Most human immunodeficiency virus (HIV) vaccine trials have lacked efficacy and empirical vaccine lead targets are scarce. Thus far, the only independent correlate of reduced risk of HIV-1 acquisition in humans is elevated levels of V2-specific antibodies identified in the modestly protective RV144 vaccine trial. Ten years after RV144, human and non-human primate vaccine studies have reassessed the potential contribution of V2-specific antibodies to vaccine efficacy. In addition, studies of natural HIV-1 infection in humans have provided insight into the development of V1V2-directed antibody responses and their impact on clinical parameters and disease progression. Functionally diverse anti-V2 monoclonal antibodies were isolated and their structurally distinct V2 epitope regions characterized. After RV144, a plethora of research studies were performed using different model systems, immunogens, protocols, and challenge viruses. These diverse studies failed to provide a clear picture regarding the contribution of V2 antibodies to vaccine efficacy. Here, we summarize the biological functions and clinical findings associated with V2-specific antibodies and discuss their impact on HIV vaccine research. Can vaccine efficacy and immune correlates be reproduced in other human and non-human primate (NHP) vaccine trials?How much did the results depend on the study population, infecting subtypes, and immunogens?Were the correlates of reduced risk of infection, and particularly the high levels of V2-specific antibodies (Abs) as the only independent variable, causally linked with protection or were they solely markers for unrelated protective responses?How do V2 Abs cooperate with other Abs, other adaptive, and innate immune responses?The almost 2 million human immunodeficiency virus (HIV) infections per year worldwide are an incentive to drive progress in the development of a preventive HIV vaccine . The lacAside from the few human vaccine efficacy trials , the majIn addition, studies of natural infection have enabled the investigation of V2 responses that are derived “naturally” after infection with replication-competent primary viruses in the human system. These studies have helped to identify similarities between V2 Ab responses in natural infection and after vaccination. Also, they have enabled to distinguish the requirements for V2 high and V2 low immune responses, to recapitulate the development of functionally potent V2 Abs, to isolate key monoclonal Abs (mAbs), and to identify how V2 Ab responses correlate with other immune responses and clinical outcome ,20,21,22Structure/function studies have illuminated structural requirements in defined V2 epitope regions for the binding of at least four different V2 Ab classes ,26,27,28The variable V1V2 region is located at the apex of the HIV-1 Env trimer and contributes to trimer stability, cellular attachment, and viral entry, e.g., via interaction with α4β7 and the CCR5 or CXCR4 co-receptors ,41. V1V2V1V2 forms a five-stranded β-barrel structure at the apex of the native closed HIV Env trimer  46,47].,47.46,47V2i Abs (such as 830A) bind a conformational epitope overlapping the integrin binding site and preferentially detect the β-barrel conformation. In contrast, V2p Abs (such as CH58) bind linear V2 peptides corresponding to the C-strand of V1V2 (centered on aa 170–176) and are specific for the α-helix (or helix-coil/coil-helix) conformation. The glycan-dependent, quaternary V2q Abs (such as PG9) preferentially detect the closed native Env conformation with V1V2 in β-barrel conformation, whereof the trimer-dependent Ab subclass V2qt (such as PGT145) is exclusive for closed native trimers  22,49].,49.22,49One of the major challenges towards the development of an effective HIV vaccine is the enormous viral sequence diversity ,56. HIV-Compared to other Env regions such as V3, V1V2 is far less immunogenic as evident by the weaker plasma binding levels of V2 Abs in human cohorts of HIV-1 infection and the absence of V2 Ab responses in a fraction of the participants ,59,60,61Different structural binding modalities of the V2 Ab classes are associated with particular biological and antiviral features. Neutralization plays a primary role in vaccine-mediated protection against most viruses, and animal challenge experiments with passive administration of bnAbs indicate that bnAbs can confer protection against HIV if appropriate levels are reached ,77,78. HThe majority of vaccine-induced Abs are V2i mAbs that weakly neutralize mostly tier 1 pseudoviruses  31,34,3,65,80 wiBnAbs occur in a small percentage of HIV-1 infected individuals (10%) after 1–3 years of continuous antigenic stimulation and maturation . A largeV2 mAbs can mediate phagocytosis, which correlated with protection in a rhesus macaque study , but theWhile the antiviral functions of vaccine-inducible V2i and V2p Abs are mostly limited in strength, concerted actions of poly-functional immune responses might provide additive functions ,112,113.The six completed human HIV-1 efficacy (Phase III) and proof of concept (Phase IIb) vaccine trials indicated that the development of an Ab-based vaccine might be more likely than a T-cell-based vaccine . Both T-+ T cell responses have been associated with inferred risk. Specifically, Env-specific poly-functional CD4+ effector memory T cells with the capacity to produce multiple cytokines, such as CD40L, IL-2, IL-4, IFN-γ and TNF-α were identified as a strong beneficial factor. Dominant IFN-γ responses were identified in CD4+ T cells stimulated with CRF01_AE V2 peptides [One of the most puzzling findings of RV144 has been the identification that high Env-specific plasma IgA levels were associated with an enhanced risk of infection. Plasma IgA Abs were shown to interfere with functional IgG Abs directed against key epitope regions in RV144, such as C1 ,125,132.peptides . These dMore mechanistic insights into correlates of protection are expected to come from the running vaccine trials in South Africa, i.e., HIV Vaccine Trials Network (HVTN) 702 and HVTN 705/HPX2008 . HVTN 70In view of the upcoming vaccine trials, the viral diversity in a study population may have a significant impact on vaccine efficacy. As such, the CRF01_AE diversity in Thailand was less pronounced than the clade C diversity in South Africa. Interestingly, the similarity between the clade C protein boost sequences (1086 and TV-1) in HVTN 100 and HVTN 702 and the circulating strains in South Africa is ~8% more distant from each other than the respective RV144 immunogens and prevalent strains in Thailand . On the The passive transfer of Abs to animals followed by virus challenge(s) is a well-established method to determine their antiviral functions. Such experiments have demonstrated that Abs against HIV-1 Env are critical for the protection against HIV-1 ,144,145,BaL.P4 challenge [50 ranging from 0.4 to 36 µg/mL [To test the protective function of weakly-/non-neutralizing anti-V2 mAbs, a single V2i mAb was administered to rhesus macaques with subsequent mucosal SHIVhallenge . The tes36 µg/mL . Passive36 µg/mL . SF162P3 vaginal transmission, the non-neutralizing gp41 mAbs 246-D and 4B3 reduced plasma viral load but had no impact on SHIV acquisition [SF162P4 challenge in seven of seven macaques, while the non-neutralizing CD4bs mAb b6 did not protect any of the five studied animals. Non-neutralizing anti-gp41 mAb F240 achieved protection in two of five passively immunized animals, thus yielding a limited but overall non-significant reduction of infected animals [BaL virus infection, contrary to control mAb CH22 (anti-V3), which prevented infection in 4 of 6 animals [These results are similar to three passive immunization experiments in rhesus macaques that tested the protective functions of non-neutralizing mAbs. Whereas a combination of neutralizing mAbs, 2G12, 2F5 and 4E10, specific to gp120 high mannose carbohydrates and gp41, prevented SHIVuisition . Another animals . The thi animals . These fBaL.P4 at 5 mg/kg, despite a mean neutralization IC50 of 0.06 mg/mL against the same virus, whereas VRC01 and 10E8 were fully protective at the same Ab concentration [325c was achieved at low serum Ab concentrations 0.75 mg/mL for V2qt mAb CAP256-VRC26.25-LS [In contrast to non-neutralizing Abs, many bnAbs effectively protected rhesus macaques from SHIV infection in a dose-dependent manner, including cases of sterilizing immunity with a single intravenous dose. High efficacies have been reported for engineered bi- and tri-specific bnAb constructs and for bnAbs targeting the CD4bs, the MPER region, the outer domain glycans, the V3 glycan, and the V2 apex region ,161,162.ntration . In contDespite the promising results of many passive bnAb SHIV protection studies and a report on vaccine-induced protection from homologous SHIV challenges, which depended on serum nAb titers , it is nProtection experiments in rhesus macaques are important pre-clinical studies to test the efficacy of candidate HIV vaccines. Experiments using SHIV as a challenging virus are the closest model for evaluating the potential of candidate HIV vaccines to generate protective Ab responses in NHPs. The SIV model is more distant from the human system but provides useful data for immune correlate analyses. Some of these NHP experiments analyzed the relationship between the level or titer of vaccine-induced anti-V2 Abs and the outcome of viral infection. SF162, Env-specific CD4+ T cell responses, ADCC, phagocytosis, and MIP-1β in NK cells  vector (40% protection), but not in the arm using non-replicating Ad4 (30% protection) . SeveralNK cells . Though esponses . Comparahallenge .There is [BaL.P4 . Four of+ monocytes and mucosal NKp44+IL17 cells . Surprisingly, only vaccinations with alum reduced the risk of infection (44% vaccine efficacy), which correlated with mucosal anti-V2 Ab levels, whereas vaccinations with MF59 did not achieve significant viral control . Notably+ T cell subsets to the gastrointestinal tract, which is one of the primary sites of HIV and SIV pathogenesis [+ T cells predicts HIV acquisition and disease progression as measured by the increased rates of CD4+ T cell decline and higher viral loads [+ T cells correlated with susceptibility to rectal SIV infection [+ T cells in an HIV attachment-independent manner [V2p, and to a lesser extent V2i Abs, have the unique feature of binding an epitope region, which encompasses the α4β7 integrin binding site. The integrin α4β7 mediates homing of immune cells, including highly HIV-susceptible CD4ogenesis ,189,190.ogenesis , presumaogenesis ,193,194.ogenesis . Consideogenesis ,196,197,al loads . Furthernfection . α4β7 int manner .+ T cells, a steric blockade of the α4β7-gp120/V2 interaction was suggested as a promising target for drug and Ab therapies, and active vaccinations via induction of blocking V2 Abs [+ T cell numbers [Based on the physiological functions and clinical relevance of the attachment and signaling factor α4β7 on CD4g V2 Abs ,201,202.g V2 Abs ,191,203.g V2 Abs . A primag V2 Abs . When ad numbers . In anot numbers . SF162 infection of cells in vitro or in humanized mice [By contrast, a humanized version of the same anti-α4β7 mAb (Vedolizumab) neither prevented nor controlled HIVzed mice . More rezed mice . In a smzed mice . These dzed mice . New genzed mice  and upcozed mice ,197,212 Studies of natural infection provide the opportunity to analyze the development of V1V2 immune responses after sustained viral “priming and boosting” in humans ,213,214.Natural HIV-1 infection is characterized by generally dominant V3 and gp120 Ab responses with subordinate V1V2 responses, caused by the lower immunogenicity of V1V2 compared to V3 ,63. MutaCompared to V2i, V2p Ab responses are less frequent in natural infection  19,20,6,62,226. + T cell decline, which partly explains why bnAbs have no impact on disease progression in natural infection [In contrast to V2i and V2p Abs, which can be readily induced in natural infection and vaccinees, broadly neutralizing V2q or V2qt Abs have only been identified in a small percentage of HIV-1 infected individuals . Neutralnfection . The gennfection ,216,217 nfection . Slower disease progression in natural infection has been associated primarily with host factors, whereas vaccine efficacy is assumed to be associated with humoral responses ,231. RecBased on the high plasticity and inter-subtype diversity in the V1V2 region, V2 Ab responses are complex and regionally different. These features may affect the development of an HIV-1 vaccine. V2q/V2qt bnAbs are highly potent in neutralizing a broad variety of HIV-1 strains, but it has not been possible to induce such bnAbs by vaccination. While germline targeting approaches with sequential boosting are still too far