The platelets count was lower in SIP than in NEC infants (p < 0.001). Glucose levels strongly correlated with lactate levels (p = 0.01) only in the NEC group. The odds of being diagnosed with SIP decreased as lactate levels increased . Our results suggest that a combination of laboratory markers, namely glucose and lactate, could help differentiate SIP from NEC at early stages so that, in the presence of an elevated blood glucose, an increase in blood lactate was associated with a decrease in the odds of being diagnosed with SIP.The incidence of spontaneous intestinal perforation (SIP) increases up to 10% with decreasing gestational age (GA). We aimed to explore early biomarkers for predicting SIP in preterm infants. In this case–control study, neonates born at ≤34 weeks GA diagnosed with SIP were compared with GA and/or birth-weight-matched neonates diagnosed with necrotizing enterocolitis (NEC). Laboratory markers assessed prior and adjacent to the day of SIP or NEC diagnosis were evaluated. The cohort included 16 SIP and 16 matched NEC infants. Hyperlactatemia was less frequent in SIP than in NEC infants (12% vs. 50%,  Spontaneous intestinal perforation (SIP) of the preterm infant is defined as an isolated intestinal perforation typically found at the terminal ileum ,2. SIP oYet, there is a paucity of data on laboratory markers for predicting SIP. A large prospective multicenter study found only a moderate ability to distinguish the isolated intestinal perforation from NEC entities pre-operatively . MoreoveThis is a retrospective case–control study of neonates, born at ≤34 weeks gestational age (GA) diagnosed with SIP or NEC, between 1 January 2010 and 31 March 2021, at the Lis Maternity Hospital of the Tel Aviv Medical Center and admitted to the Neonatal Intensive Care Unit of this tertiary referral hospital. SIP infants were matched, in a 1:1 ratio, with medical or surgical NEC infants, by GA (GA ± 2 weeks) and/or birth weight (BW ± 15%). We excluded infants at GA > 34 weeks and infants with an intestinal pathology other than NEC or SIP. The study was approved by the local institutional review board of the Tel Aviv Medical Center (0028-21-TLV). Informed consent was waived due to the retrospective nature of the study.A single investigator (J.H.) reviewed the extracted hospital medical records using the “intestinal perforation” keyword, to confirm infants’ prematurity and diagnosis of either SIP or NEC. NEC cases were defined by modified Bell’s criteria of stage 2 or greater with radiographic evidence of pneumatosis intestinalis . Physicap values were 2-sided and p < 0.05 was considered statistically significant.IBM SPSS Statistics for Windows, version 25, was used for statistical data analyses. Categorical variables were reported as frequencies and percentages. Normality of continuous variables was assessed by Shapiro–Wilk tests. Data were expressed as mean ± standard deviation (SD) and range for normally distributed variables and median and interquartile range (IQR) for skewed distributions. Chi square tests or Fisher’s exact tests were applied to compare categorical variables and the Mann–Whitney U test to compare continuous variables, between the SIP and the NEC groups. Pearson’s correlation coefficient was used to evaluate the relationship between the assessed variables. Adjusted odds ratios (ORs) were calculated with 95% confidence intervals (CIs) for the association of laboratory parameters  with the diagnosis of SIP, in logistic regression. All As depicted in n = 9), primary closure (n = 3) or drainage (n = 4) as opposed to NEC infants (3 stoma and 1 explorative laparotomy) (p < 0.001). The majority of NEC infants (75%) were managed expectantly. Five neonates died in the SIP group, all at GA ≤ 28 week, two of them shortly after diagnosis and surgical treatment (drainage or stoma), the other three due to complications of prematurity. In the NEC group, three out of the four deaths occurred following surgical treatment. Platelets count  was lower in the SIP group (130.5 [97.2–184.8]) than in the NEC group (347.5 [186.8–510.2]), (p < 0.001). Hence, the incidence of thrombocytopenia (platelet count < 150 K/µL) was significantly higher in the SIP group (56.3%) than in the NEC group (12.5%), (p = 0.009). Median nucleated red blood cells (NRBC) count was higher in SIP infants than in NEC, but did not reach significance. Hyperglycemia (Glucose level > 120 mg/dL) was present in most of the SIP (88%) and NEC (81%) infants, whereas hyperlactatemia (Lactate level > 4 mmol/L) was less frequent in the SIP group than in the NEC group . However, recorded blood lactate levels were not statistically different between the groups (p = 0.109). SIP was diagnosed within the first days of life as opposed to NEC .Descriptive characteristics of mothers and infants in the SIP and NEC groups are presented in r = 0.612; 95% CI: 0.168–0.850, p = 0.012) and an absence of correlation in the SIP group  where only glucose levels increased (2(3) = 21.175, p < 0.001. The model explained 64.5% of the variance in SIP diagnosis and correctly classified 78.1% of cases. Every one-unit increase in lactate was associated with a 39% decrease in the odds of being diagnosed with SIP, and every 1 K/µL increase in platelet was associated with a 2% decrease in the odds of being diagnosed with SIP . NEC can be lethal in VLBW and ELBW infants. There are continuing improvements in neonatal care and surgical outcomes for abdominal pathologies. Early detection is critical to that process. NEC and SIP might have overlapping clinical signs at their initial presentation ,10. SrinOur study showed that a combination of laboratory markers, namely glucose and lactate, could help differentiate SIP from NEC, at early stages, so in the presence of an elevated blood glucose, an increase in blood lactate was associated with a decrease in the odds of being diagnosed with SIP. The sudden increase in blood glucose in SIP infants occurred within the 24 h prior to perforation. Elevated blood lactate levels are usually a marker for tissue alteration . All SIPThe study has a few limitations. First, it is limited by its retrospective nature and therefore prone to sampling bias. Secondly, the study is limited by a small sample size of the SIP group collected over 10 years at our institution. In addition, the data extraction procedure has shown that there were some discrepancies between indicated diagnosis  and clinical reports . Finally, despite a limited available data set, a low matching ratio, and our belief that surgical NEC infants (with perforation) would be a more suitable control group in the comparison with SIP infants, this pilot study provides valuable insights to warrant further studies.Our results suggest that as opposed to NEC where lactate levels increased along with glucose levels, in SIP infants, prior to intestinal perforation as the onset of disease, hyperglycemia was not coupled to hyperlactatemia. We propose that a sudden hyperglycemia within the first week of life along with lactate levels within the normal range in VLBW and ELBW infants presenting with a distend belly can reveal a first clue on potential SIP development, similarly to what has been advocated by Swanson et al. .


Few multi-ethnic dietary culture-sensitive food frequency questionnaires (FFQ) have been developed due to the complexity and diversity of cooking methods and styles. This study aimed to develop and validate a specific FFQ among multi-ethnic groups in Northwest China.Han nationality or Ethnic minority).In the reliability study, 139 participants aged 20–65 completed two identical FFQs separated by 3 months. The relative validation of the FFQ was assessed by three 24-h recalls (24HR) employed in the interval of two FFQs, as a reference. Stratified analyses were also conducted by the major ethnic groups  of Spearman’s correlation coefficients (SCC) was 0.71 (0.43–0.84) for nutrients. The intra-class correlation coefficients (ICC) covered a spectrum from 0.39 to 0.78 (median: 0.64). Meanwhile, the weighted kappa values ranged from 0.11 to 0.64. For validity, the median (range) of Pearson’s correlation coefficients derived from the energy unadjusted and the adjusted values between FFQ and 24HR were 0.61 (0.12–0.79) and 0.56 (0.12–0.77), respectively. The results of correlation coefficients were similar between the two ethnic groups. Moreover, the Bland–Altman plots likewise demonstrated a satisfactory level of agreement between the two methods.The FFQ showed acceptable reproducibility and moderate relative validity for evaluating dietary intake among multi-ethnic groups in northwest China. It could be a credible nutritional screening tool for forthcoming epidemiological surveys of these populations.The online version contains supplementary material available at 10.1186/s41043-023-00452-9. Noncommunicable and chronic diseases (NCDs) are increasing in prevalence and now giving rise to a tremendous burden on the global healthcare system . DietaryHan, Uygur, and Kazak nationality . According to surveillance data across the Xinjiang multi-ethnic cohort study (XMC), the prevalence of NCDs substantially varies by ethnic group but is higher than the national average in 2018 [Xinjiang province locates in northwest China, as a representative area of multi-ethnic populations gathered, whose population structure is mainly composed of  in 2018 . Ethnica in 2018 , 15 base in 2018 . Further in 2018 . The repn = 5), diet change (n = 1), voluntarily withdrew (n = 1), and participants who had extreme total energy intake values ≤ 600 or ≥ 4500 kcal [n = 1; FFQ2, n = 3), and loss to follow-up (n = 2) in the diet administrations  who required strict dietary control, and all participants were older than 18 years and no more than 65 years old. A total of 152 participants were recruited from September 2021 to October 2022. Thirteen individuals were excluded for personal reasons including incomplete data . Secondly, the first FFQ (FFQ1) was performed by two trained interviewers face-to-face. The same participants were invited to participate in the second interview (FFQ2) approximately three months later at the selected location of their convenience. During the study period, all three datasets of the 24HRs were conducted over the course of three non-consecutive days (two weekdays and one weekend day) through a phone interview. The sequence of dietary assessment measurements is shown in Fig. Dietary intake was measured via the interviewer-administered FFQ and three 24-h recalls (24HR). The first interview lasted around 30–40 min conducted at the medical examination site and was divided into two parts. In the first step, demographic and anthropometric data were collected for all participants. Demographic and lifestyle variables of age, gender, ethnicity, smoking status, alcohol consumption, education levels, and monthly income were obtained by a structured questionnaire. Besides, anthropometric measurements were obtained from physical examinations. The height and weight were obtained from physical examinations, and body mass index (BMI) was calculated as a ratio of weight (kg) to the square of height (mt FFQ FFQ was perfn = 109) included in this FFQ  , 23. TheHan nationality or Ethnic minorities). Chi-squared test was used to compare the categorical variables, and parametric continuous variables of characteristics  were compared across groups with independent t tests.Firstly, the continuous variables with a