Background: The Special Supplemental Nutrition Program for Women, Infants, and Children (WIC) provides nutrition assistance to half of infants born in the United States. The nationally representative WIC Infants and Toddler Feeding Practices Study-2 (ITFPS-2) reported a caloric deficit at 7 months among infants receiving WIC mixed feeding packages, suggesting these infants may be at risk for growth deceleration/faltering.Methods: Longitudinal administrative data collected prospectively from WIC participants in Southern California between 2010 and 2019 were used . Infant lengths and weights were used to calculate weight-for-length (WLZ), weight-for-age (WAZ) and length-for-age (LAZ) z-scores at different time points. Growth deceleration/faltering was determined at 9, 12, 18, and 24 months by the change in z-score from the last measurement taken ≤ 6 months of age. Infant feeding was categorized by the food package  infants received from WIC at 7 months. Poisson regression models were used to evaluate the association between WIC infant package at 7 months and deceleration/faltering at 9, 12, 18, and 24 months.Results: The proportion of infants displaying decelerated/faltering growth was low for all infant food package groups. Receiving the WIC mixed feeding package at 7 months of age was not associated with WLZ, WAZ, and LAZ deceleration/faltering growth.Conclusions: Growth deceleration/faltering rates were very low among WIC participating children in Southern California, highlighting the critical role of nutrition assistance in supporting adequate growth in early childhood. The first 1,000 days of a child's life represent a critical period, with adequate nutrition imperative for healthy growth and development  until 1 The Special Supplemental Nutrition Program for Women, Infants and Children (WIC) is a nutrition assistance program of the federal government of the United States and is administered by the United States Department of Agriculture, serving over 6.2 million participants monthly in 2020 . For infThe nationally-representative WIC Infant Toddler Feeding Practices Study-2 (ITFPS-2) reported a substantial caloric intake deficit at 7 months of age among infants receiving mixed feeding packages, compared to infants receiving the fully breastfeeding or fully formula feeding packages . The repAdministrative data collected in Southern California between 2010 and 2019 by Public Health Foundation Enterprises (PHFE) WIC, the largest WIC local agency in the United States, were used in this study. Children were included in the analysis if they: were enrolled in WIC continuously from birth (within 42 days) to after 1 year of age, had complete WIC infant package data for 12 or more of the first 13 months of life; had at least two length and weight measurements separated by more than 3 months before 6 months of age and at least one length (or height) and weight measurement between 7 and 26 months of age, and; had complete sociodemographic covariate data . Infants born pre-term, with developmental delays, or with genetic or congenital conditions that may affect feeding or metabolism and consequently growth, including Down syndrome and cleft palate, were excluded from the sample.The WIC food package received at 7 months of age was the independent variable of interest in this study. It differed according to whether the infant was fully breastfed (“breastfeeding” package), partially breastfed (“mixed feeding” package), or fully formula-fed (“formula feeding” package). The breastfeeding package did not include infant formula; the mixed feeding package included some formula, with quantities varying based on the amount of breastfeeding reported; and the formula feeding package contained enough formula to meet the infant's age-specific nutritional needs, taking into account nutritional needs expected to be met by complementary foods beginning at 6 months of age .Covariates available from WIC administrative data include the child's age, birth year, sex, and caregiver-reported race/ethnicity ; maternal education  and preferred language ; and household income .Growth deceleration/faltering was the outcome of interest and was assessed using length (or height) and weight measurements collected by WIC during service delivery . Anthropometric indices- length-for-age (LAZ), weight-for-age (WAZ), and weight-for-length (WLZ)- were calculated from these measurements taken from birth to <39 months of age, using sex-specific CDC growth curves , 16. As n = 16,255) is shown in the To focus on the impact of the WIC infant food package received at 7 months on growth deceleration/faltering, infants who showed evidence of poor growth during the first 6 months of life, namely infants with any anthropometric index <2nd percentile or who showed a decrease in any anthropometric index percentile category between two measurements at ≤ 6 months of age, were excluded . The selP-values < 0.05 were considered statistically significant. All analyses were conducted using SAS 9.4 .The characteristics of children receiving breastfeeding, mixed, or formula packages from WIC at 7 months of age were described using means and standard deviations or frequencies and percentages, as appropriate. The associations between the WIC infant package received at 7 months and growth deceleration/faltering were evaluated in modified Poisson regression models with robust standard error estimation, which were used to calculate adjusted risk ratio (RR) estimates accommodating repeated measurements for children , 20. TheThe analysis was conducted on 16,255 children with 116,398 unique length (or height) and weight measurements. At 7 months of age, 17.0% of children received the breastfeeding package, 22.6% of children received the mixed feeding package, and 60.3% of children received the formula feeding package. Most children in each infant package type were Hispanic . ChildreAcross all ages of follow-up in this study (7 to <39 months), and for all infant package groups, a large majority of children demonstrated normal (−1 < z-score <1), high (1 ≤ z-score < 2) or very-high (2 ≤ z-score) values for all anthropometric indices . A smallDecelerated/faltering growth between ≤ 6 months of age and 7 to <10, 10 to <14, 14 to <18, 18 to <22, and 22 to <26 months of age was observed among 1.1–3.3% of children, depending on the age range and anthropometric index: for WLZ = 1.5, 1.3, 1.8, 1.9, and 2.0% in each age range, respectively; WAZ = 1.1, 2.3, 3.3, 3.0, and 2.4% in each age range, respectively; and LAZ = 1.5, 2.8, 2.6, 2.3, and 1.9% in each age range, respectively. A majority of growth deceleration/faltering for each anthropometric index was observed among children who were between the 25th and 75th percentile at ≤ 6 months of age .Risks of WLZ, WAZ, and LAZ growth deceleration/faltering were low for children who received each type of WIC infant package at 7 months of age. Risks of WLZ, WAZ, and LAZ deceleration/faltering were significantly higher among children who received the breastfeeding WIC package relative to children who received the mixed feeding WIC package . At 9 moPrompted by a reported caloric deficit at 7 months among mixed-fed infants participating in WIC , this stTo our knowledge, no prior evaluation of growth deceleration/faltering by food package provided by WIC has been undertaken. Moreover, no similar study comparing the impact of infant feeding on growth deceleration/faltering among a general  infant population was identified in the United States. However, a population-based study conducted among a cohort of all children in the catchment area of the Avon Health Authority in England identified failure to thrive between 8 weeks and 9 months of age in 4.2% of sampled children, which was more prevalent among children breastfed longer than 6 months . The metThe proportion of children who experienced growth deceleration/faltering was in line with previous estimates ; howeverChildren who received the mixed feeding package from WIC at 7 months of age had a lower risk of each type of growth deceleration/faltering at 9, 12, 18, and 24 months relative to children who received the breastfeeding package at 7 months. The reported caloric deficit among mixed-fed children from a nationally representative sample of WIC participants in 2013–2014 . PreviouDairy products, a component of most infant formulas, are associated with linear growth, which may contribute to differences in growth patterns between breastfed, mixed-fed and formula-fed infants . The conStrengths of this study include a large, well-characterized study population, with prospectively collected data for exposures and outcomes. The analysis was restricted to infants with stable growth trajectories at ≤ 6 months of age, and children below the 2nd percentile at ≤ 6 months of age were excluded from the analysis. Infants with known medical conditions which could contribute to nutritional risk and growth deceleration/faltering were also excluded from the analysis. The analysis controlled for potential confounding factors by sample restriction and statistical adjustment; however, there are also limitations. Data on infant diet and caloric intake were not available, so we were unable to evaluate the association between infant package and caloric intake; however, WIC infant package assignment has been identified as a valid proxy for infant feeding patterns . ChildreCaloric intake insufficient for growth maintenance leads to growth deceleration/faltering. Nutrition assistance from WIC aims to alleviate food insecurity and provide children in low-income households access to nutritionally adequate diets that will support growth. The low rates of growth deceleration/faltering identified among all children studied highlight the critical importance of nutrition assistance among WIC participants, and demonstrate the need for the provision of education and foods to support adequate nutrition in the early months of life. Breastfeeding provides many health benefits and represents the best infant feeding practice from a public health perspective . Additiodatamining@phfewic.org.The data analyzed in this study is subject to the following licenses/restrictions: the authors used confidential administrative data for this article, and are unable to make these data publicly available due to a memorandum of understanding with the California Department of Public Health WIC Division. Requests to access these datasets should be directed to the Data Mining Project, The studies involving human participants were reviewed and approved by University of California Los Angeles Institutional Review Board. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.MC, CA, and SW designed the study. MC, CC, and CA conducted the research and statistical analysis. SW provided essential materials. CA drafted the manuscript and CC, MC, MW, and SW edited the manuscript. All authors were involved in the interpretation of the results, approved the final version for submission, and agree to be responsible for its contents.Funding from a research partnership with First 5 LA (Contract 07030) supported the analysis time of CA.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.


