Synergistia and the genus TG5, which are related to periodontitis, were statistically more frequent in the control group. The similar microbiome profiles of medicated diabetics and the control group indicates that the relationship between the T2DM and the oral microbiome might be more related to either the lifestyle/diet rather than diabetes per se. Moreover, this study provides, for the first time, insights into the oral microbiome of a population with a high prevalence of diabetes.Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease that is becoming a significant global health care problem. Several studies have shown that people with diabetes are more susceptible to oral problems, such as periodontitis and, although the causes are still inconclusive, oral microbiota is considered to play a major role in oral health. This study aimed to characterize the oral microbiome of a sample representing T2DM patients from Portugal and exploit potential associations between some microorganisms and variables like teeth brushing, smoking habits, average blood sugar levels, medication and nutrient intake. By sequencing the hypervariable regions V3-V4 of the 16S rRNA gene in 50 individuals belonging to a group of diabetes patients and a control group, we found a total of 232 taxa, from which only 65% were shared between both groups. No differences were found in terms of alpha and beta diversity between categories. We did not find significant differences in the oral microbiome profiles of control and diabetes patients. Only the class  Type 2 diabetes mellitus (T2DM) is a metabolic disease characterized by chronic hyperglycemia caused by defects in insulin secretion, which can contribute to the development of resistance to its action . T2DM isA growing number of studies have been reporting a close association between diabetes and susceptibility for some oral illnesses, such as periodontitis , derivedFirmicutes, Actinobacteria, Fusobacteria, Proteobacteria, and Bacteroidetes dominate the oral microbiota, accounting for 80–95% of the total species  were associated with lower diabetes risk, as they were less abundant among diabetic subjects compared to normal-weight controls. Differences in taxa proportions were also found comparing diabetics and non-diabetics in subjects with caries in Indians  and were focused on the subgingival microbiome  and twenty-five healthy individuals (average age 60) participated in this study . The stuVolunteers were ineligible if they presented less than one-third of the dentition, were under 18 years old, or had taken antibiotics less than 2 months before. All participants were instructed not to brush their teeth after their last meal before the saliva sample collection. In addition, each volunteer was submitted to a questionnaire, through face-to-face interviews, regarding their lifestyle, including information on smoking and oral hygiene habits , food reA validated semiquantitative food frequency questionnaire  was usedDNA was extracted from saliva following To amplify the V3-V4 hypervariable regions, the 341F/805R universal primers were used . A two-sTM dsDNA BR Assay Kit (Thermo Fisher Scientific). All samples along with two negative controls were normalized to 9 nM and pooled with 5 μl of each sample. The two negative controls correspond to an extraction blank and a library blank.The amplicons’ size was checked in 2% agarose gel and purified using the AMPure XP kit according to the manufacturer’s instructions. A second PCR was performed using two indices (i5 and i7) with 7 bp each based on PRJNA679485.A TapeStation 2,200 (Agilent Technologies) was used for the precise sizing and library quantification of the pool, followed by a library validation through a quantitative PCR. Finally, the pool was sequenced in an Illumina MiSeq sequencing platform, using the MiSeq v2 500-cycle sequencing kit (Illumina Inc.), with a 2 × 250 bp paired-end configuration at Novogene facilities. Raw metagenomic data are available from the SRA database with accession number Reads were analyzed using the Quantitative Insights into Microbial Ecology pipeline (QIIME) version 2-2019.7 . The paiFor the taxonomic assignment, we used the Naïve Bayes classifier trained on the Greengenes (version 13.8) . The ASVt-test , with a 5% level of significance, followed by a Bonferroni correction for multiple tests.The taxonomic abundance and ASV relative frequencies were calculated for each sample with QIIME2, for the phylum, class, genus, and species level. The differential taxa abundance between the two groups was evaluated through the Analysis of Composition of Microbiomes (ANCOM) and the Mann-Whitney U-test in SPSS v.25, which was also used to compare the relative frequencies of bacteria associated with the periodontitis between the control and diabetes groups. To perceive if potential differences between the microbiome of both groups could be due to the diet, nutrients consumption and energy intake were compared between groups with the Mann-Whitney U-test or a Student’s Microbiome diversity was evaluated within individuals  with the ASVs abundance and the Shannon diversity index , and betSpearman’s correlation test was used to investigate the correlation between the diabetics’ HbA1c levels and the obtained taxa using the SPSS V.25.Lastly, to investigate the microbiome composition profiles of diabetics and controls, we explored the grouping patterns with: (i) a principal coordinate analysis (PCoA) based on the Bray-Curtis matrix, performed through EMPeror , and ii in R v3 based onA total of 12,754,645 raw reads were obtained with a mean of 255,092.9 reads per sample . No reads were generated from the library blank or extraction blank. After quality filtering and mitochondria and chloroplast removal, 752,526 reads remained for further analyses, with an average of 15,053 reads per sample.Considering the 46 samples included in the analysis, the high-quality reads were assigned to 10,746 ASVs with a total absolute frequency of 605,211. The median ASV frequency per sample was 12,437, with a mean of 13,144.59 . The ASVs were assigned to 232 taxa . The taxWe detected a total of 13 phyla, 21 classes, 37 orders, 60 families, 86 genera, and 51 species. At the phylum level, the oral cavity of all the 46 samples was dominated by Firmicutes (45%), Bacteroidetes (22%), Proteobacteria (16%), Actinobacteria (9%), and Fusobacteria (6%), constituting 98% of the total oral microbiome.Bacilli and Bacteroidia are the dominant classes in both groups, accounting for 50%. Gammaproteobacteria had a higher abundance, not significant (p = 0.241), in the control (9.9%) than in the diabetes group (5.4%) (Betaproteobacteria was higher in the diabetics group (p = 0.033), and Deltaproteobacteria (p = 0.013), Spirochaetes (p = 0.035), Mollicutes (p = 0.043), and Synergistia (p < 0.001) were higher in the control group, nevertheless, after Bonferroni correction, only Synergistia class remained significantly  . Five clficantly .Streptococcus (29%) is the most abundant genus, present in all subjects, followed by Prevotella (14%) and Neisseria (5%).When focusing on the taxa abundance at the genus level, all individuals displayed an average of 32 genera. The frequency distribution of the 10 most abundant taxa, up to genera is shown in TG5 genus remained significant (p < 0.001). TG5 genus belongs to the Synergistia class, the only class that was statistically significant.The 10 most frequent genera accounted for approximately 77% in both groups . Some ofStreptococcus spp. and Prevotella melaninogenica were the most frequent species in both. For the two studied groups, 20 taxa were significantly different .At the species level, an average of 16 different taxa per individual was observed (most taxa were not identified up to species level). The 10 most-frequent taxa (only four were assigned to species level), accounted for 67% in both groups . Streptoifferent , but aftThe Shannon index was 7.03 ± 0.75 for the control group and 6.97 ± 0.66 for the diabetes group, whereas the ASVs abundance was 306 ± 111 and 271 ± 86 for the control and the diabetes group, respectively . We did F = 1.092; p = 0.231) indicating that there is no differentiation in the microbiome composition of the diabetes group compared to the control one. Similar results were obtained with the Jaccard distance values .Bray-Curtis dissimilarity values were calculated to measure the differences between individuals, in terms of taxonomic structure. Streptococcus abundance and secondly by Prevotella melaninogenica and Veillonella dispar and to a lesser extent possibly by Neisseria and Rothia mucilaginosa abundances. Both clusters contain individuals from both groups.The differences between the microbiome profiles of the individuals were visualized through a Principal-Coordinates Analysis (PCoA), from the Bray Curtis dissimilarity matrix, that did not reveal a clear clustering pattern . The hiePrevotella intermedia, Campylobacter rectus, Porphyromonas endodontalis, Treponema socranskii, and TG5 spp.)  , being vol group .We evaluated how these habits affected the oral microbiome of the individuals studied. Regarding the alpha diversity , our resConcerning smoking habits, smokers showed a higher Shannon index, as well as ASVs, when compared to non-smokers.As to the variable age, those from the percentile 33–67% presented the highest Shannon index value, however, the percentile 67–100% was the one with higher ASVs abundance.Females have a higher Shannon index as ASVs abundance than males. Additionally, considering the BMI variable, the obese category has a higher Shannon index as ASVs abundance, followed by the preobese category.However, none of the comparisons between the above-mentioned categories were statistically significant.The PCoA plot colored by the teeth brushing and smoking habits’ categories , S5 did Streptococcus , Granulicatella  and Lautropia  genera and negatively correlated with Oribacterium , and Catonella  genera. After the Bonferroni correction test, only Lautropia and Oribacterium genera remained statistically significant.HbA1c levels were positively correlated with We did not find significant differences in the distribution of the Shannon index for the HbA1c categories  nor for The PCoA plot dyed by the glycemic level categories  and by tWe measured nutrient intake to be able to identify differences in the oral microbiome of diabetes and controls that could be related to diet. However, no significant differences were found regarding nutrients consumption and energy intake between both groups after Bonferroni correction .Prevotella, Streptococcus, and Veillonella genera were responsible for about 50% of the total salivary microbiome, which is similar to our findings. Although at very low frequencies (0.25–0.87%), we found the Gluconacetobacter genus in nine individuals. Interestingly, this genus is not present in the Human Oral Microbiome Database  than the diabetics (183), a trend that is in line with previous studies using saliva samples . Other sActinobacteria was slightly more frequent in the control group, although not statistically significant. This tendency is consistent with Actinobacteria was associated with a decreased risk of diabetes. We did not find differences between both groups for the most abundant classes, Bacilli, and Bacterioidia, in contrast to Gammaproteobacteria abundance was higher but not significant in the healthy individuals, whereas Betaproteobacteria was significantly higher in the diabetes group (p = 0.03). An interesting result was that the TG5 genus (Synergistia class), the only one that showed significant differences between groups, has been related to periodontitis (TG5 just slightly higher in controls. As at the moment of sampling we did not conduct a detailed medical evaluation of the individuals’ periodontal/oral health, and only inquired the individual on the last year history of its periodontal/oral condition, we could not conduct association test between oral health and the presence/absence of a specific microorganism.When we focus on the abundance differences of particular taxa, our results showed that dontitis , failed dontitis , and smodontitis . In our dontitis , are vesConcerning the association between T2DM and the oral microbiome, we did not find clear differences between the microbiome composition of both groups, even though it was possible to identify some taxonomic dissimilarity. The work of https://www.ncbi.nlm.nih.gov/, PRJNA679485.The datasets presented in this study can be found in online repositories. The names of the repository and accession number can be found below: The studies involving human participants were reviewed and approved by the Centro Hospitalar de Vila Nova de Gaia/Espinho’s Ethics Committee. The patients/participants provided their written informed consent to participate in this study.AA-S, AB-P, MG-V, LP-P, and DM-M conceived and designed the study. AA-S and DM-M collected the samples. AA-S and MG-V performed laboratory assays. AA-S and LP-P performed bioinfomatic analysis. AA-S performed statistical analysis and wrote the draft of the manuscript. AA-S, MG-V, and LP-P interpreted the results. MG-V, LP-P, and AB-P supervised the work and revised and contributed to the final manuscript. AB-P contributed with resources and funding. All the authors read and approved the final 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.


