Wnt signaling is a highly conserved pathway crucial for development and homeostasis of multicellular organisms. Secreted Wnt ligands bind Frizzled receptors to regulate diverse processes such as axis patterning, cell division, and cell fate specification. They also serve to govern self-renewal of somatic stem cells in several adult tissues. The complexity of the pathway can be attributed to the myriad of Wnt and Frizzled combinations as well as its diverse context-dependent functions. In the developing mouse inner ear, Wnt signaling plays diverse roles, including specification of the otic placode and patterning of the otic vesicle. At later stages, its activity governs sensory hair cell specification, cell cycle regulation, and hair cell orientation. In regenerating sensory organs from non-mammalian species, Wnt signaling can also regulate the extent of proliferative hair cell regeneration. This review describes the current knowledge of the roles of Wnt signaling and Wnt-responsive cells in hair cell development and regeneration. We also discuss possible future directions and the potential application and limitation of Wnt signaling in augmenting hair cell regeneration. Drosophila models  are a set of secreted factors that together with the Frizzled receptors make up the basis of the Wnt signaling pathway. Components of the pathway were discovered 30 years ago by different researchers working simultaneously in mouse and Wnt genes in the human and murine genome, 15 in the zebrafish and 8 in Drosophila , the utricle, and the saccule, also contain sensory hair cells in order to detect head position and motion. Both auditory and vestibular signals are in turn relayed centrally via the spiral and vestibular ganglion neurons, allowing for sound and balance perception. Although the mature mammalian cochlea is a terminally differentiated, non-regenerating organ, regeneration has been described in the auditory systems of lower vertebrates and additionally to a limited extent in the mammalian vestibular organs. Numerous studies have characterized the multiple roles of the Wnt signaling during cochlear development. Moreover, recent studies have highlighted potential applications of Wnt signaling in promoting hair cell regeneration. We will begin by summarizing the work done on Wnt signaling in inner ear development.Pax2-expressing ectoderm, which constitutes the otic-epibranchial placodes  signaling originating from the mesendoderm and the hindbrain instruct the preplacodal field surrounding the neural plate to form a patch of Wnt8c (mouse Wnt8a) in otic ectoderm specification . Lastly, Wnt-responsive cells in the early otic epithelium was found to contribute to several different parts of the inner ear, including the majority of the vestibular system and parts of the cochlea. A similar finding on the effect of Wnt3a in dorsal-ventral patterning of the inner ear was recently described in the zebrafish , using a hair cell specific Cre driver, hair cell maturation appeared unaffected, suggesting that Wnt/β-catenin signaling is required for proliferation of prosensory cells and for differentiation of hair cells but not for their subsequent maintenance. In support of the previous in vitro results, stabilization of β-catenin before the onset of hair cell differentiation increased cell divisions in the prosensory domain and induced the formation of supernumerary hair cells in a disorganized cochlea  cochlea and found that inhibition of Wnt signaling results in decreased proliferation of prosensory cells , which in turn is strictly organized along a basal-apical axis , is expressed on the opposite side of the developing organ of Corti, possibly creating a gradient of active Wnt5a signaling across the sensory domain. In contrast to Wnt7a deficient mice, Wnt5a null mice display clear PCP defects with a slightly shortened and broader cochlea consisting of additional rows of hair cells along its entire length, especially towards the apical end. However, this phenotype is only observed in about one third of the mutant animals  present during cochlear development, is expressed in the greater epithelial ridge cells  G-coupled family of receptors, was recently linked to Wnt signaling , a secreted Wnt inhibitor, further reduced this baseline level of proliferation. Conversely, pharmacologic inhibition of GSK3β (using 1-azakenpaullone) increased supporting cell proliferation in a β-catenin dependent manner, indicating that the effects are mediated by canonical Wnt signaling. After neomycin-induced hair cell loss, Wnt activity as indicated by the TOP;GFP reporter transgene increases, and this rise coincides with the increase in proliferation observed in supporting cells in the neuromasts  and its overall activity as measured by a TCF/LEF-GFP reporter were downregulated during the early post-damage period. Subsequently, an increase in these components precedes active Wnt signaling  in the late phase of regeneration. In parallel, microarray data generated by Steiner et al., who isolated mantle cells after having used copper to lesion hair cells, showed a complicated damage response consisting of upregulation of a subset of the Wnt pathway components and target genes  but downregulation of others  immediately after damage  did not exhibit significant changes prior to proliferation. As was performed in the zebrafish lateral line, an examination of the timing of mRNA expression reveals a much more complex picture of Wnt signaling during regeneration. This complexity is likely attributed to the dynamic interplay of several major pathways with canonical Wnt signaling to stepwise regulate the initiation of regeneration, proliferation, and conversion of progenitor cells towards a hair cell fate. In particular, Notch signaling has been demonstrated to limit both proliferation and hair cell differentiation via the process of lateral inhibition in zebrafish lateral line  mutants (van den Heuvel et al., A major hurdle in dissecting the functions of individual Wnt ligands and Frizzled receptors is their redundancy. This was illustrated previously by the lack of inner ear phenotypes in mice deficient in either The identity of the cells targeted by Wnt secretions present additional obstacles. The majority of work on downstream Wnt signaling has so far focused on manipulating β-catenin through genetic approaches or by means of small molecules targeting its destruction complex. While this has garnered substantial information on where active canonical Wnt signaling is required during development and regeneration, much of the identity of Wnt secreting cells and what combinations of Wnt and Frizzled members are responsible for activating Wnt/β-catenin signaling in specific developmental and regenerative processes still remain unknown Figure . How canLgr5-positive cells in the neonatal cochlea in vitro (Bramhall et al., Lgr5-positive cells, as the auditory and vestibular organs age. Such an understanding and the development of additional genetic and molecular tools to facilitate a combinatorial approach are critical next steps towards further facilitating mammalian hair cell regeneration.As previously mentioned, much work is still needed to improve our understanding of the relationships among multiple signaling pathways. The studies undertaken in species where hair cells naturally regenerate as part of homeostatic turn-over or in response to damage have begun to paint a picture of the spatio-temporal activity of pathways during supporting cell proliferation and hair cell differentiation. Some parts of the interaction of Wnt signaling with other pathways have already begun to unravel. For example, Wnt activation has been discovered to result in the upregulation of Jagged1, a member of the Notch pathway with a prosensory function (Morrison et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A previous study showed that in patients infected with HTLV-1, the activity of regulatory CD4+ T cells and their consequent expression of inflammatory and anti-inflammatory cytokines are altered. In this review, we discuss the mechanisms underlying changes in cytokine release leading to the loss of tolerance and development of autoimmunity.Human T-lymphotropic virus type-1 (HTLV-1) infection is associated with adult T-cell leukemia/lymphoma (ATL). Tropical spastic paraparesis/HTLV-1-associated myelopathy (PET/HAM) is involved in the development of autoimmune diseases including Rheumatoid Arthritis (RA), Systemic Lupus Erythematosus (SLE), and Sjögren’s Syndrome (SS). The development of HTLV-1-driven autoimmunity is hypothesized to rely on molecular mimicry, because virus-like particles can trigger an inflammatory response. However, HTLV-1 modifies the behavior of CD4 The etiology of autoimmune diseases is unknown, but it is clear that the interaction between genes and the environment is an important step in breaking immune tolerance to self-antigens. This can lead to inflammation and destruction of specific tissues and organs. Although host genetic background contributes to autoimmunity, research indicates that infectious agents are one of most important environmental factors responsible for the development of autoimmune diseases ,2,3. Chr+ T lymphocytes, and can modify the cell function. CD4+ T lymphocytes are the central acquired immune response regulators. Changes in their behavior can trigger inflammatory reactions that can break immune system tolerance, leading to autoimmunity. In this review, we discuss immunological changes in HTLV-1 infection and its association with autoimmune diseases.Human T-lymphotropic virus type-1 (HTLV-1) is associated to a number of diseases, such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), and autoimmune diseases such as the Sjögren’s Syndrome (SS), arthropathies, and uveitis, which are often related to changes in the immune response ,7,8,9. TDeltaretrovirus, family Retroviridae, and subfamily Orthoretrovirinae [.Human T-lymphotropic virus type-1 (HTLV-1) is classified as a complex type C retrovirus belonging to the genus ovirinae ,11.The morphological structure of this virus is similar to other retroviruses; the capsid contains two simple RNA strands together with the reverse transcriptase and integrase enzymes. These enzymes are important for insertion of the virus into the host genome, resulting in a provirus ,13,14,15gag, pro/pol, and env that are flanked by two long terminal repeat (LTR) regions. Additionally, the pX region was identified in the region between the env gene and the 3′-LTR region. The pX region codes for the Tax (p40), REX (p27), p12, p13, p21, and p30 regulatory proteins that are involved in viral infection and proliferation. Tax is a phosphoprotein that exerts an essential role in viral transcription and cell behavior transformation [The virus genome contains structural and functional genes, such as the ormation ,17,18,19ormation ,21,22.hbz (HTLV-1 b-ZIP factor), that codes for a protein involved in the pathogenesis of the virus together with Tax. HBZ can function in two different molecular forms, mRNA and protein. In mRNA form, it can promote cell proliferation by positive regulation of E2F1. HBZ protein can down-regulate Tax expression and can interact directly with c-Jun and c-Jun-B [Recently, another gene was identified,  c-Jun-B ,24.