Background: Breastfeeding is recommended due to its beneficial effects on human health. However, the effect of breastfeeding on health differs, resulting in various childhood diseases. Objective: Our purpose was to investigate the association between breastfeeding at least in the first 4 months and the subsequent development of 15 certainly defined childhood diseases until 10 years of age, the all-cause hospitalization rate and growth at 6–7 years of age. Methods: Participants included propensity-score matched 188,052 children born between January 2008 and December 2009, who were followed up till 10 years of age. Data were taken from the National Investigation of birth Cohort in Korea study 2008 database. Risk ratios were obtained using a modified Poisson regression and weighted risk differences using binomial regression. Results: Compared to formula feeding, breastfeeding was associated with decreased risks of febrile convulsion, attention deficit hyperactivity disorder and autism spectrum disorder, pneumonia, acute bronchiolitis, hypertrophic pyloric stenosis, asthma, all-cause hospitalization, overweight/obesity and short stature. Exclusive breastfeeding at 4 to 6 months of age had similar results to exclusive breastfeeding over 6 months of age. Conclusions: Breastfeeding in early infancy reduces the risk for various childhood diseases, all-cause hospitalization rate, obesity, and short stature during childhood. Breastfeeding is the prerequisite for human evolution in terms of infant nutrition and subsequent health effects in later life. The health effects of breastfeeding results in various childhood diseases at different ages. Breastfeeding in early life is known to have beneficial effects on various childhood diseases, including infectious diseases such as respiratory tract infections , whereasThe inconclusive results on the effects of breastfeeding on childhood health might be partially attributable to the differences in the characteristics of the study population, follow-up duration, mechanisms underlying the association of breastfeeding and specific childhood disease and/or duration of breastfeeding ,7,8,9,10Although the World Health Organization (WHO) guidelines recommend exclusive breastfeeding for 6 months, the recommendation strategies of breastfeeding and its duration for the prevention of allergic diseases, including food allergy or atopic dermatitis, have changed in the last decade according to the results of previous studies [Therefore, in the present study which includes a large number of children born between 2008 and 2009, we evaluated the association of breastfeeding in early life with common and important childhood diseases. The primary purpose of the present study was to investigate the association between breastfeeding and 15 childhood diseases in the nationwide cohort. The secondary purpose was to elucidate the association between breastfeeding and the risk of admission and need for intensive care during childhood. We also identified the association between exclusive breastfeeding in the first 4–6 months and over the first 6 months with childhood diseases.Information regarding the feeding type until the first 4 to 6 months of age was obtained from the National Investigation of birth Cohort in Korea study 2008 (NICKs-2008) in South Korea . For theICD-10) procedure and prescription codes at the time of inpatient, outpatient, and emergency department visits, as well as admission to the intensive care unit (ICU), were obtained  and special milk due to underlying diseases (n = 1966) were excluded from the study. The flow diagram for the cohort is shown in Among the 917,707 infants born between January 2008 and December 2009, 467,880 infants, whose data on feeding type until the first 4 to 6 months of age could be accessible, were enrolled in the present study. Infants who received mixed feeding  breastfeeding alone, (2) formula alone, and (3) mixed feeding with breastfeeding and formula. To classify children according to the duration of exclusive breastfeeding, questions on complementary feeding practices were obtained using a parent-answered questionnaires at 9 to 12 months of age. The question included “When did you start complementary feeding?” The answers were as follows: (1) before 4 months of age, (2) between 4 months to 6 months of age, (3) after 6 months of age, (4) not yet. Breastfed infants were further classified, (1) exclusively breastfed either in the first 4–6 months or (2) over the first 6 months.19 ICD-10 codes based on these diseases. The diseases whose definitions have been validated using administrative data in a previous study are termed certainly defined diseases (The primary outcome was to investigate the associations between breastfeeding in the first 4 to 6 months and subsequent development of childhood diseases from 6 months till 10 years of age. diseases . The secThe propensity score (PS) was estimated using multivariable logistic regression with 26 covariates chosen a priori. The demographic and perinatal clinical variables in p < 0.05 was considered statistically significant.All analyses were performed using SAS version 9.4 . Two-sided The baseline socioeconomic characteristics of the study population are shown in The prevalence of each disease was comparable to that previously reported . Figure However, the breastfeeding group had a higher risk of atopic dermatitis (RR (95% CI), 1.13 (1.10 to 1.16) and 1.10 (1.06 to 1.14), respectively) and chronic urticaria (RR (95% CI), 1.13 (1.01 to 1.26) and 1.14 (1.01 to 1.28), respectively) diagnosed after 6 months and 24 months of age than the formula-feeding group. In addition, the breastfeeding group had no association with epilepsy, intussusception, Kawasaki disease, and malignancy.ICD-10 based , −0.03 (−0.05 to −0.01)) and obesity (risk difference (95% CI), −0.11 (−0.14 to −0.08)) at the age of 6–7 years . In addiIn subgroup analysis stratified by gender, the association of breastfeeding with overweight/obesity and short stature was consistent. In subgroup analysis into the duration of exclusively breastfeeding, only the group of exclusive breastfeeding during 4 to 6 months of age had significant associations with overweight, obesity, −1.64 < HFA z score ≤ −1.03, and short stature .We investigated the effect of breastfeeding an infant during the first 4 to 6 months of age on childhood diseases, hospitalization, ICU admission, and mortality, regardless of the causes, and growth until 10 years of age in a large population-based nationwide cohort. Breastfeeding in the first 4 to 6 months of age was associated with decreased risks of various diseases, including neurological diseases , respiratory infectious diseases (pneumonia and acute bronchiolitis), gastrointestinal diseases (hypertrophic pyloric stenosis and irritable bowel syndrome), asthma, and alopecia areata . BreastfWe summarized the association of breastfeeding with childhood diseases in previous studies . The preIn this world, nothing can replace breastmilk with regard to nutrients and emotional connection. Breastmilk contains diverse macronutrients, micronutrients, microbial communities, hormones, growth factors, and microRNAs ,44. The The increased risk of childhood diseases, such as iron deficiency anemia in infants with breastfeeding might be associated with deficient components in breastmilk. Breast milk does not carry enough iron to meet estimated needs , and theThe association of breastmilk feeding with atopic dermatitis is inconclusive . A recenIn a subgroup analysis of the duration of exclusively breastfeeding, we identified both groups of exclusive breastfeeding during 4 to 6 months of age and over 6 months of age had similar effect on most childhood disease and all-cause hospitalization compared to formula feeding. However, only exclusive breastfeeding during the first 4–6 months had beneficial associations with ASD, asthma, overweight, obesity, and short stature, while exclusive breastfeeding over 6 months was not. Until now, there has been no consensus on when to start complementary feeding. WHO guidelines recommend exclusive breastfeeding for first 6 months , but ESPThe results of the present study are significant in that this study has identified the overall childhood health effect of breastfeeding in the first 4 to 6 months of age in the general population-based nationwide cohort covering > 90% of children born during 2008–2009 in South Korea combined with national claims, which also covers > 90% of the total population. In addition, this study included the health effects of breastfeeding during the important period of childhood for a long period follow-up of 10 years. To reduce bias, we performed PS-matching analysis in the national health insurance service claims data and adjusted for important confounding factors, including socioeconomic status and perinatal factors, which have a significant impact on children’s health , by imprICD-10 codes at the time of outpatient, inpatient, or emergency department visits, which may have led to overestimation of the diagnosis. However, the prevalence or incidence of each disease in the present study was comparable to that reported in the previous studies. Furthermore, to improve the accuracy of each disease definition, we combined the number of claims during specific periods in some chronic diseases, which required recurrent hospital visits, in conjunction with ICD-10 codes. In addition, if certain diseases required disease-specific procedure or medication, we combined the ICD-10 codes with disease-specific procedure or prescription codes to improve the accuracy of the specific diseases. Second, information regarding the duration of breastfeeding and disease severity was lacking. Thus, the effect of the duration of breastfeeding on diverse childhood diseases could not be analyzed in the present study. Third, this study was an observational study, because of which causality cannot be established. In addition, information on the possible confounding factors, such as immunization and nutrition, could not be obtained, although the confounding factors might be limited to specific diseases. The health effects of breastfeeding may be affected by income levels [Nevertheless, the present study has some limitations. First, the ascertainment of childhood diseases was identified using e levels , and theBreastfeeding in early life has beneficial effects against the subsequent development of various childhood diseases, including obesity, as well as risk of hospitalization rate during childhood. In addition, this study shows that exclusive breastfeeding in the first 4–6 months was not inferior to exclusive breastfeeding in the first 6 months in decreasing the risk of childhood diseases, hospitalization, obesity and short stature compared to formula feeding. Our results will improve understanding regarding the health benefits of breastfeeding in children, thereby contributing in the establishment of breastfeeding guidelines to improve global childhood health.


