Lifelong regular physical activity is associated with reduced risk of type 2 diabetes (T2D), maintenance of muscle mass and increased metabolic capacity. However, little is known about epigenetic mechanisms that might contribute to these beneficial effects in aged individuals. We investigated the effect of lifelong physical activity on global DNA methylation patterns in skeletal muscle of healthy aged men, who had either performed regular exercise or remained sedentary their entire lives (average age 62 years). DNA methylation was significantly lower in 714 promoters of the physically active than inactive men while methylation of introns, exons and CpG islands was similar in the two groups. Promoters for genes encoding critical insulin-responsive enzymes in glycogen metabolism, glycolysis and TCA cycle were hypomethylated in active relative to inactive men. Hypomethylation was also found in promoters of myosin light chain, dystrophin, actin polymerization, PAK regulatory genes and oxidative stress response genes. A cluster of genes regulated by GSK3β-TCF7L2 also displayed promoter hypomethylation. Together, our results suggest that lifelong physical activity is associated with DNA methylation patterns that potentially allow for increased insulin sensitivity and a higher expression of genes in energy metabolism, myogenesis, contractile properties and oxidative stress resistance in skeletal muscle of aged individuals. A physically active lifestyle is associated with a number of physiological, biochemical and transcriptional changes contributing to reduced risk of developing type 2 diabetes (T2D) and cardiovascular diseases during aging3. Among the beneficial effects of regular exercise is maintenance of muscle mass through enhanced proteostasis of structural proteins such as actin and myosin4. In addition, exercise activates several signaling cascades that ultimately initiate transcriptional regulation of genes encoding proteins involved in mitochondrial function and other pathways associated with energy metabolism6.Regular physical activity promotes health and longevity. The Harvard Alumni Health Study revealed that regular exercise reduces mortality in elderly men7. Although patterns of DNA methylation are mainly established early in development and traditionally have been thought to be largely maintained in the differentiated cells, several environmental factors have been shown to affect DNA methylation8. Hence, physical activity has been found to induce changes in DNA methylation patterns as well as expression of corresponding genes in skeletal muscle, both in response to a single exercise bout9 and after prolonged exercise training11. In addition, the cellular aging process seems to be tightly linked with complex epigenetic changes. Aging has been associated with global hypomethylation of the genome12 although a recent study in aging humans suggest that DNA in muscle is generally hypermethylated as we age13.However, little is known about epigenetic mechanisms that might contribute to the beneficial effects of regularly performed exercise in aged individuals. Epigenetic modifications are known to constitute an important regulatory layer of transcription and DNA methylation is a major epigenetic modification that governs chromatid structure and gene expression. Methylation of promoter regions is associated with repression of transcription, and DNA methylation will therefore typically cause gene silencingThe aim of the present study was therefore to investigate the impact of lifelong physical activity on global DNA methylation patterns in muscle tissue of 16 healthy aged men with an average age of 62 years. Eight of these subjects had a physically active lifestyle with regular exercise their entire lives and eight subjects were healthy but with a physically inactive lifestyle. Our data reveal physical activity level dependent epigenetic modifications in genes involved in muscle structural dynamics, energy metabolism, and predisposition to T2D. This suggests that the level of physical activity throughout life affects DNA methylation of genes within cellular pathways and metabolic processes in skeletal muscle.To test how lifelong endurance exercise training influences epigenetic imprinting, we measured global DNA methylation in muscle tissue of two groups of healthy 60–65 year old men. One group of 8 men reported high levels of physical activity through regular exercise training their entire lives, while another group of 8 men reported low lifelong levels of physical activity. While height and weight were similar between the two groups, body fat percentage was 39% higher with a corresponding 13% lower percentage of lean mass in the inactive than the active men  of 28857 genes Fig. . MoreoveGYG2), glycogen synthase 1 (GYS1), as well as the glycogen degrading amylase alpha 2B (AMY2B). The result supports a model in which DNA-methylation patterns allow a higher capacity for expression of genes involved in glycogen storage and metabolism in active than inactive men , a functional homologue of hexokinase that catalyzes the phosphorylation of glucose to glucose 6-phosphate using ADP as substrate, and the muscle specific pyruvate kinase (PKM), which mediates the last step in glycolysis. Similarly, promoter methylation of the gene encoding 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB) was lower in the active relative to inactive subjects, which could potentially increase the capacity for regulation of glycolysis. Also, the promoter regions of genes encoding the pyruvate dehydrogenase alpha 1 subunit (PDHA1) that links glycolysis and the TCA cycle, and the rate limiting enzyme in TCA cycle flux, isocitrate dehydrogenase (IDH3A), were found to be hypomethylated in the active compared with inactive subjects.Among the 714 genes with significantly less methylated promoters in the active group were several genes encoding enzymes involved in energy metabolism Fig. . This liPDHA1 product, pyruvate dehydrogenase E1 alpha, was higher at the protein level in active than inactive men  family, which catalyzes the exchange of ATP produced in the mitochondria with ADP from the cytosol. Western blots revealed that protein content of both ANT1 and ANT2 were indeed higher in active than inactive men  were all hypomethylated in the active relative to the inactive men Fig. . The mitive Fig. .Figure 318. This led us to further analyze promoter methylation of genes involved in myogenesis and muscle structural dynamics. Indeed, the promoter region of the MYOD1 gene was relatively hypomethylated in the active versus the inactive group  proteins that crosslink actin filaments with the plasma membrane, mDIA, which participates in formation of actin stress fibers important for establishment of cellular tension, the ADF/cofilin gene, SSH1, involved in actin dynamics and the dystrophin gene, DMD, which links sarcolemma to the contractile apparatus. A number of genes  involved in the CDC42 signaling pathway, which regulates actin/myosin cross-linking and actin polymerization19, also exhibited promoter hypomethylation in the active relative to inactive subjects. In addition, the promoter of the smooth muscle repair protein thymosin-4 peptide, TMSB4, was relatively hypomethylated in active subjects. Overall, the results show that promoters of numerous genes involved in myogenic, contractile, structural and reparation properties of skeletal muscle were hypomethylated in the active compared with inactive men  were significantly higher in muscle tissue of physically active men than inactive men . To test if the oxidative stress response in trained men rendered muscle tissue more resistant to oxidative stress, we measured protein carbonylation as an indicator of oxidative damage. Carbonylation of proteins was 29% lower in tissue of active men, supporting a higher tolerance to oxidative stress in this group (Result in20).In general, promoter regions of genes involved in the oxidative stress response was hypomethylated in active men. Microsomal glutathione S-transferase 1 gene, men Fig.  in accor22. A negative regulator of the MAPK p38 pathway, PTPRR, displayed reduced promoter methylation, suggesting a potential for reduced p38 pathway activity in the tissue of active men. Recent studies of muscle stem cells from aged mice revealed that inhibition of the p38 pathway, activated muscle stem cells, which thereby improved the potential for regeneration of damaged muscle23.Mitogen activated protein kinases, MAPKs, can be activated by ROS and are directly involved in induction of antioxidant genes24. However, we found no difference in the protein content of PAX7 between trained and untrained muscle (data not shown), suggesting that the number of stem cells was similar in muscle from the two groups of men, although this does not exclude that the stem cell potential is higher in tissue from physically active men than from inactive men.Because target genes of the stem cell regulating GATA3 and MYC1 were also found to be hypomethylated in active men  in physically active men relative to inactive men, suggesting increased capacity for transcription of this methyltransferase gene and hence higher de novo methyltransferase capacity in skeletal muscle with lifelong physical activity. However, the levels of DMNT3B protein were similar in the physically active and inactive men, indicating that the capacity for higher DNMT3B expression was apparently not evident at the protein level (data not shown).To understand how differential DNA methylation may arise in the muscle tissue of active compared with inactive men, the list of hyper- and hypomethylated genes for DNA methyltransferases and demethylases was examined. The promoter of the DNA methyltransferase gene This study presents a library of changes in global DNA methylation patterns in skeletal muscle that occurs during lifelong physical activity which serves as a resource for future research. Of particular interest, we show that lifelong physical activity was associated with differential promoter DNA methylation levels in skeletal muscle with effects on rate limiting and insulin sensitive steps of glycolysis and TCA cycle as well as muscle regeneration.26, but how this occurs at the epigenetic level in skeletal muscle is unclear. The observed methylation profiles suggest that the capacity for glycolytic as well as TCA cycle flux is higher in muscle from physically active than inactive men through promoter hypomethylation of several key genes in the active subjects .It is well established that exercise prevents or delays the loss of muscle mass, insulin sensitivity and the ability to repair muscle damage during agingcts Fig. . The pro27. This study identified 600 promoters that were hypermethylated in response to exercise training. While these hypermethylations are in contrast to the general observation in the current study, there is a general trend in both studies for altered promoter methylation of genes involved in T2D via the insulin- and Wnt-sensitive transcription factor TCF7L2.A six month exercise invention was reported to increase the absolute level of methylation in DNA from adipose tissue of younger menTCF7L2 gene is, to date, the strongest genetic marker associated with T2D28. While TCF7L2 was shown to be hypermethylated in adipose tissue upon a six month exercise training invention27, we did not find a significant association between lifelong exercise training and promoter methylation of TCF7L2 in muscle tissue. However, the finding, that the protein level of the upstream regulator, GSK3β, was significantly higher in the group of physically active than in the inactive men, supports a role of insulin- and/or Wnt-mediated GSK3 signaling in harnessing of the promoter potential of metabolic genes  had lived a lifelong sedentary lifestyle with physical activity at most one time per week throughout their life, while the physical active