Kampo medicine, a traditional Japanese medicine, is widely used in Japan, especially in the field of menopause medicine. However, few studies have shown evidence-based effects. This study aimed to confirm the effects of kamishoyosan on menopausal symptoms with a randomized, placebo-controlled, double-blind clinical trial. n = 101) or a placebo resembling kamishoyosan (n = 104). The primary outcomes were the change in the number of hot flashes, depression scores, improvements of anxiety, quality of life (QOL), and menopausal symptoms before and 4 and 8 weeks after initiation of treatment with the study drug. The secondary outcome was drug safety.  Subjects were randomly allocated to groups that received either kamishoyosan ( After 8 weeks, the number of hot flashes decreased after treatment in both groups, but there was no significant difference between the two groups. The changes in SDS scores showed the same results. Moreover, no significant differences were observed between the two groups in assessments with the STAI, SF-36, and JSOG menopausal index. No serious adverse effect was reported. 000006042. This first placebo-controlled double-blind randomized trial with kamishoyosan demonstrated that it was safe and had some effects on climacteric symptoms, but not significant compared with placebo. Some problems, such as placebo effects, in the study of Kampo therapy for menopausal symptoms, were revealed. This trial is registered with the trial registration number. UMIN  Menopausal symptoms are characterized by a wide range of variations and have been known to significantly and negatively affect the quality of life (QOL) of women during menopause . They arAmong drug therapies, it is well known that hormone therapy (HT) is pertinent to and has an extremely high response rate for hot flashes, sweating, and sleep disturbances. However, the effects of HT on other symptoms are somewhat limited, especially those involving the neuropsychiatric system . The risγ-aminobutyric acid A (GABAA)-benzodiazepine receptor in male mice [Among Kampo formula, kamishoyosan is frequently prescribed to relieve various menopause-related symptoms. It reportedly improved anxiety and depression in midlife women with psychological symptoms  and imprale mice  and sociale mice . Moreoveale mice .To determine the efficacy and safety of kamishoyosan, in this study, we conducted a randomized, placebo-controlled, double-blind clinical trial with patients who had menopausal symptoms.Among patients aged 40–60 years with a chief complaint of climacteric disorders who visited one of the 13 outpatient gynecological clinics consisting of four university hospitals and 9 related hospitals which the authors are affiliated to, we selected those with hot flashes, insomnia, headache, and/or neurological symptoms and those who were diagnosed with menopausal symptoms by the authors during the period spanning April 2011 through February 2013.Patients who suffered from the aforementioned symptoms in their daily life were recruited. The menopausal status was not taken into consideration.Patients with serious comorbidities  and a history of HT or Kampo therapy within 4 weeks prior to treatment initiation were excluded from this study. Following the informed-consent procedure, the depression status of all patients was assessed using the Zung's self-rating Depression Scale (SDS) . To exclhttps://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000006042). The study was conducted in accordance with the ethical guidelines for clinical studies, considering patients' human rights and privacy. The study protocol was approved by the Institutional Review Board of each institution.This study is of a multicenter, randomized, double-blind, placebo-controlled design to examine the pharmacological efficacy and safety of kamishoyosan therapy in menopausal patients at 13 sites in Japan, and is registered in UMIN Clinical Trials Registry in Japan (UMIN 000006042). A summary of this study is on UMIN Website , we assumed the changes of the SDS score to be −4.7 and −2.2 in the kamishoyosan group and placebo group, respectively, with a common standard deviation of 6.0. A sample size of 92 per group was calculated to obtain a power of 80% and a level of significance of 0.05. Taking into account 10% dropout, the total sample size was set at 204.Subjects were randomly assigned to receive 7.5 g tid kamishoyosan (kamishoyosan group) or 7.5 g tid placebo (placebo group) for 8 weeks according to a computer-generated randomization list provided by a statistician from the site management organization . The placebo was produced to have a similar smell and taste to kamishoyosan. Granulated powders of kamishoyosan and placebo appeared identical and were identically packaged and labeled by the CRO to maintain blinding. Subjects, investigators, and all other personnel involved in conducting the study were blinded to the treatment allocation. During the baseline period (0 weeks), subjects were assigned a randomized number. These numbers were allocated in sequential order and registered in the patient enrollment center . The allocation was concealed. Emergency envelopes containing the randomization code were kept under lock and key by the CRO and were examined at the end of the trial to ensure that blinding had been maintained.Bupleurum falcatum), 13.3% Paeoniae Radix (Paeonia lactiflora), 13.3% Atractylodis Rhizoma (Atractylodes ovate), 13.3% Angelicae Radix (Angelica acutiloba), 13.3% Hoelen (Poria cocos), 8.9% Gardeniae Fructus (Gardenia jasminoides), 8.9% Moutan Cortex (Paeonia suffruticosa), 6.7% Glycyrrhizae Radix , 4.4% Zingiberis Rhizoma , and 4.4% Menthae Herba (Menthae arvensis).After the 2-week pretreatment observation period, subjects initiated treatment with the study drug or placebo. Subjects were administered either kamishoyosan (Tsumura-Kampo kamishoyosan Extract Granules) or placebo having a similar appearance, smell, and taste to kamishoyosan  at a dose of 7.5 g/day divided into three portions and taken before or after each meal. Kamishoyosan was used in the form of a powdered extract obtained by spray drying a hot extract mixture of the following 10 crude herbs: 13.3% Bupleuri Radix  was prohibited in general. Supplements, such as soy supplements, were allowed if the usage had been the same throughout the study period.The primary outcome was the efficacy of kamishoyosan, the number of hot flashes, which was investigated every day using a self-reported diary and shown by the mixed effect model for repeated measures, and changes in SDS scores, anxiety assessed using the State-Trait Anxiety Inventory (STAI) , QOL witTo evaluate drug safety for secondary outcome, blood chemical examinations were performed, and weight and blood pressure were monitored before and after 4 weeks of treatment, as well as after treatment discontinuation.Safety and tolerability were assessed by recording all adverse events which were based on declarations by subjects and the result of blood sample analysis. Adverse events were defined as “adverse drug reactions” if a causal relationship with the study drug could not be ruled out, and the frequency of occurrence of adverse events and adverse drug reactions was tabulated. “Serious” adverse events were defined as (1) any event that leads to death, (2) any event that is life-threatening, (3) any event that requires inpatient hospitalization or prolongation of existing hospitalization for treatment, (4) any event that results in persistent or significant disability/incapacity, and (5) any event that results in congenital anomaly or birth defect.t-test.In consideration of missing data, changes in the number of hot flashes were calculated by a mixed effect model using values described in patient diaries in the Full Analysis Set. For analyzing the number of hot flashes, the logarithmic conversion was employed because it showed a skewed distribution. When the number was 0, it was calculated as log (number + 1). For comparing the changes over time, a repeated-measures analysis of variance for changes from the baseline value was performed with the group as the factor , results before initiation of treatment as covariates, and results after completion of treatment as dependent variables. Also, analysis with ANOVA was done for sensitivity analysis. The analysis of the SDS score, STAI, and SF-36 was performed with ANOVA. Summary statistics were calculated for differences in results before and after initiation of treatment as well as rates of change between the two groups. Comparisons between the kamishoyosan and placebo groups were performed using the For safety evaluation, the frequency and proportion of subjects who developed any adverse events or adverse drug reactions in the kamishoyosan and placebo groups were determined along with 95% confidence intervals (CIs) and examined using Fisher's exact test.Analyses were carried out using SAS 9.2 software . The significance level was set at 0.05.Among registered patients who provided consent, the 205 who scored <62 on the SDS assessment were enrolled as subjects during the trial period. Of these 205 subjects, 101 were randomly allocated to the kamishoyosan group and the remaining 104 to the placebo group . There were 11 who subjects discontinued treatment for reasons such as low compliance (1 subject), no visit (1 subject), withdrawal of consent (5 subjects), withdrawal due to worsening of symptoms (2 subjects), and adverse drug reactions (2 subjects). Accordingly, the full analysis set  for efficacy assessments consisted of 194 subjects  .Subject characteristics for each group are shown in The mean number of hot flashes before treatment was about 1.7 times/day in the kamishoyosan group and 1.6 times/day in the placebo group, respectively. The mean number of hot flashes was calculated by a mixed effect model using values described in patient diaries and was significantly decreased in both groups .P < 0.001). However, we did not find differences between the groups in terms of a decrease in SDS score relative to baseline at each assessed time point (SDS scores before initiation of treatment were 43.7 (standard deviation (SD) 7.8) in the kamishoyosan group and 44.2 (SD 7.8) in the placebo group. We did not find differences between the groups. SDS scores after 4 and 8 weeks of treatment were 42.5 (SD 8.1) and 40.6 (SD 8.8) in the kamishoyosan group and 40.4 (SD 8.4) and 39.3 (SD 8.4) in the placebo group, respectively. In both groups, these scores were significantly decreased compared to before treatment .The STAI assessment before initiation of treatment revealed state anxiety and trait anxiety scores of 44.3 (SD 1.1) and 48.8 (SD 1.2), respectively, in the kamishoyosan group, and 47.0 (SD 1.1) and 48.6 (SD 1.1) in the placebo group, and we did not find differences between the two groups. Compared to pretreatment scores, both state anxiety and trait anxiety scores were significantly decreased at 4 and 8 weeks after initiation of treatment in both groups. Also, we did not find differences between the two groups .In a health related QOL analysis, kamishoyosan improves mental health scores in SF-36 but not physical health scores . In eighAdverse events were noted in 15 subjects (18 events) in the kamishoyosan group and 7 subjects (8 events) in the placebo group, and 11 subjects (14 events) in the kamishoyosan group and 2 subjects (3 events) in the placebo group could not be denied a causal link to the treatment. In the kamishoyosan group, the serious adverse event was anxiety symptoms in one subject. However, other 7 gastrointestinal complaints, 2 vertigo, 2 articular pain, one palpitation, and one common cold, were not serious. On the other hand, eruption, constipation, and gastric discomfort were found, but not serious, in the placebo group. No subject dropped out due to adverse events.This study is the first report on the use of the double-blind methodology to verify the effects of Kampo therapy on vasomotor and neuropsychiatric symptoms in menopausal women. Menopausal symptoms are characterized by a wide range of pathologic manifestations and diverse underlying factors. Among factors of menopausal symptoms, decreased ovarian function causes hot flash, sweating, and insomnia . AdditioKampo therapy is characteristic in that few adverse drug reactions are observed, and that it is well accepted by patients. Many studies have reported on its significance and effects on menopausal symptoms , 9, 19. Several Kampo medicines are indicated for menopausal symptoms in Japan. In this study, we selected kamishoyosan for the following reasons. Kamishoyosan contains herbal medicines having sedative effects on the central nervous system, such as bupleurum root, ginger, rhizomes of atractylodes lancea, and moutan bark. So, it is indicated for menopausal symptoms and is appropriate for women who get tired easily or suffer from hot flashes, stiff shoulder, and neuropsychiatric symptoms such as dysphoric mood and irritability, which are common in Japan . In addiThe primary objective of the present study was to investigate the efficacy of kamishoyosan. A hot flash is a typical symptom related to estrogen deficiency, and observing its occurrence as well as changes brought about by treatment is useful in determining the effects of kamishoyosan on menopausal symptoms . The numIn patients with menopausal symptoms, psychological symptoms, as well as physical symptoms, interfere with QOL . KamishoThere were some limitations of this study. The present study did not consider so-called “patterns” as they are used in the context of Kampo medicine. This was because the “pattern” evaluation is strongly influenced by the subjectivity of the attending physician and is not suitable for a multicenter study. Moreover, many women who complain of menopausal symptoms are considered to belong to the “medium pattern,” which likely matches the pattern of kamishoyosan. Furthermore, since the present study used a placebo drug, the distribution of patterns was considered equivalent. So, the influence of patterns, if any, would have been diminished.The findings of the present study reassured the safety of kamishoyosan, but did not prove the usefulness of kamishoyosan as a therapeutic drug for patients diagnosed with hot flashes. Nevertheless, our results do not necessarily rule out the effects of kamishoyosan on a variety of symptoms experienced by women during menopause. It is important to note that our evaluations did not assess the direct effects of kamishoyosan on the various symptoms of menopause. Rather, we comprehensively evaluated the secondary symptoms that develop due to the presence of those symptoms. Previous open trials found that kamishoyosan was effective in treating mental and neurological symptoms such as dysphoric mood and irritability, and indeed, kamishoyosan has been shown to be effective in relieving symptoms such as anxiety, irritability, insomnia, dizziness, and headache . Women dIn conclusion, this first placebo-controlled double-blind randomized trial with kamishoyosan demonstrated that it was safe and had some effects on climacteric symptoms, but not significant compared with placebo. Some problems, such as placebo effects, in the study of Kampo therapy for menopausal symptoms, were revealed.


