AbstractOculogryphus is described and figured as Oculogryphus bicolor sp. n. from Vietnam. The definition of the genus is slightly modified with consideration of newly detected morphological variation from this species. According to a comprehensive phylogenetic analysis including nearly 80% of documented lampyrid genera, Oculogryphus is the putative sister group to Stenocladius s. str. within the paraphyletic group of Ototretinae-Ototretadrilinae.The classification of Stenocladius is briefly discussed in this context.A second species of the enigmatic lampyrid genus  Oculogryphus was established for a single male specimen of a peculiar species, Oculogryphus fulvus Jeng, from Vietnam . A second species of the genus, also from Vietnam, was recently identified while examining material in the American Museum of Natural History (AMNH). Herein we describe and figure this new species and discuss some morphological variations between it and Oculogryphus fulvus, requiring a slight modification to the generic diagnosis. In addition, comments are provided regarding the phylogenetic position of Oculogryphus in the light of a comprehensive phylogenetic analysis of Lampyridae conducted by the senior author (Jeng 2008).The genus The methodology and morphological terminology used herein follows that of Jeng et al. (2007). The body length (BL) is the sum of the pronotal and elytral lengths  plus length of those exposed portions of the head from the pronotum; while body width is considered as twice the elytral width (BW = 2EW). Pronotal width was abbreviated as PW. The nomenclature of the hind wing venation follows that of Kukalová-Peck and Lawrence (2004) rather than that of Kukalová-Peck and Lawrence (1993) which was employed by Jeng et al. (2007). In reporting label data the symbol “/” indicates separate lines on a single labe, while “//” denotes material located on separate labels.GenusJeng, Engel & Yang, 2007http://species-id.net/wiki/OculogryphusOculogryphus  Jeng, Engel & Yang, 2007: 4.Oculogryphus fulvus Jeng, 2007, by original designation.Oculogryphus fulvus. The genus is characterized by its large and ventrally approximate compound eyes which are clearly emarginate posteriorly, filiform antennae, unmodified mandibles (sensu Green 1959), considerably exposed head from pronotum, absence of tibial spurs and lanterns, progressively shortened tarsomeres from I–IV, eight abdominal ventrites which are not lobed, dorsal abdominal spiracles not enclosed by parasternites, symmetrical aedeagal sheath and aedeagi, and several other charactersPageBreak . The new species demonstrates some variations in hind wing venation (bifurcate MP3+4). The following pairs contrast the different hind wing venations under the two nomenclatural systems used between Jeng et al. (2007) and the current work: CuA = Cu; CuA1+2 = CuA; AA = AA3+4; CuA3+4 + AA = AA3. Otherwise the original definition of the genus remains unchanged. These modifications do not affect the key to Oriental genera provided by Jeng et al. (2007).The original diagnosis of the genus provided by Jeng et al. (2007) was based strictly on urn:lsid:zoobank.org:act:649F93C0-8F6F-46A8-A163-040DB4AFA3CEhttp://species-id.net/wiki/Oculogryphus_bicolor18°22'N, 106°13'E/ 900m, April 20–28, 1998/ Malaise, AMNH, Carpenter/ Grimaldi, Herman, Silva, Long”. Deposited in the Division of Invertebrate Zoology (Entomology), American Museum of Natural History, NY, with eventual deposition in the Institute of Ecology and Biological Resources Collection (IEBR), Hanoi, Vietnam.♂, “VIETNAM: Ha Tinh, Huong/ Son, 4 ♂♂, with identical data as holotype; 1 ♂, with identical data except collected on 18 May 1998; 1 ♂, with identical data as holotype except collected at 600m above sea level on 7–14 April 1998 by K. Long; 1 ♂, with identical data as holotype except collected on 5 May 1998 by K. Long. All deposited in the AMNH.18°22'N, 106°13'E.Vietnam, Ha Tinh Province, Huong Son, The species has several diagnosable characters separating it from the type species: 1) body size slightly larger (6.2–8.2 vs. 6.0 mm); 2) more vivid light brown-tan bicoloration ; 3) sligOculogryphus fulvus except: aedeagal sheath has length of 0.9 mm and width of 0.6 mm; abdominal tergites IX and X clearly recognizable individually  for a complete account of this species.A second specimen of ♂, “Vietnam: Cuc Phuong/ Ninh binh, 3-10.V.1966. Exp. Gy. TOPÁL// Nr. 261/ beaten from bushes”.Oculogryphus has a mosaic of features intermingling those of Rhagophthalmidae , Luciolinae , Ototretinae , Ototretadrilinae , and Lampyrinae  . Based on their morphological comparison across 80 documented genera of Lampyridae, Jenget al. (2007) inferred that the genus was either a member of Ototretinae or a stem taxon linking Luciolinae and some basal lineages of Lampyridae. It now appears that this inference was quite accurate. In the most comprehensive phylogenetic analysis of Lampyridae conducted to date , Oculogryphus was found to be the sister group of Stenocladius Fairmaire within the paraphyletic group of Ototretinae-Ototretadrilinae complex (both subfamilies were defined by Crowson 1972). Morphologically this position appears intuitive, and seems further supported by the discovery of Oculogryphus bicolor which shows an even more similar morphology with that of Stenocladius, especially in the aedeagal sheath and male genitalia (cf. Kawashima 1999). However, Stenocladius is a problematic taxon. The type species, Stenocladius davidis Fairmaire from China, has pectinate antennae bearing branches arising from the base of flagellar articles I–VIIPageBreakI, and with flagellar articles that are not abbreviated. All Stenocladius species from Japan, Taiwan, and adjacent islands share a more-or-less uniform male genital structure (Kawashima 1999). By contrast, some species of Stenocladius are questionably assigned to this genus, especially those from India and Sri Lanka . We have termed the former group as Stenocladius s. str. so as to differentiate them from those atypical group(s) within the genus. Several significant differences have been detected among these atypical species, such as the position of the abdominal spiracles (enclosed by parasternites rather than adjacent to them), a different kind of pectinate antennae (abbreviated flagellar articles and branches arising from the article apex rather than the base), and male genitalia . It is likely that many of the Oriental Stenocladius do not truly belong to this genus but are allied to some other ototretines. Further differentiation of Stenocladius is under investigation by the senior author. Regardless, the discovery of enigmatic new taxa like Oculogryphus bicolor and the recognition of significant difficulties in the current classification of some diverse genera, highlights the need for continued exploration and taxonomic study of Asian Lampyridae.


M. tuberculosis infection (LTBI).Association studies have been employed to investigate the relationships between host single nucleotide polymorphisms (SNPs) and susceptibility to pulmonary Tuberculosis (PTB). However, such candidate genetic markers have not been widely studied in Chinese population, especially with respect to the disease development from latent M. tuberculosis infection) from Zhengzhou, China. The two groups of controls were frequency matched on gender and age with PTB patients. Genotyping was carried out by the Illumina Golden Gate assay.In this case–control study, 44 candidate SNPs were examined in a total of 600 participants . A combined effect of the genotypes associated with increased risk of PTB (i.e. TLR9 1174G/G and IFNG 2109 A/A) was found when comparing PTB patients with LTBI controls (p=0.004) but not with healthy controls without infection (p=0.433).When comparing PTB patients with LTBI controls but not healthy controls without Potential associations between TLR9 and IFN-γ genetic polymorphisms and PTB were observed in a Chinese population which supports further study of the roles played by TLR9/IFN-γ pathway during the development of PTB. Mycobacterium tuberculosis , however, only a minority (10%) of those infected will develop tuberculosis (TB) during their lifetime [It is estimated that one third of the world’s population is infected with minority 0% of tholifetime . This cllifetime -5. Assoclifetime -9. Most minority % of thoslifetime . Howeverminority % of thoslifetime .Treatment of Tuberculosis: Guidelines for National Programmes. World Health Organization, 2011). High-risk population identification is crucial for diseases prevention and early diagnosis. Besides to assess potential genetic markers, association study also contributes to clarify potential mechanisms underlying host defense to the disease development [In 2010, there were about 8.8 million incident cases of TB around the world, and China has the world’s second largest number of incident cases (0.9–1.2 million) . Written informed consent to participate in this study and to provide 8 mL blood for research was obtained from each study participant before the interview and testing.In this case–control study, participants were recruited between September 2010 and February 2011 in Zhengzhou , China. Two hundred participants were recruited in each of the following three groups: patients with PTB, LTBI controls and healthy controls without MTB infection.Newly diagnosed PTB patients, since September 2010, were enrolled from the outpatient tuberculosis clinic in Henna Provisional Infectious Disease Hospital (Zhengzhou). Eligibility criteria for case patients included: 1) ≥ 18 years old; 2) registered residence in Zhengzhou; 3) diagnosis of PTB based on clinical evaluation and laboratory tests  according to the Guidelines for Implementing the Prevention and Control of Tuberculosis in China . Exclusion criteria included malignancy and diagnosis of extra-pulmonary TB cases.Healthy controls without MTB infection and LTBI controls were registered residence in Zhengzhou and above 18 years of age. Both groups of controls were selected from participants of the general health examination and frequency matched with PTB patients on gender and 5-year age group. Healthy controls without MTB infection were tuberculin skin test (PPD test) negative (< 10 mm induration) and without evidence of active TB and history of TB. LTBI controls, without evidence of active TB and history of TB, should have a positive PPD test with diameter of the induration ≥15 mm to minimize the misclassification cased by vaccination with Bacille Calmette Guerin (BCG) . ExclusiA questionnaire .The 44 target SNPs from 32 genes . Selected SNPs were genotyped by the Illumina GoldenGate Genotyping Assay on an Illumina BeadXpress System. All of the assessed SNPs qualified for inclusion. Genotyping efficiency for the SNPs was between 97% and 100%. Duplicate SNPs were assessed in 27 samples and no errors were detected.Differences in the distribution of demographic variables and lifestyle factors between the three groups were assessed by chi-square tests. Hardy-Weinberg Equilibrium (HWE) in healthy controls without MTB infection was tested using asymptotic Pearson’s chi-square tests for each SNP. SNPs not in HWE were excluded in the data analyses. The chi-square tests were used to compare genotype frequencies of PTB patients with