For a significant proportion of patients with inflammatory bowel disease (IBD), primary non-response and secondary loss of response to treatment remain significant issues. Anti-tumor necrosis factor therapies have been licensed for use in IBD. Other disease-related pathways have been targeted as well, including the interleukin 12/23 axis and lymphocyte tracking. However, the need for parenteral administration and the associated costs of dispensing and monitoring all biologics remain a burden on healthcare systems and patients. Janus kinase inhibitors are small-molecule drugs that can be administered orally and are relatively inexpensive, thus offering an additional option for treating IBD. They have been shown to be effective in patients with ulcerative colitis (UC), but they are less effective in those with Crohn’s disease (CD). Nonetheless, given the immune-system-based mechanism of these drugs, their safety profile remains a cause for concern. This article provides an overview of Janus kinase (JAK) inhibitors and new trends in the treatment of IBD.  Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn’s disease (CD), is caused by chronic inflammation of the gastrointestinal tract . TargeteThe JAK family comprises four intracellular tyrosine kinases, JAK1, JAK2, JAK3, and tyrosine kinase 2 (TYK2), and seven transcriptional activators, which include signal transducers and intracellular transcription factors (STATs). The binding of these factors activates the JAK-STAT pathway via different cytokine receptors and leads to changes in the levels of immune mediators, such as interferons and interleukins ,5. SpeciAn online search was performed in PubMed/MEDLINE in June 2021 to identify articles reporting JAK inhibitors for IBD treatment. The searched keywords used were “inflammatory bowel disease”, “IBD”, “ulcerative colitis”, “UC”, “Crohn’s disease”, “CD”, “small molecule”, “janus kinase inhibitor”, and “JAK inhibitor”. The reference list of the selected articles was manually investigated to confirm relevant studies. This list includes not only published studies but also e-pub or online ahead of print. Articles that cannot be interpreted because they are not written in English are excluded. The authors checked and reviewed the manuscripts of all selected studies. The relevant or comprehensive studies were finally included in our descriptive review article.Tofacitinib is an oral JAK inhibitor  that, inIn 2017, Sandborn et al. conducted three randomized, double-blind, placebo-controlled, phase 3 trials to evaluate the efficacy of tofacitinib in the treatment of UC . Their OSands et al. also published the results of the OCTAVE Sustain trial, which enrolled 593 patients with a clinical response to induction therapy, defined as a reduction in the total Mayo score of at least 3 and a ≥ 30% decrease in the rectal bleeding subscore to at least 1, or in the absolute rectal bleeding subscore to 0 or 1. These patients were randomized to receive maintenance therapy with tofacitinib (5 or 10 mg twice daily) or placebo for 52 weeks. The primary endpoint was remission at 52 weeks. The results showed that, at 52 weeks, remission was achieved in 34% of patients in the 5 mg group and 41% of those in the 10 mg group, compared to 11% in the placebo group.In 2020, Sands et al. concluded OCTAVE Open, a long-term extension, open-label study of the OCTAVE trials that included patients already in remission on 10 mg tofacitinib twice daily; efficacy endpoints  and remission) were assessed at baseline and 2, 12, and 24 months after the conclusion of OCTAVE Open. In most patients, remission was maintained following the de-escalation of tofacitinib therapy (10 mg twice daily) after 52 weeks [In 2021, Sandborn et al. assessed the safety and efficacy of tofacitinib 5 and 10 mg twice daily in patients who had previously failed to respond to TNF inhibitors . EfficacData from the clinical use of tofacitinib in patients with UC have been reported in several retrospective studies. In 2019, Weisshof et al. conducted a retrospective observational study of the use of tofacitinib in IBD patients . Those wIn 2020, Honap et al. published the results of a retrospective observational cohort study performed at four centers in the UK and consisting of 134 UC patients treated with tofacitinib. The patients were enrolled from October 2018 to October 2019 and, initially, orally administered the standard tofacitinib induction dose of 10 mg twice daily; after at least 8 weeks, the dose was reduced to 5 mg twice daily [In 2021, Berinstein et al. performed a retrospective case-controlled study to evaluate the efficacy of tofacitinib induction in biologically experienced patients hospitalized for acute severe UC requiring intravenous corticosteroids . Forty pIn 2021, Straatmijer et al. published a retrospective cohort study that included 36 UC patients who received tofacitinib . The medIn 2017, Panés et al. conducted two randomized, placebo-controlled, multicenter, phase 2b studies that evaluated the efficacy of tofacitinib as induction and maintenance therapy for CD . In bothIn 2020, Fenster et al. conducted a retrospective cohort study to examine the real-world efficacy and safety of the off-label use of tofacitinib in patients with CD or unclassified IBD (IBD-U) . SeventyUpadacitinib is an oral JAK1-selective inhibitor with a 74-fold selectivity for JAK1 over JAK2. It has a half-life of 4 h and is metabolized in the liver (80%) and eliminated by renal excretion (20%) . The enrThe authors also conducted a double-blind, phase II trial that included adult patients with moderate to severe CD and an inadequate response to or intolerance of immunosuppressive agents or anti-TNF inhibitor . The patFilgotinib is an oral JAK1 selective inhibitor. Its half-life of 6 h for the parent compound and 23 h for the active metabolite allows once-daily dosing . In 2021p = 0.0077). In the pooled analysis of all filgotinib and placebo exposures for 20 weeks, serious treatment-emergent adverse events developed in 14 of 152 (9%) patients treated with filgotinib and 3 of 67 patients treated with placebo. The authors concluded that filgotinib therapy could induce clinical remission in CD patients and that the safety profile was acceptable.In 2017, Vermeire et al. conducted a randomized, double-blind, placebo-controlled phase II study that recruited patients from 52 centers in nine European countries . EligiblPeficitinib, an oral pan-JAK inhibitor with moderate selectivity for JAK3, was approved in Japan in March 2019 for the treatment of rheumatoid arthritis in patients with an inadequate response to standard therapy. Peficitinib is metabolized in the liver, with ~57% of the dose excreted in the feces and 37% in the urine [The safety and efficacy of pepicitinib were studied in a phase 2b dose range trial that included 219 adult patients with moderate to severe UC who were randomized to receive placebo or peficitinib 25, 75, or 150 mg or peficitinib 75 mg twice daily . AlthougBecause the immunosuppressive effects of JAK inhibition are broad, much attention has been paid to the safety profile of these drugs. Integrated safety analysis of clinical studies of tofacitinib in 1157 UC patients who were treated for up to 4.4 years, revealed that tofacitinib was globally well tolerated . Safety In the OCTAVE Induction 1 and 2 trials, severe infections occurred in six patients (1.3%) and in one (0.2%) patient in the tofacitinib 10 mg group, but in none of the patients in the corresponding placebo group. In the OCTAVE Sustain trial, severe infections occurred in two patients (1.0%) in the 5 mg tofacitinib group, in one patient (0.5%) in the 10 mg tofacitinib group, and in two patients (1.0%) in the placebo group. In the OCTAVE Induction 1 and 2 trials, herpes zoster infection occurred in three patients (0.6%) and two patients (0.5%) in the tofacitinib 10 mg group, but in only one patient and in none in the respective placebo group. Moreover, in the OCTAVE Sustain trial, herpes zoster infection occurred in three patients (1.5%) in the 5 mg tofacitinib group, in ten patients (5.1%) in the 10 mg tofacitinib group, and in one patient (0.5%) in the placebo group. The herpes zoster infections involved one dermatome or two adjacent dermatomes and did not lead to serious adverse events or drug discontinuation. In the OCTAVE Induction 2 trial, one patient in the 10 mg tofacitinib group had cytomegalovirus colitis. Tuberculosis was not reported in any of the three trials . In the In addition, from a systematic review in 2020, the association between tofacitinib use and infection was analyzed . SeriousPulmonary cryptococcosis, histoplasmosis, and cytomegalovirus hepatitis were included as non-herpes zoster opportunistic infections in the Global UC trial . Severe A large amount of data is available concerning the risk of deep vein thrombosis and pulmonary embolism in patients with IBD receiving tofacitinib, including a pooled analysis of 1157 patients with UC treated with tofacitinib who were enrolled in three induction studies, one maintenance trial, and one ongoing long-term extension study. In both the OCTAVE induction and maintenance (OCTAVE Sustain) studies, deep vein thrombosis (DVT)/pulmonary embolism (PE) events occurred in two placebo-treated patients. In the overall cohort, DVT/PE events occurred in five patients (one DVT and four PE), all of them in patients receiving tofacitinib 10 mg twice daily. Two patients had discontinued tofacitinib 4–5 days prior to the event, although one of them later resumed treatment with tofacitinib. All patients received a predominant dose of tofacitinib 10 mg twice daily during the entire UC clinical program . To dateIn the overall tofacitinib trials in UC patients, during the OCTAVE Open, 11 patients had malignancies, excluding those with non-melanoma skin cancer (NMSC). These consisted of one case each of cervical cancer, hepatovascular sarcoma, cholangiocarcinoma, cutaneous leiomyosarcoma, Epstein-Barr virus-associated lymphoma, renal cell carcinoma, essential thrombocythemia, acute myeloid leukemia, colon adenocarcinoma, lung cancer, and breast cancer. A total of 8 of the 11 patients had been previously treated with TNF inhibitors and all had prior treatment with thiopurine . No pattAmong UC patients in the tofacitinib trials, there was an increase in lipid parameters, but there were no significant changes in other laboratory parameters .In the overall tofacitinib trials for patients with UC, the OR of major adverse cardiovascular events (MACE) was 0.2 , based on four patients with events , includiIn the total tofacitinib trial in UC patients, the incidence rate of gastrointestinal tract perforation was 0.2, based on three patients with an event . In all For many years, CD and UC have been treated mainly with corticosteroids, aminosalicylates, immunosuppressants, and TNF inhibitors. However, the recent development of small-molecule drugs and biologics has improved the treatment and thus the prognosis of IBD patients. JAK inhibitors usually can be administered orally and exert fewer adverse effects on the immune response. Therefore, the use of this drug enhances the convenience of the user. Tofacitinib was approved for patients with UC, and its efficacy has been good regardless of previous TNF inhibitor use . HoweverIn the treatment of IBD, the use of anti-TNF agents had superior effects compared to conventional treatment. However, despite the use of biologics in many patients, problems, such as loss of response and development of drug tolerance, are appearing. Since JAK inhibitors are small molecules, they have little autoantibody formation compared to macromolecular proteins, so the possibility of response loss is expected to be relatively low, and rapid therapeutic effects are expected in patients with acute severe UC. IBD is an autoimmune disease that requires long-term treatment. Therefore, stability and continuity of drug effect are very important in treatment choice. JAK inhibitors, such as tofacitinib, open up a new horizon for small molecule drugs in IBD treatment strategies that now result in biologics. In the future, more comprehensive and head-to-head trials comparing various JAK inhibitors with existing biologics are needed. Based on the studies currently in progress and scheduled to be conducted in the future, it is expected that a new era of JAK inhibitors in the treatment of IBD will come.