Salmonella is a leading human pathogen and a significant public health concern worldwide. Massive food production and distribution have contributed to this pathogen dissemination, which, combined with antimicrobial resistance (AMR), creates new control challenges in food safety. The development of AMR is a natural phenomenon and can occur in the bacterial evolutionary process. However, the overuse and the misuse of antimicrobial drugs in humans and in animals have increased AMR selective pressure. In Brazil, there is an accuracy lack in AMR frequency in Salmonella because too many isolates are under-investigated for genetic and phenotypic AMR by the Brazilian health authorities and the research community. This underreporting situation makes the comprehension of the real level of Salmonella AMR in the country difficult. The present study aimed to use bioinformatics tools for a rapid in silico screening of the genetic antimicrobial resistance profile of Salmonella through whole-genome sequences (WGS). A total of 930 whole-genome sequences of Salmonella were retrieved from the public database of the National Biotechnology Information Center (NCBI). A total of 65 distinct resistance genes were detected, and the most frequent ones were tet(A), sul2, and fosA7. Nine point mutations were detected in total, and parC at the 57 position (threonine → serine) was the highest frequent substitution , followed by gyrA at the 83 position   and at the 87 position (aspartic acid → asparagine) . The in silico prediction of resistance phenotype showed that 58.0% (540/930) of the strains can display a multidrug resistance (MDR) profile. Ciprofloxacin and nalidixic acid were the antimicrobial drugs with the highest frequency rates of the predicted phenotype resistance among the strains. The temporal analysis through the last four decades showed increased frequency rates of antimicrobial resistance genes and predicted resistance phenotypes in the 2000s and the 2010s when compared with the 1980s and 1990s. The results presented herein contributed significantly to the understanding of the strategic use of WGS associated with in silico analysis and the predictions for the determination of AMR in Salmonella from Brazil. Salmonella is a frequent human pathogen, and salmonellosis is a global public health concern. Among the diarrheal and/or invasive agents, including bacteria, viruses, protozoa, helminths, and chemicals, Salmonella is among the 31 pathogens with a high capability of causing intestinal or systemic disease in humans  in pre-existing genes and then transfer them transfer . The acqtransfer  and impltransfer . Althougtransfer . In addie agents . The cure agents .In this context, it is important to acquire accurate information regarding the level of antimicrobial resistance as well as the data on antimicrobial use, employing harmonized strategies for sampling, management of data and metadata, and analysis. For example, the accurate knowledge of the resistance level present in the bacteria population associated with livestock animals is essential to plan strategies to control the overuse and the indiscriminate use of antimicrobials in animal production centers. As well known, instigating better practices in antimicrobial drug use avoids both the emergence and the spread of antimicrobial resistance in bacteria among multiple sources, like humans, animals, and the environment . The surSalmonella because too many isolates are under-investigated for genetic and phenotypic AMR by the Brazilian health authorities and the research community. This underreporting situation makes the comprehension of the real level of Salmonella AMR in the country difficult. Several national studies have focused on analyzing the phenotypic antimicrobial resistance in Salmonella  database in the National Biotechnology Information Center (NCBI) website. The genomes and their metadata information were retrieved for all SRA runs available as of July 2019 from Brazil. Four highly relevant metadata fields were originally kept from the source to standardize the contained terms and highlight the value of the sequence record: serovar, collection date, strain name, and isolation source. To find all genomes available from Brazil, we first search through the keyword “Salmonella and Brazil,” and then we use the “Run Selector” SRA tool to select only the genomes sequenced by the Illumina platform. In addition to maintaining the original information for each record, curated fields were created and can be accessed in A total of 1,077 De novo genome assembly was performed by using the SPAdes 3.10.0 software with –careful option activated   was usedctivated . Finallyctivated  was usedSalmonella genomes isolated from feces, biological fluid or tissue, blood, lymph nodes, and others. The clinical-human group included isolates from blood, brain abscess, feces, coproculture, hip secretion, and others. In the food group, the non-animal-based foods, such as beans, papaya, tomato, potatoes, vegetables in general, and others were considered. The animal-based food group included foods that have chicken, eggs, pork, beef, milk, or seafood in the composition or raw cuts. The environment group included animal box swab, flock environment, truck after cleaning, chicken cage after cleaning, disposable shoe covers of the farm’s workers, and slaughterhouse environment. These five source groups, after genome assemblies and quality filtering exclusion, comprised 930 out of the 1,077 Salmonella genomes initially downloaded from NCBI. The number of genomes per group were as follows: animal-based food, n = 374; clinical-human, n = 249; clinical-animal, n = 133; environment, n = 110; and food, n = 64 . The R software3 was used, combined with the Complex Heatmap Package, from Bioconductor, to construct the frequency heat maps  and the total number of genomes assessed in the study. The frequency heat maps were constructed to represent each antimicrobial resistance mechanism in all sources studied  and/or in the four most frequent serovars . Finally, one last supplementary temporal graph was constructed to display the temporal frequency of the four more frequent Salmonella serovars along with all serovars detected through the decades. For all these analyses, we considered the information about the year of isolation as supplied in the original records of the NCBI metadata. To reduce the variability in the isolation date of the bacterial strains, we only considered information about the year of isolation, which was from the last four decades ranging from 1980 to 2018. The frequencies of the genus Salmonella or each Salmonella serovar were calculated by the ratio between each type of antimicrobial resistance and the total number of genomes from strains collected in each decade. The serovars’ temporal frequency was calculated by the ratio between each serovar and the total number of genomes from the strains collected in each decade.Three main graphs were constructed to display the temporal analysis of the nine more frequent resistance genes detected, the three more frequent chromosomal mutations, and the predicted AMR resistance within the 930 genomes of the genus Salmonella enterica analyzed, 46 distinct serovars were detected in silico , S. Typhimurium , S. Heidelberg , S. Dublin , and others   (Among the 930 complete genomes of n silico . The ser Yoruba) .in silico detected serovars  were clustered in three different groups according to their similarity between the resistance gene frequency , fosA7, sul2, and blaCMY–2. Lastly, cluster 3 displayed that Salmonella Typhimurium has an intermediate to high frequency rate of resistance genes and showed the highest diversity of antimicrobial resistance genes . This class of antimicrobials also showed the largest gene diversity, comprising 18 different genes conferring resistance to this class . The second highest frequency class of AMR genes detected was tetracycline , with four different genes . The gene tet(A) was the most frequent, detected in 20.1% genomes (187/930). β-Lactamases producer genes also presented a high frequency , with 16 different genes . The sulfonamide resistance genes  were sul1, sul2, and sul3. Genes conferring resistance to chloramphenicol (catA1 and cmlA1), trimethoprim , florfenicol (floR), fosfomycin (fosA3 and fosA7), lincosamide [lnu(G)], colistin (mcr-1.1 and mcr-9), macrolide [mph(A)], fluoroquinolone , quinolone , and macrolide/lincosamides/streptogramins [erm(42)] were also found but in lower frequencies (tet(A), the genes sul2 and