Classical and emergent serum, urinary, and kidney tissue (gene and protein expression) markers were assessed. Renal lesions were analyzed in hematoxylin and eosin, periodic acid-Schiff, and Masson's trichrome stains. SRL-treated rats presented proteinuria and NGAL (serum and urinary) as the best markers of renal impairment. Short CsA treatment presented slight or even absent kidney lesions and TGF-β, NF-κβ, mTOR, PCNA, TP53, KIM-1, and CTGF as relevant gene and protein changes. Prolonged CsA exposure aggravated renal damage, without clear changes on the traditional markers, but with changes in serums TGF-β and IL-7, TBARs clearance, and kidney TGF-β and mTOR. Conversion to SRL prevented CsA-induced renal damage evolution (absent/mild grade lesions), while NGAL (serum versus urine) seems to be a feasible biomarker of CsA replacement to SRL.Protocols of conversion from cyclosporin A (CsA) to sirolimus (SRL) have been widely used in immunotherapy after transplantation to prevent CsA-induced nephropathy, but the molecular mechanisms underlying these protocols remain nuclear. This study aimed to identify the molecular pathways and putative biomarkers of CsA-to-SRL conversion in a rat model. Four animal groups ( Calcineurin inhibitors, such as cyclosporin A (CsA), are clinically important immunosuppressive drugs for prevention of allograft rejection after organ transplantation and also for several autoimmune disorders, such as psoriasis, rheumatoid arthritis, systemic lupus erythematosus, and inflammatory bowel disease, among other indications , 2. DespComplete avoidance of CNIs, in particular of CsA, from transplantation immunotherapy, has been viewed as an invalid option by almost all the transplantation centres worldwide, particularly because of the risks in acute rejection. The main long-term goals of immunosuppressive therapy are to reduce drug exposure while maintaining a well-functioning graft, keeping efficacy and minimizing drug-induced serious side-effects, including infections and nephrotoxicity . The recEarly diagnosis of nephropathy can greatly improve patient diagnosis, but the initial stages of CsA-induced nephropathy are largely asymptomatic, making early diagnosis difficult . Since tThe mechanisms underlying CsA-induced nephropathy have been debated for the last decades and are clearly viewed as having a multifactorial nature  –20, as eWe have previously shown, in a preliminary study using an experimental model for comparing CsA and SRL, that distinct mechanisms and players are involved in the effects of these drugs on the kidney tissue, in a moment when renal lesions are almost absent . In addiMale Wistar rats (aged 11 weeks) were purchased from Charles River Laboratories  and housed  in IVC racks, subjected to 12 h dark/light cycles and given standard laboratory rat chow  and free access to tap water. Forty-two animals were divided into two groups, according to the period of the treatments. Eighteen animals were used in a protocol of 3-week treatments and divided in 3 groups: control (vehicle), cyclosporin A , and sirolimus . Twenty-four animals were used in a protocol of 9-week treatments and divided into 4 groups: control (vehicle), cyclosporin A, sirolimus, and conversion (cyclosporin A during 3 weeks and sirolimus during the last 6 weeks). Treatments were performed by oral gavage and body weight was monitored daily. Animal experiments were conducted according to the European Council Directives on Animal Care and to the National Authorities.The rats were anesthetized (i.p.) with 2 mg/Kg BW of a 2 : 1 (v : v) 50 mg/mL Ketamine  solution in 2.5% chlorpromazine . When the animal did not present response to stimulus, blood samples were immediately collected by venipuncture from the jugular vein in needles with no anticoagulant for serum samples collection. Then, the rats were sacrificed by cervical dislocation, and the kidneys were immediately removed, weighted, divided, and stored according to the next procedure: RNA-stabilizer reagent for gene expression determinations, frozen in nitrogen for lipid peroxidation assays, prefixed with formaldehyde for histopathological analysis and immunohistochemical detections.β (IL-1β), interleukin 2 (IL-2), vascular epidermal growth factor (VEGF), and transforming growth factor beta 1 (TGF-β1) were measured by ultrasensitive Quantikine ELISA kits . High sensitivity CRP (hsCRP) was detected by using an ELISA kit . Interleukin-7 (IL-7) was measured through an ELISA kit obtained from Wuhan EIAAB Science Co .Serum creatinine and blood urea nitrogen (BUN) contents were evaluated by automatic validated methods and equipment . Serum levels of interleukin 1 ad libitum. The urine concentration of creatinine, BUN, and protein was assessed in 24-hour urine , and the urine volumes were measured in order to calculate creatinine and BUN clearance and glomerular filtration rate, as previously described [The animals were housed in metabolic cages during 24 hours and received tap water and foodescribed .μL of kidney tissue supernatant, serum, or urine (previously centrifuged to remove particulates) was incubated 1 hour in a TBA solution. Samples incubated at 90°C for 60 min. In this test, one molecule of MDA reacts with two molecules of TBA with the production of a pink pigment producing maximal absorbance at 532 nm. The concentration of MDA was calculated with respect to a calibration curve using 1,1,3,3-tetramethoxypropane as the external standard (range: 0.1–83.5 μM) and results were expressed as μM/g of kidney tissue and μM of serum or urine.Lipid peroxidation was determined by assaying the malondialdehyde (MDA) production by means of the thiobarbituric acid (TBA) test. Briefly, 100 μL of RLT lysis buffer was added, and tissue disruption and homogenization for 2 min at 30 Hz were performed using a TissueLyser . Tissue lysates were processed according to the RNeasy Mini Kit protocol . Total RNA was eluted in 50 μL of RNase-free water . In order to quantify the amount of total RNA extracted and to verify RNA integrity , samples were analyzed using a 6000 Nano Chip kit, in the Agilent 2100 Bioanalyzer  and the 2100 expert software, following manufacturer's instructions. The isolation yield was from 0.5 to 3 μg; RIN values were 6.0–9.0 and purity (A260/A280) was 1.8–2.0.The kidneys were stored in RNA later solution . For RNA extraction, 10 mg of tissue was weighted, 450 μL (2 U) RNase H for 20 min at 37°C and, finally, cDNA was eluted to a final volume of 50 μL and stored at −20°C.RNA was reverse transcribed with SuperScript III First-Strand Synthesis System for RT-PCR . One microgram of total RNA was mixed with a 2x First-Strand Reaction Mix and a SuperScript III Enzyme Mix (Oligo (dT) plus random hexamers). Reactions were carried out in a thermocycler Gene Amp PCR System 9600 , 10 min at 25°C, 30 min at 50°C, and 5 min at 85°C. Reaction products were then digested with 1  Rattus norvegicus  and geNorm software  to select optimal housekeeping genes for this study [−ΔΔCt method [Gene expression was performed using a 7900 HT Sequence Detection System . A normalization step preceded the gene expression quantification, using geNorm Housekeeping Gene Selection kit foris study . Real-tit method . The resμm thick sections were mounted on glass slides and stained for routine histopathological diagnosis with haematoxylin and eosin (H&E).Samples were fixed in Bock's fixative and embedded in paraffin wax, and 4 μm thick sections were immersed in water and subsequently treated with a 1% aqueous solution of periodic acid, then washed to remove any traces of the periodic acid, and finally treated with Schiff's reagent. All samples were examined by light microscopy using a Zeiss Microscope Mod. Axioplan 2. The degree of injury visible by light microscopy was scored in a double-blinded fashion by two independent pathologists. Lesions were evaluated on the total tissue on the slide.Periodic acid of Schiff (PAS) was used to evaluate and quantify the renal lesions. Samples were fixed in 10% neutral formalin, embedded in paraffin wax, and 4 Glomerular damage was assessed by evaluating mesangial expansion, the glomerular basement membrane and the Bowman's capsule thickenings, nodular sclerosis, and vascular pole hyalinosis. The analysed tubulointerstitial lesions comprised inflammatory infiltration, presence of hyaline cylinders, tubular basement membrane irregularity, tubular calcification, tubular vacuolization, and the association of interstitial fibrosis and tubular atrophy (IFTA). The evaluation of vascular lesions was concentrated on vascular congestion and hyperemia, arteriolar vacuolization, arteriolosclerosis, and arteriosclerosis. A semiquantitative rating for each slide ranging from normal  to severe (extensive damage) was assigned to each component. Severity was graded as absent/normal (0), mild (1), moderate (2), and severe (3). Scoring was defined according to the extension of the lesion (number of capsules): normal: 0%; mild: <25%; moderate: 25–50%; severe: >50%. The final score of each sample was obtained by the average score observed in the individual glomeruli, in the considered microscopic fields. Tubular calcification was evaluated and graded by the same semiquantitative method. Regarding vascular lesions, arteriosclerosis was scored as 0 if no intimal thickening was present, as 1 if intimal thickening was less than the thickness of the media, and as 2 if intimal thickening was more than the thickness of the media and considering the worst artery on the slide. Using PAS, the rating was set for intensity and extension of staining, ranging from 0 (no staining) to 3 (intense and extensive staining), respecting tissue specificity scoring when adequate.Deparaffinise and rehydrate through 100% alcohol, 95% alcohol, and 70% alcohol. Wash in distilled water. After that, refix in Bouin's solution for 1 hour at 56°C to improve staining quality and rinse in running tap water for 5–10 minutes to remove the yellow colour. Stain in Weigert's iron hematoxylin working solution for 10 minutes and rinse in running warm tap water for 10 minutes. Wash in distilled water. Stain in Biebrich scarlet-acid fuchsin solution for 10–15 minutes and wash in distilled water. Differentiate in phosphomolybdic-phosphotungstic acid solution for 10–15 minutes and transfer sections directly to aniline blue solution and stain for 5–10 minutes. Rinse briefly in distilled water and differentiate in 1% acetic acid solution for 2–5 minutes and wash in distilled water. Finally, dehydrate very quickly through 95% ethyl alcohol, absolute ethyl alcohol, clear in xylene, and mount with resinous mounting medium. All samples were examined in a blind fashion by expert personnel (pathologists) by light microscopy using a Zeiss Microscope Mod. Axioplan 2.