Research on HTLV in Colombia is limited; despite being an endemic country there are few studies on the magnitude of this infection. The aim of this study was to determine the seroprevalence of HTLV I/II and its associated factors in donors to a blood bank of Medellín Colombia, 2014–2018.This is a cross-sectional study of 52,159 donors with a secondary information source. Seroprevalence of HTLV I/II was determined with its confidence interval and the population characteristics were described by frequency and summary measures. To explore the associated factors, Pearson’s Chi square test, Mann-Whitney U test, crude odds ratios were used and they were adjusted by logistic regression in SPSS 25.0.88% of the population lived in the metropolitan area, 68.5% belonged to the University. 76.2% were altruistic donors (unpaid donors who did not donate to a specific patient). 24.5% were repetitive (paid) donors. 75% of the donors were under 41 years old. The seroprevalence of HTLV I/II was 0.176% (95% CI = 0.139% -0.213%), being statistically lower in repetitive donors and men.The seroprevalence of HTLV I/II infection in the studied blood bank is lower than that reported in other blood banks at the departmental and national levels. In Medellín, it was associated with the frequency of donation and gender, which is useful information for the hemovigilance programs of the city. HTLV is the first human pathogenic retrovirus discovered in the laboratories of Dr. Gallo in 1979 . The rouStrongyloides stercoralis [After decades, HTLV-I chronic infection can trigger oncogenic diseases such as leukemia or T-cell lymphoma (ATL), characterized by its aggressiveness, resistance to treatment and high lethality. It can also generate neurological pathologies such as tropical spastic paraparesis (HAM/TSP); inflammatory diseases, for instance uveitis and osteoarthritis derived from the reactivity of monoclonal T lymphocytes; as well as the increase of opportunistic infections such as infective dermatitis and infection by rcoralis . Those drcoralis .All HTLV illnesses involve long and costly treatments that affect people’s quality of life. In the case of leukemias/lymphomas of T-lymphocytes, poor prognosis is usually caused by the disease per se and those disorders triggered by immunosuppression. In addition, HTLV infections also represent an important cost for health systems .The number of infected people in the world ranges between 10 and 20 million . ColombiWorldwide there are many studies on the sero-prevalence of HTLV in blood donors. In Japan from 2006 to 2007 a prevalence of 0.317% was reported in 1.2 million samples . In AustIn Costa Rica from 2002–2004, a study found, in 107 thousand samples, a reactivity of 0.54% by ELISA and a prevalence of 0.03% with the confirmatory test (WB) . In CubaIn Colombia there are several studies showing the exposure to the virus (reflected in the positivity of HTLV-I/II antibodies) in blood donors. In Antioquia (at the state level) HTLV-I/II antibodies were screened between 2001 and 2014 in 1.3 million donors showing a seroprevalence of 0.54% . In CaliIn addition to the frequencies reported, other factors associated with HTLV infection should be considered in blood donors, for instance replacement donors, women, people with a high number of sexual partners, users of intravenous psychoactive substances, children of mothers with HTLV who were breastfed and young people between 18 and 33 years old ,24,25. HThe information presented above shows the heterogeneity in the prevalence of HTLV in blood donors, as well as a low number of studies in Medellin (at the city level). Therefore, the objective of this study was to determine the seroprevalence of HTLV I/II and its associated factors in serum samples from donors to a blood bank in Medellín-Colombia, during the 2014–2018 period.We conducted a cross-sectional study of 52,159 donors to the blood bank of the School of Microbiology of the University of Antioquia in Medellín city, screened for HTLV I/II between the years 2014 and 2018.The inclusion criteria were: age between 18 and 65 years, weight greater than 50 kg, hemoglobin greater than 12.5 g / dL in women and 13.5 g / dL in men, blood pressure between 100/60 mmHg and 180/90 mmHg.The exclusion criteria were: having more than one sexual partner in the last 6 months, being or having been a sex worker or consumer of psychoactive substances intravenously, being pregnant or having given birth or suffered an abortion in the past months, having been ill in the last 15 days, having systemic autoimmune diseases such as lupus, previous infections transmissible by transfusion; having had piercings, tattoos, permanent makeup or acupuncture procedures in the last 12 months.We used a secondary source based on the records of the Hexabank software blood bank database license 1.28.30.50. From each donor, demographic information and the results of the HTLV I/II screening tests were collected, based on a chemiluminescent test that detects antibodies against HTLV-I /II with 99.5% specificity. All positive samples were confirmed by specific Western Blot for HTLV1 and HTLV 2  with a sensitivity and specificity of 97%.Validated laboratory methods were used to establish sero-positivity for HTLV I/II. Internal quality control of the Architect I200 immunoassay analyzer and external equipment of the Colombian National Institute of Health (INS) was carried out. The variables taken from the blood bank database were: age, gender, recruitment place , type of donation , frequency of donation, type of extraction group, place of residence, and result of the screening and confirmatory test.The variables were described with summary measures and frequencies. Seroprevalence was determined as a proportion with a 95% confidence interval. To evaluate the assumption of normality in the quantitative variables, the Kolmogorov-Smirnov test was performed with Lilliefors correction. To explore the factors associated with the infection, Pearson Chi-square test was used for the nominal variables, Chi-square linear trend for the ordinal (age group), and Mann Whitney U for comparison with age. To adjust for the effects of confounding variables, odds ratios were adjusted using logistic regression. All analyses were performed in SPSS 25.0.The project complies with the guidelines of Helsinki, is a risk-free study according to Resolution 8430 of the Ministry of Health of Colombia 1993, and meets the provisions of Resolution No. 1995 of 1999 "By which standards are established for the Management of the Clinical History". In addition, the project has endorsement from the director of the Blood Bank, and all personally identifiable data were removed from the data set prior to access and analysis.Of the study participants, 56% ranged between 22 and 41 years of age, 53.2% were women, 68.5% were extramural (from companies and universities), 58% lived in Medellín, 76.2% were altruistic donors, 45.6% were donors for the first time and 91.2% of the blood samples collected were whole blood .The overall seroprevalence was 0.176% (95% CI = 0.139% -0.213%). Of the total of positive samples, the presence of HTLV 1 was confirmed in 12 donors, of HTLV 2 in four individuals, and in a woman of 21 years the presence of both was confirmed.Age was not related to seropositivity (p> 0.05 U of Mann Whitney). The seroprevalence of HTLV in women was 83% higher than in men, regardless of the frequency of donation. In first-time and non-repetitive donors, it was 2.25 and 2.31 times, respectively, that registered in repeat donors, independent of gender .The groups with the highest frequency of donation were women, people under 41 and first-time donors and altruists; these results agree with reports from the National Institute of Health between 2014 and 2017. An exception was that the altruistic donation was lower than that reported in Antioquia (at the state level) with data of 82.3% in 2014 and 86.1% in 2017 ,28. ThisAdditionally, some demographic and cultural factors related to the type of donation predominant in each blood bank should be considered. A previous study showed a greater number of intramural donations from adults, while in the extramural donations the proportion of adolescent donors was higher (18–20 years). In addition, the donation by altruism or "social commitment" is more frequent from women, while in men the "own benefit” prevails ,30.The seroprevalence found in this investigation was lower than that reported from the departmental average of 0.30% and the national average of 0.54% ; althougIn the study population, there was little association of HTLV infection with age, as in the blood bank of the Pablo Tobón Uribe Hospital located in the same city . Most otIn this study, the prevalence of HTLV-I/II was higher in women, which coincides with other studies in blood banks conducted in Medellín, Cali and Tumaco ,31,35. HWe also found higher seroprevalence in first-time donors and non-repetitive donors, which is consistent with reports in the literature that relate being a repetitive donor with lower HTLV-I /II seroprevalences in the United States and the United Kingdom. This has been explained by having better lifestyles and better quality of information from self-exclusion surveys in repetitive donors –43.HTLV-1 genomic RNA can be detected in the plasma of a minority of patients but not at a level or frequency to be useful clinically or diagnostically. Lack of transmission of HTLV-1 by plasma is due to the rare presence of HTLV-1 virions, regardless of any other factor" [It must be remembered that donors must meet specific inclusion criteria and requirements that make them a low-risk group for infection, for this reason, the results shown are a good proxy for the level of exposure of the healthy general population in the area near the blood bank. However, it is important to highlight that HTLV infection in the general population may differ according to the behavior of each subgroup with different risk factors, such as the use of psychoactive substances or promiscuous behavior, from those without any risk factors. Additionally, according to guidelines of Colombian blood banks, viral RNA detection was not performed; in this sense, the study of the Demontis group concludes that " factor" .Finally, the importance of this type of study should be highlighted, because it is aligned with the recommendations of experts from the Global Virus Network’s HTLV task force, HAM-net, and HTLV Aware suggesting as research priorities: epidemiological studies, mechanisms of HTLV-1 persistence, replication, pathogenesis, treatments, and prophylactic and therapeutic vaccines ,46. PartIn this study, the seroprevalence of HTLV I/II infection in the blood bank was lower than that reported in other banks at the departmental and national levels, and in the case of Medellín, it was only associated with the frequency of donation and gender, which is useful information for the hemovigilance programs of the city.S1 Database(SAV)Click here for additional data file.S2 Database(SAV)Click here for additional data file.


