As uterine rupture may affect as many as 11/1000 women with 1 prior cesarean birth and 5/10.000 women with unscarred uterus undergoing labor induction, we intended to estimate the prevalence of such rare outcome when PGE2 is used for cervical ripening and labor induction.ClinicalTrials.gov and the Cochrane library up to September 1st 2020. Retrospective and prospective cohort studies, as well as randomized controlled trials (RCTs) on singleton viable pregnancies receiving PGE2 for cervical ripening and labor induction were reviewed. Prevalence of uterine rupture was meta-analyzed with Freeman-Tukey double arcsine transformation among women with 1 prior low transverse cesarean section and women with unscarred uterus.We searched MEDLINE, We reviewed 956 full text articles to include 69 studies. The pooled prevalence rate of uterine rupture is estimated to range between 2 and 9 out of 1000 women with 1 prior low transverse cesarean . The prevalence of uterine rupture among women with unscarred uterus is extremely low, reaching at most 0.7/100.000 .Uterine rupture is a rare event during cervical ripening and labor induction with PGE2. Approximately 1 in 5 women in the USA, UK, and Europe undergo labor induction . As the Uterine rupture is a rare obstetric complication that continues to be associated with a high rate of perinatal and maternal morbidity and mortality , 7. The Studies on the relationship between cervical ripening agents and induction of labor are generally small, underpowered to detect relevant differences, and difficult to use for definitive conclusions ; furtherCurrently, the practice of labor induction continues to increase, PGE2 is widely used for cervical maturation, and concerns for the safety of mother and infant are prominent factors in decisions about childbirth, especially among at risk categories such as women with a history of cesarean birth; therefore, we conducted a systematic review and meta-analysis to determine the prevalence of uterine rupture among women with 1 prior low transverse cesarean and among those with an unscarred uterus receiving PGE2 for cervical ripening and labor induction at term.ClinicalTrials.gov , and the Cochrane Collaboration databases from inception up to September 1st 2020, using a combination of text words including “induction of labor”, “induced labor”, “prostaglandin E2”, “dinoprostone”, “vaginal birth after cesarean”, “trial of labor after cesarean”, “prior cesarean”, and “uterine rupture” . Pooled prevalence rates of uterine rupture were estimated using Freeman-Tukey double arcsine transformation, which stabilizes the variances of the included studies . Between-study heterogeneity was accounted for using random-effects meta-analyses.Two reviewers (GC and ALT) independently evaluated the titles and abstracts of all citations produced by the electronic searches. The full text of the potentially eligible studies was retrieved and independently assessed for eligibility by two review authors (GC and ALT). Discordance between reviewers was resolved by discussion, and if not a third author (FF) was involved. When multiple publications addressed the same cases, the main study report was used as reference. Data were extracted in a standardized manner by 3 authors .To better describe our study population, we investigated the different PGE2 pharmaceutical formulations utilized in the included studies, along with the induction agents PGE2 was compared to (if the study design required a comparator). Moreover, maternal characteristics , obstetric variables , and neonatal features  were reported. Tachysystole was defined as more than five contractions per 10 min . UterineContinuous variables presented as means in the original studies, were summarized as means of the means reported in the original publications. Categorical variables presented as proportions in the original studies, were summarized as means of the proportions specified in the original publications. All statistical analyses were performed with the Stata software package version 15.1 . Dataset was included among the Supporting Information to the manuscript.This systematic review was conducted according to the MOOSE  and PRISMA  statements. Although no RCT intended to address our study question, information about uterine rupture was often provided when PGE2 effects were investigated with this study design; therefore, MOOSE reporting guidelines were followed even when prevalence of uterine rupture was inferred from RCTs . A risk The flow diagram of the electronic search details and selection process are shown in 2 47.7%, 122/9000). Rates are similar across all study designs, being 5/1000  according to retrospective cohort studies, 6/1000  considering prospective cohort studies, and of 6/1000  including only RCTs , reaching the highest value of 22/100.000 according to retrospective cohort studies, 22/100.000 considering prospective cohort studies, and 58/100.000 analyzing RCTs only.The prevalence of uterine rupture among women with unscarred uterus is extremely low. Only 7 out of 24 studies included in our meta-analysis reported at most 2 cases of uterine rupture, totaling 8 instances  among 17.684 women receiving PGE2 for cervical ripening and labor induction . The characteristics of the entire study populations are shown in When confronted with the risk of bias tool for prevalence studies , all theAs uterine rupture is a rare event when PGE2 is used for cervical ripening, its prevalence needs to be estimated in studies with large populations to limit publication bias. We estimated that the pooled prevalence rate of uterine rupture is 5/1000 among women undergoing TOLAC and at most 0.7/100.000 among those with an unscarred uterus.Women with history of low transverse cesarean birth are at higher risk of uterine rupture, as reported by several studies showing a clinically determined uterine rupture rate after TOLAC of approximately 5–9/1000 –87, thatCervical ripening protocols, that differ in terms of PGE2 dose, pharmaceutical formulation and timing of administration, contribute to determine the safety and the success of TOLAC together with many other maternal and obstetric characteristics, such as history of prior vaginal deliveries, gestational age, fetal weight, labor augmentation, maternal age and BMI. Such wide range of contributing factors and a variety of different study designs led us to use the random effects model, which accounted for study heterogeneity. The contribution of different PGE2 dosages, pharmacological formulations and timing of administration was not further explored with meta regression due to the insufficient number of studies; instead, the role of study design was addressed with subgroup analyses.Spontaneous rupture of the unscarred uterus is an extremely uncommon event. No cases were reported by two observational studies respectively analyzing 21,998 and 30,874 primigravid labors , 93. WheWhen PGE2 is administered for cervical ripening, we found a cesarean birth rate of 34.1% among women with 1 prior CD, and 22.8% in case of unscarred uterus . Our finStrengths of our study include the comprehensive search strategy with no language restrictions, as well as the low risk of bias of the included surveys. Limitations primarily relate to the underlying data that was available on this topic. Primary studies were not specifically designed to address our study question, and they were mostly observational in nature. As labor induction is a complex multi-step process, it becomes challenging to single out the role played by PGE2; moreover, a high degree of heterogeneity characterized the studies including women with 1 prior low transverse cesarean. Subgroup analyses on PGE2 formulation , dose, and route of administration  were not performed due to the rarity of the outcome and the heterogeneity in study designs. For the same reasons, the potential additional risk from oxytocin infusion could not be addressed.Despite our findings may reassure clinicians on cervical ripening with PGE2, a multicenter prospective cohort study or national registry would offer the best opportunity to assess the safety of such agent, especially among women with a prior low transverse cesarean. Different PGE2 dosages and administration protocols could be compared to other induction strategies, while accounting for confounding. In the meantime, the use of PGE2 should continue in case of no prior uterine surgery, and it should be considered among women undergoing TOLAC who are comfortable with the increased risks of uterine rupture associated with induction of labor.S1 Checklist(DOCX)Click here for additional data file.S1 Dataset(DTA)Click here for additional data file.S2 Dataset(DTA)Click here for additional data file.S1 File(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Vascular aging has been closely associated with various cardiovascular disorders; however, its molecular mechanism remains poorly understood. In our study, RNA sequencing was utilized to explore the expression profiles of long non-coding RNAs (lncRNAs) and mRNAs in the thoracic aortas of young (3 weeks) and old (16 weeks) rats. Functional categorization of differentially expressed mRNAs was evaluated using the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases, and lncRNA–microRNA–mRNA networks was constructed using Cytoscape software. In addition, three upregulated and three downregulated lncRNAs were further confirmed by quantitative reverse transcriptase-polymerase chain reaction. A total of 36 lncRNAs and 922 mRNAs were differential expression in the thoracic aortas of young and older rats. In addition, we found differentially expressed mRNAs that were enriched in multiple biological processes and signaling pathways associated with angiogenesis, such as extracellular matrix–receptor interaction and adenosine 3ʹ,5ʹ-monophosphate-activated protein kinase (AMPK) signaling. Moreover, AABR07013558.1, AABR07014823.1, and AABR07031489.1 were upregulated and ABR07053849.3, AABR07067310.2, and AC111292.1 were downregulated in the thoracic aortas of older rats compared with the young ones. Therefore, our findings provide several potential lncRNAs and mRNAs and signaling pathways related to vascular aging, which provide new clue for underlying the improvement of vascular aging.  