These results suggest that both psychopharmacotherapies with a low dose of amitriptyline and aripiprazole are effective for the very elderly patients with BMS. Furthermore, aripiprazole may have some advantages in the adverse events compared to amitriptyline; however, the low dose amitriptyline monotherapy may have more benefit in the effectiveness and tolerability over prudent collaboration with primary physicians.Burning mouth syndrome (BMS) is defined by chronic oral burning sensations without any corresponding abnormalities. Besides amitriptyline, aripiprazole has been reported as a possible medication to manage BMS. However, especially for elderly patients, the adverse events of these medications would be a problem. The aim of the present study was to investigate the differences in the effectiveness and adverse events of amitriptyline and aripiprazole in very elderly patients with BMS. This is a retrospective comparative study of 80 years old and older patients with BMS who were initially treated with amitriptyline or aripiprazole and who were new outpatients of our department from April 2017 to March 2020. All clinical data, including sex, age, comorbid physical diseases, comorbid psychiatric disorders, the prescribed doses , prognosis, and adverse events, were collected from their medical charts. Each medication was selected considering their medical history. Amitriptyline was prescribed in 13 patients  and aripiprazole was prescribed in 27 patients . There were no significant between-group differences in sex, age, duration of illness, pain intensity, salivation, and psychiatric comorbidity at the first examination. Amitriptyline clinically improved more patients  with the effective dose of 10  mg than aripiprazole  of which the effective dose was 1.0  mg, although there were no significant between-group differences. The adverse events of amitriptyline were found in 9 patients (69.2%) and most patients had constipation (46.2%). For aripiprazole, 7 patients (25.9%) showed adverse events, most of them reported sleep disorder (11.1%). Amitriptyline had significantly longer duration taking medication ( Burning mouth syndrome (BMS) is defined by chronic burning sensations in the oral cavity without any corresponding abnormalities . For theThe aim of this study was to investigate the differences in the effectiveness and the adverse events of amitriptyline and aripiprazole focusing on very elderly patients primarily 80 years of age and over with BMS to facilitate the appropriate and effective treatment of BMS.The patients with BMS who first visited our departments between April 1, 2017, and March 31, 2020, were involved in this study. The diagnoses of BMS were according to the criteria of the International Classification Headache Disorders-3 , the salivation (the results of Saxon test), comorbid physical diseases, psychological questionnaires, the prescribed doses of amitriptyline or aripiprazole , clinical global improvement (CGI), and adverse events, were obtained from their medical charts.t-test, Mann-Whitney U-test, and Kaplan-Meier  were performed by using IBM SPSS Statistics version 25.0 . Statistical significance was set at p < 0.05.The elderly patients often have a comorbidity of somatic symptoms, i.e., other chronic pain, which may make BMS symptoms more complicated. To assess the comorbid somatic symptoms, a somatic symptom scale-8 (SSS-8) was used . The scoAll participants provided written informed consent. This study was approved by the Ethical Committee of Tokyo Medical and Dental University Dental Hospital .n = 73), atypical odontalgia (n = 14), phantom bite syndrome (n = 2), oral cenesthopathy (n = 27), and so on. In 73 patients with BMS, 57 patients underwent psychopharmacotherapy, including 18 patients who were prescribed amitriptyline and 34 patients who were prescribed aripiprazole. After exclusion of the duplicated prescriptions from other clinic and withdrawal, 13 patients  who were prescribed amitriptyline and 27 patients  who were prescribed aripiprazole were included in this study (p = 0.242), age (p = 0.052), the duration of illness , the initial VAS , and salivation . Almost all patients had comorbid physical diseases . While the patients prescribed amitriptyline mostly had comorbidity of orthopedic diseases  followed by hypertension , cataract , and gynecological diseases , the patients prescribed aripiprazole had orthopedic diseases , digestive diseases , hypertension , and cataract . Moreover, there was no psychiatric comorbidity in 9 patients (69.2%) with amitriptyline and 16 patients (59.3%) with aripiprazole without a significant between-group difference (p = 0.730). Benzodiazepines had been prescribed to 11 patients (84.6%) with amitriptyline and 18 patients (66.7%) with aripiprazole by their primary physicians at the first examination in our clinic.Of 1,568 new patients to our clinic between April 2017 and March 2020, 120 patients (7.7%) were 80 years old and above diagnosed with BMS (is study . As showp = 0.042), while no between-group significance was observed in the short intolerance of uncertainty scale , Zung's self-rating depression scale , and subgrouping for targeted treatment generic .The scores of pain catastrophizing scale (PCS) were significantly higher in the patients of aripiprazole compared to the one in the patients of amitriptyline (p = 0.097). While the most common comorbid somatic symptom was “trouble sleeping” in the patients treated with amitriptyline, “stomach or bowel problems” and “feeling tired or having low energy” were the most common in the patients treated with aripiprazole  mg] and the median of the maximum doses was 15  mg. The effective dose of amitriptyline 10  and the median duration of taking amitriptyline until “much improvement” was 105  days. For aripiprazole, the initial prescribed dose was 0.3  mg and titrated up to a maximum dose of 0.75  mg. The effective dose of aripiprazole was 1.0  mg, and the duration until clinical improvement was 80  days. While amitriptyline represented clinically improvement in 7 patients (53.9%), aripiprazole represented in 11 patients (40.4%) without a significant between-group difference . In 16 pn = 6) followed by dizziness (n = 5), dry mouth (n = 4), drowsiness (n = 3), and dysuria (n = 2). On the other hand, 7 patients (25.9%) reported adverse events with aripiprazole, and most were sleep disorders (n = 3). Aripiprazole represented significantly fewer adverse events than amitriptyline (p = 0.015).The adverse events of amitriptyline were observed in 9 patients (69.2%) with the most complains of constipation  days vs. 89  days, This is the first comparative study that investigated the effectiveness and adverse events of amitriptyline and aripiprazole for patients with BMS, focusing on very elderly patients. While amitriptyline clinically improved 53.8% (7/13) patients, aripiprazole was effective in 40.7% (11/27) patients with low initial doses and slow titration. Amitriptyline showed significantly more adverse events (69.2%) than aripiprazole (25.9%); however, no withdrawal due to adverse events were observed in both groups. Moreover, the discontinuation of amitriptyline was significantly lower than aripiprazole.Amitriptyline is the first-line medication of psychopharmacotherapy for BMS  which isIn the present study, anticholinergic effects, such as constipation, dizziness, dry mouth, and drowsiness, were observed in the patients treated with amitriptyline while sleep disorders were observed in the patients treated with aripiprazole. Constipation is mostly found in patients with amitriptyline and sometimes becomes troublesome because it is often already seen in elderly people. Since no patient with aripiprazole complained of constipation, aripiprazole would be another choice for patients who have comorbid digestive system disorders. Bristol stool chart  might beAt the same time, since amitriptyline induces drowsiness, taking medication at bedtime is generally recommended. For the cases drowsiness is shown daytime, taking in the early evening would be suggested. For amitriptyline, the complaining rate of drowsiness was lower in 80 years old and older patients (23.1%) in this study than those under 65 years old patients (57.5%), 65–75 years old patients (48.1%), and over 75 years old patients (36.4%) in our previous study . In the While amitriptyline is prescribed in depressed patients with a dose of 150–300 mg, it is prescribed in patients with chronic pain with 10–25 mg initially and titrated to 150 mg . The effFurthermore, amitriptyline has been reported its problematic adverse events with higher withdrawal than other antidepressants while aripiprazole has been reported as a useful antipsychotic with better tolerability. Interestingly, the present study revealed that amitriptyline has lower discontinuation than aripiprazole despite the higher occurrence rate of adverse events. The low dose amitriptyline monotherapy may be effective for BMS in safety with better tolerability over the collaboration with primary physician despite some adverse events. In addition, 59.3% of patients with aripiprazole quit or switched to other medication or augmentation with other antidepressants requiring more improvements in the early period of treatments similar to our previous report . AripiprThere are a few limitations to this study. First, the sample size is small without a placebo arm. Further investigation with a larger sample size considering a placebo arm is required to analyze the effectiveness and adverse events of antidepressants. Second, the quantitative examinations were lacking. During the follow-up duration, blood tests that include the concentration of medication in plasma level and salivation were not constantly performed by practitioners. However, all participants had been kept attending their physicians and regularly performed blood tests if necessary, and the collaboration between physicians and dentists had also kept throughout the treatment and the effects on comorbid physical diseases were not observed. Third, the bias at the decision for the first prescription of amitriptyline or aripiprazole might exist since this is a retrospective study.In conclusion, for the very elderly BMS patients of 80 years and over, both treatments with the low initial doses and slow titration of amitriptyline and aripiprazole were effective. Moreover, amitriptyline showed significantly lower discontinuation despite a higher occurrence rate of adverse events than aripiprazole. Therefore, aripiprazole may have some advantages in the adverse events compared to amitriptyline; however, the low dose amitriptyline monotherapy may have more benefit in the effectiveness and tolerability over prudent collaboration with primary physicians.The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by the Ethical Committee of Tokyo Medical and Dental University Dental Hospital. The patients provided their written informed consent to participate in this study.MW participated in data acquisition, analysis, and writing the manuscript. CT, ZL, GN, TS, CH, TT, TY, and MT participated in collecting data. HM, TN, and AT developed the theory and supervised the research. AT designed the research and administrated project. All authors read and approved the final manuscripts.This research was funded by KAKENHI from the Japanese Society for the Promotion of Science (JSPS), grant number 19K10328 to AT and grant number 19K19211 to MW. The funder had no role in study design, data collecting, data analysis, the decision of publishing, and preparation of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