from achieving sufficient neutralization breadth and potency, passive vaccination or combinational treatment options with V2 bnAbs may be realistic goals for the next years. In contrast, V2i and V2p Abs that are readily induced by vaccination or natural infection have not yet proven to be equipped/linked with sufficient antiviral activity to confer independent protection from HIV-1 infection. The controversy after RV144 regarding the role of anti-V2 Abs is still sustained. While several NHP studies identified that V2i and/or V2p Abs correlate with protection from infection, most of these studies used SIV as challenge virus, which is more constrained and accessible in V1V2 based on additional disulfide bonds. The majority of SHIV studies did not provide such correlations. Notably, preliminary data of a current NHP study showed 45% protection from SHIV infection after a V1V2-focused DNA prime/protein boost vaccination; however, neither high V2 Ab levels, nor neutralization, ADCC, or ADCP correlated with protection. This and other studies underline the still incomplete picture. It seems V1V2 immunogens can induce partially protective immune responses; however, it is possible that non-specific/innate immune responses are the driving force while V2 Abs serve as a surrogate marker. The ability of V2i and V2p Abs to block α4β7 has attracted much attention. Animal studies and preliminary clinical data confirmed α4β7 signaling as a biologically important pathway; however, V2 Abs did not consistently block the acquisition of HIV-1 infection. Based on the currently available data, an independent role for V2i or V2p Abs in conferring vaccine efficacy seems unlikely. Ongoing studies will need to address whether concerted effects of V2 Abs with other Abs and Ab-mediated or innate immune responses influence vaccine impact. Since the antigenic and immunogenic features of V1V2 differ according to clade, vaccine design may need to include regional adaptations. For example, CRF01_AE strains were suggested to inherit a naturally more open Env conformation as compared to most other clades, and thus may be more prone to Ab-mediated clearance. Additionally, CRF02_AG infected individuals were reported to exhibit a similar immunologic pattern of V1V2 responses to those observed in RV144 participants to be associated with slower disease progression. In contrast, V1V2 were shown to be less immunogenic in clades B and C. While SHIV challenge experiments in NHP, including both active vaccinations and passive transfer of V2 Abs, did not provide evidence for a protective role of these Abs, further vaccine studies in humans currently underway will help to determine whether V2-specific Abs can contribute to protection from HIV-1 acquisition and which regional/clade-specific differences should be taken into account for the design of a presumably regional HIV vaccine.


Heart failure with preserved ejection fraction (HFpEF) is one of the largest unmet clinical needs in 21st-century cardiology. It is a complex disorder resulting from the influence of several comorbidities on the endothelium. A derangement in nitric oxide bioavailability leads to an intricate web of physiological abnormalities in the heart, blood vessels, and other organs. In this review, we examine the contribution of cardiac and noncardiac factors to the development of HFpEF. We zoom in on recent insights on the role of comorbidities and microRNAs in HFpEF. Finally, we address the potential of exercise training, which is currently the only available therapy to improve aerobic capacity and quality of life in HFpEF patients. Unraveling the underlying mechanisms responsible for this improvement could lead to new biomarkers and therapeutic targets for HFpEF. Heart failure (HF) is a complex clinical syndrome that results from a structural or functional impairment of contraction or filling of the heart. It is a very common condition: 1–3% of the adult population has HF, and the prevalence rises with age . The chiCurrent guidelines divide HF patients according to left ventricular (LV) ejection fraction (defined as LV stroke volume over LV end-diastolic volume) . Signs aIn patients with HF and preserved ejection fraction (HFpEF), LV ejection fraction is normal, although contractile dysfunction is often present and only detected with advanced imaging techniques. End-diastolic pressure elevation and congestion are as severe as in HFrEF . In HFpEHF symptoms are more subtle in HFpEF than in HFrEF and often only present on exertion, a fact that often delays diagnosis. Prognosis is, however, equally grim as in HFrEF: 5-year mortality is around 75%, which is worse than most cancers . NeurohuAge-standardized incidence rates of HF are more or less stable. Still, due to aging of the population and increasing presence of cardiovascular risk factors, the prevalence of HFpEF will steadily increase in the coming decades . These eIn this review, we give an overview of HFpEF pathophysiology. First, we outline the underlying causes of exercise intolerance in HFpEF. Three players in this process are highlighted: the endothelium, comorbidities, and microRNAs. We critically review existing evidence and address gaps in our current knowledge for each section. Finally, we anticipate on the potential effects of exercise training in HFpEF.Several pathophysiologic mechanisms lead to an increased LV end-diastolic pressure in HFpEF, and thus cause HF symptoms . Most paAll patients with HFpEF exhibit some degree of diastolic dysfunction, and diastolic dysfunction is considered a precursor of symptomatic HFpEF. The diastole or filling phase of the cardiac cycle can be divided in two parts. First, the LV changes from a contracted to a fully relaxed state. This is called the “active relaxation” phase, because cardiomyocyte relaxation is an energy-consuming process. The second phase of diastole is called “passive stiffness.” No energy is consumed, but the LV passively stretches under the influence of further filling . A landmIn the absence of mitral valve disease, left atrial pressure reflects LV end-diastolic pressure. Long-standing left atrial hypertension leads to left atrial dilation, which is used as a marker of long-term diastolic dysfunction. HFpEF patients are characterized by an increased left atrial volume at rest and reduced left atrial filling on exertion .Despite a normal ejection fraction, HFpEF patients often have subtle signs of systolic dysfunction. Global longitudinal strain, a very sensitive parameter of LV contraction, is reduced at rest in HFpEF patients . More drApart from the cardiac abnormalities mentioned above, the blood vessels also play a vital role in HFpEF pathophysiology. Increased stiffness is not only seen in the LV, but also in large arteries such as the aorta. Invasive measurement of arterial waveforms shows reduced arterial compliance and higher arterial elastance at rest in HFpEF patients, independent of blood pressure . Arteria2) demands of skeletal muscles. This reactive vasodilation is regulated by shear stress on the endothelial cells and is impaired in almost half of HFpEF patients  and its second messenger cyclic guanosine monophosphate (cGMP), producing relaxation of the vascular smooth muscles and widening of the blood vessel .−) and reducing NO bioavailability. ROS also uncouple endothelial NO synthase, which starts to produce highly reactive superoxide , endothelial homeostasis is disturbed . These runction” .Endothelial dysfunction is considered the first step in the atherosclerotic process and a precursor to overt cardiovascular disease . Clinica+ cells form tube-like structures and express endothelial markers in vitro  . Mice wiBoth epidemiological studies and randomized trials consistently showed that most HFpEF patients are women (50–84%) . This seWhile we do not fully understand these divergent responses, perhaps the underlying molecular mechanisms leading to HFpEF differ according to sex. Endothelial dysfunction and arterial stiffness are less prominent in women , but on Also, the comorbidity profile differs between male and female HFpEF patients. Recent “big data” approaches to datasets of HFpEF risk factors have shown that different phenotypes can be identified, some of which exhibit a sex-specific dominance . These pObesity, arterial hypertension, and diabetes mellitus are common in HFpEF patients and often coexist . Arteria2)  .Diabetes mellitus can contribute to the development of HFpEF through several pathways. First, diabetes mellitus is associated with a systemic inflammatory state and increased oxidative stress, causing microvascular dysfunction and LV hypertrophy . Second,Arising from a systemic inflammatory state and increased oxidative stress, patients with hypertension, obesity, or diabetes also have impaired endothelial function and reduced EPC and TA levels .Most data on anemia and iron deficiency in HFpEF are extrapolated from studies in HFrEF patients. Iron deficiency is the most frequent cause of anemia in HF patients, and both anemia and iron deficiency without anemia predict mortality . BesidesWhile we know that abovementioned comorbidities contribute to endothelial dysfunction and diastolic dysfunction, the underlying mechanisms leading to these changes are unknown. Most animal models of HFpEF are young, male animals subjected to an acute event  which does not accurately reflect HFpEF pathophysiology. Also, the relation between comorbidities and vascular dysfunction has never been examined specifically in HFpEF patients. Finally, an important barrier in identifying sex-specific underlying mechanisms of HFpEF is the predominance of male animals in experimental studies .Unlike HFrEF, in which several monogenic subtypes have been defined, little is known about potential genetic determinants of HFpEF . Some geAlthough about 75% of the human genome is transcribed, less than 2% is translated into proteins. However, the remaining “noncoding” transcripts do participate in regulation of biological processes, through interaction with coding RNA . MicroRNMicroRNAs can be secreted in the circulation, packed in exosomes and microparticles, or bound to lipoprotein complexes or RNA-binding proteins. These circulating microRNAs are stable in plasma and thus form attractive biomarkers . Cells rIn HF, microRNAs have been investigated as possible biomarkers to aid in diagnosis. Several microRNAs provide benefit over traditional biomarkers to differentiate HFrEF from HFpEF . Also, sAs active participants in cellular cross talk, microRNAs are also attractive therapeutic targets. Inhibiting a microRNA or mimicking its activity potentially influences dozens of genes, which could lead to larger treatment effects compared to standard drugs . While mAs microRNA research is still emerging, little evidence exists, and many questions remain. Is there a mechanistic link between microRNAs and exercise intolerance in HFpEF? Or between microRNAs and vascular function? Is microRNA expression influenced by exercise training? What are the downstream targets of these deregulated microRNAs and can they become a novel therapeutic target? Can microRNAs be used as biomarkers to identify HFpEF phenotypes, or HFpEF patients with certain traits ?2 and/or quality of life , a noncardiac factor , or both. In HFrEF patients, both cardiac and noncardiac factors are improved by exercise  is still unknown. Moreover, the effects of exercise training on microvascular endothelial function and cellular endothelial repair in HFpEF are unknown. The recently completed multicenter Optimizing Exercise Training in Prevention and Treatment of Diastolic Heart Failure (OptimEx) and Ex-DHF 2 trials will soon shed more light on the benefits of exercise training in HFpEF.Evidence regarding the cause of improvement in peak VOHFpEF is one of the largest unmet clinical needs in cardiology. It is a complex disorder with a central role for endothelium dysfunction induced by several comorbidities. Endothelial dysfunction leads to an intricate web of physiological abnormalities in the heart, blood vessels, and other organs. Several microRNAs have been identified as crucial regulators of pathophysiological processes underlying HFpEF, and could form interesting future diagnostic and therapeutic tools. Exercise training is currently the only available therapy to improve aerobic capacity and quality of life in HFpEF patients. Unraveling the underlying mechanisms responsible for this improvement could lead to new biomarkers and therapeutic targets for HFpEF.AG and EV contributed to the conception and design of the paper. AG drafted the manuscript. JB, VS, and EV contributed to the critical revision of the manuscript. VS and EV obtained funding and supervised the work.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.


