Hypoxic training is believed to be generally useful for improving exercise performance in various athletes. Nowadays, exercise intervention in hypoxia is recognized as a new therapeutic modality for health promotion and disease prevention or treatment based on the lower mortality and prevalence of people living in high-altitude environments than those living in low-altitude environments. Recently, resistance training in hypoxia (RTH), a new therapeutic modality combining hypoxia and resistance exercise, has been attempted to improve muscle hypertrophy and muscle function. RTH is known to induce greater muscle size, lean mass, increased muscle strength and endurance, bodily function, and angiogenesis of skeletal muscles than traditional resistance exercise. Therefore, we examined previous studies to understand the clinical and physiological aspects of sarcopenia and RTH for muscular function and hypertrophy. However, few investigations have examined the combined effects of hypoxic stress and resistance exercise, and as such, it is difficult to make recommendations for implementing universal RTH programs for sarcopenia based on current understanding. It should also be acknowledged that a number of mechanisms proposed to facilitate the augmented response to RTH remain poorly understood, particularly the role of metabolic, hormonal, and intracellular signaling pathways. Further RTH intervention studies considering various exercise parameters  are strongly recommended to reinforce knowledge about the adaptational processes and the effects of this type of resistance training for sarcopenia in older people. Hypoxia refers to a condition in which oxygen saturation of arterial blood decreases and the amount of oxygen supplied to tissues is reduced, and is used for altitude/low oxygen training in natural or simulated high oxygen environments ,2. The hIO2 < 15%) compared to sea level, and effectively increases energy consumption of various movements, such as low intensity intermittent and aerobic exercise [Nowadays, based on the lower mortality and prevalence of people living in high-altitude environments than those in low-altitude environments, hypoxia is being used to improve diabetes, cardiovascular diseases, hypertension, obesity, and age-related diseases . Hypoxiaexercise . Overweiexercise ,12. As aexercise ,11,12,13Recently, resistance training in hypoxia (RTH), a new therapeutic modality combining hypoxia and resistance exercise, has been attempted to improve muscle hypertrophy and muscle function ,15,16,17Therefore, this review would like to explain the possibility of RTH with low exercise intensity and mechanical tension as a novel and valuable treatment that can be effectively applied to the sarcopenia population, along with recent previous studies. We can discuss the clinical and physiological aspects of sarcopenia and RTH for muscular function and hypertrophy.Involuntary loss of skeletal muscle mass and function, defined as sarcopenia, is one of the most notable corollaries of aging and is a predictor of physical disability/mortality ,26. SarcIn people with an inactive lifestyle, muscle mass loss usually progresses gradually from age 40 and accelerates after age 60, resulting in a loss of approximately 2% of muscle mass per year . Muscle In this section, various clinical and physiological points related to sarcopenia are described in relation to physical fitness, inflammation responses, antioxidants, and mitochondrial function, hormones, biochemical properties of muscles, and metabolic and cardiovascular diseases. The mechanism for various clinical and physiological points related to sarcopenia is shown in The decrease in muscle mass in the process of degeneration of the human body is due to the decrease in the number and FCSA . This phDecrease in muscle strength and obesity in the elderly are highly correlated with chronic inflammatory conditions . de LucaAs one of the important causes of metabolic abnormalities associated with sarcopenia, reactive oxygen species produced in tissues are receiving much attention . Free raAging is known to cause changes in the expression and sensitivity of growth hormones (GH), insulin-like growth factor (IGF)-1, cortisol, testosterone, and female hormones ,71,72. TIn the balance between protein synthesis and degradation for the maintenance of skeletal muscle tissue, the activated phosphatidylinositol-3-kinase (PI3K)/Akt/mTOR signaling pathway plays an important role in anabolic metabolism induced by protein synthesis ,79. IncrMyostatin is a muscle growth regulatory factor produced by skeletal muscle and is regulated by the transcription factors for small mothers against decapentaplegic (SMAD) 2 and 3, which induce IGF-1/Akt signaling . MyostatConsequently, it can be explained that these various biochemical pathways regulate protein synthesis and degradation, thereby affecting sarcopenia.Sarcopenia reduces daily energy consumption, leading to obesity and type 2 diabetes ,91,92. OAdditionally, the loss of muscle mass with increasing age induces obesity, type 2 diabetes, inflammation, and oxidative stress, which in turn increases the risk of cardiovascular disease ,99,100. Exercise intervention is the most commonly used method to improve sarcopenia . Among tIO2 on the strength function of an elderly population. As a result, the WBVT showed a significant enhancement in the maximal strength of knee extensors with a small effect size. They concluded the WBVT under hypoxia did not have a remarkable effect on strength function in healthy elderly subjects. Like this, it cannot be definitively argued that RTH brings great advantages over traditional resistance exercise, but it is considered to have a great advantage in envisioning a more efficient and effective training strategy. Therefore, it is very important to interpret the effect of RTH on muscle function and hypertrophy as a new therapeutic modality for sarcopenia.However, such high intensity can cause problems with the musculoskeleton for inactive elderly people to perform high-intensity resistance training to slow the development of sarcopenia and improve muscle function ,103. In pO2). As a result, the RTH group showed a 3.2% increase in the sum of the FCSA of the knee flexors and flexors compared to the traditional resistance training group. Nishimura et al. [IO2 = 0.12) for 4-week.First, with regard to the morphological change via RTH, Manimmanakorn et al.  designeda et al.  reporteda et al.  reportedThese discrepancies can be explained by differences in RTH program duration, intensity, repetition, and rest between sets ,123. Mann = 7) that performed exercise resistance under normoxia and an experimental group (n = 9) that conducted exercise resistance under hypoxia (FIO2 = 14.4%), and then all subjects performed 8 weeks of resistance training in each environmental condition. Consequently, the experimental group that performed RTH showed a significant improvement in muscle endurance, but no difference in muscle strength. Park and Lim [n = 10) and a hypoxic group (n = 10) for training at 526 mmHg hypobaric hypoxic condition. They reported a greater improvement in muscle function  and anabolic hormone  in the hypoxic training group than in the normoxic training group. Further, Manimmanakorn et al. [pO2). Consequently, the RTH group showed significantly improved muscle strength and endurance than the traditional resistance training group. They also reported that the improvement of muscle functions  via RTH can enhance skill-related fitness . Törpel et al. [pO2) with matched designed resistance training under normoxia on normal muscular strength capacity and body composition after 5 weeks in young (n = 42) and older people (n = 42). They reported that the RTH design used had no superior effect on the tested parameters in young and older people over matched designed RT under normoxia after a 5-week intervention period. Based on this, they explained that it can be assumed that the expected higher effect of RTH can be achieved by changing the exercise parameters .RTH has been reported to be effective in improving muscle functions such as muscle strength and endurance, as well as muscle morphological changes such as FCSA ,20,121.  and Lim  verifiedl et al.  comparedIn summary, to utilize RTH as a new therapeutic modality for sarcopenia, further RTH intervention studies considering various exercise parameters  are strongly recommended to reinforce knowledge about the adaptational processes and the effects of this type of resistance training in older people.The most representative potential mechanisms for morphological changes  and muscle function  via RTH are the accumulation of metabolites, hormonal response, and intramuscular signaling pathways. The proposed mechanism for muscle morphological changes and muscle function via RTH is shown in +) are known metabolites that play a pivotal role in muscle hypertrophy [IO2 = 0.13) and a control group performing resistance exercise in normoxia (FIO2 = 0.21). Then, the metabolic response immediately after performing bench press and leg press exercises with 10 repetitions for 5 sets with 70% 1-RM was examined. Consequently, the experimental group showed 1.2 times higher blood lactate level than the control group. Kon et al. [IO2 = 0.13) and resistance exercise in normoxia group (FIO2 = 0.21). Then, they evaluated the acute effects of an RTH composed of 3 sets of 75% 1RM to muscle failure with a 2-min rest between sets to muscle failure on bench press performance. As a result, there was no difference in physical performance during bench press between the two groups, however, there were significant increases of blood lactate levels in the hypoxia group. Scott et al. [IO2 = 0.16) augments anabolic responses. As a result, they concluded moderate-load resistance exercise under hypoxia augments metabolite accumulation  and muscle activation . Ramos-Campo et al. [The accumulation of metabolites has been reported to play various intervention roles in inducing muscle hypertrophy ,126,127.ertrophy ,129. Curertrophy  equally n et al.  confirmen et al.  classifiIO2 = 0.13) were reported. However, serum IGF-1 levels increased immediately after resistance exercise in both environmental conditions, but there was no difference between the two groups. These results suggest that elevated metabolic stress via RTH induces an increase in GH and testosterone [IO2 = 0.129) on hormonal response and muscle hypertrophy in resistance trained male subjects. Consequently, 6-week of RTH induced a significant increased IGF-1 level, body mass, and fat free mass compared to training in normoxia. In addition, Kon et al. [Hormonal responses by RTH also play an important role in muscle morphological and functional changes . In the osterone ,133. Chyosterone  evaluaten et al.  reportedn et al.  reportedn et al. ,135. CurIO2) and normobaric hypoxic (12% FIO2 for 3.5 h) post-absorptive conditions. They concluded that although the phosphorylation of ribosomal S6 kinase 1 was increased, hypoxia did not reduce muscle protein synthesis over 3.5 h at rest but blunted the increased muscle protein synthesis response to acute resistance exercise to a degree dependent on extant SpO2. Moreover, they suggested that other signaling processes that are probably not currently known in hypoxia could ignore mTOR (mechanistic target of rapamycin) signaling and trigger a physiological response to resistance exercise. Usually, exposure to hypoxia has been reported to activate hypoxic inducible factor-1α (HIF-1α), which acts as a primary transcriptional response factor for adaptation to hypoxia [In the intramuscular signaling pathway, chronic exposure to hypoxia has been reported to negatively affect protein kinase B/mTOR signaling and increase myostatin expression, resulting in skeletal muscle atrophy ,137,138. hypoxia . The act hypoxia ,142,143. hypoxia ,145. The hypoxia . This au hypoxia . The sig hypoxia ,147. In  hypoxia . As a reA recent Britto et al.  study reResistance training provides a strong mechanical stimulus to the human body, through which metabolic changes, hormonal changes, and changes in intracellular signaling pathways appear, thereby inducing muscle hypertrophy and improving muscle function. However, traditional resistance training with high-intensity can cause problems with the musculoskeleton for inactive elderly people. Therefore, in recent years, RTH, which combines low- to moderate-intensity resistance exercise and hypoxia, that can induce physiological and biochemical changes related to muscle morphology and function more strongly has attracted much attention. However, there are very few studies that have applied RTH to the elderly with sarcopenia, and previous studies demonstrating that RTH has a greater effect on sarcopenia-related clinical and physiological points than traditional resistance training are also very scarce. Therefore, it is difficult to argue strongly that RTH is a more effective method for improving muscle function and hypertrophy in the elderly than traditional resistance training based on the review of previous studies. This is even more persuasive in that the potential mechanisms for the improvement of muscle function and hypertrophy that appear during RTH are not yet clearly identified. Further, RTH intervention studies considering various exercise parameters  are strongly recommended to reinforce knowledge about the adaptational processes and the effects of this type of resistance training in older people for utilizing RTH as a new therapeutic modality for sarcopenia.