From the mineral analysis of the ethnic foods, we found out that Chinese cuisines contain significantly higher sodium (average of 238 mg/100 g) than Malay cuisines (p = 0.006) and Indian cuisines (p = 0.03). Knowing the caloric density and nutrition content of local ethnic foods may aid hawkers and government officials in developing healthier options to tackle Singapore’s obesity epidemic.Singapore is a multi-ethnic country with a great variety of traditional ethnic cuisines. In this modern society where there is an increasing prevalence of obesity, it is important to know the nutritional content and energy density of our foods. However, there have been little data on the nutritional content of our local foods. The energy density and nutrient content of 45 commonly consumed meals by three ethnic groups in Singapore  were assessed in this study. Chinese, Malay, and Indian cuisines had an average energy density of 661, 652, and 723 kJ/100 g, respectively. Moreover, the macronutrient content is different between the different ethnic groups. Compared to Chinese and Malay cuisines, Indian cuisine contained lower protein but higher fat and carbohydrate content ( In modern society, there has been an exponential increase in obesity due to the changes in diet and “nutrition transition”. This consists of the increase in consumption of fat, meat, added sugars, and bigger portion sizes, as well as the decrease in physical activity . This glSingapore is a microcosm of Asia where three broad ethnicities corresponding to the major population centers in Asia are present, namely the Chinese (East Asians), the Malays (Southeast Asians), and the Indians (South Asians) . Street TM to determine the calorie content of various foods [However, there is insufficient information on the caloric content of local ethnic foods, with the majority of the information dating back many years and based on bomb calorimetry and Atwater conversion factors . We receus foods ,8. Our rus foods .Obesity is recognized to be a major risk factor for a variety of ailments, including cancer, heart disease, and diabetes mellitus, which are among the top 10 diseases afflicting Singaporeans ,10. Diet23, Mg24, Al27, K39, Ca40, Mn55, Fe56, Cu63, Zn66) of 45 local ethnic cuisines commonly consumed in Singapore.Data on the nutritional content of these local ethnic foods are critical for determining nutrient intake, giving dietary recommendations, and avoiding illnesses. Therefore, the objective of the study is to analyze the energy density, macronutrient content , and mineral content (Nan = 15), Malay food (n = 15), and Indian food (n =15). To achieve a smooth, consistent texture, all of the samples were homogenized in a kitchen blender . The samples were then put into 6 tubes, 3 for Calorie Answer analysis, and 3 for inductively coupled plasma mass spectroscopy (ICP-MS) analysis. For the ICP-MS analysis, the samples were kept in a −20 °C freezer. The samples were analyzed using the procedure and techniques described previously. The foods are described in Local food samples were purchased from a local food court . There were, in total, 45 local food samples from different ethnic groups that were used for analysis: Chinese food (v/v) HNO3 solution, then rinsing with ultrapure water and drying. The dilution of 10 µg/mL  Internal Standard Mix solution  was required for the internal standard solution. The calibration curves were made using a 10 µg/mL and 100 µg/mL custom mix multi-element ICP-MS standard , as well as a 1000 µg/mL mineral element ICP-MS standard solution  .Analytical and trace-metal grade reagents were utilized throughout. The Mili-Q IQ-7000  water purification system was used to provide high quality deionized water (resistivity > 18.2 M). Prior to use, all glassware and plasticware were cleaned by soaking for 48 h in a 10%  was used to collect near-infrared (NIR) spectra of homogenized samples throughout a wavelength range of 1100–2200 nm, with a resolution of 7.5 nm and a data interval of 2 nm. A halogen lamp served as the radiation source, while an acousto-optic tunable filter (ATOF) served as a wavelength sensor and light-receiving sensors served as light detectors. To increase accuracy, the integrated computer program  was programed to scan each triplicated component 10 times, then averaged to obtain a mean spectrum. After converting the data to log 1/R, the calorie density for each sample was determined using regression formulas preprogramed in the software. Each measurement took roughly 5 min to analyze . The procedures used were elaborated intensively by Lau, et al. [The homogenized samples were then put in cylindrical sample cells  and tested in triplicates. For all samples, the Prepared Food settings were used. For solid samples, the reflectance mode was applied, and the reference reflectance data were analyzed using a calcium-carbonate-filled cell. Calorie Answer3 . The mixture was heated to 210 °C in 15 min and then held for another 15 min. After cooling, sample solutions were diluted to 50 mL with 5% HNO3 and 0.5% HCl in decontaminated 50 mL skirted centrifuge tubes. Of these 12 vessels, ten were samples, one was blank, and one was a spiked sample from the same digestion batch. In each digesting operation, this arrangement was retained. Duplicates of each sample were digested. The 7900 ICP-MS analyzer  with the Ultra High Matrix Introduction (UHMI) option was used to perform inductively coupled plasma-mass spectrometry (ICP-MS) analysis. A MicroMist nebulizer, quartz spray chamber, and quartz torch with a 2.5 mm internal diameter injector were utilized during the experiment. The interface cones used had a platinum tip. The plasma was created using high quality (99.9997%) argon . The following were the parameters of the ICP-MS instrument: 1550 W RF power, 10 mm sampling depth, 0.90 L/min carrier gas. The samples, which were kept in 50/15 mL tubes, were delivered by an Agilent SP4 auto-sampler. Internal standards were used: Sc45, Ge72, Y89, In115, and Bi209. Using the tuning solution  for the ICP-MS , the instrument was set for optimal signal sensitivity and stability. Triplicates of each analysis were performed.A CEM MARS6 Microwave Digestor System with an iPrep 12 vessels rotor  was utilized to digest the local food samples. Each Teflon vessel contained 0.25 g of sample, followed by 10 mL of HNOThe ICP-MS analytical technique was derived from an Agilent application note . To vali44 were converted to Ca40 using the atomic abundance of the Ca element. The formula is as follows:The values of CaThe energy density and the macronutrient content of the selected foods were determined and reported for 100 g edible portion, as presented in The foods were listed from the highest to the lowest energy density for each ethnic group. The average energy density of Chinese, Malay, and Indian cuisines was 661, 652, and 723 kJ/100 g, respectively, as in p < 0.05). This may be due to Chinese cuisines using seasonings, such as soya sauce and monosodium glutamate (MSG), to season their food.Local ethnic foods are the main meals consumed in Singapore, and with more Singaporeans eating out, this may contribute to higher energy intake and higher sodium intake, which increases the risk of obesity and associated diseases. Since the research relating to diet and health is constantly evolving, appropriate dietary guidelines have been implemented to help Singaporeans adopt healthier food eating habits. The dietary guidelines include having a varied diet, and the foods chosen should be low in fat, especially saturated fat, low in salt, and low in sugar, replacing refined grains with whole grains, eating more fruit and vegetables each day. In recent years, there is also an increasing culture of dining out among Singaporeans from a recent 2018 Nielsen survey. The percentage of Singaporeans eating out is constantly increasing, from 51% in 2015 to 55% in 2019, on a weekly basis. Moreover, dining out meals generally deliver large portions that can lead to a substantially higher energy intake. Therefore, reducing portion size is one of the key requirements of moderating food intake. The first prerequisite for such action is the need to know the individual energy density of various local ethnic foods. In meeting this requirement, we are presenting for the first time a comprehensive list of energy density and nutrient content of various local ethnic foods.The recommended daily allowances for the different macronutrients and micronutrients are shown in Local ethnic foods in Kopitiam or hawker centers can be considered “unhealthy” foods due to their high amount of sodium, MSG, and fat. Mineral contents were assessed in addition to macronutrients, since they are known to have a significant role in metabolism and tissue function. The local ethnic foods consumed in Singapore were found to be high in macro-elements, like sodium, potassium, magnesium, and calcium, but deficient in trace elements, like copper, iron, manganese, and zinc, according to this study. Sodium is essential for the control of blood pressure and stimulation of muscles and nerves. It is an electrolyte that controls the extracellular amount of fluid in the body and is needed for hydration. Excessive consumption of dietary salt and sodium-containing substances, like monosodium glutamate (MSG), has been linked to high blood pressure, making it a risk factor for cardiovascular illnesses . The daiCalcium is important to prevent osteoporosis and for bone development ,22. The Iron insufficiency is the most frequent micronutrient deficit worldwide ,26. It iThe energy density and the nutrition composition of 45 popular consumed local ethnic cuisines in Singapore were assessed. Indian ethnic cuisine has the highest average energy density of 723 kJ/100 g. This is due to the higher fat and carbohydrate content in Indian cuisine as compared to the Chinese and Malay cuisines. From the ICP-MS analysis, the mineral content of the local ethnic cuisines differs greatly. Overall, Chinese cuisine has the highest amount of sodium in their local ethnic foods. This may be due to the seasonings used to season the food. In addition to the dietary advice and guidelines by the government agencies, the validated data of energy density and macronutrient contents of the local ethnic foods will serve as an important tool in reviewing or setting new dietary guidelines in mitigating health disorders, as well as maintaining sustainable human health in Singapore.


