To compare data obtained by Scheimpflug camera (Pentacam) from both eyes of unilateral keratoconus patients and normal controls.This study was performed by retrospective chart review of 919 keratoconus patients. From these patients, 31 keratoconus eyes of 31 patients with unilateral keratoconus (Group 1), 31 normal fellow eyes of these patients (Group 2), and 30 right eyes of 30 normal controls (Group 3) were included in the study. Detailed ophthalmologic examination and Pentacam parameters at initial examination were analyzed and relationships between Groups 1, 2, and 3 were statistically evaluated. ROC curve analysis was also performed to determine the sensitivity and specificity of parameters that could be used to differentiate Group 2 from Groups 1 and 3.The mean age was 30.07±11.00 (15-60) in Group 1-2 patients and 32.33±9.30 (18-45) in Group 3 patients (p=0.392). In comparison of Pentacam data, there were statistically significant differences between Groups 1 and 2 in all parameters except corneal volume (p<0.05). Group 1 and Group 3 were significantly different in all evaluated parameters (p<0.05). Steep keratometry, flat keratometry, mean keratometry, and posterior elevation (PE) were statistically similar between Groups 2 and 3 (p>0.05), while the other evaluated parameters differed significantly (p<0.05). ROC curve analysis showed that the difference in corneal thickness between the apex and thinnest point, progression index, index of surface variance, index of height asymmetry and inferior-superior had the highest sensitivity and specificity in differentiating Group 2 from Group 3, while CCTapex, CCTmin, PE, and minumum radius had the highest sensitivity and specificity in differentiating Group 2 from Group 1.In patients with unilateral keratoconus, fellow eyes appear to not be completely normal. Thus, it is recommended that fellow eyes also be evaluated in every examination of unilateral keratoconus patients. The incidence of keratoconus is approximately 1/2000 and its prevalence is 54.5/100,000. The disease is caused by both genetic and environmental factors.7In addition to clinical examination, various auxiliary instruments are used in the diagnosis of keratoconus. In the past, keratoconus was diagnosed using Placido-disc based topographers, which are only able to evaluate the anterior surface of the cornea. The development of the Scheimpflug camera system  also enabled evaluation of the posterior cornea surface. This device allowed the detection of early changes originating in the posterior cornea in clinically normal patients, which was a major breakthrough in the diagnosis and monitoring of the disease.8 determined that 50% of patients initially diagnosed with unilateral keratoconus also developed keratoconus in the apparently normal fellow eye. However, Imbornoni et al.9 emphasized that keratoconus was not observed during long-term follow-up in any of the fellow eyes in a series of 5 cases. Therefore, different terms such as preclinical keratoconus, forme fruste keratoconus, and keratoconus suspect are used instead of unilateral keratoconus.11 Although different rates have been reported for unilateral keratoconus, the proportion generally ranges between 0.5% and 4.5%.16 Various keratoconus studies have demonstrated abnormalities in the Pentacam data of fellow eyes considered unaffected.19Keratoconus is usually progressive and bilateral. Even if one eye is not affected initially, the fellow eye is eventually affected as well in the majority of patients. Holland et al.The aim of this study was to compare anterior segment parameters of the apparently normal fellow eyes of patients who presented to our center with unilateral keratoconus with those of keratoconus eyes and the eyes of healthy control subjects.This study was carried out with the approval of the Ege University Faculty of Medicine Ethics Committee (129362). The medical data of patients with keratoconus who presented to the Cornea Unit of the Ege University Faculty of Medicine Department of Ophthalmology were retrospectively analyzed. Patients who had a history of trauma or corneal surgery for keratoconus and those from whom reliable measurements could not be obtained were not included in the study. In addition, patients who used contact lenses at time of presentation and those with a history of allergic conjunctivitis were excluded from the study. Of the remaining 919 patients, 31 patients (3.3%) who had been evaluated as having unilateral keratoconus at initial presentation were included in the study. The patients’ best corrected visual acuity, intraocular pressure measurements, and anterior and posterior segment examination findings were evaluated. In addition, the patients’ keratometric parameters, topometric parameters, posterior elevation, corneal pachymetry, and pachymetric index values obtained with Pentacam were analyzed.20 According to this classification, stage 1 is defined as eccentric steepening, myopia and/or astigmatism <5 D, and/or central keratometry value <48 diopter (D); stage 2 involves myopia and/or astigmatism of 5-8 D, central keratometry value <53 D, and minimum corneal thickness >400 µm; stage 3 is defined as myopia and/or astigmatism of 8-10 D, central keratometry value >53 D, and minimum corneal thickness 300-400 µm; and in stage 4, refraction is not measurable, central keratometry value is >55 D, there is central corneal scarring and minimum corneal thickness is <200 microns. The groups were compared in terms of demographic and Pentacam  data. Measurements were repeated until a reliable measurement was obtained according to the Pentacam device’s software. Our analysis included the following Pentacam data: the anterior corneal surface keratometric parameters steep keratometry (Ks), flat keratometry (Kf), mean keratometry (Km), and the inferior-superior (I-S) difference at 4 mm; the topometric parameters index of surface variance (ISV), index of vertical asymmetry (IVA), keratoconus index (KI), central keratoconus index (CKI), index of height asymmetry (IHA), index of height decentration (IHD), and minimum radius (Rmin); and posterior elevation (PE), corneal thickness at the apex and the thinnest point , corneal volume (CV), and mean pachymetric progression index (PPI).The eyes were divided into 3 groups: keratoconus eyes , fellow eyes considered clinically and topographically normal , and the healthy right eyes of control subjects . The Amsler-Krumeich classification was used when diagnosing keratoconus.Statistical analyses were done using the SPSS software package version 20 . The Shapiro–Wilk test was used to test all parameters for normal distribution. Comparisons between groups were done with one-way ANOVA with post-hoc Bonferroni test. Chi-square test was used to compare demographic data. ROC curve analysis was done to determine the sensitivity and specificity of the parameters. A p value below 0.05 was considered statistically significant.Mean age was 30.07±11.00 (15-60) years in Groups 1/2 and 32.33±9.30 (18-45) years in Group 3 (p=0.392) . The femComparison of Pentacam data between Groups 1 and 2 showed that Ks, Kf, Km, PE, I-S difference, ISV, IVA, KI, CKI, IHA, IHD, and PPI values were significantly higher in Group 1 (p<0.05) . In contIn comparisons of Groups 1 and 3, Group 1 had significantly higher Ks, Kf, Km, PE, I-S difference, ISV, IVA, KI, CKI, IHA, IHD, and PPI (p<0.001) and significantly slower Rmin, CCTapex, and CCTmin (p<0.001). CV was also significantly lower in Group 1 than in Group 3 (p=0.009).Comparisons of Groups 2 and 3 revealed similar Ks, Kf, Km, and PE . The other analyzed parameters  all differed significantly between the two groups (p<0.05).In ROC curve analysis to identify parameters that could be used to differentiate Groups 2 and 3, the parameters with the largest areas under the curve (AUC) were ISV  and I-S . In addition, PPI, IHA, and CCTdiff, which is the difference between CCTapex and CCTmin, also had significantly high AUC values . ROC cur1 The corneal thinning seen in keratoconus is not central, but usually occurs in the inferonasal region. The Pentacam has a key role in the early diagnosis and monitoring of keratoconus due to its ability to evaluate the anterior and posterior corneal surfaces together. Abnormalities emerge in the posterior surface of the cornea in early keratoconus. Therefore, development of the Pentacam led to a significant increase in diagnostic sensitivity in keratoconus.4Keratoconus is a chronic, usually bilateral, non-inflammatory corneal ectasia.21 compared CV in keratoconus patients, subclinical keratoconus patients, and normal control subjects and showed that this parameter is helpful in distinguishing keratoconus eyes from normal eyes but not in differentiating between keratoconus and subclinical keratoconus. In their study of patients with keratoconus, Emre et al.22 found that CV decreased with disease progression. The results of our study also support these data. We found that the keratoconus eyes in Group 1 differed significantly from the eyes in Groups 2 and 3. We also determined that the Pentacam data of the eyes in Group 2 were statistically closer to the results in Group 3, especially in terms of keratometry readings. In another study on this subject, Bae et al.17 compared the affected and unaffected eyes of patients with keratoconus and reported a significant difference, with normal fellow eyes being more similar to the eyes of healthy volunteers. Hashemi et al.23 found that the normal fellow eyes of patients with keratoconus were not significantly different in terms of average keratometry values, but did show significant differences in topometric indexes. These findings are consistent with the data obtained in the current study. These results may be due to evaluating the patients’ fellow eyes before emergence of the disease or to the patients having true unilateral keratoconus. However, in contrast to these studies, Muftuoglu et al.11 found that the fellow eyes of patients with keratoconus were significantly different from healthy controls. Further studies are needed to be able to clearly differentiate these eyes.In this study, we attempted to identify differences between the apparently normal fellow eyes of patients with unilateral keratoconus and the patients’ keratoconus eyes and the healthy eyes of controls. Comparison of Pentacam data revealed significant differences between Groups 1 and 2 in all parameters except CV. Çağıl et al.11 found that I-S and PPI had high sensitivity and specificity for discriminating keratoconus patients from healthy controls. Bae et al.17 mentioned the importance of I-S and PE in evaluating the fellow eyes of patients with keratoconus in their study, while Mihaltz et al.24 emphasized that PE was the most sensitive parameter for diagnosing keratoconus. Hashemi et al.23 reported that in addition to pachymetric indices, IVA and ISV showed high accuracy rates in identifying cases of subclinical keratoconus. The results obtained in the present study are also consistent with the aforementioned studies. However, none of these values alone is sufficient for the diagnosis or discrimination of keratoconus. Combining them with other clinical data may increase their diagnostic value.In this study, we also performed ROC curve analysis to enable the discrimination of clinically unaffected fellow eyes of keratoconus patients from keratoconus eyes and eyes of healthy subjects. According to our results, CCTdiff, PPI, ISV, IHA, and I-S showed high sensitivity and specificity for distinguishing Group 2 from Group 3, while CCTapex, CCTmin, PE, and Rmin values showed high sensitivity and specificity for differentiating between Group 2 and Group 1. In their study, Muftuoglu et al.Limitations of this study include its retrospective nature, the limited number of subjects, and the fact that conclusions were based solely on measurements made at the time of presentation.To summarize, the results of this study indicate that although the fellow eyes of patients diagnosed with unilateral keratoconus did not exhibit measurement anomalies great enough to be considered keratoconic at the time of diagnosis, they were also not completely normal. However, based on the data obtained, it does not seem possible to diagnose these eyes with keratoconus using available diagnostic tests. In patients with unilateral keratoconus, it is particularly important to monitor fellow eyes evaluated as normal at presentation for development of keratoconus in the long term and to advise patients to avoid mechanical trauma.