Vascular aging refers to the dysfunctional alterations of blood vessels that occur during the aging process. Its common features are structural and functional changes, such as dysregulation of vascular homeostasis and vascular dyshomeostasis, enhanced vascular cell senescence, and vascular remodeling . In contPrevious reports showed that the progression of aging in blood vessels is mediated by complex networks of cellular and molecular events ,8. For iLong non-coding RNAs (lncRNAs) are a recently characterized group of non-coding RNAs, usually comprising >200 nucleotides, and are recognized as essential components of epigenetic regulation system ,14. ExteIn this study, we explored characterization of the differentially expressed lncRNAs and mRNAs for the first time in the thoracic aortic tissues of young and older rats by high-throughput RNA deep sequencing. The results may provide new insights into molecular mechanism driving vascular aging, which might be further explored as a basis to develop new treatments for aging-related vascular disorders.2 [Healthy male Sprague Dawley (SD) rats were purchased from Chengdu Dashuo Biotechnology Co., Ltd.  and maintained in a pathogen-free barrier facility at 27°C at the Laboratory Animal Center of the Nanfang Hospital of Southern Medical University. All experimental procedures on SD rats in this study was approved in advance by the Experimental Animal Care and Use Committee of the Nanfang Hospital, Southern Medical University and performed following the Guide for the Care and Use of Laboratory Animals . To explore the lncRNA and mRNA expression profiling related to vascular aging, three healthy male rats aged 3 weeks with an average body weight of 52 g were used as the young group and another three healthy male rats aged 16 weeks with a body weight of approximately 480 g were selected as the older group. When SD rats reached the humane end point of euthanasia, they were euthanized in the euthanasia chamber with 20% chamber volume/min of CO2 . Death wg at 4°C. The resulting pellet was washed with 1 mL 75% ethanol and resuspended in 50 µL RNase-free water. The RNA samples were analyzed by NanoDrop™ 1000 spectrophotometer (Thermo Fishier Scientific), and samples with a A260/A280 ratio of ~2 were stored at −80°C for subsequent assays.SD rats were sacrificed at designated ages through anesthesia with chloral hydrate, and the rat thoracic aortic tissues were surgically collected and immediately stored in liquid nitrogen. The extraction of total RNA from rat aortic tissues for RNA sequencing was performed using Invitrogen™ TRIzol™ Reagent  following the manufacturer’s instructions. In brief, approximately 0.1 g rat aortic tissues were homogenized in 1 mL TRIzol™ Reagent with a homogenizer, which was then incubated with 0.5 mL isopropanol for 10 min at 4°C, followed by centrifugation for 10 min at 12,000 × The quantitation of lncRNA and mRNA expression profiles in rat aortic tissues were performed by RNA deep sequencing as previously described with minor modifications . In briehttp://www.genome.jp/kegg/). Finally, the interaction networks between lncRNA, microRNAs, and mRNAs were constructed using Cytoscape software.The numbers of lncRNAs within different size ranges and encoded by DNA sequences on each rat chromosome were counted separately. Hierarchical clustering of lncRNA or mRNA differential expression was presented using heatmaps created in R language software. Moreover, the differential expression of mRNA of the young and older rat groups was also evaluated using a scatter plot constructed using the transcripts per million (TPM) values and a false discovery rate of <0.01. Functional categorization of differentially expressed mRNA of the young and older rat groups was performed using the Database for Annotation, Visualization and Integrated Discovery system based on the following Gene Ontology (GO) terms: molecular functions, biological processes, and cellular components. Moreover, the signaling pathway enrichments of these differentially expressed mRNAs were assessed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database . Primers used for the qRT-PCR method are provided in  method. In brief, total RNA samples were extracted using Invitrogen™ TRIzol™ Reagent  as described above. The cDNA library was then established using TransScript® Reverse Transcriptase kit  as instructed by the manufacturer. Subsequently, the qRT-PCR assay was then performed to measure the expression levels of lncRNA between groups using the TransStart® Green qPCR SuperMix  following the manufacturer’s instructions. The relative expression levels of lncRNAs were finally calculated via the 2vided in .Table 1.All experiments were performed with at least three replicates. Quantitative data were expressed as means ± standard deviation and analyzed using SPSS 18.0 software. Differences between the two groups were evaluated using the Student’s t-test, and statistically significant differences were defined by a P value of <0.05.LncRNAs perform vital regulatory roles in various biological and pathogenic processes due to their potent ability of modulating gene expression at both transcriptional and post-transcriptional levels, such as sponging microRNA, regulating RNA splicing, mRNA processing, and acting as cis-acting components. In this study, we explored characterization of the differentially expressed lncRNAs and mRNAs in the thoracic aortic tissues of young and old rats for the first time. Then, GO and KEGG analyses were performed for the differentially expressed mRNAs, and lncRNA–microRNA–mRNA interaction networks were established based on the differentially expressed lncRNAs and mRNAs. Finally, six lncRNAs in the networks were verified to test the reliability of RNA sequencing.To explore the potential involvements of lncRNAs in vascular aging, we performed a general characterization of lncRNAs that were differentially expressed in the thoracic aortas of the young (3-week-old) and older (16-week-old) rats by deep sequencing, with three biological replicates in each group . Our traFor insights into the molecular mechanisms underlying vascular aging progression, we also characterized the significantly differentially expressed mRNAs in the thoracic aortas of young and older rats during RNA deep sequencing. The expression levels of 922 mRNAs were significantly altered between the young and older rat’ thoracic aortas, including 474 upregulated and 448 downregulated mRNAs in the thoracic aortas of older rats compared with those of young rats . In addiTo obtain a comprehensive view of biological processes possibly linked with vascular aging, we functionally categorized the differentially expressed mRNAs in the thoracic aortic tissues of young and older rats. Based on GO analysis of biological processes, we found that these differentially expressed genes were significantly enriched in single-organism process, biological regulation, metabolism, responses to stimulus, development, cellular component organization, signaling, localization, the immune system, reproduction, growth, locomotion, cell killing, biological adhesion, and others . In addiIn addition, we further analyzed the enrichment of these differentially expressed mRNAs in KEGG signaling pathways . Using KPrevious reports showed that lncRNAs could induce gene expression alterations by sponging microRNAs . For a dTo test the reliability of our RNA sequencing results, we analyzed the expression of six lncRNAs by qRT-PCR method, which were shown to be differentially expressed in the thoracic aortas of young and older rats . Among tMolecular mechanisms underlying vascular aging have been a major research interest during the past decades because of the high incidences and mortalities caused by CVD, atherosclerosis, and aging-related pathogenic conditions ,3,4,24. Non-coding RNA molecules have emerged as key components of epigenetic regulator networks, with potent capabilities of modulating gene expression in almost every biological and pathogenic process . In receThe main biological and pathogenic roles of lncRNAs are mediated by their modulation of functional gene expression through various molecular strategies at the transcriptional and post-transcriptional stages . For a pMicroRNAs are a large group of non-coding RNAs with a common size of 22 bp, which are known to post-transcriptionally repress gene expression and mediate vascular aging and several vascular pathologies . By contin vivo, we can adopt adeno-associated viruses for gene interference in specific tissues or import exogenous RNA through methods, such as exosome encapsulation. At the same time, we are focusing on whether different foods and drugs have stimulating or inhibiting effects on the corresponding lncRNAs and mRNAs.Next-generation RNA sequencing and bioinformatics analysis provide comprehensive information of vascular aging from a new perspective and direction. While the method of next-generation RNA sequencing has certain error rate inevitably as all sequencing method have, which could be avoided by further validation. To test the reliability of our RNA sequencing results, we analyzed the expression of six lncRNAs by qRT-PCR method. However, the present study still has several limitations. For example, the expression changes and functions of several lncRNAs and mRNAs need to be further verified in the rats and cell lines. In addition, the prognostic analysis of the differential lncRNAs and mRNAs in old rats need to be further explored. In future studies, to further apply differential lncRNAs and mRNAs for improving vascular aging, detailed animal experiments need to be further performed. According to studies on the effects of exogenous RNA on tissues We characterized a large number of lncRNAs and mRNAs that were differentially expressed in thoracic aorta of young and older rats that covered multiple biological processes and signaling pathways, such as ECM–receptor interactions. The differential expression of several collagen genes in rat aorta may be attributed to the sponging of microRNAs by lncRNA AC111292.1. According to these results, we can further explore the treatment of diseases and the delay of aging from a new perspective and direction. In addition, verification of differential lncRNAs in vascular aging can lay a foundation for future research on mechanism in vascular aging.Click here for additional data file.


