Onobrychis viciifolia) is rich in condensed tannins (CT). CT function includes inhibiting bacterial and fungi activity during the ensiling process. We used polyethylene glycol (PEG) to deactivate tannin activity to find out the effects of CT. The results show that the addition of PEG increased dry-matter loss  after 60 d of ensiling, and also increased lactic acid  and acetic-acid content  after 30 d of ensiling. The PEG-treated group increased its Pediococcus relative abundance  during the ensiling process, increased its Gibellulopsis relative abundance after 3 d of ensiling (5.96% vs. 19.52%), increased its Vishniacozyma relative abundance after 3 d and 7 d of ensiling , and increased its Aspergillus relative abundance after 7 d, 14 d and 60 d of ensiling . However, the PEG-treated group decreased its Alternaria relative abundance during entire ensiling process (14.00–25.21% vs. 3.33–7.49%). These results suggest that condensed tannins inhibit lactic-acid bacteria fermentation though reducing Pediococcus activity, and inhibiting fungi activity depending on different strains.Sainfoin ( Lespedeza cuneata Dum.-Cours.), crown vetch (Coronilla varia L.), hedysarum , birdsfoot trefoil (Lotus corniculatus L.) and sainfoin (Onobrychis viciifolia). Among these plants, CT from sainfoin has the highest capacity to bind protein and causes the least inhibition of cellulose digestion by rumen bacteria [Escherichia. coli O157:H7 [E. coli strain. It is not clear how the CT in sainfoin affect the bacterial and fungi community and the quality of silage. Polyethylene glycol (PEG) is a commonly used CT-inactivating agent that can be used to study the effect of tannin on silage because it forms stable PEG-CT complexes, attributed to hydrogen bonding between PEG and CT [Dalea purpurea Vent.) silage, and that CT decreased bacterial and fungi diversity during ensiling [A large number of agricultural scientists have studied condensed tannins (CT) over more than 50 years. A major focus is interactions of CT with proteins that can generate important agricultural effects, such as protecting forage protein during ensiling and rumen digestion. This protection can improve silage quality and increase the flow of undegraded proteins into the abomasum. CT is also beneficial for the environment, as it leads to reduced greenhouse-gas (methane) emission from ruminants . Variousbacteria . Sainfoibacteria . Sainfoibacteria . Some ofbacteria . It is wbacteria . Limitedbacteria . Several O157:H7 ,10; howeG and CT . Previouensiling .To clarify the bacteria and fungi community during ensiling would illuminate the fermentation process of ensiled forage . As far Onobrychis viciifolia) was planted in Shihezi City, Xinjiang Province, China. Whole-plant sainfoin was harvested in July 2021 at the early flower stage. After wilting for 12 h to a dry-matter (DM) content of approximately 240 g/kg fresh weight, the sample was chopped into 1–2 cm pieces. The sample was then sprayed with a solution of 640 g/L PEG  at A rate of 217 mL/kg DM to achieve a CT:PEG ratio of 1:2 in the samples [Sainfoin  *2(two treat-ment) *3(replicates) of each silage were collected. Silage samples were dried and ground, using 1.0 mm in preparation for obtaining the DM content. The water-soluble carbohydrates (WSC) were determined by using boiling water for extraction according to the anthrone method . Total n2HPO4 (1 mM) was used for the mobile phase with a flow rate of 0.6 mL·min−1, with an injection volume of 20 μL and oven temperature of 50 °C [For fermentation characteristics, the sample from each different fermentation day and treated (control and PEG) silage was taken at 20 g, then combined with 180 mL distilled water and thoroughly blended in a homogenizer . After filtering the mixture of sample solution by four layers of cheesecloth, the supernatant was collected for volatile fatty acids (VFA) and AN content analysis. The pH was measured using a portable pH meter . AN content was determined by the phenol-hypochlorite colorimetric method . For meaof 50 °C .Microbial count was carried out according to methods previously described . Briefly® Spin Kit for Soil, MP Biomedicals, New York, NY, USA). Primers targeting the V3-V4 regions of 16S rDNA  and primers targeting ITS1 regions  of fungi were used to conduct PCR amplification according to [Total DNA of each sample was extracted with a commercial DNA Kit .The fermentation characteristics of sainfoin silage data were subjected to Two-way ANOVA, 2 × 5 factorial Complete Randomized design (PEG and control treatments × five ensiling-time days). Data were analyzed using SPSS software . Significant differences between each treatment were determined by Tukey’s test when The DM, CP and WSC content of wilted sainfoin was 236.50 g/kg, 223.12 g/ kg DM and 101.43 g/kg DM, respectively .The characteristics of sainfoin silages are shown in p < 0.05). AN content showed the same results as DM loss during ensiling, which were 1.60 g/kg DM and 2.64 g/kg DM after 60 d of ensiling, respectively. The pH dropped to 4.3 for both the control and PEG-treated groups after 60 d of ensiling. There was no difference in pH between the control and PEG-treated groups during the entire ensiling (p > 0.05). The significant difference was found only in LA and acetic acid (AA) content between the control and PEG-treated groups after 30 d of ensiling (p < 0.05), which were 108.99 g/kg vs. 158.89 g/kg and 73.15 g/kg vs. 138.47 g/kg, respectively. The AA content in both treatments was high  after 60 d of ensiling. There was no difference of LA to AA ratio between control and PEG-treated groups during the entire ensiling (p > 0.05), and this ratio was below 3 during ensiling. The LAB counts were highest in both the control and PEG-treated groups after 3 d of ensiling, which were 9.04 Log10 cfu/g FM and 9.19 Log10 cfu/g, respectively. The PEG-treated group showed a higher trend in LAB count compared with the control group during ensiling, and this trend became a significant difference after 7 d, 30 d and 60 d of ensiling (p < 0.05). The mold count had the same trend as the LAB count during ensiling, which showed a significant difference after 3 d, 30 d and 60 d of ensiling (p < 0.05). The yeast count showed significant difference between the control and PEG-treated groups after 3 d of ensiling (p < 0.05), which were 3.82 Log10 cfu/g FM and 4.33 Log10 cfu/g FM, respectively.The extractable CT in sainfoin dropped to 1.96% (on a dry-matter basis) after 3 d of ensiling, and then maintained a constant level during the ensiling process. The protein-bound tannins increased to 6.97% after 3 d of ensiling, and then maintained a constant level during the ensiling process. After 60 d of ensiling, DM losses were 8.32% and 14.15%, respectively. The highest DM loss of the PEG-treated group was observed compared to the control group during the entire ensiling . The Good’s coverage index was higher than 0.99, indicating that the degree of sequencing was sufficient for the bacterial community analysis. With or without PEG, the richness of the microbial community in sainfoin decreased during ensiling. The richness and diversity of the microbial community increased in sainfoin with PEG treatment at 30 d. However, there was no difference in the microbial community richness and diversity between silage with or without PEG at 60 d of ensiling. In addition, using a weighted uniFrac distance to assess the bacterial communities, a clear distinction was found between the bacterial communities in the control and PEG-treated silage at 30 d of ensiling  was the predominant phylum, followed by Proteobacteria (9.85%), Firmicutes (1.2%), and Actinobacteria (1.05%). In the wilted sainfoin, Weissella, Lactobacillus, Enterobacter and Pediococcus were the four richest bacterial communities on the genus level. During the ensiling process, the Lactobacillus relative abundance increased as the days of ensiling increased. The Lactobacillus relative abundance of the control and PEG groups was 42.06–90.56% and 27.4–69.92%, respectively. The Enterobacteria relative abundance decreased with ensiling days, with a relative abundance for the control and PEG groups at 1.04–11.92% and 2.78–12.66%, respectively. The Weissella relative abundance slightly decreased after 7 d of ensiling, then stabilized until 30 d of ensiling in the control silage. After 60 d of ensiling, the Weissella relative abundance dropped considerably to 3.85% in the control silage. The Weissella relative abundance in the PEG silage decreased slightly for 7 d but stabilized for the remaining ensiling. Pediococcus showed the most significant difference in relative abundance of the LAB bacteria (p < 0.05), with the Pediococcus relative abundance for the control and PEG group at 0.37–3.38% and 7.82–23.5%, respectively.Analyzing the bacterial community on the genus level  showed tLactobacillus was the main genus (42.06%) in the control silages at 3 d of ensiling, followed by Weissella (31.83%), and Enterobacter (17.96%), with the Pediococcus relative abundance being the lowest (0.50%). For the PEG group, Weissella became the main genus in silages at 3 d of ensiling (42.57%), followed by Lactobacillus (27.4%), Enterobacter (12.66%) and Pediococcus (7.92%). However, there was no significant difference (p > 0.05) between these four bacteria on the genus level between the control and PEG silage at 3 d of ensiling. During ensiling, a significant difference (p < 0.05) in Pediococcus relative abundance between control and PEG silages was found after 7 d of ensiling, with the control and PEG silage Pediococcus relative abundance at 0.37% and 14.2%, respectively. This same significant difference for Pediococcus relative abundance between control and PEG silages was no longer present at 60 d of ensiling. This suggests that CT affected the activity of Pediococcus primarily in the early and middle ensiling stages. Alpha diversity of fungi community show in p < 0.05) the diversity of the microbial community after 3 days of ensiling. There was no difference (p > 0.05) in microbial-community diversity between the control and PEG treatments during ensiling days 7 to ensiling days 60. Addition with PEG decreased (p < 0.05) the richness of the microbial community during 30 days of ensiling. In addition, weighted UniFrac distance was used to assess fungi communities, as shown in As a result, the richness of the microbial community decreased in both the control and treated groups with prolonged ensiling. Addition with PEG, however, increased .Analysis of the fungi community on a genus level is shown in Cladosporium dominated in wilted sainfoin with a relative abundance of 34.81%, followed by Filobasidium (21.28%), Alternaria (12.39%), Gibberella (11.55%), Gibellulopsis (7.95%) and Vishniacozyma (4.95%). Cladosporium dominated in control groups during entire ensiling with a relative abundance of 20.68–41.30%, followed by Alternaria (14.00–25.21%). Cladosporium dominated in PEG groups during 30 days of ensiling. Addition with PEG increased Gibellulopsis relative abundance after 3 days of ensiling (5.96% vs. 19.52%) and Vishniacozyma relative abundance after 3 days and 7 days of ensiling . Addition with PEG increased Aspergillus relative abundance after 7 days, 14 days and 60 days of ensiling . Addition with PEG decreased Alternaria relative abundance during 60 days of ensiling (14.00–25.21% vs. 3.33–7.49%), Gibberella relative abundance after 3 days and 7 days of ensiling (10.07% vs. 3.22% and 15.05% vs. 3.77%) and Cladosporium relative abundance after 3 days of ensiling (41.30% vs. 22.05%).