colon, with the subtitle presenting a more typical serious title. Titles were categorized as amusing if one part of the title (before or after the colon) conveyed little or no information about the content of the paper but instead appeared (to CWF and CSB) to be a metaphor, a pun or double entendre, or a play on famous quotes, titles, or other phrases in common parlance.Callosobruchus or Callosobruchus maculatus) versus more general titles that did not reference a particular genus or species by their taxonomic name; the latter category includes papers with no organismal reference and those that reference organisms by higher taxonomic groupings  or use common names. One special case is the group Drosophila (the most common group of organisms referenced in titles), which is both the taxonomic name (genus) and the common name of a group of fruit flies; because we cannot identify author intent from their titles, we treated Drosophila as a genus and thus categorized titles including this name as referencing a particular genus or species.One question of interest is whether papers written to be understandable by a narrow versus broad readership fare better during peer review and have more impact post-publication. A proxy for this is whether a title includes reference to a specific study organism by its taxonomic name rather than reference to more widely recognizable common names or higher level taxonomic categories. We thus scored whether titles had a specific organism indicated in the title by genus or species name  and then, if a paper is sent for peer review, whether it was rejected or not . Both models were of the form Reject - SubmissionYear + Question + Subtitle + SpeciesNames + TitleWordCount, with SubmissionYear, Question, Subtitle, and SpeciesNames as categorical variables (0 vs. 1), and TitleWordCount as a covariate (SAS PROC LOGISTIC). Because amusing titles generally contained subtitles, and subtitles were predictive of manuscript fate, we compared manuscript decisions for amusing versus nonamusing titles for the subset of titles that had subtitles, Reject - SubmissionYear + TitleWordCount + Amusing.We used logistic regression to analyze how title structures changed over time , for example, Subtitles - SubmissionYear. We also examined the fate of submitted manuscripts using logistic regression, but in two sequential analyses, first examining whether a paper is rejected How the word count of paper titles changed over time, and whether the number of reviewers invited (ScholarOne dataset) and citation counts were influenced by title structure, were all analyzed using standard general linear models (GLM). Word counts met the assumption of GLM and were not transformed. However, citation counts deviated substantially from the assumptions of GLM, with most papers having few citations and few papers having many citations  first identifying the terms included in the best fit model (SAS PROC GLMSELECT using AICc as our selection criterion), with the starting model Citations - PublicationYear + PageCount + TitleWordCount + Question + Subtitle + SpeciesNames, with PublicationYear, Question, Subtitle, and SpeciesNames as categorical variables, and + PageCount and TitleWordCount as covariates, then (2) individually adding terms not included in the best fit model (SAS PROC MIXED) to confirm they were nonsignificant and that the significance of terms in the best fit model did not change. Additional GLMs presented in the Results section were one-, two- or three-factor GLM models as described in the Results where presented.Functional Ecology increased substantially from 2004 (425 submissions) to 2013 (937 submissions), a total increase of 120% and an average increase of 9.2% per year over the time period included in the peer-review aspect of this study  to 138 in 2013. Because submissions have been increasing much faster than the journal page allocations, editors have necessarily accepted a smaller proportion of submissions; in 2004, editors accepted 24% of submissions (excluding invited papers), whereas they accepted just 15% of submissions in 2013. Most of this difference in acceptance rate is due to stricter pre-review screening; editors sent 87% of submissions out for peer review in 2004, but sent only 50% out for review in 2013. Of papers sent for peer review, 27% were accepted in 2004, whereas 30% of papers sent for review were accepted in 2013 .Submissions of papers for consideration for publication in Functional Ecology changed over the course of the study (2004–2013) . In contrast, the proportion of submissions that had amusing titles increased over time (P < 0.001), although this effect was largely in just the last few years . There was also no statistically significant change in average word count of titles over the period of the study . However, titles were on average longer for papers that included a genus or species name  or had subtitles . After removing these effects, we observed that title word counts increased slightly but significantly over time, a total change of ∼0.6 words (based on least-squares means) over the period of the study .Titles of