fosA7 also were considered as highly frequent, being detected in 16.9% (157/930) and 14.9% (139/930) of the WGS, respectively , aac(6’)-Ib-cr, qnrS1, qnrB2, fosA3, mcr-9, tet(D), mph(A), erm(42), blaOXA–17, blaOXA–4, blaTEM–229, blaTEM–220, blaTEM–116, blaCTX–M–2, blaCMY–33, blaCMY–18, dfrA25, dfrA15, dfrA17, dfrA7, aadA22, aadA12, aadA5, aac(6’)-Ib, and ant(2”)-Ia]. Cluster 2 was composed by genes showing lower frequency rates in strains isolated from clinical-human, clinical-animal, environment, and food sources and intermediate rates from animal-based food . Cluster 3 was composed solely by genes displaying intermediate frequency rates such as blaCMY–2, tet(B), aph(3”)-Ib, aph(6)-Id, blaTEM–1B, and sul1. Lastly, cluster 4 joined genes that showed the highest frequency rates in strains isolated from animal-based food and environment   and two at the 83 position ]. In gyrB, two distinct amino acid substitutions were found [at the 466 and 464 positions ]. In pmrA and parC, only one amino acid substitution was found in each [at the 53 and 57 positions ]. The three top amino acid substitutions that may result in resistance against nalidixic acid and ciprofloxacin were in parC at the 57 position (threonine → serine) with 26.7% (249/930), followed by gyrA at the 83 position  with 20.0% (186/930), and at the 87 position (aspartic acid → asparagine) with 15.7% (146/930) of frequency. A single genome presented a mutation that may result in colistin resistance (in pmrA at the 53 position glycine → glutamic acid). All the remaining WGS point mutations detected may result in resistance to ciprofloxacin and/or nalidixic acid.In the 930 WGS analyzed for chromosomal mutations, we identified nine distinct mutations that would result from an amino acid substitution . In gyrAparC at the position 57 (threonine → serine) was the most frequent in WGS from animal-based food and environmental sources; gyrA at the position 83  was the most frequent in the clinical-animal source; gyrA at the position 87 (aspartic acid → asparagine) was the most frequent in food and clinical-human sources  of the strains are possible pan-susceptible to all analyzed antimicrobial classes and 59.2% (551/930) might present phenotypical drug resistance to one or more antimicrobial classes , with 66 different profiles. The highest frequency of predicted MDR profile was against six antimicrobial classes . The environment, clinical-animal, clinical-human, and food sources comprised the predicted MDR strains, with 6.6% (61/930), 2.9% (27/930), 2.6% (24/930), and 0.4% (4/930) of frequency, respectively .For the temporal analysis, we used all of the 928 WGS, of which the year of isolation was provided in the metadata . The fretet(A), sul2, fosA7, and blaCMY–2 increased in the Brazilian Salmonella spp. strains after the 2000s. The frequency rates of the genes tet(B), aph(3”)-Ib, aph(6)-Id, blaTEM–1B, and sul1 remained constant throughout the last four decades  decreased very less throughout the last decades. On the other hand, gyrA at the 83 position  and parC at the 57 position (threonine → serine) considerably increased after the 1990s , fosA7, sul2, and parC at the 57 position (threonine → serine) and gyrA at the 83 position  is closely related to the increased frequency of Salmonella Heidelberg (aph(3”)-Ib, aph(6)-Id, tet(B), sul1, and gyrA at the 87 position (aspartic acid → glycine) are closely related to resistance in Salmonella Typhimurium  technologies has been beneficial in detecting those bacteria. However, NGS produces a high amount of complex raw data that have to be analyzed by appropriate bioinformatics tools to turn the raw reads into meaningful data  and S. Typhimurium  were the most commonly found serovars among the WGS deposited from Brazil. Salmonella Enteritidis and S. Typhimurium are the most common serovars reported in human sources in Latin America between 2001 and 2014  by the in silico analysis of Salmonella WGS. The highest frequency of antimicrobial resistance genes was against the aminoglycoside class, which also showed the highest diversity of resistance genes (n = 18). The aac(3)-IIa, aac(6’)-Ib, ant(2”)-Ia, aac(3)-IId, aac(3)-IV, aac(3)-VIa, and ant(3”)-Ia genes belong to the aminoglycoside N-acetyltransferase group, which are a chromosome-encoded superfamily of aminoglycoside acetyltransferase proteins  in animals (87.7%) and animal feed isolates (91.3%)  1 or genomic island 2 (SGI2) and usually are associated with MDR gene clusters (We found a high diversity of resistance genes (proteins . In Braz (91.3%) . This hicrobials  can alsolmonella , 2016. Aclusters . Those gtet(A) and sul2 that may confer resistance against tetracycline and sulfonamide, respectively, were also highly frequent among the analyzed WGS, with 20.1% (187/930) and 16.9% (157/930) frequency, respectively  and topoisomerase IV (parC and parE), making them less susceptible to quinolone binding  involved in DNA binding . The ami binding . Particulmonella . Our in pulation . Nowadaypulation . The plance 2000 .qnrB19, qnrB2, qnrS1, and qnrE1 were detected in all WGS analyzed. Interestingly, qnrE1 was detected only in Salmonella Typhimurium WGS obtained from isolates from animal-based food and clinical-animal sources. However, previous studies have reported the presence of qnrE1 in other Salmonella serovars in Brazil  displays a decreased susceptibility to certain cationic agents that bind to lipopolysaccharide  is globally distributed , which may result in resistance to polymyxin. Moreover, three analyzed Salmonella WGS showed mcr genes (one WGS showed mcr-1.1 and the other two showed mcr-9). The announcement of the first report of mcr genes in Salmonella from Brazil has been recently published , they quickly can be horizontally transferred to other bacterial species of animal and human origin. The presence of mcr-like genes should not be neglected in zoonotic pathogens such as Salmonella. Our results highlight the importance of the controlled use of colistin in livestock and agriculture to curb the spread of colistin resistance since, in humans, colistin is used as a last-resort antimicrobial, treating extensively antimicrobial-resistant pathogens  and sul2 and the predicted AMR against sulfonamide and tetracycline notably increased during the last decades, with the highest increase after the 2010s  resistance has been noted in different orldwide . One of actamase . The resosporins . Althougosporins , ceftiofosporins . In Canaidelberg . Our resn Brazil . Althougn Brazil . The usen Brazil . The evihe 2010s . The broSalmonella (n = 66). The emergence of MDR among Salmonella contaminating the animal-based food is a global public health concern. Although intensive antibiotic therapy can select associated AMR pathogens and the misuse and the overuse of antimicrobials in animal husbandry may cause resistance in bacteria in the treated host, the correlation between resistance in animal-based food and the antimicrobials used in husbandry is poorly determined (Animal-based food was the most common source of the predicted antimicrobial-resistant lmonella . The strtermined . The emein silico analysis and the predictions for the determination of AMR in Salmonella from Brazil. Point frequency surveys and prediction analysis are imperfect and far from the best approach for epidemiological surveillance networks. However, in the absence of systematic surveillance, in silico studies seem to be useful to guide interventions against AMR increase. Furthermore, more detailed studies on resistance genes carried by mobile genetic elements are necessary to reveal how the AMR interchange occurs between different sources.It is of concern that little epidemiological surveillance data of AMR is publicly available in Brazil. The establishment of strategies to monitor the genetic and the phenotypic AMR is of utmost importance to supply the underestimation of AMR frequency, particularly in pathogenic bacteria from different sources such as food, animal-based food, clinical-human, clinical-animal, and environment. The results presented herein contributed significantly to the understanding of the strategic use of WGS associated with All datasets presented in this study are included in the article/PP conceived and designed the study. GR, PP, and AS retrieved data and performed bioinformatics and temporal analysis. GR performed the statistical analysis and wrote the first draft of the manuscript. All other authors contributed to manuscript revision and read 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.