Objectives: To screen and identify ideal leading compounds from a drug library (ZINC15 database) with potential inhibition of aminopeptidase N(CD13) to contribute to medication design and development.Results: Two novel natural compounds, ZINC000000895551 and ZINC000014820583, from the ZINC15 database were found to have a higher binding affinity and more favorable interaction energy binding with CD13 with less rodent carcinogenicity, Ames mutagenicity, and non-inhibition with cytochrome P-450 2D6. Molecular dynamics simulation analysis suggested that the 2 complexes, ZINC000000895551-CD13 and ZINC000014820583-CD13, have favorable potential energy, and exist stably in the natural circumstances.Conclusion: This study discovered that ZINC000000895551 and ZINC000014820583 were ideal leading compounds to be inhibitions targeting to CD13. These compounds were selected as safe drug candidates as CD13 target medication design and improvement.Materials and Method: Potential inhibitors of CD13 were identified using a series of computer-aided structural and chemical virtual screening techniques. Structure-based virtual screening was carried out to calculate LibDock scores, followed by analyzing their absorption, distribution, metabolism, and excretion and toxicity predictions. Molecule docking was employed to reveal binding affinity between the selected compounds and CD13. Molecular dynamics simulation was applied to evaluate stability of the ligand-CD13 complex under natural environment. Cancer is a lethal condition ranking the second among all factors that cause human death in the world. The treatment has evolved to include different modalities including surgery, chemotherapy, radiotherapy and immunotherapy , 2. DespCD13, also known as the aminopeptidase N(APN), belongs to the M1 metalloprotease family, and it is common and widespread. CD13 can residue from the peptide hormone angiotensin III (AngIII) to generate angiotensin IV (Ang IV), which has been shown to cause vasodilatation, hypertrophy, and activation of NF-κB.  CD13 plaOver all, CD13 is related to the metastasis, adhesion, vascular hyperplasia and cell apoptosis, which are all related with tumorigenesis. Tumor angiogenesis and metastasis can be suppressed by inhibiting its activity. Many studies have revealed that CD13 is related with various kinds of cancers such as melanoma, colon cancer, prostate cancer, lung cancer, liver cancer, leukemia, thyroid cancer, etc. –14 ThereNowadays, natural products and their derivatives play a decisive role in pharmacologic market, because of their unique molecular structures and potential biologic function, which have made a great contribution to the design and improvement of medicine , 18. SevThe S1 pocket is an important regulatory site of CD13, as the Val (P1) side chain in the Ang IV complex binding to this active site could cause vasodilatation, hypertrophy, and stimulate NF-κB pathway, and then neovascularization occurs. Therefore, this pocket region was selected as a reference site. A total of 17,931 biogenic-for sale-named product molecules were taken from the ZINC15 database. Chemical structure of the receptor protein, CD13, was selected to test the pharmacologic properties of other compounds. One inhibitor, Bestatin, was chosen as the reference compound. After screening, 4761 compounds were found to be eligible to bind stably with CD13; among those, 318 compounds were found to have higher LibDock scores than Bestatin. (LibDock score: 97.2164), which are listed in Pharmacologic properties of all the 20 selected ligands with Bestatin were predicted using the ADME module of Discovery Studio 4.5, including aqueous solubility level, blood-brain barrier level, CYP2D6 binding, hepatotoxicity, human intestinal absorption level, and plasma protein binding properties . The aquSafety and toxicity should also need full consideration in this study. To confirm the safety of the top20 compounds, different kinds of toxicity indicators of these compounds and Bestatin, including rodent carcinogenicity , developmental toxicity potential properties, Ames mutagenicity were predicted by using a computational method in the TOPKAT module of Discovery Studio 4.5 . Results28 of the compound with ALA819:O of CD13, H32 of the compound with GLU364:OE2 of CD13, the O20 of the compound with ARG305:HD1, the H25 of the compound with GLU364:OE1, the H30 of the compound with SER307:OG and the H31 of the compound with THR820:O. Besides, 4 π-related interactions were formed in the complex, including the π-Orbitals between ZINC000000895551 and the ALA308, LEU368, LEU821, and VAL822 of CD13. ZINC000014820583 formed 6 pairs of hydrogen bonds with CD13, by the H34 of the compound with ALA819:O of CD13, the O9 of the compound with ASN823:HN of CD13, the H24 of the compound with LEU368:O of CD13, the H30 of the compound with GLU364:O of CD13, the H36 of the compound with SER307:OG of CD13 and the H37 of the compound with THR820:O of CD13. It also formed 5 pairs π-related interactions in the complex, consist of 2 pairs between ALA308 of CD13 and the compound, ARG305 of CD13 with the compound, LEU368 of CD13 with the compound, and LEU821 of CD13 with the compound. As to the reference compound Bestatin, it formed 2 hydrogen bonds with CD13, by the H38 of the compound with THR820:OG1 of CD13, and H36 of the compound with THR820:O of CD13. Also three π-related interactions formed between Bestatin and CD13, including ALA308, LEU368 and LEU821 of CD13 with Bestatin.To identify ligand binding mechanisms of these compounds and Bestatin, ZINC000000895551 and ZINC000014820583 were docked into the molecule structure of CD13 using CDOCKER module, and CDOCKER potential energy was calculated and displayed in In order to estimate the stabilities of the ligand-CD13 complexes on natural environment conditions, the molecular dynamics simulation module test was conducted. According to the original conformations from the molecular docking experiment through CDOCKER module, RMSD curves and potential energy chart of each complex were shown in Cancer is the second most frequent cause of death worldwide in spite of significant improvements in cancer therapies during past decades. Research on the prevention and treatment of cancer has attracted worldwide attention . Past re1/2, no more than 20 min; while administered by i.v. administration, Bestatin has a high initial serum level [Though CD13 inhibitors have therapeutic possibility in anticancer field, there are significant limitations of Bestatin, currently being tested as potential anticancer compound. Relevant research showed that concentration level of serum Bestatin depends on its administration; when administered via the i.p., s.c., i.m., or p.o. routes, Bestatin had short serum tum level . This stIn this study, 17,931 biogenic-for sale-named product molecules taken from the ZINC15 database for virtual screening, was followed by ADME, TOPKAT, CDOCKER, and molecular dynamics simulation. LibDock scores unfolded degree of energy optimization and stability of the conformation. High LibDock score compounds illustrated better energy optimization and a stable conformation than lower score achievers. After calculation by the LibDock module, 4761 compounds showed to be capable to bind stably with CD13. Besides, among these ligands, 318 compounds had higher LibDock scores than Bestatin (LibDock score: 102.86), indicating that these 318 compounds could form a more stable complex with CD13 with better energy optimization compared with Bestatin. Based on LibDock score, the top 20 natural compounds were selected and pooled for further study.ADME and toxicity predictions of the selected compounds were carried out to assess the pharmacologic properties of these compounds. The result suggested that ZINC000000895551 and ZINC000014820583 were confirmed as ideal candidacy compounds because of their good solubility in water and good intestinal absorption level. Furthermore, ZINC000000895551 and ZINC000014820583 were predicted to be no inhibitors of CYP2D6, no Ames mutagenicity, and less rodent carcinogenicity and developmental toxicity potential by comparison with other compounds. This information illustrates that these 2 compounds have preferable safety, and they are enough to be considered as potential ideal leading compounds. All in all, ZINC000000895551 and ZINC000014820583 were reasonably identified as relatively high quality drug candidacies. Results data above elucidated their potential use in drug designing. In spite of the toxicities or negative effects of the remaining drugs on the list, they also had prospective application in drug development by adding or deleting particular functional groups and atoms to reduce toxicity. Combining the above results, ZINC000000895551 and ZINC000014820583 were identified as ideal leading compounds, followed by further analysis was performed.Following study revealed Ligand binding mechanism and chemical bonds of candidate compounds with CD13. CDOCKER module computation confirmed that CDOCKER interaction energy of ZINC000000895551 and ZINC000014820583 was obviously lower than the reference ligand Bestatin, which demonstrates why these 2 compounds may have a higher binding affinity with CD13 compared with Bestatin. Then, the chemical structures of the 2 compounds were analyzed by molecular structure inspection. The 2 complexes that CD13 combined with 2 candidacies have more chemical bonds than Bestatin, which again indicates that these 2 compounds could bind with CD13 at active site S1 pocket more stably. So they may contribute to competitive inhibition of activity of CD13 and act in improved the effect of cancer treatment.Finally, their stabilities in the natural environment were assessed performing molecular dynamics simulation. Computational results of RMSD and potential energy of these ligand-CD13 complexes showed that the trajectories of complexes reached equilibrium after 18 ps. RMSD and potential energy of these complexes tends to stability with time, which suggested that these 2 complexes could exist in the natural environment stably. Based on these results, drug designation and development, such as modification and refinement, could be prospectively carried out to make combination of ligand and receptor more stable. Moreover, it is also noteworthy that agonist and inhibitor always share a similar chemical skeleton structure, and adding or deleting different groups or atoms leads the opposite effects. As a result of their significantly pharmacologic properties, highly binding affinity and stabilization combining with CD13, natural compounds identified in this research could provide a valuable resource for CD13-related drug development.In recent years, design and development of oncology drugs are hot research topics worldwide. However, overall progress has not seemed encouraging. This study elucidated that the first and the most important step in drug designation was to identify ideal leading compounds. In this research, 5 modules of Discovery Studio 4.5, including LibDock, ADME, TOPKAT, CDOCKER, and molecular dynamics simulation, were applied to screen and analyze the biochemical structure properties of novel potential compounds. Molecule conformation, pharmacologic properties, binding affinity, and stability of each selected compound were also fully tested and calculated to determine their advantages compared with the reference compound, Bestatin. A series of high-tech computational studies showed that these 2 compounds may have potential effect in drug therapy of multiple types of cancer. However, it is noteworthy that no single natural compound could be directly marketed without thousands of refinement and improvement. On the basis of the identification of these 2 leading compounds with the most potential, further research could directly concentrate on improvement and refinement of them. Furthermore, our study provides a guideline for screening leading compounds with potential therapy effect. Through this high-tech method, more leading compounds could be screened, which could improve current drug development and bring increase of efficiency.Although this study was conducted by elaborate design, it still has some limitations. Further experiments, such as animal testing, need