Bacteroides, Prevotella, Escherichia coli, Klebsiella, DNA molecules, and ARGs, as well as antimicrobials. Azithromycin and doxycycline removal rates were >99% immediately after treatment, and levofloxacin and vancomycin removal rates remained between 90% and 97% for approximately one month. Clarithromycin was more readily removed than the other antimicrobials (81–91%), and no clear removal trend was observed for ampicillin. Our findings provide a better understanding of the environmental management of hospital wastewater and enhance the effectiveness of disinfection wastewater treatment systems at medical facilities for mitigating the discharge of pollutants into aquatic environments.Antimicrobial resistance (AMR) is becoming a global concern. Recently, research has emerged to evaluate the human and environmental health implications of wastewater from medical facilities and to identify acceptable wastewater treatment methods. In this study, a disinfection wastewater treatment system using an ozone-based continuous flow system was installed in a general hospital located in Japan. The effectiveness of antimicrobial-resistant bacteria (ARB) and antimicrobials in mitigating the environmental impact of hospital wastewater was evaluated. Metagenomic analysis was conducted to characterize the microorganisms in the wastewater before and after treatment. The results demonstrated that ozone treatment enables effective inactivation of general gut bacteria, including  The Lancet, an internationally renowned medical journal, annual deaths attributable to AMR are anticipated to nearly double in five years to 1.27 million in 2019 [In recent years, antimicrobial resistance (AMR) has become dangerously close to our daily lives, raising concerns about sustainable human development ,2,3. A s in 2019 . In JapaPrevious research has indicated the impact of AMR originating from wastewater entering the environment, despite the fact that the origins of AMR detected in aquatic environments are diverse ,10,11. SE. coli, which are widely distributed in the environment, by encouraging the development of new AMR, even though the direct health effects of ARGs are considered minor, as is the case with viable bacteria [Human environmental risks from AMR environmental contamination include the health impacts associated with direct inoculation exposure to viable bacteria surviving in the environment ,28,29. Fbacteria ,31,32. Mbacteria ,34,35. Ubacteria ,37,38. Fbacteria ,40.3) was installed in the hospital to evaluate the inactivation effect of ozone treatment on ARB and antimicrobials. Both ARB and residual antimicrobials were reduced to ≤1% after 20 min of treatment [With the recent remarkable development of science and technology, wastewater treatment systems that are effective in treating hospital wastewater have been developed ,43,44,45reatment . If the reatment ,53,54. Freatment ,56,57.As part of the efforts to implement a hospital wastewater treatment system in society, a pioneering trial has begun to verify the effectiveness of measures utilized to reduce the environmental burden of the hospital wastewater treatment system by conducting continuous treatment of the entire hospital wastewater before discharging it into the public sewer system. Therefore, in the present study, a continuous treatment system that can effectively treat hospital wastewater without interfering with hospital operations was developed. To achieve more effective treatment, we additionally tested an ultraviolet light-emitting diode (UV-LED) ,59, whic3. The annual daily inflow of the wastewater tanks was 50 m3/day, which was approximately equal to the volume of the wastewater tanks. The supernatant was pumped directly into the public sewage system at 2 m3 per discharge with an average frequency of 25 discharges per day (50 m3/day as outflow). First, hospital wastewater from one of the two storage tanks was introduced into wastewater treatment tank 1 (ozone) at a flow rate of 20 L/min, with an effective volume of 1 m3 for ozone treatment. The ozone-treated wastewater was then flowed into wastewater treatment tank 2 (UV-LED) with an effective volume of 1 m3, which was connected to a UV disinfection unit for further inactivation owing to overflow inflow at a flow rate of 20 L/min. Finally, the treated wastewater was returned to the original storage tank on one side using a return pump (flow rate 150 L/min) for circulation to the original storage tanks (influent). The appearance and configuration of the ozone treatment system used in this study are shown in An ozone inactivation system for bacteria and antimicrobials present in hospital wastewater based on a continuous flow system was installed at the University Hospital, Ohashi Medical Center , with a capacity of 319 beds, in Toho University, Tokyo, Japan, as previously reported . Various® TOGC45X, Suez Environment, Paris, France) equipped with a Pressure Swing Adsorption (PSA) oxygen generator. The hospital wastewater in the wastewater treatment tank was circulated using a circulation pump  at a flow rate of 5 L/min, and ozone gas was fine-bubbled through fine-bubble generating nozzles   of the solution in this tank was collected 0, 1, 4, 6, 8, 15, and 29 days after the start of the experiment. Basic water quality parameters , chemical oxygen demand (COD), suspended solids (SS), and total nitrogen (TN)) during treatment in this investigation, along with DNA concentration and total reads of metagenomic DNA sequences, are shown in Ozone was generated using an ozone generator  and β-lactam-resistant bacteria, an aliquot (100 µL) of influent or treated wastewater sample was 10-fold serially diluted with phosphate-buffered saline, followed by spreading on non-selective media BTB  agar and CHROMagar ESBL plates  for extended-spectrum β-lactamases (ESBL) producing bacteria. Colony-forming units per mL (CFU/mL) were determined at the appropriate dilution for each treatment time point.To determine the efficacy of ozone treatment in inactivating potential 2, 0.2 µm polyethersulfone membranes. The membranes were removed from the bottles and stored at −30 °C until DNA extraction. One-fourth of the collected membrane, corresponding to 25 mL of influent or treated water, was cut into small pieces and placed in ZR-96 BashingBead Lysis Tubes . Bacterial lysis buffer  was added to the bead tube, which was then frozen at −30 °C and thawed at 23 °C. The tube was subjected to bead-beating (1500 rpm for 10 min) using a GenoGrinder 2010 homogenizer. After brief centrifugation (8000× g for 3 min), 400 µL of the supernatant was collected. The DNA in the supernatant was purified using a Roche MagNa Pure Compact instrument . DNA concentration and purity were measured using a Qubit DNA HS kit .To collect organisms larger than bacteria, ozone-treated wastewater samples were passed through TPP Rapid Filtermax Vacuum Filtration systems  in 100 mL bottles fitted with 49 cmhttps://github.com/najoshi/sickle) considering the following parameters: average quality threshold “−q 20” and minimum length threshold “−l 40.” Metagenomic DNA-seq analysis was performed using clean reads for homology searches without de novo assembly for all subsequent analyses. Detailed scripts and databases are described below.Metagenomic DNA-seq libraries were prepared using the QIAseq FX DNA Library Kit , followed by short-read sequencing using the NextSeq 500 platform (2 × 150-mer paired-end) . Adapter and low-quality sequences were trimmed using Sickle version 1.33  against the NCBI nt database using MePIC2 [Taxonomic classification of every read from the metagenomic analysis was performed using mega-BLAST , new quinolones (levofloxacin), macrolides (azithromycin and clarithromycin), tetracyclines (doxycycline), and glycopeptide (vancomycin) (>98%), were examined in the present investigation on the basis of a previous report on their concentrations and detection frequencies in hospital effluent, wastewater, and river water, both in Japan and around the world [A total of six antimicrobials grouped into five classes, he world ,76, as whe world ,78.2 gas at 37 °C. The residue was solubilized in 200 μL of a 90:10 (v/v) mixture of 0.1% formic acid solution in methanol, and 10 μL of this solution was analyzed using an ultra-performance liquid chromatography–tandem mass spectrometry (UPLC) system coupled to a tandem quadrupole mass spectrometer . Quantification was performed by subtracting the blank data from the corresponding data yielded by the spiked sample solutions to account for matrix effects and losses during sample extraction [The concentrations of the target antimicrobials in the wastewater were analyzed as previously described ,25. Brietraction ,80. The traction ,82.Hospital wastewater was treated with ozone followed by UV-LED irradiation in a continuous-flow pilot plant  for 0, 1Although it did not exhibit significant inactivation of the general parameters apart from the visible color, the treated water collected from the ozone or UV-LED tanks showed >90% inactivation of viable bacteria on BTB agar after 1 d of treatment B,C, and Bacteroides and Prevotella, showed 90% less detection following ozone treatment compared with the original storage on day 1  and Pseudomonas sp. LTGT-11-2Z was isolated from the roots of Alhagi sparsifolia Shap. in the Taklamakan Desert, China (BioSample: SAMN10219285), suggesting that neither were identified as clinical specimens; however, their pathogenicity remains to be investigated.To characterize the actual susceptibility of bacterial genera to ozone, membrane-trapped bacteria in the treated sample (corresponding to the 25 mL water sample) were subjected to genomic DNA extraction , and meton day 1 . In addireatment . On the o genera . Further onwards . Acinetosul1 and qacEdelta), β-lactamase GES variants , aminoglycoside acetyl transferase (aac(6′)-31), and tetracycline resistance (tet(39) and tet(36)) were mainly detected in sewage samples. Most of these were significantly inactivated to less than 10% of the sequencing reads on day 1, consistent with the results of the CFU  among Bacteroides, Prevotella, Pseudomonas, Bifidobacterium, and Ruminococcus families, and partial effects on the other bacterial families were also suggested  and limits of quantification (LOQs) are summarized in Ozone treatment was effective in removing all the targeted antimicrobials. More than 99% of azithromycin and doxycycline was removed immediately after treatment and was not detected in the ozone treatment tank throughout the experiment. The removal rates for levofloxacin and vancomycin remained between 90% and 97% during treatment. Clarithromycin was more readily removed than the other antimicrobials (81% to 91%), and no clear removal trend was observed for ampicillin. In the wastewater treatment tank where UV-LED irradiation followed ozonation, ampicillin and levofloxacin additionally decreased from their levels in the ozone treatment tank by an average of 15% and 53%, respectively. However, no notable changes in the concentration were observed for the other compounds. Finally, the concentration of antimicrobials before discharge into the public sewage system decreased during continuous-type treatment. The average removal rates of antimicrobials in the present study were 71 ± 24% for levofloxacin, 82 ± 16% for azithromycin, 88 ± 10% for doxycycline, and 44 ± 42% for vancomycin. However, no clear removal trends for ampicillin and clarithromycin were observed during the experiment, which was largely consistent with the removal trend of antimicrobials in the ozone treatment tank.The detection of ARB and antimicrobials in hospital wastewater throughout the period of this treatment test suggests that a certain amount of these pollutants is released from every hospital into the public sewage system, and that it is vital to implement social countermeasures on a large scale. The original storage tank (influent) showed an increase/decrease in the detected concentrations of both ARB and antimicrobials, which was associated with the continuous inflow of untreated raw hospital wastewater into the original tank.Bacteroides, Prevotella, E. coli, and Klebsiella, were significantly inactivated by ozone treatment  were consistent for the antimicrobials and for ampicillin and clarithromycin, which were not adequately removed by the sequential treatment in this study; the removal rate was 96–100% for these antimicrobials at 40 min after treatment. These data suggest that it is essential to upgrade the treatment system and conduct demonstration tests and evaluations at an actual plant scale when developing from a batch  system to the actual treatment of hospital wastewater.