normal distribution were described by the mean and standard deviation (SD) according to the Kolmogorov–Smirnov tests. Data with non-normal distribution were reported as median and the 25th/75th percentile . Analyses were stratified by ethnicity  because most values were not normal distributions. For reproducibility, differences in nutrients intake between the FFQ1 and FFQ2 were evaluated by Spearman’s correlation coefficient (SCC). The SCC of less than 0.20, 0.20–0.49, and more than 0.50 represent poor, acceptable, and good, respectively [, P75 becP-value ≤ 0.05 was considered statistically significant.To assess the validation, Pearson correlation coefficients were assessed for unadjusted and adjusted nutrients between the average intakes of FFQs and 24HRs, respectively. Before regression analysis for energy adjustment, all dietary intake data were log-transformed to accord with normal distribution. Using the residual method of Willett  to decren = 77) than Han groups (n = 62) were male (58.4% vs. 53.2%) and drinkers (46.8% vs. 40.3%). Additionally, there was no statistical difference between demographic characteristics  between Han and ethnic minority groups.There were a total of 152 patients who agreed to take part in this study. During the measurement, 8.6% of participants dropped out. Therefore, the final statistical sample consisted of 139 participants (61 women and 78 men), who completed two FFQs and three 24HRs were included in the following analysis. The baseline characteristics of the participants are shown in Table κw) was 0.39. All correlations were statistically significant (P < 0.05).The median intake of micronutrients and macronutrients derived from FFQ1 was approximately equal to those from FFQ2, which are presented in Table P < 0.05), except for iron and copper (P > 0.05).As shown in Table Han groups, for reliability, SCC ranged from 0.35 to 0.86 (median = 0.64). For validations, the Pearson correlation coefficients between the two methods were from 0.11 to 0.79 (median = 0.53). Among ethnic minority group, SCC ranged from 0.33 to 0.79 (median = 0.62), the Pearson correlation coefficients between FFQ and 24 HRs ranged from 0.12 to 0.78 (median = 0.57)  and fats (0.68). A possible factor was that native residents prefer high-calorie foods such as roast meat, beef, and mutton  due to tHan group (median: 0.53) tended to have a lower validity than the ethnic minority group (median: 0.57). Different from the explanation of education differences caused lower validation [Han groups tended to have more proportion of the higher education level across total groups. However, those who were well educated may be affected by greater social desirability bias [Among different ethnic groups, the lidation , Han groity bias . This miDue to the variety of local cuisines in China, especially in multi-ethnic settings of Xinjiang province, it is harder to estimate the portion size of dietary intake information precisely using FFQ than in other populations with single diet background . Cade etThis study has strengths and limitations that need to be described. To our knowledge, it was the first comprehensive validation study of dietary assessment among randomly assembled multi-ethnic adult populations in northwest China, setting the criteria for adults under 65 years of age, and the actual age distribution of the included population ranged from 20 to 65 years. Therefore, we believe that there is a certain representation of adults in the Xinjiang region. Our FFQ was adapted to include a comprehensive list of both traditional and novel foods, providing an accepted evaluation of relative dietary intakes. In summary, the results on the agreement and effectiveness of our FFQ were generally satisfactory and not inferior to other investigations among more homogeneous groups. Whereas, further validation should be conducted on bigger scales of populations derived from diverse multi-ethnic backgrounds.The study still has some limitations. Indeed, selection bias cannot be avoided in hospital-based studies. However, the study objects are random hospital physical examination population, most of which are similar to the normal population structure in the community, so the selection error is small, and the results may be representative of a wide range of people. Dietary sodium intake is low compared to other studies. Over- and underestimations in sodium intake have been similarly reported by various studies. On the one hand, there may be several errors related to the FFQ such as errors in individuals’ reports, different sodium content in food items, and daily alterations in diet . BesidesWillett  has recoIn this study, we developed an FFQ that provides a novel, comprehensive, and culture-sensitive method for dietary assessment. The result of acceptable reproducibility and moderate validity indicates that this FFQ is an appropriate tool for nutrition epidemiological exploration among the multi-ethnic populations in northwest China.Additional file 1. Table S1. List of main food items and food groups of FFQ. Table S2. Daily nutrient intakes and FFQ reproducibility and validity among Han group. Table S3. Daily nutrient intakes and FFQ reproducibility and validity among ethnic minority group.


Both BP and PF were similar (average of 0.48 mmol*g−1 of substrate) and lower than for TP, with CP having the lowest VFA concentration (0.21 mmol*g−1 of substrate) among all treatments. Additionally, when comparing molar concentrations, AP and TP (average of 0.0476 mmol*g−1 of substrate) had greater butyrate concentrations than did PF (0.0344 mmol*g−1 of substrate). The AP, BP, and TP treatments had both linear and quadratic relationships  for acetate, propionate, and butyrate concentrations across time. CP only demonstrated a linear relationship for propionate (P < 0.05), whereas acetate and butyrate had quadratic relationships with time. PF only demonstrated quadratic relationships between acetate, propionate, and butyrate concentrations and time (P < 0.05). Overall, the fiber substrates evaluated were marginally to moderately fermentable when incubated for up to 12 h with canine fecal inoculum.Many fiber ingredients are used by the pet food industry; however, little data are available regarding the fermentation characteristics of alternative fibers currently being used. The objectives of this study were to determine organic matter disappearance (OMD) and postbiotic production from various fruit and vegetable fiber sources using an in vitro dog fecal inoculum model. Apple pomace (AP), blueberry pomace (BP), cranberry pomace (CP), tomato pomace (TP), and pea fiber (PF) were used as experimental treatments. Inoculum was prepared using freshly voided feces under anaerobic conditions. Predigested fibers were inoculated and incubated for 1, 3, 6, and 12 h at 39 °C. Short-chain fatty acids (SCFA), branched-chain fatty acids (BCFA), total volatile fatty acids (VFA), and OMD were determined for each fiber source and time point in triplicate. After 12 h of incubation, OMD was similar  among treatments. Proportionally, acetate was greater for BP and AP  than for the other treatments (68.3% to 71.2%). Molar proportions of propionate was greatest (P < 0.05) for CP (26.8%) compared to the remaining treatments (13.6 to 20.7%). Butyrate was proportionally greater for PF  than for BP and CP (average of 4.8%) and was lowest for AP (3.8%); however, TP was not different from PF . Total VFA concentration was highest for AP (P < 0.05) followed by TP (1.17 and 0.75 mmol*g This work evaluated substrate degradation and postbiotic production of several alternative fiber ingredients that are currently used in the pet food industry. This data will provide valuable information to nutritionists and formulation scientists in the selection of cost-effective alternative fiber ingredients for the development of weight management and gut health diets. Under the U.S. Food and Drug Administration’s Code of Federal Regulations, dietary fiber is defined as “nondigestible soluble and insoluble carbohydrates (with three or more monomeric units), and lignin that are intrinsic and intact in plants; isolated or synthetic nondigestible carbohydrates (with three or more monomeric units) determined by FDA to have physiological effects that are beneficial to human health” . Fiber iFor the last few decades, the use of fruit and vegetable byproducts in extruded foods has become a growing area of research . The resA single source of apple pomace (AP), blueberry pomace (BP), cranberry pomace (CP), tomato pomace (TP), and pea fiber (PF) were obtained from a local mill  and used as experimental treatments. These fiber sources were selected based on their prevalence in diet formulations within the pet food industry but with limited reported data on potential benefit to gut health for companion animals. Prior to the in vitro protocols, fiber treatments were analyzed for dry matter , organic matter (AOAC 942.05), crude protein (AOAC 990.03), insoluble dietary fiber and total dietary fiber  according to AOAC Int’l approved analytical methodologies. Additionally, soluble fiber was calculated by the difference between TDF and insoluble fiber content. Crude protein of the residual samples remaining after the digestion procedure were also analyzed (AOAC 990.03) for the estimation of protein content entering fermentation.The medium composition and the anaerobic dilution solution were prepared according to To prepare treatments for inoculation, fiber sources underwent an in vitro digestion procedure using α-amylase, protease, and amyloglucosidase enzymes supplied from standard assay kit  to simulate small intestinal digestion adapted from the enzymatic TDF procedure described by 2 to maintain anaerobic conditions. Tubes were sealed with rubber stoppers equipped with one-way valves and allowed to hydrate overnight at 4 °C.Residual samples remaining after enzymatic digestion were ground through a 1 mm screen in a fixed blade laboratory mill . For each treatment, 300 ± 0.1 mg of predigested and ground substrate was placed into a 50-mL polypropylene centrifuge tube  in triplicate. Additionally, three blank tubes were used during each time point for correction of background postbiotic concentrations present in fecal inoculum. Twenty-six milliliters of media solution were added to all tubes while purged with COn = 12, neutered/spayed, average age 5.4 ± 0.3 yr old, average weight 12.7 ± 1.5 kg) were group-housed in indoor kennels located at the Large Animal Research Center of Kansas State University in Manhattan, KS. Dogs were fed a commercial maintenance diet twice a day  at 39 °C and was immediately inoculated into tubes to begin incubation.Beagle dogs were used as donors for the preparation of fecal inoculum in this study. Dogs  and 78% ethanol. When all sample volume had been filtered, residue was rinsed with two 10 mL volumes of 95% ethanol followed by two 10 mL volumes of acetone. After filtration, residues and filter paper were dried in a convection oven overnight at 105 °C (AOAC 930.15). Dry residue weight was recorded the following day and placed in muffle furnace at 450 °C overnight for determination of organic material (AOAC 942.05). Organic matter disappearance (OMD) was calculated using the following equation:Prior to fecal collection, tubes containing treatments and media were placed in a water bath at 39 °C for at least 1 h to equilibrate temperature to 39 °C prior to inoculation. All treatments were incubated at four timepoints: 1, 3, 6, and 12 h. Tubes were inoculated with 4 mL of fecal inoculum starting from the longest time point (12 h) and ending with the earliest time point (1 h). During inoculation, tubes remained under COwhere OM residue is the organic matter remaining in sample after fermentation and filtration in g, OM blank is the organic matter remaining in blank after fermentation and filtration in g, and initial OM is the organic matter of the raw sample in g.