Adequate levels of vitamin D are important for women of childbearing age as vitamin D helps maintain the pregnancy and ensures proper maternal and fetal bone metabolism and fetal skeletal development. However, vitamin D deficiency is a health problem prevalent in women of all ages, worldwide. This study aimed to determine the current status of serum vitamin D levels and the risk factors for vitamin D deficiency among South Korean nurses of childbearing age.Serum levels of 25-hydroxyvitamin D (25[OH]D) were measured in 1594 registered nurses aged 20 to 45 years who are participants in an ongoing prospective cohort study of the Korean Nurses’ Health Study initiated in 2013. The participants completed surveys about demographic and occupational characteristics and physical and psychological health. We examined associations with vitamin D deficiency through multivariable logistic regression analysis.The average blood 25(OH) D concentration of the participants was 12.92 ng/mL (4.0–63.4 ng/mL), while the prevalence of vitamin D deficiency (< 20 ng/mL) was 89% (1419/1594). Multivariable logistic regression showed that significant risk factors for vitamin D deficiency included month of sampling , age (women in their 20s had a lower vitamin D level than those in their 30s and 40s), and stress symptoms. Vitamin D levels were not associated with body mass index, physical activity, and depressive symptoms.This study showed a high prevalence of vitamin D deficiency in Korean female nurses. Serum levels of vitamin D were associated with age and season. Vitamin D deficiency should be recognized as one of the primary health concerns among young women. More proactive actions, such as vitamin D supplements and food fortification, are needed to improve vitamin D deficiency in high-risk groups. Vitamin D deficiency is very prevalent worldwide, though the prevalence varies according to age, region, culture, and lifestyle . It has Pregnancy, obesity, reduced exposure to sunlight, and being a person of color are all factors that can increase the risk of vitamin D deficiency. In pregnant women, low serum vitamin D levels have been associated with gestational diabetes, preterm birth, and poor health outcomes for premature newborns . In SoutVitamin D is primarily synthesized in the skin during exposure to sunlight . The monRecent studies that examined the relationship between vitamin D levels and mental health have found that low 25(OH) D levels caused a pro-inflammatory state and could be associated with depression . StudiesAs demonstrated in these previous studies, inadequate vitamin D levels are related to many factors, including sunlight exposure, ethnicity, gender, indoor and shift work, and physical and mental health. Considering the high prevalence of vitamin D deficiency in South Korean women and its negative impact on women’s health as well as the high rate of chronic diseases in this population, low serum 25(OH) D levels in women of childbearing age require attention. However, previous studies primarily investigated the commonalities of vitamin D deficiencies in male and female adults and seniors. Therefore, studies to identify specific characteristics related to low vitamin D levels in women of childbearing age are crucial. In this study, we focused on the impact of working patterns and health characteristics on serum vitamin D levels in women of childbearing age.This study was designed as a cross-sectional study that analyzed data from the Korean Nurses’ Health Study (KNHS) . The KNHIn order to recruit survey participants for Modules 3 and 5, information on study participation was sent using the mobile phone numbers and email addresses captured at baseline. Volunteers participated in the survey by accessing the KNHS website (either online or via the mobile phone). The top 30 hospitals were chosen based on the number of participants at baseline. Of those, 12 hospitals cooperated and indicated their availability. These were selected as sites for blood testing. Promotional materials were posted at each of the 12 hospitals to recruit more participants from the baseline data group for blood testing. In addition, email and text messages with information on the blood test were sent out to all eligible participants to request voluntary participation.After applications were completed by the nurses who had volunteered to participate, instructions on how to prepare for the blood draw, including fasting beforehand, were delivered to participants in collaboration with the nursing department of each hospital. A research team comprising 5–6 members  visited the designated hospital for 3–10 days. The phlebotomist drew blood samples by venipuncture of the median antecubital vein using a vacuum system in volunteers who had fasted for 8 h. The collected blood was stored and transported according to the process followed by certified laboratories . The serum 25(OH) D level was analyzed using an autoanalyzer .The 25(OH) D levels were classified as deficient or non-deficient using a 25(OH) D concentration of 20 ng/mL as a cutoff [Further, the survey questioned participants on their weekly average of overtime hours and whether they worked on a shift. In this study, shift work was defined as “day shift and evening shift and/or night shift in a sequential basis.”2). In order to determine the level of physical activity, the participants were asked how much of each of the following activities they performed on average over the past year: walking, jogging, running, cycling, tennis/squash/racquetball, lap swimming, dancing, skiing, aerobic exercise , yoga, simple workouts , and intense physical activities . Metabolic equivalent of task (MET) values (hours/week) for each physical activity were calculated using the criteria suggested by previous studies [The height and weight of participants reported in Module 5 were used to calculate the participant’s body mass index, which is calculated by dividing the weight (kg) by the height in meters squared   which isThe South Korean version of the Perceived Stress Scale-10 , composeSPSS version 23  was used to analyze data. Data were expressed as frequencies and percentages or averages and standard deviations following a descriptive statistical analysis. In contrast, the chi-square test and t-test were used to analyze the differences between the participants who had been classified as having deficient and non-deficient vitamin D levels. These were further grouped by general and occupational characteristics and physical and psychological health. The statistical significance of vitamin D deficiency was confirmed using a multivariable logistic regression analysis. The timing of blood tests, demographic factors, work environment factors, and physical and psychological factors were input into Models 1–4.The mean concentration of serum 25(OH) D was 12.92 ng/mL, ranging from 4 to 63.4 ng/mL A total of 1419 participants (89%) had a vitamin D concentration of < 20 ng/mL, and 175 participants (11%) had vitamin D levels of ≥ 20 ng/mL. There were 1127 (70.7%) shift workers, with an average of 5.26 (SD = 4.71) overtime working hours. The chi-square tests indicated that the levels of vitamin D level differed significantly based on the sampling month, age, marital status, education, and shift work  METs (hours/week). Further, 750 (47.1%) participants exhibited minimal levels of depressive symptoms. The stress symptoms scores showed a significant difference based on vitamin D levels  as shown in Table The multivariable logistic regression model analysis revealed that the timing of the blood test was a significant factor influencing vitamin D levels . Compared with the participants in their 20s, those in their 30s  and 40s  were more likely to have vitamin D levels over 20 ng/mL. Of the physical and psychological factors, stress symptoms (p = .009) were a significant factor influencing vitamin D levels: among participants with higher stress symptoms scores , there were fewer participants with vitamin D levels over 20 ng/mL.Compared with participants who underwent blood tests in December–February, those who underwent blood tests in March–May , June–August , and September–November  presented with higher ORs for vitamin D levels greater than 20 ng/mL. Among the general characteristics, age was a significant factor influencing vitamin D levels  D concentrations, which is consistent with the results of a previous study which demonstrated that 88% of the women in their 20s and 30s were diagnosed with vitamin D deficiency [In this study, the sampling month, age, and stress were identified as factors influencing vitamin D levels. Vitamin D levels were lowest in participants who had their blood drawn between December and February. Given that vitamin D synthesis in the skin is affected by exposure to sunlight, individuals are likely to have relatively low concentrations of vitamin D during the winter months . A previThe participants with the lowest vitamin D levels were those in their 20s. This is consistent with the high vitamin D deficiency rates reported among Australian women aged 25 to 34 , which iWe also found that stress levels affect vitamin D deficiency in women of childbearing age, consistent with the results of a study by Gwon et al. which showed that perceived stress was a risk factor for low 25(OH) D levels in older women . Abu-SamOur results showed that factors that had no effect on vitamin D deficiency included level of education, shift work, physical activity level, and depression level, which is contrary to reports demonstrating that those with higher levels of education show a higher prevalence of 25(OH) D deficiency , 30. ThiIncreased physical activity is known to play an important role in preventing low serum vitamin D levels; vitamin D synthesis increases with sun exposure during outdoor activity, which reduces sedentary activity and strengthens muscles , 28, 30.Our study has several limitations. First, as the study had a cross-sectional design, we cannot yet determine the causality of associations with diseases that may occur owing to vitamin D deficiency. Further longitudinal studies will need to be conducted on the prevalence of diseases associated with vitamin D deficiency. Second, bone densitometry measurement was not included in this study; thus, we could not assess the current bone health of participants. As bone densitometry is a basic tool for assessing bone health directly, simultaneous measurements of serum vitamin D levels and bone density are suggested. Third, there were gaps in time points of collecting data because physical activity was measured in M3 during November 2015–2017 and vitamin D level was measured in M5 during November 2016–2017. Physical activity will, thus, not reflect the levels of vitamin D directly.Despite these limitations, our study has a major strength. Although we analyzed serum vitamin D levels of only 1594 samples, this is the first nationwide cohort study that included more than 10,000 registered nurses in South Korea. This study administers surveys to participants every year. Therefore, conducting further longitudinal studies to explore the association between the prevalence of diseases and general characteristic and clinical factors is promising. The study analyzed factors associated with vitamin D deficiency using self-reported data on job-related characteristics of South Korean nurses, thereby providing basic information for an intervention plan for female nurses of childbearing age.This study aimed to determine the current status of serum vitamin D levels and the risk factors for vitamin D deficiency among South Korean nurses of childbearing age. Our study results were consistent with the results of other cohort studies conducted in different parts of the world; hence, vitamin D deficiency appears to be a common health problem across the globe. However, this is the first nationwide study to investigate the current status of vitamin D levels of South Korean nurses of childbearing age. Therefore, our contribution to this field of study can be significant. Further studies should focus on investigating causal relationships between vitamin D deficiency and comprehensive risk factors such as sunlight exposure, lifestyle , adiposity, diseases, and demographics as well as long-term effects of untreated vitamin D deficiency.