LTBI controls and healthy controls without MTB infection, respectively. Unconditional logistic regression analysis was used to estimate OR and corresponding 95% confidence intervals (CI) for disease development using co-dominant, dominant and recessive models for each SNP, respectively, adjusting for age and sex. In additional sensitivity analyses, the associations of the target SNPs were additionally adjusted for demographic factors which significantly related to TB in the study participants. Linkage disequilibrium (LD) was assessed by Linkage Disequilibrium Analyzer software . To estiAs shown in Table Genotyping data for all SNPs but one rs2280788) were successfully obtained for ≥ 95% of the subjects. The minor allele frequency was < 5% for 8 assessed SNPs  and genotype distribution of another 5 selected SNPs was found to be not in HWE in our study population . Therefore, after excluding these 14 SNPs, our following analyses were based on genotyping data of 30 SNPs as shown in Table 280788 weTLR9 1174 A/G, TLR9 1635 A/G and IFNG 2109 G/A. As shown in Table TLR9 1174 A/G with an adjusted OR of 1.87 (95% CI: 1.08–3.23) (p = 0.027) for G allele carriers when compared with the more frequent AA genotype carriers. A significantly increased risk of PTB development was observed for TLR9 1635 AA genotypes carrying cases compared with G allele carriers, with an adjusted OR of 1.90 (95% CI: 1.09-3.31) (p = 0.025). These two loci in TLR9 were in LD in our study population . In addition, the GG genotype of IFNG 2109 G/A was found to be associated with decreased risk of disease development with an adjusted OR of 0.54 (95% CI: 0.30-0.98) (p = 0.042) when compared with AA genotype. After additional adjustment for demographic factors which significantly related with the development of TB as shown in Table Associations between each SNPs and the development of PTB were assessed in co-dominant, dominant and recessive models after control for age and gender. When comparing PTB patients with LTBI controls but not healthy controls without MTB infection, significant associations with developing disease were observed for TLR9 1174 A/G and IFNG 2109 G/A were investigated as shown in Table TLR9 1174 G/G, IFNG 2109 A/A) was found to contribute to disease development.The potential combined effect of TLR9 1174 A/G, TLR9 1635 A/G and IFNG 2109 G/A both individually and in a combined effect model.To our knowledge, this is the first large-scale molecular epidemiological study from China that evaluated host genetic susceptibility to PTB development from LTBI. A total of 44 selected SNPs in 32 genes were examined for PTB patients, LTBI controls and healthy controls without MTB infection, respectively. When comparing PTB patients with LTBI controls but not healthy controls without MTB infection, significant associations were observed for It is becoming clear that innate immunity plays an important role in the host defense against MTB infection, and the first step in this process is recognition of MTB ,20. SeveIFNG 2109 G/A polymorphism influences transcription levels of IFN-γ but the underlying mechanism is still unclear [TLR9 and IFNG further supports a potential role of TLR9/IFN-γ pathway in active PTB development. Previous studies provided intriguing information about TLR9 SNPs and Malaria severity [IFN-γ is one of the most widely studied candidate gene potentially associated with TB susceptibility. Positive associations with PTB susceptibility have been observed for multiple SNPs as assessed by individual studies or by means of meta-analysis in different populations including +874 T/A ,28, 2109 unclear . The intseverity ,36, the severity . HoweverTLR9/IFNG SNPs with PTB by combining LTBI controls and healthy controls without MTB infection together. The results showed that no statistically significant relation was found . Such correction might not be needed since our study mainly addressed moderately well-characterized SNPs with previous suggestions of association with PTB. However, considering this study was conducted in a different population and the strength of the previous data is modest, a possibility of false-positive results in our study could not be completely excluded. Fifth, as the controls were selected from participants of the general health examination, although from the same city, risks of exposure to MTB might be different between the two groups of controls and between PTB patients and controls. Therefore, potential bias caused by selection of controls should be kept in mind when interpreting our results. Further and larger studies, preferably large collaborative studies conducted in different populations, are needed to verify our findings.In the interpretation of our results, some limitations have to be considered. First, the definition of latent infection based on tuberculin skin test (TST) could not be claimed to be perfect especially among the subjects with BCG vaccination despite our study participants were adults. As reported that on average 30%-35% will have BCG-related positive TST results even after an interval of more than 10 to 15 years . TherefoIn conclusion, potential associations were observed between TLR9 and IFN-γ genetic polymorphisms and PTB development from LTBI in a Chinese population. These epidemiological findings provided clue for further studies to explore the potential role of TLR9/IFN-γ pathway in human immune responses to MTB infection and active disease development.BCG: Bacille Calmette Guerin; GWAS: Genome-wide association studies; TB: Tuberculosis; HWE: Hardy-Weinberg Equilibrium; IGRA: Interferon-gamma release assays; LTBI: Latent M. tuberculosis infection; MTB: Mycobacterium tuberculosis; PTB: Pulmonary tuberculosis; SNPs: Single nucleotide polymorphisms; TLRs: Toll-like receptors; TST: Tuberculin skin test.The authors declare that they have no competing interests.LG and LJM conceived and designed the study; YY and XWL carried out the study participants’ enrollment, samples collection and tests; JML, FZ, CG, LG, QH and MFL participated in the questionnaire collection and data management quality control; WC, XJS and QH participated in study coordination; YY, LG and JML performed the statistical analysis and wrote the manuscript; All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/511/prepubThe questionnaire used in the study.Click here for fileList of selected single nucleotide polymorphisms.Click here for fileThe association of selected SNPs with the development of pulmonary tuberculosis.Click here for fileThe association of TLR9 and IFN-γ SNPs with TB (PTB patients v.s. combined controls).Click here for file


Birth by cesarean delivery (CD) as opposed to vaginal delivery (VD) is associated with altered health outcomes later in life, including respiratory disorders, allergies and risk of developing type I diabetes. Epigenetic gene regulation is a proposed mechanism by which early life exposures affect later health outcomes. Previously, type of delivery has been found to be associated with differences in global methylation levels, but the sample sizes have been small. We measured global methylation in a large birth cohort to identify whether type of delivery is associated with epigenetic changes.DNA was isolated from cord blood collected from the University of Michigan Women’s & Children Hospital and bisulfite-converted. The Luminometric Methylation Assay (LUMA) and LINE-1 methylation assay were run on all samples in duplicate.p =0.01). LUMA measurements were significantly lower for total CD and planned CD, but not emergency CD when compared to VD . However, this association did not persist when adjusting for maternal age, maternal smoking and infant gender. Furthermore, total CD deliveries, planned CD and emergency CD deliveries were not associated with LINE-1 measurements as compared to VD . This lack of association held when adjusting for maternal age, maternal smoking and infant gender in a multivariable model.Global methylation data at CCGG sites throughout the genome, as measured by LUMA, were available from 392 births , and quantitative methylation levels at LINE-1 repetitive elements were available for 407 births . LUMA and LINE-1 methylation measurements were negatively correlated in this population (Spearman’s r = −0.13, Type of delivery was not associated with global methylation in our population, even after adjustment for maternal age, maternal smoking, and infant gender. While type of birth may be associated with later health outcomes, our data suggest that it does not do so through changes in global genomic methylation. Infant stress during labor is advantageous in preparation for extrauterine life; however, the stress encountered by infants varies vastly between delivery types. The progression of labor involves a surge in stress hormones, including catecholamines and cortisol, in the infant to promote lung maturity for gas exchange, increase blood flow, activate the central nervous system and mobilize fuel ,2. This Surges of stress hormones during labor progress development of essential pathways that will enable the infant to survive outside the womb, thus implicating changes in the genome in response to stress. However, these changes may not occur if the infant does not undergo such stress, such as during a CD. The increasing number of CDs has led to scrutiny of the impact that type of delivery has on infant health and predisposition to disease outcomes. Birth via CD is associated with a higher risk of developing diseases such as allergic rhinoconjunctivitis and asthma ,11, typeThe molecular mechanisms associated with these phenomena remain unknown; however, emerging evidence suggests that when an infant does not develop essential defense mechanisms, he or she may be predisposed to developing disease later on in life. The developmental origins of health and disease (DOHaD) hypothesis posits that increased susceptibility to disease following early life experiences is shaped by epigenetic modifications such as DNA methylation and chromatin modifications . EpigeneA disease outcome likely results from aberrant activity of many different molecular pathways, and thus it is likely that epigenetic modifications occur at multiple genes or genic regions. Measurement of global DNA methylation patterns is a practical means of identifying differential epigenetic effects in neonates. Furthermore, recent research has shown differential global methylation between CD and VD infants, although the sample size was small .The objectives of this study are to (1) confirm the apparent difference in global DNA methylation between infants born via CD as compared to VD in white blood cells from cord blood in a large population, using two separate global DNA methylation measurements and (2) determine if methylation differences are associated with type of CD  as compared to VD.IRB approval was obtained for the purpose of collecting excess cord blood samples from our study population, which consisted of 408 infants (> 37 weeks) delivered at the University of Michigan Women’s & Children Hospital. Only pregnancies occurring between 8 a.m. and 2 p.m. were included to provide more time for sample processing, and included scheduled and emergency CDs as well as VDs. Because this study collected excess clinical