μm thick sections with sagittal orientation of kidney fixed in Bock's fixative and embedded in paraffin wax. The samples were processed by indirect immune detection technique with mouse and rabbit specific HRP/DAB detection IHC kit  using the primary antibody mammalian target of rapamycin  (dilution 1 : 250). The protocol was executed according to the manufacturer's instructions. In this study, we employed primary antibodies against CTGF , TGF-b , mTOR , NF-κβ p50 , and KIM-1 . For KIM-1 detection the secondary antibody was anti-goat . After testing the different antigen-retrieval methods and negative controls, immunohistochemical procedures were optimized. To identify PCNA protein we used a standard kit . An appropriate positive control was used in each staining run, and each slide was stained with a negative control obtained by omitting the primary antibody. Standard procedures were used for visualisation and the staining was quantified using a semiquantitative scale (1–4) that evaluated both the intensity and area of staining. Intensity was graded as very low (1), low (2), moderate/mild (3), and high (4); staining area was graded as <25% (1), 25–50% (2), 25–75% (3), and >75% (4). All slides were reviewed independently by 2 investigators blinded to the data. In this evaluation a quantitative immunohistochemical score (QIC) was calculated. QIC = % of staining area * staining intensity * 0.1.Immunohistochemical analyses were performed in 4 P less than 0.05.Statistical analyses were performed using the GraphPad Prism for Windows (version 5.00). The results are presented as means ± S.E.M. Comparisons between groups were performed using one-way ANOVA test, followed by the post hoc Bonferroni's multiple comparisons. The association between categorical variables was analyzed using Pearson's test in the IBM Statistical Package for Social Sciences (SPSS) for Windows, version 20.0 . Significance was accepted at P < 0.05) and tubules (P < 0.01) after the first 3-week period ). Long-term CsA treatment (9 weeks) promoted relevant changes on the kidney  structure, which are viewed as clear signs of nephrotoxicity. The main changes encountered compared with the normal controls are represented in P < 0.05). The conversion protocol does not promote any advantage in the vascular field when compared with the CsA treatment alone; two rats of the group presented arteriosclerosis (grades 1 and 2).Nephrotoxicity was confirmed by two independent pathologists, which have characterized the lesions through the attribution of degrees to each vascular, glomerular, and tubular lesion, examining kidney slices stained with H&E and PAS. After 3 weeks of CsA treatment, only slight morphological changes on the tubules (tubular vacuolization) were found when compared with the control . However Figures 1 and 2(cP < 0.01), hyalinosis of vascular pole (P < 0.001), and thickening of Bowman's capsule (P < 0.001) when compared with the control rat kidneys (P < 0.05 versus CsA). The global glomerular score clearly showed that SRL is less toxic than CsA (P < 0.01) and this was reflected in the lower score found in the conversion group.Regarding the glomerular field after 9 weeks of CsA treatment, the major lesions found were mesangial expansion ( kidneys 1–4). OneP < 0.001, both), versus the normal profile found in the control rats  and inflammatory infiltration were absent in the kidneys of the conversion group rats. 1) gives an idea about the treatments influence on the tubules. Clearly, CsA promotes more tubular damage than SRL, and CsA conversion to SRL revealed less total lesions grade in glomerular and tubular fields.CsA induces tubular damages and the main lesions identified were tubular vacuolization and calcification . HowCollagen is the major insoluble fibrous protein in the extracellular matrix and in connective tissue and is clearly marked with Masson's trichrome; modifications of collagen production reflect cellular changes and consequent kidney dysfunction. In the kidneys from vehicle-treated rats, collagen staining was rare in the glomeruli, and a small amount of blue Trichrome staining appeared in the outer borders tubules and around the vessels Figures 4-5, 2b44-5). AftP < 0.05). The main change found for the SRL treatment after 3 weeks was increased urinary protein, with additional increment after 9 weeks, suggesting a time-dependent effect. The conversion protocol revealed no significant change on serum creatinine and BUN levels and clearances; moreover, GFR remains unchanged as well as urinary protein and TBARs ;nd TBARs .P < 0.05), without affecting urine and clearance values. The rats treated with SRL presented increased serum and urine NGAL contents, as well as augmented clearance. The use of SRL to replace CsA leads to increased NGAL levels in serum and urine compared to the CsA group (P < 0.05) , which was even more evident and statistically significant, in the conversion group .NGAL has been described as a putative biomarker of nephrotoxicity. Long-term CsA treatment only promoted serum NGAL increment . Identical reduction (P < 0.01) was found at 9 weeks for all the treated groups  versus the control one .Unchanged values of serum hsCRP were found for both immunosuppressive drugs when compared with the control group. However, serum IL-1 Figures . Serum Itrol one . Serum ctrol one . InteresP < 0.001), CRP (P < 0.01), TNF-alpha (P < 0.05), and VEGF (P < 0.01) was found  and tumor protein p53 (TP53) mRNA (P < 0.001), accompanied by a slight increase (P < 0.05) in the expression of TGF-β1 and NF-κB. On the other hand, the mTOR inhibitor only stimulated the expression of TP53 gene and downregulated some inflammatory markers . In the long-term CsA treatment, almost all the genes presented normal mRNA expression, when compared with the control group. However, a significant upregulation of IL-2, mTOR, and Mki67 was encountered. In the SRL and conversion groups only mTOR and Mki67 remained overexpressed, in contrast to what was observed with the short-term exposures.Several markers of proliferation, fibrosis, inflammation, and angiogenesis were evaluated in terms of kidney mRNA expression in the weeks 3 and 9 for the three immunosuppressive protocols in comparison to control group . After 3as found . Howeverκβ1, and TGF-β (P < 0.001) protein expression, when compared to control, while SRL treatment was unable to promote changes on the expression of these proteins  when compared to the short-term (3 weeks) CsA treatment. Kidney KIM-1, NF-κβ1, and PCNA expression were unchanged in the CsA-treated rats when compared with the control animals. SRL treatment promoted, after 9 weeks, a decreased KIM-1 expression and overexpression of TGF-β. In the conversion protocol, CTGF and PCNA protein overexpression was obtained, but mTOR and TGF-β expression were significantly reduced when compared with the CsA group ; the lesions were further aggravated with prolonged CsA exposure. This data complements the information that the first CsA pathologic events are related to afferent arteriolar vasoconstriction, thrombotic microangiopathy, and isomeric tubular vacuolization . In addiAfter 3 weeks of CsA exposure very slight changes on tissue structure were found, with absent or only mild lesions; however, after the long-term CsA exposure, significant glomerular, tubular, and vascular lesions were observed. In spite of that, at week 9 renal markers used in clinical practice  appeared only modestly changed. Additionally, we observed an interesting variation of MDA clearance levels between 3 and 9 weeks of CsA treatment, when renal lesions were clearly noted. Knight et al. detected high MDA levels in urine of transplanted patients, but they were unable to explain their importance . Our datThe development of noninvasive biomarker that could diagnose renal dysfunction early and also monitor the response to therapy, as well as the ability to predict severity and outcome, would be very valuable. It is also important to recognize that changes in serum creatinine and BUN concentrations primarily reflect functional changes in filtration capacity and are not genuine injury markers . During β1 was found in the CsA-treated rats, starting after just 3 weeks and aggravating with prolonged exposure, suggesting this factor as a putative good biomarker of nephrotoxicity progression. Interestingly, similar pattern was encountered for serum IL-7 levels, showing significant correlation with serum TGF-β1 contents in the short- and long-term treatments. IL-7 is produced constitutively by stromal cells and consumed by the available pool of resting T cells, all of which express the IL-7 receptor (IL-7R) at high levels except for CD4+ CD25+ regulatory T cells. Circulating IL-7 levels increase during periods of lymphopenia to maintain naïve T-cell homeostasis and support the thymic-independent peripheral expansion and maintenance of mature T cells [In our study, a trend to increased serum and kidney TGF- T cells  because  T cells , 46. CsAκβ activation and induces dendritic cell maturation and T-lymphocyte infiltration into the graft, with both pathways ultimately resulting in interstitial inflammation and interstitial fibrosis that contributes to chronic nephropathy [κβ gene (RT-qPCR) and protein (immunohistochemistry) overexpression after 3 weeks of CsA treatment, an effect that is then downregulated with prolonged exposure. SRL treatment reduced mRNA levels, resulting in normal protein expression, when compared to control, both in short- and long-term treatments, indicating that this nuclear factor could have an important impact in the development of nephrotoxicity. At the end of the conversion protocol (CsA replaced by SRL), no difference was found in gene expression but protein overexpression remained in the tubulointerstitial region, most probably because of the previous CsA exposure. In our animal model, the short-term CsA treatment was mainly associated with upregulation of TGF-β1 and PCNA in the kidney tissue, which has been identified as the key mediator of fibrosis and proliferation [α, and CRP), as well as of cellular proliferation (MKi67) and angiogenesis (VEGF), were downregulated, perhaps responsible for the attenuation of the cytotoxic effects of CsA in the short term. The overexpression of NF-κβ and TP53 might be included in this compensatory response, since they inhibit mTOR [α, COX2, IL-1β, TGF-β1, NF-κβ, and mTOR. Nevertheless, during prolonged CsA exposure, nephrotoxicity evolves, as viewed by the degree of increased histological lesions, which seems to be associated with other molecular pathways and mediators. In fact, there was a significant overexpression of MKi67, contrary to what was observed after the short-term treatment, suggesting a depletion of counter-regulatory responses, which was accompanied by a parallel increase in mTOR expression, a serine/threonine protein kinase, important in regulating cell growth, proliferation, motility, survival, protein synthesis, and transcription [Changes in messenger RNA expression are considered to be one of the earliest events, which may occur in response to cellular and tissue damage; it has been speculated that these biomarkers might help to predict adverse effects before damage is indicated by the current gold standard markers . Current theories point that renal damage is caused by nonimmunological factors, such as ischemia, which lead to activation of various proinflammatory and profibrotic mediators. A parallel concept of how CsA might induce renal injury was described by Li and Yang, suggesting that kidney damage involves activation of the innate immune response that causes NF-hropathy . In agreferation , 48. Howbit mTOR . Short-tcription . Moreovecription –52. In oIn the last years, some toxicological studies showed hypothetical biomarkers that could predict acute nephropathy , 37, 54.β1 downstream modulator) and has been pointed as a possible biomarker of CsA-evoked damage. In our model, kidney CTGF expression increased after short- and long-term treatment with CsA, in agreement with the kidney overexpression of TGF-β1 viewed by immunohistochemistry. After a longer CsA exposure the kidney expression of CTGF was slightly reduced, which might be explained by an increased urinary elimination, as previously suggested by O'Connell et al. in another experimental study [Connective tissue growth factor (CTGF) is a polypeptide implicated in the extracellular matrix synthesis that belongs to a profibrotic signalling  seems to be a feasible indicator of substitution to the mTOR inhibitor.This experimental study demonstrated that CsA-induced nephrotoxicity is significantly aggravated over time and distinct mechanisms seem to underlie short- and long-term renal toxicity. The currently used clinical techniques and biomarkers, namely of biochemical impairment (such as serum and urine creatinine and BUN contents and clearance), if coupled with genetic and protein analysis in different samples, will bring more accuracy to early detect and follow up the appearance and development of nephrotoxicity. Conversion to SRL prevented CsA-induced renal damage evolution, which is better viewed by nontraditional, emergent biomarkers including serum TGF-