Channa striata extract toward the acceleration of tuberculosis treatment. The study used true experiment, in which the intervention of the study was Channa striata supplementation to respondent. In addition, Chi-square was used to analyze the data with SPSS version 22. The result is the proportion of respondent classified in negative category in intervention group within week 0, week 1, week 2, week 3, and week 4 being 10.7%, 35.9%, 56.3%, 70.9%, and 90.3%, respectively. Besides, the proportion of respondent classified in negative category in control group within week 0, week 1, week 2, week 3, and week 4 was 13.4%, 23.7%, 37.1%, 49.5%, and 68%, respectively. Based on Chi-square test, the P value of Channa striata supplementation toward the acceleration of tuberculosis treatment week 1, week 3, and week 4 is 0.045, 0.019, and 0.005 , respectively. It means that there were differences between Channa striata supplementation and acceleration of tuberculosis treatment among respondent. Therefore, Channa striata treatment was significantly related to the acceleration of tuberculosis recovery.Tuberculosis is international health problem, which is classified in Global Emergency disease since 1992. The objective of the study is to determine the effect of Mycobacterium tuberculosis [Tuberculosis (TB) is an international health problem, which is classified as Global Emergency Disease since 1992. Based on WHO report 2004, there are 8.8 million of new tuberculosis cases in 2002 [rculosis . In 2016rculosis . TherefoTuberculosis is deadly disease worldwide , which c Mycobacterium tuberculosis infection and (2) the risk of the progressivity of tuberculosis infection [The risk factors of the development of tuberculosis are (1) the risk ofnfection , 9. Thosnfection . Furthernfection  toward infection . Besidesnfection .The nutrient supplement can improve the recovery of tuberculosis patient . The micChanna striata (Ikan Gabus) is one of essential fatty acid and essential amino acid (protein) sources that is cheap and comprehensive. The extract of Channa striata contains 16 kinds of amino acids and 8 kinds of essential amino acid such as arginine, threonine, valine, methionine, isoleucine, leucine, phenylalanine, and lysine [ Channa striata has 8 kinds of fatty acid and two kinds of essential fatty acid that is classified in omega-6 group such as linoleate acid (C18:2) and arachidonic acid (C20:4) [d lysine . Further (C20:4) . Channa striata supplement related to clinical aspect has been conducted twice in Indonesia. The first research that was conducted among patients in Wahidin Sudirohusodo Hospital was written by Nurpudji Astuti [The study ofi Astuti . The resi Astuti . Channa striata (Ikan Gabus) extract toward the acceleration of TB patient treatment, in which the acceleration of tuberculosis treatment in this study is shortening the tuberculosis treatment's duration by supplementation of Channa striata. The result of study is useful to the government to create appropriate policy related to accelerate the treatment of tuberculosis in Jember regency and Situbondo Regency, especially in East Java Province-Indonesia.The objective of the study is to determine the effect ofThe study was conducted in work area of primary health care in Jember Regency and Situbondo Regency. Jember and Situbondo had 49 units and 17 units of primary health care, respectively. The study was held in May-December 2017. The sputum test of tuberculosis patient was examined in Jember Chest Hospital (Rumah Sakit Paru Jember), in which Jember Chest Hospital was the only government hospital in 7 regencies , East Java-Indonesia, where the service focused on lung disease, particularly tuberculosis. Channa striata (ikan gabus) extract to respondent (intervention or treatment group), in which control group was administered placebo. In addition, the respondent's sputum was examined in Jember Chest Hospital to recognize the availability of Mycobacterium tuberculosis each week within a month. The design of the study used randomized pretest-posttest only control group design that was showed by  Channa striata supplementation before and after administered to respondent.The study was truly experimental, in which all of influential variables except intervention can be controlled. The author used true experiment to get the valid result, and the intervention of the study can be managed randomly. The treatment of the study was by supplementation of Channa striata is not appropriate in Indonesia, where there is no policy that regulated this issue. Therefore, this study used the policy of Health Ministry of Indonesia related to tuberculosis diagnosis and treatment as guidelines, in which the new tuberculosis patient must get standard antibiotic treatment for tuberculosis.The study was conducted on an ambulatory basis, where all respondents performed antibiotic treatment for tuberculosis in their house. The procedure of this study referred to the policy of Health Ministry of Indonesia, in which all new tuberculosis patients must get 6-month-full antibiotic treatment for tuberculosis in Indonesia. The medication for tuberculosis patient only with Channa striata extract was as complementary supplement to accelerate tuberculosis treatment by increasing the nutritional status, and the Channa striata extract of this study is permitted to consume by Health Ministry of Indonesia (Registered Number: P-IRT: 202350901620).The prescription of tuberculosis antibiotic treatment was classified into two categories, namely, (a) primary drug  and (b) secondary drug . The function ofThe population of the study was all of new positive pulmonary tuberculosis patient that performed standard antibiotic treatment for tuberculosis in primary health care of Jember and Situbondo Regency, and respondent agreed to participate in research voluntarily. The total of pulmonary tuberculosis patients was 2,733 cases, in which the distribution of patients in Jember and Situbondo was 2,176 patients and 557 patients, respectively. Based on Kelsey, the total samples of control and intervention group are 100 respondents, respectively ; therefore the total sample of the study was 200 respondents. In addition, the proportionated to size method was used to distribute the sample. The total samples of control and intervention group in the study is 97 respondents and 103 respondents, respectively. Channa striata supplementation within the acceleration of tuberculosis treatment by shortening the tuberculosis duration recovery. The exclusion criteria of this study are (a) HIV/AIDS patients, (b) diabetes mellitus patients, (c) MDR patients, (d) respondent who did not take Channa striata supplement or placebo regularly (3x1 day), and (e) tuberculosis patients who fail drug treatment. Those respondents will be drop out of study or lost to follow-up.There were exclusion criteria in this study, where the exclusion criteria were used to control the potential confounding variables, such as tuberculosis similar disease. Therefore, the authors can ensure the positive effect of Channa striata supplementation. Meanwhile, the control group performed standard antibiotic treatment for tuberculosis and placebo supplementation. The tuberculosis antibiotic drug, Channa striata supplement, and placebo were administered to respondent by nurse in primary health care, in which the supplementation of Channa striata was 3 times a day during a month.All respondents of this study were divided into 2 group by randomization, namely, intervention group and control group, where all respondents did not know their status in the group (blinding process). The intervention group performed not only standard antibiotic treatment for tuberculosis but also Channa striata that was registered in Health Ministry of Indonesia was administered to intervention group, in which the Channa striata supplement contains 90% Channa striata extract and 10% others. In addition, the supplement of Channa striata had several nutrients such as protein (80.9%), albumin (12.5%), and polyphenol bioflavonoid (6.6%). During study, the authors also used field research assistant to supervise and to monitor antibiotic drug-, Channa striata-, and placebo-used among respondent daily, where the respondent will be a dropout if they did not take standard tuberculosis antibiotic treatment and Channa striata extract or placebo regularly. In addition, the field research assistant also asked the respondent about the effect or complaint after taking Channa striata, in which respondent who got negative effect after administered with Channa striata will be referred to hospital under specialist doctor's control.The 500 mg of extract or supplement of Channa striata supplement. If there is an acid fast bacilli (AFB) in the smear, the smear is positive Mycobacterium tuberculosis.The process of collection, handling, and microscopic examination of sputum was conducted by trained staffs, where the collection and handling process was performed by nurse in primary health care and the microscopic examination was conducted by health staff in Jember Chest Hospital. The sputum of respondent was collected every week (week 0-4) in a month or Day-0 (week-0), Day-7 (week-1), Day-14 (week-2), Day-21 (week-3), and Day-28 (Week-4), in which respondent should check the sputum in health primary care in Jember and Situbondo. The sputum was collected and putted on safety container (cylinder-form container) and directly delivers to Jember Chest Hospital. Therefore, this procedure can prevent the sputum damaged. The steps of microscopic examination of respondent's sputum consist of sputum culture, sputum-coloured culture, and finally the microscopic examination of sputum's smear. The process of sputum-coloured culture was conducted based on ziehl-neelsen technique, and the authors used International Union against Tuberculosis and Lung Disease (IUATLD) guideline to determine the availability of acid fast bacilli on the smear with 1,000x magnitude of microscopic examination. In addition, the authors used the number of acid fast bacilli in smear as indicator of acceleration of tuberculosis treatment after administered by Mycobacterium tuberculosis availability in this study consists of negative, positive 1, positive 2, positive 3, and positive >3. The value of positive Mycobacterium tuberculosis in sputum was based on 3 stage of sputum examination, namely, sputum examination during a visit, in morning, and sputum collection. If the AFB was found within 3 stages, it is classified as positive 3. Meanwhile, positives 2 and 1 mean AFB was found in 2 stages and 1 stage of sputum examination, respectively. In addition, the sputum was classified as negative if the AFB was not found in these 3 stages.The indicator of Channa striata supplementation toward the acceleration of tuberculosis treatment. The significance level of the study is 5% (α= 0.05), and the confidence level is 95%.The study used Chi-square to analyze the data in SPSS version 22. Chi-square was used to determine the effect ofBased on α; α=0.05). H0 is accepted, which means that there are no differences between intervention and control group related to sputum test result among tuberculosis patient at week 0.Based on statistical test, the P value is 0.401 . Therefore, H0 is rejected, which means that there are differences between Channa striata supplementation and the acceleration of tuberculosis treatment among patients at week 1.Based on statistical test, the P value is 0.045 . Therefore, H0 is accepted, which means that there are no differences between Channa striata supplementation and the acceleration of tuberculosis treatment among patients at week 2.Based on statistical test, the P value is 0.086 . Therefore, H0 is rejected, which means that there are differences between Channa striata supplementation and the acceleration of tuberculosis treatment among patients at week 3.Based on statistical test, the P value is 0.019 . Therefore, Ho is rejected, which means that there are differences between Channa striata supplementation and the acceleration of tuberculosis treatment among patients at week 4.Based on statistical test, the P value is 0.005  is communicable disease caused by bacteria callede, brain , nerve, e, brain . M. tubedirectly . Tubercudirectly . Based odirectly . Tubercudirectly  that hasdirectly . The majdirectly .The sputum of tuberculosis respondent is collected periodically by primary health officer of Jember and Situbondo on week 0, week 1, week 2, week 3, and week 4, in which the sputum is examined in Jember Chest Hospital. Jember Chest Hospital (Rumah Sakit Paru Jember) is one of chest-concerned hospitals in East Java Province that the work area of the hospital is Eastern Pasuruan, Probolinggo, Lumajang, Jember, Situbondo, Bondowoso, and Banyuwangi—Indonesia. In addition, the sputum is examined to determine the level of the tuberculosis. Channa striata supplementation is shown by α; α=0.05). It shows that there are no differences between intervention and control group related to sputum test result of tuberculosis patient at week 0.The distribution of tuberculosis level of intervention and control group without Channa striata supplementation of intervention and control group is shown by Tables 3The distribution of tuberculosis level subsequent to Channa striata supplementation in this study is significantly related to the acceleration of tuberculosis recovery. It is caused by the elevating distribution of respondent classified in negative category of tuberculosis in intervention group on week 1, week 2, week 3, and week 4 with 35.9%, 56.3%, 70.9%, and 90.3%, respectively. Furthermore, based on statistical test, the P value of Channa striata supplementation toward the acceleration of tuberculosis treatment week 1, week 3, and week 4 is 0.045, 0.019, and 0.005 , respectively. Therefore, there are differences between Channa striata supplementation and the acceleration of tuberculosis treatment among respondent.Generally,Channa striata is cheap resource in Thailand [ Channa striata is vulnerable to aquatic Mycobacterium [ Mycobacterium tuberculosis is not founded in Channa striata [ Channa striata through chloroform solvent contains several amino acids  and fatty acid  [ Channa striata extract is glycine  and alanine . Besides, the major fatty acid found in Channa striata extract is palmotoleic acid , oleate acid , stearate acid , and linoleate acid  [ Channa striata on male mice (25-30 g) possessed a concentration-dependent antinociceptive activity [Thailand , which iThailand . Channa acterium , 22. How striata . The extic acid) . The majd total) . Based oactivity . Channa striata was administered orally to osteoarthritis-induced rabbits (OA), in which the result is that there was a significant improvement in the density of Protein Gene Product (PGP) of 9,5-immunoreactive nerve fibers in the synovial membrane of treated animals [ Channa striata possesses antifungal activities in restricted spectrum [ Klebsiella pneumoniae, Pseudomonas aeruginosa, and Bacillus subtilis [ Channa striata extract can heal the wound in Sprague-Dawley rats (250-300 g) [ Channa striata is also formulated in spray form to heal the wound [ Channa striata without bone is unable to decrease the blood sugar and HDL (High Density Lipoprotein) level toward Sprague-Dawley rat and mice.The extract of animals . Furtherspectrum ; howeversubtilis . The cre0-300 g) . Besideshe wound , 30. Thehe wound  noted th Channa striata among multidrug-resistant (MDR) tuberculosis patients. Therefore, future research is needed to find the effect of Channa striata supplementation with standard antibiotic for MDR patients related to accelerating the MDR TB recovery.The limitation of study is that the authors cannot provide the information about the effect of test, the P value of Channa striata supplementation toward the acceleration of tuberculosis treatment week 1, week 3, and week 4 is 0.045, 0.019, and 0.005 , respectively. Therefore, there are differences between Channa striata supplementation and the acceleration of tuberculosis treatment among respondent, in which Channa striata treatment in this study is significantly related to the acceleration of tuberculosis recovery.Based on the result of the study, the proportion of respondent classified in negative category in intervention group within week 0, week 1, week 2, week 3, and week 4 is 10.7%, 35.9%, 56.3%, 70.9%, and 90.3%, respectively. Besides, the proportion of respondent classified in negative category in control group within week 0, week 1, week 2, week 3, and week 4 is 13.4%, 23.7%, 37.1%, 49.5%, and 68%, respectively. Furthermore, based on Chi-square


PeAQPs in bamboo have been reported, the aquaporin-led mechanism of maintaining diurnal water balance in bamboo shoots remains unclear. In this study, a total of 63 PeAQPs were identified, based on the updated genome of moso bamboo (Phyllostachys edulis), including 22 PePIPs, 20 PeTIPs, 17 PeNIPs, and 4 PeSIPs. All of the PeAQPs were differently expressed in 26 different tissues of moso bamboo, based on RNA sequencing (RNA-seq) data. The root pressure in shoots showed circadian rhythm changes, with positive values at night and negative values in the daytime. The quantitative real-time PCR (qRT-PCR) result showed that 25 PeAQPs were detected in the base part of the shoots, and most of them demonstrated diurnal rhythm changes. The expression levels of some PeAQPs were significantly correlated with the root pressure. Of the 86 sugar transport genes, 33 had positive co-expression relationships with 27 PeAQPs. Two root pressure-correlated PeAQPs, PeTIP4;1 and PeTIP4;2, were confirmed to be highly expressed in the parenchyma and epidermal cells of bamboo culm, and in the epidermis, pith, and primary xylem of bamboo roots by in situ hybridization. The authors’ findings provide new insights and a possible aquaporin-led mechanism for bamboo fast growth.Water supply is essential for maintaining normal physiological function during the rapid growth of bamboo. Aquaporins (AQPs) play crucial roles in water transport for plant growth and development. Although 26  Phyllostachys edulis) can grow up to more than 1 m in 24 h and reach more than 20 m over one-and-a-half months in spring [Bamboo plays a very important role in reducing soil erosion and enabling land restoration and water conservation . Fast grn spring ,4. Plantn spring . A stronn spring . Bamboo n spring . The supn spring . Moreoven spring ,9.Unlike trees, bamboos have a hollowed culm and they lack secondary growth, suggesting that the vascular conduits in bamboo xylem have to remain functional throughout their life . In othePhyllostachys edulis [Arabidopsis thaliana [PeAQP family have been performed, actual functional identification was rare. Until now, only one aquaporin gene, PeTIP4;1-1, has been cloned and functionally analyzed [PeTIP4;1-1 in A. thaliana showed enhanced tolerance under drought and salinity stresses. The functions of the other bamboo AQPs remain unknown.As water channel proteins, aquaporins (AQPs) can maintain water and hydraulic conductivity balance in plants . AQP acts edulis  and Arabthaliana . With ththaliana , a totalthaliana . Those ethaliana . Althouganalyzed . Its expAQPs (PeAQPs) from 26 to 63, based on the updated annotation information from the Genome Atlas of Bamboo and Rattan (GABR) Project, using both Illumina  and PacBio  sequencing platforms [PeAQPs were further analyzed, and their expression profilings in the rhizome bud, as well as the top, middle, and base parts of shoots with heights of 0.2, 1.5, 3, and 6.7 m, were generated. In addition, the diurnal variation of root pressure in ~6 m shoots was measured. A correlation between the root pressure and the expression levels of PeAQPs was analyzed. Furthermore, a co-expression network of PeAQPs and sugar transport genes was constructed. The expression of two PeAQPs that were positively and negatively correlated with root pressure was further analyzed by in situ hybridization. Finally, the relationship of the PeAQPs and root pressure with bamboo’s rapid growth were proposed and discussed.In this study, the authors expanded the members of moso bamboo latforms . The phyhttp://gigadb.org/dataset/100498, 1 October 2018) [Based on the updated data of moso bamboo (er 2018) ,22,23,24http://gsds.cbi.pku.edu.cn/). The subcellular localization of the PeAQPs was predicted with the online tool Plant-mPLoc  (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/). Transmembrane (TM) helices were predicted using the online tool TMpred  .The distribution of the introns and exons were analyzed, following the reference method . The resSugar transport genes were re-identified using the keywords of sugar transport, sucrose transporter, SWEET, monosaccharide transporter, hexose transporter, polyol transporter, myo-inositol transporter, plastidic glucose transporter, monosaccharide-sensing protein, sugar facilitator, and so on. The sequences of each sugar transport gene are listed in P. edulis, Zea mays, and Oryza sativa was constructed in MEGA6.0  using the following parameters: the neighbor-joining tree method, partial deletion, and bootstrap with 1000 replicates. The sequences of ZmAQPs and OsAQPs were downloaded from the MaizeGDB  [http://www.ricedata.cn/gene/) [PtAQPs in Populus trichocarpa were downloaded to investigate the presence of XIP aquaporins in bamboo [To investigate the phylogenetic relationship of aquaporins, a clustering analysis of AQPs in NY, USA)  and Ricen/gene/) , based on/gene/) ,28. In an bamboo .PeAQP genes in the different tissues and developmental stages of shoots, transcriptome data generated from 26 tissues of rhizome, roots, shoots, leaves, sheaths, and buds during different development stages were downloaded from the NCBI Short Read Archive (SRA) [2 and loaded into Matrix2png  (http://www.chibi.ubc.ca/matrix2png/) to generate a heat map.To investigate the expression of ve (SRA)  and usedRoot pressure dynamics were monitored at the base of six different moso bamboo shoots at ~6 m height from 7:00 p.m. on 20 April 2017 to 7:00 p.m. on 22 April 2017. The measurement experiments were undertaken in a field site belonging to the Jiangxi Academy of Forestry , Nanchang, Jiangxi Province, which is a research partner of the authors’ institute. During the experiment, the selected bamboo shoots were crosscut at the base part, 10 cm above the ground. The shoot cross-section was polished with a fresh razor blade, then covered with a plastic tube made by a Le buckle cup filled with distilled water. The cup was connected to a digital pressure transducer  linked to a data logger ; the pressure data were collected every 10 s according to the method described in previous studies ,11.PeAQPs and the root pressure in the base part of the bamboo shoots was constructed by calculating pairwise Pearson correlation coefficients (PCC) based on the transcriptome data of PeAQPs across the 26 moso bamboo samples, which were then loaded into Java TreeView 1.1.6r4  [PeAQPs and sugar transport genes were constructed using Cytoscape 3.5.1 , based on the PCCs of the PeAQPs and sugar transport genes in the 26 moso bamboo samples [The correlation between the gene expression level of the gow, UK)  to gener samples . The thrPeAQPs, the internodes at the base parts of the bamboo shoots were collected from the shoots that were used for root pressure measurements and