+ lymphocytes, an increase in the migratory capacity of circulating leukocytes, and increased production of inflammatory cytokines—particularly neurotoxic cytokines such as IFN-γ and TNF-α—in affected regions along the spinal cord [+ T cell cross the blood-brain barrier and destroy HTLV-1 infected glia cells by cytotoxicity or cytokine production. The last theory suggests that autoimmunity mechanism can cause lesions by molecular mimicry. A host neuronal protein seems to be similar to Tax protein from the virus, which can cause immune cross-reaction, leading to CNS inflammation [HTLV-1 infection is associated with several diseases, primarily with adult T-cell lymphoma (ATL) and HAM/TSP. HAM/TSP patients present a series of immunological dysfunctions, including spontaneous proliferation of HTLV-infected T CD4nal cord ,26,27,28nal cord ,30,31. Anal cord ,26,32,33ammation ,34,35.+ T lymphocytes by HTLV-1.Patients infected with HTLV-1 may develop a number of associated diseases, such as HAM/TSP, or other autoimmune diseases such as the Sjögren’s Syndrome (SS), arthropathies, and uveitis, which are often related to changes in the immune response ,8,9,35. + T lymphocytes exhibit altered signaling cascades and transcription factor activation, leading to changes in cell behavior [HTLV-1-infected CD4behavior ,16,36,37Many studies have reported that the HTLV-1 Tax protein affects several transcription factors including CREB/ATF, NF-κB, AP-1, SRF, and Nuclear factor of activated T-cells (NFAT), as well as a number of signaling cascades involving PDZ domain-containing proteins such as Rho-GTPases and Janus kinase (JAK)/signal transducer and activator of transcription (STAT), thus altering the transforming growth factor-β (TGF-β) cascades. These factors are involved in cell proliferation and activation, including expression of cytokines and activation of viral proteins ,39,40,41The expression of forkhead/winged helix transcription factor (FOXP3), which is an important transcription factor, has also been reported to be altered in patients infected with HTLV-1. FOXP3 is an essential transcription factor for the differentiation, function, and homeostasis of regulatory T cells (Tregs). Irregularities in the expression of FOXP3 may lead to loss of immune tolerance and the probable development of autoimmune diseases ,42.et al. [+CD25+FOXP3+ T cells that possess a suppressive function and an accumulation of CD4+CD25+FOXP3- T cells that can exacerbate the pathogenic process of HAM/TSP. The authors demonstrated that in patients with HAM/TSP, there was an increase in the sub-population of IFN-γ-producing T cells with a CD4+CD25+FOXP3- phenotype and that this increase correlated with the clinical severity of HAM/TSP.Previous studies have demonstrated that an increase in FOXP3 expression in patients that developed ATL leads to an exacerbated Treg function, resulting in increased production of IL-10 and TGF-β, which in turn triggers the immunosuppression phenotype observed in these patients. In contrast, studies performed with patients that developed HAM/TSP showed a decrease in FOXP3 expression and in the production of the IL-10 and TGF-β cytokines responsible for suppression of the immune response ,43,44,45et al.  showed tChanges in signaling pathways and transcription factor activation caused by viral proteins play an important role in modifying immune response homeostasis, resulting in a cytokine environment that may influence the immune cells phenotype. The effect of the environment may lead to autocrine and paracrine activation and thus influence T cell differentiation and homeostasis. IFN-γ can stimulate the production of Th1 cells, while the presence of IL-4 leads to the differentiation of Th2 cells. Several studies have shown that the presence of these cytokines triggers the activity of known suppressors of cytokine signaling (SOCS) family proteins, which are able to inhibit the recruitment of STATs or the activation of JAKs. SOCS play a role in the maturation, differentiation, and maintenance of T lymphocytes ,47,48. S+ T lymphocytes of HAM/TSP patients exhibit spontaneous proliferation, in addition to an increased production of proinflammatory cytokines such as IFN-γ, TNF-α, IL-1, and IL-6; neurotoxic cytokines such as IFN-γ and TNF-α, in particular, are found at high concentrations in the spinal fluid of HAM/TSP patients [The HTLV-1-infected CD4patients ,16,50,51patients ,31,45.et al. [Toulza et al.  observedin vitro and regulate the activity of CD4+ and CD8+ T cells in vivo. There are two major types of Treg cells, naturally occurring cells and cells produced in the periphery [HTLV-1 infection leads to changes in the systemic immune response even in asymptomatic patients ,55,56,57eriphery .During the development of autoimmunity, there is a loss of tolerance to self-antigens, causing an inflammatory response that attacks organs and tissues of the individual. The pathogenesis of autoimmune diseases is generally studied in the context of T helper cells and the balance between the Th1 and Th2 responses. Thus, it has been observed that some diseases such as Rheumatoid Arthritis (RA) fit the Th1 profile, in which a cell-mediated response occurs, while others such as Systemic Lupus Erythematosus (SLE) fit the Th2 profile, involving antibodies and immunocomplexes in their physiopathology . However+ T lymphocytes and alters their behavior  and xerophthalmia (dry keratoconjunctivitis) due to the lymphocytic infiltration of the salivary and lachrymal glands, which in turn leads to the destruction of the ducts. Furthermore, antinuclear antibodies (ANA) and other self-antibodies, such as anti-SS-A (Ro) and SS-B (La), are also found in these patients. Disease development is also associated with genetic and hormonal factors ,81. Seveet al.  reportedet al. ,83,84.SLE is a systemic autoimmune disease of unknown etiology. This disease progresses with polymorphic clinical manifestations and periods of exacerbation and remission. Disease development is associated with a genetic predisposition and environmental factors, such as exposure to sunlight and viral infection ,86,87,88in vitro culturing. This indicates the occurrence of viral replication in SLE patients, which could explain the high seropositivity for HTLV-1 and HTLV-2 observed in these patients [The association between HTLV-1 and SLE is still controversial ,90,91. Opatients .Several studies have described HTLV-1 infection in the context of autoimmune diseases, although this subject is still under debate. Molecular mimicry has been hypothesized as a possible mechanism; however, HTLV-1-promoted altered cytokine release is critically involved in breaking tolerance. HTLV-1-induced changes in the activity of regulatory CD4 T-cell molecules affect the homeostasis of cytokines, including IFN- γ, TNF-α, TGF-β and IL-10, and disrupt the balance in inflammatory and anti-inflammatory responses, leading to the loss of tolerance and the development of autoimmunity.


The Human T lymphotropic virus type 1 (HTLV-1) infection is neglected mainly due to the concept that it is associated with a low morbidity. However, a few reports have shown that a large percentage of HTLV-1 infected subjects have signs and symptoms of a variety of diseases. Nevertheless the relationship between the cause and effect needs to be proven. The HTLV-1 associated myelopathy (HAM/TSP) and adult T-cell leukemia (ATL), the main disease associated to HTLV-1, are characterized by high proviral load and lymphocyte activation. Moreover the exaggerated inflammatory response and proviral load are markers of diseases associated with HTLV-1. In this study we compare the frequency of sicca syndrome, chronic periodontal disease (CPD), HTLV-1 associated over reactive bladder (HTLV-1 OAB), artropathy and erectile dysfunction (ED) in HTLV-1infected subjects who did not have definitive HAM/TSP with that observed in seronegative controls. The proviral load was measured by real time PCR and production of interferon–γ and TNF in supernatants of lymphocyte culture by ELISA. Of the 180 individuals participants of the study, 106 (50.8%) were female, 56 (31.1%) had sicca syndrome, 37 (20.5%) had CPD, 32 (17.7%) had HTLV-1 OAB, 29 (16.6%) had HTLV-1 associated arthropathy. Moreover of the 74 males 31 (41.8%) had ED. All these manifestations were higher (P<0.001) in HTLV-1 infected subjects than in controls. Proviral load in sicca syndrome, CPD, HTLV-1 OAB and ED patients were higher (P < 0.05) than in 78  HTLV-1 carriers. Patients with OAB, CPD and sicca syndrome had higher TNF and IFN-γ than HTLV1 carriers. The majority of the patients with diseases associated to HTLV1 had at least two of the above diseases. Overall 56,7% of HTLV-1 infected subjects without definitive HAM/TSP or ATL had diseases associated with HTLV1 infection indicating that HTLV-1 is associated with high morbidity.