−1 DM is the common threshold level for achieving well-preserved silage [−1 DM, which is sufficient for fermentation. LAB counts in the silage were higher than those of the spoilage organisms in wilted sainfoin, such as molds and yeasts, which might be favorable to the establishment of a dominant LAB community during fermentation.Levels of CT greater than 6% have shown a strong ability to inhibit bacterial activity and protein degradation in silage . In addid silage . In thisPrevious studies found that protein-bound CT increased because the extractable CT decreased after ensiling . The pre10 CFU g−1 FM, respectively. The results suggest that AA was not the sole inhibitor of yeast in these silages. Studies have found that CT-inhibited yeast grow during the ensiling process in a species-dependent manner [Yeast activity in silage usually causes nutrient loss during ensiling. Yeast activity is enhanced by ensiling during the beginning stage due to the aerobic environment, then decreased as the silage pH and oxygen is reduced. AA is recognized as an important yeast inhibitor. Here, yeast counts decreased with the accumulation of AA during the ensiling process for both silage groups. Similar levels of AA were found in the control and PEG silages for the duration of the ensiling process, except at 30 d of fermentation. However, a significant difference between yeast counts was only found at 3 d of fermentation, when control and PEG silage yeast counts were 3.82 and 4.33 logt manner . It can Firmicutes became the dominant phylum in both groups, with a relative abundance of 94.52–97.97%; however, the Cyanobacteria relative abundance dropped to 0.12–0.49%. Similar results were observed in [After 60 d of ensiling, erved in , except Lactobacillus were the main genus in the control and PEG groups during ensiling. The Lactobacillus relative abundance (90.56%) was much higher after 60 d of ensiling in the control silages. This is because other bacteria in the community dropped to their lowest level at this stage. Some species of Lactobacillus, such as Lactobacillus plantarum NBRC15891, Lactobacillus fermentum NBRC15885 and Lactobacillus delbrueckii NBRC3073 have a strong tolerance for catechins [Lactobacillus activity in the ensiling process.atechins . Since CPediococcus are facultative heterofermentative LAB, and produce LA and AA by fermenting hexoses and pentoses, respectively [Pediococcus than the control silages from 7 d to 30 d of ensiling (p < 0.05). Similar results were found with purple prairie clover silage with added PEG [Pediococcus species isolated from forage can grow in a 3.5–6.0 pH environment [Pediococcus species growth and proliferation are well under a pH range of 4.3–4.9 in sainfoin and alfalfa-mixture silages [pediococcus relative abundance may not be affected by pH during ensiling. During the ensiling process, acidity, substrate availability, aerobiosis, moisture, or temperature could affect the fermentation and LAB succession during the ensiling process [Pediococcus.The various ectively . In thisdded PEG . Some stironment . Further silages . In this process . In thisEnterobacteria relative abundance decreased with increasing ensiling days, consistent with the pH decrease from 5.2 to 4.3. The critical pH for Enterobacteria was 4.5 [Enterobacteria were inhibited [Enterobacteria relative abundance compared to control silages. However, the lowest AN level was found in control silage throughout the silage-fermentation process. This seems to disagree with the general assumption that the production of AN in silage usually results from the activity of Enterobacteria if Clostridia are not detected  [ was 4.5 . Once thnhibited . The PEGetected) . In addietected) . Thus, AFusarium and Alternaria are often considered as field fungi; Penicillium and Aspergillus are storage fungi. Those fungi are usually considered toxin producers. Fusarium, Alternaria and Cladosporium can produce mycotoxins during their growth by infections of grass, resulting in forage crops already containing toxins before being ensiled or grazed [Cladosporium species from silages [Cladosporium were dominant fungi in sainfoin silages, which is in agreement with others observed [Gibberella is a sexual form of Fusarium. Fusarium is an important toxin-producing fungus; however, several studies observed that mycotoxins produced by Fusarium were already present at the harvest and contents were stabled in silage [Fusarium is trichothecenes, which is involved in the plant-infection process. Thus, these traits are not needed for Fusarium growth on harvested silage [Gibberlla relative abundance; however, those impacts may not affect mycotoxin contents in silage. Vishniacozyma exists in plants such as alfalfa, maize and wheat crops [Vishniacozyma activity during 30 d of ensiling.r grazed ; however silages . The Claobserved ,37. Gibbn silage ,39. Furtd silage . As resuat crops . The preAlternaria relative abundance decreased; however, Aspergillus relative abundance increased in maize silage [Alternaria in PEG treatment during the entire ensiling. The results suggested that CT probably promotes Alternaria activity during sainfoin ensiling. However, the mechanism of CT promoting Alternaria activity during sainfoin ensiling needs further study. As a storage fungus, Aspergillus can grow under different environments of silage, such as lower oxygen and pH levels [Mimosa tenuiflora (Willd.) bark can inhibit Aspergillus flavus growth. The present study showed that relative abundance increased in both groups with prolonged ensiling; however, addition with PEG increased its relative abundance after 7, 14 and 60 days of ensiling. The results suggested that CT might inhibit Aspergillus growth in sainfoin silages.In general, field fungi activity decreased with prolonged ensiling; however, storage fungi activity increased during ensiled. A previous study observed that e silage . The preH levels ,44. A prPediococcus. CTs in sainfoin have little inhibitive effect on Vishniacozyma activity; however, CT showed strong inhibit effect on Aspergillus activity during sainfoin ensiling. On the contrary, CTs have no inhibit effect on Alternaria activity, but CT probably promotes its growth. This study indicated that CT from sainfoin can inhibit LAB fermentation by reducing Pediococcus activity and inhibiting fungi activity, depending on different strains.The CT in sainfoin significantly decreased DM loss, content of LA, AA and AN during ensiling. At the same time, CT inhibited bacterial richness and diversity, particularly for


Human T-lymphotropic viruses 1 and 2 (HTLV-1/2) have a worldwide distribution. HTLV-1 has been associated with several diseases, including an aggressive malignant disease known as adult T-cell leukemia/lymphoma and a chronic inflammatory neurological disease called HTLV-1-associated myelopathy, while HTLV-2 has not been definitively associated with diseases. HTLV-2 is most prevalent in specific groups such as injecting drug users and the indigenous population. In Brazil, most studies about HTLV in indigenous are carried out in indigenous communities from the north of the country. Mato Grosso do Sul (MS), Central Brazil, has the second-largest indigenous population in Brazil. However, there is no available data about HTLV infection in this group. We conducted the first investigation of HTLV-1/2 infection prevalence in the indigenous population from Jaguapiru and Bororó villages in Dourados City, MS, to provide the prevalence and molecular characterization of HTLV. For that, a total of 1875 indigenous participated in the study. All the serum samples were screened by an enzyme-linked immunosorbent assay commercial kit for the presence of anti-HTLV-1/2 antibodies. Positive samples were confirmed by HTLV-1/2 Western Blot assay. The HTLV-1 5’LTR region was detected by nested PCR amplification and sequenced by Sanger. Most of the study population declared belonging to Guarani-Kaiowá ethnicity (69.18%), 872 (46.51%), and 1003 (53.49%) were from Jaguapiru and Bororó villages, respectively. The median age of participants was 31 years, and 74.24% were females. Two individuals were detected with HTLV-1 . The phylogenetic analysis revealed that isolates belong to the Cosmopolitan subtype and the Transcontinental subgroup (HTLV-1aA). The low HTLV-1 prevalence found in this study is similar to that observed among blood donors, and pregnant populations from Mato Grosso do Sul. The absence of HTLV-2 infection among these Brazilian indigenous communities would suggest a distinct behavior pattern from other indigenous populations in Brazil. Lower socioeconomic and educational levels associated with cultural aspects of some ethnicities as cross-breastfeeding, polygamy, unprotected sex, and interaction with non-indigenous society, may expose them to risk factors that lead to an increase in the detection rate of infectious diseases6. Mato Grosso do Sul (MS), Central Brazil, is the second state with the largest indigenous population in Brazil7. The Guarani-Kaiowá, KiniKinawa, Kadiwéu, Terena, Guató, Atikum and Ofaié are the ethnicities present in the state8. This indigenous reserve, with about 18,000 people and located in Dourados city, 3474.50 ha, is represented mainly by the Guarani-Kaiowá and Terena ethnicities living in the Bororó and Jaguapiru villages10.The indigenous population in Brazil shows a high prevalence of sexually or vertically transmitted infections and a complex epidemiological profile12. It is estimated that 5–10 million people are infected by HTLV-1 worldwide. South Japan, sub-Saharan Africa, Caribbean islands, Melanesia, and South America are considered endemic regions for HTLV-1 infections13. HTLV-2 has no clinical manifestations well-defined and has been observed as prevalent in specific groups such as American indigenous populations and people who inject drugs (PWID)15. HTLV-1/2 are transmitted by sexual relations, transfusion/transplantation of contaminated blood/organs, and from mother to child, mainly by breastfeeding16.The Human T-lymphotropic virus 1 (HTLV-1) is a retrovirus associated with adult T-cell leukemia/lymphoma (ATL), HTLV-1-associated myelopathy (HAM), and other inflammatory diseases19. However, HTLV-1 infection can be found in this group in lower prevalence or in isolated cases20.HTLV-2 is predominant in the Brazilian indigenous population, which the prevalence can reach rates greater than 30%, mainly in the Kayapo Indigenous communities from the north of the countryOnce remaining gaps in the knowledge about HTLV infection in the country, especially among these indigenous people, this cross-sectional study aimed to investigate the prevalence, risk factors, and molecular characterization of HTLV among this second-largest indigenous reserve in Brazil located in Mato Grosso do Sul.The study population was conducted among indigenous individuals in Dourados City, MS, Central Brazil, from Bororó and Jaguapiru villages Fig. .Figure 1From September 2017 to March 2020, 2190 indigenous persons were invited, and 1875 agreed to participate in the study. Participants