group  had exercised more than three times per week throughout their life. The reported physical activity differed over the years for several of the participants and included soccer, bicycling, hiking, running, gymnastics, handball, badminton, military training, fitness, swimming, tennis, boxing, track and field and ice hockey. Moreover, the lifelong physically active subjects trained for at least 5 hours/week at the time of the experiment. Alcohol and smoking are unlikely to be confounding factors, although three physically inactive subjects and one physically active subject were smokers, while all other subjects were nonsmokers, while the alcohol consumption varied more within groups than between groups. The diet of the subject was not recorded and diet can therefore not be ruled out as a potentially confounding factor. Ethical approval was granted by the Committee of Copenhagen and Frederiksberg communities, reference number H-7-2014-001 and the research was conducted in accordance with the guidelines of Measurements of percent body fat by DEXA scanning confirmed the group differences . An incremental ergometer cycling challenge was performed consisting of cycling at 120 watts for 5 minutes, followed by 20 watt increments every second minute until reaching perceived exertion of 18 on the Borg scale, at which participants continued until exhaustion. The cycling test confirmed the difference in endurance exercise performance between groups, while no significant differences were documented for age, height, weight or blood levels of cholesterol, testosterone, inflammatory markers or leukocyte count , transferred into liquid nitrogen and stored longterm at −80 °C. The same group of men was also tested for presence of extrachromosomal circular DNA in muscle and lymphocytes which is described in20.On the day of the experiment, subjects arrived at the laboratory late afternoon (5 PM) having fasted at least 2 hours prior to experiment. Under local anaesthesia, muscle biopsies from The genome-wide mapping of 5-methylcytosine in DNA from muscle was carried out by whole genome bisulphite sequencing. For this purpose, 10–20 mg of muscle tissue was homogenized with the TissueRuptor (QIAGEN) in 360 µl ATL buffer (QIAGEN) on ice. DNA was subsequently extracted with the DNeasy Tissue Mini Kit according to protocol (QIAGEN) treating homogenized tissue sample with proteinase K for 6 hours. The bisulphite conversion was performed on 500 ng input DNA using EZ DNA Methylation Kit (Zymo). Promega unmethylated control Lambda DNA was spiked in the DNA samples to serve as a control for measuring bisulphite conversion rate. After bisulphite conversion, the library preparation was done by Illumina Epicenter methylSeq kit according to the manufacturer’s instructions. Subsequently each individual DNA library sequenced in three lanes of HiSeq2500 machine achieving approximately on average a base coverage more than 20X.http://www.bioinformatics.babraham.ac.uk). Low quality base calls were removed from the 3′ end of the reads before trimming the adapter sequences. Our cut off for base quality control was Phred quality score of 20. Subsequently, Cutadapt detected and trimmed the adapter sequences from the 3′ end of reads. BSMAP aligner34 was used for mapping reads against the human genome hg19. For post data analyses purposes, we used an R library package methylKit35 and custom scripts implemented in R and Python. We used the MethylKit package to calculate methylation percentages per each single CpG. Percent methylation values for CpG dinucleotides were calculated by dividing the number of methylated Cs by the total coverage on that base. CpGs with at least 8X read coverage and at least a Phred score threshold of 20 were retained for calling CpG methylation.The quality of the reads were measured by FASTQC (version 0.10.1) program as implemented in the trime galore wrapper  include CpG islands, CpG shores (defined as 2000 bp flanking regions on each side of the CpG island), promoters , and gene body (introns and exons). For scoring DNA methylation in our analyses, each individual CpG was required to pass a minimum Phred base quality score of 20 and be covered by at least 8 reads. To score DNA methylation over a region, the region had to contain at least three CpGs each covered by at least 8 reads. In order to identify differentially methylated regions (DMRs) in gene promoters, each region was scored for being DMR using the Fisher’s exact test. The p-values were adjusted for multiple testing using the sliding linear model (SLIM) method that corrects pFor principal component analysis (PCA), methylation per CpG nucleotide was collected as a matrix format; each row represented samples, that is 16 subjects; eight for active men and eight for physically inactive men. Each column represented a CpG feature. Mean, standard deviation, p-value and q-value were computed for each CpG locus of the active group and inactive group. Methylated CpGs with q-value < 0.001 were selected to be used for PCA analysis. During feature preprocessing, missing data either in the active group or inactive group were separately imputed by a weighted mean of the k nearest-neighbor algorithm (k = 4). Given the matrix, mean and standard deviation were computed for each CpG locus of the group of physically active and inactive men. We further removed CpG nucleotides that were not covered in each group. CpG loci were next sorted by the mean differences between the groups. CpG loci that had over 0.15 methylation level difference and standard deviation less than 0.01 between the two groups were considered significantly altered. A total of 26 CpG loci were selected after feature preprocessing. The data are standardized to z-score and were plotted using “svd” algorithm as implemented in Matlab software.37 to examine enriched histone marks (Epigenomics Roadmap HM ChIP-seq) and transcription factor binding sites TFBS (ENCODE_TF_ChIP-seq_2015) in ENCODE human cell lines based on the program Enrichr. For histone marks enrichment analyses (Epigenomics Roadmap HM ChIP-seq), we used data only for muscle tissues. It is of note that we used all hypo-methylated and hyper-methylated DMRs as input for EnrichR program. Moreover, we performed TFBS enrichment analyses based on ENCODE human cell lines that may not necessarily reflect the behavior of TFBS in human muscle tissues.We used Enrichr38. Supernatant was collected following centrifugation for 20 min at 16,000 g (4 °C) and total protein content in the lysates was measured using the bicinchoninic acid assay (Thermo Fisher Scientific). Target proteins were analyzed by SDS‐PAGE and western blotting with equal total amount of protein loaded in each well of the SDS-gels. Primary antibodies were used to detect OXPHOS subunits (Abcam 110413), SOD2 (Millipore 06–984), Catalase (SC 50508), ANT1 (Abcam 102032), ANT2 (Abcam 118125), GSK3beta , PDH-E1alpha . Membrane imaging and band quantification were performed using ImageQuant LAS 4000  and ImageQuant TL v8.1 software . Citrate synthase activity was measured with acetyl-CoA and oxaloacetate as substrate39 and protein carbonylation was measured with the OxiSelectTM ELISA-kit (Cell Biolabs).Muscle lysate was prepared from ~10 to 15 mg dry weight of muscle as previously describedt test was applied to test the effect of lifelong physical activity (untrained versus trained subjects) on basal in vivo and molecular parameters in the muscle biopsies. Significance was accepted at P < 0.05. The statistical tests were performed using Sigmaplot 13.0 .Values are presented as means ± standard error. A Student paired allDataset 1Dataset 2Dataset 3Dataset 4Dataset 5


Tissue engineering approaches to regenerate myocardial tissue after disease or injury is promising. Integration with the host vasculature is critical to the survival and therapeutic efficacy of engineered myocardial tissues. To create more physiologically oriented engineered myocardial tissue with organized cellular arrangements and endothelial interactions, randomly oriented or parallel-aligned microfibrous polycaprolactone scaffolds were seeded with human pluripotent stem cell-derived cardiomyocytes (iCMs) and/or endothelial cells (iECs). The resultant engineered myocardial tissues were assessed in a subcutaneous transplantation model and in a myocardial injury model to evaluate the effect of scaffold anisotropy and endothelial interactions on vascular integration of the engineered myocardial tissue. Here we demonstrated that engineered myocardial tissue composed of randomly oriented scaffolds seeded with iECs promoted the survival of iECs for up to 14 days. However, engineered myocardial tissue composed of aligned scaffolds preferentially guided the organization of host capillaries along the direction of the microfibers. In a myocardial injury model, epicardially transplanted engineered myocardial tissues composed of randomly oriented scaffolds seeded with iCMs augmented microvessel formation leading to a significantly higher arteriole density after 4 weeks, compared to engineered tissues derived from aligned scaffolds. These findings that the scaffold microtopography imparts differential effect on revascularization, in which randomly oriented scaffolds promote pro-survival and pro-angiogenic effects, and aligned scaffolds direct the formation of anisotropic vessels. These findings suggest a dominant role of scaffold topography over endothelial co-culture in modulating cellular survival, vascularization, and microvessel architecture. Cardiovascular disease is the leading cause of death in the US, with over 16 million people suffering from coronary heart disease and 0.7 million new myocardial infarcts per year  scaffolds have been capable of providing provide spatial guidance cues that direct cellular rearrangement. Some of the approaches to fabricate parallel-aligned scaffolds include mechanical loading  and ECs (iECs) from embryonic stem cell or induced pluripotent stem cell at high efficiency for cardiovascular tissue engineering  and PEO  were simultaneously electrospun from separate syringes at 3.5 ml/h and 1.0 ml/h. Both polymers were co-electrospun using an electrospinner  at a voltage of 25.0 kV, mandrel speed of 200 rpm, and a distance from spinneret to mandrel of 100 mm. Aligned scaffolds was generated from the randomly oriented scaffold using heat-based mechanical rearrangement of fiber organization, where segments of the scaffold sheet (12 × 25 × 0.1 mm) were stretched uniaxially to 300% of initial length using a 1 kg weight for 1 h at 55°C. Circular discs were cut from electrospun scaffold sheets using either a 6 mm or a 12 mm biopsy punch (Medex Supply). The scaffold discs were submerged in double distilled water at 37°C overnight with agitation to dissolve and remove the PEO from PCL. The scaffold discs were disinfected in 70% ethanol with agitation for 18 h. The scaffolds were then coated with Geltrex basement membrane ECM proteins  with agitation for 18 h to facilitate cell attachment onto the scaffolds.Electrospun microfibrous polycaprolactone (PCL) scaffolds were fabricated by electrospinning as previously described  tool in ImageJ.Burkholderia cepacia, Sigma) and incubated at 37°C for up to 14 days. As a control for enzyme-mediated degradation, separate samples were included in phosphate buffered saline. At specified intervals , the scaffolds were taken out the lipase enzyme solution and placed in distilled water for 24 h. The lipase enzyme solution was then replaced with fresh enzyme solution at each interval for the duration of the experiments. At each interval, the scaffolds were then dried for 24 h and weighed at room temperature. The scaffold weight loss percentage was calculated below (n = 5), where W0 denotes the original weight and WT denotes weight at the designated time interval:Lipase, a water-soluble enzyme that hydrolyzes ester bonds of water-soluble