–1 g of substrate on a DM-basis. For the purpose of this work, acetate, propionate, butyrate, and valerate were collectively referred to as SCFA. The volatile iso-acids, isobutyrate and isovalerate, were referred to as BCFA. Total volatile fatty acid (VFA) refers to the sum of them all.The duplicate supernatant subsamples removed from each tube at the end of each time point were used for postbiotic analysis. Supernatants were extracted from gas chromatography vials and placed into a gas chromatograph  via direct liquid injection using a 10:1 split ratio with injection volume of 1 µL. The gas chromatography system used helium as a carrier gas with a flow rate of 3.5 mL/min and the volatile organic compounds were separated using a capillary gas chromatography column . A flame ionization detector was used for the determination of volatile compounds and was configured with nitrogen as the makeup gas with a flow rate of 25 mL/min to clarify peak resolution. Peak area of chromatograms was determined using integrative software . Short-chain fatty acids  and branched-chain fatty acids  were quantified by comparing the sample peak area to a known standard of 10 mM concentration . The mean value of the duplicate subsamples was used as concentration for each tube and concentrations were reported as mMP < 0.05.Data were analyzed as a completely randomized design with triplicate centrifuge tubes as experimental units . Treatment least squares means within each timepoint for OMD and postbiotic compounds were compared using the general linear model procedure from statistical analysis software . Pairwise treatment comparisons were conducted using Fisher’s Least Significance Difference to minimize type I error. Linear and quadratic trends across time within treatments were evaluated for concentrations of acetate, propionate, butyrate, and their sum using single degree of freedom orthogonal polynomial contrasts. Contrast co-efficients were estimated using the interactive matrix language procedure in SAS. For all statistical analyses, differences were considered significant at The nutrient composition of experimental fiber treatments is presented in P > 0.05) on OMD ; however, by the end of incubation (12 h), AP and BP had higher proportions of acetate  than the other treatments. Propionate was the second most abundant compound at all treatment and time point combinations and molar proportions differed (P < 0.05) among all treatments after 12 h of incubation. At the end of incubation, CP had the largest molar proportion of propionate (P < 0.05). Molar proportions of butyrate were smaller compared to acetate and propionate for each treatment and time point. After 12 h of incubation, PF had greater molar proportions of butyrate  than AP, BP, and CP, but was similar (P > 0.05) to that of TP (average 7.2%).After 12 h of incubation, there was no treatment effect (P < 0.05) for AP (1.1647 mmol*g–1 of substrate) followed by TP (0.7530 mmol*g–1 of substrate), BP and CP (average 0.4804 mmol*g–1 of substrate), and lowest for PF (0.2049 mmol*g–1 of substrate). Butyrate concentrations were greater (P < 0.05) for AP and TP (average of 0.0476 mmol*g–1 of substrate), followed by PF (0.0344 mmol*g–1 of substrate), BP (0.0249 mmol*g–1 of substrate), and lowest for CP (0.0093 mmol*g–1 of substrate). Branched-chain fatty acid concentrations were greater for TP  compared with the remaining treatments (ranging from –0.0101 to 0.0057 mmol*g–1). The AP, BP, and TP treatments had both linear and quadratic relationships (P < 0.05) for acetate, propionate, and butyrate concentrations across time. CP only demonstrated a linear relationship for propionate (P < 0.05), whereas acetate and butyrate had quadratic relationships with time. PF only demonstrated quadratic relationships between acetate, propionate, and butyrate concentrations and time (P < 0.05).Results for molar concentrations presented here will be exclusively focused on the 12 h time point. Total VFA concentrations were greatest , propionate was the only compound that demonstrated a linear relationship with time, whereas acetate, butyrate, and the sum of the three all demonstrated a quadratic relationship with time. tructure . Human ftructure .–1 of substrate, respectively). While the molar proportion of butyrate from PF was higher compared to other sources, AP and TP produced the highest molar concentrations of butyrate. Pea fibers contain large amounts of insoluble cellulose (Generally, PF had the largest proportion of butyrate during incubation; however, at 12 h pea fiber was not different from TP. Butyrate proportions were greater than that previously reported for PF 4.2%;  and pea ellulose , which iellulose  and may Total VFA concentration of TP was about 65% that of AP with most of the difference represented by acetate. TP had similar concentrations of butyrate to that of AP, but proportionally butyrate made a greater contribution to the total VFA pool. Thus, TP contributed both greater concentrations and relative proportions of butyrate compared to other treatment fibers. However, TP also produced the largest concentration of BCFA. The presence of BFCA is a direct indicator of protein fermentation, whereby isobutyrate and isovalerate are formed by the metabolism of the branched-chain amino acids valine and leucine, respectively . ProteinSome interesting and useful points of data were observed during this study; however, there were a few limitations identified here. First, for each fiber treatment, only a single sample was sourced for substrate preparation. A composite sample composed from multiple sources of the same ingredient would have provided a better representation of each ingredient and helped account for batch-to-batch variability. However, treatments that have been previously evaluated demonstrated relative consistency in terms of nutrient composition as well as fermentation patterns. Second, while the postbiotic analysis showed good consistency among treatment replicates, the OMD values between replicates varied widely. Incomplete homogenization of fecal material during inoculum preparation was likely a source of error here, potentially masking any detectible differences in substrate degradation between treatment fibers. Additional replication would be necessary to draw inference on substrate degradation. Third, reference standards including a completely fermentable and a nonfermentable standard such as crystalline cellulose and isolated pectin, respectively, would have provided an upper and lower limit for treatment comparisons. However, PF and TP are commonly used within the pet food industry and have been previously evaluated using the same in vitro procedures. Thus, bridging previous reports to this current study. Finally, differences in fermentation patterns were likely a function of the unique monosaccharide composition in the cell walls of fiber treatments. Unfortunately, this was beyond the scope the current study. Future work should characterize the composition and disappearance of monosaccharides in these and other fibers to help elucidate the mechanisms driving differences in postbiotic production. Despite these limitations, this data provides valuable knowledge to the limited published research evaluating novel fiber ingredients in companion animal diets.The fruit and vegetable fibers evaluated here AP, BP, CP, PF, and TP were low to moderately fermentable with AP being the most fermentable and CP being the least. All fiber treatments had unique patterns of postbiotic production across time. However, there were no large shifts toward butyrate production demonstrated for any of the evaluated fiber sources. Potential remains for these alternative ingredients in companion animal diets to promote gastrointestinal health, weight management, and digestive regularity.


Prunus domestics) and Japanese  plums caused by the hemibiotrophic fungus Apiosporina morbosa. After infection, the appearance of warty black knots indicates a phytohormonal imbalance in infected tissues. Based on this hypothesis, we quantified phytohormones such as indole-3-acetic acid, tryptophan, indoleamines , and cytokinins  in temporally collected tissues of susceptible and resistant genotypes belonging to European and Japanese plums during of BK progression. The results suggested auxin-cytokinins interplay driven by A. morbosa appears to be vital in disease progression by hampering the plant defense system. Taken together, our results indicate the possibility of using the phytohormone profile as a biomarker for BK resistance in plums.Black Knot (BK) is a deadly disease of European ( Apiosporina morbosa causes a tumor-like fungal disease called ‘Black knot’ (BK) in both of the commercially important plum species, i.e., European (Prunus domestica) and Japanese  plums [The fungus a) plums . The disa) plums . Once esa) plums . BK causa) plums ,4. What A. morbosa was considered to be a hemibiotrophic fungus because of its biotrophic behavior at the early stage of infection and then conversion into necrotrophic phage at the later stage [Plant-invading pathogens based on nutrient acquisition are classified as necrotrophs, biotrophs, and hemibiotrophs . Hemibioer stage . The defer stage . Moreoveer stage . A commoer stage . Phytopaer stage .The knot-forming nature of the BK disease points to the possible involvement of phytohormones like auxin and/or cytokinins in symptom development. Pathogen-induced hormones such as auxins probably could enhance the plant’s susceptibility to hemibiotrophic pathogens by directly or indirectly impacting the plant’s defense-related hormones ,11. BaseBK is a very slow disease taking almost 2 years for a full manifestation of the knots. Due to its extremely slow nature and environment-dependent infection process, consistent controlled infection is futile. Thus, one must rely on the natural infection of susceptible genotypes in a genetically diverse population over an extended period of at least 7–8 years before a phenotypic judgment on resistant genotypes can be passed. This perhaps is the single most important reason for this disease being minimally researched. Based on multiple years of observations, we developed a scoring system for disease progression depicting five clear stages of black knot development in plums . BrieflySusceptible plum genotypes (European and Japanese) were analyzed for changes in phytohormones titers during the progression of BK disease in comparison with unaffected/resistant genotypes. Phytohormones titers were affected by BK infection in susceptible plum genotypes irrespective of their origin. In general, TRP A,B, BA E,F, and In general, resistant European plums had higher levels of TRP than the rest A,B. At sNAS peaked in stage 3 of BK development and then dropped rapidly in both plum varieties E,F. InteSince IAA is a natural auxin, we measured IAA levels during BK development. Auxin levels were significantly higher in European plum knots in all stages of disease progression than