The ability of the extracts to protect human red blood cell membranes against free-radical-induced damage and their antifungal activity was also determined. The results showed that the concentration of flavonoids in the propolis extracts was dependent on the solvent used in the extraction process and pinocembrin, chrysin, galangin, and coumaric acid were the most abundant phenols. All extracts exhibited high antioxidant potential and significantly protected human erythrocytes against oxidative damage. On the other hand, the antifungal activity of the propolis extracts depended on the solvent used in extraction and the fungal strains tested. It needs to be stressed that, to the best of our knowledge, there is no study relating the effect of solvent used for extraction of Polish propolis to its phenolic profile, and its antifungal, antioxidant, and cytoprotective activity.Propolis is a natural bee product with various beneficial biological effects. The health-promoting properties of propolis depend on its chemical composition, particularly the presence of phenolic compounds. The aim of this study was to evaluate the relationship between extraction solvent  and the antifungal, antioxidant, and cytoprotective activity of the extracts obtained from propolis. Concentrations of flavonoids and phenolic acids in the propolis extracts were determined using ultrahigh-performance liquid chromatography. The antioxidant potential of different extracts was assessed on the basis of 2,2-diphenyl-1-picrylhydrazyl (DPPH·) free-radical-scavenging activity, Fe Propolis is a natural product collected by honeybees from various plant sources growing around hives, including flowers, buds, or tree exudates ,2,3. ExtAccording to the literature data, the biological activity of propolis is mainly attributed to the presence of phenolic compounds, especially flavonoids, phenolic acids, and their esters ,6,10. FoOver the past few years, the biological activities of Polish propolis were studied intensively, and its chemopreventive, antioxidant, antibacterial, antiproliferative, and antifungal activity was observed ,31,32,33The identification of individual compounds responsible for the biological activity of propolis extracts would be a significant contribution to the standardization process of this material.The extraction efficiency of propolis varied depending on the solvent used for the extraction process as follows: acetone—58.15%, 70% EtOH—59.10%, and 96% EtOH—66.70%. The concentrations of selected phenolic compounds, namely, flavonoids and phenolic acids, in the propolis extracts are presented in Flavonoid concentrations in the tested extracts varied depending on the solvent used in the extraction process. Statistical analysis of flavonoid concentrations indicated that only two of the eight detected compounds, namely, naringenin and pinobanksin, were found in all extracts at the same statistical level. Pinocembrin was the most abundant flavonoid in all the extracts and its concentration was the highest when 70% EtOH was used as the solvent (EEP70). Galangin and chrysin were also detected in all propolis extracts at high concentrations. In addition, EEP96 contained a relatively high concentration of kaempferol. The propolis extract obtained using acetone as a solvent (EPA) was characterized by the highest sum of flavonoids (95.25 mg/g of propolis extract) among the tested extracts. On the other hand, the sum of flavonoids in EEP70 was equal to 97.77% of that obtained for EPA and it was almost 20% higher than obtained for EEP96. The galangin concentration was the highest in EPA, whereas chrysin and pinocembrin concentrations were the highest in EEP70, and the highest concentration of kaempferol was detected in EEP96. The concentrations of rutin, epicatechin, catechin, genistein, pinostrobinm and myricetin were below the detection limits of ultra high-performance liquid chromatography (UPLC) with photodiode array detection (PDA) and triple-quadrupole mass spectrometry (TQD) in all propolis extracts.The highest concentration in all propolis extracts among phenolic acids was observed for coumaric acid, and its concentration was statistically solvent-dependent. The concentration of cinnamic acid was also high and at the same statistical level in all the extracts. Concentrations of five out of seven phenolic acids detected differed statistically depending on the solvent used in the extraction process. In EEP96, the sum of phenolic acids was only about 4% higher than that detected in the other two extracts. Chlorogenic and sinapic acids were detected in none of the tested propolis extracts.3+-reducing power, and ferrous ion (Fe2+)-chelating activity. The results obtained are presented in The antioxidant potential of the propolis extracts was evaluated using standard cell-free antioxidant assays, namely, 2,2-diphenyl-1-picrylhydrazyl (DPPH·) free-radical-scavenging activity, Fe3+-reducing power, and ferrous ion (Fe2+)-chelating activity. The values of the antiradical activity of the propolis extracts toward DPPH· ranged from 73.05 (EEP70) to 77.70% (EPA) of the activity determined for the standard antioxidant Trolox, and they were over 90% of the level for another standard, butylated hydroxyltoluene (BHT). All the propolis extracts were characterized by a statistically similar reducing potential equivalent to approximately 62% of the value for Trolox and over 75% of BHT activity. The extract obtained with 96% EtOH as a solvent (EEP96) was characterized by a lower ferrous-ion-chelating activity and differed statistically from the two other extracts. Moreover, all the extracts exhibited a capacity to chelate Fe2+ corresponding to 43% and to 55% of ethylenediaminetetraacetic acid (EDTA) activity, used in this study as the ferrous ions standard chelator.All the propolis extracts exhibited a high and statistically similar free-radical-scavenging activity, FeAs shown in To study the cytoprotective activity of the propolis extracts under oxidative stress conditions, in vitro human RBC-based assays were applied. As presented in Simultaneously to the oxidative hemolysis assay, a microscopic analysis of the RBC shape was performed. As shown in p-values of the F test are summarized in p < 0.05) and, therefore, these flavonoids were not included in this comparative analysis.One-way analysis of variance (ANOVA) was used to determine the effect of a single flavonoid concentration on the antioxidant potential of extracts. The corresponding 3+-reducing power, while they showed an effect  on the ferrous ion (Fe2+)-chelating activity of the propolis extracts. In addition, flavonoid concentrations had no influence on the cytoprotective potential of the extracts against oxidative hemolysis.Statistical analysis showed that the flavonoid concentrations determined in the extracts did not affect the DPPH· free-radical-scavenging activity and the FeThe effect of Polish propolis extracts on several molds was examined, and the results expressed as minimal inhibitory concentration (MIC) are presented in Aureobasidium pullulans being the most sensitive fungal strain toward all propolis extracts. Moreover, EPA and EPP70 showed higher activity (MIC = 0.5 mg/mL) against A. pullulans compared to the reference compound (MIC = 1.0 mg/mL). The antifungal effect of extracts was also noticeable in Penicillium cyclopium. The acetone propolis extract showed a higher inhibitory effect toward Paecilomyces variotii in comparison to the ethanolic extracts and a lower activity against Penicillium funiculosum than the other extracts. Aspergillus niger was the most resistant fungal strain to all the propolis extracts, with an MIC of 7.5 mg/mL.The tested mold strains exhibited various sensitivities to the propolis extracts. In this experiment, the values of the minimum inhibitory concentration (MIC) ranged from 0.5 mg/mL to 7.5 mg/mL. The intensity of the antifungal activity depended on the type of solvent and the species of fungi, with p-values of the Wilks test are given in p < 0.05)).In order to assess the effect of flavonoid concentrations in the propolis extracts on the antifungal activity, multivariate analysis of variance (MANOVA) was applied. The The statistical analysis indicated that the concentrations of apigenin, chrysin, galangin, kaempferol, and pinocembrin in the propolis extracts significantly affected their antifungal activity.Pinocembrin, galangin, and chrysin , which wThe qualitative and quantitative analyses of the phenolic profile in the propolis extracts indicated that the solvent used in the extraction process affected concentrations of flavonoids  and, to 2+-chelating activity of all the extracts (34.00–43.33%) was comparable to that of propolis extracts from central Poland (35.41–38.85%) [3+-reducing power of all the extracts. However, literature data showed that the solvent had an impact on the antioxidant properties of propolis extracts [2+)-chelating activity of our extracts was statistically dependent on the solvent (3+-reducing power (2+)-chelating activity of the propolis extracts. Therefore, taken together, it can be assumed that the beneficial activity of extracts is a result of a specific cooperation of different phenolic compounds.The examined propolis extracts showed a high antiradical potential, which was also characteristic of propolis extracts originating from different geographical regions, including samples from New Zealand, Brazil, Morocco, Hungary, Slovenia, India, or various parts of Poland ,44,45,46–38.85%) ,34. More–38.85%) . In turn–38.85%) . The resoland 35.–38.85% [extracts ,46,48. Mextracts  stated t solvent . These r solvent , which png power . The conHuman RBCs are used as a cell model for screening of the antioxidant activity of bioactive compounds ,34,51,52As indicated by data presented in To estimate the protective efficiency of the propolis extracts against the discoid RBC shape alternation, simultaneously to the AAPH-induced hemolysis measurements, a scanning electron microscope analysis of the RBC shape was performed. As shown in Aspergillus niger, Penicillium chrysogenum, Candida albicans, C. glabrata, Rhodotorula mucilaginosa, Fusarium solani, Botrytis cinera, P. notatum, and Saccharmyces cerevisiae [A. niger, which was comparable to the MIC values previously reported for propolis extracts from Poland [A. niger, Trichoderma viride, and Penicillium chrysogenum than the aqueous extract. Mavri et al. [The values of the minimal inhibitory concentration (MIC) of fungal strains  indicaterevisiae ,7,31,57.m Poland . The resm Poland ,48. Garei et al. ,45,59. Ap-hydroxybenzoic acid) and chemicals  used in UPLC analysis were purchased from Sigma Aldrich . Chemicals for the determination of antioxidant activity and cytotoxicity of the propolis extracts in human erythrocytes (dimethyl sulfoxide (DMSO), potassium ferricyanide, trichloroacetic acid, iron(III) chloride, iron(II) chloride, ferrozine, glucose, paraformaldehyde, glutaraldehyde) were purchased from Avantor Performance Materials . Butylated hydroxyltoluene (BHT), Trolox, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azobis-(2-methylpropionamidine) dihydrochloride (AAPH), poly-l-lysine, ethylenediaminetetraacetic acid (EDTA), phosphate-buffered saline ), and potato dextrose agar (PDA) were purchased form Sigma Aldrich .Acetone and ethanol used to prepare propolis extracts were purchased from Avantor Performance Materials . All phenol standards , and raw propolisW is the weight of raw propolis (g).Raw propolis was purchased from a beekeeping company  and was originally collected from May to September 2014 in an apiary located in the Warmia-Masuria Province in northeastern Poland. Ground propolis samples (15 g) were extracted with 150 mL of acetone and ethanol at two different concentrations, i.e., 70% and 96%. The extraction was carried out under shaking  for 5 days at ambient temperature. The propolis extracts were then filtered through a Watman no. 4 filter paper  and solvents were evaporated using a rotary evaporator . Then, the extraction yield of propolis was calculated according to the following equation:The obtained residues were next used in chemical and biological