Salmonella spp. and Listeria monocytogenes), Enterobacteriaceae and Pseudomonas spp., even if a limited presence of Staphylococci was observed. No biogenic amine histamine was detected and negligible levels of total volatile basic nitrogen (TVB-N) were revealed. Total bacterium, yeast and mold counts were negligible or undetectable by conventional accredited methods, and conversely the results were higher when optimized saline conditions were used. This study, for the first time, established a set of quality and safety parameters necessary for first-operations and subsequent processing of jellyfish as novel food. Highlights: Jellyfish can represent a novel food in Europe. Identification of safety and quality parameters for jellyfish food products. Saline conditions are essential for improving safety and quality assessment of jellyfish as food.Edible jellyfish are mainly consumed and marketed in Southeastern Countries, generally produced by a multi-phase drying process, using mixtures of salt and alum. Recently, jellyfish have become very attractive also for Western food markets. They are novel food in Europe and no recognized handling/processing steps have been set up yet. Moreover, no specific food safety and quality parameters are available. In this study, we identified a set of safety and quality parameters for jellyfish, based on standards and process hygiene criteria used in Europe for other products. These assays were tested on three different jellyfish preparations that can be used as raw materials for subsequent food processing. All jellyfish samples revealed the absence of pathogens ( In Southeast Asia, the fishery and market of edible jellyfish (JF) is around 1 million tons of JF landings and the corresponding estimated business exceeds 100 million US $ ,3,4. CurVery recently, the Food and Agriculture Organization of the United Nations (FAO) has considered that the introduction of JF as new seafood products for human consumption, and as feed for aquaculture, can represent a possible strategy to improve the overall sustainability of the global fishery sector . Indeed,In Europe, the lack of food tradition makes edible JF market still virtually absent or possibly restricted to the Asiatic immigrant community. Currently, JF and JF-derived food products are not explicitly mentioned in the European regulation on microbiological criteria for foodstuffs ,15, evenRhopilema, Nemopilema, and Stomolopus [Commercially exploited JF belong to the phylum Cnidaria, class Scyphozoa and particularly to Rhizostomeae species of the genera omolopus ,11,18. Aomolopus ,19,20. Homolopus . Then, aomolopus .At the present time, a production chain both for edible JF and JF-derived foods does not exist in Europe and handling/processing steps together with associated critical control points have to be determined.Rhizostoma pulmo, from the Ionian Sea Italian coasts has been assessed as model for human food preparations alternative to traditional ones. Moreover, the necessity to develop new specific and dedicated assays for JF and JF-derived products, mainly due to the possible presence of pathogens and spoilage microbiota and/or possible toxic metabolites, has been here demonstrated.In this study, the main safety and quality parameters suitable for the future risk assessment of JF, as a novel food in Europe, were identified. These parameters were tested on three different JF preparations that can be used as raw materials for subsequent food processing. The barrel JF, Rhizostoma pulmo were collected offshore in Ionian sea  in four sampling periods , with a nylon landing net with 3.5 cm mesh size, from an open boat. Three different types of samples were produced (Specimens of jellyfish (JF) species produced : 1) wholEnterobacteriaceae and Pseudomonas spp.All the three types of JF samples were delivered to the external accredited laboratory  stored in sterile plastic bags on ice in a box. The accredited laboratory performed the analyses, with the exception of assays for enumeration of Escherichia coli [Staphylococci [Salmonella spp. [Listeria monocytogenes [Ten grams of each sample were added to 90 mL of Buffered Peptone Water  as diluent (1:10) and homogenized for 2 min in a Stomacher, in accordance with specific standard methods for total bacterial count , coliforhia coli , coagulaylococci , yeast aylococci ,28. For lla spp.  and Listytogenes , 25 g ofStaphylococci was checked on Baird Parker Agar with Rabbit Plasma Fibrinogen  supplement at 37 °C for 24 h, whereas diluted samples were plated by pour plating method on ColiID Agar , and incubated at 37 °C for 24 h for the enumeration of coliform bacteria (blue colonies) and E. coli (red colonies).For the total bacterial count (TBC), samples were diluted and plated by pour plate technique on Plate Count Agar (PCA)  pH 7.0 and incubated at 30 °C for 72 h; enumeration of yeast and molds were performed by incubation at 25 °C for 5 days on Dichloran Rose–Bengal Chloramphenicol Agar . The presence of coagulase-positive Salmonella spp. was performed according to UNI EN ISO 6579:2017 methods [Salmonella Shigella (SS) agar  and Xylose-Lysine-Desoxycholate (XLD) Agar  at 37 °C for 24 h. Colonies grown on these media were streaked on nutritive agar at 37 °C for 24 h and finally identified by API  and specific antiserum test.Determination of  methods . The staListeria monocytogenes was identified by ISO 11290-1:2017 method [7 method . AccordiEnterobacteriaceae enumeration by incubation at 37 °C for 18–24 h; Pseudomonas Agar Base  supplemented with Cetrimide, Fucidin, Cephaloridine (CFC) selective agar supplement  was used for Pseudomonas spp. identification by incubation at 30 °C for 18–24 h.Further microbiological analyses were applied by testing diluted samples in peptone water on agar slants containing the following media: Violet Red Bile Glucose Agar  was used for The total volatile base nitrogen (TVBN) was determined by treating each jellyfish sample (100 g) with 0.6 M perchloric acid . After alkalization, the extract was exposed to steam distillation and an acid receiver absorbed the volatile base components. The TVBN concentration was determined by titration of the absorbed bases following the method EC Regulation N. 2005/2074 .Histamine concentration was determined according to the method AOAC N° 021402 (2014)  .2, 0.54% MgSO4, 0.1% CaCl2, w/v [JF samples were homogenized with a sterilized blender and 25 g of each sample were added to peptone seawater . All san = 3).All data represent the mean of three independent replicates (p < 0.05). All statistical comparisons were performed using Sigma-Stat, version 3.11 .Statistical analysis was based on one-way analysis of variance. Tukey’s post-hoc method was applied to establish significant differences among means  whoSalmonella spp. and L. monocytogenes were not detected in all the four sampling times (June-July-August-October 2018). Total counts of Staphylococci showed a similar trend in all water and JF samples. Indeed, they ranged from 0 to 1.4 × 102 CFU/g in the untreated samples (WJ), from 0 to 3.7 × 102 CFU/g and from 0 to 2.6 × 102 CFU/g in SJ and SFJ samples, respectively. These data can be correlated to the Staphylococci presence in seawater samples (C), where total counts ranged from 0 to 1.5 × 102 CFU/g. Escherichia coli and coliforms were undetectable in WJ, SJ and C samples during the entire period of sampling. Relatively low level of E. coli and coliforms contamination were detected in SFJ. It was up to 5.5 × 101 CFU/g and up to 7.7 × 102 CFU/g, respectively. However, TBC in all JF samples and in all sampling times ranged from 2.7 × 101 CFU/g to 8.5 × 102 CFU/g. Yeast and moulds were undetectable in all JF and seawater samples. In addition, no histamine was revealed in any tested samples during the samplings, revealing that microorganisms detected in some samples were not able to produce this biogenic amine in these conditions. In our study, TVBN ranged between 0.22 and 1.39 mg/100 g in WJ samples, between 0 and 1.39 mg/100 g in SJ samples and between 3.51 and 5.56 mg/100 g in SFJ samples. Among all microbiological and chemical parameters applied to the different JF treatments and to seawater samples along the four sampling times, statistically significant differences were observed only for TVBN values  did not contain common pathogens, except for Staphylococci in few cases, which can probably derive from the specific marine environment. Moreover, no quality loss was detected since TVBN was very low in all samples. In addition, the absence of Enterobacteriaceae and Pseudomonas spp. was verified in all JF and in seawater samples.These analyses revealed that JF . Total counts of bacteria, yeasts and molds on saline media are reported in 2–4.5 × 103 CFU/g to 2 × 101–6.2 × 102 CFU/g moving from WJ to SFJ samples, respectively.In saline PCA medium, total bacteria counts were almost similar to the values observed using the conventional accredited method. Instead, the counts in R2A and Marine Agar media resulted at least one order of magnitude higher than the corresponding values for total bacteria in non-saline medium . However1–1.5 × 103 CFU/g in WJ samples to 2.8 × 101–5.5 × 101 and 0–2.5 × 101 CFU/g in SJ and SFJ samples, respectively  were useful to produce a statistically significant reduction of the presence of yeast and molds from 9.8 × 10ectively .The first goal of this study was the identification of a set of main relevant quality and safety parameters to be applied for the possible consumption of JF-based foods in Europe. In the absence of specific indications, we considered market safety standards in force in Europe for food products comparable to JF ,15,31, tStaphylococci detected in all tested JF samples and in seawater