Clostridioides difficile infection (CDI). Fidaxomicin has been used as part of the standard treatment for nonsevere and severe CDI according to the guidelines by the Infectious Diseases Society of America and the Society for Healthcare Epidemiology of America, but not in severe fulminant CDI due to lack of randomized clinical trials supporting its use. We present the case of a patient who developed severe fulminant colitis initially refractory to treatment with oral vancomycin and intravenous metronidazole that had an impressive improvement within 24-hour of starting fidaxomicin. The patient had a complete resolution of the symptoms at the end of the therapy without requiring a surgical approach. There are few case reports of fulminant CDI treated with fidaxomicin as a salvage therapy for fulminant CDI. In this challenging scenario, information about the use of fidaxomicin is still limited and more clinical trials are needed to support its widespread use.Few studies have demonstrated the efficacy of fidaxomicin in the treatment of fulminant  Clostridium, Clostridioides difficile is a gram-positive, anaerobic, toxin-producing bacteria that colonize the human intestinal tract after the normal gut flora has been disrupted. It is the leading cause of hospital-acquired diarrhea with increasing morbidity and mortality with a total incidence of 130.2 per 100,000 patients according to the Centers for Disease Control and Prevention in the year 2017 [C. difficile infection (CDI) can cause a large spectrum of manifestations ranging from an asymptomatic carriage to fulminant disease with toxic megacolon symptoms. Its cardinal symptom is diarrhea, usually defined as three or more episodes of loose stools or 200 g output within a 24-hour period, that can be associated with lower abdominal pain and cramping, low-grade fever, nausea, and anorexia in a setting of a recent history of hospitalization and antibiotic use as well as due to advanced age and proton pump inhibitor suppression of gastric acid [Formerly ric acid .C. difficile toxins A and B. Less commonly, CDI can be confirmed by cell culture cytotoxicity assay and selective anaerobic culture. EIA for GDH has high sensitivity and poor specificity as detection cannot distinguish between toxigenic and nontoxigenic strains, whereas EIA for toxins A and B has poor sensitivity and high specificity. Although NAAT (which includes polymerase chain reaction) can detect one or more genes specific to toxigenic strains, of which tcdB is the most important gene, which encodes for toxin B, it cannot test for active toxin protein production and is capable of detecting asymptomatic carriers of C. difficile [CDI can be diagnosed by several diagnostic methods, most commonly by nucleic acid amplification testing (NAAT) alone but also in combination with tests using an enzyme immunoassay (EIA) for glutamate dehydrogenase (GDH) and enzyme immunoassay for ifficile .Management of CDI is determined based on its severity that is categorized into nonsevere, severe, and fulminant colitis infection. The clinical practice guidelines of the Infectious Diseases Society of America (IDSA)/Society for Healthcare Epidemiology of America (SHEA) define severe disease as the clinical criteria for CDI infection associated with white blood cell (WBC) count of >15,000 cells/µL and/or creatinine >1.5 mg/dL. The fulminant disease is defined by the presence of decompensating clinical features such as hypotension, shock, ileus, or megacolon associated with the criteria for severe disease . FulminaThe following case reports a patient with fulminant CDI who was admitted to the intensive care unit (ICU) with septic shock and renal insufficiency requiring urgent dialysis. He displayed a fast response to fidaxomicin use with clinical improvement after having failed to improve with parenteral metronidazole and oral vancomycin use.Staphylococcus aureus, and he was completing his six-week treatment of intravenous (IV) cefazolin in a skilled nursing facility after hospitalization. Nearly 10 days after hospital discharge, the patient started developing diffuse abdominal pain, decreased appetite, poor oral intake, and diarrhea. He tested positive for C. difficile and was started on oral vancomycin. Two days after starting the antibiotic, his clinical status rapidly declined with worsening diarrhea, oliguria, and hypotension. Although he was initially transferred to a regular floor in another hospital, he was admitted to our ICU two days later with septic shock, respiratory failure, and acute kidney injury.Here, we report the case of a 60-year-old Caucasian male patient with a past medical history of diabetes mellitus type 2, coronary artery disease, atrial fibrillation, hypertension, obstructive sleep apnea, chronic kidney disease stage 3, and nonalcoholic steatohepatitis. The patient had a recent hospital admission due to a surgical C2-C3 epidural abscess which was treated with drainage and spinal C2-C5 laminectomy and fusion, initially manifesting as quadriparesis. Abscess cultures were positive for methicillin-sensitive 2 28 mmHg, PO2 127 mmHg, HCO3 7.9 mEq/L, creatinine 4.54 mg/dL, blood urea nitrogen 152 mg/dL, potassium 4.4 mEq/L, lactate 9.0 mmol/L, WBC 14.35 × 109/L with a neutrophil count of 88%. C. difficile stool antigen and toxin genes were positive confirming the results of the external facility. Admission CT of the abdomen and pelvis showed a marked diffuse colonic wall thickening with adjacent fat stranding, suggestive of colitis without dilated bowel (maximum right colon dilation of 4 cm) and nodular contour of the liver, suggestive of hepatic cirrhosis . Fidaxomicin was started, and within 24 hours of treatment, the patient had a rapid clinical stabilization with a reduction in diarrhea volume and a significant reduction in the use of vasopressors. The patient was extubated and transferred to the regular floor in four days. He completed a total of seven days of IV vancomycin and piperacillin/tazobactam and 15 days of IV metronidazole; fidaxomicin was continued for 10 days.In the ICU, his workup at admission showed pH 7.061, PCOOral vancomycin and parenteral metronidazole are the recommended therapy of choice for fulminant CDI according to the IDSA/SHEA 2017 guidelines. The strength of recommendation is considered to be strong based on moderate-quality evidence for both medications . In our C. difficile colonization, and potentially reducing drug resistance [Vancomycin is a bacteriostatic drug, whereas fidaxomicin is a bactericidal drug with an 18-membered-ring macrolytic antibiotic that inhibits RNA polymerase sigma subunit resulting in the inhibition of protein synthesis and bacterial death [sistance ,10. A stsistance . In addisistance ,7. Anothsistance -14.Our patient had a poor prognosis due to his presentation with fulminant CDI and renal insufficiency according to a German retrospective study in patients with CDI and severe medical conditions. The resolution of diarrhea (67.5% and 68.0%) and mortality (24.6% and 27.6%) for patients with fulminant CDI and severe renal impairment were lower compared to 78% of diarrhea resolution and 30-day mortality rate of 17% for other severe medical conditions . Our patThe use of fidaxomicin in CDI has been reported to be advantageous over vancomycin in different settings. Our case shows the successful use of fidaxomicin as a salvage treatment for a patient with fulminant CDI that failed to respond to the initial treatment with oral vancomycin and IV metronidazole. The impressive clinical improvement is encouraging; however, more studies are required to support the use of fidaxomicin as a salvage therapy for patients who fail to respond to first-line antibiotic treatment.