To explore whether daytime napping and sleep duration are linked to serum C reactive protein (CRP), a pro-inflammatory marker, in an older aged British population.Cross-sectional study.European Prospective Investigation into Cancer and Nutrition (EPIC)-Norfolk study.A total of 5018 men and women aged 48–92 years reported their sleep habits and had serum CRP levels measured.CRP was measured (mg/L) during 2006–2011 in fresh blood samples using high-sensitivity methods. Participants reported napping habits during 2002–2004, and reported sleep quantity during 2006–2007. Multivariable linear regression models were used to examine the association between napping and log-transformed CRP, and geometric mean CRP levels were calculated.After adjustment for age and sex, those who reported napping had 10% higher CRP levels compared with those not napping. The association was attenuated but remained borderline significant (β=0.05 (95% CI 0.00 to 0.10)) after further adjustment for social class, education, marital status, body mass index, physical activity, smoking, alcohol intake, self-reported health, pre-existing diseases, systolic blood pressure, hypnotic drug use, depression and in women-only hormone replacement therapy use. The geometric means (95% CI) of CRP levels were 2.38 (2.29 to 2.47) mg/L and 2.26 (2.21 to 2.32) mg/L for those who reported napping and no napping, respectively. A U-shaped association was observed between time spent in bed at night and CRP levels, and nighttime sleep duration was not associated with serum CRP levels. The association between napping and CRP was stronger for older participants, and among extremes of time spent in bed at night.Daytime napping was associated with increased CRP levels in an older aged British population. Further studies are needed to determine whether daytime napping is a cause for systemic inflammation, or if it is a symptom or consequence of underlying health problems. Our study benefits from a large population-based sample, measures of daytime as well as nighttime sleep, and a wide range of covariates available for adjustment.Sleep habits were self-reported.The purpose and duration of napping was not reported, and sleep apnoea was not measured.Recently there has been growing interest in studying the health implications of daytime napping. Although the benefits of napping on wakefulness performance have been widely documented, especially among shift workers,et alet alOne important proposed pathway for the association between sleep and health outcomes is through inflammation.Despite the growing interest into the inflammatory correlates of nighttime sleep, there has been limited evidence on the physiological effects of daytime napping. Experimental study has reported a beneficial effect of napping on immune cells following acute sleep restriction.The design and study methods of EPIC-Norfolk have been described previously.Habitual daytime napping was ascertained during 2002–2004, by asking participants the question “Do you normally take a nap during the day?”‘Nighttime sleep duration’ and ‘Time spent in bed at night’ were ascertained during 2006–2007, by asking participants “On average, about how many hours have you slept each night?”; “At what time do you normally get up?” and “At what time do you normally go to bed?”. ‘Time spent in bed’ was derived from the differences between rise time and bedtime, and as a weighted mean measure of weekday and weekend times (5/7×(time on a weekday)+2/7×(time on a weekend day)).CRP was measured (mg/L) at the third health examination (2006–2011) in fresh blood samples using high-sensitivity methods using the Siemens Dimension clinical chemistry analyser , with between-batch coefficient of variation (CV) values of 3.1% at 7.86 mg/L and 3.7% at 88.75 mg/L.Covariates measured closer in time to each measure of sleep were chosen and included in the models for the corresponding sleep measures. The covariates were chosen as a priori based on their link with CRP and sleep.Analysis was restricted to those participants with complete data on all covariates. A total of 5018 participants had complete data on CRP and at least one of the sleep measures, and were included in the analysis.e CRP) to approximate a normal distribution. Owing to the U-shaped relationship reported by previous studies on sleep duration and health risk,e CRP as the outcome were conducted to examine the associations between each sleep measure (daytime napping (N=4712), time spent in bed (N=4476) and sleep duration (N=4795)) and CRP levels. For each sleep variable, results were presented: (A) adjusted for age and sex; (B) further adjusted for social class, education and marital status, BMI, physical activity, smoking and alcohol intake, depression, self-reported health, pre-existing diseases, SBP, hypnotic drug use and in women-only postmenopausal HRT. The sleep measures were mutually adjusted in model B. Since the associations for sleep duration and time spent in bed might be non-linear (approximately U-shaped), the β coefficients (with 95% CIs) were calculated with 6–8 h being the reference groups for the examination of sleep quantitative measures. For analysis by napping habit, the no napping category was chosen as reference group. Results were presented for the whole sample and for men and women separately.The CRP values were natural-log transformed . These analyses were adjusted for age, sex, social class, education, marital status and BMI. The two-by-two interactions between napping habit, time spent in bed and sleep duration were examined using the likelihood ratio test. Sensitivity analysis: all statistical tests were two-sided, and a p value <0.05 was considered statistically significant. Analyses were implemented in STATA V.12.0.Geometric—mean (95% CI) of serum CRP values (mg/L) were calculated by exponentiating crude and adjusted-least square mean (95% CI) logCharacteristics of the study sample (2267 men and 2751 women) are shown in e CRP according to the report of napping. After adjustment for age and sex, daytime napping was associated with 10% higher CRP levels , p<0.001). With further adjustment for socioeconomic status, health-related behaviours, physical health and nighttime sleep, the association was attenuated but remained borderline significant , p=0.048). The unadjusted geometric means of CRP were 2.53 (2.44 to 2.63) and 2.20 (2.15 to 2.26) mg/L for those who reported napping and no napping, respectively. e CRP was not significant. The geometric means of CRP levels among those who spent >10 and <6 h in bed were 2.57 (2.30 to 2.87) and 3.00 (2.04 to 4.40) mg/L, respectively, higher than those who spent 6–8 h in bed (2.28 (2.19 to 2.38) mg/L). Nighttime sleep duration was not associated with loge CRP. Repeating the analyses restricted to individuals with CRP levels below 10 mg/L did not change the results.The unadjusted geometric means of CRP for those who reported spending <6, 6–8, 8–10 and >10 h in bed at night were 3.70 (2.48 to 5.53), 2.25 (2.15 to 2.35), 2.29 (2.23 to 2.35) and 2.84 (2.52 to 3.19) mg/L, respectively. After adjustment for age and sex, spending <6 and >10 h in bed were associated with 51% and 17% increase in CRP levels, respectively . After fSubgroup analysis investigated differences in these associations by age, sex, social class, smoking, BMI, physical activity and pre-existing diseases . These dOur findings from more than 4000 middle to older aged English adults suggest that daytime napping was independently associated with higher CRP levels. Those who reported napping had 10% higher CRP levels after adjustment for age and sex. The association was attenuated but remained statistically significant after adjustment for all the covariates. A U-shaped association was observed between time spent in bed at night and CRP levels, though only the effect of long time spent in bed was statistically significant. The napping–CRP associations were more pronounced among extremes of time spent in bed at night. No association was found between sleep duration and CRP levels.To the best of our knowledge, this is the first study to report an association between habitual daytime napping and inflammation. This gives biological insights to the health impact of daytime napping proposed by previous studies.The present study suggested that daytime napping and long time spent in bed at night were associated with higher CRP levels. The observed effect size was similar to previous report on sleep duration and CRP levels,Time spent in bed was rarely studied in relation to CRP concentrations, but long sleep duration has been associated with increased CRP levels.It has been suggested that sleep deprivation is related to the activation of the autonomic nervous system and increased catecholamines, and subsequently stimulate the release of inflammatory mediators.In the UK, daytime napping is not part of the cultural norm. It is plausible that those who reported daytime napping and long time spent in bed may represent a population with underlying ill health, and inflammation could be an early sign of these health problems. Although the association between daytime napping and CRP concentrations remained after adjustment for pre-existing diseases, we did find it more pronounced among the older participants, and there might be more existing health problems among this population. Further studies are needed to determine whether daytime napping is a cause for systemic inflammation, or if it is a symptom or consequence of disturbed sleep at night or underlying health problems.In summary, we found for the first time that daytime napping was associated with increased CRP levels in a population-based middle to older aged English cohort. The association between daytime napping and CRP levels was more pronounced among older people, and among those who spent extremes of time in bed at night. While the present study provides new insights into the biological effects of daytime napping, causality cannot be established from an observational study. Daytime napping might be useful as an independent indicator of people who are at underlying health risk or who suffer from disturbed sleep at night. Future physiological studies are needed to gain better understanding of the association.


