Engraftment occurred in all experimental animals examined. Cells from endometriotic lesions are capable of migration to and engraftment of multiple organs outside of the peritoneal cavity. Micrometastasis of endometriosis is a novel and frequent phenomenon. These data suggest that widespread dissemination of endometriosis may be common, clinically unrecognized and contribute to the diffuse clinical manifestations of this disease.Endometriosis is an inflammatory gynecological disorder among reproductive-aged women caused by the engraftment and proliferation of endometrial cells outside the uterus, most commonly in the pelvis. It is thought that the disease arises primarily from retrograde menstruation where cells from the endometrium travel through the fallopian tubes to the peritoneal cavity. However, migration of endometriosis-derived cells to distant organs outside of the peritoneal cavity have not been explored. In the present study, we developed and validated a mouse model of disseminated endometriosis using syngeneic DsRed endometrial tissue introduced into the peritoneum of immunocompetent mice. Flow cytometry and immunofluorescence analysis, demonstrated the presence of endometriosis-derived cells in multiple organs  in the murine endometriosis model. Immunostaining revealed the presence of DsRed Endometriosis is a common gynecological disorder among reproductive aged women 6–10%) [0% [1] duDespite being one of the most common gynecologic diseases, the pathogenesis of endometriosis is poorly understood , 8. The Endometriosis is most commonly found in the pelvis. However, it is also found in organs not contiguous with the peritoneal cavity; these include the pericardium, lung, brain, and skin , 17–19. In this study, we used a murine model of surgically induced endometriosis to determine whether endometriosis-derived cells are capable of migration or micrometastases to distant organs, including the lung, spleen, liver, or brain. Micrometastasis is a form of metastasis where the spread and engraftment of a small number of cells shed from a primary location to another part of the body are too minuscule to be detected by the conventional detection methods . Previou+ cells were found in the lungs of all DsRed+ positive control mice. No DsRed+ cells were observed in the lungs of the negative controls that underwent sham surgery. Small numbers of DsRed+ cells were identified in the lungs of all animals at eight weeks after induction of endometriosis. Experiments were carried out 3 times with different samples and each time in duplicate. . As expected, DsRed. Figure . The mea+ cell population from the spleen in each group  were evaluated using FACS  and spleen (p = 0.001) in the endometriosis model  mice (20) from Jackson Laboratories  and female C57BL/6 mice (20) were kept under controlled conditions  with free access to water and chew. Animals were treated in accordance with a protocol approved by the Yale University Institutional Animal Care and Use Committee (IACUC), and the U.S. Principles for Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training were followed. Briefly, the female transgenic DsRed mice were collected from litters of the same age from different dams of C57BL/6 background. Both parents have construct containing a Red Fluorescent Protein gene (DsRed.MST) under the control of a chicken beta actin promoter.n = 10) or sham control group (n = 10). Experimental endometriosis was created in ten female C57BL/6 mice with intact ovaries as previously described [n = 10).Twenty C57BL/6 mice were randomly assigned to one of two groups, endometriosis group . The remaining tissue was stored in 4% paraformaldehyde overnight, transferred to 70% ethanol, and then embedded in paraffin for immunofluorescent analysis.+cells using FACS. The specimens were finely minced with surgical scalpels, and subsequently digested with a solution of Hank’s Balanced Salt Solution  containing HEPES (25 mM), collagenase B , and deoxyribonuclease  for 60–90 min at 37°C. All samples were filtered using 70-μm cell strainers and centrifuged at 3,000 rpm at 4°C for 5 minutes. Cell pellets were suspended in FACS buffer and then sorted via fluorescence activated cell sorting (FACS) on a Beckman Coulter MoFlo machine . Acquired data were analyzed using the FlowJo V10 software  for three independent experiments.Lung, spleen, liver, and brain tissue were assessed to determine the presence of DsRedParaformaldehyde-fixed specimens were paraffin embedded, cut into 3-μm sections, mounted on glass slides, and stained for DsRed and CD45, a hematopoietic cell marker mainly found on leukocytes. Six sections (one/slide) were prepared from each organ of interest of every animal. Three random regions from each organ of the lung, liver and brain were selected in two independent experiments. Sections were deparaffinized and rehydrated through a series of 15-minute xylene and 25-minute ethanol washes and rinsed in Phosphate-Buffered Saline (PBS). The slides with sections were then placed in boiling sodium nitrate (pH = 6) for 10 min and then treated for 15 min with ammonium chloride in PBS to protect against auto-fluorescence. Sections were incubated in blocking buffer using 5% donkey serum (Invitrogen) for 30 min at room temperature followed by primary antibodies: polyclonal goat anti-DsRed IgG antibody , and rat anti-CD45 antibody  diluted 1/200 in 5% donkey serum and kept in a humidified chamber overnight at 4°C. Then, the sections were rinsed three times for 20 min respectively in PBS on an orbital shaker and incubated with the following secondary antibodies: donkey anti-goat Alexa Fluor 568 , and donkey anti-rabbit Alexa Fluor 488  diluted 1/500 in PBS for one hr, away from light, at room temperature. Sections were mounted with Vectashield mounting medium with DAPI  for 15 min and immunofluorescence signals were observed using a laser scanning confocal microscope . A minimum of 100 high-power microscopic fields were analyzed from triplicate slides in each sample, in three independent experiments. The investigators who performed the tissue investigations were blinded to the experimental groups.p < 0.05.Data were analyzed using GraphPad Prism 5 software  and are presented as the Mean ± SEM from at least three independent experiments. One-sided analysis of variance (ANOVA) was used to analyze differences in multiple organs comparisons, followed by Tukey’s post hoc test. Values were considered to be statistically significantly different if


Silybum marianum (L.) Gaertn, is primarily composed of flavonolignans, with silibinin (SB) as its major active constituent. The present study aimed to evaluate the antigenotoxic activities of SM and SB using the alkaline comet assay in whole blood cells and to assess their effects on the expression of genes associated with carcinogenesis and chemopreventive processes. Different concentrations of SM or SB  were used in combination with the DNA damage-inducing agent methyl methanesulfonate  to evaluate their genoprotective potential. To investigate the role of SM and SB in modulating gene expression, we performed quantitative real-time PCR (qRT-PCR) analysis of five genes that are known to be involved in DNA damage, carcinogenesis, and/or chemopreventive mechanisms. Treatment with SM or SB was found to significantly reduce the genotoxicity of MMS, upregulate the expression of PTEN and BCL2, and downregulate the expression of BAX and ABL1. We observed no significant changes in ETV6 expression levels following treatment with SM or SB. In conclusion, both SM and SB exerted antigenotoxic activities and modulated the expression of genes related to cell protection against DNA damage.Silymarin (SM), a standardized extract derived from Silybum marianum (L.) Gaertn, popularly known as milk thistle, is one of the most widely used herbs worldwide. S. marianum has been well-known since ancient times and has been mostly used in traditional European and Asian medicine for treatment of liver disorders [According to the World Health Organization, 70% to 95% of the world's population rely on traditional medicine for primary health care, and most health practices involve the use of plant extracts or their active components . Silybumisorders . S. marianum are attributed to its ability to accumulate bioactive flavonolignan complexes, which are referred to as silymarin (SM). S. marianum contains approximately 70% to 80% flavonolignans (silymarin complex), small amounts of flavonoids, 20% to 30% fatty acids, and other polyphenolic compounds. The flavonolignan mixture present in S. marianum mainly consists of silibin (SB), also known as silibinin, the major bioactive component of the extract. Milk thistle extract is currently marketed worldwide as silymarin and silibinin under various trade names, such as Siliphos, Silipide, and Legalon [The medicinal properties ofThe effects of SM and SB have been investigated in mice, rats, rabbits, and dogs and results demonstrated that their acute, subacute, and chronic toxicities are very low. SM and SB are primarily used for the treatment of various liver disorders that are characterized by degenerative necrosis and functional impairment, such as chronic inflammatory diseases, cirrhosis, and toxic liver damage . In addi in vitro and in vivo. However, little is known about their protective effects on the genetic material. Numerous phytochemicals have been reported to interfere with specific stages of carcinogenesis, and multiple mechanisms have been shown to account for the anticarcinogenic properties of dietary constituents [The pharmacological activities and toxicological safety of SM and SB have been extensively studied bothtituents . ETV6 and PTEN, the cell death regulators BCL2 and BAX (pro/antiapoptotic processes), and the protooncogene ABL1 can reveal the molecular basis underlying the effects of SM and SB.Analysis of genes related to DNA damage, carcinogenesis, and/or chemoprevention can help elucidate the mechanisms by which dietary supplements can exert protective effects on DNA –13. ThusConsidering the biological activities presented by SM and SB, as well as their widespread use as herbal medicines, the present study aimed to evaluate the antigenotoxic activities of SM and SB using the comet assay and to evaluate the expression patterns of genes that are known to be associated with carcinogenesis and chemopreventive processes.2EDTA, Tris base, and Tris-HCl were purchased from Bio-Rad Laboratories .Silymarin , silibinin , RPMI 1640 medium, methyl methanesulfonate (MMS), ethidium bromide, dimethyl sulfoxide (DMSO), NaCl, and Triton X-100 were purchased from Sigma-Aldrich . Low melting point agarose and normal melting point agarose were obtained from Thermo Fisher Scientific . NaμM MMS and subsequently incubated for 3 h at 37°C and 5% CO2 in RPMI medium containing 15% (v/v) fetal calf serum. The positive (MMS) and negative (DMSO) control groups were included. The experiment was performed in triplicate. The MMS concentration of 800 μM used in the present study was selected based on its previously demonstrated effectiveness in inducing DNA damage [Peripheral blood was obtained through venous puncture from three young and healthy volunteers who had no history of smoking or drinking. Our work was approved by the Human and Animal Research Ethics Committee of the Universidade Federal de Goiás (CEPMHA/HC/UFG n. 016/2011). Whole blood was treated with varying concentrations of SM or SB  in combination with 800 A damage .μl of blood and 130 μl of low melting point agarose (0.5%) and incubated at 37°C. The mixture was spread on the slides with coverslips and placed in a cold chamber. Afterwards, the coverslips were carefully removed, and the slides were immersed in lysis solution protected from light  at 4°C for 4 h. Subsequently, the slides were incubated with freshly prepared alkaline solution buffer  at 4°C for 20 min to unwind the DNA. Samples were then subjected to electrophoresis in the same buffer at 1 V/cm and the current of 300 mA for 30 min in the dark. After electrophoresis, the slides were placed on a staining tray, covered with neutralizing buffer , and kept in the dark for 5 min. For analysis, the slides were stained with 20 μl of ethidium bromide solution (0.02 mg/ml) and covered with a cover slip. A total of 50 nucleoids were analyzed per slide, corresponding to 100 nucleoids per sample.The alkaline version of the comet assay was performed according to the protocol described by Singh and coworkers  with sliThe analysis was performed on a fluorescence microscopy system Axioplan-Imaging®  using the Isis software with an excitation filter of 510-560 nm and a barrier filter of 590 nm under 20× magnification. To assess genomic damage, we used the TriTek Comet ScoreTM software (version 1.5), in which pixels intensities are used to estimate the degree of genomic damage and are given as arbitrary units. The nucleoids with completely fragmented heads were not included in the analysis.From the 17 parameters provided by the software, we selected the percentage of DNA in the tail for assessing DNA damage. This parameter has been proposed by several authors to be the most useful parameter because it provides a quantitative measure of DNA damage (from 0 to 100%) .For antigenotoxicity assessment, the percentage of reduction in MMS-induced damage by SM and SB was calculated according to Waters et al., 1990 , using twhere A corresponds to the DNA damage observed following treatment with MMS (positive control), B represents the group treated with SM or SB plus MMS, and C represents the negative control.