may be attributable to hygienic conditions and pollution of marine environment, since these microorganisms are generally from both human and animal sources.These parameters worked efficiently when applied to different steps of JF preliminary treatment, traditionally used in Asiatic processing methods . Indeed,Salmonella spp., Listeria monocytogenes, Vibrio spp. and Aeromonas hydrophila tests, together with heavy metal contaminants and allergenic assays on raw material and umbrella product snack derived by edible JF Catostylus tagi [In this study, the direct application of accredited available microbiological and chemical assays seems to reveal JF cannot represent a source of severe microbiological hazards for humans. Recently, Raposo et al. have reported similar evidence by applying some safety important parameters, such as lus tagi .The need to increase knowledge on JF applicability as novel food for humans can be demonstrated by the presence of other potential microbiological safety hazards associated with them, such as discrete counts of microorganisms with halophilic/halotolerant characteristics not revealed by the regulated criteria and counting methods for foods in force in Europe. Although preliminary in their application, the saline media can be useful in the detection of both bacteria and yeast and molds during treatment steps of JF derived products. Even though traditional culture-dependent methods, generally applied in accredited methods, provide information regarding only the cultivable part (1–5%) of the total microbial community , this laAspergillus, Cladosporium, Purpureocillium, and Tilletiopsis were isolated as JF-associated microorganisms in Nemopilema nomurai [Cyanea capillata and C. lamarckii, has been suggested by Schuett and Doepke [Aurelia aurita [Stenotrophomonas spp., Alteromonas spp., Pseudoalteromonas spp. and Vibrio spp.) in terms of tissue residence has not been determined [Fungi belonging to different genera  nomurai , whereasd Doepke . The prea aurita . Howevertermined ,45. At ttermined ,47,48,49Experiments are now in progress for the identification and characterization of bacteria and fungi isolated from JF together with their possible toxic or beneficial metabolites, in order to discover their possible negative  or positive  role for humans. Future studies should consider metagenomics and metabolomics approaches for the analysis of raw and processed JF for obtaining information on total microbiota associated to JF and qualitative and quantitative data on microbial metabolites.Rhizostoma pulmo (known as sea lung or barrel JF) was used here as model organism, since it is considered edible in some Asian countries [Aurelia aurita, Cotylorhiza tuberculata, Rhopilema nomadica, etc) suitable to be used as novel foods in Western countries.ountries  and it hountries ,12,13. TIn this study, a collection of quality and safety parameters together with “process hygiene criteria” were identified and applied for assessment of JF as novel food in Europe. The results here reported indicate the importance of basic JF treatments by separating the umbrella and oral arms, by eliminating gastric contents followed by extensively washing the material with fresh water in order to maintain good quality and safety levels before processing to obtain a final product. These findings provide experimental data on the thousand-year old practice of JF pre-treatment in Asian Countries.The use of optimized saline media is proposed here as an important emerging tool for going beyond the limits of conventional accredited methods for safety risk assessment of JF as human food. The present results, although preliminary, are in line with Commission Regulation (EC) N. 2073/2005 , where i


ADAMs perform proteolytic processing of the ectodomains of diverse transmembrane molecules into bioactive mediators. This review summarizes on their most well-known members, ADAM10 and 17, focusing on the kidneys. ADAM10 is expressed in renal tubular cells and affects the expression of specific brush border genes, and its activation is involved in some renal diseases. ADAM17 is weakly expressed in normal kidneys, but its expression is markedly induced in the tubules, capillaries, glomeruli, and mesangium, and it is involved in interstitial fibrosis and tubular atrophy. So far, the various substrates have been identified in the kidneys. Shedding fragments become released ligands, such as Notch and EGFR ligands, and act as the chemoattractant factors including CXCL16. Their ectodomain shedding is closely correlated with pathological factors, which include inflammation, interstitial fibrosis, and renal injury. Also, the substrates of both ADAMs contain the molecules that play important roles at the plasma membrane, such as meaprin, E-cadherin, Klotho, and CADM1. By being released into urine, the shedding products could be useful for biomarkers of renal diseases, but ADAM10 and 17 per se are also notable as biomarkers. Furthermore, ADAM10 and/or 17 inhibitions based on various strategies such as small molecules, antibodies, and their recombinant prodomains are valuable, because they potentially protect renal tissues and promote renal regeneration. Although temporal and spatial regulations of inhibitors are problems to be solved, their inhibitors could be useful for renal diseases.A disintegrin and metalloproteinases (ADAMs) are a Zn ADAMs are approximately 750 amino acids long and evolutionarily conserved, and 22 ADAM genes have already been identified in humans. ADAMs cleave a variety of transmembrane proteins at the plasma membrane, a process which is known as ectodomain shedding , a superfamily of Znshedding . ADAM10 shedding . Althougshedding .ADAM10 is ubiquitously expressed in various mammalian cells and reacts with more than 40 substrates . ADAM10 de novo.ADAM17, also named TACE (TNF-α converting enzyme), is the most widely studied, and releases the ectodomain of various substrates from their transmembrane preforms to produce active soluble ligands. After ectodomain shedding, these ligands bind to receptors, which lead to downstream signaling. ADAM17 is widely expressed in various tissues including the kidney, and its expression changes during embryonic development and adult life . Especiain vitro studies, it may be found that CADM1 ectodomain shedding could contribute to the development of chronic kidney disease (CKD).Cell adhesion molecule 1 (CADM1) is an intercellular adhesion molecule that belongs to the immunoglobulin (Ig) superfamily, and it is localized on the lateral cell membrane and mediates neighboring cell–cell binding . It funcPkd1 (an ADPKD responsible gene) mutation or deletion promotes the maturation of ADAM10 via Gα12 activation, which increases E-cadherin shedding and results in the cystogenesis of renal TECs.E-cadherin forms adherens junctions between areas of cell–cell contact through its ectodomain, and it plays crucial roles in the integrity of cellular polarity and cell–cell adhesions . It can CXCL16 not only functions as an adhesion molecule for CXCR6, but also plays an important role as a scavenger receptor for oxidized low-density lipoprotein (oxLDL) . The humProinflammatory tumor necrosis factor (TNF)-α belongs to a family of both soluble and cell-bound cytokines, and it is produced by immune cells and vascular endothelial cells, but also renal TECs and mesangial cells . TNF-α aTwo epidermal growth factor receptor (EGFR) ligands, heparin-binding (HB)-EGF and transforming growth factor (TGF)-α, are involved in proliferative, migratory, and fibrotic responses of tubular cells. Elevated ADAM17 activity causes sustained EGFR activation and fibrosis after kidney injury . The incAngiotensin-converting enzyme 2, highly expressed in renal PCTs, degrades the vasoconstrictor angiotensin II (ANG II) to ANG-(1-7) . It is sKidney injury molecule-1 is a receptor for phosphatidylserine, an efferocytosis signal on the surface of apoptotic cells that labels them for phagocytic clearance. Its expression is induced on PTECs in ischemic AKI, and KIM-1 ectodomain shedding generates a soluble fragment that serves as an important biomarker for AKI. Oxidative stress accelerated KIM-1 shedding . Of noteNotch is a critical regulator of renal development, and its signaling is involved in both acute and chronic kidney injuries . Its ove2+-dependent metalloproteinases that are highly expressed at the brush-border membranes of the kidney and evolutionarily related to other proteases, MMPs and ADAMs  can overcome the problems of hydroxamate-based compounds. The 8C7 mAb masked the ADAM10 recognition pocket and was more efficient than GM6001, a broad-spectrum metalloprotease inhibitor . BecauseAlso, ADAM17 antibodies were developed. D1(A12) antibody binds to both catalytic and non-catalytic domains of ADAM17 . HoweverThe recombinant mouse ADAM10 prodomain is a potent competitive inhibitor of human ADAM10 activity with higher selectively .ADAM17 prodomains could also be valuable inhibitors. A stable form of the auto-inhibitory TPD (TACE prodomain) inhibits ADAM17, but does not prevent the related ADAM10 activity . FurtherSome natural compounds reduce ADAM10 activity. Rapamycin suppresses ADAM10 activity  and prevBecause targeted inhibition of active ADAM10 and/or 17 is expected to become a potential therapy for associated diseases, these strategies have been advanced. However, ADAM10 and 17 have many substrates with diverse functions; therefore, it is important for the temporal and spatial regulation of inhibitors to avoid undesirable side effects.TK conceived the idea and wrote the manuscript. MH and AI edited the manuscript and helped to improve the quality of this review paper.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.