HTLV-1 infection is considered as highly endemic in central Africa. Thirty years ago, a first epidemiological study was performed in Gabon, central Africa, and revealed that the prevalence varied from 5.0 to 10.5%. To evaluate current distribution of HTLVs in Gabon, 4.381 samples were collected from rural population living in 220 villages distributed within the 9 provinces of country. HTLVs prevalence was determined using two ELISA tests and positive results were confirmed by Western Blot. The overall HTLV-1 seroprevalence was of 7.3% among the rural Gabonese population; with 5.4% for men and 9.0% for women. Prevalence of HTLV-1 differed by province, ranging from 2.3% to 12.5% into the rain forest. Being a woman older than 51 years represented a high risk for HTLV-1 acquisition. Hospitalization, operation/surgery, transfusion and medical abortion or fever, arthritis and abdominal pain are also significant risk factors. In addition, 0.1% of samples were found as HTLV-2 positive, while 12.0% had an indeterminate HTLV serological pattern. HTLV-3 and HTLV-4 were not found. Phylogenetic analysis was performed on 87 samples and demonstrated that HTLV-1 present in Gabon belongs mostly to subtype B, however the rare subtype D was also found. Altogether, our results demonstrate that almost thirty years after the first epidemiological study prevention of HTLVs infection is still an issue in Gabon. HTLV-1 is endemic in Africa where it infects millions of individuals and causes Adult T Leukemia. Thirty years ago, an epidemiological study was performed in Gabon, where HTLV-1 is endemic. This revealed a high prevalence of this retroviral infection. Thirty years later, we performed a larger study in the general Gabonese population. This reveals that this oncogenic retrovirus is still highly frequent in this country and that prevalence remains unchanged. It would be extremely important to inform the general population about this infection in order to be able to decrease this retrovirus transmission. Human T-cell leukemia virus type 1 (HTLV-1) and -2 (HTLV-2) belong to the Primate T Lymphotropic Virus group (PTLV), sharing common epidemiological and biological properties, and in particular T-lymphocytes tropism . It has HTLV-1 is not ubiquitous but endemic in some areas and among some ethnic groups. HTLV-1 is highly endemic, in southern Japan, sub-Saharan Africa and the Caribbean Basin as well as parts of South America or Middle East . The gloHTLV-1 and HTLV-2 infection is spread through sexual transmission mainly, from man to woman; parenteral transmission as by needle sharing, blood transfusion or organ transplants and mother-to-child transmission associated with prolonged breast-feeding –25. DiseMother-to-child transmission has been reported as predominant route in endemic areas. HTLV-1 seroprevalence increase with the age, especially among women . In GaboTo determine the situation and evolution of HTLV-1 infection in Gabon thirty years after the first epidemiological survey, we performed a new epidemiological study and determined the prevalence of HTLVs throughout the country among rural adult population. HTLV-1 proviral load, a major factor for the viral transmission and disease evolution , was alsThe study protocol was reviewed and approved by the Gabonese Ministry of health (Research authorization n°00093/MSP/SG/SGAQM). The Health Director and the Governor of each province received written information from the traditional chief of each village. Likewise, future research studies were orally described to all participants and an individual written consent was signed before blood sampling. Parents’ written consent was obtained for the participating children.2 for about 1.5 million of inhabitants (5.6 inhabitants/km2), including 73% living in urban areas. Gabon is divided into 9 provinces for a total of 2.048 villages. Villages are predominately located along roads and rivers. Few have more than 300 inhabitants. The main population activities are subsistence farming, hunting, gathering and fishing. Geographically, the country is constituted of four ecosystems: the Forest composed of deep forest generally characterized by presence of “Okoumé” trees ; the Grassland with mountains and plateaus (i.e. between Coastline and Mountain Forest); the dry Savannah  and the wide “Lakeland”, a coastal and continental marine ecosystem (i.e. in West of Gabon).Gabon is localized in central Africa, crossed by the equator and covered by nearly 80% of rain forest. This country has a surface area of 267.667 kmThe survey covered 220 randomly selected villages (range from 10 to 41 villages per province) located in the different ecosystems. It focused on rural villages with fewer than 300 inhabitants located in the 9 administrative regions of Gabon. Besides, all the villages were geo-located. A multidisciplinary team including a doctor from the Gabonese Ministry of Health, a nurse, an epidemiologist, a virologist and laboratory technicians took place during nine field missions between June 2005 to September 2008. Healthy volunteers older than 15 years and who had been living in the village for more than one year were eligible for the study.Each volunteer participant answered to an anonymous questionnaire for subsequent epidemiological investigation on their geographical location, sociodemographic characteristics or lifestyle. Participants were also interviewed on their history of medical past. Only age and gender were collected for children. In addition, a free medical examination and basic medicine including clinical neurological examination were provided to all participants and non-participants. Blood smears for malaria diagnosis and field blood typing were also proposed.g) on a daily basis. Plasma and Buffy-coat were stored at -20°C until the end of field mission then, transferred on dry ice to the “Centre International de Recherches Medicales de Franceville” (CIRMF) and kept at -80°C until analysis.Blood samples were usually collected in the villages’ local healthcare centers. They were stored into 7 mL Vacutainer tubes containing EDTA . Tubes were transported to the field laboratory for centrifugation  were first used for HTLV serology. HTLVs positive or borderline-positive samples were tested by western blotting  to differentiate HTLV-1 and HTLV-2 status from HTLV indeterminate serological status.DNA was extracted from 200μl of Buffy-coat with the QIAamp DNA mini kit  according to the manufacturer’s recommendations. HTLV-1/2 proviral load was measured by a multiplex real-time PCR assay involving a molecular beacon probe for simultaneous detection, differentiation, and quantification of HTLV-1, -2, and -3 and STLV-1 and -3, as previously described . Samplesenv) sequence, as previously described [Polymerase chain reaction (PCR) amplification was performed with HTLV-1 or HTLV-2 specific primers located within the long terminal repeat (LTR) or the envelope glycoprotein (escribed –39. PCR http://tree.bio.ed.ac.uk/software/tracer/), and all estimated sample sizes were greater than 545 [env sequences (accession numbers JF270595 to JF270597) confirmed the results obtained with the LTR sequences (accession numbers JF270592 to JF270594).For phylogenic HTLV-1 analysis, LTR sequences were analyzed and aligned with MEGA package, the editor program . Trees wthan 545 . Phylogeenv gene of HTLV-1/-2 were assigned GenBank accession numbers JF270481 to JF270597.Nucleotide sequences obtained from the LTR and i.e. prevalence and proviral load) and the sociodemographic characteristics , a trend test across ordered groups was used. HTLV-2 statistical analysis could not be performed because of a low number of infected samples (n = 5). All statistical analyses were performed using STATA software version 10 . Statistically significance was assumed at p≤0.05.To describe our rural Gabonese population, we assessed crude HTLVs seroprevalence. In order to reflect the linear association between HTLV-1 infection  and risk factors  using Chi-square test (χ2) or Fisher’s exact test when appropriate. We performed two separate binary logistic regression models HTLV-1 vs. negative status or HTLV-1 vs. indeterminate status as outcome. In univariate analysis, strength association between serological status and risk factors was estimated as odds ratio (OR) with 95% confidence interval (95%CI).For the explanatory univariate analysis, we examined potential associations between HTLV-1 seroprevalence .For explanatory multivariate analysis, a multinomial logistic regression model was built using all variables significantly associated with serological status in both previous explanatory univariate analysis. In this model, the referent group was HTLV-1 compared to negative status 4,381 participants were enrolled throughout the whole country, representing 1.1% of the rural Gabonese population (n = 387 670 inhabitants). Blood samples and sociodemographic data were collected for all participants. Male/Female sex ratio was of 0.90 and mean age was 47±14 years old (range from 15 to 90 years old). There was no mean age significant difference in the different location. Participants were living in Lakeland , Savannah , Grassland , and Forest , subdividing into Mountain Forest ; Interior Forest  and North-East Forest . The investigated villages were randomly selected, covering 10.7% of all villages in Gabon , right.HTLV-1 and HTLV-2 prevalence was 7.3% (n = 320) and 0.1% (n = 5), respectively . HTLV-1 p<0.001). The odd ratio identified a strong risk for HTLV-1 acquisition when living in province of Haut-Ogooué , Ogooué-Ivindo  and Ogooué-Lolo  compared with the other Gabonese provinces.Thus, overall HTLV-1 seroprevalence was 7.3% among rural Gabonese population; ranging from 2.3% to 12.5% by province . HTLV-1 p<0.001) among women than among men (9.0% vs. 5.4%) , including 8.3% of HTLV Gag-indeterminate pattern with reactivity to p24 and p19 proteins and a lack of reactivity against gp21 and gp46. The highest frequency of HTLV indeterminate serological status was found in Haut-Ogooué, province where elevated HTLV-1 seroprevalence was also observed (11.6%). The odd ratio was especially higher in this province . The lowest HTLV indeterminate serological status was found in Ogooué-Maritime, province where HTLV-1 seroprevalence was also low (3.9%). HTLV-1 status was statistically higher (s. 5.4%) . HTLV-1 A multiplex real-time PCR assay involving a molecular beacon probe for simultaneous detection, differentiation, and quantification of HTLV-1, -2, and -3 and STLV-1 and -3 was used on samples showing HTLV indeterminate WB profile. Neither HTLV-1, nor HTLV-2 or HTLV-3 could be found in any of these HTLV WB indeterminate samples.3 ± 1.6x104 copies per 106 cells , with no statistical difference between province (p = 0.06). Although no significant correlation was found between proviral load and sex or age, it is still noteworthy that average magnitude of HTLV-1 proviral load was higher among the <35 years old , while hunting and consuming bush meat were not statistically significant risks.Consuming non-human primate food was a risk factor associated with the indeterminate serological status compared with the HTLV-1 serological status (p≤0.009). Fever, arthritis, abdominal pain and skin lesion were significantly more frequent among HTLV-1 infected people (p≤0.036). Excepted medical abortion and skin lesion, risk factors observed among indeterminate persons were identical to those of HTLV-1 infected people (p≤0.025).Hospitalization, surgery, transfusion and medical abortion represented risk factors for HTLV-1 acquisition . This finding is a well-known characteristic of HTLV-1 infection. Epidemiologic evidences suggest that sexual transmission of HTLV-1 is one of the predominant modes of transmission and occurred mainly from male to female. Sexual activity at an earlier age could be also a risk factor for HTLV-1 transmission. The highest prevalence in women might be due to an accumulation of sexual exposures with age [Consistent with other reports, prevalence of HTLV-1 is significantly higher in women than in men .p = 0.006) throughout this country.A previous report performed in Franceville described the fact that blood transfusion was a major risk for getting HTLV-1 in hospitals in Gabon . It is uCercopithecus nictitans [HTLV-1/2 infections with different HTLV subtypes, including rare ones  have beeictitans . These dp = 0.03), although 5 times lower than being hospitalized. Medical examination and basic medicine including clinical neurological examination in the field were provided to all participants of the study. No clinical manifestation related to HTLV-1 such as clinical neurological dysfunction could be detected. However, there is an urgent need for a large survey and clinical investigations to evaluate exactly public health consequences of HTLV-1 infection in this highly endemic area of the world.Recent reports have shown that, similar to foamy virus infection, being exposed to non-human primate and non-human primate food was a risk factor for HTLV-1 acquisition , 56. OurP. falciparum exposition as previously reported remains to be determined. Our multiplex real-time PCR primers allowed amplification of all PTLV-1/3 strains. Given that PTLV-4 is present in Gabon and because HTLV-4 carriers have an HTLV indeterminate western-blot pattern with very faint K55 and MTA-1 and GD21 envelope reactivity as well as a faint gag p24 reactivity [As previously reported with African sera/plasma samples, we observed a high number and various HTLV indeterminate patterns that were not HTLV-1/2 PCR positive –60. Whetactivity , an alteIn conclusion, our study demonstrates that HTLV-1 seroprevalence did not change 30 years after the first report, and that HTLV-1 subtype B is widely present in Gabon. We also showed that Gabon is one of the most highly endemic areas in the world for HTLV-I infection. Therefore, preventive measures to decrease spread and transmission of this human retrovirus are urgently warranted. These include: (1) systematic HTLV-I screening of blood donors ; (2) systematic screening of pregnant women in order to counsel them concerning the risk of HTLV-I transmission by prolonged breastfeeding ; and (3) prevention of sexual transmission of HTLV-I by educational programs emphasizing the importance of using condoms to prevent all sexually transmitted diseases, including HIV infection, which is still spreading in this central African country.Thus, it would be important to inform more efficiently the Gabonese population about this virus and its mode of transmission in order to allow a decreased transmission and a decreased prevalence in the future.S1 Checklist(DOC)Click here for additional data file.