Food addiction is currently not an official diagnosis  in the Diagnostic and Statistical Manual of Mental Disorders, 5th Edition (DSM-5). To best of our knowledge, there is no previous research on differences between addictive-like eating behavior regarding its associations with psychological distress, eating behaviors and physical activity among individuals with obesity. The objective of the present study was to distinguish psychological and behavioral patterns of individuals with obesity concerning food addiction using a cluster analysis. We determined the profiles of the participants in terms of psychological distress, eating behaviors and physical activity and evaluated their association with food addiction. A cross-sectional study was conducted between September and November 2020, during the lockdown period imposed by the government for the COVID-19 pandemic. A sample of 507 individuals with obesity aged between 18 and 65 years participated in the present study by filling in the online questionnaire, including the validated Arabic version of the modified version of the Yale Food Addiction Scale, the Arabic version of the Depression, Anxiety and Stress Scale, the Three-Factor Eating Questionnaire, and the short version of the International Physical Activity Questionnaire. A cluster analysis was performed using the K-mean method to identify and group participants according to their patterns/profiles. A stepwise linear regression was conducted, taking the food addiction score as the dependent variable. Higher levels of uncontrolled eating, emotional eating and stress were significantly associated with higher food addiction score. Belonging to cluster 2  (B = 14.49) or cluster 3  (B = 6.67) compared to cluster 1  was significantly associated with higher food addiction score. The present research revealed that food addiction is significantly associated with higher psychological distress and maladaptive eating behaviors. Higher levels of uncontrolled eating, emotional eating and stress as well as belonging to clusters 2 and 3 were found to be predictors of food addiction in individuals with obesity in the present study. This knowledge could be useful in regard to psychological treatment of obesity and addictive-like eating behavior. Inappropriate dietary patterns, low levels of physical activity combined with high levels of sedentary behaviors and an increasingly obesogenic environment promoting poor dietary behaviors  are regaFood addiction is defined as hedonic eating behavior including the consumption of highly palatable foods in quantities beyond homeostatic energy requirements . Food ad2). Several studies have also demonstrated the relationship between food addiction and disordered eating [Obesity is comorbid with food addiction. Previous findings  have shod eating , compulsd eating , emotiond eating ,19, moodd eating ,21 and dd eating ,23,24. Id eating . Whereasd eating . Existind eating . Contrard eating .The scientific validity of food addiction as a mental disorder and addictive behavior is still under investigation . PreviouThe current study was part of a large cross-cultural project (conducted in Lebanon and Poland) focusing on the Multidimensional Approach to Eating and Obesity (the MATEO study). The present study was carried out between September and November 2020, during the SARS-CoV-2 pandemic, when lockdown procedures were implemented at different instances, and the measures taken by the Government of Lebanon used to change on a regular basis according to the severity of the COVID-19 situation. A sample of 507 individuals with obesity aged between 18 and 65 years participated in the present study by filling in the online questionnaire. The sample was recruited through a snowball technique, from different demographic backgrounds from the five governorates of Lebanon . Respondents were briefed about the objective of the study and assured of the anonymity of the response. Participation in this study was voluntary. The participant could withdraw from participating in the study at any time.According to the G-power software, and based on an effect size f2 = 2%, an alpha error of 5%, a power of 80%, and taking into consideration 20 factors to be entered in the multivariable analysis, the results showed that a minimal number of 395 was needed.Sociodemographic characteristics information. Data were collected using a background information sheet. The background information sheet asked for information on age, gender, educational level , marital status . Information about self-reported anthropometric measurement (height and weight) of the participants was also collected, which allowed us to calculate the body mass index.Assessment of food addiction. The validated Arabic version of the modified version of the Yale Food Addiction Scale (mYFAS)  was usedAssessment of psychological distress. The Arabic version of the Depression, Anxiety and Stress Scale (DASS-21)  was usedAssessment of eating behaviors. The Three-Factor Eating Questionnaire (TFEQ-R18)  was used−1) were calculated as MET intensity × duration × frequency per week [−1) = 3.3 × walking minutes × walking days; moderate (MET·min·wk−1) = 4.0 × moderate-intensity activity minutes × moderate days; vigorous (MET·min·wk−1) = 8.0 × vigorous-intensity activity minutes × vigorous-intensity days . The weighted MET-minutes per week were summed across three activity categories to produce a weighted estimate of total physical activity from all reported activities per week (MET·min·wk−1) [Assessment of physical activity. The short version of the International Physical Activity Questionnaire (IPAQ-SF)  was usedper week : walkingin·wk−1) . The Arain·wk−1) ,33.t-test and ANOVA test were used when comparing two and three or more means. Effect sizes were calculated for each association; in terms of effect size/correlation, coefficient values of │0.1–0.23│, │0.24–0.36│, and >│0.37│ indicated small, moderate, and large effect sizes/correlations, respectively [p < 0.05.Statistical package for the Social Sciences (SPSS) v.25 was used for the data analysis. The normality of distribution of the mYFAS score was confirmed via a calculation of the skewness and kurtosis; values for asymmetry and kurtosis between -1 and +1 are considered acceptable in order to prove normal univariate distribution . These cMBMI = 30.35 ± 5.36 kg/m2). The mean age of the participants was 29.86 ± 13.32 years. The sociodemographic characteristics of the participants as well as their scores in food addiction (assessed using mYFAS), psychological distress (using DASS-21), eating behaviors (using TFEQ-R18) and physical activity (using IPAQ-SF) are summarized in n = 128).The total sample was composed of 507 individuals with class I obesity  compared to those belonging to cluster 1  and 3   .Moreover, higher psychological distress  and maladaptive eating behaviors  were significantly associated with a higher food addiction score. Whereas, higher levels of physical activity were significantly associated with a lower food addiction score .The results of the linear regression , Model 1The results of the linear regression , Model 2The present study explored the typology of Lebanese adults with obesity based on psychological and behavioral patterns in regard to food addiction. In our study, three clusters emerged, consisting of “psychological well-being, appropriate physical activity levels and eating behaviors” (cluster 1), “psychological well-being and cognitive restraint” (cluster 2) and “moderate psychological distress and cognitive restraint” (cluster 3). Our findings showed that food addiction score was higher in individuals belonging to the cluster with lower levels of depression, anxiety and stress and higher levels of restrictive dietary intake to control weight (cluster 2) compared with the other two clusters. It is worth pointing out that, in a recent review  aimed toGiven the association of clusters 2 and 3 with food addiction, the presence of cognitive restraint might make a difference, more than the psychological well-being/moderate psychological distress. Restraint measures an individual’s cognitive effort to control their food intake to manage their body weight , and to Our results are consistent with the previous studies, which suggested that food addiction (both diagnosis and symptom score) was positively associated with both emotional and external eating ,16,19. AOur findings showed that a higher level of physical activity was significantly associated with lower food addiction score in individuals with obesity. Previous findings showed that participants reporting a high level of physical activity displayed more symptoms of food addictive behavior than those with low and moderate physical activity . ContrarHealthcare providers may gain insight from the findings of our study and help foster appropriate and effective programs to prevent excess weight problems among adults . For example, underlying problems  may be tackled by the individuals through assistance from healthcare providers. In addition, it can be thought that performing awareness raising and encouraging studies in terms of the importance of healthy eating and regular exercise habits in the fight against obesity will decrease the incidence of obesity. Furthermore, food addiction is both an individual and collective health problem, and should be addressed at the societal level with broad policy interventions.Some limitations of our study need to be mentioned. In the present study, we used the mYFAS—a substitute for the YFAS (the most widely used measure of food addiction) . Thus, pOur findings showed that the prevalence of food addiction according to the mYFAS criteria was 25.2% in Lebanese individuals with obesity. Our study has revealed that food addiction is significantly associated with higher psychological distress and maladaptive eating behaviors, while higher levels of physical activity were related to low levels of food addiction. Higher levels of uncontrolled eating, emotional eating and stress, as well as belonging to the cluster named “psychological well-being and cognitive restraint” and the cluster named “moderate psychological distress and cognitive restraint”, were found to be predictors of food addiction in individuals with obesity in the present study. This knowledge could be useful concerning the psychological treatment of obesity and addictive-like eating behavior.To sum up, our study provides insight into psychological and behavioral patterns of individuals with obesity concerning food addiction using a cluster analysis. These findings would allow the inclusion of psychological treatment among the cornerstones of obesity treatment, thus achieving a multidisciplinary approach in accordance to the multifactorial association of the obesity.