Arterial bypass graft implantation remains the primary therapy for patients with advanced cardiovascular disease, but most lack adequate saphenous vein or other conduits for bypass procedures and would benefit from a bioartificial conduit. This study aimed to produce human endothelial cells (hECs) in large scale, free from xenogeneic antigens, to develop a small diameter, compatible vessel for potential use as a vascular graft. Human adipose-derived stromal cells (hASCs) were isolated, cultured, and differentiated in the presence of human serum and used for the reendothelization of a decellularized rat aorta. hASC derived ECs (hASC-ECs) expressed VEGFR2, vWf and CD31 endothelial cell markers, the latter in higher levels than hASCs and HUVECs, and were shown to be functional. Decellularization protocol yielded aortas devoid of cell nuclei, with preserved structure, including a preserved basement membrane. When seeded with hASC-ECs, the decellularized aorta was completely reendothelized, and the hASC-ECs maintained their phenotype in this new condition. hASCs can be differentiated into functional hECs without the use of animal supplements and are capable of reendothelizing a decellularized rat aorta while maintaining their phenotype. The preservation of the basement membrane following decellularization supported the complete reendothelization of the scaffold with no cell migration towards other layers. This approach is potentially useful for rapid obtention of compatible, xenogeneic-free conduit. Approximately one-third of the 500.000 coronary bypass patients operated on in the USA every year  lack suf in vitro [ in vitro, constituting an excellent option for large-scale production of compatible and functional hECs. However, current cell culture supplements contain xenogeneic proteins, which may be source of infections [One of the strategies adopted so far to prevent graft failure is to cover graft interface with primary human endothelial cells (hECs), even though this procedure is hindered by low cell yield, limited cell plasticity, and loss of differentiation capacity of hECs following prolonged expansionin vitro . In suchfections , 6 and gfections . in vitro, it maintains hASCs multipotency and induces higher proliferation rates without promoting cell senescence or loss of differentiation capacity [Recently, our group has shown that human allogeneic serum (aHS) is a suitable cell culture supplement for hASCs . In addicapacity –10. TherIn the present study, we aimed to produce functional hECs and use them to promote the reendothelization of rat decellularized aortas. We propose that the association of hECs originated from human adipose-derived stromal cells (hASC-ECs) and a xenogeneic extracellular matrix (ECM) is feasible and may potentially constitute a fast and efficient strategy to develop a compatible vessel free from xenogeneic soluble antigens for potential use as a small-diameter vascular graft.The aHS was obtained from the whole blood of healthy donors from all blood group types, as previously described by our group . BrieflyBasal Medium (BM) consisted of Dulbecco's modified Eagle medium-high glucose (DMEM) (Sigma-Aldrich) supplemented with 5 mM sodium bicarbonate (Cinética Química Ltda), penicillin (100 U/mL), streptomycin (0.1 mg/mL), amphotericin B , gentamicin , and 10% aHS.Endothelial medium (EM) was composed of DMEM supplemented with 5 mM sodium bicarbonate (Cinética Química Ltda), penicillin (100 U/mL), streptomycin (0.1 mg/mL), amphotericin B (0.25 mg/mL), (Sigma-Aldrich), gentamicin , 1% aHS, 1 mg/mL VEGF (Invitrogen), and 0.1 mg/mL bFGF (Invitrogen).2 and a humidified atmosphere. After 12–28 h of incubation, plastic adherent cells were isolated with media change and those were termed hASCs. BM was changed every 2-3 days. Subculture was performed every time cell population achieved 80–90% confluency, with incubation of the cells in 0.05% Trypsin-EDTA (Invitrogen) solution for 3–5 min, when cells began to detach from tissue culture flasks. Trypsin solution was then inactivated by adding the same volume of BM supplemented with human serum to flasks, and the resulting cell suspension was replated in new cell culture flasks. The cells were expanded this way until passage 4, when they were used in the assays.Human adipose tissue was harvested from healthy patients, which had abdominal reduction surgery for aesthetic reasons. This study was approved by the Ethics Committee of the Federal University of Minas Gerais (number ETIC 21176413.9.0000.5149). The isolation and culture of hASCs were performed as described, with minor modifications , 11. BriPhenotypic characterization of isolated hASCs was performed as recommended by the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy  and perf4 cells in endothelial medium. Briefly, hASCs were detached from tissue culture flasks, counted, and seeded in BM at a density of 103 cells/cm2 in T75 cell culture flasks (Sarstedt). Twenty-four hours later, the BM was removed and EM was used thereafter up to 21 days. EM was changed every 2-3 days.Endothelial differentiation was performed with the culture of 7.5 × 10The differentiation of hASCs towards endothelial cell fate was assessed according to gene expression of the endothelial markers VEGF receptor type 2 (VEGFR2) and Von Willebrand Factor (vWF). In order to do that, at the end of the induction period, total RNA was extracted using TRIzol and reverse transcripted into cDNA using RevertAidTM H Minus M-MuLV RT. PCR was performed for VEGFR2 (forward primer (FP): 5′GGAATACCCCTTGAGTCC3′, reverse primer (RP): 5′CCTCCAACTGCCAATACC3′), vWF , GAPDH . mRNA was also isolated from human umbilical vein endothelial cells (HUVECs) as positive control. PCR cycles were as follows: 94°C for 2 min, 94°C for 30 s, 56°C (for vWF and VEGFR2) and 60°C (for GAPDH) for 45 s and 72°C for 45 s (30 cycles), and 72°C for 10 min. The RT-PCR products were analyzed through 1% agarose gel electrophoresis and visualized with ethidium bromide.At the end of the differentiation period, cells were stained for endothelial markers. Immunofluorescence was performed following cell fixation with 4% paraformaldehyde. Afterwards, cells were washed, permeabilized, and incubated with primary antibodies overnight at 4°C. Cells were then washed and incubated with secondary antibodies for 1 h at room temperature. Finally, cell nuclei were stained with DAPI and slides were mounted using Hydromount. Primary antibodies used were mouse anti-vWF  and rabbit anti-VEGFR . Secondary antibodies used were Alexa Fluor goat anti-mouse 488 , and Alexa Fluor goat anti-rabbit 555 . HUVECs were stained and considered positive control.The same antibodies were also used for tissue immunostaining of the recellularized aortas. Tissue sections were washed in water and protocol was similar to described for cells. in vitro assay to assess endothelial cell function [4 cells were seeded on each well of a 24-well plate coated with 225 µL of previously solidified Matrigel (BD Biosciences) and incubated overnight at 37°C in 5% CO2 incubator. Cells were then incubated in 3 µg/mL Calcein-AM (Invitrogen) diluted in PBS for 30 min at 37°C in 5% CO2 incubator, protected from light. Finally, tube formation was observed, quantified and tube area was measured both under light microscopy and under fluorescence microscope, using the ImageJ 1.47 software.The capacity of hASC-ECs to form tubes in matrigel was assessed as previously described as anfunction . For thiLewis LEW-Tg (EGFP) F455.5/Rrrc rats 6–8 weeks old, which express enhanced fluorescence green fluorescent protein (EGFP), were obtained from the Rat Resource and Research Center, Missouri, USA. The animals were housed in a climate-controlled environment under a 12 h light/dark cycle with free access to rat chow and water. All experimental protocols were performed in accordance with the guidelines for the humane use of laboratory animals established at our Institution. This study was approved by the Committee of Ethics in Research at the Federal University of Minas Gerais (Protocol number 140/2011).Aorta decellularization was performed according to the protocol published by , with mi6 cells were then perfused into the decellularized matrix with an initial static period overnight, followed by 7 days of continuous perfusion with EM.hASC-EC were evaluated according to their potential of reendothelizing a decellularized aorta in the presence of EM with human serum. In order to do so, endothelial cells were harvested and counted. 2.5 × 10μm tissue slices were cut for histology and immunostaining. Hematoxylin and Eosin staining was performed. ECM proteins laminin and collagen I were stained following tissue slice production, following the same steps of immunofluorescence cell staining. Primary antibodies used were mouse anti-rat collagen I and rabbit anti-rat laminin . Secondary antibodies were the same used for cell staining.Following fixation in 4% paraformaldehyde, aortas were embedded in paraffin or OCT . 5 Isolated hASCs cultured in BM were positive for CD29 (98.3%) and CD44 (99.6%) and lacked the expression of hematopoietic stem cell-associated markers CD34 (0.38%) and CD45 (0.72%). In addition, hASCs expressed HLA-ABC (99.8%) and lacked HLA-DR (0.15%) .Functionally, isolated hASCs proved their multilineage differentiation potential and were capable of generating osteoblasts, chondrocytes, and adipocytes following differentiation (data not shown). Those were capable of mineralizing the ECM, depositing proteoglycan in ECM, and storing lipid droplets in their cytoplasm, respectively. Therefore, isolated hASCs attended to the criteria of mesenchymal stromal cell (MSC) characterization both phenotypically and functionally.http://dx.doi.org/10.1155/2015/652474) and indicated that hASC-ECs had approximately 11 times higher CD31 expression compared to both hASCs and HUVECs, which had statistically similar expression of this marker, even though the latter had a slightly higher CD31 expression level then hASCs.Endothelial differentiation of hASCs in the presence of aHS was confirmed by gene and protein expression of endothelial markers; therefore, they were regarded as hASC-ECs. First, expressions of VEGFR2 and vWF were assessed by PCR. As expected, hASC-ECs were capable of expressing both markers related to the endothelial lineage. Undifferentiated hASCs also expressed such genes in discrete smaller levels, similar to HUVECs, which were used as positive control . In a quThe expression of both VEGFR and vWF was assessed at protein level by immunofluorescence. Our results show once again the consistent differentiation of hASC-ECs into the endothelial phenotype, as both hASC-ECs and HUVECs exhibited VEGFR and vWF proteins. hASCs presented no expression of those proteins, evidentiating the difference between undifferentiated hASCs and their hASC-ECs derivatives .μm2. HUVECs were closely followed by hASC-ECs, which were also able to generate tubes under the tested conditions, with 14 tubes per field (average size: 8599 μm2). Tube formation by hASCs was not observed and considered virtually absent.One of the functional assays to assess endothelial differentiation consists on investigating the capacity of hECs of forming tubes when cultured in Matrigel in the presence of stimulus. Since the undifferentiated hASCs express VEGFR and vWF in discrete levels, this assay is necessary to differentiate hASCs from functional hECs. To do so, first, we cultured HUVECs in such condition and showed their capacity to form tubes. Following, hASCs and hASC-ECs were also used in the experiment and only differentiated hASCs (hASC-ECs) and HUVECs were capable of forming tubes once stimulated in Matrigel . Even th in vivo. First, aorta decellularization was verified by histology and DNA isolation. As expected, decellularized aorta was shown to be deployed of cell nuclei and DNA. Additionally, decellularized aortas presented laminin preserved along their layers, including the basement membrane . hASCs may be obtained from the patient in times of health, in order to be used when needed, or from healthy donors. Therefore, the isolation of hASCs and further differentiation towards endothelial lineage constitute faster and more reliable approach for tissue engineered vascular graft (TEVG). in vitro is consistent and reproducible [ in vivo angiogenesis and contributes with matrix-metalloproteinase to the formation of capillary-like structures seen in vitro [hASCs endothelial differentiationoducible –27. Mostoducible , 27. Eveoducible , 28. In oducible , 30, andoducible . Therefoin vitro . The latin vitro , which iThe seeding of hASC-ECs in biomaterials and decellularized matrices has been performed with exciting results , 31. It The production of the aforementioned TEVG requires the investigation of ways to exclude soluble animal antigens from cell culture, before performing hASCs differentiation. Use of animal origin reagents is controversial due to the possibility of zoonosis disease transmission. The risk of prion infection can be avoided, but the use of an average of 20% FBS in cell culture, regardless of the type, results in MSC carrying between 7 and 30 mg of bovine serum proteins, resulting in specific immune reaction in humans .Animal-free alternatives have emerged for immortalized cell cultures, but few products exist for primary adult human cells, and they typically provide suboptimal results. Previously, our group has described that allogeneic human serum is a suitable supplement for proliferation and differentiation of hASCs . In thisSupplemental File 1 presents the difference in expression of CD31 among hACSs, hASC-ECs and HUVECs, as assessed by qPCR.


