As defined by some of the founders of the field, Barkow et al. , “evolutsequalae. However, since at least Descartes and, perhaps as far back as Plato, a mind-body dualism has existed whereby the mind  has been treated as distinct from the body and there is a tendency to treat humans as distinct from “animals” in some form of implicit anthropocentrism which has led to psychological theories generally being developed in parallel deafness to biological theories  in nature, evolutionary models must apply to the brain and its Adopting an evolutionary framework to the study of human behavior and psychology has been incredibly fruitful. I cannot hope to do it justice here but, instead, I will highlight some of the major areas that evolutionary psychology has provided novel insights. Even work using genetic or hormonal assays, on their own, are merely descriptive in nature indicating that, for instance, being high in sensation-seeking is heritable , misunderstandings (Buss and Schmitt, Despite this rather simple premise and extensive/impressive research, the field is mired in controversy (e.g., Satoshi Kanazawa's redacted How might the field begin to approach addressing these issues? There are a few types of submissions that will help in this effort. First, theory/commentary papers that respond to papers published elsewhere along with more expansive theory papers that better articulate the utility of evolutionary models. For example, researchers might publish a paper in another journal that claims to refute evolutionary predictions. A response in the form of a note or commentary might be warranted to lay out why the target article does not actually refute evolutionary models on theoretical or methodological grounds. Alternatively, notes or commentary that further develop theoretical issues are warranted and even present modern updates of “old” theories like sexual strategies theory (Buss and Schmitt, Second, replications of “big” papers in evolutionary psychology are especially warranted. As researchers and lay-people have observed in the last five years, the field of psychology has gone through a crisis of faith. Many of the most famous findings in psychology at large have been cast into doubt or even downright refuted (e.g., facial feedback hypothesis; Buck, Third, while direct replications of “big” papers in evolutionary psychology are useful, extending this research is warranted as well. This allows us to test the boundary conditions of the findings as well as the robustness to, for example, methodological and sampling differences. For instance, testing the cross-cultural robustness to life history models of personality (Jonason et al., Fourth, there is a long tradition in the legal profession. When two parties disagree on something, they engage in an “adversarial” process. Each party puts forth their argument and evidence in hopes of testing which holds more weight. In contrast, to the legal profession, however, science has a different burden of proof. That burden of proof is based on who has the data that best fits its model and, ideally, refutes alternative models. As such, another way to redress the existential threats to evolutionary psychology is to engage in “adversarial” papers whereby researchers pit two or more psychological theories in accounting for phenomena against each other. Importantly, papers that derive contradictory hypotheses from competing theories are especially useful here as they can simultaneous support one model and refute another (see Li et al., Fifth, and last, I propose that meta-science papers Webster,  might fuDarwin saw new fields of inquiry opening from his theory of natural selection, one of which is psychology. Modern evolutionary psychologists attempt to answer that call. The field faces many existential threats that warrant direct attention. As such, I consider these to be the grand challenge of evolutionary psychology now and welcome papers that attempt to answers those challenges.This is a review paper requested as part of the journals' editorial policies.PJ is solely responsible for the content of this manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Environmental drug resistance constitutes a serious impediment for therapeutic intervention in multiple myeloma. Tumor-promoting cytokines, such as tumor necrosis factor (TNF), induce nuclear factor-κB (NFκB)- driven expression of pro-survival factors, which confer resistance in myeloma cells to apoptotic insults from TNF-related apoptosis-inducing ligand (TRAIL) and other chemotherapeutic drugs. It is thought that RelA:p50 dimer, activated from IκBα-inhibited complex in response to TNF-induced canonical NFκB signal, mediates the pro-survival NFκB function in cancerous cells. Myeloma cells additionally acquire gain-of-function mutations in the non-canonical NFκB module, which induces partial proteolysis of p100 into p52 to promote RelB:p52/NFκB activation from p100-inhibited complex during immune cell differentiation. However, role of non-canonical NFκB signaling in the drug resistance in multiple myeloma remains unclear. Here we report that myeloma-associated non-canonical aberrations reinforce pro-survival TNF signaling in producing a protracted TRAIL-refractory state. These mutations did not act through a typical p52 NFκB complex, but completely degraded p100 to reposition RelB under IκBα control, whose degradation during TNF signaling induced an early RelB:p50 containing NFκB activity. More so, autoregulatory RelB synthesis prolonged this TNF-induced RelB:p50 activity in myeloma cells harboring non-canonical mutations. Intriguingly, TNF-activated RelB:p50 dimer was both necessary and sufficient, and RelA was not required, for NFκB-dependent pro-survival gene expressions and suppression of apoptosis. Indeed, high RelB mRNA expressions in myeloma patients correlated with the augmented level of pro-survival factors and resistance to therapeutic intervention. In sum, we provide evidence that cancer-associated mutations perpetuate TNF-induced pro-survival NFκB activity through autoregulatory RelB control and thereby exacerbate environmental drug resistance in multiple myeloma. TNF-related apoptosis-inducing ligand (TRAIL) and other chemotherapeutic drugs induce Caspase-8 dependent apoptosis in various cancerous cells with negligible toxicity for healthy cells.2 Preclinical studies accordingly advocated for TRAIL-based interventions in multiple myeloma.5 However, subsequent clinical trials revealed a rather moderate benefit with only a small subset of patients responding satisfactorily to TRAIL.6 Indeed, certain patient-derived myeloma cells were found to be resistant to drug-induced apoptosis owing to increased level of cFLIP and other pro-survival factors, which counteract caspase activation.4 As drug resistance constitutes a serious impediment for myeloma therapy, it is important to understand fully the underlying molecular mechanisms.Multiple myeloma, an incurable plasma cell malignancy, accounts for ~13% of all hematological cancers.9 Bone marrow stromal cells provide paracrine TNF signals in myeloma.10 Serum level of TNF was correlated with disease severity in multiple myeloma,12 and provided for a predictive indicator of high symptom burden for patients undergoing maintenance therapy.13 Clinical trials also demonstrated that thalidomide analogs, which inhibit TNF production in the tumor microenvironment, enhance overall response in TRAIL-based therapy.15 These studies implicated TNF in environmental-mediated drug resistance in multiple myeloma.In myeloma cells, tumor necrosis factor (TNF) induces expression of the pro-survival factors, which are known to confer resistance to apoptotic insults, including TRAIL.16 In a negative feedback loop, RelA:p50 transcriptionally induces synthesis of IκBα, which ensures post-induction attenuation of RelA:p50/NFκB activity. TNF stimulates transcription of pro-survival factors from their cognate κB-driven promoters. It is commonly believed that RelA:p50 mediates this pro-survival NFκB function in myeloma cells.7 Importantly, IKK inhibitors were shown to sensitize myeloma cells to apoptotic death.18At the molecular level, TNF engages NEMO -IKK2  kinase complex, which promotes phosphorylation and degradation of inhibitory IκBα (inhibitor of NF-κBα), thereby, liberating RelA:p50 dimer into the nucleus via the canonical NFκB pathway.19 In resting cells, cIAP1/cIAP2 and TRAF3-dependent ubiquitination promotes degradation of NIK (NFκB inducing kinase). Induction of the non-canonical pathway rescues NIK from constitutive degradation resulting in activation of IKK1 . IKK1 phosphorylates Nfkb2-encoded precursor p100, which retains RelB in the cytoplasmic complex in unstimulated cells. Subsequent partial proteolysis or processing of p100 removes the C-terminal inhibitory domain to generate mature p52 subunit, thereby liberating RelB:p52/NFκB activity into the nucleus. Myeloma cells acquire gain-of-function mutations in this non-canonical NFκB pathway with separate studies reporting ~10–15% multiple myeloma patients harbor such genetic aberrations.21 Myeloma-associated mutations either inactivate negative regulators of NIK, such as cIAP1/cIAP2 and TRAF3, or augment expression of upstream inducers of the pathway, including CD40, LTβR as well as NIK itself.23 Moreover, mutations in Nfkb2 that produce dysfunctional p100 lacking the NFκB inhibitory domain has been reported.24 It was proposed that NIK activates the canonical IKK2-RelA pathway in a subset of human myeloma cell lines (HMCLs) to promote cell survival.25 However, a role of NIK-induced non-canonical RelB/NFκB activity in imparting drug resistance in multiple myeloma has not been explored.In addition, a distinct non-canonical NFκB pathway is activated by cell-differentiating cues in physiological settings.19 Interestingly, a low level of NIK-independent, constitutive RelB:p52/NFκB activity has been identified in myeloma cells.26 Surprisingly, constitutive RelB:p52 activity was shown both to promote and suppress NFκB-dependent gene expressions in HMCLs and patient-derived myeloma cells.27 As NFκB target genes encode important pro-survival factors, we asked if mutational activation of the non-canonical RelB/NFκB pathway modulates TNF response, and thereby modifies resistance of myeloma cells to apoptotic insults.The RelB/NFκB-mediated gene expressions is thought to have limited physiological functions in immune cell differentiation.Here, we demonstrate that non-canonical NFκB mutations collaborate with TNF signaling in producing a protracted TRAIL-refractory state in myeloma cells. In an interdisciplinary approach, we identify that depletion of p100, owing aberrations in the non-canonical module, triggers an autoregulatory RelB pathway, which perpetuates an alternate RelB:p50 NF-κB activity in response to TNF. Intriguingly, pro-survival TNF response is attributed to RelB:p50, and not RelA:p50, NFκB activity in myeloma cells harboring non-canonical mutations. Finally, our database analyses indicate a possible role of autoregulatory RelB/NFκB pathway in resistance of myeloma patients from therapeutic intervention. In sum, we provide evidence that cancer-associated mutations perpetuate TNF-induced pro-survival NFκB response through autoregulatory RelB control and thereby exacerbate environmental drug resistance in multiple myeloma.Traf3, and JK6L contains an inactivating mutation in Nfkb2/p100.28 As a control, we used OciMy5 cells that lack non-canonical mutations but bear an amplification of Nfkb1. However, cell treatment using 100 ng/ml of TRAIL led to similar ~45-55% cell death at 12 h post-treatment in these HMCLs irrespective of mutations in non-canonical module  and supershift analyses revealed that TNF induced comparable nuclear RelA NFκB DNA-binding activity, which was similarly attenuated by 8 h in OciMy5, KMS28PE, JK6L and OciMy1 cell lines regardless of non-canonical mutations . TNF sig6L cells , we exam6L cells . Consistse HMCLs . We alsose HMCLs , which lNext, we investigated role of individual NFκB dimers