Obstructive sleep apnoea–hypopnea syndrome (OSAHS) and multimorbidity are common in elderly patients, but a potential link between the two conditions remains unclear. This study aimed to assess the prevalence of OSAHS, chronic multimorbidity and their relation in older adults in primary care settings. A screening study was performed in a cross-section of 490 elderly adults  receiving home care services in Thessaly, central Greece. The Berlin Questionnaire was employed to assess the likelihood for OSAHS and the Epworth Sleepiness Scale to assess daytime sleepiness. Multimorbidity was defined as a documented history of at least two chronic diseases. The prevalence of high risk for OSAHS, excessive daytime sleepiness and multimorbidity was 33.5, 11.6 and 63.9 %, respectively. None of the study subjects had a confirmed diagnosis for OSAHS prior to this study. A marked dose–response association between a high pre-test likelihood for OSAHS and multimorbidity was noted in patients with two [adjusted odds ratio (OR) 3.13; 95 % confidence interval (CI) 1.85–5.30) and three or more  chronic morbidities, independently of age, sex and smoking status. This association persisted across different levels for OSAHS risk in the Berlin questionnaire, was insensitive to varying definitions of multimorbidity and more pronounced in patients with excessive daytime sleepiness. These findings point out that primary care physicians who care for elderly patients who present with several, common and burdensome, chronic diseases should expect to find this multimorbidity often coinciding with undetected, and therefore untreated, OSAHS. Thus it is crucial to consider OSAHS as an important co-morbidity in older adults and systematically screen for OSAHS in primary care practice. Obstructive sleep apnoea–hypopnoea syndrome (OSAHS) is a common disorder that affects least 2–4 % of the adult population .We employed the Berlin Questionnaire to screen for OSAHS  is a common complaint in OSAHS patients, we used the Epworth Sleepiness Scale (ESS) to assess daytime sleepiness Johns . ESS quaTo record morbidity data, we checked for confirmed diagnoses of chronic diseases in the medical records of each study subject and also assessed current medications. We sought to include common and burdensome chronic diseases based on the main causes of disease burden in primary healthcare in Greece  ≥30 kg/mReliabilities of the Berlin questionnaire and the ESS were assessed using Cronbach’s coefficient. 95 % confidence intervals (CI) for proportions were calculated using Wilson’s Score method. The strength and direction of associations between a high pre-test likelihood for OSAHS and the various chronic morbidities were presented using odds ratios (OR) and corresponding CI as calculated by binary logistic regression. The Extended Mantel-Haenzsel Chi square test for linear trend was used to assess the dose–response relationship between the level of multimorbidity and a high pre-test likelihood for OSAHS.To adjust for potential confounding effects by gender, age and smoking status, multiple logistic regression analysis was performed with a high risk for OSAHS in the Berlin questionnaire constituting the dependent variable and multimorbidity being the main independent variable. Multimorbidity was examined both as a binary (yes/no) and as an ordinal variable .Because hypertension and obesity are accounted for when assessing the likelihood for OSAHS in the Berlin questionnaire, we performed a sensitivity analysis by excluding these two conditions from the definition of multimorbidity. We also examined the persistence of our findings in the subgroup of subjects with EDS.P values were two-tailed, and a P value <0.05 was considered statistically significant. Data were analysed using IMB-SPSS version 22.All reported This study has a written ethical approval by the Scientific Council of the Department of Medicine at the University of Thessaly . All study participants were advised about the voluntary nature of the study and gave informed consent.Of the 518 elderly adults solicited, 490 were interviewed and completed fully the study questionnaires (95 % response rate). Study participants had an average age of 77.5 ± 6.9 years , and 51 % were male. None of the study subjects had a confirmed diagnosis for OSAHS prior to this study. Main characteristics of the study participants are presented in Table Both the Berlin questionnaire and the ESS demonstrated good degrees of internal consistency . One-third of the study subjects  were classified as being at a high risk of having OSAHS according to the Berlin questionnaire. Presence of EDS, as assessed by the ESS, was observed for 57 subjects .The most prevalent chronic morbidities overall were hypertension, obesity, cardiovascular disease, diabetes mellitus and mental health disorder Table . More thA high pre-test likelihood for OSAHS was noted more frequently for study subjects who had a history of stroke, cardiovascular disease, diabetes mellitus or mental health disorder, compared to those with no history of chronic disease Table . Defined2(1) = 33.34; P < 0.001 for linear trend). This association persisted across different levels of risk for OSAHS in the Berlin questionnaire  and three or more  chronic diseases (χP = 0.017) and retained a dose–response feature .The association between multimorbidity and high risk for OSAHS was more pronounced in the subgroup of the 57 subjects who presented with EDS . When multimorbidity was treated as an ordinal variable, the observed dose–response relationship also retained statistical significance  = 4.37, P = 0.037 for linear trend).Following exclusion of hypertension and obesity from the definition of multimorbidity, the association between multimorbidity and high pre-test likelihood for OSAHS retained statistical significance, but was weaker  with four or more medical conditions were more likely to have sleep problems  than those with no medical conditions  and patients with three or more  chronic morbidities and was more pronounced in patients with EDS . This association persisted across different levels of OSAHS risk and was insensitive to varying definitions of multimorbidity. Therefore this study adds evidence to a pool of results which suggest that OSAHS is common in older adults and often coincides with their multimorbidity.Compared to the general population, patients with OSAHS have been shown to use 23–50 % more resources  in the five years prior to their diagnosis (Smith et al. As expected we found strong associations between a high pre-test likelihood for OSAHS and the presence of hypertension and obesity in our study subjects. High risk for OSAHS was also more prevalent in patients with stroke, cardiovascular disease and diabetes mellitus in our sample, but these differences were not statistically significant probably because our study was underpowered to detect relatively small effect sizes. There are accumulating evidences suggesting that OSAHS may be an independent risk factor for metabolic syndrome and cardiovascular disorders Vijayan . HoweverIt has been suggested that sleep-related symptoms such as snoring and excessive daytime sleepiness are associated with anxiety and depression (Bouscoulet et al. This study has limitations that should be acknowledged. First, the cross-sectional nature of our study does not allow conclusions to be drawn regarding cause-effect relationships. Second, measurement error is possible in our assessment of multimorbidity because chronic morbidities were documented from medical files and we cannot exclude the possibility of undocumented or undiagnosed medical conditions. Third, performing a full sleep investigation including overnight in-laboratory polysomnography was not feasible in this large population-based study, as it is too costly, not possible for all study subjects, and portable equipment is unreliable. Instead we used two highly sensitive screening questionnaires to assess sleep apnoea, but these may be less specific for the diagnosis of OSAHS in older adults (Sforza et al. The present study adds to an emerging pool of information regarding two newly developing areas of research, i.e. the epidemiology and clinical importance of OSAHS and those of multimorbidity. Comorbidity should be regarded as a patient characteristic that may affect the occurrence and relevant outcomes of another disease. Instead of focusing mainly on specific chronic diseases and their specific outcomes, this study points out that future research may need to consider OSAHS as an important co-morbidity in older adults.This burden needs to be addressed with policies aiming at improving the identification of undiagnosed OSAHS via population screening in primary care (Young et al. Multimorbidity may also be an important methodological issue in studies which assess the effects of new treatments or other interventions on clinical outcomes of elderly patients with OSAHS. Randomised controlled trials that exclude patients with multimorbidity may have severely limited external validity. Observational studies should adjust for potential confounding effects of multimorbidity on outcome variables in patients with OSAHS.OSAHS is prevalent and largely undiagnosed in older adults and often coincides with multiple, common and burdensome, chronic diseases. This is a crucial incentive to consider OSAHS as an important co-morbidity in older adults and systematically screen for OSAHS in primary care practice.


The mechanisms regulating viral gene expression from the provirus are a key to understanding the persistent/latent infection with HTLV-1 and its pathogenesis. In this article, we would like to review our current understanding on this topic.Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus associated with human diseases, such as adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/Tropic spastic paraparesis (HAM/TSP). As a retrovirus, its life cycle includes a step where HTLV-1 is integrated into the host genomic DNA and forms proviral DNA. In the chronic phase of the infection, HTLV‑1 is known to proliferate as a provirus via the mitotic division of the infected host cells. There are generally tens of thousands of infected clones within an infected individual. They exist not only in peripheral blood, but also in various lymphoid organs. Viral proteins encoded in HTLV-1 genome play a role in the proliferation and survival of the infected cells. As is the case with other chronic viral infections, HTLV-1 gene expression induces the activation of the host immunity against the virus. Thus, the transcription from HTLV-1 provirus needs to be controlled in order to evade the host immune surveillance. There should be a dynamic and complex regulation  In the case of vertical transmission, infected individuals acquire the virus during infancy and need to carry the virus for decades, before they are able to transfer the virus to their children. Also, infected women need to remain healthy for decades in order to become pregnant, in spite of the persistent HTLV-1 infection in their bodies. To achieve such a long-term persistent infection without having severe health problems, HTLV-1 seems to have developed a strategy to achieve an asymptomatic condition by minimizing the effect of viral infection on our vital systems. At the same time, as with other viruses that cause persistent infections, HTLV-1 needs to evade the host immune surveillance [It has been estimated that Human T-cell leukemia virus type 1 (HTLV-1) has been infecting humans for several thousand years [ed cells ,3,4. Theeillance .de novo HTLV-1 infection are cell-to-cell transmission and/or extracellular biofilm-like-structure-mediated transmission [de novo infection. This implies that the virus makes use of a reversible system to switch from the latent phase of infection to the active phase, where viruses are produced. For example, when the infected cells are transferred from mother to child, proviruses in the infected lymphocytes contained in the breast-milk would be transcriptionally reactivated, and producing infectious viral particles or inducing cell-to-cell transmission. Since an anti-virus immunity has not been established yet in the new host during the initial phase of infection, the virus would be able to spread via de novo infection  in the sense orientation of the LTR, but not in the anti-sense orientation. Promoters containing a TATA-box structure generally exhibit high-plasticity in their promoter activity, whereas TATA-less promoters show low transcriptional plasticity ,21. In lvice versa [A recent interesting study has further extended our understanding on how both sense and antisense transcriptions are regulated within the provirus. They showed that sense transcription from the 5′-LTR did not interfere with antisense transcription from the 3′-LTR and ce versa . They fuce versa . On the other hand, there are several negative regulatory systems acting on the 5′-LTR during transcription, translation, and even post-translation phases. HTLV-1 p30 has the potential to inhibit the interaction between Tax and p300, resulting in suppression of the 5′-LTR ,29. p30 In summary, there is convergent