Regardless of the disease burden of human papillomavirus (HPV), the vaccine has not been included in the Indonesia National Immunization Program. Since 2017 there was a demonstration program of the HPV vaccination in Yogyakarta Province. This vaccine was given free to female primary school students in the 5th and 6th grades (11–13 years old). This study aimed to assess whether a structured-educational intervention focus on HPV increases the parental awareness, knowledge, and perceptions toward HPV and the vaccine acceptability.We conducted a pre-post structured-educational intervention study from July to August 2017 before the implementation of the HPV vaccination demonstration program, in Kulon Progo District, Yogyakarta Province, Indonesia. Parents of female primary school students grades 5th and 6th were selected using a school-based proportional random sampling. A pediatric resident provided a structured-educational intervention, which consists of the burden and risk of HPV disease, as well as the benefit and safety of the vaccine. Parents were required to complete validated self-administered questionnaires before and after the structured-educational intervention.p < 0.001). HPV vaccine’s acceptability increased from 74.3 to 87.4% (p < 0.001). There was a significant correlation between increasing HPV vaccine acceptability with the improvement of awareness, knowledge, and perception toward HPV infection, cervical cancer and HPV vaccination . After the structured-educational intervention, better knowledge and positive perceptions of HPV vaccination were predictive of HPV vaccine’s acceptability with OR 1.90 (95%CI:1.40–2.57) and OR 1.31, respectively.A total of 506 parents participated. Before receiving the structured-educational intervention, parents’ awareness of HPV infection and the vaccines were low. Only 49.2% of parents had heard HPV infection, and 48.8% had heard about the vaccine. After the structured-educational intervention, there were significant improvements in parent’s awareness, knowledge, and perceptions of HPV infection, cervical cancer, and HPV vaccination (all .A structured-educational intervention may improve parental awareness, knowledge, and perceptions toward HPV and the acceptability of the vaccine. Further study, a randomized control trial with longer follow-up are needed to evaluate the long-term and actual effectiveness of improving parents’ knowledge, perceptions and HPV vaccine acceptability Globally, cervical cancer ranks the third most common cancer in women, with a total estimated 569,847 diagnosed cases of invasive cervical carcinoma with 311,365 deaths from the disease annually . CervicaAs one proactive way to increase HPV vaccine coverage, the Global Alliance for Vaccines and Immunization (GAVI) supports low-middle income countries in Southeast Asia, including Indonesia, to conduct the HPV Demonstration Program .  The HBecause children in this age need parental consent for the vaccination, the acceptance of the HPV vaccine is highly dependent on the knowledge, perceptions, and approval of their parents , 8, 9]..9]. A syThe WHO recommended countries which are introducing the HPV vaccine into their NIP should invest in a comprehensive communication plan to first build community awareness and acceptance for the vaccine and the program. This plan includes clear program and communication objectives, understanding community knowledge, attitudes and practices, and defined target audiences. Communication efforts should reach all key target audiences, especially parents . ParentsTo our knowledge, no study in Indonesia has assessed the impact of a structured-educational intervention on parental knowledge, perceptions, and acceptability of HPV vaccination. This study aimed to evaluate whether a structured-educational intervention about HPV risks and the HPV vaccine increases the parental awareness, knowledge, and perceptions regarding HPV and the HPV vaccination acceptability.We conducted a one group pre-test post-test design study in KulonProgo District, one of districts in Yogyakarta Province, Indonesia, where the demonstration program was implemented. The study was conducted from July to August 2017, before the implementation of the HPV vaccination demonstration program. This research was part of a larger study assessing knowledge, perceptions, and acceptability of parents and teachers regarding HPV related to the disease and the vaccine. Parents of female primary school students grades 5th (11–12 years old) and 6th (12–13 years old) were selected using a school-based proportional random sampling. A study information statement was sent to the parents, and they were invited to participate in the study. We excluded parents who cannot read and write.After collecting socio-demographic and baseline data on knowledge, perceptions, and acceptability on HPV infection and the vaccine, a structured-educational intervention was provided to groups of parents in the classroom. Each group consisted of 40–50 parents. The researcher developed the intervention based on literature review and using the framework of the health belief model (HBM), which emphasize that a person’s perception, such as the perception of disease severity, susceptibility, barriers and benefits, influence the intention and actual health-seeking behavior . The intData were collected using validated self-administered questionnaires, which consisted of awareness (4 questions), knowledge and perceptions about HPV infection (14 and 6 questions), knowledge and perceptions about cervical cancer (3 and 2 questions), and knowledge and perceptions about HPV vaccination (5 and 2 questions). For the assessment of knowledge scores, if the answer was correct then it was given a value of 1 and if the answer was incorrect then a value of 0. The total score of awareness ranged from 0 to 4, the score of knowledge on HPV infection ranged from 0 to 14, the score of knowledge on cervical cancer ranged from 0 to 3, and the score of knowledge on HPV vaccine ranged from 0 to 5. Questions about perceptions used a 5-point Likert scale, ranging from strongly disagree  to strongly agree . The total score of perception on HPV infection ranged from 6 to 30, the score of perception on cervical cancer ranged from 2 to 10, and the score of perception on HPV vaccine ranged from 2 to 10. Acceptability was measured by a dichotomous item asking whether they are willing their daughters to get the HPV vaccine, free of charge (yes or no answer options). The full item questioner was presented in supplementation File , seven parents were excluded due to inability to read and write, resulting in a total data set of 506 participants. The majority of the respondents are mothers (92.8%), with a median age of 40 years, and the majority are Moslem (98.2%). Forty-three participants (8.8%) experienced a history of any type of cancer in their family, and 4.4% mentioned that their daughter had already received the HPV vaccination  72.7%) and cervical cancer (73.7). However, only 46.2% of them had ever heard about HPV infection, and 46.2% of them mentioned that HPV caused cervical cancer. The majority (76.5%) of respondents already knew that the vaccination could prevent infection, but only 44.1% of parents had ever heard of the HPV vaccination , and every woman was at risk of having cervical cancer (72.6%). However, the perception of the risk of having HPV infection was low, with only 40.3% strongly agree or agree that they are at risk of having HPV infection. The perceptions of the benefit and safety of the HPV vaccine were high; 78.6% of respondents strongly agree/agree that they believe HPV vaccination is useful for preventing cervical cancer, and 69.7% of them strongly agree/agree that they believe HPV vaccine is safe  and OR 1.31(95%CI:1.05–1.63), respectively , scientifically safe (97.0) and effective in preventing the disease (97.7%). The three most important considerations were free of charge, the vaccine is halal, and the vaccine is effective.Considerations for accepting HPV vaccinations were free of charge (according to 95.5% of respondents), the vaccine was included in NIP (according to 97.1% of respondents), the vaccine is considered safe by Moslem standards or p < 0.001(Table p < 0.001). There was a significant correlation between increasing vaccine acceptability with improvement of awareness , knowledge of HPV infection , cervical cancer  and HPV vaccination  and perceptions of the HPV infection , cervical cancer  and HPV vaccination .After the structured-educational intervention, there were significant improvements in parents’ awareness, knowledge, and perceptions of HPV infection, cervical cancer, and the vaccine, all 001Table . ParentaThis research was a school-based study to assess whether a structured-educational intervention can improve parental awareness, knowledge, and perceptions about HPV infection, cervical cancer, and HPV vaccination as well as acceptability of HPV vaccination. Before the intervention, most of the parents had poor baseline knowledge and some negative perceptions of HPV. After the intervention, there was a significant improvement in parents’ HPV-related knowledge and perceptions as well as their intention to permit the HPV vaccine for their children.At the baseline, we found that most of our participants had heard about STIs and cervical cancer, but less than half of them had heard about HPV infection and the vaccine. A previous qualitative study assessing parental knowledge and perceptions of HPV and cervical cancer prevention in rural Central Java, Indonesia 2015) found that most respondents have limited knowledge of HPV and the vaccine as well as the relationship between HPV and cervical cancer  found th. Since tThere were several factors related to the acceptability of HPV vaccination. From the regression analysis, we found that better knowledge and positive perceptions of the HPV vaccination were significantly associated with higher acceptability. In line with our study, previous reports have shown that parents’ knowledge about HPV was positively correlated with HPV vaccination acceptability , 12. Perhalal (permissible under Islamic Shariah Law) were important considerations for accepting the vaccine. Including HPV vaccine in the NIP and providing health insurance could eliminate the cost barriers. Because the majority of our study population is Muslim, the halal issue is an important consideration for accepting the vaccine. Previously, the non-halal status was reported as a barrier to the rotavirus vaccine acceptability in Yogyakarta, Indonesia. Engagement of religious leaders is needed to facilitate the bridge between immunization programs and the community, particularly during the introduction of a new vaccine [We found that besides the vaccine’s benefits and safety, free of charge, the vaccine being included in NIP, and the vaccine considered  vaccine .Earlier studies found that parents need HPV-related information to decide whether to accept or refuse the HPV vaccination , 13, 30.Previous studies conducted in more developed countries showed the effectiveness of digital media/ website to educate parents. A randomized cluster trial, parents of 11–17 years old girls watched a digital video outlining the risks and benefits of the vaccine was conducted in Indiana, United