Fermented feed has been of wide concern in livestock and poultry production because of its many advantages. In this study, the nutritional quality of the feed before and after fermentation was assessed, and four supplemental levels of fermented feed were used to replace unfermented feed to study the influence of fermented feed on the gut health of the laying hens during the laying peak period. The results suggest that fermented feed can improve the intestinal morphology and barrier functions of laying hens, possibly by altering the cecal microbiome.p < 0.05). Moreover, fermentation in the feed significantly increased the crude protein content (p < 0.05). Supplementation with fermented feed significantly reduced the crypt depth and increased the villi height:crypt depth ratio of duodenum and jejunum (p < 0.05). Meanwhile, fermented feed increased the secretory immunoglobulin A content and MUC2 mRNA expression of jejunum (p < 0.05). These beneficial effects were exhibited at the addition level ≥6% and microbial composition of caeca in the control, and so 6% fermented feed groups were analyzed. The structure of the gut microbiota was remarkably altered by additions, characterized by increased abundances of some health-promoting bacteria, such as Parasutterella, Butyricicoccus and Erysipelotrichaceae (p < 0.05). In summary, fermented mixed feed modulated cecal flora, subsequently contributing to improvements in intestinal morphology and barrier functions in laying hens.This study aimed to evaluate the effects of fermented corn–soybean meal mixed feed on intestinal barrier function and cecal microbiota in laying hens. A total of 360 Jingfen No.6 laying hens (22 wk-old) were assigned to 4 dietary treatments, which were offered basal diets (without antibiotics) containing 0, 4, 6 and 8% of fermented mixed feed respectively. The results showed that the pH value and anti-nutritional factor concentrations in fermented mixed feed were lower than those in unfermented feed ( To achieve resistance-free breeding, a growing number of studies has been conducted to evaluate non-antibiotic alternatives and their effects on the health and production performance of animals . FermentGut integrity and function are essential factors in maintaining animal health and production performance. Intestinal morphology is an important criterion for evaluating intestinal health of animals. Villus height and crypt depth were related to nutrient digestion and poultry performance . The comIn China, fermented feed mainly refers to the fermented single feed material, such as soybean meal , rapeseeBacillus 2 × 109 CFU/g, Lactobacillus 3 × 109 CFU/g, Saccharomyces cerevisiae 5 × 108 CFU/g. The probiotic powder was dissolved in 1 L sterile water at 37 °C, and stirred evenly to make a probiotic solution. The basal substrate (12 kg) included 60% corn, 20% soybean meal and 20% wheat bran, which was mixed and inoculated in probiotics with probiotic solution. The fermentation process according to the method of Shi et al. [The probiotic purchased from Baide Biotechnology Co., Ltd. , was a lyophilized powder containing i et al. , with apn = 3) were tested to determine their crude fiber , crude protein  and ether extract  contents according to AOAC International guidelines [g for 5 min, the supernatant pH was tested using a probe style-pH meter . Protein extraction and SDS-polyacrylamide gel electrophoresis (PAGE) of the feed samples were performed according to the method described in reference [Fermented and unfermented feed were collected and dried at 65 °C for 48 h. Feed samples . A total of 360 Jingfen No.6 layers aged 22 weeks were randomly assigned to four numerically equal groups, with 6 replicates per treatment and 15 birds per replicate. The layers had a similar body weight and good health. The feeding period lasted 10 weeks, commencing when the layers were 25 weeks of age and ending when they were 35 weeks of age, with an addition of 3 weeks for feed adaptation. During the experiment, three laying hens were kept in each cage and had ad libitum access to feed and water. Proper indoor temperature (15~22 °C) and humidity (30%~50%) were maintained.According to the NRC (1994) layer feeding standard, the corn–soybean meal basal diet (antibiotic-free) was designed based on the actual situation of layer feeding in the experimental chicken farm. Unfermented corn, soybean meal, and wheat bran were replaced in the basic diet with increasing levels of fermented mixed feed . The ingredients were mixed in a mixer for 15 min. Feed composition and nutrient content of the experimental diets are shown in At 35 wk, 24 chickens (one with an average body weight from each replicate pen) were selected to collect samples. All birds were killed by cervical dislocation and small intestinal  mucosa was scraped off at the forepart of individual small intestinal segments with a glass microscope slide on ice and frozen and stored immediately at −80 °C liquid nitrogen tanks for further analysis. About 3 cm of the mid-portion of the small intestines were excised carefully and washed in saline solution before storing in formalin (10%). The cecal contents were gathered using sterile spatulas into sterile plastic tubes, immediately refrigerated (maximum 2 h) and stored at −80 °C refrigeration until the DNA extraction. Three-centimeter lengths from the medial portions of duodenum, jejunum and ileum from chickens were fixed with 10% formalin for 24 h. Tissues were later embedded in paraffin wax blocks, mounted onto glass slides, and then stained with Haematoxylin & Eosin (H&E). The fixed segments were sectioned and observed under the light microscope, and the villus height VD, crypt depth CD and the ratio of VD and CD evaluated.Secretory immunoglobulin A (sIgA) content of mucous membrane samples of the duodenum, jejunum, and ileum were measured by ELISA kits  according to the kit instructions. Total RNA was extracted from the snap-frozen jejunal tissue samples with a RNeasy mini kit , following the instructions. Complementary DNA (cDNA) was synthesized from 1 μg of total RNA using MMLV reverse transcriptase . Then, the transcriptional changes were identified by quantitative PCR using Premix Ex TaqTM with SYBR Green  and the Bio-Rad CFX 96™ Real-Time Detection System . The thermocycle protocol lasted for 30 s at 94 °C, followed by 40 cycles of 5 s denaturation at 94 °C, 34 s annealing/extension at 60 °C, and then a final melting curve analysis to monitor the purity of the PCR product. The 2−ΔΔCt method was used to estimate mRNA abundance . Relative gene expression levels were normalized using β-actin as an internal control. The primers were synthesized by the Xi’an Qingke Biological Company, and the sequences are shown in ® High-Fidelity PCR Master Mix  and primers F341 and R806. The 30 μL reaction system was used in PCR reactions with 15 μL Mix, 0.2 M of forward and reverse primers, and about 10 ng template DNA. Thermal cycling consisted of initial denaturation at 98 °C for 1 min, followed by 30 cycles of denaturation at 98 °C for 10 s, annealing at 50 °C for 30 s, elongation at 72 °C for 30 s and a final elongation at 72 °C for 5 min. The amplicons were examined using 2% agarose gel electrophoresis, and then the GeneJET Gel Extraction Kit was used  to purify the excised target fragments. Finally, 16S rRNA gene sequencing was performed using the Illumina NovaSeq 6000 PE250  with the MiSeq Reagent Kit from Novogene Bioinformatics Technology Co., Ltd., Beijing, China.Total genomic DNA was extracted from samples using the CTAB/SDS method and then stored at −80 °C until sequencing analysis. The V3 + V4 fragments of the 16S rRNA gene were amplified using the Phusionhttp://qiime.org/index.html). Data regarding alpha diversity indices, intestinal morphology, total sIgA concentration and relative mRNA expression levels among all treatments in this study were analyzed by one-way ANOVA with SPSS 22.0. Differences among groups means were determined by Duncan’s multiple comparison test. The significance of differentiations in microbial structure among groups was assessed by ANOSIM using R package “vegan”. t-test was used to compare the chemical composition of feed and cecal microflora at the phylum and genus level. The correlations between the cecal microbial composition and gut health were assessed by Spearman’s correlation analysis using GraphPad Prism version 8.00. p < 0.05 was considered statistically significant and 0.05 < p ≤ 0.10 a trend.Alpha diversity (Shannon and Simpson indices) and abundance (Chao1 and ACE indices) were analyzed withQiime v.1.7.1. , crypt depth (CD) and the villi height:crypt depth ratio (VH/CD) are displayed in Secretory IgA (sIgA) acts as the first immune defense for intestinal epithelium and maintains the homeostasis of the gut. Therefore, we assessed the intestinal immune function by measuring the intestinal sIgA contents. As shown in p > 0.05; p < 0.05) to the control group and were larger than those of the inner-group (R > 0.05). Hence, to illuminate how fermented mixed feed served to improve the gut health of laying hens, the 6% group was selected as the representative group among the 3 treated groups to identify the roles of fermented mixed feed in regulating the cecal microbiota.As the microbial community plays an important role in the intestinal health and barrier function of laying hens, the composition of the cecal microbial community was analyzed using the 16S rRNA gene amplicon sequence. After filtering, an average of 60,788 ± 6481 reads were obtained for each sample. In the 16S amplicon data analysis, the Shannon and Simpson indices were used to assess the community diversity, and the ACE and Chao1 indices reflected the community richness. Therefore, alpha diversity was measured using these four indices, and there were no significant differences in cecal feces microbiome