transcription, in the sense- and antisense-orientations, and various viral transcripts with alternative splicing within HTLV-1 provirus. Growing evidence so far indicates that HTLV-1 maintains an equilibrium between viral antigen expression and the host immune surveillance by controlling both sense and antisense transcription, viral regulatory and accessory proteins’ expression, and host cellular mechanisms, such as cellular-signaling pathways and cell cycling.in vitro and in vivo. Even among cell lines infected with HTLV-1, there is a wide variation in transcription level. The distinct viral gene expression should be the consequence of different cellular transformation processes occurring during in vitro culture or in infected individuals. In vitro T-cell immortalization by HTLV-1 is induced by a high expression level of Tax protein. As shown in previous studies, high level of tax expression is sufficient to induce immortalization of T cells [in vitro cell culture, viral antigen expression does not confer survival disadvantage on infected cells. In contrast, adult T-cell leukemia (ATL) cell lines derived from ATL patients are generated after a long latent period in vivo in the presence of anti-virus immune surveillance [in vivo, the process of transformation depends not only on HTLV-1 infection but also on several other factors, such as escape from the host immune surveillance [It has been reported that there is clearly a distinct level of proviral expression  T cells ,37,38. Beillance . In the eillance ,41, as weillance ,48,49,50eillance ,41.in vivo is its low immunogenicity. A previous report demonstrated that the amino acid sequence of HBZ possesses substantially low immunogenicity [ex vivo culture induces expression of viral proteins [in vivo silencing, it is generally accepted that HTLV-1 expression is silenced but the virus keeps its capacity to re-start production of viral particles. Recent interesting studies have shown the kinetics of the sense and antisense proviral transcription during ex vivo culture, suggesting the existence of a time-dependent regulatory mechanism of HTLV-1 provirus, which should be important at the initial phase of infection [Transcripts with sense orientation from the 5′-LTR are generally barely detectable, if not at all, in PBMCs freshly isolated from infected individuals ,51. In cgenicity . Anotherproteins ,55,56. Anfection ,58,59.et al. published the results of a comprehensive and genome-wide sequencing analysis of fresh ATL cells from several hundreds of ATL cases in Japan [Recently Kataoka in Japan . The RNAin Japan ,52. Minuin Japan ,34,60. Ain Japan ,62. Intein Japan ,63. AlthHTLV-1 provirus is integrated into the cellular DNA, chromatinized, and affected by genomic and epigenomic circumstances nearby the integration site ,65,66,67DNA methylation is a key mechanism to control gene expression of the host cells. Based on sequence analyses, HTLV-1 LTRs contain CpG islands, which are defined based on their high percentage of GC content (a GC percentage greater than 50%) and the length of the region (more than 200 bp) . CpG islin vitro cell cultures, where there is abundant Tax expression. The Tax-mediated strong transcription from the 5′-LTR is the case in vitro but not in vivo, as discussed above.The nucleosome is the fundamental unit of chromatin, and it is composed of an octamer of the four core histones . Around 147 bp of DNA wrap around each histone octamer. The histone tails are modified by acetylation, methylation, phosphorylation, and ubiquitylation . These min vivo, we have to analyze fresh ATL cells without doing any ex vivo culture. There are some reports on histone acetylation and methylation of ATL cells. The previous data suggested that histone acetylation is detectable both in 5′- and 3′-LTRs [In order to characterize the pattern of histone modifications  3′-LTRs ,81. We r 3′-LTRs ,67. Anotex vivo culture.In general, there are two categories of promoters, active and poised promoters . Active Most studies on histone modifications of HTLV-1 have, so far, been limited to the promoter regions, 5′- and 3′-LTRs. However transcription is a biological event with multiple steps, including transcriptional initiation, elongation, and termination . To undeAs mentioned above, there is a significant difference in the transcriptional activity and the epigenetic characteristics of the 5′ and the 3′ regions of HTLV-1 provirus. Since the size of the provirus is just about 9000 bp, there should be a positive regulatory mechanism to maintain such distinct transcriptional activity within the provirus . We havein vitro and in vivo pattern of viral gene transcription [Another function of CTCF is chromatin loop formation by homodimerization . CTCF, icription . A previcription . EBV is cription ,94.In principle, each infected clone has a different integration site (IS). IS data has been used to identify clonality of HTLV-1-infected cells. According to technological advances in DNA detection and sequencing, current analysis of clonality of infected cells is becoming far more accurate and quantitative than before ,97,98,99i.e., initial integration-targeting and preferential survival of infected clones in vivo. There is clear difference in HTLV-1 IS between in vitro-infected samples and in vivo-infected clinical samples [in vivo, the infected clone that has an advantage in escaping from the host immune surveillance would be able to live for a long time. Although there is no clear hot spot of IS associated with development of ATL, there are some tendencies as listed below [The distribution of HTLV-1 integration sites in the infected individuals is determined by two key factors,  samples . This id samples . The dated below ,65.(i)HTLV-1 tends to be integrated into open chromatin.(ii)Infected clones with ISs within a gene and with the same orientation as the host gene tend to expand.(iii)ex vivo culture.Presence of a transcription factor- or histone modifier-binding site, such as Brg1 and STAT-1, near the IS can affect the frequency of spontaneous Tax expression in ex vivo culture is inversely correlated with clonal abundance of infected clones, suggesting that Tax expression is not necessarily related with clonal expansion of infected cells in vivo [The data has also demonstrated that spontaneous Tax expression in  in vivo . Taken tde novo infection with viral particles, in which error-prone reverse transcriptase generates genetic mutations in the viral genome [et al. showed the presence of point mutations with a premature stop codon in tax gene in ATL patients [et al. performed a comprehensive analysis and found that there are mutations in various viral genes but not in the HBZ gene [Compared with HIV-1 provirus, the HTLV-1 genome is highly conserved, which is evidence for the idea that HTLV-1 maintains its viral copy number via clonal expansion of infected cells rather than l genome . Even sol genome ,104,105 patients . Fan et HBZ gene . Such deRecent HTLV-1 research has made substantial progress in proviral regulation, but there are several issues that remain to be addressed. For example, the evidence we have so far is the result of analyzing a population of infected cells or ATL cells, so we cannot exclude the possibility that a part of the infected cells sporadically expresses plus-strand transcripts like Tax at the single-cell level. Also, it is still unclear how much the maintenance of ATL cells depends on HTLV-1 provirus. It is obvious that genetic and epigenetic alterations associated with oncogenesis in the host genome play a central role in the leukemogenesis of ATL ,48,49,50


Human T-cell Lymphotropic Virus type-1 (HTLV-1) is the causative agent of an aggressive malignancy of CD4+ T lymphocytes. Many studies have shown that the constitutive activation of NF-kB pathway by the viral transactivator Tax-1 is crucial for T-cell transformation. We previously identified the cellular factor Class II transactivator (CIITA), the master regulator of Major Histo compatibility Complex Class II gene transcription, as a restriction factor inhibiting HTLV-1 replication by blocking Tax-1-mediated activation of the viral LTR promoter (1). Here we show that CIITA suppresses also the activation of the canonical NF-kB pathway by Tax-1 and mapped the region of CIITA mediating this effect. CIITA affects the subcellular localization of Tax-1, which is mostly retained in the cytoplasm and this correlates with an impaired migration of Rel Ain to the nucleus. By using nuclear and cytoplasmic deletion mutants of CIITA, we demonstrate that CIITA suppresses the activation of NF-kB by Tax-1 in both the subcellular compartments. Interestingly, CIITA binds to Tax-1 in vivo without preventing the binding of Tax-1 to both IKKg and RelA. Nevertheless, Tax-1-induced IKK kinase activity is affected in the presence of CIITA as demonstrated by impaired phosphorylation of IkB inhibitor, which is responsible for the impaired migration of RelA into the nucleus. Thus, the inactive p50/RelA heterodimer is trapped in the cytoplasm and this results in the suppression of the activation of NF-kB responsive genes by Tax-1. Overall, these findings indicate that CIITA has evolved as a versatile molecule that, besides inhibiting viral gene expression promoted by Tax-1, it might counteract also Tax-1 transforming activity. Thus, unveiling the molecular basis of CIITA-mediated Tax-1 inhibition may be important in defining new strategies to control HTLV-1 spreading and on cogenic potential.


Due to their low hydrophilicity, triterpenes were considered to be inactive for a long period of time; however, evidence regarding their wide range of pharmacological activities is emerging, and elegant studies have highlighted these activities. Several triterpenic skeletons have been described, including some that have presented with pentacyclic features, such as oleanolic and ursolic acids. These compounds have displayed incontestable biological activity, such as antibacterial, antiviral, and antiprotozoal effects, which were not included in a single review until now. Thus, the present review investigates the potential use of these triterpenes against human pathogens, including their mechanisms of action, via in vivo studies, and the future perspectives about the use of compounds for human or even animal health are also discussed.Triterpenoids are the most representative group of phytochemicals, as they comprise more than 20,000 recognized molecules. These compounds are biosynthesized in plants via squalene cyclization, a C The triterpenoids are the most representative group of phytochemicals; they comprise more than 20,000 recognized compounds and are biosynthesized in plants through squalene cyclization . The triThe diversity of triterpenes is highly associated with their broad range of pharmacological effects. In Asian countries, triterpenes are traditionally used as anti-inflammatory, analgesic, hepatoprotective, cardiotonic, and sedative agents . Other sOleanolic acid (OA) and its isomer, ursolic acid (UA), are triterpenoid compounds that widely occur in nature in free acid form or as an aglycone precursor for triterpenoid saponins . These tβ-hydroxyolean-12-en-28-oic acid) is a pentacyclic triterpenoid with widespread occurrence throughout the plant kingdom. This compound and its derivatives possess several interesting pharmacological activities, such as anti-inflammatory, antioxidant, anticancer, and hepatoprotective effects. OA was previously isolated from almost 2000 plant species 50 of 8 μg/mL). OA and its derivatives were also capable of inhibiting HIV-1 protease, with an IC50 of 4–20 μg/mL [ ex vivo experiments showed that peripheral blood mononuclear cells (PBMC) from HIV-infected patients, which were incubated with different doses of OA, presented significant reduction of viral replication, which was comparable with the drug, azidothymidine (AZT). Similar results were found when PBMC from healthy donors were infected with HIV-1, yielding an effective concentration (EC)50 of 22.7 μM and 24.6 μM, respectively [The antiviral properties of OA and UA have been studied since the 1990s, specifically those used against human immunodeficiency virus (HIV) and the hepatitis virus. HIV belongs to the Retroviridae family and the genus,nfection . One of nfection  dealing 20 μg/mL . The inh20 μg/mL ; this wiectively . Moreoveectively  demonstrβ-mediated apoptosis than were the control cells. In vivo experiments showed that HBV-induced tumors were significantly lower in UA-treated animals when compared to controls [ Ligustri lucidi seems to be very effective at eliminating intracellular HCV with an IC50 of 5.5 μg/mL and a high selectivity index (SI) of 30.8. Otherwise, the IC50 found for UA activity was higher than that determined for OA (IC50 of 33.8 μg/mL), and the latter featured a lower SI (6.7). In addition, one possible mechanism of action of OA was related to the suppression of the viral NS5B RdRp enzyme, which is a central enzyme responsible for HCV RNA replication [The potential of OA and UA was also determined against hepatitis B and C viruses . These viruses are of serious concern for human populations, since