to be performed to verify our results more firmly, and more indicators, such as half-maximal inhibitory concentration and half-maximal effective concentration, should be assessed in future studies to test more compounds properties.This study performed a series of computer-aided structural and chemical analysis technology  to screen and identify the ideal leading compounds with functions to inhibit CD13. Two compounds, ZINC000000895551 and ZINC000014820583, were selected as safe candidacies, and they had great significance in CD13 inhibitor medication development. Furthermore, this study provided a list of drug candidates and their pharmacologic properties, which may contribute to CD13 or other proteins in medication design and improvement.Discovery Studio 4.5 software  is a suite of software for simulating small molecule and macromolecule systems; it is a new generation of molecular modeling and environmental simulation software for the life sciences field, which aims to provide protein modeling, optimization, and drug design tools by applying protein structure and structural biologic computation. Numerous leading compounds and drug candidates were identified and refined through this method. LibDock module of Discovery Studio was employed for virtual screening; CDOCKER module was used for docking study; and ADME module was analyzed for pharmacologic properties. The Natural Products database in the ZINC15 database was selected to screen STING agonists. The ZINC15 database is a free database of commercially available compounds provided by the Irwin and Shoichet Laboratories, Department of Pharmaceutical Chemistry, University of California, San Francisco .Ligand-binding pocket region of CD13 was selected as the binding site to screen potential compounds to inhibit CD13. Virtual screening was carried out using the LibDock module of Discovery Studio 4.5 . LibDockThe ADME module of Discovery Studio 4.5 was employed to calculate absorption, distribution, metabolism, and excretion (ADME) of selected compounds, including their aqueous solubility, blood-brain barrier penetration, cytochrome P-450 2D6 (CYP2D6) inhibition, hepatotoxicity, human intestinal absorption and plasma protein binding level. TOPKAT module of Discovery Studio 4.5 was employed to calculate the toxicity and other properties of all the potential compounds, such as U.S. National Toxicology Program rodent carcinogenicity, Ames mutagenicity, developmental toxicity potential, and rat oral median lethal dose (LD50) and chronic oral lowest observed adverse effect level (LOAEL). These pharmacologic properties were fully considered in screening proper probable drug candidates for CD13.CDOCKER module of Discovery Studio 4.5 was used for molecular docking study. CDOCKER is a molecular docking method based on CHARMM force field, which can produce high-precision docking results. The CHARMM force field was used for both receptors and ligands. Receptor is held rigid, while ligands are allowed to flex during the docking process. For each complex pose, the CHARMM energy and the interaction energy, which indicated ligand binding affinity, were calculated. Crystal structure of CD13 was obtained from the protein data bank. The crystal water molecules were generally removed in a rigid and semi-flexible docking process, causing the fixed water molecules to possibly affect the conformation of the receptor-ligand complex , 24. TheThe best binding conformations of the ligand-CD13 complexes among the poses predicted by the molecule docking program were selected and prepared for molecular dynamics simulation. The ligand-receptor complex was put into an orthorhombic box and solvated with an explicit periodic boundary solvation water model. To simulate the physiologic environment, sodium chloride was added to the system with the ionic strength of 0.145. Then the system was subjected to the CHARMM force field and relaxed by energy minimization (500 steps of steepest descent and 500 steps of conjugated gradient), with the final root mean square gradient of 0.227. The system was slowly driven from an initial temperature of 296 K to the target temperature of 302 K for 2 ps, and equilibration simulations were run for 5 ps. Molecular dynamics simulation (production module) was performed for 25 ps with time step of 1 fs. The simulation was performed with the normal pressure and temperature system at a constant temperature of nearly 300 K during the process. The particle mesh Ewald algorithm was used to calculate long-range electrostatics, and the linear constraint solver algorithm was adapted to fix all bonds involving hydrogen. With initial complex setting as a reference, a trajectory was determined for RMSD, potential energy, and structural characteristics through the Discovery Studio 4.5 analysis trajectory protocol.Supplementary Table 1


IL4 and IL13 genes in asthma susceptibility and their participation in plasma cytokine levels depending on genotypes Methods: We include 486 subjects, divided into asthma patients  and clinically healthy subjects . We genotyped three SNP, two in the IL4 and two in the IL13 gene; also, two CNVs in IL4. The IL-4, IL-13 and IgE plasma levels were quantified. Results: Biomass-burning smoke exposure was higher in the AP group compared to CHS . No statistical differences were found in the genetic association analysis. In both CNV, we only found the common allele. For the analysis of IL-4, IL-13, and IgE measures stratified by genotypes, no significant association or correlation was found. Conclusion: In the Mexican-mestizo population, SNPs neither CNVs in IL4 nor IL13 are associated with asthma susceptibility or involved serum cytokine levels. Biomass-burning smoke is a risk factor in asthma susceptibility.Background: Asthma is a complex and chronic inflammatory airway disease. Asthma’s etiology is unknown; however, genetic and environmental factors could affect disease susceptibility. We designed a case-control study aimed to evaluate the role of single-nucleotide polymorphisms (SNP), and copy-number variants (CNV) in the  Asthma is a chronic inflammatory disease characterized by airway hyperresponsiveness, causing recurrent dyspnea, cough, and wheezing. These events are generally associated with an extensive and variable obstruction of the lung airflow, often irreversible with or without treatment .In the last four decades, there has been a sharp increase in the global prevalence, morbidity, and mortality associated with asthma, and it has calculated that approximately 300-million people worldwide undergo asthma, having world prevalence between 1 and 18%. In México, between 5 to 15% of people suffer it , affectiDifferent extrinsic and intrinsic factors are recognized and, even when it is a common chronic illness, asthma etiology is at day unknown. It has described that the interaction between genetic background and environmental factors, as the pollution and tobacco smoke in intrauterine life and during childhood ,5, have loci and genes associated with asthma and clinical phenotypes; for example, ADORA1 [hCLCA1 [CTLA4 [TNF [Several association studies in diverse populations have reported a high number of different , ADORA1 , hCLCA1  [hCLCA1 ,8, CTLA41 [CTLA4 , and TNFLA4 [TNF ,11,12. BLA4 [TNF , IL-8 [1LA4 [TNF , IL-6 [1LA4 [TNF ,16, eNOSLA4 [TNF  and FcR1LA4 [TNF  due to tIL4 and IL3, previously described in genetic susceptibility with asthma [Other closely related genes to this inflammatory process are h asthma ,20,21. Fh asthma ,22,23,24Recent investigations have shown IL-4 increased in serum, bronchoalveolar lavage fluid (BALF), and sputum in asthma patients and animal models, as well as different asthma phenotypes, as rhinitis, severity or atopy ,25,26. TOn the other hand, IL-13 has been associated with polarization to Th2 immune response, promoting the resistance to corticosteroids and the production of chemokines as CCL13 , and incA meta-analysis has revealed the association of different single nucleotide polymorphisms (SNPs) in the IL4 gene associated with susceptibility to asthma in Caucasian-European populations, especially the rs2070874 (C-33T) and rs2243250 (C-589T), located in the promotor region . InteresOther studies have explored the copy-number variations (CNVs) and variable number tandem repeats (VNTR)  as possiIL4 , and IL13 (rs1800925 C/T and rs20541 G/A) genes in the asthma susceptibility, and the possible effect in the serum levels of IL-4, IL-13, IgE and the potential effects implied in the risk environmental exposure factors.Based on all mentioned above, we aimed to describe the effect of genetic variants type SNPs and CNVs in In this case-control study, we included 492 Mexican-mestizo subjects over 18 years-old divided into two groups; 147 asthma patients (AP) and 345 clinically healthy subjects (CHS). Patients were recruited from clinical services from Instituto Nacional de Enfermedades Respiratorias Ismael Cosío Villegas (INER), with a previous asthma diagnosis. The diagnosis was based on clinical history, physical examination, spirometry data, as well as the criteria established by the American Thoracic Society (ATS); also, no history of respiratory viral or bacterial infections in the last three months. Subjects with other respiratory diseases were excluded. In the control group were included healthy subjects from blood bank service from INER, without asthma family background, and neither food nor drug allergies.Demographical and exposure to risk factors data were collected by a personal questionnaire. Data on arterial blood gas measurements, routine blood markers, blood cellularity, lung function, and pharmacological treatment were obtained from clinical records. Cases were classified by severity based on the Global Initiative for Asthma (GINA) 2006. The Strengthening the Reporting of Genetic Association Studies (STREGA) guidelines were taken into account in the design of this genetic association study .All participants fulfilled a hereditary-pathology background survey. Exclusion criteria included being of non-Mexican ancestry and having chronic respiratory diseases other than asthma and/or inflammatory disorders. After being given a detailed description of the study, patients who met the inclusion criteria were invited to participate. All participants signed a written informed consent and were provided with a privacy statement describing the protection of personal data. The research protocol was reviewed and approved by the Ethics in Research Committee of the INER in Mexico City (approbation code: B38-11). After signing the informed consent, a blood sample was drawn by venipuncture of the forearm of each patient. From each sample, we proceed to separate serum and plasma for storage at −80 °C until use.The DNA extraction was carried out from EDTA tubes from the plasma pellet with the commercial BDtract DNA isolation kit  and then rehydrated in TE buffer . Subsequently, the extracted material was quantified using a Nanodrop 2000 system  and stored at −80 °C until use.www.hapmap.org) were included. For CNVs selection, we consult the database of genetic variants (DGV) to localize two CNVs structural variants in the IL4 gene; one of these variants is a deletion between exon 3 and 4. SNPs and CNVs molecular information is presented in The SNPs were chosen by bibliography research. Those variants reporting a significant association, gene location, possible post-translational changes in the protein, and minor allele frequency (MAF) in Mexican from the Los Angeles population (Mex-LA), reported in the HapMap database  and commercial TaqMan DNA probes . Briefly, a DNA template, a probe sequence-specific for each SNP, and TaqMan Universal PCR Master Mix  arranged into 96-well microplates. Besides, three wells without a template included for each genotyping plate (contamination controls). For ~5% of the samples included in the study, genotyping in duplicate for control allele assignment was done.n = 15) and subjects with Amerindian ancestry n = 10 (five Otomíes and five Mazahuas). These groups were only used in the genotyping of the CNVs as a population control group, looking for samples with probable different copy-number in the analyzed variants.TaqMan specific sequence probes  were selected for each CNVs . The