β-lactam antimicrobials such as ampicillin, are attenuated in water within a few hours [The results of the present investigation show that ozone treatment removes antimicrobials, and the treatment time required for removal differs for each compound, supporting the results reported in previous evaluations of ozonation of pharmaceuticals in environmental water ,88. Antiew hours ,94. Clarew hours ,96. Levoew hours . Previouew hours ,99,100. ew hours ,102. Whoew hours ,104,105.ew hours ,106,107.It is possible to reduce or eliminate the inflow of ARB and antimicrobials as environmental pollutants into the aquatic environment through the advancement of wastewater treatment systems ,65,108. As studies have revealed the antimicrobial-resistant nature of hospital wastewater and the significant impact of the loads discharged into the environment, more attention is being paid to how hospital wastewater should be treated ,56. HoweThe limitations of this study are as follows: The first is the optimization of wastewater treatment systems. An ideal wastewater treatment system involves the continuous direct discharge of treated water into a public sewage system. However, there are still issues that have not been fully covered in the present investigation in terms of practical aspects, such as technology, funding, and the need to reconstruct the entire hospital facility to continue treating the entire hospital wastewater while maintaining a balance with the constant inflow of untreated wastewater ,115. AnoSecond, the inactivation effects of ARB and antimicrobials were investigated. Neither ARB nor antimicrobials were completely inactivated during the trial of hospital wastewater treatment. Under these conditions, microorganisms remain viable in the treated hospital wastewater. It is essential to elucidate the potential of these microorganisms to form biofilms in treatment tanks  and to eFinally, the treatment effectiveness for the basic general water quality parameters was noted. For the hospital wastewater treatment in this study, the treatment system was found to be capable of inactivating ARB and antimicrobials, which have become new environmental pollutants of concern in recent years, although some improvements, including water quality, have not yet been achieved. Improving the treatment to include these water quality items can be achieved by increasing the amount of ozone injected, but in some respects, this treatment strategy is not practical or energy-efficient and it would be more effective in combination with other treatments ,119,120.In the present study, an ozonation-based continuous-flow disinfection wastewater treatment system was implemented in a core hospital located in the center of Japan, and its effectiveness in mitigating the environmental impact of AMR associated with hospital wastewater was evaluated. The results showed that both ARB and antimicrobials that would have an impact on the environment were effectively removed and maintained at a low level during treatment, which would be an effective countermeasure to mitigate the environmental impact caused by AMR. These findings are significant for implementing feasible and effective countermeasures to address AMR in the environment. The overall results facilitate a comprehensive understanding of the AMR risk posed by hospital wastewater and provide insights for devising strategies to eliminate or mitigate the burden of ARB and flow of antimicrobials into aquatic environments. Our findings could help enhance the effectiveness of introducing wastewater treatment systems, not only in wastewater treatment plants, but also in medical facilities, to reduce the discharge of pollutants into rivers, thereby contributing to environmental and human health safety.


Digital behavioral weight loss programs are scalable and effective, and they provide an opportunity to personalize intervention components. However, more research is needed to test the acceptability and efficacy of personalized digital behavioral weight loss interventions.In a 6-month single-arm trial, we examined weight loss, acceptability, and secondary outcomes of a digital commercial weight loss program (WeightWatchers). This digital program included a personalized weight loss program based on sex, age, height, weight, and personal food preferences, as well as synchronous  and asynchronous  elements. In addition to a personalized daily and weekly PersonalPoints target, the program provided users with personalized lists of ≥300 ZeroPoint foods, which are foods that do not need to be weighed, measured, or tracked.We conducted a pre-post evaluation of this 6-month, digitally delivered, and personalized WeightWatchers weight management program on weight loss at 3 and 6 months in adults with overweight and obesity. The secondary outcomes included participation, satisfaction, fruit and vegetable intake, physical activity, sleep quality, hunger, food cravings, quality of life, self-compassion, well-being, and behavioral automaticity.2. Participants had an average weight change of −4.25% (SD 3.93%) from baseline to 3 months and −5.05% (SD 5.59%) from baseline to 6 months. At 6 months, the percentages of participants who experienced ≥3%, ≥5%, and ≥10% weight loss were 63.4% (97/153), 51% (78/153), and 14.4% (22/153), respectively. The mean percentage of weeks in which participants engaged in ≥1 aspects of the program was 87.53% (SD 23.40%) at 3 months and 77.67% (SD 28.69%) at 6 months. Retention was high , and more than two-thirds  of the participants reported that the program helped them lose weight. Significant improvements were observed in fruit and vegetable intake, physical activity, sleep quality, hunger, food cravings, quality of life, and well-being .Of the 153 participants, 107 (69.9%) were female, and 65 (42.5%) identified as being from a minoritized racial or ethnic group. Participants’ mean age was 41.09 (SD 13.78) years, and their mean BMI was 31.8 (SD 5.0) kg/mThis personalized, digital, and scalable behavioral weight management program resulted in clinically significant weight loss in half  of the participants as well as improvements in behavioral and psychosocial outcomes. Future research should compare personalized digital weight loss programs with generic programs on weight loss, participation, and acceptability. Digital behavioral weight loss programs are effective , and thesurface level, including age, height, weight, and sex, and deep level, including food preferences, diabetes status, breastfeeding status, physical activity habits, and water consumption. The app uses user-entered data regarding the factors that influence energy needs, including age, height, weight, and sex, to personalize the PersonalPoints budget. This is further personalized based on breastfeeding status and physical activity level, which also directly affect energy needs. Users receive a personalized list of ZPFs that includes foods that form the basis of an overall healthy dietary pattern and can reduce hunger and cravings, given their macronutrient composition . This minimizes hunger because, as ZPFs do not need to be tracked, even if no PersonalPoints remain in a user’s budget for the day, they may eat foods on their ZPF list. The program reinforces physical activity by allowing users to receive additional PersonalPoints for being active. The personalization is also dynamic such that participants can change their food preferences, physical activity habits, nonstarchy vegetable consumption, and water intake at any time, which then changes their PersonalPoints daily and weekly goals as well as their ZPF list (which includes >300 options). The purpose of the PersonalPoints program is to provide dynamic, personalized targets to help members lose weight while improving overall dietary patterns, reducing hunger and cravings, encouraging physical activity, and simplifying diet tracking by not requiring all foods consumed to be tracked.The WeightWatchers (WW) program is a well-validated commercial weight management program that now leverages a personalized eHealth approach -10. WW’sOur first aim in this 6-month single-arm trial was to examine the effect of this personalized program on percentage weight loss from baseline to 3 and 6 months. Our second aim was to describe participation and acceptability as indicators of program satisfaction. Our third aim was to examine the impact of this program on fruit and vegetable intake, physical activity, sedentary behavior, hunger, and food cravings. Our fourth aim was to examine the impact on sleep, quality of life, self-compassion, overall well-being, and behavioral automaticity at 6 months. This is the first trial to evaluate the WW PersonalPoints program.2, Wi-Fi connectivity at home, having an iPhone (because the app was only available on iOS at the time), English proficiency, self-reported desire to lose weight, and US residence. The exclusion criteria were being pregnant, lactating, or planning to become pregnant during the study period; severe mental illness ; eating disorders; hospitalization for psychiatric disorders during the past 12 months; type 1 or type 2 diabetes; taking medications that affect weight; currently in a structured weight loss program; had bariatric surgery or plans to have any surgery during the study; unable to walk one-fourth of a mile unaided without stopping; smoker or uses nicotine vape daily; medical condition that precludes ability to make dietary changes or increase physical activity; weight loss of ≥5 kg in the last 6 months; WW membership in the last 12 months; major surgery within the previous 6 months; implanted cardiac defibrillator or pacemaker; history of cancer within past 5 years or current treatment for cancer; and unable to attend any virtual workshop meeting times.This study was a 6-month single-arm trial evaluating the effectiveness of a commercially available WW PersonalPoints weight management and wellness program on percentage weight loss at 3 and 6 months. We recruited adults with overweight or obesity who were interested in losing weight by posting recruitment advertisements on Facebook, Instagram, Twitter, Reddit, Craigslist, and Research Match, which connects volunteers to research studies throughout the United States ,13. The Participant screening involved an initial web-based survey, followed by a telephone interview. Eligible participants then completed a web-based baseline survey. Subsequently, they were mailed a WW Conair Bluetooth Scale, and they completed an onboarding process, which included emailed instructions on how to set up the app and Bluetooth scale, including an option to schedule a call with study staff to assist with setup. Study staff checked that all scales were set up before the program commenced. Participants then took part in the 6-month WW PersonalPoints program. Data collection occurred at 3 and 6 months, which involved a web-based survey and a weigh-in on the scale. Participants were compensated with Amazon e-gift cards at baseline (US $20), 3 months (US $50), and 6 months (US $50) and were allowed to keep the Bluetooth scale after the study. The study period was from April 5, 2021 to September 19, 2021. This trial is registered at ClinicalTrials.gov (NCT04302389).All work was approved by the University of Connecticut Institutional Review Board .The 6-month intervention included WW’s mobile app , weekly group virtual workshops, weekly one-on-one virtual check-ins, and an invitation to a private members-only digital community. The PersonalPoints program guides members toward their weight and wellness goals through a weekly curriculum that is complemented by specific behavioral goals each week across 4 main pillars  to drive healthy habits. The WW program is based on recommendations by national and international guidelines to form the foundation for a healthy pattern of eating . FurtherThe personalized aspects of the program are as follows. Participants receive a daily PersonalPoints budget based on their age, height, weight, sex, and breastfeeding status. The PersonalPoints budget is dynamic to the extent that these factors not only change, but the participants’ daily PersonalPoints budget also increases when they consume nonstarchy vegetables and water. The personalized dietary plans include >28,000 options, depending on user inputs. Participants also receive a weekly PersonalPoints budget that gives them extra points to use throughout the week. The weekly budget is dynamic, based on changes in age, weight, and the amount of physical activity. Once a member has spent all their daily PersonalPoints, they may spend their weekly PersonalPoints. Up to 4 unused daily PersonalPoints are automatically rolled over into the weekly PersonalPoints budget, but each participant has the option to turn off rollovers to expedite weight loss. Participants also receive a personalized ZPF list based on their food preferences, which they may change at any time. WW has >300 ZPFs. Food preferences are assessed with the questions presented in Participants were asked to complete their profile and log their food intake and exercise daily, weigh in at least once a week but no more than daily, and attend the weekly virtual workshops and individual check-ins. When participants hit a weight loss milestone, they were sent small keychains to commemorate the milestone they achieved .Virtual workshops were conducted via videoconferencing software and occurred weekly for 6 months. Links to the virtual workshops were emailed weekly to the participants. WW offered meeting times on several different days and times. Study staff assigned each participant a workshop based on their preferred days and times. The workshops, which ranged in size from 16 to 19 participants and lasted between 30 and 60 minutes, were led by a trained WW coach. During the workshops, the coach led a conversation on a topic related to effective weight management and behavior change. The participants also had the opportunity to receive group support by discussing their successes and setbacks as well as help to troubleshoot challenges .Virtual check-ins with a WW coach were conducted over Zoom  and occurred weekly for 6 months. Participants scheduled their weekly check-ins via Calendly, a web-based appointment