underwent an interview with a standardized questionnaire containing sociodemographic and sexual behavior information. Blood samples were collected from all subjects to perform serological tests. The inclusion criteria were being an indigenous community member from Bororó or Jaguapiru villages and aged over 18 years, unless unable to give informed written consent. All subjects gave their written informed consent to participate in the study. This study was approved by the Comissão Nacional de ética em Pesquisa (CONEP) under protocol number 2.000.496. All research was performed following relevant guidelines and regulations.All the serum samples were screened by an enzyme-linked immunosorbent assay (ELISA) commercial kit for the presence of anti-HTLV-1/2 antibodies (GOLD ELISA HTLV-I/II—REM), following the manufacturer’s instructions. Positive samples were repeatedly tested and confirmed by HTLV-1/2 Western Blot (WB) assay (MP Diagnostics HTLV BLOT 2.4–Singapore). HTLV infection was defined as a repeatedly positive ELISA and positive WB.21. Furthermore, the amplicons were purified using a QIA Quick Purification kit , according to the manufacturer’s instructions. The fragments were sequenced using BigDye Terminator Cycle Sequencing Ready Reaction Kit and ABI 3500XL  by Sanger’s method.The HTLV molecular characterization of ELISA and WB positive samples was performed by nested polymerase chain reaction (nested PCR). To this end, DNA was extracted from the whole blood of the anti-HTLV positive participants using the QIAamp DNA Blood mini kit (QIAgen), according to the manufacturer’s instructions. Then, the amplification of a 646 bp fragment of the HTLV-1 5’LTR region was performed by nested PCR as previously reportedThe HTLV-1 isolates were subjected to an analysis in the BLAST  after nucleotide sequencing to identify the subtype and subgroup. Nucleotide sequences were aligned and compared with 50 published HTLV-1 sequences available from GenBank with reference sequences used as outgroup, and sequences were searched with filter HTLV-1 and Transcontinental and Brazil using MAFFT v.7 (Multiple Alignment Program for Amino acid or nucleotide sequences). Phylogenetic analysis was performed using Maximum likelihood with 1000 bootstrap replicates with RAxML program and Bayesian method based on appropriate models determined by JModelTest (TIM2 + G model) with MR-base program on CYPRES Science Gateway. The phylogenetic tree was constructed by use of the FigTree v.1.4.4 program. The haplotype network was developed with NETWORK v.10.2 using the Median Joining algorithm.The GenBank nucleotide sequences accession number included in the phylogenetic analysis: Z32527, L02534, L76310, Y17014, KM023757, KM023765, D13784, AF033817, DQ235699, DQ235698, Y16481, AF054627, J02029, KY510690, KY510691, KY490575, KY490581, GQ443755-57, L36905, JF271836-38, JF271840-42, AY499185, AY920503, DQ471187-97, DQ70891-92, EU392159-60, FJ853490-91, OK247616-17, KM023763-62.The variables of interest were analyzed using STATA 13.0 software . Prevalence for serological markers of HTLV infection was estimated with 95% confidence intervals. Categorical variables were presented by absolute and percentage frequency. Continuous variables were expressed as mean, standard deviation, median, and range. The chi-square test was used to evaluate differences between proportions.The study involving human participants was reviewed and approved by the Comissão Nacional de Ética em Pesquisa (CONEP), an ethics Committee on Human Research, protocol number 2.000.496. The participants provided their written informed consent to participate in this study.A total of 1875 individuals were enrolled in the study. The median age of participants was 31 years, including 483 males (25.76%) and 1392 females (74.24%). Regarding the village, 872 (46.51%) and 1003 (53.49%) were from Jaguapiru and Bororó, respectively. Most of them declared belonging to Guarani-Kaiowá ethnicity (69.18%). In relation to formal education, 66.28% had more than 5 years of study.History of alcohol, drug, and sexually transmitted infections was reported by 27.68%, 3.73%, and 2.88% of the participants, respectively. None of them reported injectable drug use history. Other risk behaviors such as multiple sexual partners, irregular condom use, history of blood transfusion, tattoos, sharing sharp objects, and history of surgery are shown in Table Anti-HTLV was detected in two individuals . The two ELISA reactive samples were further tested by WB assay and confirmed anti-HTLV reactivity with complete profiles in the WB test for HTLV-1. The test results were subsequently referred to the health authorities responsible for this study population.The HTLV-1 5’LTR region was detected by nested PCR amplification in the two anti-HTLV-1 positive samples, which were successfully sequenced. The phylogenetic analysis revealed that both isolates were classified as belonging to the Cosmopolitan (1a) subtype and the Transcontinental (A) subgroup Fig. . The GenTable The other positive individual was an 81-year-old man (ID-137). Both individual ID-137 and ID-763 did not report any risk behaviors such as injection drug use, unprotected sexual intercourse, and multiple sexual partners. Both positive individuals were born in Dourados city, lived in Bororó village, and declared themselves Guarani-Kaiwá ethnicity.23. However, the results of this present study contrast with most of the studies conducted among indigenous populations in Brazil.This is the first investigation of HTLV-1/2 infection prevalence carried out among the indigenous population from Central Brazil. The prevalence of HTLV infection found in this study  is similar to the prevalence observed in the blood donors, and pregnant population from Mato Grosso do Sul 25. The high HTLV-2 prevalence in the indigenous population in Brazil usually differs between communities (from 1.4% in the Wayampi community on Amapá to 57.9% in the Kayapo community on Pará)26. Unlike these studies conducted in Brazil, the present study, no individuals infected with HTLV-2 were found.Several studies carried out among the indigenous population from the Brazilian Amazon region were developed, and HTLV-2 infection was shown to be prevalent, which is a change in the epidemiological pattern of this HTLV type in this specific group29. A prevalence study conducted among several Brazilian indigenous populations has already identified HTLV-1 infection, and all of them were from the North region of the country. Ishak et al.17 observed HTLV-1 infection prevalence of 2.94%, 0.67%, and 0.48% among Yanomami, Galibi, and Aukre communities, respectively. Besides, a prevalence of 0.62% was found in the Wayampi community in 2002, and one case was reported in the same community in 200730. The absence of more recent HTLV-1 studies in a Brazilian indigenous group may be due to the cultural characteristics of the studied communities since most studies are carried out in closed communities27. The geographic isolation of these villages limits contact with urban centers or other indigenous groups24. However, the Jaguapiru and Bororó villages are located 5 km from Dourados city, the second-largest city of Mato Grosso do Sul state, therefore geographically accessible to the urban center32. HTLV-1 infection was recently reported among Warao indigenous refugees living in urban area of Belém city33. Also, HTLV-1 is already reported in Indigenous people from other South American countries, such as Chile and Venezuela35. There was no difference of positivity for anti-HTLV  between female and male participants, respectively. These results are in accordance with studies conducted among the indigenous population in which no difference between genders was found36.The introduction of HTLV-1 in the Mato Grosso do Sul State probably occurred during the human migratory process from the slave trade from Africa and Japanese migration in the previous centuries23. The positive female individual (ID-763) reported that she was evaluated during her youngest daughter's pregnancy by the antenatal screening. She was diagnosed with positive anti-HTLV-1/2 and was recommended the interruption of breastfeeding since that time. The Brazilian Ministry of Health recommends the interruption of breastfeeding and the provision of milk formula substitutes feeding for those babies born from HTLV-1/2 seropositive mothers to avoid mother-to-child transmission16. Although these measures, adequate counseling with information about HTLV-1 transmission or clinical evaluation to identify signs or symptoms of HTLV-1 associated diseases was not offered to the ID-763 participant. This demonstrates the need to spread knowledge about HTLV infection and the public health policies that are already implemented in Brazil to properly provide counseling for positive patients.HTLV-1 testing has been included in the routine antenatal screening of the state of Mato Grosso do Sul by the Program for Protection of Pregnant Woman since 2002The HTLV-1aA sequences present in this study diverge from the previous studies conducted in Brazil among indigenous populations that found mainly HTLV-2. The sequences obtained from the present study grouped next with other isolates from various non-indigenous populations and geographical regions in Brazil, such as people who use drugs, Afro-descendants, pregnant women, blood donors, and people who live with HIV (PLHIV), suggesting a possible HTLV-1 transmission from these non-indigenous individuals. Unfortunately, sequences from other worldwide indigenous populations belonging to the Transcontinental subgroup (HTLV-1aA) were not found registered on Genbank.Some limitations must be acknowledged. First, underreporting of risk behaviors and reporting bias are some of the limitations of this cross-sectional study. Because some of them may have been omitted during the interviews due to the distrust of discrimination and stigma. In addition, individuals from the micro-areas of indigenous health care were enrolled by convenience sampling, with greater acceptance of the study among residents of the Bororó village, resulting in unequal sample distribution between them. Because of the low number of HTLV infected individuals in this study, regression analysis could not be performed. Despite these limitations, this study is representative of the indigenous population of Central Brazil since we used a large sample size. Moreover, our study has important information which would contribute to closing the gaps in HTLV epidemiology and controlling public policies.37. Also, the absence of HTLV-2 infection among these Brazilian indigenous communities would suggest a behavior pattern distinct from other indigenous populations in Brazil.The results of the study show the presence and circulation of HTLV-1 in indigenous populations from Jaguapiru and Bororó villages in Dourados City, MS. A better understanding of the patterns of occurrence of HTLV-1 in the indigenous population of Mato Grosso do Sul is critical to guiding the development of public health initiatives to reduce transmission. Although a higher risk of infection has been supposed for this specific study population, the prevalence rate of HTLV-1 infection found in this study was similar to that observed among Brazilian blood donors (0.48%)Supplementary Figure S1.