For decades, the glial function has been highlighted not only as the ‘structural glue’, but also as an ‘active participant’ in neural circuits. Here, we suggest that tumor necrosis factor α (TNF-α), a key inflammatory cytokine, alters the neural activity of the cerebellar Purkinje cells (PCs) by facilitating gliotransmission in the juvenile male rat cerebellum. A bath application of TNF-α (100 ng/ml) in acute cerebellar slices elevates spiking activity of PCs with no alterations in the regularity of PC firings. Interestingly, the effect of TNF-α on the intrinsic excitability of PCs was abolished under a condition in which the type1 TNF receptor (TNFR1) in Bergmann glia (BG) was genetically suppressed by viral delivery of an adeno-associated virus (AAV) containing TNFR1-shRNA. In addition, we measured the concentration of glutamate derived from dissociated cerebellar cortical astrocyte cultures treated with TNF-α and observed a progressive increase of glutamate in a time-dependent manner. We hypothesised that TNF-α-induced elevation of glutamate from BGs enveloping the synaptic cleft may directly activate metabotropic glutamate receptor1 (mGluR1). Pharmacological inhibition of mGluR1, indeed, prevented the TNF-α-mediated elevation of the intrinsic excitability in PCs. Taken together, our study reveals that TNF-α triggers glutamate release in BG, thereby increasing the intrinsic excitability of cerebellar PCs in a mGluR1-dependent manner. The gliotransmission that primarily occurs between neurons and astrocytes contributes to Hebbian and non-Hebbian synaptic plasticity in a variety of brain regions6. In the cerebellum, PCs are surrounded by the BGs, a particular type of astrocyte in the cerebellar cortex, with processes extending into the molecular layer where parallel fibres (PFs) contact the dendrites of PCs, thereby enveloping synaptic connections7. Depolarising stimulation of BGs via photostimulation of BGs induces release of glutamate, thereby mediating long-term depression (LTD) at the synapse between PFs and PCs upon a mGluR1-dependent manner2. In addition, strong stimulation at PFs induces calcium transients within BGs, which is linked to the direct activation of mGluR1 and the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) expressed on BGs. Collectively, glutamatergic signalling from BGs into PCs is strongly implicated in cerebellar synaptic plasticity and the cerebellum-mediated behaviours10.Under physiological and pathological conditions, glial cells are involved in the dynamic and/or sustained modulation of neural circuits by releasing excitatory and inhibitory gliotransmitters, such as glutamate and γ-aminobutyric acid (GABA), respectively14. Of interest, glial TNF- α participates in synaptic adjustments against prolonged activity-deprivation in the hippocampal neurons, which is mainly mediated by the internalisation and/or insertion of AMPARs and GABA receptors (GABARs) in neurons15. In addition, exogenous application of the TNF-α triggers glutamatergic gliotransmission, resulting in the strengthening of the synaptic weight in the hippocampus17. In the cerebellum, the basal level of TNF-α is constitutively altered during the cerebellar development and abnormal changes in the concentration of TNF-α result in the manifestation of cerebellar dysfunction or diseases, indicating that TNF-α is necessary for the development of cerebellar neurons18. However, it is yet to be addressed what roles of TNF-α plays in modulating the intrinsic excitability of cerebellar PCs.Immune-related cytokines such as TNF-α, interleukin-1β (IL-1β), and IL-6 have been reported to be considered as modulators in controlling the functional interactions between neurons and glial cellsIn the present study, we investigated the physiological effects of the TNF-α on the intrinsic excitability of PCs and whether BGs are involved in TNF-α-mediated changes of PC activity in the cerebellar circuits. Notably, TNF-α treatment in acute cerebellar slices potentiated the firing rates of the cerebellar PCs, which is thought to be mediated via the facilitation of glutamate release from the BGs. The elevation of intrinsic excitability in PCs was fully abolished by pharmacological blockage of type 1 metabotropic glutamate receptor (mGluR1), further suggesting that TNF-α-mediated an increase of excitability in PCs is required for the activation of mGluR1 in PCs.ARs were inhibited by 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione  and picrotoxin (100 μM), respectively, in order to exclude the unexpected involvement of excitatory and inhibitory inputs in the intrinsic properties of PCs. Treatment of TNF-α significantly increased the spontaneous firing rates of the cerebellar PCs compared to baseline for 10 minutes  in BGs in a cell-type specific manner using a molecular genetic strategy. A viral vector approach with an AAV using the pSicoR system  containing a TNFR1-shRNA was generated19 and then delivered into a cre recombinase expressing mice line in PCs (PCP2-cre)  = 75.8 ± 15.0, n = 9; TNFR1-shRNA = 9.6 ± 19.4, n = 4, p = 0.03, t-test; Fig. It has widely been reported that TNF-α modulates neural activity via the activation of glial cellsre) Fig. . In thisn PCs PCP-cre , a potent blocker of the excitatory amino acid transporter, to the cerebellar slices and then monitored the excitability of PCs under the presence of excitatory and inhibitory synaptic transmission inhibitors. Notably, the excitability of PCs was not changed by pharmacological inhibition of glutamate transporters  in the cerebellar astrocyte cultures following application of TNF-α. Our data revealed that glutamate concentration was increased in a time-dependent manner and a significant change was detected in 30 minutes after TNF-α treatment [0.52 ± 0.10 μM  vs. 2.28 ± 0.81 μM , q = 3.925, p < 0.05, one-way ANOVA on Ranks post-hoc Tukey test; Fig. Previous reports have described that neurons communicate with surrounding glial cells through gliotransmitter such as glutamate and ATP10. However, the underlying mechanism are less understood. Here, we provided insights into how TNF-α regulates the excitability of PCs in cerebellar circuits. In this study, we found that TNF-α facilitates glutamatergic gliotransmission, leading in turn to the potentiation of intrinsic excitability in cerebellar PCs. When expression of TNFR1 in BGs was inhibited in a cell-type specific manner, the TNF-α-induced intrinsic plasticity of PCs was abolished, implying an involvement of BG activity in potentiation of PC excitability after TNF-α treatment. Strikingly, in a cerebellar astrocyte culture, incubation of TNF-α gave rise to an increase of glutamate, thus we suggest that TNF-α-induced intrinsic plasticity of PCs is facilitated via glutamate release from BGs. Furthermore, the alterations in active properties and frequency of PC firing following TNF-α application was prevented by pharmacological inhibition of mGluR1 and the mGluR1-mediated current was augmented after TNF-α treatment. Taken together, our results propose a plausible mechanism by which BGs contribute to the modulation of the intrinsic excitability of PCs in the cerebellum.Neuro-glia interactions are known to be crucial for the functional modulation of cerebellar behaviours24. Photostimulation of BGs can trigger glutamate release onto PCs, giving a rise to mGluR1-dependent LTD between PF-PC synapses and induction of the horizontal optokinetic reflex upon repetitive visual stimuli2. Our observation that genetic suppression of TNFR1 in BGs does not induce TNF-α-mediated elevation of intrinsic excitability in PCs  channels could also be a contributors to the mGluR1-dependent intrinsic plasticity by TNF-α based on the literatures reporting that mGluR controls the BK gating and modulate the pacemaking activity35. Small conductance Ca2+-activated potassium (SK) channels could be one mechanism for the TNF-α-induced intrinsic plasticity because activity-dependent intrinsic plasticity is modulated by SK channel activity36. In addition, considering our observations from this study, including the reduction of spike onset time, and latency, D-type potassium channels might also be required for the TNF-α-induced intrinsic plasticity in PCs39. Collectively, TNF-α-induced intrinsic plasticity might involve synergistic alterations of several ion channels that modulate neural excitability40, nevertheless detailed mechanisms by which activation of the BG affects the PCs activity need to be further investigated.Activation of mGluR1 enables te persistent change in neuronal excitability as well as the induction of synaptic plasticity through the conductance alterations in a variety of ion channels42. For example, pro-inflammatory cytokines including TNF-α have been reported to be elevated in psychiatric disorder patients43. The concentration of TNF-α we used in this study (100 ng/ml) was quite high, which is likely to reflect a more pathophysiological circumstance. This concentration could lead to an imbalance of AMPA/GABA receptor activity at dendrites of neurons and give rise to inappropriate expression of Ca2+-permeable AMPA receptors not only at synapses but also in extrasynaptic sites45 which in turn could result in excitotoxicity. In addition, local inflammation can triggers abnormal strengthening of synaptic weight through TNF-α, consequently causing an impairment in memory function46. Therefore, the TNFR1 gene has been considered to be an attractive therapeutic target for reducing AMPA receptor-mediated excitotoxicity47. In contrast, TNF-α serves as protective and adaptive cytokine for regulating the homeostasis of the neural network activity49. Of interest, ablation of TNF signalling shows more severe neurological impairment and demyelination with high mortality in animal models such as autoimmune diseases and brain injury50. Although it remains unclear whether the amount of TNF-α (100 ng/ml) used in our study represents physiological conditions, a certain amount of TNF-α is indeed necessary for stabilising neural excitability in the brain. Thus, further investigation on how TNF-α affects cerebellar network activity has to be addressed. Taken together, TNF-α may act as a functional switch that not only determines the magnitude of basal neural activity, but also regulates the functional homeostasis of the cerebellum.Previous implications have shown that the alterations of the levels of TNF-α in the serum and/or in the cerebrospinal fluid are associated with various brain diseases in human52. The data presented here shows electrophysiological outcomes of TNF-α elevation in cerebellar circuits. Alteration of PC spiking activity indeed has been implicated in the underlying mechanisms of neurodegenerative diseases and seizure generation54. Thus, the TNF-α-induced potentiation of excitability might reflect several cerebellum-related diseases. In a different pathological viewpoint, TNF-α-induced elevated excitability in PCs may undergo homeostatic adjustments of the cerebellar network by decreasing a potential increase in the deep cerebellar nuclei against neuronal hyper-excitability upon brain inflammation. We previously reported that chronic alteration of cerebellar