reaThe IL-4 and IL-13 measurements were made in de plasma samples through Human IL-4 and Human IL-13 Standard ABTS ELISA Development Kits  and following supplier instructions. The standard curve and sample measurement were carried out by triplicate.Quality control and Hardy-Weinberg equilibrium (HWE) were carried out using Plink v. 1.0.7  and De Fp < 0.01, OR = 3.4, 95%CI = 2.25–5.22); in the opposite, among subjects in the CHS group, tobacco-smoking in most frequent than in the AP group. We found ~30% of AP group had no pharmacological treatment; the rest of the patients had at least bronchodilator treatment (~69%); 43% of the treated patients had inhaled corticosteroids as therapy, while 9% was oral glucocorticoid. In the clinical laboratory testing, we found differences in the five cellular populations among groups. The leukocyte and neutrophils are increased in the AP group; while, lymphocytes, eosinophils, and basophils are decreased in the same group , while in the CHS group, men were slightly higher (57.9%). In the exposure to environmental risk factors, the AP group had a higher percentage of exposed to biomass-burning smoke (BBS) than in the CHS group , and two in IL13; rs20541  and rs1800925 .We include three SNPs localized within two genes related to immune response; one in For rs20541, the G allele frequency reaches 65%, while A is around 35%, considering cases and control. In the case of rs1800925, the C allele frequency is ~69%, and T is about 31%. Once rs2070874 is analyzed, the frequency for common and minor allele frequency are similar, while in the AP group, the C variant is the common allele (50%), but in the CHS group, T is the common allele (52%). No significant differences were found in the AP vs. CHS comparison. .Next, we calculate the genotype frequencies for each SNPs. In the rs20541, the homozygous for the common allele (GG) was found in around ~42%, heterozygous (GA) in approximately 46%, and homozygous to minor allele (AA) was almost 35%, including cases and controls; to note, GA is the most common genotype in our population. For the rs2070874, the most frequent genotype is the heterozygous , followed by CC (~27%) and at last TT (~25%). For the last SNP, rs1800925, the most frequent genotype is heterozygous CC (around 48%), next CT (~39%), and in the final, TT (~11%). After all, we compare genotype frequencies applying the co-dominant model, but no significant association was found for any SNP .Besides, in those asthma patients who have a complete medical record, a stratified analysis based on phenotypes associated with asthma, as rhinitis, allergies, and cellularity, was performed. No significant associations were found for any of the stratified association groups. Data are presented in We decided to apply different genetic models to compare genotyping frequencies; results for full-genotype, dominant, and recessive models for each SNP are described in Since the three SNPs were localized in the same chromosome, LD and HB were calculated. No significant LD or HB was found in any possible combination for the SNPs A. We incIn all included subjects for case-control comparison, was detected the common allele (one-copy per chromosome), no alleles harboring copy-numbers different to the common allele were found. The same behavior happens in the subjects included as a populational reference; only the common allele was discovered .p = 0.15) using Kruskal-Wallis test and after applying the post hoc test. The IL-13 levels were stratified by the rs20541 and rs1800925 in full-genotype model comparing by the Kruskal-Wallis test and post hoc analysis. No differences were found for any SNP by this model , but this was used to organizing a second analysis based on dominant (rs20541) and recessive models (rs1800925) and comparing with Mann-Whitney’s test. No significant differences were found for the applied models   .Asthma is a complex disease consequence of the interaction of intrinsic and extrinsic factors; the current study focused on the possible relation between the genetic component and the risk of developing the disease or clinical characteristics in a Mexican mestizo population. We found significant differences in some demographical variables, like sex, weight, and height, but no in the BMI. In the exposure variables, we observed a higher percentage of subjects exposed to BBS in the AP group (47.5%) with considerable exposition time (~13.5 years). The socioeconomic status in the population could explain differences in the exposition factors; unfortunately, we do not count with the corresponding data to adjust for this potential confounder. Most of the participants attend the INER asthma clinic; INER is a third-level hospital; most of the attending people come from different places of the country, especially from rural and suburban zones. Several authors have described the continuous exposure to BBS in these zones and especially in the female and children populations ,56,57,58Other changes that could be observed are the decrement of eosinophils and the increased percentage of neutrophils. The most studied cells described in asthma are eosinophils ,65,66, nIn the case of neutrophils, there is no evidence about the direct effect of corticosteroids/glucocorticoids in these cells. On the other hand, neutrophils could be stimulated by environmental pollution or smoking ,82,83,84Regarding pro-inflammatory cytokines, we did not find significant differences in IL-4 nor IL-13 levels. IL-4 is a cytokine related to the polarization of inflammatory profile to Th2 in lymphocytes; it is produced mainly by eosinophils, basophils, and mast cells ,89. IL-1p = 0.08; OR = 0.4, CI = 0.12-1.33). In a previous report in the Asian population, the rs2070874 was associated with the risk of severe forms of asthma [For the genetic association study, we did not find significant results with the illness or any clinical phenotype. Our report is the first in the Mexican population, including these variants; for that reason, we conducted a complete analysis stratifying the genotypes by co-dominant, full-genotype, recessive, and dominant models, but with any of the models found significant associations. Possibly due it is necessary to increase the sample size; when we compare the highest vs. normal levels of leukocytes, we found a trend with the TT genotype of rs1800925  was found, but prospective studies with a most robust sample size are needed to confirm this hypothesis.Following a similar procedure in the analysis of the continuous clinical variables , we applied the models above described, but no significant associations or correlations were found. Also, in the correlation analysis, a bordering correlation between years of exposition and neutrophils (%) with the CC genotype of rs1800925 (For CNVs, we did not find variation in this kind of polymorphism for any of the two included variants. To the best of our knowledge, there are no studies related to these two CNVs. The inclusion of CNVs aimed to enrich the asthma study with different polymorphisms that could explain the disease complexity.Among the potential limitations that must be contemplated are the variability of phenotypes, and the lack of adjustment by treatment, as well as not including biological samples that can reflect better the lung environment, as sputum.In the current study, no association was found between the SNPs/CNVs studied, the disease susceptibility, nor in the plasma levels of the clinical markers. Association studies with a larger sample size are necessary, considering other inflammatory markers related to asthma and clinical phenotypes.IL4 nor IL13 are associated with asthma susceptibility or involved in plasma cytokine levels. Biomass-burning smoke is a risk factor for asthma susceptibility.In the Mexican-mestizo population, SNPs neither CNVs in


Female patients with psychosexual disorders were associated with the risk of psychiatric disorders. This finding could be a reminder for clinicians about the mental health problems in patients with psychosexual disorders.We aimed to investigate whether females with psychosexual disorders were associated with the risk of affective and other psychiatric disorders. A total of 2240 enrolled individuals, with 560 patients with psychosexual disorders and 1680 subjects without psychosexual disorders (1:3) matched for age and index year, from the Longitudinal Health Insurance Database, retrieved from the National Health Insurance Research Database (NHIRD), between 2000 and 2015 in Taiwan. The multivariate Cox regression model was used to compare the risk of developing psychiatric disorders during the 15 years of follow-up. There were 98 in the cohort with psychosexual disorders  and 119 in the non-cohort without psychosexual disorders  that developed psychiatric disorders. The multivariate Cox regression model revealed that the adjusted hazard ratio (HR) was 9.848 (95% CI = 7.298 — 13.291, The online version contains supplementary material available at 10.1186/s12888-021-03060-1. Psychosexual disorders could be classified into sexual dysfunctions, paraphilias, and gender identity disorders , 2, and Several researchers have shown the neurodevelopmental interlinks between the psychosexual and psychiatric disorders: Sex differences in the microglial function might partially explain the differences observed in susceptibilities and outcomes of the neuropsychiatric disorders in men and women . RajkumaPrevious studies have found that depressive disorders are frequently associated with sexual dysfunction, across all the phases of sexual responses , and theThe National Health Insurance (NHI) Program was launched in Taiwan in 1995, and as of June 2009, including contracts with 97% of the medical providers, with approximately 23 million beneficiaries, or more than 99% of the entire population . The NatPatients with newly diagnosed psychosexual disorders were selected from the 2 million Longitudinal Health Insurance Database (LHID), randomized retrieved from the NHIRD, which covers 99% of the entire population of Taiwan, between January 1, 2000, and December 31, 2015. The patients with psychosexual disorders before 2000 were excluded. Besides, the patients diagnosed with psychiatric disorders before 2000, or before their first visit for any psychosexual disorder, were also excluded. In Taiwan, the legal age of full civil competency is 20 years of age, according to Taiwan’s Civil Code , therefoThe covariates included age groups , geographical area of residence , urbanization level of residence (levels 1 to 4), and monthly income . The urbanization level of residence was defined according to the population and various indicators of the level of development. Level 1 was defined as a population of > 1,250,000, and a specific designation as political, economic, cultural, and metropolitan development. Level 2 was defined as a population between 500,000 and 1,249,999, and as playing an important role in the politics, economy, and culture. Urbanization levels 3 and 4 were defined as a population between 149,999 and 499,999, and < 149,999, respectively.We assessed the comorbidities by using the Charlson Comorbidity Index (CCI), which categorizes comorbidities using the ICD-9-CM codes, and scores each comorbidity category –39. The Enrolled individuals in these two cohorts were tracked for 15 years, starting from the index date, to identify those who developed psychiatric disorders, comprising dementia, anxiety disorders, depressive disorders, bipolar disorders, eating disorders, sleep disorders, and psychotic disorders, withdrew from the NHI program, or reached the end of 2015. All the ICD-9-CM codes of psychiatric disorders are as listed in Table 2 and t-tests were used to appraise the distributions of the categorical and continuous variables, respectively. The multivariate regression model was used to determine the risk of psychiatric disorders since death can act as a competing risk factor for psychiatric disorders  vs 3.15 [SD ± 3.97]), without a statistical difference (1) Table .p < 0.001) in the association between the psychosexual disorders and psychiatric disorders, and the female patients with psychosexual disorders had a 9.8-fold increase in the risk of psychiatric disorders, after the adjustment of