The increased cell membrane permeability and porosity, stimulated transcription and translation of competence, SOS response, universal stress response and ATP synthesis-related genes are also associated with antidepressants-enhanced transformation. This study demonstrated that some antidepressants can speed up the spread of antibiotic resistance by promoting the transformation of ARGs, which emphasizes the necessity to assess the potential risks of antidepressants in spreading antibiotic resistance during clinical antidepressant applications.The development of antibiotic resistance as an unavoidable consequence of the application of antimicrobials is a significant concern for human health. Antidepressants are being increasingly consumed globally. Human gut microbial communities are frequently exposed to antidepressants, yet little is known about the interaction between antidepressants and antibiotic resistance. This study aimed to investigate whether antidepressants can accelerate the dissemination of antibiotic resistance by increasing the rate of the horizontal transfer of antibiotic resistance genes (ARGs). Results demonstrated that some of the commonly-prescribed antidepressants  at clinically relevant concentrations can significantly ( Among tn months . Therefon months , 8, leadeumoniae .Escherichia coli, which led to bacterial mutation conferring multiple drug resistance against fluoroquinolone, aminoglycoside, β-lactams, tetracycline and chloramphenicol [It is well recognized that exposure to non-lethal concentrations of antibiotics have accelerated the development and HGT process of antibiotic resistance , 12. Comphenicol . HoweverAcinetobacter baylyi ADP1 and plasmid pWH1266 which encodes antibiotic resistance against tetracycline and ampicillin. We exposed the transformation assay to six antidepressants at clinically relevant concentrations to test whether the transformation ratio could be significantly enhanced. The underlying mechanisms of promoted transformation ratio were revealed by transformation assays under aerobic or anaerobic conditions, flow cytometry analyses, and genotypic analyses on transcriptional (whole-genome RNA sequencing) and translational  responses. Our findings provide insights into the mechanisms that govern HGT associated with antidepressants, in terms of whether and how they can facilitate the spread of antibiotic resistance via transformation.To evaluate the potential roles of antidepressants in prompting the HGT of ARGs, we simultaneously investigated whether commonly-prescribed and widely-detected antidepressants, including Serotonin-norepinephrine Reuptake Inhibitor (SNRI): Duloxetine; Selective Serotonin Reuptake Inhibitor (SSRI): Sertraline, Escitalopram and Fluoxetine; Norepinephrine-dopamine Reuptake Inhibitor (NDRI): Bupropion; and Atypical Antidepressant: Agomelatine, can promote HGT through transformation. A transformation assay was established based on naturally competent recipient bacterium A. baylyi ADP1 and a competence-deficient mutant ∆comFEBC strain (lifO-lipB::aphA3′ΔcomFEBC::DHFR-1) [tetA) and ampicillin (blaTEM-1) was chosen as the carrier of extracellular ARGs. E. coli DH5ɑ cells containing donor plasmid pWH1266 were incubated in LuriaBertani (LB) medium supplemented with 50 mg/L ampicillin shaking (150 rpm) overnight at 37 °C. InvitrogenTM PureLink® Quick Plasmid Miniprep Kit  was used to extract plasmid pWH1266 from overnight E. coli DH5ɑ culture.A transformation assay was established based on naturally competent recipient bacterium :DHFR-1) . PlasmidA. baylyi ADP1 (ATCC 33305) was incubated in LB broth with shaking (150 rpm) overnight at 30 °C to reach the stable phase. Then, 1% of overnight cell culture was transferred into fresh LB broth and further incubated for 6 h at 30 °C with 150 rpm shaking. After, the culture was precipitated by 5 min 6000 × g centrifuge and washed twice using phosphate buffer solution . The bacteria were resuspended in PBS containing 40 mg/L sodium acetates as the organic carbon source to reach a OD600 of 1.1 for the transformation assay.The recipient All antidepressants including duloxetine, sertraline, escitalopram, fluoxetine, bupropion and agomelatine were purchased from Sigma-Aldrich (USA). Ampicillin was purchased from Gold Biotechnology (USA) and tetracycline was purchased from Sigma-Aldrich (USA).A. baylyi ADP1 as the recipient. In brief, 500 μL transformation assays containing 108 cfu/mL of the recipient (OD600 of 1.1) were mixed with donor plasmid to obtain a final concentration of 0.8 ng/μL. Then, duloxetine, sertraline, escitalopram, fluoxetine, bupropion and agomelatine were dosed into each of the transformation assays to reach final concentrations  and incubated without shaking at 25 °C for 6 h (n = 9). In order to evaluate the correlations between ROS overgeneration and antidepressants-promoted transformation, two parallel transformation assays as described above with the presence of six antidepressants at designated final concentrations  were individually established with the addition of ROS scavenger thiourea (100 μM) and under anaerobic conditions (n = 9). LB agar plates containing 5 μg/mL tetracycline and 100 μg/mL ampicillin were used to quantify the number of transformant cells after a 48 h incubation, while plain LB agar plates were used to quantify the total recipient cell number. The transformation ratio was calculated by dividing the number of transformants by the total number of recipients. The changes in transformation ratios in antidepressant-dosed groups were normalized to the untreated controls to obtain the fold change of transformation ratio.The effects of antidepressants in mediating the transformation of ARGs were evaluated on a model using pWH1266 plasmid as extracellular ARGs, and 90 against tetracycline and ampicillin of A. baylyi recipient and transformants were measured according to a previously described method [A. baylyi by a CytoFLEX flow cytometer  in both aerobic (with and without ROS scavenger) and anaerobic mating systems. Briefly, the cellular ROS detection was conducted by using 2’,7’ –dichlorofluorescin diacetate (DCFDA) kit , following the manufacture’s protocol. Cell membrane permeabilities were measured by staining with 2 mM of propidium iodide . A parallel detection was conducted under anaerobic conditions as mentioned above. The fold change of ROS production or cell membrane permeability was calculated by normalizing the ROS level or membrane permeability of antidepressants-treated samples to those of the corresponding untreated control. The test was conducted at least as biological triplicate.In order to confirm the successful transformation, the ICd method . The effA. baylyi, the percentages of live, damaged, and dead cells after 2 h of various fluoxetine, duloxetine, sertraline, escitalopram, bupropion and agomelatine dosages were quantified by CytoFLEX after duo-stained by LIVE/DEAD® BacLight™ Bacterial Viability Kit . Detailed testing methods are described in Supporting Information.To evaluate the inhibitory effects of antidepressants on blaTEM-1 gene and tetA gene encoded on the pWH1266 plasmid were amplified by using long amplicon PCR primers  and subjected to strand-specific cDNA library construction and Illumina paired-end sequencing  at Novogene. The bioinformatics pipeline was reported in our previous study [To investigate the transcriptional response upon antidepressants exposure, transformation assays were established as described above, but in a larger volume (5 mL). Biological triplicated transformation assays containing antidepressant dosages of 0 mg/L (control), 10 mg/L duloxetine, 1 and 10 mg/L sertraline, 50 mg/L escitalopram, 10 mg/L fluoxetine, 100 mg/L bupropion and 50 mg/L agomelatine were incubated for 2 h at 25 °C. Afterward, the total RNA was extracted by the RNeasy Mini Kit (QIAGENus study  and descn = 3) were extracted using the B-PER method, Digested protein was then analyzed by liquid chromatography -tandem mass spectrometry  (LC-MS/MS). Raw sequencing data were processed by applying Thermo Proteome Discoverer (version 2.2.0.388) towards the database of E. coli MG1655 (received from Uniprot on 12th of July 2019) as described in the Supporting Information. Abundance ratios of each protein between the experimental and the control sample were calculated. A stringency cut-off of false discovery rate  less than 0.01 was applied to identify the proteins with significantly different expression levels.To investigate the translational response upon antidepressants exposure, another set of transformation assays was established the same as described above in RNA sequencing. After static mating for 4 h, proteins from each antidepressant-treated group . Significant differences were analysed by Analysis of variance (ANOVA). A. baylyi’s acquisition of ARGs via transformation, from the concentrations range used in clinical applications (0.01–10 mg/L), to lethal/sub-lethal dosages  against A. baylyi ADP1 (50 and 100 mg/L) as confirmed by live/dead staining  enhance the transformation ratios of A. baylyi ADP1 by ARG-carrying plasmid pWH1266 after 6 h mating. In contrast, all tested dosages of escitalopram and agomelatine could not increase the plasmid uptake ratio of A. baylyi ADP1  under 1 and 10 mg/L duloxetine exposure, when compared to the spontaneous transformation ratio of the control. By comparison, the transformation ratios of A. baylyi ADP1 by plasmid pWH1266 decreased significantly  under the exposure to sub-lethal/lethal dosages of sertraline, fluoxetine and escitalopram (50 and 100 mg/L), as well as duloxetine (100 mg/L)  were tested for their abilities to stimulate the ing Fig.  and IC90ng Table  results.tions 0.0–10 mg/L,DP1 Fig. . For dul/L) Fig. .Fig. 1An90 against Amp and Tet for all the transformants were 15 and 5 folds higher than the recipient, which suggested the acquisition of tetA and blaTEM-1  enhance the generation of ROS in A. baylyi ADP1  out of all six tested antidepressants could significantly , superoxide dismutase (ACIAD0193) and Fe-S cluster  and bacterioferritin  have been significantly  upregulated with a maximum 6.9-fold increase  and the other set dosed with ROS scavenger—thiourea  [n = 9; padj < 0.05) reduce the ROS levels to almost the baseline level of the control group, indicating that all tested antidepressants could not induce excessive oxidative stress to A. baylyi ADP1 with scavenger added or under anaerobic conditions. Meanwhile, the results of antidepressant-mediated transformation experiment under anaerobic conditions  , to elimons Fig.  and withons Fig.  showed nA. baylyi ADP1. Firstly, flow cytometer results showed concentration-dependent increases of membrane permeability on A. baylyi under all antidepressants exposure, except bupropion (only 100 mg/L of bupropion increased the membrane permeability)  Fig. . For dulges Fig. , which cA. baylyi ADP1 showed further evidence of antidepressants-promoted membrane permeability. In transcriptional levels, the majority of the porin component genes was upregulated by exposure to antidepressants  in the translation of outer membrane protein assemble and porin-related proteins under escitalopram and agomelatine exposure  when compared to those levels under aerobic conditions, to approximately the level of the control group without antidepressant exposure.The antidepressants-increased cell membrane permeability and porosity could be associated with the oxidation of membrane lipids due to over-generation of ROS . To inveons Fig. , the memcomEFLN, pilIR), recombination genes  and SOS response-related genes  were up-regulated under the exposure to duloxetine, sertraline, fluoxetine and bupropion, while the corresponding up-regulation was limited under escitalopram or agomelatine exposure , ACIAD2005, DnaK