activity leads to homeostatic changes in the intrinsic excitability of PCs, suggesting that an increase of TNF-α could serve adaptive functions in the cerebellum30. TNF-α also plays a neurotrophic role in the differentiation of PCs at the early developmental stages18. In addition, given the note that prolonged optic-stimulation of PCs facilitates elimination of climbing fibres (CFs)55, elevation of excitability in PCs through TNF-α could be involved in the fine-tuning of cerebellar development. Lastly, the fact that glutamate released from BGs is involved in PF-PC LTD and regulation of motor behaviour suggests that glutamatergic gliotransmission may be critical for neural plasticity in the cerebellum10. Here, we demonstrated that TNF-α facilitates glutamate spillover which enables the activation of mGluR1 leading to induce the neural plasticity in PCs. Further studies should address how elevation of TNF-α in the cerebellum contributes to synaptic and intrinsic plasticity in the cerebellar cortex and influences cerebellum-associated behaviours.What is the functional consequence of TNF-α-induced intrinsic plasticity of PCs in the cerebellar cortex? Elevation of TNF-α concentration in the cerebellum has been implicated in cerebellar pathophysiological dysfunction2, 2 CaCl2, 1.25 NaH2PO4, 26 NaHCO3, 10 glucose bubbled with 95% O2 and 5% CO2. For recovery, slices were incubated at 32 °C for 30 minutes and further 1 hour further at room temperature.All animal use was in accordance with protocols approved by the Institution’s Animal Care and Use Committee of Seoul National University College of Medicine. Cerebellar parasagittal slices were dissected into 250 μm by vibratome  from anaesthetized juvenile rats (P17–25) and 6 weeks old male C57BL/6 mice in ice-cold standard artificial cerebrospinal fluid (aCSF) containing the following (in mM): 125 NaCl, 2.5 KCl, 1 MgCl2, 10 HEPES, 4 NaCl, 4 Na2ATP, 0.4 Na3GTP and 17.5 sucrose (pH 7.25) for current clamp recordings. The membrane potential was held at −70 mV in voltage-clamp (VC) and current-clamp mode (CC). When the series resistance (Rs) varied by >15% and the injection current for the holding potential exceeded 500 pA, recordings were discarded. To evaluate the PC excitability, we monitored the spontaneous spiking activity with no current injection from the membrane potential of each neurons and/or measured the spike counts elicited by the injection of square-wised a series of current steps ranging from +100 pA to +600 pA with increments of 100 pA, and with a step interval of 4.5 s for 500 ms from a membrane potential of −70 mV in CC mode.Slices were put onto a submerged recording chamber on the stage of an Olympus microscope  and perfused with aCSF, and kept in place with a nylon-strung platinum anchor. All recordings were performed using the multiclamp 700B patch-clamp amplifier (Axon Instruments) with a sampling frequency of 20 kHz and signals were filtered at 2 kHz. To exclude the influence of the synaptic activity on the intrinsic properties of neurons, excitatory and inhibitory synaptic inputs were totally blocked with 10 μM NBQX and 100 μM picrotoxin, respectively. Patch pipettes (3-4 MΩ) were borosilicate glass and filled with internal solution containing the following: 9 KCl, 10 KOH, 120 K-gluconate, 3.48 MgCl22. Next, U6-loxP-CMV-mCherry-loxP sequence was amplified by PCR with XbaI(forward) or BglII(reverse)-containing specific primers, and then it was sub-cloned into the pAAV-MCS vector(Stratagene). Recombinant AAV was produced by co-transfection into HEK293A cells using AAV-DJ Helper-Free Packaging System (Cellbiolabs).The TNFRSF1A nucleotides from 385 to 405 were selected for the target region of TNFR1 shRNA. For the suppression of TNFR1 expression in BG cells in a cell-type specific manner, we first cloned TNFR1 shRNA into pSicoR-mChe-empty lentiviral vector(addgene #21907) as previously reported2.Cerebellar cortices were dissected from P0–P1 postnatal mice, cleared of adherent meninges, minced, and dissociated into a single-cell suspension by trituration. Dissociated cells were plated onto 12-mm glass coverslips coated with 0.1 mg/ml poly D-lysine. Cells were grown in DMEM supplemented with 25 mM glucose, 10% heat-inactivated horse serum, 10% heat-inactivated fetal bovine serum, 2 mM glutamine, and 1000 units/ml penicillin-streptomycin. Cultures were maintained at 37 °C in humidified atmosphere of 5% COPrior to HPLC assay anlaysis, astrocytes were washed 3 times with PBS. Cells were incubated with TNF-α (100 ng/ml) or PBS in time course . The amino acid content was derivatized with o-phthaldialdehyde (OPA) and quantified with UV (DAD) detection. OPA-derivatized samples were collected with a programmed autosampler and injected onto a Zorbax Eclipse Plus C18 column with detection at 338 nm . Mobile phase A was 40 mM Na2HPO4 (pH 7.8) and phase B was acetonitrile–methanol–water . The flow rate was 2 ml/min with a gradient condition that allowed for 1.9 min at 0% B and a rise to 26% B over a 12.5-min step. Subsequent washing at 100% B and equilibration at 0% B was performed within a total retention time of 15 min. Reagents for OPA derivatization and all equipment for HPLC analysis were obtained from Agilent Technologies.Sampling. Sagittal slices of the cerebellar vermis (300 µm thick) were obtained from juvenile rats . For extracting total RNA, we used RNeasy Mini Kit (QIAGEN) and followed manufacturer instructions. DNase I (QIAGEN) was treated. A total of 350 ng of total RNA per sample was reverse transcribed into cDNA using SuperScript III First-Strand (Invitrogen). qPCR was performed using the CFX Connect system (Bio-Rad) with SYBR Premix (TaKaRa). qPCR reactions with three individual pairs of primers were specific for: rat GLAST, forward primer, TGTCTTCTCCATGTGCTTCG, reverse primer, CAAGAAGAGGATGCCCAGAG, rat EAAT4, forward primer, GGAGACTGTGCCTGTACCTGG, reverse primer, GCAGAGCTGGAAGAGGTACCC, rat GAPDH, forward primer, CCACGAAGGGTGGAGCCAAA, reverse primer, GTCTTCTGGGTGGCAGTGATGG.57. The mRNA expression levels of TNF-α-treated neurons relative to control were normalized according to the ∆Ct method. All ∆Ct values were normalized by Ct value of GAPDH.All primers were previously usedS was calculated by fitting a single exponentials to the voltage responses of the test pulse (−5 mV); Input resistance (Rin) was determined from negative peak voltage deflection in response to brief hyperpolarising current injection ; Voltage threshold (Vthreshold) of AP was defined as the voltage where the dV/dt first exceeds 30–60 mV/ms); Fast afterhyperpolarization (fAHP) and medium afterhyperpolarization (mAHP) were calculated the difference between voltage threshold and hyperpolarised negative peak voltage after the first AP or depolarising square current injection, respectively. The AP waveform, including AP amplitude, half-width (FWHM) and 10–90% rise time was analysed from the first evoked AP of the firing train when +300 pA of the depolarising current was injected; AP amplitude was determi ned as difference between peak amplitude and the voltage threshold of the AP. Half-width and 10–90% rise time were the time duration at the half-maximal voltage, elevation time from 10 to 90% of the maximal AP voltage, respectively. The spike onset time was determined as the delay from the beginning point of the depolarising current injection to the voltage threshold where the upstroke phase of the first spike was initiated when the 300 pA of current was injected. The first spike latency was evaluated by measuring the delay from beginning point of ramp current injection (400 pA s−1 for 1 s).All data were acquired by the Clampex software (Molecular Devices) and analysed by IgorPro 8.1 (Wavemetrics). Electrical properties of the neurons were monitored with following parameters: Rt-test, One-way repetitive measured (RM) ANOVA and Two-way RM ANOVA with post hoc tukey test by Origin 8.5, SigmaPlot 12.0 and Prism 7.0 software. The sample size was approved by power analysis using the G*power 3.1.9.2.Data were presented as the mean ± SEM and statistical evaluations were performed using the normality test, equal variant test, independent Supplemental information


These data are related to the research “The accumulation and distribution of arsenic species and association with arsenic toxicity in rats after 30 days of oral realgar administration”  [1]. These data include the rat body weights, haematology, electrolytes, coagulation and biochemical parameters, and relative organ weights after 30 days of oral administration of realgar, which was consistent with the current OECD guideline “Repeated Dose 28-Day oral Toxicity Study in Rodents”. The data also include the content of arsenite (As(III)), arsenate (As(V)), dimethylarsinic acid (DMA), monomethylarsonic acid (MMAV), arsenic betaine (AsB) and arsenic chrome (AsC) in rat blood, liver, kidneys, brain and urine after single-dose and 30-day oral administration of realgar.The provided data are intended to demonstrate whether realgar has short-term toxicity and the role of accumulated arsenic species in realgar toxicity. Specifications tableValue of the data•The data show the repeated-dose 30-day oral toxicity of realgar in rats, which provides a reference for the safe dose range for repeated oral administration of realgar for 2 weeks in humans.•The accumulation and distribution of arsenic species in rat tissues not only helps in understanding the metabolism of arsenic in animals but also helps elucidate the main arsenic-accumulating organs in the body.•The total arsenic content is not sufficient for the safety evaluation of realgar. The data revealed a possible link between arsenic species and the sub-chronic toxicity of realgar, which can help people study the toxicity of realgar more scientifically and find reasonable prevention and treatment.1The content of arsenic species in rats after a single administration of realgar is shown in 22.1The rats were fasted for 16 h and then given a single realgar dose of 0 mg/kg (control) or 170 mg/kg . Then, a blood sample was taken from the medial canthus vein of each mouse at 0.25, 0.5, 1, 2, 4, 8, 16, 32, and 48 h. Blood samples were anticoagulated and centrifuged to obtain plasma samples. Finally, rats were euthanized and their livers, kidneys and brains were removed.2.2In total, 40 rats were randomly and equally divided into 4 groups as follows: ① the control group; ② the realgar 10.6 mg/kg/d group; ③ the realgar 40.5 mg/kg/d group; and ④ the realgar 170 mg/kg/d group. All rats were orally administered either 0.3% carboxymethylcellulose sodium (CMC-Na) in ultra-pure water (control group) or realgar  once daily for 30 consecutive days. At 31 days, the rats were anaesthetized using an intraperitoneal injection of phenobarbital sodium. Blood samples were then collected from the abdominal aorta for haematology, coagulation, biochemical and electrolyte analyses. Finally, the rats were sacrificed, and all organs were excised, observed, and weighed.2.3An Agilent 7700ce ICP-MS instrument coupled with the Agilent 1200 Series HPLC system was used for quantitative analyses of the tAs and As species content in rats plasma and organs.