in HMCLs bearing distorted non-canonical module. We found that shRNA-mediated knockdown of RelA expression does not impact TRAIL resistance of TNF-primed KMS28PE cells in our cell-death assay . Intact 31 To further understand RelB/NFκB control in inflammatory settings, we rewired p100- and IκBα-mediated regulations in a mathematical model, termed the NFκB Systems Model v2.0  in TNF-treated MEFs of the following genotypes, namely WT fibroblasts, which primarily elicits RelA:p50 activity, ubgroups . Abrogatd RANTES , as welld RANTES , in WT Mactivity . Intrigu−/− MEFs . shRNA-m−/− MEFs . However−/− MEFs . Finally−/− MEFs . Therefo34 Sensitivity of Rela−/− MEFs to TNF-induced apoptosis underscored the importance of RelA/NFκB-driven pro-survival gene expressions in the cell-death pathway.35 Given capacity of the RelB:p50 dimer in stimulating expression of these pro-survival factors, we asked if alternate RelB/NFκB activity suppresses apoptosis in Rela−/−Nfkb2−/− MEFs. As a control, we utilized Rela−/−Nfkb1−/− cells, which lack RelA as well as p50, thus resulting in the deficiency of both RelA:p50 and RelB:p50 dimers , which was resistant to NIK-mediated degradation,19 in KMS28PE cells led to a drastic reduction in TNF-induced RelB:p50 NFκB activity . Our analyses revealed that Bcl2 and cFLIP mRNA levels were augmented in the patient group with high RelB mRNA expression as compared with those expressing RelB at a low level . MoreoveActivation of canonical NFκB signaling in myeloma cells by tumor microenvironment derived TNF has been implicated in environmental drug resistance. Recurrent gain-of-function mutations in the non-canonical NFκB module indicated its possible involvement in the resilience of myeloma cells to chemotherapeutic agents. We found that while TNF induces an early NFκB activity, mutational activation of the non-canonical module prolongs TNF-induced pro-survival NFκB response, albeit composed of the RelB:p50 dimer, in exacerbating TRAIL resistance .Relb synthesis  activity in T-cell acute lymphoblastic leukemia.40 Except for Rela, genes encoding the NFκB subunits per se also bear κB sites at their promoters. In this study, we extended evidence for the involvement of autoregulatory NFκB control in neoplastic diseases. Genetic analyses and mathematical modeling studies substantiated that depletion of p100 in myeloma cells triggers this autoregulatory loop to promote perpetuating RelB:p50/NFκB response to TNF. Strikingly, this TNF-induced, persistent RelB:p50 activity was impervious to RelA inhibitions  from GP2-293 cells co-transfected with pBabe.puro construct and Ampho envelope vector (Clonetech). Following shRNA-expressing lentiviral particles  were used for knockdown in HMCLs: Rela#1-TRCN0000353629, Rela#2-TRCN000029875, Relb#1-TRCN0000280360, Relb#2-TRCN0000280361, Nfkb2#1-TRCN0000356047 and Nfkb2#2-TRCN0000356005, and control-SHC202V. For knockdown studies in fibroblasts, lentiviral constructs expressing control shRNA (#RHS4346) or RelB-shRNAs (RMM#4431-101264262 and RMM#4431-200408572) purchased from GE-Dharmacon  were used for generating lentiviral particles.49Wild-type or gene-deficient C57BL/6 mice were housed at NII and used in accordance with the IAEC guidelines. MEFs, immortalized using 3T3 protocol, were generated from E13.5 embryos, as described.50 Antibody against p50 (BB-AB0080) was from BioBharati Life Sciences . Antibodies against Caspase-8 (#4790), Caspase-3 (#9665) and PARP1 (#9542) were from CST .MEFs and HMCLs stimulated using 1  and 10 ng/ml of TNF , respectively, were harvested, nuclear, cytoplasmic or whole-cell extracts were analyzed by EMSA, supershift analysis, immunoblotting or subjected to immunoprecipitation.50 using cDNA synthesis kit from BioBharati. For microarray analysis, labeling, hybridization to Illumina MouseRef-8 v2.0 Expression BeadChip, data processing and quantile normalization were performed by Sandor Pvt Ltd . Differential expression of genes between different genotypes were analyzed by k-median clustering implemented in the MEV program.51 The MIAME version of the microarray data is available on NCBI-GEO (accession GSE68615). For ChIP, 3 × 106 MEFs stimulated with 10 ng/ml of TNF were fixed with 2% formaldehyde, nuclei were isolated, sonicated and lysates were subjected to immunoprecipitation using indicated antibodies . Subsequent to reverse cross-linking, genomic DNA fragments were extracted from immunopellet and analyzed by qPCR using specific primers.Total RNA was isolated from cells stimulated with 10 ng/ml of TNF and quantitative reverse transcriptase–PCR was conducted, as described,52 TRAIL-induced  cell death in HMCLs was assessed in trypan blue dye exclusion assay. HMCLs were primed with 10 ng/ml of TNF. Apoptosis was scored at 4 h post-stimulation using annexin-V/FITC and propidium iodide (BD Biosciences) staining. Flow cytometry was performed using VERSE  at NII Central facility with the help from A Das, NII, and data were analyzed using FlowJo.TNF-induced (1 ng/ml) cell death in MEFs was quantified at 18 h post-stimulation using crystal violet staining, as described.v2.0 was developed based on previously published models,50 refined using our experimental data and was simulated in Matlab. A detailed description including parameter values has been provided in The NF-κB Systems Model t-test and the P-values have been mentioned in the respective figures. For analyses of patients data obtained from Multiple Myeloma Research Foundation, Welch's t-test was used.Error bars are shown as s.e.m. of 3–5 biological replicates, which was determined according to the standard practices for cell-culture-based analyses. Exact numbers of replicates for each experiment have been indicated in the legends of the corresponding figures. Statistical significance between groups was calculated using two-tailed Student's


Neorickettsia are a group of intracellular α proteobacteria transmitted by digeneans . These endobacteria can also infect vertebrate hosts of the helminths and cause serious diseases in animals and humans. Neorickettsia have been isolated from infected animals and maintained in cell cultures, and their morphology in mammalian cells has been described. However, limited information is available on the morphology and localization of Neorickettsia in the trematode host.Neorickettsia-infected strain of the model trematode Plagiorchis elegans to infect Syrian Golden hamsters to produce adult worms. Ultrastructure of Neorickettsia was assessed by transmission electron microscopy of high pressure freezing/freeze substitution fixed specimens. A Neorickettsia surface protein from P. elegans (PeNsp-3) was cloned and antibodies against the recombinant protein were used to localize Neorickettsia by immunohistochemistry.We used a P. elegans showed many endobacteria at the host-parasite interface such as the oral and ventral sucker, the tegument and the excretory pore.Ultrastructural analysis revealed moderate numbers of pleomorphic endobacteria with a median size of 600 × 400 nm and characteristic double membranes in various tissue types. Endobacteria showed tubular membrane invaginations and secretion of polymorphic vesicles. Endobacteria were unevenly localized as single cells, or less frequently as small morula-like clusters in the ovary, Mehlis’ gland, vitelline follicles, uterus, intrauterine eggs, testis, cirrus-sac, tegument, intestine and the oral and ventral sucker. Examination of hamster small intestine infected with P. elegans trematodes carry Neorickettsia endobacteria in varying numbers in many tissue types that support vertical transmission, trematode to trematode transmission via seminal fluid, and possibly horizontal transmission from trematodes to vertebrate hosts. These means appear to be novel mechanisms of pathogen transmission by endoparasitic worms.We conclude that adult The online version of this article (doi:10.1186/s13071-017-2123-7) contains supplementary material, which is available to authorized users. Rickettsiales. For example Wolbachia endosymbionts are crucial for development and reproduction of some filarial nematodes and are a target for novel anti-filarial chemotherapy in humans and domestic animals [Neorickettsia are a different group of endosymbionts that infect some digenean trematode species. They are similar to Wolbachia in terms of genome reduction, number of protein-coding sequences and biochemical pathways. Both, Wolbachia and Neorickettsia, lack the ability for lipopolysaccharide (LPS) synthesis [Neorickettsia are not obligatory endosymbionts (like Wolbachia), they can infect mammalian cells, and may have mammalian vertebrate reservoirs. Furthermore, some Neorickettsia species have been shown to be highly pathogenic causing Sennetsu fever in humans (N. sennestu), Potomac horse fever (N. risticii) or salmon poisoning disease in dogs (N. helminthoeca) [A number of parasitic worm species of medical or veterinary relevance contain intracellular α proteobacteria of the order  animals . Neorickynthesis , 3. Howenthoeca) .Neorickettsia have been detected in a considerable number of trematode species by PCR [Neorickettsia have been repeatedly described morphologically using mammalian cell cultures and a typical ehrlichial growth cycle with elementary body, initial body and morula has been suggested [Neorickettsia in its trematode host. Vertical transmission has been described and granular, bacteria-like structures have been detected in eggs of the bat trematode Acantharium oregonense using an antiserum of a horse infected with N. risticii [Neorickettsia in different life-cycle stages of the rodent flatworm Plagiorchis elegans [Neorickettsia, the exact mechanisms for vertical transmission within trematode generations and horizontal transmission to the mammalian hosts are not clear.s by PCR , but weruggested –8. Althouggested , 10 littristicii . In a re elegans . AlthougPlagiorchis (Plagiorchiidae) are intestinal parasites of a variety of reptiles, birds and mammals and only accidentally infect humans [Plagiorchis elegans is abundant in some areas in North America and Europe and they naturally carry Neorickettsia. Plagiorchis elegans can be maintained in the laboratory in Syrian golden hamsters (Mesocricetus auratus) [Neorickettsia in trematodes. In the present study we have infected hamsters with P. elegans, and studied the ultrastructure and localization of Neorickettsia in adult trematodes. We cloned and expressed a Neorickettsia surface protein and raised antibodies against the recombinant protein. These antibodies were used to localize Neorickettsia not only at the attachment sides of the trematodes, but also in the tissue surrounding the intestinal mucosa of the hamster.Digenean trematodes of the genus t humans . Plagiorauratus) , 14, 15,Lymnaea stagnalis snails infected with the digenean P. elegans, were collected in October of 2015 from a pond in Pennington County, Minnesota, USA. Cercariae shed by snails were screened for the presence of Neorickettsia endobacteria following a real-time PCR protocol targeting a 152-bp portion of the 3′ end of the heat shock protein coding gene, GroEL as described by Greiman et al. [P. elegans with high level of Neorickettsia infection and it was selected for isolation of sporocysts as described previously [P. elegans containing numerous infective metacerariae were fed directly to two outbred Syrian golden hamsters . After 20 days hamsters were humanely euthanized and their small intestines were examined for adult P. elegans  for 24 h and washed afterwards for 3–5 days using 70% ethanol. For electron microscopy, 10 worms were chemically fixed and 6 worms were physically fixed by high pressure freezing/freeze substitution (HPF/FS) as described below. Adult P. elegans were chemically fixed using 2% paraformaldehyde/ 2.5% glutaraldehyde  as described previously [P. elegans worms were directly snap-frozen in PBS at -80 °C.Adult eviously . FurtherNeorickettsia in hamster tissue, pieces (~20 mg) of the small intestine (about 1 cm distance from an accumulation of worms), the