The very-low-calorie ketogenic diet (VLCKD) is effective and safe for obese individuals, but limited information exists on its impact on the intestinal barrier. This study analyzed the effects of 8 weeks of VLCKD on 24 obese patients (11M/13F). Carbohydrate intake was fixed at 20–50 g/day, while protein and lipid intake varied from 1–1.4 g/kg of ideal body weight and 15–30 g per day, respectively. Daily calorie intake was below 800 kcal. The lactulose–mannitol absorption test assessed small intestinal permeability. Multiple markers, such as serum and fecal zonulin, fatty acid-binding protein, diamine oxidase concentrations, urinary dysbiosis markers (indican and skatole), and circulating lipopolysaccharide levels, were analyzed. Inflammation markers  were also evaluated. The results showed significant reductions in weight, BMI, and waist circumference post-diet. However, the lactulose–mannitol ratio increased by 76.5%, and a significant increase in dysbiosis markers at the end of the diet occurred. This trend was particularly evident in a subgroup of patients. Despite initial benefits, the VLCKD might negatively affect the intestinal barrier function in obese patients, potentially worsening their compromised intestinal balance. Obesity is a metabolic disorder characterized by long-term, low-grade inflammation, leading to significant impairments in health status with severe comorbidities . In patiThe intestinal barrier represents a functional unit that allows the absorption of nutrients and prevents the penetration of unwanted, often dangerous, macromolecules . In addiAccording to animal studies, the connection between the gut’s adipose tissue and the gut’s brain is crucial for preserving energy balance and is hampered by metabolic abnormalities. There is growing evidence that the intestinal microflora causes the low-grade inflammation that results in intestinal barrier dysfunction, increases its permeability, and permits endotoxemia, which is one of the main factors in the emergence of metabolic inflammation and insulin resistance ,4. The aIn this respect, it is known that particular foods and excessive caloric intake can determine changes in the intestinal microbiota  and epitConcerning the diet as a treatment to reduce body weight, the ketogenic diet (KD), with its drastic carbohydrate decrease, is now common for weight loss and, in particular, a very-low-calorie ketogenic diet (VLCKD) is regarded as an efficient and secure therapeutic intervention for people affected by obesity ,9.Many studies have also shown the benefits of VLKD on body composition, metabolic profile, inflammation and oxidative stress gene expression in people with obesity ,11. Moreover, unaffected by changes in the symptoms of Non-alcoholic Fatty Liver Disease (NAFLD) such as obesity, fat mass, insulin resistance, lipids, and blood pressure, VLCKD is an effective treatment for this condition . HoweverIn humans, intestinal barrier function can be evaluated using non-invasive methods such as the urinary assay of non-absorbable sugars of various sizes . Lac is a disaccharide that provides information on the paracellular pathway and tight junction (TJ) integrity, while Man is a monosaccharide that is thought to reflect the transcellular route. These sugars are degraded by bacteria, mostly those present in the colon. Therefore, their 0–5 h urinary fractions are currently used as markers for small IP (s-IP), and the Lac to Man ratio represents humans’ most-used functional measurement of s-IP, as it eliminates factors related to uptake, distribution, or excretion. The third sugar, Suc, is a disaccharide hydrolyzed by the enzyme sucrase in the jejunum, and it can represent an index of gastroduodenal permeability. This sugar absorption test (SAT) is now widely used for diagnosing “leaky gut” syndrome, defined as a gut mucosal barrier dysfunction that results in abnormally increased intestinal permeability .The evaluations of fecal and serum levels of zonulin, a protein that reversibly modulates s-IP by altering the TJ interaction , are amoTo our knowledge, no research has previously been performed to evaluate, in humans, the possible modifications occurring in the gut barrier homeostasis following a VLCKD. Based on these premises, the present study aimed to assess, in patients with obesity without impaired small intestinal permeability, the effects of 8 weeks of VLCKD on the integrity and function of the intestinal barrier. For these purposes, the circulating and urinary markers mentioned above were investigated. Additionally, the inflammatory status was assessed by measuring the circulating levels of interleukin 6, 8, and 10 , and tumor necrosis factor-α (TNF-α). Finally, the urinary markers of dysbiosis indican and skatole and the lipopolysaccharide (LPS) circulating levels were also evaluated.The Center of Nutrition for the Research and the Care of Obesity and the Metabolic Diseases together with the Functional Gastrointestinal Disorders Research Group afferent to the National Institute of Gastroenterology IRCCS “Saverio de Bellis”, Castellana Grotte (Ba) Italy, performed this pilot study. 2, underwent a check of medical history, an anthropometric assessment, physical examination, and laboratory tests. To obtain a homogenous group, only patients with obesity responding to the exclusion criteria (see below) and without IBS and impaired small intestinal permeability, defined as Lac to Man ratio ≥ 0.030 [Patients with obesity, aged 18–65 yrs., with a body mass index (BMI) higher than 30 kg/m ≥ 0.030 , were enDuring the medical history collection, specific questions were asked concerning the smoking habits and the daily alcohol consumption, according to the American and European recommendations, that is, two glasses of alcohol per day for male patients and one glass of alcohol per day for female patients ,20, defiThe exclusion criteria included the contraindications to begin a VLCKD ,9,10,11,0) and eight weeks after the beginning of the diet (T1)  questionnaire was administered during the recruitment visit as an initial screening . Follow-iet (T1) . Before After eight weeks of the diet program, the patients had to repeat blood withdrawal. Lastly, the patients had to undergo the permeability and dysbiosis tests again within three days. ClinicalTrials.gov attributive of the study is NCT05477212. The study protocol was approved by the internal Medical Ethical Committee (Prot. n. 170/CE De Bellis). The study was performed according to the Helsinki Declaration (1964). Before starting their participation in the study, each patient gave written consent. The The protocol has been previously published in our studies  and was Protein and lipids intakes were 1–1.4 g/kg of ideal body weight and 15–30 g per day, respectively. Patients were instructed to consume at least 2 L of water each day. There were fewer than 800 calories consumed each day. In order to avoid nutritional shortages, micronutrient supplements were mandated during the whole dietary therapy . During The protein dish consisted of fish, white meat, red meat, eggs, and bresaola. As variance, the products from Penta comprised mostly whey proteins as proteins and oleic acid, omega 3 fatty acids, and other polyunsaturated fats as fatty acids; carbohydrates were very little represented, and the type of carbohydrate changed according to the product type. The semi quantitative concentration of acetoacetic acid was measured in the first-morning urine at baseline and every week until the end of the study by the patients .2), body weight and height were measured in fasting subjects wearing light clothing, barefoot, and with an empty bladder. All patients were measured using the same calibrated scale and stadiometer. Blood samples were taken between 8:00 and 9:00 a.m. after overnight fasting. Standard laboratory procedures were used to measure the biochemical characteristics before and after the VLCKD. To measure BMI (kg/mw/v) chlorohexidine was added to every collection as a preservative, regardless of the final volume. The total volume of urine excreted was measured and recorded. After the mixture was thoroughly combined, a sample of 2 mL was removed and stored at −80 °C until analyzed. In urine, the detection and measurement of the three sugar probes, Lac, Man, and Suc, were accomplished via chromatographic analysis, as previously described by our group [All participants in the study were evaluated by SAT after an overnight fast. The laboratory’s staff collected a pre-test urine sample to assess the presence of endogenous sugars. After that, patients consumed a solution that contained 10 g of Lac, 5 g of Man, and 40 g of Suc in a volume of 100 mL. Urine was collected for up to 5 h following administration. A 1 mL sample of 20% . According to the manufacturer’s instructions, serum and stool levels below 48 ng/mL and 107 ng/mL were considered normal zonulin levels. I-FABP and DAO serum levels were assessed using ELISA kits  and . Circulating IL-6, IL-8, IL-10, and TNF-α were measured using ELISA kits . Lipopolysaccharide (LPS) was measured using the Cloud-Clone Corp. ELISA kit . Urine samples were taken in the morning from all patients. According to the manufacturer’s instructions, a typical colorimetric assay kit  was used to measure urine indican levels. Thermo Scientific’s Dionex high-performance liquid chromatography (HPLC) equipment was used to detect and analyze urine skatole using the 3-methylindole kit , as was previously described . Urinaryp < 0.05 was considered statistically significant.No studies of the effect of VLCKD on gut barrier homeostasis have been performed previously, and the present research represents a pilot study. Thus, based on these premises, the statistical power calculation was unnecessary. Statistics were performed using Sigma Stat 11.0  and GraphPad Prism 8 . The Wilcoxon matched-pairs signed rank test was used to detect differences before and after the VLCKD. The Mann–Whitney test was used to