States. It showed that adolescents whose parents watched the video had a 3-times higher odds of receiving a dose of the HPV vaccine .. Anothehow to facilitate an interactive discussion”.In developing countries such as Indonesia, which internet access is not always available, providing a more “conventional” education would be more feasible and acceptable. Our study found that a structured-education intervention using a standard PPT presentation followed by an interactive discussion focused on HPV disease and the vaccine improved parents’ knowledge, perception, and acceptability. This result emphasized the effectiveness of “conventional” educational interventions to target consumers/ parents in improving the acceptability of vaccines proven by previous studies. A hospital-based, brief, HPV-focused intervention, using a PPT presentation, improved the awareness and acceptability of HPV vaccine among rural Chinese woman’s . Another. Another systematic review conducted by Radisic et al. (2017) found that uptake of HPV vaccination may be facilitated by encouraging health care provider endorsement [The structured-educational intervention conducted in our study, in the form of a presentation and interactive discussion by professional health educators, was shown to significantly improve the parents’ knowledge and perceptions about HPV. Giving educational campaigns could increase parental knowledge and perceptions about HPV, concurrently with physician recommendations and nationally funded vaccination programs , 28. A sorsement .This research has some limitations. This one group pre-test -post-test design study without a control group did not allow us to confirm any causal relations between the intervention and outcomes. A pre- and post-test without follow-up could not determine the long-term effectiveness of the educational intervention on HPV vaccination acceptability. Further study, a randomized control trial with longer follow-up are needed to evaluate the long-term and actual effectiveness of improving parents’ knowledge and perceptions about the HPV vaccination as well as the vaccine acceptability. The strength of this study is that it assessed the effectiveness of an education intervention before the implementation of the HPV vaccination demonstration program, which included a representative sample of parents in Kulon Progo, a rural District. The results of this study can provide important evidence to the policy-makers about the importance of health education.A structured-educational intervention may improve parental awareness, knowledge, and perceptions toward HPV as well as the acceptability of the vaccine. There was a significant correlation between improving the parental knowledge and perceptions of HPV vaccine with increasing acceptability of the HPV vaccine. A structured-educational intervention designed for parents may have important implications for improving vaccine acceptability. Further study, a randomized control trial with longer follow-up are needed to evaluate the long-term and actual effectiveness of improving parents’ knowledge, perceptions and HPV vaccine acceptability.Additional file 1: Table 6 The full item questioner


To identify risk factors associated with intubation and time to extubation in hospitalized patients with coronavirus disease 2019 (COVID-19).Retrospective observational study.Ten hospitals in the Chicago metropolitan area.Patients with laboratory-confirmed COVID-19 admitted between March 1 and April 8, 2020, were included. We evaluated sociodemographic and clinical characteristics associated with intubation and prolonged intubation for acute respiratory failure secondary to COVID-19 infection.P < .001) and rate of diabetes  as compared with nonintubated patients. Multivariable logistic regression analysis identified age, sex, respiratory rate, oxygen saturation, history of diabetes, and shortness of breath as factors predictive of intubation. Age and body mass index were the only factors independently associated with time to extubation.Of the 486 hospitalized patients included in the study, the median age was 59 years ; 271 (55.8%) were male; and the median body mass index was 30.6 . During the hospitalization, 138 (28.4%) patients were intubated; 78 (56.5%) were eventually extubated; 21 (15.2%) died; and 39 (28.3%) remained intubated at a mean ± SD follow-up of 19.6 ± 6.7 days. Intubated patients had a significantly higher median age (65 vs 57 years, In addition to clinical signs of respiratory distress, patients with COVID-19 who are older, male, or diabetic are at higher risk of requiring intubation. Among intubated patients, older and more obese patients are at higher risk for prolonged intubation. Otolaryngologists consulted for airway management should consider these factors in their decision making. Existing literature has described the clinical presentation of COVID-19 to be similar yet distinct from prior comparable coronaviruses, such as Middle East respiratory syndrome and severe acute respiratory syndrome.5 However, risk factors associated with the need for mechanical ventilation among those infected with COVID-19 are unclear. While preliminary reports from the Centers for Disease Control and Prevention suggest that >70% of hospitalized patients have ≥1 underlying health conditions, only 5.8% of reported cases contain information on comorbidities.6 Moreover, the relative importance of different underlying health conditions and sociodemographic factors in the disease course have yet to be determined from currently available studies due to inadequate adjustment for possible confounding factors.7In the United States, the rapid transmission of the virus, which can also be spread by asymptomatic individuals, has led to a sharp increase in infections in a short period, straining the health care system. Of significant national concern is the limited supply of mechanical ventilators and the number needed to adequately satisfy the demand from the US population.As COVID-19 infections continue to rapidly consume the US health care system’s limited resources, identifying populations at risk of a more severe disease course is critical for otolaryngologists, who may be called on to manage the airway of patients with COVID-19 in the hospital or decide which intubated patients are candidates for a tracheostomy. In this study, we aimed to identify individual risk factors associated with intubation among hospitalized patients with laboratory-confirmed COVID-19 in the Chicago metropolitan area, as well as time to extubation among intubated patients.8 A confirmed case was defined as a positive result on a reverse transcriptase polymerase chain reaction assay of a specimen collected by a nasopharyngeal swab. Patients were included if they were aged ≥18 years and were admitted to any of the 10 hospitals in the Northwestern Memorial HealthCare system spread across the Chicago metropolitan area  between March 1 and April 8, 2020. Hospitalized patients with documented “do not resuscitate and do not intubate” (DNR/DNI) orders and those who left the hospital against medical advice were excluded from the study cohort. Patients who had missing data on investigated predictor variables and did not reach a clinical endpoint of intubation or discharge from the hospital were also excluded. Clinical outcomes, such as mortality, discharge, intubation, and extubation, were last recorded on April 18, 2020.After approval by the institutional review board at the Northwestern University Feinberg School of Medicine, data on hospitalized patients with laboratory-confirmed COVID-19 infection during or prior to admission were identified and compiled from the Northwestern University Enterprise Data Warehouse, an integrated repository of clinical data for Northwestern-affiliated health care centers.Sociodemographic information was collected, including age, sex, race, ethnicity, and history of tobacco use. Symptom and clinical history obtained on the day of admission, including recent travel and contact with individuals infected with COVID-19, was manually extracted from the medical record. Vital signs included systolic and diastolic blood pressure, heart rate, respiratory rate, maximum temperature, lowest documented oxygen saturation, and body mass index (BMI) measured in the emergency room. Medical history included all comorbidities documented on admission.Comorbid disease was classified into diabetes, hypertension, cardiovascular disease, organ transplantation, immunosuppression, cancer, obstructive sleep apnea, pulmonary disease, and chronic kidney disease. Cardiovascular disease included myocardial infarction, cerebrovascular accident, congestive heart failure, valvular heart disease, and arrhythmias. Pulmonary disease included asthma, chronic obstructive pulmonary disease, and interstitial lung disease. Patients with no history of the aforementioned comorbidities were classified as “none of the above.”Chest radiographic imaging results were reviewed on the day of admission and classified as having positive findings if the radiologist documented any evidence of ground-glass opacity, consolidation, or infiltrates.The primary outcome of this study was intubation with an oral endotracheal tube and attachment to a mechanical ventilator during the hospitalization. The secondary outcome was time to extubation, defined as the number of days from intubation to extubation.P values were considered significant at <.05. See the online appendix for details regarding the statistical methods.Univariate analysis was performed with Wilcoxon rank sum tests for continuous variables and chi-square or the Fisher exact test for categorical variables. A random forest model was used to predict intubation by entering all collected variables into the model via repeated cross-validation with internal down-sampling to account for imbalance between cohorts. Logistic regression of selected variables of importance from the random forest model was performed to report odds ratios (ORs). Leave-one-out cross-validation was performed to assess the discrimination of the final logistic regression model. For analysis of time to extubation, the Kaplan-Meier method and log-rank tests were used to explore the relationship between baseline variables and outcome. A multivariable analysis was performed with Cox proportional hazards regression models stratified by location of hospital, in which (1) demographics and medical history were considered covariates and (2) deaths and patients still intubated at the time of last follow-up were censored. All statistical analyses were conducted with version 3.6.0 of the R programming language . All reported This study included 564 unique hospitalized patients admitted between March 1 and April 8, 2020, who tested positive for COVID-19 on a polymerase chain reaction assay. After review of the medical records, 78 of these patients were excluded because they had a DNR/DNI order, left the hospital against medical advice, had missing BMI or smoking data, or had not been intubated or discharged at the time of censoring Figure 1P < .001), male percentage , and prevalence of diabetes . Of the other comorbidities queried, there were no significant differences between intubated and nonintubated patients.Among these hospitalized patients, 138 (28.4%) were intubated