samples, samples were obtained from all deliveries during this time period. Samples were collected with few exclusion criteria, only requiring singleton pregnancy and clinically excess tissue available immediately after delivery in order to maximize sample quality. After delivery of the placenta, the umbilical cord was clamped, washed thoroughly to prevent maternal blood contamination, and whole venous blood was drawn via venipuncture into PAX gene Blood DNA tubes , kept on ice and transported to the laboratory.Genomic DNA was extracted using the PAX gene Blood DNA kit . Leukocyte DNA was isolated following the manufacturer’s protocol with some minor modifications for dense blood. For complete digestion of dense samples, the initial 65°C incubation period was increased from 10 min to 1 h. The final incubation of the solution was repeated a second time in order to completely dissolve the DNA containing pellet in elution buffer. Both the quality and quantity of the extracted DNA were assessed using a ND1000 spectrophotometer . For bisulfite conversion, the addition of sodium bisulfite selectively deaminates unmethylated cytosine nucleotides into uracil whereby a subsequent polymerase chain reaction (PCR) incorporates thymine, leaving methylated cytosines unchanged. Complete bisulfite modification of genomic DNA results in methylation-dependent genome-wide alterations in DNA sequences. Approximately 1 μg of genomic cord blood DNA was bisulfite converted using the EpiTect Bisulfite Kit  and QIAcube® purification system.MspI (Biolabs) and of methylation-sensitive HpaII (Biolabs) enzymes were separately added to 300 ng of genomic DNA along with the internal standard EcoRI (Biolabs) enzyme and 10× buffer.The Luminometric Methylation Assay (LUMA) is a restriction enzyme-based procedure that detects methylation at 5’-CCGG-3’ sequences throughout the genome, irrespective of methylation at repetitive elements. The procedure for LUMA analysis via pyrosequencing has been previously described ,21. BrieTango™with BSA (Fermentes) was used for differential digestion. Samples were incubated at 37°C for 4 h. Twenty μl of annealing buffer was added prior to pyrosequencing in AQ mode using the SNP PyroMarkMD software with the following dispensation order: GTGTCACATGTGTG. The ratio of normalized product signal between methylation-sensitive and -insensitive digestions from an individual sample was measured to calculate global DNA methylation using the following equation: 1 − [(HpaII/EcoRI)/(MspI/EcoRI)] × 100. Individual samples were run in duplicate along with lowly and highly methylated human genomic standards (EpigenDx).The LINE-1 assay interrogates promoter DNA methylation of repetitive elements, specifically long interspersed (LI) retrotransposons, throughout the genome . Fifty np < 0.05. Generalized linear models were run in R version 2.12.0 , and mixed effects models were run in SAS 9.2 .Duplicate LUMA and LINE-1 global methylation measurements were averaged for all analyses, and duplicates resulting in a greater than 15% difference were deleted before statistical analysis, leading to 16 subjects deleted for LUMA and 1 deleted for LINE-1. Due to departures from normality, data are expressed as median values and ranges. Bivariate nonparametric tests  were used to test the association between global methylation (LUMA and LINE-1) and covariates . Significance of correlations between covariates was tested using Spearman’s correlation. Generalized linear models were used to test the association between type of deliveries with each methylation measurement, adjusting for maternal age, maternal smoking status and infant gender. Mixed effects models were used to test the association between each specific LINE-1 site  to account for covariance between measurements. Statistical significance was set at rho = 0.2, 0.1, p < 0.001, p = 0.03, respectively).Data were available for 408 subjects. Thirty-six percent were VDs, while 64% delivered via CD. Of the CD deliveries, 80% were planned CD deliveries, and 20% were emergency CD deliveries. Maternal age ranged from 18–48 years, with approximately equal male and female infants . The majority of mothers did not have a history of smoking (60%), while 11% were past smokers and 6% were current smokers; for 93 of the 408 subjects, smoking status was unknown (Table p = 0.03); however, this association did not persist after adjustment for maternal age, maternal smoking and infant gender , and mean LUMA measurement was 73.8% Table . In comper Table .p = 0.02]. There was no difference in methylation between VD and emergency CDs . When adjusting for maternal age, maternal smoking and infant gender, LUMA methylation measurements in infants born via planned CD were still lower than those found in infants born via VD; however, this result was not significant (p = 0.08) in the multivariable model. There was no difference in methylation between emergency CD and total CD with VD infants after adjusting for covariates (p = 0.28) , median planned CD = 74.2 (46.3-81.6), .6, mediap = 0.19). This result did not change after adjustment for maternal age, maternal smoking and infant gender (p = 0.27) (Table p = 0.19] and via emergency CD  compared to those born through VD [median = 82.2 (73.6-97.4)] (Table The median LINE-1 methylation for the sample population was 82.0% range: 73.6-97.4), and the mean was 81.6% . Type of delivery was not associated with methylation of any individual LINE-1 site (Table p = 0.02). This was seen in both crude and adjusted models of smoking.Comparing individual sites within LINE-1, sites 2, 3 and 4 all had significantly lower methylation than site 1 (p = 0.01). The coefficient of variation for LUMA was 5.86%, and the coefficient of variation for LINE-1 was 2.82%. When statistical analyses were restricted to individuals with both measures (n = 392) similar results were observed (data not shown).LUMA and LINE-1 measurements were negatively correlated : luminometric methylation assay; (CD): cesarean delivery; (VD): vaginal delivery; (DOHaD): developmental origins of health and disease.The authors declare that they have no competing interests.VP serves as the principal investigator of this birth cohort and the IRB protocol. VP, SED and SH identified subjects and collected cord blood immediately after birth. VP, DCD and LSR conceived of the study question and assisted in data analysis, interpretation and conclusions. SV analyzed data and drafted the manuscript. DCD and LSR supervised laboratory and statistical analyses. TRJ isolated DNA and conducted LINE-1 methylation assays. MSN conducted LINE-1 and LUMA assays. All co-authors assisted in data interpretation and manuscript revisions. All authors read and approved the final manuscript.


Although evidence indicates that fatty acids (FA) can affect insulin resistance (IR), not all FA contribute equally to the process. Indeed, monounsaturated FA (MUFA) and polyunsaturated FA (PUFA) are reported to reduce IR, whereas saturated FA (SFA) and trans FA appear to increase IR. However, it is not yet clear how individual FA are associated with markers of IR, and whether these relationships are influenced by ethnicity and/or sex. Therefore, the goal of this study was to examine the ethnic- and sex-specific relationships between plasma FA and markers of IR in a cohort of healthy young Caucasian, East Asian, and South Asian adults.Gas chromatography was used to quantify fasting plasma FA from young Canadian adults (22.6 ± 0.1 yrs) of Caucasian (n = 461), East Asian (n = 362), or South Asian (n = 104) descent. Linear regression models were used to investigate associations between plasma FA and markers of IR  according to ethnicity and sex.P < 0.05, adjusted for multiple testing) were identified between individual FA and markers of IR, with the majority identified in Caucasians. For SFA, positive associations were found between 14:0 and fasting insulin and HOMA-IR in Caucasian and East Asian populations, and 18:0 and fasting glucose in Caucasians only. Several positive associations were also found for specific MUFA  and PUFA (18:2n6 with fasting glucose and 18:2c9t11 with HOMA-IR) in Caucasian adults only. Most of the aforementioned associations were stronger in males compared to females. Interestingly, no significant associations were found between FA and markers of IR in South Asian adults.Numerous significant associations (We report numerous associations between plasma FA and markers of IR in Caucasian and East Asian populations, but not in South Asian individuals. Furthermore, these associations appeared to be more robust in men. This demonstrates the importance of investigating associations between FA and markers of IR in an ethnic- and sex-specific manner in order to better understand the contribution of plasma FA to the development of IR and type-2 diabetes. Type-2 diabetes (T2D) is a health concern affecting 285 million individuals worldwide, and is expected to affect 439 million individuals by 2030. T2D is et al. also demonstrated that individuals of South Asian, African-Caribbean and European origins differ significantly in their habitual dietary intake  and that these characteristics are associated with differences in basal insulin sensitivity and secretion[While several excellent cross-sectional studies have examined the relationships between FA and markers of cardiometabolic risk-15, it iecretion. Such etFurthermore, evidence also demonstrates that plasma FA profiles vary between men and women,20. Thes2, did not complete a food frequency questionnaire (FFQ), or were diagnosed with diabetes or cancer. The TNH study was approved by the Research Ethics Boards at the University of Toronto and the University of Guelph. All subjects provided informed written consent.Participants recruited from the University of Toronto campus (2004–2009) for the Toronto Nutrigenomics and Health (TNH) study were used for the current investigation,24. Parthttp://www.dtu.ox.ac.uk/homacalculator/index.php).Plasma samples were collected following a 12 hour overnight fast, and were used for analysis of glucose and insulin at LifeLabs Laboratories . The homeostatic model assessment of insulin resistance (HOMA-IR) was calculated using the HOMA Calculator v2.2.2 (Subjects were asked to complete a one-month 196-item food frequency questionnaire (FFQ), adapted from the Willet questionnaire,25. This3-MeOH and hexane were added to samples, prior to methylation, for 1 hour at 100°C. After samples had cooled, double-distilled water was added, and samples were centrifuged at 1000 rpm for 10 minutes. The upper-layer was extracted and dried under nitrogen gas, prior to reconstitution in hexane. Samples were analyzed using an Agilent Technologies 7890A GC system . All FA peaks were identified by comparison to retention times of FA methyl ester standards. Only those FA that were consistently detected across all plasma samples were considered for the current study (i.e. 24 of the 62 FA that can be measured by GC). Absolute FA values were calculated by comparing individual FA peaks to the internal standard and are reported as μg FA / mL plasma ± SE.Fasting plasma samples were assessed using gas chromatography (GC) as previously described. BrieflyP-value < 0.05 was considered statistically significant.All