in Japanese plum knots G,H. IAA Both zeatin A,B and iSignificantly high levels of GA were observed in all five of the stage equivalents in the resistant genotypes of both plums G,H. ResiCorrelation among the phytohormone levels was determined using PCA of the susceptible genotypes of European plum, where approximately 50% variability was explained by PCA I and approximately 40% by PCA II . HoweverIn Japanese susceptible plum genotypes, except for SER and BA, all other analyzed compounds were positively correlated . TRP, NAOverall, our results suggest that higher endogenous levels of TRP, GA, and SER contribute to BK resistance while auxin and cytokinins promote susceptibility. In particular, higher levels of TRP seem to be a strong indicator of BK resistance. However, it must also be noted that the extremely high levels of IAA produced by the knots are suggestive of this auxin being produced by the fungus, rather than fungus–host interaction. Such high levels of auxin and cytokinins, probably produced by the fungus along with the utilization of gibberellic acid from the host plants results in uncontrolled cell division at the infected sites leading to black knots. Although both auxin and cytokinins (zeatin and ip) are higher in susceptible genotypes, the levels of cytokinins are only a fraction of IAA, indicating a possible antagonism than synergism between the two hormones.A. morbosa, during BK disease progression. Overall, the susceptible genotypes exhibit significantly higher levels of auxin and cytokinins and lower levels of indolamines than the resistant genotypes. This opens up the question of identifying the possible mechanisms of how plums counter a fungal infection through phytohormones and whether the contents of phytohormones can be used as biomarkers. Pathogen-induced hormones such as auxin probably could enhance the plant’s susceptibility by impacting plant defense [Phytohormones perturbance is certainly triggered by  defense ,9,13.P. spinosa and P. cerasifera [In general, European plums had significantly higher phytohormone titers, which could be related to the hexaploidy nature of the European plum . As a herasifera , which cIn resistant plums, the TRP is stored as a part of dormancy acquisition , while tBotrytis, Rhizoctonia, and Alternaria in different plants like arabidopsis, rice, and lupin [Phytophthora parasitica in arabidopsis [Magnaporthe grisea, Pseudomonas syringae, and Xanthomonas oryzae in rice [The most common form of auxin in plants is indole-3-acetic acid (IAA). The involvement of auxins in plant defense response is a subject of recent interest and, for a long time, auxins were not considered a primary line of plant defense . The rolnd lupin ,22,23,24bidopsis , and Mag in rice . IAA promotes disease in many plant–pathogen interactions by acting as a plant hormone and as a trigger of the microbial signal. As a plant hormone, IAA increases host susceptibility by modulating host signaling and physiology, and as a microbial signal, IAA directly boosts pathogen virulence . To takeAgrobacterium tumefaciens and parasitic nematodes interfere with host auxin physiology to encourage hypertrophy and hyperplasia which leads to gall/knot formation. Further, pathogens can either synthesize auxin themselves [A. morbosa will be a subject of interest for future research. Knots reach their maximum size during the 3rd developmental stage  and Japanese  plum trees were selected for the current study. Phytohormonal analysis was performed using genotypes ‘Vision’ and ‘Veeblue’ from the European plum and ‘Vampire’ and ‘Shiro’ from the Japanese plum, which are extremely susceptible to the black knot disease, and resistant genotypes V982014 and V911415 from the European plum and ‘Underwood’ and ‘Redcoat’ from the Japanese plum. The resistance and susceptibility of these genotypes were established based on the assessment of BK incidence and progression over 10 years, with limited disease control in a diverse population consisting of ~150 genotypes of each Japanese and European plum [From the University of Guelph’s plum breeding program, 15-year-old European  with liquid nitrogen.Freeze drying of all the woody samples was carried out for 48 h with a FreeZone 4.5L −50 °C Benchtop Freeze Dryer  immediately after sample collection from the field. Then, the freeze-dried samples were stored at −80 °C until they were ground using an IKA® HLB 1cc (30 mg), Waters Canada, Mississauga, ON, Canada) was deployed to concentrate the samples before eluting them in 200 μL methanol. Later, the eluant was filtered through a 0.22 μM centrifuge filter . All standards were analytical grade and purchased from Sigma Aldrich, Oakville, ON, Canada. Phytohormones were separated by reverse-phase liquid chromatography (ultra-performance liquid chromatography system (UPLC); LC-40D XS, Shimadzu, Tokyo, Japan) with an injection of a 5 μL aliquot of sample onto the Shim-pack Scepter LC column . Metabolites were separated with a gradient of solvents A (0.1% formic acid) and B (100% methanol) with initial conditions at 95% A (5% B) increased to 5% A (95% B) over 4 min using a curve of 0. The column temperature was 40 °C and the flow rate was 0.2 mL/min. Metabolite peaks were identified by comparison to standards and quantified by a standard curve generated using a similar separation method and gradient conditions. Phytohormones were detected using a single-quadrupole mass spectrometer  in single ion recording mode (SIR). The majority of the compounds were detected in positive mode with a cone voltage of 10 for mass to charge (m/z) of 205 (tryptophan (TRP)), 177 (serotonin (SER)), 219 (N-acetylserotonin (NAS)), 176 (indole-3-aceic acid (IAA)), 204 (2-isopentenyladenine (ip)), 220 (zeatin), 226 (6-benzylaminopurine (BA)), and in negative mode with a cone voltage of 10 for mass to charge (m/z) of 345 (gibberellic acid (GA)). In all cases, the probe temperature was set to 250 °C with a gain of 5; capillary voltage (positive and negative) was set to 0.5 kV. The linear range for all eight compounds was 1.53 ng/mL–6.25 μg/mL. All the phytohormones from ground woody samples were extracted using the methanol double extraction method. Briefly, 100 mg of freeze-dried, powdered woody samples were extracted with a solvent mixture (methanol: formic acid: milli-Q water = 15:1:4). Samples were then held at −20 °C for 30 min and spun down  and the supernatant was removed. A second extraction was performed on the same sample using similar conditions to those described above and the supernatants were pooled. Solid phase extraction  in SAS v9.4 . Shapiro–Wilk normality tests and studentized residual plots were used to test error assumptions of variance analysis including random, homogenous, and normal distributions of error. Outliers were removed using Lund’s test. Means were calculated using the LSMEANS statement, and significant differences between the treatments were determined using a post hoc LSD test α ≤ 0.05 and are mentioned in each figure.The phytohormonal contribution in BK disease development was analyzed using principal component analysis (PCA) at the 1st and 5th stages. Here, PCA was conducted separately on phytohormonal data from resistant and susceptible genotypes of European and Japanese plums, using PROC PRINCOMP in SAS v9.4 .


Wolbachia—infect a broad range of phylogenetically diverse invertebrate hosts in terrestrial ecosystems. Wolbachia has an important impact on the ecology and evolution of their host with documented effects including induced parthenogenesis, male killing, feminization, and cytoplasmic incompatibility. Nonetheless, data on Wolbachia infections in non-terrestrial invertebrates are scarce. Sampling bias and methodological limitations are some of the reasons limiting the detection of these bacteria in aquatic organisms. In this study, we present a new metagenetic method for detecting the co-occurrence of different Wolbachia strains in freshwater invertebrates host species, i.e., freshwater Arthropoda (Crustacea), Mollusca , and water bears (Tardigrada) by applying NGS primers designed by us and a Python script that allows the identification of Wolbachia target sequences from the microbiome communities. We also compare the results obtained using the commonly applied NGS primers and the Sanger sequencing approach. Finally, we describe three supergroups of Wolbachia: (i) a new supergroup V identified in Crustacea and Bivalvia hosts; (ii) supergroup A identified in Crustacea, Bivalvia, and Eutardigrada hosts, and (iii) supergroup E infection in the Crustacea host microbiome community.The infamous “master manipulators”—intracellular bacteria of the genus  Wolbachia, belonging to the α-Proteobacteria  ; foot tis, 1771)   gene sequences. The same DNA isolates as for the microbiome analysis were used. The barcode sequences of Crustacea, Bivalvia and Eutardigrada were amplified using the universal invertebrate primers: HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) and LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) . The PCR species ). Conseq species , with th species .The obtained sequences were checked for the quality and manually aligned in the BioEdit ver. 7.2.5 . The comAll laboratory procedures were performed using sterile equipment and all steps were carried out in a sterile laminar flow hood to avoid cross-contamination of the samples. Moreover, the “RIDE” checklist, i.e., a set of minimal experimental criteria, to improve the reliability of samples with low microbial biomass  was applied . When a Wolbachia was performed using PCR screening and Sanger sequencing. We applied Wolbachia-specific WF (forward: 5′–CGGGGGAAAATTTATTGCT–3′) and WR (reverse: 5′–AGCTGTAATACAGAAAGGAAA–3′) primers according to the PCR protocol provided by Singh et al. [Wolbachia gene fragment with sequences deposited in the NCBI database. We accepted only the results which indicated query cover near 100%, high identity ˃95% and an E value near 0.0. All obtained sequences have been deposited in GenBank under the accession numbers provided in Preliminary detection of h et al. , as wellh et al. . The PCRWolbachia 16S rRNA sequences in the microbiome community. In addition, to test whether Wolbachia can also be detected in the same samples using commercial primers, a simultaneous amplification was performed using 341F (forward: 5′–CCTACGGGNGGCWGCAG–3′) and 785R (reverse: 5′–GACTACHVGGGTATCTAATCC–3′) primers [We performed high-throughput sequencing using the new isolates of invertebrates , Mollusca , and water bears (Tardigrada)) and those described in our previous papers ,95,105; . Metagen primers , indicat primers  as the m2, and 0.08 mM of dNTP; Sigma-Aldrich, Germany), 0.4 µM of 341F and 785R primers and about 5 ng of DNA. The 16S rRNA gene fragment was amplified under the following conditions: initial denaturation at 95 °C for 3 min followed by 25 cycles of 95 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s and ending with 72 °C for 5 min. The PCR products were separated in 1% agarose gel in a 1× SB buffer and it was visualized using Midori Green Advance DNA Stain (Genetics) under UV light (Vilber Lourmat V01 7107). The PCR amplification of the bacterial fragment of the 16S rRNA gene using WOLBSL and WOLBSR primers was performed under conditions described by Mioduchowska et al. . In turnWolbachia using our and Wolbachia-specific primers or for which poor Sanger sequences were obtained using the designed primers  despite good