Systemic inflammation plays an important role in the initiation, promotion, and progression of lung carcinogenesis. In patients with non-small cell lung cancer (NSCLC), fibrinogen levels correlate with neoplasia. Here we compared the effects of pulmonary rehabilitation (PR) with chest physical therapy (CPT) on fibrinogen and albumin levels in patients with LC and previous inflammatory lung disease awaiting lung resection.We conducted a randomized clinical trial with 24 patients who were randomly assigned to Pulmonary Rehabilitation (PR) and Chest Physical Therapy (CPT) groups. Each group underwent training 5 days weekly for 4 weeks. All patients were assessed before and after four weeks of training through clinical assessment, measurement of fibrinogen and albumin levels, spirometry, 6-minute Walk Test (6MWT), quality of life survey, and anxiety and depression scale. PR involved strength and endurance training, and CPT involved lung expansion techniques. Both groups attended educational classes.A mixed between-within subjects analysis of variance (ANOVA) revealed a significant interaction between time (before and after intervention) and group (PR vs. CPT) on fibrinogen levels  = 0.57, p < 0.0001) and a significant main effect of time  = 0.68, p = 0.004). Changes in albumin levels were not statistically significant relative to the interaction effect between time and group  = 0.96, p = 0.37) nor the main effects of time  = 1.00, p = 1.00) and group  = 0.59, p = 0.45). A mixed between-within subjects ANOVA revealed significant interaction effects between time and group for the peak work rate of the unsupported upper limb exercise  = 0.77, p = 0.02), endurance time  = 0.60, p = 0.001), levels of anxiety  = 0.60, p = 0.002) and depression  = 0.74, p = 0.02), and the SF-36 physical component summary  = 0.83, p = 0.07).PR reduced serum fibrinogen levels, improved functional parameters, and quality of life of patients with LC and inflammatory lung disease awaiting lung resection.RBR-3nm5bv.Current Controlled Trials  Malignant neoplasms and chronic degenerative diseases replace infectious and parasitic pathologies as the main causes of death in Brazil as well as worldwide, particularly in the United States and Europe . Lung caSurgery without adjuvant treatment is most frequently indicated to treat patients with early stages (I and II) of non-small cell lung cancer (NSCLC) , and surFew studies assessed the impact of pulmonary rehabilitation on postoperative complications of patients who undergo lung resection for lung cancer -11, One We conducted a randomized clinical trial in two teaching hospitals in Fortaleza, Northeastern Brazil. The study included patients with NSCLC who were eligible for lung resection and experienced previous pulmonary disease with impaired respiratory function identified using spirometry. After selection, six groups of four patients each were randomly assigned  to either the Pulmonary Rehabilitation  or the Chest Physical Therapy  groups. Training was conducted five times weekly for 4 weeks. After receiving approval from the Ethics Committee of the Hospital de Messejana (Opinion No. 540/80), all subjects granted their informed written consent.Thirty-one patients were recruited between March 2008 and March 2011, and seven patients were excluded, because five refused to participate and two did not meet inclusion criteria because of normal pulmonary function. Twenty-four patients were randomly assigned (12 each) to the PR and 12 CPT groups  to forced vital capacity (FVC) ratio >0.70 and FVC and FEV1 > 80% of the predicted value. Physical capacity was measured using the following tests: unsupported upper limb exercise test (UULEX), endurance testing, and the 6-min walk test (6MWT). The 6MWT was performed twice over a 30-m corridor according to the American Thoracic Society (ATS) guidelines [Before and after training, all participants were assessed demographically and clinically , as well as by laboratory analysis of serum levels of fibrinogen and albumin and by pulmonary function tests. Physical capacity, quality of life, levels of anxiety, and depression were also assessed. Serum levels of fibrinogen and albumin were measured using a blood sample collected in disposable Vacutainer tubes ,13. Pulmidelines ,17. Qualidelines , which cidelines , which aThe group of patients undergoing PR was submitted to upper limb (UL) and lower limb (LL) stretching exercises, warm-up exercises, UL strengthening exercises, aerobic conditioning, and inspiratory muscle training. UL strengthening started at 50% of peak work capacity during preliminary testing, and it was performed using Proprioceptive Neuromuscular Facilitation (PNF) diagonal movements with barbells. Aerobic conditioning was performed on a treadmill at 80% of peak work capacity determined by endurance testing. The group of patients who underwent CPT followed the routine protocol of the hospital comprising lung expansion techniques . Both groups attended educational sessions informing them of the importance of pre- and postoperative care, the surgical process, energy conservation techniques, as well as relaxation, stress, and nutritional management techniques.Data analysis was performed using the Statistical Package for the Social Sciences 13.0 and GraphPad Prism 5.0. The Kolmogorov-Smirnov test was used to test for normality. Data are presented as absolute or relative frequency, or both, represented by the mean ± standard deviation of normally distributed data or median  for data not normally distributed. The variation (delta) between pre- and postoperative periods is presented as the mean . The Mann–Whitney U test was used to compare continuous variables between the two groups at baseline. The values of continuous variables as a function of time were compared between groups using a mixed between-within subjects ANOVA. P <0.05 indicates statistical significance.All 24 participants successfully completed the training assignments. Nineteen patients were diagnosed with chronic obstructive pulmonary disease (COPD), two with interstitial lung disease, and three with bronchiectasis. Their characteristics are presented in Table Both groups comprised mostly women, older people, smokers, and patients with Stage I or II lung cancer. The two groups were equivalent regarding the presence of COPD and severity of the disease. Of the patients in the PR and CPT groups, 50% and 60% were classified as Stage III of the Global Initiative for Chronic Obstructive Lung Disease, respectively (p = 0.1). The groups did not differ systematically at the outset.Fibrinogen levels before and after each intervention are presented in Figure Table In the present study, we show that 4 weeks of PR reduced serum fibrinogen levels in patients with lung cancer and previous inflammatory respiratory disease who were eligible for lung resection. However, the change in the albumin levels of this group was not statistically significant,As recently as 2006, the ATS and European Respiratory Society (ERS) did not mention the effect of PR on the inflammatory status of patients with LC, in contrast to studies on inflammation in COPD patients , using aVogiatzis et al.  studied A study of 635 patients undergoing lung resection has shown that male gender, preoperative fibrinogen level > 400 mg/dL, and duration of surgery are significantly associated with pulmonary complications . BecauseThere are few studies on the effects of exercise interventions designed to reduce inflammation in patients with NSCLC ,22. PatiThere are limitations of the present research. For example, the number of subjects was small but is similar to others studies involving exercise and lung cancer. The study by Jones et al.  also obsA presurgical short-term, high-intensity pulmonary rehabilitation program reduced serum fibrinogen levels in patients with NSCLC and previous inflammatory pulmonary disease. This exploratory study provides preliminary evidence to support the vigorous pursuit of future studies to investigate the role of exercise in inflammation and cancer.1: Forced expiratory volume in the first second; FVC: Forced vital capacity; GOLD: Global initiative for obstructive lung disease; PR: Pulmonary rehabilitation; CPT: Chest physical therapy; BMI: Body mass index; PNF: Proprioceptive neuromuscular facilitation; ERS: European respiratory society; UULEX: Unsupported upper limb exercise; 6MWT: 6-minute walk test; SF-36: Short form health survey; PCS: Physical component summary; MCS: Mental component summary; HADS: Hospital anxiety and depression scale; UL: Upper limb; LL: Lower limb; ATS: American thoracic society; ICAM-1: Intracellular adhesion molecule; CRP: C- reactive protein; TNF-alpha: Tumor necrosis factor; IL-6: Interleukin-6.LC: Lung cancer; NSCLC: Non small cell lung cancer; FEVThe authors declare that they have no competing interests.EDBP contributed as the principal investigator, to the study concept and design, analysis of the results, and writing of the manuscript. MTAPM, GPFS, ASA, AGN, CMSV contributed to execution of the study and acquisition of data. RM was responsible for the statistical analysis of the data. JMdSP. and MOdMF contributed to the data analysis and interpretation and editing of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2466/14/121/prepub


Influence of antibiotics on surface-active properties of exogenous surfactant was assessed using the modified Pattle method. Effects of exogenous surfactant on antibacterial activity of antimicrobials against Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa were evaluated using conventional microbiologic procedures. Addition of amikacin or cefepime to surfactant had no significant influence on surface-active properties of the latter. Obvious reduction of surface-active properties was confirmed for surfactant/colistimethate composition. When suspended with antibiotics, surfactant either had no impact on their antimicrobial activity (amikacin) or exerted mild to moderate influence . Considering favorable compatibility profile, the surfactant/amikacin combination is advisable for subsequent investigation of joint surfactant/antibacterial therapy in animals with bacterial pneumonia.Owing to its unique surface-active properties, an exogenous pulmonary surfactant may become a promising drug delivery agent, in particular, acting as a vehicle for antibiotics in topical treatment of pneumonia. The purpose of this study was to assess a mutual influence of natural surfactant preparation and three antibiotics   The success of pneumonia treatment in large part is defined by effectiveness of antimicrobial therapy. In turn, efficiency of antibacterial therapy depends on formation of sufficient antimicrobial drug concentration in the site of infection . Direct It was hypothesized that use of exogenous pulmonary surfactant as a vehicle for antibacterial agents could enhance the efficiency of local antimicrobial therapy in pneumonia , 7. PulmDespite the theoretical feasibility for use of exogenous surfactant as a vehicle for antimicrobials, this method is still not realized in clinical settings. Scientific achievements in this area are limited to several preclinical studies , 10–13.  in vitro and, consequently, to determine a suitable combination(s) for subsequent investigations of surfactant/antibiotic experimental mixtures intended for local treatment of pneumonia. The study consisted of two parts: (1) assessment of influence of the antibiotics on surface-active properties of the exogenous surfactant and (2) assessment of influence of the exogenous surfactant on antibacterial properties of the antimicrobials.The purpose of this study was to evaluate potential interactions between exogenous surfactant and three antibiotics Sterile natural pulmonary surfactant preparation  was used. This emulsion includes purified phospholipids  isolated from porcine lung homogenates and physiological solution as an excipient. Suzacrin contains surfactant-associated proteins B and C but is free of surfactant-proteins A and D.The following antibiotics were utilized: amikacin , cefepime , and colistimethate sodium . These antibacterial agents are currently recommended to treat pulmonary infections, caused by multidrug-resistant pathogens.Solutions of antibiotics, emulsion of surfactant, and experimental mixtures of antibiotics in surfactant were freshly prepared in aseptic conditions using sterile 0.9% NaCl solution immediately before testing.Specific requirements were established to standardize experiments and to provide a balanced calculation of antibiotic and surfactant amount in experimental mixtures: quantitative ratio of antibiotic and surfactant should correspond to the ratio of mean daily doses recommended for adult male patient with a body mass of 70 kg, that is, surfactant phospholipids—700 mg, cefepime—2,000 mg, amikacin—1,000 mg, and colistimethate sodium—4,000,000 IU (320 mg). Staphylococcus aureus ATCC25923, Klebsiella pneumoniae 3534/51, and Pseudomonas aeruginosa ATCC27853. Hottinger broth  and meat-peptone agar  were used as culture media to investigate susceptibility of bacteria to antibiotics, surfactant, and surfactant-antimicrobial mixtures.The following test strains were provided by Crimean Sanitary-Epidemiological Station :The commercial surfactant preparation was diluted (reemulsified) in sterile 0.9% NaCl solution (control) or similar volume of antibiotic solution (three test series) to produce equal final phospholipid concentration of 20.6 mg/mL. The total volume of prepared experimental mixtures was 34 mL . Final concentrations of antibiotics in surfactant-containing mixtures were as follows: amikacin—29.4 mg/mL, colistimethate sodium—117,647 IU/mL, and cefepime—58,8 mg/mL.Apart from the experimental series, five subsidiary series were investigated: undiluted surfactant preparation (phospholipid concentration—50 mg/mL), solutions of amikacin, colistimethate sodium, and cefepime in concentrations recommended for clinical use , and diluent (0.9% NaCl solution).