statistical analyses were conducted with JMP Genomics software Version 5.1 . The Shapiro-Wilk test was used to assess the distribution of all variables. Fasting insulin was subsequently log transformed for all analyses. Fourteen outliers were identified by performing a Jackknife test and removed prior to the analyses. Baseline anthropometric, clinical, and FA data were examined using a Kruskal-Wallis analysis of variation (ANOVA) test followed by a post-hoc Mann–Whitney-Wilcoxon test. Individual associations between FA and markers of IR  were examined using mixed-effects linear regression models, which accounted for both fixed effects  per day) and one random effect (date of GC processing). For analyses of sex-specific associations, sex was removed from the aforementioned list of variables. We used a two-step process to determine statistical significance: (1) ethnic-specific associations between FA and markers of IR were considered significant only if they satisfied a Bonferroni correction for multiple testing; followed by (2) an investigation of sex-specific differences for those FA that were statistically significant in the previous step. A Anthropometric and clinical characteristics of the 927 study participants are shown in Table Our GC platform is able to detect 62 distinct FA; however, for the current study we only considered FA that were consistently detected in all plasma samples. Using this judicious approach, we investigated associations between 24 individual FA  and HOMA-IR . Similar associations were also seen in the East Asian population, where 14:0 was positively correlated with both fasting insulin  and HOMA-IR ; however, these associations were borderline significant when adjusting for multiple testing. After separating the Caucasian and East Asian populations by sex, 14:0 remained positively associated with insulin in Caucasian men and women  and East Asian men . The relationship between 14:0 and insulin was borderline significant in East Asian females . For HOMA-IR, 14:0 remained positively associated in both Caucasian men and women  and East Asian men and women . No associations were seen with 14:0 in the South Asian group.Several SFA were examined  for associations with markers of IR Tables ,4 and52 = 0.10; P = 0.0007) in the Caucasian population. Furthermore, this association remained significant after separating Caucasians into men  and women . No significant associations were found between 18:0 and markers of IR in the East Asian or South Asian populations.We also found that 18:0 was positively associated with fasting glucose levels . Separating Caucasians by sex revealed that the positive association between 18:1t11 and HOMA-IR remained significant in both men  and women . No significant associations were detected between 18:1t11 and markers of IR in East Asians or South Asians. Additionally in the Caucasian population, 18:1t6-8 was positively associated with HOMA-IR , and this relationship also remained significant in both Caucasian men  and women . Similarly, this relationship was not observed in East Asians or South Asians.Numerous MUFA were examined for associations with markers of IR, including 16:1c9, 18:1t6-8, 18:1t9, 18:1t10, 18:1t11, 18:1c9, 18:1c11 and 18:1c12 Tables ,4 and52 = 0.09; P = 0.0017); however, this relationship remained significant only in men , and not women , when separated by sex. No associations were found between 18:1c9 and markers of IR in East Asians or South Asians.Next, we found that 18:1c9 was positively associated with fasting glucose in the Caucasian population , while 18:2c9t11 was positively associated with HOMA-IR . Further sex-specific investigations demonstrated that the positive association between fasting glucose and 18:2n6 remained significant in both men  and women . The positive associations between 18:2c9t11 and HOMA-IR also remained significant in both men  and women . No associations with these PUFA were found in South Asians or East Asians.Twelve PUFA were examined for their associations with markers of IR  . This finding seems to agree with past work in which differences in glucose metabolism between South Asian and European individuals residing near San Francisco  or southeast England were reported[Of additional interest, we noted that South Asian subjects had significantly higher levels of fasting insulin and glucose concentrations compared to Caucasian subjects despite having a similar BMI  and phospholipid (PL) fractions of red blood cells (RBC) and markers of IR  in 38 young men and women[et al. suggest that, across multiple ethnicities and within both genders, 14:0 appears to be strongly correlated with markers of IR.Of the SFA examined, 14:0 (myristic acid) was positively associated with fasting insulin and HOMA-IR in the Caucasian population, and with fasting insulin in the East Asian population. Furthermore, these associations remained significant when examining men and women separately within each ethnicity. This agrees with past work by Lovejoy nd women. Lovejoynd women. Togetheet al. who demonstrated that the relative abundance of 18:0 in RBC was positively associated with fasting glucose[et al. found that 18:0 in CE and PL fractions were positively associated with the development of diabetes in middle-aged adults[We also found that 18:0 (stearic acid) was positively associated with fasting glucose and HOMA-IR in men and women in our Caucasian population. Similar results were reported by Ebbesson  glucose. Additiod adults. Althouget al. demonstrated that the relative abundance (expressed as % FA) of 16:1n9  in the PL fraction of plasma was positively associated with markers of IR in men , but not women, in a European-majority multi-ethnic population[Previous work by Mozaffarian pulation. We coulet al. demonstrated that diets rich in 18:1c9 can reduce IR in T2D adult males[et al. revealed a positive association between dietary 18:1c9 and IR[et al. and Hekmatdoost et al., who both reported no evidence of a relationship between 18:1c9 and markers of IR[We observed a positive association between 18:1c9 (oleic acid) and fasting glucose in both Caucasian and East Asian populations; however, this association appeared to be stronger in Caucasians. Interestingly, previous studies examining the relationship between 18:1c9 and IR have generated inconsistent results. For example, Ryan lt males, while M9 and IR. Even furs of IR,35. A noInterestingly, we observed associations between 18:1t6-8  and HOMA-IR in the Caucasian population, which to our knowledge has not been previously reported. Furthermore, this association appeared to be stronger in men compared to women. Although we adjusted our regression models to account for total fat intake, we acknowledge that FFQ can introduce inaccuracies in self-reporting. Therefore, we cannot exclude that this sex difference in 18:1t6-8 may be related to differences in dietary habits (e.g. ruminant milk and meat consumption) that weet al. recently reported that healthy young Japanese men and women (22.8 ± 3.0 yrs) supplemented with 0.6% trans-FA for 4-weeks demonstrated no effect on fasting measures of insulin and glucose[We also found that 18:1t11 (trans-vaccenic acid) was positively associated with HOMA-IR in Caucasians, but not in East or South Asian populations. This finding is in agreement with a previous study reporting that vaccenic acid levels isolated from serum triglycerides were positively associated with fasting insulin and HOMA-IR in Caucasian males classified as either normoinsulinaemic/normoglycaemic or hyperinsulinaemic/hyperglycaemic. Convers glucose. While tet al. observed that 18:2n6 in serum CE was lower in diabetic patients compared to healthy controls[In the PUFA class, we found that 18:2n6 (linoleic acid) was positively associated with fasting glucose in our Caucasian population, but not in the East Asian or South Asian groups. Previous studies investigating the relationships between 18:2n6 and markers of IR have often generated inconsistent results. For example, several previous studies have found no evidence of an association between 18:2n6 and markers of IR in diabetic Japanese adults or overwcontrols. Our datWe also found that 18:2c9t11  was positively associated with HOMA-IR in Caucasians. These results appear to conflict with previous findings demonstrating a beneficial effect of 18:2c9t11 on insulin sensitivity in middle-aged healthy adults; howeverWe acknowledge that there are several limitations to consider with the present study. Firstly, we did not separate total FA into individual lipid classes, which would provide further insight into dietary habits (i.e. namely FA consumption) in relation to ethnicity and sex. Thus, we were unable to determine whether a FA in a specific lipid fraction was driving the associations with markers of IR. Future investigations examining FA and markers of IR should consider separating total FA into distinct lipid classes, as well as considering different biological materials (e.g. serum or erythrocytes) in order to better understand the relationships between FA and IR. Secondly, the smaller sample size for the South Asian population may have limited our ability to detect potential associations in this ethnic group, and further studies in larger populations are still required. Thirdly, it is important to note that the current study was performed in young healthy adults. As such, it remains to be seen whether these relationships also exist in at-risk (e.g. obese and T2D) populations. Finally, our cross-sectional analysis did not allow us to draw conclusions on cause and effect. To address this, prospective studies are required to examine whether the plasma FA profile in young adults can predict risk for developing T2D later in life.In summary, we have demonstrated that circulating levels of specific FA appear to be positively associated with markers of IR. Moreover, we have revealed potential ethnic-differences regarding these relationships . Our data also suggests that the associations identified may be stronger in men compared to women. This suggests that underlying sex-specific differences  may be influencing these associations. Taken together, our results illustrate the significance of investigating associations between FA and IR in an ethnic-specific and sex-specific manner in order to fully understand whether specific plasma FA could contribute to the development of IR and T2D.ALA: α-linolenic acid; BMI: Body mass index; CE: Cholesterol ester; CLA: Conjugated linoleic acid; DHA: Docosahexaenoic acid; EPA: Eicosapentaenoic acid; FA: Fatty acid; FFQ: Food frequency questionnaire; HOMA-IR: Homeostatic model assessment of insulin resistance; IR: Insulin resistance; LA: Linoleic acid; MUFA: Monounsaturated fatty acid; PA: Physical activity; PL: Phospholipid; PUFA: Polyunsaturated fatty acid; RBC: Red blood cells; SFA: Saturated fatty acid; T2D: Type 2 diabetes; TNH: Toronto nutrigenomics and health (study).The authors declare that they have no competing interests.JCR, MAZ, and DMM designed the study, conducted the analyses, and wrote the manuscript. DEN and AES established the TNH study cohort and collected DNA samples from all participants. SC and DWLM carried out plasma fatty acid analyses. All authors read and approved the final manuscript.