analyses.m/z values obtained by MS and MS2 with the mass spectra of the corresponding standards tested under the same conditions. The mass-to-charge ratios (m/z) of the parent and daughter ions, as well as the mode of ionization and the best collision energy, are presented in The residues of propolis extracts obtained in 3+-reducing power, and (iii) ferrous ion (Fe2+)-chelating activity, according to the methods described in our previous work [The antioxidant activity of the propolis extracts was determined on the basis of three cell-free assays: (i) the DPPH· free-radical-scavenging activity, (ii) Feous work . In thisAbs0 is the absorbance of the control samples, and Abs1 is the absorbance of the tested samples. Every sample tube was prepared in triplicate, and every experiment was repeated three times. The results (n = 9) are presented as the mean value ± standard deviation (±SD).The solution of (0.1 mM) of DPPH· in ethanol (0.2 mL) was added to 0.2 mL of propolis extracts and vortexed . The reference antioxidants Trolox and BHT were used to determine the DPPH· free-radical-scavenging activity. The samples of reference compounds and propolis extracts were incubated in the dark for 30 min at room temperature. After this time, the absorbance (Abs) was measured at 517 nm, using an EPOLL 2000 ECO spectrophotometer . The percentage DPPH· scavenging effect was calculated using the following equation:n = 9) are presented as the mean value ± standard deviation (±SD).The propolis extracts (0.06 mL) were mixed with 0.1 mL of 0.2 M PBS and 0.1 mL of 1% potassium ferricyanide. Trolox and BHT were used as reference compounds. The samples were vortexed  and incubated for 20 min at 50 °C. After incubation, 10% trichloroacetic acid (0.1 mL) and 0.6 M chloride irons (III) (0.04 mL) were added to each sample. Finally, the absorbance (Abs) was measured at 700 nm in an EPOLL ECO spectrophotometer . Every sample tube was prepared in triplicate, and every experiment was repeated three times. The results  are presented as the mean value ± standard deviation (±SD).The propolis extracts (0.2 mL) were mixed with a solution of 0.6 mM chloride irons (II) (0.05 mL). EDTA was used as the standard metal chelator. The reaction was started by the addition of 5 mM ferrozine (0.05 mL) in ethanol, and the mixture was immediately vigorously shaken . The samples were stored for 10 min at room temperature and, after this time, the absorbance (Abs) of the samples was measured at 562 nm using an EPOLL ECO spectrophotometer . Every sample tube was prepared in triplicate, and every experiment was repeated three times. The percentage of inhibition of ferrozine–Fen = 15) are presented as the mean value ± standard deviation (±SD).The hemolytic activity of the propolis extracts and their effects on the RBC morphology were studied on the propolis extracts at a concentration of 100 µg/mL. The analyses were carried out on fresh human erythrocyte concentrates purchased from the Blood Bank in Poznań, Poland, according to the methods described by Woźniak et al. . The cyt4 for 30 min at RT. Following the next washing, the RBCs were dehydrated in a series of ethanol solutions  in RT, critical-point-dried, and gold-sputtered. A large number of RBCs were examined using an EVO 40  scanning electron microscope.The RBCs treated as above were fixed in 5% paraformaldehyde (PFA) and 0.01% glutaraldehyde (GA). Following washing three times with PBS buffer (by gentle exchanging the supernatant with PBS), cells were fixed in 1% GA for 60 min at room temperature (RT). Following washing, RBCs were post-fixed with 1% OsOAspergillus niger van Tiegen ATCC 6275, Aspergillus versicolor ATCC 11730, Paecilomyces variotii ATCC 18502, Penicillium funiculosum ATCC 11797, Aureobasidium pullulans ATCC 9348, Penicillium cyclopium Westling, and Trichoderma virens. The species were provided by the BAM Federal Institute for Materials Research and Testing collection or by the Institute of Chemical Wood Technology (Poznań University of Life Sciences). The bioassay investigating the propolis extracts was prepared in a 96-microdilution tray using the broth microdilution method. The MIC  value was defined as the lowest concentration of the antifungal agent which resulted optically clear. The 96-well plates were prepared by dispensing 100 μL of double-concentrated potato dextrose agar culture medium into the each well. Then, 100 μL from their serial dilutions with different amounts of the propolis extracts (0.01–10 mg/mL) were transferred into the subsequent wells. To each well, 10 µL of freshly made fungal spore suspensions in concentrations of colony-forming units (CFU) from 10−5 to 10−6 CFU/mL were added using a microtiter. Those fungal spore suspensions were obtained from two-week agar slants. The results for each extract were compared to the control wells with a commercial fungicide 3-iodo-2-propynylbutylcarbamate (IPBC) as Preventol® MP100 . The last well contained 200 μL of PDA with the fungal suspension to confirm the cell viability (viability control). Next, the plates were mixed on a plate shaker (300 rpm) for 30 s and incubated aerobically for 3–5 days in a moist chamber with relative humidity (RH) above 95% at 28 ± 1 °C in the dark. For reproducibility and accuracy evaluation of the microtiter plate method, the compounds were tested in triplicate.The following fungal strains were used: The results were analyzed using multivariate analysis of variance (MANOVA) applying the Wilks test and one-way analysis of variance (ANOVA) with Tukey’s honestly significant difference (THSD) test. All of the statistical analyses were performed using TIBCO Software Inc. Statistica version 13 .3+-reducing power, and Fe2+-chelating activity. Moreover, the cytoprotective activity against free-radical-induced human erythrocyte hemolysis was observed for all the propolis extracts. Statistical data showed no correlation between the content of individual flavonoids in the propolis extracts and their DPPH· free-radical-scavenging activity and Fe3+-reducing power. Concentrations of flavonoids, except for galangin and quercetin, statistically affected the ferrous ion (Fe2+)-chelating activity of the extracts. All the propolis extracts showed antifungal properties, which were dependent on the type of solvent used and the fungal strains. According to the statistical results, the concentrations of apigenin, chrysin, galangin, and pinocembrin significantly affected the fungistatic activity of these propolis extracts.The qualitative and quantitative analysis of the phenol profile in the extracts of Polish propolis indicated that the solvent used in the extraction affected the concentration of flavonoids and, to a limited extent, the concentrations of phenolic acids. In all the propolis extracts, the most abundant phenolic compounds were pinocembrin, chrysin, galangin, and coumaric acid. The results of antioxidant assays indicated that all the extracts exhibited high DPPH· free-radical-scavenging activity, FeIn summary, this is the first study presenting the dependence between the solvent used in Polish propolis extraction and the extract composition, as well as its biological properties, including in vitro effects on human erythrocyte shape and membrane permeability under both physiological and oxidative-stress conditions. On the basis of our results, it can be stated that the solvent-dependent Polish propolis extracts possess antioxidant and antifungal activities that can be attributed to presence of phenolic compounds. The statistical analysis showed that chrysin, galangin, pinocembrin, apigenin, and kaempferol are phenolic compounds that significantly affect the antifungal activity of Polish propolis extracts in a dose-dependent manner. It can be concluded that the identification of compounds responsible for the biological activity of Polish propolis will contribute to the development of procedures for its standardization, thus ensuring high quality of this product. Moreover, it is very important to search for new bioactive substances in different solvent-extracted propolis extracts, especially in the context of increasing antibiotic resistance of pathogenic microorganisms.


P. aeruginosa produces two siderophores and is also able to use many siderophores produced by other bacteria. We investigated the phenotypic plasticity of iron-uptake pathway expression in an epithelial cell infection assay in the presence of two different siderophore–antibiotic conjugates, one with a hydroxamate siderophore and the second with a tris-catechol. Proteomic and RT-qPCR approaches showed that P. aeruginosa was able to sense the presence of both compounds in its environment and adapt the expression of its iron uptake pathways to access iron via them. Moreover, the catechol-type siderophore–antibiotic was clearly more efficient in inducing the expression of its corresponding transporter than the hydroxamate compound when both were simultaneously present. In parallel, the expression of the proteins of the two iron uptake pathways using siderophores produced by P. aeruginosa was significantly repressed in the presence of both conjugates. Altogether, the data indicate that catechol-type siderophores are more promising vectors for antibiotic vectorization using a Trojan-horse strategy.Iron acquisition pathways have often been considered to be gateways for the uptake of antibiotics into bacteria. Bacteria excrete chelators, called siderophores, to access iron. Antibiotic molecules can be covalently attached to siderophores for their transport into pathogens during the iron-uptake process.  Acinetobacter, Pseudomonas, and various enterobacteria . The bottleneck in the development of new antibiotics for Gram-negative bacteria is the need for these drugs to cross the outer membrane. The use of nutrient-importation pathways as gateways for the uptake of antibiotics has often been proposed to bypass the low permeability of the Gram-negative cell wall [Antibiotic resistance is a complex and growing problem for human health. In 2017, the World Health Organization published a list of highly resistant bacteria for which new antibiotics are urgently needed. The most critical pathogens on this list are Gram-negative bacteria, such as Iron is a key nutrient, as this metal is involved in many crucial biological processes, and is therefore essential for bacterial growth and virulence. To access iron, bacteria produce siderophores, small molecules with a very high affinity for ferric iron . These iP. aeruginosa carries genes within its genome for at least 20 different iron-uptake strategies [2+) iron uptake pathway, (ii) three heme acquisition pathways, (iii) ferric iron (Fe3+) uptake pathways by the two siderophores pyoverdine (PVD) and pyochelin (PCH), produced by the pathogen itself, and (iv) at least 10 different “siderophore piracy” strategies to uptake Fe3+ using xenosiderophores (siderophores produced by other bacteria). Indeed, most bacteria can use siderophores produced by other bacteria in multispecies communities and during infections by expressing compatible transporters that are able to capture and import ferric complexes they cannot synthesize themselves [One major bottleneck limiting the development of siderophore–antibiotic conjugates is that all bacteria are equipped with several iron-uptake pathways. Consequently, bacteria can switch off the expression of the targeted iron uptake pathway and express an alternative one, possibly leading to the rapid development of resistance strategies. For example, rategies : (i) oneemselves ,14,15,16c iron Fe+ uptake emselves . Consequc iron Fe+ uptake P. aeruginosa cells with an analogue of the siderophore enterobactin (ENT) [P. aeruginosa more efficiently than tris-hydroxamate siderophores, such as ferrichrome [P. aeruginosa to the presence of two different siderophore–antibiotic conjugates in an epithelial cell infection assay using proteomics and RT-qPCR. We used the natural sideromycin albomycin (ALBO) , as well as that of virulence factors. Finally, when bacteria were simultaneously in the presence of both ALBO and TCVL6, the proteins of the ferri-TCVL6 uptake pathway were more highly expressed than those of the ferri-ALBO uptake pathway, indicating that between the two compounds, P. aeruginosa preferentially uses TCVL6 to access iron.In contrast to the large amount of genomic data generated by bacterial genome sequencing, almost no data are available concerning the phenotypic plasticity associated with the expression of these various iron-uptake pathways. Uptake pathways are considered to be generally expressed at a basal level and pathogens only induce the expression of the most efficient pathway(s) for iron acquisition depending on the bacterial environment . It has in (ENT) . Recentlrichrome . The datrichrome . Such phn (ALBO) a, for whn (ALBO) a. Both c pathway b and TCV pathway c. Both cP. aeruginosa PAO1 cultures as previously described [m-chlorophenylhydrazone) was purchased from Sigma-Aldrich. 