assess differences between the two groups. For categorical variables, comparisons were performed using Fisher’s exact test. Unless otherwise specified, all results were expressed as means ± SEM. p < 0.05) after VLCKD. Before entering the study, a sugar absorption test with three sugar probes  was performed in all patients. The Lac/Man ratio value had to be <0.03 as an inclusion criterion, indicating the absence of altered s-IP. The same test was repeated in all patients after eight weeks of VLCKD. p = 0.009) higher than the baseline percentage (0.345 ± 0.034 vs. 0.244 ± 0.023), whereas Man% was significantly (p = 0.002) lower than the starting value (11.40 ± 0.62 vs. 14.76 ± 1.07). The Lac/Man ratio increased significantly . Moreover, the Suc% increased significantly at the end of the diet . In percent terms, Lac% increased by 41.4% and Man% decreased by 22.8%. The Lac to Man ratio increased by 76.5%. Finally, also the Suc% increased by 49.6% at the end of the diet. At the end of the diet, Lac% was significantly (p = 0.0006), the ratio increased significantly, with values above the cut-off, indicating an alteration of the s-IP . Oppositely, the Lac to Man ratio was not significantly affected by diet in the other 14 patients (7M/7F) and even tended to increase, although not significantly . The differences between the two groups relating to the markers of the intestinal barrier, dysbiosis, and bacterial translocation are reported in the following paragraphs.Interestingly, not all the subjects in the study showed the same behavior of the Lac to Man ratio following VLCKD. As shown in p = 0.983), reaching values below the cut-off limit in none of the patients. Similarly, the diet did not affect serum zonulin values . At the start of the study, mean fecal zonulin concentrations in patients with obesity were above the cut-off level (239.20 ± 23.86 ng/mL). The diet did not modify its concentrations . Likewise, serum DAO levels were unaffected by treatment .Regarding the integrity of the intestinal barrier, the serum I-FABP concentrations were not different between the start and the end of the study . Notably, TNF-α circulating levels decreased significantly at the end of the treatment . On the contrary, the anti-inflammatory IL-10 significantly increased its circulating levels at the end of the treatment compared to baseline values . Also, after the diet, the subgroup of patients with altered Lac to Man ratio showed serum IL-10 levels significantly increased compared to levels before the diet . On the contrary, serum concentrations of the other markers of inflammation did not differ between the start and the end of the study in subgroups of patients with either altered or normal s-IP. Moreover, there were no significant differences in values recorded at the beginning of the VLCKD between the two groups. The same occurred at the end of the diet. As for the interleukin circulating levels, both IL-6 and IL-8 were not significantly affected by the diet  increase in urinary concentrations occurred (57.50 ± 3.83 mg/L) , suggesting the presence of fermentative dysbiosis. At the end of the diet, a significant (83 mg/L) A. p = 0.255) (p = 0.009) at the end of the diet (0.046 ± 0.012 ng/mL) than at the start of the study (0.028 ± 0.006 ng/mL), suggesting an increase in the bacterial translocation . On the contrary, the urinary indican and serum LPS levels were not different between the start and the end of the study in the subgroup of patients with normal s-IP. The urinary skatole concentrations were below the level of 20 μg/L, and they were unaffected by diet  B. Finalllocation C. IntereObesity is a major health problem affecting millions of persons worldwide, and diet often represents a therapeutic approach for managing these patients. VLCKD is now widely used for weight loss and is regarded as an efficient and secure therapeutic intervention for people affected by obesity . PresentInterestingly, obesity has often been associated with impaired intestinal barrier function, which may facilitate the entry of dietary or microbial antigens, resulting in chronic inflammation, tissue damage, and allergies ,26. ConcIn this pilot study, we evaluated s-IP, the urinary; serum, and fecal markers of GI barrier function and integrity; and dysbiosis in subjects with obesity and analyzed the effect of eight weeks of VLCKD on these parameters. IP evaluation, particularly the s-IP, is a diagnostic measure of intestinal barrier function . In animAt the start of the study, although all patients showed normal s-IP, as evaluated by Lac to Man ratio, the mean fecal zonulin and urinary indican levels, which indicate fermentative dysbiosis, were twice as high as the cut-off values. Our results agree with recent research performed in a group of professional athletes that showed no statistically significant association between fecal zonulin and increased IP . AdditioMoreover, it has been suggested that intestinal permeability changes in obesity may be a late event in older individuals ,33. All An important finding of our study shows that, after eight weeks of VLCKD, considering the patients as a whole group, the Lac to Man ratio increased significantly compared to baseline values. However, this behavior was not common for all the patients since, at the end of the diet, 10 out of 24 had an increased Lac to Man ratio above the threshold value for altered intestinal permeability, while 14 out of 24 patients still showed normal values. Interestingly, the other intestinal barrier function and integrity markers were unaffected by VLCKD, since they did not significantly change after the diet in the whole group or subgroups divided by altered intestinal permeability. Nonetheless, in the whole group, the VLCKD diet decreased TNF-α and increased IL-10 levels, confirming its anti-inflammatory properties, as reported by other researchers ,35,36.Additionally, the whole group and the subgroup of patients with impaired s-IP had urinary indican and serum LPS levels significantly higher after the diet than baseline. On the contrary, the urinary indican and serum LPS levels did not change significantly after diet in the subgroup of patients with normal s-IP. The last two parameters indicate a modification of the balance conditions between the intestinal lumen and microbiota.The dietary components may significantly influence the gut microbiota, affecting its richness and diversity in terms of composition, according to some research. On the one hand, a high diet of animal proteins, saturated fat, sugar, and salt could promote the proliferation of harmful bacteria at the expense of helpful bacteria, perhaps causing changes to the intestinal barrier. On the other hand, ingesting complex polysaccharides and plant protein may be linked to an increase in the number of beneficial bacteria, which in turn stimulates the creation of SCFA, which is essential for maintaining the integrity of the intestinal barrier .Akkermansia or E. coli, can increase during a ketogenic diet, adversely affecting gut health.Rondanelli et al.  describeAlthough it did not fall within the aims of the present research, the results of this study allow us to hypothesize that the increased levels of indican and LPS reflect the effects of diet on microbiota and s-IP. However, it is conceivable that this effect may not occur to the same extent in all obese patients. Future studies will be performed by profiling the intestinal microbiota of these patients to shed light on this issue. Accumulating evidence suggests that dysregulated microbiota and their metabolites caused by a diet with a higher percentage of fat can lead to intestinal mucosal barrier dysfunction by affecting the programmed death of intestinal epithelial cells, reducing the secretion of goblet cells and Paneth cells, impairing intercellular connections, and disrupting the immune balance .While the VLCKD diet can show a positive impact on the weight of patients with obesity, as well as represent a helpful approach for NAFLD , its colThus, for future research, it will also be mandatory to assess whether these changes in intestinal barrier function and integrity persist over time, as they may only be temporary. If so, they should be considered a small price to pay for the immediate beneficial effects of the ketogenic diet on the weight and metabolism of patients with obesity.This study has some weaknesses. Firstly, the present results are derived from a pilot study, so the size of the tested population prevents us from drawing firm conclusions. Secondly, the onset of GI symptoms, impaired s-IP, and the role of the indices of mucosal integrity need further study. Third, the suggested fermentative dysbiosis found with urinary indican evaluation was not supported by data from other, more appropriate methods because bacterial populations in the GI tract have not been adequately analyzed . Another weakness of this study is that we did not measure the ketogenic ratio of the diet, as suggested by Zilberter et al. . HoweverOverall, the evidence provided here suggests a relationship between the intestinal barrier, the ketogenic diet, and its impact on the gut microbiota in patients with obesity. The main finding of this study shows that obese patients, after 8 weeks of the VLCKD diet, presented a compromised intestinal barrier, dysbiosis, and elevated serum levels of LPS. However, this behavior was not common for all the patients. Therefore, while the VLCKD diet can show a positive impact on the weight of patients with obesity, its effects on the gut microbiota composition and function, as well as the integrity of the gut barrier, may raise concerns. VLCKD may likely represent a triggering factor capable of worsening a compromised intestinal balance, such as that in patients with obesity. Further research is needed by recruiting more patients and extending the observation time to fully understand the role of the intestinal barrier and gut microbiota variations during VLCKD.