These results suggest that the Kidney-Tonifying Recipe is capable of repairing asthma-associated alterations in endocrine function and the ultrastructure of AMCC.Traditional Chinese medicine suggests that renal deficiency is a causative factor of asthma, and tonifying kidney drugs are believed to be an appropriate and beneficial treatment. The adrenal medullary chromaffin cells (AMCC) transition to the neuronal phenotype is known to occur in asthma, as evidenced by degranulation of chromaffin granules, decline of epinephrine (EPI) and phenylethanolamine-n-methyl transferase (PNMT), and obvious alterations in cellular architecture. In this study, rats were sensitized and challenged with ovalbumin, then treated with Kidney-Tonifying Recipe (KTR) to evaluate the therapeutic effect. Tissues were evaluated for changes in pathology and EPI, PNMT, and peripherin expression. Degranulation of chromaffin granules and appearance of neurite-like process were found in AMCC from asthmatic rats, and these changes were corrected by KTR treatment. EPI and PNMT expressions were decreased in asthmatic rats and increased by KTR treatment. Peripherin expression was increased in asthmatic rats and decreased in the KTR-treated group. Morphological changes and decreases in EPI were observed when cultured AMCC were exposed to sera from asthmatic rats  Traditional Chinese medicine (TCM) considers renal deficiency as a principal cause of asthma. A tonification strategy for kidney is often used to treat asthma in TCM, and many consider this to be a highly effective therapeutic approach , 2. ReceAsthma attacks are induced by the dysfunction of cholinergic and adrenergic nerves and their cognate receptors. Since there is a lack of adrenergic nerves innervating the human airway smooth muscles, the relaxation of airways is mainly regulated by epinephrine (EPI) binding to adrenergic receptors. Adrenal medullary chromaffin cells (AMCC) are the primary cells that secrete EPI , 7, and The role and mechanisms underlying the beneficial effects that are observed upon use of tonifying kidney drugs are unclear. We hypothesized that tonifying kidney drugs affect the structure and function of AMCC. In this study, asthmatic rats were treated with a classic TCM formula known as Kidney-Tonifying Recipe (KTR), and the effects of the kidney-tonifying drugs on the alteration of structure and function of AMCC in asthma were investigated.Rehmannia glutinosa (Shudi), oriental water plantain rhizome (Zexie), monkshood (Fupian), medicinal Indian mulberry root (Bajitian), Common Yam Rhizome (Huaishan), Indian Buead (Fuling), cortex cinnamomi (Rougui), salvia chinensis (Danpi), cistanche (Roucongrong), and Cornus officinalis (Zaopi). All of these natural drugs were provided by the Department of Pharmacy, Xiangya Hospital, Central South University. These drugs were mixed at a ratio of 10 : 10 : 3 : 10 : 10 : 10 : 10 : 10 : 10 : 6, and then boiled for 30 minutes three times in distilled water at a volume of 10-fold the dry weight of the herb mixture. The solution was heated until the drugs reached a concentration of 1.33 g/mL (crude drug/solution volume).KTR is composed of All animals used in this study were conventionally bred 6- to 8-week-old male Sprague-Dawley rats . This study was carried out in strict accordance with the recommendations from the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health, USA. The protocol was approved by the Ethics Committee at the Asthma Research Institute, Hunan Province.n = 8 per group): control rats (control), asthmatic rats (asthma), and asthmatic rats treated with KTR (KTR). The rats were treated as follows: on day 0 and day 7, the asthma and KTR groups were sensitized with an intraperitoneal injection of 100 mg of ovalbumin (OVA) , 200 mg of aluminum hydroxide (Sigma), and 6 × 109 heat-killed Bordetella pertussis  in 1 mL of sterile saline. The control rats were sham sensitized by using an intraperitoneal injection of sterile saline without OVA. The sensitized rats were exposed to 30 minutes of 1% OVA (wt/vol) aerosol every day from day 14 to day 28, while the control rats were exposed to aerosolized sterile saline only. KTR-treated rats received a gastric gavage with the KTR solution (10 g/kg) 40 minutes prior to exposure. Control and asthmatic rats received a gastric gavage with the same volume of distilled water into the stomach at 40 minutes before exposure.Twenty-four rats were divided into three groups using a random digits table . Rats were treated for 2 minutes with aerosolized saline or increasing doses of histamine generated by an ultrasonic nebulizer, and airway resistance was measured. Histamine-induced bronchoconstriction was calculated as the index of percent increase in airway resistance as compared to the peak of the reaction with baseline airway resistance [sistance .μm filter and stored in −20°C until use. The left adrenal glands were excised and fixed in 4% paraformaldehyde or in 2% glutaraldehyde stored at −80°C until use. After pulmonary artery perfusion, the right middle lung lobe from each rat was also fixed in 4% paraformaldehyde and stored at −80°C.After determination of bronchial responsiveness, blood from each rat was obtained by cardiac puncture, and the serum was inactivated by heating at 56°C for 30 minutes, then filtered through a 0.22 Medium A was prepared as a mixture of 85% DMEM serum-free medium  and 15% serum from experimental rats. According to the origin of serum, three Medium A subgroups were generated: serum from control rats (control serum), serum from asthmatic rats (asthma serum), and serum from KTR-treated rats (KTR serum). Medium B was prepared as a mixture of 15% fetal bovine serum  and 85% DMEM serum-free medium.6/mL and incubated at 37°C. Medium was changed after 24 hours, and then every 48 hours thereafter, to remove dead cells and cell debris.Adrenal glands were obtained from conventionally bred 6- to 8-week-old male Sprague-Dawley rats. Medullae were carefully freed from the capsular and cortical tissue under sterile conditions and incubated with Hanks' balanced salt solution. Subsequently, medullae were dissociated with 0.1% type I collagenase dissolved in glucose-enriched phosphate-buffered saline for 40 minutes. After harvesting by centrifugation and resuspending in culture medium B, the cells were adjusted to a cell concentration of 5-6 × 10The cultures in medium B were maintained for a total of 5 days and divided into the three experimental groups for treatment with one of the different medium A subgroups described above. Seventy-two hours after the AMCC cells were treated with medium A , the cells and the supernatants were collected for subsequent analysis.μm) were stained with HE, and the morphological changes of lung and adrenal medulla were observed under a light microscope. The airway wall thickness was quantified by pathology image analysis system.The tissue of lung and adrenal medulla were fixed in 4% paraformaldehyde and then embedded in paraffin. Tissue sections  from the different specimens were stained with uranyl acetate and lead citrate and examined with an H-7500 transmission electron microscope . Ultrastructural changes were assessed by pathologists who were blinded with respect to treatments.Adrenal medulla and AMCCs were fixed with 2% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2. Three hours later, specimens were postfixed in buffered 1% OsOThe levels of EPI, corticosterone, and NGF in serum or cellular supernatant were quantified by commercially available ELISA kit and antibodies, according to the protocol provided by the supplier . Immunoreactivity was determined by an ELISA reader at 450 nm.Total RNA was extracted from tissues or cells using Trizol reagent  according to the manufacturer's instructions. For RT-PCR reactions, primer sequences were designed that yielded 137 and 307 bp products of peripherin and GAPDH, respectively. The gene-specific primer sequences were as follows: 5′-GGTGGAGGTAGAGGCAACA-3′ (forward) and 5′-TCGGACAGGTCAGCGTATT-3′ (reverse) for peripherin, and 5′-TGAACGGGAAGCTCACTGG-3′ (forward) and 5′-TCCACCACCCTGTTGCTGTA-3′ (reverse) for GAPDH. The PCR amplification program consisted of preheating at 94°C for 5 minutes and 35 cycles of denaturing , annealing , and extension , followed by a final extension at 72°C for 10 minutes. The PCR products were electrophoresed on a 2% agarose gel to confirm size. Results were normalized to GAPDH, which was used as an internal control.μm) and mounted on slides. The tissue sections were then deparaffinized in toluene and rehydrated in a decreasing ethanol series. Endogenous peroxidase activity was blocked with 3% H2O2 for 10 minutes. The sections were then incubated with polyclonal rabbit antibodies against rat (anti-phenylethanolamine N-methyl transferase (PNMT), 1 : 2500; antiperipherin, 1 : 150; both from Millipore, USA) at 4°C overnight and followed by incubation with biotin-labeled goat anti-rabbit immunoglobulin (Ig)G  for 20 minutes. The sections were then treated with the streptavidin-peroxidase kit  and developed with DAB substrate. Counterstaining was performed with hematoxylin. Afterwards, the sections were incubated with nonimmune rabbit sera for negative controls. The results were observed under a light microscope (100x) and the average optical density was calculated by using the Image Pro Plus 6.0 software (IPP6.0) for subsequent statistical analysis.After embedding in paraffin at 4°C overnight, the tissues were sectioned . Thirty micrograms of protein were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were then electrotransferred to polyvinylidene fluoride (PVDF) membrane at 120 V for 1.5 hours. Nonspecific binding was blocked by incubating with 0.05 g/mL skim milk powder at room temperature (20°C) for 2 hours, and then membranes were incubated at 4°C overnight with rabbit anti-rat peripherin polyclonal antibodies . After washing with PBST, the membranes were incubated at 37°C for 1 hour with secondary antibodies  and visualized by using the enhanced chemiluminescence reagent system . Results were normalized to GAPDH, which was used as an internal control.P value of less than 0.05 was considered statistically significant.Data are presented as mean ± standard deviation P < 0.05). KTR treatment attenuated the airway resistance in asthmatic rats (P < 0.05)  .μm2/μm) was thicker than that in control rats (18.88 ± 3.36 μm2/μm) (P < 0.05), and this effect was alleviated in KTR-treated rats   .Compared with the control rats, asthmatic rats presented with vacuolar degeneration, tissue edema, and increased lipid in AMCC, as observed under light microscope. However, these lesions were reversed by KTR treatment Figures .Electron microscopy revealed a regular morphology of AMCC in control rats, which contained a clear structure of mitochondria and abundant chromaffin granules. Lesions were evident in asthmatic rats, including swollen cytoplasm and mitochondria, and decreased chromaffin granules. These pathological changes were relieved in the KTR-treated rats Figures .P < 0.05), as evidenced by ELISA. Compared with asthmatic rats, the EPI levels increased significantly in KTR-treated rats (P < 0.05) (P < 0.05), and corticosterone levels were further increased in asthmatic rats treated with KTR (P < 0.05) (P < 0.05), but this response was attenuated in asthmatic rats by KTR treatment (P < 0.05) (EPI levels in asthmatic rats were lower than in control rats ( < 0.05) . Cortico < 0.05) . NGF lev < 0.05) .P < 0.05), while a further enhanced expression was found in asthmatic rats treated with KTR (P < 0.05) . However, KTR treatment inhibited the expression of peripherin in asthmatic rats (P < 0.05) , as evidenced by RT-PCR and western blot, respectively. Compared with the asthmatic rats, peripherin mRNA and protein expression was inhibited by KTR treatment (P < 0.05) , as evidenced by ELISA. This was reversed by KTR serum treatment (P < 0.05)  .P < 0.05), as evidenced by RT-PCR and western blot, respectively. This increase was inhibited by KTR serum (P < 0.05)  plays an important role in cellular architecture through regulating axon formation, thus giving nerve cells their unique morphology . The impin vivo and inhibit the transformation of AMCC into neurons [PNMT, the rate-limiting enzyme that catalyzes nor-EPI to EPI, is mainly expressed in adrenergic chromaffin cells, rather than in nerve cells. Decreases in PNMT mediate the transition from AMCCs to neurons. Glucocorticoids are known to promote the expression of PNMT in the adrenal medulla –28. In o neurons , 29, 30. neurons , 31.In the current study, PNMT expression was found to be decreased in asthmatic rats, consistent with decreased EPI and increased NGF levels, and KTR treatment inhibited these changes. Despite increased corticosterone levels in asthmatic rats, there remained a trend for NGF-induced transformation of AMCC. The further increase in corticosterone in asthmatic rats treated by KTR may act as a compensatory mechanism, as supplementation with exogenous glucocorticoids is a highly effective treatment in asthma patients. To some extent, these data suggest that the effect of KTR in treating asthma is dependent on maintaining the phenotype of AMCC, which may be ascribed to correcting the imbalance of NGF and glucocorticoids; as a result, EPI concentrations would be expected to be increased, thus relieving bronchospasms.in vitro and treated them with sera from control, asthmatic, or KTR-treated rats. The results demonstrated that treatment with sera from asthmatic rats caused a decrease in the density of chromaffin granules and development of a synapse-like cellular structure. In addition, asthmatic sera also caused a concurrent decrease in EPI and increase in peripherin. These changes were attenuated in AMCCs treated with the sera from KTR-treated rats.To test this hypothesis, we cultured AMCC In summary, it can be concluded that administration of KTR to asthmatic rats leads to upregulation of EPI and prevention of AMCC transformation to neurons, thus allowing for normal endocrine function.