and required invasive mechanical ventilation. Intubated patients had a higher median age  or contact with a confirmed individual with COVID-19 infection (26.7%). The most commonly reported symptoms were cough (77.4%), fever (75.7%), shortness of breath (70.6%), and fatigue (55.8%).P < .001), respiratory rate >24/min , temperature >100.4 °F , and oxygen saturation <90% . Intubated patients were also more likely to have evidence of opacities, infiltrates, or consolidation on chest radiographs .As compared with nonintubated patients, intubated patients were more likely to present with shortness of breath , antibiotics (67.7%), and hydroxychloroquine (55.1%); 161 (33.1%) were transferred to the intensive care unit (ICU); 399 (82.1%) were discharged; and 22 (4.5%) died during their admission. Among patients who were not intubated, 45 (12.9%) had a hospital stay >10 days; 347 (99.7%) were discharged; and 1 (0.3%) patient died.The first intubation for a patient infected with COVID-19 occurred on March 7, 2020 , which regulates inflammation and is already present in lower levels in the elderly, has been proposed as a possible mechanism leading to higher disease severity.16 ACE2 is also a component of Leydig cells within the male testes, leading to a potential site of infection and viral safe harbor, which may contribute to the higher percentage of male patients with COVID-19 as compared with females.17 However, further investigation is necessary to elucidate how age and sex influence clinical presentation.In our study, advanced age was strongly associated with intubation and time to extubation, after adjusting for confounding factors. Age was reported as a predictor of mortality in Chinese and Italian populations. Patients at least 65 years old in Wuhan, China, were identified as being at higher risk of mortality in a prospective cohort of 179 patients.18 The United States has one of the highest rates of obesity in the world, with 40% of adults having a BMI ≥30 and 9.2% having a BMI ≥40.20 Initial reports from New York City have suggested that obese patients may have a higher likelihood of being admitted and placed on mechanical ventilation.21 The impact of obesity on severity of disease is not surprising. Obesity is already associated with decreased pulmonary function, and with the addition of COVID-19 injuring lung tissue, adequate ventilation would inevitably become more difficult.22 Biologically, obesity is also associated with higher levels of inflammatory cytokines, which may be exacerbated by the release of TNF-α and IL-6 from infected pneumocytes and pulmonary cells.24BMI was also a significant risk factor predictive of time to extubation among intubated patients with COVID-19. In the literature, BMI had not been initially discussed as extensively as other risk factors in the COVID-19 pandemic, likely due to obesity not being as prevalent in the Italian and Chinese populations as in the United States.11 Similarly, in a national Chinese study, chronic obstructive pulmonary disease, hypertension, and diabetes were identified as risk factors associated with admission to the ICU, placement on invasive mechanical ventilation, or death.25 Diabetes had a higher prevalence in intubated patients in our study and, after adjustment for confounding factors, was the only comorbidity found to be independently associated with intubation in our study population. While the pathophysiology of the interaction between diabetes and COVID-19 disease is unknown at this time, metabolic disorders have been associated with the impairment of macrophages and lymphocytes.26 As a result, diabetic patients may be at higher risk of an inadequate immune response to a COVID-19 infection. Further research is necessary to explore this possible relationship.The most common comorbidities among patients in this study were hypertension, diabetes, and cardiovascular disease, which are consistent with 2 case series of hospitalized patients with COVID-19 in New York.27 Our cohort of intubated patients had a lower case fatality rate of 15.2%, which is similar to a study from New York City, which had a case fatality rate of 14.6% among those requiring intubation and 10.2% among admitted patients.4 In our study, overall mortality of admitted patients was noticeably much lower than other studies at 4.5%, but that is likely attributable to our exclusion of patients with DNR/DNI status, who in general had a high mortality rate due to baseline poor clinical status and refrainment from aggressive care.The overall case fatality rate in the literature ranges from 1.4% to 2.3%, while ICU mortality varies from 26% to 67%, although current reports likely underestimate overall inpatient mortality, given the unknown outcome of a high percentage of reported cases, which applies to our study as well.2830 However, the optimal timing of tracheostomy in the COVID-19 population and whether there is any benefit to an early tracheostomy as seen in other diseases are unknown at this time.31 In our study, most deaths occurred within 2 weeks of intubation, and many patients continue to remain intubated beyond 2 weeks. A portion of these patients will very likely need tracheostomies for long-term ventilation and weaning from ventilation. The timing and outcomes of tracheostomy in the COVID-19 population require further study.The outcomes of tracheostomy for intubated patients with COVID-19 are unclear at this time. Open tracheostomy has a high risk of transmission of COVID-19 to health workers, as it is an aerosol-generating procedure. Several guidelines and checklists have been developed to protect otolaryngologists from infection during these procedures.There are several limitations to this study that should be considered. First, several patients in this study were still hospitalized at the time of last follow-up; as a result, some clinical outcomes were not known, such as mortality. Second, deciding when to intubate a patient is a complex clinical decision based on many factors, and different health care providers may have different criteria for deciding when to intubate patients with COVID-19. While this study included patients treated at several hospitals, the results might not be generalizable to other institutions. Third, laboratory results were not collected and could contribute as additional predictive factors for intubation and prolonged intubation. Fourth, although data were derived directly from medical records, they were originally collected for clinical care and thus suffer from rapidly evolving practice guidelines, which may lead to bias. We utilized several statistical methods to reduce bias and confounding effects. Last, the retrospective design of this study precludes the ability to draw causal conclusions.Age, male sex, and a history of diabetes were independent risk factors associated with intubation in hospitalized patients with COVID-19 in the Chicago metropolitan area. Time to extubation was influenced by age and obesity only. Further research and data from other institutions are warranted to more accurately characterize this novel disease.Click here for additional data file.Supplemental material, Appendix_5.1.20 for Factors Associated With Intubation and Prolonged Intubation in Hospitalized Patients With COVID-19 by Kevin Hur, Caroline P. E. Price, Elizabeth L. Gray, Reeti K. Gulati, Matthew Maksimoski, Samuel D. Racette, Alexander L. Schneider and Ashoke R. Khanwalkar in Otolaryngology–Head and Neck Surgery


Despite immense advancements in treatment modalities, cancer remains a dreadful disease until the present. The major influencing factors behind the increased mortality rate of cancer are increased drug resistance and severe adverse effects caused by conventional cancer therapies. To overcome these limitations, the current medical field is focusing more on natural phyto-derived molecules to mitigate cancer. Mangosteen is a phytotherapeutic with potent anti-inflammatory and antioxidant properties. In the present study, we investigated the anticancer potential of the crude ethanolic extract of mangosteen against two dreadful forms of cancers, namely, oral cancer and cervical cancer, Garcinia mangostana or mangosteen was removed, air-dried, ground to fine powder, and macerated with ethanol. The extract obtained was then filtered and extracted with water for 48 h. The aqueous fraction thus obtained was then concentrated with a rotary evaporator at 40°C and dried with a freeze dryer. The anticancer efficacy of these extracts was investigated in human tongue squamous cell carcinoma (H357) cells and cervical cancer cells (HeLa) using the MTT assay, TUNEL assay, western blotting, and flow cytometry techniques.The pericarp of The crude mangosteen pericarp extract (MPE) significantly inhibited the growth of H357 and HeLa cells in a dose-dependent manner. Moreover, mangosteen induced early apoptosis in these cells after 48 h of incubation. Mangosteen also upregulated the expression of pro-apoptotic proteins, including caspases and Bax, and downregulated the expression of anti-apoptotic protein Bcl-2.The MPE exerted significant cytotoxicity against the H357 and HeLa cells in a dose-dependent manner and promoted their apoptosis. Hence, this natural phytoextract can be considered a potent anticancer agent for treating oral cancer and cervical cancer.  Oral cancer is widespread in the Indian subcontinent, with India contributing 7.8% to the global cancer burden and 8.3% of global cancer deaths . Oral cancer is the sixth most common cancer in the world  and the second most common cancer in men after lung cancer . The majority of oral cancers (75%) are linked to excessive use of tobacco and alcohol consumption. Cervical cancer in women continues to be the leading cause of death worldwide, despite efforts for the early detection of cervical cancer . It is the fifth most common cancer in the world. In India, cervical cancer is the second most common cancer and accounts for 1/4 of all cervical cancer deaths . Oral cancer and cervical cancer thus continue to cause increased levels of mortality and contribute to the economic burden in the Indian subcontinent. Cancer still remains a disease that is difficult to treat, with only a 20% cure rate after treatment. India contributes to about 1/3 of global cancer deaths. The conventional modes of treatments include surgery, radiation, and chemotherapy . These treatment modalities induce a significant amount of morbidity in treated individuals with the additional drawback of frequent recurrences. In addition, these treatments are too expensive and cause severe adverse effects, thus lowering the quality of life after treatment . There is hence a dire need to explore novel herbally derived molecules that can act as supplements in treating oral cancer and thus can overcome the drawbacks. Garcinia mangostana, a fruit native to South East Asia, is used in classical ayurvedic formulations. The pericarp of the fruit is recommended for the treatments of diarrhea, dysentery, and skin infections. It is also used as an astringent in the treatment of gum diseases . The fruit rind or pericarp of mangosteen possesses anticarcinogenic property based on in vitro studies on liver, breast, colorectal, lungs, cervical, and, prostate cancer cell lines . Mangosteen exerts its anticancer