substrates, has been shown to degrade PCL  was generated by reprogramming of healthy human peripheral blood mononuclear cells using Sendai virus-mediated transduction of Sox2, Oct3/4, KLF4, and c-myc , supplemented with forskolin , vascular endothelial growth factor , and polyvinyl alcohol . After 7 days, the cells were washed twice with PBS, and then cultured in endothelial growth media  supplemented with additional VEGF (100 ng/ml) for 7 more days. For indicated in vivo experiments, the human iECs derived from induced pluripotent stem cells were further lentivirally transduced between day 11–14 of differentiation using a double fusion reporter construct consisting of the ubiquitin promoter driving firefly luciferase (luc) and green fluorescence protein (GFP)  for iCMs and CD31 for iECs. The cell-seeded scaffolds were fixed in 4% paraformaldehyde (Electron Microscopy Sciences), permeabilized in 0.1% Triton X-100 (Sigma-Aldrich), and then blocked with 1% bovine serum albumin , and then placed in a prone position to expose the upper back. After disinfecting the skin of the mice using 70% ethanol and a betadine solution, incisions were made to the skin of the back and expanded into subcutaneous pockets using sterile cotton swabs. One of the following cardiac engineered myocardial tissues was inserted into each pocket: (1) randomly oriented scaffold seeded with iCMs; (2) randomly oriented scaffold seeded with iECs; (3) randomly oriented scaffold seeded with iCMs + iECs; (4) aligned scaffold seeded with iCMs; (5) aligned scaffold seeded with iECs; (6) aligned scaffolds seeded with iCMs + iECs; (7) acellular randomly oriented scaffold; or (8) acellular aligned scaffold (n ≥ 3). After engineered myocardial tissue transplantation, 5–0 sutures were used to close the pockets. Slow releasing buprenorphine (1 mg/kg) was given subcutaneously for analgesia.NOD SCID mice  were used in subcutaneous implantation studies. The mice were anesthetized with 2% isoflurane  and then expressed over time as the percent difference relative to day 0.For non-invasive imaging of cell survival En face tissue sections (5-μm-thick) were placed on silanized slides for immunohistochemistry or immunofluorescence staining. These studies were approved by the Institutional Animal Care and Use Committee at the Veterans Affairs Palo Alto Health Care System.At 2 weeks after transplantation, the animals were euthanized, and a skin incision was made to reveal the subcutaneous scaffolds. The scaffolds within the subcutaneous pockets were explanted and processed for routine paraffin-embedding. TM, VetOne), intubated with a 16G angiocatheter, and maintained on a ventilator . The heart was exposed through a left thoracotomy, and the left coronary artery was permanently ligated using a 6-0 polypropylene suture placed 3 mm below the left atrial appendage to produce a relatively small anterolateral infarct. To ensure consistently-sized infarcts, the suture was snared prior to ligation and the area of resulting pallor was assessed. Immediately after induction of myocardial injury by arterial ligation, the animals were randomized to receive one of the following treatment groups: (1) randomly oriented scaffold seeded with iCMs; (2) randomly oriented scaffold seeded with iCMs + iECs; (3) aligned scaffold seeded with iCMs; (4) aligned scaffold seeded with iCMs + iECs; (5) acellular randomly oriented scaffold (n ≥ 3). The engineered myocardial tissues were secured to the left ventricular epicardium by a single suture. The thorax was then suture closed, and the animals were allowed to recover for 1 month. Echocardiography was performed pre-operatively, as well as post-operatively on days 4 and 28 for assessment of ejection fraction . Buprenorphine was provided for analgesia (0.1 mg/kg). After 1 month of transplantation, the rats were euthanized and the myocardium was explanted for routine paraffin embedding. Transverse tissue sections (5 μm-thick) were placed on silanized slides for routine hematoxylin and eosin (H&E) staining, immunohistochemistry and immunofluorescence staining. These studies were approved by the Institutional Animal Care and Use Committee at the Veterans Affairs Palo Alto Health Care System.Immunocompromised RNU rats  were anesthetized with 2% isoflurane , according to the manufacturer's instructions . Capillaries were identified as a single layer of CD31+ cells with flattened morphology. For each sample, four images for each sample were acquired using 20X objectives (n ≥ 3). From each image, the total number of capillaries was manually counted and expressed in the form of capillary density (#/mm2)  was used to quantify the global angle of vessel orientations from range of 0–90°, where 0° denotes parallel alignment relative to the axis of the aligned microfibers, and 90° denotes orthogonal orientation with respect to the aligned microfiber axis. For randomly oriented scaffolds, an arbitrary axis was selected. From tissue cross sections of engineered myocardial tissues that were transplanted onto the rat epicardium, arterioles within the engineered tissues were identified having visible lumen with a diameter of 10–100 μm and by the co-expression of SMA and CD31. The average number of arterioles out of four representative images was quantified per sample, and then expressed in the form of arteriole density (#/mm2)  with Bonferroni adjustment was performed for comparisons of three or more treatment groups. A repeated measures ANOVA with Bonferroni adjustment was used for analysis of the same treatment group at multiple time points. A value of P < 0.05 was considered statistically significant.Data are shown as mean ± standard deviation. A Student's The electrospun scaffolds with parallel-aligned or randomly oriented microfiber organization were characterized by their spatial organization and degradation . Both alin vitro hydrolytic degradation studies were conducted in the presence of lipase enzyme , resulting in complete degradation of the scaffolds after 12 days . To verify the viability of iECs, the engineered myocardial tissues were explanted after 14 days for immunofluorescence and immunohistochemical staining of human specific CD31 expression , transverse (capillaries organized transversely with respect to the en face sections), or other . Among the engineered myocardial tissue derived from aligned scaffolds, the percentages of total capillaries with longitudinally orientation were within the range of 36–56%, suggesting a relative high percentage of longitudinally oriented vessels , compared to those composed of randomly scaffolds seeded with iCMs+iECs (75 ± 6/mm2), or those composed of aligned scaffolds seeded with iCMs alone (76 ± 47/mm2) or iCMs+iECs (63 ± 7/mm2) . The engineered myocardial tissues were instead repopulated by infiltrating cells from the host, based on H&E staining . Host miin vitro  anisotropic scaffolds can guide the organization of iCMs and iECs along the direction of the aligned microfibers in vitro ; (2) in in vitro , althougin vitro ; and (3)in vitro .in vivo. Our results suggest that scaffold anisotropy plays a larger role than endothelial co-culture in modulating cell survival and revascularization.Cardiac muscle is generally comprised of parallel-aligned CMs, interspersed with parallel-aligned microvessels (Kaneko et al., in vivo.To engineer myocardial tissues that mimic the ordered physiological cellular organization of the native myocardium, we employed anisotropic biomaterials to guide the assembly of the iECs and iCMs. Despite the ability of aligned microfibrous scaffolds to orient the iCMs along the direction of the aligned microfibers , the surin vitro studies suggest that iEC retention on scaffolds declined when in co-culture with iCMs, independent of scaffold anisotropy. As shown in + iECs was markedly lower on both aligned and randomly oriented scaffolds, suggesting that iCMs may impart mechanical perturbations to neighboring iECs that reduce their cell attachment over time. This is consistent with the observed decline of iEC survival in vivo when co-cultured with iCMs on randomly oriented scaffolds, compared to that of iECs alone (In contrast to the iCMs, the retention of iECs on the scaffolds appears to be affected by both scaffold topography as well as by intercellular interactions. Based on bioluminescence imaging, iECs alone seeded on randomly oriented scaffolds had significantly higher cell survival, compared to iECs on aligned scaffolds , suggestCs alone . The difin vitro owing to topographical patterning cues from aligned scaffolds is in agreement with previous reports (Kim et al., in vitro, creating anisotropic network-like structures with abundant expression of paxillin, a focal adhesion protein that mediates cellular attachment to the ECM, along the primary axis of the elongated iECs. It was also reported that spatial patterning of vascular networks in vivo by physical constraints not only led to organized vessel formation, but also alterations in the proportion of vessel subtypes, than when compared to in the absence of spatial patterning (Chang et al., in vivo microvasculature to mimic the anisotropic microvascular architecture of native myocardium (Kaneko et al., In order to support cell survival and integration, vascularization of the engineered myocardial tissue through the formation of microvasculature is critically important (Huang et al., In the myocardial injury model, randomly oriented myocardial tissues containing iCMs alone significantly promoted arteriogenesis in an epicardial transplantation model. It is likely that randomly oriented scaffolds support more degrees of freedom in the formation of microvessels, and thereby led to a higher density of microvessels. This reasoning is supported by prior publications showing that the formation of iEC vascular networks within randomly oriented scaffolds were characterized by a greater number of vascular-like branch points, compared to those within aligned scaffolds (Kim et al., in vitro studies in which scaffolds seeded with iCMs alone promoted a more mature iCM phenotype and higher magnitudes of contractility, compared to scaffolds seeded with iCMs+iECs (Wanjare et al., Our studies also examined the effects of co-culturing iECs with iCMs on the engineered myocardial tissues. According to our results, co-cultures were not superior to monocultures with regards to iECs survival and host revascularization. These results concur with In conclusion, we demonstrated that engineered myocardial tissues derived from randomly oriented scaffolds seeded with iCMs could promote revascularization in a myocardial injury model, but engineered myocardial tissues formed from parallel-aligned scaffolds preferentially directed the formation of capillaries along the direction and plane of the microfibers. Randomly oriented scaffolds also conferred pro-survival effects to iECs, based on significantly higher cell survival upon subcutaneous transplantation. Based on these findings, we conclude that the scaffold microtopography imparts differential effects, in which randomly oriented scaffolds promote pro-survival and pro-angiogenic effects, and aligned scaffolds directed the formation of anisotropic vessels. Our findings suggest a dominant role of scaffold topography over endothelial co-culture in modulating cellular survival, vascularization, and microvessel architecture. Future studies warrant further elucidation of the signaling pathways underlying topography-mediated effects on revascularization, host cell infiltration, and cell survival.The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.MW, CH, CA, and NH designed and carried out experiments and analyzed data. MW and NH interpreted the results. MK, HW, and YW provided technical support for animal surgeries. MW and NH wrote and organized the manuscript, with editorial input from HW.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.