Wasmannia auropunctata (Roger) (Hymenoptera: Formicidae), both employing the pheromone attractant 2,5-dimethyl-3-(2-methylbutyl)pyrazine (2-MeBu-diMePy), were compared with peanut butter based detection, in order to evaluate differences in species specificity and detection reliability. Trapping was conducted using a transect through a macadamia orchard on the island of Hawaii. The transect consisted of a series of three-tree plots, each plot contained a peanut butter coated stick (the most common detection method used for W. auropunctata in Hawaii), a one—way trap treated with 2-MeBu-diMePy, and a piece of double-sided tape treated with 2-MeBu-diMePy. While there were no differences in the number of W. auropunctata counted with each detection method, and no differences in detection reliability (detecting the known presence of W. auropunctata in a plot), the pheromone—incorporating methods showed greater species specificity, retaining W. auropunctata almost exclusively. These results demonstrate the potential of pheromone—detection methods to selectively capture target ant species even when other ants are present and abundant. Combined data from all three detection methods and a previous visual survey along the transect showed a marked difference in the frequency of cohabitation among ant species. Of the 10 ant species collected, W. auropunctata was found as the sole ant species on a given tree at a significantly higher frequency than all other ant species except Pheidole fervens. 94% percent of the trees with W. auropunctata had only W. auropunctata, supporting previous observations that this species tends to displace other ant species. In addition, W. auropunctata microhabitat preferences were investigated using one—way traps containing 2-MeBu-diMePy, which were placed in three arboreal and three non—arboreal locations. While the number of ants captured did not differ by trap placement, when grouped, captures were significantly higher in arboreal than non-arboreal microhabitats.Two detection methods for the little fire ant,  Wasmannia auropunctata (Roger) (Hymenoptera: Formicidae) has negatively impacted both biodiversity and agriculture pyrazine (2-MeBu-diMePy) and 3-methyl-2-(2-methylbutyl)pyrazine (2-MeBu-MePy)  . In fielBu-MePy) . The strW. auropunctata were observed to arrest around the adhesive area for up to eight days  with some means of capture  . In a prght days . Ants prW. auropunctata. It is expected that other small ants will not be attracted by the pheromone and will have little reason to explore the trap while larger ants will be excluded from the trap by the small hole size in the weedmat. The combination of a one— way trap and an attractant has previously been used to trap tephritid fruit flies in Hawaii, demonstrating the feasibility of this trapping technique , from 9 June 2010 to 6 July 2010 . AverageS)-2-methylbutyl)pyrazine (S2MeBu-diMePy) was used in Experiment 3. This compound was prepared from (S)-1-bromo-2-methylbutane and 3-chloro-2,5-dimethylpyrazine  using previously described methodology . Lures used in Experiments 3 and 4 were made by loading 1 mg of pheromone in 100 µL CH2Cl2 onto rubber septa with a glass syringe. Controls were loaded with 100 µL CH2Cl2 only.A single enantiomer of 2-MeBu-diMePy, 2,5-dimethyl-3- with a 5 × 5 cm square cut from the middle. Conically—perforated weed mat  was affixed over the opening with hot—glue. A push pin was glued onto the outside of the Petri dish to secure the trap to trees at test sites. The top part of the trap consisted of a second Petri dish bottom, with a rubber septum  glued onto the inside surface, which fit snugly onto the bottom half of the trap. The rubber septa were treated with pheromone in the field, and trap halves were fitted together immediately before each experiment.One—way traps were assembled as follows . The botwww.3m.com) with a rubber septum stuck to the center of the outward—facing surface. The tape was precut and rubber septa were applied in the laboratory. The rubber septa were treated with pheromone in the field immediately before each experiment.The sticky trap was made from a 3.5 × 3.5 cm square of double—sided outdoor carpet tape  of the number of ants stuck to each piece of tape was recorded. Evaluated tapes were: duct tape (3M), electrical tape (3M), blue painters tape (3M), outdoor carpet tape (3M), clear string reinforced tape (3M), label tape , scotch tape (3M), brown packing tape (3M), masking tape (3M), double—sided poster tape (3M), and double—sided scotch tape (3M).Experiment 1 assessed different sticky catch materials for possible use in combination with a pheromone attractant. Specifically, 10 different types of tape were compared for their effectiveness in capturing W. auropunctata within the transect, additional plots were selected every third row in areas with observed W. auropunctata populations. In areas without W. auropunctata, plots were chosen from every 10th row. This increased the number of plots with W. auropunctata to roughly one—third of the total test plots. During the survey, an isolated population of W. auropunctata was found (plot 30). In this instance, the three trees selected were positive for W. auropunctata, and adjacent plots were 10 rows apart. GPS coordinates were recorded for all selected trees in each plot. Ants were identified to species in the laboratory based on adult morphology using several keys , double—sided tape with a pheromone attractant, and a peanut butter—coated popsicle stick. These detection devices were deployed, one per tree, within the plots previously selected in Experiment 2. The order in which the detection devices were deployed was rotated within each sequential plot. Detection methods were deployed in the early morning and were collected in plastic bags after 24 hours. After retrieval, traps were frozen and ants were identified and counted.Experiment 3 compared the species specificity and detection reliability of the three potential W. auropunctata microhabitat preferences in order to determine optimal one-way trap placement. Replicate plots were selected in areas containing W. auropunctata. Each replicate plot contained arboreal placements in macadamia trees, which were subdivided into branch, crook (branch/trunk junction), and the foot of the tree, and non—arboreal, which were subdivided into ground (under tree canopy), grass , and scrub (overgrown areas adjacent to the orchard). Traps were deployed in six replicate plots for 24 hours before being collected in plastic bags and frozen. Ants were then counted in the laboratory.Based on the success of the one—way trap detection method used in Experiment 3, Experiment 4 aimed to investigate W. auropunctata (based on data from Experiment 2) without capturing other species. Differences in species specificity between detection methods were analyzed using Fisher's exact test. Ant species diversity (cohabitation) within plots was analyzed using Fisher's exact test, since many of the species had small sample sizes. Initially, W. auropunctata cohabitation was compared to all other ant species by collapsing the ant species categories into a 2 × 2 contingency table. Cohabitation for all ant species were then compared in 2 × 2 contingency tables with Fisher's exact test. Multiple hypothesis testing error was measured by false discovery rate (FDR) using the False Discovery Rate Tool . The number of W. auropunctata captures with each detection method (Experiment 3) were analyzed using ANOVA followed by Tukey's HSD test (α = 0.05) to compare means. The number of other ant captures with each detection method (Experiment 3) were not homogeneous (Levene's test) and were analyzed by Kruskal—Wallis test followed by pair—wise comparisons with Mann—Whitney U test to compare medians. The number of W. auropunctata trapped in different microhabitats (Experiment 4) was not homogeneous (Levene's test) and wasanalyzed by Kruskal—Wallis test. Additionally, for the vegetation survey, arboreal and non—arboreal placements were grouped and compared with a Mann—Whitney U test. All analyses of significance were made at the p < 0.05 level. Statistical analyses other than FDR were performed using SPSS version 15.0 .Data from transect visual survey (Experiment 2) and trapping comparison (Experiment 3) were mapped using GIS software. For detection method comparisons, species specificity is defined as detecting the known presence of W. auropunctata. Tape types that retained ants (2–500 ants per piece of tape) were clear string reinforced, outdoor carpet, colored label, duct, masking, and electrical (ranked highest to lowest). Of these, the double—sided outdoor carpet tape was chosen for further testing based on the comparatively high number of ants captured and the ease of field placement for testing. Tapes that did not retain ants were scotch tape, blue painter's tape, double—sided poster tape, double—sided scotch tape, and brown packing tape. In several instances, ants were observed to walk unimpeded across this latter group of tapes.Experiment 1 compared 10 types of tape for capturing and retaining Brachymyrmex obscurior Forel, Cardiocondyla obscurior Wheeler, Leptogenys falcigera Roger, Monomorium floricola (Jerdon), Pheidole fervens Smith, Solenopsis papuana Emery, Tapinoma melanocephalum (Mayr), Technomyrmex sp. (vitiensis (Emery) and/or albipes Mann), Tetramorium bicarinatum (Mayr), and W. auropunctata (Roger). With the exception of plots 30 and 39, W. auropunctata was found exclusively in the western end of the transect   .W. auropunctata detection methods , the number of ants caught per tree did not vary significantly between detection methods    were recovered from the ground, some of which no longer had peanut butter. Presumably they were knocked down by vertebrate wildlife  and licked clean. The one—way trap and double—sided tape captures were 98% and 83% species specific (detecting the known presence of W. auropunctata without capturing other species), respectively. While there were no significant differences between the species—specificity of captures with the one—way trap or double—sided tape captures (p = 0.0571), they were both significantly more selective than peanut butter (39%) (p < 0.01 for both).Experiment 3 compared three  methods  and 6. I= 0.558) . In plot < 0.01) . In one punctata . There wW. auropunctata microhabitat preferences with one-way trap placement . When trap placements were grouped as arboreal or non—arboreal, the tree-associated traps caught significantly more W. auropunctata than those located further from the tree .Experiment 4 investigated lacement . WasmannW. auropunctata detection (Experiment 1) suggested that tested tapes fall into two groups; materials which W. auropunctata will traverse and sometimes become stuck to, and those which they will traverse without becoming stuck. A third group of materials are those that deter W. auropunctata from traversing, presumably because they are highly adhesive and are perceived as such by the ants. An example of this third group of sticky materials is Tanglefoot, which acts as a barrier that W. auropunctata will not cross or become entangled in when 2-MeBu-diMePy is present as an attractant  supports previous research describing the ability of  species . Of all ven tree . The majW. auropunctata detection methods (Experiment 3) showed increased species specificity when the pheromone 2-MeBu-diMePy was used in combination with a one—way trap or double—sided tape  made field testing problematic. Similar problems were encountered in W. auropunctata surveying attempts using peanut butter in the Galapagos  to aid in control. Surveys for ant taxa diversity commonly rely on pitfall traps, baited traps, and leaf—litter extractions (or some combination thereof) for collections . This reW. auropunctata monitoring. From a design standpoint, the simplicity of the double—sided tape is appealing. However, over longer test periods the sticky surface is likely to become covered with detritus from the environment, which would decrease the capacity to retain attracted W. auropunctata. The one—way trap has the advantage of excluding insects ∼1 mm and much of the environmental detritus, which might affect the tape. Additionally, even though species specificity was not significantly different between these two detection methods, it is our opinion that the one—way trap has the potential to be more selective long—term. The one—way trap also has the potential to capture many more ants given the larger volume inside the trap. Beyond use with W. auropunctata, this trap has the potential to be used with other ant pheromones, providing a model for species specific monitoring of small (1–2 mm) ants.While the one—way trap and the double—sided tape performed similarly in the field, there are differences between the two methods that should be considered in assessing which is better suited for W. auropunctata, one—way traps in trees or near the foot of macadamia trees caught more ants than those on the ground or in grass or shrub thickets. This speaks to the near ubiquity of W. auropunctata in infested areas, and the preference of W. auropunctata populations for tree-based microhabitats in the macadamia orchard.Results from the assessment of several microhabitats within the macadamia orchard (Experiment 4) suggested that the optimal location for one—way trap placement is in trees. Although all traps caught