quality PCR products. All samples for which non-target Wolbachia sequences have been obtained, e.g., uncultured bacteria [We decided to analyze samples of taxa for which we detected bacteria , were reD. polymorpha and three taxa of Crustacea, i.e., A. salina, A. parthenogenetica, and Eulimnadia sp. when primers designed by us were used. However, library concentrations of the remaining samples  were below the detection limit, despite the good quality of the PCR reaction products. Paired-end (PE) sequencing was performed with an Illumina MiSeq platform .Indexing PCR reactions, which were performed using Q5 Hot Start High-Fidelity 2× Master Mix, was the next step prior to NGS. Reaction conditions were used according to the manufacturer’s recommendations. All PCR products obtained using commercial primers passed the final Quality Control (QC). Libraries of appropriate quality were obtained for one species of Bivalvia, i.e., https://www.illumina.com/systems/sequencing-platforms/miseq/products-services/miseq-reporter.html; URL accessed on 16 March 2018.Automatic preliminary data analysis was performed using a MiSeq apparatus and MiSeq Reporter (MSR) v2.6  removing adapter sequences using the cutadapt program , (2) analysis of read quality and removal of low-quality sequences , using cutadapt program , (3a) commercial primers: paired sequences were joined using the fastq-join algorithm , (3b) designed primers: due to the amplicon size >450 bp, paired readings were treated as individual reads and not as pairs, (4a) commercial primers: for clustering based on the selected GreenGenes v13.8 reference sequence database, the uclust algorithm  was used(4b) designed primers: clustering and taxonomy assignment were carried out based on the GreenGenes v13.8 reference sequence database, without the possibility of forming new clusters (closed-reference OTU picking), which allowed to study two regions simultaneously ; the taxonomy to the selected reference sequence database was assigned using the uclust algorithm with a sequence similarity limit of 97%,http://www.geneious.com, URL accessed on 18 January 2022), and(5) visualization of the microbiome profile obtained as a result of 16S rRNA fragment amplification using two pairs of primers was performed in Geneious 2022.0.2 ((6) the comparison of bacterial community structure, i.e., the Venn diagram; OTUs common to the microbiome from all stations were visualized using R 4.0.3  with theWolbachia sequences from the microbiome community based on Hamming distance (pairwise distance) values. Hamming distance values were calculated using the Wolbachia operational taxonomic unit (OTU) detected in our study as the reference sequence  score between the two aligned sequences. The local alignment is adjusted using Biopython’s pairwise2.align.localxx  implementation of the dynamic programming local alignment algorithm. The local alignment is required, as the Hamming distance is defined for sequences of equal length. When calculating this pairwise distance, we assumed 0 for the match, 1 for the mismatch, and the gaps were treated as mismatches. Afterwards, the amplified sample sequences are returned and sorted according to their Hamming distance  and according to their local alignment scores . The returned outputs are two .csv files (per pair of fastq files) with lists of amplified sample sequence identifiers and their scores (Python script has been posted on GitHub (17 May 2023): https://github.com/krzbar/Wolbachia_Peekaboo). The bioinformatics pipeline is shown in In principle, our script first reads in all available fastq files in its working directory. It is assumed that each amplified sample is present in two files (from reverse and forward primers separately), following each other alphabetically. Then, it reads the target Wolbachia are deposited in the GenBank database due to the application of various primers for the amplification. Therefore, the data for H and L supergroups of the Wolbachia strains could not be used in phylogenetic analysis. We downloaded the data from GenBank on 16 supergroups of Wolbachia strains from different invertebrate hosts species and aligned them in our dataset. The quality of all the sequences obtained was then checked and trimmed to the same length in BioEdit v. 7.2.5 [Wolbachia supergroups was derived from a phylogenetic analysis and uncorrected p-distances calculated in MEGA X [Different fragments of the 16S rRNA gene of the genus v. 7.2.5 . The aliv. 7.2.5  with defv. 7.2.5 . The φ tn MEGA X .Wolbachia sequences obtained from the microbiome community of freshwater invertebrate hosts and sequences representing different Wolbachia phylogenetic supergroups were tested using Bayesian inference (BI) analyses using MrBayes v.3.2.6 [Ehrlichia chaffeensis Anderson, Dawson, Jones and Wilson, 1991 ([Wolbachia sequences identified in Tardigrada until now: (i) twelve sequences from our study; (ii) three Wolbachia OTUs from Mioduchowska et al. [Wolbachia OTU from Tibbs-Cortes et al. [The phylogeny of the 16S rRNA  v.3.2.6  implemenn, 1991 (, order Ra et al.  and (iiis et al. . As an os et al.  and Tibbs et al. . The moss et al.  for sequs et al. .


Parathyroidectomy is associated with improved survival in patients with end-stage kidney disease. Protein-energy wasting (PEW) is common in patients with kidney failure and predicts poor outcomes. Recent clinical trials have linked hyperparathyroidism to PEW. The present retrospective cohort study examined whether parathyroidectomy was associated with improvement in nutritional status in maintenance hemodialysis patients.One hundred twenty-nine maintenance hemodialysis patients who had successful parathyroidectomy during 2012–2018 were identified (PTX group) and matched 1:1 to 479 patients with parathyroid hormone (PTH) levels ≤1,000 pg./mL (non-PTX control group) and 187 patients with PTH levels >1,000 pg./mL (pre-PTX control group) by propensity score. The matchings yielded 120 matched pairs from PTX and non-PTX groups (cohort 1) and 76 matched pairs from PTX and pre-PTX groups (cohort 2). Baseline and follow-up nutritional parameters associated with PEW were compared over the 12-month study period.2 (low Cr/BSA) were observed in the PTX group. These parameters improved significantly after parathyroidectomy. Total lymphocyte count (TLC) was comparable at baseline but the percentage of patients with TLC <800 cells/mm3 (low TLC) decreased substantially after parathyroidectomy. At follow-up, serum albumin, serum Cr/BSA and proportions of patients with low albumin and Cr/BSA became comparable with the non-PTX control group. The percentage of patients with low TLC became lower in the PTX group. Mixed-models analysis confirmed significant differences in the changes in serum albumin, serum Cr/BSA, and proportions of patients with low albumin and TLC between the two groups. In cohort 2, nutritional parameters were comparable at baseline. At follow-up, serum Cr/BSA was higher and proportions of patients with body mass index ≤18.5 kg/m2, low TLC and low Cr/BSA were lower in the PTX group. Weight gain was more frequent and of greater magnitude in the PTX group in both cohorts. A substantial reduction in blood pressure was also observed in the PTX group.In cohort 1, substantially lower serum albumin and serum creatinine/body surface area (Cr/BSA) and higher proportions of patients with serum albumin ≤38 g/L  and serum Cr/BSA ≤380 μmol/L/mSevere hyperparathyroidism was associated with nutritional impairment which improved considerably after parathyroidectomy. Parathyroidectomy continues to play an important role in the treatment of severe hyperparathyroidism in patients with end-stage kidney disease (ESKD) especially in countries where accessibility to calcimimetics is limited. In addition to increasing bone mass and reducing fracture risk, improvements in quality of life, cardiovascular and overall survival have also been reported in several large observational studies from different parts of the world –4. The bProtein-energy wasting (PEW) is prevalent in ESKD and is a powerful predictor of outcomes. Multiple mechanisms are involved including inadequate intake, inflammation, acidosis, hormonal imbalance, and dialysis procedure that lead to protein and fat breakdown . Until rThe aim of the present study was to explore a relationship between parathyroidectomy and improvement in nutritional status in patients receiving maintenance HD. The changes in nutritional parameters associated with PEW in parathyroidectomized patients were compared with two non-parathyroidectomized control groups. The first control group represented HD patients whose PTH levels were in the recommended range or mildly elevated. The second control group consisted of patients with a more severe degree of hyperparathyroidism or awaiting parathyroidectomy.The present study is a propensity-score matched retrospective cohort study of patients receiving maintenance HD at Ramathibodi hospital, Mahidol university, Bangkok, Thailand. The study was approved by the Human Research Ethics Committee of Faculty of Medicine, Ramathibodi Hospital, Mahidol University . Written informed consents were waived. The study was performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.The selection process of cases and controls are illustrated in The two control groups consisted of HD patients who did not undergo parathyroidectomy during the study period. They had to have at least 4 sets of laboratory and nutritional data available for the consecutive 12-month period. Six hundred eighty non-parathyroidectomized patients were identified and 666 patients met these criteria. The first control group included patients with PTH levels within the target range or mild hyperparathyroidism (non-parathyroidectomy or non-PTX group). To be eligible for the non-PTX control group, all PTH values during the 12-month period had to be ≤1,000 pg./mL. The second control group consisted of patients with a more severe degree of hyperparathyroidism or those on the waiting list for parathyroidectomy (pre-parathyroidectomy or pre-PTX group). To be eligible for the pre-PTX control group, at least one PTH value during the 12-month period had to be >1,000 pg./mL. The cut-off PTH levels used in this study were adapted from the findings from our previous published study that showed no differences in nutritional parameters between the group of patients with PTH between 200–599 pg./mL and 600–1,500 pg./mL, whereas substantial deterioration of nutritional status was observed in the group of patients with PTH >1,500 pg./mL . Due to 2. The dialysate sodium concentration ranged between 136–140 mEq/L, potassium 2–3 mEq/L, calcium 2.5–3.5 mEq/L, magnesium 0.7 mEq/L and bicarbonate 32–36 mEq/L.To minimize the differences in baseline demographic data, 1:1 propensity score matchings were performed between PTX and non-PTX groups and between PTX and pre-PTX groups according to age, diabetes mellitus and dialysis vintage. These factors were chosen because they were important predictors of nutritional status and the degree of hyperparathyroidism in dialysis patients , 13. ThePrimary outcomes were differences in nutritional parameters at baseline and follow-up between the parathyroidectomy and the two control groups. Secondary outcomes were between group differences in the changes of nutritional parameters from baseline and factors associated with nutritional impairment.Demographic and laboratory data were obtained from the electronic