55FeCl3 was obtained from Perkin Elmer Life and Analytical Sciences , at a concentration of 71.1 mM, with a specific activity of 10.18 Ci/g. RPMI was purchased from Thermo-Fisher.Enterobactin (ENT) and ferrichrome (FERRI) were obtained from Sigma-Aldrich. Albomycin δ2 (ALBO) was purchased from EMC Microcollection. Pyoverdine (PVD) was purified from escribed . TCV wasescribed  and TCVLP. aeruginosa strains used in this study are all listed in P. aeruginosa strains were grown at 30 °C in LB broth. When P. aeruginosa strains were grown in iron-restricted conditions, CAA medium was used , 1.46 g L−1 K2HPO4 3H2O, and 0.25 g L−1 MgSO4 7H2O).The Compounds were prepared in solution at 10 mM in DMSO (TCV and TCVL6) or water . The competition assay with PVD-Fe was carried out as described by our team previously . For theP. aeruginosa growth assays in microplate, a first overnight culture was carried out at 30 °C in 10 mL of LB broth; afterwards, bacteria were washed and a second overnight culture was carried out in 20 mL CAA medium at 30 °C. Following that, bacteria were washed, resuspended in CAA medium at 0.02 OD at 600 nm, and distributed in the wells of a 96-well plate  in the absence or presence of 10 µM of FERRI, ALBO, ENT, TCV, or TCVL6. The plate was incubated at 30 °C, with shaking, in a Tecan microplate reader  and bacterial growth was monitored at OD600 nm. We calculated the mean of three replicates for each measurement.For 55Fe complexes were prepared at 55Fe concentrations of 50 µM, with a ALBO:iron (mol:mol) ratio of 20:1. P. aeruginosa strains were first grown overnight at 30 °C in LB broth at 30 °C, and afterwards overnight in CAA medium at 30 °C, and at last, overnight in CAA medium with 10 µM ALBO at 30 °C. The bacteria were then used for 55Fe uptake kinetics as described previously [ALBO-eviously , in the ® CCL-185™) human pulmonary epithelial cells were grown as previously described [P. aeruginosa PAO1 cells grown in LB medium and with a multiplicity of infection of 50; 10 µM FERRI, ALBO, ENT, TCV, or TCVL6 were added to the A549 cells at the same moment as P. aeruginosa cells. After 3 h of infection at 37 °C, 5% CO2, the plates were washed with cold PBS 1X buffer and the A549 cells and bacteria still adhering to the bottom of the plate harvested, and prepared for RT-qPCR and proteomic analyses, as described previously [A549  relative to the conditions without conjugates or siderophores.A549 cells were infected as described above, in the absence or presence of FERRI, ENT, TCV, ALBO or TCVL6. The sample preparation and RT-qPCR analyses were carried out as previously described by our team . The priA549 cells were infected as described above, in the absence and presence of conjugates, FERRI, ENT, or TCV. The pellets of A549 cells and bacteria  in PBS were prepared for proteomic analyses, as described by our team previously . The resP. aeruginosa produces two siderophores, PVD and PCH, with PVD having a higher affinity for iron than PCH , a proton motive force inhibitor [fiuA (FERRI OMT) resulted in 40% inhibition of 55Fe uptake at 2 h. These data show that ferric iron-loaded ALBO uptake occurs via FiuA and at least one other OMT.Previous studies have shown that FERRI and ENT are both able to transport Fe into bacteria via the OMT FiuA for FERRI and PfeA for ENT ,14. NothA strain ) with ALnhibitor , indicatnhibitor ,37. The 55Fe uptake assays could be carried out because of small precipitation of the conjugates generating a significant background noise in the radioactivity signal. Consequently, we used an indirect approach to show that TCVL6 is able to cross the bacterial outer membrane. Indeed, previous proteomic and RT-qPCR analyses of P. aeruginosa grown in iron-restricted medium have shown that TCV is able to enter P. aeruginosa cells via the OMT PfeA, as the presence of this compound induces the expression of PfeA and PfeE, two proteins of the ENT-Fe uptake pathway [P. aeruginosa PAO1 cells were grown in the presence of TCVL6 and the transcription of pfeA and pfeE analyzed by RT-qPCR  in iron-restricted CAA medium, in the presence or absence of 10 µM ENT, TCV, TCVL6, FERRI, or ALBO . The baxpressed . Only th2 fold change of 5.68 for proteomics and 3.03 for RT-qPCR for ALBO) and no other OMT, suggesting that the other unidentified OMT that is involved in iron acquisition by ALBO . The expression of PfeE  had a similar effect on the P. aeruginosa phenotype, confirming that TCVL6 is able to enter the bacterial periplasm, like ENT and TCV, and bind to PfeS, the sensor of the two-component system involved in the regulation of the expression of the ENT-dependent iron-uptake pathway in P. aeruginosa cells [Proteomic and RT-qPCR data  showed tnd TCVL6  with a lsa cells ,23, and 2 fold change of −3.16 for the proteomic data) as well as the enzymes involved in PCH biosynthesis   d. The exenzymes) e. As mosenzymes) , this phnjugates a. The laP. aeruginosa is able to detect the two siderophore conjugates used in this study (ALBO and TCVL6) in its growth environment, even in a complex system such as an epithelial infection assay, and clear phenotypic adaption is observed. Both compounds were able to induce the transcription and expression of their corresponding OMT in P. aeruginosa cells: FiuA for ALBO and PfeA for TCVL6. The phenotypic adaptation of P. aeruginosa in this infection assay also involved repression of the transcription and expression of the proteins of the PCH and PVD pathways.In conclusion, P. aeruginosa and Piv  endoprotease [P. aeruginosa cells during the infection of A549 cells  ,40, a maprotease ,42. The protease . Thus, w49 cells a. Howeve or OxyR b, as welaprA and toxA, as the transcription of these virulence factors is regulated by PvdS. TCVL6 also repressed the transcription of aprA (more strongly than ENT) and toxA, as well as the transcription of piv and phzA2. Finally, ALBO repressed the transcription of aprA, exoY, piv, and toxA.Proteomic analyses showed repression of the expression of many virulence factors, mostly in the presence of ALBO, but also FERRI, ENT, and TCV, and, to a lower extent, TCVL6 . The lowP. aeruginosa cells infecting epithelial A549 cells increase the transcription and expression of catechol siderophore-dependent iron-uptake pathways, and not those of hydroxamate siderophores, in the presence of a mixture of four different siderophores [pfeA (ENT OMT), with a log2 fold change of 5; fiuA (FERRI OMT) transcription being almost not induced . In the presence of a mixture of ALBO and TCVL6, only pfeA transcription was strongly induced (log2 fold change of 5.2), that of fiuA almost not, and that of fptA, fpvA, and hasR repressed, with a of 0.5–0.9 log2 fold change. As the various genes involved in iron acquisition by a given siderophore are always in an operon, a change in the transcription and expression of the gene encoding an OMT implies a similar change for the transcription and expression of the other genes present in the operon. In conclusion, the data show that conjugates with a catechol-type siderophore moiety have more chances to induce the transcription and expression of their corresponding OMT, and consequently, their corresponding uptake pathway than hydroxamate vectors.We have previously shown that rophores . Here, t induced a. In addP. aeruginosa, via a Trojan-horse strategy using siderophores as vectors is a promising approach. However, there are still several bottlenecks in its development. The high antibiotic activity of natural sideromycins clearly shows that the strategy works and the fact that cefiderocol has been approved by the FDA for use in the treatment of urinary infections is a sign that antibiotic vectorization by siderophores should be explored further by pharmaceutical companies. However, developing such compounds requires extensive knowledge of the molecular mechanisms and regulatory network involved in bacterial siderophore-dependent iron acquisition. Most bacteria carry genes encoding several iron uptake pathways in their genome, allowing very high phenotypic plasticity, depending on the presence or not of xenosiderophores or sideromycins. Such phenotypic plasticity of P. aeruginosa has been investigated in various growth media and in the presence of several xenosiderophores [Introducing antibiotics into Gram negative bacteria, such as rophores . The datrophores .P. aeruginosa on the concentrations used. The bacteria sense the presence of both conjugates in their environment and adapt their phenotypes to access iron via the sideromycins that are present. This occurs via auto-loop regulating systems: the two-component system PfeS/PfeR for TCVL6 and the sigma and anti-sigma factors FiuI/FiuR for ALBO. Comparison of the transcription induction levels between FERRI and ALBO and between TCV and TCVL6 for their corresponding OMTs shows that the presence of the antibiotic group on each sideromycin had no inhibitory effect on the regulatory mechanism: the presence of the conjugates was sensed by P. aeruginosa with the same efficiency as the vector or corresponding siderophore alone.Here, we show that this also occurs in the presence of the two siderophore–antibiotic conjugates ALBO and TCVL6 in a complex system  with rapid kinetics (3-h infection with the bacteria having been previously grown in iron-rich media). Both compounds have no antibiotic activities on Such induction of FiuA and PfeA expression occurred in parallel with a decrease in the transcription and expression of the proteins involved in PVD and PCH biosynthesis and ferri-PVD and ferri-PCH uptake. In such a context, the bacteria use the conjugates to get access to iron and less their own siderophores. Moreover, the phenotypic effect induced by the presence of the siderophore conjugates was not only limited to the expression or not of the iron uptake pathways but also that of virulence factors, especially those regulated by PvdS, the sigma factor that regulates the transcription of the genes involved in PVD production The ability of siderophore conjugates to repress the transcription and expression of virulence factors is clearly an asset that has to also be considered in such a Trojan-horse strategy using siderophores as vectors.a of 1049 M−1 for ENT [29 M−1 for FERRI [30.8 M−1 for PVD [When the infection assay was carried out in the simultaneous presence of both sideromycins, ALBO and TCVL6 , the catechol-type vector was clearly more efficient in inducing the expression of its corresponding transporters, PfeA, than that for ferrichrome, because of its strong affinity for iron. Catechol-type compounds are clearly more efficient in competing for iron with PVD than hydroxamate molecules , consist for ENT , 1029 M−or FERRI , and 103 for PVD . BecauseThe ability of siderophore conjugates to be detected by the pathogen in its growth environment, even in a complex system, such as an epithelial infection assay, is a great advantage in Trojan-horse strategies, because it ensures the expression of the needed uptake pathway and suggests that such induction should occur as well in the host during an infection. Moreover, the data show that conjugates with a catechol-type siderophore moiety have a greater chance to induce their corresponding OMT, and consequently, their corresponding uptake pathway, than hydroxamate vectors and are potentially more efficient Trojan-horse vectors.