We conducted this meta-analysis for a precise estimation of the association between BsmI (rs1544410), ApaI (rs7975232), TaqI (rs731236), and FokI (rs2228570) polymorphisms and acute pancreatitis.Several studies have been performed to investigate the association between Appropriate studies were retrieved by searching Web of Science, PubMed, Scopus, and Google scholar databases, until January 31, 2021. Two reviewers independently conducted data extraction and literature quality evaluation. Odds ratios and 95% confidence intervals were calculated to evaluate the strength of the association.All of the data were analyzed with Stata 16.0.The results of this meta-analysis will be submitted to a peer-reviewed journal for publication.This meta-analysis will summarize the association between BsmI, ApaI, TaqI, and FokI polymorphisms and the risk of acute pancreatitis.Ethical approval was not required for this study. The systematic review will be published in a peer-reviewed journal, presented at conferences, and shared on social media platforms.DOI 10.17605/OSF.IO/83W7R. The imbalance of active VD level is a major cause of hypercalcemia that is a risk element for AP. Due to polymorphisms in the VDR gene, the impairment of VDR can lead to a significant effect on the equilibrium of VD levels in the circulation and on its activity in the body. Moreover, Serum 25-hydroxyvitamin D3(25-(OH) D3) level was inversely related to the severity of AP and inflammatory markers such as C-reactive protein. VD also affects the innate and adaptive immune responses and reduces oxidative stress, which could initiate AP.Vitamin D (VD) dysregulation was proposed to be associated with the production of AP.VDR polymorphism and the risk of AP, but the results of these studies are not consistent.,11 In this study, we performed a systematic review and a meta-analysis to evaluate the association between different genetic variants of VDR gene and AP, and to evaluate the association of BsmI, ApaI, TaqI, and FokI polymorphisms within different ethnic subgroups. The results were pooled together to obtain reliable conclusions.In recent years, there are more and more studies on the relationship between 22.1The protocol of this review was registered in OSF (OSF registration number: DOI 10.17605/OSF.IO/83W7R). It was reported to follow the statement guidelines of preferred reporting items for systematic reviews and meta-analyses protocol.2.2Qualified studies have to meet the following criteria:(1)Predefined diagnosis criteria for AP;(2)Case-control studies evaluated the association between BsmI, ApaI, TaqI, and FokI polymorphism and AP risk;(3)Provided detailed genotype frequencies;(4)If multiple reports based on the same study population were available, the most recent or largest study will be selected.2.3The main exclusion criteria are as follows:(1)Insufficient data offered to calculate Odds ratio (OR) estimate;(2)Non-case-control studies;(3)Duplicate data from a same cohort.2.4vitamin D receptor, VDR, polymorphism, BsmI, rs1544410, ApaI, rs7975232, TaqI, rs731236, FokI, rs2228570, pancreatitis, acute pancreatitis.” Furthermore, studies were identified through manual searches on reviews and retrieved studies. The search strategy for PubMed is displayed in Table Two investigators independently performed a systematically computerized search for English studies through Web of Science, PubMed, Scopus, and Google Scholar databases up to January 31, 2021. The keywords for searching included the combination of “2.52.5.1Two reviewers complete the screening process independently, and any differences are decided by a third reviewer. The screening process of the article includes reading the title, the abstract, and the full text, so as to determine whether it meets the inclusion criteria. The researchers record the reasons to exclude each study in light of the preferred reporting items for systematic reviews and meta-analysis guidelines and report the screening results as well. The flowchart is exhibited in Figure 2.5.2P value for Hardy–Weinberg equilibrium (HWE) of controls, single nucleotide polymorphism detection method and so on. If the extracted data are inconsistent, discussion should be made to solve it.The collected information from each article are as follows: The first author and year, country, region, race, gene frequency of case group and control group, source of control group, 2.5.3 This scale uses a star rating system to judge the methodological quality and consists of three parts, namely selection, comparability, and ascertainment of exposure. The full score is 9 stars, and the score of 5 or more is regarded as “high quality.” Otherwise, the study is regarded as “low quality.” Any disagreements on the Newcastle-Ottawa scale score of the studies were resolved through a comprehensive reassessment by other authors.Two authors independently assessed the quality of the included studies based on Newcastle Ottawa Scale.2.5.4The reason for the loss of data in the period of data screening and extraction is identified here. If the data of potential studies are insufficient, missing, or vague, we would attempt to contact the authors. These studies would be excluded only if the data are not available through the method described above.2.5.5Z test and P < .05 was considered as statistically significant. In the meta-analysis, the overall pooled ORs were calculated for four models, including the allelic, dominant, recessive, and homozygous models. Deviation from HWE was examined by carrying out Chi-square test and P value < .05 indicated a departure from HWE. The between-study heterogeneity was assessed by the I2 statistic, which was calculated from the Q statistic. If the heterogeneity was statistically significant (P < .05 for Q-test or I2 > 50%), the random-effects model (based on DerSimonian-Laird method) was adopted to get the pooled estimates. Otherwise, the fixed-effects model (based on Mantel–Haenszel method) was adopted. All of the statistical analyses were conducted by STATA 16.0 , and the P values were 2-sided.Crude Odds ratio (ORs) with corresponding 95% confidence intervals were used to estimate the strength of the association between the BsmI, ApaI, TaqI, and FokI polymorphism and AP risk. The statistical significance of the pooled ORs was examined by performing 2.5.6We will conduct subgroup analyses based on age, ethnicity, and disease severity2.5.7The eligible study was sequentially removed to perform the sensitivity analysis.2.5.8P < .05.,16Publication bias was assessed by Begg rank correlation and Egger linear regression. The publication bias was regarded as statistically difference when 2.5.9The content of this article does not involve moral approval or ethical review and would be presented in print or at relevant conferences.3In recent years, studies on the relationship between inflammatory mediators and the risk of AP have proved that the severe systemic inflammatory response triggered by AP is not mediated by a single vector, but has complex pathophysiological pathways. Among these pathways, oxidative stress and cytokines are particularly important. Kim et al discovered that the concentration of 25-(OH) D3 in AP dogs was significantly lower than that in healthy dogs. Active VD metabolite, and 1,25-dihydroxyvitamin D selectively bind to specific VDR. Afterward, the VDR regulates the transcription of genes that are involved in calcium metabolism, cell proliferation and differentiation, senescence, and t-cell-mediated immune response.–28VDR is also expressed in pancreatic β cells and may play an important role in maintaining normal insulin levels consistent with glucose concentration and glucose tolerance.–31 The polymorphism of VDR may have key effects on the balance of circulating VD concentrations and the activity of final metabolites throughout the body, because VD works through VDR.The 25-(OH) D3 participates in human immune and inflammatory response.VDR polymorphism and AP susceptibility, the cumulative evidence of this association has not been systematically evaluated. In this study, we will conduct a systematic review and meta-analysis to combine the results of numerous studies and generate more reliable risk association estimation to guide the prevention and treatment of AP. The advantages of this study are as follows:So far, although many researchers have focused on the relationship between (1)Large data sets from all eligible latest studies will be merged;(2)In order to explore heterogeneity, we will try to avoid post-subgroup analysis;(3)Sensitivity analysis will be conducted on each genetic model for the improvement of the reliability of the results. Therefore, the release protocol will avoid potential biases related to data mining as much as possible and help to obtain convincing evidence.Conceptualization: Mengqin Li.Data curation: Xiaofeng Li, Xianghai Gan.Formal analysis: Mengqin Li, Xianghai Gan, Junzuo Gong.Methodology: Xianghai Gan, Tianyi Mou.Project administration: Mengqin Li.Resources: Junzuo Gong.Software: Junzuo Gong, Hua Zhou.Supervision: Mengqin Li.Validation: Tianyi Mou, Hua Zhou, Xiaofeng Li.Visualization: Xiaofeng Li, Hua Zhou.Writing – original draft: Mengqin Li, Xiaofeng Li.Writing – review & editing: Mengqin Li, Xiaofeng Li.