quickly frozen into liquid nitrogen for RNA isolation. Total RNA isolation and qRT-PCR were carried out using previously described methods [PeAQPs were designed, and they are listed in TIP41  and NTB  were used to analyze the expression levels of the PeAQPs. Both NTB and TIP41, which were found to be homogeneously expressed and which were adequate for normalization purposes, were used for gene expression analysis in moso bamboo [For the diurnal variation expression analysis of the  methods . Primerso bamboo ,19,31. Ao bamboo .PeTIP4;1 and PeTIP4;2) showing positive and negative correlations with the root pressure, respectively, were selected for tissue localization of expression analysis in moso bamboo culm and root cells. The gene-specific probe of PeTIP4;1 was 5′-AGCCGCCCCCGGCAACTCCGGCGACCCG-3′, and that of PeTIP4;2 was 5′-GGCCCATGATCGTATCGGTGCCGCCG-3′, which were complementary to the corresponding messenger RNAs (mRNAs) of PeTIP4;1 and PeTIP4;2. The specific oligonucleotide probes were labeled with fluorescein isothiocyanate (FITC). A blue-fluorescent DNA stain of 4′,6-diamidino-2-phenylindole (DAPI) was used to stain double-stranded DNA (dsDNA) as a control. The images were captured with an IX71 fluorescence inverted microscope . To investigate aquaporin gene expression in moso bamboo, the two-year-old seedlings were used for in situ hybridization. Moso bamboo culm and root tissue preparation and in situ hybridization were conducted according to the method described in . Two canAQP genes in bamboo [PePIPs, 20 PeTIPs, 17 PeNIPs, and 4 PeSIPs. Detailed information, including BambooGDB  [http://gigadb.org/dataset/100498, 1 October 2018) [PeAQPs is shown in With the help of the updated genome and the transcriptomic data of moso bamboo , the autn bamboo . Of thesch 2014)  and GABRPeAQPs become bigger after the dramatically improved bamboo genome assembly, but the number of subgroups was also increased. For example, five subgroups of PeTIPs are found in the updated bamboo genome database, of which PeTIP3 and PeTIP5 subgroups were absent in the previous study [Not only did the total members of the us study . HoweverP. edulis, Z. mays, and O. sativa) and one dicot (Populus trichocarpa), was constructed. The results showed that they were clustered into five clades representing five distinct AQPs subfamilies. The XIPs in P. trichocarpa were clustered into a single clade. This further indicated that XIPs do not exist in moso bamboo or in other monocots. Consequently, the PeAQPs were distributed in four subfamilies, including 22 PePIPs, 20 PeTIPs, 17 PeNIPs, and 4 PeSIPs , five TIPs  and one NIP , showed an increasing trend with the continuous elongation of roots. Among these, the expression of PePIP2;1, PePIP1;6, and PePIP2;6 in 10 cm roots was significantly up-regulated to 17.7, 13.7, and 12.9 times, respectively, compared to that in 0.1 cm roots. Additionally, no or weak transcript accumulation (fragments per kilobase of exon per million fragments mapped (FPKM) value <1) of PePIP2;3-1, PePIP2;4, PePIP2;10, PeTIP2;1, PeTIP2;2, PeTIP2;4, and PeNIP2;3 was found in 0.1 cm roots, whereas they were all up-regulated significantly with root elongation. Accordingly, the results indicated that these genes might be involved in water absorption in roots.The recent transcriptome data generated from 26 different tissue samples collected from six main moso bamboo-producing areas in China were used to investigate the expression patterns of PeAQPs . Since mPIPs , eight TIPs , and one NIP  had an increasing expression trend with the growth of bamboo shoots. In the middle part of the shoots, most of genes first showed a rise, followed by a decline, including six PIPs , seven TIPs , and two NIPs . In the base part of different heights of shoots, six PIPs , four TIPs , and two SIPs  were highly expressed in 0.2 m shoots, whereas most of them demonstrated a decreasing expression trend with an increase of shoot height. Moreover, some genes were found to have the characteristics of tissue-specific expression. For example, PeNIP2;4 showed a higher expression level in leaf sheaths; PePIP2;7 was exclusively expressed in leaves. These tissue-specific PeAQPs might have specific functions in those tissues.Water is essential to sustain bamboo shoot growth in the boosting stage. In the top part of the shoots, five The measurement of root pressure was conducted; the pressure displayed diurnal rhythm changes, which were negative in the daytime and positive at night. The root pressure of bamboo shoots presented a continued upward trend during the period of 7:00 p.m. to 3:30 a.m. and remained positive until 7:30 a.m. The pressure decreased sharply to negative at 7:45 a.m., which was two hours after sunrise. Those values remained negative, which showed a decreased trend at first, and then they gradually recovered to zero and finally rose to positive values at 7:00 p.m. The maximum value of the root pressure was approximately 69.9 kPa at 3:00 a.m., which could pump water up to 7 m in height . As is kPeAQPs, with FPKM values in any part of the shoot that were greater than 10, were considered as taking part in the regulation of root pressure, and these were selected for further analyses. The combined analyses of the gene expression and root pressure changes in bamboo shoots with diurnal rhythm were conducted. The results indicated that all of the selected genes were detected in the bamboo base part of the shoots, except PePIP1;3, and most of them showed diurnal rhythm changes , such as sugars and starch, provided a temporary source of carbon for the bamboo shoots, since their photosynthesis could not meet the immediate carbon demands. Almost all of the NSCs were transferred to new “explosively growing” bamboo shoots from the mature bamboos via underground rhizomes, for the structural growth and metabolism of shoots . TherefoThe results indicated that a total of 86 sugar transport genes were identified in the updated genome of moso bamboo, including 7 putative monosaccharide sensing protein (AZT/MSSP) genes, 4 myo-inositol transporter (ITR/MIT) genes, 5 hexose transporter (STP/HXT) genes, 5 plastidic glucose transporter (pGlcT) genes, 19 polyol transporter (PLT) genes, 2 sugar facilitator (SFP) genes, 8 sucrose transporter (SUC/SUT) genes, and 36 sugars which will eventually be exported transporter (SWEET) genes . ConversPeAQPs and sugar transport genes for bamboo shoot development. The 86 sugar transport genes and 63 PeAQPs genes were used to build a co-expression network for inferring their function in bamboo shoot development. A total of 33 sugar transport genes and 27 PeAQPs were found to have positive features of co-expression  and Panicum virgatum (68), and one dicot, Populus trichocarpa (58). It has been validated that the genome of moso bamboo is 2.05 Gbp, and that it consists of 24 pairs of chromosomes (2n = 48) and that it is characteristic of a diploid. It was the whole-genome duplication event of moso bamboo during evolution that led to the generation of more gene duplicates in gene families [PeAQPs were still classified into four subfamilies. Although the number of gene members was significantly increased, members of the XIP subfamily were still not found in the updated moso bamboo genome, which further confirmed that XIPs are not present in moso bamboo and other monocots. Additionally, members of TIP3 and TIP5 were found in the genome, which were absent in a previous study [PeAQPs showed lower-expressed levels, indicating that a number of lower expression genes were found in the dramatically improved genome assembly.In this study, the authors expanded the members of irgatum 6, and onePeAQPs, the authors found that some genes have very long intron regions, such as in PeTIP4;3 and PeNIP3:3-1, whereas the corresponding genes in the same subfamily, such as PeTIP4;3-1 and PeNIP3:3, have small intron regions. From the transcription expression profile, they found that PeTIP4;3 has a higher expression level in roots, and a lower expression level in the top part of the shoots, compared to that of PeTIP4;3-1. PeNIP3:3 has a higher expression level in almost all tissues, compared to that of PeNIP3:3-1. The two homologous genes with different intron lengths in the same subfamily have different expression patterns, which might be explained by the introns regulating the gene transcripts in different ways. Although the intron regions are spliced out during transcription, introns have increasingly been shown to serve important biological functions. It has been confirmed that introns can improve the expression of exogenous genes in transgenic plants [PeAQPs in bamboo still needs further study.By comparing the gene structures of the c plants ,41. The PeAQPs in different tissues, as well as those at different developmental stages, may be a cue to reflect their physiological functions. Almost all of the PeAQPs were detected in all samples, which indicated that they might be essential genes in different tissues, and in different development stages. For example, with the elongation of bamboo roots, nine PIPs , five TIPs , and one NIP  showed increasing expression trends, which indicated they might have main functions in the transportation and absorption of water in the process of root maturation. In addition, with the development of bamboo shoots, the expression of five PIPs , eight TIPs , and one NIP  was increased in different parts of shoots. All of those genes might be involved in water transportation and absorption in bamboo shoots. Most of the water supply in bamboo needs to be absorbed by roots from the soil, and then transported upwards, for the growth of bamboo shoots. The expression levels of PeAQPs exhibited significant positive correlations with the root pressure, and some PeAQPs presented significant negative correlations with root pressure, indicating that these genes might play crucial roles in the regulation of water supply for cell division and elongation during the fast growth of bamboo shoots.The previous study of bamboo showed that a decline in nocturnal xylem pressure, as measured from the base to the apex of individual culms, was very close to the gradient that was expected by hydrostatic pressure, which confirmed that the origin of the nocturnal xylem pressure was at the root . In addiPeAQPs was very important for water transport during the bamboo fast-growth stage.It has been reported that the maximum size in bamboo species is determined below ground by the maximum pressure that can be generated in roots overnight, and that water transport in bamboo is dependent on root pressure to repair the hydraulic dysfunction that is sustained during normal diurnal gas exchange . Sugars PeAQPs were co-expressed positively with sugar transport genes, such as PeTIP4;1-2 and PeTIP4;2-1 co-expressed with SUC/SUT genes, PeNIP1;1 co-expressed with SWEET genes, and PePIP1;6 and PePIP2;7 co-expressed with both SUC/SUT and SWEET genes in moso bamboo shoots, indicating that aquaporins and sugar transporters might work together for the regulation of water and carbon source supply during the period of shoot rapid growth, through gene co-expression.New bamboo shoots have little photosynthetic capability, and most of the required carbon source for fast growth must be transferred to the shoots by the sugar transporter from the mother plant . SucrosePeTIP4;1 and PeTIP4;2, were selected to investigate their expression in the culms of moso bamboo seedlings by in situ hybridization. The results showed that they were all mainly expressed in the parenchyma cells of the culm, which agrees with evidence that parenchyma cells serve as a large storage site for water (∼52% of the bamboo culm) [PeTIP4;1 and PeTIP4;2 