spleen, the kidney and the heart were collected and snap-frozen. As negative control respective biopsies of an uninfected hamster were used.For PCR detection of P. elegans (qPCR) containing Neorickettsia were used. For conventional PCR primers targeting Neorickettsia 16 rDNA (n16S-25 F and n16S-1500R) were used as described previously [Neorickettsia suface protein or P. elegans 28S rDNA  according to the instructions of the manufacturer. Each sample was tested in duplicate and 1 μl of template DNA was used. As positive controls either snails infected with trematodes  or adult 20, stained with uranyl acetate , rinsed again in dH20, dehydrated and embedded in Eponate 12 resin (Ted Pella Inc.). Sections of 95 nm were cut with a Leica Ultracut UCT ultramicrotome , stained with uranyl acetate/ lead citrate, and viewed on a JEOL 1200 EX transmission electron microscope  equipped with an AMT 8 megapixel digital camera . Figure plates were assembled using Microsoft Powerpoint 2010 and Adobe Photoshop Elements 8.0. Images were not computationally enhanced except for adjustments for brightness and contrast.Following chemical fixation samples were washed, postfixed in osmium tetroxide (Polysciences Inc.), rinsed in dH20) under liquid nitrogen and placed in the Leica AFS, automatic freeze substitution system (Leica Microsystems). For freeze substitution, the samples were brought to -90 °C for 1 h, remaining at -90 °C for10 h, and subsequently warmed to -20 °C for18 h. Samples were placed at 4 °C for 30 min prior to washing with anhydrous acetone at room temperature, embedded in Eponate 12 resin, and sectioned and stained as described above.Individual live flukes were placed in specimen planchettes containing 20% bovine serum albumin and processed as described previously . BrieflyWolbachia endosymbionts in Brugia malayi worms by immunohistology using the Wolbachia surface protein 1  we used its amino acid sequence to search the genome of N. risticii (GenBank accession number CP001431) [N. risticii surface protein 3 (Nsp-3) as a member of the porin subfamily (GenBank accession number gi|340806186) and designed primers (Nsp3-F: 5′-CAC CAT GAT AAA TAG AAA GTT CCT AGT GGG TG-3′ and Nsp3-R: 5′-GAA TTA TGC ATT CAT AGG TAC CAT C-3′) to amplify an open reading frame of Nsp-3 from genomic DNA extracted from Neorickettsia-infected P. elegans. The PCR product was cloned into the pET Directional Topo Expression vector  and the open reading frame of the plasmid insert was confirmed by DNA sequencing. BL21 cells were transformed with the plasmid and the protein was expressed according to the instructions of the manufacturer. The recombinant His-tagged P. elegans Nsp-3 protein (rPeNsp-3) was purified using His-Select Cobalt Affinity Gel (Sigma) chromatography and used for further studies.Because of our experience to detect PeNsp-3 protein in complete Freund’s adjuvant and boosted with 20 μg rNSp3 in incomplete Freund’s adjuvant 14 days later. Immune sera were collected 21 days post initial immunization. Pre-immune sera of the same mice were collected before the initial immunization and used as negative controls.Two BALB/c mice were each immunized using 20 μg purified rP. elegans protein was extracted and electrophoresed using 4–12% NuPAGE Bis-Tris minigels (Invitrogen) as described previously [Neorickettsia infected P. elegans total worm protein extract or rPeNsp-3 antigen. Briefly, 10 μg of antigen per cm was separated on a 412% reducing gel, blotted onto a nitrocellulose membrane, blocked at room temperature (RT) for 30 min using 5% non-fat dry milk  in PBS, and incubated with animal sera at a dilution of 1:100. Sera and secondary antibodies were diluted in PBS-tween and incubated at 37 °C for 1 and 2 h, respectively. After incubation with a secondary antibody , blots were washed with PBS-tween, and antibody binding was detected using nitro-blue tetrazolium/5-bromo-4-chloro-3′-indolyphosphate substrate (Sigma).Total adult eviously . WesternPeNsp-3 were used as primary antibodies. Sera were tested at dilutions (in PBS/0.1% triton-X) of 1:50, 1:100, 1:200, 1:250, 1:500 and 1:1,000. The dilution 1:250 was determined to deliver the best signal/background relationship and all further experiments were performed at that dilution. Rabbit-anti mouse IgG  was applied as secondary antibody and was bound to the APAAP complex. As substrate for alkaline phosphatase the chromogen Fast Red TR salt (Sigma) was used and hematoxylin  served as counter-stain. Sections were examined using an Olympus-BX40 microscope  and photographed with an Olympus DP70 microscope digital camera.The alkaline phosphatase anti-alkaline phosphatase (APAAP) technique was applied for immunostaining according to the recommendations of the manufacturer  and as described earlier . Pre-immhttp://micro.imaging.wustl.edu/).For fluorescent analysis Alexa Fluor 488 conjugated goat anti-mouse IgG (H + L)  was used as a secondary antibody. Alexa Fluor 633 conjugated wheat germ agglutinin  was used as membrane stain at 200 μg/ml for 10 min prior to mounting. For mounting Prolong Gold Antifade medium (Thermo Fisher Scientific) with DAPI was used. Wide field fluorescence microscopy  with plan-apochromat 100× oil, 63× or 40× objectives was performed as described previously . Wide-fiP. elegans  with adapters for directional cloning in the expression vector. The recombinant, His-tagged, PeNsp-3 is comprised of 296 AA with a predicted molecular weight of 31.1 kDa, while the native protein has only 260 AA with a predicted MW of 27 kDA. These predictions were confirmed by western blot using either rPeNsp-3 or native total P. elegans extract as antigens and either a monoclonal antibody against the His-tag or a polyclonal mouse antibody against rPeNsp-3 as antibodies , endobacteria were detected in somatic tissues as well as in female and male reproductive tissues. Although endobacteria appear to be randomly scattered throughout the worms, densities were highly variable in different tissue types but a clear distribution pattern was recognized.The antisera directed against rund Fig. , and theund Fig. . Table 1Neorickettsia in vitelline tissue is high. High densities of endobacteria were observed in the Mehlis’ gland  were needed to detect Neorickettsia DNA compared to worm DNA. For the intestine of infected hamsters almost the same number of cycles (28.93 vs 28.18) was needed to detect Neorickettsia and worm DNA. These results can be interpreted as further evidence that the bacteria are not only residing in a few P. elegans eggs but are also infecting the intestine.The presence of endobacteria in the suckers and the intestinal tract Figs.  could inder Fig.  or via sent Fig. . We haveNeoricketttsia in adult trematodes on the ultrastructural level. Neorickettsia have been shown to infect a broad variety of trematode parasites of wildlife [Neorickettsia endobacteria have been shown to infect the common liver fluke Fasciola hepatica [Neorickettsia endobacteria, that may constitute important pathogens themselves, is urgently needed.The present study is the first detection of wildlife . Only rehepatica . This imNeorickettsia in P. elegans. Almost all previous studies on the ultrastructure of Rickettsiales used exclusively chemical fixation, and only few studies used physical fixation for TEM [Neorickettsia in adult P. elegans described in the present study was in general similar to the ultrastructure of N. risticii and N. sennetsu described from mammalian cell cultures [Neorickettsia in P. elegans appears to be more similar to N. sennetsu that rarely forms large morulae with many reticulate or dense-cored cells like N. risticii [P. elegans. Although related tick-transmitted Ehrlichia species may show different morphology in mammalian and tick cell cultures, the number of bacterial cells per morula is usually similar in vertebrate and vector cells [Neorickettsia in adult P. elegans is predominantly of the N. sennestu-type.High pressure freeze/freeze substitution fixation was well suited for TEM detection of  for TEM , 21. Whicultures . For exacultures . Howeverristicii , 8. In oor cells , 24. ThePeNsp-3 and showed that polyclonal mouse antisera raised against PeNsp-3 are suitable for sensitive and specific detection of Neorickettsia in P. elegans. With this antibody endobacteria show the typical ‘donut’-shaped labeling pattern of a surface protein  of Ehrlichia chaffeensis [E. chaffeensis it has been shown that P28 Omp expression is tightly regulated with peaks of expression in the mid- and late-exponential growth phases at 28 and 37 °C [Neorickettsia may not show maximum expression of PeNsp-3 in all life-cycle stages of P. elegans. However, adult trematodes live at mammalian body temperature where PeNsp-3 may show its maximum expression level. Recently, a serum of a horse infected with N. risticii has been used to localize Neorickettsia in different life-cycle stages of P. elegans [PeNsp-3 antisera confirm most of the results for adult trematodes from this previous study, minor differences were recorded that may be explained by the higher specificity and lower background staining observed in our staining using antisera against a single specific surface protein. For example, we unambiguously detected small numbers of endobacteria in periphery of the ovary.We have successfully expressed ein Fig. , while iffeensis , 25. In  elegans . While oNeorickettsia in entire histological worm sections in a larger number of P. elegans specimens. Transmission electron microscopy and IHC provided similar results  has been described previously from several other invertebrates [P. elegans may not only act as vectors for Neorickettsia, but also function as reservoir with circulation of endobacteria between adult trematodes. On the other hand, some Neorickettsia species infect more than one digenean host species. Our findings do not explain how Neorickettsia are transmitted between digeneans belonging to different species. This is likely to involve horizontal transmission via tissues of either intermediate or definitive hosts.Immunohistochemistry was used to localize  in eggs . We haveematodes  the numbtebrates , 27. AduN. risticii was horizontally transmitted to bats by its trematode host Acantharium oregonense or that N. helminthoeca was transmitted to dogs by adult Nanophyetus salmincola trematodes [N. helminthoeca transmission by trematode eggs, dogs were injected either with fluke eggs homogenized in a glass tissue grinder or with intact eggs. Only homogenized eggs were infectious to dogs, suggesting that infectious Neorickettsiae were localized within the interior of the unembryonated N. salmincola eggs but not outside the egg shells [It has been reported that ematodes , 28. In P. elegans reside in the small intestine, produce eggs with thick egg shells like many other trematodes, and the eggs need the operculum to be opened to release miracidia. It appears unlikely that a considerable number of the sturdy and acid-resistant eggs burst and release Neorickettsia that will infect the mammalian host. Therefore, we do not favor the hypothesis that Neorickettsia are transmitted horizontally by trematode eggs. Tick-transmitted Rickettsiales are usually localized in large densities within the salivary glands of the vectors and transmitted with the saliva during the blood meal [P. elegans are actively attached to the host tissue by their suckers (frequently causing a hemorrhage at the attachment site) and that Neorickettsia can be found in the suckers and the intestinal epithelium it is possible that they are actively transmitted at the attachment site. Other potential routes of horizontal transmission are during shedding of tegumental material or via the excretion pore. This first study indicates that 20 days after exposure of hamsters to Neorickettsia-positive P. elegans metacercariae, Neorickettsia infection outside the trematode is mainly restricted to the small intestine, the mucus and the connective tissue within the villi. Further studies have to elucidate the detailed mechanism of horizontal