Several studies show that chronic opioid dependence leads to higher in-hospital mortality, increased risk of hospital readmissions, and worse outcomes in trauma cases. However, the association of outpatient prescription opioid use on morbidity and mortality has not been adequately evaluated in a critical care setting. The purpose of this study was to determine if there is an association between chronic opioid use and mortality after an ICU admission.A single-center, longitudinal retrospective cohort study of all Intensive Care Unit (ICU) patients admitted to a tertiary-care academic medical center from 2001 to 2012 using the MIMIC-III database.Medical Information Mart for Intensive Care III database based in the United States.Adult patients 18 years and older were included. Exclusion criteria comprised of patients who expired during their hospital stay or presented with overdose; patients with cancer, anoxic brain injury, non-prescription opioid use; or if an accurate medication reconciliation was unable to be obtained. Patients prescribed chronic opioids were compared with those who had not been prescribed opioids in the outpatient setting.None.The final sample included a total of 22,385 patients, with 2,621 (11.7%) in the opioid group and 19,764 (88.3%) in the control group. After proceeding with bivariate analyses, statistically significant and clinically relevant differences were identified between opioid and non-opioid users in sex, length of hospital stay, and comorbidities. Opioid use was associated with increased mortality in both the 30-day and 1-year windows with a respective odds ratios of 1.81  and 1.88 , respectively.Chronic opioid usage was associated with increased hospital length of stay and increased mortality at both 30 days and 1 year after ICU admission. Knowledge of this will help providers make better choices in patient care and have a more informed risk-benefits discussion when prescribing opioids for chronic usage. Opioid dependence is a significant contributor to the global burden of disease . The opiStudies show that chronic opioid dependence leads to higher in-hospital mortality, increased risk of hospital readmissions, and worse outcomes in trauma cases –10. HoweWe performed a single-center, longitudinal retrospective cohort study of all Intensive Care Unit (ICU) patients admitted to a tertiary-care academic medical center from 2001 to 2012 using the MIMIC-III database .MIMIC III is a widely used, reliable, de-identified electronic database created by MIT using EMR information from patients at the Beth Israel Deaconess Medical Center. It is open access and available for research purposes. Informed consent and ethical approval were not required due to the anonymization. Patients age 18 years and older were included in the study, and we defined chronic opioid use by the presence of an outpatient opioid prescription on admission to the ICU. Exclusion criteria included patients who expired during their hospital stay or presented with overdose; as well patients with cancer, anoxic brain injury, non-prescription opioid use, or if an accurate medication reconciliation was unable to be obtained. Patients on chronic opioids were compared with those who had not been prescribed opioids in the outpatient setting. The selection criteria and procedure are shown in There was no intervention in this study, as this was on observational study using ICU admission data from an electronic health record. The major outcomes assessed in our study were mortality and hospital length of stay. Mortality was determined at the end of the follow-up period based on the data in MIMIC III. Hospital length of stay was recorded from the date the patient was admitted to the hospital until the date of discharge.After ICU encounters were obtained from MIMIC-III, a Chi-Square Test was used to determine whether 30-day mortality and 1-year mortality were independent of the use of these medications. As a secondary outcome, we assessed total hospital length of stay (LOS) with the two tailed T-Test. All statistical analyses were performed in R version 4.0.0.We performed a logistic regression with propensity score weighting to assess both 30-day mortality and 1-year mortality. To assess hospital LOS in days, we built a generalized linear model with an underlying Gaussian distribution and also weighted based on the propensity scores. In each case, the alpha level was 0.05. In the preliminary models, we adjusted the outcomes for chronic opioid use, Sequential Organ Failure Assessment (SOFA) score, age, sex, the number of comorbidities, as well as the disease status for Coronary Artery Disease (CAD), Congestive Heart Failure (CHF), Chronic Obstructive Pulmonary Disease (COPD), Diabetes, End Stage Liver Disease (ESLD), End Stage Renal Disease (ESRD), obesity and stroke. The SOFA score is used to track patients’ status during the stay in an intensive care unit (ICU) to determine the extent of organ dysfunction . The SOFTo determine whether specific opioids in particular are associated with 30-day mortality, 1-year mortality, and hospital LOS, the same models were used and the covariate for opioid use expanded to adjust for hydromorphone, hydrocodone, oxycodone, morphine, fentanyl, tramadol, methadone, and meperidine.Lastly, a third set of models was built to determine the association between outcomes and SOFA score, to assess how opioid use is related to mortality after adjusting for improvement in clinical condition. SOFA score between day 1 and day 3 (SOFA D1-D3) represents the improvement in the clinical condition of the admitted patients in each group. This model adjusted for the same variables as the preliminary models along with the effects of each opioid as above.Table 1.A total of 32,384 records were screened. The final sample for the first two sets of models included 22,385 patients with 2,621 (11.7%) patients in the treatment group (mean age 62.37 ± 0.24) and 19,764 (88.3%) in the control group (mean age 61.16 ± 0.60). The demographic distribution of initial patient data is represented in In regards to demographic distribution data, there are significant differences between those who were prescribed opioids and those who were not prescribed opioids across all the variables except in the number of comorbidities. However, the majority of these differences were not clinically relevant. Clinically relevant differences between opioid and non-opioid users were found in sex, discharge location, length of stay, and comorbidity type.Table 2.The comorbidities of interest for this study were coronary artery disease, congestive heart failure (both systolic and diastolic), chronic obstructive pulmonary disease, depression, diabetes mellitus, end stage liver disease (ESLD), end stage renal disease (ESRD), obesity and stroke. There was a statistically significant difference across every comorbidity of interest except for obesity which appears in 3.86% and 3.89% of the non-opioid using and opioid using populations respectively as noted in Fig 2)Another feature of interest is the type of opioid prescribed. Among patients with an opioid prescription the most frequently prescribed opioid was oxycodone (45%). There were 3 times as many prescriptions of oxycodone as the next most prescribed opioid, hydrocodone.  and 1.88 . Moreover, patients on opioids had a significantly greater hospital LOS (p<0.001) with a mean hospital stay of 11.62 days (SD = 0.43) versus 10.02 days (SD = 0.13) for patients not on opioids prior to admission. Opioid use was associated with an increased LOS by 1.54 days , while controlling for the other covariates. The results of this preliminary model are shown in S1 Table. In addition, those who use fentanyl have a longer length of stay by 3.42 days  compared to those who did not use fentanyl.A second set of models was created to account for the specific type of opioid. In these models, nearly all of the opioids showed a statistically significant change in their 30-day or 1-year mortality Using the second set of models for 30-day mortality, 1-year mortality, and hospital length of stay, it was found that patients with obesity have a 71%  and 62%  decrease in the risk of 30-day and 1-year mortality, respectively. In addition, people with ESLD have 4.62  and 5.10  times the risk of 30-day and 1-year mortality, respectively. Lastly, a one unit increase in SOFA scores is associated with an 8%  increase in 30-day mortality and a 3%  increase in 1-year mortality while adjusting for the other covariates.S2 Table. In addition, those who used meperidine spent 5.93 more days in the hospital  compared to non-meperidine users. An association of a decreased risk of 30-day mortality for those with CAD was also noted.For the third set of models that adjusted for SOFA D1-D3 as well as the other covariates, there were a total of 8,711 total participants for whom a SOFA D1-D3 score could be calculated. Of these, 7,598 (87.2%) were in the non-opioid using group and 1,113 (12.8%) were in the opioid using group. The results showed that opioid use was associated with increased mortality in both the 30-day and 1-year windows with a respective odds ratio of 1.84  and 1.75  In this study, we report that even after adjusting for severity of illness, chronic opioid users have worse outcomes in the ICU compared to those not on chronic opioids. Chronic opioid usage was associated with increased hospital length of stay and increased mortality at both 30 days and 1 year after ICU admission. All models had comparable rates of increased 30-day and 1-year mortality rates with respect to SOFA score with highest risk at 1 year implying the long-term effects of critical illness and possible role of chronic opioids. This study highlights the relationship between chronic opioid use and clinical outcomes in critical care settings, to help equip providers with information when engaging in risks-benefit discussions with patients.Similar findings were demonstrated in a study  where boOne explanation for increased hospital stay and mortality among chronic opioid users is the potential for greater infectious complications with chronic opioid abuse. Studies have shown that opioid abuse causes immunosuppression by disrupting both innate and adaptive components of the immune system , 16. OpiOpioids are also known to have central adverse effects of oversedation, respiratory depression, and cough suppression . These eHydrocodone, oxycodone, and tramadol are the most commonly prescribed outpatient opioids in the USA. Our dataset showed the oxycodone was the most prescribed, followed by hydrocodone and Tramadol. While historically, hydrocodone was the most prescribed opioid in the outpatient settings, the amounts of oxycodone dispensed from retail pharmacies in the U.S. increased by 678% between 1997 and 2006. That increase, along with the known geographical variation in opioid prescription , is likeIt is unclear by what mechanism this effect is generated, whether baseline functional status, modestly poorer in patients on long term opioids , and chrWe attempted to address the effect of confounding variables by extensively adjusting for presence and type of opioid use, demographic differences, differences in illness severity and comorbidities as described in the statistical analysis section, but we have not accounted for a the possible impact of opioid tolerance, difference in dose, chronic use of other neuroleptics or other hidden confounding variables. The dataset, while providing a comprehensive list of home medications, does not capture the dose and duration of using these medications. Regardless, our study still shows a qualitative effect of chronic opiate use regardless of dose and duration of therapy.There is also the concern of this study being a product of a single database and of patients with an average SOFA score of 7, which may limit its generalizability to other centers and sicker populations. We hope these questions can be answered by future studies.Lastly, there is the concern of coding bias from using electronic health records. Studies using electronic health records share many common limitations such as coding errors, inaccurate reporting of past medical history, and underdiagnosis . HoweverThis study is unique in its attempt to explore the relationship between chronic opioid use and clinical outcomes in critical care settings. While outpatient opioid use has been decreasing over the last decade, the US has witnessed 153,260,450 opioids prescriptions in 2019, with 46.7 Opioid Dispensing Rate Per 100 Persons [The findings of this study are that chronic opioid usage was associated with a statistically significant increased hospital and ICU length of stay, as well as increased mortality at both 30 days and 1 year after an ICU admission after controlling for age, sex, delta sofa, the number of comorbidities, presence of CAD, CHF, COPD, diabetes, ESLD, ESRD, obesity and stroke. This is key information when making a risk-benefit decision when prescribing opioids for chronic usage.