Galeus melastomus) from various locations in the Mediterranean Sea. This research focuses provides insights into the distribution and accumulation patterns of these elements in G. melastomus and sheds light on the potential risks posed by chemical contamination in the Mediterranean Sea. The study underscores the significance of investigating the impacts of pollutants on marine organisms, particularly sharks, to develop effective conservation and management strategies. The data contribute to our understanding of trace element bioaccumulation in elasmobranch species, thus highlighting the importance of protecting these key ecological players in marine ecosystems.This study examines the bioaccumulation of 17 trace elements  in the muscle tissue of the blackmouth catshark  from different areas in the Mediterranean Sea. Trace elements are of interest due to their persistence, toxicity, and potential for bioaccumulation. This research aims to assess the distribution and accumulation of trace elements in the muscle tissue of G. melastomus and investigate their potential impact on the deep-sea environment of the Mediterranean. The focused areas include the Ligurian Sea, the northern and central Tyrrhenian Sea, the southern Tyrrhenian Sea, the Ionian Sea, the Pantelleria Waters, and the Gela Waters. Samples were collected following established protocols, and trace element analysis was conducted using inductively coupled plasma mass spectrometry. The study provides data on the concentrations of 17 trace elements, namely aluminum, arsenic, cadmium, cobalt, copper, manganese, molybdenum, nickel, zinc, selenium, strontium, lead, chromium, iron, barium, bismuth, and uranium. The findings contribute to a better understanding of trace element bioaccumulation patterns in elasmobranch species, specifically G. melastomus, and highlight the potential risks associated with chemical contamination in the Mediterranean Sea. This research emphasizes the importance of studying the impacts of pollutants on marine organisms, particularly those occupying key ecological roles, like sharks, to support effective conservation and management strategies.Environmental pollution, particularly in the marine environment, has become a significant concern due to the increasing presence of pollutants and their adverse effects on ecosystems and human health. This study focuses on the bioaccumulation of trace elements in the muscle tissue of the blackmouth catshark ( Environmental pollution is an issue of particular interest to the scientific community due to the drastic increase of pollutants in the environment observed in the last decade . PollutiTrace elements are frequently assessed due to their high persistence, toxicity, and bioaccumulation affinities . The WorTrace elements adsorbed onto aeolian particles and river particulates  are capaThe tissue accumulation of trace elements depends on an organism’s water uptake. It can occur either via direct contact  or diet . SeveralSharks are mostly long-lived elasmobranch species and occupy apex–meso-predator roles in their respective food webs . TherefGaleus melastomus  in the South of Sicily [G. melastomus feeds mainly on shelf-living species and natantian and reptantian crustaceans, as well as with teleosts and occasionally galley leftovers [Available information regarding the bioaccumulation of pollutants in elasmobranchs is currently limited to either specific locations of the world or to a restricted number of species ,33,34. Df Sicily ,41,42. Tf Sicily ,44, evenf Sicily ,43,45,46eftovers  and eveneftovers . Given tThe study area is the western–central Mediterranean Sea, which includes the northern Tyrrhenian and Ligurian Sea , the central and southern Tyrrhenian Sea (GSA 10), the Ionian Sea (GSA 19), and the South of Sicily (GSA 16) . The norG. melastomus were collected following the Italian law “Decreto Legislativo 4 marzo 2014, n. 26”, which implemented the “European Directive 2010/63/UE” and is recognized as using the guidelines put forward in Ebert et al. [The specimens of the GSA9 and GSA16 were collected during the international bottom trawl survey in the Mediterranean (MEDITS), while the samples of the GSA10 and GSA19 were collected via commercial bottom trawling. In t et al. .The specimens—previously stored at −20 °C—were thawed at room temperature for 24 h prior for further manipulation . Each sa4) to “chemically scrub” polyatomic or isobaric species from the ion beam before they enter the analyzer, resulting in improved detection limits for traditionally difficult elements, including As, Cr, Fe, Se, and others. The plasma torch, however, is powered using Argon with a purity of 5.00. The strength of this equipment is its fast multiplexing, allowing the acquisition of the data of several elements at once with high precision.The analysis provided the concentrations, expressed in ppb (parts per billion), of 17 trace elements: aluminum (Al), arsenic (As), cadmium (Cd), cobalt (Co), copper (Cu), manganese (Mn), molybdenum (Mo), nickel (Ni), zinc (Zn), selenium (Se), strontium (Sr), lead (Pb), chromium (Cr), iron (Fe), barium (Ba), bismuth (Bi), and uranium (U). This analysis was conducted using an inductively coupled plasma mass spectrometer . The DRC consists of a quadrupole placed inside an enclosed reaction chamber. This quadrupole eliminates polyatomic interferences caused by the combination of plasma gases and sample matrix constituents before they can enter the analyzing quadrupole. This ion-molecule chemistry uses a reaction gas (CH3 (64–69%) was added to the tissue, the vessels  were installed on a carousel and placed inside an EMC microwave oven (Mars 6). A program that provides a rising temperature ramp up to 180 °C was then applied for 20 min, followed by 20 min of cooling. After cooling, the vessels were worn out. (d) Evaporation—the content was transferred from the digestion vessel into larger vessels, taking care to rinse any residue with ultra-pure water (UPW). These vessels were then placed on a hot plate at 200 °C for about 2–3 h (t changes according to the starting volume) in order to eliminate acid fumes and to avoid damage to the instrumentation used for analysis. (e) Make up to the mark—the concentrated solution obtained by evaporation was transferred into glass matrasses and diluted to the volume of 100 mL using ultra-pure water. The solution was then homogenized inside the matrasses by shaking  in order to avoid the uneven distribution of the elements at the bottom of the flask. (f) Storage—the solution was temporarily stored in sterile containers at 4 °C until analysis.Prior to analysis, in order to solubilize the samples, muscle tissue samples were chemically digested, according to the protocol of De Donato et al. , and is 3:10 mL UPW). The internal standard (Re) was added to both blank, standard, and other solutions. The ICP-MS was calibrated using the ICP multi-element standard solution VI for ICP-MS (Merck). The accuracy of the method was evaluated using the Lobster Hepatopancreas  as the certified reference material (CRM). The same preparation procedure was used on the CRM and these solutions were used as unknown samples during the analytical sequence. During the analysis, the blank and the CRM were processed by the ICP-MS at the beginning and at the end and were also interspersed with every six samples . Accuracies were better than 8%, with most elements within ±5%. The detection limit was set at three times the standard deviation of the blanks and its value oscillated around 0.2 mg/Kg.During phases (a) and (b), the muscles’ water percentage was also measured, thus allowing the conversion of the dry weight values, returned by the ICP-MS, to wet weight (mg/Kg), using the equation of Gaion et al. :WW = DW p > 0.05), not quite significant , significant , very significant , and extremely significant . In addition, the trace element pollution index (TEPI) was calculated as stated by Richir and Gobert [n is the normalized mean concentration of the trace element—calculated as the mean concentration of each TE per location divided by the overall mean of that TE—and n is the total number of examined potentially toxic elements [For the comparison of trace elements mean values between sexes and among areas, the Mann–Whitney U-test and Kruskal–Wallis test were used, respectively, with Dunn test as the post hoc test. To investigate a possible correlation between trace element concentration and the growth of the species, the bioaccumulation of each area was compared to the TL of the specimens by means of linear regression. All statistical analyses were carried out using the Instat 3.0 program. We considered five significance levels: no significance  and 10 in the sub-adult size class (23 cm > TL > 34 cm), according to Ebert et al. . The meaThe results obtained for 17 trace elements were grouped by sex and areas. The average values of DW concentrations (mg/Kg) were summarized in p < 0.01)  . The spep < 0.0001), while the specimens from Gela Waters (GW) showed a statistically significant difference in six elements . Moving to the Tyrrhenian Sea areas (GSA 9 and 10), specimens from ST showed no statistically significant differences, whereas those caught in CT, NT, and LS areas displayed statistically significant differences in five , three , and six  elements, respectively. Finally, the