Tumor necrosis factor receptor (TNFR) associated factor 1 (TRAF1) is a signaling adaptor first identified as part of the TNFR2 signaling complex. TRAF1 plays a key role in pro-survival signaling downstream of TNFR superfamily members such as TNFR2, LMP1, 4-1BB, and CD40. Recent studies have uncovered another role for TRAF1, independent of its role in TNFR superfamily signaling, in negatively regulating Toll-like receptor and Nod-like receptor signaling, through sequestering the linear ubiquitin assembly complex, LUBAC. TRAF1 has diverse roles in human disease. TRAF1 is overexpressed in many B cell related cancers and single nucleotide polymorphisms (SNPs) in TRAF1 have been linked to non-Hodgkin's lymphoma. Genome wide association studies have identified an association between SNPs in the 5′ untranslated region of the TRAF1 gene with increased incidence and severity of rheumatoid arthritis and other rheumatic diseases. The loss of TRAF1 from chronically stimulated CD8 T cells results in desensitization of the 4-1BB signaling pathway, thereby contributing to T cell exhaustion during chronic infection. These apparently opposing roles of TRAF1 as both a positive and negative regulator of immune signaling have led to some confusion in the literature. Here we review the role of TRAF1 as a positive and negative regulator in different signaling pathways. Then we discuss the role of TRAF1 in human disease, attempting to reconcile seemingly contradictory roles based on current knowledge of TRAF1 signaling and biology. We also discuss avenues for future research to further clarify the impact of TRAF1 in human disease. Tumor necrosis factor receptor (TNFR)-associated factors (TRAF) proteins play important roles in the immune system as key intracellular signaling molecules in TNFR, Toll-like receptor (TLR), cytokine, and antigen receptor signaling pathways . While TTRAF1 was originally identified along with TRAF2 in immunoprecipitates of TNFR2  and as sTraf1−/− mice are viable and fertile and have normal numbers of lymphocytes  pathways. For TNFRI, TRAF proteins are recruited indirectly through TRADD Figure , whereas2TRAF1 coiled coil heterotrimer, is more efficient in recruitment of cellular cIAPs than the TRAF2 homotrimer, and thus TRAF1 provides an NF-κB induced positive feedback loop to enhance TRAF2-dependent signaling (Traf1−/− dendritic cells (DC) show increased apoptosis and marked deficiency in classical NF-κB activation after CD40 stimulation, implicating TRAF1 in sustaining TRAF2-dependent signaling through CD40 , and the EBV-encoded TNFR family member latent membrane protein (LMP)-1, by enhancing classical NF-κB and MAPK activation , 23–29. ignaling . Traf1−/ugh CD40 . Similarugh CD40 . The absugh CD40 –13, 28.7, and thBeyond its demonstrated role in collaboration with TRAF2 in recruiting cIAPs, TRAF1 can prevent proteasome-dependent degradation of TRAF2 downstream of CD40, 4-1BB and TNFR2 signaling, thereby sustaining TRAF2 dependent signaling , 25, 26.TRAF1 and LUBAC can be co-immunoprecipitated with the TES domain of the EBV-encoded TNFR family member LMP1, when TRAF1, and the TES construct are overexpressed in HEK293 cells . ConsistThe protein kinase C related kinase, PKN1 binds to and phosphorylates TRAF1 on serine 146 in human and serine 139 in mouse, but does not phosphorylate TRAF2, 3 or 5 , despiteTraf1−/− T cells to anti-CD3 was later shown to be dependent on NIK and was associated with excessive cytokine production  to the active p52 form. In resting cells, NIK is constitutively degraded due to its ubiquitination by cIAP1 and/or 2. TRAF2 and TRAF3 play non-redundant roles in this process, with TRAF2 bringing in cIAPs to the complex, and TRAF3 bringing in NIK, thereby inducing NIK degradation and preventing constitutive NF-κB activation. Accordingly, mice lacking TRAF2 or 3 have constitutive non-canonical NF-κB signaling and die of lethal inflammation , 36. Micoduction . In T ceoduction . The roloduction .TNFR family members induce activation of the alternate NF-κB pathway by inducing degradation of TRAF2 or TRAF3, usually with delayed kinetics compared to the activation of the classical NF-κB pathway , 35, 36.Traf1−/− cells hyper-proliferated in response to anti-CD3 alone or in response to TNF and the response of the activated T cells to TNF was specifically blocked by antibodies to TNFR2, leading to the suggestions that TRAF1 is a negative regulator of TNF signaling , thereby preventing linear ubiquitination of NEMO, and thus limiting downstream NF-κB activation after TLR or NLR signaling  Figure . TRAF1 bIn another study, TRAF1 was identified in a yeast 2-hybrid screen that used the TLR signaling molecule TIR-domain-containing adapter-inducing interferon-β (TRIF) as bait. The TRAF-C domain of TRAF1 was found to bind to the TIR domain of TRIF. TRAF1 overexpression blocked TRIF-dependent NF-κB reporter activation in 293 cells, dependent on the caspase-sensitive cleavage site in TRAF1 . HoweverIn summary, TRAF1 contributes to signaling in the TNFR signaling pathway as part of a complex with TRAF2, where it can promote classical NF-κB activation through cIAP recruitment and possibly through stabilization of TRAF2. Later, TRAF1 may also contribute to induction of the alternate NF-κB pathway, again through cIAP recruitment. TRAF1 can also contribute to NF-κB activation independently of cIAPs downstream of LMP1 signaling, through becoming a substrate for linear ubiquitination, possibly contributing to recruitment of IKKγ/NEMO. Conversely, during TLR or NLR signaling, TRAF1 can sequester LUBAC to negatively regulate NF-κB activation. How these opposing roles of TRAF1 in different contexts impact human disease will be discussed in the remainder of the article.There is extensive evidence for altered expression of TRAF1 in lymphoid malignancies –46. ManyIn vitro, these B cells exhibit comparable proliferation rates to wild-type B cells but have markedly increased survival and resistance to apoptosis induced by dexamethasone and chemotherapeutic agents. The histopathologic features of these B cells are consistent with mouse small B cell lymphoma progressing to leukemia and exhibit many similarities to human chronic lymphocytic leukemia , TRAF1 can be found in other cancers besides lymphomas and CLL, including head and neck, melanoma, pancreatic, and thyroid cancers. In addition, as discussed below, recent evidence shows that human squamous cell carcinoma and non-small cell lung carcinomas can show overexpression of TRAF1.According to the Human protein atlas  in human patients -specific CD8nfection . As withnfection .It has been long established that latent membrane protein-1 (LMP1) is essential for Epstein-Barr virus (EBV)-mediated lymphocyte transformation , 91, 92.in vivo. TRAF1 interacts with LUBAC as both a substrate in the LMP1 signaling pathway and as an inhibitor in the TLR signaling pathway, with opposite effects on NF-κB activation. How the TRAF1-LUBAC interaction results in distinct outcomes in different signaling complexes will require a precise understanding of the protein-protein interactions involved. Moreover, the fact that TRAF1 can be M1-ubiqutinated by LUBAC and phosphorylated by PKN1 suggest that post-translational modifications will be important in this regulation and need further study.TRAF1 has diverse roles in human health and disease. TRAF1 contributes to control of chronic viral infection and can limit inflammation. This suggests that enhancing TRAF1 expression could be beneficial for both chronic infection and inflammatory diseases. Blocking TGFβ or stimulating IL-7 offer possible interventions for achieving higher TRAF1 in chronically stimulated T cells. Conversely, TRAF1 is dysregulated in cancer, where it likely contributes to a positive feedback loop that perpetuates NF-κB signaling and survival of cancers of mature B cells. TRAF1 also contributes to survival of EBV-dependent cancers through enhancing LMP1- mediated survival signaling. Interfering with TRAF1 in this process could break the cycle of NF-κB activation in these cancers. Human variations in TRAF1 correlate with increased incidence of rheumatic disease, increased mortality from sepsis in RA patients, and increased incidence of NHL. The role of TRAF1 as both a positive and negative regulator of immune responses can be attributed to its participation in diverse signaling pathways. TRAF1 is important in TNFR superfamily signaling as a complex with TRAF2 and in TLR/NLR signaling independently of TRAF2. What happens in a monocyte responding to both a TLR and a TNF signal? Does TRAF1 limit one and enhance the other simultaneously, and/or are there separate pools of TRAF1 in the cell that engage in these different functions? More work is required to understand how the diverse roles of TRAF1 play out in complex biological systems ME and TW wrote the manuscript with editorial input from AA-S. ME prepared the figures.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.