Early-life adversity is an important risk factor for major depressive disorder (MDD) and schizophrenia (SCZ) that interacts with genetic factors to confer disease risk through mechanisms that are still insufficiently understood. One downstream effect of early-life adversity is the activation of glucocorticoid receptor (GR)-dependent gene networks that drive acute and long-term adaptive behavioral and cellular responses to stress. We have previously shown that genetic variants that moderate GR-induced gene transcription (GR-response eSNPs) are significantly enriched among risk variants from genome-wide association studies (GWASs) for MDD and SCZ. Here, we show that the 63 transcripts regulated by these disease-associated functional genetic variants form a tight glucocorticoid-responsive co-expression network (termed GCN). We hypothesized that changes in the correlation structure of this GCN may contribute to early-life adversity-associated disease risk. Therefore, we analyzed the effects of different qualities of social support and stress throughout life on GCN formation across distinct brain regions using a translational mouse model. We observed that different qualities of social experience substantially affect GCN structure in a highly brain region-specific manner. GCN changes were predominantly found in two functionally interconnected regions, the ventral hippocampus and the hypothalamus, two brain regions previously shown to be of relevance for the stress response, as well as psychiatric disorders. Overall, our results support the hypothesis that a subset of genetic variants may contribute to risk for MDD and SCZ by altering circuit-level effects of early and adult social experiences on GCN formation and structure. Above all, early-life experiences, such as maternal stress during pregnancy and child abuse but also supportive parenting, can cause long-lasting changes in neural circuit function and stress hormone regulation that may moderate risk for major depressive disorder (MDD) and schizophrenia (SCZ)4. Importantly though, the impact of early-life adversity on disease risk is moderated by genetic variation5 and later life experiences through biological mechanisms, which are insufficiently understood at present.Social experiences shape brain structure and function by inducing plastic changes from the early prenatal period until the end of life6. GRs reside in the cytoplasm and translocate into the nucleus upon cortisol binding to regulate gene expression through sequence-specific DNA binding or protein interactions with other DNA-bound transcriptional regulators7. GR-regulated gene networks coordinate acute and long-term adaptive responses to stress, as well as timely termination of the stress response once the threat has been mastered. Failure to turn-on and -off GR responses efficiently has been proposed to result in “wear and tear” of the body and the brain and facilitates the development of associated pathologies8.Adverse early experiences typically activate the stress hormone system leading to increased glucocorticoid secretion and the activation of glucocorticoid receptors (GRs) by cortisol9. We previously hypothesized that genetic variation in GR-regulated gene networks could contribute to the risk for psychiatric diseases10. Therefore, we measured glucocorticoid-induced changes in gene expression in genetically diverse individuals to infer GR expression quantitative trait loci (eQTLs). Genetic variants significantly associated with differential GR-induced expression preferentially mapped to long-range enhancer elements consistent with GR’s major mode of transcriptional regulation11. Furthermore, these GR-response eQTLs were significantly enriched in brain-specific enhancers and among genetic variants identified in genome-wide association studies (GWASs) for MDD and SCZ10. In additional independent samples, these variants also predicted major depression and amygdala (AMY) reactivity to threat in a cumulative manner.Studies on the genetics of gene expression provide a unique opportunity to link DNA sequence variation to phenotypes and diseaseFocusing on MDD risk genes in our initial analyses, we showed that the transcripts moderated by these variants are expressed in the brain, are regulated by stress, and form a tight co-expression network that comprised basic biological processes such as ubiquitination, proteasome degradation, and inflammation. We hypothesized that the cumulative genetic scores for these functional disease variants could change the response of the whole co-expression network to different qualities of social experience and by this influence disease risk. A first step towards testing this hypothesis would be to assess whether indeed exposure to different qualities of social experience can alter the co-expression network structure of these transcripts. This can be tested in animal models exposed to different qualities of social support and stress throughout development and beyond for which the expression levels of the transcripts within these gene networks is measured in different brain regions.12.The main intent of our experiment was to explore the dynamics of this glucocorticoid-responsive co-expression network (GCN) (capturing the information from GWAS risk variants) in response to extreme differences in social experiences during early and or adult life, modeling epidemiological risk and protective factors for MDD/SCZ. We have chosen an animal model that contrasts different qualities of social support and stress during different developmental stages to explicitly capture the complexity of social life trajectories10. A co-expression network of those genes was predicted by GeneMANIA13 without the addition of related genes and attributes. To estimate the null distribution, we calculated the gene network for 10 same size sets of randomly chosen GR-response transcripts . Hub genes were detected by calculating the node degree distribution using the Network Analyzer tool within Cytoscape 3.5.114. The functional annotation was performed using Enrichr (http://amp.pharm.mssm.edu/Enrichr/enrich)16 focusing on KEGG 201617 and Wikipathways 201618 for pathway enrichment and the gene ontology (GO) terms molecular function, biological processes, and cellular component, all in the version 2017b. The significance tests for these analyses are described in detail in16. Briefly, Enrichr implements three approaches to compute an enrichment: (1) Fisher’s exact test, (2) permutation-based Fisher's exact test (generates multiple random gene lists and computes a z-score for deviation from the expected rank), and (3) a combined test  × (z-score of 2)). We report the permutation-based p-value .For this study, 63 genes  was explicitly omitted to test only two opposing rearing environments. At adulthood (12 weeks of age), animals of each group were then either housed with an ovariectomized female  or underwent chronic social defeat stress  for three consecutive weeks giving rise to four experimental groups exposed to different qualities of social experience   encoding the GR as described in19. From each treatment group, eight mouse brains were analyzed. The animals were sacrificed under basal conditions at 2 h after lights on during the circadian nadir on P100, 12 days after the end of the adult manipulations. Micropunches from desired brain regions were collected under histological control by Cresyl Violet staining according to the Mouse Brain Atlas21 and immediately stored at −80 °C. Twelve different brain regions including AMY, bed nucleus of the stria terminalis, cerebellum (CER), dorsal hippocampal Cornu Ammonis (CA) 1 region (dCA1), dorsal hippocampal CA3 region (dCA3), dorsal dentate gyrus (dDG), prefrontal cortex, nucleus accumbens (Nac), paraventricular nucleus (PVN), ventral hippocampal CA1 region (vCA1), ventral hippocampal CA3 region (vCA3), and ventral dentate gyrus were collected  or a caring environment . LM was performed by placing the animals from postnatal day (P) 2 to P9 in a cage with a metal grid instead of bedding material and reduced nesting material, as described previouslyAll procedures on animals were approved by the Government of Upper Bavaria and were in conformity with European Union Directive 2010/63/EU. Standard laboratory animal housing conditions were maintained throughout the experiments (unless otherwise stated) and 12-h daily illumination (lights on at 06:00 a.m.).Frozen micropunches were lysed quickly in 500 µl Trizol (QIAzol) reagent and homogenized by repeated passing through an insulin syringe (29G). Subsequently, samples were kept at room temperature for at least 3 min before adding 100 µl chloroform. The remaining steps were conducted according to the miRNeasy Mini Kit  protocol. Elution was performed with 30 µl RNAse-free water with a second round using the eluate from the first round.TruSeq® Targeted RNA sequencing  was carried out according to the manufacturer’s protocol.n = 55; see Supp. Table As a first step, mouse orthologous of GR-response MDD- and/or SCZ-related eQTL genes were mapped  from 12 brain regions, which summed up to a total number of 384 samples. During RNA extraction, 24 samples were extracted at once, whereas the library preparation was performed in four 96-well plates. Each 96-well plate contained three brain regions, which were distributed randomly into the columns of the plate. Each plate column contained two samples each from the four experimental groups randomly distributed over the rows. Three columns of a 96-well plate were assigned to one RNA extraction from each of the three brain regions on that plate.22. Low-quality reads with phred scores lower than 20 or a length smaller than 25 bp were removed using PRINSEQ (PReprocessing and INformation of SEQuence data) lite Version 0.20.423. Subsequently, the reads were aligned with Burrows-Wheeler Aligner (BWA) Version 0.7.1224 with default settings  and the manifest file provided by Illumina as reference.After sequencing with the MiSeq Sequencer (V3 chemistry for 150 cycles), sequencing quality was analyzed with FastQCn = 5). The data were then normalized using DESeq225. Finally, surrogate variable analysis (SVA) from the R/Bioconductor package sva26 was applied to identify hidden technical batches. To identify outliers, a linear model (LM) was calculated with the normalized expression counts and the five significant SVs . Subsequently, the residuals were clustered and samples more than four standard deviations away from the mean in either of the first two principal components were excluded (n = 2 samples). Gene expression values of 50 genes in 382 samples passed the preprocessing.Next, the gene expression quantification was inferred from the alignment. Genes with low overall coverage, i.e. <2000 reads across all samples, were excluded from further analysis q-values in the results and supplemental