μg of total RNA in a 10 μL sample volume using RT2 First Strand Kit®  as recommended by the manufacturer. Amplified cDNA was stored at -20°C.After treatment, human blood samples were transferred to tubes provided by GeneJET™ Whole Blood RNA Purification Mini Kit . RNA extraction was performed according to the manufacturer's instructions. Afterwards, the final RNA concentration was determined using a spectrophotometer NanoVuePlusTM . RNA purity and integrity were assessed via 3% agarose gel electrophoresis. To ensure the purity of the RNA, the A260/A230 and A260/A280 absorbance ratios were evaluated. cDNA synthesis was performed using 1 ETV6, PTEN, ABL1, BAX, and BCL2) using GAPDH as reference gene. In addition, the plates contained a genomic DNA control, a reverse-transcription control, and a positive PCR control.Customized qRT-PCR assay was performed using 96-well-plates. We analyzed five target genes . Gene expression analysis was carried out using the comparative ΔΔCt method.The qRT-PCR using cDNA derived from treated human whole blood cells was performed using the RT2 SYBR Green Master Mix Kit®  according to the manufacturer's instructions. Array-based qRT-PCR analysis was performed in 28 http://dataanalysis.sabiosciences.com/pcr/arrayanalysis.php). First, gene expression levels for each sample were normalized against those of the reference gene GAPDH (ΔCt). Ct data was used as an input, and the web-based software will automatically perform quantification using the ΔΔCt method (using positive control MMS treatment as standard sample). The fold change was calculated for each gene for all group samples.The cycle threshold (Ct) values were exported to the PCR Array Data Analysis Web Portal  were compared relative to the positive control (MMS) by performing the Student'sAssessment of the antigenotoxicity of SM and SB via the alkaline comet assay demonstrated reduced DNA damage (% DNA in tail) in cells cotreated with SM or SB and MMS relative to cells treated with the positive control MMS alone Figures  and 2.Treatment with the standardized extract (SM) significantly reduced DNA damage for all tested concentrations of the compound when combined with MMS relative to treatment with MMS alone. SB, the major active constituent of SM, also exerted significant protective effect against DNA damage induced by the genotoxic agent MMS, except at a lower concentration 1.0 mg/ml) , and the protooncogene ABL1.We evaluated the expression levels of the following five genes associated with DNA damage, carcinogenesis, and/or chemoprevention mechanisms: the tumor suppressors ETV6 gene  and the highest SB concentration (7.5 mg/ml), corresponding to fold change values of 2.71, 3.07, and 2.33, respectively   that are generated inside the cell . Several TP53 and CDKN1A and downregulating the expression of BCL2 [To evaluate the chemopreventive effects of SB and SM on the DNA, human whole blood cells were treated with SB or SM in combination with methyl methanesulfonate (MMS). MMS is an alkylating agent that induces damage to genetic material and forms monoadducts with the nucleophilic centers of DNA . Damage  of BCL2 .γ-radiation-induced strand breaks in plasmid DNA, reduce DNA damage and micronuclei formation in human lymphocytes and rat leukocytes, and reduce mouse mortality and DNA damage in blood leukocytes following whole-body γ-exposure in mice [Our results showed that both the complex (SM extract) and its main active constituent (SB), cotreated with MMS, exerted protective effects by reducing the amount of DNA in the comet tail in 42.49% and 34.20% respectively, when compared to positive control (MMS). Previous studies also demonstrated the protective effects of SM and SB. In particular, SM and SB significantly decreased point mutations based on the Ames test and reduced the proportion of micronucleated polychromatic erythrocytes based on the mice bone marrow assay . Further in mice , 30.In addition, the current findings revealed that the extract complex (SM) exerted slightly stronger antigenotoxic activity compared to the primary active constituent (SB); however, the observed difference was not statistically significant. Our current findings were consistent with those of a previous study, which demonstrated that “high purity” milk thistle extracts exerted weaker antioxidant activity relative to the complex extract . The aboTo elucidate the effects of cotreatment with MMS and SM or SB, we evaluated the expression levels of five genes that are specifically known to mediate chemoprevention and response to DNA damage. ETV6 (ets variant gene 6) encodes a protein that functions as a transcriptional regulator by binding to a specific DNA sequence. Our current findings revealed that the ETV6 expression patterns were not significantly altered following treatment with any tested concentration of SM and SB when compared to the positive control, indicating that SM and SB did not influence ETV6 expression.The tumor suppressor gene PTEN (phosphatase and tensin homolog) gene is a tumor suppressor involved in cell migration and proliferation inhibition and participates in the modulation of cell growth and apoptosis. The essential role of nuclear PTEN in maintaining chromosomal stability has been demonstrated in both mouse and human systems [ PTEN leads to a high proportion of double-strand breaks. Our results demonstrated significant upregulation of PTEN expression following treatment with high concentrations of SM or SB, what could contribute to explain the anticytotoxic and antigenotoxic properties of SM and SB. Also, these data allow us to infer that these protective effects may also help in the regulation and preservation of DNA in cells that are in the active process of cell division as shown in a study using mice bone marrow cells, in which SM and SB reduced the frequency of micronucleated cells [The systems . Accordi systems , nucleared cells . BCL2 expression at all tested concentrations. MMS is known to downregulate BCL2 expression [ BCL2 promoter, which harbors several putative responsive sites, or through an indirect pathway. In addition, treatment with SM or SB significantly downregulated the expression of BAX, thereby suggesting that SM and SB can prevent apoptosis and act as chemoprotective agents. Previous studies demonstrated the modulatory effects of SM and SB on cell survival and apoptosis via interference with the expression of cell cycle regulators and proteins involved in apoptosis [The Bcl-2 family comprises proapoptotic and antiapoptotic proteins. BCL2 (B-cell lymphoma protein 2) is associated with programmed cell death inhibition in various cell types. The antiapoptotic function of BCL2 appears to be mediated by its ability to heterodimerize with other Bcl-2 family members, especially BAX (BCL-2 associated X protein). BCL2 prevents the oligomerization of BAX, which normally causes the release of several mitochondrial apoptogenic molecules . Our respression ; howeverpoptosis , 35–37. ABL1 gene is a protooncogene that encodes a protein tyrosine kinase known to be involved in various cellular processes, including cell division, adhesion, differentiation, and stress response. Nuclear ABL proteins modulate cellular responses induced by DNA damage and are known to participate in cell growth inhibition and apoptosis promotion [ ABL1 expression relative to treatment with MMS alone, although the lower SM and SB concentrations did not significantly alter ABL1 expression patterns. The observed downregulation of ABL1 expression can be associated with the repair of DNA lesions.Theromotion . In the The decrease in DNA damage and the modulation of gene expression to protect cells against lesions suggested the roles of SM and SB in the DNA repair system . Thus, t


Qm, mg−1) was larger onto GPP20 (202 mg g−1 ≤ Qm ≤ 273 mg g−1) than onto the polymeric resin (7 mg g−1 ≤ Qm ≤ 18 mg g−1). With respect to the parameter KLF (mg g−1 (mg L−1)−1/n), which points to the adsorbent-adsorbate affinity, greater values were determined for the pharmaceuticals adsorption onto GPP20 than onto Sepabeads SP207. For both adsorbents and pharmaceuticals, neither Qm or KLF were affected by the aqueous matrix.The performance of a polymeric resin  was compared with that of an activated carbon  in the adsorption of acetaminophen and ibuprofen from either ultrapure or waste water. Kinetic and equilibrium adsorption experiments were carried out under batch operation conditions, and fittings of the obtained results to different models were determined. The kinetic experimental results fitted the pseudo-first and -second order equations, and the corresponding kinetic rates evidenced that the pharmaceuticals adsorption was faster onto GPP20 than onto Sepabeads SP207, but was mostly unaffected by the aqueous matrix. The equilibrium results fitted the Langmuir-Freundlich isotherm model. The corresponding maximum adsorption capacity ( Emerging contaminants (ECs) may be defined as compounds that are not currently covered by existing water regulations but are thought to be a threat to environmental ecosystems and human health . AmongstECs may enter the environment by different routes, depending on their usage pattern and on their application mode. In the specific case of pharmaceuticals that come from human consumption and/or excretion, municipal waste water treatment plants (WWTPs) are main sources in the aquatic environment . These pRemoval of pharmaceuticals at WWTPs before discharge would be possible by the inclusion of an appropriate tertiary treatment . Among tAnti-inflamatory drugs are frequently detected in natural waters due to their widespread consumption . In factSeveral authors have reported the utilization of different types of adsorbents to remove acetaminophen ,24,25 anIn this context, the aim of the present work was to compare the performance of a polymeric resin and an activated carbon in the removal of two pain relievers, viz. acetaminophen and ibuprofen, both with large global consumption and occurrence in waste water. In view of the practical application of these adsorbents in the tertiary treatment of waste water, the adsorption of the considered pharmaceuticals was studied both from ultrapure water and from the secondary effluent of a municipal WWTP.The activated carbon used in this study was pulverised GPP20, which was kindly provided by Chemviron Carbon . The utilization of GPP20 was recommended by Chemviron Carbon for the adsorption of pharmaceuticals, since it is an activated carbon suitable for waste water treatment applications, namely, for the removal of aromatic compounds. The polymeric resin Sepabeads SP207  was gently offered by Resindion . Resindion pointed to Sepabeads SP207 as an appropriate solution for the adsorption of aromatic compounds, including pharmaceuticals. On the other hand, according to the manufacturer , and due to its high specific gravity, this resin is appropriate not only for fixed-bed, but also for batch adsorption operation. The adsorbent materials here used were selected for this work because they have already been used for the removal of pharmaceuticals and/or other organic pollutants from water (including waste water) ,10,31,32Acetaminophen (≥99%) was purchased from Sigma–Aldrich , while ibuprofen sodium (≥98%) was purchased from Fluka . The main properties of these pharmaceuticals are depicted in v/v) for the analysis of acetaminophen and a mixture of methanol:water:orthophosphoric acid (75:25:0.3) for the analysis of ibuprofen. For the preparation of the mobile phase mixtures, HPLC quality acetonitrile (CH3CN) and methanol (CH3OH) were acquired from Sigma-Aldrich , orthophosphoric acid (H3PO4) was purchased from Panreac , and ultrapure water obtained by a Milli-Q purification system from Merck Millipore . Before use, each mixture was passed through a Millipore 0.45 µm pore size filter and degasified in an ultrasound bath for 30 min. For the chromatographic determination of