specimens originating from the GSA 19 (IS) differed in two elements .As for the comparison of the trace elements between different areas , specimeRegarding the relationship between trace elements and the growth of the species, As was the only significant element in every area, with a positive trend , while five elements  showed a negative trend or no trends across all sites. Specimens from CT showed the greatest statistical significance—in nine elements—and the greatest number of negative trends. In general, despite slight differences between areas, more negative correlations were observed. Statistical results are summarized in Scales of abundance for each sex, sampling areas, and overall amounts  are obtaTaking into account the most abundant elements found  and the two elements within the Regulations EC No 1881/2006 (Cd and Pb), we calculated the mean concentrations for the WW and determined the percentages of contribution for each area . The 100The water percentage in muscles was found to be 70.45% (average), then the average values of the concentrations for the WW were obtained.TEPI was calculated and reported in G. melastomus in the Mediterranean Sea are indeed present in the literature, e.g., [Although previous studies on bioaccumulation for e, e.g., ,60,61, oMales showed a trend for higher mean values for the concentration of trace elements than females, supporting previously published findings that described a lower bioaccumulation in females of many species.Energy investment during reproduction, the size at which they reach sexual maturity, the energy cost of gamete synthesis, and the transfer of contaminants during oocyte development, have all been suggested as putative explanations for the observed sexual dimorphic variation ,62.Interestingly, females showed a very high bioaccumulation of As, which is one of the most controversial elements. According to the WHO, As is classified as potentially essential. Its presence in the water can originate from both natural and anthropogenic sources, and numerous studies investigating human toxicity are underway. Storelli et al.  had alreBoth sexes showed an identical bioaccumulation of Pb with no significant differences. This non-essential element can be toxic to marine organisms as well as humans, even at low concentrations . The accIn terms of areas, CT and LS specimens showed the highest bioaccumulation (35.3% each), followed by IS specimens, then NT, ST and finally PW. These differences could be explained by the hydrodynamic conditions that characterize the various areas, as observed in other contexts ,69. NeveThe correlation between trace elements and growth has showed an overall negative trend, probably due to a change in diet (in cases where food was the main source), a possible “moving away” dispersal from the source or to the mechanisms of metabolization and the expulsion of pollutants that developed during growth . It is worth noting that while samples from most sites do not have a wide variety of lengths (TL > 34 cm), only the central Tyrrhenian Sea batch included sub-adults (23.6 < TL < 51.0 cm). The results of the present study showed values for Cd and Pb equal to 0.023 mg/Kg (WW) and 0.029 mg/Kg (WW), respectively. Both values exhibited below maximum levels for certain contaminants in foodstuffs  set by the “Commission Regulation (EC) No 1881/2006 of 19th December 2006”, suggesting that the levels of this trace element in blackmouth catshark in the western–central Mediterranean Sea are safe for human consumption.By comparing the As content in the blackmouth catshark with previous studies, we found that the mean value at our sites (2020–2021) is 27.004 mg/Kg WW (91.383 mg/Kg DW), as opposed to the quantified maximum value in the Aegean Sea of 13.07 mg/Kg WW reported in Storelli and Marcotrigiano  in otherFocusing only on the northern Tyrrhenian Sea, the elements As, Cd, Pb, and Cu appeared in amounts of 121.07 mg/Kg, 0.14 mg/Kg, 0.16 mg/Kg, and 1.75 mg/Kg, respectively. Previously, Gaion et al.  found grG. melastomus—one in the Mediterranean Sea (Gulf of Lions) and one in the Atlantic Ocean (Bay of Biscay). The comparison between the mean values of their populations (Atlantic and Mediterranean) and our seven areas from the western–central Mediterranean Sea showed higher concentrations for the specimens of the present study, i.e., Cd, Pb, Zn, and Ni, while the average of our values for Cu was lower than the average they obtained in the Mediterranean Sea.Mille et al.  analyzedFurthermore, the trace elements pollution index (TEPI) for the western–central Mediterranean Sea (0.873) estimated in the present study yielded results that were lower than those reported by Mille et al.  in the nIn conclusion, these results enrich our knowledge about this deep-water species and provide more insights into the condition of the Mediterranean Sea’s environment in terms of pollution by trace elements. This study, nevertheless, represents a starting point for further studies, aimed at deepening our understanding of what occurs in different tissues and what the consequences for human health could be.


We systematically reviewed the literature and performed a meta-analysis on the effects of speech therapy and phonosurgery, for transgender women, in relation to the fundamental frequency gain of the voice, regarding the type of vocal sample collected, and we compared the effectiveness of the treatments. In addition, the study design, year, country, types of techniques used, total therapy time, and vocal assessment protocols were analyzed.We searched the PubMed, Lilacs, and SciELO databases for observational studies and clinical trials, published in English, Portuguese, or Spanish, between January 2010 and January 2023. The selection of studies was carried out according to Prisma 2020. The quality of selected studies was assessed using the Newcastle–Ottawa scale.p = 0.6254). The type of sample collected significantly influenced the post-treatment voice frequency gain (p < 0.01). When the vocal sample was collected through vowel (p < 0.01) and reading (p < 0.01), the gain was significantly more heterogeneous between the different types of treatment. Phonosurgery is significantly more effective in terms of fundamental frequency gain compared to speech therapy alone, regardless of the type of sample collected (p < 0.01). The average gain of fundamental frequency after speech therapy, in the /a/ vowel sample, was 27 Hz, 39.05 Hz in reading, and 25.42 Hz in spontaneous speech. In phonosurgery, there was a gain of 71.68 Hz for the vowel /a/, 41.07 Hz in reading, and 39.09 Hz in spontaneous speech. The study with the highest gain (110 Hz) collected vowels, and the study with the lowest gain (15 Hz), spontaneous speech. The major of the included studies received a score between 4 and 8 on the Newcastle–Ottawa Scale.Of 493 studies, 31 were deemed potentially eligible and retrieved for full-text review and 16 were included in the systematic review and meta-analysis. Six studies performed speech therapy and ten studies phonosurgery. The speech therapy time did not influence the post-treatment gain in voice fundamental frequency .The online version contains supplementary material available at 10.1186/s13643-023-02267-5. Studies show that pitch is one of the main markers of gender in the voice of TW and that TW whose voices present a higher fundamental frequency  are on average 20% higher than in men [Changes in vocal resonance can contribute to the perception of a female voice in TW because, in cis women, the frequencies of formants , alone or combined . Howeverfo, those most cited in the literature are open techniques such as the cricothyroid approach and the modified laryngoplasty technique known as feminization laryngoplasty [2laser techniques [Among several surgical procedures available for altering goplasty , which achniques , 6. Theschniques . Howeverchniques . One stuchniques .fo; however, the satisfaction of individuals with their voices is not necessarily related to pitch. Other characteristics, such as intonation pattern, articulation, resonance, loudness, Fmpatterns, tongue placement in the oral cavity, airflow, pragmatics, and the way of speaking, are mentioned as important gender markers and are prescribed in VFT [Vocal complaints are common in the transgender, nonbinary, and gender-nonconforming (TNG) population . For mand in VFT , 4.When VFT is performed without the assistance of a specialized practitioner, the attempt to raise the pitch in a vocal apparatus with male anatomy can cause mild to moderate dysphonia or vocal tension. In addition, other aspects, such as vocal health, breathing exercises, and relaxation must be addressed, because TW is exposed to the same vocal risk behaviors of cisgender people, such as incorrect vocal use .fo(mean increase 106 Hz), as well as significant improvements in the degree of voice feminization and self-reported satisfaction [fo, and voice quality outcomes, as well as which vocal techniques are most effective alone or in conjunction with vocal surgery.The benefits of VFT before and after surgery combined with the Wendler glottoplasty technique were reported in a study with 10 TW. All experienced significantly increased sfaction . HoweverResearchers report that VFT and behavioral changes are of great relevance in the TW voice transition