were highly expressed in the epidermis, pith, and primary xylem of the root. PeTIP4;1-1 could increase the length of taproots in transgenic A. thaliana under osmotic stresses, and it could enhance the tolerance of transgenic plants to osmotic stresses [PeTIP4;1-1 has the ability to regulate the water osmotic balance in roots. Taking this into consideration, the authors find that PeTIP4;1 and PeTIP4;2 might not only participate in the formation of root pressure, but they may also play important roles in water absorption and transport in the root and culms. Therefore, they believe that PeTIP4;1 and PeTIP4;2 must be the key genes for the formation of root pressure, water absorption, and transportation in bamboo. However, the regulation of bamboo fast growth is a complex network, and many studies need to be carried out for a further understanding of the functions of PeAQPs.Although woody bamboo had excellent mechanical strength, the bamboo lacked secondary growth and had a hollowed culm. Bamboo root pressure was used to recharge the culm water storage, specifically the culm parenchyma cells surrounding all vascular bundles . Two roooo culm) . The expstresses . This inPeAQPs exhibited significant correlations with the root pressure, and many sugar transport genes were positively co-expressed with multiple types of PeAQPs. These findings indicate that aquaporins and sugar transporters might work together for the regulation of water supply through gene co-expression in moso bamboo, which increases the understanding of the aquaporin-led mechanism for bamboo fast growth.Water and energy supply are essential for cell division and elongation, resulting in the rapid growth of woody bamboo, which will affect the yield and quality of bamboo. AQPs have the function of regulating the rapid flow of water in cells, whereas sugar transport genes are responsible for the transport of soluble sugars, providing both energy and a low water potential for cells during the explosive growth of new bamboo shoots. The expression levels of many


Tamarindusindica L.) (TTI) seeds in satiety and obesity. In the present study, TTI nanoencapsulation (ECW) was performed to potentialize the effect of TTI and allow a controlled release in the stomach. The impact on glycemia, insulin, and lipid profile was evaluated in Wistar rats overfed with a high glycemic index diet (HGLI). Characterization of the nanoparticles and in vitro stability in simulated gastrointestinal conditions, monitored by antitrypsin activity and HPLC, was performed. ECW and empty nanoparticles (CW) were administered by gavage, using 12.5 and 10.0 mg/kg, respectively. Both nanoformulations presented a spherical shape and smooth surface, with an average diameter of 117.4 nm (24.1) for ECW and 123.9 nm (11.3) for CW. ECW maintained the antitrypsin activity (95.5%) in the gastric phase, while TTI was completely hydrolyzed. In Wistar rats, the nanoformulations significantly reduced glycemia and HOMA IR, and ECW increased HDL-c compared to CW (p < 0.05).Pancreas histopathology of animals treated with ECW suggested an onset of tissue repair. Thenanoencapsulation provided TTI protection, gradual release in the desired condition, and improvement of biochemical parameters related to carbohydrate metabolism disorders,without compromising insulinemia.In vivo studies show the benefits of the trypsin inhibitor isolated from tamarind ( Diets with a high glycemic index, with low intake of fruits and vegetables, and the high consumption of fat, directly influence the nutritional status leading to the development of obesity and related complications, such as diabetes mellitus (DM), further intensifying the metabolic risks ,2,3,4. Studies with molecules that have bioactive properties to combat metabolic disorders related to inadequate diets, such as overweight or obesity and associated complications, are reported. Regarding diabetes mellitus type 2 (DM2), the pharmacological resources are used when it is not possible to control the blood glucose with diets and physical exercises. Medications available for diabetes therapy include oral hypoglycemic agents and insulin . Metabolic disordersdue to high glycemic levels may also be associated with the presence of other factors, such as insulin resistance (IR) and, in this case, anti-hyperglycemic drugs are more appropriate. These drugs improve the performance of endogenous insulin, with better metabolic control .Whey protein is a rich source of bioactive peptides that may play a hypoglycemic rolein the treatment of chronic diseases. These diseases arouse interest in peptides that can cross the gastrointestinal barrier with bioactive properties, modulating nervous, immune, cardiovascular, and digestive functions ,7. Chitosan is a cationic biopolymer obtained from the deacetylation of chitin, present mainly in shells of crustaceans and exoskeleton of insects , with seTamarindusindica L.) (TTI) was studied by Carvalho et al.  using probity regression, with the TTI concentrations transformed to log 10 base and the response frequency as the percent inhibition. Therefore, the amount of ECW used was proportional to its composition of 1:4, and the amounts  were used to the determination of IC50. The evaluation was also performed in the CW and encapsulating agents (purified chitosan and isolated whey protein).Thus, after obtaining these data, the efficiency of incorporation was determined, according to Queiroz et al. , based o2(H2O)2 added on the day of use, to avoid precipitation. Triplicates were performed for each condition. The assay was accomplished by using TTI (30 mg/mL), ECW (150 mg/mL), CW (120 mg/mL), and distilled water as a control.The formulations prepared were tested under simulated oral, gastric, and intestinal conditions, in order to evaluate their stability to digestive conditions. Simulate fluids that mimic the physiological environment of the mouth (SSF), stomach (SGF), and intestine (SIF) were prepared . The stoECW and CW were used as positive controls, without subjection to the physiological conditions of the gastrointestinal tract. The following concentrations were used in the evaluated phases: ECW in the oral phase—75 mg/mL, in the gastric phase—50 mg/mL and, in the intestinal phase—20 mg/mL; CW in the oral phase—60 mg/mL, in the gastric phase—40 mg/mL, and in the intestinal phase—16 mg/mL. This standardization was performed with consideration to the concentrations of ECW and CW submitted to physiological conditions.2(H2O)2, and 975 mL of Milli-Q water and incubated in a thermomixer , at 500 rpm (37 °C), for 10 min.Five milliliters of sample was added to 4.0 mL of SSF, 25 μL CaCl2(H2O)2, 200 μLof HCl 1 mM, and 0.695 mL of Milli-Q water. The pH was adjusted to 3.0 (using HCl 1 mM) and left in constant agitation in the thermomixer, at 500 rpm (37 °C), for 2 h.The gastric phase was prepared by adding to the previous mixture 7.5 mL of lecithin (10 mg/mL in SGF), 1.6 mL of pepsin (10 mg/mL in SGF), 5 μL of CaCl2(H2O)2, 150 μLNaOH, and 1.31 mL of Milli-Q water. The pH was adjusted to 7.0 (using NaOH 1 mM) and left in constant agitation in the thermomixer, at 500 rpm (37 °C), for 2 h, and then the samples were centrifuged at 14,000× g.The intestinal phase was prepared by adding to the previous mixture 11 mL of simulated intestinal fluid (SIF), 5.0 mL of pancreatin (70 mg/mL in SIF), 2.5 mL of bile salts (40 mg/mL in SIF), 40 μL of CaClAfter each phase, 2 mL of sample was withdrawn for follow-up analyses. All aliquots were evaluated for antitrypsin activity . ChromatWistar rats were obtained from Potiguar University (UnP), kept at room temperature (25 °C), and exposed to light for 12 h. During the course of 17 weeks, the animals were overfed with an experimental diet of the high glycemic index (77.6) and glycemic load (38.8) (HGLI diet), according to Carvalho et al. [Male o et al. .n = 5), and they were acclimated and adapted to gavage for five days. During this period, the animals were fed as described in ®), nutritionally adequate, was also used for one of the groups of animals previously overfed for 17 weeks with an HGLI diet. The experimental diet HGLI [After that, the rats were divided into four groups  was used. Insulin dosage was performed by using the Rat/Mouse Insulin Elisa kit (Millipore EZRMI-13K).Their blood was used to determine fasting glucose, insulin, triglycerides (TG), total cholesterol (TC), VLDL-c, HDL-c, and LDL-c. Automated colorimetric–enzymatic  was used based on the formulas (1) proposed by Matthews et al. [β = (20 × Insulin μIU/mL)/(Glucose mg/dL − 3.5). Wistar ratswith normal nutritional status (320–380 g), acclimated under the same conditions of the experiment, consuming Labina® diet. All values used were in agreement with Guimarães et al. [To measure insulin resistance (IR) and pancreatic s et al. : HOMA-IRs et al. .The analysis was performed according to Martins et al. , with son = 4.36 animals, that is, five animals per group. Data were analyzed for normality, using the Kolmogorov–Smirnov test. The Kruskal–Wallis test was necessary to verify if total cholesterol differed between the groups, using Dunn’s posthoc test to detect significant differences (p < 0.05). The other study variables were considered parametric and were evaluated by ANOVA and Tukey’s posthoc test, in order to determine significant differences between groups (p < 0.05).The sample size was calculated, considering a simple and random sampling (Cochran model) and according to the 3Rs principle. A physiologically significant difference of the parameters evaluated was assumed when the treatment exerted a biological effect of 25% or more in relation to the group that did not receive the treatment. Furthermore, the coefficient of variation was adopted, with a probability of error <5% and power of 90%, resulting in TTI isolated by affinity chromatography in trypsin–sepharose A was eva−1, and 1400 cm−1 (folding of the hydroxyl groups –OH) are displaced in the ECW (e) (1446 cm−1 and 1401 cm−1), and with the attenuated stretches, an indication that the encapsulating agents are protecting the active agent. Furthermore, the presence of new vibrational bands formed in the region of 1071 cm−1  for ECW, while the other materials analyzed  did not present antitrypsin activity.w/w/w)  . ECW preThe aliquots of TTI A, ECW, aAfter the oral phase, TTI and ECW maintained their antitrypsin activity, showing resistance to hydrolysis by oral phase enzymes. However, after the gastric phase, only ECW maintained the inhibitory activity, demonstrating the susceptibility of TTI to digestion conditions and, consequently, to gastric enzymes. The antitrypsin activity of the intestinal phase was evaluated in controls of TTI and ECW, in the same concentrations used in the digestion phases. These controls were not submitted to digestion. The antitrypsin activity of the intestinal phase for TTI and ECW was not evaluated, due to the presence of trypsin, which is the target TTI’s inhibitory activity.The chromatographic profile of TTI in the oral phase shows a protein peak at 10.3 min, corresponding to TTI. However, this peak disappears in the gastric and intestinal phases A and, inHDL-c increased in the animals overfed with the HGLI diet when compared to the CW group C. There Wistar rats overfed with the HGLI diet when compared to the untreated animals (p < 0.05). Lower concentrations of TTI in ECW were able to significantly reduce fasting glucose. CW also significantly reduced insulin, HOMA IR, and HOMA-β (p < 0.05).Wistar rats of all groups presented chronic pancreatitis. In the group of rats that received no treatment, consuming only the HGLI diet, the main changes observed were related to hyperemia and acinar destruction, as