age, monthly income, urbanization level, geographic region, and comorbidities. The Kaplan-Meier analysis demonstrated that the cohort with psychosexual disorders had a significantly higher 15-year psychiatric disorders cumulative incidence than the comparison cohort. To the best of our knowledge, this is the first study on the topic of an association between female patients with psychosexual disorders and the risk of psychiatric morbidity. This finding could serve as a reminder for the clinicians to pay much more attention to these patients because of the issues about psychiatric disorders.The adjusted HR was 9.848 (95% CI = 7.298—13.291, Previous studies have shown the association between psychosexual disorders and psychiatric disorders that included antidepressant-related sexual dysfunctions in patients with depressive or anxiety disorders , 45–47, Previous studies revealed that the prevalence of female sexual dysfunctions was 30—60%, in different countries –52, but In the present study, female patients with sexual dysfunctions were associated with psychiatric disorders. There are several neurodevelopmental, endocrine, and psychological factors related to the linkage between these two groups of disorders. The stress from the suffering of sexual dysfunction , 62, parEvidence suggests that female and male brains are different in the mean volumes of the hippocampus, amygdala, and thalamus , the conPsychological, social, and cultural factors might also contribute to both psychosexual disorders and psychiatric disorders. Previous studies have shown that patients with paraphilias might suffer emotional distress, social embarrassment , and stiThe present study has several limitations that warrant consideration. First, similar to previous studies using the NHIRD on psychosexual disorders , 81–83, Female patients who suffer from psychosexual disorders have a 9.8-fold increase in the risk of psychiatric disorders, and this finding should serve as a timely reminder for the clinicians to pay much more attention to these patients because of their mental health issues.Additional file 1: Table S1. ICD-9-CM codes of Psychosexual disorders.Additional file 2: Table S2. Frequency of psychiatric service.


In T1 burger, the decrease of lipid content was mitigated during the cooking process, because the beta-glucans added had a fat-retaining effect. Compared to CTRL, replacing fat led to a softer texture of cooked burgers evaluated by Texture Profile Analysis. The differences in color, significant in raw burgers, were smoothed with cooking. The consumer evaluation, carried out according to the duo-trio test, highlighted significant differences between CTRL and T2 burgers in terms of odor, taste, color and texture. The consumers expressed a higher preference for the T2 burger, probably due to its softer texture and greater juiciness.Low-fat beef burgers with high beta-glucan content was obtained using a gel made from an oat-hull-based ingredient as fat replacer. Two levels of fat substitution were considered: 50% (T1) and 100% (T2). The nutritional composition, cooking yield, textural properties, color characteristics and consumer preference were evaluated, in comparison with a burger without fat replacer (CTRL). After cooking, T2 burger showed a significant increase in the cooking yield and a very low lipid content (3.48 g 100 g Meat and meat products play an important role in human nutrition, constituting a rich source of proteins with high biological value, vitamins A, B1, B3 and B12), as well as iron, zinc and other micronutrients [ and B12,In this context, researchers and private companies alike are strongly engaged in trying to improve the nutritional value of meat products by lowering the cholesterol and lipid content, as well as decreasing saturated and increasing polyunsaturated fatty acids. Fats, however, play an important role in meat products, ensuring optimal rheological and textural properties  and confAmong dietary fibers, beta-glucans from cereal grains have been recently studied in relation to the health benefits associated with their consumption such as the reduction of cholesterol level and a chemo-preventive effect as reported by Ho et al. .Moreover, beta-glucans show several technologically useful properties , which make them suitable ingredients in health-promoting functional foods . The majIn this frame, the aim of this study was the production of low-fat burgers with a beta-glucan content very close to the recommended daily intake and with good textural and sensorial characteristics.An oat-hull-based ingredient  containing, as declared by the producer, 55% beta-glucans, <10% proteins, <2% fat, was used to prepare a gel by mixing 27.27 g of flour with 72.73 mL of distilled water for 5 min at 13,500 rpm by means of a T25 Ultraturrax . The gel was then cut into small pieces to be used, freshly prepared, as a fat replacer in burgers.The ratio flour:water was defined in preliminary tests to obtain a gel: (i) able to mimic as much as possible the consistency and homogeneity of the beef fat conventionally used to prepare meat burgers; (ii) having a beta-glucan concentration able to achieve, when added to burgers as total fat replacer, a beta-glucan content as near as possible to the daily intake recommendation (3 g per day) .−1 fat content) and the adipose tissue  were separately ground using a grinder equipped with a 4 mm plate . Adipose tissue and lean meat, both ground, were then mixed manually. During the mixing step, three batches were prepared, according to three different formulations at increasing levels of fat: control (CTRL), with 15% of beef adipose tissue added; T1, with a partial (50%) substitution of beef adipose tissue ; and T2, with a total substitution of beef adipose tissue . With the exception of salt, no other spices or ingredients were added. The formulations of the three burgers are reported in Beef meat, purchased at a local butcher’s shop, was manually sectioned with a sharp knife to separate the lean meat from the visible adipose and connective tissues. Then, the lean meat  and after cooking.Protein content , ash, and moisture content were determined, according to the AOAC International methods, to be 928.08, 920.153 and 950.46, respectively . The lipv/v) following the method proposed by Folch et al. [The fatty acid composition was determined by gas-chromatographic (GC) analysis of fatty acid methyl esters. The lipid fraction was cold-extracted with methanol/chloroform  and Thrombogenic (TI) indices were calculated according to the following equations :AI = [C1The cooking yield of beef burgers was determined by measuring the weight (w) of the burgers before and after cooking according to the following equation:−1, with 5 s of pause between the two compressions, up to 70% of recorded deformation. The following parameters were assessed: hardness (N), indicating the maximum force recorded during the first compression; cohesiveness, measured as the area of work during the second compression divided by the area of work during the first compression; gumminess (N), calculated as hardness × cohesiveness; springiness, measured by the distance of the detected height during the second compression divided by the original compression distance; chewiness (N), calculated as gumminess × springiness. Ten different burgers per formulation were considered, and each burger was subjected to one measurement by TPA.Texture profile analysis (TPA) of beef burgers was performed according to Afshari et al.  with somL*, a*, and b* parameters were recorded: lightness (L*), red index (a*) and yellow index (b*), together with ΔE [Instrumental determination of the surface color of both raw and cooked burgers was carried out by using the CM-600d colorimeter  supported by SpectraMagic NX software . The CIE   with ΔE .∆E = [.Data were subjected to one-way ANOVA followed by the Tukey’s HSD test. Significant differences were determined at p < 0.05, p < 0.01 and p < 0.001, respectively [The results of duo–trio test were expressed as number of correct answers considering thirty-nine, forty-one and forty-four as minimum correct answers to identify statistically significant differences at ectively .The addition of the fat replacer significantly influenced the chemical composition of cooked burgers . An incrOn the contrary, the protein content of beef burgers (on fresh matter), showed a progressive and significant decrease when the fat replacement increased. Piñero et al.  and Afsh−1. After cooking, the CTRL burger showed a lipid content of 8.42% (6.04 g of fat in 71.82 g of cooked burgers); therefore, an estimated fat loss of 56% occurred. The lipid content of the raw T1 burger could be estimated at 8.17 g 100 g−1, whereas the cooked burger had a 7.25% fat content (5.45 g of fat in 75.14 g of cooked burger), with a fat loss of 34%. Therefore, even if considering estimated values, cooking induced a more limited fat loss when fat was replaced by the beta-glucan based gel than in the CTRL burger. This phenomenon could be imputable to the ability of the beta-glucans to form a tri-dimensional network which entraps fat and water within the meat protein system [Compared to CTRL, the addition of the fat replacer resulted in a slight but significant fat decrease in T1 formulation, whereas the T2 burger showed a more marked decrease. Considering the lipid content of the beef adipose tissue (accounting for 71.5%) used in CTRL and T1 formulations, and the contribution of the residual intramuscular fat of the lean fraction (3.5%), the lipid content of the CTRL raw burger could be estimated at 13.6 g 100 gn system . TherefoThe use of the fat replacer caused, as expected, a slight but significant increase in the carbohydrate content of T1, even if no significant differences were observed comparing T1 and T2. This was imputable to the presence of carbohydrates in the oat-hull-based ingredient. The addition of vegetable fat replacer in burgers is reported to be influential on the chemical composition of the product . The con−1 (CTRL) to 146.24 kcal 100 g−1 (T2). In particular, the T2 formulation allowed the research to obtain a product with lower fat content and, consequently, lower energy value compared to the products proposed by other studies [The fat substitution resulted in a significant decrease in energy value, from 203.44 kcal 100 g studies ,18,19. AThe fat replacement caused an increase in cooking yield. The difference, compared with the control burger, became significant in the T2 formulation. These findings agreed with previous studies ,36 in wh−1 of burger and g 100 g−1 of fatty acids. The nutritional value of beef burgers is also related to the composition of the lipid fraction, which usually is dominated by saturated fatty acids, palmitic and stearic acids in particular, whereas oleic acid was the most abundant unsaturated acid. The fatty acid composition of cooked burgers agreed with other studies carried out on the same category of products [−1 of burger) of all fatty acids due to the general decrease of lipid content. Moreover, a different level of reduction was observed as a function of the unsaturation rate. In particular, T2 showed a content of palmitic acid 60% lower than the CTRL. The reduction was slightly lower for oleic acid (−57%), whereas linolenic, the most abundant polyunsaturated fatty acid, decreased by 45% comparing T2 with CTRL. This aspect could be better explained considering the composition of fatty acids expressed as percentage. In particular, comparing the T2 with the other formulations, we observed a significantly (p < 0.05) lower percentage of saturated fatty acids and a higher percentage of the polyunsaturated fatty acids, whereas the monounsaturated fatty acids remained constant across the formulations. Previous studies report significant differences in the fatty acid composition of subcutaneous and muscular beef fat, with the latter characterized by higher polyunsaturated and lower saturated fatty acids [Fatty acid composition of burgers is reported in products ,37. Owinty acids ,39. This20:4 n-6) eicosapentaenoic (C20:5 n-3) and docosapentaenoic acids (C22:5 n-3), without significant differences among the formulations. The amount of these important fatty acids was lower than that reported