medical record system. Body weight, height and blood pressure were obtained from the records of outpatient clinic visit on non-dialysis days. To qualify for using phosphate binders , active vitamin D  or calcimimetics , the medications had to be prescribed for at least two consecutive months. To ensure that calcium was prescribed for phosphate binding, the dose of elemental calcium had to be at least 1,000 mg/day and the frequency at least twice daily. To ensure that active vitamin D was prescribed for lowering PTH, the dose had to be at least 1.75 μg/week for calcitriol or 3 μg/week for alfacalcidol. Neutrophil-to-lymphocyte ratio (NLR) was selected as a marker of inflammation because the data were available in all patients . NLR ≥3.2, respectively 0.425. Thailand Renal Replacement Therapy Registry provided the data on nPCR and adequacy of hemodialysis (Kt/V). Electronic records of HD treatment were available from 2016 onward and the data on Kt/V and nPCR were available in 77 and 40 patients in cohort 1 and cohort 2, respectively. Total lymphocyte count (TLC) was also included as another mean of nutritional assessment. The cut-off value for lymphopenia at ≤800 cells/mm3 was selected based on its association with an increase in mortality in HD patients  represented body mass, serum creatinine/body surface area (Cr/BSA) represented skeletal muscle mass, and normalized protein catabolic rate (nPCR) represented protein intake. The cut-off values for serum albumin and serum Cr/BSA were 38 g/L and 380 μmol/L/mectively , 18. Thepatients , 20. BSApatients . Serum cp-value <0.05 was considered statistically significant. All statistical analyses were performed using the IBM SPSS Statistics for Windows, version 26 .Data are presented as mean ± standard deviation, median (interquartile range) or mean . Propensity score matching was performed with a matching tolerance of 0.05, without replacement, and by giving priority to exact matches. The differences between the two groups were compared using Student’s T-test, Mann–Whitney U test or Chi-square test. Linear fixed and mixed-effects regression models were used to analyze the differences between baseline and follow-up biochemical data within the same group and between two groups over the study period. Linear regression analysis was used to analyze factors associated with an increase in the BMI. The relationships between two continuous variables were evaluated by Pearson correlation. 2 and TLC ≤800 cells/mm3. Hemoglobin, serum albumin and Cr/BSA were lower, whereas proportions of patients with serum albumin ≤38 g/L and Cr/BSA ≤380 μmol/L/m2 were higher in the PTX group. At follow-up, systolic and pulse pressure were lower in the PTX group. Serum albumin, Cr/BSA and proportions of patients with serum albumin ≤38 g/L and Cr/BSA ≤380 μmol/L/m2 became equivalent to the non-PTX control group. Moreover, hemoglobin and BMI became higher and the percentage of patients with TLC ≤800 cells/mm3 became lower in the PTX group at follow-up. 3, serum albumin ≤38 g/L and Cr/BSA ≤380 μmol/L/m2 decreased substantially after parathyroidectomy. These parameters were largely unchanged in the non-PTX control group. There were no changes in the NLR, BMI, nPCR and proportion of patients with BMI ≤18.5 kg/m2 in either group. Between group comparisons confirmed significant differences in the alterations in serum albumin, Cr/BSA, and proportions of patients with TLC ≤800 cells/mm3 and serum albumin ≤38 g/L during the study period.2, TLC ≤800 cells/mm3 and Cr/BSA ≤380 μmol/L/m2 were observed in the PTX group. Within group comparisons showed similar findings for the PTX group as in cohort 1, whereas all parameters were largely unchanged in the pre-PTX group. Between group comparisons revealed significant differences in the alterations in pulse pressure and proportions of patients with TLC ≤800 cells/mm3 and serum albumin ≤38 g/L.The biochemical data of patients in cohort 2 at baseline and follow-up are shown in p = 0.009) and Cr/BSA , p = 0.02) and positive associations between log PTH level , p = 0.001) and parathyroidectomy , p = 0.006) with ≥10% increase in the BMI among patients in cohort 1. There were no relationships between age, sex, diabetes, baseline BMI, dialysis vintage, hemoglobin and NLR with ≥10% increase in the BMI (p < 0.001) and Cr/BSA   and creatinine . Similar findings were observed among patients in cohort 2 (data not shown).Increases in the BMI ≥5% and ≥ 10% occurred more frequently in the PTX group compared with the non-PTX (24.1% vs. 7 and 13.8% vs.1.8%) and the pre-PTX (25% vs. 8 and 12.5% vs. 0%) control groups . Univari the BMI . The cha= 0.001) . There wThis is the largest study with appropriate control groups that demonstrated an improvement in nutritional parameters after parathyroidectomy in patients receiving maintenance HD with severe hyperparathyroidism. The main findings included substantial nutritional impairment among patients with severe hyperparathyroidism, which improved considerably after parathyroidectomy. Several nutritional indices became equivalent to those with PTH levels in the recommended range or mild hyperparathyroidism and better than those with severe hyperparathyroidism. Weight gain was also more frequent and more substantial among patients who underwent parathyroidectomy. Mixed-model analyses confirmed significant differences in the changes of nutritional parameters between the PTX group and the two control groups. Incidentally, a reduction in blood pressure was also observed in the PTX group.Patients with severe hyperparathyroidism had lower serum albumin and Cr/BSA compared with patients with PTH levels in the target range or mild hyperparathyroidism. Dietary intake as indicated by nPCR and inflammation as indicated by NLR were comparable between the two groups. These data suggested the presence of PEW and sarcopenia in patients with severe hyperparathyroidism that were unlikely to be due to decreased protein intake or inflammation. Inflammation and retention of uremic toxins are two main factors contributing to PEW in patients with kidney failure. The relationship between increased PTH level and inflammation has been somewhat inconsistent, however, in a large cohort of over 8,000 adults from United States, increased PTH level showed a positive correlation with the levels of inflammatory markers . Since cIncreased resting energy expenditure among patients with severe hyperparathyroidism compared with patients with mild to moderate hyperparathyroidism has been reported previously . Later sAfter parathyroidectomy, nutritional indices improved considerably. These findings supported the idea of hyperparathyroidism causing PEW and removal of excess PTH could improve nutritional status. Other small uncontrolled observational studies have reported similar observation –11, 30. A considerable improvement in hematologic parameters including anemia and lymphopenia were observed after parathyroidectomy. Suppression of erythropoiesis in secondary hyperparathyroidism as a result of bone marrow fibrosis has been well documented . An adveThe strengths of the present study were the number of patients, the presence of two matched control groups with different degrees of hyperparathyroidism and the analysis of multiple nutritional parameters associated with PEW and malnutrition. Heavy smoking and alcohol drinking could impact nutritional status, but these data were either missing or incomplete in the medical records in most patients. The missing nPCR data prior to 2016 also prevented an accurate assessment of dietary protein intake especially among patients in cohort 2.In conclusion, maintenance HD patients with severe hyperparathyroidism showed nutritional impairment which improved considerably after parathyroidectomy. These findings should emphasize the importance of early and proper management of hyperparathyroidism in patients with ESKD.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Faculty of Medicine, Ramathibodi Hospital, Mahidol University. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.SD conceived and designed the study. SD, SS, SK, and RS acquired the data. SD analyzed and interpreted the data. SD, RS, YW, and JT wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


Idiopathic membranous nephropathy (IMN) is a common type of nephrotic syndrome, and is associated with acute kidney injury (AKI). We investigated the association of multiple variables with AKI in patients with IMN.The data of 187 patients with biopsy-proven IMN were examined. Renal outcome was defined as progression to end-stage renal disease (ESRD). Binary logistic regression and Kaplan–Meier’s analysis were used for statistical analysis.p < .01). The AKI group had higher uric acid, lower serum PLA2R antibody positive, and worse baseline kidney function . Most patients in the AKI group had stage I (71.74%) or stage II (21.74%). The AKI group had higher renal tubular injury score and chronicity index (both p < .05). Binary logistic regression indicated that uric acid and baseline estimated glomerular filtration rate (eGFR) were independent risk factors for AKI in patients with IMN (p < .05). The optimal cutoff value of serum uric acid for predicting AKI was 402.50 μmol/L and the baseline eGFR was 96.83 mL/min/1.73 m2. Kaplan–Meier’s analysis showed that the cumulative renal survival rate was lower in the AKI group (p = .047).During follow-up, 46 (24.6%) patients developed AKI. The incidence of AKI was greater in males than females (AKI increases the risk of poor prognosis in IMN patients and the high uric acid and low baseline eGFR were considered independent predictors for developing AKI in patients with IMN. Idiopathic membranous nephropathy (IMN) is a common cause of primary nephrotic syndrome (PNS) in nondiabetic adults worldwide . Xu et aAcute kidney injury (AKI) is a relatively common complication in patients with IMN, and patients who present with PNS are more likely to develop AKI. A recent study reported that AKI is the most serious complication of PNS, and patients who were elderly and had more underlying diseases had a poorer prognosis . FailureThe mechanism of secondary AKI in patients with PNS is not completely clear. It is believed that AKI in these patients may be related to intrarenal ischemia, renal interstitial edema, glomerular lesions, renal tubular necrosis, drug-related interstitial nephritis, and certain other conditions ,6. HowevThis retrospective cohort study initially examined 208 patients with clinical manifestations of PNS who were diagnosed with MN based on renal biopsy and received follow-up for at least one year from September 2015 to August 2019 in the Department of Nephrology, The First Affiliated Hospital of Fujian Medical University. Patients were excluded if they had chronic renal insufficiency; secondary MN (SMN) due to systemic lupus erythematosus, hepatitis B viral infection, or allergic purpura; concomitant diseases, such as diabetic nephropathy, IgA nephropathy, focal segmental glomerular sclerosis (FSGS), proliferative glomerular nephritis, cryoglobulinemia, or tumor; or incomplete clinical data. This research was performed in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of the First Affiliated Hospital of Fujian Medical University . Informed written consent was obtained from all participants.The clinical data of enrolled patients were collected at the time of renal biopsy, including sex, age, body mass index (BMI), systolic blood