transmission.Adult ood meal . Little Neorickettsia are not obligatory endosymbionts of digeneans, since neither all strains nor all individuals within a host species are usually infected [Neorickettsia-positive sporocysts and the examined adult worms were all infected with Neorickettsia. Our study showed that endobacteria are probably interacting with trematode tissues and organelles by releasing vesicles. However, it is not known whether Neorickettsia cause certain phenotypes such as Wolbachia endobacteria in insects. Since Neorickettsia are susceptible to some common antibiotics further studies may show whether removal of endobacteria by antibiotic treatment causes a phenotype in previously infected digeneans [It is generally agreed that infected , 15, 30.igeneans .Neorickettsia in its trematode host, it establishes a novel detection method for Neorickettsia using PeNsp-3 antisera and describes in detail the localization of Neorickettsia in adult trematodes. The gathered information is crucial for proper explanation of the transmission mechanisms of Neorickettsia and to further analyze the relationship of Neorickettsia and their trematode host.Taken together our study uncovers for the first time the ultrastructure of Neorickettsia in adult P. elegans reveals a high degree of pleomorphy. Polyclonal anti-sera raised against recombinant PeNsp-3 protein are helpful for the localization of Neorickettsia. These endobacteria are localized in varying numbers in many tissue types that support vertical transmission, trematode to trematode transmission via seminal fluid, and possibly horizontal transmission from trematodes to vertebrate hosts. These means appear to be novel mechanisms of pathogen transmission by endoparasitic worms.Transmission electron microscopy of Additional file 1: Figure S1.P. elegans anatomy, showing the proportions of major organs and tissues. a Representative longitudinal section. b Whole, unstained, living trematode. c Magnification of the proximal part (boxed area from b) showing many anatomical details. Abbreviations: ate, anterior testis; cs, cirrus-sac; g, gut; me, metraterm; Mg, Mehlis’ gland; os, oral sucker; ov, ovary; ph, pharynx; pr, prostate gland; pte, posterior testis; sv, seminal vesicle; ut, uterus; vf, vitelline follicles; vs, ventral sucker. Scale-bars: 1 mm. (TIF 5591 kb)Overview of adult Additional file 2: Table S1.Neorickettsia DNA and P.elegans DNA in various tissue samples by qPCR. (DOCX 15 kb)Primers and probes used to detect Additional file 3: Figure S2.P. elegans. a Intense labeling of electron dense areas of the nucleus of a subtegumental cell. b DNA of endobacteria (arrows) is extensively labeled by gold particles, while mitochondria that contain only about 1% of the amount of DNA are not labeled. c Three strongly labeled endobacteria (arrows) in a vacuole (arrow heads). d Small, more electron-dense endobacterium labeled for DNA in the cytoplasm (arrow) and close proximity inside and outside of the vacuole membrane (arrowhead). Abbreviations: m, mitochondrion; n, nucleus. Scale-bars: a-c, 500 nm; d, 100 nm. (TIF 6472 kb)Immunogold labeling using an anti- DNA antibody which detects double stranded DNA in the nucleus and endobacteria, but not in mitochondria of Additional file 4: Figure S3.P. elegans. a Overview of a cross-section of the tegument showing a lose cluster of endobacteria (arrows) in one area, but no endobacteria in other areas. b Large endobacteria (arrows) are localized in the wall of the gut, while endobacteria are mostly absent in adjacent tissues. Note the cluster of small structures (boxed area) in the parenchyma. c Magnification of boxed area from b shows a few small endobacteria (arrows) with typical membrane structures among electron dense structures without pronounced membranes. Abbreviations: m, mitochondrion; sgc, shell globule cluster. Scale-bars: a, b, 5 μm; c 200 nm. (TIF 6120 kb)TEM of HPF/FS fixed Additional file 5: Figure S4.P. elegans. a Large clusters of endobacteria in the tegument (circles) and the cytons. b Large clusters of endobacteria (circles) in the subtegument. c Magnification of b showing pleomorphic endobacteria without well preserved membranes. d Three endobacteria with multiple and well preserved membranes. e Magnification of boxed area of d showing the well preserved membrane structure. Abbreviation: tsp, tegumental spine. Scale-bars: a, b, 2 μm; c-e, 500 nm. (TIF 6604 kb)TEM of chemically fixed Additional file 6: Figure S5.P. elegans using polyclonal antisera against rPeNsp-3. a Longitudinal section near the ventral sucker showing diffuse red staining for endobacteria in many tissue types. b Light pink background around the vitelline follicles , notice the red staining outside of the ovary (arrow). d Intense red staining for endobacteria in the Mehlis’ gland where density of Neorickettsia is high APAAP staining for endobacteria in Additional file 7: Figure S6.Neorickettsia outside P. elegans . a Adult P. elegans in the intestine of a hamster, pre-immune serum. b Consecutive section to a but stained with anti-sera against PeNsp-3. c Magnification of b showing connective tissue of the gut within the pharynx of P. elegans. Note single red cells (arrow) positive for PeNsp-3. d Another magnification of b showing single red cells (arrow) positive for PeNsp-3 in connective tissue within the hamster villi. e Green labeling for PeNsp-3 of bacteria (arrow) within the intestinal mucus or connective tissue within the villi. f High resolution, donut-shaped labeling for PeNsp-3 of bacteria (arrow) in the mucus. g Conventional PCR detection of Neorickettsia targeting 16S rDNA. Lane M: Marker; Lane 9: no template; Lanes 10, 11: Neorickettsia positive trematodes in field-collected snails; Lanes 1–4: P. elegans-infected hamster 1; Lanes 5–8: P. elegans-infected hamster 2; Lanes 12–15: uninfected hamster. Lanes 1, 5, 12: heart tissue; Lanes 2, 6, 13: kidney tissue; Lanes 3, 7, 14: spleen tissue; Lanes 4, 8, 15: small intestine tissue. Abbreviations: ct, connective tissue; os, oral sucker; ph, pharynx; ts, tegumental syncytium; vil, villi of the intestine. Scale-bars: 100 μm. (TIF 7014 kb)Detection of Additional file 8: Table S2.P.elegans DNA and Neorickettsia DNA from 20 mg of various tissues. Both targets were only detected in isolated P. elegans worms and in the small intestine from hamsters infected with P. elegans (but without adult trematodes present). Significantly more Neorickettsia DNA relative to trematode DNA was found in the intestine. (DOCX 14 kb)Comparison of qPCR amplification  of


The norovirus genogroup II genotype 4 (GII.4) variants have approximately 5% divergence in capsid amino acid identity and have dominated over the past decade. The precise reason(s) for the GII.4 emergence and persistence in the human population is still unknown, but some studies have suggested that chronically infected patients might generate novel variants that can cause new epidemics. We examined GII.4 noroviruses isolated from an immunocompromised patient with a long-term infection. Numerous norovirus capsid quasi-species were isolated during the 13-month study. The capsid quasi-species clustered into two genetic and antigenic types. However, the HBGA binding profiles were similar between the two antigenic clusters, indicating that the amino acid substitutions did not alter the HBGA binding interactions. The isolated sequences represented two new GII.4 variants, but similar sequences were not found in the database. These results indicated that chronically infected patients might not generate novel noroviruses that cause outbreaks. Typically, human noroviruses cause symptoms of acute gastroenteritis for 2 to 4 days. Often, the virions are shed in stool for several days after the symptoms recede, which in turn can lead to further contamination and transmission. Moreover, a number of reports have considered that chronic norovirus infections, i.e., lasting months and years, might even function as reservoirs for the generation of novel strains that can escape the herd immunity or have modified binding interactions with histo-blood group antigens (HBGAs). In this study, we analyzed noroviruses isolated from a patient who has presented a chronic infection for more than 6 years. We found that the isolated capsid sequences clustered into two main genetic types (termed A and B), despite a plethora of capsid quasi-sequences. Furthermore, the two genetic types corresponded well with distinct antigenicities. On the other hand, we showed that numerous amino acid substitutions on the capsid surface of genetic types A and B did not alter the HBGA binding profiles. However, divergent binding profiles for types A and B were observed with human milk oligosaccharides (HMOs), which structurally mimic HBGAs and may act as natural antivirals. Importantly, the isolated capsid sequences only had approximately 90% amino acid identity with other known sequences, which suggested that transmission of these chronic noroviruses could be limited.IMPORTANCE The norovirus genogroup II genotype 4 (GII.4) variants have approximately 5% divergence in capsid amino acid identity and have dominated over the past decade. The precise reason(s) for the GII.4 emergence and persistence in the human population is still unknown, but some studies have suggested that chronically infected patients might generate novel variants that can cause new epidemics. We examined GII.4 noroviruses isolated from an immunocompromised patient with a long-term infection. Numerous norovirus capsid quasi-species were isolated during the 13-month study. The capsid quasi-species clustered into two genetic and antigenic types. However, the HBGA binding profiles were similar between the two antigenic clusters, indicating that the amino acid substitutions did not alter the HBGA binding interactions. The isolated sequences represented two new GII.4 variants, but similar sequences were not found in the database. These results indicated that chronically infected patients might not generate novel noroviruses that cause outbreaks. Human noroviruses are the dominant cause of outbreaks of acute gastroenteritis. The disease is typically self-limiting, with most symptoms lasting two to four days. However, noroviruses can be shed in stool for several weeks after an infection and asymptomatic individuals can transmit infectious particles to other individuals, which can also lead to additional outbreaks . In geneHuman noroviruses are genetically and antigenically diverse , yet a s8–Several reports have identified chronic norovirus infections lasting months to years, especially in immunocompromised individuals –16. Some–The norovirus genome is divided into three open reading frames (ORFs), where ORF1 encodes the nonstructural proteins, including the RNA-dependent RNA polymerase (RdRp), ORF2 encodes the capsid protein (VP1), and ORF3 encodes a small structural protein. The expression of the norovirus capsid protein in insect cells leads to the self-assembly of virus-like particles (VLPs) that are morphologically similar to the native virion . The norThe norovirus capsid interacts with host histo-blood group antigens (HBGAs). This interaction with HBGAs is known to be important for virus entry and replication , 22, andDespite their discovery more than four decades ago, there are still no approved vaccines or antivirals available for human noroviruses. However, human milk oligosaccharides (HMOs), which mimic HBGA structures, were recently shown to block norovirus VLPs from binding to HBGAs , making In this study, we analyzed noroviruses isolated from an immunocompromised patient presenting a long-term infection in order to examine the hypothesis that chronically infected individuals are reservoirs for clinically relevant noroviruses. We identified a plethora of novel capsid quasi-species that showed varied cross-reactivities and binding interactions with HBGAs and HMOs. Importantly, similar capsid sequences are not yet reported, suggesting that these viruses were not easily transmitted to other individuals.A