in other studies carried out on the raw beef lipid fraction [Albeit in low amounts, we detected also some polyunsaturated fatty acids important from a nutritional point of view, such as the arachidonic  and yellowness (b*) was observed with fat replacement, while redness (a*) was not significantly influenced , whereas the number of correct answers increased when considering odor, texture and taste, with highly significant results (p < 0.001). The consumer test confirmed the results of textural and colorimetric evaluations (see for example the ΔE parameter). Szpicer et al. [p > 0.05); therefore, the addition of beta-glucan gel did not cause a significant decrease in the sensorial acceptability of the burgers. Both texture and taste, in fact, are known to influence the acceptability of meat products, especially the juiciness and the tenderness [As shown in r et al.  also repr et al.  highlighnderness . Moreovenderness , a low w−1 in the formulation with a total fat substitution) and with a 2.96 g 100 g−1 content of beta-glucans, almost reaching the recommended daily intake per single portion of burger. With a partial substitution, the decrease of lipid content in the raw product was mitigated during the cooking process (34% and 56% of estimated fat loss in T1 and CTRL respectively). This could be related to the fat-retaining effect of beta-glucans added. Compared to CTRL, replacing fat by the oat-hull-based gel caused a significant decrease in hardness and other textural parameters of cooked burgers. Conversely, the differences in color, significant in raw burgers, were smoothed with cooking. The consumer evaluation, carried out according to the duo–trio test, highlighted significant differences between CTRL and T2 burgers in terms of odor, taste, color and texture. The consumers expressed a higher preference for the T2 burger, probably due to its softer texture and greater juiciness.The use of an oat-hull-based gel as fat replacer allowed us to obtain a beef burger with a very low lipid content (3.48 g 100 gThese results are a step forward for the improvement of the nutritional characteristics of meat products and indicate that the use of the oat-hull-based ingredient, rich in beta-glucans, as gel is an effective strategy for a complete fat substitution.


Constipation is one of the most frequent non-motor problems in older adults. As constipation is commonly ignored by dementia patients, it is not usually reported on time. Constipation has a serious impact on the activity of daily living and quality of life in dementia patients. The relationships between constipation, demographic variables, and the nutritional status of patients with dementia remain unknown. This study aimed to assess the possible factors associated with constipation. This cross-sectional study was conducted at the Kaohsiung Chang Gung Memorial Hospital from January to November 2019. This hospital is a medical center and the main referral hospital of southern Taiwan, serving 3 million inhabitants. In total, 119 patients with dementia were evaluated using the Rome III diagnostic criteria for functional constipation. There were 30 patients with dementia included in the constipation group and 89 patients with dementia included in the no constipation group. Mini-Nutritional Assessment and 3-day diet diary records were employed. The clinical dementia rating score was used to evaluate the severity of dementia in patients of the outpatient clinic. Approximately 25.2% of dementia patients had constipation. Patients in the dementia with constipation group were older, had severer dementia, and displayed a lower water intake. After multivariable adjustment, low liquid consumption was the predictor of constipation among patients with dementia. The findings support the clinical recommendations to treat constipation with an increased liquid intake, but not exercise, in dementia patients. Dementia is a neurodegenerative disease with a progressive, irreversible, and long-term course. Its recent high prevalence rate in Western and Asian countries has a significant impact on modern society due to global aging . The greToileting, which includes bowel movements (stool passage) and urination, is a basic need, and constipation and urinary incontinence are common health problems among older adults, including those with dementia. Constipation is one of the most common health problems encountered in primary healthcare settings and occurring in the general population, as well as among older adults, especially those with dementia ,4,5. ConThe pathogenesis of constipation is multifactorial, with a focus on dietary behavior, such as low fiber consumption, a lack of adequate fluid intake, a lack of mobility, disturbance in the hormone balance, the side effects of medications with anti-cholinergic actions, and hemorrhoids ,10,11,12A total of 119 patients with dementia in the Dementia Outpatient Clinic of Kaohsiung Chang Gung Memorial Hospital were enrolled in the study. Patients or primary caregivers who could not record all food consumed within a 72 h period were excluded from this study. All procedures performed in the study were in accordance with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. This study was approved by the Chang Gung Medical Foundation Institutional Review Board, and informed consent was obtained from all participants (CGMH-IRB No 201600385B0).In the International Classification of Diseases, Tenth Edition (ICD-10) , dementiThe criteria included in the study were that the patients, supported by their caregivers, could receive all examinations and complete the records. The main caregiver needed to stay with the patient every day and have no subjective cognitive decline/complaint. The exclusion criteria for all subjects in the study were patients who had colorectal cancer or colorectal inflammation disease. The other exclusion criteria for enrolled subjects included end stage renal disease, tumor under hospice care, chronic pain with regular painkillers in use , and hematologic, endocrine, and autoimmune diseases. All patients underwent general physical examinations and clinical interviews. Patients’ clinical data were obtained, including their age, sex, and comorbidities, including diabetes mellitus, hypertension, hyperlipidemia, coronary artery disease, and chronic kidney disease. A trained neuropsychologist administered the neurobehavioral tests, including the Mini-Mental State Examination (MMSE)  to assesBlood samples were collected between 8 and 10 a.m. after an overnight fast and analyzed by the central laboratory of Kaohsiung Chang Gung Memorial Hospital to determine the serum levels of hemoglobin, total cholesterol, triglycerides, glycated hemoglobin (HbA1c), homocysteine, vitamin B12, and folate. The blood samples were centrifuged at 3000 rpm for 10 min to obtain serum samples, and all circulating biochemical markers were immediately analyzed, as reported previously ,18,19.Height and weight were measured, with patients wearing a disposable gown given to them. The body mass index (BMI) was calculated as the weight in kilograms divided by the height in meters squared. The waist circumference (narrowest diameter between the xiphoid process and iliac crest) was measured using a Lufkin measuring tape. The hip circumference was measured at the level of maximum extension of the hip. With regard to the Mini-Nutritional Assessment (MNA) (maximum score: 30) , a scoreWe analyzed the macronutrients according to the information from the Taiwan Food and Drug Administration website . The daiWe modified a previous question that was used for a survey on exercise habits . The patThe following mandatory information on constipation symptoms was collected by the case manager: loose stools rarely present without the use of laxatives; less than three bowel movements per week; manual maneuvers necessary to facilitate defecation; hard or lumpy stools; straining during defecation; the need to use laxatives and stool pattern; the sensation of incomplete evacuation; and the sensation of anorectal obstruction. Constipation is mainly diagnosed using the Rome III criteria, according to the patient and caregiver’s report ,22,23. p < 0.05), between two groups and the factors previously reported to be associated with constipation, including age, fiber, and exercise, for the binary logistic regression analysis. Multiple binary logistic regression was used to determine the factors associated with constipation in patients with dementia. Statistical analysis was performed using the SPSS version 22.0 software package . A p value of <0.05 was considered significant.All values were expressed as the mean ± standard deviation. The chi-square test was used to compare categorical variables. Non-parametric methods were used when continuous variables were non-normally distributed. The Mann–Whitney U tests were used to compare the differences between two groups. We chose those factors with significant differences, including the MMSE, MNA, and water intake , and water intake amounts . The MNAp = 0.028). The medication, which included acetylcholinesterase inhibitors (p = 0.152) and antipsychotic medications (p = 0.788), displayed no significant differences in the two groups. There were no significant changes in the clinical and demographic data, including diabetes mellitus, hypertension, coronary artery disease, hyperlipidemia, and chronic kidney disease, and biochemical data such as hemoglobin, total cholesterol, triglyceride, HbA1c, homocysteine, vitamin B12, and folate levels. MNA was statistically higher in patients without constipation (24.2 ± 3.5) than in those with constipation (22.6 ± 2.9) (p = 0.029). The number of patients at risk of malnutrition was higher in the no constipation group (60%) than in the constipation group (38.2%) (p = 0.029). In the daily intake analysis, the total caloric, carbohydrate, lipid, protein, calcium, fiber, and salt (sodium) levels were not significantly different in patients with constipation and in those without constipation (p = 0.001).We divided the patients into two groups, according to the status of constipation, and compared their basic demographic characteristics. No significant differences were found for age, gender, CDR, BMI, or the number of exercise habits between the two groups. However, MMSE was statistically higher in patients without constipation (20.4 ± 5.9) than in those with constipation (16.9 ± 7.7) (tipation . Howeverp < 0.05). Therefore, we chose the MMSE, MNA and water intake, and common risk factors associated with constipation, including age, fiber, and exercise, for the analysis (p = 0.002). However, fiber was not associated with constipation (p = 0.051).The MMSE, MNA, and water intake were significantly lower in the constipation group  (25.2%) in patients with dementia was higher than its overall prevalence. Constipation symptoms were defined based on stool consistency and frequency. Dementia patients with a lower MMSE score, risk of malnutrition, and lower water intake had a higher tendency to develop constipation. After controlling for other known factors for chronic constipation in adults, the only modifiable factor that may improve constipation was increasing the dietary intake of liquids, instead of fiber intake or exercise. Chronic constipation is a common geriatric syndrome. The prevalence rates of chronic constipation are as follows: In patients older than 60 years, 15–20% ; in patip < 0.05). This finding indicates that severe dementia may be associated with the risk of constipation. The correlation analysis revealed a positive correlation between dementia prevalence and constipation prevalence [In this study, the constipation group had lower MMSE scores . In older adults, constipation can be the result of multiple factors, including common diseases that can cause chronic constipation, functional or organic diseases of the colorectum and anus, and drugs [Our study showed that patients with constipation (77.2 ± 8.3) were older than those without constipation (74.5 ± 7.4)  the fluid intake ; (2) the dietary fiber intake (>25 g/d); (3) activity ; a lack of exercise for a long time; and slow intestinal peristalsis . We alsop = 0.029). The number of patients with a risk of malnutrition was higher in