pressure (SBP), diastolic blood pressure (DBP), serum creatine (SCr), serum albumin, estimated glomerular filtration rate (eGFR), uric acid, total cholesterol (TC), triglycerides (TGs), serum phospholipase A2 receptor (PLA2R) status, 24 h urinary protein, urinary red blood cells, D-dimer, prothrombin time (PT), international normalized ratio (INR), use of a renin–angiotensin–aldosterone system (RAAS) inhibitor, use of diuretics, use of a glucocorticoid, and use of an immunosuppressant .The following five parameters were assessed during histopathology of the renal biopsy specimens: global sclerosis; glomerular segmental sclerosis; severity of tubulointerstitial changes; vascular lesions. Four semi-quantitative grades were used to classify serum PLA2R antibody titer: 0 (negative), 1+ 1:10), 2+ (1:32), 3+ (1:100), and 4+ (≥1:320). Tubulointerstitial changes, including tubular atrophy, interstitial fibrosis, and mononuclear cell infiltration, were expressed as a percentage of the area of cortical kidney tissue covered by lesions. These lesions were classified as none, mild (˂25%), moderate (25–50%), or severe (>50%). Semi-quantitative scores for the chronicity index (CI) and tubular interstitial lesion (TIL) were determined according to Austin et al. [, 2+  level of each patient at the time of outpatient visit or readmission, and evaluated whether AKI or endpoint events occurred.2 or the initiation of renal replacement therapy. AKI was diagnosed if any one of the following conditions were present during follow-up: an increase of SCr of at least 26.5 μmol/L within 48 h; an increase of SCr of at least 1.5 times above the baseline value within seven days; or hourly urine output below 0.5 mL/kg for more than 6 h. In addition, some patients had elevated SCr on the first day of admission; the baseline SCr values of these patients were assumed to be lower than on the first day based on the subsequent clinical course. Thus, based on the 2012 KDIGO Clinical Practice Guidelines for AKI [Baseline SCr level and baseline eGFR level were defined as SCr value and eGFR value that measured at the time of renal biopsy. The endpoint event in the present study was defined as ESRD, as indicated by an eGFR below 15 mL/min/1.73 m for AKI , these p for AKI : AKI stat-test or a nonparametric test (as appropriate) or with the Chi-square test . Survival curves for the AKI and non-AKI groups were generated using the Kaplan–Meier method, and were compared using the log-rank test. Binary logistic regression analysis and receiver operating characteristic (ROC) curve analysis were performed to evaluate the risk factors for AKI. A two-sided p value below .05 was considered statistically significant in all analyses.All data were analyzed using SPSS version 25.0 . Continuous variables were presented as means ± standard deviations or medians with interquartile ranges (as appropriate). Differences between groups were assessed using the We initially examined the records of 208 patients who were diagnosed with MN based on renal biopsy from September 2015 to August 2019. A total of 190 patients (91.3%) had IMN and 18 patients (8.7%) had SMN. Eleven of the patients with SMN (5.3%) had hepatitis B virus associated glomerular nephritis .After excluding the patients with SMN and the three patients lost to follow-up, we included 187 IMN patients in the analysis. Overall, there were 130 males (69.52%) and 57 females (30.48%), corresponding to a male-to-female ratio of 2.28:1 . The ovep = .003, p < .05). However, the two groups had no significant differences in age, BMI, hematuria, 24-h urine protein, serum TC, serum TGs, serum albumin, PT, D-dimer, INR, serum PLA2R antibody grade, and use of different therapies .The AKI group had 40 males and six females, corresponding to a male-to-female ratio of 6.67:1, significantly higher than the ratio in the non-AKI group , followed by stage II , stage III , and stage IV . Most patients in the AKI group were in stage I  and stage II , but the AKI and non-AKI groups did not significantly differ in pathological stages (p > .05). The TIL and CI were significantly higher in the AKI group (both p < .05). However, the two groups had no statistically significant differences in glomerulosclerosis, mesangial proliferation, tubular atrophy, interstitial inflammatory infiltration, interstitial fibrosis, crescent formation, renal microangiopathy, kidney IgG4 deposition, and kidney PLA2R staining .We examined the pathological stage and other features of all patients based on renal biopsy results . Overallp = .006) and baseline eGFR (p = .003) were independent risk factors for AKI in these IMN patients (2 with high specificity (68.1%) and sensitivity (69.6%)  . These rp = .047; At the end of follow-up, five (10.9%) patients in AKI group and five (3.5%) patients in non-AKI group reached renal endpoint events, separately. Kaplan–Meier’s analysis of renal survival in the AKI and non-AKI groups showed that cumulative renal survival was significantly lower in the AKI group (log-rank test: IMN is a common pathological cause of PNS, and these patients are generally sensitive to glucocorticoid therapy. IMN patients usually present with hypertension, lower extremity edema, hematuria, and renal dysfunction , and cerWe found that the incidence rate of AKI in our IMN patients was 24.6% during follow-up. By comparison, two recent studies ,16 of PNOur results showed that patients in the AKI group had significantly higher TIL and CI scores than the non-AKI group. Smith and Hayslett  studied A meta-analysis showed that lower eGFR is associated with higher risks of AKI among individuals with or without either diabetes or hypertension . Recent In addition, we found that IMN patients with high levels of serum uric acid had an increased risk for AKI, in agreement with previous research ,23. OtheMany studies showed that patients who had AKI with an underlying disease accelerated progression to ESRD ,30. In aThere are several limitations to the present study. First, we excluded patients who likely had IMN but did not receive renal biopsies, and this group presumably had a lower incidence of AKI than the patients we studied. Second, our study was in lack of contemporaneous non-AKI control, pathological and therapeutic change data during the course of AKI development meant that we could not infer causal relationships with a dynamic perspective. Third, more detailed subgroup analysis could not be performed on the prognosis of IMN patients with different recovery conditions after AKI due to the insufficient sample size. In addition, all patients were from a single center in China. Therefore, a prospective multi-center study with a large sample is required to validate our conclusions.In summary, we found that the incidence of AKI in IMN patients was 24.05% during follow-up and IMN patients with AKI were more likely to progress to ESRD. Our results also indicated that the severity of serum uric acid and baseline eGFR could be considered independent predictors for developing AKI in patients with IMN. Therefore, when examining patients with IMN, we suggest inclusion of serum uric acid and baseline eGFR in risk scoring systems that are used to predict AKI.Click here for additional data file.


Daily kiwifruit (KF) consumption has been associated with improved sleep quality, but underlying physiological mechanisms are unknown. This study examined acute effects of fresh and dried green KF, compared with a water control, on sleep quality, mood, and urinary serotonin and melatonin metabolite concentrations.2) with poor (n = 12) or good (n = 12) sleep quality participated in a randomized, single-blind crossover study. One of three treatments was consumed with a standardized evening meal; (1) the flesh of two fresh green KF, (2) dried green KF powder  mixed with water, or (3) a water control, in their own home. Subjective and objective sleep quality, mood, waking urinary 5-hydroxyindoleacetic acid (5-HIAA), 6-sulfatoxymelatonin (aMT6s), vitamin C and B-vitamin concentrations were determined.24 men  after dried KF consumption. Compared to control, both fresh and dried KF treatments tended (p < 0.1) toward improved esteem and total mood disturbance. Both KF treatments increased  urinary concentration of the serotonin metabolite 5-HIAA compared to the control (4.32 ± 0.4 ng/g). In poor sleepers, ease of awakening improved by 24% after dried KF consumption (p = 0.005) and tended to improve by 13% after fresh KF intake (p = 0.052) compared to the control. Good sleepers tended toward 9% improved ratings of getting to sleep with fresh KF (p = 0.053) compared to the control. Poor sleepers had lower amounts of some B-vitamins compared to good sleepers (p < 0.05).Regardless of sleep quality group, compared to control, morning sleepiness, alertness upon awakening, and vigor were improved (Consumption of dried or fresh KF with a standard evening meal, was associated with improved aspects of sleep quality and mood, possibly mediated through changes in serotonin metabolism.www.anzctr.org.au], identifier [ACTRN12621000046808].[ Inadequate sleep is associated with increased health risks such as inflammation, diabetes, hypertension, and obesity. –5. InsufThe circadian clock in the hypothalamus, which is affected by the light and dark cycles, controls sleep–wake cycles in humans. The neurohormone melatonin is secreted from pinealocytes in the pineal gland, and its precursor serotonin plays a vital role in regulating this. The urinary melatonin metabolite 6-sulfatoxymelatonin (aMT6s) is related to subjective and objective sleep quality measures  and the shumeji and shiitake mushrooms)  , tomatoeshrooms) , and cheshrooms) , which ashrooms) –16 and ishrooms) –17. Consshrooms)  and a Jeshrooms)  increaseshrooms) , 20 and shrooms) . Additioshrooms) . KF is hshrooms) . HoweverThe aim of this study was to determine the effect of consuming the flesh of two fresh green KF (without the skins), whole freeze-dried green KF , or a water control with a standardized evening meal on urinary concentrations of metabolites of melatonin, serotonin, vitamin C and B-vitamins as well as objective (actigraphy) and subjective sleep quality and mood measures in healthy young men. We hypothesized that sleep quality and mood would improve and urinary metabolite concentrations would increase after the KF interventions compared to the control intervention.2.2.1.2) were recruited by advertisement from the Massey University campus and community in Palmerston North, Manawatu, New Zealand, between January 2021 and May 2021 amid the COVID-19 pandemic. The Pittsburgh Sleep Quality Index (PSQI) was used to determine participants subjective sleep quality. Participants were classified as a ‘good sleeper’ when their global PSQI score was ≤ 5 and as a ‘poor sleeper’ when their global PSQI score was > 5  18.5–30 kg/m was > 5 . ExclusiThe study protocol was approved by the Massey University Human Ethics Committee (Massey University HEC: Southern A application-20/52), and the study was conducted according to the guidelines in the Declaration of Helsinki. The study was registered as a clinical trial with the Australian New Zealand Clinical Trial Registry.2.2.Actinidia deliciosa cv. Hayward) , (2) freeze-dried whole (flesh and skin), green KF  with 200 ml of water, or (3) a control of 200 ml of water. The nutritional composition of each treatment is shown in The intervention was a randomized, single-blind, cross-over study. The