potential by promoting cell cycle arrest, inhibiting cell proliferation, inducing apoptosis, and abrogating tumour cell adhesion, migration, and metastasis . The pericarp of mangosteen is rich in bioactive agents, such as xanthones, flavonoids, procyanidin, and benzophenones, which are said to contribute to its anti-inflammatory, antibacterial, and anticancer effects . The percentage of xanthones is high in the pericarp rather than the pulp of the fruit . Among the xanthones, alpha-mangostin, beta-mangostin, gamma-mangostin, mangostinone A, mangostinone B, and mangostenol are present abundantly in the pericarp. These xanthones are responsible for the anticancer action of mangosteen on leukaemia, prostrate, breast, and colon cancer cell lines .in vitro. The pro-apoptotic potential of MPE on the HeLa and H357 cell lines was evaluated by TUNEL  assay, annexin / propidium iodide (PI) flow cytometry, and western blot analysis of proteins involved in the mitochondrial apoptotic pathway. Although the chemoprotective potential of the mangosteen pericarp extract (MPE) has been widely studied in different types of cancer, the cytotoxic effects of this herbal medicine against the most dreadful forms of cancers, including oral cancer and cervical cancer, are not yet known. Hence, the present study explored the role of MPE on the human oral squamous cell carcinoma cell line of the tongue (H357) and cervical cancer cell line (HeLa) Preparation of MPEG. mangostana or mangosteen was botanically authenticated by a pharmacognosist from Siddha Central Research Institute (G17120701M). The pericarp of the fruit was removed, air-dried, and ground to a fine powder in a lathe. Then, 100 g of the pericarp powder was macerated with ethanol and allowed to stand for two days. The ethanolic extract was then filtered, and the dried residue was extracted with distilled water for 48 h and stored in a dark closed glass container. The obtained aqueous fraction was then concentrated with a rotary evaporator at 40°C and dried with a freeze dryer. The dried fraction was weighed and stored in Teflon-capped tubes at -20°C.The study protocol was submitted to the Institutional Review Board and Institutional Ethical Clearance of MAHER University, and approval for the study (MAHER-MU-004-IEC/2016) was obtained. Chemicals and reagentsThe chemical and reagents used for this study were of molecular biology grade and were procured from HiMedia and Sigma Aldrich, USA. Dulbecco’s modified eagle medium , fetal bovine serum , streptomycin and penicillin, and phosphate-buffered saline  were procured from HiMedia, France. PI , APO-DIRECT™ Kit , and FITC Annexin V Apoptosis Detection Kit I , were procured from BD Biosciences, India.Phytochemical analysisThe mangosteen pericarp samples were analysed to detect the presence of bioactive compounds, such as tannins, saponins, flavonoids, alkaloids, proteins, steroids, quinones, terpenoids, and cardiac glycosides.Cell culture 2 until they reached 80% confluence. H357  and HeLa  lines were obtained from the American Type Culture Collection  and maintained in DMEM supplemented with 10% FBS, 100 µg streptomycin, and 100 µg penicillin at 37°C in a humidified incubator under 5% of COMTT assay6 cells were seeded into a 96-well plate and allowed to grow for 48 h. Then, the cells were treated with varying concentrations of the MPE  and incubated for 24 h at 37°C in a 5% CO2 atmosphere. After the incubation period, the effect of the MPE on cell viability was assessed as per cent cell viability and compared with vehicle-treated control cells, which were arbitrarily assigned 100% viability. The lowest concentration required to cause 50% cell growth inhibition (IC50) was determined via an interpolation from the dose response curve. The IC50 was employed as an optimal dose for studying the anticancer properties of MPE in HeLa and H357 cell lines. This experiment was performed in triplets, and the average values were estimated.The effect of mangosteen on the viability of H357 and HeLa cells was assessed by 3-4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay . Briefly, 1 × 10Detection of DNA fragmentation by TUNEL assay6 cells were treated with the MPE for 48 h. After the incubation period, the supernatant and adherent cells were collected in a centrifuge tube and washed twice with 1× PBS. The cells were fixed with 70% ice-cold ethanol by incubating at -20°C for 30 min. The cells were resuspended in 1 ml of wash buffer and washed twice via centrifugation. 50 μl of the DNA labelling solution was added, and the cells were incubated at 37°C for 60 min. The cells were rinsed with 1.0 ml of the rinse buffer and centrifuged, and the supernatant was removed. The cell pellet was resuspended in 0.5 ml of a PI/RNase staining buffer and incubated for 30 min at room temperature. Controls were included in the study; for the negative control, TdT was omitted. Flow cytometry was performed to detect the DNA fragmentation.TUNEL assay identifies DNA breaks by labelling the 3’-OH terminal of DNA with modified nucleotides to TdT or terminal nucleotidyltransferase. Briefly, 1 × 106 cells/well) were seeded in a 6-well plate and treated with MPE for 48 h. After the incubation period, the cells were harvested, washed twice with PBS, gently resuspended in 100 μL annexin V-FITC binding buffer (1×), and incubated with 5 μL annexin V-FITC in dark for 10 min at 25°C. The cells were then centrifuged at 2,000 rpm for 5 min and gently resuspended in 500 μL annexin V-FITC binding buffer (1×), and 5 μL PI was added in an ice bath. The tagged cells were immediately analysed via flow cytometry through the CellQuest software (BD Biosciences). Flow cytometry: HeLa and H357 cells  at 4°C. The membranes were then incubated for 1 h with respective secondary antibodies at room temperature. Finally, the membranes were incubated with chromomeric substrate 3,3′-diaminobenzidine (DAB), and the bands were visible within 3–5 minutes of incubation.Statistical analysis The Kolmogorov–Smirnov and Shapiro–Wilks normality test results reveal that all variables do not follow a normal distribution. Therefore, to analyse the data, non-parametric methods were applied. To compare values between the H357 and HeLa groups, the Mann-Whitney test was performed. To analyse the data, SPSS  was used. The significance level was fixed as 5% (α = 0.05). The Mann–Whitney test for MTT showed that the MPE significantly inhibited the H357 and HeLa cells at varying concentrations of 0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, 100, 200, and 250 μg. The results of the TUNEL assay were also statistically significant for the MPE-treated H357 and HeLa cell lines. In the apoptotic assay using annexin V and PI, the results were statistically significant only in live cells, early apoptosis, and necrotic cells in the H357 and HeLa cell lines.Phytochemical analysis: Phytochemical analysis showed that the MPE is rich in tannins, flavonoids, proteins, steroids, and cardiac glycosides .50 value was found to be 9.8 µg/ml. Hence, 9.8 µg was used for further investigation. Similarly, in HeLa cells, mangosteen exhibited an IC50 value of 7.5 µg/ml. Hence, 7.5 µg was used for further studies on HeLa cells , and the ICLa cells .Detection of DNA fragmentation by TUNEL assay TUNEL assay measures the DNA strand breakdown and provides information on cell apoptosis. Flow cytometry analysis: Annexin V-FITC and PI flow cytometry assay were conducted to evaluate the pro-apoptotic potential of MPE. Here, the exposure of the phospholipid layer of the cell membrane in H357 and HeLa cancer cells after treatment with MPE were visualized by its binding to annexin V. Annexin V/PI dual staining showed that the MPE could trigger early apoptosis, late apoptosis, and necrosis in HeLa cells . HoweverWestern blottingWestern blot analysis was performed to study the effect of mangosteen pericarp on the expression of caspase 3, caspase 9, Bcl-2, and Bax proteins in H357, and HeLa cells . There wDespite the efficient mitigation of cancer cells by conventional cancer drugs, chemotherapy is always associated with inadvertent damage to adjacent normal cells . Hence, the search for an ideal anticancer therapy that selectively kills the tumour cells without causing any adverse effect to the host cells still continues. Naturally obtained products are considered a boon for cancer treatment due to their effective action on cancer cells and minimal cytotoxicity to normal cells . In this regard, the mangosteen extract, which is rich in xanthones, such as alpha-mangostin, gamma-mangostin, mangostinone, and gartanin, has been proven to be an effective anticancer agent . The therapeutic property of mangosteen is attributed to the presence of xanthones, a flavonoid present in the pericarp. Interestingly, our phytochemical analysis also shows that the MPE is rich in tannins, flavonoids, steroids, proteins, and cardiac glycosides. The study investigated the chemoprotective effects of ethanolic crude MPE on H357 and HeLa cells. Mangosteen has been found to severely affect the viability of these cells in a dose-dependent manner. The treatment with the MPE resulted in a steady decrease in the count of viable cells as the extract concentration was increased from 1.56 to 250 μg/mL. The findings of the study were in accordance with those in the studies performed by Kwak et al. wherein alpha- mangostin, a mangosteen derivative, altered the cell viability in human oral squamous cell carcinoma cell lines, namely, HSC2, HSC3, and HSC4, in a dose- and time-dependent manner.17G. mangostana with cellulose) and GME-EC/MC (G. mangostana with ethyl and methyl cellulose) polymeric nanoparticles on HeLa cell lines was studied. This finding was also confirmed to be dose dependent. As an added advantage, introducing mangosteen as a nanoparticle also improved the bioavailability of mangosteen and increased its cellular uptake . Compared with the effect of the MPE on the H357 and HeLa cell lines, the MPE at concentrations between 1.56 and 250 μg/mL was significantly more cytotoxic to the HeLa cell line than to the H357 cell line.As observed in H357 oral cancer cells, the cytotoxicity of the ethanolic MPE on HeLa cell lines has also been shown to increase dose dependently from 1.56 to 250 μg/mL. The same results have also been obtained with alpha-mangostin and, the standard anticancer drug cisplatin on HeLa cell lines was evaluated. Cell lines were preincubated with alpha-mangostin for 24 h, followed by coincubation with cisplatin for 24 h. In both cases, the cell viability showed a cytotoxic effect in a concentration-dependent manner. In addition, the alpha-mangostin was protected from cytotoxicity at an extremely low concentration of 5–15 μM . In another study, mangosteen extract in the form of GME-MC (The TUNEL assay results showed that the MPE can trigger apoptosis in H357 and HeLa cell lines (p < 0.0495). To further monitor the progression of apoptosis and identify the cells in various stages of apoptosis, annexin V/PI assay was performed. Our results show a significant increase in cells showing early apoptosis in H357 and HeLa cells when compared with the control cells. This finding, in turn, provides direct evidence that MPE can induce early apoptosis, which can be attributed to the increase in membrane