μm) was measured for the first and final (obtained after 20 min) images only. Totally, 1,200 bubble measurements were performed during this experiment. Stability index (SI) was calculated for each replication using the formuladx is a result of final measurement in a series (41st image) and dy is a result of first measurement (1st image).The experiment was conducted at a temperature of 26°C. Test suspensions, as well as control and subsidiary samples (five samples for every series), were foamed in sterile tubes with single-use medical syringes equipped with 23G injection needles. Foam was formed by triple aspiration and return of test material into a tube. The modified Pattle method , 15 was 4 CFU/mL. Obtained mixtures were incubated within 24 hours at 37°C. Then, MIC was determined as a concentration of test substance in the last tube with clear broth in the presence of bacterial growth in control tube (free of test substances).Minimum inhibitory concentrations (MIC) of test substances were determined using conventional technique of serial dilution in Hottinger broth. Each series of dilutions was investigated in two replications. The volume of liquid culture medium in each tube amounted to 1 mL. During the antimicrobial activity assessment of antibiotics and mixtures of surfactant with antibiotics the quantity of surfactant and antibacterials in the initial tubes of series corresponded to aforecited mean daily dose ratios. When assessing surfactant's inherent influence on the bacterial cultures, surfactant concentration in the initial tube of series was 5,000 mg/L. Test substances were introduced into the tubes in a volume of 0.170 mL (0.100 mL of amikacin and colistimethate sodium solutions and 0.070 mL of surfactant suspension) or 0.135 mL (0.100 mL of cefepime solution and 0.035 mL of surfactant suspension). Then, these tubes were filled up with Hottinger broth till the final volume of 1 mL. Maximum test concentration matched 1,000 mg/L for amikacin and cefepime, and 320 mg/L for colistimethate sodium. Maximum test concentration of surfactant in mixtures with antibiotics was 700 mg/L (for amikacin and colistimethate sodium) or 350 mg/L (for cefepime). Sequential twofold dilution of initial tube contents has been performed using the Hottinger broth. Then, the bacterial suspensions were introduced in all tubes in the volume of 0.1 mL. The bacterial suspensions were prepared from diurnal bacterial cultures using the same broth. The density of introduced bacterial suspension amounted to 5 × 10In order to determine a minimum bactericidal concentration  0.1 mL aliquots of test material from the tubes with transparent broth  were transferred with a loop to MPA-containing plates. Subsequent to incubation within 24 hours at 37°C, MBC was determined as a minimum concentration of test substance that corresponded to the last plate in a series where microbial growth was completely absent.t-test. A probability level of P < 0.05 was considered to be statistically significant. Statistical analysis was performed using STATISTICA software version 10.0 .Most data are presented as a mean ± standard error. Data were compared using the unpaired Student's The assessment of foaming properties of the officinal surfactant preparation, antibiotics (in therapeutic concentrations), and solvent demonstrated a wide range of surface activity characteristics : from alP < 0.001) and at the end of the experiment (P < 0.001). The diameter of colistimethate sodium and surfactant bubbles decreased during the experiment by 21% and 8%, respectively (P < 0.001). SI values also suggested significantly higher surface activity of surfactant as compared with colistimethate sodium (P < 0.001).Bubbles of colistimethate sodium solution were significantly larger than bubbles of surfactant suspension both in the beginning (P > 0.05). However, the diameter of bubbles of diluted surfactant was significantly higher as compared with diameter of original surfactant suspension bubbles both in the beginning and at the end of the experiment (P < 0.001), thus suggesting a trend for reduction of surface activity.Comparison of surfactant properties before and after dilution with normal saline Tables  and 2 shP < 0.001), as well as significantly lower tendency for reduction of bubble size in 20 min (P < 0.001), whereas there were no substantial changes of SI (P > 0.05).As compared with control, experimental surfactant/amikacin mixture demonstrated smaller diameter of bubbles  both in P > 0.05). However, this mixture differed from the control by greater bubble diameter in the beginning of the experiment (P < 0.01) and somewhat more pronounced, or accelerated, reduction of bubble size (P < 0.01).The surfactant/cefepime mixture also retained high surface activity as per SI values (P > 0.05), whereas experimental mixture bubbles had substantially smaller diameter than surfactant bubbles at the end of the experiment (P < 0.001). Therefore, a trend for bubble diameter decrease was more pronounced after antibiotic addition (P < 0.001). Relevant SI decrease was observed in this experiment as compared with control (P < 0.01), original surfactant preparation (P < 0.001), or colistimethate sodium solution (P < 0.05)  . S. aureus, K. pneumoniae, and P. aeruginosa cultures.The exogenous surfactant preparation was devoid of apparent intrinsic antimicrobial effects as for S. aureus and P. aeruginosa in 256-fold dilution, as well as growth of K. pneumoniae—in 526-fold dilution, that is, in concentration of 3.9 and 1.9 mg/L, respectively  higher than MBC of amikacin, whereas antipseudomonal MICs of these antibiotics were equally substantial. K. pneumoniae demonstrated full resistance to cefepime . Suspending of exogenous surfactant in cefepime resulted in slight decrease of bactericidal activity against S. aureus and P. aeruginosa cultures.Cefepime exhibited most pronounced antistaphylococcal activity . However, when tested against S. aureus). The ability of colistimethate sodium to inhibit growth of K. pneumoniae and P. aeruginosa was moderate but higher than in cefepime. When combined with surfactant, bacteriostatic effect of colistimethate increased 4- and 2-fold for S. aureus and P. aeruginosa, respectively, though remaining on relatively low level (20 mg/L and 10 mg/L), while bactericidal activity was unchanged.Colistimethate sodium had significantly lower antibacterial activity as compared to amikacin  and cefepime (against in vitro and to identify appropriate combination(s) for subsequent investigations of topical surfactant/antibiotic therapy in experimental acute bacterial pulmonary infection. Potential limitations of this study include restrictions as regards extrapolation of the results to other exogenous surfactant preparations due to differences in chemical composition of currently available surfactant products.It has been previously shown that interactions between antibiotics and surfactant may exist, and these interactions may influence efficiency of either substances , 10. TheIn the first part of the study it has been revealed that addition of the amikacin or cefepime to the surfactant had no significant influence on the surface-active properties of the latter, whereas obvious reduction of this feature was detected when mixing surfactant with colistimethate sodium. Considering the ability of colistimethate sodium to disrupt phospholipid bilayer of bacterial cell envelope , suppose S. aureus and P. aeruginosa.The results of microbiological part of the study suggest that the surfactant preparation was devoid of intrinsic antibacterial activity. When mixed with antibiotics, the surfactant either had no impact on antimicrobial activity of the latter  or exerted mild to moderate influence. Specifically, there was a trend for decrease of cefepime bactericidal activity and increase of colistimethate sodium bacteriostatic activity against3 buffer [Interaction between exogenous surfactant and antibiotics was previously studied by van't Veen et al. , 10. The in vitro [ K. pneumoniae, P. aeruginosa, S. aureus, and Streptococcus pneumoniae by the ability of aminoglycosides to bind negatively charged phospholipids contained in surfactant preparation and assumed that an excess of phospholipids can inactivate almost all the aminoglycosides [An ability of exogenous surfactant to suppress activity of certain antibiotics was demonstratedin vitro  in termsycosides , 7.In contrast to the aforecited tobramycin/surfactant compatibility issues , 10, the S. aureus, K. pneumoniae, and P. aeruginosa appears to be unreasonable. Conversely, it is advisable to use the surfactant/amikacin composition for experimental studies in animals infected with given strains of S. aureus, K. pneumoniae, and P. aeruginosa, and the surfactant/cefepime composition for experimental staphylococcal infection in laboratory animals.Assuming relatively low activity of the colistimethate sodium against tested bacterial cultures, as well as noticeable reduction of surfactant surface activity in the surfactant/colistimethate sodium mixture, the use of this composition in subsequent studies in animals infected with in vitro, whereas significant suppression of both surfactant and colistimethate sodium surface-active properties is evident when mixed in one volume. Addition of the surfactant suspension to amikacin had no influence on high antimicrobial activity of the latter, whereas suspending surfactant with cefepime or colistimethate sodium resulted in a trend for decrease of cefepime bactericidal activity and increase of colistimethate sodium bacteriostatic activity against S. aureus and P. aeruginosa. Obtained data confirm the necessity of surfactant-antibacterial interaction assessment before utilization of any surfactant/antibiotic mixtures in clinical settings and in preclinical animal studies and show that further investigation of combined surfactant/antibacterial therapy should include an evaluation of the surfactant/amikacin composition in animals with bacterial pneumonia.in summary, the study showed that an addition of therapeutic concentrations of the amikacin and cefepime to the exogenous surfactant preparation does not cause a decrease of surfactant surface activity