study involved two green KF interventions of different forms and a control. The two KF interventions were: (1) flesh of two fresh green KF , ∼720 kcal, P 26.5 g, C 75.0 g, F 33.5 g] Enrolled participants were asked to refrain from eating KF from enrolment until the completion of the study period. On the day of the study, participants were asked to collect a data collection pack . The pacThe participant consumed the standardized evening meal 4 h before their usual sleep time, followed immediately by the allocated intervention taking note of the time. Participants were asked not to eat any food or drinks, except water, until the following morning. Immediately before going to bed, participants were asked to rate their sleepiness level using the Stanford Sleepiness Scale (SSS). The participant was asked to activate the phase marker on the actigraphy watch when they were in bed and ready to sleep. Upon waking the following day, participants were asked to activate the phase marker, collect the whole first-morning urine sample noting the time, and complete a set of surveys including another SSS, Leeds Sleep Evaluation Questionnaire (LSEQ), and the abbreviated Profile of Mood States (POMS). The urine sample and surveys were delivered to the laboratory within 2 h of waking . Arrange2.2.1.Two questionnaires to measure subjective sleep quality were used. The SSS measures sleepiness, consisting of a one-item scale; the participants select one of seven statements that best represent their current sleepiness. For example, a score of one indicates feeling active, vital, and alert, while a score of seven indicates no longer fighting sleep, sleep onset soon . The LSEMood was assessed using the abbreviated POMS questionnaire . This fo2.2.2.Participants were provided with an Actiwatch Spectrum Plus  to wear on the wrist of their non-dominant arm during the study evenings. The medium threshold setting was used due to its use in studies using actigraphy to assess sleep , and 30 2.2.3.Urine samples were collected in 500 ml containers with 1.0 g of ethylenediaminetetraacetic acid (EDTA). Participants were provided with two containers. Upon arriving at the laboratory, urine samples were weighed, and their volume was recorded. The urine was aliquoted into 1.5 ml Eppendorf tubes and stored at -80°C until analysis of aMT6s and vitamin C. Ten milliliters of urine were acidified with 6 M HCL and aliquoted and stored at -80°C until analysis of 5-HIAA. Urinary aMT6s and 5-HIAA analyzes were analyzed according to the manufacturer’s instructions using an enzyme-linked immunosorbent assay (ELISA) kit . Assays were performed in duplicate and averaged. The average intra-assay coefficient of variance for the aMT6s assay was 5.2%, and the inter-assay was 2.9%. The average intra-assay coefficient of variance for the 5-HIAA assay was 7.5%, and the inter-assay was 3.5%. The ascorbate content of the samples was determined by reverse-phase high-performance liquid chromatography (HPLC) with electrochemical detection . B-vitam2.3.A power analysis was performed using ease of getting to sleep VAS from LSEQ as the dependent variable to estimate the required sample size. Estimates of variance components were conducted based on data from a study examining caffeine ingestion’s effect on VAS getting to sleep in healthy subjects . When tht-test was used to compare nutritional compositional and demographic data. Effects of sleeper type and treatment and their interaction effect were determined using a repeated measures mixed-effects model. An unstructured covariance structure was used to account for the repeated treatments by the subject. Post hoc comparisons, adjusted for multiple comparisons using Bonferroni’s correction, were performed when there were significant main or interaction effects. Residual plots were inspected to confirm that the normality and constant variance model assumptions were met. Statistical significance of the mixed effects models was accepted at a probability inferior to 0.05 (p < 0.05). A trend was noted at a probability lower than 0.10 but higher than 0.05. To assess the within-subject correlations between urinary metabolites and sleep quality and mood measures, a univariate model with sleep and mood measures as the dependent variable, urinary metabolites as the covariate, and subject as the fixed factor were performed . No outliers for any outcome measures were noted by examination of studentized residuals for values greater than ±3. A paired erformed . Correlanificant . All dat3.3.1.2) and 12 with poor sleep quality . The demographics between the groups were similar except for PSQI which was significantly lower in good sleepers (3 ± 1) compared to poor sleepers   = 5.71 p = 0.026], getting to sleep (LSEQ) [F = 4.57 p = 0.044] and quality of sleep (LSEQ) [F = 7.91 p = 0.010]. Regardless of treatment, good sleepers rated themselves sleepier in evening  compared to poor sleepers . Good sleepers rated themselves as harder getting to sleep and had worse sleep quality  compared to poor sleepers . All subjects completed and tolerated the study protocol. All participants had not been diagnosed with COVID-19.Twenty-four young men (18–35 years) completed the study: 12 with good sleep quality  . Main ef3.2.3.2.1.F = 4.81, p = 0.018] and ease of awakening following sleep (LSEQ) . Within good sleepers, ratings of ease of getting to sleep tended to increase after the fresh KF treatment (54.1 ± 4.1) compared to the control  and tended to be higher after the fresh KF treatment , compared to the control  = 5.47, p = 0.012] and alertness upon awakening (LSEQ) . Irrespective of sleeper type, morning sleepiness ratings were higher after the control  and fresh KF  interventions compared to the freeze-dried KF treatment  and tended to be higher following the fresh KF treatment  compared to the control  , vigor , and total mood disturbance . Furthermore, main trend effects of treatment for fatigue  and confusion  were identified. Irrespective of sleeper type, ratings for esteem-related affect tended to be higher following freeze-dried KF  and fresh KF  compared to the control , but not fresh KF  treatments compared to the control  and fresh KF  treatments compared to the control  = 4.06, p = 0.032]. Within poor sleepers, the number of awakenings tended to be lower after the fresh KF treatment  than the control  compared to the control  = 3.74, p = 0.040]. Irrespective of sleeper type, the number of awakenings was higher after the freeze-dried KF treatment  but not the fresh KF treatment  compared to the control (37.0 ± 4.3) (There were main effects of treatment for wake after sleep onset  with post hoc comparisons identifying freeze-dried KF  and fresh KF  treatments as showing significantly higher 5-HIAA concentration, as compared to the control (4.9 ± 0.4 mg/g creatinine)  = 6.11, p = 0.022], biotin , riboflavin , pyridoxamine , thiamine  and was near significant for pyridoxal . The post hoc comparison revealed that good sleepers had significantly higher concentrations of all measured B-vitamins than poor sleepers  and latency . A full within correlation table is presented in A summary of urinary measures is in atinine) . A signisleepers . No inte4.The current study shows that irrespective of the sleep quality participant experience, acutely supplemented freeze-dried KF resulted in feeling less sleepy and more alert. Both fresh and freeze-dried KF increased urinary excretion of 5-HIAA. Furthermore, poor sleepers felt it was easier to wake up in the morning after consuming freeze-dried KF in the evening and this was also moderately felt when consuming the fresh KF. Good sleepers rated themselves as getting to sleep easier when consuming the fresh KF. Interestingly, poor sleepers tended to wake more often in the night after consuming freeze-dried KF with the evening meal, while good sleepers woke less often during the night after consuming fresh KF with the evening meal.via potentiating the bioaminergic responses. This has been demonstrated in in vitro studies , pyridoxine and pyridoxamine (forms of B6) are required as co-factors in metabolizing neurotransmitters  or KF flesh eaten fresh without the skin (solid). Including skin in the freeze-dried powder altered the polyphenolic content, and as mentioned previously, KF skin extracts potentiate sleep induction in a pentobarbital-induced sleep mouse model . Thus, tNevertheless, it is worth noting that the food structure may influence stomach emptying rate and satiety and cause differential interactions with other food matrices, consequently impacting gut comfort. For example, consumption of a carbohydrate-rich meal with KF lowered postprandial hunger compared to consumption of a carbohydrate meal only . Also, cContrary to the previous KF and sleep quality studies, this study was the first to assess the acute and separate effects of a fresh KF or dried (containing skin) KF treatment on sleep quality and mood in a healthy male cohort with good or poor-quality sleep. The urinary 5-HIAA and aMT6s concentrations suggest a novel potential mechanism underpinning the relationship between KF and sleep. Additionally, the results provide evidence for the repurposing of KF skin and lower-quality fruit, which would otherwise be wasted, into products that can easily be stored with extended shelf-life.This study is not without limitations. One of the limitations was the low subject numbers, the study was powered using a different intervention to this , as thisAlthough actigraphy was used here and in other acute settings , results5.Overall, this study is the first to demonstrate that a single evening meal with KF, whether fresh or freeze-dried, improved sleep quality and mood in males with good or poor-quality sleep. This effect may have been mediated through increased serotonin metabolism. Further studies should be conducted to elucidate the impact of freeze-dried KF on those who experience poor sleep and the potential mechanisms by measuring other biomarkers in urine, plasma, and saliva. Nonetheless, as beneficial effects of sleep were identified following supplementation with KF in young men, these data help provide additional evidence for the role of KF in facilitating healthy sleep regulation.The original contributions presented in the study are included in the article/The study protocol was approved by the Massey University Human Ethics Committee study (Massey University HEC: Southern A application-20/52), and the trial has been registered at the Australian New Zealand Clinical Trials Registry (ANZCTR) (ANZCTR12620000411943). Participants provided written, informed consent for participation.AK, CG, NR, WM, and SH conceptualized the study and contributed to editing the manuscript. AK designed and conducted the study and prepared the manuscript with input from the other authors. All authors contributed to the article and approved the submitted version.This study was funded by the Riddet Institute, a Centre of Research Excellence (CoRE) funded by the NZ Tertiary Education Commission, and the High-Value Nutrition National Science Challenge (UOAX1902) with in-kind support from the partner organization: Zespri International and Alpha Group Holdings. The funders had no role in the design of this study and did not have any role during its execution, analyses, interpretation of the data, or decision to submit results. AK was supported by a PhD stipend from the Riddet Institute.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.