scheduling software, after which links to the virtual check-in meetings were emailed to each participant. Check-ins lasted approximately 5 minutes and were conducted one-on-one. During check-ins, coaches queried participants about their progress and setbacks, answered questions, and set goals for the week. Check-ins allowed participants to receive individual support to complement the group support from weekly workshops.All participants were invited to an optional private members-only digital community group where they could interact with, and support, each other. Participants could share their journey via posts, photographs, videos, and comments. The group also was a place for participants to ask questions as well as to give motivational support to other participants and receive motivational support from them. The group moderator was a WW coach who posted in the group 3 to 4 times per week to share recipes and ask questions to foster conversations among participants regarding healthy lifestyle changes.App Academy to provide interested participants with a more detailed review of the functionalities and features of the app and to answer participant questions about the app. These workshops are led by a WW coach, last up to 60 minutes, are held twice, and were optional. Of the 153 participants, 27 (17.6%) attended these sessions.WW offers additional workshops coined the At baseline, 3 months, and 6 months, weight values were extracted directly from the WW beta app, which synchronized to the WW Bluetooth scale. In rare events where a participant was unable to get their scale to pair with the WW app, participants uploaded a screenshot of their weight through REDCap  using a private link. Participants were asked to step on the scale in the morning, undressed and after voiding. Weight was recorded in pounds directly from the Bluetooth scale mailed to participants. Weight change was defined as the difference between baseline weight and the weight at 3 months or 6 months. Percentage weight change was calculated as weight change divided by baseline weight and multiplied by 100.Participation was calculated as both the percentage and mean number of weeks out of the 24 total weeks in which participants attended virtual workshops, percentage and mean number of weeks in which they attended weekly wellness check-ins, percentage and mean number of weeks in which they engaged in the private group , and percentage and mean number of days in which participants tracked food, physical activity, and weight in the WW app. If a participant tracked at least 1 food in a day, they were given credit for a day of tracking. Given that we offered participants myriad ways to engage, the mean number of weeks in which the participant carried out ≥1 of each of these forms of participation was also calculated.At the 6-month follow-up, participants were asked 14 questions about how satisfied they were with their experiences in the program. These 14 items were scored using a 5-point Likert scale ranging from 1=strongly disagree to 5=strongly agree. The responses were then dichotomized to strongly disagree, disagree, or neither agree nor disagree (1-3) and agree or strongly agree (4-5).At baseline, 3 months, and 6 months, participants rated their hunger over the past week using a slider on a visual analog scale ranging from 0=not at all hungry to 100=extremely hungry.At baseline, 3 months, and 6 months, participants completed the Food Craving Inventory, which measures food craving via 28 items. In addition to providing a total score , it includes 4 subscales, including high fats (8 items), sweets (8 items), carbohydrates or starches (8 items), and fast-food fats (4 items) rated on a 5-point Likert scale ranging from 1=never to 5=always .At baseline, 3 months, and 6 months, participants completed the Five-Factor Screener, which contains 19 items that assess the consumption frequency  of 21 food groups, including fruits and vegetables. For the analysis, we converted all reported frequencies to daily frequencies based on the Five-Factor Screener scoring guide .Physical activity was measured at baseline, 3 months, and 6 months using the Global Physical Activity Questionnaire (GPAQ). Physical activity was categorized as either moderate or vigorous. We defined vigorous  and moderate  physical activities as activities that cause large and small increases in breathing or heart rate for at least 10 minutes continuously, respectively. We asked participants whether (yes or no) they were engaged in vigorous or moderate physical activity, the number of days in a week in which they were engaged in this activity, and the amount of time (in h and min) they spent on a typical day engaging in this activity at work, during travel and during leisure time. We reported total metabolic equivalent (MET) minutes per week for each participant where 1 minute of moderate activity and 1 minute of vigorous activity are equivalent to 4 MET minutes and 8 MET minutes, respectively ,18. SedeThe Pittsburgh Sleep Quality Index is a 19-item questionnaire that assesses sleep quality and disturbances . It has The Impact of Weight on Quality of Life-Lite questionnaire is composed of 31 items rated on a 5-point Likert scale ranging from 1=never true to 5=always true, and it assesses the perception of how weight affects day-to-day life. It has 5 subscales: physical function (11 items), self-esteem (7 items), sexual life (4 items), public distress (5 items), and work (4 items) .The Self-Compassion Scale is a 26-item measure of self-compassion and consists of 6 subscales: self-kindness, self-judgment, common humanity, isolation, mindfulness, and overidentification. These were scored on a scale ranging from 1=almost never to 5=almost always. A total self-compassion score was obtained by reverse scoring the negative subscale items  and computing a grand mean of all 6 subscale means .Well-being was measured at baseline, 3 months, and 6 months using the World Health Organization-5 Well-Being Index, which assesses subjective psychological well-being with 5 items: “I have felt cheerful in good spirits,” “I have felt calm and relaxed,” “I have felt active and vigorous,” “I woke up feeling fresh and rested,” and “My daily life has been filled with things that interest me.” The 5-point Likert scale items range from 0=at no time to 5=all the time and were summed to create a total score .Behavioral automaticity for 11 behaviors, of which 7 were healthy behaviors and 4 were unhealthy behaviors, was measured at baseline, 3 months, and 6 months using the Self-Report Behavioral Automaticity Index . For eact test. Our final sample included 153 participants, with 140  completing the 6-month follow-up.With α=.05, assuming an SD of 6% based on our previous trial  and 15% t test to evaluate weight change from baseline to 3 months and from baseline to 6 months. We also calculated the proportion of participants who achieved weight loss of ≥3%, ≥5%, and ≥10%. Using the same analytic approach as examining weight, we assessed changes in hunger, food craving, dietary intake, physical activity, and well-being from baseline to 3 months and from baseline to 6 months. Missing values in these secondary measures were imputed by baseline observation carried forward.We used REDCap for data collection and monitoring the completion of study assessments. Data analysis was performed using SPSS software for Windows . We calculated weight change and percentage weight change from baseline to 3 months and baseline to 6 months using the intention-to-treat approach. Given that weight at 3 months and weight at 6 months are likely not missing completely at random , we used the multiple imputations approach to impute the missing weight at 3 months and 6 months ,26. SpecIn total, 759 individuals completed the initial screening survey, of whom 153 (20.2%) were enrolled in the study and 606 (79.8%) were excluded from the study . Among tP values <.001), equivalent to a weight loss of −4.25% (SD 3.93%) at 3 months and a weight loss of −5.05% (SD 5.59%) at 6 months (Participants lost an average of −8.19 (SD 7.51) lb at 3 months and −9.72 (SD 10.78) lb at 6 months  during the first 3 months and 40.33% (SD 29.83%) over 6 months . The meaParticipants tracked food on a mean of 53.62% (SD 33.48%) of the days from baseline to 3 months and 39.12% (SD 30.76%) of the days from baseline to 6 months. The mean percentage of days in which participants tracked physical activity was 66.95% (SD 42.63%) at 3 months and 63.31% (SD 43.59%) at 6 months. On average, participants tracked weight on 24.22% (SD 20.28%) of the days from baseline to 3 months and 19.32% (SD 18.24%) of the days from baseline to 6 months.Of the 140 participants who completed the 6-month follow-up, 116 (82.9%) agreed or strongly agreed that the new WW program made them more mindful of their choices , 102 (72.9%) agreed or strongly agreed that the program made them feel healthier, 96 (68.6%) agreed or strongly agreed that the program made healthy living simple, and 95 (67.9%) agreed or strongly agreed that the program helped them to lower their risk of health conditions (such as diabetes or high blood pressure). Furthermore, 67.9% (95/140) of the participants agreed or strongly agreed that the WW app helped them to stay on track, and 67.9% (95/140) agreed or strongly agreed that the WW app simplified weight loss. Of the 140 participants, 95 (67.9%) agreed or strongly agreed that the workshops were welcoming and that they felt part of the group, and 95 (67.9%) agreed or strongly agreed that the tools and information provided in the program were relevant to people who were similar to them .P values <.001).There was a significant decrease in hunger from baseline to 3 months  and from baseline to 6 months . From baseline to 6 months, we also observed significant decreases in craving for sweets , high fats , carbohydrates or starches , fast foods , and total food craving , high fats , carbohydrates or starches , fast foods , and total food craving  and 6 months . Salad intake increased significantly at both 3 months  and 6 months . Vegetable intake increased significantly among participants at 3 months  and 6 months  and 6 months . Sedentary behavior also significantly decreased at 3 months  and 6 months  but not at 3 months  and 6 months . We did not observe a significant change in self-compassion at 3 or 6 months. Participants’ well-being increased significantly at 3 months  and 6 months  and 6 months . As hypothesized, negative behavioral automaticity significantly decreased at both 3 months  and 6 months .The total impact of weight on quality of life significantly improved at both 3 months  resulted in a mean weight loss of nearly 10 (SD 10.78) lb or 5.05% (SD 5.59%) of the baseline weight at 6 months, with 51% (78/153) of the sample losing ≥5%. A recent meta-analysis of 10 digital weight loss interventions revealed a mean weight loss of 5.3 (range 2.86-13.7) lb [Previous trials of personalized weight loss interventions have focused personalization on feedback ,29, educThe PersonalPoints program was designed to encourage healthy diet choices, including intake of fruit, vegetables, and legumes, all of which have positive impacts on appetite and satiety . Indeed,4 pillars—food, activity, sleep, and mindset—as the foundation of this program, and each is addressed in all aspects of the program. We also observed a significant improvement in sleep quality, which is evidence that the sleep-related content in the program had an impact on participants.We also observed effects of the personalized program on physical activity, sedentary behavior, and sleep. The WW program allows users to add extra weekly PersonalPoints for the exercise they perform, with higher-intensity forms of exercise adding more points, which may motivate participants to exercise. Time spent in sedentary activities declined by an average of nearly 90 minutes per day, which is further evidence that the program nudged people toward more active lifestyles. WW describes The mindset component, the fourth pillar of the WW program, encompasses techniques from cognitive behavioral therapy and positive psychology to help users cope with setbacks, approach the process of weight loss with self-compassion, and stay focused on lifestyle changes that have a positive impact on overall well-being and happiness . We founThis study can be considered in the context of trials of previous versions of the WW program. Tate et al  conducteWorkshop attendance varied across our trial as well as the 2 aforementioned trials using WW workshops ,36. In oThis study has limitations to consider. First, this was a single-arm trial, which means that the absence of a comparison group prevents comparisons with no treatment or other programs. That said, no-treatment control groups in weight loss trials produce negligible weight loss  . Second,In conclusion, this personalized, fully digital, and scalable WW program, which emphasized 4 pillars  and leveraged >300 ZPFs, personalized ZPF lists, and the PersonalPoints plan, resulted in statistically and clinically significant weight loss in half  of the participants. PersonalPoints and ZPFs are features designed to simplify the task of dietary self-monitoring while nudging people toward a high-quality diet. WW first offered this program to the public in November 2021. Future studies should examine how different forms of personalization in digital weight loss programs affect weight loss outcomes.