process and that surgery can be indicated as an additional . In addifo, cost, and complications of the procedures. The authors did not analyze the types of vocal samples collected. Another study [In a meta-analysis , concluder study  report tfo, fo range, vocal satisfaction, self-perception and listener perception of vocal femininity, voice-related quality of life, and social and professional participation. However, there is a lack of randomized controlled trials, small sample sizes, inadequate long-term follow-up, lack of control groups, and control of confounding variables. In addition, the last edition of the Standards of Care [Authors  cited se of Care  recommenfo and vocal tract resonance in voice and communication feminization/masculinization training programs, and provide preliminary evidence for more emphasis on vocal intensity and speech rate and for the importance of non-verbal communication targets in voice training programs and gender-affirming communication [Current studies , 15 provnication . In addinication , 19.fo gain and type of sample voice?” Does the VFT time influence the fo gain?Within this context, the underlying research questions of this systematic review were as follows: “What are the methods used in VFT for TW? What are the effects of VFT on the voice? What is the most effective approach (VFT or phonosurgery) concerning This paper is part of the project “A Voz na Disforia de Gênero”, approved by the ethics committee of Hospital de Clínicas de Porto Alegre, Brazil, under number: 04075/2014. The study protocol was registered at PROSPERO (CRD42017078446).In January 2023, two independent raters (KS and APVB) carried out a search of the PubMed, Lilacs, and SciELO databases for articles on the topic. The search strategy consisted of a combination of Medical Subject Headings (MeSH) descriptors and relevant keywords. The keywords used for the search were as follows: ((Transgender Persons) or  or  or (Gender Identity)) and ((Voice Training) or (Voice) or ). The searches were adjusted to meet the requirements of each electronic database. The following filters used: studies published in the last 13 years (2010.01.01 to 2023.01.01).fo and type of the sample voice.According to PRISMA guidelines (2020) Fig. , the selfo gain after the treatments and described the fo collection method.The inclusion criterion was articles in Portuguese, English, or Spanish, from the last 13 years, and, according to the PICO questions, having the theme VFT or VFTC, in TW, which contained the result of the fo of the voice post-treatment or when it did not mention the type of vocal sample collected; and (4) when the study sample was not composed only of TW.The study exclusion criteria were as follows: (1) when individuals in the treatment groups did not undergo speech therapy, only surgery; (2) when not all individuals in the study group or control group underwent VFT; (3) when the study did not describe the gain of the The selection of studies consisted of four stages, with the following hierarchy of eligibility: 1) We removed the reviews and books, (2) duplicates were removed, (3) screening of articles according to the PICO question by reading the title and abstract, and (4) full-text reading by two study researchers (KS) and (APVB) and application of exclusion criteria. The flowchart in Fig.  We removfo, pre- and post-fo data, and final fo gain, according to the type of the sample collected.A descriptive analysis of the selected articles began with the application of a protocol prepared by the researchers, designed to collect the following data: authors, year of publication, country, study design, sample size and age, type of procedure, VFT methods, time-course variables of therapy, evaluations performed, results, means of measuring The methodological quality of the studies was assessed by three previously trained, independent reviewers using the Newcastle–Ottawa Scale (NOS) . The methodological quality score of the cohort and case–control studies was calculated in three components: selection of the groups (0–4 points), quality of adjustment for confounders (0–2 points), and evaluation of exposure after the outcome (0–3 points). The maximum score is 9 points, which represents high methodological quality.fo gain, with a 95% confidence interval (CI), was estimated using a random effects model. We assessed heterogeneity between studies with I2 > 50% suggesting moderate heterogeneity and p < 0.10 on Cochran’s Q test indicating significant heterogeneity [fo gain, in relation to the type of vocal sample collected , as well as the effectiveness between treatments, subgroup analysis was used. To evaluate the influence of treatment time (in sessions) concerning voice fo gain, meta-regression analysis was used. A p value of less than 0.05 was considered statistically significant. Statistical analyses were performed using R version 4.2.0 (http://www.r-project.org). The meta package (version 5.2–0) for doing meta-analysis was used within the R environment.The mean difference of ogeneity . The folTable Of the 16 studies included  [As for vocal assessment, some studies performed an acoustic voice analysis through a collection of the e (TWVQ) , 31, 32 fo and the type of vocal sample collected . As for the gain of fo, one study about anterior glottic web formation assisted by temporary injection augmentation [Figures entation  obtainedentation  reportedfo (p < 0.05), regardless of the type of vocal sample collected. The study [fo 24 Hz in the control group), used spontaneous speech [Among the studies that performed phonosurgery fo , with less heterogeneity in gain between studies when collected with spontaneous speech  was found in two studies [Two studies , 21–24 tch Figs. , and 7.  studies , 23, in  fo gain result. Meta regression p = 0.6254, coeficient: − 0.1280 .Figure Regarding the assessment of methodological quality, Table fo of the TW voice; however, phonosurgery provides a significantly greater gain. In both treatment approaches, the smallest gains occurred in the spontaneous speech sample, and even so, the individuals reached the frequency range where the voice could be perceived as female. In addition, the gain results are more homogeneous when spontaneous speech is used. These results provide important guidelines for clinicians who work with the transgender population since vocalizations with reading, automatic sequences, or vowels are situations in which TW can maintain greater self-control of vocal behavior than during spontaneous speech , and report the evaluation protocols, instruments, and techniques used for the feminization of TW’s voices and the quality of selected studies by Newcastle–Ottawa Scale. Both VFT and phonosurgery resulted in a significant increase in the ch Figs. , and 7.fo in a spontaneous speech reported by the different studies analyzed. Thus, it is suggested that isolated or post-surgical speech therapy focuses on prolonging or intensifying learning and the domains of vocal behaviors that facilitate the increase and maintenance of an fo closer to that obtained during automatic speech.In spontaneous speech, attention may be more focused on the message and interaction with the listener than on sustaining vowels, automatic speech, or reading; even after phonosurgery, several behavioral adjustments are necessary to ensure vocal adequacy. The focus on the message can divert attention from vocal production and justify the smaller gains in fo gains, fovalues at follow-up, and perceptual analyses concerning femininity and masculinity corroborate the indication of VFT. One study notes that the literature supports both VFT and phonosurgery, depending on the magnitude of pitch gain desired by the client, costs, and possible complications. In addition, the authors concluded that VFT provides high vocal satisfaction to customers, is effective at increasing the pitch, and is not invasive [While studies of VFT were generally of small sample size and not randomized, the positive results in post-VFT invasive . Studiesinvasive , 15, 24 invasive .fo gain was not significant , VFT was performed only before surgery, and the duration of therapy was not mentioned  and post-treatment ant Fig. . Nonethesessions . Anotherions [fo 10 Hz, VFRegarding vocal assessment, the studies were uniform in terms of auditory-perceptual assessment through the application of the CAPE-V and GRBAS protocols Table . In thisfo  and measurement of the mean frequency range, perturbation, and noise . However, differences in study results and difficulties in comparing the analysis of VFT effects concerning those of phonosurgery can also be related to the various programs used in data extraction, forms of recording, environmental noise, or cultural factors that can affect amplitude and fo [fo, to compare results and the use of spontaneous speech in the sample collection, as it better translates the patient’s speech and contributes to the generalization of the highest fo gain to spontaneous speech ; and /i/ from the selected semi-spontaneous Q/A set included Fmanalysis [Fm based on the source/filter theory of voice production. Thus, it is important to include an investigation of Fm through spectrographic vocal analysis in future protocols for voice evaluation in TW.In a study that set out to identify which are the predictive parameters of