table Significance is reported after controlling for multiple testing with a false discovery rate (FDR) lower than 10% for all detectable transcripts within each brain region and across the four conditions and FDR-corrected significance is reported as 28 and deposited as GSE25219. Of the 63 genes within the tested GCN, 40 had data within GSE25219. Dimension reduction using principal component analysis (PCA) was performed on the expression levels of these 40 genes and the first principal component (PC1) was extracted. PC1 was then associated with developmental stages using a linear regression model.For analysis of GCN gene expression in human hippocampus, we used the data described in Kang et al.Using the residuals of the normalized and batch corrected gene expression values, the pairwise correlation coefficients between all gene pairs were calculated and adjusted for the expression levels of all other genes using Gaussian Graphical Model implemented in the R package GeneNet.n = 12) and condition (n = 4) and controlled for the expression levels of all other transcripts, resulting in 48 networks. Partial correlation coefficients are not directly comparable to Pearson's correlation coefficients as they control for confounding effects of all other transcripts. The pairwise partial correlation between transcripts A and B will be calculated correlating A – (Z1 to Zn) and B – (Z1 to Zn), so that partial correlation coefficient will be numerically lower than Pearson's correlation coefficients. Statistical significance of the partial correlations was assessed using the empirical Bayes local FDR statistic29 and partial correlations at an FDR < 0.2 were considered as significant according to Schäfer et al.30. Networks were visualized with the R Bioconductor package Rgraphviz, where each gene corresponds to a node and edges represent the dependencies, i.e., partial correlation coefficient, between them. The network properties, i.e., number of edges, number of nodes, absolute sum of partial correlation coefficients, and link density were analyzed using iGraph and visualized in pheatmaps. Edges were defined as pairwise partial correlations with an FDR < 0.2 and node degree as the number of such significant edges connected to a node. A node was defined as a transcript with at least one edge.Partial correlation calculation was performed separately for each brain region . Given the additional significant enrichment of GR-eQTLs among schizophrenia risk loci and the significant SNP-based co-heritability31 between these two disorders, we now investigated if the 63 GR-response eQTL transcripts (see Supplementary Table n = 20) or SCZ (n = 39) or both (n = 4), form a common transcription network structure. GeneMANIA was used to generate such a GR-eQTL/GWAS gene network . Of these, 58 transcripts formed a tight network with 423 edges. Only three transcripts were isolated nodes  was the top connected gene with a node degree of 30. This gene was followed by CCNDBP1 (cyclin D1 binding protein 1) (28 nodes) and HMBS (hydroxymethylbilane synthase) (27 nodes)  = 17–25), in the above-described human GCN network, whereas C7orf25 (orthologue of AW209491) had a very low degree of 4 and HIST2H2AA3, d = 0, falls on an edge , CCNDPD1 (d = 28), as well as ADHD5 (d = 18) and HIGD1A (d = 19).The univariate analyses are summarized for each brain region in Supplemental Table C7orf25 (orthologue of AW209491) in the Nac would remain significant after correction for multiple testing. Collectively, these data thus indicate that robust differential expression of single GCN risk genes with large effects is a rare event in response to different social stress experiences.Taking into account additional tests related to the number of brain regions, only the effect of CD on n = 30 vs. mean = 5.9), well-structured (number of edges n = 38 vs. mean = 5.3), and dense (link density n = 2.53 vs. mean = 0.8) GCN in the absence of any stress history (EHOX). The co-expression network in this brain region was completely absent in mice exposed to both, early and late social stress  and to a lesser degree in mice exposed to early adversity only . On the other hand, chronic adult stress without a history of early-life adversity  did not alter paraventricular network formation when compared with stress-free mice (EHOX). This suggests stronger effects of early adversity on the GCN in PVN than adult chronic stress, as well as a cumulative effect of stress exposure on the strength of this network.Given the absence of strong single gene effects, we next performed a comprehensive co-expression analysis of the 50 orthologue GCN genes across the 12 different brain regions and the four conditions. All pairwise correlations were adjusted for the expression levels of all other genes in the GCN, by calculating partial correlation. Figure 32 did not express the GCN  in the absence of any stress history (EHOX), but GCN network strength emerged with exposure to adult stress  and was further strengthened by combined exposure to early and adults stress . However, exposure to early-life adversity alone (LMOX) was not associated with network formation.The brain region with the second largest dynamic network changes across the four conditions was the vCA1. In contrast to the PVN, the vCA1 region, sharing a major role in stress regulationIt is important to note that changes in GCN formation were not confined to the PVN and hippocampal vCA1 region, but observed also in other brain regions although to a much lower degree see Fig. . These fBrain region-specific social environment-responsivity raises the question whether the network properties of the underlying risk genes are preserved across brain regions and conditions. Therefore, we next investigated the number of edges per GCN gene in the PVN and the vCA1 in those conditions exhibiting GCN formation , and also included data from the dCA1 , the third most connected region  for comn = 16) transcripts with the EHOX network. Interestingly, six of the eight genes with more than four connections in the EHOX network were also included in the EHCD network, namely Slc7a7, Rbm4, Zap70, St6galnac4, Zfp641, and Ociad2 and the majority of them (n = 4), again had four or more connections. This suggests that while these two PVN networks are not identical, they share a number of key features, supporting that adult stress also does not have a major impact on the network topology. However, one has to note that the centrality and connectivity pattern of some transcripts were affected by the additional stress condition. For example, Psd5a with two edges in the EHOX condition, moved to a more central position in the EHCD condition, with nine edges. As described above, this paraventricular co-expression network is disrupted with exposure to early adversity, especially when followed by adult chronic stress.We then explored the exact topology of the stress-dependent GCNs in the PVN and the vCA1 see Fig. . We firsOciad2 and Zfp641). This suggests that while a substantial number of transcripts are shared between the low early stress PVN network and the combined stress vCA1 network, the topology of this network differed more than between the two PVN networks. Interestingly, Psd5—the most connected gene in the PVN adult stress network (eight edges) was also part of the vCA1 combined stress network and showed four connections, suggesting that connectivity of this gene may more specifically relate to chronic adult stress.Contrary to the PVN, the vCA1 GCN was mainly apparent in the combined stress condition (LMCD). Here 30 of the 50 transcripts formed a correlated expression network, with 16 genes shared with low stress PVN network (EHOX) and an additional six, which were only present in the adult only stress (EHLM) PVN network. Of the eight more highly connected genes in the PVN EHOX condition, four were also included in the vCA1 LMCD network, however, only two of them still showed a high connectivity . This raises the question whether this GCN could respond to differences in social experiences, especially early-life stress, an important risk factor for MDD and SCZ. When analyzing the effects of different qualities of social stress in contrast to different qualities of social support on GCN formation across distinct brain regions during distinct developmental phases in mice, we observed that these different social experiences substantially affect GCN formation and structure. These changes were highly brain region-specific and apparent mainly in two functionally interconnected regions, the ventral hippocampus and the hypothalamus (see below). The fact that the transcripts within this network are highly regulated across human hippocampal development suggests a role in neuronal development and their developmental trajectory may serve as a substrate for early-life experiences to influence differential GCN formation.Drosophila alter endocytic trafficking: mutations disrupting the formation of signaling endosomes cause reduced synaptic growth, while mutations altering traffic to the recycling endosome or lysosome cause synaptic overgrowth due to enhanced signaling33. Endosomal regulation of signaling pathways in synaptic growth and development appears critical for activity-dependent circuit refinement and are proposed to contribute to risk for MDD and SCZ35. In support of this hypothesis, postmortem brain studies showed these pathways to be altered in both SCZ and MDD36. This suggests that alterations in stress-induced changes of endocytosis could contribute to disease risk by altering neuronal activity. For example, glutamate receptor turnover has been shown to be altered by glucocorticoid-induced enhancement of ubiquitin/proteasome-mediated degradation of receptor subunits37, a process localized to late endosomes and multivesicular membranes39. Pathway analyses revealed enrichment for cytoplasmic ribosomal proteins; ribosomal protein have been shown to be altered both in postmortem gene expression studies for MDD and SCZ36, as well as in pluripotent stem cell-derived neural progenitor cells40 or olfactory-derived neuroepithelial cells41 from patients with SCZ.When annotating the 58 transcripts connected within this GCN, we found enrichment of the GO cellular components “late endosome membrane” and “multivesicular membrane”, both contributing to endocytosis. Regulation of endosomal traffic is a critical component of synaptic growth and development. Many of the synaptic growth mutants identified in 43 but also from recent cross-disorder annotations of GWASs including both disorders44.The second enriched pathway was inflammation, a system not only strongly implicated in both disorders by a large body of experimental evidenceSnrnp70, Rps2, Rps12, Mrpl54, Rbm4 for ribosomal processes; Zap70 and Rfx5 for immune-related functions, and Anx2 for endocytosis  and MDD-associated