There was a significant improvement in UCLA activity scale scores from 4.78 to 6.05 at follow-up, p = 0.045. Pain levels decreased from a mean VAS score of 4.73 prior to surgery to a mean score of 2.73 at follow-up, p = 0.047. The functional FFI-R scores showed a significant improvement from 140 points prior to surgery to 97.75 points at follow-up, p = 0.017. Comparison of the preoperative and follow-up values of the range of plantar flexion, adduction, and abduction in the operated limb also showed no significant changes in those individual parameters. The mean values of dorsiflexion, plantar flexion, adduction, and abduction at the ankle joint at follow-up, compared individually between the operated and non-operated foot showed no statistically significant differences. Conclusions: The use of a talar screw in the treatment of symptomatic pes planovalgus helps reduce pain and improve functional outcomes after treatment. Foot function assessments showed diminished pain, improved levels of physical and sport activity, and no effect on the range of motion after surgery in comparison with preoperative data. Arthroereisis with a talar screw is a valid surgical technique for the treatment of symptomatic pes planovalgus.Background: Pes planovalgus, or flexible flatfoot, deformity is a common problem in pediatric orthopedic patients. There is no consensus on using the technique of arthroereisis in the treatment of symptomatic pes planovalgus. The aim of our study was to prospectively assess the functional outcomes following symptomatic pes planovalgus treatment with the use of the Spherus talar screw. Methods: Twenty-seven patients , at a mean age of 10.5 years (7–14 years) were included in the prospective study. We assessed the level of physical activity (including sports) based on the University of California, Los Angeles (UCLA) activity scale, a 10-point level-of-activity VAS scale, and the Grimby physical activity scale. Pain was assessed based on a VAS pain scale; foot function was assessed with the revised Foot Function Index (FFI-R); and ankle joint mobility was measured. Results: The mean follow-up period was 18 months (14–26 months). There was a significant improvement in VAS-measured physical activity scores from 5.47 to 7 at follow-up,  Pes planovalgus, or flexible flatfoot, deformity is a common problem in pediatric orthopedic patients ,20,21,22There are many known techniques for surgical treatment used in cases of symptomatic pes planovalgus ,20,21,22Since it is a minimally invasive, inexpensive, and time-efficient procedure with few complications, arthroereisis is an accepted method of treatment for symptomatic pes planovalgus, yielding high rates of patient satisfaction with treatment ,9,19,22.In our previous study we assessed short-term and medium-term radiological and clinical arthroereisis outcomes achieved with a talar Spherus screw, in the same group of patients included in the current study . We repoTo the best of our knowledge, there have been no studies assessing foot function, range of motion in the ankle joint, pain severity, and patients’ physical and sport activity following talar screw insertion as part of arthroereisis treatment of symptomatic pes planovalgus. Some studies on arthroereisis are retrospective in nature ,14,15,19We hypothesized that arthroereisis with the use of a talar implant would improve functional outcomes.The aim of our study was to prospectively assess the functional outcomes following symptomatic pes planovalgus treatment with the use of a Spherus talar screw. This was a prospective study conducted at a single center. We analyzed every patient with pes planovalgus who underwent arthroereisis with a Spherus talar screw in the period between 2021 and 2022. All patients underwent physical and radiological examinations and were diagnosed with symptomatic pes planovalgus based on the following signs and symptoms: foot pain, flexible flatfoot deformity, a talocalcaneal angle (kite angle) of >40 degrees measured on a weight-bearing foot radiograph ,5,19. AlThe study inclusion criteria were age between 7 and 14 years, symptomatic flexible flatfoot, failure of conservative treatment, arthroereisis with a Spherus talar screw , The exclusion criteria were ages under 7 years and over 14 years, history of lower limb injury, radiographic evidence of synostosis in the foot or the ankle joint, neurological comorbidities, a follow-up period of less than 14 months, incomplete medical, radiographic, or functional assessment records. All patients and their legal guardians were informed of the voluntary nature of their participation. The study, which was conducted in accordance with the Declaration of Helsinki, had been approved by an ethics committee.In the period from the year 2021 to 2022, there were 35 arthroereisis procedures with the use of a Spherus talar screw conducted at our clinic. Application of the inclusion and exclusion criteria yielded 27 patients , at a mean age of 10.5 years (7–14 years) who were ultimately included in the study. Patients with foot dorsiflexion limited to 5–10 degrees ,14,18 unAs part of the study, we assessed the level of physical activity (including sports) based on the 6-point University of California, Los Angeles (UCLA) activity scale , a 10-pop < 0.05.Data were statistically analyzed using Statistica 13.1. The Shapiro–Wilk test was used to check for normality of distribution. The Mann–Whitney U test was used to compare quantitative variables. The level of statistical significance was set at p = 0.045; see The mean follow-up period was 18 months (14–26 months). Detailed results of the physical activity, functional, and range-of-motion assessments have been presented in p = 0.048, p = 0.047; see Grimby activity scores showed an increase in physical activity from 3.26 prior to treatment to 3.94 at long-term follow-up; however, the difference was not statistically significant; see p = 0.017; see The functional scores assessed with the FFI-R scale also showed significant improvement from a mean score of 140 points prior to surgery to 97.75 points at follow-up, Dorsiflexion increased, though non-significantly, from a mean of 22° prior to surgery to a mean of 25.33° at follow-up; see Importantly, the mean values of dorsiflexion, plantar flexion, adduction, and abduction at the ankle joint at follow-up, compared individually between the operated and non-operated foot, showed no statistically significant differences; see The aim of our study was to assess whether the use of a talar screw for arthroereisis would improve functional parameters of the foot in symptomatic pes planovalgus. We observed improvement in the UCLA and VAS physical activity scores following treatment. There was also improvement in the FFI-R scores at follow-up and the VAS showed pain reduction following treatment, with no postoperative decrease in the range of motion at the ankle joint. The obtained results support our initial hypothesis. Arthroereisis, which is used in the treatment of symptomatic pes planovalgus, is minimally invasive, inexpensive, produces few complications, and ensures high rates of patient satisfaction ,15,17,19To date, there has been no consensus among orthopedic surgeons regarding which type of arthroereisis implants to choose in patients with symptomatic pes planovalgus ,2,5,7,8.In our previous study we assessed short-term and medium-term radiological and clinical arthroereisis outcomes achieved with a talar Spherus screw in the same group of patients included in the current study . We repoMartinelli et al. assessed the level of sport activities in 49 patients following arthroereisis . Twelve The main purpose of arthroereisis is to reduce pain ,10,12,13Vogt et al. assessed 73 patients after various arthroereisis procedures with the use of various implant types . Those ap = 0.047. All joint range of motion measurements before and after surgery were performed in the same way by the same investigators. These results indicate good ranges of motion after treatment with a talar screw. Unlike wedge osteotomy and arthrodesis procedures, arthroereisis is a reversible treatment method that helps keep some range of motion at the subtalar joint [Our study group showed no significant differences between the operated and non-operated limb in mean degrees of postoperative dorsiflexion, plantar flexion, adduction, or abduction at the ankle joint. Nor were there significant differences between the preoperative and postoperative ranges of motion at the ankle joint. The exception was the pre-operatively larger range of dorsiflexion in the unoperated limb (36.25°) compared to the operated limb (22°), ar joint . On the ar joint . In addiar joint . In our ar joint ,14, whicThirty-seven percent of our study group underwent simultaneous Achilles tendon lengthening. Other authors also reported performing additional procedures in 17–100% of patients undergoing arthroereisis ,16,17,18In our group, none of the patients had the screw removed.The use of a talar screw allows for other foot or ankle surgeries to be performed in the future. Tendon lengthening or transfer, osteotomy, or arthrodesis procedures may be performed without limitations.The limitations of our study are the relatively short follow-up period and a relatively small sample size. Both are due to a prospective nature of the study, limited number of patients with indications for arthroereisis, our eagerness to present an evaluation of the novel type of implants, and the limited capacity for orthopedic surgery procedures at our hospital. However, other authors also reported a similar follow-up period ,14,19. OThe strengths of our study include its prospective nature, identical rehabilitation protocol, and the surgeries being conducted by one of only two orthopedic surgeons. Some studies on arthroereisis are retrospective ,14,15,18The use of a talar screw in the treatment of symptomatic pes planovalgus helps reduce pain and improve functional outcomes after treatment. Foot function assessments showed diminished pain, improved levels of physical and sport activity, and no effect on the range of motion after surgery in comparison with preoperative data. Arthroereisis with a talar screw is a valid surgical technique for the treatment of symptomatic pes planovalgus.