permeability and mitochondrial dysfunction. The regulation of apoptosis is performed by complex interactions between various proteins. Some of these proteins, such as Bax, promote apoptosis, and others, such as Bcl-2, are antiapoptotic in function. Caspases are key enzymes controlling apoptosis. Caspase 3 has a critical role in the apoptotic process and is activated by caspase 9. The expressions of Bax, Bcl-2, caspase 3, and caspase 9 were detected by the western blot analysis. The MPE induced an increased level of expression of pro-apoptotic protein Bax and decreased level of expression of antiapoptotic protein Bcl-2 in a concentration-dependent manner in both cell lines. The levels of caspase 3 and 9 also increased with an increase in the concentration of the MPE from 6.25 and 12.5 µg to 25 µg/ml. In a previous study, it was inferred that alpha-mangostin induces cell death through the generation of ROS, which activates the ASK/p38 signaling pathway leading to the rupture of the mitochondria, increased release of Bax and cytochrome C, and decreased level of Bcl-2. This causes the activation of the caspase-9/caspase-3 cascade, causing apoptosis of cervical cancer cells .Although various studies have evaluated the anticancer effects of mangosteen on HeLa cell lines, this is the first study to investigate the chemotherapeutic potential of the MPE on H357 oral cancer cells. Our study has shown that crude alcoholic MPE is effectively cytotoxic to H357 cell and HeLa cell lines. The MPE causes DNA fragmentation in both cell lines, indicating the intrinsic pathway of apoptosis to be involved in cell death. In this study, an increase in early apoptosis can be attributed to membrane permeability and mitochondrial dysfunction. Furthermore, an increased level of Bax, caspase 3, and caspase 9 with a decreased level of Bcl-2 confirm the involvement of the intrinsic and extrinsic pathways of apoptosis. In conclusion, the present study demonstrates that ethanolic crude MPE is cytotoxic and can induce apoptosis on oral and cervical cancer cells. These results prove that mangosteen pericarp can act as a potential therapeutic anticancer agent. However, further research into the molecular mechanism involved and on in vivo models will provide substantial evidence to the anticancer potential of MPE.


For the taxonomic assignment using 16S rRNA, three major 16S rRNA databases are independently used since the lineage of certain bacteria is not consistent among these databases. On the basis of the NCBI taxonomy classification, we re-annotated inconsistent lineage information in three major 16S rRNA databases. For each species, we constructed a consensus sequence model for each hypervariable region and determined homologous species groups that consist of indistinguishable species in terms of sequence homology. Using a k-nearest neighbor method and the species consensus sequence models, the species-level taxonomy was determined. If the species determined is a member of homologous species groups, the species group is assigned instead of a specific species. Notably, the results of the evaluation on our method using simulated and mock datasets showed a high correlation with the real bacterial composition. Furthermore, in the analysis of real microbiome samples, such as salivary and gut microbiome samples, our method successfully performed species-level profiling and identified differences in the bacterial composition between different phenotypic groups.With the emergence of next-generation sequencing (NGS) technology, there have been a large number of metagenomic studies that estimated the bacterial composition via 16S ribosomal RNA (16S rRNA) amplicon sequencing. In particular, subsets of the hypervariable regions in 16S rRNA, such as V1–V2 and V3–V4, are targeted using high-throughput sequencing. The sequences from different taxa are assigned to a specific taxon based on the sequence homology. Since such sequences are highly homologous or identical between species in the same genus, it is challenging to determine the exact species using 16S rRNA sequences only. Therefore, in this study,  Metagenomics has been widely used to analyze microbial communities without cultivating strains . MoreoveUsing 454 pyrosequencing  and IlluThe 16S rRNA analysis pipeline involves preprocessing, clustering [operational taxonomic units (OTU) picking], assigning taxonomy, and estimating the bacterial composition. Although most of the sequencing errors are filtered out at the preprocessing step, there are still some sequencing errors that remain. To overcome these errors and strain variations, processed reads are clustered into OTUs using a 97 or 99% sequence similarity threshold. Since sequences belonging to the same OTU are considered to be derived from the same clade, OTU clustering directly affects the estimation of bacterial composition. Therefore, several clustering algorithms have been developed to overcome strain variation and sequencing errors. For example, the UPARSE algorithm  clusterstaxonomic separability was measured for the V1–V2 and V3–V4 hypervariable regions at the genus and species level. For each species, we constructed consensus sequences for each hypervariable region and determined indistinguishable species. By comparing the consensus sequences of each species, homologous species groups in which the species share high similarity were constructed for each hypervariable region, which was then used for the species-level taxonomy assignment. The evaluation performed using simulated datasets and mock datasets showed a high correlation with the real bacterial composition. Moreover, when analyzing real microbiomes, such as the salivary and gut microbiome, our method successfully performed species-level profiling to identify differences in bacterial composition between different phenotypic groups.In this study, we re-annotated the inconsistent or mislabeled taxa in the three 16S rRNA databases on the basis of the NCBI taxonomy classification. The 16S rRNA sequences were combined from the re-annotated GreenGenes, SILVA, and NCBI databases to include species that exist exclusively in each database or were recently annotated. In the evaluation of taxonomy classification, the classifier trained with all three databases showed the best accuracy in terms of precision and recall rates. Moreover, To investigate the taxonomy consistency, GreenGenes v13.5, SILVA v132, and RDP v11.5 databases were used. As quality control, sequences whose length range in three times the standard deviation from the mean without any ambiguous nucleotide  were used. Out of 1,242,330, 1,861,373, and 3,196,041 sequences obtained from GreenGenes, SILVA, and RDP databases, respectively, 1,191,315, 1,779,305 and 1,559,121 sequences were retained for the re-annotation after quality control process .To apply the latest version of NCBI taxonomy, NCBITaxa class in the ete3 python package  was usedEscherichia and Shigella species have essentially identical 16S rRNA sequences, sequences labeled as Escherichia or Shigella were collectively labeled as Escherichia.Shigella.Each 16S rRNA sequence was re-annotated using the taxon at the lowest taxonomy rank in the database . To iden1. The information for each genome is listed in the assembly_summary_refseq.txt file . In the generic feature file (GFF), the regions where the feature is described as “rRNA” and the product as “16S ribosomal RNA” were identified as the 16S rRNA sequences and extracted from the genome. Thus, we obtained 78,270 16S rRNA sequences from the 16,337 genomes analyzed. As quality control, the same filtering step was performed for the sequences extracted and 77,803 16S rRNA sequences were retained to train the classifier and generate consensus models.The genomes assembled at the complete-level or chromosome-level were downloaded from the ftp site of the NCBI RefSeq databaseThe 27F/308R and 337F/806R primer pairs are widely used to target the hypervariable regions V1–V2 and V3–V4, respectively, for Illumina MiSeq amplicon sequencing. The fragment sequences of the hypervariable regions were simulated by extracting the sequences between the forward and backward primers from the 16S rRNA sequences using cutadapt . MoreoveTo determine which species are distinguishable by their 16S rRNA sequences, sequence similarities between species belonging to the same genus were calculated. A consensus sequence of the strains belonging to the same species was obtained using the “cons” function in EMBOSS v6.6.0  with thehomologous species groups that shared 99% or higher sequence similarity were constructed. The species in a homologous species group were considered indistinguishable by their 16S rRNA sequences. To name the homologous species group, the species in the group with the largest number of strains were selected and extended with a “+” sign.On the basis of the sequence similarity of the consensus sequences, Amplicon sequences for the V3–V4 hypervariable region were simulated from the bacterial reference genomes using Grinder . To targThe 16S rRNA genes were sequenced using the Illumina MiSeq sequencer, and paired-end reads were generated and merged on the basis of their overlapping region. Each read pair was assembled using FLASH  with theThe classification of the 16S rRNA sequence was performed according to the conventional classification approach . PreprocMycobacterium was annotated as Actinomycetales in the GreenGenes and RDP databases but as Corynebacteriales in the SILVA database. The order of Corynebacterium was also annotated as Actinomycetales in the GreenGenes and RDP database but as Corynebacteriales in the NCBI taxonomy classification. Taxonomy reclassification also resulted in inconsistent taxonomic lineage annotation among the three databases. For example, Propionibacterium was originally identified as Bacillus but was later renamed as Propionibacterium , 907,944 (51%), and 1,275,668 (82%) sequences were re-annotated in this study, respectively . As a reAfter the re-annotation, the sequences from GreenGenes and SILVA databases were used in our classification method . The RDPFor the genus-level taxonomy assignment, the RDP naïve Bayesian classifier was retrained with the sequences re-annotated in this study. Classifiers were tested using the V3–V4 region sequences extracted from the NCBI database. In the evaluation, the classifier trained with our combined database showed the best performance in terms of precision and recall rates from the phylum to genus level . NotablyMuribaculum and Duncaniella were first reported in the NCBI repository in July 2016 and March 2018, respectively. Notably, the classifier trained in our study correctly predicted the sequences as Muribaculaceae at the family rank, suggesting that the relative abundance of Duncaniella is similar to that obtained via metagenomic analysis. Although Duncaniella was well classified, Muribaculum was still reported with a low confidence score. This result suggests that there might be some genera belonging to the Muribaculaceae family that are still unknown.To evaluate the classification performance for the newly annotated bacteria, the gut microbiome of mice  were protaxonomic separability  using the V1–V2 region, the V3–V4 region, and the entire 16S rRNA gene. OTUs were created using a 99% sequence