The Hedgehog (Hh) signaling pathway is crucial for vertebrate embryonic development, tissue homeostasis and regeneration. Hh signaling is upregulated in basal cell carcinoma and medulloblastoma and Hh pathway inhibitors targeting the Smoothened (SMO) protein are in clinical use. However, the signaling cascade is incompletely understood and novel druggable proteins in the pathway are in high demand. We describe the discovery of the Hh‐pathway modulator Pipinib by means of cell‐based screening. Target identification and validation revealed that Pipinib selectively inhibits phosphatidylinositol 4‐kinase IIIβ (PI4KB) and suppresses GLI‐mediated transcription and Hh target gene expression by impairing SMO translocation to the cilium. Therefore, inhibition of PI4KB and, consequently, reduction in phosphatidyl‐4‐phosphate levels may be considered an alternative approach to inhibit SMO function and thus, Hedgehog signaling. Cell‐based screening identifies a thienopyrimidine derivative as an inhibitor of Hedgehog signaling by selective inhibition of phosphatidylinositol 4‐kinase IIIβ (PI4KB), reduction of PI4P levels and prevention of Hedgehog‐mediated ciliary localization of Smoothened. Thus, PI4KB may be a relevant target protein for inhibition of Hedgehog signaling. Gli1, Ptch1 and Ptch2.The Hedgehog (Hh) signaling pathway is essential during vertebrate embryonic development as well as for tissue homeostasis and regeneration in adults. Crucial events during pathway activation include binding of the Hh ligand to the Patched (PTC) receptor, transduction of the signal to the seven‐pass transmembrane protein Smoothened (SMO) and subsequent relocalization of SMO from intracellular vesicles to the cilium.Hh signaling is upregulated in cancers of the skin We monitored Hh‐induced osteogenesis as a cell‐based screening for inhibitors of the Hh pathway. Subsequent target deconvolution and validation led to the discovery of the Hh pathway inhibitor Pipinib, which selectively targets PI4KB over the other three PI4‐kinases. Inhibition of PI4KB by Pipinib reduces intracellular PI4P levels, in particular at the Golgi apparatus, and impairs translocation of SMO to the cilium as well as subsequent pathway activation. Our results indicate that PI4KB may be a relevant target protein for pharmacological inhibition of Hh signaling.1  or cyclic substituents  and introduction of heteroatoms  or of tertiary amines (Table 9–11) leads to reduction of bioactivity. In two cases, moderate cytotoxicity was observed (Table 5 and 11). Replacement of the methyl ester in R2 by a nitrile group (Table 12–14) and wider variation of the substitution pattern of the aromatic ring (Table S1) also yielded compounds with lower activity. Introduction of a substituent into the thiophene ring was not beneficial either (Table S1).In order to identify novel inhibitors of Hh signaling, 336,639 compounds were subjected to an osteoblast differentiation assay that monitors differentiation of multipotent C3H10T1/2 mesenchymal progenitor cells to osteoblasts upon activation of the Hh pathway by treatment with the SMO agonist Purmorphamine.1  to which the full‐length protein is proteolytically converted upon pathway inactivation  revealed eight potential targets (Table S3), of which phosphatidylinositol 4‐kinase IIIβ (PI4KB) showed the highest inhibition (76±2 %). Pipinib did not inhibit the enzymatic activity of Syk and PRKD1 and PRKD2 (Table S2). Of the potential targets, GRK7 was excluded because mouse cells were used in the osteoblast differentiation assay and mouse orthologs of GRK7 do not exist  but not the isoenzymes PI4KA (phosphatidylinositol 4‐kinase IIIα), PI4K2A (phosphatidylinositol 4‐kinase IIα) or PI4K2B (phosphatidylinositol 4‐kinase IIβ) up to a concentration of 50 μm . Kinetic measurements and analysis demonstrated that Pipinib is an ATP‐competitive inhibitor of PI4KB activity  identified PI4KB as the only target protein of Pipinib  and GT‐CFP led to reduction of the Golgi intensity of YFP‐PH(FAPP1) within 10 min . Using the CRISPR‐Cas9 system, we attempted to disrupt the Pi4kb gene. Unfortunately, Pi4kb knockout cells could not be generated, most likely because PI4KB is an essential gene.Ptch1 and Gli1 after pathway stimulation with Purmorphamine  at the same concentration reduced gene expression only by 44 and 17 %  was proposed as small‐molecule mediator of signal propagation from PTC to SMO. We demonstrate that phosphatidylinositol 4‐kinase IIIβ (PI4KB) generates PI4P that is relevant for Hh signaling. The mechanism by which the Hh signal is transduced from PTC to SMO and by which PTC controls SMO in mammalian cells has been described as “the major mystery of the Hh field”.Drosophila, loss‐of‐function mutations have shown that the lipid kinase SST4, the ortholog of mammalian PI4KA, is necessary for SMO translocation to the membrane upon Hh pathway activation.In Our results demonstrate the relevance of PI4KB for Hh signaling as previously indicated by genetic evidence.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.


Human T-lymphotropic virus type 1 (HTLV-1) is the etiologic agent of both adult T-cell leukemia/lymphoma and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HAM/TSP is clinically characterized by chronic progressive spastic paraparesis, urinary incontinence, and mild sensory disturbance. Given its well-characterized clinical presentation and pathophysiology, which is similar to the progressive forms of multiple sclerosis (MS), HAM/TSP is an ideal system to better understand other neuroimmunological disorders such as MS. Since the discovery of HAM/TSP, large numbers of clinical, virological, molecular, and immunological studies have been published. The host-virus interaction and host immune response play an important role for the development with HAM/TSP. HTLV-1-infected circulating T-cells invade the central nervous system (CNS) and cause an immunopathogenic response against virus and possibly components of the CNS. Neural damage and subsequent degeneration can cause severe disability in patients with HAM/TSP. Little progress has been made in the discovery of objective biomarkers for grading stages and predicting progression of disease and the development of molecular targeted therapy based on the underlying pathological mechanisms. We review the recent understanding of immunopathological mechanism of HAM/TSP and discuss the unmet need for research on this disease. Human T-lymphotropic virus type 1 (HTLV-1) is the first human retrovirus discovered and infects 10–20 million people worldwide . Highly Deltaretrovirus genus of the Orthoretrovirinae subfamily of retroviruses. HTLV-1 integrates a single copy of the provirus into the genome of the host cell   after culture without any exogenous stimulation (tax mRNA was significantly higher in HAM/TSP patients than in ACs (+ T-cells (CTLs)  . The virpathways . Tax is pathways . Ex vivomulation , and then in ACs . Tax is s (CTLs) . The nums (CTLs)  and shows (CTLs) . Though ll level . HBZ plall level . As HBZ ll level . HBZ is ll level , and proll level . HBZ intll level . The levpatients . HBZ is patients  and HBZ-patients . Antibodpatients . The lowHTLV-1 proviral load, which is strongly related to the risk of developing HAM/TSP, remains relatively stable within each subject while HTLV-1 drives a strong proliferation of infected T-cells . The genOne of the hot topics in HTLV-1 is the high prevalence in Central Australia, where more than 40% of Indigenous adults in some remote communities are HTLV-1c infected . HTLV-1 + T-cells  is the main risk factor for developing HAM/TSP in HTLV-1 infected subjects. The HTLV-1 PVL is measured as the number of HTLV-1 DNA copies per PBMCs and is usually expressed as the percentage of infected PBMCs. The PVL is 16-fold higher in HAM/TSP patients than in carriers and associated with an increased risk of progression to disease . This hi T-cells , which a T-cells . Further T-cells  and corr T-cells . The PVL T-cells , which i T-cells . Althoug T-cells , genetic+ T-cell response and consequently has a large impact on the proviral load and the risk of developing HAM/TSP. Possession of the HLA-class I gene HLA-A*02 and Cw*08 was associated with a significant reduction in both HTLV-1 PVL and the risk of HAM/TSP  and non-HLA gene polymorphisms affect the occurrence of HAM/TSP . The HLA HAM/TSP , while i HAM/TSP , 2000. A HAM/TSP  and the  HAM/TSP  were ass HAM/TSP , stromal HAM/TSP  were rel HAM/TSP . IL28B g HAM/TSP . Familia HAM/TSP , 2017. CHBZ mRNA expression (HTLV-1 is segregated into seven major genetic subtypes (1a–1g) based on the nucleotide diversity of its LTR region, including a cosmopolitan subtype (subtype 1a), a Melanesian/Australian subtype (subtype 1c), and five African subtypes  . The cospression  and highpression . Differepression , which a+ and CD8+ cells were evenly distributed in active inflammatory lesions, while the predominance of CD8+ cells and high levels of interferon (IFN)-γ were observed in the chronic stage of disease   , and HTLem (CNS) . HTLV-1  response has been considered to play an important role in the development of HAM/TSP. One of the most prominent features of the cellular immune response in HAM/TSP patients is that the number of HTLV-1-specific CTLs is greatly elevated in PBMCs compared with ACs 22), which is produced by Tax (+CD25+CCR4+ T-cells was increased and correlated with disease activity and severity (HTLV-1 preferentially and continuously infects CD4 in vivo . CD4+CD2phocytes . In HAM/ng INF-γ . HTLV-1- T-cells . In HAM/controls . Furthery donors . HTLV-1 d by Tax . In HAM/severity . Anti-CCseverity . Clinicaseverity .Robust humoral immune responses against HTLV-1 antigens have been reported , and it + T-cells, are deregulated in HAM/TSP and contribute to a variety of immunological abnormalities characteristic of this disorder (+ T-cells expressed high IL-15Rα (in vitro immune activation in PBMCs from HAM/TSP patients (Elevated cytokine expression and production have been demonstrated in the peripheral blood and CNS lesion of patients with HAM/TSP . Inflammdisorder . HTLV-1  IL-15Rα  and indu IL-15Rα . Elevatepatients . Monoclopatients , and a cpatients . A selecpatients . The incin vitro (Exosomes are unilamellar extracellular vesicles originated from the endosomal compartment in various cell types . Severalin vitro . Tax appin vitro , which win vitro ,b. Recenin vitro . Exosomain vitro . HTLV-1 HAM/TSP is clinically characterized by chronic progressive spastic paraparesis, urinary incontinence and sensory disturbance. The mean age at onset is 43.8 years, and like many other autoimmune diseases, the frequency of cases of HAM/TSP is greater in women than in men . Weaknes+ T-cell frequency, and CSF PVL in HAM/TSP patients (The diagnosis of HAM/TSP is based upon a combination of characteristic clinical features and confirmation of HTLV-1 infection, along with exclusion of other disorders presenting spastic paraparesis . The diapatients . ReliablMost therapeutic trials in HAM/TSP have aimed to suppress or modulate the immune response, or to reduce the HTLV-1 proviral load in an attempt to decrease the risk or modify the course of the disease. Corticosteroids are most commonly used to reduce the inflammation in the spinal cord, particularly in the early stage. Motor disability might be improved with steroids , but impRecent advances in research on HTLV-1 provide better understanding of the molecular pathogenesis and mechanisms of HAM/TSP, and several clinical trials of novel therapies for patients with HAM/TSP have been initiated. However, long-term improvement of motor disability and quality of life still have not been achieved in HAM/TSP patients, and the clinical management remains challenging. Given that HAM/TSP is characterized by activated T-cells in both the periphery and CNS, studies in HAM/TSP will be highly informative for clarifying the pathogenesis of other neuroinflammatory disorders such as multiple sclerosis. Novel approaches will be required to better define host-virus interactions and host immune response underlying the pathogenesis of HAM/TSP.SN wrote the manuscript, and SJ supervised and contributed to the discussion and writing.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.