This study aimed to identify the mechanism of Yiqi Huayu Decoction (YQHY) induced ferroptosis in gastric cancer (GC) by using network pharmacology and experimental validation.The targets of YQHY, ferroptosis-related targets, and targets related to GC were derived from databases. Following the protein–protein interaction (PPI) network, the hub targets for YQHY induced ferroptosis in GC were identified. Furthermore, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were used to analyze the hub targets from a macro perspective. We verified the hub targets by molecular docking, GEPIA, HPA, and the cBioPortal database. Finally, we performed cell viability assays, quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, lipid peroxidation, and GSH assays to explore the mechanism of YQHY induced ferroptosis in GC.We identified the main active compounds and hub targets: Quercetin, DIBP, DBP, Mipax, Phaseol and TP53, ATM, SMAD4, PTGS2, and ACSL4. KEGG enrichment analyses indicated that the JAK2-STAT3 signaling pathway may be a significant pathway. Molecular docking results showed that the main active compounds had a good binding activity with the hub targets. The experimental results proved that YQHY could induce ferroptosis in AGS by increasing the MDA content and reducing the GSH content. qRT–PCR and Western blot results showed that YQHY can induce ferroptosis in GC by affecting the JAK2-STAT3 pathway and the expression of ACSL4.This study indicated that YQHY can induce ferroptosis in GC by affecting the JAK2–STAT3 pathway and the expression of ACSL4, and induction of ferroptosis may be one of the possible mechanisms of YQHY’s anti-recurrence and metastasis of GC. The incidence of GC in China accounts for nearly half of the world , 2. At pin vitro and in vivo can restore intracellular glutathione levels and prevent ROS production, thus inhibiting ferroptosis in GC cells induced by erastin . Especially for patients with stage III disease, the percentage of recurrence and metastasis risk is decreased to 34.7% (P = 0.0072) . Traditihttp://lsp.nwu.edu.cn/tcmsp.php). To better screen the active compounds of YQHY, OB ≥30% , the screened active compounds were transformed into the SMILES structural formula, and the SMILES structural formula was imported into the Swiss Target Prediction website, setting Homo sapiens, to predict all potential targets of the active compounds in YQHY. The relevant targets in GC were obtained by using the DisGeNET, GeneCards, and OMIM databases. We screened targets related to ferroptosis from the FerrDB (http://www.zhounan.org/ferrdb/) database , Cellular Component (CC), and Molecular Function (MF). GO enrichment analysis was performed according to P <0.05 and FDR <0.05, and KEGG enrichment analysis was performed according to P <0.01 as the filtering criterion. Top20 were plotted into bubble charts using R language.Molecular docking is mainly used for structural docking of small molecules with target proteins and evaluating their binding affinities with defined binding sites . A negathttp://gepia.cancer-pku.cn/index.html) (https://www.proteinatlas.org/)  (The hub targets were input into the online tool GEPIA (ex.html)  to verifas.org/) . The cBial.org/)  was usedThe YQHY decoction consists of Huangqi, Dangshen, Chenpi, Banxia, Baizhu, Baishao, Danggui, Sanleng, Ezhu, Sheshecao, Shijianchuan, Fulin, Muxiang, Sharen, and Gancao. YQHY medicine was dried for 24 h, pulverized, and sieved with an 80 mesh sieve. The decoction was mixed, filtered, and concentrated to 250 ml. The concentrated solution was added to a special freeze-drying bottle, quickly frozen with liquid nitrogen, and dried at −50°C in FreezeDryers. The lyophilized powder was removed at 48 h and stored under cryogenic drying after crushing.Different concentrations of YQHY were added to the experimental group, and F12K culture medium was added to the zeroing group and control group. The reaction was carried out at 37°C and 5% carbon dioxide for 24 h. After CCK8 solution was added to each well to react for 1 h, the OD of each sample was measured by setting up the microplate reader at 490 nm. IC50 was calculated using GraphPad Prism software.The MDA and GSH contents in cell lysates were assessed using lipid peroxidation and GSH assay kits, respectively, according to the manufacturer’s (Abcam) instructions.According to the results of network pharmacology, we selected ACSL4, JAK2, and STAT3 to explore the mechanism by which YQHY induces ferroptosis in GC. Total RNA was extracted from cultured cells using TRIzol and phenol–chloroform phase separation according to the manufacturer’s instructions and then reverse transcribed using the RevertAid RT Reverse Transcription Kit . The primer sequences used for qRT-PCR are listed in The protein expression of JAK2, STAT3, p-STAT3, and ACSL4 was detected by Western blot. Total proteins were obtained using RIPA buffer, and protein concentrations were measured using a BCA protein assay kit. The protein samples were blotted onto PVDF membranes. Next, the membranes were incubated at 4°C overnight with primary antibodies against JAK2, STAT3, p-STAT3, ACSL4, and β-actin, followed by HRP-conjugated secondary antibodies. The grayscale values were quantified by Image Lab software.The experimental results were statistically analyzed by GraphPad Prism software. Single-factor ANOVA was used to compare the differences in experimental data among groups. A P-value less than 0.05 was considered significant.Through the screening of the TCMSP database, a total of 360 active compounds were obtained, namely, 23 Huangqi, 27 Dangshen, 10 Chenpi, 20 Banxia,16 Baizhu, 17 Baishao, 22 Danggui, 10 Sanleng, 21 Ezhu, 7 Sheshecao, 4 Shijianchuan, 16 Fulin, 30 Muxiang, 54 Sharen, and 83 Gancao.After the prediction of the database and deletion of duplicate values, a total of 1,373 potential targets of active compounds, 15,842 targets of GC, and 205 targets of ferroptosis were obtained. A total of 87 common targets were obtained by combining the three target sets. At the same time, a “YQHY compound-target” network was constructed Figure 2According to the ranking of BC, CC, and degree calculated by the CytoNCA plug-in, TP53, ATM, SMAD4, PTGS2, and ACSL4 were identified as the hub targets of YQHY induced ferroptosis in GC. Meanwhile, a PPI network diagram of hub targets was constructed.GO enrichment analysis showed that the biological functions mainly involved positive regulation of transcription from the RNA polymerase II promoter, negative regulation of apoptotic processes, and oxidation–reduction processes. Cell components mainly included the cytoplasm, cytosol, and nucleus. Molecular functions mainly included protein binding, identical protein binding, and DNA binding. KEGG functional enrichment analysis showed that the hub targets were mainly enriched in microRNAs in cancer and the JAK2-STAT3 signaling pathway Figure 4−1 indicates good binding activity. The results of molecular docking were plotted as a heatmap  had genetic mutations in these five targets  . After the addition of liproxstatin-1, the mRNA expression of JAK2, STAT3, and ACSL4 was decreased compared with that in the YQHY group (P <0.05). The mRNA expression of JAK2, STAT3 and ACSL4 in the liproxstatin-1 group was significantly decreased relative to the control group (P <0.05) . The levels of ATM changed significantly with pathological stage and increased significantly in stage IV. The cBioPortal tool showed that 278 of 434 patients (64%) had genetic mutations in these five targets. The results of the above analysis were mostly consistent with previous literature reports.To further clarify the relationship between YQHY and ferroptosis, we selected ACSL4, which is closely related to ferroptosis, as the target of this topic, and the JAK2–STAT3 pathway as the pathway of this topic to explore whether YQHY could induce ferroptosis in GC by influencing the expression of ACSL4, JAK2, and STAT3. The results showed that YQHY had a toxic effect on AGS, and with increasing concentration, the inhibition rate of AGS showed a gradually increasing trend. The increase in MDA and the decrease in GSH synthesis are the main characteristics of ferroptosis. We found that the MDA content in the YQHY group was the highest compared with the other three groups (P <0.05), and YQHY reversed the ferroptosis inhibition induced by the ferroptosis inhibitor liproxstatin-1. The GSH content in the YQHY group was the lowest compared with the other three groups (P <0.05), and the GSH content in the YQHY + liproxstatin-1 group was lower than that in the control group, indicating that YQHY can reduce the intracellular GSH content and induce ferroptosis in AGS and that YQHY can reverse ferroptosis due to liproxstatin-1 inhibition.The qRT-PCR results showed that the mRNA expression levels of JAK2, STAT3, and ACSL4 were higher in the YQHY group than in the control group. After the addition of liproxstatin-1, the mRNA expression of JAK2, STAT3, and ACSL4 was decreased compared with that in the YQHY group. The mRNA expression of JAK2, STAT3 and ACSL4 in the liproxstatin-1 group was significantly decreased relative to the control group.Western blot analysis confirmed that compared with the control group, the expression of JAK2, STAT3, p-STAT3, and ACSL4 protein in the YQHY group was higher than those in the other three groups, while the expression level of each protein in the liproxstatin-1 group was lower. It could be concluded that YQHY can induce ferroptosis in AGS by regulating the JAK2-STAT3 pathway and the expression of ACSL4. This illustrates that influencing the JAK2-STAT3 signaling pathway and expression of ACSL4 is one of the mechanisms of inducing ferroptosis in GC, and induction of ferroptosis may be one of the possible mechanisms of YQHY’s anti-recurrence and metastasis of GC.The material basis of YQHY induced ferroptosis in GC is based on active compounds such as Quercetin, DIBP, DBP, Mipax, and Phaseol. The related mechanism was characterized by multiple targets and pathways. YQHY can induce ferroptosis in GC by affecting the JAK2–STAT3 pathway and the expression of ACSL4, and induction of ferroptosis may be one of the possible mechanisms of YQHY’s anti-recurrence and metastasis of GC.The original contributions presented in the study are included in the article/PS designed the research. SS analyzed the data and wrote the paper. FW, SG, WH, JL, JZ, PG, SR, XC, and YL selected the materials. All authors read and approved the submitted version.This work was funded by the National Natural Science Foundation of China (nos. 81673918). Pilot GC project of clinical collaboration of traditional Chinese medicine and Western medicine on major difficult diseases in the state administration of traditional Chinese medicine; the 2019” Construction Project of Evidence-based Capacity for Traditional Chinese Medicine” (2019XZZX-ZL003) in state administration of traditional Chinese medicine; the Open Program of the Third Phase of the Program of Traditional Chinese Medicine (TCM) Advantageous Subjects (ZYX03KF020); and the Science and Technology Project of Jiangsu Provincial Administration of Traditional Chinese Medicine (ZD201803).