and DnaJ have been up-regulated under the exposure to antidepressants. Meanwhile, the transcription and translation levels SOS response-related genes mostly increased under the exposure to antidepressants, with a maximum increase of 9.9-fold of RuvB protein translation levels when under 100 mg/L bupropion dosage  three orders of magnitude lower than the wild-type A. baylyi  significantly increased the ROS generation and cell membrane permeability of comFEBC- knocked out A. baylyi ADP1  in the transformation ratio of comFEBC- knocked out A. baylyi by pWH1266 plasmid  within the clinical concentration range (0.01 to 10 mg/L) , were foormation , which saumannii . In addinfection .Oxidative stress as one of killing mechanisms of antimicrobials has been associated with the development of antimicrobial resistance . Some anA. baylyi ADP1 recipient increased significantly with the increased concentrations of four antidepressants , which is in correspondence with antidepressants-enhanced transformation. The upregulated transcription and translation of antioxidant, superoxide dismutase and redox active centre-related genes as a defense response to oxidative stress [A. baylyi ADP1. In addition, as indicators against Fe2+-induced oxidative damage, the transcription and translations of Fe-S cluster-related genes and ferric uptake regulator (Fur) genes were also provoked under antidepressant exposure. The causation between antidepressants-induced oxidative and antidepressant-mediated transformation was further verified by eliminating the antidepressants-induced oxidative stress, via: (i) a parallel transformation with the addition of a ROS scavenger, and (ii) a parallel transformation experiment conducted under the anaerobic condition to exclude oxygen. Evidently, when the overproduction of ROS is abolished, previously effective antidepressant dosages could no longer enhance the plasmid transformation. Together, flow cytometry ROS detection, RNA sequencing, proteomics and ROS-precluding experiments results indicate that oxidative stress is essential in antidepressant-mediated transformation increase.In this study, we found that the ROS production in the e stress , furtherA. baylyi ADP1, and upregulated transcription and translation levels of the outer membrane protein assemble-related genes and porin component genes were observed under the exposure to four antidepressants . These increases are associated with the enhancements in antidepressants-induced oxidative stress, upregulated transcription and translation of competence genes and antidepressant-promoted plasmid transformation. When the excessive ROS generation is abolished under the anaerobic condition, both the plasmid transformation ratio and cell membrane permeability return to the control level. Therefore, the enhanced cell membrane permeability was primarily caused by antidepressants-induced oxidative stress.In addition, ROS-caused oxidation of membrane lipids may alter membrane functions and increase the permeability of the membrane . A concecomFEBC- knocked out A. baylyi to conduct a parallel transformation assay. This mutant strain deleted the active DNA uptake machinery of A. baylyi [∆comFEBC A. baylyi, which is similar to the response of wild type A. baylyi. Secondly, the transformation ratio of ∆comFEBC A. baylyi by pWH1266 plasmid was significantly three orders of magnitude lower than the wild-type A. baylyi, further validating that comFEBC operons are essential for the competence of A. baylyi species. However, antidepressants-induced cell membrane damages could not promote the transformation of comFEBC- knocked out A. baylyi by plasmid pWH1266, when the competence machinery is absent. Since ROS overgeneration is the cause of increased membrane permeability, and competent bacteria express a higher membrane permeability for biomolecules like plasmid DNA [In order to further evaluate whether ROS-induced membrane damage is essential for the increase in antidepressants-promoted transformation, we further applied a . baylyi , donor Dsmid DNA , 22, antA. baylyi. Compared to the non-competent state cells, the competent cells can uptake DNA to repair the ROS-induced DNA damages and they can maintain stationary during the competence state [During the exponential growth period of competent bacteria, the competence only develops and expresses by a subpopulation of the cells , 30. Upoce state . In contce state . Consequ90 did not promote the transformation of plasmid due to excessive damage. Under extensive ROS exposure, lipid peroxidation can cause excessive damage to the properties of the membrane and may lead to the increase of cytoplasmic Ca2+ concentration, finally to cell death [Although transformation can be promoted under certain antidepressant stress conditions, our results showed that antidepressants at near-ICll death . TherefoA. baylyi consists of three competence pathways: (i) type IV transformation pilus systems (pil gene family)-mediated exogenous DNA capture; (ii) DNA translocase complex (com gene family)-mediated internalization of single-stranded DNA through the cytoplasmic membrane; and (iii) recombination of exogenous DNA after the homology search [umuD [A. baylyi. In addition, our results identified increased transcription and translation levels of ATP-related genes  [Oxidative stress could also affect the development of competence states in bacteria . The tray search , 34, which [umuD , 36. Thech [umuD . Under tization) , 33 undeA. baylyi ADP1. As effective medical interventions, antidepressants are commonly prescribed to treat the major depressive disorder which has the highest regional occurrence rate of 19% [Our research demonstrated SNRI category antidepressant-duloxetine, SSRI category antidepressant-sertraline and fluoxetine, and NDRI category antidepressant-bupropion at clinical relevant concentration can promote the natural transformation of plasmid-borne ARGs into an opportunistic pathogen e of 19% . The dure of 19% , even poe of 19%  to prevee of 19%  and the e of 19%  and the e of 19%  are knowClostridium difficile infection among 4047 adult patients [n = 63,579; OR = 1.36, 95% CI 1.29–1.44) [C. difficile infection and urinary microbiome resistome.In clinical aspects, antidepressants can circulate in the human body after being absorbed, with half-life ranging from 1 to 2 h for agomelatine, to up to 144 h for fluoxetine  before bpatients . For the29–1.44) . Collect29–1.44) , and medFurthermore, in environmental aspects, antidepressants will be released into wastewater after human consumption and are frequently detected in aquatic environments , 44. WasConclusively, our research demonstrates that antidepressant drugs can significantly promote horizontal gene transfer via transformation, which are associated with antidepressant-induced ROS overgeneration, membrane damage, SOS and stress response. Our findings offer molecular insights into antibiotic-like properties of antidepressants, and recommend further assessing the risk of spreading antibiotic resistance associated with antidepressant therapies and environmental release.Supporting information


The receptors that mediate neurotransmission were organized by the end of the 1970′s into ionotropic and metabotropic receptors by the Nobel laurate Jhon Eccles and his colleague Patrick McGeer . In the American Society for Biochemistry and Molecular Biology (ASBMB) sponsored the first symposium on flux-independent signaling by ionotropic receptors: unforeseen roles, complexities, and challenges. This symposium was held virtually on the 21st of June given the current severe acute respiratory syndrome coronavirus 2 pandemic conditions. The main goal of this event was to gather those independent and scattered efforts carried out by different groups through decades that have demonstrated the mechanism of flux-independent signaling by ionotropic receptors. This in turn should enhance the awareness of its relevance in different scientific fields beyond neuroscience including cellular and molecular biology, biochemistry, and immunology, among others, in which flux-independent channel function may open new perspectives beyond electrophysiology to understand cell function. This is due to the ubiquitously expression of channel receptors in cells that do not elicit action potentials, within and beyond the CNS. At the same time, these fields could provide novel approaches to further investigate this mechanism that has been mostly disregarded but that could be critical to expand our understanding of cell physiology and pathology.Last summer, in 2021, the In this report, the most relevant aspects of this symposium are briefly highlighted, referring the most recent published works and including the research by some of the scientists who did the first observations with glutamate ionotropic receptors and have continued their investigations. Also, recent findings by young investigators are included, regarding how flux-independent signaling by glutamate ionotropic receptors mediate neuronal function in health and disease. In addition, it is depicted how voltage-gated calcium channels (VGCCs) are proposed to mediate different excitation coupled mechanisms through flux-independent signaling. Finally, it is stressed how other non-neuronal cells also express different ionotropic channels such as the nicotinic acetylcholine receptor, the voltage-gated potassium channels (Kv), or the NMDAR that through flux-independent signaling regulate cell function.2+ binding and functional outcomes , who has authored one of the first comprehensive reviews on noncanonical signaling by ion channels , recapitoutcomes . The thioutcomes . The lasoutcomes . Dr Doreoutcomes .2+ flux through pannexins that occurs at resting membrane potentials  explained his findings regarding how flux-independent signaling by the NMDAR is involved in excitotoxicity, how it modulates Src kinase activity that in turn modulates Catentials . He alsoophrenia . In the ophrenia . In the 2+ release from intracellular pools after sensing acidic extracellular pH and how it modulates, in combination with flux-dependent signaling, mitochondria membrane potential through the establishment of plasma membrane–mitochondria bridges that allow mass transfer from plasma membrane to mitochondria, mechanisms that could be critical for astrocyte activation in brain pathology but also probably for cell physiology at discrete domains  explained his findings that described how flux-independent signaling by the nicotinic acetylcholine receptor in monocytes modulates interleukin-1 secretion through alternative agonists such as phosphocholine. She also showed some of the intracellular mediators that are involved in this mechanism . The sec domains .Finally, to close the symposium, conclusions and final comments were made to underpin the most relevant aspects related to flux-independent signaling by ionotropic receptors. Perhaps the most relevant assumption that stems form these findings is the paradigm shift in which ionotropic receptor–mediated signaling is not a linear phenomenon depending necessarily upon ion-flux; instead, it is suggested that ion-flux is one of different possible outcomes from receptor conformational rearrangement. In addition, some molecular mechanisms relevant for this signaling were summarized and include the receptor interactome, posttranslational modifications, or modulators, among others . One of Hopefully, this symposium will also cooperate to setup a new framework to understand ionotropic receptors, their biology, biochemistry, biophysics, and signaling, etc., that in turn will help to boost our comprehension of the cell in health and disease, in the CNS but also beyond the CNS, where channels are ubiquitously expressed but are poorly considered, despite they are there.