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Sphingomonas, which was the predominant genus in the majority of tested samples, was more than 85–90% among the total identified bacterial community in most samples. Among these identified genera, 13 low abundance genera were potential opportunistic pathogens, including Acinetobacter, Bacillus, Staphylococcus, Clostridium, Klebsiella, Mycobacterium, Ochrobactrum, Chryseobacterium, Stenotrophomonas, and Streptococcus. Of these 13 genera, 13 major opportunistic pathogenic species were validated using polymerase chain reaction. The pathogens were not detected in the samples of different stages and the final products of fermentation, except in one sample from the first stage of fermentation in which S. aureus was detected. This finding was consistent with that of ASV-based taxonomic classification according to which S. aureus was detected only in the sample from the first stage of fermentation. However, S. aureus was not significantly correlated with the human disease pathways. These results indicated that fermentation is a reliable and safe process as pathogenic bacteria were not detected in the fermentation products. The hybrid method reported in this study can be used simultaneously to evaluate the bacterial diversity, their functional predictions and safety assessment of novel fermentation products. Additionally, this hybrid method does not involve the random detection of pathogens, which can markedly decrease the time of detection and food safety verification. Furthermore, this hybrid method can be used for the quality control of products and the identification of external contamination.Fermented fruits and vegetables play an important role in safeguarding food security world-wide. Recently, robust sequencing-based microbial community analysis platforms have improved microbial safety assessment. This study aimed to examine the composition of bacteria and evaluate the bacterial safety of fermented fruit products using high-throughput 16S-rRNA metagenomic analysis. The operational taxonomic unit-based taxonomic classification of DNA sequences revealed 53 bacterial genera. However, the amplicon sequencing variant (ASV)-based clustering revealed 43 classifiable bacterial genera. Taxonomic classifications revealed that the abundance of  Listeria monocytogenes, Clostridium spp., Salmonella spp., Staphylococcus spp., Bacillus spp., and Escherichia coli survive and escape during fermentation and remain infectious [Fermentation is a cost-effective method to preserve perishable and seasonal fruits, which play a vital role in safeguarding food security world-wide ,2. Fermefectious ,10,11,12fectious ,14. Addifectious .Enterococcus and Enterobacteriaceae, produce toxic metabolites, such as biogenic amines during the fermentation process [Enterococcus, Staphylococcus, and Lactobacillaceae can harbor transmissible antibiotic resistance genes [Recently, there is increased consumer awareness on food safety and health benefits, including food quality and microbial traceability and safety ,17. The  process . Thus, t process . Some fece genes  Hence, tce genes . Traditice genes ,23. Addice genes . Moleculce genes ,26. Furtce genes . The advce genes ,29. Recece genes ,24,30,31ce genes .This study aimed to investigate the total bacterial community and their functional prediction along with the identification of potential pathogens in fermented fruits at different stages of fermentation. In this study, a metagenomics approach was used to target the V3 and V4 regions of the 16S rRNA gene to determine the change in bacterial diversity in the different stages of fermentation and the final fermentation products. The identification of unique bacterial communities, including pathogens, and their predicted functions can aid in improving food safety. This study characterized the bacterial community structures and their safety parameters, which can increase the consumer acceptance of fermented fruit-based products.In this study, sampling was performed during various stages of the fruit fermentation process using the intermediate and finished products from a commercial fermented beverage production company  located in Chiayi County, Taiwan. The fruit fermentation of pineapple, orange, and banana is carried out into two stages, which ultimately lead to the final product. In the 1st stage of fermentation, these fruits are washed and cut in fermentation tanks followed by the addition of sugar, yeast, lactic acid bacteria (LAB), and acetic acid bacteria (AAB), then followed by mixing vigorously for 24 h. The concentration of yeast, LAB, and AAB additives was 0.0025%, whereas, the concentration of sugar was 23.2% in a total of 1 metric ton of fermented fruit. However, pineapple samples without starter culture did not contain yeast, LAB, and AAB in the fermentation tanks. Finally, these fermentation tanks were kept at room temperature under anaerobic conditions to accomplish the 1st stage of the fermentation process. The total fermentation duration of pineapple, banana and orange in the 1st stage was approximately 180, 150, and 365 days, respectively. The sampling time was based on 1/3 and 2/3 of the fermentation duration for pineapple and banana, whereas orange was collected at 1/2 and 3/4 of the fermentation duration. The 2nd stage of fermentation was performed by mixing the above fermented products from the 1st stage in a fermentation tank and kept for approximately one year at room temperature under anaerobic conditions. Real-time temperature detection was used to ensure the range of room temperature (in between 24–28 °C) during fermentation. The final product of fermentation was a mixture of liquid products from the 2nd stage of fermentation. In total, 28 samples (40 mL of fermented broth for each sample) were aseptically collected from their respective fermentation tanks and final product into sterilized containers and transported to the laboratory under controlled temperature. Description of the samples, fermentation processes and sampling time are provided in g for 3 min. The supernatant was discarded and the bacterial cells were lysed to extract gDNA using a commercial auto DNA extractor kit , following the manufacturer’s instructions. The purity and concentration of the extracted gDNA were determined using a Nanodrop 2000 spectrophotometer  at 230–280 nm. The quality of gDNA was examined using gel electrophoresis (1.5% gel in Tris-acetate ethylenediaminetetraacetic acid buffer) at 110 V for 30 min. DNA bands were visualized under ultraviolet light. The purified gDNA was stored at −20 °C for further analysis.The fermented broth (2 mL) was thoroughly mixed, aseptically transferred to a sterilized tube, and centrifuged at 12,000× t-test (p < 0.05).The libraries for the amplified V3–V4 regions of 16S rRNA hypervariable regions were constructed using the 341F and 805R primer sets (with some modifications) with the Illumina adapter overhang sequence attached at the 5′ end of the primers. The sequences of forward primers used in this experiment were as follows: (16S_341F) 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWG CAG-3′, (16S_341F _N) 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGAC AGnCCTACGGGNGGCWGCAG-3′, (16S_341F _N N) 5′-TCGTCGGCAGCGTCAG ATGTGTATAAGAGACAGnnCCTACGGGNGGC WGCAG-3′, and (16S_341F _NNN) 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGnnnCCTACGGGNGG CWGCAG-3′. The reverse primer sequences were as follows: (16S_805R) 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTA TCT AATCC-3′, (16S_805R _N_) 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGA CAGnGACTACHVGGGTATCTAATCC-3′, (16S_805R _NN) 5′-GTCTCGTGGGC TCGGAGATGTGTATAAGAGACAGnnGACTACHVGGGTATCTAATCC-3′, and (16S_805R _NNN) 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG nnnGACTACHVGGGTATCTAATCC-3′. The amplification was performed in triplicates, following the previously reported method . The opthttps://github.com/picrust/picrust2/, accessed on 15 November 2020) using KEGG (Kyoto Encyclopedia of Genes and Genomes). All ASVs with nearest-sequenced taxon index  were removed by default for reliable annotation of metabolic functions using the KEGG reference database as previously described [p < 0.05) with 95% confidence intervals. Pearson correlation analysis was performed using IBM SPSS Statistics 24  to evaluate the significant correlations between bacterial diversity and potential functional prediction considering p ranging 0.01–0.05.To examine the potential metabolic function of microorganisms at different stages of fermentation, the representative sequence and denoised ASV abundance table were used. These tables were input into the phylogenetic investigation of communities’ reconstruction of Unobserved States (PICRUSt2) pipeline  PCR analysis as described previously [Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Gardmerella vaginalis, and Chryseobacterium indologenes were validated using 6S-23S ribosomal DNA region PCR analysis as described previously [nuc, bal, recA, tyrB and pneumolysin genes PCRs were used to validate Staphylococcus aureus, Bacillus cereus sensu lato s.l, Ochrobactrum anthropic, Klebsiella pneumoniae, and Streptococcus pneumoniae, respectively [Clostridium difficile was validated by two genes  PCRs [In this study, species identification of suspected pathogens in fermented samples were validated by various specific genes by targeting PCRs. eviously . Pseudomeviously ,41,42,43ectively ,46,47,48dB) PCRs . The conThe 16S rRNA amplicon sequencing targeting the V3 and V4 regions was performed to examine the bacterial community diversity and composition in each fermentation stage sample. After quality filtering and the removal of chimeric sequences, a total of 785,339 sequence reads  ranging from 4595–75,976 were obtained from 28 samples  and the The bacterial diversity and richness among the fermentation stages and final