An 18-year-old man presented with altered mental status. He was found to have diabetic ketoacidosis. Chest X-ray showed pneumomediastinum. After intubation for air-way protection, an oral-gastric tube was placed. A chest computed tomography scan showed the tip of the oral-gastric tube to be in the right hemithorax. The patient underwent a thoracotomy and was managed in the intensive care unit. Both diabetic ketoacidosis and Boerhaave's syndrome should be considered as possible causes of pneumomediastinum in a patient with similar presentation. Boerhaave's syndrome should be ruled out prior to the insertion of an oral-gastric tube to avoid further morbidities. An 18-year-old man was brought to the emergency department (ED) because of altered mental status. Family reported that he had nausea and vomiting before presenting to the ED. Past medical history was significant for diabetes, diabetic ketoacidosis (DKA), and hypothyroidism. Past surgical and social histories were unremarkable. Home medication was insulin glargine.Physical exam revealed a lethargic patient. Vitals were blood pressure 130/70 mmHg, pulse 96/minute, respiration 28/minute, temperature was normal. Lung auscultation showed left basal crackles. 2 16 mmHg (35–45), PO2 138 mmHg (81–110), and serum bicarbonate 3 mmol/L (22–26); serum electrolytes: sodium 129 mmol/L (136–144), potassium 6.4 mmol/L (3.3–5.1), and chloride 100 mmol/L (101–111); glucose 1087 mg/dL (60–250), blood urea nitrogen 40 mg/dL (8–26), and creatine 2.68 mg/dL (0.70–1.20). Blood counts were normal.Chest radiograph (CXR) showed lower lobe infiltrates and pneumomediastinum (PM). Laboratory results: Arterial blood gas: PH 6.930 (7.350–7.450), PCOThe patient was intubated for airway protection because of altered mental status. A postintubation CXR showed PM, lower lobe infiltrates, and an air-filled distended stomach. An oral-gastric (OG) tube was placed easily, to decompress the air-filled distended stomach after bagging prior to intubation and in view of nausea and vomiting. A chest computed tomography scan showed multilobar infiltrates suggestive of aspiration pneumonia, PM, and the tip of the OG tube to be in the right hemithorax. The patient underwent a right thoracotomy with the intention to repair the esophageal rupture. Intraoperatively, the tip of the OG tube was found to be retracted into the esophageal lumen. Inspection of the esophagus did not reveal a clear rupture. An OG tube was placed intraoperatively, and a chest tube was placed into the right pleural space.The patient was given intravenous (IV) fluid for hydration, IV insulin drip, IV metronidazole, and IV levofloxacin. Serial CXRs showed clearing of pneumonic infiltrates and PM. The chest tube was discontinued, and the patient was successfully extubated. After extubation, an esophagogram was performed and showed no extravasation of contrast.PM is a rare comorbidity of DKA. Other reported pneumo-complications in patients with DKA are pneumothorax , pneumopPauw et al. conducted an extensive review of the literature and found that esophageal rupture (Boerhaave's syndrome) was not detected in any of the reported cases and suggested that the mechanism of PM is alveolar rupture . The PM This case illustrates the need to consider both of DKA and Boerhaave's syndrome as possible causes of PM in a patient who has a similar presentation. If the clinical scenario necessitates placement of an OG tube, Boerhaave's syndrome should be excluded prior to the insertion, to avoid displacing the OG tube through the esophageal wall into the pleural space. Esophageal gastroendoscopy is only indicated when other pathologies are being considered and after healing of the rupture is documented with a water-soluble contrast esophageal swallow .


Escherichia coli and Klebsiella pneumoniae. The clinical presentation is nonspecific and ranges from asymptomatic urinary tract infection to urosepsis and septic shock. The diagnosis is made by abdominal imaging. Treatment consists of broad-spectrum antibiotics, bladder drainage, and management of the risk factors. Surgery is reserved for severe cases. Overall mortality rate of emphysematous cystitis is 7%. Immediate diagnosis and treatment is necessary because of the rapid progression to bladder necrosis, emphysematous pyelonephritis, urosepsis, and possibly fatal evolution.We report the atypical case of a nondiabetic 66-year old male with severe abdominal pain and vomiting who was found to have emphysematous cystitis. Of all gas-forming infections of the urinary tract emphysematous cystitis is the most common and the least severe. The major risk factors are diabetes mellitus and urinary tract obstruction. Most frequent causative pathogens are  Emphysematous cystitis (EC) is a rare but occasionally severe clinical problem. Aetiology is multifactorial and pathogenesis is poorly understood. Most patients diagnosed with EC are elderly diabetic females. Clinical presentation varies individually and may not correlate with the degree of inflammation. Rapid recognition and treatment is necessary to prevent progression to bladder necrosis, emphysematous pyelonephritis (EP), and urosepsis. We present an atypical case of EC in a nondiabetic 66-year old male.A 66-year old schizophrenic male was brought to the emergency department because of severe abdominal pain and vomiting for three hours. He was pale, sweating, and tachypneic. Autoanamnesis was impossible because of his psychiatric comorbidity but heteroanamnesis told us he had not mentioned any other complaints. Besides schizophrenia no other pathology was retained in his medical history. His daily medication consisted of an antipsychotic drug, an anticholinergic drug, and a benzodiazepine. Physical exam revealed a body temperature of 37.8°C, heart rate of 145 beats per minute, blood pressure of 130/55 mmHg, and oxygen saturation of 91%. Oxygen was administered with a quick increase of the oxygen saturation to 99%. Palpation of the abdomen was extremely painful with diffuse muscular defence. No other abnormalities were found. Physical examination was very difficult since patient was uncooperative.μL (4.50–11.00 × 10³/μL), prothrombin time of 59% (75–100%), serum creatinine of 6.0 mg/dL (0.70–1.20 mg/dL), BUN (blood urea nitrogen) of 217 mg/dL (8–50 mg/dL), and an eGFR (estimated glomerular filtration rate) of 9 mL/min (>60 mL/min). PSA (prostate specific antigen) measured 141 ng/mL (<6.9 ng/mL). Transaminases were augmented: ASAT (aspartate aminotransferase) 190 U/L (5–34 U/L), ALAT  118 U/L (5–41 U/L). Furthermore an LDH (lactate dehydrogenase) of 605 U/L (240–480 U/L) and CK (creatine kinase) of 2206 U/L (<200 U/L) were found. Blood glucose level was 113 mg/dL (70–110 mg/dL).Laboratory evaluation resulted in a CRP (C-reactive protein) of 49.47 mg/dL (0.00–0.50 mg/dL), white blood cell count of 6.52 × 10³/Macroscopically urine was cloudy. Microscopic urinalysis showed >100 RBC (red blood cell)/HPF (high power field), 50–100 WBC (white blood cell)/HPF and some bacteria. Despite painkillers, the patient continued complaining of abdominal pain. Because of the severity of his pain, computed tomography scan of the abdomen was performed. This revealed air in the bladder and the thickened bladder wall, associated with air intra- and retroperitoneally. Furthermore, bilateral pleural fluid, some free fluid in the peritoneal cavity, and a few dilated jejunal segments without characteristics of bowel ischemia were seen Figures . The diaBroad-spectrum antibiotics (piperacilline-tazobactam) were empirically started. Intravenous fluid and analgesics were associated. A transurethral catheter was placed and patient was admitted to the surgical department. A few hours later he was transferred to the intensive care unit (ICU) since he became more septic  and developed a reddishness of the suprapubic region . There w6 colonies/mL of Escherichia coli. Intravenous piperacilline-tazobactam administration was continued.The following days the patient became less septic, renal function improved and the abdominal pain decreased. Contrary to the clinical improvement, the zone of reddishness expanded up to the flanks bilaterally . After tDespite continuous intravenous antibiotics and bladder catheterization, patient developed fever (38°C) one week after admission. A control computed tomography scan of the abdomen showed necrosis of the bladder wall, predominantly the bladder dome and right bladder wall, with associated peritonitis signs Figures . The samA few hours postoperative patient became hemodynamically unstable. Chest X-ray and electrocardiogram were normal. Arterial blood sample showed a haemoglobin level of 4.7 g/dL. Although abdominal drains evacuated no blood a postoperative bleeding was suspected and patient was brought to the operating room for an urgent second look. A venous bleeding of the right iliac vein was visualised and sutured. Afterwards patient was sedated, intubated, and ventilated for several days on the ICU. His situation gradually improved and ten days later he was again admitted to the surgical department with the transurethral catheter in situ. Antibiotics were switched to quinolones (levofloxacin). A cystography three weeks postoperatively showed some contrast extravasation into the space of Retzius Figures . ConsequLevofloxacin administration was ceased after a sterile urine culture 25 days postoperatively.Two weeks later patient left the hospital with the transurethral catheter in situ, which he himself promptly removed on the third day following his discharge. Afterwards no voiding problems were seen so no new drainage was provided. A control cystography one week later revealed no contrast leakage anymore . PatientPathology report of the partial cystectomy specimen showed only necrotic tissue, Gram- and PAS staining (periodic acid-Schiff) were negative.EC is an infection of the bladder associated with gas production, usually occurring in elderly diabetic females. Thomas et al.  identifi2 (hydrogen) and CO2 (carbon dioxide) gas. In all cases it concerns an inappropriate host response to the causative microorganism. Urinary tract obstruction, and consequently urinary stasis, is another major risk factor besides diabetes. All patients with recurrent urinary tract infections, indwelling urethral catheter, neurogenic bladder, immunosuppressive comorbidity, and others. are predisposed to complicated UTIs (urinary tract infection) such as EC. Grupper et al. [The aetiology and pathogenesis of EC remain poorly understood. In diabetic patients an elevated tissue and urinary glucose level is a plausible source of fermentation for gas-forming bacteria. This process is possibly favoured by associated impaired renal perfusion, diabetic nephropathy, bladder dysfunction secondary to neuropathy, and impaired leukocyte function. In non-diabetic patients higher levels of urinary albumin, lactose, or tissue proteins can result in the formation of Hr et al.  found anClinical features of EC are nonspecific and vary individually. Some patients are asymptomatic or report only minor voiding problems or abdominal pain, while others present with septic shock. Thomas et al.  found thSince clinical presentation of EC is very different and nonspecific, EC is diagnosed radiologically. Most accurate examination is the computed tomography scan of the abdomen/pelvis –3. BecauEscherichia coli, Klebsiella pneumonia, Enterococcus, Candida, Clostridium perfringens, and many others were all isolated in urinary culture, with Escherichia coli being the most prevalent followed by Klebsiella pneumonia [Multiple gas-producing microorganisms can cause EC. neumonia –3. The sneumonia , 7. MostThe management of EC generally consists of broad-spectrum antibiotics, bladder drainage, correction of the blood glucose level, and treatment of any underlying comorbid disorders. In most cases broad-spectrum antibiotics are started. Once the sensitivities of the isolated urinary pathogens are known, antibiotics should be switched to more specific ones. No consensus exists about the duration of antibiotic treatment. Grupper et al.  found a Thomas et al.  publisheEmphysematous cystitis is an uncommon infectious condition of the bladder caused by gas-producing micro-organisms Because of the possible insidious clinical presentation it is of utmost importance that emergency physicians are aware of this clinical entity, especially in high risk patients. Immediate diagnosis and treatment is necessary because of the rapid progression to bladder necrosis, EP, urosepsis, and possibly fatal evolution.