concentration, four replicated injections were carried out under a mobile phase flow rate of 1 mL min−1.The target pharmaceuticals were analysed by High Performance Liquid Chromatography (HPLC), using a Waters HPLC 600 equipped with a 2487 Dual λ Absorbance Detector, a phenomenex C18 column , a Rheodyne injector and a 50 μL loop. The wavelengths of detection were 246 and 220 for acetaminophen and ibuprofen, respectively. The mobile phase consisted of a mixture of acetonitrile:water , which was sourced by a Milli-Q purification system. Then, aiming to assess the possible utilisation of the considered adsorbents in tertiary waste water treatment, experiments on the adsorption of pharmaceuticals from real waste water were carried out. For this purpose, the secondary effluent from the municipal waste water treatment plant (WWTP) of León (Spain) was collected. This WWTP consists of a primary stage, followed by a secondary stage treatment. The primary stage comprises screening, sand removal, fat removal, and primary clarification treatments. Then, the secondary stage involves a plug-flow activated sludge with nitrification/denitrification followed by a secondary clarification. After the secondary clarification, the effluent is directly discharged at the Bernesga river, which has a length of 77 km, and is a tributary of the Esla river. The WWTP was designed to treat the waste water of 330,000 equivalent inhabitants, and has an inflow of 123,000 m3 day−1, with a hydraulic retention time (HRT) of about 6 h.In this work, adsorption experiments were first carried out in ultrapure water (conductivity = 55 μS·cm5), chemical oxygen demand (DQO), NTK, N–NH4, N–NO3, N–NO2, total P–PO4, were determined according to Standard Methods )−1 ≤ Qm ≤ 273 mg g−1) than the polymeric resin (7 mg g−1 ≤ Qm ≤ 18 mg g−1), which was especially remarkable in the case of acetaminophen. Still, the adsorption capacity of these pharmaceuticals onto the employed adsorbents did not decrease in waste water with respect to ultrapure water. This is a very important finding, which points to the practical application of GPP20 and Sepabeads 207 in the tertiary treatment of waste water.Both the polymeric resin Sepabeads SP207 and the activated carbon GPP20 were able to adsorb acetaminophen and ibuprofen from ultrapure and waste water. Adsorption kinetics were faster onto GPP20 than onto Sepabeads SP207, but were mostly unaffected by the aqueous matrix. Experimental results fitted both the pseudo-first and the pseudo-second kinetic models, the latter giving slightly higher correlation coefficients. Equilibrium results onto both the considered adsorbents and for both pharmaceuticals fitted the Langmuir-Freundlich isotherm model, either from ultrapure or from wastewater. The activated carbon displayed larger adsorption capacity values at the equilibrium (202 mg g


However, the ability to stabilize the nanoparticles depended not only on the Mw of the polymer but particularly on the polymer pattern which was determined by the enzyme used to depolymerize the parent chitosan. Low Mw chitosan samples produced by lysozyme were more effective than those produced by chitosanase to stabilize the AgNPs and smaller and less polydisperse nanoparticles were visualized by transmission electron microscopy (TEM). With some polymer sets, more than 80% of the AgNPs produced were lower than 10 nm which correspond to quantum dots. The preparation method described in this paper is general and therefore, it may be extended to other noble metals, such as palladium, gold or platinum.Silver nanoparticles (AgNPs) are of great interest due to their antimicrobial, optical and catalytical properties. Green synthesis of AgNPs is fundamental for some applications such as biomedicine and catalysis. Natural polymers, such as chitosan, have been proposed as reducing and stabilizing agents in the green synthesis of AgNPs. Physico-chemical properties of chitosan have a great impact on its technological and biological properties. In this paper, we explore the effect of chitosan molecular weight (Mw) on the thermal AgNPs production using two sample sets of low Mw chitosans  produced by enzymatic depolymerization of a parent chitosan with chitosanase and lysozyme. Both polymer sets were able to effectively reduce Ag On the other hand, the physical methods avoid these toxic products but they require the use of expensive technologies. Therefore, there is a need to synthesize nanoparticles using eco-friendly and clean processes. Several methods to produce silver nanoparticles assisted with microorganisms and natural products such as plant extracts or polymers such as chitosan, starch, agar, chondroitin sulfate, or heparin have been reported [+ to Ag0 but they also are able to stabilize the nanoparticles avoiding their aggregation. Among natural polymers, chitosan is a very promising molecule due to its technological and biological properties. Chitosan is a copolymer composed by N-acetylglucosamine  and glucosamine  in variable proportion. Chitosan biological and technological properties depend on the physicochemical characteristics of the polymer, mainly its acetylation degree, Mw, and polymer pattern [Metallic nanoparticles (NPs)  are of interest due to their unique optical, electrical, biomedical, or chemical properties among others . Differereported ,9,10,11. pattern . Chitosa pattern . In TablN-acetylglucosamine and glucosamine) along the polymer chains differs depending on the selected enzyme. Previous studies have shown pronounced differences between the low Mw chitosan samples generated either by lysozyme or chitosanase [Low Mw chitosan samples can be produced by chitosan depolymerization using different methodologies such as chemical depolymerization, radiation, sonolysis, or enzymatic depolymerization . In the tosanase .+ to Ag0 and to stabilize the AgNPs. Our hypothesis is that not only chitosan Mw or acetylation degree but also the polymer pattern plays a fundamental role in AgNPs production and stabilization. We have depolymerized chitosan with lysozyme and chitosanase to produce low Mw chitosan samples with different patterns and the ability of these polymers to produce and stabilize AgNPs has been evaluated. The growth of nanoparticles has been monitored by ultraviolet–visible (UV–Vis) spectroscopy and the formed nanoparticles have been characterized by transmission electron microscopy (TEM).The aim of this paper is to evaluate the ability of low Mw chitosan samples produced by enzymatic degradation to reduce AgStreptomyces griseus (EC 3.2.1.132) were provided by Sigma-Aldrich , dialysis membranes  were provided by Medicell International, Ltd. . Milli-Q water was used in all experiments.Chitosan was provided by InFiQus, S.L. , silver nitrate (purity ≥ 99%), lysozyme from chicken hen (EC 3.2.1.17) and chitosanase from w/v) (pH 5.7) or in 0.2 M acetic/acetate buffer at 0.5% (w/v) (pH 4.5) and depolymerized using a commercial chitosanase or lysozyme, respectively. One milliliter of enzyme  was employed per 10 mL or 100 mL of substrate (lysozyme reaction or chitosanase reaction) and the reaction was carried out at 37 °C in an orbital Lab Therm LT-X shaker  at 100 rpm for four days (chitosanase) and seven days (lysozyme) [Chitosan was dissolved in either 0.2 M acetic/acetate buffer at 0.5% (ysozyme) .The depolymerized chitosans were separated by tangential ultrafiltration using Vivaflow Crossflow Cassettes connected to a Vivaflow 200 system  with 30, 10, and 5 kDa cut-off polyethersulfone membranes. Three fractions were isolated from each process: fraction F1 (Mw > 30 kDa), fraction F2 (Mw 30–10 kDa) and fraction F3 (Mw 10–5 kDa). Fractions were dialyzed against distilled water in membranes with Mw 12–14, 7 and 3.5 kDa cut-off, respectively, until complete salt elimination. Dialyzed fractions were freeze-dried and a white powder was recovered.A) was determined by the first derivative UV spectrophotometric method using a Specord 205 spectrophotometer  [Polymer acetylation degree (FGermany) . Polymerw/v under magnetic stirring (150 rpm) overnight. The pH of parent chitosan dissolved in acetic acid was 3.6 while the pH of the depolymerized chitosan samples dissolved in water ranged between 6.2 and 6.5 . To one milliliter of each polymeric solution, 200 µL of AgNO3 (5 mM) dissolved in water were added, the mixture was vortexed for 30 s. Finally, the solutions were heated at 90 °C for 5 h without stirring.Parent chitosan was dissolved in acetic acid 0.1 M and depolymerized chitosan samples were dissolved in distilled water at a polymer concentration of 1% The formation of AgNPs within the chitosan solutions was followed by the appearance of the characteristic Ag exciton peak around 400 nm. Transmission electronic microscopy was used to determine AgNPs size and morphology. Micrographs were analyzed with ImageJ  and Origin  software’s to produce particles histograms . The crystalline nature of the AgNPs was determined by selected area electron diffraction (SAED) and X-ray diffraction (XRD) . Silver nanoparticles particle size and zeta potential were determined by dynamic light scattering (DLS) and laser doppler electrophoresis, respectively. Determinations were carried out in triplicates in a Nano-Zetasizer system .Parent chitosan sample was enzymatically depolymerized using chitosanase and lysozyme and the resulting products were fractionated into three samples; fraction F1 (Mw > 30 kDa), fraction F2 (Mw 30–10 kDa), and fraction F3 (Mw 10–5 kDa). All samples were characterized in terms of their physicochemical properties  as shown in 3 were heated at 90 °C for 5 h. The color of the solutions changed from colorless to light yellow (FiL fractions) or yellowish brown (FiQ fractions). The AgNO3 solution of the control sample (parent chitosan) changed to a greyish color. The color change confirmed the synthesis of AgNPs  agglomerate into clusters and finally colloidal AgNPs are formed. Due to the tendency of AgNPs to aggregate, the use of stabilizing agents is mandatory to produce small nanoparticles.Silver nanoparticle synthesis occurs in a multi-step process; firstly AgN-acetylglucosamine and D denoted glucosamine units, respectively) [n − mAm, n = 4–24, m = 0–3, n + m < 25) [It has been described that chitosan can served simultaneously as a reducing agent for silver cation and a stabilizing agent for AgNPs. In this paper, we have evaluated the ability of a series of low Mw chitosan samples to reduce silver ions to silver atoms and to stabilize the formed AgNPs. A chitosan sample was depolymerized with chitosanase or lysozyme and the reaction mixture was separated into three fractions. As seen in ctively) . A previ m < 25) . As obse m < 25) ,14,15,16+ to Ag0 no matter their physico-chemical properties. However, chitosanase fractions were less effective to stabilize the nanoparticles than the lysozyme ones. In fact, only the nanoparticles produced with the fraction F1Q (>30 kDa) were stable while those produced with the lower Mw fractions (F2Q and F3Q) tended to aggregate forming large clusters observed by the naked eye.The presence of the silver exciton in all samples (parent chitosan and depolymerized fractions) indicates that all fractions were able to effectively reduce AgOn the contrary, AgNPs formed in the presence of chitosan fractions depolymerized by lysozyme showed a good ability to stabilize the nanoparticles. In fact, fraction F3L was able to stabilize the nanoparticles with a small size (lower than 10 nm) and monodisperse nanoparticles were observed. Interestingly, in long-term experiments, F1Q was the most effective fraction regarding nanoparticles stabilization since the SPR was almost unaltered after 30 days.N-acetylglucosamine and glucosamine units are distributed along the polymer chain—have effect on chitosan technological and biological properties [It is generally accepted that chitosan Mw and acetylation degree affect the AgNPs formation although a systematic study has not been carried out. Moreover, due to poor or inexistent polymer characterization, it is not trivial to determine how these properties affect AgNPs formation. Results point that medium and low Mw polymers are more effective to stabilize AgNPs and some authors point to the role of primary amino groups in the AgNPs stabilization . Some stoperties ,18,23.When fractions F1Q and F1L are compared strong differences regarding size, polydispersity, and stability are observed. These fractions are quite similar regarding Mw and acetylation degree; therefore, we consider that the strong differences, regarding AgNPs production and stabilization found between these samples are likely due to the different polymer pattern. Samples F2Q and F2L as well as F3Q and F3L exhibited similar Mw but different acetylation degrees so differences cannot be ascribed solely to polymer pattern. However, it is very remarkable the lack of ability of samples F2Q and F3Q to stabilize the AgNPs and the ability of other chitosan samples with similar acetylation degrees to stabilize AgNPs .This work has demonstrated that AgNPs size and stability can be tailored by selecting the appropriate chitosan sample. These results indicate that not only chitosan acetylation degree or Mw as previously reported have a strong influence on the green AgNPs synthesis and stabilization. Polymer pattern also plays a fundamental role on AgNPs synthesis and stabilization. Taking into account that a large number of depolymerization methods are available and that the physicochemical properties of the obtained depolymerized samples will depend on both the physicochemical properties of the parent chitosan and the depolymerization method it is necessary to carry out systematic research on this issue.Further research must be carried out to study how to balance mixtures of chitosan with different physicochemical properties in order to produce AgNPs for specific purposes. Moreover, thermal reduction of noble metal ions in the presence of natural polymers to produce metallic nanoparticles is a general method; therefore, these findings are likely to be useful in the green synthesis of other metallic nanoparticles.