well as the presentation of inflammatory, infiltrate in the pancreatic islet . These characteristics associated with the observed attenuated stretches of TTI also indicate the entrapment of TTI in ECW. CW was characterized to ensure that a nanoformulation with similar physical and chemical characteristics to the ECW would be administered in rats, for comparison between the results.The behavior or susceptibility of TTI, ECW, and CW under digestive conditions  was performed in the in vitro stability assessment in conditions simulating the gastrointestinal tract. Three phases were considered: oral, gastric, and intestinal. TTI, ECW, and CW were monitored at each stage of the digestive process, according to Pagels and Prud’homme  and EFSAIn view of the ECW susceptibility in acidic pH and consequent gradual release of TTI observed by Costa , and conDue to the interaction of TTI with the encapsulating agents, visualization in HPLC would be unlikely, as observed B, once TSince chitosan is a polysaccharide, the inability to visualize the protein peaks in HPLC corresponding to TTI in the intestinal phase reinforces the hypothesis that TTI present in ECW is strongly interacting with chitosan, as observed by Costa .The results indicate that, in the intestinal phase, there was a mixture of released TTI, and TTI interacted with chitosan particles. This mixture was conditioned by acid pH, which promoted the digestion of the isolated whey protein in the stomach phase B. The saRegarding the in vivo analyses, the increase in total cholesterol in the group treated with ECW A is justWistar rats with metabolic syndrome; however, no statistical difference was observed between the groups regarding HDL-c. In the present study, an increase in HDL-c was observed compared to the untreated group, although not significant (p > 0.05). On the other hand, the ECW-treated group significantly increased the HDL-c concentration compared to the group receiving nanoparticles without TTI (CW). This fact, associated with the nonsignificant increase in triglycerides (p > 0.05) promoted by CW, shows the beneficial effects supported by encapsulated TTI (ECW).There was no statistical difference between the treated groups for LDL-c, triglycerides, and VLDL-c B,D,E. CaConsidering the glucose results, the standard diet is considered the conventional treatment, and ECW and CW were responsible for the reduction of fasting glucose, supposedly by different mechanisms. However, ECW presented improved TTI effect through encapsulation. This improvement in effect can be evidenced by comparing our results with the study conducted by Carvalho et al. , which fMomordica charantia (mcIRBP) [Costa et al.  suggeste(mcIRBP) . It acts(mcIRBP) ,35,36.Another hypothesis to explain this effect could be related to the probable probiotic action of TTI, or even its digestion peptides, when reaching the large intestine, which could indirectly influence the lowering of glucose in the blood,  and furtβ. Furthermore, whey protein isolate also exhibits, after digestion, high postprandial concentrations of specific amino acids that may contribute to glucose homeostasis [Regarding the reduction of fasting glucose concentrations in the CW group A, this ceostasis .β, and HOMA IR, when compared to the groups that received the HGLI diet.In contrast, ECW did not change insulin, HOMA-β greater than 100 indicates β-pancreatic-cell dysfunction [Once all groups of animals were overfed, for 17 weeks, a high glycemic index diet before treatments, glucose concentrations increased as a response to the HGLI diet, which stimulated the release of insulin. Consequently, insulin resistance (IR) induced by this diet reduced insulin sensitivity from effectively metabolizing glucose in the untreated groups. HOMA-IR ranging from 2.5 to 3.9 ,43,44 mafunction .Considering the effect of ECW on the parameters analyzed in this study, ECW may be a candidate for the treatment of metabolic disorders as an adjuvant therapy. There are no reports of these joint effects on fasting glucose and lipid profile components for trypsin inhibitors.In the present study, under physiological conditions simulating the gastrointestinal tract, not only TTI’s hydrolysis products from the gastric phase were in the intestinal phase, but also ECW. Thus, the bioactive effects observed in the study could be, in part, promoted by the interaction with chitosan. These results are innovative and unprecedented, considering the isolated bioactive effects of TTI and chitosan, for which previous research have used higher concentrations than those in this ,46,47,48Wistar rats, as well as their physiological mechanisms. To understand the results obtained regarding glycemia and insulinemia, it is necessary to investigate other aspects, such as the pancreatic morphology. The pancreas releases several hormones, including insulin (β-cells) and glucagon (α-cells), which are both released directly from the bloodstream through a vascular network and have a relevant role in the regulation of blood glucose concentrations [Thus, analyses that evaluate the direct cause–effect relationship of TTI or ECW and their bioactivities are necessary to understand the results regarding the improvement in biochemical parameters in overfed trations ,50, factβ-cells degeneration, and damage of the Langerhans islets. In the present study, all groups presented chronic pancreatitis due to the HGLI diet administered in all groups of animals.Alterations in the pancreas were reported in a study using the HGLI diet . MorpholWistar rats (Wistar rats treated with CW contribute to the reduction of insulin concentrations. Thus, the pancreatic morphology presented may be directly related to the low concentrations of insulin presented by the group of CW-treated rats (At the same time, ECW gave better results when compared to other treatments, considering that chronic pancreatitis was presented by all tar rats C. The CWtar rats D aggravated rats B.β-cells. Therefore, we emphasize the importance of the present study, which was the first preclinical study to evaluate the effect of chitosan-based nanoparticles with an average diameter of approximately 100 nm, which promoted harmful impact on the organ evaluated, compromising insulinemia. This might be related to the increased chitosan reactivity provided by the smaller particle size. The results suggested that this effect was minimized in ECW due to the presence of TTI, which, through chemical interactions with chitosan, reduced its reactivity.Seo et al.  evaluateThis study demonstrated that nanoencapsulation improved the effect of TTI onfasting-blood glucose levels. The encapsulation was accomplished by using a low TTI concentration, without worsening chronic pancreatitis presented nor compromising insulinemia by the animals studied.


CHRM1-5 genes. The mAChRs are G-protein-coupled receptors, which can be divided into the following two subfamilies: M2 and M4 receptors coupling to Gi/o; and M1, M3, and M5 receptors coupling to Gq/11. However, despite the fundamental roles played by these receptors, their evolution in vertebrates has not yet been fully described. We have combined sequence-based phylogenetic analyses with comparisons of exon–intron organization and conserved synteny in order to deduce the evolution of the mAChR receptors. Our analyses verify the existence of two ancestral genes prior to the two vertebrate tetraploidizations (1R and 2R). After these events, one gene had duplicated, resulting in CHRM2 and CHRM4; and the other had triplicated, forming the CHRM1, CHRM3, and CHRM5 subfamily. All five genes are still present in all vertebrate groups investigated except the CHRM1 gene, which has not been identified in some of the teleosts or in chicken or any other birds. Interestingly, the third tetraploidization (3R) that took place in the teleost predecessor resulted in duplicates of all five mAChR genes of which all 10 are present in zebrafish. One of the copies of the CHRM2 and CHRM3 genes and both CHRM4 copies have gained introns in teleosts. Not a single separate (nontetraploidization) duplicate has been identified in any vertebrate species. These results clarify the evolution of the vertebrate mAChR family and reveal a doubled repertoire in zebrafish, inviting studies of gene neofunctionalization and subfunctionalization.The family of muscarinic acetylcholine receptors (mAChRs) consists of five members in mammals, encoded by the  Despite their pivotal physiologic role, the evolution of the muscarinic acetylcholine receptors (mAChRs) has not yet been resolved. By investigating the genomes of a broad selection of vertebrate species and combining three different types of data, namely sequence-based phylogeny, conserved synteny, and intron organization, we have deduced the evolution of the mAChR genes in relation to the major vertebrate tetraploidizations . Our analyses show that all vertebrate mAChR gene duplications resulted from the tetraploidizations. Interestingly, following 3R, zebrafish doubled its gene number, resulting in the 10 mAChR genes present. By knowing how and when the mAChR genes arose, studies of receptor subtype specialization and possible neofunctionalization or subfunctionalization can follow.CHRM1-5 genes. The structures of the muscarinic receptors follow the typical GPCR structure with the extracellular N terminus followed by seven transmembrane (TM) domains (TM domains 1–7), which are separated by three intracellular loops , three extracellular loops , and finally the intracellular C terminus. The orthosteric binding site for acetylcholine consists of a hydrophobic pocket formed by the side chains of TM domains 3–7. The crystal structures of the M2 and M3 receptors have been reported  are G-protein-coupled receptors (GPCRs) involved in a variety of CNS processes such as cognition, learning, and memory. They are also present in the peripheral nervous system and smooth muscle tissue. The mAChR family consists of five different receptor subtypes named M1–M5, which are encoded by the reported , showingreported . In the nogaster . In the nogaster , 2014. TThe mAChRs are widely expressed in the nervous system, the cardiovascular system, and the gastrointestinal tract, as well as elsewhere. In the peripheral nervous system, the mAChRs play a major role in the parasympathetic system stimulating smooth muscle contraction and glandular secretion as well as slowing the heart rate . In the Although the muscarinic receptors have prominent roles in various nervous system functions, the evolution of the mAChR gene family has not yet been fully resolved. It is important to deduce evolutionary relationships to distinguish orthologs (species homologs), paralogs (gene duplicates), and ohnologs , especially when studying species that belong to evolutionarily distant groups, for instance the commonly used experimental animals mouse/rat, chicken, and zebrafish. Furthermore, the time points of the gene duplication events are important for studies of evolutionary change between orthologs and paralogs as well as ohnologs. It is now well established that the vertebrate predecessor underwent two rounds of whole-genome duplication  before the radiation of jawed vertebrates . These tCHRM1 has not been found. Furthermore, the teleost 3R event doubled the repertoire once more, resulting in the 10 genes present today in zebrafish, albeit some teleosts seem to lack both copies of CHRM1. This long-lived multiplicity invites further studies of the roles of each of the subtypes.As the availability of high-quality genome assemblies is continuously increasing, it is now possible to perform a more extensive analysis of the evolution of the mAChR family. We have used an