total of 186 full-length capsid genes from the patient were isolated and sequenced. All sequences belonged to GII.4. Four of 186 sequences contained premature stop codons near the C terminus, while 182 were of the putative full-length size. Interestingly, an earlier study showed that norovirus capsids with C-terminal deletions produced VLPs with larger diameters but reduced stability compared to those of native-size particles . TherefoGenetic analysis showed that a plethora of capsid quasi-sequences were generated over the 13-month period. However, the 182 capsid sequences clustered into two main genetic types (termed A and B), which encoded 540 and 539 amino acids, respectively . The nucInterestingly, type A sequences were isolated in all months, whereas type B sequences were only detected in 9 months . Haploty5 and 9.4 × 106 virus cDNA copies/g of stool  and B  were plotted onto P domain homology models in order to illustrate sequence variations . For typ31–In order to better describe the capsid phenotypes, the antigenic variations were analyzed with norovirus-specific antisera  and six different VLPs that represented genetic types A and B . These r10.1128/mSphere.00352-16.1FIG S1 490 of 0.2) is shown with dashed lines. Saga-1 VLPs  were used as a positive control in all experiments (black line). MAb-1, MAb-3, and MAb-6 cross-reacted with all VLPs. MAb-4 and MAb-9 cross-reacted with type A VLPs, i.e., VLP2, VLP5, and VLP7. MAb-2, MAb-8, and MAb-10 did not cross-react with any of the VLPs except the positive control. Download FIG S1, PDF file, 0.2 MB.Cross-reactivities of the six VLPs against GII.4 antisera. The VLPs were normalized to the same starting concentration. The cutoff , saliva samples, and synthetic HBGAs. We found that the type A and B VLPs bound to PGM at similar cutoff levels  and with similar binding profiles . All VLP50s) were 60 mM, 116 mM, and 82 mM for type A VLPs . For type B VLPs , the inhibition was considerably stronger (~10-fold), such that the 3FL IC50s were 6 mM, 6 mM, and 3 mM, respectively. Overall, these data showed that 3FL interacted differently with type A and B VLPs. Moreover, these results showed that 3FL’s inhibition of the binding of chronic and GII.10 VLPs was equivalent, whereas 2′FL’s inhibition of the binding of chronic VLPs was noticeably different than its inhibition of the binding of GII.10 VLPs (Our previous data indicated that the HMOs 2′-fucosyllactose (2′FL) and 3FL inhibited GII.10 VLPs from binding to HBGAs . In orde.10 VLPs .The main aim of this study was to investigate whether a chronically infected individual generated novel norovirus variants that were further transmitted into the community. For this purpose, we analyzed norovirus variants in an immunocompromised patient with long-term infection. We isolated a plethora of GII.4 capsid quasi-species during the 13-month period. The sequences clustered into two main genetic types (A and B) that also corresponded with two antigenic types. We found that the capsid sequences isolated had approximately 90% amino acid identity to other known sequences in the database. Therefore, these capsid sequences were assumed to represent novel GII.4 variants that have not yet been reported for any outbreaks.Interestingly, both the patient and his parents exhibited antibodies that were reactive against the chronic noroviruses. Remarkably, the parents had not presented a norovirus infection during the study period, and yet, the viral loads in the chronic patient were equivalent to those found in acute infections. On the other hand, a recent study has also shown that chronically infected patients had not transmitted noroviruses to other individuals . These rNumerous molecular epidemiological studies have indicated that the GII.4 noroviruses evolve into new genetic variants every other year, leading to six major pandemic outbreaks since 1996 –41. We p–As mentioned above, our enzyme-linked immunosorbent assay (ELISA) data showed that the type A and B VLPs interacted with HBGAs in a similar manner. On the other hand, we found that 3FL’s inhibition of binding was different with types A and B, while 2′FL showed little inhibition with both type A and B VLPs. This result suggested that amino acid substitutions located near the HBGA pocket altered specific HMO binding interactions. We previously showed that 2′FL and 3FL bound to GII.10 P domains with various residues and different orientations, although the fucose moiety was held similarly . AlthougIn summary, our data indicated that the chronically infected patient produced numerous norovirus capsid quasi-sequences over the span of an infection. This is the first reported case of an individual cocontaminated with two GII.4 variants for such a long period. Similar capsid sequences are not yet reported in the database, suggesting that these viruses are not easily transmitted to other individuals. More importantly, since outbreaks with closely matching sequences have also not been reported, it could be that these viruses were less pathogenic than other GII.4 variants, although direct evidence is lacking.The male patient suffers from a complex X-linked syndrome and is under immunosuppressive therapy. During the study, the patient lived at home together with his parents and a healthy brother who was three years older. The patient was admitted to hospital within the study period only once for 5 days due to pneumonia. He had no severe diarrhea during the study period. He attended a school for disabled children, and no outbreaks of norovirus gastroenteritis occurred there within the 13-month study. In August 2009, the patient suffered from an acute norovirus infection that lasted approximately 6 days. Thereafter, norovirus could be isolated repeatedly, leaving him as a chronic norovirus carrier. However, the patient reported only a few symptoms of acute gastroenteritis that were directly norovirus associated during the past 6 years. In 2009, 2 days after the patient was hospitalized, his mother, who accompanied him in the hospital, suffered from an acute gastroenteritis infection that lasted 3 days. The visiting father also became ill with mild gastroenteritis for 2 days. At this time, neither parents’ stool was tested for norovirus. Subsequently, no further symptoms of gastroenteritis have occurred in the parents up to the present time. The elder brother had no reported cases of gastroenteritis. For this study, stool specimens were collected from the patient  and both parents (March 2015), with whom he resides. Additionally, sera from the patient and his parents, as well as saliva from the patient, were collected in July 2015. All materials were obtained after written informed consent and in agreement with the Declaration of Helsinki, federal guidelines, and the Local Ethics Committee (2015-589N-MA).−6 errors/bp/PCR cycle) (4OOX and 5IYN) in order to display amino acid substitutions.Initially, the partial capsid gene was amplified with broadly reactive primers using RT-PCR , and quaR cycle)  with genR cycle) . The virR cycle) . Genetico-phenylenediamine (OPD) and H2O2 in the dark for 30 min at room temperature. Finally, the reaction was stopped with 3 N HCl, and absorbance was measured at an optical density of 490 nm (OD490). All experiments were performed in triplicate. The binding cutoff was set to an OD490 of 0.2, as previously determined for norovirus VLPs  and saliva was measured using a method previously described . BrieflyMaxiSorp 96-well plates were coated with 15 μg/ml VLPs overnight at 4°C, washed with PBS-T, and blocked with 5% SM. Synthetic HBGAs  conjugated with phosphonoacetic acid (PAA)-biotin were dissolved in distilled water to 100 μg/ml. Twofold serial dilutions of synthetic HBGA were added to the plates and incubated for 2 h at 37°C. The plates were then washed, incubated with streptavidin-HRP for 1 h at 37°C, and processed as described above.490 of the untreated VLPs was set as the reference value corresponding to 100% binding. The percentage of inhibition was calculated as follows: [1 − (mean treated-VLP OD490/mean reference OD490)] × 100. The half-maximal inhibitory concentration (IC50) was determined using Prism software (version 6.0)  were pretreated with serially diluted HMOs (2′-fucosyllactose [2′FL] or 3FL) for 1 h at room temperature. PBS was used as a blank, and untreated VLPs were used as a binding reference for each plate. The ODion 6.0) .KX514170 to KX514370.The nucleotide sequences determined in this study were deposited in GenBank under the following accession numbers:


Fanconi Anemia (FA) complementation group D2 protein (FANCD2) is the center of the FA tumor suppressor pathway, which has become an important field of investigation in human aging and cancer. Here we report an overlooked central player in the FA pathway, FANCD2 variant 2 (FANCD2-V2), which appears to perform more potent tumor suppressor-function compared to the known variant of FANCD2, namely, FANCD2-V1. Detailed analysis of the FANCD2 gene structure indicated a proximal and distal polyadenylation site (PAS), associated with V2 and V1 transcripts accordingly. RNA polymerase II Chromatin immunoprecipitation (ChIP) targeting the two PAS-regions determined lesser binding of RNA pol II to DNA fragments in the distal PAS region in non-malignant cells compared to malignant cells. Conversely, the opposite occurred in the proximal PAS region. Moreover, RNA immunoprecipitation (RIP) identified that U2 snRNP, a major component of RNA splicing complex that interacts with the 3′end of an intron, showed greater binding to the last intron of the FANCD2-V1 transcript in malignant cells compared to the non-malignant cells. Importantly, our data showed that in human tissue samples, the ratio of V2 /V1 expression in lung, bladder, or ovarian cancer correlates inversely with the tumor stages/grades. Therefore, these findings provide a previously unrecognized central player FANCD2-V2 and thus novel insights into human tumorigenesis, and indicate that V2/V1 can act as an effective biomarker in assisting the recognition of tumor malignance. Fanconi Anemia (FA) is one of aging diseases, characterized with many developmental abnormalities and an extremely high incidence of both hematological and non-hematological malignancies –6. At thAlthough FA genes do not share sequence similarity, their corresponding protein products are closely related . Among 2The functions of the FA pathway are heavily depending upon each FA gene encoded protein. How FA protein product is produced depends upon the multiple layers of regulatory processes, especially those involved in RNA processing , 37. PolThrough sequence similarity searching from NCBI, we found a 60 bp longer version of FANCD2 coding cDNA, encoding a protein carrying more than 95% of homology with the known “FANCD2” protein. We named the newly recognized version of FANCD2, −“FANCD2-V2”−(V2), and −“FANCD2-V1”− (V1) for the long- known form. As this distinction has not been previously described, many of the reported functions of FANCD2 could reflect the properties of either V1, V2, or both. In this study, we are among the first to report an overlooked representative of the FA signaling pathway, which we termed FANCD2-V2. Our data show that this variant of FANCD2 exhibits greater association with non-malignant cells compared to malignant cells and properties of a potent tumor suppressor. Most importantly, we found that PAS plays a critical role in regulating the expression of different variants of FANCD2.Considering the importance of FANCD2 in the FA pathway, we investigated its potential homologs and found that, besides the long-known form of FANCD2 , there is an overlooked alternative variant (NCBI RefSeq accession# NM_033084.4). This newly discovered variant, which we named FANCD2-V2, has not been previously characterized. Henceforth, in order to both recognize and distinguish the two variants we have named the long-known form of FANCD2, FANCD2-V1. As shown in Figure To understand how FANCD2-V2 expression is regulated, we thoroughly analyzed the gene