similarity threshold to measure the proportion of OTUs that were assigned to multiple taxa  was also observed (Staphylococcus aureus and Staphylococcus epidermidis (labeled in blue and red) were connected. Such strain-level heterogeneity could be caused by either distinct strains in a specific species or incorrect annotation.In the tococcus . Notablyobserved . For exak-nearest neighbor method, the species was determined by considering the most k homologous species. In this study, k was set to 1 among the sequences with >97% sequence similarity and an e-value of <1.0e-10. When no hit met the criteria, it was reported as unclassified. If the assigned species were from the homologous species groups, the query sequence was labeled as the name of the homologous species group.To assign a species-level taxon to the OTUs, the representative sequence of each OTU was searched against our species consensus sequence database using BLAST search . SimilarCandida albicans ATCC MY-2876 was not included since it is a fungus. Four simulated datasets were generated with the abundance models of uniform, linear, and power-law parameters with 1 and 2 (Methanobrevibacter and Propionibacterium (Cutibacterium) were excluded from the simulation since the 806R primer could not extract the region sequences from their genomes. The simulated datasets were analyzed using our species-level profiling method.To test the performance of our species-level profiling method, simulated datasets were generated using a set of bacteria reported as the constituents of the Human Microbiome Project (HMP) gut microbiome . The rep 1 and 2 . MethanoBacillus cereus, Bacteroides vulgatus, Clostridium beijerinckii, Escherichia coli, Lactobacillus gasseri, Listeria monocytogenes, Neisseria meningitidis, Pseudomonas aeruginosa, S. aureus, S. epidermidis, and Streptococcus pneumoniae created the homologous species groups with other species. For instance, the V3–V4 region of B. cereus was identical to that of Bacillus mobilis. These species are technically indistinguishable in terms of their V3–V4 region. Similarly, the V3–V4 sequence of S. pneumoniae differs by only one nucleotide from that of Streptococcus infantis. Notably, our method based on the homologous species groups was able to accurately estimate the species-level composition in the simulated datasets  under the accession number ne index . Howeverne index . Notablys groups . With thStreptococcus OTUs were identified from the sample data by considering the size of the OTUs : two OTUs with the S. pneumoniae group, three OTUs with the Streptococcus gordonii group, one OTU with the Streptococcus sanguinis group, one OTU with the Streptococcus thermophilus group, and one OTU with the Streptococcus parasanguinis group  assigned to the S. gordonii group also showed a similar pattern: they were equally similar to three species in the S. gordonii group. As shown in this case study, many OTUs were indistinguishable among the species in the species group but were distinguishable among the species group. Most of the Veillonella OTUs were assigned to the Veillonella parvula, Veillonella dispar, or Veillonella atypica group (V. parvula group, OTU 853 and OTU 276 were equally similar to multiple species in the group. These results might be inevitable when hypervariable regions are used at the species level. In addition, some novel species that are not stored in the 16S rRNA database but are equally similar to multiple known species could exist in the microbiome.In total, eight major is group . Two OTUca group . In the SRP131074. Bacteroides, Escherichia, and Prevotella were reported as the most abundant genera in the previous study that analyzed these samples (Megamonas, Pseudomonas, Morganella, Aeromonas, Megasphaera, Fusobacterium, Veillonella, Roseburia, Sutterella, Subdoligranulum, and Eubacterium was found to differ by threefold between any two samples from the control, adenoma, and cancer groups (Pseudomonas, Veillonella, Fusobacterium, and Aeromonas OTUs were assigned to the homologous species groups. Notably, Aeromonas and Fusobacterium were the most abundant in the samples from the cancer group. For the Aeromonas OTUs, most of the dominant OTUs in the cancer group were assigned to either the Aeromonas veronii or Aeromonas caviae group (Fusobacterium mortiferum, Fusobacterium necrophorum, and Fusobacterium nucleatum were found to be abundant in samples from the cancer group, whereas Fusobacterium ulcerans was abundant in samples from the adenoma group (homologous species groups could produce more specific and reliable information, which is higher resolution than the genus-level, to show differences in bacterial composition among patient groups.In total, 105 gut microbiome samples, consisting of 35 samples each from control, adenoma, and cancer patients, were downloaded from the SRA under the accession number  samples . Our res samples . Among tr groups . Althougae group . Moreovema group . Therefohomologous species groups, which was used for the species-level taxonomy assignment. The evaluation using simulated datasets and mock datasets showed a high correlation with the real bacterial composition. When analyzing real gut microbiomes, our method successfully performed species-level taxonomic assignment and identified differential abundance between different phenotypic groups.In the microbiome studies, one of the important tasks is profiling of the bacterial composition, which helps understand the biological functions of the microbiome. The species-level taxonomic assignment is critical, but an optimal solution has not been available thus far since the 16S rRNA sequences are highly homologous between the species in the same genus in many cases. We combined all the sequences from the GreenGenes, SILVA, and NCBI databases to include species that exist exclusively in each database. Even in the evaluation of genus-level taxonomy classification, the classifier trained with the sequences combined showed the best accuracy in terms of precision and recall rates. For each species, we constructed a consensus sequence model and determined https://sourceforge.net/projects/reannotated-16s-rrna-databases/files/.The original contributions presented in the study are included in the article/H-JG designed and performed the data analysis and wrote the manuscript. MR designed and supervised the data analysis and wrote the manuscript. Both authors critically reviewed the manuscript and approved the final version.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.


Mindfulness is connected to positive outcomes related to mental health and well-being. However, the psychological mechanisms that account for these relationships are largely unknown. A multiple-step multiple mediator structural equation modeling (SEM) model was tested with mindfulness as the independent variable; purpose in life and behavioral activation as serial mediators; and happiness, anxiety, and depression as outcome measures. Data were obtained from 1267 women. Higher mindfulness was associated with higher levels of happiness and lower anxiety and depression symptoms. The association of mindfulness with the outcome variables could be partially accounted for by purpose in life and behavioral activation. The SEM model explained large proportions of variance in happiness (50%), anxiety (34%), and depression (44%) symptoms. Mindfulness is associated with both a sense of purpose in life and engagement in activities, which are also connected with positive outcomes. Moreover, having purposes in life is linked to higher levels of behavioral activation. The positive effects of mindfulness have been highlighted by a growing number of publications. Mindfulness-based interventions have demonstrated efficacy in the treatment of depression, anxiety, and stress-related disorders, among many other psychological problems ,2,3. In Essentially, mindfulness involves the self-regulation of attention so that it remains focused on the present moment. Along with this self-regulation, mindfulness also requires a focus on the immediate experience with an attitude of curiosity, openness, and acceptance . How canPurpose in life may be defined as an elementary aim that serves to self-organize and inspire concrete goals, trigger behaviors, and provide meaningfulness assessments . Having Interestingly, the presence of meaning and purpose in life has been found to be positively associated with mindfulness. For instance, increased mindfulness has been found to be positively associated with greater presence of meaning in life, a relationship that could be explained by increased self-awareness , and greSupport for the connections between mindfulness and increased purpose in life comes not only from correlational research but also from experimental studies. For instance, Jacobs et al.  found thPurpose in life has been proposed to be a central construct to explain well-being- and health-related outcomes . People Having purposes in life is somehow considered a motivation to greater behavioral activation. The construct of meaning in life entails a motivational component ,18, whicBehavioral activation may somewhat explain the relationship between purpose in life and potential mental health and well-being-related outcomes. First, life purposes operate as a motivational component . IndividOn the basis of previous literature on mindfulness, purpose in life, behavioral activation, and well-being and mental health-related outcomes, our study aimed to test a model that integrates the complete patterns of relationships among these variables. This model entails the following hypotheses:Hypothesis 1 (H1). Mindfulness is expected to be positively correlated with happiness (H1.1) and negatively associated with anxiety (H1.2) and depression (H1.3) measures.Hypothesis 2 (H2). Higher levels of mindfulness will be connected with a greater sense of purpose in life (H2.1) and behavioral activation (H2.2).Hypothesis 3 (H3). Purpose in life will be positively connected with happiness (H3.1) and negatively associated with anxiety (H3.2) and depression (H3.3) measures.Hypothesis 4 (H4). Higher behavioral activation is expected to be associated with higher levels of happiness (H4.1) and lower scores in anxiety (H4.2) and depression (H4.3).Hypothesis 5 (H5). Higher levels of purpose in life are expected to correspond to higher levels of behavioral activation (H5).Taken as a whole, the abovementioned hypotheses suggest some possible mediating effects that will be tested:Hypothesis 6 (H6). The associations between mindfulness and happiness (H6.1), anxiety (H6.2), and depression (H6.3) may be accounted for, to some extent, by both purpose in life and behavioral activation.Hypothesis 7 (H7). The link between mindfulness and behavioral activation may be accounted for, to some extent, by purpose in life (H7).Hypothesis 8 (H8). The associations between purpose in life and happiness (H8.1), anxiety (H8.2), and depression (H8.3) may be accounted for, to some extent, by behavioral activation.SD = 14.85) and ranged from 18 to 70 years. Eighteen Latin American Spanish-speaking countries and Spain were represented. Most of the participants came from Venezuela (44.2%), Nicaragua (10.3%), Bolivia (8.4%), Paraguay (5.6%), the Dominican Republic (5.4%), and Argentina (5.2%). Other