Salvador, Bahia (northeastern Brazil), has been identified as the epicenter of Human T-cell leukemia virus Human T-cell leukemia virus (HTLV) type 1 infection in the country. This study aims to estimate the rate of HTLV infection and the geographical distribution of this virus in this state.All HTLV tests (chemiluminescence/ELISA assays/Western Blotting) performed in the Central Laboratory of Public Health of Bahia (LACEN) from 2004 to 2013 were included. Data was extracted from LACEN’s database using high volume extract, transformation and load throughput. Infection rate was expressed as the number of infected individuals per 100,000 inhabitants considering municipalities grouped in microregions and/or mesoregions as the unit of analysis.A total of 233,876 individuals were evaluated. Individuals were from 394 out of 417 municipalities of Bahia (94.5%). HTLV chemiluminescence/ELISA assay was found to be reactive for 3,138 individuals from whom 2,323 had WB results . Out of 1978 reactive samples, 1,813 (91.7%) were positive for HTLV-1, 58 (2.9%) for HTLV-2 and 107 (5.4%) were for both HTLV-1 and HTLV-2. The cumulative mean rate of HTLV-positive cases in Bahia was 14.4 per 100,000 inhabitants. Three microregions presented rates >20 HTLV-positive cases/100,000 inhabitants: Barreiras , Salvador , and Ilhéus-Itabuna .HTLV infection is disseminated in the state of Bahia, with an overall moderate rate of infection. Further studies should be conducted to characterize the epidemiological and clinical profile of HTLV-infected individuals better and to propose effective prevention measures. Strongyloides stercoralis (Human T-cell leukemia virus (HTLV) type 1 was the first human oncogenic retrovirus to be identified in 1979 . Subsequrcoralis  and Norwrcoralis . The trarcoralis , as wellrcoralis . Recentlrcoralis .It is estimated that at least 5–10 million people are infected with HTLV-1 worldwide, with the highest prevalence occurring in Japan, on the African continent, in the Caribbean, the Melanesian Islands and South America . HTLV-2 Brazil is home to around 800,000 HTLV-1-infected people and represents one of the largest endemic areas for the diseases associated with HTLV-1 . The virBahia is the largest state in the Brazilian Northeast, with a total of 417 municipalities. Sparse studies have indicated that HTLV-1 infection may be present in some regions of the state. However, these studies have investigated specific population groups, such as pregnant women, blood donors or drug users . Large-sThe Institutional Review Board (IRB) for Human Research at the Gonçalo Moniz Institute of the Oswaldo Cruz Foundation  provided ethical approval to conduct this study (CAAE number 22478813.7.0000.0040).2. The state is comprised of 417 municipalities, which have been grouped into 32 microregions and seven mesoregions by the Brazilian National Institute of Geography and Statistics (IBGE) in accordance with the economic and social similarities among them (2 (http://www.ibge.gov.br).This study was performed in the state of Bahia, Brazil, the fourth largest state in terms of population size, and the fifth largest in terms of area: 565,733 kmong them . AccordiA retrospective ecological study was conducted using data obtained from the Central Laboratory of Public Health of Bahia (LACEN-BA), which is responsible for infectious disease surveillance throughout the state via laboratory analysis. The target population was comprised mainly of blood donors, pregnant women and individuals exhibiting symptoms of infectious disease, referred by blood banks, prenatal physicians or clinicians in the public health system.®, Singapore) was used throughout the study period for confirmation. HTLV-negative samples were defined as lack of reactivity to HTLV-specific proteins; HTLV-1-positive samples were defined as reactive to GAG (p19 with or without p24) and two ENV (GD21 and rgp46-I) proteins; HTLV-2-positive samples were those reactive to GAG (p24 with or without p19) and two ENV (GD21 and rgp46-II) proteins; HTLV seropositive samples were defined as samples reactive to GAG (p19 and p24) and ENV (GD21). The samples that indicated co-infection of HTLV -1/2 demonstrated reactivity for specific markers of both HTLV-I and HTLV-II: GD21, p19, p24, rgp46-II and rgp46-I. Samples were considered indeterminate when no specific bands for HTLV-I, HTLV-II, or HTLV were detected. All serological tests for HTLV that lacked confirmatory results were excluded.All serological tests for HTLV were included among the 32 Bahia microregions from 2004 to 2013. From 2004 to 2008, testing was carried out by ELISA using the Murex HTLV-1/2 immunoassay , from 2009 to 2010 using the anti-HTLV-1/2 Sym Solution kit  and, from 2011, the CLIA chemiluminescence assay  was used. When HTLV seropositivity was detected, Western blotting  intervals were calculated for the age variable, and individuals were grouped accordingly. Absolute and relative frequencies were calculated for all categorical variables . Geographic information system techniques and spatial analysis tools were employed to determine the geographical distribution of HTLV throughout the state of Bahia. Population data were acquired from IBGE, supported by national census data from 2010 and official estimates for all other years2. Spatial distribution maps based on moving averages were constructed by applying 3-year intervals to data from 2004 to 2013. This method was also used to minimize the effects of random fluctuation for a time-series of infection rates calculated for rare events. Annual incidence rates were calculated for each of the 32 micro-regions and the entire state using standardized methods. To assess the relative risk of HTLV in each microregion, maps were constructed using the moving average for the first 3-year rate in Bahia as the denominator assuming no changes in time and space.Infection rate was expressed as the number of infected individuals per 100,000 inhabitants. Microregions and/or mesoregions were adopted as the unit of analysis in order to obtain improved accuracy concerning differences among regions. Digital maps were obtained from the IBGE cartographic database in shapefile (.shp), which were subsequently reformatted and analyzed using TerraView version 4.2, open source software freely available from the National Institute for Space ResearchA total of 249,869 serologies for HTLV were performed during the studied period. Due to multiplicity , 15,993 samples were excluded . The finOf the positive samples, 1,813 (91.7%) were positive for HTLV-1 , 58 (2.9%) for HTLV-2  and 107 (5.4%) were positive for both HTLV-1 and HTLV-2 . A total of 394 (94.5%) of the 417 municipalities in the state reported at least one positive result during the study period. Information regarding the municipality of sample origin was missing for 1.4% of the HTLV-positive samples. The cumulative mean rate of HTLV-positive cases in Bahia was 14.4 per 100,000 inhabitants. Considering the grouping of HTLV-positive samples into microregions , three dThe microregions of Barreiras, Salvador and Porto Seguro were all notable for high rates of HTLV-1 and HTLV-2 infection, as well as HTLV-1/2 coinfection. HTLV-1 was also predominant in Ihéus-Itabuna and Itapetinga microregions. Higher rates of HTLV-2 infection were also found in the Livramento do Brumado and Juazeiro microregions. HTLV-1/2 coinfection was most prevalent in the Entre Rios and Valença microregions .The median age of the studied population was 31 years (interquartile range: 25–39 years) and the female:male ratio was 8:1. The median age of HTLV-infected persons was 46 years (IQR: 33–59 years), and 75% were women. Overall, our analysis of HTLV-positivity according to age and sex showed much higher frequencies in individuals aged 31 years or older for both males (85.7%) and females (79.3%) . Our strIn two mesoregions, Extremo Oeste Baiano and Nordeste Baiano, higher frequencies of positive cases were observed among females aged 15–30 years as compared to males. Conversely, a higher frequency of positive cases among males aged >30 years was found in the Vale do São Francisco da Bahia mesoregion.Salvador, the capital of the Brazilian state of Bahia, has been considered the epicenter of HTLV-1 infection in Brazil. A population-based study conducted in 2003 estimated a total of 40,000 infected individuals in this city . ScatterNotably, our results identified one important cluster of HTLV infection comprising four microregions surrounding the Salvador microregion . This cluster consists of 43 cities and concentrates almost a third of the state’s population. Geographically, the municipalities in this cluster are mainly located in the area surrounding the Baía de Todos os Santos, which is considered to have a subtropical climate characterized by Atlantic Forest. The population is largely of similar ethnic origin (mainly of African descent) with uniform socialdemographic characteristics. As expected, the Salvador microregion which includes 10 municipalities, presented a high overall rate of HTLV-infection . This finding confirms previous reports indicating that Salvador is highly affected by this virus . Cruz daUnexpectedly, the present results also identified three other endemic clusters: one located in the southernmost , one in the central region  and another in the westernmost region of the state . Scarce studies identified the presence of HTLV-1 infection in some cities located in these regions: a recent study evaluating pregnant women in Ilhéus and Itabuna, cities in the southern region of Bahia, found 1.05% were infected with HTLV-1 , while aWhile higher rates of HTLV-1/2 co-infection were mainly present in some microregions identified as HTLV-1 clusters, including Salvador, Barreiras and Porto Seguro, the highest rates of HTLV-2 were present in Livramento do Brumado microregion, located in the center of the state, and Juazeiro, located in the north. In Brazil, few studies have reported on the prevalence of HTLV-1/2 coinfection . HTLV-2 Of note, a progressive increase in the incidence of HTLV infection was observed throughout the study period. This is likely due to an expansion in the number of municipal primary health clinics provided by the federal Family Health Program, as well as increases in testing and counseling centers  in the countryside of the state. In addition, awareness surrounding HTLV infection increased during the study period and HTLV was included on the list of compulsory disease notifications for the state of Bahia in 2011.With respect to HTLV infected individuals profile, the mean age was 46 years with a predominance of female (75%). HTLV infection is known to be more prevalent in women, and its prevalence increases with age . Herein,By contrast, in the Vale do São Francisco and Centro Norte Baiano mesoregions, men were found to be predominantly infected by HTLV in the age range of 31–50 years. Both mesoregions are highly productive agricultural zones containing municipalities of high population density. In addition, the Centro Norte Baiano mesoregion attracts young men to work in the mining industry. Accordingly, it is theoretically possible that the higher prevalence observed in men of this age range could be due to increased exposure to illicit drugs, as well as to an increased incidence of STDs, such as HIV, gonorrhea and syphilis. In addition, lower levels of formal education and income have been attributed to a higher risk of acquiring HTLV infection . Moreove3. A diffuse distribution is observed throughout the microregions, especially with respect to white/mixed-race and black, which corresponds to the distribution of HTLV-1 cases, while native Brazilians are more concentrated in the Porto Seguro microregion, which also had a notable rate of HTLV-2 infection. However, the information available prevents us from making direct correlations between ethnic profile and the distribution of HTLV types. Interestingly, the data herein indicate that the microregions with the highest rates of infection in the state of Bahia were areas where communities recognized as quilombos predominated. Originally, quilombos were places of refuge for the African and Afro-descendant slaves who fled slavery. Their descendants often remained in these areas and were then termed quilombolas. Bahia is one of the states with the highest number of quilombolas recognized4, Brazil is considered an endemic area for HTLV . HoweverOne limitation of the present study was the non-random sample, which was predominantly female. However, our analysis considered samples from 94% (394/417) of the municipalities in Bahia, a state with an estimated population of 15,126,371 inhabitants; i.e., just 5.5% of the municipalities  did not send any samples to LACEN during the study period. This was especially relevant in two microregions (Santa Maria da Vitória and Jeremoabo), from which no information regarding HTLV testing was obtained. To explain this, we can only speculate that perhaps local health authorities chose to outsource testing to private laboratory services instead of sending samples to LACEN, or that authorities in these municipalities did not properly implement actions designed to increase HTLV detection. In fact, the municipality of Jeremoabo has, to date, not reported a single case of HTLV to the Information System on Diseases of Compulsory Declaration (Sistema de Informação de Agravos de Notificação—Sinan), while Santa Maria da Vitória reported a single case in 2013 and another in 2018 (data not shown). Accordingly, we surmise that health professionals in these areas faced challenges in identifying patients suspected of HTLV infection, and/or did not recognize HTLV infection as a sexually transmitted infection, and therefore did not request appropriate serological testing. While population-based studies are considered the gold standard for estimating infection prevalence, high cost and methodological difficulties can present significant challenges . AccordiIn conclusion, the present study serves to highlight previously unreported data regarding clusters of HTLV infection throughout the state of Bahia, which is considered to be the Brazilian state with the highest HTLV infection rate. HTLV infection cannot be considered as restricted to the area of Salvador, the capital of Bahia, as the data reported herein identified new endemic areas. Considering the overall prevalence of HTLV in Bahia to be 0.84% , it follows that ∼130,000 individuals would be infected with HTLV. However, it is important to note that this number could be underestimated, since 26% of the HTLV reactive samples were not submitted to Western blotting, and were thusly excluded. Had 85.2% of these samples been confirmed, and subsequently included in our analysis, the overall prevalence would increase to 1.18%. Further studies are needed to better describe the epidemiological profile of the infected population and to reinforce public policies designed to prevent HTLV transmission, especially in pregnant women.As the present study has a retrospective design, evaluating HTLV serologies performed in a 10 years-period in the Public Health Laboratory of Bahia, the Institutional Research Board of Fiocruz approved a study, dispensing the signing of written informed consent.All authors read and approved the final version of the manuscript, analyzed and interpreted the data, and drafted the manuscript. FP, MC, MG, and BC conceived and designed the study. FP, MC, CR, RC, and MG acquired the data. FP, MG, FS, and BC critically revised the manuscript.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.