variants (OCIAD2 and ATP5F1)  also used here. The same group had previously reported differential changes of co-expression networks and changes in network strength, suggesting that changes in co-expression strength has the potential to impact cellular function in animals susceptible or resilient to this stress paradigm in a coordinated manner across brain regions, including the ventral hippocampus50. In fact, among the 30 genes constituting the network emerging in the ventral CA1 in the combined early and late stress group in our experiments, five genes  also showed significant changes in gene expression in the ventral hippocampus between resilient and susceptible animals and control animals.In fact, recent data from postmortem brain gene expression studies support a role for changes in co-expression network strength in both MDD and SCZ51.Overall, our data support the hypothesis that genetic risk variants for MDD and SCZ could influence coordinated gene network properties across brain regions in response to different qualities of social experiences. In human studies, differences in social experiences ranging from early adversity to supportive social networks have been associated with differences in risk or resilience to MDD and SCZ. Targeting multifaceted dysregulation of risk gene co-expression networks within interconnected brain regions in MDD and SCZ may offer an interesting entry point for therapy and may lead to more effective treatments than simply modulating single differentially expressed risk genesLegend for supplementary filesSupplemental Figure 1Supplemental Figure 2Supplemental Table 1Supplemental Table 2Supplemental Table 3Supplemental Tables 4 and 5


Uncaria tomentosa is a woody vine with a long history of use in traditional Peruvian medicine and nowadays supplements containing this vine as ingredient are available. Immunomodulating, anti-inflammatory and anticancer properties of Uncaria tomentosa have been suggested and attributed mainly to the presence of tetracyclic or pentacyclic oxindole alkaloids. However, the synergic action of different compounds occurring in extracts and modulation of redox processes may significantly influence the anticancer activity of Uncaria tomentosa. The aim of the present study was to investigate for the first time the cytotoxic effects of the tetracyclic alkaloids free aqueous extract (decoction) of dried Uncaria tomentosa leaf blades in normal and cancer cells, and to assess the effect of the tested extract on cisplatin (CDDP) cytotoxicity. Tested Uncaria tomentosa extract was not cytotoxic for NHDF cells, but demonstrated cytotoxic effect against HepG2 cells. The extract increased ROS production in HepG2 cells, which resulted in decreased GSH level, leading to apoptosis of these cells through activation of caspase-3 and caspase-7. A reduction of NF-κB active form was observed in cancer cells. In normal cells the extract did not affect ROS production, GSH level and NF-κB activity, and maintained cell viability. HepG2 cells incubation with Uncaria tomentosa decoction and simultaneously with CDDP resulted in an increase in CDPP cytotoxic activity against HepG2, while under the same conditions Uncaria tomentosa prevents NHDF cell viability reduction due to CDDP. The results indicate that Uncaria tomentosa leaves decoction modulates differently cancer and normal cells oxidative metabolism and, enhanced cytotoxicity of CDDP against cancer cells and at the same time increased normal healthy cells resistance to cisplatin. Further studies are needed to confirm our observations and to describe underlying molecular mechanism, and the potential usefulness of Uncaria tomentosa decoction in adjuvant therapy for cancer. Uncaria tomentosa, a Peruvian plant of the Rubiaceae family, contain biologically active substances, including immunostimulating pentacyclic oxindole alkaloids [Previous studies have revealed that different organic and aqueous extracts of lkaloids , ursane lkaloids  and inhilkaloids . These elkaloids , as welllkaloids . NowadayUncaria tomentosa bark extracts on various cell lines, including human leukemia cells K562 and HL60, EBV-transformed B lymphoma cells [Uncaria tomentosa in HL60 cells caused changes in mitochondrial membrane potential, release of cytochrome c to the cytosol and caspase-3 activation [In vitro studies have demonstrated antiproliferative and proapoptotic effects of ma cells . Ethyl ativation .Uncaria tomentosa [Uncaria tomentosa may be related to a synergic action of different compounds [Although, in in vitro studies, organic extracts containing large amounts of oxindole alkaloids ,10 and pomentosa ,12 inhibompounds  and moduompounds .Uncaria tomentosa leaf blades, and its effect on ROS production in human hepatoma, HepG2 cells and normal human dermal fibroblast, NHDF. We assessed also the influence of this extract on cisplatin cytotoxicity against cancer cells and in normal healthy cells to give an insight in potential usefulness of Uncaria tomentosa in adjuvant treatment for cancer. Previous reports focused on the effects of bark and root extracts of Uncaria tomentosa on cancer cells, while leaves contain similar active compounds and appropriate material can be collected without significant plant damage [Uncaria tomentosa by heating dried plant leaves in water.The aim of the present study was to investigate for the first time the cytotoxic activity of tetracyclic alkaloid free and tannins rich aqueous extract (decoction) of dried t damage . The extPlant extracts are extensively tested for anticancer activity, which may be a result of their antioxidant properties and apoptosis induction capacities, and in a future such extracts could be useful as an adjuvant treatment for cancer ,16Uncaria tomentosa leaves contains pentacyclic oxindole alkaloids (13% of dry extract mass) and is free of teracyclic alkaloids (The decoction (water extract) from lkaloids . Pentacylkaloids , includiUncaria tomentosa leaves was recognized to be not toxic for NHDF cells, while significantly decreased cell viability was observed only when high extract concentrations >1 g/mL were used. In the present study treatment of HepG2 cells with this extract caused a significant concentration dependent decrease in cell viability (50 (50% viability inhibition concentration) of 580 µg/mL was determined.The extract from iability , and basp < 0.05). In fluorescence images in NHDF cells incubated under similar conditions as HepG2 cells no significant changes in cell morphology and in quantity of red stained cells were noted  in the number of late apoptotic (Annexin positive and PI positive) or also necrotic cells (Incubation of HepG2 cells with the ic cells .Uncaria tomentosa decoction at the concentrations of 72.5 µg/mL and 145 µg/mL on the number of Annexin-positive as well as Annexin-positive and PI positive cells was found (data not shown).Under similar conditions in NHDF cells no effect of The changes in HepG2 cells viability might be associated with functional changes in the mitochondrial electron transport chain and development of oxidative stress, therefore, the production of ROS in HepG2 and NHDF cells was determined.Uncaria tomentosa concentrations. This indicates an increase in cellular ROS production caused by the Uncaria tomentosa extract. The highest rate of ROS release was observed during the initial 0.5 h of incubation, while after 1 h, ROS levels became stabilized. Uncaria tomentosa extract, increased ROS formation, and a statistically significant increase (* p < 0.05) was seen. Under similar experimental conditions in NHDF (Uncaria tomentosa extract on ROS production. All tested doses resulted in significantly (* p < 0.05) lower levels of fluorescence intensity as compared to controls indicating lower ROS production by Uncaria tomentosa treated NHDF cells than by control (untreated) NHDF cells.In HepG2 cells the control plot (for cells without the extract) as shown in b lies be in NHDF a cells wUncaria tomentosa extract may induce cytotoxic effect in cancer cells by stimulation of ROS overproduction. Glutathione (GSH) plays a significant role in maintaining oxidative-reductive balance in cells, and its level is an indicator of response to oxidative stress. We have shown that in HepG2 cells the Uncaria tomentosa extract caused a reduction of GSH levels and significant (** p < 0.001) GSH decrease was observed when Uncaria tomentosa concentration of 145 µg/mL was used  of NF-κB activity (p < 0.05) in NF-κB activity was noted.Oxidation-reduction potential of a cell has a direct impact on NF-κB regulation, which is involved in various processes, including cell proliferation and death . Inconsireported ,24. The activity , while i® a standardized Uncaria tomentosa bark formulation on NF-κB and apoptosis induction in neoplastic cells [Uncaria tomentosa extracts on human leukemia cells (THP-1) indicated inhibition of the classical NF-κB activation pathway [The importance of the lack of enhanced NF-κB modulation for effectiveness of cancer therapy was indicated before  and receic cells . Anticanic cells . Inhibit pathway . Both ox pathway  and proc pathway  have beeUncaria tomentosa extract decreased cancer cells viability (measured by MTT assay). Therefore, it can be hypothesized that it may enhance cytotoxic effect of anticancer drugs such as CDDP commonly used in clinical practice in cancer treatment. To test this hypothesis HepG2 and NHDF cells were 72 h incubated with tested extract and the last 48 h simultaneously with CDDP (at concentration corresponding to IC50 of 11.25 µM in HepG2 cells as recognized in our laboratory). Results of MTT assay (Uncaria tomentosa extract increased CDPP cytotoxic activity against HepG2 cells as indicated by 10%–15% decrease in cell viability.The water TT assay  show thap < 0.05) increase in cell viability by 30% as compared to NHDF cells treated only with CDDP was observed. This data suggests that tested Uncaria tomentosa decoction may enhance cytotoxic activity of CDDP against cancer cells and at the same time may protect normal cells against the harmful effects of cisplatin. Recently, it was reported that the use of Uncaria tomentosa capsules (300 mg/day) in patients with colorectal cancer. Uncaria tomentosa showed positive effects in reduced neutropenia and thrombocytopenia as well as in the repair of immune response [However, when normal NHDF cells were treated with this extract (at concentrations of 72.5 and 145 µg/mL) and CDDP a significant , through Phytotherapy Center of Wilcaccora . Comminuted dried leaf blades of Uncaria tomentosa (4 g) were extracted with warm water  for 20 min. The decoction was subsequently centrifuged at 4000 rpm for 20 min. For cell culture experiments samples were separated by filtration. To analyze alkaloids and phenolic content the aqueous extract was lyophilized and as a result of extraction an