papers submitted to he study 004–2013 ears Fig.. There wFunctional Ecology  , as did the frequency of amusing titles . In contrast, the frequency of genus and species names decreased over time (P < 0.001). As with journal submissions, title word counts were greater for papers with subtitles  and for papers including genus or species names (by ∼2.2 words) than for papers without . Titles containing questions did not differ in length from titles written as statements . The overall word count of titles did not change across time  but, when controlling for the change in frequency of subtitles and genus/species names in the analysis, we see that word counts of titles increased slightly but significantly, by approximately one word over 25 years .The structure of manuscript titles has changed over the course of the study for the subset of papers actually published by 014) Fig.. The freP - 0.02) and were more likely to be accepted if sent for review (P - 0.001) , although there was no difference in rejection rate between these two types of papers if they were sent for peer review (P - 0.95)  and more likely to be rejected if sent for review (P - 0.02) compared to papers with no organismal reference in their title. Finally, title word count was predictive of a paper's fate. Papers with overly long titles fared especially poorly, although the effect was not linear; papers with intermediate length fared the best through the peer-review process .Three features of titles were predictive of the fate of a paper throughout the editorial process. Papers with subtitles were more likely to be sent for review than papers without subtitles (001) Fig.. Papers .95) Fig.. This neFunctional Ecology manuscripts has become substantially more difficult for editors over the 10 years of the study. In 2004, editors invited an average of 3.53 reviewers per paper, of which 2.05 agreed to review, and 1.94 submitted their reviews. In 2013, editors needed to invite on average 5.18 reviewers (47% more than in 2004) to have on average 2.07 agree to review and obtain 2.00 reviews per paper. This difference in the number of reviewers invited is driven entirely by an increase between 2004 and 2013 in the proportion of invited reviewers that decline to review; in 2004, just 0.89 invited reviewers on average declined per paper whereas 2.45 declined per paper in 2014 .Finding reviewers for 2014 Fig. , paper length  and the presence/absence of a species name in the manuscript title . Title word count and whether the paper title is a question or contains a subtitle did not influence the number of citations a paper received; these terms were not present in the best fit model and were nonsignificant when added individually to the best fit model . Of papers with subtitles, there was no evidence that papers with amusing titles were cited differently from papers without amusing titles .The best fit statistical model describing the number of citations a paper received  included only the year of publication , the usage of humor increased, and the inclusion of specific names (genus and species names of study organisms) in titles decreased over the period of the study. The frequency of subtitles in submissions did not change between 2004 and 2013 (the period covered by our peer-review dataset), but subtitles did increase in frequency among published papers throughout the longer history of the journal (starting in 1987). Papers with subtitles fared better than papers without through editorial and peer review, whereas papers with specific taxonomic names fared poorly during editorial (but not peer) review. Papers with very short titles, and especially those with very long titles, fared more poorly during editorial review than did papers with intermediate length titles. We found no evidence that the structure of titles affects the ability of editors to recruit reviewers for peer review. Only one feature of titles – the presence of specific names – was predictive of manuscript impact; papers containing specific names were less well cited, on average, than were papers lacking specific names.We found that titles of manuscripts submitted to and/or published by Functional Ecology almost certainly reflects the growing competitiveness of high impact factor ecology journals. Submissions have been increasing more quickly than page allocations for many of the top impact journals and thus the criteria for acceptance have gotten more stringent. As a consequence, papers that do a poor job of placing work in a broad conceptual or comparative context probably fare poorly at most top impact journals, and certainly do at Functional Ecology, because these journals preferentially publish papers expected to have broad impact on the field. It is thus likely that the decline in frequency of specific names in titles reflects a growing understanding by authors that papers, or at least titles, need to emphasize broader issues to appeal to editors and likely also to attract readers.The most striking result of our analysis is that papers containing a genus or species name in their title – those referencing a specific study organism by its