Several studies have often reported low testosterone and SHBG to be associated with type 2 DM and the metabolic syndrome (MetS). Our objective was to determine the impact of metabolic syndrome and diabetic parameters on testosterone and SHBG in both MetS subjects and type 2 DM patients. 2 that served as control, 30 MetS, 30 type 2 DM without MetS, and 30 type 2 DM with MetS according to IDF criteria.  In this study, 120 Yemeni male aged 30–70 years old were enrolled, 30 of whom were healthy subjects with BMI < 25 kg/m Testosterone  and SHBG were significantly lower in MetS subjects and modestly reduced in type 2 DM with and without MetS. Stepwise linear regression showed free and total testosterone to be negatively affected by waist circumference, and univariate analysis shows this significant difference to disappear when adjusted for waist circumference. On the other hand, stepwise linear regression showed SHBG to be positively affected by testosterone and age and negatively affected by FBG and TG. Univariate analysis shows this observed significant difference to disappear when adjusted for testosterone.  Abdominal obesity is a major determinant of low testosterone levels irrespective of diabetes status. Thus, supporting evidence suggesting that the causative relationship between the often low testosterone and type 2 DM might be bidirectional or even multidirectional and interrelated with obesity, MetS, and IR. Type 2 diabetes mellitus (DM) is the predominant form of diabetes and accounts for at least 90% of all diagnosed cases. International diabetes federation estimates that 9% of the world's population 415 million) has diabetes in 2015, and this number is predicted to rise to 10% (642 million) by 2040, of whom 91% are diagnosed with type 2 DM 5 million. MetabolThe association between late-onset hypogonadism and type 2 DM has been demonstrated in numerous studies , 6, indiThe contribution of the diabetic state per se relative to the effects of obesity and comorbidities to lowered testosterone in type 2 DM remains unclear with consequent implications for the therapeutic approach. We hypothesize that testosterone levels are related to the metabolic syndrome state and are not prevalent in type 2 DM. The present study compares the plasma levels of testosterone and SHBG between obese and nonobese type 2 DM versus nonobese normoglycemic controls, as well as determines the impact of metabolic syndrome factors and diabetic parameters on testosterone and SHBG in both MetS subjects and type 2 DM patients.2 that served as controls, 30 subjects with metabolic syndrome (nondiabetic), 30 subjects with type 2 DM without metabolic syndrome, and 30 subjects with type 2 DM with metabolic syndrome according to IDF criteria. Subjects excluded include those with type 2 DM treated with insulin , with hypogonadism or treated for hypogonadism (since those treated for hypogonadism will affect testosterone levels), as well as with liver and renal diseases. The study protocol was approved by the institutional review board (IRB) of the Faculty of Medicine and Health Sciences, Sana'a University. Informed consent was obtained from all individuals after explaining the purpose and nature of the study.This case-control study was performed on 120 Yemeni male subjects aged 30–70 years who were recruited from the Endocrine Unit in Al-Thawra Hospital, Sana'a. Thirty were healthy subjects with body mass index (BMI) < 25 kg/m2). Waist circumference was measured midway between the lower rib margin and the superior iliac spine at the end of gentle expiration in a standing position. Blood pressure (BP) measurements were taken from each patient's right arm in the seated position by using an automatic blood pressure monitor after 10 min of rest in a quiet room. Two to three successive BP readings were obtained at 5-minute intervals and averaged.Body weight and height were measured, and BMI was computed as weight in kilograms (kg) divided by height in meters squared (mFasting venous blood samples (6 ml) were collected from each subject in the morning after 12-hour fast using Vacutainer plain tubes and separated by centrifugation within 10 minutes of collection at 3000 rpm for 10 minutes. The separated serum from each sample was divided into two separate Eppendorf tubes; one of them was stored at −20°C for testosterone, SHBG, and insulin measurements. Glucose and lipid profiles were determined immediately after sample collection.β-cell function were calculated using the homeostasis model assessment (HOMA 2) calculator v2.2 which is available from Oxford Centre for Diabetes, Endocrinology and Metabolism. Free and total testosterone was calculated from serum total testosterone and SHBG using an online calculator .Plasma glucose (FPG), triglyceride (TG), and HDL cholesterol (HDL-c) were measured by chemistry autoanalyzer . Testosterone, SHBG, and insulin were measured by electrochemiluminescence immunoassay (ECL) on cobas E-411 . Insulin resistance and β, and HOMA-IR levels were screened with bivariate correlation (Pearson correlation), to be included in stepwise linear regression analysis. The stepwise linear regression was applied to assist the impact of metabolic syndrome factors and diabetic parameters on testosterone, SHBG, HOMA-β, and HOMA-IR. General linear model with univariate analysis (ANCOVA) was applied to compare testosterone, SHBG, HOMA-β, and HOMA-IR among different groups adjusted for cofactors confirmed by stepwise linear regression analysis. The accepted level of significance was set below 0.05 (p < 0.05).The statistical analyses were performed on Social Package of Social Sciences (SPSS) version 11.5 . ANOVA was used for comparing the means of parameters under study between normal subjects, metabolic syndrome, and type 2 DM with and without metabolic syndrome. The cofactors that affect testosterone, SHBG, HOMA-β). However, free testosterone was only negatively correlated with BMI, waist circumference, SBP, and DBP, respectively. Plasma SHBG levels were negatively correlated with metabolic syndrome factors  and diabetic parameters (insulin and HOMA-IR).The general characteristics of the study population are listed in p = 0.01, p = 0.0002, and p = 0.009) in all tested groups as shown by ANOVA but were only significantly  lower in MetS by 22.4%, 32.8%, and 27.5% as compared to healthy normal subjects. SHBG was nonsignificantly lower in type 2 DM by 20% and that of total testosterone was nonsignificantly lower in type 2 DM with MetS by 16.4% with respect to the health normal subjects. On further usage of the general linear model with univariate analysis (ANCOVA) adjusting for waist circumference, free testosterone was not significantly different in all tested groups, whereas on adjusting for SHBG, total testosterone remained significantly (p = 0.017) lower in the MetS subjects as compared to the other groups. However, on comparing MetS and type 2 DM with and without MetS, the total testosterone was lower in MetS subjects  (data not shown). On further adjustments for both SHBG and waist circumference, the significant difference previously observed in the total testosterone levels disappeared. Similarly, on adjusting for age, FBG, and TG, the level of SHBG remained significantly (p = 0.002) lower in the MetS group. However, on comparing MetS and type 2 DM with and without MetS, SHBG was lower in MetS subjects  (data not shown). On further adjusting for testosterone, the significant difference in the SHBG levels disappeared.The impact of metabolic syndrome on free and total testosterone and SHBG was investigated in this study. Abdominal obesity was a major determinant of low testosterone levels irrespective of diabetes status. Both testosterone  and SHBG were significantly lower in MetS subjects, with a modest but not significant decrease in type 2 DM with and without MetS with respect to healthy normal subjects. These results are in line with other studies , 26. OurMeta-analyses of case-control studies have reported type 2 DM to be associated with a modest but significant decrease in testosterone levels  , 32. ThiCurrent evidence suggests that the causative relationship between the often low testosterone and type 2 DM might be bidirectional or even multidirectional and interrelated with obesity, MetS, SHBG, and other factors , 38. Theβ, which is in line with a number of studies [The results presented in this study also showed testosterone to be negatively correlated with insulin, HOMA-IR, and HOMA- studies , 44, 45. studies , 34, 46. studies . A recen studies , suggestOn using stepwise linear regression and controlling for the parameters that previously showed significant correlation, waist circumference was an independent factor that decreases free and total testosterone levels, whereas SHBG increases total testosterone. The general linear model with univariate analysis (ANCOVA) showed that on adjustment for SHBG, testosterone remained lower in the MetS subjects as compared to the other groups. However, on further adjustments for both SHBG and waist circumference, the significant difference disappeared, thus suggesting a role for abdominal obesity in the association of testosterone with both MetS and type 2 DM. This is in line with the cross-sectional survey from the National Health and Nutrition Examination Survey (NHANES) III  reportinin vitro evidence in which insulin reduces SHBG secretion directly and also inhibits the stimulatory action of thyroxine and estradiol on its synthesis in human HepG2 cells [in vitro study has shown that SHBG is not regulated by insulin but rather repressed by monosaccharide-induced lipogenesis in human HepG2 cells [in vivo, but rather the improvement of insulin resistance elevates SHBG after intensive insulin hypoglycemic therapy [Moreover, in accordance with several reports , 51, ourG2 cells , 53. HowG2 cells . MoreoveG2 cells , suggestOn comparing MetS and type 2 DM with or without MetS, the improved (higher) levels of testosterone and SHBG observed in type 2 DM with and without MetS may be the consequence of the diabetic treatment that improves the insulin sensitivity and insulin concentration and subsequently improves testosterone and SHBG levels. An earlier study in nondiabetic men has shown that SHBG is negatively correlated to insulin secretory pulse frequency , thus fuOur study is based on a single measurement of hormones and SHBG and lack information on symptoms of hypogonadism and/or gonadotropin levels. Hormonal and SHBG measurements were also conducted using immunoassay, which is less reliable and less specific than more recent methods such as the liquid chromatography tandem mass spectrometry that is used to determine steroidal hormone levels.In conclusion, this case-control study demonstrates that abdominal adiposity is a major determinant of low testosterone, irrespective of diabetes, by examining the impact of metabolic syndrome factors and diabetic parameters on testosterone and SHBG using stepwise linear regression and confirmed by ANCOVA. Our study, unlike many previous studies, examined the testosterone and SHBG levels in both nonobese and obese type 2 DM patients and showed type 2 DM patients to be associated with a modest but not significant decrease in testosterone with respect to healthy normal subjects. This raises the question whether testosterone therapy decreases insulin resistance independent of visceral fat reduction.