approach that combines amino acid sequence-based phylogeny and analyses of chromosomal locations for comparison of synteny and duplicated chromosome regions. We report here that the 1R and 2R genome-doubling events duplicated the two ancestral mAChR genes to the five genes that are present today in all tetrapods investigated except birds, where Homo sapiens; Hsa), mouse , opossum , chicken , anole lizard , frog , coelacanth , spotted gar , Japanese eel , European eel , zebrafish , stickleback , medaka , tunicates , and nematode . The amino acid sequences from the species listed were retrieved from the Ensembl genome browser  or NCBI  databases. If sequences were not found in either of the databases, the sequence of a closely related species was used as a query sequence in a TBLASTN search . The Japanese and European eel genome assemblies are not annotated. Therefore, spotted gar mAChR gene sequences were used as templates to run a TBLASTN search and retrieve mAChR orthologs present in the Japanese eel; thereafter, Japanese eel sequences were used as templates in TBLASTN search for mAChR orthologs present in the European eel. Due to a high degree of sequence conservation between all mAChR genes in the Japanese eel and European eel, only the European eel sequences were included in sequence-based phylogenetic analysis as this species contained a more complete mAChR gene repertoire than the Japanese eel.Species sequences included in the analysis of the mAChR family were the human (SCR_006459). If the amino acid sequences were aligning poorly and the predictions appeared questionable, the genomic sequences were investigated and the sequences were manually edited, by comparing sequence homology and consensus donor and acceptor splice sites. Manual corrections were made where the alignment appeared shifted due to a low degree of conservation, such as the first part of the sequence or the IL3 loop region between TM5 and TM6. However, these adjustments were kept to a minimum. All sequence details are included in SCR_014629; SCR_008515), rooted with C. elegans.The retrieved amino acid sequences were aligned using Jalview 2.10.3b1, with Muscle default settings , rooted with C. elegans. The tree topology is supported by a nonparametric bootstrap analysis with 100 replicates. This tree is based on the complete multiple sequence alignment . In the sequence names, the species is followed by the chromosome or genomic scaffold at which the gene is located . If several genes are located on the same chromosome or genomic scaffold, their order is indicated by an additional number. Cin, Ciona intestinalis; Csa, Ciona savigny. Download Fig. 1-3, TIF file.PhyML tree of the mAChR genes . For the CHRM2/CHRM4 genes, lists of gene families were also retrieved in a similar manner in the chicken genome, due to the small number of neighboring gene families in the genomic regions in the spotted gar. Thereafter, families containing two members or more were phylogenetically analyzed by retrieving the amino acid sequences for human, chicken, coelacanth, spotted gar, and zebrafish. The region surrounding the CHRM1, CHRM3, and CHRM5 genes contained a higher number of families; therefore, families with at least three gene family members in the spotted gar were analyzed. As outgroups tunicates, amphioxus, drosophila, C. elegans and in some cases other human gene sequences were included. aLRT SH-like trees were constructed using the PhyML 3.0 web server , and the proposed gene names were submitted to ZFIN. The details of the gene family sequences included in the analysis are provided in CHRM2 and CHRM4 paralogon and in CHRM1, CHRM3, and CHRM5 paralogon and the aLRT SH-like trees are included in For synteny and paralogon analyses, the neighboring regions of the mAChR genes were investigated in human, chicken, and spotted gar. Lists of gene families in the genomic regions 10 Mb upstream and 10 Mb downstream of the CHRM2b, CHRM3b, CHRM4a, and CHRM4b teleost genes, additional teleost species included in the analysis were the Amazon molly  and fugu . Sequences were analyzed and aligned as described above. Intron positions were determined by manual investigation of amino acid and nucleotide sequences. Transmembrane domain regions were predicted by consulting the TMHMM Server version 2.0 . For comparative analyses and confirmation of intron positions, the Japanese eel was also analyzed.To analyze specific intron gains in the CHRM1, 87% for CHRM3, and 90% for CHRM5. In the other subfamily, the identity is even higher with 96% for CHRM2 and 95% for CHRM4, displayed by Jalview 2.10.3b pairwise alignment. Overall, CHRM2 displays the highest degree of conservation, whereas the CHRM1 displays the lowest. The CHRM2/CHRM4 subfamily displays a higher degree of conservation than the CHRM1/CHRM3/CHRM5 subfamily. This is also confirmed in human–chicken ortholog comparisons and human paralog comparisons. However, when including the complete amino acid sequences in the pairwise alignment, the percentage of identity drops considerably. For instance, the well conserved CHRM2 subtype decreases from 96% to 75% identity when including the complete sequence. One reason for this is that IL3 located between TM5 and TM6 is highly variable between receptor subtypes as well as between species for each subtype  tree of the predicted mAChR proteins is displayed in M4 genes . The CHRM5 genes . The CHRtic tree .CHRM1/CHRM3/CHRM5 subfamily has a few tunicate sequences as its closest relatives , a period that coincides with the timing of the 1R and 2R events. This is further supported by the species distribution of these three subtypes. Although a tunicate group is missing for the CHRM2/CHRM4 subfamily, the timing of the duplication events, as well as the species distribution of the two subtypes, coincides with the duplication events in the CHRM1/CHRM3/CHRM5 subfamily, supporting the 1R and 2R expansion hypothesis also for this subfamily. Hence, from this phylogenetic analysis we conclude that the mAChR family most likely expanded from two ancestral members present before 1R and 2R, to five members present following the vertebrate tetraploidizations. Additionally, duplicates found in the group of teleosts coincide with the timing of the teleost-specific tetraploidization, hence suggesting that those gene duplicates are paralogs resulting from 3R and can thus be called ohnologs (see below). The PhyML tree resulting from the analysis of the complete multiple sequence alignment, also including the IL3 region, is shown in The positioning of the tunicates basally to the To explore the hypothesis that two ancestral mAChR genes expanded to five as a result of the basal vertebrate tetraploidizations, analyses of the mAChR neighboring genomic regions were conducted. If the members of each of the two mAChR subfamilies are located in chromosomal regions that also contain representatives from several other gene families, this would strongly indicate that a large block of genes, or even a chromosomal region or an entire chromosome, had been duplicated. The related genes resulting from these events are named ohnologs, as described in the Introduction. On the other hand, if the members of an mAChR subfamily are in completely different chromosomal neighborhoods, this would indicate independent duplications of an mAChR gene and insertion into unrelated chromosomal regions. Related chromosomal regions that arose as a result of the 1R and 2R tetraploidizations (or any other tetraploidization event) are referred to as comprising a paralogon . Thus, tCHRM2/CHRM4 genes in the spotted gar were retrieved, and the gene families with at least two members present were analyzed. Following the exclusion criteria at the preliminary analysis stage stated in the Materials and Methods, five gene families were included in the final analysis, namely ARHGAP, NAV, NELL, PPFIA, and SHANK. However, due to the low number of gene families found in spotted gar, the genomic regions surrounding the CHRM2/CHRM4 genes were investigated in the chicken and six additional gene families were identified. The reason why they were not found in the neighboring regions in spotted gar could be chromosomal rearrangements. The additional neighboring families are ABTB2, CREBL, CRY .The genomic regions surrounding the lication , red. Thlication , brown s10.1523/ENEURO.0340-18.2018.f2-1Figure 2-1CHRM2 and CHRM4 genomic regions. There is one neighboring gene family per page in alphabetical order. Orthologs are color coded according to paralogon member. Gray color means that those orthologs are not included in paralogon analysis due to exclusion based on results from the phylogenetic analysis, or that there is no human, chicken, or spotted gar sequence present. Assigned sequence names and sequence information details are provided in Fig. 2-1, TIF file.Phylogenetic aLRT SH-like tree of the neighboring gene families included in analysis of the 10.1523/ENEURO.0340-18.2018.f2-2Figure 2-2CHRM2 and CHRM4 genes and their neighboring gene families included in the paralogon analysis in www.egosumdaniel.se). Download Fig. 2-2, TIF file.Analysis of chromosomal positions of the 10.1523/ENEURO.0340-18.2018.f2-3Figure 2-3CHRM2 and CHRM4 genes. First, the genome assembly versions used are stated, followed by information about the neighbor gene families included in the analysis in the following order: species, HGNC/ZFIN/Flybase symbol name, chromosome or genomic scaffold position, Ensembl ID or NCBI accession number, assigned sequence name, and additional comments regarding the sequence update date on NCBI or whether there have been manual edits of the original Ensembl or NCBI sequence. Download Fig. 2-3, DOCX file.Information about the neighboring gene family sequences included in the analysis of the genomic regions surrounding the MYBP, PPFIA, and SHANK ohnologs (on chromosomes 3 and 24). However, it appears that additional translocations have occurred in zebrafish, most likely following the 3R event. These genes are located in a paralogon that has been studied previously, where the focus was on a region containing numerous other gene families including the visual opsins, transducin alpha subunits and oxytocin/vasopressin receptors . Download Fig. 3-2, TIF file.Analysis of chromosomal positions of the 10.1523/ENEURO.0340-18.2018.f3-3Figure 3-3CHRM1, CHRM3, and CHRM5 genes. First, the genome assembly versions used are stated, followed by information about the neighbor gene families included in the analysis in the following order: species, HGNC/ZFIN/Flybase symbol name, chromosome or genomic scaffold position, Ensembl ID or NCBI accession number, assigned sequence name, and additional comments regarding sequence update date on NCBI or whether there have been manual edits of the original Ensembl or NCBI sequence. Download Fig. 3-3, DOCX file.Information about the neighboring gene family sequences included in the analysis of the genomic regions surrounding the LTBP3, PROX, and SPTBN2; SPTB family member SPTBN2 (located on a scaffold). As mentioned previously, the CHRM1 gene is absent in the chicken, as well as in other birds. The second and third paralogon members in human are located on three and two different chromosomes, respectively . However, one member of the TGFB family, the TGFB1 gene, is located on chromosome 32, which is a very short chromosome in the chicken, consisting of only ∼78 kb.The first paralogon member , green iCHRM1/CHRM3/CHRM5 neighboring gene repertoire and chromosomal locations in zebrafish, presented in The synteny analysis in human, chicken, and spotted gar shows that these genomic regions have undergone a number of rearrangements such as translocations, and several ohnologs