Rp1-D21 confers a spontaneous HR response in the absence of pathogen recognition. Previous work identified a set of loci associated with variation in the strength of Rp1-D21-induced HR. A polygalacturonase gene homolog, here termed ZmPGH1, was identified as a possible causal gene at one of these loci on chromosome 7.The hypersensitive defense response (HR) in plants is a fast, localized necrotic response around the point of pathogen ingress. HR is usually triggered by a pathogen recognition event mediated by a nucleotide-binding site, leucine-rich repeat (NLR) protein. The autoactive maize NLR gene ZmPGH1 inhibited the HR-inducing activity of both Rp1-D21 and that of another autoactive NLR, RPM1(D505V), in a Nicotiana benthamiana transient expression assay system. Overexpression of ZmPGH1 in a transposon insertion line of maize was associated with suppression of chemically-induced programmed cell death and with suppression of HR induced by Rp1-D21 in maize plants grown in the field.Expression of ZmPGH1 functions as a suppressor of programmed cell death induced by at least two autoactive NLR proteins and by two chemical inducers. These findings deepen our understanding of the control of the HR in plants.The online version of this article (10.1186/s12870-019-1897-5) contains supplementary material, which is available to authorized users. The plant innate immune system is often thought of a two inter-related systems . The firRp1 locus carries a number of CNL R-gene paralogs that are tandemly-repeated and that have the capacity to recognize and mediate resistance to certain races of the fungus causing common rust, Puccinia sorghi  with iCycler iQ Real-Time PCR Detection System (Bio-Rad). The reaction was run under the following cycle: 10 min at 95 °C, 40 cycles of 15 s 95 °C, 1 min 60 °C. Dissociation analysis was performed after each reaction. The relative expression of the polygalacturonase (GRMZM2G135763) gene in different genotypes was calculated by ΔCt method. The Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) housekeeping gene was used to standardize the results. Primers sequences are provided in Additional file qRT-PCR was performed in a 20 μl volume [10 μl SYBR Green PCR Master Mix (Applied Biosystems), 1 μl cDNA, 0.5 μl Primer 1 (10 μM), 0.5 μl Primer 2 (10 μM) and 8 μl Hmaizegdb.org) that was highly associated with variation in this trait  [Zea mays polygalacturonase homolog 1 or ZmPGH1.The maize gene GRMZM2G135763 was previously identified as a candidate gene for modulation of HR induced by Rp1-D21  as it spam.org/)  identifiam.org/) . Additioam.org/)  also ideZmPGH1  ~ 1.8 Kbp upstream of the predicted ATG site of 2 families we identified 5 families homozygous for the mu1029380 insertion and 6 families in which the mu1029380 insertion was absent. We then used quantitative real time PCR (qRT-PCR) to quantify the mRNA levels of ZmPGH1in plants homozygous present and absent for insertion mu1029380. Surprisingly, a substantially enhanced level of ZmPGH1 expression was detected in plants homozygous for mu1029380 compared to plants without the insertion. The four mu1029380 homozygous plants assessed accumulated 1.6-, 2.2-, 4- and 12.7-fold more ZmPGH1 transcript than plants from the same family that were homozygous absent for the mu1029380 insertion  induced HR in a leaves . We havea leaves .Fig. 3ZmIn this study, we co-expressed ZmPGH1-EYFP  with Rp1-D21-HA to determine if ZmPGH1 can affect Rp1-D21-induced HR. As the β-glucuronidase (GUS) reporter gene does not affect Rp1-D21-induced HR , a GUS-EN. benthamiana leaves. Each combination was infiltrated into no fewer than 30 sites in no fewer than 4 leaves. The number of sites displaying HR was assessed every day at the same time for four days. As expected, very few sites co-expressing Rp1-D21 and HCT showed HR, while almost all of sites co-expressing GUS and Rp1-D21 showed HR  gene has not been previously investigated. All these previously-characterized polygalacturonases were shown to be strongly expressed in maize pollen where their role was assumed to be in facilitating pollen growth through the stigma. The maize gene expression atlas [maizegdb.com  shows that GRMZM2G135763 is expressed throughout development and throughout the plant but that expression is particularly strong in the anthers and developing roots .The cell wall plays a central role in plant-pathogen interactions –37. It ion atlas , 44 avaiZmPGH1 gene in maize suppressed the effects of Rp1-D21 and the cell death caused by SA and 10-OPEA while the transient overexpression of ZmPGH1 in N. benthamiana suppressed Rp1-D21- and RPM1(D505V)- induced HR. The measurement of plant height to assess the effect of the Rp1-D21 phenotype was a somewhat indirect measurement, however we have used this approach previously with some success [Rp1-D21.ZmPGH1 appears to function as a general suppressor of PCD plants. The enhanced expression of  success , 45. Hei success . This maZmPGH1 enhanced accumulation of ZmPGH1 message at least 1.5- fold. This expression enhancement may have been caused by the insertion abolishing the binding site of a transcriptional repressor or the Mu transposon might itself drive downstream gene transcription [pericarp color 1 gene allele P1-wr that increased expression. We have also previously observed a Mu insertion in the 3′ UTR that increased mRNA levels [The Mu transposon insertion mu1029380, ~ 1.8 Kbp upstream of the predicted ATG site of cription . Robbinscription  characteA levels . While tCCoAOMT2 and HCT1806/HCT4918 (two tightly linked and highly homologous HCT genes) were both initially associated with variation in Rp1-D21-mediated HR in the same mapping study in which ZmPGH1 was identified [ZmPGH1, HCT1806/HCT4918 and CCoAOMT2, were induced by Rp1-D21-induced HR  and by treatment with 10-OPEA  which suggests they are associated with a response to PCD [In previous studies we demonstrated that two other enzymes predicted to be important in plant cell wall synthesis were also associated with variation in the severity of HR mediated by Rp1-D21. Hydroxycinnamoyltransferase (HCT) and caffeoyl CoA O-methyltransferase (CCoAOMT) catalyze sequential steps in the lignin biosynthesis pathway. Genes encoding these enzymes, entified . We demoentified , 17. In entified  and thatentified . In thise to PCD , 21.Polygalacturonases and polygalacturonase inhibiting proteins as well as other enzymes involved in pectin degradation have previously been implicated in plant disease resistance and response –54, as hZmPGH1 functions as a suppressor of programmed cell death induced by at least two autoactive NLR proteins and by two chemical inducers. These findings deepen our understanding of the role of the cell wall in mediting disease resistance and the defense response, including control of the HR in plants.Additional file 1:Table S1. PCR primers used in this study (PDF 213 kb)Additional file 2:Figure S1. Chemical-Induced Cell Death Assay. This assay has been described previously [eviously . A. The


HFpEF), it remains unknown whether central hemodynamic alterations inherently present in this condition do affect blood pressure and blood volume (BV) regulation. The present study sought to determine hemodynamic and endocrine responses to prolonged orthostatic stress in HFpEF patients. Central venous pressure (CVP) assessed via the internal jugular vein (IJV) aspect ratio with ultrasonography, arterial pressure and heart rate were determined at supine rest and during 2 hours of moderate (25–30°) head‐up tilt (HUT) in 18 stable HFpEF patients (71.2 ± 7.3 years), 14 elderly (EC), and 10 young (YC) healthy controls. Parallel endocrine measurements comprised main BV‐regulating hormones: pro‐atrial natriuretic peptide, copeptin, aldosterone, and erythropoietin (EPO). At supine rest, the IJV aspect ratio was higher (>30%) in HFpEF patients compared with EC and YC, while mean arterial pressure was elevated in HFpEF patients (98.0 ± 13.1 mm Hg) and EC (95.6 ± 8.3 mm Hg) versus YC (87.3 ± 5.0 mm Hg) (P < 0.05). HUT increased heart rate (+10%) and reduced the IJV aspect ratio (−52%), with similar hemodynamic effects in all groups (P for interaction ≥ 0.322). The analysis of endocrine responses to HUT revealed a group×time interaction for circulating EPO, which was increased in YC (+10%) but remained unaltered in HFpEF patients and EC. The EPO response to a given reduction in CVP is similarly impaired in HFpEF patients and elderly controls, suggesting an age‐dependent dissociation of EPO production from hemodynamic regulation in the HFpEF condition.Despite growing research interest in the pathophysiology of heart failure with preserved ejection fraction ( The condition of heart failure with preserved ejection fraction (HFpEF) entails prominent hemodynamic alterations challenging the pressure gradient driving venous return as well as the regulation of blood volume (BV) Miller . A fairlThe present study experimentally assessed for the first time whether central hemodynamic alterations inherently present in the HFpEF condition alter blood pressure and BV regulation during an orthostatic challenge. Specifically, the purpose of this study was to assess the impact of prolonged moderate HUT on central hemodynamics and major BV‐regulating hormones in stable HFpEF patients and healthy controls including age‐matched elderly (EC) and young (YC) individuals. We hypothesized that hemodynamic and endocrine responses to reduced CVP will be impaired in HFpEF patients compared with control individuals, independently of the age status.The study was approved by the Ethical Commission of Zurich  and conducted in accordance with the declaration of Helsinki. Prior to the start of the experiments, informed oral and written consents were obtained from all participants.−1), relevant structural/functional cardiac alterations, and no history of iron‐deficiency anemia. Healthy age‐ and sex‐matched individuals (EC) were recruited from the community and excluded if they presented any chronic medical illness, were taking daily prescription medications, had current medical symptoms, or were performing aerobic exercise on a regular basis. An additional group of healthy young control individuals (YC) enrolled in a parallel investigation  = 55.7 ± 4.9%) were recruited from the HF outpatient clinic of the University Hospital of Zurich. Inclusion criteria comprised: signs and symptoms of HF  for 120 min. A climbing harness (bicycle saddle for YC) gently held the body weight of individuals at the hip level throughout the HUT protocol. Hemodynamic variables and blood samples were obtained at supine rest and discrete time points  during HUT. Blood samples at the last time point (120 min) were not available in YC due to specific protocol characteristics. All blood samples were centrifuged and stored at −80°C until analyzed.The internal jugular vein (IJV) aspect ratio, a surrogate marker of central venous pressure (CVP), was determined at the level of the cricoid cartilage using the method described by Keller et al. . In brieD is arterial diameter, ∆D is distension, and PP is pulse pressure. DC represents the reciprocal value of arterial stiffness.where ® DxC 600/800 and Synchron® Systems Multi Calibrator .Blood samples (5 mL) from the antecubital vein were collected anaerobically in heparinized glass syringes. Serum creatinine was measured via the kinetic Jaffe reaction  and glomerular filtration rate (eGFR) was calculated by the CKD‐EPI Creatinine Equation (2009) according to guidelines from the National Kidney Foundation  . Data were tested for normal distribution with the Kolmogorov–Smirnov test, homogeneity of variances with Levene's test, and sphericity with Mauchly's test. Data not‐normally distributed, violating the assumptions of homogeneity of variances and/or sphericity were logarithmically transformed before parametric testing. Fisher's exact test and one‐way ANOVA with Tukey post hoc tests were used to compare baseline variables in HFpEF patients and control individuals. Tilt‐table experiments were analyzed with two‐way repeated measures ANOVA with “time” and “group” as within‐ and between‐subject factors, respectively, along with the interaction among these factors. Pairwise comparisons were performed using Student's paired ) Durand  was consP < 0.05) in HFpEF patients compared with EC and YC. Sex distribution was similar among groups. Kidney function, determined by eGFR, was mildly reduced (P < 0.05) in HFpEF patients relative to EC (data not available in YC). Comorbidities comprising hypertension (78%), coronary artery disease (44%), and diabetes mellitus (28%) were the most prevalent among HFpEF patients. With respect to medication, beta‐blockers (67%), loop diuretics (61%), and angiotensin‐converting‐enzyme inhibitors/angiotensin II‐receptor blockers (61%) were common and similarly prevalent.Table P < 0.05) in HFpEF patients compared with EC and YC, while mean arterial pressure was augmented (P < 0.05) in HFpEF patients and EC versus YC. No difference was detected for aortic augmentation index and carotid artery distensibility between HFpEF and EC (data not available in YC). With respect to endocrine variables, all BV‐regulating hormones were elevated in HFpEF patients compared with EC and/or YC.Hemodynamic and endocrine variables at supine rest are reported in Table P for time < 0.001), with a similar decline observed in all groups (P for interaction = 0.322). No definite pattern was detected for systolic blood pressure (SBP) , whereas PP was reduced with HUT . Conversely, heart rate was increased during HUT . None of the hemodynamic effects induced by HUT differed among groups. No sign/symptom of orthostatic hypotension was observed in any individual.Figure P for time < 0.001, P for interaction = 0.908). Likewise, HUT increased copeptin  and aldosterone (P for time = 0.003 P for interaction = 0.520) (data not available in YC). A group × time interaction was detected for EPO (P for interaction = 0.061) in that EPO was only increased with HUT in YC. No change in EPO during HUT was noted in HFpEF patients and EC.The effects of HUT on BV‐regulating hormones in HFpEF patients and control individuals are presented in Figure This study assessed hemodynamic and endocrine responses to HUT in stable HFpEF patients and healthy controls comprising age‐matched elderly (EC) and young (YC) individuals. The main findings are: (1) HUT elicits similar reductions in IJV aspect ratio and compensatory increases in heart rate in HFpEF patients and control individuals, irrespective of the age status; (2) circulating EPO augments with HUT in YC but remains unaltered in HFpEF patients and EC.Since the inception of current concepts in cardiovascular physiology in the mid‐20th century, the regulation of the cardiovascular system has been intrinsically linked to its internal volume, that is, BV Guyton . A clearBeyond swift hemodynamic responses driven by neural reflexes, endocrine responses are capital to cope with sustained orthostatic challenges. As aforementioned, afferent signals from central baroreceptors regulate the production of BV‐regulating hormones, thereby modulating kidney function according to the “fullness” of the intrathoracic compartment , a variable regulating EPO production independently of central hemodynamics, was not assessed. Nonetheless, the main determinant of kidney PO2, that is, glomerular filtration rate, was not altered during HUT in HFpEF patients and control individuals. Third, smoking may have influenced EPO levels (Eisenga et al. First, findings arose from a moderate sample size of stable HFpEF patients, mainly males, thus our conclusions should be taken with caution. Likewise, control individuals did not present comorbidities or risk factors. Therefore, a clear separation between HFpEF and additional conditions cannot be ascertained. Larger prospective studies might buttress the present findings and contrast different HFpEF phenotypes, comorbidities, risk factors, and pharmacotherapies (Montero and Flammer The current study demonstrates that prolonged HUT elicits comparable central hemodynamic responses in HFpEF patients and control individuals. In contrast, endocrine responses to reduced CVP comprising increases in circulating EPO are absent in HFpEF patients and age‐matched elderly individuals, suggesting that the hemodynamic regulation of EPO production is age‐dependent. The pathophysiological mechanisms underlying these findings are speculative at present and will have to be characterized in future studies.None declared.