To date, social and nonsocial decisions have been studied largely in isolation. Consequently, the extent to which social and nonsocial forms of decision uncertainty are integrated using shared neurocomputational resources remains elusive. Here, we address this question using simultaneous electroencephalography (EEG)-functional magnetic resonance imaging (fMRI) in healthy human participants (young adults of both sexes) and a task in which decision evidence in social and nonsocial contexts varies along comparable scales. First, we identify time-resolved build-up of activity in the EEG, akin to a process of evidence accumulation (EA), across both contexts. We then use the endogenous trial-by-trial variability in the slopes of these accumulating signals to construct parametric fMRI predictors. We show that a region of the posterior-medial frontal cortex (pMFC) uniquely explains trial-wise variability in the process of evidence accumulation in both social and nonsocial contexts. We further demonstrate a task-dependent coupling between the pMFC and regions of the human valuation system in dorso-medial and ventro-medial prefrontal cortex across both contexts. Finally, we report domain-specific representations in regions known to encode the early decision evidence for each context. These results are suggestive of a domain-general decision-making architecture, whereupon domain-specific information is likely converted into a “common currency” in medial prefrontal cortex and accumulated for the decision in the pMFC.SIGNIFICANCE STATEMENT Little work has directly compared social-versus-nonsocial decisions to investigate whether they share common neurocomputational origins. Here, using combined electroencephalography (EEG)-functional magnetic resonance imaging (fMRI) and computational modeling, we offer a detailed spatiotemporal account of the neural underpinnings of social and nonsocial decisions. Specifically, we identify a comparable mechanism of temporal evidence integration driving both decisions and localize this integration process in posterior-medial frontal cortex (pMFC). We further demonstrate task-dependent coupling between the pMFC and regions of the human valuation system across both contexts. Finally, we report domain-specific representations in regions encoding the early, domain-specific, decision evidence. These results suggest a domain-general decision-making architecture, whereupon domain-specific information is converted into a common representation in the valuation system and integrated for the decision in the pMFC. Most strategic decisions occur under considerable uncertainty. For example, when investing in the stock market, a trader may use only purely probabilistic models to estimate risk in the market's fluctuations. In contrast, when negotiating a deal in person, the trader's risk assessment may rely instead on how trustworthy the other party appears . SimilarIn standard economic utility models , the rulWhile these studies add valuable insight into the extent of shared value representations across social and nonsocial contexts, the domain-generality hypothesis has not been fully established, with some studies providing evidence in support of largely dedicated brain networks for encoding social and nonsocial forms of decision uncertainty and for assigning value to different choice alternatives . MoreoveWe argue that these are important considerations in providing a unified framework for integrating social and nonsocial decisions. Correspondingly, to characterize the full cascade of constituent neural processes both the algorithmic  and implementational  levels of social and nonsocial choices need to be considered. This approach, in turn, will offer an opportunity to further ascertain whether the distinction between social and nonsocial choices in the brain is categorical or could be better understood in terms of a shared relationship .To address these issues, here, we design a novel task in which decision evidence in social and nonsocial contexts varies along comparable scales. Importantly, in our task, the social information is carried by the social interpretation of facial features  rather than self-versus-other or group decisions . Across In doing so, we identify, in both contexts, centroparietal EEG signals exhibiting decision dynamics consistent with a common process of EA. Consistent with such domain-general mechanisms, the trial-by-trial temporal variability in these accumulating signals is reflected in the fMRI data in the region of the posterior-medial frontal cortex (pMFC) previously implicated in other types of decisions . MoreoveForty participants were recruited through The University of Glasgow subject pool. Since facial perception may depend on one's race and racial history , particit test on β coefficients from a single-trial regression analysis to examine the effects of task difficulty on choice [reaction time (RT) data] with an α level of 0.05, power of 0.95 to inform our estimated effect size (0.847). This power analysis indicated a minimum sample size of 21 participants. We effectively doubled this estimate (N = 40) to account for the potential removal of participants on the basis of poor behavioral performance and noisy neuroimaging data, given the added challenges of the simultaneous EEG-fMRI acquisition. Our eventual sample size of 31 participants is comparable to previous EEG-fMRI studies in which reliable brain differences were observed in the EEG/fMRI data . We presented all stimuli centrally via an LCD projector (frame rate = 60 Hz) on a screen placed at the rear opening of the bore of the MRI scanner, and viewed through a mirror mounted on the head coil (distance to screen = 95 cm), using Presentation software . These face images were assigned to the five levels of reward probabilities given a “Play” choice , yields a one-dimensional projection  that discriminates between the two contexts:D represents the number of channels, indexed by i, and T indicates the transpose of the matrix. We applied this method to identify w for short (60 ms) overlapping time windows centered at 20-ms interval time points, between −100 and 800 ms relative to the onset of the decision stimulus. This procedure was repeated for each subject and time window separately. By integrating information spatially across the multidimensional sensor space, we increase signal-to-noise ratio while simultaneously preserving the trial-by-trial variability in the relevant discriminating component. More specifically, applied to an individual trial, spatial filters (ws) obtained in this way produce a measurement of the discriminant component amplitude for that trial, which we treat as a neural surrogate of the relevant decision variable that is being integrated.More specifically, our method estimates an optimal combination of EEG sensor linear weights . We replaced the condition-wise covariance matrices with regularized versions of these matrices to counteract potential estimation errors: iS . Note that λ = 0 yields unregularized estimation and λ = 1 assumes spherical covariance matrices. Here, we optimized λ for each participant using leave-one-trial-out cross validation with the following λ values ∈  (λ mean ± SE: 0.067 ± 0.072).To estimate the optimal discriminating spatial weighting vector zA value), using a leave-one-trial-out cross-validation procedure. Specifically, for every iteration, we used N–1 trials to estimate a spatial filter (w), which we then applied to the remaining trials to obtain out-of-sample discriminant component amplitudes [y(t)]. We used these out-of-sample amplitudes to compute the zA. In addition, we determined participant-specific zA significance thresholds (rather than assuming an zA = 0.5 as chance performance) using a subsequent bootstrap analysis whereby trial labels were randomized and submitted to the leave-one-trial-out test described above. This randomization procedure was repeated 500 times, producing a probability distribution for zA, which we used as reference to estimate the zA value leading to a significance level of p < 0.05 (b).To quantify the performance of the discriminator for each time window, we computed the area under a receiver operating characteristic (ROC) curve ], we applied the spatial filter w of the window associated with the highest discrimination performance  across the entire stimulus-locked window (−100 to 800 ms poststimulus) and separately for each of the social and nonsocial contexts as well as the three difficulty conditions . The times course of these discriminating components were then Z-scored separately for each participant and for each of the social and nonsocial contexts.To produce the full temporal profile of the relevant discriminating components  and response times (RT) on individual trials b, we pery(t)] would correlate with sequential sampling model estimates of drift rate that were derived purely from participants' behavior .To offer further validation that the EEG signals we identified through our LDA analysis  were related to the process of EA, we tested the extent to which the build-up rate of the EEG EA signals ; (2) a parametric regressor with amplitude modulation based on individual trial RTs; (3) a parametric regressor with amplitude modulation based on the individual trial task difficulty, not accounted for by the EEG-derived regressor ; and (4) an unmodulated regressor  to account for any additional unaccounted variance in the data, which specifically aimed to capture activity in early evidence representations independent of any other task-related manipulations (a).Our GLM included four regressors of interest for each of the social and nonsocial contexts . More specifically, for each of the social and nonsocial trials, we included (1) an EEG-informed regressor with a parametric amplitude modulation based on the trial-by-trial fluctuations in the rate of EA .Participants' fraction of “Play” choices  correlatncreased a. More ifunction  fit the b), consistent with a positive relationship with task difficulty  = 10.024, p < 0.001; Nonsocial: t(30) = 10.692, p < 0.001). In other words, we observed the longest RTs for the most difficult trials (reward probabilities 0.4–0.6), the shortest RTs for the easiest trials (reward probabilities 0–0.2 and 0.8–1) and intermediate RTs for medium difficulty trials (reward probabilities 0.4–0.6 and 0.6–0.8). The overall RTs showed a small (41.637 ms), albeit significant difference between the social and nonsocial trials (paired t test: t(30) = −3.274, two-sided p = 0.003), with social trials  being on average faster than nonsocial ones .The mean response times (RTs) as a function of the overall reward probability exhibited an inverted V relationship, across both the social and nonsocial trials b, consisHaving established comparable behavioral performance across the social and nonsocial contexts, we asked whether these share a common underlying mechanism for integrating relevant decision evidence. To this end, we aimed to identify correlates of a general process of EA in our EEG data by looking for a reliable ramp-like activity with a build-up rate that is proportional to the amount of decision difficulty .b). On average, the classifier's performance began increasing after 400 ms poststimulus  and peaked several hundred milliseconds later.We used a single-trial multivariate linear classifier  designedFig. 