structure of FANCD2. Inspection of the FANCD2 gene showed two potential polyadenylation sites, a proximal or distal site, associated with V2 and V1 respectively Figure  42]. Ac. Ac42]. To test this, we performed RNA Pol II ChIP with two sets of ChIP PCR primers as illustrated in Figure As illustrated in Figure http://firebrowse.org/). The ratio of V2/V1 expression was higher in stage I, II and III tumors compared to stage IV tumors. Additionally, the V2/V1 ratio in stage IV tumors were significantly lower than that in lower-stage tumors, even when combined  shown in Figure d Figure . This sud Figure . In loweSince 1971, FA signaling has been argued to be responsible for various tumor suppressor roles by Dr. Swift . HoweverIn living cells, transcriptional variants, which differ in length and production are common, especially in human malignant cells. Importantly, intricate regulatory mechanisms underpin the diversity observed amongst these variants. At the DNA level, the local DNA structure, such as the B-B or A-A premeltons , a varieCompared to genetic, epigenetic, and other processes in RNA processing, PAS influences a fate of a gene function was under-reported. However, PAS is shown here to be an essential factor in determining the tumor suppressor function of the FA pathway, because the newly recognized version of FANCD2 (V2) appears to have tumor-suppressor function more potent than the version we have known for decades (V1) Figure  and 4. OAll cell lines were obtained from the American Type Culture Collection (ATCC).Cellular RNA was extracted by TRIzol Reagent (Life technologies). Reverse Transcription was performed using the Bio-Rad iScript Reverse Transcription kit as per the manufacturer's instruction.Ovarian cancer tissue cDNA samples were provided by Dr. Herbert Yu , 47. ReaThe following PCR primer sequences were used: FANCD2 V1 F, GAT GGT GAA GAA GAC GA and FANCD2 V1 R, GGT CTA ATC AGA GTC ATC A; FANCD2 V2 F, GTA TCT CTA CAA AAC CCA C and FANCD2 V2 R, GCT GTT ATG GAG GGA ATG.A ChIP assay was performed as previously described . The priDistal forward primer: TGC CTC AGT TGC CTC ATT TAT, (reverse primer): GCA CCA GTC TCC ATA ATT TCT CT;Proximal forward primer: GGC TCA CAC CTG TAA TCG TAG, (reverse primer): GTC TCG GGT TGG TCT TGA AA;Cells were harvested using trypsin and suspended into well isolated and subsequently crosslinked with 1% formaldehyde at room temperature for 10 min. After the reaction was quenched with 125 mM Glycine, the cells were washed 3 times in PBS and then resuspended in buffer A  and 50 μl/mL SUPERase In RNase inhibitor (Life technologies) and incubated on ice for 10 min. The pelleted crude nuclei fractions were centrifuged at 2500 g for 15 min at 4°C and then washed once in buffer A without NP40. The pellet was resuspended in RIP buffer , and the solution was incubated on ice for 10 min. The lysate was sonicated in a Bioruptor Plus , two times for 10 min each at high power while cycling for 30 seconds on and 30 seconds off. Following sonication, the lysate was pelleted via centrifugation at 13,000 RPM for 10 min to remove insoluble debris. The supernatant was diluted in IP buffer  at a 1:1 ratio. After the solution was IgG pre-cleaned, a 5% aliquot of each sample was preserved in 1 mL of TRIzol Reagent (Life technologies), and this aliquot was used as the input control. The rest of the lysate was divided into two equal parts to which 2 μg of the SF3A1 antibody  or the control rabbit IgG (Sigma-Aldrich) was added. All tubes were rotated at 4°C overnight, and then 50 μl of rec-Protein A-Sepharose 4B (Life Technologies) beads were added to each eppendorf tube, and then rotated for an additional 2 h at 4°C. The beads were washed 3 times using wash buffer , and then 1 mL of TRIzol Reagent was added to each tube. RNA was isolated from these samples, including the input controls, according to the manufacturer's instructions. RT-qPCR was performed using a SensiFAST SYBR Hi-ROX One-Step Kit (Bioline) in a StepOnePlus Real-Time PCR System (Life Technologies). The primer sequences that were used in these experiments are listed below.Proximal (forward primer): TGT GTA TCC TCT AGG AGC TGT AT, (reverse primer): CTT CGT CTT CTT CAC CAT CCT AA;Distal (forward primer): TGC CTG TAA ACT CAA CCT TCT C, (reverse primer): GGG ACT TAA TCT CTT CAC CCT AAA T.t-test.Ovarian cancer RT-qPCR results were tested by Distribution of Wilcoxon Scores. The rest data were examined using two-tail student's


TNF and IL17 genes are associated with SpA, ankylosing spondylitis (AS), and psoriatic arthritis (PsA) in a mixed Brazilian population.  Spondyloarthritis (SpA) represents a heterogeneous group of immune-mediated inflammatory diseases that have overlapping clinical features, genetic predisposition, and pathogenic mechanisms. Hence, we investigated, through a case-control study, whether single-nucleotide polymorphisms of TNF-308 (rs1800629), TNF-238 (rs361525), IL17A (rs2275913), IL17F (rs763780), and HLA-B27 polymorphisms was performed in 243 patients with SpA and 210 controls from Southern Brazil using SSOP-Luminex (One Lambda) and PCR-SSP assays.  Genotyping of TNF-308 (rs1800629) AA/GA, IL17A (rs2275913) AA/GA, and IL17F (rs763780) CC/TC genotype frequencies were associated, in the dominance inheritance model, with SpA and AS, regardless of gender, the presence of HLA-B27, TNF-238 (rs361525) GA/AA, IL17A (rs2275913) AA/GA, and IL17F (rs763780) genotypes was associated with PsA.  Significant associations were confirmed between the HLA-B27 marker and SpA, AS, and PsA diseases. While TNF and IL17 gene polymorphisms responsible for the expression of important inflammatory cytokines were associated with overall SpA, and, specifically, with AS and PsA, regardless of gender and HLA-B27. However, future larger studies with different ethnicities may be necessary to confirm these genetic associations. In this Brazilian population,  Spondyloarthritis (SpA) represents a heterogeneous group of immune-mediated inflammatory diseases that have overlapping clinical features, genetic predisposition, and pathogenic mechanisms. The disease may be undifferentiated or manifest as reactive arthritis, psoriatic arthritis (PsA), arthritis associated with inflammatory bowel disease, or ankylosing spondylitis (AS) , 2.HLA-B27 and AS [HLA-B27-positive people develop the disease, and not all patients with the disease possess the HLA-B27 antigen, suggests that other genes may be involved in the development of the pathology [The pathogenesis of SpA is partly attributed to the interaction between genetic factors and the environment. Males are affected more frequently than females according to results from two meta-analysis studies , 4, and athology , 6.TNF) and interleukin (IL) 17A and IL17F genes, encoded on the same chromosome 6 of the HLA genes, and their clinical importance in the pathogenesis of SpA have not been fully elucidated [α and IL17 may be elevated in patients with SpA and may contribute to the pathogenesis of the disease  [α, IL1β, and IL6, which can cause generalized tissue damage [Polymorphisms in genes encoding cytokines that interfere on cytokine level production have been associated with rheumatologic chronic inflammatory diseases –10. Howeucidated –13. Proi et al.) . These ce damage .TNF, IL17A, and IL17F genes are associated with SpA and its clinical forms, AS, and PsA, in a mixed population from the South of Brazil.Thus, the aim of the present study was to investigate whether the single-nucleotide polymorphisms (SNPs) of the HLA-B27. In patients with PsA, we also used the CASPAR criteria [In this case-control study, 243 unrelated patients were diagnosed by the ASAS Criteria 2009  for axiacriteria , to compIn addition, 210 individuals were selected to form the control group, following the criteria for inclusion: no autoimmune and/or rheumatic diseases, unrelated to the patient group, and belonging to the same ethnic group as the patients.The population of Paraná is predominantly of European origin (80.6%), with a small but significant contribution of African (12.5%) and Amerindian (7.0%) genes . Both thThe project was approved by the Ethics Committee of the Maringa State University (number 687.222/2014). After terms of consent were signed, blood samples were collected, centrifuged, and frozen at −20°C until use.TNF-238 (rs361525), TNF-308 (rs1800629), IL17A (rs2275913), and IL17F (rs763780) SNPs was performed with DNA samples by PCR-restriction fragment length polymorphism (RFLP) [Genotyping of m (RFLP) , 20. PriGenotyping of HLA-B27 was performed using PCR-SSP according to the method published before by our research group .http://biostats.usc.edu/Quanto.html) was used to calculate the sample size using the less frequent allele (0.095 for TNF-238), population risk (1.5%), and OR (2.0–4.0).The software Quanto (http://www.openepi.com/Menu/OE_Menu.htm). The statistical comparisons between groups were realized, and the estimated risk in individuals who hold genetic polymorphisms was calculated by the determination of OR (odds ratio) with 95% of confidence interval, adjusted for gender and age. All statistical analyses were performed by the software SNPStats (http://bioinfo.iconcologia.net/index.php), which was also used for detecting the Hardy-Weinberg (HW) and odds ratio balance, with a 95% confidence interval (CI). The P ≤ 0.05 values were considered statistically significant to the chi-square test with Yates' correction and logistic regression. The association tests were realized to the codominant, dominant, recessive, overdominant, and log-additive genetic inheritance models.The allele and genotype frequencies in patients and controls were estimated and compared by chi-square distribution tables using the OpenEpi 3.01 software  and controls (n = 210) regarding age, gender, and genetic marker HLA-B27 are presented in Baseline characteristics of patients  = 1.7 (1.1–2.5), 1.7 (1.2–2.6), and 4.5 (2.6–7.6), respectively  in AS patients . Then, wTNF and IL17 genotypes associated with these diseases, independent of these factors, which could provide more powerful evidence of association between these polymorphisms and SpA, AS, and PsA susceptibility.In our current study, we found TNF-308 and TNF-238 polymorphisms were considered to be factors involved in SpA pathogenesis, although other groups failed to confirm this theory [In numerous studies, s theory , 23. PotHence, in this study, care was taken with the pairing of patients and controls, regarding age, sex, and ethnicity. The control group included individuals of the minimum age at which disease develops and with the same proportion of men and women, since some forms of the disease may be more common in a specific gender. To minimize stratification errors due to differences in allelic frequencies in different ethnic groups, only patients and controls belonging to the mixed ethnic groups  were included.TNF, IL17A, and IL17F genes, mapped on the same chromosome at position 6p12, several polymorphic sites, including microsatellites and promoter polymorphisms, have been detected, which may influence the expression of TNF-α, IL17A, and IL17F. The substitution of G for A at the −308 position of the TNF gene promoter leads to increased production of TNF-α, whereas the substitution of G by A at position −238 results in decreased expression [IL17A G197A and IL17F T7488C affect the production of IL17A and IL17F, respectively [In pression –26. Prevectively , 28.TNF-308 GA/AA genotype was associated with the development of overall SpA and AS, independent of gender and HLA-B27 allelic group. This finding is in agreement with that of Höhler et al. who demonstrated an association of the TNF-308 A/A genotype with AS in German patients [TNF-308 was increased in Columbian patients with SpA, AS, and rheumatoid arthritis [TNF-308A could be a susceptibility factor for this disease.Our analysis showed that the patients  and thatTNF-238 GA/AA genotypes and PsA in concordance to Rahman et al. [TNF promoter gene polymorphisms and psoriatic arthritis in two well-characterized