patients without constipation (60%) than in those with constipation (38.2%) (p = 0.029). MNA is a questionnaire used for whole-body health evaluation, including body weight change, appetite, and body figure. In our study, we found that patients at risk of malnutrition according to their MNA scores were also at risk of constipation, and this finding was similar to what was reported for those with Parkinson’ s disease [The MNA has recently been designed and validated to provide a single, rapid assessment of the nutritional status in older adults and to identify people at risk of malnutrition . MNA was disease .An increased intake of dietary fiber is widely considered to protect against constipation, and several studies have found an inverse relationship between dietary fiber intake and constipation. Fiber is important for stool passage. However, almost all older individuals in this study were unable to achieve the standard fiber intake. Studies have indicated that a high-fiber diet can increase the stool weight, resulting in a decreased colon transit time, while a poor-fiber diet induces constipation . Howeverp = 0.015). Furthermore, after adjusting for all possible factors, a low water intake was an independent risk factor associated with constipation in patients with dementia (p < 0.05). We further analyzed 14 patients with constipation (46.7%) who drank >500 mL and 66 (74.2%) without constipation who drank >500 mL per day (p = 0.006). A few studies have reported an association between the fluid intake and intestinal constipation. Epidemiological evidence has indicated an association between a lower fluid intake and intestinal constipation [In our study, the water intake was significantly lower in patients with constipation 584.11 ± 277.60) than in those without constipation 788.60 ± 422.42) (tipation . An inadtipation . Increas ± 277.60tipation ,39,40. Atipation . The stotipation ,40,41. I2.42 (tiptipation . No randtipation  and decrtipation ,45,46. TThe major strength of this investigation is that it is the first study to investigate the risk factor of constipation in patients with dementia who were diagnosed by two neurologists or a psychologist’s confirmation. Some risk factors were found. However, there were some limitations in this study. The first limitation of our study was the cross-sectional design of the study; we cannot infer the cause and outcome relationship. The second limitation of the study is that the incidence could be underestimated because of dementia and information from the caregiver. Third, this study did not consider the amount of water present in food, but only in the form of liquids. The fourth limitation is the dietary data collection having possible recall bias, despite the double confirmation by caregivers. The fifth limitation is that we did not measure serum osmolality for dehydration. The final limitation is the small number of participants. This indicates that a large sample size is needed to show the effect of fluid intake on constipation. In future studies, prospective randomized clinical trials with water ingestion to prevent dehydration should be performed to confirm the results.A low fluid intake was associated with constipation. An increased adequate water intake could improve constipation. However, randomized prospective clinical trials are still needed to confirm these results.


Erannis ankeraria belongs to the subfamily Ennominae of Geometridae. The mitogenome (GenBank accession number: MN046105) of E. ankeraria was sequenced, the new representative of the mitogenome of the subfamily. The nearly complete mitogenome is 15,250 bp totally, consisting of 13 protein-coding genes, two rRNAs, and 22 transfer RNAs. All genes have the similar locations and strands with that of other published species of Geometridae. The nucleotide composition biases towards A and T, which together made up 79.3% of the entirety. Bayesian inference analysis strongly supported the monophyly of Bombycoidea, Geometroidea, Noctuoidea, Papilionoidea, Pyraloidea, and Tortricoidea were strongly supported. This result also suggested that the Geometroidea was the sister to Bombycoidea, and then Noctuoidea was assigned to the sister group to the clade of Geometroidea + Bombycoidea, and then Pyraloidea was the sister group to the clade that contains Geometroidea, Bombycoidea, and Noctuoidea, and then Papilionoidea was the sister group to the clade that contains these four superfamilies mentioned above.The measuring worm  Lepidoptera is one of the world largest insect orders, second only to Coleoptera, with approximately 157,424 described species  of the specimen using the QIAamp DNA Blood Mini Kit  and stored at −20 °C until needed. The mitogenome was sequenced in Allwegene technology limited company with the sequenced number: AWGT19042503. The nearly complete mitogenome of E. ankeraria is 15,250 bp (GenBank accession number: MN046105). It encoded 13 PCGs, 22 tRNA genes, two rRNA genes and were similar with related reports before  initiated with ATT codons, and six PCGs  initiated with ATG codons, NAD1, NAD6, and NAD3 initiated with ATA as a start codon, start codon of CO1 not determined. Twelve PCGs used the typical termination codons TAA except NAD4 used T in E. ankeraria.The specimens of E. ankeraria could provide the important information for the further studies of Lepidoptera phylogeny.Phylogenetic analysis was performed based on the nucleotide sequences of 13 PCGs from 23 Lepidoptera species. Bayesian (BI) analysis generated the phylogenetic tree topologies based on the PCGs matrices . Accordi


TM). BC residents ≥30 years old at the time of diagnosis from January 2000 to January 2019 were included. The relevant International Classification of Disease codes was used to identify patients with GCA, HZ, and HSV. Comparisons were made using two-sample Z tests. There were 4315 GCA diagnoses, from a total population of 3,026,005 subjects. The prevalence of GCA was 143 per 100,000 people. In terms of herpetic infections, 850 GCA cases were identified in 249,900 subjects with HZ versus 310 diagnoses of GCA in 163,170 subjects with HSV. The prevalence of GCA in subjects with HZ (0.340%) was significantly higher than the prevalence of GCA (0.143%) in the general population (p < 0.00001). The prevalence of GCA in HSV subjects (0.190%) was also significantly higher (p < 0.00001) than the population prevalence but lower than (p < 0.00001) the GCA with HZ prevalence. The likelihood of GCA appears to increase with herpetic infections, more significantly with HZ.Recent data suggests that herpes zoster (HZ) and herpes simplex virus (HSV) may be one of the underlying immunological triggers for giant cell arteritis (GCA). However, there is limited population-based data to support this. Our goal was to determine if herpetic infections increase the likelihood of GCA in the British Columbia (BC) population. The background prevalence of GCA was compared to the prevalence of GCA in subjects with HZ and HSV using diagnostic billing code data from an online BC database (BC Data Scout Chlamydia pneumoniae, cytomegalovirus, or parvovirus [Giant cell arteritis (GCA), also known as temporal arteritis, is a chronic granulomatous medium and large vessel vasculitis which can have devastating vision and life threatening consequences . The exarvovirus ,2,3. Relrvovirus ,5,6, thervovirus . Antigenrvovirus ,8, althorvovirus ,10,11,12rvovirus .Outside of these histopathological analyses, there is limited population-based studies evaluating this hypothesis. One recent study from the UK highlighted a possible relationship with multiple infections and to a lesser degree, HZ, and GCA . AnotherTM  [We conducted a comparative descriptive study using a population database analysis. Research protocols were reviewed and approved by the Research Ethics Board of the University of British Columbia. Data collection and analysis were in adherence with the guidelines of the Declaration of Helsinki. An online British Columbia (BC) database, BC Data Scout 474094) , was useData extraction included all BC residents ≥30 years old by 1 January 2000. This range was selected to include patients diagnosed with HSV, HZ, or GCA. Diagnoses made from 1 January 2000 to 31 January 2019 were included. The background prevalence of GCA in this population was compared to the prevalence of GCA in subjects with HZ and HSV. All comparisons were made using two-sample Z tests using Microsoft Excel 2013 and the significance level was set via the simple Bonferroni correction for multiple comparisons to α = 0.025. The effect of gender on the diagnosis of GCA, HSV and HZ was also investigated using a separate 2-by-2 Chi-Square test for each diagnosis compared to the background population. The significance level was set as α = 0.0125 via the simple Bonferroni correction.TM showed a total population of 3,026,005 British Columbia (BC) residents who were ≥30 years old by January 1, 2000. There were 4315 probable giant cell arteritis (GCA) diagnoses within this group. The prevalence of GCA was 143 per 100,000 population ≥30 years of age. Using only the diagnostic code for GCA , the prevalence was very similar, at 136 per 100,000. To determine if there were any GCA cases before the age of 50, which would be uncommon [p < 0.00001) predominance of females in each of the study groups when compared separately to the background gender distribution.The population-level data extracted from BC Data Scoutp < 0.00001; p < 0.00001) than the population prevalence of GCA, but lower than (p < 0.00001) the GCA with HZ prevalence  as compared with 310 diagnoses of GCA in 163,170 subjects with previous herpes simplex virus (HSV). The prevalence of GCA in subjects with previous HZ  was significantly higher than the prevalence of GCA in the general population  is triggered by an infectious source ,3. The eOverall, we identified a GCA prevalence of 143 per 100,000 people ≥50 years of age. Although this is higher than previous studies which generally ranged from 1 to 30 cases per 100,000 people . A previOur study also found the prevalence for HZ and HSV, 82 per 1000 people and 53 per 1000 people, respectively, with a female predominance of approximately 1:2 in HSV and 2:3 in HZ. Previous studies have reported varying numbers for the prevalence of HZ from 80 per 1000 people to 238 per 1000 ,28,29,30After considering the relationships and prevalence data, it cannot be ignored that many histopathological studies did not find HZ or HSV correlations in their studies ,10,11,12There are relevant weaknesses in our study. First, there is lack of detail about our patient cohorts and temporality. As compared with other epidemiological and population-based studies, the available data we were able to extract from our database was limited. This made it impossible to complete age-matched or cohort-matched analyses used in other studies , and limFurthermore, it was not possible to extract data on subjects diagnosed with GCA only after having had HZ or HSV as BC Data Scout did not release this type of temporal information. We suggest that, given the relative ages of common diagnosis, there is a presumption that HZ and HSV was diagnosed prior to GCA. From the available dataset, we were able to confirm at least 44.7% of the HZ and 68.4% of the HSV diagnoses occurred prior to the GCA diagnosis. We recognize weaknesses to our assumptions. First, if HZ and HSV indeed occurred prior to the diagnosis of GCA, this could be many years earlier to the GCA occurrence, especially given the high seroprevalence of both in elderly populations ,36. ThisTaking these weaknesses into account, our study has several strengths and findings that are valuable for moving our current knowledge forward. Our study is the first and largest epidemiological study, to date, to identify a link between HSV and GCA, whereas previous studies have focused only on VZV ,15. FurtOverall, our study suggests the likelihood of developing GCA seems to increase with herpetic infections, especially HZ. Further study is required to identify an exact causative relationship between the two. If a link is identified, the therapeutic implications of using antivirals for treatment and prevention of GCA would be an exciting avenue for future clinical research.