Psychiatric disorders are often characterised by cognitive impairment. The Screen for Cognitive Impairment in Psychiatry (SCIP) was developed for routine screening of psychiatric patients and is available in several languages.Using the German version (SCIP-G), 3 studies were conducted: 1. feasibility, reliability, and validity of the SCIP-G were investigated , 2. a confirmatory factor analysis was performed , and 3. patients with psychotic, bipolar affective, and depressive disorders were assessed before and after standard inpatient treatment including cognitive remediation.Study 1 included patients with schizophrenia or schizoaffective psychosis and thirty healthy controls matched for sex, age, and education. Data were collected at the Medical University of Vienna, Department of Psychiatry and Psychotherapy. In studies 2 and 3, patients from the Klinik Hietzing, 1st Department of Psychiatry and Psychotherapeutic Medicine, Vienna, Austria, were studied. In study 3, all patients received modern pharmacotherapy plus cognitive remediation using the COGPACK® software package version 6.06; based on the ICD-10 criteria for research, 54 patients received an F2 diagnosis , 39 patients met criteria for bipolar disorder (F30 and F31), and 50 for depression (F32 and F33).In Study 1, significant differences in cognitive performance were found between patients and healthy controls on both versions of the SCIP. The SCIP effectively discriminated between patients and the control group. In Study 2, a two-factor solution in which the Verbal Learning Test-Immediate Recall subtests, Delayed Recall Test of the VLT, and Working Memory Test loaded on the first factor and the Verbal Fluency Test and Psychomotor Speed Test subtests loaded on the second factor yielded good model fit . In Study 3, SCIP total score showed significant improvement after treatment in all three diagnostic groups (p < .001), with no statistically significant interaction between SCIP total score and diagnostic groups (p = .860).Our data indicate that the SCIP-G is a valid and reliable instrument for assessing cognitive impairment. Good model fit can be achieved with a two-factor solution for the SCIP. Our study is the first to perform a confirmatory factor analysis with the German SCIP version and to test its dimensional structure with a hypothesis-testing approach. Inpatient treatment consisting of pharmacotherapy and cognitive remediation improved cognitive deficits. This improvement in cognitive performance was observed to a similar extent in patients with psychotic disorders, bipolar disorder, and depression.None Declared


Petaloidy leads to a plump floral pattern and increases the landscape value of ornamental pomegranates; however, research on the mechanism of petaloidy in ornamental pomegranates is limited. In this study, we aimed to screen candidate genes related to petaloidy. We performed transcriptomic and proteomic sequencing of the stamens and petals of single-petal and double-petal flowers of ornamental pomegranates. Briefly, 24,567 genes and 5865 proteins were identified, of which 5721 genes were quantified at both transcriptional and translational levels. In the petal and stamen comparison groups, the association between differentially abundant proteins (DAPs) and differentially expressed genes (DEGs) was higher than that between all genes and all proteins, indicating that petaloidy impacts the correlation between genes and proteins. The enrichment results of transcriptome, proteome, and correlation analyses showed that cell wall metabolism, jasmonic acid signal transduction, redox balance, and transmembrane transport affected petaloidy. Nine hormone-related DEGs/DAPs were selected, among which ARF, ILR1, LAX2, and JAR1 may promote petal doubling. Sixteen transcription factor DEGs/DAPs were selected, among which EREBP, LOB, MEF2, MYB, C3H, and trihelix may promote petal doubling. Our results provide transcriptomic and proteomic data on the formation mechanism of petaloidy and a theoretical basis for breeding new ornamental pomegranate varieties. Petunia hybrid [Gentiana scabra [Camellia japonica [DAD1 and the homologous gene AG jointly regulate the stamen development of Arabidopsis thaliana [ARF regulates stamen petaloidy in Rosa hybrida by inhibiting AG transcription [Hyacinth orientalis flowers by homologous gene HAG1 [Nelumbo nucifera [Malus spp. [Lagerstroemia speciosa [Increasing the number of petals makes flowers plumper and improves their ornamental value. Therefore, petaloidy is a popular topic in flower pattern research ,2. Currea hybrid , Gentiana scabra , and Camjaponica . Howeverjaponica ,13,14. Pthaliana , the auxcription , and cytene HAG1 . The samnucifera , Malus slus spp. , and Lagspeciosa .Punica granatum), a species of Punica in the Lythraceae family, is widely planted in courtyards, roads, and parks because of its beautiful flowers and fruits. Petaloidy mainly occurs during the flower bud and flowering periods, including stamen petaloidy and transitional petal formation and growth. Transcriptomics and proteomics are the studies of total RNA and total proteins, respectively, in cells or tissues under certain conditions [N. nucifera through transcriptome and proteome association analysis, and Huang et al. [Phalaenopsis using genomics and transcriptome association analysis. However, there are no reports on petaloidy molecular mechanisms during the flowering period of ornamental pomegranate based on transcriptomics and proteomics.Ornamental pomegranate  screen the candidate genes involved in the transformation from stamen to petal stamen and from transition petals to normal petals in ornamental pomegranate by using transcriptome and proteome analyses and (ii) elucidate the molecular mechanism of petaloidy and lay a foundation for cultivating new varieties of ornamental pomegranate.When compared with the single-petal flower, the double-petal flower of the ornamental pomegranate had a larger flower diameter, more petals, and a different flower shape a,b. The The 12 libraries resulted in 78.19 Gb of clean data and 75.68 Gb of high-quality reads (Q > 20). Each sample having more than 6.31 Gb of clean data was retrained and used for further analysis . ApproxiIn total, 10,373 and 5331 differentially expressed genes (DEGs) were identified in the PeSi vs. PeDo and StSi vs. StDo groups, respectively, of which 3207 were common DEGs . BrieflyARFs) and one indole-3-acetic acid-amido synthetase (GH3.17). ARFs was highest in PeSi and lowest in StSi, while that of GH3.17 was highest in StSi, indicating that ARFs and GH3.17 may have a reverse regulatory effect on the floral organ development of pomegranate. Fourteen TF DEGs belonged to the APETALA2-ethylene-responsive element binding protein (EREBP), C3H, LIM, LOB, MADS, MYB, trihelix, and WRKY families. The TF DEGs had different spatial expression patterns (Previous studies showed that plant hormones and transcription factors (TFs) are involved in stamen petaloidy and petal variation . In thispatterns .To validate the reliability of the RNA sequencing (RNA-Seq) data, 12 genes were chosen at random for quantitative real-time polymerase chain reaction (qRT-PCR) analysis. The correlation coefficient between the qRT-PCR data and transcriptome data fell between 0.815 and 0.996, which is indicative of the high reliability of RNA-seq data .After protein extraction, 2,178,185 secondary spectra were obtained, of which 411,677 were available and the utilization rate was 18.9%. In total, 49,696 peptides were identified through spectral analysis, including 43,127 unique peptides. The peptide length distribution diagram showed that most of the peptides were distributed in the range of 1–20 amino acids, which conformed to the general rule of the trypsin enzyme and higher energy collisional dissociation . A totalIn total, 419 and 642 DAPs were identified in PeSi vs. PeDo and StSi vs. StDo, respectively, of which 101 were common DAPs . A totalThree hormone-related DAPs were screened from the common DAPs: the IAA-amino acid hydrolase protein (ILR1), jasmonic acid-amido synthetase protein (JAR1), and abscisic acid stress ripening-induced protein (ASR). Among these, the expression of ILR1 and JAR1 in PeDo and StDo was higher than that in PeSi and StSi, whereas the expression pattern of ASR was the opposite . Two TF In total, we identified 24,567 genes and 5865 proteins in PeSi, StSi, PeDo, and StDo, 5721 of which could be quantified at both the transcriptional and translational levels . The corAdditionally, 223 and 277 genes/proteins were expressed in PeSi vs. PeDo and StSi vs. StDo, respectively, and the Pearson correlation coefficients (PCC) between the genes and proteins were 0.324 and 0.492, respectively a. There LAX2) and one JAR1 were found among the DEGs-DAPs with the same expression trend in PeSi vs. PeDo. The expression of LAX2 in StSi, StDo, and PeDo increased in sequence and was highly expressed in PeDo. The expression of JAR1 in StDo and PeDo was higher than that in StSi and PeSi. One auxin induced in root culture protein 12 (AIR12) and one GH3.17 were found in StSi vs. StDo. Their expression levels were significantly higher in StSi than in StDo, PeDo, and PeSi , three hormone-related DAPs from the proteome , and four hormone-related DEGs-DAPs from the association analysis of the transcriptome and proteome   leads to structural variation in the inflorescence [LAX2 in StSi, StDo, and PeDo increased, and it was the highest in PeDo, indicating that LAX2 might promote stamen petaloidy and transitional petal development in ornamental pomegranates. Previous studies have found that ARF mutants lead to changes in the number of stamens and petals [ARF17 regulates anther dehiscence by regulating the lignification of the anther endothecium cells [ARF increased in StSi, StDo, PeDo, and PeSi, indicating that it might promote ornamental pomegranate petaloidy. However, the expression of the auxin response factor AIR12 in StSi was higher than that in other flower organs, indicating that auxin signal transduction-related genes have complex regulatory mechanisms.Previous studies have shown that hormone-related genes are involved