product was elevated using various alpha diversity indices as shown in p < 0.05) in the bacterial beta diversity among the fermentation stages and final product.The beta diversity analysis based on Bray–Curtis distances revealed high variation among the bacterial diversity associated with the fermentation stages and product as the result showed a distinct clustering pattern F indicatSphingomonas was the most predominant bacteria, which accounted for more than 80% of the total bacteria, in most fermentation samples. The second most abundant bacterial genus was Oribacterium (18%), which was the predominant genus in Ch3_2nd and Ba5_1st samples with maximum relative abundances of >42% and >98%, respectively. The resolution of low abundance genera, which accounted for less than 10% of the total genera, was limited due to the increased relative abundance of Sphingomonas. Therefore, this genus was excluded to visualize and interpret the relative abundance of the remaining 51 genera . Among these pathogenic bacteria, Acinetobacter exhibited a high abundance and was detected in all samples. Bacillus, which was detected in 99% of samples, was the second most predominant genus. Stenotrophomonas, Streptococcus, and Staphylococcus were detected in more than 95% of samples irrespective of their relative abundance. In contrast, Clostridium was detected in almost 67% of samples. Klebsiella, Chryseobacterium, Ochrobactrum, Mycobacterium, and Deutzia were detected in more than 10% of samples. Among the 28 samples, Ba1_1st and Ba3_1st exhibited a high distribution of bacterial genera with 15 genera detected in each sample.Genus level OTU based classification revealed a total of 52 classifiable genera as shown in 1 genera B. Of theSphingomonas spp., which accounted for 85% of all bacteria was the most predominant bacterial genus in 98% of samples. The second most predominant bacteria were Nesterenkonia and Lactobacillus with maximum relative abundances of more than 3%. Acetobacter (2.9%) was the third most abundant genus, which was predominant in the Ba_1st sample and accounted for more than 85% of bacteria in the sample. Similarly, Lactobacillus (more than 65%) was the predominant genus in the Pi1_1st sample. Among the potential opportunistic pathogens, the relative abundance of Bacillus, Clostridium, Dietzia, Gardnerella, and Pseudomonas was less than 1%  whereas, these bacteria were absent in non-starter culture containing pineapple samples. Contrarily, Achromobacter, Anaerobacillus, Delftia, Kocuria, Ochrobactrum, and Staphlococcus were only present in non-starter culture containing pineapple samples and these bacteria were absent in starter culture containing pineapple samples. Two genera including Lactobacillus and Nesterenkonia showed higher abundance in starter culture containing pineapple samples whereas, Sphingomonas abundance was higher in non-starter containing pineapple samples.To compare the bacterial diversity in each fermentation sample, the Qiime2 pipeline was used. After quality filtering, 193,323 sequence reads were obtained, which were classified into 43 identifiable genera C. Sphing than 1% D. DietziGardnerella and Pseudomonas, whereas the OTU-based clustering approach identified Acinetobacter, Klebsiella, Mycobacterium, and Streptococcus. All these bacteria were detected only in the samples from the first stage of fermentation, except Acinetobacter, which was detected in the samples from the first stage of fermentation and the final fermentation product. Other pathogenic genera commonly identified in both clustering approaches included Bacillus, Staphylococcus, Clostridium, Ochrobactrum, Cryseobacterium, Dietzia, and Stenophomonas, which were detected only in the samples from the first and second fermentation stages, whereas, Bacillus and Clostridium were also detected from the final fermentation products.Both OTU and ASV taxonomic classifications generated a total of 13 opportunistic pathogenic genera associated with the fermentation processes and products, as shown in Dietzia, Devosia, Lactobacillus, Nesterenkonia, Paracoccus, and Pelomonas was significantly different between the first and second stages of fermentation with the abundance of most genera enriched in the first stage of fermentation  was significantly different between the second stage of fermentation and the final product. This genus was predominant in the second stage of fermentation, whereas the mean proportion of this genus decreased in the final product.The changes in bacterial diversity and abundance at the genus level in the samples from different fermentation stages and the final fermentation products were examined using STAMP software. The analysis revealed that the abundance of entation . Among tStaphylococcus infection was detected in all samples with a maximum relative abundance of 0.6%.Functional prediction analysis revealed 5322 KOs, which were grouped into 155 KEGG pathways (level 3). These data were further processed to filter the low variant and extremely low abundant pathways (<4 abundance) using MicrobiomAnalyst. In total, 125 high-quality level 3 KEGG pathways were obtained. The level 2 KEGG pathways were presented in the form of a bubble plot for clear visualization and interpretation . These pp < 0.05) in the samples from the first and second stages of fermentation. However, environmental adaptation and infectious disease were significantly abundant in both stages of fermentation. Moreover, the mean proportion of all significant functions decreased in the second stage of fermentation. Glycan biosynthesis and metabolism, translation, replication and repair, nucleotide metabolism, environmental adaptation, cell growth and death, and folding, sorting, and degradation were significantly enriched between the first stage of fermentation and the final product.The STAMP software was used to further investigate changes in the metabolic functions, especially during the fermentation stages and in the final product . The comSimilarly, the mean proportion of all enriched pathways decreased at the end of fermentation. Additionally, metabolism of terpenoids and polyketides, amino acid metabolism, and membrane transport were significantly enriched in the second stage and final products of fermentation. Furthermore, the abundance of these pathways in the final product of fermentation was lower than that in the second stage of fermentation.p < 0.05 and < 0.01, respectively  with all the 25 predicted functions related to fermentation processes and products. Additionally, Paracoccus and Pelomonas were significantly and positively correlated (p < 0.05) with only the endocrine system. In contrast, most bacterial taxa, except Acetobacter, were negatively and non-significantly correlated with all predicted functions. Acetobacter was significantly and negatively correlated (p < 0.05) with the endocrine system. Similarly, Gardnerella and Kocuria were significantly and positively correlated (p < 0.05) with all predicted functions, except cell mobility, transport and co-metabolism, and infectious disease. Additionally, Deleftia, which was the fourth most predominant bacterial taxa, was significantly and positively correlated with more than 50% of the metabolic pathways (p < 0.01). In contrast, only Lactobacillus was significantly and negatively correlated (p < 0.05) with cell mobility, cellular community-prokaryote, signal transduction, and digestive system pathways.The correlation analysis revealed that among all the observed bacterial taxa, Sphingomonas, were significantly and positively correlated with the predicted pathway related to infectious disease, which was the focus of this study. However, Staphylococcus, a potential pathogen, was not significantly and positively correlated with the pathways related to infectious diseases. Staphylococcus was significantly and positively correlated with the endocrine system.None of the identified genera, except Acinetobacter baumannii, Bacillus cereus sensu lato s.l, Clostridium difficile, Chryseobacterium indologenes, Dietzia maris, Gardnerella vaginalis, Klebsiella pneumoniae, Pseudomonas aeruginosa, nontuberculous mycobacteria, Ochrobactrum anthropi, Staphylococcus aureus, Streptococcus pneumoniae, and Stenotrophomonas. The PCR analysis revealed absence of all these pathogens except S. aureus was detected only in the Pi4_1st sample, which was from the first fermentation stage. The genus Staphylococcus was also detected in Pi4_1st sample in 16S amplicon sequencing analysis.The 16S rRNA amplicon sequencing using ASV and OTU based taxonomic classifications revealed 13 genera belonging to potential opportunistic pathogens associated with the fermentation stages and the final product. To confirm their presence or absence in the fermentation stages and product we targeted the following 13 bacterial species using PCR: Globally, fermented foods are consumed as ready-to-eat foods. However, the lack of microbial characterization of these food products has raised consumer concerns for food safety, which has led to decreased consumption of these essential food items . Modern Sphingomonas was the predominant bacterial genus in almost 90% of samples and accounted for 80–85% of all identified bacteria. Other studies have reported that Sphingomonas is associated with pulque fermentation (80%) and that this was the predominant genus in fermented products of fruit (78%) among all identified genera. Sphingomonas, which is considered to be the indigenous flora of soil and plants [Sphingomonas was the major bacterial genus in the final product [Oribacterium was the second most predominant genus in the OTU-based clustering, whereas Nesterenkonia was the predominant genus in ASV-based clustering. Oribacterium and Nesterenkonia are frequently associated with the surface of fruits, water, and soil. A previous study also reported that these two approaches yield different taxonomic levels, which explains the difference in the discriminatory power of these approaches [Among the genera detected in both clustering approaches, d plants  and is md plants . A recen product . Additioproaches . Moreoveproaches . Similarproaches ,59. The proaches . Other fproaches . These fParacoccus, Dietzia, Devosia, and Pelomonas were significantly enriched in the samples from the first stage of fermentation, whereas their abundance significantly decreased in the samples from the second stage of fermentation. Similarly, Dietzia, Devosia, Lactobacillus, Nesterenkonia, Paracoccus, Pelomonas, and Stenotrophomonas were enriched in the samples from the first stage of fermentation but their abundance significantly decreased in the final product. The increased abundance of these bacteria at the initial stages of fermentation can be attributed to the availability of sufficient nutrients and the suitability of environmental conditions, which promote the proliferation and growth of these bacteria. However, these nutrients are depleted and the culture conditions are unfavorable as the fermentation progresses due to decreased pH caused by organic acid producers, such as Lactobacillus. These changes decrease the abundance of other sensitive microorganisms [Lactobacillus was the most abundant genus in the first stage of fermentation, whereas Nesterenkonia was the abundant genus in the second stage of fermentation. Lactobacillus and Nesterenkonia are beneficial for the fermentation of sugars, which leads to acid production and consequently decreased pH