Punica granatum L.), a well-known Rasayana, were tested on Drosophila melanogaster (fruitfly) model. Supplementation of standard corn meal with 10% (v/v) pomegranate juice (PJ) extended the life-span of male and female flies by 18 and 8%, respectively. When male and female flies were mixed and reared together, there was 19% increase in the longevity of PJ fed flies, as assessed by MSD, the median survival day (24.8). MSD for control and resveratrol (RV) groups was at 20.8 and 23.1 days, respectively. A two-fold enhancement in fecundity, improved resistance to oxidative stress (H2O2 and paraquat induced) and to Candida albicans infection were observed in PJ fed flies. Further, the flies in the PJ fed group were physically active over an extended period of time, as assessed by the climbing assay. PJ thus outperformed both control and RV groups in the life-span and health-span parameters tested. This study provides the scope to explore the potential of PJ as a nutraceutical to improve health span and lifespan in human beings.Exploring innovative ways to ensure healthy aging of populations is a pre-requisite to contain rising healthcare costs. Scientific research into the principles and practices of traditional medicines can provide new insights and simple solutions to lead a healthy life. Rasayana is a dedicated branch of Ayurveda (an Indian medicine) that deals with methods to increase vitality and delay aging through the use of diet, herbal supplements, and other lifestyle practices. The life-span and health-span enhancing actions of the fruits of pomegranate ( Global statistics predict 223% increase in the number of elderly aged >60 years) accounting for about 1.2 billion between 1970 and 2025 . Countri0 years aRasayana is a special branch of Ayurveda, which deals with methods of rejuvenation such as dietary recipes and regimen, herbal and mineral supplements, and health-promoting lifestyle that are said to enhance quality of life and delay aging (Agni), leading to better bio-availability, (ii) increasing the micro-circulation of nutrients through the body channels (Srotas), and (iii) enhancing the nutritive value of the plasma that gets generated (Rasadhatu)  , 9. Sevesadhatu) .Phyllanthus emblica), Pomegranate (Punica granatum), Drumsticks (Moringa oleifera), Long pepper (Piper longum), etc., as Rasayanas  fruit with several properties including tissue generation and development and strength promotion  radiation, infant brain ischemia, prevention of cancer, Alzheimer’s disease, male infertility, arthritis, and obesity –13. Pomeromotion . It is promotion .Saccharomyces cerevisiae, Caenorhabditis elegans, D. melanogaster, and rodents (Drosophila (commonly known as fruit fly) has many advantages for studying the biology of aging. For example, flies show physiological, genetic, and anatomical similarities with human , and pomegranate juice (PJ) supplemented Drosophila melanogastor wild type, Canton-S strain, was used for this study. The basal media for culturing the flies consisted of corn flour (60 g), sugar (20 g), d-glucose (20 g), dry yeast (15 g), and agar (8 g) per liter. After cooking, media was allowed to cool to room temperature and then propionic acid (4 ml), benzoic acid (0.7 g in 5 ml of ethanol), orthophosphoric acid (0.6 ml) were added and mixed thoroughly. Media cooked as slurry was poured into the bottles or vials and was allowed to solidify before transfer of adult flies or pupae. Stock flies were maintained in bottles while culture vials were used for maintenance of smaller populations or experimental groups. Flies were allowed to feed ad libitum and grown at 25°C with 60% humidity in 12 h day–night cycle.Pomegranate juice was prepared by hand crushing the arils of fresh fruits through sterile muslin cloth. The juice was passed through a filter  and stored as aliquots of different volume  in screw capped tubes at −80°C until use. Freezing did not significantly alter the high-performance liquid chromatography (HPLC) profile (data not shown). RV  stock (100 mM) was prepared by dissolving 22.8 mg in 1 ml of ethanol. Both PJ and RV were added to the cooked and warm basal media before dispensing into vials.Age matched (2–3 days) flies were generated by removing all adult flies in the bottles. Once a synchronous culture was generated, sex separated flies were transferred into vials containing basal medium supplemented with 0.1, 1, 5, 10, and 15% (v/v) of PJ. Flies were transferred to fresh media every third day. Dead flies were counted and removed daily until the death of the last fly in each vial. PJ at 15% v/v could not be used since at that concentration the medium would not set.Synchronous cultures of 2–3-day-old files were obtained as described earlier and transferred into vials containing basal medium supplemented with 10% of PJ (v/v) or 200 μM RV and control. Each group including the control had 10 vials each with 14–20 flies per vial with equal sex ratio. Flies were transferred to fresh media every third day. Dead flies were counted and removed daily throughout the experiment. New flies emerging in the emptied vials were counted daily for 10 days from the day of the emergence of the first fly in each experimental group.The method described by Lee et al.  was adop2O2  and paraquat . H2O2 generates hydroxyl radical (OH*) while paraquat produces superoxide (O*) radicals. Twenty days old male and female flies from control, RV (200 μM), and PJ (10%) fed were starved for 2 h and transferred to separate vials  containing 5% H2O2 or 20 mM paraquat prepared with 5% sucrose on saturated tissue paper mat. Dead flies were counted every 4 h until the death of the last fly.The method described by Peng et al.  was usedC. albicans SC5314 in sterile distilled water . Flies (20 days old) were anesthetized with CO2. Infection was induced with a sterile tungsten needle (0.01 mm diameter) dipped in the C. albicans suspension by gently pricking in the thoracic region. Flies were returned to their respective food vials and were incubated at 25°C with 12 h day–night cycle until 50% of flies died in any of the groups. Control flies were pricked with needle dipped in sterile distilled water.The method described by Apidianakis and Rahme  was follOn day 20 and 40, locomotor function of fruit flies was assessed using the climbing or negative geo-taxis assay as reported by Bahadorani and Halliker  with slit-test was used for comparison of means. ANOVA was employed while comparing more than two groups. p-Value <0.05 was considered as significant.The day when 50% of the total flies in each of the experimental group survived was calculated as the median survival day. Log rank testDrosophila, we supplemented the basal media with 0.1, 1, 5, 10, and 15% of PJ. Substitution of the media with 10% PJ significantly enhanced the median lifespan by 18.51 and 8% in male and female flies, respectively   more number of flies throughout the reproductive phase. Also, the new fly emergence data indicated that PJ extended the reproductive viability phase  to 24.8 days (PJ), i.e., PJ enhanced median life-span by 19.23% more than the control , particularly in the male flies  in food intake between the male and female flies in both PJ and RV groups  increase in the post H2O2 and paraquat exposure survival time was observed in both PJ and RV supplemented flies when compared to the control  potential  protective effect of PJ until 144 h post-infection. Even though, RV too protected the flies (>50%) from C. albicans infection, the magnitude of protection by PJ (>70%) was significantly higher. The control flies had only 40% survival  of organisms. The physical performance of the flies was measured using the climbing or negative geo-taxis assay. This assay was performed with 20 and 40 days old male and female flies fed on media supplemented with PJ or RV. Even though, significant difference among the groups was not observed in the percentage of flies climbing on 20th day, the PJ group retained the physical activity even on day 40 (aged flies), while the other groups showed severe decline (p < 0.05)  in the fly fat body , i.e., the organism is satisfied with lesser intake of food when supplemented with PJ (The current study corroborates earlier reports that RV enhances lifespan in osophila , 31, 41.% Figure A. RV sub with PJ , 16. WhiFree radical induced stress tolerance, improved infection survival, and climbing performance assays show that, pomegranate supplementation helps in reducing age-related functional decline in the flies. This study justifies the Ayurvedic claims that pomegranate is a Rasayana by being a multi-functional health promoter in fruit fly model. Given the exploratory nature of our study and the large number of comparisons made, the results are certainly very interesting; and it has opened up avenue for further research. Further research needs to be done to study the effectiveness of PJ supplementation in human beings at the community setting. Scientific exploration of other Rasayanas as quality-of-life enhancers is a potential area for research. Studying the mode of action of PJ, identification of bioactive components and molecular targets would be of value to understanding aging and anti-aging process.Drosophila has been a strong candidate as a model in contemporary aging research. D. melanogaster has also been used as a model by researchers to study Rasayanas. An insect specific Rasayana was developed that was found to increase lifespan by 50% in Drosophila model (la model . Dwivedila model  reportedAging population and the associated physical, physiological, and neurological deficiencies are being recognized as the major challenges for healthcare in the twenty-first century. While people are living longer; they are aging prematurely . ScientiDrosophila based experiments, analyzed results, and edited manuscript. Subramani Paranthaman Balasubramani performed the experiments, analyzed, interpreted results, prepared, and edited the manuscript. Jayaram Mohan maintained the Drosophila stocks and assisted Subramani Paranthaman Balasubramani in experimentation. Esha Patnaik and Chatterjee Arunita standardized the infection and stress experimentation protocol and analysis of results. Subrahmanya Kumar Kukkupuni provided the Ayurveda knowledge.Padmavathy Venkatasubramanian conceptualized the transdisciplinary research methodology, obtained fund support, and edited the manuscript. Upendra Nongthomba designed 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.


Background. Central neurocytoma and oligodendroglioma are rare tumors of the central nervous system. However, diagnosis between these two types of tumors is challenging due to their many cytological and histological similarities. Death-associated protein kinase (DAPK) is a calcium/calmodulin-regulated serine/threonine protein kinase involved in many apoptosis pathways, and repressed expression of DAPK by promoter hypermethylation has been found in a variety of human cancers. The purpose of this study was to assess DAPK protein expression and promoter hypermethylation in central neurocytoma and oligodendroglioma. Method. Central neurocytoma and oligodendroglioma samples were obtained from age- and sex-matched patients. DAPK protein expression was performed using immunohistochemical assays in formalin-fixed, paraffin-embedded sections. DAPK promoter hypermethylation was carried out using bisulfite-modified genomic DNA in methylation-specific PCR followed by separation in agarose gels. Findings. A statistically significant difference (P = 0.021) in DAPK promoter hypermethylation between central neurocytoma (76.9%) and oligodendroglioma (20%) was observed. High levels of DAPK protein expression were generally found in oligodendroglioma (90%), compared with 38.5% in central neurocytoma . There was an inverse correlation between DAPK protein expression and DAPK promoter hypermethylation in the cohort of 23 patients (P = 0.002). Conclusions. The results show that DAPK promoter hypermethylation and repressed expression of DAPK protein were more common in central neurocytoma than in oligodendroglioma. Thus, DAPK promoter hypermethylation could be useful for differential diagnosis between these two types of tumors, whereas DAPK protein expression might be less predictive. The role of DAPK promoter hypermethylation in the pathogenesis of central neurocytoma warrants further study. Central neurocytomas are rare tumors of the central nervous system, comprising only 0.1–0.5% of all brain neoplasms . GeneralOligodendrogliomas occur primarily in the cortex and white mater of the cerebral hemispheres of adults in their fourth and fifth decades of life, while projection into the ventricles has also been found . HistoloOligodendroglioma cells can actively induce neuronal damage by releasing molecules able to inhibit neurite sprouting and to eventually cause apoptotic neuronal death , 24. As This study was approved by the Kaohsiung Medical University Hospital Review Board. Central neurocytomas and oligodendrogliomas were obtained from age- and sex-matched patients  treated at the Kaohsiung Medical University Hospital. The specimens were diagnosed by H&E stain under light microscopy and immunostaining of synaptophysin and glial fibrillary acidic protein. All the 13 central neurocytomas are located intraventrically. All the 10 oligodendrogliomas are low grade tumors. The diagnosis was confirmed by physicians. Consents were received from all patients. Each tissue was divided into two equal parts, one for DNA extraction and the other for immunohistochemical staining.