Opioid-induced constipation (OIC) is one of the most common complications of analgesic therapy for cancer pain patients who suffer moderate to severe pain. Acupuncture as an effective treatment in constipation has been widely applied. But its efficacy has not been assessed systematically. Thus, the purpose of this study is to provide a protocol to explore the efficacy and safety of acupuncture for OIC.Randomized Controlled Trials (RCTs) of acupuncture treatment for OIC in 4 Chinese electronic databases  and 3 English electronic databases  will be searched from their inception to September 31, 2020. RevMan 5.3 software and Stata 14.0 software will be used for meta-analysis, EndNote X9.2 and Cochrane Risk of Bias Tool will be used for literature screening and quality assessment.This study will present an assessment of the efficacy and safety of acupuncture treatment for OIC patients through summarize high-quality clinical evidence.The conclusion of our systematic review and meta-analysis may provide evidence of whether acupuncture treatment is beneficial to patients with OIC.INPLASY registration number: INPLASY2020100026. Though the WHO three-step ladder has been recognized and used widely around the world for many years, Side-effects are widespread, and among the most troublesome are those linked to opioids-induced bowel dysfunction, which particularly includes opioids-induced constipation (OIC).,4 OIC has a negative influence on work productivity, quality of life, and increased national health expenditures. The Rome IV standard defines OIC as new or upgraded symptoms of constipation that appear at the beginning, change, or increase of opioid therapy, and have further clinical features, such as a feeling of incomplete emptying and less than 3 spontaneous bowel movements per week.Opioids are powerful analgesics used for the treatment of acute and chronic pain.,8 It has been reported that about 215 million prescriptions for opioids in the United States in 2019. OIC occurs primarily related to μ-opioid receptor activation in the gut that reduces rectal sensation, decreases peristalsis and increases colonic fluid absorption. This results in harder stools. The National Comprehensive Cancer Network (NCCN) guidelines referred that the prevention and treatment of adverse reactions are an important part of the analgesic therapy plan. Once opioids are used, the prescription laxatives should be implemented to treatment OIC. However, laxatives do not target the underlying cause of opioid binding to the μ-receptors in the enteric system and as such are not very effective at managing OIC.,13 Accordingly, it is essential to find an alternative treatment.OIC is the most common and bothersome problem for patients with chronic taking opioids therapy, it affects 60% to 90% of cancer patients with opioids.–16 However, acupuncture as an adjunctive therapy exists a doubt in mainstream oncology. Therefore, it is necessary to evaluate the efficacy and safety of acupuncture in treating OIC through systematic review and meta-analysis, which intend to offer a reliable basis for clinical practice.Acupuncture is highly valued in traditional Chinese medicine and has a long historical source of more than 2500 years, and maybe a utility non-drug therapy option for OIC.22.1This study has been registered on INPLASY and the registration number is INPLASY2020100026 and the protocol follows the Cochrane Handbook for Systematic Reviews and the Preferred Reporting Items for Systematic Reviews and Meta-Analysis Protocols (PRISMA-P) statement guidelines.2.22.2.1All randomized controlled trials (RCTs) of acupuncture therapy for OIC will be included in the study, while animal experiments, cluster RCTs, reviews, and case reports will be excluded.2.2.2The study will include patients who were clinically diagnosed with OIC. There is no restriction on age, gender, or nationality. Besides, the diagnostic criteria are based on the Rome III criteria.2.2.3The patients in the intervention group adopt acupuncture and related treatments, regardless of needle material, acupoint selection, duration of treatment, acupuncture manipulation, while the patients in the control group are treated with drugs, placebo, sham acupuncture, or other conventional therapy, either.2.32.3.1The primary efficacy outcomes measure will be as follows: changes in the Bowel Function Index (BFI) score or Cleveland Constipation Score (CCS).2.3.2The secondary outcome measures will include the Patient Assessment of Constipation Quality of Life (PAC-QOL) questionnaire, adverse effects linked to interventions.2.4The following conditions in the literature will be excluded: repeated literature; incomplete data; inappropriate design method.2.5We will search for PubMed, Embase, Cochrane Library, CNKI, WF, VIP, CBM literature databases from its inception to September 2020 with a language restriction on Chinese or English. The details of the search strategy for PubMed are shown in Table 2.62.6.1Two of the researchers (PY and YCX) will be extract data independently by reading all titles and abstracts. The screen results are inconsistent will be settled through discussion between the above 2 authors. If their discussion still cannot reach accordance, another author (JRH) will make a final decision of eligible study selection. We will adopt EndNote X9.2 software to conduct a preliminary elimination of duplicate literature, then according to the inclusion and exclusion criteria, a brief screening will perform by reading the titles, abstracts, and keywords of the literature. Besides, we will review the full text to determine the final eligible literature based on details in the articles. The selection procedure of studies is summarized in the following PRISMA flow diagram Fig. .2.6.2All information will be extracted by 2 of the independent authors (QLM and RHM) according to predetermined criteria form. Disagreement will be resolved by consulting a third author (YCW), and the extracted data as following: first author, publication date, country, sample size, gender, mean age, details of interventions, treatment courses, follow-up, outcomes, and adverse event. If the information on the papers is unclear, we will contact the author by sending an email.2.6.3The risk of bias assessment of the included RCTs will be evaluated by using the risk of bias assessment tool of the Cochrane Handbook, version 5.1.0, which includes 7 items as following: random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, selective reporting, other bias. This evaluation will be conducted by 2 independent reviewers (JM and PY) according to a judgment for literature that will be categorized as low bias, unclear bias, or high bias.2.6.4RevMan 5.3 software will be used for data synthesis and analysis. When the outcome data is a binary variable, select the relative risk (RR) as the effect scale; when the outcome data is a continuous variable, use the mean difference (MD) and standardized mean difference (SMD) as an effect scale, both calculated by 95% confidence interval (CI).2.6.52 test, and I2 statistic will be used to evaluate heterogeneity. If P > .1 and I2 < 50%, the fixed effects model will be used. If P ≤ .1 and I2 ≥ 50%, the random-effects model will be used.The heterogeneity test adopts the χ2.6.6If significant heterogeneity is detected between a group of studies, subgroup analysis will be performed based on acupuncture types, countries, treatment courses, the control group intervention measures.2.6.7If the heterogeneity is significant, we will conduct a sensitivity analysis according to eliminating each of the included studies one by one, and changing the effect scale of studies to evaluate the robustness and quality of the conclusion in the studies.2.6.8First, if there are more than 10 studies are included, we will draw a funnel plot to analyze publication bias via RevMan 5.3 software, after Egger test and Begg test will be carried out to explore the potential publication bias of studies by using Stata 14.0 software if the funnel plot is asymmetric.2.6.9This meta-analysis and systematic review protocol will not involve ethical approval because where there not contain individual patient data. We will publish this study in peer-reviewed journals and conference presentations, which provide evidence of the efficacy and safety of acupuncture treatment for OIC.3 OIC is caused by the action exerted on opioid receptors in the gastrointestinal tract, in which the mechanism differs from idiopathic constipation. A study has shown that lifestyle changes and over-the-counter drugs are first-line treatments.  But in fact, there is still no satisfactory effect and effective alternative therapy in some cases. In recent years, studies of animal experiments show that acupuncture can improve gastrointestinal motility and expression of 5-HT by adjusting nerve stimulation.–22 However, the efficacy has not been recognized by the clinical guidance and medical organizations, meanwhile, there is not a systematic review about acupuncture for OIC to investigate the clinical efficacy and safety. So we conduct this study to provide a basis of evidence-based medicine and help clinicians make decisions in practice.Constipation is the most common and long-term intolerant adverse reaction of opioids, it seriously affects patient quality of life and cannot currently be effectively treated.As far as we know, it is the first time to conduct a systematic review and meta-analysis of acupuncture treatment for OIC, it demonstrates acupuncture has better results in clinical outcomes than non-acupuncture therapy. On the other hand, this study has some limitations, involving the quality of included literature, the inconsistency of acupuncture types, the methodology of studies, language limitation, which may lead to the high heterogeneity.Conceptualization: Pu Yang.Methodology: Pu Yang, Yuanchun Wang.Formal analysis: Yingchun Xiao, Qiaolin Ma, Runhong Ma, Jing Mi.Supervision: Jianrong Hui.Writing – original draft: Pu Yang, Yuanchun Wang.Writing – review & editing: Pu Yang, Yuanchun Wang, Jianrong Hui.