levels. Compared with those at the second stage of fermentation, the production of organic acids was upregulated and the pH level was lower in the final product as shown in Lactobacillus is a vital probiotic that produces several types of short-chain fatty acids, which are beneficial to human health [The change in microbial communities during fermentation must be considered to understand the functionality of beneficial microorganisms and the survival of opportunistic pathogens. The results of this study demonstrated that most differentially abundant genera, such as rganisms . Addition health .To examine the role of microorganisms during fermentation, PICRUSt2 was used to analyze the 16S rRNA sequencing data. Six major functional categories (KEGG level 1) , environmental information processing (3.2%), genetic information processing (9.6%), human disease (0.8%), metabolism (78.4%), and organismal systems (3.2%)) were significantly associated with the fermentation process. The upregulated function of metabolism suggested the active involvement of microorganisms during fermentation. These results are consistent with those of previous studies, which reported that metabolism was the predominant pathway among the six main KEGG pathways ,62. AmonAdditionally, the metabolism of other amino acids and glycan biosynthesis and metabolism were the predominant functions between the first and second stages of fermentation. Most functions were significantly enriched in the first stage of fermentation. The functions related to bacterial growth and proliferation, such as replication and repair were the dominant function in the final product. However, amino acid metabolism and metabolism of terpenoids and polyketides were significantly enriched in the second stage and the final product of fermentation. The mean proportion of all significantly enriched pathways decreased in the following order: first stage > second stage > product of fermentation. These results suggest that the bacterial community has varied metabolic capacity and preferential functions in each stage of the fermentation process depending on the fermentation environment. The decline in the mean proportion of these significantly enriched pathways during the fermentation process might be due to the enzymatic activity of various microorganisms. Alternatively, the decline in the number of potential microorganisms responsible for the production of these predicted functions may also contribute to the downregulation of these functions. A similar pattern of decline in the mean proportion of bacterial community was observed in significant microbial shift analysis with the passage of the fermentation process. However, for further confirmation of the presence of these bacterial metabolites and their potential role, we strongly suggest using metabolomics and transcriptomic analyses.Staphylococcus infection (KEGG level 3) was detected in all samples with a maximum relative abundance of 0.6%. This proportion is consistent with the results of PCR analysis but less than that reported in the recent study on Chinese traditional fermented food reports. Approximately 2% of bacterial genes in the fermentation process were associated with the human disease, which indicated pathogenic contamination or enrichment during the fermentation process. However, further categories of KEGG pathways and microbes associated with the human disease were not reported in their study [S. aureus was only detected in the pineapple sample at the first stage of fermentation in ASV-based taxonomic classification and PCR validation assay.Bacterial function prediction revealed only one crucial predicted human disease pathway associated with ir study . MoreoveSphingomonas was correlated with all the predicted functions as this was the predominant genus in fermentation samples and accounted for 80–85% of all observed genera. Additionally, Sphingomonas can metabolize various carbon compounds and toxic compounds that are present in low concentrations of nutrients [Delftia and Gardnerella were significantly and positively correlated (p < 0.05) with most of the observed functions. In contrast, Lactobacillus was significantly and negatively correlated with cell mobility, cellular community-prokaryotes, signal transduction, and digestive system. Delftia and Gardnerella, which are soilborne bacteria, are commonly found on the surface of fruits. A previous study has demonstrated that these bacteria can metabolize various carbon sources and are correlated with the fermentation processes [Staphylococcus and infectious disease was not observed. However, this bacterium was significantly and positively correlated with the endocrine system. This may be due to the limited number of sequences (0.6%) representing this particular function associated with infectious disease. A previous study demonstrated that differences in the relative abundance of metagenomic data and PICRUSt inferences might not allow an accurate representation of functional traits [Pearson correlation analysis was performed to examine the correlation between predicted functions and bacterial community of fermentation. The analysis revealed that utrients . Additiorocesses . Additiol traits . Thus, tS. aureus was detected in one sample. This indicated that human pathogenic species are not the predominant species in the fermented samples. In this study, the fermented samples were obtained from fruits associated with soil environments. Soil microbes are not considered to be pathogenic. In the genera Acinetobacter and Bacillus, A. calcoaceticus, A. johnsonii, B. subtilis, and B. megaterium are commonly detected in plants and soil [C. difficile is the major pathogenic species in the genus Clostridium. However, more than 250 species have also been reported to be beneficial to human health [S. aureus was detected in the Pi4_1st sample in the PCR assay, it was not detected in the sample from the second stages and the final product of fermentation. Therefore, this hybrid method could ensure that the pathogen was inactive in the fermentation process and the final fermentation product did not contain pathogenic microorganisms. Additionally, the genus Staphylococcus was identified in the fermentation stages and the final product using OTU-based taxonomical classification. However, Staphylococcus was only identified in the first stage of fermentation using the ASV-based clustering approach. Hence, we propose that compared with the PCR assay, the ASV-based taxonomical classification analysis is more suitable for pathogen prediction.In this study, the 16S rRNA amplicon was sequenced by targeting the V3–V4 regions of DNA for bacterial identification. The identification of bacteria at the species level based on NGS of 16S rRNA targeting the specific conserved regions of DNA is not reliable as it can yield false-positive results . Howeverand soil . C. diffn health . AlthougSphingomonas was predominantly detected during all fermentation stages and in the final product. Additionally, Lactobacillus was significantly enriched with a higher abundance in the 1st stage of fermentation, whereas Nesterenkonia was significantly enriched with a higher abundance in the 2nd stage of fermentation. Functional prediction revealed carbohydrate metabolism was the most abundant metabolic pathway throughout the fermentation process. Moreover, Staphylococcus aureus associated with the KEGG human disease pathway was not significantly correlated with opportunistic bacterial pathogenic genera. To confirm the presence of opportunistic bacterial pathogens, PCR assays were performed which revealed absence of these opportunistic pathogens in different fermentation stages or the final products, except in one sample from the first stage of fermentation. Thus, the hybrid method employed in this study can be used to verify the bacterial safety and functions of microorganisms during fermented food production and avoid random detection of opportunistic bacterial pathogens. This hybrid method could provide an effective and high-resolution determinant of the types of bacteria and their functional profile influencing the properties of fermented foods and ensure that no harmful bacteria are present in the final fermentation products.This study revealed the correlation between bacterial community composition and functional characteristics during the two stages of fruit fermentation and the final product using high-throughput sequencing and powerful statistical tools.


Immunoglobulin light chain amyloidosis (AL) is an indolent plasma cell disorder characterized by free immunoglobulin light chain (FLC) misfolding and amyloid fibril deposition. The cytogenetic pattern of AL shows profound similarity with that of other plasma cell disorders but harbors distinct features. AL can be classified into two primary subtypes: non-hyperdiploidy and hyperdiploidy. Non-hyperdiploidy usually involves immunoglobulin heavy chain translocations, and t is the hallmark of this disease. T is associated with low plasma cell count but high FLC level and displays distinct response outcomes to different treatment modalities. Hyperdiploidy is associated with plasmacytosis and subclone formation, and it generally confers a neutral or inferior prognostic outcome. Other chromosome abnormalities and driver gene mutations are considered as secondary cytogenetic aberrations that occur during disease evolution. These genetic aberrations contribute to the proliferation of plasma cells, which secrete excess FLC for amyloid deposition. Other genetic factors, such as specific usage of immunoglobulin light chain germline genes and light chain somatic mutations, also play an essential role in amyloid fibril deposition in AL. This paper will propose a framework of AL classification based on genetic aberrations and discuss the amyloid formation of AL from a genetic aspect. Immunoglobulin light chain amyloidosis (AL) is an indolent plasma cell disorder characterized by the misfolding of free light chains (FLC) and amyloid fibril deposition in different tissues . The annPlasma cells in AL are usually indolent, but AL is regarded as a dangerous disease because only about 20% of patients could survive over 10 years, and about 25% of patients would die within the first 6 months mainly due to related organ complications –11.In the past decades, significant progress has been made in understanding the molecular and genetic pathogenesis of AL. Concepts of precise treatments based on the disease’s genetic properties have emerged, which hopefully would lead to a new era of therapies in the future. This review article will focus on the pathogenesis, clinical features, and prognostic impacts of different genetic aberrations present in AL. We will also briefly discuss the influences of specific usages of germline genes and mutations on the immunoglobulin gene in amyloid formation.The cytogenetic landscape of AL has profound similarity with that of monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM). Several studies have indicated that the cytogenetic concepts applied in MM may be transferable to AL.MM can be classified into two distinct genetic branches: non-hyperdiploidy and hyperdiploidy based on chromosome contents, and they can further form various subclones through secondary mutations –14. SimiPatients with AL also show a high frequency of chromosome instability like other plasma cell disorders. Data shows that 60–90% of AL patients harbor at least one