taxon abundance and diversity  on the phylum and genus  and Enterococcus were significantly decreased in the 6% fermentation group when compared with the 0% control group (p < 0.05).Since pairwise comparisons (0 and 6%) and the data were in accordance with normal distribution, nd genus . Compareunidentified_Lachnospiraceae, unidentified_Spirochaetaceae, Barnesiella Helicobacter, Parasutterella and Synergistes. In contrast, the intestinal morphology was negatively correlated with Bacteroides, Megamonas, Desulfovibrio, Alistipes, Tyzzerella, Fournierella Succinatimonas, Gallibacterium and Elusimicrobium significantly. In addition, the abundance of genus Faecalibacterium and Desulfovibrio were negatively correlated with the jejunal sIgA contents, and the abundance of genus Sutterella and Lachnoclostridium were negatively correlated with the duodenal sIgA content. Moreover, Fournierella was positively correlated with ileal sIgA contents. In order to explore the specific bacteria related to gut health, the Spearman correlation coefficient  was usedThe corn–soybean meal diet is the most widely applied in poultry production in China. However, ordinary corn–soybean meal diets contain some anti-nutritional factors, for instance, phytic acid, soybean antigenic protein and soy oligosaccharides, which may compromise the nutrient bioavailability and exhibit negative effects on animal health . Solid-sAmong a variety of parameters, pH is an important reference to estimate the quality of fermented feed, because lower pH may favor the digestion of intestinal nutrients and inhibit the growth of pathogenic microorganisms in feed . Our resThis study found that fermented mixed feed could partially improve the intestinal morphology of laying hens. This was consistent with the past study of Missotten et al. , who notThe gut barrier can prevent the intestinal tract from the colonization of pathogens, and sIgA is the critical component of the immune barrier, limiting epithelial contact with pathogens and other antigens . In thisParasutterella, Butyricicoccus, unidentified_Erysipelotrichaceae and Mailhella were identified as the main microbes with increased abundances in response to the addition of fermented feed. The increased proportion of Parasutterella has been reported to be beneficial to intestinal mucosal homeostasis [Parasutterella abundance and villus height VH and VH/CD ratio. Butyricicoccus, as butyrate producers, are assumed to improve growth performance, inhibit the proliferation of pathogens and relieve intestinal inflammation in broilers [Erysipelotrichaceae may be associated with the degradation of feed ingredients and the production of short-chain fatty acids [Alloprevotella, Gallibacterium, Romboutsia and Enterococcus were observed in 6% fermented feed group compared to the control. Gallibacterium has been recognized as a main cause of peritonitis and salpingitis in laying hens [Alloprevotella is known as an opportunistic pathogen; however, previous studies have noted that the increased abundance of Alloprevotella genera was linked to better intestinal health [Romboutsia is a valuable intestinal biomarker maintaining host health, and Enterococcus with natural antimicrobial probiotic properties could prevent diarrhea in animal production [Gallibacterium is directly associated with poultry intestinal disease, which was also confirmed in this study by a negative relationship between abundance and villus height [To further explore the underlying mechanism of its modulation on gut health, we have used Illumina MiSeq sequencing to analyze the cecal microbiota. The present study revealed no significant effect on the α-diversity of the cecal microbiota, whereas the β-diversity analysis showed significant clustering between the 6% and 8% groups and controls, indicating that the cecal microorganism community profiles in the 6% and 8% fermented groups could be altered following fermented feed addition. This was in line with the results on the intestinal morphology and the barrier-related gene expression, indicating that the 6% and 8% fermented feed could significantly improve intestinal health with no remarkable difference between the two groups. It was possible that fermented mixed feed at levels above a certain threshold (which was 6% in this study) could result in changes of the gut microflora and subsequently improve intestinal morphology and barrier functions. Therefore, control and 6% groups were selected for further analysis. At the genus level, eostasis . Similarbroilers . Erysipety acids . On the ing hens , which ll health . Besidesoduction . Howevers height . TherefoIn conclusion, fermented mixed feed could improve the morphology and barrier functions of the intestine, and alter the cecal microflora. This study demonstrated that fermented mixed feed could be used as a novel feed ingredient for laying hens, and that their favorable effects could be exhibited at addition levels of ≥6%. The specific mechanism of fermented feed-changing of cecal microflora of laying hens needs further study.


Bacteria are vital components of lake systems, driving a variety of biogeochemical cycles and ecosystem services. Bacterial communities have been shown to have a skewed distribution with a few abundant species and a large number of rare species. The contribution of environmental processes or geographic distance in structuring these components is uncertain. The discrete nature of lakes provides an ideal test case to investigate microbial biogeographical patterns. In the present study, we used 16S rRNA gene metabarcoding to examine the distribution patterns on local and regional scales of abundant and rare planktonic bacteria across 167 New Zealand lakes covering broad environmental gradients. Only a few amplicon sequence variants (ASVs) were abundant with a higher proportion of rare ASVs. The proportion of locally abundant ASVs was negatively correlated with the percentage of high productivity grassland in the catchment and positively with altitude. Regionally rare ASVs had a restricted distribution and were only found in one or a few lakes. In general, regionally abundant ASVs had higher occupancy rates, although there were some with restricted occupancy. Environmental processes made a higher contribution to structuring the regionally abundant community, while geographic distances were more important for regionally rare ASVs. A better understanding of the processes structuring the abundance and distribution of bacterial communities within lakes will assist in understand microbial biogeography and in predicting how these communities might shift with environmental change. Many ecological studies have overarching goals to understand the biodiversity and spatial distribution (biogeography) of organisms and to determine the drivers underlying these patterns. Microbes are ubiquitous and amongst the most abundant organisms on Earth, playing vital roles in biogeochemical and metabolic processes . The disIt has been postulated that local environmental processes and geographic distances  factors are potential mechanisms in structuring microbial biogeography . EnvironWith the development of molecular techniques such as high throughput sequencing, the composition of microbial communities is increasingly being investigated. A common feature in microbial biospheres is a skewed distribution of species abundance with communities composed of a small number of species that are highly abundant, and a large number of rare species  which isThe biogeography of regionally abundant and rare microbial taxa in lakes has been investigated with differing results. In this study, we used DNA metabarcoding to explore the distribution of planktonic bacteria in lake surface waters, termed bacterioplankton, at a national scale in 167 New Zealand lakes encompassing broad environmental gradients. We hypothesized that (i) there would be few abundant Amplicon Sequence Variants (ASVs) with rare ASVs accounting for the majority of the observed richness on both a local and regional scale; (ii) environmental variables would be different for structuring the composition of locally abundant and rare ASVs; (iii) the taxonomic makeup of regionally abundant and rare ASVs would differ; (iv) regionally abundant ASVs would have high occupancy across the lakes with rare ASVs having a restricted occupancy; and (v) environmental processes would be strong drivers of the composition of regionally abundant communities, while geographic distance would contribute more in rare communities.A total of 167 lakes, which were not hydrologically connected, were sampled across New Zealand between 23 January 2019 and 15 February 2021 . Lakes rin situ at each lake. Satellite imagery available in the Land Cover Database Version 5 1 was used to derive seven broad land cover variables: (1) Native vegetation, (2) Urban, (3) Non-native vegetation, (4) Forestry, (5) High Production Grassland , (6) Low Production Grassland , and (7) Other. Specific land cover variables for each category are detailed in Altitude and lake depth were assessed 3 Multi-Channel Logger .Prior to sampling, a side scan sonar survey  or depth sounder  was used to find the deepest part of the lake. Subsequent sampling was undertaken at this site except for lakes deeper than 100 m where sampling was undertaken in shallower bays . Secchi a was collected at the deepest point in the lake and kept on ice until further processing. A subsample (up to 600 mL) was filtered , and the volume filtered was recorded. Filters were placed in aluminum foil and stored in the dark (−20°C). Chlorophyll a analysis was undertaken following the APHA 10200 H method at Watercare Laboratories  with a reporting limit of 0.0006 mg L–1.Surface water (1 L) for chlorophyll 3 (Water samples (1 L) were collected using an integrated tube sampler through the surface mixed layer as determined by