nationalities represented less than 5%. Concerning education, 14% of the participants reached the postgraduate level, and 52.2% completed graduate studies. Professional training and high-school education represented, respectively, 15.4% and 16.3%, with elementary studies being reported by 1.9% of the sample. More than one-third of the participants (38.8%) were active workers, 14.1% were unemployed, 32% were students without a job allowing for economic autonomy, 7.7% were retirees, and 7.3% reported other labor situations. With respect to the participants’ attitudes toward religion, most (56.2%) characterized themselves as “nonpracticing believers”; 27.7% reported being believers involved in religious practice; and 16.1% defined themselves as atheists, agnostics, or indifferent concerning religious belief.Our sample comprised 1267 women. Their mean age was 33.76 years . In total, 1528 questionnaires were received. However, 155 of them were discarded because they were duplicated submissions (57 questionnaires) or responses out of the age range of this research, i.e., younger than 18 years (82 questionnaires) or older than 70 years (16 questionnaires). Questionnaires from male individuals (106 submissions) were not considered in this study in order to avoid potential problems due to the unequal distribution of gender among respondents. Therefore, female respondents participating in this study represent 82.92% of the received questionnaires.Mindfulness. The participants’ capacity for paying attention to experiences and being fully aware of internal and external stimuli while being focused in the present moment was measured by means of the Mindful Attention Awareness Scale (MAAS), developed by Brown and Ryan  , developed by Kanter et al.   is a 4-item scale used to measure the global level of perceived happiness ; SpanishAnxiety and depression symptoms. The Hospital Anxiety and Depression Scale (HAD), developed by Zigmond and Snaith, was used  (Spanishr bivariate correlations were obtained to assess the associations among variables.Descriptive statistics (means and standard deviations) were calculated for the study variables. Pearson’s BC) bootstrap confidence intervals (percentile method) for the indirect (mediated) effects were calculated using 5000 bootstrap samples. A statistically significant mediated effect, different from zero with 95% confidence, is obtained if zero is not between the lower and upper bound of the BC confidence interval for the indirect effects. The ratio of the indirect effect to the total effect (MP) was calculated as an effect-size measure for mediation effects. Although this ratio cannot be properly interpreted in terms of the proportion of the explained variance in total effects that is due to the indirect effect [Following Hayes ,67, boott effect , it is uM1) and behavioral activation (M2), and 3 outcome variables: happiness (Y1), anxiety (Y2), and depression (Y3). Therefore, a multiple-step multiple mediator model was used [a, b, and/or c’ paths were omitted [Our hypotheses entailed 2 mediators, purpose in life  below 10, tolerance values higher than 0.2, and condition indexes clearly below 15, allowed us to safely conclude that there was no collinearity in the data, according to the usual criteria ,72.All analyses were carried out using the statistical package IBM SPSS 19 and AMOS 16 .SD = 4.36), which yielded a 95% CI with 8.95 and 9.43 as lower and upper limits, respectively. According to Bjelland et al. [Participants presented moderate levels of mindfulness, with average scores around the midpoint of the response scale . Similard et al. , a cut-oAge was moderately correlated with all of the focus variables of interest, yielding positive associations with mindfulness, purpose in life, behavior activation, and happiness, as well as presenting negative relationships with anxiety and depression .As presented in Finally, as expected, happiness was negatively and statistically significantly associated with anxiety and depression, whereas significantly positive correlations between psychological symptoms were observed.After controlling for the effects of age, mindfulness scores significantly predicted happiness, anxiety, and depression, thus confirming the existence of significant total effects that may be mediated . MindfulThe proposed mediators also yielded significant total effects on the outcome variables . After cBootstrap-corrected confidence intervals for the indirect effects of mindfulness through purpose in life and behavioral activation confirmed significant mediation effects on happiness (H6.1), anxiety (H6.2), and depression (H6.3) .The abovementioned significant connection between mindfulness and behavioral activation was proposed to be mediated through purpose in life (H7). Here, two additional conditions were met. As presented above, mindfulness was also associated with purpose in life. Second, purpose in life yielded a significant total effect on behavioral activation , after cAs previously mentioned, purpose in life was a significant predictor of happiness, anxiety, and depression after controlling for age and mindfulness. This relationship was hypothesized to be mediated by behavioral activation. The required conditions were met because purpose in life was a significant predictor of behavioral activation after controlling for age and mindfulness; additionally, behavioral activation significantly predicted the outcome variables , after controlling for age, mindfulness, and purpose in life. Bootstrap bias-corrected 95% confidence intervals indicated a significant indirect (mediated) effect of purpose in life on the outcome variables through behavioral activation , thus sua, b, or c’ were omitted.The saturated SEM model of the multiple-step multiple mediation  always fWith respect to the amount of variance in the outcomes variables that were accounted for by the multiple-step multiple mediator model, 50%, 34%, and 44% of variations in happiness, anxiety, and depression, respectively, were explained.Our study contributes to shedding light on the potential mechanisms that may connect mindfulness and healthy outcomes. We found that individuals who reported higher levels of mindfulness also showed higher levels of happiness and lower anxiety and depression symptoms. In this regard, the negative correlations found between mindfulness and depression and anxiety are highly consistent with correlations previously found ,56,74. AHow is mindfulness connected with positive outcomes? We hypothesized a series of sequential associations between mindfulness, having purposes in life, and increased behavioral activation. In this regard, mindfulness may lead people to clarify values around which to organize their lives and gain awareness of those things that truly matter in life, i.e., to discern their purposes and find meaning in life. Consistent with previous research, we found that individuals reporting higher levels of mindfulness also reported a greater sense of purpose in life ,43,75. HPurpose in life and the outcome variables were connected as expected. The association between having purposes and happiness was coincident with Lyubomirsky et al.  and GarcInterestingly, Pearson et al.  tested aThis research also contributes to extend previous models where meaningfulness is considered to mediate the positive effects of mindfulness  by addinIn summary, higher levels of mindfulness and purpose in life appears to be connected with greater engagement with life, which is linked to beneficial psychological outcomes.This research’s contributions should be considered in the light of its limitations. First, although relationships among variables depicted in SEM models apparently suggest causality, especially when mediation analyses are tested, such causal inferences cannot be properly derived from cross-sectional data, as used in this research. Therefore, assertions that may suggest a possible direction of causality should be taken cautiously. In this regard, longitudinal designs and cross-lagged analyses would be advisable in future research. Research exploring the effects of mindfulness-based interventions would offer interesting possibilities to longitudinally analyze whether changes in mindfulness-related attitudes may lead to changes in meaningfulness and changes in engagement with life, which would be expected to be associated with changes in wellbeing and mental health across time. A second limitation comes from the sample participating in this study. As stated, we used a convenience sample, recruited among internet users , which may compromise the generalization of the results. In particular, our study used a sample of women. Previous research has found that women usually report higher levels of anxiety  and deprFinally, we considered two mediating mechanisms that, along with mindfulness, could account for the great proportions of the variance of happiness, anxiety, and depression. However, our mediating variables do not exhaust other potential explanatory mechanisms that may be involved in the relationship between mindfulness and psychological outcomes. Similarly, additional mediating and/or moderating variables could be explored concerning the relationships among mindfulness and purpose in life and the connections between purpose and behavioral activation. In this regard, research focused on integrating new possible explanatory variables into the proposed model would be worth doing. For instance, it would be interesting to analyze the role played by personality traits, and eventually include this variable in the model, since previous research has found that part of the variance in meaningfulness can be explained by personality factors . In addiOur results may contribute to provide some rationale for intervention approaches that include the training of mindfulness skills. As presented, mindfulness is associated with positive psychological outcomes. Moreover, this study contributes to shed light on how such association may be accounted for. Having a sense of purpose in life and behavioral activation appeared to play a relevant role on the connections between mindfulness and happiness, anxiety, and depression. This point yields important possibilities for clinical practice. For instance, clinicians using mindfulness-based approaches could presumably strengthen the links between mindfulness and positive outcomes by focusing on promoting a sense of purpose and fostering involvement in meaningful activities, as such variables are associated to lower anxiety and depression symptoms and higher happiness. In fact, evidence-based approaches such as acceptance and commitment therapy  and the Mindfulness, meaningfulness, and behavioral activation are connected with enhanced happiness and reduced anxiety and depression symptoms. Moreover, mindfulness is linked to increased sense of purpose in life, which was revealed to be associated with greater activation and engagement with valued activities. To some extent, the relationships between mindfulness and salutary outcomes could be accounted for by factors such as meaningfulness and behavioral activation, which were identified as mediating variables. Here, the findings may contribute to suggest psychological pathways aiming to reduce mental health disturbances and promote a happy life.