masculinity-femininity ratings by presenting auditory and visual cues for transgender and cisgender, the authors found statistical significance to re level . Indeed,re level  report tanalysis , 23. Oneanalysis . A reseaanalysis  used resThe most used self-assessment questionnaire among the included studies was the Trans Woman Voice Questionnaire (TWVQ) followed by the Transgender Self-Evaluation Questionnaire (TSEQ) and Voice Handicap Index (VHI) Table . The TSEBoth studies focusing on VFT , 23 and fo target for habituation and facilitation of oral resonance. A target fo was chosen for each person based on age, vocal range, and initial fo measurements. Afterward, words starting with /n/, /l/, and /r/ were introduced, followed by phrases with the objective of training intonation, pitch, and vocal quality, and, finally, multiple sentences involving descriptions of figures, roleplaying, and open-ended questions. Two studies [Real-Time Pitch Software from Multi-Speech for immediate feedback on the appropriate frequency and production of smooth voice quality  method was used in two studies , 23. Ste studies , 23 alsoty Table .fo, and practicing with phrases, sentences, and paragraphs [Among the six studies that performed VFT, three used intonation as one of the focuses of therapy. The authors showed that intonation was the target of VFT in 68% of cases; during VFT, the client should demonstrate an appropriate vocal variety and increase in rising intonation, regardless of the objective of increasing ragraphs . Researcragraphs , but it As for resonance exercises, the three studies of VFT and two of phonosurgery recommended this modality. The authors  reportedFm, in addition to an increase in fo [Fm  of vowels /a/, /i/, and /℧/ and fo increased after VFT, with the change in F3being statistically significant. In addition, most TW were perceived as more feminine by others and through self-assessments and were more satisfied with their voices [Fmdiffers between genders; however, further research is needed to prove the effectiveness of the method for TW. One study recommended the use of vocabulary, pragmatic [The results of one prospective study found that the TW voice was perceived as female for vowels that had increased frequencies in the se in fo . In a stse in fo  on the er voices . Oral reragmatic , and tworagmatic . There iragmatic .fo gain of up to 107 Hz [fo (110 Hz) with pre-surgery VFT and anterior web formation with injection augmentation. The lowest gain of fo was collected through spontaneous speech, using VFT (15 Hz) [fo when combined with surgery (107 Hz with VFT vs. 76 Hz without VFT), corroborating the results of this study, which showed that phonosurgery is more effective than speech therapy alone, in relation to the gain in fo. One research reported that endoscopic shortening was more effective in raising fo, with an increase of more than 70 Hz, versus a change from 26 to 40 Hz achieved by VFT and other surgical options [As for surgical techniques combined with VFT, Wendler glottoplasty was the most cited, in seven studies with an o 107 Hz  (see Addo 107 Hz  obtained (15 Hz) . A reser7 Hz [fo 0 Hz with options .fo was collected, which acoustic analysis software was used; sample sizes are small, and there are biases in the selection of control groups, as well as the lack of detailed information about the speech therapy steps and techniques used. This made it difficult to compare results across the included studies.The authors  state thfo analysis methods of each study. As we verified differences between the different types of collected voice samples, it is not possible to group all the results without considering these differences.The limitations of the present review are the small number of published articles, their small sample sizes, and the differences in the fo of the voice, regardless of the type of vocal sample collected, with phonosurgery having presented a significative greater gain of fo. The type of vocal sample collected significantly influenced the result of fo gain after treatment. The speech therapy time did not influence the post-treatment gain in voice fo. The quality of evidence of the studies was low, given the lack of randomized controlled trials, the small sample sizes, and the different methods of collecting fo. Thus, this study may provide additional evidence on the role of VFT and phonosurgery on voice feminization and encourage further research in the field.Both VFT and phonosurgery showed an increase in the Additional file 1. Effects of voice feminization therapy on fo in transgender women Legends: NA: not available; SD: standard deviation; fo: fundamental frequency of voice. * Statistical result found by the authors of the study cited in the comparison of fo gain between the pre and post treatment or Significant effects for therapy time versus fo significant effects for time versus fo 15.Additional file 2. Effects of phonosurgery on fo in transgender women Legends: SD: standard deviation; fo: fundamental frequency; * Statistical result found by the authors of the study cited in the comparison of f0 gain between the pre and post treatment. * Wendler glottoplasty (WG) and its modification, the vocal fold shortening, and retrodisplacement of the anterior commissure (VFSRAC) associated with laser assisted voice adjustment (LAVA) cordotomy. VFT: Voice Feminization Therapy.


Anorexia nervosa (AN) is a severe and often chronic eating disorder that leads to alterations in the gut microbiome, which is known to influence several processes, such as appetite and body weight regulation, metabolism, gut permeability, inflammation, and gut–brain interactions. Using a translational activity-based anorexia (ABA) rat model, this study examined the effect of chronic food starvation, as well as multistrain probiotic supplementation and refeeding, on the structure of the gut and gut-associated lymphatic tissue (GALT). Our results indicated that ABA had an atrophic influence on intestinal morphology and increased the formation of GALT in the small bowel and colon. Higher formation of GALT in ABA rats appeared to be reversible upon application of a multistrain probiotic mixture and refeeding of the starved animals. This is the first time that increased GALT was found following starvation in the ABA model. Our results underscore a potential role of gut inflammatory alterations in the underlying pathophysiology of AN. Increased GALT could be linked to the gut microbiome, as probiotics were able to reverse this finding. These results emphasize the role of the microbiome–gut–brain axis in the pathomechanisms of AN and point to probiotics as potentially beneficial addendum in the treatment of AN. Anorexia nervosa (AN), a severe eating disorder, is mostly prevalent in girls and young women. It is characterized by restricted energy intake associated with extreme underweight, disturbed body perception, and anxiety about gaining weight . The cliTo study AN, the activity-based anorexia (ABA) animal model is most commonly used and well established because it mimics the main characteristics of AN, such as loss of body weight, increased physical activity, amenorrhea, and brain volume loss, by combining food starvation with running-wheel activity ,9.In recent years, several studies have provided evidence that periods of starvation are associated with alterations in the gut microbiome and significant shifts in its diversity in patients with AN and ABA animals ,12,13,14Over the past 40 years, there has been increasing research, suggesting that probiotics can influence the immune system both in vivo and in vitro. According to the World Health Organization, probiotics are defined as “live microorganisms which when administered in adequate amounts confer a health benefit on the host” .This interaction appears to be linked to the gut microbiota, as well as polysaccharide antigens and metabolites produced by these bacteria. For instance, Bifidobacteria has been found to compete with pathogenic bacteria in vitro, while a probiotic mixture comprising Lactobacilli, Bifidobacilli, and Streptococcus thermophilus has been shown to ameliorate ulcerative colitis, an inflammatory bowel disease ,32. FurtBased on these studies, we raised the hypothesis that administration of probiotics could potentially be beneficial as add-on treatment for AN, affecting the gut microbiome, intestinal morphology, formation of GALT, and thereby intestinal permeability . Some hu2, Polysulfone, Tecniplast). The cages were enriched with sawdust bedding, cotton wool pads as nesting material, and an in-cage running wheel. The room was maintained at a constant temperature (22 °C) and humidity (55%) and under a 12-h light/dark cycle (light on at 7 a.m.). During a daily handling procedure until the end of the protocol, the body weight, food intake, and running wheel activity  were recorded for each rat; 31 days delineated the chronic starvation protocol, and 48 days delineated the refeeding protocol  were commercially purchased and arrived in the animal facility with an average body weight of 103.98 g ± 8.14 g. The rats were single-housed under specific pathogen-free conditions according to DIN ISO 9001:2008 in type IV cages , activity-based anorexia , and activity-based anorexia treated with probiotics (ABA + P n = 12) see A. AnotheThe animal trial was approved by the Governmental Animal Care and Use Committee . All experiments were performed in accordance with the German legislation governing animal studies, following the Guide for the Care and Use of Laboratory Animals  and the 2010/63/EU Directive on the Protection of Animals Used for Scientific Purposes .