To characterize the intake of macronutrient and fiber in adolescents with type 1 diabetes (T1D) and examine their association with health indicators. n = 257, mean age 12 ± 1.2 years, 49.4% girls) reported dietary intake via two separate 24-hour recall interviews during a two-week period. Demographic and medical variables were abstracted from questionnaires and medical charts.  Baseline data from an RCT were examined. Adolescent-parent dyads ( Controlling for demographic and diet variables, a higher percentage of daily energy intake from fats was associated with poorer HbA1c. In contrast, an association between higher percent of energy intake from proteins and carbohydrates was found with higher systolic and diastolic BP, respectively.  Many early adolescents with T1D did not meet diabetes nutritional guidelines. Lower adherence to nutritional guidelines, specifically more than recommended energy intake from fats, was associated with poorer HbA1c. Addressing nutritional guidelines and increasing adherence as part of treatment may improve health outcomes for youth with T1D. Type 1 diabetes (T1D), one of the most common childhood chronic illnesses, requires adherence to a complex daily routine for proper management . Poorer Adhering to diabetes-specific guidelines for percentage of daily energy intake of macronutrients , as well as intake of dietary fiber, is especially important for individuals with T1D to promote good ambient glycemic control and to prevent long-term consequences including cardiovascular disease –9. AdoleThe dietary composition of adolescents with T1D is not likely to significantly differ from the general population, which is generally related to high fat consumption ; howeveNutritional guidelines based on both the American Diabetes Association  and theFew studies have explored specific macronutrient and fiber intake in adolescents with T1D. Initial evidence of suboptimal nutrition and its adverse relation to metabolic control has been demonstrated –23. AlthGiven the association of dietary intake with health outcomes, the first aim of the current study is to describe the macronutrient and fiber intake of early adolescents with T1D. The second goal is to evaluate the association of macronutrient and fiber intake with important health indicators including glycemic control , obesity as indicated by body mass index, dyslipidemia as indexed by levels of plasma LDL, and hypertension as indexed by systolic and diastolic blood pressure (BP). It is hypothesized that only a small percentage of early adolescents with T1D will meet diabetes-specific nutritional guidelines and that macronutrient and fiber intake, as well as adherence to guidelines, will be associated with health indicators.Early adolescents, aged 11 to 14, and their parents participated in a randomized controlled trial (RCT) at two mid-Atlantic children's hospitals. Participation in the RCT entailed completion of four brief sessions of behavioral intervention or diabetes education in conjunction with quarterly diabetes clinic visits over the course of 1 to 1 and 1/2 years. The intervention and outcomes are described in greater detail in a previously published paper . The datEligibility requirements of the RCT included diabetes duration of at least 1 year, absence of severe complications  or other medical diagnoses , and English fluency. The sample consisted of 257 dyads composed of one adolescent with T1D (49% girls) and one parent (91% mothers). Mean age at baseline was 12.8 years (SD = 1.2), mean illness duration was 5.1 years (SD = 3.1), and mean HbA1c was 73 mmol/mol (SD = 4). Two-thirds (64%) of the samples were prescribed a flexible insulin regimen, i.e., ≥4 injections or basal/bolus injections, or continuous subcutaneous insulin infusion/pump. Participant characteristics are summarized in Institutional review boards from each institution approved the larger study. Eligible families were identified from clinic lists and were mailed an informational letter. These families then received a follow-up telephone call from trained research assistants. At regularly scheduled diabetes clinic visit, each participant and his/her parent provided consent and assent, completed self-report questionnaires, and participated in separate parent and child 24-hour nutrition and diabetes self-care interviews. A second 24-hour retrospective dietary interview was completed over the telephone within two weeks of the baseline assessment. Completion of questionnaires took approximately 45 minutes, and each interview was approximately 15 minutes. Families received a $25 gift card in appreciation of their time. Of the 404 eligible families successfully contacted, 285 consented to participate (71%). Those who declined consent primarily cited lack of interest or time as the reason. Completed baseline data were provided by 257 parent-adolescent dyads (89%).Demographic and medical information was obtained via questionnaire and a retrospective review of the medical charts. Socioeconomic status (SES) was calculated based on parental occupation and education , with hiTo assess macronutrient and fiber intake, youth and one parent separately completed the 24-hour diabetes interview (DI) . The DI Parent- and youth-reported dietary intake was compared to current nutritional recommended guidelines  for adolp ≤ 0.10 in Step 1. Step 2 added nutrition variables associated with a health indicator at p ≤ 0.10 to Step 1 (model 2). Finally, a fully saturated model was tested that included demographic and nutrition variables associated with any health indicator at p ≤ 0.10 (model 3). Health indicators not associated with any nutrition variables were not included in the model testing. Continuous nutrition variables were given preference over categorized variables; however, significant categorized variables were explored in secondary models. Variables associated with health indicators at p ≤ 0.05 were considered significant in all models, and R2 was used to assess the amount of variance accounted for by the addition of nutrition variables to the model.Analyses were conducted using SAS software, version 9.3 . One of the 257 parent-adolescent dyads who completed baseline questionnaires was excluded from dietary analysis because they did not report dietary intake. To quantify the portion of the sample that met nutritional guidelines, each nutritional variable was recoded into dichotomous variables based on the cutoff recommended level for each nutrient . Next, bivariate associations between dietary intake, demographic variables, metabolic control, blood pressure, and lipid profile were assessed with general linear regression. Nonlinear relationships between significant nutrition variables and health indicators also were explored by including exponential variables in linear models. Following bivariate analyses, in order to evaluate the overall and unique contributions of hypothesized predictors to outcomes, multivariable linear regression models were conducted by including demographic variables associated with a health indicator at χ2(1) = 5.23, p = 0.02).The proportion of participants who met nutritional recommendations by category and by type of regimen is summarized in χ2(1) = 13.65, p < 0.001).Approximately half of parents (51.7%) reported that their child followed their nutrition plan less than 75% of the time. Using 75% of the time as a cutoff, parents of youth on a flexible regimen were significantly more likely to report that their child followed their nutrition plan the majority of time than those on a conventional regimen (χ2(1) = 4.03, p = 0.05). Of the health outcomes evaluated, only 21.3% achieved an HbA1c below 58 (mmol/mol) and was within the glycemic goal target range. Consideration of weight revealed 64.8% had a BMI percentile within the normal range, although 21.4% were classified as overweight and 13.6% had obesity. Moreover, 64.5% of participants had measured LDL cholesterol levels less than 100 mg/dL (2.59 mmol/L). Approximately 90% had measured blood pressure below 130/80 mmHg. These health indicators did not differ significantly by type of insulin regimen .Targets for HbA1c were met more frequently by those participants on a flexible regimen versus conventional regimen  and higher LDL . Lower SES category was related to poorer HbA1c , as was a conventional insulin regimen . Non-Caucasian ethnicity and older age were each associated with every poorer health indicator except BMI percentile, which was not statistically significantly related to either non-Caucasian ethnicity or age. A higher percentage of energy intake from proteins was the only nutrient associated with higher systolic BP , whereas a higher percentage of energy intake from carbohydrates was significantly associated with higher diastolic BP . The majority of nutritional variables were associated with HbA1c including fats , carbohydrates , and dietary fiber . No significant nutrition associations were noted for BMI percentile or LDL, and no evidence of nonlinear associations were seen.Bivariate associations between demographic and nutrition variables and all health indicators are presented in β = 2.09, p < 0.001) and longer diabetes duration  significantly contributing to the model predicting systolic BP. The addition of nutritional variables in Step 2 increased the variance explained by 2% points. Specifically, controlling for relevant demographic factors, a higher percentage of energy intake from proteins was significantly associated with higher systolic BP . Although nutritional variables were not significantly associated with diastolic BP controlling for relevant demographic factors, inclusion of nutritional variables marginally improved R2 (6% vs. 4%), showing contribution to the variance in diastolic BP. In Step 3, a higher percentage of energy intake from carbohydrates was associated with higher diastolic BP  The health indicator most highly associated with study variables was HbA1c, as 20% of the variance in outcome was accounted for by the model including relevant demographic and nutrition variables. A higher percentage of energy intake from fats was associated with higher HbA1c controlling for all other variables in the model . Fully saturated models did not alter these conclusions or improve percentage of variance explained.Results of regression analyses are shown in The current study reveals that many primarily middle-class early adolescents with T1D do not meet national nutritional guidelines set by the American Diabetes Association . NeverthA higher percentage of energy intake from fats was related to poorer glycemic control after accounting for other study demographic, disease, and nutritional factors. Taken together, demographic