Salmonella infection cycle, and asymptomatic animals can act as key component in the dissemination of the disease by horizontal, vertical, and cross-species transmission. Our study aimed and was able to confirm evidences of a cross-species transmission of Salmonella Agona between asymptomatic buffaloes and pigs. Also, we described Salmonella infection within the pig production phases, involving serotypes Agona, Senftenberg and Schwarzengrund. Rectal samples were collected from Jafarabadi buffaloes (n = 25) and Piau pigs (n = 32), located on a single farm. Salmonella Agona was isolated from lactating buffaloes, gilts, pregnant sows, and weaned pigs, Salmonella Schwarzengrund from lactating sows and Salmonella Senftenberg from gilts, pregnant sows, lactating sows, and weaned pigs. Pulsed-field Gel Electrophoresis protocol (PFGE) was performed and revealed four different profiles. Profile 1 , isolated from a pregnant sow, a gilt and two lactating buffaloes, revealed a indistinguishable PFGE pattern, confirming evidences of potential cross-species transmission. Profile 2 , 3 , and 4 , isolated from pigs, revealed important indistinguishable PFGE patterns, evidencing Salmonella infection within the pig production phases. Considering the epidemiological relevance of buffaloes and pigs in the cycle of Salmonella infection, confirmation of a potential cross-species transmission of Salmonella Agona and potential Salmonella infection within the pig production phases highlights the importance of the correct establishment of preventive health strategies in farms, in special the importance of avoiding contact between buffaloes and pigs, since cross-species transmission can occur, increasing the risk of spreading the disease.Buffaloes and pigs play an important epidemiological roll in the  Salmonella is one of the most important pathogens in livestock animals, and is a matter of concern, as it may be responsible for great economic losses within the herd, as well as being a zoonotic agent linked to foodborne illness and therefore a major public health concern worldwide .Samples were collected from Jafarabadi lactating buffaloes and five categories of Piau pigs within the production chain inside the farm . These animals were located on a single farm, in São Paulo State, Brazil. The lactating buffaloes were raised in a semi-intensive system, with a diet based on roughage and chopped sugar cane, supplemented with protein concentrate. The animals were housed during the night and released to graze in paddocks during the day, after the morning milking was performed. The pigs were raised in a semi-intensive system, with a diet based on a ration containing corn and soybean meal, supplemented with mineral core. The pigs were housed during the night and released to graze in paddocks during the day. Although the categories of pigs should be allocated into different structures, appropriate to each stage of the production chain, this did not occur on this farm. Thus, animals from all stages shared the same installation, in an inadequate breeding system where all animals had direct contact. Also, buffaloes and pigs, although housed in separate facilities during the night, were released in the same paddocks during the day, and therefore had direct contact. While in the paddocks, pigs could not access buffaloes feeders and water system because they were placed to high for the pigs. Therefore, the pigs had access to feeders and water through an adapted creep feeding system located inside the paddocks, where the buffaloes had no access.Salmonella. Rectal swab samples were collected from different categories of pigs within the production chain inside the farm: pregnant sows (n = 5), lactating sows (n = 5), gilts (n = 8), boars (n = 3), and weaned pigs (n = 11). Samples were collected at only one timepoint and on the same day.A total of 25 rectal swab samples from Jafarabadi lactating buffaloes and 32 rectal swab samples from Piau pigs were collected for microbiological isolation of Salmonella, three fecal samples were collected from the rectum of each animal, with a cotton swab, that was immediately transferred into tubes containing 10 ml of the selective enrichment broths selenite cystine (SC), Muller-Kauffmann tetrathionate (MKT) and Rappaport-Vassiliadis (RV). All samples were then transported to the laboratory in thermal boxes containing ice (time range from 2 to 3 h), where all microbiological procedures were performed.To evaluate the presence of Salmonella genus  were seeded on plates containing modified-brilliant green agar and xylose lysine tergitol 4 (XLT4) agar and incubated . From each plate, three colonies with morphologic characteristics that suggested la genus  were inoSalmonella antisera . Positive samples in slide agglutination tests were inoculated in tubes containing nutrient agar and sent to the laboratory of Enterobacteria of the Instituto Oswaldo Cruz – IOC/FIOCRUZ  for further serotyping.After biochemical confirmation, slide agglutination tests were performed using somatic and flagellar polyvalent −1 and an included angle of 120°. The gels were electrophoresed for 18 h.Positive samples in slide agglutination tests were also inoculated in tubes containing nutrient agar and sent to the “Laboratory of Veterinary Bacteriology, FCAV, UNESP, Jaboticabal Campus, Brazil,” where they were subtyped by a standardized rapid Pulsed-field Gel Electrophoresis protocol used by laboratories in PulseNet, as described previously . ChromosThe health status of the lactating buffaloes was verified by physical examination, performed at the same time that rectal swab samples were collected. Their feces were analyzed for signs of diarrhea, blood, and mucus. Fecal consistency scores were determined as 0, normal (firm); 1, mild diarrhea (soft); and 2, and moderate to severe diarrhea (liquid) . The degHemogram, including red blood cell count (RBC), hemoglobin concentration (HGB), packed cell volume (PCV), and total white blood cell count (WBC) was performed using automated hematology pocH-100iV Diff analyzer . Differential WBC count was performed on blood smear stained with modified Rosenfeld dye by optical microscopy . NormaliS. Agona  of the 25 rectal samples collected from Jafarabadi lactating buffaloes were positive for S. Agona .Salmonella. From the positive samples, 3 (15.8%) were S. Agona, 14 (73.7%) were S. Senftenberg, and 2 (10.5%) were S. Schwarzengrund. Additionally, Salmonella was detected in more than one category of a pig inside the production cycle of the farm, being positive in 3/5 pregnant sows (60%), 4/5 lactating sows (80%), 3/8 gilts (37.5%), 2/3 boars (66.7%), and 7/11 weaned pigs (63.6%)  of the 32 rectal samples collected were positive for  (63.6%) .S. Agona, one profile (profile 3) of S. Senftenberg and one profile (profile 4) of S. Schwarzengrund. Profile 1 of S. Agona revealed a indistinguishable PFGE pattern between the isolates identified in two categories (pregnant sow and gilt) of pigs. This same profile was also identified in the two lactating buffaloes. Profile 2 of S. Agona was only identified in one animal, a weaned pig  of aned pig .S. Senftenberg (profile 3) revealed a indistinguishable PFGE pattern between the isolates identified in all animal categories  of pigs. The single profile identified for S. Schwarzengrund (profile 4) also revealed a indistinguishable PFGE pattern between the isolates identified in two lactating sows  and Salmonella-negative (n = 23) animals, did not present clinical signs of salmonellosis, as well as did not present alterations in the hemogram  and Salmonella-negative (n = 13) animals, was verified by visual inspection and by feces inspection. In this sense, all animals were asymptomatic  at the time of swab collection.The health status of the pigs, both S. Agona was isolated from two lactating buffaloes, a pregnant sow and a gilt, that generated an identical PFGE pulsetype (Profile 1), revealing a indistinguishable PFGE pattern between these isolates , revealing a indistinguishable PFGE pattern between these isolates (S. Agona isolated from the weaned pig generated a different PFGE pulsetype (Profile 2)  . Since wterature , this calmonella , and is S. Senftenberg, isolated from four sows, two gilts, two boars, and six weaned pigs, generated an identical PFGE pulsetype (Profile 3), revealing a indistinguishable PFGE pattern between these isolates , also revealing a indistinguishable PFGE pattern between these isolates . Written informed consent for participation was not obtained from the owners because at the time of the execution of the project, the written consent was not a requirement of the Ethics Committee on Animal Use of “Faculdade de Ciências Agrárias e Veterinárias, UNESP”. Therefore, only a verbal consent was given.AS was responsible for the conception of the study, experimental design, collection of samples in the farm, part of laboratory work, data analysis and interpretation, and manuscript writing. DS and JF were responsible for the conception of the study, experimental design, part of laboratory work, data analysis and interpretation, and manuscript revision. RM was responsible for the conception of the study and part of the laboratory work. LP and KS were responsible for the collection of samples on the farm. CS and DR were responsible for part of the laboratory work. SR was responsible for data analysis and interpretation and manuscript revision. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.