Enhancing athletic performance is a great desire among the athletes, coaches and researchers. Mint is one of the most famous natural herbs used for its analgesic, anti-inflammatory, antispasmodic, antioxidant, and vasoconstrictor effects. Even though inhaling mint aroma in athletes has been investigated, there were no significant effects on the exercise performance.Twelve healthy male students every day consumed one 500 ml bottle of mineral water, containing 0.05 ml peppermint essential oil for ten days. Blood pressure, heart rate, and spirometry parameters including forced vital capacity (FVC), peak expiratory flow rate (PEF), and peak inspiratory flow (PIF) were determined one day before, and after the supplementation period. Participants underwent a treadmill-based exercise test with metabolic gas analysis and ventilation measurement using the Bruce protocol.2 , and VCO2 .The FVC , PEF , and PIF  significantly changed after ten days of supplementation. Exercise performance evaluated by time to exhaustion (664.5 ± 114.2 vs. 830.2 ± 129.8 s), work (78.34 ±32.84 vs. 118.7 ± 47.38 KJ), and power (114.3 ± 24.24 vs. 139.4 ± 27.80 KW) significantly increased (p < 0.001). In addition, the results of respiratory gas analysis exhibited significant differences in VOThe results of the experiment support the effectiveness of peppermint essential oil on the exercise performance, gas analysis, spirometry parameters, blood pressure, and respiratory rate in the young male students. Relaxation of bronchial smooth muscles, increase in the ventilation and brain oxygen concentration, and decrease in the blood lactate level are the most plausible explanations. Mint (mentha) is a herb which is well known for its antispasmodic, painkilling -3, anti-int oil) . MentholExternal application of peppermint extract raised the pain threshold in human . PeppermThe effect of supplementation with oral peppermint extract was also studied on the perceived lower leg muscular pain and blood lactate levels one hour before a 400-m running test . In thisUsing a Peak Flow Meter device showed an improvement in the lung capacity and inhalation ability after inhalation of peppermint aroma . After iThe possible ergogenic effect of aromas, has certainly received much publicity in recent years. However, there is very little scientific evidence to support or refute the claims made by merchants, practitioners, and manufacturers . Hence, age = 25.9 ± 1.38 yrs; Mweight = 69.9 ± 5.58 kg; Mheight = 177.0 ± 4.2 cm) randomly selected among 40 volunteers to take part in a quasi experiment by using the one-group pre-test, post-test design. Participants tested before, and after ten days of peppermint essential oil consumption. Ethical approval to conduct this study obtained from the University Human Ethics Committee.Twelve (12) healthy male university students  and the best of three forced efforts such as forced vital capacity (FVC), peak expiratory flow rate (PEF), and peak inspiratory flow (PIF) were recorded.-1), respiratory gas-exchange ratios, and other parameters recorded every ten seconds. Exercise performance parameters consist of time to exhaustion (TE), total work , maximal power (Pmax), vertical distance, and horizontal distance computed by the treadmill’s software considering the slope angle, speed and duration of each stage.Finally, participants underwent a standard treadmill exercise test (Bruce protocol), controlled by a computer program. A heart rate transmitter belt  was attached to the chest to transmit the heart rate signals to the receiver. Respiratory gas and ventilation were measured with calibrated PowerCube Gas Analyzer . Gas exchange variables including: oxygen uptake (L/min), carbon dioxide production (L/min), ventilation (L/min), breathing rate  per day, containing 0.05 ml peppermint essential oil for ten days. All the tests repeated after ten days of supplementation. Participants were asked to refrain from any medium to vigorous exercise and their diet was controlled during the study.Normal distribution was tested using the Kolmogorov-Smirnov and Shapiro-Wilk tests. Paired t-test used to examine differences between pre-test and post-test. To calculate the magnitude of the difference between pre-test and post-test, a Cohen’s d calculated, using the following formula :Cohen’s d of 0.20 considered a minor, 0.50 a medium, and 0.80 a major difference. The statistical analysis performed using the Statistical Package for Social Sciences software .After ten days of supplementation with peppermint essential oil, the exercise performance evaluated by changes in the physiological parameters  and functional indicators of exercise performance. The Kolmogorov-Smirnov and Shapiro-Wilks tests revealed the normality of the data. The parameters obtained from the gas analyzer during Bruce test presented in the Table 2/Kg, VCO2/Kg, MET, and energy expenditure were observed showing enhancement in the respiratory efficiency and energy expenditure during the exercise. An increase in the breathing rate, normally leads to a lower alveolar and arterial PCO2 and therefore, decrease in the end-tidal carbon dioxide tension (PETCO2) and fractional end-tidal CO2 concentration (FETCO2) expected , VOed Table .Time to exhaustion, vertical distance, horizontal distance, maximum work, and power compared and presented in the Table Functional indicators of exercise performance showed significant increase in the time to exhaustion and distance Table . In the Lung function tests significantly increased after ten days of supplementation. Peak inspiratory flow (PIF) shows maximum changes whereas forced vital capacity (FVC) had least changes and effect size.Both resting and exercise heart rates were significantly decreased during post-test. Similarly, the chest circumference during maximum exhale and blood pressure in the post-test significantly decreased.Previous studies have shown that various kinds of mint were effective in reducing muscle pain ,20, muscIn the current research, improvement in the spirometric measurements  and ventilation during treadmill exercise, as well as an increase in the maximum chest circumferences observed. These results can be justified by decreasing the airway and bronchial smooth muscle tonicity with or without effect on the pulmonary surfactant. Previously, reported a significant increase in the respiratory muscle strength after four-week inspiratory and expiratory muscle training on the respiratory muscle strength and time to exhaustion in healthy people . In the 2-adrenergic receptor agonist and with bronchodilator effect on the cycling performance. However, there was no significant difference in any variables related to aerobic endurance or cycling performance [In an in-vitro study, menthol vapour lowered the surface tension on synthetic surfactant films . It may formance . In yet formance . It can The result of the current study supports the theory that delaying fatigue may be related to physiological changes by decreasing blood lactate level similar to the recent finding . FurtherThe stimulating effect of peppermint on the CNS  may alsoTable It seems peppermint has a lowering effect on the heart rate and the systolic blood pressure. Reduction in the arterial smooth muscle tonicity is a possible explanation for these effects. One study administered peppermint aroma by nose and failed to find any significant effect in both heart rate and blood pressure. The differences in the peppermint essential oil method of administration and supplementation period, can justify the effectiveness of peppermint in the current study.To our knowledge, this is the first study that explored the effect of oral supplementation with peppermint essential oil on the exercise performance. Our results strongly support the effectiveness of peppermint essential oil on the exercise performance, respiratory function variables, systolic blood pressure, heart rate, and respiratory gas exchange parameters. Differences in duration of study and oral supplementation instead of inhalation of peppermint aroma could be the important characteristics of this study compare to the previous researches. Further investigations are required to unravel the mechanism underlying the effectiveness of peppermint on the exercise performance and respiratory parameters.2; EQCO2: Ventilatory equivalents for CO2; VT: Tidal volume; VA: Alveolar ventilation; METS: Metabolic equivalent; EE: Energy expenditure; FETO2: Fractional end-tidal O2 concentration; FETCO2: Fractional end-tidal CO2 concentration; CHO: Carbohydrate.FVC: Forced vital capacity; PEF: Peak expiratory flow rate; PIF: Peak inspiratory flow; Wtotal: Calculated total work; Pmax: Maximal power output; CNS: Central nervous system; VO2: Oxygen uptake; VCO2: Carbon dioxide elimination; VE: Minute ventilation; RR: Respiratory rate; PETO2: End-tidal oxygen tension; PETCO2: End-tidal carbon dioxide tension; RER: Respiratory exchange ratio; EQO2: Ventilatory equivalents for OAuthors of this paper have not received any financial remuneration for preparing this paper. The authors declare that they have no competing interests.The authors’ responsibilities were as follows–A.M. is responsible for research design, conducting laboratory tests, statistical analysis and manuscript preparation. A.R. was responsible for subject recruitment and laboratory tests assistance. Both authors read and approved the final manuscript.Dr. Abbas Meamarbashi is Associate Professor and Head of the Department of Physical Education and Sport Science at the University of Mohaghegh Ardabili. He has been published in many peer-reviewed journals. Sport nutrition is one of his fields of interest. Mr. Ali Rajabi is an MSc student in sport physiology.