taxonomic name – fared more poorly during editorial review Fig. and wereFunctional Ecology increased only very slightly over time, an effect only observable after controlling for changes in other features of titles. Increasing word counts of titles over time may reflect increasing complexity of scientific disciplines , but papers with very short titles also fared more poorly during editorial review  titles are preferable to longer titles  were more highly cited than were shorter papers. This is consistent with a variety of previous studies , which was predictive of whether a paper was sent for peer review, did not influence the willingness of reviewers to agree to a review request. This is likely because reviewers are a narrow subset of all scientists specifically because they have expertise and interest in the specific topic and/or organisms examined in the paper.One particularly novel element of our study is our examination of how manuscript titles influence the ability to recruit reviewers for peer review. Like many scientific journals  was predictive of the fate of the paper Fig.; the morFunctional Ecology paper titles have changed over time. Despite a growing body of literature suggesting that specific features of titles are more appealing to authors, or aid in information retrieval, there is little agreement among studies about whether features of titles affect readership or impact of paper. When studies do find relationships between title features and impact, observed effects are quite small. Our data agree with this generalization. One exception seems to be that titles written to focus on narrow aspects of a study – the specific geographic region or population studied  or the specific study species  – are less impactful than papers with titles that emphasize the broader conceptual or comparative context of a study. However, it remains unclear whether this reduced impact reflects cause-and-effect – title structure affects paper impact – or, as we suspect, that titles simply reflect the content of the paper and thus narrowly versus broadly focused titles accurately predict whether a paper will interest a broader readership.The use of subtitles, questions, word count, and other features of


The use of etanercept, a tumor necrosis factor (TNF) inhibitor, has revolutionized the treatment of juvenile idiopathic arthritis (JIA). TNF is a key cytokine implicated in the pathogenesis of inflammatory arthritis and etanercept, which is a soluble TNF receptor fusion protein, binds and inactivates TNF-α and lymphotoxin-A.The aim of this study was to profile serum levels of TNF-α in a large cohort of adolescent patients with JIA.Serum TNF-α was measured in samples derived from 200 adolescent and young adult patients with JIA attending the adolescent and young adult rheumatology clinic at University College London Hospital using a commercial enzyme linked immunosorbent assay (ELISA) kit (eBioscience). Samples were tested in duplicate. Median age at sampling and median disease duration were 18 years and 8 years 9 months, respectively. Male:female ratio was 1:1.2. Equal numbers of patients with polyarticular (n=64) and enthesitis related arthritis  were tested in addition to 48 with oligoarticular, 16 systemic onset, and 8 psoriatic arthritis. Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) measurements were also collected. Furthermore, an L929 cell viability bioassay was used to determine if the addition of etanercept abrogates the cytotoxic effects of TNF-α in L929 cells.Surprisingly, TNF-α serum levels were shown to be markedly elevated in patients on etanercept . TNF-α levels were also higher in patients on etanercept compared to those on other biologics  or disease modifying anti-rheumatic drugs alone , p<0.0001. In addition, ESR and CRP levels had a negative correlation with high TNF-α levels in patients on etanercept (p=0.0018 and p=0.0034 respectively). Etanercept was included at its therapeutic serum concentration (2.4ug/ml) to ensure there was no cross reactivity with the assay. Finally, we showed that the addition to TNF-α to human serum leads to cytotoxicity in a TNF-α sensitive cell line, while adding etanercept at its therapeutic concentration along with TNF-α significantly reduces cell death (p = 0.0277).in vitro experiments in which the cytotoxic effect of TNF-α was abrogated upon addition of etanercept. Our hypothesis is that etanercept prolongs the half-life of circulating TNF-α. Further studies are needed to confirm these findings and dissect the mechanisms involved. As the association between high TNF-α and etanercept treatment is so strong, we hypothesise that it may be possible to measure TNF-α levels as a surrogate marker of adherence to this drug in this cohort of patients where adherence to medication can be a significant problem. This is a hypothesis that warrants further investigation.Patients treated with etanercept have higher levels of TNF-α. As the majority of patients with elevated TNF-α on etanercept were in remission, it is likely that this circulating TNF is biologically inactive. This is supported by our None declared.