®: Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium infantis, Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei, and Lactobacillus delbrueckii subsp. bulgaricus) [9 living bacteria) on two consecutive days, as soon as the target weight was reached. For refeeding studies, ABA rats received two weeks of ad libitum access to food after the chronic starvation protocol. At the end of the respective protocol the rats were anesthetized with an overdose of isoflurane  until reflexes (tail pinch and paw pinch reflex) were no longer measurable. Body weight and running wheel activity were averaged per group and separated into the different phases of the experimental protocol ). Both parameters were normalized to the baseline measured during the habituation period. For a graphical illustration of the study design, see A chronic version of the ABA model was used, as previously described . In briegaricus) . All ratAfter transcardial perfusion with artificial cerebrospinal fluid (pH = 7.4), intestinal tissue from the small intestine and the colon was extracted. Small intestine samples were taken from the duodenum, right behind the stomach. Duodenum and colon samples were dissected with scissors and washed with a 3.7% paraformaldehyde solution (pH 7.4) using a gavage syringe. For staining, the tissue specimens were rinsed with 3.7% paraformaldehyde solution (pH 7.4) for postfixation, dehydrated by an increasing alcohol/xylol series, and embedded in paraffin . Finally, samples were cut on a microtome into 5 µm sections. From each animal, four sections of the duodenum and four sections of the colon were stained with hematoxylin and eosin. After deparaffinizing, sections were incubated in 0.1% hematoxylin for 4 min, followed by washing with running tap water for four minutes. Afterwards, sections were incubated in 0.2% eosin for 30 s, dehydrated in an ascending alcohol/xylol series, and mounted with DePeX .Stained slices were digitalized under a light microscope (Nikon Eclipse 80i) connected to a camera (Nikon DS-2Mv). The depth of the crypts, thickness of the lamina mucosae, and the thickness of the lamina muscularis of the colon, as well as the length of the villi and lamina muscularis of the duodenum and the surface area of GALT, were traced with the selection tool in ImageJ software . Moreover, the incidence of GALT in each slice was counted. If a GALT plaque was present, it was additionally recorded whether the plaque reached the lumen by disrupting the lamina mucosae in the colon.g for 15 s, RNA was isolated by phenol-chloroform extraction using peqGold RNA TriFast , as previously described [For molecular analysis, intestinal tissue samples were snap frozen in liquid nitrogen and kept at −80 °C until further processing. After tissue collection in cryo-tubes containing 1.4 mm ceramic beads and homogenization in a FastPrep  at 5000× escribed . The purt-test for comparison between two groups (C_ref and ABA_ref). Differences in running wheel activity before starvation and during starvation for the ABA group were also calculated by a Student’s t-test.Differences in body weight and gut morphology  between three groups  were evaluated in a one-way analysis of variance (ANOVA) test with a Bonferroni post hoc test and Student’s The incidence of GALT was analyzed in a linear mixed model with the assumption that missing values occurred at random, as the tissue slices could not be defined as independent. All statistical analyses were performed with SPSS version 25 . The significance level was set to 5%. The results are presented as the means and standard errors of the mean (±SEM).p = 0.0001, p = 0.0001, As expected, the average body weight (p = 0.003). A similar pattern was observed during starvation in ABA animals in the refeeding group (p = 0.0001). Toward the end of the chronic starvation, RWA somewhat decreased, probably to prevent exhaustion, but settled on a still increased level. At the beginning of refeeding, RWA returned to baseline during the first few days, and then continuously grew again and on average increased compared to baseline (p = 0.0001) ( 0.0001) C. These p = 0.0001 and p = 0.0012, respectively; All morphological parameters in both the colon and duodenum tended to be decreased in the ABA group due to starvation . In partp = 0.057, p = 0.042). Administration of probiotics led to significantly reduced GALT (incidence) in the duodenum  with a similar effect in the colon . This frequency of breakthroughs was significantly lower after probiotic application in the ABA + P group (p = 0.016), again pointing to a potential positive effect of probiotic supplementation  due to atrophy caused by food restriction and running wheel activity with a consequential increase in bacterial parts or products traversing the gut wall and leading to local immune reactions. Probiotic applications associated with a reversal of gut morphological aberrations and possible reduction in immune reaction might thus be a promising route to explore for future additions to AN treatment.Food starvation-induced gut atrophy and increased gut permeability were previously observed in male ABA mice by Jésus et al. 2014), suggesting that intestinal barrier dysfunction, influenced by alterations in tight junction protein expression, might be involved in the pathophysiology of AN . Another14, suggeGenton et al. (2015) also determined an increased intestinal permeability due to caloric deprivation both in animals by measuring FITC-dextran fluxes, as well as in human probes by measuring their urinary recovery . InteresIn our chronic starvation model, we detected gut atrophy to a higher extent in the colon than in the duodenum, which mirrors the findings of Jésus et al. (2014). This might be because of a significantly higher bacterial number and diversity in this region of the gut. Recent studies have postulated that the gut microbiome plays an important role in AN, as alterations in its diversity were found in patients with AN, as well as in ABA animals compared to controls . A possPotentially related to alterations in inflammatory processes and increased permeability, our results showed more and larger aggregated lymphatic tissue in ABA animals compared to controls. The assumption that RWA and food starvation have an influence on the formation of lymphatic plaques was already raised in a historic study from 1977 by Pare. Rats increased their physical activity due to food restriction, and Adan et al. (2010) postulated that this hyperactivity in acutely starved animals is associated with foraging behavior . FurtherOur present research showed that administration of probiotics was associated with a significant reduction in aggregated lymphatic plaques in the small bowel in ABA animals. A systematic review by Hungin et al. (2018) proposed the use of probiotics as a therapeutic option in somatic diseases due to their beneficial effects on gastrointestinal functions . In addiBecause weight gain, however, is often problematic and difficult to achieve in patients with AN, supplementation with probiotics might be an interesting additional option to help normalize the intestinal system.L. bulgaricus, S. thermophilus) has shown improvement in immune response in adolescents with AN or malnourished children [Currently, only preliminary evidence exists on the use of probiotics in AN. Supplementation with probiotic yogurt  currently examines the clinical efficacy of the administration of multistrain probiotic mixture, similar to that used in this study, and omega-3 polyunsaturated fatty acids in adolescent patients with AN, hypothesizing an improvement in neuropsychological functioning in these patients compared to controls receiving placebos ,72. OtheThe ABA animal model is a well established experimental model that mimics psychological and physiological characteristics of AN and was successfully applied in the present study . NeverthFood restriction and increased locomotor activity had an influence on gut atrophy and the formation of GALT in this animal study, which fits well with previous research data and might help to explain potentially increased gut permeability. For the first time, we showed increased formation of GALT in the ABA model, which could be induced by more bacterial parts and products traversing the gut wall. These lymphatic plaques might also play a role in the dysregulated balance of pro- and anti-inflammatory cytokines found in patients with AN. Furthermore, our refeeding study supports the importance of weight restoration as a key factor in the treatment of AN, as morphological parameters and growth of aggregated lymphatic plaques seem to be reversible due to refeeding. The observed alterations in the lymphatic plaques were further improved by administration of multistrain probiotics. These results help to explain important parts of the underlying pathophysiology regarding gut permeability and inflammation in the ABA model and potentially AN, and they might help to develop microbiome-targeted interventions, such as probiotics, to be added to AN treatment.