in the petaloidy of various plants ,36. We snd JAR1) . These Dthaliana ; promotethaliana . In thisthe cell , which ithe cell ; the mutrescence . The expd petals , and ARFum cells . AdditioJAR1 regulates the opening, closing, and cracking of rice anthers [JAR1 in StDo and PeDo was higher than that in StSi and PeSi, indicating that it may promote stamen petaloidy. Nonetheless, in a transcriptome study of Malus spectabilis, the expression of JAR1 in single-petal flowers was higher than that in double-petal flowers, which may be owing to species specificity [GH3.17 and JAR1 regulated the petaloidy of ornamental pomegranate, which was consistent with their functions in hormone metabolism. Jasmonic acid affects the development of flower organs and participates in stress response. It is one of the main hormones involved in late stamen development and petal formation. Jasmonic acid-amido synthetase protein catalyzes the formation of active jasmonate isoleucine conjugates which in turn activates the downstream signal transduction pathway . Xiao et anthers . We founcificity . Notablycificity . In thisClematis patens [ASR and TF genes are involved in flower development; however, their specific functions in flower organogenesis have not been reported [The level of abscisic acid, a stress hormone, rapidly increases under stressful conditions, promoting flower bud differentiation and other developmental processes. Previous studies have found that the expression of abscisic-acid-related genes in single-petal flowers was higher than that in double-petal flowers of s patens . Abscisis patens . Previoureported . We founLOB, one MEF2, one MYB, and two EREBP genes were expressed at their highest levels in StDo, indicating that they might promote stamen petaloidy, and one C3H and one trihelix gene increased successively in StSi, StDo, and PeDo, indicating that they might promote stamen petaloidy and transitional petal growth. The EREBP transcription factor is mainly involved in plant stress response [LOB transcription factor participates in pollen development [MEF2 transcription factor belongs to the MADS family and participates in flower development [MYB transcription factor regulates stamen and petal formation [C3H encodes coumarin-3-hydroxylase, which is a key enzyme in the lignin synthesis pathway, and trihelix regulates the morphogenesis of floral organs, such as sepals and petals [In this study, fourteen TF DEGs were selected from the transcriptome, and two were selected from the proteome . Among tresponse , but itselopment , the MEFelopment , and theormation . Additiod petals . Hormoned petals .The pomegranate varieties ‘Nanlindanbanhong’, with single-petal flowers, and ‘Nanlinchongbanhong’, with double-petal flowers, were planted in the nursery of Nanjing Forestry University . The two varieties had the same plant type, branch type, and flower color. The number of petals and petalized stamens in the two varieties was counted, and the flower morphology was observed. StSi  and PeSi  on pomegranate varieties ‘Nanlindanbanhong’, and StDo  and PeDo  on pomegranate varieties ‘Nanlinchongbanhong’, were collected for transcriptome and proteome sequencing, with three biological replicates for each group. All the samples were immediately frozen in liquid nitrogen and kept at −80 °C.http://ccb.jhu.edu/software/stringtie; accessed on 10 January 2020), and Cuffmerge software  was used to integrate the reconstructed information from all the samples. The screened-out new transcripts were then compared to transcripts with reference annotations. Subsequently, CPC  was used to predict the protein-coding potential of the novel transcripts. Additionally, the software getorf  was used to detect the open reading frame (ORF) of the unigene program, hmmsearch  was used to align ORFs to the protein domain of the transcription factor, and transcription factor capacity was identified based on the Plant Transcription Factor Database.The RNA integrity was assessed by choosing an RNA integrity number that was greater than 7.0 for all samples. RNA reagent  was used to extract total RNA from the samples. Isolated mRNA was spiked with oligo beads (dT) and fragmented into short fragments. The complementary DNA fragments (cDNA) were then sequenced with a platform BGISEQ-500 . The reads that contained adapters, poly N, or low-quality sequences were excluded. The high-quality reads were mapped to the reference transcriptome of pomegranate ‘Dabenzi’  using HISAT  and the −ΔΔCT method.The RNA-seq results were validated using qRT-PCR and the expression of 12 randomly selected genes. The cDNA was obtained by reverse transcription using the extracted total RNA as a template. The reverse transcription reagent for qRT-PCR was the Hiscript II Q RT Super Mix . Then, the designed qRT-PCR primers  were useThe samples were ground to powder with liquid nitrogen and then added to a four-volume cracking buffer followed by ultrasonic treatment on ice. An equal volume of Tris-Balanced phenol was added to remove residual debris by centrifugation. Five volumes of 0.1 M ammonium acetate/methanol were then added to precipitate the protein overnight. The protein was redissolved in 8 M urea, and the protein concentrations were detected with a BCA kit .The protein solution was reduced with 5 mM dithiothreitol at 56 °C for 30 min, followed by alkylation with 11 mM iodoacetamide under dark conditions for a further 15 min. The protein sample was then diluted to a urea concentration of no more than 2 M by the addition of 100 mM TRAB. Lastly, trypsin was added at a 1:50 mass ratio of trypsin to protein and incubated for 4 h at a mass ratio of 1:100 trypsin to protein.The peptides were dissolved in solvent A (0.1% formic acid) and separated using a gradient of solvent B (0.1% formic acid in 98% acetonitrile), 6–23% solvent B for 30 min, 25–35% solvent B for 8 min, and 80% solvent B for 6 min. They were then subjected to segregation using the ultra-high performance liquid phase NanoElute system  and injected into the NSI source followed by tandem mass spectrometry (MS/MS) in an Ultra High Resolution Liquid Mass Spectrometer  coupled in-line with the UPLC system. An electrospray voltage of 2.0 kV was used. For the full scan, the m/z scan ranged between 350 and 1800, and the intact peptides were detected using an ion trap analyzer  at a resolution of 70,000 s. Peptides were selected for MS/MS analysis using the NCE setting as 28, and fragments were detected using Orbitrap at a resolution of 17,500 s. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans with 15.0 dynamic exclusion was conducted. The automatic gain control was set to 5E4. The first fixed mass was set to 100 m/z, while the scanning range of the secondary mass spectrometry was set to 100–1700 m/z. The parallel cumulative serial fragmentation mode was used for data acquisition.http://www.maxquant.org/; accessed on 17 September 2020). The database that was used was the Punica_granatum_22663_NCBI_20190902 , which included an inverse database to compute the false positive rate that was caused by random matching as well as a common pollution database. Trypsin/P was specified as a cleavage enzyme allowing up to 4 missing cleavages. The mass tolerance for precursor ions was set to 20 ppm in the first search and 5 ppm in the main search, while the mass tolerance for fragment ions was set to 0.02 Da. Carbamidomethyl on Cys was specified as a fixed modification, and acetylation modification and oxidation on Met were specified as variable modifications. The false discovery rate was adjusted to <1% and the minimum score for modified peptides was set to >40. The peptide length and protein molecular weight distributions were determined to assess the quality of the proteome sequencing.The secondary mass spectrometry data were searched using MaxQuant  were used to compute the gene expression levels in the transcriptome, and the label-free quantitation intensity was used to calculate the relative quantitative value of the proteins in the proteome. The detection of the DEGs and DAPs was based on the Poisson distribution [https://www.omicshare.com/tools; accessed on 15 July 2022) was used to conduct the GO and KEGG classification and enrichment analyses.The fragments per kilobase of transcript per million mapped reads method and the RSEM software package (ribution . The genThe aim of this study was to perform transcriptomic and proteomic sequencing of the stamens and petals in single-petal flowers and double-petal flowers of ornamental pomegranates during the flowering period. A total of 24,567 genes and 5865 proteins were identified, of which 5721 genes were quantified at both transcriptional and translational levels. In the petal and stamen comparison groups, the association between the DAPs and DEGs was higher than that between the total genes and proteins, indicating that petaloidy has an impact on the correlation between the genes and proteins. The enrichment results of the transcriptome, proteome, and correlation analyses showed that cell wall metabolism, jasmonic acid signal transduction, redox balance, and transmembrane transport had important effects on petaloidy. Nine hormone-related DEGs/DAPs were selected, among which ARF, ILR1, LAX2, and JAR1 may promote petal doubling. Sixteen transcription factor DEGs/DAPs were selected, among which EREBP, LOB, MEF2, MYB, C3H, and trihelix may promote petal doubling. This study provides transcriptomic and proteomic data on the petaloidy molecular mechanism and provides a theoretical foundation for double-flower breeding in ornamental pomegranate. Further functional verification of hormone-related and TF genes could offer more insights and allow for an in-depth understanding of transcriptional and translational involvement in regulating flower organ development. In this study, transcriptome and proteomic data were obtained for the breeding of new double-petal flower varieties of ornamental pomegranate; however, functional verification for hormone-related genes and TF genes has not been carried out. In particular, the relationship between these candidate genes and ABCDE model genes warrants further investigation.