approximately 500 million people are chronically infected with one or both viruses, resulting in fibrosis and cirrhosis of the liver, and ultimately leading to the development of hepatocellular carcinoma , 143. Alcontrols . These ilication . Mallotus peltatus and Achyranthes aspera [ M. peltatus and A. aspera, which contained UA and OA. The researchers found that both fractions presented with strong inhibitory activity against HSV-1 and HSV-2, which was comparable to the standard drug, Aciclovir. In addition, the OA-containing fraction from A. aspera triggered interleukin- (IL-) 12 production in treated peritoneal macrophages [+Th1 cell population and for eliminating intracellular pathogens [UA and OA were also assayed against the proliferation of herpes viruses in host cells. Herpes simplex viruses (HSV) cause herpes labiles, herpes genitalis, keratitis, and encephalitis. The HSV infection caused by type-1 and type-2 viruses is mainly transmitted through close personal contact. The therapy that is used against the infection has severe side effects, and drug-resistant viruses have been detected , justifys aspera , 148, prs aspera . Consides aspera  and Mukhs aspera  assayed rophages , which iathogens , 152.These works indicate that OA and UA inhibit viral spreading in different host cell lineages with high levels of sensitivity and selectivity; this mainly depends upon the virus type and the host cell. In addition, the mechanism of action of both triterpenes was related to the control of virus replication and also to the immunomodulatory effect on the host cells, suggesting that new drugs can be developed from these structures. Plasmodium falciparum, Toxoplasma gondii, Trypanosoma cruzi, and Leishmania sp.OA and UA also displayed appreciable antiparasitic effects against Plasmodium [ Plasmodium sp. is that its resistance needs to be overcome [The parasitic disease with the greatest impact is malaria; it affects around 40% of the world's population, spanning across more than 100 countries, and its etiological agent is a protozoa belonging to the genus,asmodium . Althougovercome ; this in Plasmodium falciparum was conducted by Steele et al. [ Uapaca nitida (Euphorbiaceae). UA showed antimalarial effects with an IC50 of 36.5 μg/mL and 28 μg/mL against chloroquine-resistant and chloroquine-sensitive strains, respectively. Otherwise, the IC50 that was found for OA was 88.8 μg/mL and 70.6 μg/mL for chloroquine-resistant and chloroquine-sensitive strains, respectively. Other studies have also corroborated the potential of UA, purified from Mitragyna inermis, against chloroquine-sensitive and chloroquine-resistant strains, showing an IC50 between 15 μg/mL and 18 μg/mL. In addition, infected blood cells treated with UA presented with lower parasitism than did infected controls [ Satureia parvifolia, Mimusops caffra, M. obtusifolia, and Kleinia odora were able to eliminate P. falciparum [In this regard, one of the first works to demonstrate the antimalarial properties of triterpenes against chloroquine-resistant and chloroquine-sensitivee et al. . In thiscontrols . Other slciparum –158. Leishmania sp. parasites, and then against Trypanosoma cruzi, the etiological agents of leishmaniasis and American trypanosomiasis, respectively. Leishmaniasis is a complex disease, and its symptoms range from the presence of severe cutaneous lesions to the more visceral form of the disease, which affects the spleen, liver, and bone marrow [Drugs based on pentavalent antimonials, Amphotericin B, nifurtimox, and benznidazole, are employed to treat patients with leishmaniasis and American trypanosomiasis but, unfortunately, these drugs are toxic and reports of parasite resistance to them have been constantly published, justifying the search for new active compounds. In infections caused by trypanosomatids, OA and UA were also tested, first in the use againste marrow . Salvia cilicica roots. The obtained results showed that UA was primarily active against intracellular amastigote forms of L. donovani and L. major, with an IC50 of 12.7 nM and 7.0 nM, respectively. These values were comparable to the standard drug, Pentostam, whose IC50 was 10.6 nM and 9.8 nM against the same parasite species, respectively. L. (L.) amazonensis promastigotes were shown to be highly sensitive to OA and UA, presenting an IC50 of 10 μg/mL and 5 μg/mL, respectively. In addition, both of these compounds were active against the intracellular form of L. (L.) amazonensis, showing an IC50 of 27 μg/mL and 11 μg/mL, respectively. On the other hand, an IC50 of 83 μg/mL was obtained for experimental treatment with meglumine antimoniate [ L. (L.) amazonensis, L. (L.) infantum [ L. (L.) donovani [Tan et al.  evaluateimoniate , suggestinfantum , and L. donovani . Baccharis uncinella leaves led to the identification of a bioactive fraction that contained OA and UA triterpenes. This fraction showed moderated activity against L. (V.) braziliensis and L. (L.) amazonensis promastigotes, although it was very active against amastigote forms of both parasite species; moreover, the leishmanicidal effect could be related to a direct effect on the amastigote forms. Additionally, these compounds triggered nitric oxide production in the macrophages, since infected cells incubated with the highest concentration of this fraction produced significant amounts of this effector molecule [ L. (L.) amazonensis-infected mice. Animals that were treated with 1.0 mg/kg and 5.0 mg/kg of triterpene fraction presented with reduced lesion sizes and skin parasitism, which was accompanied by a significant elevation of IL-12 and interferon- (IFN-) γ cytokines. Furthermore, the treatment did not alter the histological profile of the spleen, liver, heart, lungs, and kidneys of mice [Recently, a bioguided study conducted with extracts ofmolecule . Due to  of mice . Interes L. (L.) amazonensis promastigote forms treated with 10.96 μg of UA presented with irreversible morphological changes after 18 hours of incubation. Control parasites presented with normal membrane morphology, cytoplasm, nucleus, mitochondrion, and flagellum ( L. (L.) amazonensis, UA induces a mechanism of death associated to apoptosis or even autophagy. This is the first study that depicted the possible mechanism of action of UA on L. (L.) amazonensis promastigote forms.In addition, our group demonstrated, through ultrastructural studies, thatlagellum . Otherwilagellum  and swellagellum ; blebs wlagellum . In addilagellum , suggestBased on previous works, these triterpenes can be regarded as antileishmanial agents since these studies demonstrated that these agents can be more effective than conventional drugs. In addition, more attention needs to be paid to UA, which is the primary antileishmanial agent when compared to its isomeric derivative, OA. T. cruzi infects a broad range of cell types, preferentially, muscle cells from the gut and heart, leading to a loss of organ function [ Miconia species were shown to be active against the blood form of T. cruzi; they showed an IC50 of 80.4 μM and 21.3 μM, respectively, while the IC50 for gentamicin violet was 71.6 μM [ T. cruzi.In American trypanosomiasis, the parasitefunction , 164. Unfunction , indicat 71.6 μM , reinfor 71.6 μM . Ferreir 71.6 μM  also demSeveral triterpenes, which displayed interesting structural features, have been considered inactive for a long period of time. However, different works have since demonstrated the wide array of pharmacological activities inherent in this class of natural compounds. in vitro potency of these agents with studies dealing with their therapeutic action (in vivo); this should be a priority in this field. In addition, these types of strategies will be crucial in the development of new drugs that can be used for populations that are at risk for contracting certain diseases.Specifically, UA and OA present remarkable antimicrobial activities, and they act against important human pathogens such as mycobacteria, HIV, and different protozoal species. The present review described interesting works about the antimicrobial action of UA and OA that, in fact, could be considered drug prototypes. In spite of this, the present review also alerted us to some concerns, insofar as the majority of the works presented here have not depicted the possible mechanism of action of these triterpenoids in microorganisms. Moreover, studies have not associated the


Interestingly, UA-mediated MPO and LPO inhibition can be prevented by innate immune protein human NGAL or its murine ortholog lipocalin 2 (Lcn2), implying the complex nature of host innate immunity-microbiota interactions. Spectral analysis indicates that UA inhibits heme peroxidase-catalyzed reaction by reverting the peroxidase back to its inactive native state. In support of these in vitro results, UA significantly reduced phorbol myristate acetate (PMA)-induced superoxide generation in neutrophils, however, EA failed to block the superoxide generation. Treatment with UA significantly reduced PMA-induced mouse ear edema and MPO activity compared to EA treated mice. Collectively, our results demonstrate that microbiota-mediated conversion of EA to UA is advantageous to both host and microbiota i.e. UA-mediated inhibition of pro-oxidant enzymes reduce tissue inflammation, mitigate non-specific killing of gut bacteria, and abrogate iron-binding property of EA, thus providing a competitive edge to the microbiota in acquiring limiting nutrient iron and thrive in the gut.Numerous studies signify that diets rich in phytochemicals offer many beneficial functions specifically during pathologic conditions, yet their effects are often not uniform due to inter-individual variation. The host indigenous gut microbiota and their modifications of dietary phytochemicals have emerged as factors that greatly influence the efficacy of phytoceutical-based intervention. Here, we investigated the biological activities of one such active microbial metabolite, Urolithin A , which is derived from the ellagic acid (EA). Our study demonstrates that UA potently inhibits heme peroxidases i.e. myeloperoxidase (MPO) and lactoperoxidase (LPO) when compared to the parent compound EA. In addition, chrome azurol S (CAS) assay suggests that EA, but not UA, is capable of binding to Fe One of the major functions of gut microbiota is to metabolize dietary components to a spectrum of metabolites. Several of these microbial-derived metabolites aid in developing and controlling immune system not only in the gut but also distant organs. The balance between microbial controlled pro and anti-inflammatory activities is critical to maintain gut homeostasis and regulate gut inflammation and colon carcinogenesis [vs bad; cause vs consequence) using animal models in germ-free conditions and antibiotic-mediated microbiota ablation [Gut microbiota is considered as hidden metabolic ‘organ within an organ’ because of its immense effect on health by influencing the host nutrient acquisition, metabolism, physiology, and immune functions –4. The hogenesis –10. Seveogenesis , 11–17. ablation , 18–21. in vivo are due to gut microbiota-mediated conversion into metabolites called urolithins [in vivo. For instance, upon consumption of pomegranate juice by humans, peak plasma levels of UA could reach from 14 to 40 μM but with a large individual-variations [Gordonibacter urolithinfaciens and Gordonibacter pamelaeae DSM 19378T) that are responsible for metabolizing the EA to produce luteic acid, UroM-5, UroM-6 and UroC [The health benefits rendered by consumption of several natural plant products  have been associated with their high levels of health promoting polyphenolic compounds, specifically ellagitannins and EA –24. Thesolithins , 36. Uroolithins  and are olithins  as well olithins –40 withoolithins , but theolithins ). These riations , 41. Suc2O2) and chloride anion (Cl-) during respiratory burst of neutrophils. Heme group is critical to catalyze these actions [Myeloperoxidase (MPO) is a 150-kDa protein belonging to the heme peroxidase superfamily that includes eosinophil peroxidase, lactoperoxidase (LPO), thyroid peroxidase, and prostaglandin H synthase . It is p actions . MPO-pro actions , 45. How actions –49. Acco actions .in vitro and in vivo models. Here, we examined the effects of EA and UA on activities of MPO/LPO, interdependence of Ferric iron and host neutrophil gelatinase-associated lipocalin (human NGAL) and its murine ortholog lipocalin 2 (Lcn2). We also tested the efficacy of EA and UA in phorbol 12-myristate 13-acetate (PMA)-induced ear edema model. Altogether, our findings provide insights into interplay between microbiota-derived phytometabolites and host NGAL/Lcn2 in the complex regulation of heme peroxidases.The aim of the study is to determine the potential beneficial anti-inflammatory and peroxidase inhibitory activities of UA compared to EA in 2O2 were procured from Sigma-Aldrich . Bovine milk LPO was purchased from Worthington Biochemical Corp . Chrome Azurol S was purchased from Acros Organics. Guaiacol (2-methoxyphenol) was obtained from Alfa Aesar .Mouse recombinant (rec)-Lcn2 was obtained from Cell Signalling and human rec-NGAL