Punica granatum L.) flower.Ursolic acid (UA) is an important bioactive component in many traditional medicinal plants including pomegranate . Lower quantities of UA were determined in pomegranate fallen flowers (16.763 to 5.754 mg/g).UA values obtained from Iranian cultivars of pomegranate flowers are very significant when compared with other sources of UA. All of the analyzes suggested that the Iranian pomegranate flowers  might be an excellent UA rich source. Ursolic acid  , commonly known as pomegranate, is a small tree, extensively cultivated in the Middle East, North Africa, the Mediterranean and in parts of Asia  (RThe inter-day and intra-day variations for the determination of UA were less than 3% at concentrations of 200, 300 and 400 µg/mL (Table By mixing a suitable amount of quantified samples with the standard compound, recovery experiment was conducted in order to confirm that the method was accurate, and the average recovery of UA was 100%.In this study, the investigation of UA contents of pomegranate flowers from ten cultivars grown in Iran was performed. Among the ten pomegranate flower cultivars researched, eight cultivars including ‘Shirin Bihasteh Mehriz’, ‘Malas Daneh Zard Yazd’, ‘Shirin Siyah Saveh’, ‘Alack Zood Ras’, ‘Golnar’, ‘Zagh Sefid Yazd’, ‘Shirin Shahbar’ and ‘Ghorj Tafti Torsh are characterized by the highest content of UA and can be used as a rich source of UA for the synthesis of a wide variety of bioactive compounds such as food supplements and health products. However the results of this study showed that the UA content of pomegranate flowers is significantly reduced after falling from trees, but due to the high content of UA in pomegranate flowers and considering the fact that a large number of flowers of pomegranate trees fall and are discarded by farmers, the results suggested that the pomegranate fallen flowers are valued as a source of UA.


Introduction: Type 2 diabetes mellitus (T2DM) is a chronic inflammatory disease associated with increased cancer risk. PAK signaling is implicated in cellular homeostasis when regulated, and cancer when unrestrained. Recent reports provided a role for PAK signaling in glucose homeostasis, but the role of PAKs in the pathogenesis of T2DM is unknown. Here, we performed a mini-meta-analysis to explore if anti-diabetic drugs modify PAK signaling pathways, and provide insight regarding modulation of these pathways, to potentially reduce diabetes-associated cancer risk. Methods: PAK interacting partners in T2DM were identified using the online STRING database. Correlation studies were performed via systematic literature review to understand the effect of anti-diabetic drugs on PAK signaling. A mini-meta-analysis correlated multiple clinical studies and revealed the overall clinical response rate and percentage of adverse events in piogliazone (n = 53) and metformin (n = 91) treated patients with PAK-associated diseases. Results: A total of 30 PAK interacting partners were identified , which were highly associated with Wnt, and G-protein signaling. The anti-diabetic drug metformin activated signaling pathways upstream; whereas pioglitazone inhibited pathways downstream of PAK. Overall, clinical response upon pioglitazone treatment was 53%. Seventy-nine percent of pioglitazone and 75% of metformin treated patients had adverse events. Pioglitazone reduced molecular-PAK biomarkers of proliferation (Ki67 and CyclinD1), and metformin had the opposite effect. Conclusions: PAK signaling in T2DM likely involves Wnt and G-protein signaling, which may be altered by the anti-diabetic drugs metformin and pioglitazone. Apart from the therapeutic limitations of adverse events, pioglitazone may be promising in chemoprevention. However long-term multi-centered studies, which initiate pioglitazone treatment early will be required to fully assess the full potential of these drugs.Hypothesis: Anti-diabetic drugs modulate p-21 activated kinase (PAK) signaling.  Type 2-diabetes mellitus (T2DM) is a global epidemic that significantly reduces the quality of life of geriatric patients, especially in Western society. The etiology of T2DM is intimately linked to obesity, genetics, and a sedentary lifestyle. Disease manifestations, including blindness and neuropathy, are extremely burdensome, and long-term complications such as cardiovascular disease and renal failure, ultimately result in death .Although the pathogenesis of T2DM is multifactorial and complex, its current understanding encompasses hepatic insulin resistance, dysfunctional insulin signaling, abnormal glucose metabolism, and persistent hyperglycemia . In addiAnti-diabetic drugs such as biguanides, sulfonylureas, and glitazones have provided the current basis of understanding in the clinical management of T2DM. However, little is known regarding whether these drugs are also effective in reducing the associated cancer risk. Effective chemoprevention in T2DM will target processes involved in both glucose metabolism and carcinogenesis.Molecular targets with emerging roles in both cancer and diabetes include a family of six different kinases, the p-21 activated kinases (PAKs) . Here, whttps://www.ncbi.nlm.nih.gov/pubmed) was performed to establish upstream and downstream PAK targets. Key words included PAK signaling or p-21 activated kinases in addition to the following targets: AMPK, RAS, mTOR, PI3K/AKT, RAC1, CDC42, MAPK, p38, JNK, NF-κB, PPARγ, ROS, VEGF, and Wnt/Beta-catenin. Similar studies were screened for PAK signaling pathways involving known targets (above) of glucose homeostasis, inflammation, proliferation, survival, and angiogenesis. Both upstream and downstream PAK targets were identified in each of these pathways, and the effect of PAK signaling targets on inflammation, proliferation, survival, and angiogenesis was evaluated. Studies in the literature involving anti-diabetics drugs  were further analyzed for their effect on PAK signaling pathways  on targets upstream and downstream of PAKs.A systematic literature review and molecular analysis of pre-clinical studies using online library Pubmed , Beta-cell dysfunction (10), and obesity and insulin resistance (10) . Signalip = 0.01) and . Met altered more pathways upstream of PAK  and  , glyburide (gly), and glitazones (glit) on PAK signaling pathways . Overall = 0.11) B.Malignant PAK signaling is involved in disease pathogenesis. To further understand the role in which met, gly, and glit interfere with PAK signaling in T2DM, we asked if any of these drugs activate or inhibit inflammatory, proliferative, survival, or angiogenic pathways upstream and downstream of PAK C–F. DiffPrevious clinical trials have attempted to establish whether met and pioglitazone (pio) therapeutically have chemopreventive activity in humans, but the results remain highly ambiguous. We showed that met and pio altered the pathways both upstream and downstream of PAK, and asked whether the chemopreventive nature of these drugs was found in diseases with PAK overexpression. To investigate this, we analyzed clinical studies in diseases known to overexpress PAK in which met or pio treatment was tested .Initially, we screened hundreds of studies for diseases with PAK overexpression and either met or pio treatment. We found one observational-prospective cohort and six interventional studies (three single arm and three randomized double blind) with these criteria A.p > 0.05) and other  adverse events upon treatment with pio , which was defined as the total number of patients who had ≥50% decrease in the sum of all their lesions post-treatment. The OCRR in pio-treated patients was 53% B; 28/53 with pio B. Additiwith pio B. PiogliWe correlated how patients which had diseases overexpressing PAK responded to pio, however it was unclear whether pio or met actually interfered with PAK signaling. We therefore analyzed biomarkers downstream of the PAK signaling pathway involved in apoptosis, cell cycle, PI3K/mTOR, and PPARγ .p = 0.042; independent samples t-test).We calculated the percent change in biomarker expression in pio and met treated patients to visualize the molecular effect on PAK signaling . MoleculType 2 diabetes is a major cause of disability and death in the elderly worldwide, and in addition to its multiple disease specific complications, it also carries an increased cancer risk, such as colon cancer . PhysiolPAK overexpression drives malignant diseases such as cancer. However, in diabetes a different expression pattern has been observed by Ahn et al., who reported that diabetic stressors reduce PAK1 expression in islet cells, and this decline in total protein levels may contribute to beta cell dysfunction in diabetes . Here, wThe initial pathogenesis of T2DM involves beta cell expansion to compensate for hyperglycemia, which eventually may lead to reduced beta cell mass . Here, PWe sought to illuminate the role of PAK signaling in T2DM by analyzing the effect that three well-known classes of anti-diabetic drugs had on PAK signaling . BiguaniConsidering the role of PAK signaling in the initiation of disease , and thaWe asked whether PAK signaling was even affected in these patients by pioglitazone or metformin at the molecular level , and fouConsidering our clinical and molecular analysis of PAK biomarkers in pioglitazone treated patients, future studies should investigate the long term side effects associated with its treatment and whether PAK signaling in disease can be impeded early on, to block malignant transformation . AlthougThis was the first study to provide a mechanistic explanation of how anti-diabetics may target PAK signaling for their potential use for chemoprevention in patients with T2DM, albeit our signaling pathway analysis included multiple pre-clinical studies, which were not of high significance. Ideally, a thorough signaling pathway analysis should use PAK1 and p-PAK1 as biomarkers in multiple human studies, however this data was unavailable for analysis due to the limitations of the current literature in regard to our highly specific question. Considering the few patients analyzed here, the accuracy of this data is supported by its correlation between literature reports  and our Although not associated with diabetes, other studies of chemoprevention have shown that anti-inflammatory drugs like aspirin in colorectal cancer and mesalamine in colitis associated cancer reduce cancer-associated risk ,87. MesaAnti-diabetics like pioglitazone or metformin should be utilized as a platform for further understanding the role of PAKs as a chemopreventive target in diabetes. However, before this is possible, future studies must standardize doses specific to the associated disease in order to modulate PAK signaling appropriately and minimize adverse effects. Ideal chemoprevention, like mesalamine, will block inflammation and impede aberrant PAK signaling without altering cellular homeostasis.