4b, top) revealed comparable centroparietal topographies across social and nonsocial contexts . These similarities are suggestive of common neural generators across the two contexts, consistent with those reported previously in the perceptual domain  from participant-specific windows of peak discrimination between easy and difficult trials b, top rel domain .c, top, Social; bottom, Nonsocial). Similar to the classifier performance, the neural activity began to rise around 400 ms after stimulus presentation in both the social and nonsocial trials, with the build-up rate being proportional to the amount of decision difficulty. Note that the build-up rate from medium difficulty trials was situated between the two extreme conditions used to originally train the classifier, thereby establishing a fully parametric effect across the three levels of decision difficulty  = 16.88, p < 0.001 for the social condition, F = 26.76, p < 0.001 for the nonsocial condition, post hoc paired t tests, all p < 0.001).To formally characterize the temporal profile of the discriminating activity  for each condition separately, we applied participant-specific spatial weights from the time window of peak discrimination to an extended stimulus-locked time window and separately for social and nonsocial trials. This approach revealed a gradual build-up of activity akin to a process of EA in both social and nonsocial trials . Because of the interindividual variability in these onset times, we predicted that re-aligning the relevant signals to the participant-specific EA onset times should reveal a more pronounced depiction of the underlying process of EA at the population level, which was indeed the case .We finally extracted participant-specific EA onset times, that is the time point at which the discriminating activity began to rise monotonically after an initial dip in the data following any early evoked responses present in the data c, insetsr = 0.945, t(154) = 36.464, two-sided p < 0.001, RT: r = 0.754; t(154) = 15.154, two-sided p < 0.001; Nonsocial, Fraction “Play” Choice: r = 0.968, t(154) = 94.196, two-sided p < 0.001, RT: r = 0.765; t(154) = 14.461, two-sided p < 0.001).To further establish that our EEG signals reflect the process of EA leading up to the decision we performed two additional analyses to link these signals with our participants' behavioral performance. First, we expected the build-up rate of the EEG EA signals to correlate with drift rate estimates obtained from a sequential sampling model SSM;  fit on pa; r = 0.653, p < 0.001) and nonsocial trials . To exclude the possibility that our EEG slopes were reflective of changes in the remaining parameters, we also conducted correlations for the other (nonfixed) parameters in our model. As these parameters do not include modulations based on reward probability, we correlated the estimates with the mean EEG slopes per participant and found no significant correlations .In the SSM, we set the decision thresholds for “Play” and “Keep” choices to +1 and –1, respectively . Correspondingly, positive (negative) drift rates are associated with reward probability levels favoring a “Play” (“Keep”) choice. To align the EEG build-up rates  with this convention, we flipped the sign of the EEG slopes in the two reward probability levels which support “Keep” choices ) and nonsocial contexts (Z(max) = 4.39, MNI ). We note that in nonsocial choices the cluster extended more anterior compared with social choices, suggestive of a potential gradient organization within the pMFC associated with EA. We found no domain-specific activations surviving in the direct contrast between social and nonsocial contexts for the EA slope predictor.Consistent with a domain-general EA neural architecture, we identified a region of the posterior-medial frontal cortex (pMFC) correlating negatively with the trial-by-trial EA slope in our EEG-informed predictor c in bothb).To further establish the role of the pMFC in EA across social and nonsocial contexts, we aimed to demonstrate whether its activity exhibits a task-dependent coupling with brain regions encoding the relevant decision evidence and the extent to which this coupling arises from domain-general or domain-specific neural representations. To this end, we ran separate psychophysiological interaction (PPI) analyses for each of the social and nonsocial trials, using the context-specific pMFC clusters as seed and the trial-wise task difficulty as the psychological predictor . We hypothesized that the relevant coupling with pMFC should be negative, as easier trials decrease integration times and correspondingly the overall integrated activity  = 3.54, MNI ; Nonsocial Z(max) = 3.88, MNI ) as well as in dorso-  = 3.5, MNI ; Nonsocial Z(max) = 3.39, MNI ) and ventro-medial prefrontal cortex  = 3.19, MNI ; Nonsocial Z(max) = 3.74, MNI ), consistent with recent resting-state connectivity reports showing negative BOLD correlations between these regions and the pMFC  between the pMFC and regions of the human valuation system. More specifically, we observed activations in posterior cingulate cortex . We identified a distributed set of regions activating stronger for social compared with nonsocial trials, in the right fusiform gyrus (Z(max) = 5.18, MNI ), right amygdala (Z(max) = 4.48, MNI ) and right ventrolateral prefrontal cortex (Z(max) = 5.42, MNI ), collectively referred to as the “face network” (Z(max) = 3.39, MNI ; Fig. 7a). These findings are consistent with the processing of facial characteristics required for mentalizing and inferring the opponent's intentions in the social trials, respectively (Z(max) = 4.26, MNI ; right Z(max) = 3.63, MNI ) as well as in the left dorsolateral preforntal cortex  = 4.12, MNI ; Fig. 7a), which have both been implicated in encoding risk and reward probabilities in nonsocial contexts  we examined the contrast between the unmodulated predictors for the social versus nonsocial trials a. We idenetwork” , as wellectively . In contcontexts . We noteZ(max) = 3.75, MNI ), ventral striatum (left Z(max) = 3.54, MNI ; right Z(max) = 3.88, MNI ) and the posterior cingulate cortex (Z(max) = 3.55, MNI ; Fig. 7b). Consistent with previous reports (Z(max) = 5.09, MNI ), lateral prefrontal cortex (left Z(max) = 4.11, MNI ; right Z(max) = 3.34, MNI ) and anterior insula in both social and nonsocial contexts . We note, that though these results are consistent with a long body of previous work, they are nonetheless important to report here as further validation of the choice of our fMRI analysis design, which offered a reliable account of all relevant experimental manipulations.Task difficulty in our task was reflective of the reward probability associated with a “Play” choice. We, therefore, expected the task difficulty predictor to correlate both positively  with areas known to encode the value of a given choice and negatively  with regions of the human attentional network encoding overall task demands. Across both social and nonsocial contexts, we found positive correlations with regions of the human valuation system such as the ventromedial prefrontal cortex  analysis. This analysis allowed us to investigate the functional coupling of the pMFC with other brain regions to further probe the extent of the “common currency” account introduced above. Unlike some recent accounts, which have argued that social and nonsocial values are computed across different networks , we founWe speculate, that this discrepancy could potentially be attributed to differences in the types of social choices (i.e., choices based on a social cue as utilized here versus the moral choices used by Similarly, differences in social versus nonsocial processing based on self-versus-other decisions  can be aWhile pMFC activity covaried systematically with areas of the human reward network, we nonetheless observed some degree of spatial dissociation across contexts along an anterior/posterior axis within these areas, consistent with recent reports from human and animal work advocating for a gradient-based organization along the medial wall of the brain . SpecifiThe “common currency” account also postulates that before embedding within a domain-general valuation system, social and nonsocial choices might give rise to domain-specific early representations . Here, wConversely, the nonsocial condition was uniquely linked to activity in the lateral intraparietal cortex which has been linked to the encoding of pure reward probabilities both in primates  and humaMore importantly, the proposed “common currency” account is in line with our ability to make choices based on disparate options that ultimately require a common point of convergence in the neural code. Consistent with this idea there has been empirical evidence suggesting that the medial prefrontal cortex has the capacity to both encode the subjective values of individual options as well as re-scale these values into a common neural activity space . In turnIn conclusion, our results offer compelling new evidence that social and nonsocial choices share common neural underpinnings, whereupon domain-specific information is converted into a “common currency” in domain-general valuation areas before being accumulated for the decision in medial frontal cortex. Similarly, our multimodal research approach, including the fusion of EEG and fMRI, offers news opportunities for a more targeted characterization of the mechanistic principles and the neural systems involved in human decision-making under different forms of uncertainty. As optimal decision-making is at the heart of strategic planning, providing a mechanistic account of decision-making under risk and uncertainty could have wider, long-term, socioeconomic impact and facilitate an improved understanding of maladaptive choice behavior.