was acquired from R&D Systems; both of which are free from endotoxin, siderophore, and iron. Human MPO was procured from R&D Systems . EGCG, PMA, ferric chloride, PIPES, agar, and H3  in chlorobenzene (230 ml) was added and the reaction was refluxed for 16 hrs at 131°C. The product obtained was extracted using ethylacetate and concentrated using rotary evaporator. In a two necked round bottom flask, the demethylated product (2-bromo-5-hydroxy benzoic acid) obtained in reaction 1  along with resorcinol , sodium hydroxide  was mixed with water (45.5 ml) and reaction was refluxed for 2 hrs at 100°C. To the above mixture, 10%w/v CuSO4 (17 ml) was added and refluxed for another 2 hrs. Crude product was purified by column chromatography. The desired compound was eluted with a solvent mixture of hexane: ethyacetate (50:50) followed by recrystallization in ethylacetate to ensure high purity. The purified UA (1 g) was concentrated and characterized by TLC, NMR spectroscopy, mass spectroscopy and HPLC  and AlCland HPLC .2O2 to a chromophore 3,3’-dimethoxy-4,4’ biphenylquinone [3) for 5 min at room temperature in 20 μl reaction mixture. The reaction was initiated on the addition of 30 μL of H2O2 (6.7x10-3%) and 50 μL of 100 mmol/L guaiacol (prepared in 0.1 mol/L potassium phosphate buffer). The change in absorbance at 470 nm was recorded per minute intervals over a period of 10 min.Stock solutions of EA (10 mM) and UA (100 mM) were prepared in Dimethyl sulfoxide  and subsequent dilutions were prepared in PBS. MPO and LPO assays are based on the principle that peroxidase oxidizes guaiacol (2-methoxyphenol) in the presence of Hlquinone . The cha3 at indicated concentrations were placed on CAS plate and monitored for orange-color halo formation. The intensity of halo formation is directly proportional to the concentration of chelated iron. EGCG (Sigma-Aldrich) was used as a positive control [Chrome Azurol S (CAS) agar plates were prepared according to procedure described by Schwyn and Neilands . The pri control .CAS reagent was prepared according to the procedure described by Payne in 1994 . Briefly2O2 was added to LPO, followed by addition of either EA (50 μM), UA (50 μM) or vehicle. Spectra were recorded throughout the reaction at 300 to 500 nm every 3 seconds using CARY50BIO UV-Visible Spectrophotometer. Each spectrum represents an average of six scans. All reaction concentrations above represent final system concentrations.To perform spectral analysis, LPO (1.0 mg/ml) was reconstituted in 500 μl of 0.1 mol/l phosphate buffer (pH 6.0) and basal spectra were recorded at 412 nm for freshly reconstituted LPO. The reaction was initiated when 30 μM HMurine bone marrow cells from WT C57BL/6 mice were isolated and neutrophils were separated by density gradient centrifugation as described by Swamydas and Lionakis . BMDNs w5 cells/well) were seeded on a 96 well in triplicates with a working concentration of NBT (1 mg/mL) with and without PMA (50 nM) in presence of EA or UA (50 μM) and then incubated at 37°C for 3 hrs. Images of cells were taken after incubation and a minimum of 100 cells were observed in each well for NBT-positive cells (%). The cells were fixed using 200 proof ethanol at -20°C for 15 minutes followed by two washes in 70% ethanol. Wells were allowed to dry and 140 μL of KOH (2M) was added to lyse all cells followed by 160 μL of DMSO to solubilize the formazan. After homogenization of the contents in the wells, the absorbance was read at 620 nm [BMDN cells (2x10t 620 nm .viz. vehicle (10% glucose), UA (40 mg/kg body weight), EA (40 mg/kg body weight) and indomethacin (10 mg/kg body weight); indicated treatments were delivered orally (100 μl) 2 hrs before the application of PMA. PMA (4 μg/ear in 20 μL acetone) was applied to inner surface on right ear of each mouse while left ear of each mouse received vehicle (20 μL acetone) on inner surface. At six hours post-application of PMA, animals were sacrificed by cervical dislocation and 6 mm diameter disc from each ear was removed using metal punch. The tissues were weighed and ear edema weight was calculated by subtracting the weight of the left ear from the right ear (treatment). Percent inhibition was calculated and expressed as decrease in weight with respect to control. Tissue samples obtained from each mouse were also assessed for MPO activity using Bradley et al method [O-dianisidine and 0.005% H2O2. The change in absorbance was measured at 460 nm using Synergy HT spectrophotometer (Biotek Instruments Inc.) MPO activity results were represented as units per 6 mm of tissue. One unit of MPO activity was defined as that degradation of 1 mM peroxide per minute at 25°C.All the animal protocols were approved by IACUC at University of Louisville, Louisville, KY. C57BL/6 mice (6–8 weeks old) were purchased from Jackson laboratories. Acute PMA-induced ear edema was performed according to method described previously [l method . BrieflyAll experimental results were reproduced in at least three independent experiments performed in triplicates. All values in the results are expressed as mean ± SEM. The significance of difference between different groups was determined by unpaired Student t-test in case of two groups. P-value less than 0.05 were considered statistically significant. GraphPad Prism version 6.0  was used to calculate statistical significance.UA has been synthesized from 2-bromo-5-methoxy benzoic acid in two steps as described in Methods section. The purified UA was obtained as slight yellow color solid, which was characterized using NMR spectroscopy, mass spectroscopy and HPLC  Figs. HiEA and their microbial metabolites have been shown to have anti-inflammatory and anti-oxidative activities , althoug3+ chelation  are known to bind iron , therefohelation . Our spe+ from 1:0 to 1:1 only modestly mitigated UA inhibitory activity, whereas the increment from 1:1 to 1:3 did not result in further significant reduction of MPO activity  probably because EA bioactivity was already low (~15–20%) and could not be mitigated further.The multifaceted innate immune protein murine Lipocalin 2 (Lcn2) and its human orthologue NGAL have been demonstrated to protect MPO activity from being inactivated by bacterial siderophore enterobactin  and EGCGmax) at 412 nm due to the presence of heme moiety group in the enzyme  of LPO would interacts with H2O2 and produce redox intermediate called oxoiron with λmax at 430 nm . The addition of EA (2O2 spectra (λmax) from 430 nm back to 412 nm. These observations indicated that these compounds reverted the redox reaction and restored the native Fe3+ state of heme of LPO. Intriguingly, the UA-mediated spectral shift was noticeably more rapid and faster than the slower spectral shift mediated by EA (LPO displays maximum absorbance (λe enzyme . To detet 430 nm . The addon of EA  or UA (Fon of EA  reverteded by EA .The phorbol myristate acetate (PMA) is a well-known inducer of oxidative stress in neutrophils via induction of ROS production catalyzed by NADPH-oxidase. Hence, we next asked whether UA and EA could inhibit the ROS production in PMA-stimulated neutrophils by employing the nitroblue tetrazolium (NBT) reduction assay to detect the production of intracellular ROS. Accordingly, we observed that UA significantly mitigated PMA-induced NBT formazan production in neutrophils, suggesting that UA could also inhibits ROS production . In contin vivo effects of UA and EA, we next tested for their efficacy in treating PMA-induced ear edema in mice. In this acute inflammation model, the severity of disease is reflected by the increased ear edema weight and elevated MPO activity in ear tissue. As shown To examine the Clostridium difficile infections were highly successful in most of the cases [Bifidobacteria and Lactobacillus have been reported to have some beneficial effects in maintaining remission in IBD patients [Polyphenols-rich fruits such as berries and pomegranate have been extensively investigated for their beneficial effects in the prevention of several human diseases. However, the results across these studies are not consistent, possibly due to the large variation in the gut microbiota and their metabolites which greatly influences their bio-potency. The gut microbial dysbiosis  especially in gastrointestinal tract-related disorders such as IBD presents yet another major challenge in designing optimal dietary intervention. Several strategies have been sought to improve/alter the gut microbiota through prebiotics, probiotics, antibiotics and fecal microbial transplantations (FMT) to treat patients with IBD , 64. Thehe cases . Probiotpatients , 67. We patients . Recentlpatients  and Medzpatients . These sHere, we focused on examining one of the microbial metabolite UA for their potency in inhibiting the activity of heme peroxidases (i.e. MPO and LPO). MPO is a hemoprotein expressed in polymorphonuclear neutrophils and macrophages; its production are significantly increased in IBDs  and otheIron deficiency anaemia is one of major problem that is commonly associated with IBD. Indeed, one third of IBD patients were reported to suffer from recurrent anaemia. Unfortunately, oral iron treatment is limited by poor absorption, intolerance, and induction of oxidative stress at the site of bowel inflammation. Therefore, presence of bacteria that is responsible (currently unknown) for production UA from EA is beneficial and potentially lead to increase free iron and decrease in anaemic state in colonic disorders, including IBDs. In this study, we demonstrated that UA lacked the ability to chelate iron, unlike EA and EGCG that display iron-binding properties. We envision that the therapeutic use of EA may not be ideal since its iron-chelating property may further aggravate iron deficiency anaemia associated with IBD. In this regard, we postulate that UA may be a better therapeutic metabolite than EA, given the superior peroxidase inhibitory activity and non-iron binding properties of UA.2O2 to LPO would result in the formation of the peroxidase radical intermediate oxoiron with λmax at 430 nm. Interestingly, the treatment with UA or EA prematurely inactivates the peroxidase-catalyzed reaction and reversed the oxoiron group to its normal native state. The inactivation of peroxidase activity by UA and EA suggests that these compounds are capable of negating the production of hypohalous acid by LPO and potentially MPO as well. This outcome could be possibly be attributed to the donation of electrons by UA to the oxoiron state of the heme and thus, revert to its native state. The UA-mediated inhibition of MPO may be beneficial in mitigating hypohalous acid-mediated tissue damage in the intestines during increased inflammatory conditions.The potency of UA in inhibiting the activity of heme peroxidase prompted us to investigate the potential underlying mechanisms that are involved. In this study, we performed spectral analysis using LPO as our model heme peroxidase; however, we could not employ MPO in our spectral analysis due to technical limitation (i.e. amount supplied is inadequate). Heme peroxidase (e.g. LPO) displays absorbance spectra with λmax at 412 nm due to the presence of its heme moiety group. The addition of HLipocalin (Lcn2) and its human ortholog NGAL sequester iron-containing siderophore as an innate immune mechanism to limit the growth of bacteria, especially during infection or inflammation. Accordingly, the mice lacking Lcn2 are more susceptible to bacterial infection compare to wild type mice . Herein,2•−), hydrogen peroxide (H2O2), hydroxyl radical (•OH), and hypochlorous acid (HOCl) upon stimulation by soluble agonists, such as phorbol ester, chemotactic peptides, and calcium ionophore. The phagocyte activation leads to increase in the production of superoxide anion leading to oxidative burst. In general, stimulation of neutrophils by PMA increases the ROS formation, which can be quantified by NBT assay. In this study, we further demonstrated that UA treatment could mitigate PMA-induced ROS formation in neutrophils (in vitro), but such protective effect was not observed with EA. Instead, we found that EA alone could induce ROS formation, suggesting that EA, but not UA, may have pro-oxidant properties that need to be further investigated. Furthermore, we confirmed these observations in in vivo model, where oral treatment of UA significantly reduced PMA induced ear edema and MPO activity suggesting its potential role as an anti-inflammatory agent. In support of these observations including our unpublished data, it was demonstrated that UA is able to markedly reduce carrageen-induced paw edema as well as LPS mediated anti-inflammatory activities in other models [PMNs produce a variety of reactive oxygen species (ROS), including superoxide anion Click here for additional data file.S1 Fig1H-NMR DMSO-d6: 800MHz: δ: 10.22 , 10.15 , 8.09–8.07 , 8.00–7.99 , 7.50 , 7.31–7.30 , 6.80–6.78 , 6.72 .(TIF)Click here for additional data file.S2 Fig13C-NMR: DMSO-d6: 800MHz: δ: 160.60, 158.53, 156.93, 150.88, 126.94, 124.14, 123.77, 123.54, 120.15, 113.52, 113.02, 109.82, 102.83.(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.