could not be identified. This is further seen when analyzing the Thus, the analysis of conserved synteny and paralogons of the two mAChR subfamilies and their neighboring gene families confirms our hypothesis based on the phylogenetic analyses that the mAChR gene family expanded by the 1R and 2R tetraploidizations from two ancestral genes into the five members present today in most tetrapods and some basally diverging vertebrates, and 8-10 members in teleosts, forming two distinct subfamilies belonging to two separate paralogons.CHRM2b, CHRM3b, CHRM4a, and CHRM4b genes have independently gained at least one intron in the proximity of the region encoding TM1 and at least one intron in the region encoding the IL between TM5 and TM6, in at least one of the teleost species investigated , and therefore it was not possible to determine the exact time point when this intron was gained in zebrafish. The intron present in stickleback and medaka was also found in fugu and Amazon molly . Download Fig. 4-1, TIF file.Jalview sequence alignment of the teleost species sequences included in the analysis of teleost-specific intron gains in the 10.1523/ENEURO.0340-18.2018.f4-2Figure 4-2Fig. 4-2, TIF file.Supplementary Figure 4-2. Download 10.1523/ENEURO.0340-18.2018.f4-3Figure 4-3Fig. 4-3, TIF file.Supplementary Figure 4-3. Download 10.1523/ENEURO.0340-18.2018.f4-4Figure 4-4Fig. 4-4, TIF file.Supplementary Figure 4-4. Download 10.1523/ENEURO.0340-18.2018.f4-5Figure 4-5Fig. 4-5, DOCX file.Information about the teleost mAChR amino acid sequences included in the analysis of teleost-specific intron gains. First, the genome assembly versions used are stated, followed by information about the teleost mAChR sequences included in the analysis in the following order: species, HGNC/ZFIN/Flybase symbol name, chromosome or genomic scaffold position, Ensembl ID or NCBI accession number, assigned sequence name, and additional comments regarding sequence update date on NCBI or whether there have been manual edits of the original Ensembl or NCBI sequence. Download CHRM3b gene has gained one intron located in the beginning of the region encoding TM1 in medaka and stickleback and one additional intron in the N-terminal region in stickleback  has been identified, it is on a scaffold, perhaps indicating that additional members may be possible to identify.Thus, the repertoire of mAChR genes is quite consistent across vertebrates. This is presumably a reflection of unique functional roles for each of the gene products. The only gene that deviates from this pattern is CHRM2b, CHRM3b, CHRM4a, and CHRM4b genes. A previous study by CHRM2b, CHRM3b, and CHRM4b . This was a possibility favored by


Previous studies conducted in our laboratory have found altered adult health outcomes in animals with prenatal exposure to environmentally relevant levels of unconventional oil and gas (UOG) chemicals with endocrine-disrupting activity. This study aimed to examine potential metabolic health outcomes following a preconception, prenatal and postnatal exposure to a mixture of 23 UOG chemicals. Prior to mating and from gestation day 1 to postnatal day 21, C57BL/6J mice were developmentally exposed to a laboratory-created mixture of 23 UOG chemicals in maternal drinking water. Body composition, spontaneous activity, energy expenditure, and glucose tolerance were evaluated in 7-month-old female offspring. Neither body weight nor body composition differed in 7-month female mice. However, females exposed to 1.5 and 150 μg/kg/day UOG mix had lower total and resting energy expenditure within the dark cycle. In the light cycle, the 1,500 μg//kg/day group had lower total energy expenditure and the 1.5 μg/kg/day group had lower resting energy expenditure. Females exposed to the 150 μg/kg/day group had lower spontaneous activity in the dark cycle, and females exposed to the 1,500 μg/kg/day group had lower activity in the light cycle. This study reports for the first time that developmental exposure to a mixture of 23 UOG chemicals alters energy expenditure and spontaneous activity in adult female mice. Unconventional oil and gas (UOG) extraction combines directional drilling and hydraulic fracturing to liberate oil and gas that was previously inaccessible by traditional drilling methods, including sources of shale gas, coal bed methane, and tight gas. Across the industry, over 1,000 chemicals have been reportedly used in the hydraulic fracturing process. Varying mixtures of these chemicals are combined with millions of gallons of water to fracture underground rock. UOG extraction has been identified as a potential source of EDCs, developmental and reproductive toxicants. We have previously reported that out of 24 UOG chemicals tested, 23 exhibited antagonist activity for one or more of the estrogen, androgen, progesterone, thyroid, and glucocorticoid receptors; and a mixture of 23 of these UOG chemicals exhibited antagonistic activity for all five receptors , defined as exogenous chemicals that can interfere with normal hormone action [–6 and rein vitro  1 to postnatal day (PND) 21, and evaluated body composition, energy expenditure, activity, and glucose tolerance in adult offspring.ad libitum.This study was carried out in accordance with the recommendations of the National Research Council's Guide for the Care and Use of Laboratory Animals. The protocol was approved by the University of Missouri Animal Care and Use Committee. C57BL/6J mice (purchased from Jackson Laboratories) were housed in polysulfone cages, in a barrier facility with a 12 h light/dark cycle. Feed  and acidified water (in glass bottles) were sterilized and provided n = 14, 9, 11, 8, and 10) were exposed to the chemical mixture  for 5 weeks prior to mating  was determined by caliper measurement. At PND 21, pups were weaned and rehoused with pups of the same treatment group and sex.Female offspring at 6 months of age were transferred to an open-top conventional facility for body composition and metabolic assessments. Mice were allowed to acclimate to the new environment for a month prior to initiation of metabolic testing. This facility was temperature controlled and kept on a 12-h light/dark cycle. In this facility, the experimental animals received non-sterilized feed (LabDiet 5053) and non-acidified, non-sterilized water.Body weight was measured at PND 7, PND 21, and 7 months of age. Fat and lean mass were assessed at 7 months of age, using an EchoMRI-900 . Fat and lean percentages were calculated by dividing fat or lean mass by body weight.Energy expenditure via indirect calorimetry, activity, and behavior were measured using the Promethion from Sable Systems Int., . Oxygen consumption, energy expenditure, and activity were calculated with macros provided by the manufacturer .Total energy expenditure was measured for a 12-h cycle. Resting energy expenditure was extrapolated from the lowest average energy expenditure in a 30-min window within a 12-h cycle and calculated to be representative of the resting energy expenditure for a complete 12-h period. Non-resting energy expenditure was calculated for each 12-h cycle by subtracting 12-h calculated resting energy expenditure from 12-h total energy expenditure .Activity and meters traveled were measured by infrared beams that track movement in horizontal (X and Y plane) and vertical directions (Z plane). Spontaneous activity was defined as activity in the X, Y, and Z directions, ambulatory activity in the X and Y directions, and rearing activity in the Z direction. Meters traveled counted all meters in the X, Y, and Z direction. Food consumption was also measured in this system.n = 7, 4, 6, 6, and 6 animals in the vehicle, 1.5, 15, 150, and 1,500 μg/kg/day groups, respectively  were collected for ectively . Mice weectively .n = 16/block). Mice were weighed at 1000 h, and fasted from 1000 to 1600 h. A baseline (0 min) blood sample was collected via tail snip at 1600–1630 h, and blood glucose was determined using a glucose monitor (Accu-Chek Aviva Plus). Immediately after the baseline measurement was taken, 250 mg/mL glucose was injected intraperitoneally at 1 mg/kg body weight. Blood glucose concentrations were measured at 30, 60, and 120 min post injection, as described previously  were included as fixed effects for body weight, fat mass, lean mass, fat percent, lean percent, food consumption, and activity. For analysis of energy expenditure, body weight and size of litter were also considered as fixed effects. Data were normally distributed or transformed to achieve normality. Results are displayed in all figures as the estimated marginal means, back transformed for presentation if transformation was necessary, except for The body weights of pregnant dams were measured in order to monitor health and calculate treatment dosage. The body weights of the dams exposed to the UOG chemical mixture did not differ from those of dams exposed to the vehicle at gestation day 0 after 5 weeks of treatment . TreatmeDevelopmental exposure to the UOG chemical mixture altered the body weights of female offspring at PND 7. Body weight at PND 7 in F1 females developmentally exposed to the UOG chemical mixture was 10–26% lower in the 1.5, 15, and 1,500 μg/kg/day treatment groups relative to vehicle . At PND p = 0.054) and 150 (p = 0.054) μg/kg/day treatment groups relative to vehicle  to combine pre-conception, prenatal, and lactational exposure to assess impacts of adult maternal exposure prior to fertilization and to bracket fetal development from GD 1 to PND 21. Exposure during GD 1–11 covers development of the placenta, pancreas, and liver, and maternal high fat diet during this period has been shown to cause adverse metabolic outcomes in offspring . Also, aIn the current study, we report that a combined pre-conceptional, prenatal, and lactational exposure from GD 1 to PND 21 to a mixture of 23 UOG chemicals was associated with decreased body weights at PND 7 in females. Previously our lab has shown that a prenatal exposure from GD 11 to GD 18 to the same 23-UOG mixture resulted in the opposite–increased body weight at PND 7 and 21 –61. AlteMany factors contribute to energy balance, body composition, and body mass regulation. In this study, pre- and post-natal exposure to the 23-UOG mixture decreased total and resting energy expenditure in some UOG mix groups, but this did not result in altered body weight, lean mass, or fat mass in 7-month-old females. Although one would typically expect higher body mass or fat mass to track with lower energy expenditure, this is not always the case. For example, Wan et al. also found that AKT knockout mice displayed an increase in energy expenditure compared to control mice matched for body mass . A limitOverall, we have reported that the 23-UOG mixture can alter developmental programming and result in altered energy expenditure and activity of 7-month-old females. The results shown thus far highlight the need for additional research on metabolic health effects in humans and animals in drilling-dense regions. More studies should be aimed at understanding exposure to UOG and other environmental chemicals on metabolic health outcomes .This study was carried out in accordance with the recommendations National Research Council's Guide for the Care and Use of Laboratory Animals. The protocol was approved by the University of Missouri Animal Care and Use Committee.VB and JC-G performed animal experiments. VB analyzed the data. SN secured funding, directed experiments and assisted in data analysis and interpretation. All authors have contributed to the design of experiments, interpretation of the data, and writing the manuscript.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.