Canadian populations with the disease and carried out a meta-analysis of all TNF association studies in white populations with psoriatic arthritis.We have also found an association between the n et al.  who examTNF-308 AA and AA/AG or TNF-238 A/G polymorphisms with the disease [However, a meta-analysis of 2247 Korean patients with AS showed no association of IL17A A/G genotype and A allele and the IL17F T/C genotype and C allele. To the best of our knowledge, an association of these genetic polymorphisms with SpA has not yet been described. Our group already reported that the AA genotype and A allele for IL17A were associated with susceptibility to chronic periodontitis in South Brazil, highlighting their involvement in a chronic inflammatory condition [IL17A rs2275913 was found in the Norwegian population and more recently in the Brazilian population [IL17RA in two Spanish cohorts of patients and controls, but not involving the IL17A and IL17F polymorphisms that we have studied here [IL17A and IL17RA gene allelic variants were not associated with PsA susceptibility [In addition, we observed a higher risk of SpA in patients carrying the ondition . A weak pulation . Specifiied here . Therefotibility .In a study of 371 patients, Baeten et al. showed that treatment with anti-IL17 medication in both subcutaneous and intravenous forms led to an improvement in the clinical picture . This prHLA-B27. The risks were higher in patients with the haplotypes TNF-308/IL17A/IL17F GGC/GAC/AAT for SpA and AS and TNF-238/IL17A/IL17F GGC/GAC/AAT for PsA than in those who did not have. We may suggest that in the presence of the C allele for IL17F, the A allele for TNF-308 and IL17A is not required for a positive association with the diseases. However, if the A allele is present for both, the C allele for IL17F is not needed.Relevant result of this study was establishing risks to the development of SpA, AS, and PsA, related to the haplotype that the individual possesses for these cytokine SNPs on chromosome 6, regardless of IL17 and TNF haplotypes involved in the risk of developing obstructive coronary artery disease [Previous findings have suggested  disease , 39, whi disease . This coTNF and IL17 genes were considered in this study. Moreover, we did not evaluate the possibility of interactions between these genes and other genetic risk factors, beyond the gender and HLA-B27, known to interfere in the development of SpA.Finally, these observations should be interpreted with caution due to limitations found in this study, such as the relatively small sample size and the fact that only the TNF-308, IL17A, and IL17F gene polymorphisms were associated with SpA and AS, while TNF-238, IL17A, and IL17F gene polymorphisms were associated with PsA, regardless of gender and HLA-B27. The elucidation of the role of these biological markers together with the HLA-B27 marker, in the development of inflammatory diseases, in larger series of subjects, could provide the early diagnosis and treatment of the different clinical forms of this disease.In conclusion, in this mixed Brazilian population,


Although the burden of heart failure with preserved ejection fraction (HFpEF) is increasing, there is no therapy available that improves prognosis. Clinical trials using beta blockers and angiotensin converting enzyme inhibitors, cardiac-targeting drugs that reduce mortality in heart failure with reduced ejection fraction (HFrEF), have had disappointing results in HFpEF patients. A new “whole-systems” approach has been proposed for designing future HFpEF therapies, moving focus from the cardiomyocyte to the endothelium. Indeed, dysfunction of endothelial cells throughout the entire cardiovascular system is suggested as a central mechanism in HFpEF pathophysiology. The objective of this review is to provide an overview of current knowledge regarding endothelial dysfunction in HFpEF. We discuss the molecular and cellular mechanisms leading to endothelial dysfunction and the extent, presence, and prognostic importance of clinical endothelial dysfunction in different vascular beds. We also consider implications towards exercise training, a promising therapy targeting system-wide endothelial dysfunction in HFpEF. Heart failure (HF) is the most frequent cause of hospitalization in people over 65 years, and incidence is still increasing. Despite improved medical management, prognosis is grim, especially for heart failure with preserved ejection fraction (HFpEF) which has a 65% mortality rate at 5 years . In contAlthough HFpEF emerged as a distinct HF phenotype about three decades ago and about half of patients fall into this category, its pathogenesis remains incompletely understood. Beside advanced age, female sex, and sedentary lifestyle, HFpEF is associated with comorbidities such as arterial hypertension, diabetes, obesity, chronic obstructive pulmonary disease, and renal dysfunction . Cardiac2peak) which is determined by the product of cardiac output and arteriovenous oxygen (O2) difference. Hence, both O2 delivery mechanisms  as well as O2 utilizing factors  contribute to exercise intolerance [2 delivery, precipitating the switch to an anaerobic metabolism and thereby exacerbating fatigue and dyspnea [Clinical endothelial dysfunction (ED) is recognized as a precursor to many CV diseases including HF . Moreoveolerance . Reduced dyspnea . ED also dyspnea . This haExercise training is one of the most successful approaches to improve and even correct ED , 19. ExeIn this review, we will focus on different aspects of ED in HFpEF. First, we briefly review the underlying molecular mechanisms leading to ED. We list the existing evidence on the presence of ED in distinct vascular beds and the clinical importance relative to HFpEF. Finally, the effects of exercise training on endothelial function are discussed, portending important implications for HFpEF treatment.The endothelium was long considered a mere protective layer between the blood and different extravascular tissues. We now know that endothelial cells are dynamic, highly interacting cells regulating blood vessel function and homeostasis. The healthy endothelium prevents platelet and leukocyte adhesion and aggregation, inhibits smooth muscle proliferation, and regulates vascular tone through release of vasoactive substances, all of which are essential in organ perfusion . Nitric In the setting of CV disease risk factors , the endothelium loses these regulatory functions , 27. ReaRepair of diseased endothelium does not solely depend on proliferation of existing endothelial cells. Bone marrow-derived endothelial progenitor cells can be mobilized to sites of endothelial injury or ischemia. They are able to proliferate, exert beneficial paracrine effects through secreting vascular growth factors, and finally integrate into the endothelial layer by differentiating into endothelial cells , 29.ED is recognized as the first—but still reversible—step to overt atherosclerosis. As such, several diagnostic evaluation methods have been developed, with the goal to identify high-risk populations and start preventive therapy early. At the other end of the spectrum, presence and severity of ED is related to a negative outcome in established coronary ischemic heart disease and HFrEF .Usually, endothelial function is measured as vasodilation in response to an endothelium-specific stimulus. This includes drugs, such as acetylcholine, but a short period of local ischemia also elicits endothelium-specific hyperemia. The amount of vasodilation can be assessed invasively , although noninvasive methods are more widely used nowadays. The percentage dilation of the brachial artery in response to forearm ischemia, measured by ultrasound, is called flow-mediated dilation (FMD) . The morImpaired coronary endothelial-dependent vasodilation was found in nonischemic dilated cardiomyopathy, highlighting the implication of the endothelium in HFrEF regardless of the presence of atherosclerosis . MoreoveIn 2013, Paulus and Tschöpe hypothesized that ED plays a causal role in the development of HFpEF . They poReduced NO signaling from dysfunctional endothelium then influences adjacent cardiomyocytes and cardiac fibroblasts through the sGC-cGMP-PKG pathway . Lower mHowever, a two-way interaction between HFpEF and ED exists. Once HF develops, the syndrome maintains a vicious circle, further impairing endothelial function. HFpEF itself causes a systemic inflammatory state with high levels of circulating proinflammatory cytokines, increasing production of reactive oxygen species and exerting direct deleterious effects on eNOS expression , 43. NeuAs there is no universally accepted cutoff for defining ED, the actual prevalence of ED in HFpEF is unknown. In community studies, endothelial function declines with age and presence of CV risk factors , 46. UndIn the largest study to date, measuring endothelial function in 321 Japanese HFpEF patients, Akiyama et al. found that a RHI below the median predicted CV events . For eacperipheral ED as defined by a given RHI cutoff. In our opinion, the other 50% fail to show decreased RHI because of the following reasons. First, it takes time before microvascular inflammation is translated to clinically measureable disturbances in vasoreactivity. Second, the cutoff of RHI <1.63 reflects a value useful for clinical prognosis, but has not been correlated with pathophysiological changes such as endothelial inflammation and reduced NO bioavailability. Third, RHI and FMD show poor agreement which suggests different mechanisms are measured [Given the central role of ED in the development of HFpEF, this estimated prevalence of ED of 42–50% seems low. However, to be more precise, 42–50% of HFpEF patients have measured . Possibl2peak. This is related to adverse prognosis, since VO2peak is one of the strongest predictors of mortality in HFpEF [2 = cardiac output • arteriovenous O2 difference) states that VO2peak can be limited by either a central factor, cardiac output, or peripheral O2 extraction. The latter is influenced by oxygenation of the blood in the lungs, O2 carrying capacity of the blood, appropriate distribution of blood to the peripheral tissues, and adequate tissue O2 extraction from the blood. A key factor is the oxygen diffusion capacity (DO2), which can be a limiting factor in both pulmonary and skeletal muscle O2 kinetics. Applying Fick's law of diffusion (VO2 = DO2 • (capillary pO2 – intracellular pO2) with pO2 being partial oxygen pressure) in exercising muscle, where intracellular pO2 is very low, the capillary pO2 determines the O2 diffusion gradient. As such, capillary pO2 can limit VO2 during exercise [2 is determined by the instantaneous balance between VO2 and perfusion, ED can also limit capillary pO2 [2 both by reducing capillary blood flow and limiting O2 diffusion.Another clinical clue to the importance of ED is the relation with exercise intolerance, objectively measured by cardiopulmonary exercise testing and determination of VOin HFpEF . The Ficexercise . Adequatexercise . As capilary pO2 . In theo2peak [2 extraction during exercise is the predominant factor limiting VO2peak [2 extraction was the strongest independent predictor of VO2peak in their study. This was later confirmed using exercise hemodynamics and exercise echocardiography [2peak remains controversial, clearly peripheral elements play a role in determining exercise capacity in HFpEF. We further elaborate this finding in the next section.Reduced cardiac output on exertion was long considered the main mechanism behind exercise intolerance in HFpEF . Chronot2peak . More re2peak . Haykows VO2peak . The resiography , 59. AltTheoretically, many clinical findings related to the HFpEF syndrome could be explained by a system-wide ED, leading to alterations in several organ systems. In In what follows, we review the evidence of the presence, extent, and underlying mechanisms of ED in different vascular beds and the corresponding organs.The peripheral circulation is the preferred organ system for measuring