Copper deficiency can produce changes mimicking myelodysplasia, but its prevalence among haematology patients is unknown. This study evaluates the prevalence of deficiency in a population of patients with unexplained cytopenia(s) and dysplasia. Of 17 patients recruited, only one was found to have a low copper level. This is the first study evaluating hypocupremia in patients with isolated haematological manifestations. Our results suggest that copper deficiency is uncommon in this population, and the routine measurement of copper seems unlikely to be cost-effective. We recommend a risk-assessed approach to testing based on the identification of risk factors and of signs suggestive of deficiency.The online version of this article (10.1007/s12011-020-02203-x) contains supplementary material, which is available to authorized users. Copper is an essential micronutrient that is incorporated into copper-dependent enzymes and plays an integral role in oxidation-reduction reactions. Several risk factors for copper deficiency are reported in the literature, and hypocupremia can present with a variety of metabolic, haematological and neurological manifestations Fig. . To the There is uncertainty regarding the role of copper measurement in patients with unexplained cytopenia. The prevalence of copper deficiency among unselected patients is unknown , and theCytogenetic and molecular analysis are able to demonstrate evidence of clonal haematopoiesis in 75% of patients with MDS , and thiMDS should only be diagnosed when other causes of the haematological features have been excluded. A diagnosis of MDS has significant implications for patients, not least because of the known risk of progression to leukaemia. If an alternative cause is missed, patients will be subjected to needless investigations and hospital appointments, and will also be at risk of developing complications of their condition that could have been prevented with appropriate treatment.We designed a study to evaluate the prevalence of copper deficiency in patients with cytopenias and dysplasia who also lack a cytogenetic or molecular marker of MDS. The participants were all patients who were being monitored in our haematology clinic. The majority had bone marrow dysplasia reported in excess of 10%, and therefore fulfilled WHO criteria for a diagnosis of myelodysplasia.We evaluated our clinic and bone marrow records to identify potential participants. We wanted to exclude patients in whom a true diagnosis of MDS was highly likely. To achieve this, we excluded patients with a confirmed cytogenetic or molecular abnormality (with the exception of deletion of chromosome Y), those with a marrow blast percentage in excess of 5% on cytological examination or immunophenotyping, and patients who were receiving (or had previously received) cytotoxic treatment for MDS.We identified 17 potential patients who met our inclusion criteria ESM  and to sABL1, ASXL1, ATRX, BCOR, BCORL1, BRAF, CALR, CBL, CBLB, CBLC, CDKN2A, CSF3R, CEBPA, CUX1, DNMT3A, ETV6, EZH2, FBXW7, FLT3, GATA1, GATA2, GNAS, HRAS, IDH1, IDH2, IKZF1, JAK2, JAK3, KDM6A, KIT, KRAS, MLL, MPL, MYD88, NOTCH1, NPM1, NRAS, PDGFRA, PHF6, PTEN, PTPN11, RAD21, RUNX1, SETBP1, SF3B1, SMC1A, SMC3, SRSF2, STAG2, TET2, TP53, U2AF1, WT1, and ZRSR2. The software used for analysis was Variant Interpreter .Mutation screening in our centre is performed by targeted next generation sequencing using TruSight Myeloid panel (Illumina) using the Qiagen QIAsymphony DSP DNA Mini Kit. This assay comprises of 568 amplicons in 54 genes frequently mutated in myeloid malignancies, including mutations in The demographic data of our population and full blood count results at study inclusion are summarised in ESM All participants completed the health questionnaire. Relevant data for each patient is displayed in ESM Interestingly, six patients (33%) had copper levels which were mildly above the reference range. Inflammation has been documented to produce a reactive rise in plasma copper level , and we Measuring copper levels has been reported as an important part of the workup when investigating patients with unexplained cytopenias . The burMyelodysplasia is an uncommon condition with only 1524 new cases reported nationwide in England in 2017 . Our stuOur results suggest that copper deficiency is uncommon in this population of patients, although one must be cautious in drawing conclusions due to the small size of our population. We were not able to demonstrate a relationship between the identification of risk factors for copper deficiency and copper levels, and this is almost certainly because of the low number of positive findings. It is not likely that the low copper level in one of our patients was contributing to his haematological features because his copper level was only a fraction outside the reference range, and he had a normal CRP (0.6 mg/L) at the time of testing. This suggests his copper level was not falsely raised to mask a more severe deficiency.While copper deficiency should be considered in the differential diagnosis of cytopenias and dysplasia, our findings lead us to believe that indiscriminate testing of copper levels in patients with unexplained cytopenia may not be cost-effective. Based on data from larger studies, the identification of risk factors for the condition and signs of deficiency should improve the likelihood of a positive diagnosis , 9, 14. ESM 1(PDF 865 kb).


For further understanding the genetic control mechanisms of growth and development in Tan sheep, and culturing good traits on meat performance, which is very important to both in developing local species and improving economic efficaciously. In our study, we recruited a total of 250 Tan sheep and 174 healthy Hu sheep to detect 32 SNPs in GH, GHR, NPY, Leptin, H-FABP, MSTN, and CAST by using direct sequencing techniques, in order to explore genetic marking loci which were an association with growth characters. From the results, we found different SNPs with an obvious difference for the growth traits. In the different genetic model analysis, we found SNP12, SNP29, SNP41, SNP8, SNP34, SNP35, SNP9, SNP10, SNP36, SNP45, and SNP39 were a significantly negative association with the two kinds of sheep. And SNP46, SNP42, and SNP69 with the positive association between the different trait in sheep were analyzed. From the LD and haplotype analysis, we found three blocks with the positive association in growth traits between Tan sheep and Hu sheep. The block of SNP29, SNP32, SNP34, SNP35, SNP36, SNP39, SNP41, SNP42, SNP45, and SNP46 with the genotype “AATCTACTTA” is the most significantly association with the traits. In summary, the study initially explored the genes for growth and reproduction between Tan sheep and Hu sheep and found some statistically significant results which demonstrate that there are genetic differences. These differential molecular markers may provide a scientific theoretical basis for the preferred species of Tan sheep which with good meat performance and better utilization of species resources.The online version of this article (10.1007/s11250-019-02063-1) contains supplementary material, which is available to authorized users. As an important part of the animal husbandry industry, the sheep industry not only produces raw materials for the wool spinning industry, but also provides people with delicious lamb. Lamb is prevalent in the domestic and international markets because of its characteristics of lean meat, less fat, fresh, juicy, and easy to digest. China’s mutton sheep production needs better varieties with high meat production and good meat quality which require the continuous selection of meat quality in the breeding of sheep to improve the quality of mutton  content is a very important indicator of meat quality, mainly in the epicardium and muscle fascia as well as the endomysium, which is an important economic trait with moderately high heritability , GHR (growth hormone receptor), NPY (neuropeptide Y), Leptin, H-FABP (Heart fatty acid-binding proteins), MSTN (myostatin), and CAST , the study focused on the genetic effects of their polymorphisms on the growth and development of Tan sheep, with a view to discovering genetic markers with significant effects on growth traits and providing scientific basis for the high-quality breeding of Tan sheep and the protection and utilization of germplasm resources.As a famous suede sheep breed in China  to explore the difference. Tan sheep and Hu sheep are all raised in the Ningxia Yanchi County Tanyang Breeding Center. They are in the same ecological environment and are kept in a normally raised according to the sheep farm diet formula. Their recruitment time is from September 2015 to January 2017, and is carried out by means of whole house feeding. The lambs born in the same period were selected to measure their body weight and body size indicators. Body size indicators include body height, body length, and chest circumference. The lamb’s birth weight and body size indicators are measured before the lamb is born and eating colostrum. The selected samples kept the same husbandry and management level, and individual nutritional status in the same period. When they were born 1 year later, we took blood samples from each sheep and labeled them, and then returned to the laboratory at low temperature − 20 °C. According to the animal husbandry methods, we measured the basic growth and development trait indicators which included birth weight, weaning weight, 3 months weight, and 6 months weight which ensure that all sample nutrition levels differ by less than 5%. The animal ethics and welfare committee of NingXia Academy of Agriculture and Forestry Sciences approved the experimental procedures which is in compliance with the regulations for protection of animal research.For the association analysis, we selected these 32 validated SNPs (single-nucleotide polymorphisms) from some researches . PLINK software (http://pngu.mgh.harvard.edu/purcell/plink/) was used by the models to evaluate the association of 32 SNPs between the two kinds of sheep. Lastly, Haploview software (version 4.2) was conducted to analyses linkage disequilibrium (LD).The SequenomTyper 4.0 Software™  was used to manage and analyze the data. The univariate and multivariable logistic regression models were used to examine the association between Tan sheep and Hu sheep, and corresponding odds ratios (ORs) and 95% confidence intervals (CIs) were calculated simultaneously. All statistical analysis was carried out using SPSS19.0 statistical software  and Microsoft Excel, and a two-tailed p > 0.01). We detected the minor allele of each SNP which was assumed to be the wild-type allele. MAF (Minor Allele Frequency) in cases and controls was showed in Table χ2 test, we found the SNP12 , SNP29 , SNP41 , SNP8 , SNP34 , SNP35 , SNP9 , SNP10 , SNP36, SNP45 , and SNP39  were significantly negatively correlated with growth trait differences between the two kinds of sheep. And SNP46 , SNP42 , and SNP69  were positively correlated with these characters. These differences are consistent before and after FDR correction except the SNP61. The association of SNP61 and the trait is OR = 2.456, p < 0.05, after FDR, p > 0.05. After the χ2 test in other methods such as codominant, dominant, and recessive, we further analyzed the differences between the two kinds of sheep and found SNP12, SNP29, SNP34, SNP35, SNP9, SNP10, SNP36, SNP45, SNP39, and SNP69 as significant association with the characters of the two groups (Table p = 0.035) before FDR correction. In the dominant and log-additive models, SNP46 and SNP42 increased the growth traits difference whether or not FDR corrected. And when SNP46 is the genotype “G/A” and SNP42 with “C/T” in codominant model, we discovered the difference is increased before FDR  correction. However, SNP12, SNP29, SNP34, SNP35, SNP10, SNP36, SNP45, and SNP39 in these models, all of which, indicated their regulated trait significant difference between the two kinds of sheep  in both groups p > 0.0. We deteep Table .Table 1TWe utilized Haploview software for linkage disequilibrium analysis and build haplotypes. Through analyzing all the 32 SNP loci, we detected the existence of 6 blocks Fig. . From thIn view of the outstanding problems such as cultivar recession and quality degradation in Tan sheep, the in-depth and meticulous research on the intrinsic factors and genetic laws affecting the good traits of Tan sheep has plagued the protection of Tan sheep breeds and the healthy development of meat industrial development. We wanted to find out some molecular markers of excellent traits in Tan sheep compared with Hu sheep. Molecular marker–assisted breeding served to carry out targeted breeding of Tan sheep and builds a quality breeding system for Tan sheep. In our research, we randomly selected 32 SNPs and found SNP69 located in CAST; SNP42 and SNP46 located in H-FABP are the most significant sites with positive association with the growth trait. Regardless of the sites whether negative or positive association with the meat quality trait, all of these indicated the differences between Tan sheep and Hu sheep.p < 0.05)  can be combined with retinol or retinoic acid–binding protein (RBP or RABP) to compose intracellular lipid–binding proteins, which is an important component of intracellular lipid–binding proteins (ILBPs) and accounts for 1 to 8% of total cellular soluble proteins , indicating that the SNPs in CAST can be used as a molecular marker to promote the meat quality of Tan sheep. However, the same polymorphic site may have different effects on meat quality in different parts; the results still need further verification.Calpastatin (CAST) is an important intracellular protein with a molecular mass of approximately 120 ku that specifically inhibits calpain activity Murachi . FrekingGH and GHR have statistical significance. As the receptor and ligand exert biological functions, their importance is unquestionable. As important regulators of mammalian growth and metabolism, GH and GHR can regulate the expression of many genes by means of endocrine and nervous system conduction pathways to stimulate tissue metabolism, thereby promoting cell proliferation, bone growth, and protein synthesis ESM 2(XLSX 15 kb)