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. 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The aim of this study was to help understand the full scope of maladaptive daydreaming among medical students at the University of Khartoum, in Sudan, by displaying all maladaptive daydreaming-related statistics among studied population. An analytical cross-sectional university-based study was conducted during the academic year 2020–2021, using a total of 323 self-administered online questionnaires; the questionnaire containing the Arabic Version of Maladaptive Daydreaming Scale and other sociodemographic data were given by stratified random selection to undergraduate medical students at the University of Khartoum, in Sudan, between December and January 2021.Daydreaming is a normal cognitive phenomenon in which the individual experiences a temporary separation from reality during which contact with reality is compromised and, to some extent, replaced by a visionary fantasy. On the other hand, daydreaming can progress to a maladaptive state, known as 34.3% of medical students at the University of Khartoum have identified themselves as maladaptive daydreamers. More males were found to be maladaptive daydreamers when compared to female medical students. The average Maladaptive Daydreaming Scale score among medical students was found to be 33.6 (sd=22.3). No significant correlation was found between Maladaptive Daydreaming Scale score and gender, and there was a significant correlation between the MDS score and the academic class of medical students at University of Khartoum.Maladaptive daydreaming was discovered to have a significant impact on the population of medical students at the University of Khartoum, with some alarming rates and correlations among medical students; having more than one-third of total medical students found to be having MD must surely indicate a much larger and more widespread problem. A multifactorial holistic approach covering biological, psychological, social, and academic aspects must be used when hypothesizing the justification for these findings and planning an active intervention strategy. Daydreaming, also known as spacing out, is a normal cognitive phenomenon in which the individual experiences a temporary separation from reality during which contact with reality is compromised and, to some extent, replaced by a visionary fantasy. However, daydreaming can progress to a pathological or maladaptive state, known as maladaptive daydreaming (MD), which is defined as “extensive fantasy activity that replaces human interaction and/or interferes with academic, interpersonal, or vocational functioning” –3. DaydrThis was an analytical cross-sectional university-based study that was conducted during the academic year 2020–2021. The research was carried out virtually at the Faculty of Medicine at the University of Khartoum in Khartoum, Sudan.The research was carried out virtually at the University of Khartoum’s Faculty of Medicine (UofK-MED). UofK-MED is Sudan’s oldest medical school, having been founded in 1924. It was also the first medical school in Sudan to be accredited by the World Federation of Medical Education. Every year, thousands of students apply for admission to UofK-MED, attracted by its unique medical education and extracurricular opportunities.During the academic year 2020–2021, there are 2335 students enrolled at the college. All of the participants were undergraduate medical students, with the majority of them ranging in age from 18 to 23. In terms of inclusion criteria, all undergraduate students at the University of Khartoum–Faculty of Medicine in Khartoum in the first (batch 96), second (batch 95), third (batch 94), fourth (batch 93), fifth (batch 92), and sixth (batch 91) academic years were included in the study, and no students were excluded.A random sample of 323 students was chosen from the medical students at the University of Khartoum, Faculty of Medicine, using stratified random sampling. Sample size (n) was calculated according to the formula: n = [z2 * p * (1−p)/e2]/[1 + (z2 * p * (1−p)/(e2 * N))] where z = 1.96 for a confidence level (α) of 95%, p is the proportion , N is the population size, and e is the margin of error.The sample size (with finite population correction) is equal to 323.https://www.randomizer.org.The study population was subdivided into 6 groups according to their academic classes, and list containing all students in each academic class was obtained. Since the study sample size was 323, a proportionate random sample from each group was selected using the online sample randomizer at Fifty-two out of 325 were selected from the sixth-year students (batch 91), 51 out of 313 from the fifth-year students (batch 92), 56 out of 349 from the fourth-year students (batch 93), 58 out of 358 from the third-year students (batch 94), 54 out of 333 from the second-year students (batch 95), and 52 out of 326 from the first-year students (batch96).During the academic year, all targeted students completed online self-administered closed-ended questionnaires. Before filling out the Arabic version of the Maladaptive Daydreaming Scale, participants were asked demographic and general information questions: age, gender, marital status, and academic class. Each individual was approached separately, either directly using their personal social media accounts, or through the students’ representing bodies within UofK-MED; this process proved to be effective, yielding a 100% response rate.The Arabic Version of the Maladaptive Daydreaming Scale (MDS-16-AR) is a new 16-item scale proposed and validated in 2019 by Hisham Motkal Abu Raya, Professor Eli Somer, and Suha Meari-Amir . It consData was entered and encoded then analyzed by the computer programs by using the SPSS software (version 21). The collected data was analyzed through descriptive analysis and using the chi square and t-tests. P value <0.05 was considered statistically significant. Data were displayed and presented in form of tables.323 medical students; all 323 students were given the study questionnaire and response rate of which was 100%. Sixth-year students represented 16.1% of the sample, fifth-years represented 15.8%, fourth-years represented 17.3%, third-years represented 18%, second-years represented 16.7%, and first-years represented 16.1 students. Twenty-five percent of the studied population were males, while the females made 75% out of the total study sample . When asked about their subjective familiarity with the concept of MD “How familiar are you with the term Maladaptive Daydreaming?”, 24.1% had no familiarity with the concept at all, 18.6% had poor familiarity, 16.1% had below average familiarity, 20.1% had average familiarity, 11.5% had above average familiarity, and 9.6% had excellent familiarity  among medical students at University of Khartoum n=323) was 34.3% (CI 95%). 3 was 34.This study represents the first Sudanese study of maladaptive daydreaming, the aim of which was to investigate the full scope of MD among medical students at the University of Khartoum in Sudan, including the prevalences of daydreaming, MD among all medical students, and MD among each academic class students in separate. The study also investigated the association between MD and other factors. Since most of the college students at UofK-MED are females. In their responses, only 67% of the study participants have experienced daydreaming, which is far less than the percentage of daydreaming among the American population (96%) , 3. The 34.3% of total students were found to be MDers; on the other hand, 70% of the of students were found to be MDers in a similar study conducted on Saudi medical students [Regarding the MDS score, the mean of MDS score among the study population was found to be 33.66, which is still considered less than the 45% cutoff point measurement for MD. As mentioned in the “students , while istudents ; again, students .MD has shown some alarming rates among University of Khartoum medical students, with more than one-third of total medical students having MD, indicating a serious potential much larger and more pervasive problem. This study should provide a solid foundation for more thorough investigations into the causes and related factors of MD.