endothelial-dependent vasodilation, because of the easy, noninvasive measurement and the good correlation with “gold standard” invasive coronary vasodilation . StudiesEvidence regarding macrovascular ED in HFpEF is conflicting. The largest study to date reported no significant difference in FMD between HFpEF patients and healthy volunteers matched for age and gender . In cont2 extraction in HFpEF can also result from skeletal muscle dysfunction. HFpEF patients indeed have abnormalities in skeletal muscle mass, composition, capillary density, and oxidative metabolism. In contrast to the high prevalence of obesity, HFpEF patients have reduced lean leg mass [2peak indexed to lean body mass in HFpEF patients further confirms that abnormalities in skeletal muscle perfusion and/or metabolism contribute to exercise intolerance [2peak, emphasizing muscle mitochondrial dysfunction is likely a limiting factor to exercise capacity. Other possible underlying molecular changes could be a switch from oxidative slow-twitch type I fibers to glycolytic fast-twitch type II fibers which reduces oxidative capacity, increased muscle fatigability, and a reduction in skeletal muscle capillary density [Besides vasodilatory dysfunction of the afferent arteries to the working muscle, reduced peripheral Oleg mass . This coolerance . Mitochoolerance . These c density .2peak [2 kinetics, the authors suggest a decreased O2 diffusion to contracting muscle limits exercise capacity in HFpEF. As mentioned above, ED could play a role in this limitation of diffusive capacity by reducing the pO2 driving gradient.The latter is especially intriguing, as it links these skeletal muscle abnormalities to vascular dysfunction. Kitzman et al. demonstrated a severely reduced capillary-to-fiber ratio in muscles of HFpEF patients, related to VO2peak . A lower2peak , 71. Als2 extraction during exercise with a greater increase in blood pressure than controls [When leg blood flow is measured by ultrasound Doppler, HFpEF patients indeed have a reduced muscle blood flow during exercise compared to healthy controls, even at relatively low workloads of 10-15 W . Stroke controls . This sucontrols . Possiblcontrols , 74.2peak, including mitochondrial dysfunction, fiber atrophy, and reduced oxidative capacity. Possibly, skeletal muscle abnormalities are linked to vascular dysfunction through a reduction in muscle capillary density, which limits muscle blood flow and O2 diffusion during exercise.In summary, there is evidence for microvascular ED in HFpEF, predictive of long-term CV morbidity. Reports on macrovascular dysfunction are conflicting, and all studies suffer from methodological disparities. Also, HFpEF patients suffer numerous changes in skeletal muscles which correlate with reduced VO2peak, invasive and echocardiographic hemodynamics, and mortality [Traditionally, coronary endothelial function is measured by intracoronary infusion of a vasodilating substance such as acetylcholine. Subsequently, microvascular function can be estimated by measuring coronary flow reserve (CFR), the ratio of coronary blood flow after the vasodilating stimulus over blood flow at rest. In HFrEF patients, CFR correlates with VOortality –77. Tschortality . Furtherortality , 78, 79.ortality , 80. As As mentioned above, reduced NO bioavailability leads to both structural and functional changes in HFpEF. Structurally, HFpEF hearts are characterized by interstitial fibrosis and both macroscopic and microscopic hypertrophy . Hemodyn−), however, increase diastolic calcium content and thus delay cardiomyocyte relaxation [In the normal heart, endothelial NO bursts directly modulate relaxation in a beat-to-beat way . High lelaxation . Throughlaxation , 84. Lowlaxation .Finally, NO exerts direct antifibrotic effects in the heart by counteracting endothelin-1, angiotensin II, and aldosterone. Reduced NO bioavailability leaves profibrotic actions of these molecules unopposed, promoting proliferation of fibroblasts and myofibroblasts .In summary, microvascular cardiac endothelium modulates diastolic function and development of LV hypertrophy and fibrosis. Coronary microvascular function, as measured by CFR, is reduced in HFpEF but does not relate to peripheral ED.Pulmonary hypertension (PHT) at rest is highly prevalent in HFpEF patients, with up to 83% affected . PatientPassive transition of elevated end-diastolic pressure explains only part of the elevated pulmonary artery pressures in HFpEF . As in pPulmonary arterial endothelial function was disturbed, and pulmonary artery pressures were higher in an animal infarct model of HFpEF, while aortic endothelial function and intracardiac pressures remained unaltered . This coIn a cohort of 28 HFpEF patients with PHT that had severe macrovascular ED (FMD median 1.95%), Farrero et al. found a significant inverse correlation between FMD and pulmonary vascular resistance. No correlation was found with capillary wedge pressure . While tPHT is also induced through reactive pulmonary vasoconstriction and vascular remodeling . This prPulmonary function itself is frequently disturbed in HFpEF patients, with 59% suffering airflow limitation on spirometry . As pulm2 diffusion limitation at rest in 59% [2 diffusion in both the systemic and the pulmonary microcirculation.Pulmonary gas exchange is impaired in up to 83% of HFpEF patients, showing true Ot in 59% . At exert in 59% . These f2 diffusion in the pulmonary microcirculation and causing adverse changes in diaphragm muscle composition similar to those in skeletal muscle.In summary, PHT is a frequent and ominous finding in HFpEF patients. Vascular remodeling and reactive pulmonary vasoconstriction, caused by a reduced systemic NO bioavailability, play an important role in its development. Spirometry, diaphragm function, and pulmonary diffusion capacity are frequently impaired in HFpEF patients. Possibly, ED plays a role by impairing OHFpEF can induce renal dysfunction, and vice versa. Chronic kidney disease is highly prevalent in HFpEF patients  , 100. MoClinically, endothelial function is impaired in patients with even mild chronic kidney disease, whether measured by RHI or FMD , 103. Fuß-mediated fibroblast stimulation. Also, systemic inflammation reduces NO bioavailability as described above. Renal blood flow is dependent on systemic NO supply, which is reduced in HFpEF [before onset of HFpEF.HFpEF can cause renal dysfunction in different ways . First, in HFpEF . In a mein HFpEF . InteresOn the other hand, renal disease can also lead to HFpEF. In long-term follow-up of >8500 chronic kidney disease patients, 34% was diagnosed with new-onset HFpEF . PossiblIn summary, HFpEF and chronic kidney disease are mutually influencing conditions. ED is an important risk factor for both diseases, and interesting pathophysiological links exist.2peak and physical function scores [2peak) and high intensity interval training . In a single-center trial, high intensity training in HFrEF patients led to superior increases in VO2peak and ejection fraction compared to moderate training [2peak improvement in patients training at moderate intensity contrasts with the earlier studies.Cardiac rehabilitation programs have been a mainstay of HFrEF treatment after it was discovered that training is safe and reduces hospitalizations . The evin scores –125. Dian scores . These rtraining . Unfortutraining . Of notetraining . HoweverThe ongoing OptimEx study aims to study optimal exercise dose in 180 HFpEF patients with regard to aerobic capacity . Also, tIn the contemporary “whole-systems” approach towards HFpEF therapy, ED forms an attractive target due to its systemic nature and reversibility in early stages. Improving ED in HFpEF can be achieved through correction of comorbidities, increasing NO bioavailability, or antioxidative therapy. Sadly, none of these approaches alone has thus far been successful in decreasing HFpEF-related morbidity or mortality. Exercise training integrates all three mechanisms, forming a promising systemically oriented therapy .Both peripheral endothelial function and muscle metabolism are beneficially influenced by exercise. Exercise increases NO production by upregulating and phosphorylating eNOS through increased shear stress and vascular endothelial growth factor 2 release . ExercisCirculating progenitor cells could add to these favorable changes . EndotheMolecular determinants of exercise-induced effects specific to HFpEF are still poorly investigated. In HFpEF rats, exercise training restored endothelial-dependent vasodilation measured ex vivo in organ baths . Endotheß, 6, and 10 and tumor necrosis factor alpha) showed no change with exercise. Interestingly, levels of the growth hormone releasing peptide ghrelin, which inhibits cardiomyocyte and endothelial cell apoptosis in vitro, increased by exercise training. Clearly, molecular determinants underlying the exercise-induced benefits in HFpEF deserve further in-depth exploration.In a secondary analysis of the Ex-DHF trial, circulating cytokines and hormones were analyzed in HFpEF patients before and after training . Inflamm2 extraction. The increase in VO2peak was almost entirely attributable to an improvement in peripheral function  [2 extraction in HFpEF patients. This increase in muscle vascular function was the only significant predictor of VO2peak. Interestingly, this phenomenon was not seen in HFrEF patients, for whom improved cardiac output was the only predictor of VO2peak [Clinically, peripheral endothelial function shows improvement after exercise training in patients with CV risk factors, coronary atherosclerosis, and HFrEF –140. Of nctions) . In a st VO2peak .2peak [Conversely, Kitzman et al. could not demonstrate an improvement of FMD after training HFpEF patients, despite an increase in VO2peak . A possi2peak , 143. In2peak [There is no data regarding the effects of training on coronary, pulmonary, or renal ED in HFpEF patients. However, studies in patients with other CV diseases suggest exercise training is indeed able to improve regional endothelial function. Coronary endothelial function is improved by cardiac rehabilitation in dilated cardiomyopathy and coronary atherosclerosis , 144. In2peak . HoweverConsidering HFpEF as a multisystem syndrome rather than an isolated cardiac disease could lead us to alternative research approaches and eventually to successful therapies. The heterogeneity of the HFpEF patient population has frequently been cited as one of the reasons major trials have failed to prove a benefit for pharmacological treatment . EffortsAlso, a one-size-fits-all therapeutic approach is probably not the best strategy for the heterogeneous HFpEF population. A treatment algorithm based on presence of different comorbidities has recently been proposed . KeepingUnravelling the beneficial effects of exercise training in HFpEF could lead to patient-specific new therapies. Such a tailored approach can be useful in patients who are unable to exercise, or as add-on to a training program. Pharmacological or nonpharmacological correction of comorbidities, increase of NO bioavailability, and antioxidative therapy are possible targets, some of which are being explored in clinical trials already , 149. ThHFpEF is a multisystem pathology. Cardiac dysfunction is not the sole causative factor, but interacts with a heterogeneous range of organ dysfunctions, including pulmonary, renal, peripheral vascular, and skeletal muscle dysfunctions. Endothelial dysfunction could be a central mechanism in this system-wide CV maladaptation, as such it forms an attractive target for future HFpEF therapies. Exercise training is thus far the only therapy with proven beneficial effects in HFpEF. While exercise training does not improve macrovascular ED in HFpEF, evidence does suggest peripheral vascular and/or skeletal muscle function is enhanced. This warrants a shift in both fundamental and clinical research towards endothelial-targeted therapies, including exercise training, in the search for an effective therapeutic strategy for HFpEF.