Tumor necrosis factor (TNF) is a pleiotropic cytokine whose primary physiological function involves coordinating inflammatory and adaptive immune responses. However, uncontrolled TNF signaling causes aberrant inflammation and has been implicated in several human ailments. Therefore, an understanding of the molecular mechanisms underlying dynamical and gene controls of TNF signaling bear significance for human health. As such, TNF engages the canonical nuclear factor kappa B (NF-κB) pathway to activate RelA:p50 heterodimers, which induce expression of specific immune response genes. Brief and chronic TNF stimulation produces transient and long-lasting NF-κB activities, respectively. Negative feedback regulators of the canonical pathway, including IκBα, are thought to ensure transient RelA:p50 responses to short-lived TNF signals. The non-canonical NF-κB pathway mediates RelB activity during immune differentiation involving p100. We uncovered an unexpected role of p100 in TNF signaling. Brief TNF stimulation of p100-deficient cells triggered an additional late NF-κB activity consisting of RelB:p50 heterodimers, which modified the TNF-induced gene-expression program. In p100-deficient cells subjected to brief TNF stimulation, RelB:p50 not only sustained the expression of a subset of RelA-target immune response genes but also activated additional genes that were not normally induced by TNF in WT mouse embryonic fibroblasts (MEFs) and were related to immune differentiation and metabolic processes. Despite this RelB-mediated distinct gene control, however, RelA and RelB bound to mostly overlapping chromatin sites in p100-deficient cells. Repeated TNF pulses strengthened this RelB:p50 activity, which was supported by NF-κB-driven RelB synthesis. Finally, brief TNF stimulation elicited late-acting expressions of NF-κB target pro-survival genes in p100-deficient myeloma cells. In sum, our study suggests that the immune-differentiation regulator p100 enforces specificity of TNF signaling and that varied p100 levels may provide for modifying TNF responses in diverse physiological and pathological settings. Tumor necrosis factor (TNF) is a pleiotropic cytokine whose primary physiological function involves coordinating innate and adaptive immune responses . TNF engin vivo  activates a complex consisting of NF-κB inducing kinase (NIK) and IKK1 (or IKKα), which phosphorylates p100. Subsequently, the C-terminal inhibitory domain of p100 is removed by proteasome resulting in the release of RelB:p52 NF-κB heterodimers into the nucleus. In comparison to the canonical RelA activity, the non-canonical pathway elicits a weak but sustained RelB activity, which induces genes involved in immune cell differentiation and immune organ development. In the absence of p100, RelB appears in the nucleus as a minor RelB:p50 NF-κB activity  activities. For varying dose and treatment duration, TNF activates IKK2 with diverse kinetic profiles. Accordingly, we used theoretical IKK2 activity profiles of varying peak amplitude or duration as model inputs ., 20.in ss inputs . Short-lactivity . The prorodimers . Our comt system . TherefoNfkbia−/− cells lacking IκBα . TNFc treatment of WT cells induced a biphasic NF-κBn response comprising of an early peak, which lasted for ~1 h, and a gradually weakening second phase between 3 and 8 h . TNFp tring IκBα . TNFp innd phase . Of note/− cells . Our shi/− cells . Similarignaling , 22, 23.l by 1 h . In addimulation . Further−/− MEFs . Our stuOf note, we relied on bulk measurements of transcription factors present in the nuclear extracts. Therefore, our study does not rule out that p100 deficiency triggers an asynchronous, oscillatory RelB:p50 response to TNFp at the single-cell level. Cellular heterogeneity may also amount to two distinct cell population with only one sustaining an elevated RelB:p50 activity—this may in fact lead to an underestimation of late RelB:p50 response in our bulk measurement based analyses.Nfkb2-deficient system. We assembled the large number of model parameters into 48 distinct groups  of NF-κB-target genes activated by TNF in WT cells involving RelA:p50. Of note, previous studies also reported that RelA:p50 and RelB:p50 function redundantly in mediating the expression of certain pro-inflammatory genes, such as those encoding RANTES, as well as pro-survival genes, such as those encoding cFLIP . To. ToNfkb2response . As contactivity . Gr-II gng cFLIP , 23. IntWe then subjected these gene-groups to gene ontology (GO) analyses. Consistent with the well-articulated role of the canonical pathway in immune-activating TNF signaling, Gr-I and Gr-II comprising TNFc-induced RelA-important genes were enriched for GO terms associated with innate and adaptive immune responses . Gr-II aNfkb2−/− MEFs to TNFc treatment for 6 h and subsequently performed chromatin immunoprecipitation using anti-RelA or anti-RelB antibodies followed by deep-sequencing (ChIP-seq) analysis  analyses. TNFc treatment for 6 h triggered the RelA-dependent expression of Gr-I gene /− cells . TNFc inNfkb2−/− MEFs, we asked if TNFp triggered persistent expression of NF-κB-dependent genes in p100-deficient cells. Our time-course analyses demonstrated that TNFc induced progressive accumulation of mRNAs encoding Gr-I and Gr-II genes in WT as well as Nfkb2−/− MEFs between 1 and 6 h post-treatment  induces the sustained activity of extracellular signal-regulated kinase (Erk) for promoting cell differentiation. In contrast, transient Erk activation by epidermal growth factor (EGF) causes cell proliferation , 46. GenRela−/−Rel−/−Relb−/− 3T3 MEFs, which lacked the expression RelA, cRel, and RelB, were utilized as NF-κB-deficient cells. Relb−/−Nfkb2−/− MEFs expressing transgenic RelB from retroviral constructs were reported earlier . MEFs generated from E13.5 embryos were used subsequent to immortalization by the 3T3 protocol. Some key data have been also reproduced using primary MEFs.  earlier . Human-dIn the TNFp regime, cells were treated briefly for 30 min with 1 ng/ml of TNF . Subsequently, TNF-supplemented media was substituted with TNF-free media, and cells were harvested at the indicated times after the commencement of the TNF treatment. In certain instances, cells were subjected to repeated pulses of TNF at the specified time intervals. Alternately, cells were treated chronically with 1 ng/ml of TNF (TNFc) or stimulated with 10 ng/ml IL-1β . As described , nuclearTotal RNA was isolated from cells, stimulated either briefly or chronically with 10 ng/ml of TNF, using RNeasy kit . qRT-PCR was performed as described earlier ; see TabNfkbia-deficient system, but preserved the model behavior observed earlier in WT and Nfkb2-deficient systems (We utilized a previously published mass action kinetics-based NF-κB mathematical model  subseque systems . The mod systems . Using iSee the t-test was used for verifying statistical significance unless otherwise mentioned. Statistical tests associated with global gene-expression analyses have been detailed in the Error bars were shown as S.E.M. of 3–6 experimental replicates. Quantified data are means ± SEM, and two-tailed Student's WT and gene-deficient C57BL/6 mice were used in accordance with the animal usage guideline and recommendations of the Institutional Animal Ethics Committee of the National Institute of Immunology. The protocol was approved by the Institutional Animal Ethics Committee and the approval no is approval no. #258/11.in silico studies under the supervision of SB and JG. PR conducted cell-based analyses with the help from US, YR, and MC and the guidance from SB and RS. ChIP-seq experiments were performed by MZ and AS and analyzed by SD and BC with the guidance from RS and SB. BC and PR wrote the manuscript with SB.BC carried out 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.