Human T cell leukemia virus type-1 (HTLV-1) stimulates inflammation activity. Our previous study revealed a positive association between asymptomatic HTLV-1 infection and advanced periodontitis among elderly Japanese individuals with low levels of hematopoietic activity (reflected by reticulocyte levels). Since low hematopoietic activity has been correlated with low-grade inflammation and low-grade inflammation is associated with atherosclerosis, the status of atherosclerosis could, in turn, determine the nature of this association.To this end, a cross-sectional study of 907 elderly Japanese individuals (aged 60–99 years), who had participated in dental health check-up during the period 2016–2018, was conducted. Advanced periodontitis was defined as periodontal pocket ≥ 6.0 mm.Among the study population, 295 (32.5%) were found to have atherosclerosis defined as a carotid intima-media thickness (CIMT) of ≥ 1.1 mm. HTLV-1 infection was positively associated with advanced periodontitis in participants with atherosclerosis, but no significant associations were observed among the participants without atherosclerosis. The known risk factors’ (including reticulocyte and CIMT) adjusted odds ratio (OR) and 95% confidence interval (CI) of advanced periodontitis were OR 2.01 and 95% CI 1.06–3.81 for participants with atherosclerosis and OR 0.61 and 95% CI 0.34–1.12 for participants without atherosclerosis.This study found a significant association between HTLV-1 infection and advanced periodontitis among elderly Japanese with atherosclerosis. However, this association is absent in individuals without atherosclerosis, suggesting that atherosclerosis might act as a determinant in the association between HTLV-1 infection and advanced periodontitis among elderly Japanese. Although human T cell leukemia virus type-1 (HTLV-1), which is the earliest recognized human retrovirus, has been shown to be a causative agent of adult T cell leukemia/lymphoma , the majHowever, we have previously reported the positive association of asymptomatic HTLV-1 infection to advanced periodontitis in elderly subjects with low hematopoietic activity (and low reticulocyte levels) . AlthougIn addition, low-grade inflammation is associated with atherosclerosis  and subsSince periodontitis is a major cause of tooth loss which results in a significant decrease in the quality of life of elderly people  and HTLVTo evaluate the association between HTLV-1 infection and advanced periodontitis in relation to atherosclerosis status, we conducted a cross-sectional study involving 907 elderly people aged 60–99 years who participated in annual dental health check-ups during 2016–2018.The study population comprised of 1925 elderly Japanese (702 men and 1223 women) aged 60–99 years from Goto City in the western part of Japan who attended annual health check-ups during 2016–2018 conducted by the local government and directed by the Ministry of Health, Labor and Welfare in Japan and who underwent oral assessment.n = 4) and those without any remaining teeth (n = 920) were excluded. Advanced periodontitis is diagnosed as the maximum probing pocket depth for each gingiva. Incidentally, decayed teeth are the most frequent reason for teeth extraction and subsequent periodontal diseases in Japan  7.0) for men and 71.3 (SD 6.8) for women were enrolled in the study.Participants without data on carotid intima-media thickness (CIMT) . Body mass index (BMI) was calculated as weight (kg)/height (m)4 cells/μL) =  × RBC (× 104 cells/μL)/1000. The calculated values were 5.358 ± 2.141 [× 104 cells/μL] and 5.047 ± 1.774 [× 104 cells/μL] for male and female participants, respectively.Blood samples were collected with siliconized, sodium fluoride, and EDTA-2K containing tubes. All measurements were made following standard laboratory procedures at SRL, Inc. . Samples from the siliconized tube were used to measure high-density lipoprotein (HDL)-cholesterol and triglycerides, and sample from the sodium fluoride tube was used to measure glycated hemoglobin (HbA1c). Red blood cells (RBC) and reticulocytes in the samples collected in EDTA-2K tubes were measured. Reticulocyte levels were determined using the following formula: reticulocytes (× 10Experienced vascular technicians measured carotid intima media-thickness (CIMT) using a LOGIQ Book XP with a 10-MHz transducer . Maximum values for the left and right common carotid arteries of the CIMT were calculated with automated digital edge-detection software  following a protocol that has been described in detail elsewhere . The higTo detect human T cell leukemia virus type-1 (HTLV-1), a chemiluminescent enzyme immunoassay (CLEIA) kit  was used.n = 6) performed periodontal examination by using a modified method from the Third National Health and Nutrition Examination Survey (USA) [Trained dentists (ey (USA) . ProbingAtherosclerosis was diagnosed as having CIMT ≥ 1.1 mm, and the study population was stratified into those with and without atherosclerosis. Atherosclerosis status-specific differences in mean values or proportions of variables by HTLV-1 status were analyzed. A trend test was performed with analysis of variance (ANOVA) for continuous values, and chi-squared test was used for determining differences in proportions. Advanced periodontitis was diagnosed as a probing pocket depth of ≥ 6 mm.Logistic regression models were used to calculate odds ratios (ORs) and 95% confidence interval (CIs) to determine the association between HTLV-1 infection and advanced periodontitis. Since atherosclerosis might act as a determinant factor on the association between HTLV-1 infection and advanced periodontitis as we hypothesized in the present study, participants were stratified according to their atherosclerosis status.Periodontitis risk factors that have a direct influence on the intra-oral environment, such as smoking , and the2), smoking status (non-smoker or current smoker), number of remaining teeth, decayed teeth , HDL cholesterol (mg/dL), triglycerides (mg/dL), HbA1c (%), reticulocyte (cells/μL), and CIMT (mm). Model 3 was further adjusted for the use of glucose-lowering medication  and antihyperlipidemic medication .Three different models were used to adjust for confounding factors. Model 1 was only adjusted for sex and age. For model 2, we included other potential confounding factors, namely BMI  and atherosclerosis in the elderly , we alsop value of < 0.05 was considered statistically significant.All statistical analyses were performed with SAS system for Windows . A Of the study population, 295 and 197 were diagnosed as having atherosclerosis and advanced periodontitis, respectively.Among the participants without atherosclerosis, people who were HTLV-1 positive were older compared to those who were HTLV-1 negative. Among the participants with atherosclerosis, HTLV-1 positive individuals had significantly lower triglycerides and HbA1c levels than people who were HTLV-1 negative  as a reference group, the adjusted ORs for hypertension and atherosclerosis were 1.08  and 0.93  for the second tertiles of reticulocyte count (4.390–5.724 × 104cells/μL for men and 4.140–5.551 × 104cells/μL for women) and 1.21  and 0.89  for the highest tertile of reticulocyte count (5.725 × 104cells/μL ≤ for men and 5.552 × 104cells/μL ≤ for women) (not shown in table).By using a sex- and age-adjusted model, analysis between tertiles of reticulocytes and hypertension, and between tertiles of reticulocytes and atherosclerosis was performed. Even the power did not reach significant value; reticulocyte count is positive tendency with hypertension and inverse tendency with atherosclerosis. Using the lowest tertile of reticulocyte count  but not among elderly participants with higher hematopoietic activity (higher reticulocyte count) . Lower hAlthough in the present study the positive association between HTLV-1 infection and advanced periodontitis was observed only among participants with atherosclerosis, the biological characteristics of HTLV-1 might explain the finding. HTLV-1 possesses a characteristic of inflammation inducer since the trans-activator protein of Tax of HTLV-1 enhances inflammation , 8. The However, as shown in Table Furthermore, even the statistical power in this study could not reach a significant value among participants without atherosclerosis, compared to participants without HTLV-1 infection. Also, participants with HTLV-1 infection showed lower ORs in advanced periodontitis Table  with higLow-grade inflammation also induces hypertension . Previoun = 907), and reticulocyte count showed essentially the same tendency for hypertension and atherosclerosis. Hypertension is well known as a significant factor in endothelium injury, and atherosclerosis is a result of aggressive endothelial repair. Therefore, even a higher hematopoietic activity should have a higher tissue-repairing activity such as reduction of a marker of oxidative stress [A previous study with 2098 elderly participants reported that reticulocyte count was positively associated with hypertension, whereas it was inversely associated with atherosclerosis . In our e stress  and highe stress , 38. Thee stress . FurtherAging is a process that increases vascular aging-related inflammation . Age-relTherefore, HTLV-1 infection that stimulates inflammation activity , 8 couldThe clinical implication for the present study is that, even in non-symptomatic status, HTLV-1 infection could be associated with advanced periodontitis and atherosclerosis status might influence the association between HTLV-1 infection and advanced periodontitis among elderly people.Potential limitations of this study warrant consideration. Although the age-related endothelial dysfunction may influence the association between HTLV-1 infection and advanced periodontitis, no data on the evaluation of endothelial function was available. Thus, further analyses that include endothelial function-related data such as flow-mediated dilation (FMD) will be necessary. This study did not include confirmatory tests for detection of HTLV-1. In spite of that, we believe that the influence of HTLV-1 was limited, especially since our previous study, using real-time reverse transcription-polymerase chain reaction with the hydrolysis probe and western blotting assays, showed only a small false positive rate (1.2%) . In addiIn conclusion, we found HTLV-1 infection to be positively associated with advanced periodontitis among elderly participants with atherosclerosis but not among those without atherosclerosis. Therefore, atherosclerosis might be a crucial factor that determines the association of HTLV-1 infection with advanced periodontitis among elderly Japanese people. These results were an efficient tool to clarify the role of the possible mechanism for the association between HTLV-1 and microcirculation.