variables and diet variables explained 20% of the variance in glycemic control, consistent with existing T1D research , 37. In The current study illustrates that a sizable majority of youth did not meet ADA or ISPAD nutritional guidelines and may be at risk for cardiovascular disease and other health complications later in life. Early inklings of this risk are found in the present association between longer disease duration and higher LDL cholesterol and higher systolic blood pressure. Diabetes carries a known morbidity for hyperlipidemia, and therefore, youth may benefit from close monitoring of health status and macronutrient intake, particularly saturated fats, to prevent onset of cardiovascular disease . ParentsOur study adds to the literature by finding an association between dietary intake and health indicators of glycemic control and blood pressure in early adolescents. That is, specific dietary components in the present study had a direct relation to established health indicators, beyond the contribution of demographic factors and general nutritional status which were controlled statistically. Previous work has examined the association between nutrition and health status in Chinese adults with T1D, whose diets likely are dissimilar to those of American adolescents . NeverthA novel direct association also was found between macronutrient consumption and BP, although BP was not elevated overall in the current sample. Specifically, consumption of a higher percentage of energy from proteins related to higher systolic BP and a higher percentage of energy intake from carbohydrates related to diastolic BP. Importantly, evidence of dietary links to glycemic control and BP status based on just two samples of nutritional intake from two days is compelling and requires further study. More subtle associations may be found with more extensive sampling and other methods of assessing dietary intake. With an average disease duration in the present sample of just five years, the importance of understanding the dietary correlates of glycemic and cardiac status is underscored by the fact that longer diabetes duration was associated with higher LDL levels, a known risk factor for cardiovascular disease .Consistent with previous literature , 44, TabWhen youth with T1D are first diagnosed, especially as younger children, their care and management fall solely to their parents. At the time of diagnosis, most families receive “survival skills” training, which incorporates nutrition guidelines and medical nutrition therapy. However, early adolescence represents a period of increased independence and self-management, as well as greater freedom in food choices. Early adolescents may not be routinely included in initial diabetes education such that they may be unaware of the importance of balanced nutrition to manage their HbA1c and to prevent future health risk. The present study's findings underscore the ADA recommendation of yearly meetings with a registered dietician such that nutrition counseling is a routine and frequent part of diabetes education and management. Particularly pressing is the need to help early adolescents understand the importance of adherence to nutrition recommendations to optimize their glycemic control and to decrease their risk for future cardiovascular disease , 9.Given that education alone is not always sufficient to produce changes in behavior, for optimal efficacy, nutrition counseling could include focus on individual and cultural preferences, motivation, and self-efficacy to make healthful choices, as well as familial financial considerations. When adolescents and families have difficulty adhering to nutritional guidelines, clinical psychologists may be a critical component of care in order to promote adherence. Integration of technology may be an appealing facet of diabetes care to engage adolescents and promote self-monitoring behaviors as a means to support healthful decision-making. Technology may represent a fruitful avenue to keep nutritional education and reeducation updated and enjoyable.The current study is strengthened by the use of multimethod and multisource data at multiple time points. However, the cross-sectional nature of the study makes it impossible to determine casual relations among demographic, nutritional, and health variables. Moreover, the number of analyses increases risk for type 1 error. Future research should examine these relationships longitudinally, particularly as adolescents reach young adulthood and beyond. Moreover, although parents and youth reported their nutritional intake at two different time points using the gold standard for assessment of dietary intake, self-report of food intake is often underreported  and may Overall, many early adolescents with T1D did not meet guidelines for nutritional intake. Nonadherence to nutritional guidelines, above and beyond the influence of demographic variables, was associated with poorer HbA1c and places youth at risk for later health complications. Regular nutritional education, particularly for early adolescents, along with behavioral adherence to the guidelines may prove crucial for maintenance of better glycemic control and prevention of future cardiovascular disease.


High electrolyte wettability, thermal dimensional stability, and tensile strength are prerequisites for implementing separators in practical applications. In this study, we report on the discovery of nanofibril membranes derived from various plant fibers commonly used in the papermaking industry, for low cost and higher performances than the commercially available Celgard2325 in regard to the application of separators for lithium-ion batteries. Nanofibril membranes showed water contact angles as low as 18°, negligible size change at a heating temperature of 160 °C for 120 min, and tensile strength up to 137.6 MPa. The homogenization was found to strongly contribute to these improved performances. These findings suggest that the plant fiber-derived nanofibril membranes are anticipated to be promising candidates as separators for lithium-ion batteries. Today, lithium-ion batteries (LIBs) are one of the most promising and important energy storage technologies. LIBs can not only be used for portable devices like mobile phones, laptops, and digital cameras, but also flexible/wearable electronics, electronic vehicles, and large-scale power sources ,2,3,4. AThe separators used in modern LIBs are mainly polyolefin types such as Celgard2325, due to their good chemical stability ,11. NeveHowever, previous results showed that conventional papers seem unsuitable for LIB separators. Their thickness, pore size, and evenness could not meet the requirements because of massive size of ordinary cellulose fibers. Lee et al. obtained good results by using cellulose nanofibrils . Since tIn this work, the properties of separators derived from various types of plant fibers commonly used in paper making were compared with those of a commercially available separator . All separators were prepared via a facile papermaking process, which are used in the large-scale, low-cost production of cellulose LIB separators. It was demonstrated that the plant fiber-derived nanofibril membranes possessed excellent electrolyte wettability, thermal dimensional stability, and tensile strength, which suggested that these plant fiber-derived nanofibril membranes were anticipated to be promising candidates as separators for LIBs.6) in ethylene carbonate (EC)/dimethyl carbonate (DMC)/ethyl methyl carbonate (EMC)  , and polypropylene (PP)/polyethylene (PE)/polypropylene (PP) separator (Celgard2325)  were received from their respectively indicated suppliers.Bamboo pulp, hardwood pulp, softwood pulp, cotton pulp, and hemp pulp , 1 M lithium hexafluorophosphate . The values were obtained by averaging values from more than five drops.The thermal shrinkage behavior of cellulose nanofibril membrane and Celgard2325 was determined by an oven test ranging from 80 to 160 °C for 120 min.−1 under N2 atmosphere.The thermal stability of the separator was tested using a differential scanning calorimeter  ranging from 80 to 230 °C at 10 °C min2 atmosphere with a heating rate of 10 °C min−1 in the temperature range of 30–600 °C.The weight loss as a function of temperature was assessed by means of thermogravimetric analysis . The samples were tested in N−1 with samples 1.5 cm wide and 3 cm long.Tensile strength of cellulose nanofibril membrane and Celgard2325 was tested with a universal testing machine  at a speed of 5 mm minThe morphology of the cellulose nanofibril was observed on a scanning electron microscopy .The plant fiber is roughly cylindrical, the length of the fibers is generally 1–3 mm, and the diameter is mostly 10–20 μm. Treatments like beating, drying, pressing, and calendaring can reduce the length of fibers and the thickness of papers, but to a limited extent. For lightweight and high-energy-density batteries, the thickness of separators should be kept as small as possible. However, the thickness of commercial separators is generally 20–30 μm, which is approximately equivalent to the diameter of ordinary cellulose fibers. At this thickness, the stacking of only a few layers of fiber renders the uniformity and strength of the paper insufficient. Therefore, it is difficult to meet the development of separators by using ordinary cellulose fiber. As shown in A surface contact angle test can more accurately indicate the wettability of each separator. The results are shown in Good thermal dimensional stability of the separator is critical to the safety of batteries. TGA of Celgard2325 and the cellulose membranes with homogenization are compared in The stress–strain curves of the cellulose membranes with homogenization are shown in In summary, the separator prepared from plant fiber commonly used in papermaking industry possessed excellent electrolyte wettability, thermal dimensional stability, and tensile strength, suitable for LIBs. The contact angle of the cellulose membranes (distilled water) was less than 45°, and the membranes had no obvious change when they were placed at 160 °C for 120 min. Moreover, the homogenization effectively improved the electrolyte wettability and tensile strength of the cellulose membranes. After the homogenization, the hemp pulp and hardwood pulp cellulose nanofiber (CNF) membranes had the best wettability, with the contact angles only 18° and 21°, which were suitable for coating of composite LIB separators. Meanwhile, the separators made from softwood pulp and cotton pulp had the best tensile strength, exceeding 130 MPa, which were ideally used as the matrix material of cellulose LIB separators.