Hereditary blood diseases are widespread among the Arab population due to the high rates of consanguineous marriages; research regarding the perception of consanguineous marriage in some countries, such as Qatar, is extremely scarce. Therefore, this study aimed to investigate the prevalence of consanguineous marriage and assess the perception of consanguineous marriage among the Qatari population.t-test was used to compare the significance of the mean between the two groups with positive and negative perceptions of consanguineous marriage. Categorical data were analyzed for association using the chi-square or Fisher's exact test. Finally, a multiple logistic regression analysis was conducted to determine the significant predictors of the positive perception of consanguineous marriage. A significant level was set at p < 0.05.A cross-sectional study used a self-administered questionnaire among 395 Qatari adults aged 18–35 who attended primary healthcare institutions in Qatar. A convenience sampling technique was used to select the study participants. An independent Approximately 45% of the participants had a positive perception toward consanguineous marriage, and the most common reason stated by those participants was “habit and traditions.” The prevalence of consanguineous marriage among married couples was 62.6%, and among those with consanguineous marriage, most were married to first cousins (81.7%). Moreover, compared to the participants with negative perceptions of consanguineous marriage, those with positive ones were significantly older, married, with lower educational levels and higher monthly income levels, did not hear about glucose-6-phosphate dehydrogenase (G6PD) deficiency, did not know what kinds of diseases are being screened in the premarital test, and were married to a relative.The prevalence of consanguineous marriage is high among the Qatari population, and this requires an immediate need for community-based campaigns to raise public awareness about the problem and its potential impact. The Premarital Genetic Screening Program Services implemented in Qatar in December 2009 are available at eight primary health centers; promoting the health and couples is primarily a preventive approach, mandatory for legally certified marriages, as in many Arab countries , 2. Besiin vitro fertilization (IVF) that can assist them in birthing unaffected children  and thalassemia are among the significant health problems in Arabic countries, particularly in Qatar , 5. SickIn Qatar, there are approximately 550 Qatari patients with SCD, and most of the cases are distributed in the Northern Province and Doha, the capital of Qatar . MoreoveThalassemia is a heterogeneous grouping of genetic disorders that result from a decreased synthesis of alpha or beta chains of hemoglobin . There aA study that was conducted among 1,800 Qatari in 2004 estimated a consanguineous marriage rate of 54% . FurtherThis analytical cross-sectional study was conducted between October 2022 and December 2022 at three primary health centers under the Primary Health Care Corporation. The health services provided in those health centers are accessible to the entire registered Qatari population. Moreover, the three health centers are in different geographical areas and cover the Qatari population's different socioeconomic classes and educational backgrounds.The target population included Qatari men and women who attended Primary Health Centers for any reason during the data collection period, were willing to participate, and fulfilled the eligibility criteria.Our inclusion criteria included Qatari men and women of all educational levels, married or unmarried, aged 18 to 35 , who agreed to sign the written informed consent, and who were able to read and write either Arabic or English. In addition, we excluded adults with cognitive impairment that interfered with communication. The ethics approval was obtained from the Institutional Review Board (IRB) of the Primary Health Care Corporation (PHCC/DCR/2022/06/032).The previous prevalence of consanguineous marriage in Qatar of 54%  was utilMoreover, we added 20% of the computed sample size as the expected non-response rate; hence, the estimated sample size was 457.A multistage sampling technique was used. First, a cluster sampling technique was utilized to randomly select three health centers primarily for the Qatari population with a high attendance rate. Second, a convenience sampling technique was used to choose adults aged 18 to 35 of Qatari nationality and attending any clinic services in the selected primary health centers. An equal number of participants were selected from each health center . Finally, the selected participants were interviewed, and the orientation of the patients about the study was carried out before obtaining written informed consent with an emphasis on the subject's right to refuse participation.A structured self-administered questionnaire was developed after reviewing the relevant literature. The content and face validity were established through an extensive literature review and consultation with experts in the field of community medicine and hematology. The format of the questions varied between multiple-choice, dichotomous questions (yes/no), and open-ended questions. The questionnaire was initially prepared in English and translated into Arabic with back translation by a bilingual translator.Before data collection, a pilot study was conducted among a convenience sample of 20 participants from one primary health center to pre-test the comprehensibility and appropriateness of the questionnaire. In addition, the pilot phase provided an opportunity to measure the time needed to complete each questionnaire by the participants. As a result, some necessary adjustments were made to the questionnaire. The piloted 20 participants were omitted from the study database.The independent variables included age in years, gender of the participant, marital status, educational status, occupation, monthly family income in Qatari Riyal (QAR), personal and family history of SCD or sickle cell trait, and personal and family history of other genetic blood diseases such as thalassemia or G6PD deficiency. In addition, participants' awareness of inherited genetic blood diseases and premarital screening tests were assessed by questions such as “Have you heard about SCD, thalassemia, and G6PD deficiency?” “Is there a treatment for genetic blood diseases?” “Do you think the family suffers from psychological pressure if one of its members suffers from one of the genetic blood diseases?”, “What diseases do we screen for in the premarital screening?” and “Do you think that premarital screening limits the spread of genetic diseases?”Consanguineous marriage was assessed using the yes/no question. Then, the degree of consanguineous marriage was evaluated as an ordinal variable, and it included first cousin , first cousin once removed (parents' first cousin or first cousin's child), and second cousin (the child of a parent's cousin) . Perceptt-test was used to compare the significance of the mean between the two groups with positive and negative perceptions of consanguineous marriage. Moreover, categorical data were analyzed for association using the chi-square or Fisher's exact test. Finally, a multiple logistic regression analysis was conducted to determine the significant predictors of the positive perception of consanguineous marriage. A significant level was set at p < 0.05.The Excel and IBM SPSS statistical programs  were used to manage and analyze the data. First, continuous data were tested for normality. Second, an independent n = 320), 63% were married (n = 249), and 35.9% held a university degree or higher education (n = 142). In addition, 51.4% of the participants were currently employed (n = 203). The majority of the participants (87.6%) had a monthly family income of more than 10,000 QR.During the data collection period (October 2022–December 2022), 475 adults with Qatari nationality were approached to participate in the study. Approximately 62 refused to participate, and 395 eligible participants were included in the study, with a response rate of 83.1%. The participant's mean age (±SD) was 27.4 (±6.0) years. Most of the study participants (81%) were women  were SCD patients, and six participants (1.5%) had other genetic blood diseases such as thalassemia and glucose-6-phosphate dehydrogenase deficiency. Moreover, 20 participants (5.1%) had a family history of SCD, and 32 participants (8.1%) had a family history of other genetic blood diseases.In our sample, 45.1 % of the participants positively perceived consanguinity marriage and answered “yes” to the question, “Do you prefer to marry a relative?”. The most common reasons stated by those who preferred consanguineous marriage are “habits and traditions,” followed by “more comparability and understanding between relatives,” and “premarital test is enough to reduce the risk of the disease,” 38.2%, 28.1%, and 2.7%, respectively see . On the The prevalence of consanguineous marriage among married couples was 62.6%, as seen in When the study participants were asked about whether the test result of the premarital screening affected their decision to marry the other person, most of the participants (88.9%) answered yes; however, a minority of them (11.1%) said that they would still marry the other partner regardless of the test result, as seen in In our sample, most participants did not hear about thalassemia, sickle cell disease, or G6PD deficiency, accounting for 84.8%, 67.1%, and 81%, respectively. Moreover, most study participants (64.6%) did not know whether there was a treatment for inherited blood diseases. Most of the participants (81.8%) thought that the family would suffer from psychological stress if one of its members suffered from one of the genetic blood diseases. Although 41.4% of the participants claimed they knew about the tests on those about to get married, only 5.6% knew the diseases screened for in the premarital screening. On the other hand, almost all the participants (93.7%) agreed that premarital screening limits the spread of genetic diseases, as shown in p-value < 0.05) was found between those with consanguineous marriage (positive perception) and those against it (negative perception) in terms of age, marital status, educational level, and monthly family income.With regard to the background characteristics, a statistically significant difference . Moreover, most of the married participants preferred consanguineous marriage (67.4%); however, most of the single participants did not prefer consanguineous marriage (52.1%) .The median age (±SD) of those who preferred consanguineous marriage was 29.1 (5.8), and the median age for their counterparts who did not prefer consanguineous weddings was 26.2 (5.9) , the participants were more inclined to prefer consanguineous marriage, with a P-value of 0.013. In addition, all the study participants with SCD (eight participants) preferred non-consanguineous marriage, as shown in Furthermore, participants with educational levels up to primary school preferred consanguineous marriage compared with their counterparts with higher education qualifications . On the other hand, those with higher levels of education chose non-consanguineous marriage (9.8% vs. 78.1%), with a P-value of 0.005 and 0.05, respectively, as shown in Moreover, most study participants who preferred consanguineous marriage did not hear about G6PD deficiency (78.1%) or know what diseases are being screened in the premarital test (92.9%), with a p-value of <0.001, as shown in Furthermore, most of the participants who preferred consanguineous marriage were married to a relative (82.7% vs. 17.3%) with a P-value = 0.324, i.e., the regression model indicated no evidence of poor fit . However, after adjusting for the other variables, only four parameters have proven to be significant predictors for patients' preference to marry a relative: age , monthly family income , if patients have not heard about G6PD deficiency (p < 0.001), and if the patients have consanguinity with their spouse (p < 0.001).The parameters that showed significance in the bivariate analysis were used in the multiple logistic regression analysis to determine the predictors for the patient's preference to marry a relative. Using the Hosmer–Lemeshow test, Relatively older participants (>29 years) were more likely to have a positive perception of consanguineous marriage . Moreover, those with higher monthly family income (≥10.000QR) were approximately three times more likely to prefer consanguineous marriage than their counterparts with lower monthly payments . On the other hand, patients who have heard about G6PD deficiency were approximately 72% less likely to have a positive perception of consanguineous marriage than their counterparts who have not heard about G6PD deficiency . Finally, patients with any kinship between them and their spouses were nearly seven times more likely to have a positive perception of consanguineous marriage , as shown in This study investigated the perception of consanguineous marriage among the adult Qatari population in 2022. Approximately 45% of the study participants preferred consanguineous marriage over non-consanguineous marriage, and 62.6% were married to a close relative. A significant association was found between a positive perception of consanguineous marriage and the participant's age, marital status, educational level, monthly family income, and being a patient with SCD. Moreover, participants who positively perceived consanguineous marriage did not hear about G6PD deficiency or the diseases screened for in the premarital test..The rate of consanguineous marriage in Qatar is higher than observed in most Arab countries, including Oman (52%)  , Kuwait an (52%) , and Sudan (52%) . One posan (52%) . Similaran (52%)  and Saudan (52%) . There aan (52%) . Similaran (52%) .This study observed various patterns of consanguineous marriage in Qatar, some of which are common in other Arab countries. For example, those who are young with a lower level of education are more likely to marry their biological relative, but, again, these patterns are common in other Arab countries –30. In aLike our findings, a study conducted in Oman (11) found that the majority (72.4%) of the single participants chose to avoid consanguineous unions to prevent the chance of developing hereditary diseases 79%) and the remainder chose to avoid family problems (8%). Moreover, the awareness of SCD among our study participants was more than that of thalassemia and G6PD deficiency. However, in a later study  was used to collect the data, which hindered the generalizability of the results for the entire country.In summary, this study indicated that the prevalence of consanguineous marriage is high in Qatar, and most participants had a positive perception of consanguineous marriage. However, most of the participants needed to be made aware of the seriousness of the problem and its potential impact. Moreover, most of the study population did not hear about genetic blood diseases. These results could be changed by incorporating this information into the secondary school curriculum by raising awareness among adolescents and young adults about the negative impact of consanguineous marriage and the potential genetic problems in future offspring. Adolescents should also receive health education to change their attitudes and opinions, particularly before engagement. In addition, increasing the number of educational programs in media such as TV, radio, and newspapers is an option that should be considered for mass outreach. Finally, a qualitative study can be carried out among the Qatari population to better understand their perception and attitude toward consanguineous marriage.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The study involved human participants and was reviewed and approved by the Institutional Review Board (IRB) of Primary Health Care Corporation (PHCC/DCR/2022/06/032). The participants provided their written informed consent to participate in this study.YA: conceptualization, methodology, investigation, writing, reviewing and editing, formal analysis, and writing—original draft, data curation, and project administration. KA: conceptualization, methodology, and writing—reviewing and editing. MI: methodology, formal analysis, reviewing, and editing. MA, AA, and HA: methodology, writing—reviewing and editing, and data curation. NS and MY: methodology, writing—reviewing and editing, and supervision. All authors contributed to the article and approved the submitted version.