The subjects participated in 20 3-min cryochamber sessions (−120 °C), 1/day, 5 days/week. Body composition was analyzed before and after treatment. Blood adiponectin (ADP), leptin (LEP), and tumor necrosis factor alpha  levels, and the lipid profile were analyzed three times: at baseline and up to 2 h after 10 and 20 sessions. The 20 treatments caused significant changes in body composition. Between 10 and 20 whole-body cryostimulation (WBC) sessions, a significant decreased was observed in the LEP and TNF-alpha levels. No significant changes in the lipid profile were noted. However, a positive tendency to regain the metabolic balance in adipose tissue was apparent in the IOb group in the tested period . Collectively, for people with obesity increasing the number of treatments above the standard 10 should be recommended.Obesity is associated with chronic inflammation. While cold therapy influences the pro/antioxidative status of an individual, by affecting adipokine levels and the lipid profile, the effect of body mass index (BMI) on the response to cold exposure is unclear. We analyzed the link between BMI and the differences in effects of whole-body stimulation, depending on the number of treatments, on specific physiological parameters in men. Twenty-seven non-active men were divided into three groups: N ( In highly developed countries, the level of physical activity in a population is limited. This, among other factors, impacts the severity of diseases of civilizations, such as obesity. The number of individuals with a body mass index (BMI) over 25 is continually increasing, and today it is approximately four times higher than in the 1980s. Obesity is a chronic inflammatory disease, strongly associated with other diseases of civilizations, especially type 2 diabetes . It is cMaintenance of an appropriate adipose tissue mass is important for human health because of its participation in many processes, including thermogenesis, heat isolation, energy storage, and, especially, endocrine function . ADP andBased on the available literature, a reduction in adipose tissue mass increases serum adipokine levels , ameliorAccording to numerous recent reports, whole-body cryostimulation (WBC) has many beneficial effects on human health, including on body composition , blood cThe anti-inflammatory effect of WBC is well documented ,34,35,36Volunteers from the Association of bariatric patients at the Bariatric Clinic of Collegium Medicum UJ in Krakow, who expressed willingness to participate in the study, were subjected to medical assessment as per the inclusion and exclusion criteria (vide infra). Following anthropometric measurements, blood was collected by a qualified person for biochemical analyses. All participants received dietary instructions from a dietician, considering their body mass and nutritional energy demands. After a medical examination and the consent of the doctor to patriciate in the experiment, a series of sessions in a cryochamber began. Before each session, the blood pressure was measured. Individuals with values exceeding 160/100 mmHg were excluded from further participation in the study. Immediately after the 10th and 20th WBC session, blood was re-drawn for analysis. Then, 24 h after 20 WBC sessions, body composition and circumferences were reassessed.The project was approved by the Bioethics Committee at the Regional Medical Chamber in Krakow (Poland) (no. 122/KBL/OIL/2017) and registered in Australian New Zealand Clinical Trials Registry (ANZCTR) as a clinical trial .n = 9, BMI > 34.9, IIOb group), those with class I obesity , and those with a healthy body weight . Characteristics of the study groups are presented in Forty volunteers qualified for the study but only 27 completed the study protocol. The participants were divided into three groups: those with class II obesity . Each session lasted 3 min, and each participant underwent 1 session a day, 5 day a week. Each session was conducted with the participation of a medical doctor supervising the study. The WBC treatments were preceded by a 30 s adaptation period in an atrium at −60 °C. The stay in the cryochamber was monitored by an appropriately trained employee of the facility and contact with the study group was maintained via a camera and a voice system. Before entering the cryochamber, the subjects were informed about the recommended moving and breathing style during WBC.® system, F.L. Medical, Torreglia, Italy). Tubes containing the blood and a clot activator were centrifuged  within 15–20 min of blood collection. The obtained supernatant was pipetted, transferred to 1.5 mL microtubes, and placed in a low-temperature freezer (−60 °C) until further analysis. ADP, LEP, and tumor necrosis factor alpha  levels were determined in blood serum without traces of hemolysis using enzyme-linked immunosorbent assay (ELISA) and an ELISA microplate reader . Assay sensitivity was 0.7 ng∙mL−1 for LEP, 0.27 ng∙mL−1 for ADP, and 0.7 pg∙mL−1 for TNF-alpha. Intra and inter assay [CV%] variability was as follows: for LEP: 3.14% and 7.5%; for ADP: 5.95% and 8.7%; for TNF-alpha: 5.2% and 7.4%.To assess the initial condition of tested parameters in participants, blood samples were collected up to 1 h before the first treatment. To compare the difference between 10 and 20 treatments, blood samples were collected twice: up to 1 h after 10 and 20 treatments. Samples containing 6 mL of venous blood were collected from antecubital fossa veins by a laboratory diagnostician. For each participant, blood collection was performed at the same time of day. Since cryotherapy cannot be done on an empty stomach, blood sampling was performed at least 6 h after the last light meal. In accordance with the applicable standards, a laboratory diagnostician collected venous blood with elbow flexion into test tubes  using glycerolphosphate oxidase for TG determinations; cholesterol esterase and oxidase for TC; magnesium (II) ions, dextran sulfate, and cholesterol esterase and oxidase for HDL cholesterol (HDL-C); and a homogeneous method with cholesterol esterase and oxidase in the presence of surfactants for LDL cholesterol (LDL-C). Non-HDL cholesterol (nonHDL-C) fraction was calculated using the formula:www.graphpad.com, accessed on 19 January 2022) GPM6-277417-RLMI-DE56F). The results are presented as an arithmetic mean with standard deviation. The type of distribution was verified using the Shapiro–Wilk test and it revealed that all variables were normally distributed. Paired t-test and one-way ANOVA and post Tukey test were used to determine differences between treatments in the various groups. Differences between groups at all time points were analyzed using an unpaired t-test and a two-way analysis of variance (ANOVA) with repeated measures. For all tests, the significance level was set at p < 0.05. For statistically significant results, Cohen’s d was calculated and interpreted as follows: 0–0.2, small effect; 0.5, medium effect; and above 0.8, large effect.The data were analyzed using Excel 365  and GraphPad Prism 6.0 ((n = 5 per group). The sample sizes for IOB, IIOB, and N groups (n = 9) were determined based on the mean and SD within a group to provide 80% power and p = 0.05 .In order to calculate sample size, we used data from a pre-study, in which levels of indicators were measured before and after 10 and 20 cryotherapy treatments in both groups . Further, the analysis of the parameter differences before vs. after WBC indicated that individuals with class I obesity were the most sensitive to 20 WBC sessions.All results obtained from blood analysis were not corrected for changes in plasma volume, which were very small and statistically insignificant.LEP levels were above the health-related reference interval in the IOb and IIOb groups at baseline/rest values and increased with an increasing BMI. In the N group, the LEP levels were within the reference range, similarly to ADP levels in the N and IOb groups but were below the reference range in the IIOb group. However, there were no significant differences in the ADP levels between the three groups. TNF-alpha levels did not significantly differ between the groups at baseline and all values were within the reference range  A.TC, TG, non-HDL, and LDL levels in the IIOb group were significantly higher than those in the N group. However, all these parameters showed the tendency to increase with an increasing BMI. The TC levels in the IIOb group were above the reference range (3–5 mmol/L). At baseline, the TG values were within the reference range only in the N group. There were no differences in HDL between groups B.HDL and LDL levels in the IIOb group were outside the reference range . The mean HDL levels in the IOb group were close to those in the N group and were within the reference range (≥1.2 mmol/L). In the IIOb group at baseline, the HDL levels were significantly lower and the LDL levels were significantly higher than those in the N and IOb groups. The HDL/LDL ratio in the IIOb group was significantly lower than that in the other groups B.Finally, BAI levels in the IOb and IIOb groups were significantly higher than those in the N group C. BaseliNo differences in the effect of 10 and 20 WBC on the N group LEP levels were apparent, while a significant decrease in LEP levels was observed between 10 and 20 WBC sessions in the IOb and IIOb groups A. FurtheA significant reduction in TC levels was observed between 10 and 20 WBC sessions in the IOB and IIOb group A. In addThe tendency to decrease in nonHDL-C levels was noted between 10 and 20 WBC, especially in IOb and N groups. The nonHDL-C levels in the IIOb group after 20 WBC sessions were higher than those in the N and IOb groups C.Between 10 and 20 WBC there were no significant differences in the HDL levels, LDL levels, and the HDL/LDL ratio in any group; however, these values tended to increase between 10 and 20 WBC sessions in the IIOb and IOb groups A–C. The pFinally, BAI levels in the IOb and IIOb groups were significantly higher than those in the N group and were unaffected by 20 WBC sessions a. BaseliThe directions of changes in adipokines and lipids in tested groups after 10 and 20 WBC sessions are summarized in LEP and ADP in brown and white adipose tissues in rat. They found a significant decrease in LEP and ADP levels in brown adipose tissue, and a significant decrease in LEP levels but no changes in ADP levels in white adipose tissue. The authors postulated that LEP gene expression is inhibited by cold conditions and does not impact ADP gene expression in white and brown adipose tissue [Our results confirm a disturbed lipid metabolism in people in the IOb and IIOb groups, relying on raised levels of most parameters (except of HDL). As presented herein, rest levels of this adipokine increase with an increasing degree of obesity, with significant differences between the three groups after 10 WBC sessions. Following 10 sessions caused a further decrease in LEP, especially in the IIOb group. After 20 sessions, no significant differences between IOb, N, and IIOb groups were noted. Thus, the main finding of the current study is the significant decrease in LEP levels in the IOb and IIOB groups between 10 and 20 WBC sessions. In the N group, with a recommended LEP health related