Caffeine is routinely given to preterm infants hours after birth to treat apnea of prematurity. In view of it’s success, earlier administration in the delivery room is being considered, but little is known about how caffeine may effect the cardiovascular changes during the fetal to neonatal transition. Our aim was to determine the effect of prenatal caffeine administration on haemodynamic parameters in ventilated preterm lambs immediately after birth.Catheters (carotid artery and jugular vein) and ultrasonic flow probes (pulmonary artery and carotid artery) were implanted in preterm lambs , immediately before delivery by caesarean section. Before the cord was clamped, lambs were intubated and a caffeine  or saline (n = 5) infusion was given intravenously to the ewe and lamb over a 15-minute period. Two minutes after clamping the cord, ventilation commenced with a sustained inflation (35 cm H2O for 30 seconds) followed by ventilation for 30 minutes .Blood gas parameters and rectal body temperature were not different between the two groups. Changes in pulmonary blood flow (PBF) and carotid blood flow (CBF) did not differ significantly between groups. PBF increased significantly after ventilation onset in both groups  and remained elevated thereafter. CBF did not increase but decreased after SI in the caffeine group. Blood pressure, heart rate, and peripheral oxygen saturation did not differ between groups at any stage of the study.Prenatal caffeine infusion had no significant effect on acute haemodynamic parameters in ventilated preterm lambs during the cardiorespiratory transition. Caffeine, a methylxanthine, is one of the most widely used medications in neonatology. It acts directly on the respiratory centre to increase respiratory drive and is used as first-line therapy to treat and prevent apnea of prematurity. ,2 SeveraA receptors, mobilization of intracellular calcium and most importantly caffeine acts as a non-specific A1 and A2a adenosine receptor antagonist. At therapeutic plasma concentrations, caffeine’s physiological effects are mediated mainly by blocking A1 and A2a adenosine receptors. The location of these receptors in the body include the central nervous system, lung, heart and vascular system. [Caffeine has dose-dependent effects on various biochemical targets in the human body, such as inhibition of phosphodiesterase, blockade of GABA system. –12 In ad system. ,14Only a few conflicting studies have investigated the cardiovascular effects of caffeine in preterm infants and none were conducted during transition period. A recent study in preterm infants has demonstrated that caffeine administration within minutes of delivery significantly increases the respiratory effort.  This hasThis study, including all experimental procedures on animals, was approved by Monash University animal ethics committee.Surgery was performed on 14 anaesthetised pregnant Border-Leicester ewes, bearing single or twin fetuses, at ~126± 2 days of gestation (term is ~147 days). During surgery, fetal lambs were instrumented with catheters and ultrasonic flow probes, immediately before delivery by caesarean section as described previously.  Briefly,2) levels. The fetal trachea was intubated orally with a 4.0mm cuffed endotracheal tube, which was clamped during surgery to minimize lung liquid loss.A transcutaneous pulse oximeter  was attached to the right forelimb to measure preductal peripheral oxygen saturation , starting before the caffeine/ saline infusion commenced and continuing until the end of the experiment.2O and a fraction of inspired oxygen (FiO2) of 0.21. After the SI, lambs were connected to the ventilator  and mechanically ventilated for 30 minutes in volume guarantee mode, with a tidal volume of 7mL/kg and a positive end-expiratory pressure of 5 cmH2O. The inflation gases were heated and humidified. Ventilation parameters including FiO2 were adjusted to maintain SpO2 values at 90–95% after the first 10 minutes from birth and to target a PaCO2 between 35–55 mmHg, a PaO2 between 60–100 mmHg and an arterial pH between 7.30–7.45. Arterial blood gases (0.25mL) were collected before and after caffeine/ saline infusion and 5, 10, 15, 20 and 30 minutes after ventilation onset. To measure caffeine concentrations in plasma, 2.5 mL blood samples were collected before and after caffeine/ saline infusion and at 15 and 30 minutes after ventilation onset and replaced with an equal volume saline.Caffeine base  in a 15mg/mL solution was administered intravenously at 10mg/kg (ewe body weight) over a 15-minute period to the ewe. Just before this infusion finished, an extra 10mg/kg (estimated lamb weight) caffeine infusion was commenced into the lamb via the jugular venous catheter. The control group received the same volume of infusate, which was saline instead of caffeine. Thereafter, the lamb was fully delivered and placed on the ewe’s stomach, taking care to not obstruct catheters or twist flow probes. The endotracheal tube was unclamped and lung liquid was drained passively. Once all physiological recordings had stabilised, the cord was clamped and cut. Thereafter the lambs were closely monitored two-minutes before ventilation commenced, to mimic the time it takes to initiate respiratory support in clinical practice. Ventilation commenced with a 30 seconds sustained inflation (SI) delivered by the Neopuff  using a peak inspiratory pressure of 35 cmHAll lambs were lightly sedated  and remained apneic to allow conduction of the experiment. Antenatal glucocorticoids and postnatal surfactant were not administered to the lambs, as our primarily interest was the cardiovascular change after birth, after receiving prenatal caffeine.After the experiment the ewes and lambs were humanely euthanized using sodium pentobarbitone (100 mg/kg i.v).2 and Heart rate (HR) were averaged over 20 seconds before and after caffeine/ saline infusion, umbilical cord occlusion and applying SI and at 5, 10, 15, 20, 25 and 30 minutes after the ventilation onset.Left pulmonary artery blood flow (PBF), carotid artery blood flow (CBF), CBP , SpOAll physiological recorded data were compared over time and between groups using a two-way ANOVA for multiple comparisons with a Bonferroni post hoc test. An unpaired t test or a Fisher’s exact test was used to compare the descriptive data between the caffeine and saline group. The level of statistical significance was set at p<0.05 and data are presented as mean± SEM.Gestational age, birth weight and distribution of males and females did not differ significantly between the caffeine and saline group . SimilarThe arterial plasma caffeine concentration was similar between groups before caffeine administration and then increased significantly in ewes and lambs treated with caffeine . In caffIn both groups PBF increased significantly after the SI  and remained elevated for the duration of the experiment ; no diffNo significant differences in CBF between the groups were observed. CBF tended to increase in both groups following cord clamping, as previously described , and remMean, systolic and diastolic CBP were not different between saline and caffeine treated lambs . Cord clHR was also similar between the groups throughout the study period . While t2 was not significantly different between groups, the SpO2 decreased significantly after CC in the saline, but not in the caffeine treated lambs, reaching a minimum just before the SI. Thereafter, SpO2 was significantly increased at 5 minutes of ventilation onset, with the time course for changes in SpO2 values being very similar in both caffeine and saline treated lambs  at the end of the initial 15min infusion period. These data indicate that it is possible to preload newborn infants with therapeutic levels of caffeine prior to delivery by administering to the mother. However, as this is dependent upon transplacental caffeine exchange, any factor that disrupts this transfer, such as placental abruption or umbilical cord problems, will reduce the efficacy of this approach.The total dose administered to the lamb was estimated to be ~20mg/kg of caffeine-base , which is at the upper end of the clinically used dose range. Currently the most widely used loading and maintenance dose of caffeine citrate is 20 mg/kg  and 10 mg/kg , respectively. –23 A stu2. To some extent, these findings differ to other studies done in preterm infants and lambs after receiving caffeine. [2, they did find a significantly higher HR in caffeine-treated lambs, which increased 10 minutes after starting the infusion and remained elevated for the whole experiment. [We found that all physiological differences examined were similar in saline and caffeine-treated lambs within the first 30 minutes of the transition period. These parameters included mean, systolic and diastolic CBP, PBF, CBF, HR and SpOaffeine. ,28–30 Creriment.  In a stuA study by Hoecker et al found no meaningful changes in systolic, diastolic and mean blood pressure, HR and left ventricular output in preterm infants one and two hours after caffeine administration. Moreover, they investigated cerebral blood flow velocity and flow velocities in the carotid artery and superior mesenteric artery via Doppler ultrasound one and two hours after caffeine administration and observed a reduction in these parameters.  However,2 [2 values after caffeine treatment. [2 were observed before and after caffeine infusion and there were no differences in PaCO2 values between the two groups within the study period.We observed slightly lower CBF levels in the caffeine group than in the saline group, although the differences weren’t significant. Previous studies reported reduced blood flow velocities in cerebral arteries one to two hours after high-dose caffeine infusions, which were thought to result from vasoconstriction mediated by inhibition of adenosine receptors. ,34 Cereb2  althougheatment.  In our sHowever, we have to admit that the postnatal sedation of the lambs with Alfaxalone differs from typical conditions in humans, though any possible effect on haemodynamics would be equally distributed between both groups.Caffeine has been shown to stimulate respiratory drive and reduce the number of apneas in preterm infants due to a higher breathing frequency and an improved function of the diaphragm muscle. –3 Our exWe conclude that a high-dose prenatal caffeine infusion has no acute effect on cardiovascular function immediately after birth in ventilated preterm lambs. Arterial blood pressure and blood flow through the lungs and the brain were similar in both groups, which suggests that there is neither an obvious beneficial nor a harmful effect of caffeine on hemodynamic changes during immediate transition period.