the RBR profile. Subsamples (40 mL) were filtered (low bleed 0.45 μm) for dissolved organic carbon DOC; . Analysi3 .Surface water samples (500 mL) were collected in triplicate. For each triplicate, a subsample (up to 100 mL) of water was filtered . The volume filtered was recorded, and filters were stored in sterile tubes (1.5 mL) at −20°C until DNA extraction. All filtering apparatus were bleached between lakes.Molecular analyses  were conducted in dedicated sterile separate laboratories, with sequential workflow to reduce potential cross-contamination. UV sterilization was undertaken for 15 min before and after each procedure in the DNA extraction, amplification set-up, and template addition laboratory. The PCR set-up and template addition were undertaken in laminar flow cabinets with HEPA filtration. Aerosol barrier tips  were used throughout.Bacterial filters were cut into five pieces using sterile scissors in a laminar flow cabinet and placed into the power bead tube of a DNeasy PowerSoil Kit . DNA was extracted following the manufacturer’s instructions on a QIAcube sample preparation robot (Qiagen). A negative extraction control was included every 23 samples.via PCR in 20 μL volumes performed in triplicate as described by –1 which was sequenced on an Illumina Miseq™ platform at the Auckland Genomics Facility. Library preparation was undertaken following the Illumina 16S metagenomics library prep manual. The only exception was that after the indexing PCR, 5 μL of each sample was pooled, and a single clean-up was undertaken on the pool instead of samples being individually cleaned. The library contained extraction, PCR, and sequencing controls. A bioanalyzer was used to assess library pool quality and concentration. The library was diluted to 4 nM, denatured, and diluted to a final loading concentration of 7 ρM with a 15% PhiX spike. Raw reads are stored in the NCBI short read archive under the accession: PRJNA762383.The V3-V4 regions of the bacterial 16S rRNA gene were amplified using the primers: 341F: 5’-CCT ACG GGN GGC WGC AG-3’ and 805R: 5’-GAC TAC HVG GGT ATC TAA TCC-3’ . The priDADA2 package (8 bp of sequences were used to construct a parametric error matrix which was used to infer amplicon sequence variants (ASVs) after dereplication. Singletons were removed, and remaining paired-end reads were merged with a maximum mismatch of 1 bp and a required minimum overlap of 10 bp. Chimeric sequences were detected and removed using removeBimeraDenovo within the DADA2 package. The multiple sequencing runs were combined together before taxonomy was undertaken on the whole dataset. Taxonomy was assigned against the SILVA 138 reference database . These variables were scaled and centered using the R package caret  in caret.There were 16 measured environmental variables  available for this dataset . These wge caret . MissingThe number of observed ASVs per lake was calculated, and a Kruskal-Wallis test was used to explore differences in the number of ASVs based between the North and South Islands of New Zealand.Locally abundant ASVs were classified as having a relative abundance within a lake of >1%, while locally rare ASVs had a relative abundance of <0.01% when present in the lake.MASS  were used to assess the impact of environmental variables on total community richness and proportion of reads of locally abundant or rare ASVs in a lake using the R package MASS . A step phyloseq package.Distance Based Redundancy Analysis dbRDA;  was usedTo assess abundance patterns on a regional scale, ASVs were classified into three groups based on average relative abundance across all 167 lakes  were calculated between the three ASV Bray Curtis dissimilarity matrices  and environmental and geographical distances  within tage in R . Geograpeosphere . FourteeIn total, there were 14,763 ASVs across the 167 lakes after rarefaction and removal of ASVs that were not in at least two replicates. When the data from all lakes were combined, bacterioplankton were predominantly comprised of Gammaproteobacteria (mean 20.9 ± 12.3%), Actinobacteria (20.9 ± 10.8%), Bacteroidia (14.8 ± 8.8%), Alphaproteobacteria (12.3 ± 7.9%), and Verrucomicrobiae (9.7 ± 6.4%) . Cyanobap < 0.0003), with the South Island having a lower richness than the North Island (mean 288 ± 143 compared to 381 ± 218 ASVs).On average, 333 ASVs  were identified within each lake. There was a significant difference in the number of ASVs per lake between the North and South Islands  that had a relative abundance of >1% within a lake . There were on average 21.4 ± 4.6 locally abundant ASVs per lake. There were 6,567 ASVs (44.5%) that had a relative abundance when present of <0.01% within a lake . There were on average 65.3 ± 80.7 locally rare ASVs per lake. Locally abundant ASVs accounted for 63.9 ± 11.2% of the reads within a lake while the locally rare ASVs accounted for 0.36 ± 0.42% of reads .p = 0.012) and lake depth (p < 0.001), while a significant positive partial effect was observed for Secchi disk depth (p < 0.001) and temperature  . The genp < 0.001) in the abundant fraction, while Gammaproteobacteria, Alphaproteobacteria, Planctomycetes, and Parcubacteria were more prominent in the rare fraction (paired Wilcox test: p < 0.001).There were significant differences in classes that made up the regionally abundant and rare fraction of the community . In totaNo ASVs were shared amongst all lakes, with only five ASVs (0.03%) found in more than 75% of lakes. These ASVs belonged to Sporichthyaceae (Actinobacteria = three ASVs), Methylophilaceae (Gammaproteobacteria), and Comamonadaceae (Gammaproteobacteria). The majority  of ASVs were found in <10% of lakes, with 7,903 ASVs (52.5%) found only in a single lake. There were 191 regionally abundant ASVs and 10,212 rare ASVs. The remaining 4,377 ASVs were classified as intermediate .Occupancy of regionally rare ASVs was restricted with no rare ASVs found in more than 10% of lakes. For the regionally intermediate ASVs, the majority were found in <50% of the lakes, with only one ASV found in more than 50% of lakes. Of the regionally abundant ASVs, 16% (30 ASVs) were present in more than half the sites with a maximum occupancy of ∼90% of lakes. These 30 ASVs represented 5 classes and 12 families. The most frequent taxonomic classification of these widely distributed abundant ASVs belonged to the family Sporichthyaceae  with a further 5 ASVs belonging to the family Comamonadaceae. However, there were also abundant ASVs that had restricted occupancies, with ∼10% (18 ASVs) of abundant ASVs having an occupancy of <5% of lakes. These ASVs belonged to 12 families within 7 classes.Mantel and partial Mantel tests using all ASVs showed that environmental processes had a stronger relationship with total bacterioplankton community composition compared to geographic distance especially when using the partial mantel tests . A similThe bacterioplankton community of New Zealand’s lakes are dominated by Gammaproteobacteria and Actinobacteria, a pattern that has been observed in other lake systems globally .As we hypothesized and in agreement with other microbial studies, there was a skewed distribution to the bacterioplankton community with a low number of locally abundant ASVs and a higher proportion of rare ASVs . The meaCyanobium showed an association with lower latitudes and with increasing amounts of dissolved organic carbon which is often related to lake eutrophication, typical of lowland lakes in parts of the North Island. Previous work by Despite the low proportion of variation in community composition explained, some associations were present between the measured environmental variables and locally abundant ASVs aggregated at the genus level. The picocyanobacterial genus The locally abundant ASVs accounted for a lower proportion of the community in shallow lakes with a higher percentage of high productivity grassland in the catchment with low Secchi disk depths. In contrast, the proportion of reads accounted for by rare ASVs increased in lakes with low Secchi disk depths, which can be typical of nutrient enriched lakes. There was also a positive association of high productivity grassland in the catchment, which is often related to lake nutrient enrichment due to fertilizer use, with the overall ASV richness in a lake. Previous studies have shown that a higher diversity is observed in more eutrophic lakes  and haveMicrobial communities in a variety of habitats, including lakes, have been shown to have few regionally abundant taxa with most diversity being rare and of low relative abundance . The bacThe taxonomic composition of regionally abundant and rare communities varied, as has been previously shown in other lake systems . The phyDistribution patterns also differ between regionally abundant and rare components of the bacterioplankton. Rare ASVs had a restricted occupancy with the abundant ASVs, in general, having a broader distribution within lakes. These results are similar to other studies focusing on lake bacteria , althougTaxa that were present in the regionally abundant component did not all have high occupancy. For example, Cyanobacteria were predominantly in the regionally abundant component but had a more restricted distribution with no Cyanobacteria ASVs present in more than 50% of the lakes. This restricted distribution but high average abundance may suggest that cyanobacteria could be classified as conditionally rare taxa . These tLimnohabitans which are often abundant in freshwater environments  can be found below: JP, SAW, JH, and MV contributed to the conception and design of the study. GT-L, JT-L, LT, LR, and SW undertook the laboratory work. JP undertook the analysis of the data. JP and SAW wrote the manuscript. All authors helped with the field sampling and involved in revision and approval of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.