reference interval, no effect at the same time was observed. Many authors have reported increased LEP levels in obese subjects ,39,40,41e tissue . Furthere tissue  reportede tissue . Hence, e tissue ,46. In te tissue ,48. Moree tissue .In the current study, between 10 and 20 sessions no changes were observed in ADP levels. According to the literature, a similar effect in response to 10 WBC was reported by Ziemann et al.  and LubkMany authors postulated the anti-inflammatory properties of WBC, as demonstrated by a reduction in TNF-alpha levels in response to WBC. Our findings showed a significant decrease in TNF-alpha between 10 and 20 WBC sessions in IOb and IIOb groups. Interestingly, the changes in TNF-alpha levels were not accompanied by an increase in ADP levels. Thus, the decline in TNF-alpha levels progressed with the number of WBC sessions. According to the available literature, TNF-alpha suppresses ADP gene expression in adipocytes . TherefoIn this study we also evaluated the effect of WBC on body composition (differences at baseline and 48 h after the last WBC session). During the experiment, no significant changes were observed in, BM, PBF, HC, and WC in all groups compared to rest values, except for BMI and HC in the N group. The largest changes were observed in the IOb group. A decrease in subcutaneous fatty tissue thickness, probably associated with its metabolic activity and a reduction in adipocyte volume under cold stress conditions , was likObesity is associated with a decrease in HDL levels , and an All changes in adipokine  and TNF-alpha levels as well as in lipid profile may be associated with a response of the sympathetic nervous system. It is known that catecholamines, which inhibit production and secretion of TNF-alpha and LEP, are also modulated by the sympathetic nervous system. Moreover, animal studies showed that mutations in beta-3 adrenergic receptors lead to lowered metabolic rate in obese subjects .BAI and VAI did not change significantly in response during an additional 10 sessions WBC in participants of the current study; however, a significant decrease in VAI, indicating a reduced risk of cardiovascular disease, was apparent in the IOb group. In addition, the mean HDL and LDL levels, and the mean HDL/LDL ratio in the IOb group were close to the ones in the N group in the tested period. Interestingly, the largest changes in the adipokine levels and lipid profile in the IOb group were consistent with the largest changes in body composition in that group. It is possible that 20 WBC sessions are sufficient for individuals with healthy body mass and class I obesity with no major metabolic disturbances at baseline, however, positive changes in some parameters also occur in the IIOb group. In individuals with class II obesity, additional WBC sessions affect body composition, proinflammatory TNF-alpha and LEP levels, and HDL/LDL ratio. Our data shows a difference in response between 10 and 20 WBCs, however, the effects of greater numbers of treatments, which may be required for people with class II obesity, were not investigated in this study.Our findings indicate that between 10 and 20 WBC sessions there are many positive changes associated with adipokines, lipid profile, or body composition. The drop in LEP levels could have resulted from a decreased LEP resistance, a desired effect, especially among obese subjects. The IOb group was the most sensitive to WBC, in terms of the fact that only BAI and ADP were not affected by following 10 WBC. Overall, 20 WBC sessions had a positive effect in individuals with minor disturbances in baseline values; however, further research is needed, especially involving obese individuals undergoing a higher number of sessions than that used in the current study.Our study has some limitations. We present only differences between subjects with different BMI during tested period. We did not investigate changes in tested indicators in people who did not use WBC at the same time. Moreover, the size of the studied groups is an additional limitation.


Eunicella cavolini and the precious red coral Corallium rubrum. We tested for possible mutual influences of the two species when co-occurring. Here, we show that for both species, populations dwelling in deeper habitats show demographic features of immature or disturbed populations, possibly due to environmental and/or anthropogenic disturbances that occur locally. Moreover, the density of one species is significantly positively correlated to that of the other, but not influenced by the colony morphology  of the other species. We encourage further studies that might contribute to shedding light on inter-specific relations occurring in these vulnerable ecosystems.Octocorals are marine ecosystem engineers that can form dense patches on rocky substrates. These organisms enhance the three-dimensional complexity of the habitat and provide several ecological services within and around their canopy. Mapping their distribution and understanding the intra- and inter-specific processes that drive population dynamics is of crucial importance. In this study, we investigated via Remotely Operated Vehicle the distribution and demography of two hard-bottom octocorals that share similar habitat preferences within the Western Mediterranean: the yellow gorgonian Corallium rubrum and Eunicella cavolini are two octocorals, reported as co-occurring species in the deep rocky habitats of the Mediterranean Sea with a high hydrodynamic and moderate eutrophication. Their spatial distribution and demography in the deep sea are mainly affected by temperature and direct and indirect anthropogenic activities; however, knowledge of the factors that potentially influence their co-existence is scarce. This paper provides novel data on the distribution and demography of these two species, at depths between 50 and 290 m in the Western Mediterranean Sea, providing insights on their co-occurrence. Both species exhibited the highest population density at deeper sites (>150 m), showing an inverse size–density relation. Density values ranged from 0.03 colonies m−2 to 32 and 80 col. m−2 for yellow gorgonian and red coral, respectively. The two species co-occurred in 13% of the total frames examined, mostly dwelling between 120 and 160 m depth. Distance-based linear modeling (DistLM) emphasized that when co-occurring the variability of the two species’ densities were significantly driven by the density—rather than the morphology —of the other species. We stress the need for further studies to elucidate the possible mutual effects of suspension feeders and to test the role of different environmental factors potentially influencing inter-specific relationships. Deep-dwelling octocorals often dominate hard-bottom benthic ecosystems, where they act as ecosystem engineers and foundation species, providing three-dimensional habitats for fish and invertebrates, and also contributing to the maintenance of ecosystem functioning ,2. They The spatial distribution of benthic suspension feeders depends on settlement processes, which can influence feeding success and thus the competition with other corals dwelling within the same community . In geneCorallium rubrum  is endemic to the Mediterranean Sea and the adjacent Eastern Atlantic Ocean [The highly valuable red coral ic Ocean , dwellinic Ocean . Red coric Ocean ,12. Thisic Ocean  due to iic Ocean ,15,16,17ic Ocean ,18.−1 for deep-dwelling populations) [Intensive harvesting, along with red coral’s slow growth rate (0.26 mm yrlations)  and senslations) ,21, has lations) ,16,20,22lations) . Thus, tlations) ,25,26.Eunicella cavolini , highlighting a partially overlapping habitat [E. cavolini a potential red coral competitor for space and resources [Despite the frequent reports of monospecific patches of red coral in shallow-water ecosystems, recent studies reported its consistent co-occurrence with the yellow gorgonian  habitat ,27. Inde habitat ,29,30. Tesources . HoweverE. cavolini is a common gorgonian in the Mediterranean Sea, forming distinct facies within coralligenous assemblages [emblages . Its disemblages ,32, overemblages ,29; howeemblages .The lack of studies on deeper red coral and yellow gorgonian populations limits our understanding of their ecology and the possibility of developing an effective management plan for their conservation. These species are sensitive to environmental changes , and alsRemotely Operated Vehicles (ROVs) represent an effective and non-invasive tool, typically used to evaluate the state of the conservation of coral populations dwelling in the bathymetric range of SCUBA-diving operative limits and beyond ,34.C. rubrum; (ii) describe the population structures of E. cavolini below 50 m depth; and (iii) investigate the patterns of co-occurrence between the two species. All the collected data also constitute a baseline to evaluate the potential impacts of fishing activities and eventual populations recovery.In this study, we carried out ROV surveys from the northeastern to the southwestern coast of Sardinia (Western Mediterranean Sea) between 50 and 290 m depth in order to: (i) provide additional data on the distribution and population structures of the deep-dwelling populations of The study area is located around the island of Sardinia , which is in the central-western region of the Mediterranean Sea. The survey was conducted mostly over the shelf break of the eastern Sardinian continental margin, a passive margin of the Tyrrhenian Basin that is delimited to the north by the “Etruschi” seamount and to the south by the “Ichnusa” seamount. The narrow continental shelf that characterizes the eastern area of the island terminates at about 60–100 m depth in the southern and central areas and at around 200 m depth in the northern area. Along the entire eastern side of the island, the continental shelf and slope are both incised by profound submarine canyons and connected to inland orographic structures and river basins ,36,37. COverall, nine sites were investigated, which were a priori allocated to three geographical areas, namely: (i) North-east (NE), including Pietrame and the Canyons of Caprera, Mortorio, and Tavolara, with sites located at a greater distance from the nearest coastline (up to 12 Nm from the coast) compared to sites located along the Central-east and South-west coasts ; (ii) Central-east (CE), including the Cala Gonone Shoal and the Canyons of Orosei and Arbatax; and (iii) the South-West (SW) area, including Bancotto and Secchitella, which are located over rocky outcrops rising from the wide continental shelf that characterizes the western coast of Sardinia ; Table 1ROV surveys were conducted over a depth range between 50 and 290 m depth ; Table 1The dataset used in this study includes video and photographs collected during a ROV survey carried out in August 2013 onboard the r/v “Astrea”. Each site was explored through a variable number of ROV dives (from a min. of one up to four), to collect video material suitable to perform image analysis.The ROV “Pollux III” was equipped with a digital camera , a strobe (Nikon SB 400), a high-definition video camera (Sony HDR-HC7), track-link system, depth sensor, compass, and two parallel laser beams providing a constant 10 cm reference scale.2 . Overlaying, low-visible, poor-focus, and unsuitable frames  were discarded, while keeping only the usable ones for the analysis  as a sampling unit in each frame, hereafter referred to as frame [Frames were randomly extracted from the high-resolution ROV footage using the software “DVDVideosSoft”  density, based on the number of colonies within each frame and expressed as number of colonies m−2 [E. cavolini . In addition, the co-occurrence of red coral colonies with E. cavolini in the same frame was also recorded. In particular, we calculated: (i) the proportion of the two species when co-occurring; (ii) demographic  and morphological features  when co-occurring and not. Fishing impact was evaluated as the percentage of frames showing the presence of derelict fishing gears, which are fishing devices that are known to cause severe accidental damage to arborescent benthic fauna, such as the species under scrutiny in the present study [The substrate composition was estimated using suitable frames and was expressed as the proportion of “hard”  and “mixed” bottoms . We also evaluated the local slope of the substrate  and the presence or absence of deposited sand or silt. Then, we calculated red coral occupancy as the percentage of frames in which red coral was present over the total number of suitable frames in each site. Moreover, the following parameters for nies m−2 ; (2) mornies m−2 , colony nies m−2 , and thenies m−2 ; (3) popnies m−2 ; (5) branies m−2 , which inies m−2 ; and (6)nt study ,46. ThisE. cavolini, we used the Permutational Analysis of Variance (PERMANOVA) [p values in the PERMANOVA and pairwise tests were obtained from Monte Carlo samplings [The descriptive distribution parameters of skewness (g1) and kurtosis (g2) were estimated for the size-frequency distribution per each investigated site. Coefficients of g1 and g2 were considered significant if the ratio to their standard error was >2 . To asseRMANOVA) . The samRMANOVA) . For allamplings . The PERamplings .E. cavolini density, height, and colony orientation.We also aimed to test differences in red coral variables according to water depth, which was pooled in 4 depth categories: 90–110 m, 111–130 m, 131–150 m, and >150 m. Differences in red coral density, height, basal diameter, maximum branching level, and colonies orientation were tested through a Kruskal–Wallis routine, coupled with Mann–Whitney pairwise comparison, using the abovementioned depth categories. The same approach described above was carried out also for E. cavolini and C. rubrum density when co-occurred, non-parametric multivariate multiple regression analyses based on Euclidean distances were carried out using the routine DistLM, the forward selection procedure, and adjusted R2 and AIC as selection criteria [p values were obtained using 999 permutations of the raw data for the marginal tests , whereas, for all sequential tests, the routine used 999 permutations of residuals. The test was performed on the subset of frames where both species were present . In detail, fourth-root-transformed density values of both E. cavolini and C. rubrum were used, separately, as response variables. For each of the two response variables, the other species’ density, colony height, and depth were used as potential explanatory covariates.To identify the potential drivers of the variability of criteria . The for2) consisted of hard bottoms, except for the “Secchitella” site where the substrate was mostly (72%) formed by mixed bottom (hard bottom + sand or mud). In all investigated areas, all patches were settled on vertical and partially silted substrates, with the only exception of six and twelve colonies settled on horizontal substrates in the Mortorio Canyon and Bancotto, respectively. Red coral was found in 249 out of the 835 analyzed frames (30%). A total of 1384 colonies of C. rubrum forming 53 red coral patches were counted. Along the NE coast (n = 534 frames), red coral was recorded from a minimum of 22% to a maximum of 44% of frames, with a mean density within patches of 4.5 ± 2.4 colonies m−2. In the CE areas (n = 228), the presence of red coral dropped down to 3–33% of frames, with a mean density of 6.3 ± 3.1 colonies m−2. Finally, along the SW coast of the island (n = 73), red coral was recorded in 25–37% of the analyzed frames, with the lowest mean density recorded of 2.8 ± 1.4 colonies m−2  and diameter , respectively.p < 0.05). The maximum colony height (59 cm) was recorded in the SW area (Bancotto), while the minimum (0.8 cm) was recorded in the NE area (Tavolara Canyon). Overall, colony height did not show significant differences across the bathymetric range investigated (p = ns); however, the Mann–Whitney pairwise comparison emphasized significant differences between the shallower colonies and the deepest ones (Colony height was significantly higher in the SW area (12.6 ± 3.4 cm) than in the CE (7.8 ± 2.5 cm) and NE (6.3 ± 2.3 cm) areas , and only when co-occurring with p = <0.001; Colony orientation did not significantly change between the sampling areas: ~50% of the colonies were oriented at 135° with respect to the hard bottom surface, while only 30% were perpendicular to the walls  observed in the site Pietrame and the lowest (4%) in the Arbatax Canyon, where, consequently, the highest percentage of dead colonies was registered 96%; .The frequency of frames in which fishing gears and/or nets were present was 1.3% in the NE area, 12.5% in the CE area, and 1% in the SW area. The highest fishing impact was recorded in the Cala Gonone Shoal and Arbatax Canyon, where lost fishing gears or nets were present in 16% and 18% of the frames, respectively. Overall, a negative correlation between the presence of fishing gear and the presence of red coral was found .−2 in the CE and NE areas, respectively (−2) was recorded in the Tavolara Canyon at 143 m depth and the lowest one (0.03 colonies m−2) in the Orosei Canyon at 92 m depth. Colony density was significantly lower above 111 m depth than below  between 76 and 199 m depth. The average density ranged from 2.6 ± 3.7 to 3.3 ± 4.9 colonies mectively . The maxp = ns) despite the significant differences found through the pairwise comparison between shallower colonies (90–110) and those dwelling at greater depths (Colony height was higher in the SW (25.9 ± 10.3 cm) and CE (24.8 ± 14.6 cm) areas and lower in the NE area , whereas in the SW area, 37% of them were 0°, and 37% were oriented 135° . FurtherC. rubrum and E. cavolini was observed in 105 frames out of the total 835 analyzed frames (13%). Co-occurrence was observed throughout a wide bathymetric range  but mostly in the deepest sites (120–160 m depth), where > 80% of frames showed the co-occurrence of the two species. We here found that 42% of the frames where red coral was present, also showed the presence of the yellow gorgonian, with the highest co-occurrence rate (93%) found in the Pietrame Canyon.Overall, the co-occurrence of E. cavolini (p < 0.001). The basal diameter of red coral showed similar values with or without E. cavolini, except for the Mortorio Canyon, where it was significantly lower in the presence of the yellow gorgonian (p < 0.05). In addition, the maximum branching level significantly changed with the presence of E. cavolini (p < 0.001): red coral presented a higher proportion of colonies with the second-order branches (39% vs. 29%) and with the fifth-order branches when it co-occurred with E. cavolini.The density and height of the two species varied along the bathymetric range investigated, both in the presence and absence of the other species . Overallcavolini A,B, withE. cavolini, density was significantly higher (p < 0.01), whereas height was lower when it co-occurred with the red coral , and to a much less extent with water depth . This is a lower value compared to that found (46%) in a previous paper focused on deep-dwelling red coral colonies in the Tyrrhenian Sea ; howeverE. cavolini dwelling up to 200 m depth. Overall, density values of the yellow gorgonian were lower than those previously found in shallow-water ecosystems [E. cavolini shows high phenotypic plasticity, modulating the size, shape, and orientation of the colonies in response to water movement [Our results contribute to advancing knowledge on the distribution and demographic features of populations of osystems ,65 and mosystems . Howeverosystems . We alsoosystems . In addimovement ,68,69. Tmovement .E. cavolini and Paramuricea clavata (60% of ROV frames) [C. rubrum and the scleractinian cup coral, Leptopsammia pruvoti, usually tend to segregate one from the other at a small spatial scale, but in some cases, the latter wedged within crowded C. rubrum patches, probably due to the aggressive behavior of Leptopsammia pruvoti [In this study, we found a recurrent association  between red coral and the yellow gorgonian, especially below 120 m depth. These results indicate the two species can partially overlap their distribution in deep sublittoral areas, thus potentially competing for space and resources. Although red coral is known to form monospecific patches, its association with other gorgonians in the deep Mediterranean Sea is not completely unexpected; indeed, in the Tyrrhenian Sea, red coral has been previously found associated with  frames) . Further pruvoti .E. cavolini, with increasing density when occurring with red coral: this pattern could be likely ascribed to intra-specific self-thinning processes driven by increased density values but also to inter-specific relations, for which we acknowledge the limitation of the present dataset, which misses a temporal replication of observations that would ultimately contribute to understanding such a morphological evolution of colonies. In conclusion, the observed association could be linked to abiotic variables presumably joined with biotic ones [We report here that when the two species co-occurred, the density of both red coral and yellow gorgonian were higher and significantly influenced by the density values of the other, while colony size  or depth were less or not significant factors. Our results suggest that competition for space and settlement, likely driven by very similar larval preferences, could be the major constraint for the co-existence of these two suspension feeders. However, the higher density values found when the two species co-occurred, could also open new questions regarding the potential positive effects as foundation species, that are able to modify the peculiar habitat where they live . In addition, we also observed a decrease in the height of tic ones ,71: diffC. rubrum and E. cavolini populations (The present study advances our knowledge on the distribution and population structures of deep-dwelling ulations . Our datThis paper also reports the co-occurrence of red coral and yellow gorgonian between 90 and 200 m depth, demonstrating a partially overlapping distribution and that the variability of the two species’ densities is significantly driven by the density of the other. However, further studies are needed to better clarify the relative effects on crucial biological features, such as settlement and recruitment, and to analyze the role of different environmental factors potentially influencing these inter-specific relationships.