μg of tumor DNA was further modified by sodium bisulfite to convert unmethylated cytosines to uracils, and the modified DNA was eluted into buffer EB . This bisulfite conversion and clean-up of genomic DNA were performed using the EpiTect Bisulfite kit (Qiagen). Purified DNA was used immediately as a template for methylation-specific polymerase chain reaction (PCR) described below or stored at −70°C until use.Tissue samples from central neurocytoma and oligodendroglioma patients were digested with proteinase K at 56°C overnight, and genomic DNA was isolated by phenol-chloroform extraction using a commercially available kit according to the manufacturer's procedures. Approximately 2 μg of modified DNA was added to a PCR solution containing 1x PCR buffer, 1.25 mM MgCl2, 0.25 mM dNTP, 0.5 μM PCR primers, and 1.25 U of GoTaq DNA polymerase (Invitrogen) in a total volume of 25 μL. The forward and reverse primer sequences used for methylated DNA were 5′-GGATAGTCGGATCGAGTTAACGTC-3′ and 5′-CCCTCCCAAACGCCGA-3′, respectively, whereas the forward and reverse primer sequences used for unmethylated DNA were 5′-GGAGGATAGTTGGATTGAGTTAATGTT-3′ and 5′-CAAATCCCTCCCAAACACCAA-3′, respectively [Approximately 0.2 ectively . The CpGμm sections. They were then stained with hematoxylin and eosin and were evaluated to determine the extent of tumor cells presented in the sections using a light microscope. Subsequently, the samples were washed with PBS and incubated with anti-DAPK antibody (Santa Cruz) at a 1 : 100 dilution for 1 hr at room temperature. Afterwards, slides were washed for 30 min in PBS and incubated with secondary antibody (Dako Code K5007). Specimens were again washed with PBS, incubated with peroxidase-labeled streptavidin  for measurement of the intensity of immunoreactivity.For immunohistochemical staining, tissues were fixed in formalin, embedded in paraffin, and cut into 5 Each section was given two independent scores, namely, the extent of tumor cells in the sample and the intensity of immunoreactivity, by an investigator blinded to the experiment. A score of 0 (zero) was assigned to a section if the extent of tumor cell was <1%, whereas scores of 1, 2, and 3 were given to sections with 1%–10%, 11%–50%, and >50% tumor cells, respectively. Likewise, a section received a score of 0 (zero) when the intensity of the slide was similar to the background level. Intensity scores of 1, 2, and 3 were assigned to sections with weak, moderate, and strong intensity of immunoreactivity, respectively. The values of these two independent parameters were multiplied to generate the final score for each section (ranging from 0 to 9) according to a published procedure . A finalP value of <0.05 was considered statistically significant.All results were expressed as mean ± SEM. An analysis of variance (ANOVA) followed by Fisher's exact test was performed to determine statistical significance between two groups. A P = 0.023).DAPK promoter hypermethylation was observed in both central neurocytoma and oligodendroglioma . InteresP = 0.054).As mentioned in the methods, DAPK protein expression was assessed by both the extent of tumor cells and the intensity of immunoreactivity . These tP = 0.002).An effort was made to correlate DAPK promoter hypermethylation and DAPK protein expression in all of the central neurocytoma and oligodendroglioma samples evaluated. It was found that samples with low levels of DAPK protein expression always exhibited high levels of DAPK promoter hypermethylation. This was seen in 9 out of 9 cases  from 1 oCentral neurocytoma and oligodendroglioma are rare tumors of the central nervous system. However, diagnosis between these two types of tumors is challenging due to their many cytological and histological similarities , 16, 33.Upon analysis of data obtained from both central neurocytoma and oligodendroglioma samples, there was an inverse correlation between DAPK protein expression and DAPK promoter hypermethylation . These rBesides being localized centrally, neurocytomas have also been found extraventricularly –9. It wo


In addition to two distinct CRF01_AE clusters [cluster 4  and cluster 5 ], we identified a new CRF07_BC cluster (cluster 1) , which entered Chinese MSMs in 2004, and had been rapidly spreading since about 2004, which indicating the third wave of the HIV epidemic among the population. Moreover, two new clusters of CRF_01B recombinants were found in this study. The complexities of HIV subtypes and recombinants strongly supports the necessity for a comprehensive study about risk behaviors and their relationship with increasing HIV epidemic subtypes among the MSM group. Implementation and evaluation of comprehensive harm reduction strategies in Chinese MSM are urgently needed.To investigate the origins of HIV transmission and phylogenetic dynamics among men who have sex with men (MSM), a total of 1205 newly diagnosed HIV-infected 16–25 year-olds were recruited in 13 provinces across China between 2009 and 2014. Based on phylogenetic analyses of partial  The first Chinese HIV epidemic was reported in 1989 in Yunnan279101113144161718A total of 1205 HIV-1 nucleotide sequences of the 1.0-kb pol gene (HXB2: 2253–3278 nts) from newly diagnosed MSM between 2009 and 2014 from 13 Chinese provinces were determined and genotyped by phylogenetic tree analysis. As shown in The chi-square trend test was used to compare the changes of HIV-1 subtypes over time. The proportion of subtype CRF01_AE decreased from 55.4% to 43.5% during 2009–2014 (P = 0.044), while at the same time, the proportion of subtype CRF07_BC increased from 25.6% to 40.1% (P = 0.016), and the proportion of subtype B_EU decreased from 16.0% to 3.8% (P < 0.0001). Other subtypes and recombinant strains can be seen in As shown in Beside the five major clusters, there are two clusters of CRF_01B among the MSM population, including a distinct cluster of CRF55_01B, the strains of which were mainly found in Shenzhen, Henan, and Hunan. We also found a new URF_01B cluster, which was only detected in Beijing and Jiangsu Province in our study, and the strains were clustered with the strains from Anhui Province.We reconstructed the epidemic history of the 5 major clusters (n ≥ 45) through Bayesian analysis, using the HKY model and the Log normal relaxed clock model. As shown in In recent years, MSM have become the most significant increasing HIV-1 transmission route in China16171821222323. The CRF07_BC cluster was not discovered in the second nationwide molecular epidemiological investigation in 2002, and was only found in a limited number of male homosexuals in the third nationwide molecular epidemiological investigation in 2007131426The major finding in our study was that the new wave of the HIV epidemic among MSM was driven by the subtype CRF07_BC virus. CRF07_BC was first transmitted to MSM in about 2004, and has been the biggest cluster for over ten years. CRF07_BC originated in 1993 in China among IDUs in the western and southern provinces of China, including Xinjiang, Sichuan and YunnanOur study estimated that the subtype B_EU group viruses were first introduced into MSM in China in 1988, and then were replaced by other subtypes, such as CRF01_AE cluster 4 and cluster 5. The subtype B_EU virus might have originated in the United States and Europe, and entered China through travelers. This initial founder virus did not turn out to be the main HIV genotype in Chinese MSM.Our study found that CRF01_AE cluster 4 and cluster 5 first entered MSM in about 1994 and 1995, respectively. Then, the subtype CRF01_AE virus rapidly and widely spread over time became the main HIV genotype in China MSM. CRF01_AE cluster 4 and cluster 5 were not discovered until the second nationwide molecular epidemiological investigation in 2002, and they were found only in a limited number of male homosexuals in the third nationwide molecular epidemiological investigation in 2007212223211314In addition, we observed two newly identified clusters of CRF_01B recombinant strains. One cluster was CRF55_01B, which was first identified from MSM in China. The CRF_01B recombinant strain first entered Chinese MSM in about 2004. It includes the subtype B fragment which is related to subtype B_EU, not the Thai B’ variant. Another recombinant strain, the CRF_01AE fragment is related to Thai strains of CRF01_AE, but not to those found in other MSM in China. Our study found that CRF55_01B circulated in most cities. The other cluster of CRF_01B (URF_01B) is a region-specific cluster identified in Beijing and Jiangsu, and shows distinct mosaic models with the isolated CRF_01B strains from foreign countries. Our previous study also found a high proportion of HIV subtypes and new recombinant HIV-1 in predominantly heterosexually infected populations in a sexually driven epidemic area of Yunnan Province, China293031This study has some limitations. The study sample sizes differed between cities. Some cities such as Beijing and Zhejiang yielded large samples, whereas other sites such as Guangxi and Yunnan yielded relative few subjects. Therefore, our estimate of HIV genotype distributions may be biased. Non-participants may have different characteristics, such as demographics and risk behaviors, which could lead to a selection bias. However, our serial cross-sectional studies systematically revealed the emergence of the CRF07_BC cluster, multiple lineages of HIV viruses, and newly recombinant strains circulating in Chinese MSM. Future study needs to clarify the possible risk factors that related with changes of HIV subtypes and phylogenetic dynamics in Chinese MSM. Despite the rapid changes of subtype incidence that occurred from 2009 to 2011, there were not many changes in the distribution of any of the subtypes during 2011–2014. In order to control the fast transmission of HIV among MSM, comprehensive prevention intervention programs have been conducted since 2010, including mass education, community outreach, condom promotion, rapid scale-up of HIV testing, and ART. The impact of such programs on HIV infection and related risk behaviors among MSM urgently needs to be evaluated, which can guide scientific evidence for implementing an effective means of reducing HIV transmission.This study was approved by the China CDC Institutional Ethics Committee, and written informed consent was obtained from study participants. All experiments were performed in accordance with the approved guidelines and regulations and the experimental protocols were approved by the institutional review boards of China CDC.A serial cross-sectional study was conducted from 2009 and 2014 in 13 provinces or cities, China. The subjects were enrolled from newly diagnosed HIV cases at the local Center for Disease Control and Prevention. Subjects eligible for study were between 16 and 25 years of age, newly diagnosed HIV-1 infected MSMs, and able to provide written informed consent. All study participants completed a questionnaire administered by trained interviewers in a private room. The research staff collected 8 mL of peripheral blood samples that were anti-coagulated with EDTA-3K. Plasma was separated within 6 hours after collection, tested for antibodies and HIV-1 RNA, and frozen at −80 °C for further analysis. This study was approved by institutional review board at the National Center for AIDS/STD Control and Prevention, Chinese Center for Disease Control and Prevention.The QIAamp mini-viral RNA kit  was used to extract RNA from all plasma samples, according to the manufacturer’s instructions. The HIV-1 pol gene (protease 1–99 amino acids and part of reverse transcriptase 1–250 amino acids) was amplified, purified, and bi-directly sequenced in an ABI330XL sequencer , according to previously published methodshttp://www.hiv.lanl.gov/content/index) using the CLUSTAL X program www.mbio.ncsu.edu/bioedit/bioedit.html). All positions that contained alignment gaps were removed. To exclude experimental contamination, similarities between the pol sequences in this study and the sequence database were analyzed by applying the Los Alamos HIV Database Web tools (http://www.hiv.lanl.gov).The sequences were assembled with Sequencher 4.10.1  and then aligned with previously submitted sequences from our laboratory and other reference sequences from the Los Alamos database (http://beast.bio.ed.ac.uk). The Recombinant Identification Program 3.0 (www.hiv.lanl.gov/content/sequence/RIP/RIP.html) of Los Alamos HIV database was used to verify recombinant sequences.PhyML 3.0 was used to estimate a maximum likelihood phylogenetic tree for sequences using the GTRtItG4 nucleotide substitution modelhttp://beast.bio.ed.ac.uk/Tracer).To estimate the evolutionary rate and the time of the most recent common ancestor (tMRCA) for the CRF01_AE and CRF07_BC lineages, we used BEAST v.1.8.0 under an uncorrelated log-normal relaxed clock model, GTRtG4 substitution model, and Bayesian skyline plot demographic model3637All data were analyzed using SAS 9.2 software packages. The proportion of HIV subtypes over time was assessed using the chi-square trend test. P values < 0.05 were considered statistically significant.The sequences have been deposited in GenBank with accession numbers KR822836 - KR824040.How to cite this article: Li, Z. et al. Trends of HIV subtypes and phylogenetic dynamics among young men who have sex with men in China, 2009-2014. Sci. Rep.5, 16708; doi: 10.1038/srep16708 (2015).