The perception of painful thermal stimuli by sensory neurons is largely mediated by TRPV1. Upon tissue injury or inflammation, S1P is secreted by thrombocytes as part of an inflammatory cocktail, which sensitizes nociceptive neurons towards thermal stimuli. S1P acts on G-protein coupled receptors that are expressed in sensory neurons and sensitize TRPV1 channels towards thermal stimuli. In this study, the S1P mediated signaling pathway required for sensitization of TRPV1 channels was explored.CAPS) of sensory neurons was significantly increased after S1P stimulation within minutes after application. The potentiation of ICAPS resulted from activation of Gαi through G-protein coupled receptors for S1P. Consequently, Gαi led to a signaling cascade, involving phosphoinositide-3-kinase (PI3K) and protein kinase C, which augmented ICAPS in nociceptive neurons. The S1P1 receptor agonist SEW2871 resulted in activation of the same signaling pathway and potentiation of ICAPS. Furthermore, the mitogen-activated protein kinase p38 was phosphorylated after S1P stimulation and inhibition of p38 signaling by SB203580 prevented the S1P-induced ICAPS potentiation. The current data suggest that S1P sensitized ICAPS through G-protein coupled S1P1 receptor activation of Gαi-PI3K-PKC-p38 signaling pathway in sensory neurons.The capsaicin induced peak inward current (IThe online version of this article (doi:10.1186/1744-8069-10-74) contains supplementary material, which is available to authorized users. The perception of pain is mediated by nociceptive primary afferent neurons that are excited upon painful thermal, mechanical or chemical stimuli. These nThe perception of heat involves members of the transient receptor potential (TRP) ion channels, more specifically members of the vanilloid subfamily (TRPV). In particular activation of TRPV1 ion channels results in the excitation of nociceptors and consequently the perception of pain, 7. TRPVIn the presence of inflammatory mediators, the threshold temperature at which TRPV1 channels are activated is decreased and nociceptive neurons respond to thermal stimuli at lower temperatures and with an augmented response. The regulation of TRPV1 by inflammatory mediators released by the immune system receives extensive attention since it is clinically relevant for developing pathological and chronic pain. Activation of G-protein coupled or tyrosine kinase receptors modulate TRPV1 ion channel activity via various intracellular signaling pathways, 11.1–5) at the plasmamembrane.Tissue damage that usually coincides with damage to the blood vessels results in tissue invasion of different cells of the immune system together with thrombocytes. At the injury site, thrombocytes are activated and secrete a variety of immunomodulatory compounds including the sphingolipid sphingosine 1-phosphate (S1P). S1P can activate signaling pathways either through diffusion over the plasmamembrane or through binding to S1P specific receptors  signaling[1 and S1P3 receptors whereas the larger NF200-positive cells express S1P2 receptors. Recently it has been found that S1P enhances neuron excitability[After binding of S1P to its specific receptors, activation of the receptor subtype determines the heteromeric G-protein signaling pathway. For example, the S1Ptability and senstability.1 receptor is a main contributor to S1P-induced hyperexcitability and heat sensitization in mouse nociceptors[1 receptor signaling is restricted to Gαi-mediated signal transduction, the molecular players of TRPV1 mediated sensitization by S1P remain unclear. Here we explore the S1P-PI3K-p38 signaling pathway in sensory neurons for the potentiation of capsaicin-induced, excitatory inward currents.Converging evidence from pharmacological and genetic models suggests that the S1Piceptors–16. AlthCAPS, 0.3 μM capsaicin) from neurons isolated from mouse dorsal root ganglia (DRG). Application of S1P  caused a significant increase of ICAPS peak amplitudes  activation or rather by direct activation of the channel. In the presence of extracellular suramin (100 μM), which is generally accepted to uncouple heteromeric G-proteins from their ligand-binding receptor subunit, S1P failed to evoke an increase in I Figure A. Theref2 and S1P3 receptors couple to all three G-protein types[1 exclusively couples to Gαi/o and no activation of other heteromeric G-proteins has been reported to date[CAPS after conditioning stimulation with S1P was reduced by pretreatment with PTX in the patch-clamp recordings, suggesting the involvement of Gαi/o heteromeric G-proteins in the signaling process  isoenzymes have been identified as targets of S1P1 and S1P3 signaling in some studies[4K and PI3K by Wortmannin , S1P did not facilitate ICAPS to the same extent as in control ECS. Selective inhibition of PI3K by AS605240 (100nM), also attenutated S1P potentiation of ICAPSand the inhibition of S1P-induced potentiation of ICAPS by Wortmannin and AS605240 was comparable to the decrease in S1P-mediated TRPV1 potentiation after PTX incubation  was completely inhibited by 20 h pre-incubation with PTX and thus completely relied on the activation of Gαi  family are known to specifically bind S1P and to regulate inflammatory and regenerative processes in various systems, 36. Croammation–17, 37. 1 receptors[1 receptors are conditionally ablated in Nav1.8 expressing neurons, the S1P induced heat hypersensitivity is attenuated[1 agonist SEW2871. The augmentation of ICAPS by S1P and SEW2871 was inhibited in the presence of PTX that selectively interferes with the inhibitory G-protein Gαi. This suggests that S1P1 utilizes Gαi for signal transduction in nociceptors, just like in other tissues.A smaller portion of sensory peptidergic and non-peptidergic neurons express S1Peceptors. S1P indeceptors, 15. In 1 receptor signaling is well accepted it is largely unknown which downstream intracellular signaling pathways are activated in nociceptive neurons. In general, the S1P1 receptor has been associated with three different signaling pathways downstream of Gαi-proteins. The classical Gαi signaling involves inhibition of adenylate cyclase and reduction of cyclic AMP levels or inhibition of ERK signaling within the cell[CAPS amplitudes which would oppose our findings.Although the contribution of Gαi to S1Pthe cell, 39. Sin1 receptors regulate embryonic self-renewal[CAPS is unlikely to be explained by these two classical Gαi signaling pathways.The signaling cascade for S1P induced facilitation of capsaicin-induced currents also differs from the Gαi-mediated activation of ERK1/2 through which S1P-renewal. TrkA re-renewal–43. Howe1 signaling pathway involves Gαi-mediated activation of PI3K that has been reported to mediate chemotaxis of natural killer cells[3K signaling pathways have been associated with the regulation of TRPV1[3K signal downstream of GPCRs[CAPS through S1P1 largely depended on PI3K. Thus our results link PI3K as novel component of GPCR signaling for the regulation of nociceptor heat hypersensitivity. Downstream of PI3K several kinases of the mitogen activated protein kinase (MAPK) family can be activated. In particular activation of MAP kinase p38 is strongly associated with Gαi/PI3K signaling through S1P receptors[CAPS either by activating tyrosine kinases or protein kinases A or C. The fast changes in capsaicin sensitivity of the sensory neurons that occurred within 1 minute of S1P stimulation are likely attributable to phosphorylation of the already existing TRPV1 channels in the cell membrane. Some of the phosphorylation sites at intracellular domains of the TRPV1 channel protein do not show preference for PKC, PKA ,or CaMKII  and could be possible targets for p38 MAP kinase phosphorylation[3K/p38 and TRPV1[3K is likely and in line with ample evidence from other systems[An alternative S1Per cells. Furtherof TRPV1 and incrof GPCRs. In our eceptors, 47. Theeceptors, 49. Beseceptors–52. Actieceptors and upreeceptors. The muleceptors, 19, 55.eceptors, 26, 56.eceptors. Besideseceptors. Numerourylation. Althougnd TRPV1 do not u systems, 45, 47.1 receptor activation and downstream Gαi to PI3K to p38 signaling as important components for the S1P induced nociceptive hypersensitivity towards thermal stimuli. S1P1 to p38 mediated sensitization of TRPV1 may play a role in the early initiation phase of heat hypersensitivity in mice.Therefore we suggest that S1PAll animal breeding and experiments have been performed with permission of the Austrian BMWF ministry  and according to ethical guidelines of the IASP .Lumbar DRG containing the cell bodies of primary afferents that project into the hindpaw were harvested from adult male mice (age 8–16 weeks) as previously published, 59. Aft2, 1 MgCl2 , 10 glucose and 10 HEPES , at pH 7.3 adjusted with NaOH (Merck). Borosilicate glass micropipettes  pulled with a horizontal puller  were filled with internal solution : 148 KCl, 2 MgCl2, 2 Na-ATP, 0.2 Na-GTP, 0.1 CaCl2, 1 EGTA  and 10 HEPES (Merck), at pH 7.3 adjusted with KOH (Merck). After filling, electrode resistance was 4–6 MΩ. Currents were filtered at 2.9 kHz, sampled at 3 kHz and recorded using an EPC-9  and the Pulse v8.74 software (HEKA) without Rs compensation. Experiments were performed at room temperature and only one neuron was tested per Petri dish. An automated seven-barrel system with common outlet positioned at 100 μm distance from the recorded cell was used for fast drug administration[Using the whole-cell voltage-clamp configuration of the patch-clamp technique, ionic currents were recorded from isolated neurons at -80 mV holding potential after 18–32 hours as previously published, 58. Thestration. S1P . The phosphatase inhibitors sodium-orthovanadate (200 μM) and β-glycerophosphate  were added to the RIPA buffer to prevent protein dephosphorylation. A protease-inhibitor cocktail  was used to protect proteins from proteolysis. SDS-PAGE was performed under standard denaturing conditions using hand casted 10% polyacrylamide gels . Equal amounts of protein were loaded to each lane of the gels. Spectra