chromosome abnormalities and about 50–70% of them have translocations involving the IgH locus on chromosome 14 –17.However, although AL shares some similar genetic features with other plasma cell dyscrasia, its distribution patterns of the cytogenetic abnormalities differ significantly from that of MGUS and MM. In AL, t is the most common genetic abnormality with a frequency ranging from 40 to 60% , 18, whiUnlike other plasma cell dyscrasia, of which the prognostic roles of different genetic aberrations have been better described, less is known in AL. However, there is an increasing number of studies about this topic. Most researches about AL detected chromosome aberrations through the interphase-fluorescence in situ hybridization (iFISH) technique, using the genetic probes in MM. Different genetic aberrations involve different pathways of pathogenesis and influence prognostic outcomes and decision-making in clinical practice. The following section will focus on the basic characteristics and clinical features of the major genetic aberrations present in AL.About 10–40% of AL patients cannot be detected to harbor any chromosome aberration by the MM’s genetic probes. This patient group tends to have lower plasma cell burden, less severe cardiac involvement, longer progression-free survival (PFS) and overall survival (OS) –17, 19, IgH translocation is the most common abnormal cytogenetic event in AL. This translocation involves the juxtaposition of different chromosomes to the IgH locus on chromosome 14, which leads to the overexpression or dysregulations of various genes, especially the cyclin gene families , 20–23. CCND1) on chromosome 11, leading to the overexpression of CCND1 in plasma cells [Translocation t is the most prevalent genetic aberration present in AL. This translocation involves the juxtaposition of the IgH locus on chromosome 14 to the oncogene cyclin D1  is usually genetically stable with a low rate of subclone formation and progression both in AL and non-AL plasma cell disorders . ProbablPatients with t are associated with low plasma cell clones in the bone marrow, but they tend to be loaded with a heavy FLC burden in the serum, which is shown to be related to shorter survival in several studies –30. The Reasons for the higher free light chain level in t compared to that in non-t remain poorly understood, but the IgH locus translocation that leads to the disruption of the IgH expression or structure may partially explain that. This is supported by the evidence from a study that identified a strong relationship between t and the lack of an intact immunoglobulin in AL patients . Data frThe prognostic effect of t11;14) in AL patients is still controversial, and treatment options seem to have a significant impact on the clinical outcomes of this patient group . In patients receiving melphalan/dexamethasone as first-line therapy, a tendency of longer OS was observed in those with t compared with those without t . BesidesSelective AL patients with t also respond well to bcl-2 inhibitor, venetoclax. Bcl-2 is a protein that regulates the programmed cell death pathway in normal cells. Cells overloaded with bcl-2 fail to undergo apoptosis but accumulate in the body. Plasma cell disorders, including MM and AL, especially those with t, are associated with the overexpression of bcl-2 –39. The Daratumumab, a monoclonal CD38 antibody, has also shown promise outcome in t-positive patients as a salvage therapeutic modality, demonstrating a longer hemEFS . This maIn summary, t is associated with an increased FLC level in AL and poorer survival outcomes under bortezomib-based regimens, especially in low-risk patients. Melphalan and novel agents, including venetoclax and daratumumab, may be capable of overcoming the adverse effect of t in AL treatments.FGFR3) and multiple myeloma SET domain (MMSET), together with the formation of IGH/MMSET hybrid transcripts [Translocation t results in the dysregulation of two genes on chromosome 4, the fibroblast growth factor receptor 3  has been described in about 2% of AL, a frequency slightly lower than that of MM (~ 5%) , 27. Thito CCND2 . A closeto CCND2 . Whetherto CCND2 , 36, butCCND3 dysregulation [Other IgH locus translocations, such as t and t, are rarely found in AL , 18, 52.gulation , 53. In gulation , 54. HowHyperdiploidy is a well-recognized alternative pathogenetic pathway in plasma cell disorders. It is characterized by the presence of extra copies of multiple chromosomes, especially odd-numbered chromosome 3, 5, 7, 9, 11, 15, 19 and 21 , 55.Because of the low percentage and low proliferative rate of plasma cells, conventional karyotypic analysis methods are not suitable for the study of ploidy in AL , 57. To The application of Wuilleme’s definition of hyperdiploidy will inevitably underestimate the prevalence of this genetic aberration. A study from Granzow et al. using high-density copy number arrays detected 25% (29/118) of patients as hyperdiploidy, including six patients not identified as hyperdiploidy by Wuilleme’s criteria . AnotherOverall, hyperdiploidy seems to confer a neutral or inferior outcome in AL patients, which is contradictory to results from MM studies that indicate a favorable prognostic effect of hyperdiploidy , 13, 60.Trisomies, defined as the presence of extra copies of one or more chromosomes, include both hyperdiploidy and mono-chromosome gains. 20–30% of AL patients harbor trisomies, with trisomies 9 being the most common genetic aberration, followed by trisomies 19, 15, 5, 18, 11, 7, 3, 17, 4, 21 and 14 . High dFReasons for the adverse impact of trisomies remain unknown. One of the possible reasons is that trisomies correlate with a higher clone evolution rate, indicating a more malignant potential of this disease. Moreover, the high dFLC level and plasma cell burden in patients with trisomies may lead to more amyloid fibril formation in targeted tissues and worsen organ functions. Further studies are still needed to explore the role of trisomies in AL. Besides, it might be necessary to subdivide trisomies into hyperdiploidy group, usually regarded as the primary cytogenetic event, and mono-chromosome gain group, regarded as the secondary cytogenetic event, as the underlying mechanisms of these two groups differ and they may have different prognostic impacts.Malignant plasma cells of an individual AL patient are not usually genetically identical, but harbor some additional aberrations, forming various subclones. This intraclonal heterogeneity in AL is the consequence of secondary cytogenetic events occurring during disease progression. Studies about AL’s subclone architecture reveal that gain of 1q21, deletion of chromosome 13/13q, 17p and 8p21, secondary IgH translocations, and point mutations are frequent progression-related aberrations . HyperdiIn AL, 1q21 gain usually results from the gain of the long arm of chromosome 1, rather than trisomies 1 . 20–30% Probably, due to the low CD38 level, patients with 1q21 gain achieved less satisfactory outcomes than those without this genetic aberration when daratumumab-containing regimens were adopted , 66. OneIDP2 and MLH3, located in the minimal common region of deletions on chromosome 14q, may be responsible for the disease pathogenesis, but there is no evidence by now to validate this notion.The responsible genes underlying this chromosome aberration are unknown. The amplification of cyclin kinase subunit 1B (CKS1B) has been identified in a significant proportion of MM patients with the gain of 1q21 and is associated with an unfavorable survival outcome , 68, butRb1 gene, located on chromosome 13q, has been proposed to be responsible for disease progression in MM [Chromosome 13 abnormalities, including monosomy 13 and deletion of 13q (del13q), can be detected in about one-third of patients with AL , 25, 59 on in MM , 71. A sDeletion of 17p (del17p), specifically at the locus of 17p13, occurs at a rate of no more than 5% in AL , 26. Prop53, the dysfunction of which leads to disease progression of various tumor kinds, including plasma cell disorders [Research about the pathogenetic effect of del17p in AL is scarce. Del17p can result in the deletion of an important tumor suppressor gene, isorders –75. In Misorders , 13. Sevisorders , 77. A sisorders .Deletion of 8p21 and secondary IgH translocation occur at a low frequency in AL, so their clinical characteristics and prognostic effects are poorly understood , 31, 59,ASB15, ASCC3 and HIST1H1E are associated with inferior OS [KRAS and NRAS activated mutations, commonly found in MM, are only identified in a few cases in AL [KRAS as a recurrent mutation without survival significance [Whole exome sequencing (WES) can detect specific point mutations undetectable by the conventional iFISH method. A series of recurrent mutations in AL have been identified, but they all occur at a very low rate and there is no consistent result regarding these recurrent mutations in recent studies , 78–80. erior OS , 78, 79.erior OS , 79. KRAes in AL , 78, 79,IGLV6-57 and IGLV1-44 are linked to increased renal and cardiac involvement, respectively [IGLV6-57, IGLV3-01 and IGLV2-14, occur at a higher frequency in AL than other plasma cell dyscrasia or normal B cells [The cytogenetic landscape of AL displays profound similarity with that of MGUS and MM, confirming the concept that they are related plasma cell dyscrasia. However, these findings are insufficient to explain why plasma cells in AL are more prone to secret amyloid-generating proteins. Fortunately, research on the immunoglobulin light chains (IgL) helps shed some light on the pathogenesis of this disease. Data in the previous studies indicate that organ tropism in AL may be the function of the usage of specific IgL germline genes –88 see . For exaectively , 86–88.  B cells , 88, 91.Somatic mutations in the IgL genes, generated in the process of immunoglobulin synthesis, add complexity to the IgL structure. The burden of the somatic mutations and the specific site that a mutation occurs can influence the stability and amyloidogenicity of IgL –95. A moAltogether, these findings indicate that the formation of amyloid fibril in various organs is a multifactorial process involving chromosome aberrations, secondary mutations, specific usage of IgL germline genes, IgL somatic mutations, and some other unidentified reasons see Fig. .Fig. 2AmAL amyloidosis is a plasma cell disorder having distinct features from that of MM, although they indeed share some genetic similarities. Each genetic aberration in AL involves distinct pathogenetic pathways and displays some special clinical features. However, these are insufficient to explain the most significant feature of AL, the amyloid formation. Studies about the IgL indicate that specific usages of IgL germline genes and point mutations in the IgL locus may be capable of predicting the organ tropism and fibril formation tendency of this disease. Treatment options based on the cytogenetic features of AL and novel therapies targeting amyloid formation have a promising future. To date, due to the low incidence of AL, most studies of this disease are retrospective. Therefore, multi-center cooperations are needed to investigate specific cytogenetic abnormalities and conduct sizable clinical trials in this disease. The development of the predictive model, based on the cytogenetic properties of AL, may help improve the early diagnosis and clinical outcomes in AL patients.