Polysphondylium violaceum is a common species in both regions, while Dictyostelium sphaerocephalum and Dictyostelium mucoroides are widely distributed in Lanzhou, Gansu Province. The total number of species (seven) and the number of endemic species (three) of dictyostelids in mixed forests were higher than those in other forest types. Redundancy analysis indicated that soil pH, temperature, and elevation were the primary factors that affected dictyostelid occurrence. Nonmetric multidimensional scaling and principal component analysis indicated that the dictyostelid communities were significantly different between Gansu and Guizhou Provinces.Dictyostelid cellular slime molds (dictyostelids) are one of the major groups of eukaryotic amoeboid protists occurring in the soil microhabitat. The past few decades have witnessed an unprecedented interest in the ecology of dictyostelids, stimulated in part by the hidden soil protist diversity that has long been overlooked. However, a better understanding of the impact of various environmental factors on the biodiversity of dictyostelids is needed to understand the critical functional role of these organisms in the soil ecosystem. Species diversity of dictyostelids has been surveyed in some provinces of China, but little data exist for Gansu and Guizhou Provinces in western China. In this paper, we report data on the distribution and occurrence of dictyostelids in the soils of these two provinces. Samples were collected from 13 localities in central Gansu and the southeastern and southcentral portions of Guizhou Province. Eleven species of dictyostelids were recovered from 99 samples of soil. Six new records of dictyostelids are reported from Gansu Province and six species from Guizhou Province, respectively. Soil protists are an essential yet seriously understudied component of the soil microbiome. In this study, 11 new records of dictyostelids belonging to 2 orders, 3 families, and 4 genera were identified from 99 soil samples collected from different elevations and habitats in central Gansu and the southeastern and southcentral portions of Guizhou Province, China. We found that dictyostelid communities were significantly different between Gansu and Guizhou Provinces, apparently in response to different environmental factors. Moreover, dictyostelids were found to have the highest species diversity in mixed forests. Soil pH, temperature, and elevation were determined to be the primary factors that affect the distribution and occurrence of dictyostelids in Guizhou and Gansu Provinces. This work supplements the survey data available for dictyostelids elsewhere in China. These new findings have significant implications for our understanding of the diversity of soil microorganisms. The dictyostelids (phylum Amoebozoa) are a unique soil-dwelling group of transiently multicellular heterotrophic amoebae. Dictyostelids are widely known and studied for their unique lifestyle, in which multicellular fruiting bodies (sorocarps) bearing asexual spores are produced by unicellular amoeboids upon starvation . These oCompared with other elements of the biota in soil, the classification system used for protozoans and their ecological roles are complex. For the quantitative estimates of soil protozoans such as dictyostelid amoebae, direct observation and culture methods have been used to obtain data , 15. In Dictyostelium macrocephalum, Dictyostelium implicatum, and Dictyostelium crassicaule) known from Guizhou Province . It has a temperate continental climate, the average annual sunshine is 2,374 h, the frost-free period is 172 d, and the average annual precipitation is 300 mm (http://www.lanzhou.gov.cn/col/col5/index.html).Lanzhou  is located in northwest China and central Gansu Province . The autonomous prefecture is within the humid subtropical monsoon climate zone, the average annual sunshine is 1,068–1,296 h, the frost-free period is 270–330 d, and the average annual precipitation is 1,000–1,500 mm .Qiandongnan and Qiannan  are located in southwest China and southeastern Guizhou Province  From July to August 2020 and February 2021, 13 selected localities of Gansu and Guizhou, China, were sampled ; Table 1Escherichia coli were added to hay agar plates separately, and the plates were cultured at 23°C with a 12 h light and dark cycle under aseptic conditions. Each soil sample was set up in three parallels, each plate contained 0.02 g of soil, and the total soil of the three plates was 0.06 g. After 7 d of culture, counts were made of the number of dictyostelid clones on each plate. According to the formula “the clones of dictyostelids per gram of soil sample = the total number of clones of dictyostelids on three plates / 0.06”  that developed was marked. The characteristic stages in the life cycle, including cell aggregation and the formation of pseudoplasmodia and sorocarps, were observed under a Zeiss dissecting microscope (Axio Zoom V16) with a 1.5× objective and 10× ocular. Slides with sorocarps were prepared with water as the mounting medium. Features of spores, sorophores, and sorocarps were observed and measured on the slides using a Zeiss light microscope (Axio Imager A2), with 10× ocular and 10, 40, and 100× (oil) objectives. Photographs were obtained with a Zeiss Axiocam 506 color microscope camera. For strain preservation, a purified dictyostelid sorus was picked up with an inoculation needle and packed into HL5  medium, AACCTGGTTGATCCTGCCAG) and 18S RB (TGATCCTTCTGCAGGTTCAC)  . PCR proGGTTCAC) .In order to compare the distribution of dictyostelids with respect to elevation and habitats as a whole, the relative abundance dataset of dictyostelid species was subdivided into five elevation ranges  and five habitat types . The relative abundance (RA) (%) of each species was determined by dividing the number of clones for each species of dictyostelid by the total number of all dictyostelid clones. We used Origin 2021  to draw the bidirectional bar chart to describe the relative abundance of dictyostelid species.A histogram  was gene-diversity analysis, the R-packages “vegan”   using thA total of 71 clones of dictyostelids were recovered from the 99 collected soil samples. Soil samples came from 13 different localities representing five different habitat types, including scrub, coniferous forest, broadleaf forest, mixed forest, and farmland . The 99 Dictyostelium     were recorded  were isolated from Gansu Province, and these were new records for Gansu Province. Six species of dictyostelids  were isolated from Guizhou Province and represented new records for the province  for each species is shown in ranches) , one froolitary) , two froranches) , and onerecorded . The tax results ; Table 2province .NMDS and PCA of the dictyostelid communities revealed significant differences between soil collection sites in Gansu and Guizhou  occurred only from elevation 1,000–1,300 m; two species (H. tenuissimum and D. multiforme) occurred only from elevation 1,300–1,600 m, accounting for 18.18%; one species (D. giganteum) occurred only from elevation 1,600–1,900 m; and one species  occurred only from elevation 1,900–2,200 m, accounting for 9.09%. No species occurred in common at all five elevations . Two species  occurred only in mixed forest, accounting for 27.27%; two species  occurred only in broadleaf forest, accounting for 18.18%; and one species (D. giganteum) occurred only in farmland, accounting for 9.09%. No species occurred in common in all five habitats  occur in common from four different forest types, accounting for 56.34% of the total number of species clones in this study.The distribution of species with respect to relative abundance at different elevations and in different habitats is shown in the bidirectional bar chart . Three sP = 0.002), elevation (P = 0.002), and pH (P = 0.001) were the main impacting factors that determined the distributions of dictyostelid species in this study.Correlations of the dictyostelid species isolated from 13 sampling sites in Gansu and Guizhou Provinces with environmental factors were evaluated using RDA. Among the total environmental variance of the assemblage, 27.12% constrained was explained in RDA axis 1 (72.88%) and axis 2 25.39%; . TemperaOne of the keys to dictyostelid studies, as opposed to other types of soil-dwelling single-celled eukaryotes, is the ease with which they can be cultured, identified, and enumerated. Ecological research of dictyostelids was first initiated by Cavender and Raper , 26. A cD. firmibasis and D. multiforme were regarded as uncommon, having been recorded previously from relatively few localities in China. Cavenderia delicata was the second report in China after that of Taiwan  had significant effects on the species diversity of dictyostelids  was the important predictor that explains the 77.6% constraint axis in the community composition  was the important predictor that explains the 75.7% constraint axis; the environmental factor of temperature was significantly correlated with variations in dictyostelid communities ; this suggested that the propagules had a strong resistance to unfavorable conditions and could survive in low-temperature environments, thus maintaining their stable properties.The soil microbial community plays an important role in soil ecological function, and in the present study, temperature appeared to affect microbial activity and metabolic diversity of the dictyostelid communities. The temperature range when collecting in seven soil collection sites of Guizhou Province was 9°C–31°C, while the temperature range when collecting in six soil collection sites of Gansu Province was relatively lower, −6°C–13°C . The RDAin Gansu , includiin Gansu  recovereD. mucuroides and D. sphaerocephalum were distributed in four forest types of Gansu soil samples; these two species were the more frequent in Gansu soil samples. Other species such as D. firmibasis, C. delicata, D. giganteum, D. multiforme, D. macrocephalum, and H. tenuissimum were distributed in a single forest type, mostly mixed forest, and appeared to have a limited distribution among the localities sampled. D. mucuroides and D. sphaerocephalum are considered cosmopolitan species often encountered throughout the world  , 66. In