Heterocephalus glaber) are among the most hypoxia-tolerant mammals, but their physiological responses to acute and chronic sustained hypoxia (CSH), and the molecular underpinnings of these responses, are poorly understood. In the present study we evaluated the acute hypoxic ventilatory response and the occurrence of ventilatory acclimatization to hypoxia following CSH exposure (8–10 days in 8% O2) of naked mole rats. We also investigated the role of excitatory glutamatergic signaling in the control of ventilation and metabolism in these conditions. Animals acclimated to normoxia (control) or CSH and then exposed to acute hypoxia (7% O2 for 1 h) exhibited elevated tidal volume (VT), but decreased breathing frequency (fR). As a result, total ventilation (E) remained unchanged. Conversely, VT was lower in CSH animals relative to controls, suggesting that there is ventilatory plasticity following acclimatization to chronic hypoxia. Both control and CSH-acclimated naked mole rats exhibited similar 60–65% decreases in O2 consumption rate during acute hypoxia, and as a result their air convection requirement (ACR) increased ∼2.4 to 3-fold. Glutamatergic receptor inhibition decreased fR, E, and the rate of O2 consumption in normoxia but did not alter these ventilatory or metabolic responses to acute hypoxia in either the control or CSH groups. Taken together, these findings indicate that ventilatory acclimatization to hypoxia is atypical in naked mole rats, and glutamatergic signaling is not involved in their hypoxic ventilatory or metabolic responses to acute or chronic hypoxia.Naked mole rats ( E) that occurs in response to the detection of decreased arterial O2 tension (PaO2) by peripheral chemoreceptors  the central nervous system (CNS) responsiveness to afferent inputs from the carotid bodies increase   .nucleus tractus solitarii (NTS) in the brainstem  within the NTS  of a greater magnitude  present a neotenic phenotype in their ventilatory response to both acute hypoxia and to CSH. Specifically, E decreased ∼70% in adult naked mole rats in acute hypoxia (7% O2) and this acute decrease was mediated by an increase in inhibitory adenosinergic signaling (O2 by >70 in 7% O2 (2)  , suggest2, and days to weeks at 8% O2 (2 for 1 h), before and after glutamate receptor manipulation or sham injections of saline.Naked mole rats are the most hypoxia-tolerant mammal presently identified and tolerate minutes of complete anoxia, hours at 3% Oat 8% O2 . Adult nat 8% O2 . Furtherat 8% O2 . Given t2 in 70% humidity with a 12L:12D light cycle. Animals were fed fresh tubers, vegetables, fruit and Pronutro cereal supplement ad libitum. Animals were not fasted prior to experimental trials. All experimental procedures were approved by the University of Ottawa or the University of British Columbia Animal Care Committees in accordance with the Animals for Research Act and by the Canadian Council on Animal Care. All experiments were performed during daylight hours in the middle of the animals’ 12L:12D light cycle when the animals were awake and active. Naked mole rats that are housed within colony systems, as are our experimental animals, do not exhibit circadian rhythmicity of general locomotor activity , body temperature decreased to 30.5 ± 0.2°C, consistent with recent measurements from our laboratory  were individually placed, unrestrained, inside a 450 ml Plexiglas experimental chamber , which was in turn placed into an environmental chamber held at ∼29°C. The temperature of the animal chamber was recorded continuously throughout the experiment using multiple iButtons that recorded ambient temperature at a frequency of one measurement per minute . Body temperature was measured every 10 min from subcutaneous radio frequency identification (RFID) microchips using a RFID reader . In normoxia and at an ambient temperature of ∼29.0°C, the naked mole rat body temperature was 32.0 ± 0.2°C (data not shown). During acute hypoxia . The analyzers were calibrated prior to each trial with 20.95% O2, 1.5% CO2, balance N2, and with 100% N2 gas mixes. During experimentation, animal breathing caused pressure fluctuations due to humidity and warmth of air in each expired breath, which were compared to the pressure of an identical reference chamber. Continuous monitoring of pressure differences between these two chambers by a differential pressure transducer , connected between the animal and reference chamber, allowed detection of breaths.Animals were provided with a thin layer of bedding on the floor of the experimental chambers. The animal chamber was sealed and constantly ventilated with gas mixtures, set to the desired fractional gas composition by calibrated rotameters . The advantage of this open-flow system is that it prevents the depletion of O2 production (CO2) rates were measured by analyzing the outflowing composition of gas by a Sable Systems FC-10 O2 analyzer and a Sable Systems CA-10 CO2 analyzer, respectively, and comparing outflowing gas concentrations to inflowing gas concentrations. The 2 was calculated from the product of the constant airflow through the chamber and the difference between the inflow and outflow in the fractional concentration of O2. The CO2 was calculated from the product of the constant airflow through the chamber and the difference between the outflow and inflow in the fractional concentration of CO2. All metabolic variables are reported at STPD.Oxygen consumption and COR) was determined by counting E-induced pressure oscillations, whereas VT was determined by integrating expiratory flow and then calculated using the method described by Drorbaugh and Fenn modified for open-flow plethysmography by Jacky  into the experimental chamber at a rate similar to the respiration rate of the animal. Ventilation was calculated as the product of fR and VT. All ventilatory measurements were selected when animals were resting and are reported at body temperature and pressure, saturated (BTPS).Respiratory frequency , the ACR for CO2 , O2 delivery, and lung O2 extraction were calculated from ventilatory and metabolic measurements. Oxygen delivery to the lungs was calculated as ventilation multiplied by the fractional concentration of O2 in inspired air. The percent of O2 extracted from each breath was calculated by dividing 2 by E, multiplied by the fractional concentration of O2 in inspired air and multiplying that by 100.The air convection requirement (ACR) for O-1), or cyanquixaline . Animals were then placed back in the animal chamber and E, O2, CO2, and body temperature were monitored for 1 h to assess the effect of the injection. Next, the inflowing gas composition was switched to 7% O2 (hypoxia) and these physiological variables were measured for 1 h. Following experimentation, animals were allowed to recover in normoxia and then returned to their colonies. CNQX and MK-801 were purchased from Sigma-Aldrich .Control and CSH animals were transferred to the animal chamber under normoxic conditions and baseline recordings were obtained for 1 h. Next, animals were removed from the chamber and received ∼400 μL intraperitoneal injections of 0.9% NaCl saline alone (sham), dizocilpine  were amplified, filtered, recorded and analyzed using PowerLab data acquisition hardware and LabChart software . From the recorded signals, and calculated dependent variables, we determined average: body temperature, O2, CO2, fR, VT, E, ACRO2, ACRCO2, O2 delivery and lung O2 extraction, for the last 10–15 min of each O2 exposure  to ensure animals reached a steady state. Inflowing gas concentrations were measured before and after each O2 exposure.All signals , we log transformed the dependent variable. We entered acclimation group (control or CSH), drug treatment (sham or drug), level of inspired O2 (21% O2 or 7% O2), and body mass as fixed effects in our initial models. We tested all 2- and 3-way interactions of acclimation group, drug treatment, and level of inspired O2. We did not remove any terms from our models given the importance of all independent variables and interactions to our research objectives. When interaction terms were significant the data were separated and analyzed independently using a one-way ANOVA, followed by a Tukey-Holm post hoc analysis to determine differences between acclimation group, drug treatment, level of inspired O2, and to correct for multiple pairwise comparisons. All results are presented as mean ± SD, with statistical significance set as p < 0.05. Results from statistical tests are included in the attached supplemental table  changed significantly, but in opposite directions from one another. Specifically, and relative to pre-injection normoxic controls, fR was 26% lower in acute hypoxia  in VT, compared to control animals  decrease in f animals . In this animals . However the RER .T (and thus E) in CSH animals, the ACR of CSH-acclimated naked mole rats was also slightly smaller than that of control animals in acute hypoxia  and 8 for CSH + CNQX (closed squares); note, sham injections are shown in gray for reference]. These changes were due to a combination of mostly non-significant reductions in both fR and VT following CNQX injection, in both control and CSH groups  and 6 for CSH + MK801 (closed squares)].We next explored the potential role for excitatory glutamate receptor signaling in mediating the acute hypoxic ventilatory response and ventilatory acclimatization to hypoxia. Unlike sham injections –3, blockH groups ; only thR and VT in both groups were similar in magnitude to those of the control animals. However, while VT was still reduced in the CSH group relative to the control group in both CNQX- and MK801-treated animals in both normoxia and acute hypoxia, the difference was no longer significant.When animals in each group were then exposed to acute hypoxia, we did not observe any significant effects of either drug treatment (CNQX or MK801) on any ventilatory variable; the hypoxic changes in fO2 and CO2) was also reduced following injection of either CNQX or MK801 in both control and CSH groups during normoxia, although this effect was only significant in the CNQX-treated animals . Third, naked mole rats exhibit an atypical form of ventilatory acclimatization to hypoxia in which levels of VT and Edecrease following CSH, while metabolism is not affected by acclimation to CSH. Finally, glutamatergic inhibition reduces E through a decrease in fR, and also reduces metabolic rate in normoxia in both control and CSH groups. However, this intervention does not influence ventilatory or metabolic responses to acute hypoxia in either the control or CSH group. To our knowledge, a lack of a role for glutamatergic signaling in mediating the hypoxic ventilatory response is unique among adult mammals.In the present study we set out to re-evaluate the acute hypoxic ventilatory response and the occurrence of ventilatory acclimatization to chronic hypoxia in naked mole rats using more accurate body temperature measurements than in previous studies. We also investigated the potential role of excitatory glutamatergic signaling in the hypoxic ventilatory response with and without acclimation to CSH. Our study yielded four important findings. First, with the incorporation of more accurate body temperature measurements in our calculations of VT and therefore E when using barometric plethysmography .Our earlier studies and those from other laboratories reported that naked mole rats do not exhibit a significant decrease in body temperature in response to acute hypoxic or anoxic exposure . It now mography , 2013. W hypoxia , whereas% higher , while aher  but not E.A central finding of our study is that CSH significantly reduces Vfter CSH , not a dO2 during acute hypoxia of ∼65% relative to normoxic values. This change is consistent with measurements in similarly treated naked mole rats [65%  and thus a greater reliance on carbohydrate fuels would increase energetic efficiency during acute hypoxia. This strategy would be particularly useful in naked mole rats because they likely experience hypoxia transiently during intense exercise and when resting  and thus would have opportunities to replenish carbohydrate stores while in more normoxic regions of their burrows. It is in these normoxic regions that they rely more heavily on lipid energy stores . In CSH animals we observe a greater reliance on lipids, suggesting that some form of metabolic remodeling occurs during CSH. Endogenous carbohydrate stores are unlikely to be sufficient to sustain metabolic needs during prolonged hypoxia, likely leading to the reversal in fuel use compared to what is seen in acute hypoxia.Naked mole rats also exhibit a fuel switch toward increased reliance on carbohydrates with acute hypoxia. This response is similar to that observed from other hypoxia-adapted rodents, including murine species that live at high altitudes . The ATPE under any hypoxic condition in naked mole rats, which indicates a potential deficit of function in excitatory signaling mechanisms related to the control of breathing.We report that glutamatergic receptor inhibition reduces ventilation and metabolic rate in normoxia but does not impact the acute hypoxic ventilatory response or ventilatory acclimatization to hypoxia. These findings were surprising because excitatory glutamatergic signaling is the primary neurotransmission pathway that underlies the acute hypoxic ventilatory response and ventilatory acclimatization to hypoxia in most other adult mammalian species . ConversThe fact that we observe effects of CNQX and MK801 on breathing and metabolic rate in normoxia suggests that these drugs and the concentrations we employed in our study are efficacious in this species. However, glutamatergic receptor antagonists were injected intraperitoneally and thus their specificity of action must be interpreted with caution. Unfortunately, the anatomy of naked mole rats, and specifically the location of large muscle masses on the top of the cranium, which are critical for eating, digging, and other social functions within the colony, makes stereotaxic implantation of permanent cannulas that target the respiratory brainstem detrimental to the health and sociability of this species. However, intraperitoneal injections of these same pharmacological agents have been used previously in numerous studies in mice and rats to evaluate a wide variety of physiological and behavioral responses , which would be undesirable and energetically expensive for an organism that frequently experiences variable levels of hypoxia while engaging in highly divergent activity states.In the present study we demonstrate that naked mole rats do not alter  hypoxic . HoweverMP, AS, and WM conceived of and designed the study. MP, WM, and YD wrote the manuscript. SG, AS, DC, YD, MP, and LB performed the experiments. YD analyzed the data. All authors gave final approval of the published version and agree to be accountable for all content therein.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.