average 1.16 g of dry powder was obtained.Dried leaf blades of Uncaria tomentosa leaves was carried out according to Pilarski et al. [Determination of alkaloids in decoction of i et al. . To 100 The total phenolic content (TPC) was conducted on 96-well transparent microplates  according to the method previously described , using tv/v) AlCl3 methanol solution and filled with methanol to the volume of 200 µL. Absorbance was read at 430 nm after 30 min incubation with the solution containing methanol instead of the sample as a blank. Measurements were made using the Infinite Pro 200F microplate reader (Tecan Group Ltd.). Results are expressed as mg quercetin (Q) per 1 g of dry extract.Determination of total flavonoids was performed according to the method described by Lamaison and Carret  with somTo determine the content of tannins in our water extract (decoction), two methods were used: the vanillin assay and the protein precipitation method (precipitation of tannins with hide-powder).Analysis of tannin content was determined according to the hide-powder method described in the Polish Pharmacopoeia 6th edition . TanninsThe amount of condensed tannins was determined using vanillin assay . Absorba4 cells per well) in FGM-2 with supplements and 2% FBS.Human hepatoma (HepG2) were bought from American Type Culture Collection  and cultured according to previously described procedures ,34,35. EThe cell viability was assessed by determination of MTT salt -2,5-diphenyltetrazolium bromide, Sigma-Aldrich Chemie, Darmstadt, Germany) conversion by mitochondrial dehydrogenase according to earlier described procedure . BrieflyMicroscopic examination of changes in cell morphology was assessed in visible light as described previously ,35,36. TUncaria tomentosa extract for 72 h in 12-well plates. After the exposure, the culture medium was removed and the cells were harvested by trypsinization with 0.05% Trypsin-EDTA, and centrifuged at 2000 rpm for 10 min. The analysis by flow cytometery was performed within 1 h by counting 5000 gated events per each sample. The data were further analyzed using CELL-Quest software on FACS Calibur flow cytometer and the Cell Quest software . The results were presented as rates of early apoptotic (Annexin V-positive) and late apoptotic (Annexin V and PI-positive) cells.Apoptosis was assessed by the AnnexinV-FITC Kit . The cells were incubated with 2-DA conversion and oxidation to DCF as was described previously [2-DA, easily penetrates cells, undergoing conversion by esterase to DCFH2, which in the presence of ROS becomes oxidized to highly fluorescent DCF. Cells cultured in 12-well plates in serum-free DMEM were rinsed with PBS and incubated for 1 h in DMEM with investigated concentrations of the extract, and subsequently for 2 h with 250 µM of DCFH2-DA  in HBSS . Fluorescence measurement was started immediately after DCFH2-DA was added, using a microplate reader  equipped with Gen5 software  at an excitation wavelength of 485 nm and emission wavelength of 530 nm, with readings every 15 min.ROS production was measured by the assessment of DCFHeviously . DCFH2-DCells were incubated on 24-well plates in serum free DMEM with the tested extract at various concentrations) for 4 h. The GSH level was assessed using the protocol for the glutathione assay kit . Absorbance measurement was performed at 412 nm using a microplate reader . The results were expressed in relation to the quantity of protein in the sample using the manufacturer’s protocol Pierce BCA Protein Assay Kit .The determination of caspase activity was performed with the use of the luciferase enzyme according to the protocol for the Caspase-Glo 3/7 Assay Kit provided by the manufacturer . Luminescence measurement was performed in cell lysates prepared using cells incubating in 96-well plates for 4 h with the tested extract. Luminescence intensity is proportional to caspase activity.Chemiluminescent measurement of NF-κB active form was performed according to the protocol for NF-κB p65 ELISA Kit  in cell lysates prepared using cells incubating for 4 h with the tested extract. The results are presented in relation to the protein levels in the samples, measured by the Bradford’s method.p < 0.05 was accepted.The results are presented as experimental means and SD. Statistical significance of observed differences was assessed by one-way analysis of variance (ANOVA) with the Tukey’s post-hoc test  and as significant Plant extracts and plant derived substances, and their usefulness in patients under anticancer therapies are now attracting great attention. They have shown promising effects when combined with chemotherapy and may benefit patients with hepatocellular carcinoma .Uncaria tomentosa, also known as “cat’s claw” and “uña de gato”, is a Peruvian plant of the Rubiaceae family, whose bark, roots and leaves are commonly used by the local population in South America due to their immunomodulating, anticancer and anti-inflammatory action. Previous reports of the effects of Uncaria tomentosa on cancer cells focus on studies of bark and root extracts , while its leaves contain similar active compounds and appropriate material could be collected without significant damages of the plant. However, differences in the content of polyphenols between bark and leaves as well as between aqueous and alcoholic extracts have been reported [reported . The extUncaria tomentosa decoction enhances CDDP cytotoxicity against HepG2 cells, while incubation of NHDF cells with this extract prevents their viability reduction due to CDDP. These results indicate that Uncaria tomentosa leaves decoction modulates differently cancer and normal cells oxidative metabolism and enhances CDDP cytotoxicity against cancer cells. Every compound or combination of different compounds which is able to be more effective against cancer cells than against non-hyperplastic cells, may provide new therapeutic opportunities. Further studies are needed to describe molecular mechanisms underlying the observed effects and demonstrate the potential usefulness of the studied extract in increasing the sensitivity of cancer cells for treatment and/or in the prevention of drug resistance development. In general, the active ingredients of phytopharmaceuticals as used in ethnomedicine are mostly unknown and the plant extract is considered as active ingredient. The plant extract is a mixture of active compounds and their joint action can mediate pharmaceutical activity. Nevertheless, the isolation of active compounds should be the next step in order to elucidate the phytochemical basis of the effects observed in the current study.This extract has been found to be cytotoxic against cancer cells and to induce oxidative stress in cancer cells but not in normal cells. In cancer cells the extract activates effector caspase-3 and caspase-7, which execute apoptosis through cleavage of protein substrates that include mediators and regulators of apoptosis, structural proteins, as well as DNA repair and cell-cycle related proteins. In addition in cancer but not in normal cells a reduction in NF-κB active form has been observed. Inhibition of NF-κB activity may prevent a development of cancer cell resistance to chemotherapy. Studied


Interface fluid syndrome (IFS) is an unusual complication after laser-assisted in-situ keratomileusis (LASIK). We report the first case of IFS after uncomplicated phacotrabeculectomy in a patient who had undergone LASIK 10 years previously. This case emphasizes the importance of intraocular pressure (IOP) interpretation in eyes that have undergone LASIK.A 30-year-old woman with a history of LASIK surgery presented to glaucoma clinic due to uncontrolled IOP despite of maximally tolerable medical treatment. After receiving phacotrabeculectomy, IOP decreased to 3 mmHg on the first postoperative day, but again increased up to 21 mmHg and a diffuse corneal edema with cloudy flap interface was demonstrated by slit-lamp microscopy. Corneal edema was sustained even after the IOP was lowered to 14 mmHg. Spectral-domain optical coherence tomography scanning of the cornea revealed a diffuse, thin fluid pocket in the corneal interface. After laser lysis of the scleral flap sutures, IOP was further decreased to 9 mmHg and interface fluid was resolved.IFS should be considered as a possible cause of postoperative corneal edema despite of low IOP in the eyes that underwent LASIK surgery. Additional IOP lowering may be helpful for resolving the corneal edema. Interface fluid syndrome (IFS) is an unusual complication after laser-assisted in-situ keratomileusis (LASIK) that is characterized by diffuse fluid accumulation within the flap interface. Although elevation of intraocular pressure (IOP) is the main sign associated with the IFS , falselyA 30-year-old woman was presented to a glaucoma clinic due to uncontrolled IOP. She had undergone bilateral LASIK 10 years previously, and had been treated with oral and topical steroids as well as albendazole for 10 months for uveitis associated with ocular toxocariasis in the left eye. Sub-Tenon injection of triamcinolone acetonide (40 mg) had also been performed 5 months previously.At the first visit, her visual acuity was 20/200 and the IOP was 30 mmHg in the left eye measured by Goldmann applanation tonometry (GAT). Slit-lamp examination revealed Grade 1 posterior subcapsular opacity, and fundus examination showed glaucomatous change in the optic nerve head. Inflammatory cells were not detected in either the anterior or posterior chamber. Despite maximally tolerable medical treatment, the IOP subsequently increased up to 32 mmHg, and her visual acuity worsened to 20/500 with ongoing glaucomatous optic nerve damage and progression of posterior subcapsular opacity. Phacotrabeculectomy with topically applied mitomycin-C (0.04%) was then performed.The IOP was 3 mmHg by GAT on the first postoperative day but increased up to 21 mmHg on the following day. Her visual acuity was hand movement and could not be corrected. A diffuse corneal edema with a cloudy flap interface was noted in a slit-lamp examination. At 1 week postoperatively, the IOP had decreased to 14 mmHg after the application of brimonidine (0.2%)/timolol (0.5%) twice daily, but the corneal edema did not resolve. Spectral-domain optical coherence tomography (SD-OCT) scanning revealed a diffuse and thin fluid pocket in the corneal interface region (Fig. Lowering of IOP commonly results in the resolution of interface fluid that can appear after LASIK . HoweverIn eyes having a history of LASIK, the possibility of IFS should be considered as a possible cause of postoperative corneal edema even when the IOP is within normal range, particularly when the edema is long-standing and refractory to conventional treatment. An additional IOP lowering beyond the normal range may be helpful for resolving the corneal edema.