Multicolor Broad Range Protein Ladder (Fermentas) was used as a molecular weight standard. Gels were blotted immediately after electrophoresis onto polyvinylidene fluoride membrane . For immunodetection, membranes were blocked for 1 h with 5% (w/v) BSA and 0.1% (v/v) Tween-20 in Tris-buffered saline, pH 7.6, at room temperature. Antibodies were used according to the manufacturer’s instructions. The following antibodies were used: anti-phospho-p38 and anti-p38  and peroxidase-conjugated α-rabbit IgG  as secondary antibody. Visualization of blots was performed with enhanced chemiluminescence by using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). Membranes were scanned with LAS4000 luminescent imager . Quantification was performed using ImageJ software and relative values for phosphorylated proteins are represented as units after normalization to the non-phosphorylated form.Data are presented as mean ± SEM. For detailed statistical analysis the Sigmastat 3.0  software package was used and Mann Whitney-U test (MWU) or ANOVA on Ranks followed by Tukey post-hoc test were calculated. Differences were considered statistically significant at p < 0.05.Suramin prevents S1P-induced ICAPSsensitization. A, The augmentation of ICAPS by S1P (1.0 μM) was completely inhibited by extracellular suramin (100 μM) pretreatment. The dashed line signifies the zero current. B, The uncoupling of heteromeric G-proteins from the G-protein coupled receptors significantly inhibited S1P-induced ICAPS potentiation in sensory neurons. ***p < 0.001, **p < 0.01, MWU, numbers within the bars represent the number of individual cells recorded. (PDF 255 KB)Additional file 1:


High hydrostatic pressure (HHP) is applied on a variety of biotechnological process, including food preservation, modulation of enzymatic activity, disaggregation of proteins, vaccines development and recently it was reported its use for ethanol yield increase ,2.Saccharomyces cerevisiae strain (BT0510) submitted to sublethal pressure treatment of 50 MPa for 30 min at room temperature, followed by incubation for 5, 10 and 15 min at room pressure (0.1 MPa). The transcription of the genes involved in central carbon metabolism in response to HHP was investigated for bioinformatics tools.In this study we performed a microarray analysis in a distillery HXK1 and GLK1. Glucose-6-phosphate is a glycolysis and pentose phosphate pathway (PPP) intermediary. Glycolysis and gluconeogenesis were slightly induced by pressure, but GPD1 and GUT2, associated with the glycerol-3-phosphate shuttle, were upregulated by HHP. This mechanism transfer reducing equivalents from the cytosol to the mitochondria for the reduction of ubiquinone in ubiquinol in the electron transport chain that leads to oxidative stress. Interesting enough, HHP also induced ZWF1 and GND2 genes that encode two enzymes related to NADPH production in PPP. The PPP plays an important role in NADPH generation, which is required in oxidative stress response, in order to maintain the intracellular levels of reduced glutathione [LPD1, related to the conversion of pyruvate into acetyl-CoA in the mitochondria, and PDC1 and PDC6 correlated with the conversion of cytosolic pyruvate in acetaldehyde. Acetaldehyde is converted in acetate in the mitochondria by Ald4p and, subsequently, in acetyl-CoA by Acs1p and directed do TCA cycle; genes that encode these enzymes were also induced by HHP. Fatty acid biosynthesis related genes were not strongly affected by HHP. This evidence reinforces the hypothesis that acetyl-CoA is directed to the TCA cycle after HHP stress. HHP also upregulated the glutamate degradation I pathway genes . This pathway plays an important role in oxidative stress, and produces NADPH and succinate, which is transported into the mitochondria and participate in the TCA cycle [PDC1, PDC6, ADH1 e ADH5) were also upregulated, suggesting that anaerobic metabolism was not completely repressed during HHP stress and during the recuperation period.HHP induced genes related to the phosphorylation of intracellular glucose, tathione . PyruvatCA cycle . The indOur data showed that several genes related to the central carbon metabolism were induced by HHP, suggesting that yeast accelerates glucose consumption and, consequently, its metabolism. This data analysis allows the design of new cells for ethanol yield increase using HHP.


Single cell network profiling (SCNP) is a technology that quantifies functional immune signaling capacity and connectivity at a systems biology level. The technology is based on multiparametric flow cytometry that simultaneously quantifies in multiple and rare immune cell subsets, without the need for physical separation, both extracellular surface markers and changes in intracellular signaling proteins in response to extracellular modulators. Quantifying modulated signaling across a panel of modulators  and intracellular signaling pathways identifies the functional capacity of the signaling network which cannot be assessed by measuring basal (unmodulated) signaling alone. A signaling node is defined as the combination of the extracellular modulator with the intracellular readout. For example TLR4 - > p-Erk defines one signaling node in which TLR4 modulation is quantified through the increase in p-Erk levels as compared to the unmodulated reference. Typically 3 nodes are captured simultaneously per well across multiple immune cell subsets of interest .Understanding a patients’ immune status not only from immune cell phenotyping, but also through analysis of The application of SCNP to clinical decision-making requires the generation of high-content SCNP assays with robust, accurate, quantifiable and reproducible results across time, operators and instruments. Each of the procedural steps associated with an SCNP assay, including pre-analytical sample handling, assay execution and reagents, data acquisition and analysis and the generation of metrics, have been validated  Figs. 11 and 2.FExperimental assay setup is performed using proprietary software which enables experimental design/96-well plate layouts and data capture to be contiguously linked, ensuring that data from each well is correctly assigned. The laboratory execution can be performed on as many as 30 samples assayed for up to 40 wells (approximately 200–500 SCNP dimensions comprising modulator/inhibitor/intracellular readout/cell subset combinations) in 2 to 3 days depending on the kinetic time points. A statistical analysis plan (SAP) is drafted for all studies beyond the exploratory phase, based upon clearly stated objectives. For the identification of clinically validated classifiers the time frame for assay development and validation is comparable to that of other technologies  due to the requirements for statistical powering and for verification and validation in independent sample sets.in vitro drug exposure, are captured by metrics that are computed by comparing data for cells subject to different conditions. The “Fold” metric is applied to measure magnitude of the responsiveness of a signal in a specific cell population relative to the unmodulated reference. The proportion of a cell population that is responsive to modulation is measured by the Uu (rank based metric based on Mann–Whitney U statistic) metric. Similarly, inhibited signaling is captured using both magnitude and population-based metrics.Currently the use of 10 cytometer channels is routine, which includes 8 colors plus forward and side scatter. The biological reads labeled per well can be expanded by combining in one channel markers that are mutually exclusively expressed on immune cells, for example CD4 and CD20. SCNP further increases dimensionality of data obtained per sample via multiplexing as illustrated in Fig. For each study, node metric comma separated value (CSV) data files are generated which capture signaling across cell subsets, linked with the complete meta data annotations. These data can be integrated with data from other platforms as appropriate and analyzed using standard statistical tools. Analyses can address multiple objectives including the development of multi-variate predictors of response or prognosis, clustering, quantification of signaling network function, quantification of drug activity across nodes and immune cell subsets (including IC50 determination). More comprehensive and interactive mining of these multidimensional data sets is enabled via an interactive web-based data portal (Fig. SCNP requires live cells in suspension, and therefore is not amenable to analysis of immune cells in flash frozen or FFPE tissue samples. While broad profiling of signaling per sample has been enabled by multiplexing and an extensive array of biology has been established using this technology, the assay is dependent upon the availability of antibodies suitable for flow cytometry. Analysis of immune signaling associated with antigen specificity has not been established. It remains to be seen whether the technology can be transferred to tumor cells isolated from solid tissue.in vitro exposure, can inform on disease and drug mechanism, combination strategies, and identify biomarkers for pharmacodymanic or patient stratification that are otherwise inaccessible using less sensitive or non-functional analyses [One of the major advantages of this technology in the context of immuno-oncology is the ability to monitor cellular functional capacity without physical cell isolation. This enables the detection and monitoring of immune signaling and communication within the complex and interlocked immune system. Interrogating the frequency and signaling capacity of immune cells, and the effects of targeted therapeutics following either clinical administration or analyses –15. The Appropriate pre-analytic sample manipulation is crucial. SCNP has been used successfully with whole blood, PBMC, BMMC  lamina pOver 30 manuscripts and 70 abstracts have been published using this platform. The analytic and clinical validity is presented in many of these, with example references below , 3–16. T