Data for 82,960 patients with PV were extracted: 3852 patients were treated with hydroxyurea (HU) only, while 130 patients were treated with HU and then changed to ruxolitinib (HU-ruxolitinib). For HU-alone patients, the annualized incidence rates  decreased from 8.7 (before HU) to 5.6 (during HU) but increased markedly to 10.5 (continuing HU). Whereas for HU-ruxolitinib patients, the IR decreased from 10.8 (before HU) to 8.4 (during HU) and was maintained at 8.3 (after switching to ruxolitinib). To better understand markers associated with TE risk, we built a machine-learning model for HU-alone patients and validated it using an independent dataset. The model identified lymphocyte percentage (LYP), neutrophil percentage (NEP), and red cell distribution width (RDW) as key markers of TE risk, and optimal thresholds for these markers were established, from which a decision tree was derived. Using these widely used laboratory markers, the decision tree could be used to identify patients at high risk for TE, facilitate treatment decisions, and optimize patient management.Patients with polycythemia vera (PV) are at significant risk of thromboembolic events (TE). The PV-AIM study used the Optum Polycythemia vera (PV) is a chronic myeloproliferative neoplasm characterized by erythrocytosis and driven, in almost all cases, by mutations in the JAK2 gene . PatientRisk stratification in PV is designed to estimate the likelihood of TE and includes two risk categories based on age and prior history of thrombosis: high-risk (≥ 60 years old and/or with a history of thrombosis) and low-risk (< 60 years of age with no history of thrombosis) . Higher Hydroxyurea (HU) is a commonly used first-line cytoreductive therapy for high-risk patients with PV . However® de-identified Electronic Health Record (EHR) dataset, a large database in the United States, to investigate the incidence of TEs in patients with PV treated with HU and those who switched to ruxolitinib and apply machine learning techniques to identify individuals at risk of TE and potential markers of TE risk. Ultimately, this study aims to provide physicians with the tools to support the effective therapeutic management of individuals with PV and the potential need for timely or proactive change in therapy to reduce a patient’s long-term risk of TE.Identifying patients with PV at risk of TE and potential markers of TE risk would support the effective therapeutic management of individuals. Machine learning techniques have been used to support the focus of a differential diagnosis, the selection of therapy, and the generation of risk predictions and are increasingly being applied to different areas of hematology, including the management of hematological malignancies ,17. The ® EHR database  included pre- and post-index periods, where the index was the first date of HU prescription/administration. Study designs for the analyses are shown in ® EHR dataset, considering patients’ clinical, laboratory and therapeutic variables, and to identify novel interactions between patient variables that may act as potential drivers or markers for TE.The initial objective was the evaluation of the incidence rate (IR) in patients treated with HU  and those who changed to ruxolitinib after HU treatment (HU-ruxolitinib), which led to the key objectives that focused on using machine learning techniques to predict the occurrence of TE in patients treated with HU in the extensive and diverse Optum® EHR patient data were de-identified and, therefore, Institutional Review Board/Ethics Committee approval was not required. Approval from the Ethical Committee of the General Hospital of Sibenik-Knin County, Sibenik, Croatia was received (22 December 2020) to use patient data from an independent PV registry in Croatia (Reference number 01-22812/1-20); due to the retrospective design of the study, patient consent was waived by the Ethics Committee for this registry and was not required for Optum® EHR patient data. See All Optumhttps://cran.r-project.org/web/packages/MatchIt/index.html accessed on 21 June 2023) [Patients in the HU-alone and HU-ruxolitinib groups were matched using propensity score matching, which accounted for the treatment duration and demographics of patients using the RMatchIt package  . TE were® EHR database  model was constructed using the demographic, clinical and laboratory data extracted from the Optumdatabase  for patidatabase B. The moBased on the multiple patient variables included in the model , the inbp value) of these two-variable splits was measured by log-rank and generated four groups, or quadrants, from the combinations of “high” and “low” groups for both variables. Rather than assessing a single split threshold using the medians of the two variables only, multiple thresholds were assessed, from which a matrix of p values was generated. These matrices were visualized as “heatmaps,” such that regions of significance could easily be identified. The most significant points in this “risk landscape” were identified, and based on these outcomes, clinical “decision trees” were developed.To investigate patients’ risk of TE, pairs of variables were assessed for the “best split” based on the significance of their interactions; the significance .® EHR database were assessed in relation to thrombosis-free survival (TFS) in patients with and without a prior TE history. See Eligible patients were aged ≥ 18 years, had a diagnosis of PV (ICD-10 nomenclature), and had been treated with HU  . Key var® EHR database patient information for analysis. Probability curves were compared using Kaplan–Meier plots and log-rank tests, and variable interactions were assessed by a log-rank test . For analysis of the Optum® EHR database the ranger  and R  packages were used for survival and statistical analysis .Absolute values, yes/no or median data were extracted from Optumysis see . For the® EHR database, 82,960 patients had a diagnosis of PV (median record length of 8.4 years). Of these, 3852 HU-alone patients and 130 HU-ruxolitinib patients were eligible for analysis  cohorts were similar , the IR remained stable at 8.3; however, for those who continued HU , the IR appeared to rebound and increased markedly to 10.5 over the switch/no switch period .p = 4.6 × 10−4); physicians may have considered these patients sufficiently at risk of TE to warrant these assessments , lymphocyte percentage (LYP), and red cell distribution width  were the laboratory variables of the greatest importance for predicting TE  of NEP ≥ 72.05 and RDW < 14.3, and LYP < 19.3 and RDW < 14.05 in the Croatian dataset were predictive of inferior TFS outcomes in patients with no history of TE  [p = 0.03) [p = 0.05 and p = 0.03, respectively), and Hct was lower with ruxolitinib versus the best available treatment in patients resistant or intolerant to HU [https://clinicaltrials.gov/ct2/show/NCT04116502 accessed on 21 June 2023). Collectively, observations from these different studies highlight the benefits of ruxolitinib on TE risk in patients with PV. For patients potentially at risk of TE while receiving HU, a change in therapy may be beneficial.The IRs of patients with PV in this study were nominally higher than those observed in a meta-analysis of 3236 patients with PV receiving HU , which mutations . We cannPONSE 2) ,23 and aPONSE 2) . Indeed,PONSE 2)  and an IPONSE 2) . Similar = 0.03) . This rent to HU . A large® EHR database and novel machine learning techniques to thoroughly analyze patient demographics, history, clinical observations, and laboratory outcomes and to identify the key pre-treatment factors most predictive of TE in patients on HU treatment. In this analysis, the model exhibited strong predictive power and identified notable synergistic associations between the pairs RDW and NEP, and RDW and LYP in patients without a history of TE, as well as the optimal thresholds for patients at low and high risk of TE. Leukocytes may have a causative effect in the initiation of thrombosis, with leukocytosis increasing the risk for thrombosis in patients with PV and ET [The PV-AIM study utilized the wealth of patient information available in the OptumV and ET . LYP, hoV and ET . In our V and ET  and the V and ET . The assV and ET ,32, and V and ET  and the V and ET  have beeV and ET , which sV and ET  are consV and ET .Of note was our finding that RDW is a significant factor in predicting TE occurrence. In patients with PV, high RDW has been associated with an increased risk of venous thrombosis  and poor® EHR database could be applied to the independent Croatia PV population, and these combinations of NEP, LYP, and RDW were able to correctly identify the patients with PV in the real-life community setting that were at increased risk of future TE, which supports the broad applicability of these findings to real-world data and registries beyond the USA.The synergistic variable pairs identified in PV-AIM have greater predictive potential than individual variables or other combinations and are of particular value in patients who would be considered at low risk for TE, based on their age and history of TE alone at the start of HU therapy. Collectively, the outcomes from PV-AIM and other studies ,30,36 hi® EHR database, strict inclusion and exclusion criteria were required to obtain a focused cohort of patients for the machine learning analysis, which substantially reduced the number of eligible patients. As such, this focused analysis population may have excluded some patients of interest that may have influenced the risk of TE, such as those on different anticoagulants or antiplatelet therapies. The Optum® EHR database includes routinely collected clinical data from a wide range of sources ; therefore, data may have been entered differently at the source with the possibility of missing, invalid, unrecorded, or unknown data, inaccuracies, and/or technical errors, but also possibly as a consequence of a subjective medical judgment of diagnosis, drug, and/or procedural codes. In addition, medication use may have been overestimated as there are no guarantees of patients being dispensed their medication or using their medication as prescribed. As such, HU treatment may be different between the HU-alone and HU-ruxolitinib groups. Despite these potential limitations, the outcomes from this analysis were validated externally through the Croatian database, which corroborated the overall PV-AIM findings.As expected, given the observational and retrospective nature of this analysis and the use of real-world data, we acknowledge some limitations to this analysis. The period from which patient data was extracted was prolonged, and physician treatment practices may have changed over this period; however, sufficient patient numbers were needed, and the required pre- and post-index periods were accommodated to ensure the quality and completeness of the dataset. Although data for a substantial number of patients with PV was available within the OptumThe identification of easy-to-determine laboratory markers that are predictive for TE risk in patients with PV and the development of clinically applicable decision trees present an exciting new opportunity for physicians to identify patients who do not have a history of TE but are potentially at risk of TE and would benefit from closer surveillance and follow-up. RDW, NEP, and LYP are routine laboratory parameters and, therefore, are inexpensive and practical tools in the clinic. Ultimately, early identification of “at-risk” patients and close monitoring during treatment provide a comprehensive and personalized approach to patient management, which may promote timely changes in treatment to prevent a major cause of morbidity and mortality. Machine learning techniques have proved to be a useful tool in this study, and further studies are now needed to refine the risk for arterial or venous thrombosis.