Brugia malayi infection on Aedes aegypti flight parameters: distance, average speed, maximum speed and number of flight bursts, using a tethered flight mill. In addition, we explored whether differences in flight capacity may be due to the effect of infection on glycogen and lipid reserves. Infection with filarial worms significantly reduced flight distance but increased the number of flight bursts. Exposure to microfilaermic blood led to a significant decrease in average and maximum flight speeds even in the absence of an established infection. Mosquitoes fed on microfilaraemic blood showed reduced levels of glycogen (−37.9%) and lipids (−49.7%) compared to controls at nine days post-exposure. However, a one-hour period of flight activity caused an increase in lipid content for both infected and control mosquitoes. Consequential flight incapacitation may serve in explaining the heterogeneous distribution of lymphatic filariasis.Evidence from experimental infection studies has shown that infected mosquitoes exhibit altered host-seeking behaviours, with suppression and activation of behaviours dependent on the parasite’s development stage. The mechanisms are poorly characterised; however, infections can impact mosquito energy reserves, thereby influencing key life-history traits and behaviours. In addition, filarial infection is likely detrimental to flight due to damage caused by developing worms. This study aimed to evaluate the impacts of  Wuchereria bancrofti, Brugia malayi and B. timori2. Regarded as a Neglected Tropical Disease (NTD), recent estimates suggest that 67 million people suffer from LF3 across 73 tropical and sub-tropical countries4. Morbidity, largely in the form of elephantiasis and hydrocele, causes substantial physical, social and psychological damage7. The resulting loss of 2.8 million disability-adjusted life years (DALYs)8 places LF as a significant global health problem. Microfilariae (mf) released by gravid female worms are ingested by mosquitoes during bloodfeeding. Approximately 10 days later, following successive developmental stages in the flight muscles, the infective larvae (L3) migrate to the mouthparts of the mosquito, ready to be deposited onto vertebrate host skin in subsequent feeds. This mode of development causes substantial histological and physiological damage within the vector13. As such, filarial infection is likely to be detrimental to mosquito flight.Lymphatic filariasis (LF) is a parasitic disease caused by three nematode species: 14. Conversely, some studies suggest that filarial infection produces significant declines in continuous measures of flight ability, namely distance and time18. Comparing study outcomes is therefore difficult due to differences in measured flight variables; an issue which is further complicated considering that there are a range of tools with which to quantify flight. Therefore, while infection may indeed lead to an increased number of flight attempts but overall reduced flight outputs, no study to date has explored both aspects of flight ability and activity.Evidence of the changes in mosquito flight following filarial infection are equivocal. Previous work suggests that flight activity, when defined as the number of minutes within an hour containing at least a single flight attempt, increases three-fold during filarial infection prior to the development of L3 worms20, vitellogenesis20 and immune responses22. The relative availability of these resources may therefore influence flight ability or activity. Blood-fed and sugar-fed mosquitoes have been shown to differ in flight speed and distance20, perhaps due to the differential utilisation and availability of energy resources. Research which has shown reduced flight distance and time as a result of filarial infection18 hypothesised this was a result of depleted energy reserves, however no study to date has explored energy in infected mosquitoes.Lipids and glycogen are important sources of energy in mosquitoes, being utilised in a number of life history traits and behaviours, such as flightPlasmodium show alterations in host-seeking and host-feeding strategies depending on the stage of infection28. Similar changes in host-seeking behaviour have been observed in mosquito-filarial systems, including Aedes aegypti mosquitoes infected with B. malayi29. When harbouring the non-infective L2 stage, mosquitoes show a five-fold reduction in host-seeking following exposure to human host cues compared to uninfected controls. Once developed into L3s, this pattern is reversed, and infected mosquitoes are significantly more likely to respond to host cues than uninfected mosquitoes29. These behavioural changes offer clear advantages to the parasite in minimising mortality risk during development and increasing the chances of host-contact once transmissible. The cause of this altered host seeking behaviour remains unclear, however damage to the flight muscles caused by the developing filarial worm may inhibit or deter flight in mosquitoes prior to the L3 stage.Mosquitoes infected with 31, and modelling frameworks are likely to benefit from an increased understanding of the interaction between vector and parasite, particularly during infection32. However, the absolute fitness costs associated with infection in mosquitoes remain ambiguous33. Coevolution between obligate parasites and vectors may lead to neutral relationships to maintain effective transmission, but immune responses are costly, and parasites can cause direct physical damage to the insect13.Behavioural and physiological changes in vectors following infection can have significant implications for disease transmission-1), maximum speed (ms-1) and number of flight bursts , speed ms-, maximumAe. aegypti mosquitoes  were flown on tethered flight mills across three replicate experiments. Midgut dissections conducted 3 hours post-bloodfeeding confirmed substantial uptake of microfilariae (mf) in mosquitoes which fed on B. malayi infected blood  were infected with L1s and/or L2s at 4 to 6 days post-exposure (DPE), and 29/58 (50.0%) were infected with L3s at 11 to 13 DPE , average speed , maximum speed  and the number of flight bursts . Pairwise comparisons based on least square means found that infection and exposure both lead to declines in the distance and average speed flown, as well as an increase in the number of flight bursts when compared to controls , whereas the number of flight bursts increased significantly . A significant interaction between infection status and DPE was observed for the number of flight bursts . This indicates that the change in the number of flight bursts over time between infection groups was different. The unadjusted means for each measured parameter are shown in Fig. P = 0.001), which increased with increasing worm burden (Supplementary files S2 and S3).Generalised Linear Mixed Models (GLMMs) using likelihood ratio testing indicated that infection status had a significant effect on flight distance , decreasing it by 37.6%, and lipid , decreasing it by 49.7%. Due to the nature of content extraction, infection could not be confirmed in these mosquitoes.A total of 76 mosquitoes underwent glycogen and lipid content analysis using anthrone and vanillin reagent, respectively. Midgut dissections conducted 3 hours post-bloodfeeding confirmed uptake of mf in 2 = 2.3, P = 0.132), but did lead to significantly increased lipid content  of 34.7% 14. Thus, while high burdens of infection may influence flight at the infective stage, this has limited applicability to natural settings, where burdens of infection rarely exceed five L3 worms per host56. Flight muscle fibres can carry out post-trauma reparation if such damage is limited10, further supporting this idea that L3s only cause noticeable detriment to host flight in unnaturally high intensity infections. Previously identified stage-specific switches in host-seeking behaviour29 are unlikely to be due to the generic effects of flight capacity, however it could be partially attributed to filarial infection causing an apparent increase in the number of flight bursts. Control mosquitoes in this study otherwise saw a relative decline in the number of flight bursts over time.Physical damage from filarial worm development appeared to have little to no additive impact on the flight ability of mosquitoes. Mosquitoes undergo age-associated declines in flight activityThe distinction between infection and exposure (the absence of developing or infective larvae) on flight outputs is not obvious. While exposure appeared to match infection in its effect on flight distance and average speed, it had a significantly greater detriment to maximum speed than infection. Regardless of the reason for this difference, these results clearly highlight that exposure and clearance of filarial worms may be sufficient to cause significant changes in vector flight.57. The behavioural and physiological consequences of filarial infection on their invertebrate hosts, such as flight incapacitation, may contribute to the heterogeneous nature of LF, which can pose a challenge for elimination. Remarkable differences between mosquito species in their behavioural and physiological responses to infection warrants continued exploration with additional parasite-vector systems.This study highlights the impacts that filarial infection can bear on vector flight. Further investigations into the flight behaviour of infected mosquitoes is necessary to apply this to different ecological settings, however the interplay between infection, immunity and flight is clearly complex. Furthermore, understanding the impact of fitness costs on the ability of mosquitoes to transmit disease may help explain the heterogeneity of filariasis transmissionAe. aegypti Liverpool (LVP) strain mosquitoes which had fed on either microfilaermic blood or uninfected blood were flown at 4 to 6 days post-exposure (DPE) or 11 to 13 DPE, corresponding to the L1/L2 and L3 stages of B. malayi respectively58. Those which fed on blood containing B. malayi mf underwent subsequent dissections to confirm the prevalence and intensity of infection. The wing length of 34 infected mosquitoes was measured to determine the correlation with flight activity. Mosquitoes were subject to either (i) a one-hour flight mill assay on day 4–6 DPE followed by dissection, (ii) a one-hour flight mill assay on day 11–13 DPE followed by dissection, (iii) a one-hour flight mill assay on day 9 DPE followed by glycogen and lipid analysis, or (iv) glycogen and lipid analysis on day 9 DPE with no flight. A single energy content analysis was conducted to observe the impact of infection and flight on glycogen and lipid reserves.We tested the impact of filarial infection on various flight parameters in mosquitoes using a set of eight tethered flight mills. Ae. aegypti LVP mosquitoes took place at the Liverpool School of Tropical Medicine (LSTM) insectaries under controlled conditions . Internally sourced mosquito egg papers were floated out into plastic trays (235 × 345 × 75 mm) containing distilled water, with a larval density of approximately 200/larvae per tray. Larvae were fed on Brewers’ yeast pellets before transfer to cages (285 × 295 × 280 mm) once pupated. We maintained adults on 10% sugar solution prior to bloodfeeding. All adult females were between 2 and 5 days old when bloodfed.Rearing of the B. malayi mf at a density of 20,000 mf/ml. Mf were obtained via intraperitoneal lavage of infected gerbils by a third party, in accordance with UK Home Office requirements and following LSTM approval, before suspension in RPMI media at a dilution of 1/100. Triplicate microscopic observations confirmed the presence and mobility of mf. Uninfected control blood was diluted with equal measures of non-infected RPMI media. Approximately 3 ml of blood was then offered to mosquitoes using a Hemotek® membrane feeding system kept at 37 °C. Following successful bloodfeeding, we removed all mosquitoes that had not fed to repletion. All research red cells and plasma were supplied by National Health Service Blood and Transfusion and were mixed in a 50:50 ratio before use. To verify mf uptake, three engorged females had their midgut contents homogenised on a microscope slide, which was then scanned with phase optics at 10x magnification.On the day of bloodfeeding, female mosquitoes were split into two separate cages. We then fed cohorts on either uninfected human blood (controls) or human blood containing Ae. aegypti using tethered flight mills (provided by Dr. Lim of Rothamsted Research), which were housed under standardised insectary conditions described above  readings at 625 nm quantified contents. Triplicate OD readings were carried out using a plate spectrophotometer and recorded on software (Gen5 Version 2.04) before averaging. Standard curves of lipid and glycogen content were created using olive oil suspended in chloroform and anhydrous glucose suspended in de-ionized water, respectively. Both standard solutions included 100 mg of substrate dissolved in 100 ml of liquid. Using the standard curve equations, OD readings of glycogen and lipid content from the mosquito samples were converted to quantity readings (μg) prior to statistical analysis.Nine days post-feeding, glycogen and lipid content were analysed. Glycogen and lipids were extracted using the standardised methods as described by Van Handel62 using the lme4 (version 1.1–20) package63 and visualised using ggplot2 (version 2.2.1)64. Statistical analysis of flight ability was conducted using GLMMs. Prior to analysis, Wald test assessments of the random variables (the flight mill number and replicate) found one of the flight mills to be faulty, so individuals flown on this flight mill (n = 24) were omitted from further analysis. Individuals which flew <50 m were also not included as it was assumed that attachment to the flight mill had compromised their flight. Linear regression analysis found that wing length did not correlate with any flight parameters, and so it was removed as a random variable in models. We assessed the distribution of each flight variable by plotting a histogram of individual mosquito flights and modelled them accordingly using GLMMs. Likelihood ratio testing (LRT) between individual GLMMs tested for an effect of either infection status or DPE on each flight variable. A least square means approach for multiple comparisons with a Tukey adjustment (α = 0.05) tested for differences between infection status categories. All best fit models were assessed using residual-fitted plots and/or Normal Q-Q plots to ensure reliability. LRT between Generalised Linear Models (GLMs) tested for associations between energy reserves and flight and infection status.All statistical analysis was conducted in RStudio (version 1.1.1456)Supplementary Figures