The growth of ameloblastomas (odontogenic tumours) and odontogenic keratocyst (OKC)  is associated with the expression of proteins related to cell survival and apoptosis. Bcl-2-associated protein X (Bax) and the tumour suppressor protein p53 collectively promote p53-mediated apoptosis. This study aimed to assess the immunohistochemical expression of p53, Bcl-2 and Bax in conventional ameloblastoma (CA), unicystic ameloblastoma (UA) types, and OKC sporadic (OKC-NS/S) and syndromic (OKC-NBSCC).p<0.05.Paraffinized blocks of CA (n=18), UA (n=15), OKC-NS/S (n=18) and OKC-NBSCC (n=15) fixed in 10% formalin were used. After diagnosis, tissue specimens were stained by immunohistochemistry for p53, Bcl-2 and Bax marker. Stained cells were randomly counted in five high power fields. The data analysis was performed via Shapiro-Wilk test, ANOVA with Tukey’s multiple comparisons or Kruskal-Wallis with Dunn’s multiple comparisons. Statistical significance was defined as We did not observe differences between p53 expression in CA, mural UA (MUA), intraluminal/luminal UA (I/LUA), OKC-NS/S, and OKC-NBSCC . Similar results were recognized for Bax expression in CA, MUA, I/LUA, OKC-NS/S, and OKC-NBSCC . However, we recognized significant differences between Bcl-2 expression in OKC-NS/S vs MUA, OKC-NS/S vs I/LUA, OKC-NS/S vs CA, OKC-NBSCC vs MUA, OKC-NBSCC vs I/LUA, and I/LUA vs CA. P53, Bcl-2 and Bax levels were higher in mural morphological areas versus intraluminal and luminal morphological areas in UA.There is a tendency for an increased expression of p53, Bcl-2, and Bax proteins in CA, and mural proliferation of UA, compared to lesions with a cystic morphology, which could be associated with a local aggressive behaviour. Key words:p53, Bcl-2, Bax protein, apoptosis, odontogenic tumour, odontogenic cyst. Apoptosis represents a form of programmed cell death that plays a role in the homeostasis of tissues . HoweverThere are two main apoptotic pathways: the extrinsic and the intrinsic or mitochondrial pathway . P53, BcConventional ameloblastoma (CA), unicystic ameloblastoma (UA) and odontogenic keratocyst (OKC) are common lesions in the jaws. CA is the most frequent of the benign epithelial odontogenic tumours. UA reprThe current 2022 World Health Organization (WHO) odontogenic tumours classification identifies OKC as a nonneoplastic cyst with the potential for aggressive behaviour and a tendency for local recurrence . MoreoveThe expression of p53, Bcl-2 and Bax proteins has been studied in various pathological lesions, especially malignant neoplasms . NeverthIn this fashion, this study aimed to analyse and compare the expression of p53, Bcl-2 and Bax proteins in CA, UA, OKC-NS/S, and OKC-NBCCS as odontogenic lesions with a similar clinical-biological behaviour, but morphologically different, by immunohistochemical study.et al. . Previously, a Cohen’s Kappa (κ) coefficient was performed by two calibrated observers  for the histological diagnosis of CA, UA and OKC. The cases were distributed into four categories: CA (n=18), UA (n=15) according to histologic types: luminal and/or intraluminal UA (I/LUA) (n=7), and luminal and mural and/or intraluminal UA (MUA) (n=8), and OKC (n=33) according to OKC-NS/S (n=18) and OKC-NBCCS (n=15). Two subcategories for UA were considered according to their dissimilar clinicopathological behaviour and treatment, especially for UA with mural proliferation. The diagnosis for NBCCS was made according to Bree and Shah’s criteria  were placed on positively charged slides (CellPath Ltd.). Cases were deparaffinized in xylene for 20 minutes (min) and hydrated in graded ethanol. Next, the slides were placed on distilled water for 10 min. Deparaffinized slides were placed in PBS (pH=7.4±0.2). Subsequently, the slides were immersed in 10mM citrate buffer solution (pH=6.0) for antigen retrieval and were transferred to Oster® Food Steamer until the boiling point. The cases were maintained for 20 min and left for cooling until 21°C. Internal peroxidase activity was blocked using 3% hydrogen peroxide and Mouse/Rabbit Immunodetector Peroxidase Blocker Solution  for 20 min each at room temperature. Primary monoclonal antibodies  from BioNext, the cases were incubated with Mouse Immuno-Detector Biotin-Link and mouse/rabbit Immuno-Detector HRP Label BSB0003) for 20 min each at room temperature. PBS was used as a wash buffer for 30 min when required between each stage. Peroxidase reaction was performed with chromogen 3,3′-diaminobenzidine reagent. The cases were slightly counterstained with Harris’s haematoxylin solution for one minute. Oral squamous cell carcinoma Fig. G, non-Ho for 20 mImmunohistochemical scoring. Odontogenic epithelial cell (OEC) immunostaining was quantitatively assessed by estimating the percentage of stained cells per high-power field (HPF) with magnification of 400x. In UA, quantification of p53; Bcl-2 and Bax immunostaining considered to I/LUA: lining epithelium and intraluminal proliferation, and for MUA: exclusively proliferation mural. Moreover, areas characterized morphologically as luminal, intraluminal, and mural were quantified in each case of UA. Five photomicrographs were captured with a digital camera in an optical microscope  for each case in areas with abundant OEC by HPF. Microphotographs were taken and processed with the LEICA application suite software program (v4.7.1). All images were stored in TIF format. Cells were counted by two independent observers using the ImageJ free software. P53 immunoexpression was assessed for the nuclear staining, instead, Bcl-2 and Bax protein immunoexpression was assessed for the cytoplasmic staining. Stained and total number of OEC were counted in five HPF. The number of positive cells for immunoreactivity was counted and calculated as a percentage relative to the total cells. Previously, we performed the Bland–Altman test  to analyse the interobserver agreement of the immunoexpression count.p<0.05.Statistical analysis. Statistical analysis was performed using R Statistical Software . First, we conducted a heteroskedasticity examination. Next, we analysed the data distribution using the Shapiro-Wilk test. Consequently, we performed statistics using one-way ANOVA with Tukey’s multiple comparison test or Kruskal-Wallis with Dunn’s multiple comparisons with Holm’s correction. Last, we conducted Spearman correlation analysis to observe potential correlations. Statistical significance was defined as Clinical-demographic characteristics. The clinical-demographic features are summarized in Males and females had a similar prevalence. The mean age of prevalence of all studied lesions was 31.61 years with a median of 22.50 years. OKC-NBCSS was the earliest average diagnosis (16.47 years). The location in the mandible (n=56) was significantly greater than those observed in the maxilla (n=10), particularly in posterior areas of CA and UA. Overall, SA was the most symptoms-related lesion.P53 protein expression. We observed a higher p53 expression in CA, MUA, and I/LUA than OKC-NS/S and OKC-NBSCC; however, no statistical differences were found between these groups , A. LikeBcl-2 protein expression. Statistical differences were recognized between OKC-NS/S vs MUA, OKC-NS/S vs I/LUA, OKC-NS/S vs CA, OKC-NBSCC vs MUA, OKC-NBSCC vs I/LUA, and I/LUA vs CA. Bcl-2 immunostaining in CA was cytoplasmic and mainly located in peripheral ameloblast-like cells  or higher Bax expression (ratio <1) was similar between OKC-NS/S, OKC-NBCSS, CA, MUA, and I/LUA B. HoweveThe evasion of apoptosis induces tumourigenesis and tumour progression. The p53 gene is frequently mutated in various human tumours . P53 intThe immunodetection of the p53 protein presents the following considerations: wild-type-p53 protein has a very short half-life in normal cells and is practically undetectable by immunohistochemical staining . HoweverIncreased expression of p53 protein has been related to increased proliferative potential, which is involved in tumoural development and progression in OKC, UA and SA . In our Several studies have analysed the expression of p53 protein in UA ,16, howeIndeed, we found the highest levels of p53 in SA, MUA, and I/LUA. Other studies have reported elevated levels of p53 protein in UA compared to SA, especially for histological types of UA with mural proliferation . These dA distinctive molecular expression pattern has been described for syndromic and non-syndromic OKC . The oveIn our study, immunoexpression of Bcl-2 protein was observed mainly in the basal compartment of the epithelial lining for OKC-NS/S and OKC-NBSCC and in ameloblast-like cells for CA and UA. Higher levels of the anti-apoptotic protein Bcl-2 suggest that inhibition of apoptosis could promote increased cell proliferation in these epithelial cell populations. Likewise, greater immunoexpression of Bcl-2 protein were detected in CA and UA compared to OKC-NS/S and OKC-NBSCC. In other studies, ameloblastoma showed stronger Bcl-2 protein expression than OKC , and higet al.  and focal areas of mural ameloblastomatous proliferation for UA compared to lesions with a cystic morphology (OKC and LUA). Further investigation is required to elucidate the interactions between both proteins and their role in the pathogenesis of odontogenic lesions.