Glypican-3 (GPC3) is a cell-surface glycoprotein consisting of heparan sulfate glycosaminoglycan chains and an inner protein core. It has important functions in cellular signaling including cell growth, embryogenesis, and differentiation. GPC3 has been linked to hepatocellular carcinoma and a few other cancers, however, the mechanistic role of GPC3 in cancer development remains elusive. Recent breakthroughs including the structural modeling of GPC3 and GPC3–Wnt complexes represent important steps toward deciphering the molecular mechanism of action for GPC3 and how it may regulate cancer signaling and tumor growth. A full understanding of the molecular basis of GPC3-mediated signaling requires elucidation of the dynamics of partner receptors, transducer complexes, and downstream players. Herein, we summarize current insights into the role of GPC3 in regulating cancer development through Wnt and other signaling pathways, including YAP and hedgehog cascades. We also highlight the growing body of work which underlies deciphering how GPC3 is a key player in liver oncogenesis. Each year hepatocellular carcinoma (HCC) affects 750,000–1 million people worldwide and is projected to be the third most common cause of cancer death in the United States by 2030 , 5. WhilWnt signaling is vital in embryonic development and tissue homeostasis . In adulIn this review, we highlight the role of GPC3 in cellular signaling including Wnt and other signaling pathways such as YAP (Yes associated protein), and Hedgehog (Hh) within the current scientific milieu. Due to the importance of GPC3 in multiple signaling cascades, GPC3 could have a pivotal role as a biomarker and as a potent therapeutic target in investigational immunotherapies . TherapeGlypicans, classified among the heparan sulfate proteoglycan family, reside on the exterior cell membrane via a glycosylphosphatidylinositol (GPI) anchor and are a major part of the extracellular matrix (ECM) mediating cell-ECM and cell-cell interactions , 17. GlyNP_004475) being the most commonly expressed. A total of four alternatively spliced variants are documented , the inactive version of YAP, was greater in HCC cells treated with HN3. Overall, total YAP level was reduced in HCC cells treated with HN3. GPC3 knockdown led to lower cell proliferation and reintroduction of recombinant YAP was able to rescue the cells from apoptosis triggered by GPC3 knockdown . The obsThe hedgehog (Hh) signaling pathway  plays a The Hh signaling pathway involves recruitment of Hh ligands including: desert hedgehog (Dhh), Indian hedgehog (Ihh), and Sonic hedgehog (Shh). Any Hh ligand can initiate binding to the 12 transmembrane proteins Patched (PTCH) and various co-receptors, thus triggering Hh signaling by Smoothened(SMO) de-repression. SMO, a 7-pass transmembrane protein from the FZD family of GPCRs, mediates downstream signaling. Sufu, a cytoplasmic protein, and GLI proteins, main transcriptional effectors, cooperate to induce Hh activation and expression of Hh target genes. In the absence of Hh ligands, or in the case of Hh ligands binding to GPC3, PTCH will not be active and SMO will be repressed, thereby inhibiting Hh signaling  53, 54), 5453, 5Drosophila model demonstrated that glypicans are involved in regulation of Hh signaling  entry into hepatocytes, further entangles GPC3 with Hh and Hippo signaling. CD81 is main GPC3 binding partner and the GPC3/CD81 interaction modulates Hh signaling through hematopoietically expressed homeobox protein (Hhex), a transcriptional repressor , 59. HowGPC3 is clearly an important player Wnt, Hh, and YAP signaling cascades. However, fundamental questions regarding the GPC3/Wnt/FZD complex structure, intratumor heterogeneity of protein expression, and alternatively spliced variants of GPC3 in liver cancer have yet to be fully understood. Future work addressing the mechanism of GPC3 in the outlined signaling pathways would provide a more complete picture of its precise role in oncogenesis of liver cancer. Rigorous experimental interrogation of mechanism will be crucial in engineering therapies which can disrupt tumor progression. Nevertheless, as extensively summarized in other recent articles, the development of GPC3-targeted therapies has emerged with many clinical trials worldwide , 16, 63.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The National Cancer Institute (NCI) holds patent rights to anti-GPC3 antibodies including HN3, YP7, and HS20 in many jurisdictions, including the USA , China, Japan, South Korea, Singapore, and Europe. Claims cover the antibodies themselves as well as conjugates that utilize the antibodies, such as recombinant immunotoxins (RITs), antibody–drug conjugates (ADCs), bispecific antibodies, and modified T cell receptors (TCRs)/chimeric antigen receptors (CARs) and vectors expressing these constructs. Anyone interested in licensing these antibodies can contact MH (NCI) for additional information. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Chlorella sorokiniana UTEX 1230. C. sorokiniana produces the auxin indole-3-acetic acid (IAA), which was present in both the cell pellet and in the supernatant at a concentration of ~ 1 nM, and its genome encodes orthologs of genes related to auxin synthesis, transport, and signaling in higher plants. Candidate orthologs for the canonical AUX/IAA signaling pathway were not found; however, auxin-binding protein 1 (ABP1), an alternate auxin receptor, is present and highly conserved at essential auxin binding and zinc coordinating residues. Additionally, candidate orthologs for PIN proteins, responsible for intercellular, vectorial auxin transport in higher plants, were not found, but PILs (PIN-Like) proteins, a recently discovered family that mediates intracellular auxin transport, were identified. The distribution of auxin related gene in this unicellular chlorophyte demonstrates that a core suite of auxin signaling components was present early in the evolution of plants. Understanding the simplified auxin signaling pathways in chlorophytes will aid in understanding phytohormone signaling and crosstalk in seed plants, and in understanding the diversification and integration of developmental signals during the evolution of multicellular plants.Indole-3-acetic acid is a ubiquitous small molecule found in all domains of life. It is the predominant and most active auxin in seed plants, where it coordinates a variety of complex growth and development processes. The potential origin of auxin signaling in algae remains a matter of some controversy. In order to clarify the evolutionary context of algal auxin signaling, we undertook a genomic survey to assess whether auxin acts as a signaling molecule in the emerging model chlorophyte  Arabidopsis thaliana it is predominantly synthesized from tryptophan via the two-step TAA/YUC pathway: Tryptophan Aminotransferase of Arabidopsis 1 (TAA) converts tryptophan to indole-3-pyruvic acid (IPyA), and YUC, a family of flavin-dependent monooxygenases, converts IPyA to indole-3-acetic acid (IAA)..8].Chlorella sorokiniana UTEX 1230 to identify putative orthologs for auxin-active genes, and analyzed their evolutionary relationships with orthologs from other sequenced algae and plants. We thus identified candidate genes that mediate the synthesis, transport, and perception of IAA by this organism. Of particular note, C. sorokiniana encodes an ortholog for auxin-binding protein 1 (ABP1) in which the auxin binding residues, as well as the zinc coordinating residues essential for auxin binding, as identified in corn . The . The 8].e domain .PILS (PIN-like proteins) are recently discovered auxin transport proteins which are similar in structure and function, but phylogenetically distinct from the PIN polar auxin transporters in plants . In ordeArabidopsis as a mutant with altered indole-3-butyric acid responses [Identified in esponses , IBR5 isesponses .Zea mays ABP1 bound with napthaleneacetic acid (NAA), PDB accession 1LRH using SWISSMODEL on default settings [CsABP1 was structurally aligned to the PDB structure of settings . This alC. sorokinana UTEX 1230 culture was obtained from the University of Texas Culture Collection of Algae. Initial cultures were grown to saturation and 50 mL cultures were inoculated at a starting cell density of 5 × 106 cells/mL in Bold’s Basal Medium [2  and dissolved in 1 mL methanol. IAA was quantified via LC/MS/MS as described in [A l Medium . After tribed in . In placC. sorokiniana genome sequence revealed the presence of numerous orthologs to higher plant IAA biosynthetic enzymes, transporters, and signal transduction components, as indicated in A detailed survey of the C. sorokiniana encodes genes for the formation of IAA by three different routes . However, it does contain a highly conserved ortholog to ABP1   ChlorellChlorella. In Arabidopsis, auxin-bound ABP1 is essential for the activation of Rho-dependent GTPases (ROPs), mediated by plasma membrane associated transmembrane kinases (TMKs) [C. sorokiniana encodes both ROP and TMK orthologs for this signaling pathway  . C. soro pathway . Additiofor IBR5 , a recenndefined .The presence of ABP1 and IBR5 and the absence of AUX/IAA signaling components suggests that non-AUX/IAA mediated signaling pathways in plants may have originated in algae, and that their function broadened and diversified as plants colonized land.C. sorokiniana produces (per 50 ml culture) approximately 2 ng of IAA in the cell pellet and secretes 9 ng into the culture supernatant, resulting in a supernatant concentration of approximately 1 nM  of IAA in algae are currently poorly defined, and while our work provides a foundation upon which new studies can be built, we can currently only speculate as to the functions of auxin synthesis, secretion, and signaling in unicellular chlorophytes.The conservation of many auxin synthesis, transport, and signaling related orthologs in A number of possible functions for auxin as an inter- or intracellular signaling molecule are conceivable. First, given our demonstration that IAA is a secreted molecule, one possibility is that IAA signaling evolved as a population-density dependent mediator of quorum sensing in algae, analogous to bacterial and fungal quorum sensing molecules. Another possibility is that IAA acts as a regulator of biofilm initiation and development, whereby the molecule is constitutively secreted and maintained at a high concentration in biofilms, and at a much lower concentration when cells are not adhered to a substrate. Still another possibility is that IAA acts as an interspecies signaling molecule, allowing algae to communicate with members of mixed biofilm communities.From the perspective of the evolution of multicellularity and pattern formation in the green plant lineage, this work provides compelling evidence that the molecular machinery necessary for auxin signaling was present in the unicellular ancestors of higher plants, and was thus available for elaboration and specification of auxin signaling pathways during the evolution of land plants.


Escherichia coli 4s siphophage Shashou, which presents similarity to members of the Guernseyvirinae subfamily. Shashou is predicted to use a headful packaging mechanism for its 44,155-bp genome and to encode 77 proteins.Here, we announce the genome of the  Escherichia coli 4s siphophage Shashou, which presents similarity to members of the Guernseyvirinae subfamily. Shashou is predicted to use a headful packaging mechanism for its 44,155-bp genome and to encode 77 proteins.Here, we announce the genome of the  Escherichia coli, a Gram-negative bacterium with fast growth under diverse conditions, is one of the most studied organisms on the planet . Reads were assembled with SPAdes v3.5.0 using default parameters, yielding a single contig of 44,155 bp for Shashou and 402.6-fold contig coverage after trimming using FastX-Toolkit v0.0.14 (http://hannonlab.cshl.edu/fastx_toolkit/)  (Phage Shashou was isolated from filtered (0.2-μm filter) wastewater treatment plant influent collected in College Station, Texas, by plating on  coli 4s . Both ph coli 4s . Sequencoolkit/) . tRNA deoolkit/) –10. TMHMolkit/) ––14. HHPrlkit/) ––. The Galaxy-pub) , 18. Phaaxy-pub) .Shashou is a siphophage with a 44,155-bp genome with 77 predicted protein-coding genes. It has a 50.1% G+C content and 94.4% coding density. PhageTerm predicts that Shashou uses a pac site for packaging .Escherichia phage G AB-2017 (GenBank accession number KY295895), a Guernseyvirinae subfamily member (QEA09424) and the tape measure protein (NCBI accession number QEA09426), with no canonical slippery sequence to induce translational frameshifting. The helicase (NCBI accession number QEA09433) in Shashou contains an intein.Shashou shares 65.15% nucleotide identity and 51 proteins with y member . A hypotMK931440, BioProject accession number PRJNA222858, SRA accession number SRR8893605, and BioSample accession number SAMN11414490.The genome sequence and associated data for phage Shashou were deposited under GenBank accession number