Acridotheres melanopterus). This species is highly threatened by the illegal wildlife trade, with its wild population numbering in the low hundreds, and its continued survival urgently depending on ex-situ breeding. Its three subspecies occur along a geographic gradient of melanism and are variably interpreted as three species. However, our integrative approach revealed that melanism poorly reflects the pattern of limited genomic differentiation across BWM subspecies. We also uncovered allelic introgression into the most melanistic subspecies, tertius, from the all-black congeneric Javan Myna (A. javanicus), which is native to the same islands. Based on our results, we recommend the establishment of three separate breeding programs to maintain subspecific traits that may confer local adaptation, but with the option of occasional cross-breeding between insurance populations in order to boost genetic diversity and increase overall viability prospects of each breeding program. Our results underscore the importance of evidence-based integrative approaches when determining appropriate conservation units. Given the rapid increase of terminally endangered organisms in need of ex-situ conservation, this study provides an important blueprint for similar programs dealing with phenotypically variable species.In today’s environmental crisis, conservationists are increasingly confronted with terminally endangered species whose last few surviving populations may be affected by allelic introgression from closely related species. Yet there is a worrying lack of evidence-based recommendations and solutions for this emerging problem. We analyzed genome-wide DNA markers and plumage variability in a critically endangered insular songbird, the Black-winged Myna (BWM,  This quandary has led to conflicts among conservationists, some of whom call for taxonomic practices that err on the side of elevating distinct populations to species level, whereas others fear ‘taxonomic inflation’ is diluting the value of conservation efforts3. In the context of the question of how much biological diversity we can afford to save, the taxonomic problem has featured prominently in conservation debates5, and has generated a panoply of partly overlapping, partly conflicting streams of action, such as the consideration of phylogenetic diversity in conservation6, extensions of environmental legislation to subspecies level in various jurisdictions7, and the formulation of multiple mutually non-overlapping definitions of ‘conservation units’8.Our planet is heading into an extinction crisis, and conservation resources are finite9. However, the Next Generation Sequencing (NGS) revolution of the last 10 years has increasingly shown that some species have taken considerable ‘short-cuts’ in the generation of substantial phenotypic differentiation, by-passing hundreds of thousands to millions of years of independent evolution by appropriating novel traits through horizontal gene transfer and genetic introgression13. This transfer of traits is now well-documented across a wide variety of model and non-model organisms20. Such phenotypic appropriation has occurred even in some populations of modern humans (Homo sapiens) which are known to carry traits conferred to them through genetic introgression from ancient, now-extinct hominine lineages22. Genetic introgression can sometimes lead to great phenotypic differences between those members of a species affected by interspecies admixture and those that are not. The significance of these processes for conservation, however, has so far been widely ignored. Should special conservation action be extended to phenotypically-different populations of an endangered species, even in cases where this may be a product of introgression rather than deeper evolutionary divergence?Biological diversity is typically generated slowly and cumulatively through evolutionary processes, which is why most conservationists agree that deeply diverged or distinct lineages deserve preferential conservation attention as compared to shallow lineagesAcridotheres melanopterus; BWM), a Critically Endangered songbird endemic to Java and Bali that is almost extinct in the wild due to illicit poaching pressure23. The species has been identified as a focal target requiring urgent conservation attention by IUCN’s Asian Songbird Trade Specialist Group and the Asian Species Action Partnership. BWMs have traditionally been divided into three distinctly-colored subspecies: (1) nominate melanopterus from West and Central Java, extinct in the wild as of 2018 except for a small feral population in a commercial wildlife park , which has an almost entirely white body coloration apart from its black remiges and uppertail coverts; (2) East Javan tricolor, thought to number a few dozen individuals in the wild in two national parks, which is characterized by a greyish-black mantle with a distinctive white rump, and (3) Balinese tertius, with ~ 200 wild individuals thought to survive in Bali Barat National Park, which is the darkest subspecies with a dark-grey to black plumage coloration over its entire mantle and rump29 . This taxonomic uncertainty has generated confusion among conservation breeders as to whether it is appropriate to cross-breed the three taxa or not. To make matters worse, only two of the three taxa are held by captive breeding programs, and the levels of genomic differentiation between them are unknown.With poaching pressure unrelenting across Java and Bali, conservation breeding has been strongly recommended as one of the main strategies in preventing the BWM’s extinction39, not only by inferring evolutionary patterns of divergence, identifying unique lineages and fine-scale patterns in population structure42 or revealing the hidden impact of illegal wildlife trade44, but also by being capable of identifying kinship groups and assessing the genetic viability of individuals within breeding populations46. In the case of the BWM complex, genomic tools allow for an assessment of whether to maintain separate breeding programs for each taxon, as suggested by the taxonomic arrangement recognized by the IUCN, or a single combined breeding program for the entire species complex. The former approach risks genetic inbreeding due to bottlenecks in the captive populations, while the latter approach risks the loss of evolutionarily unique lineages due to hybridization, so the consequences of incorrect decision-making will unavoidably impact the genetic makeup of resultant insurance populations generations into the future. It has become clear that a rigorous ex-situ breeding strategy for the conservation of the BWM must be evidence-based, and should preferably include an assessment of both genomic and phenotypic levels of differentiation among subspecies, along with an evaluation whether some of the phenotypic differences may have been generated by processes such as introgression.Genomic tools play an increasingly crucial role in conservation managementex-situ breeding programs (melanopterus and tertius) are reflective of deep genomic variation. We also include single-gene data from the remaining taxon tricolor to allow for an estimation of divergence levels among all three extant taxa. Last but not least, we discuss the implications arising from the detection of differential secondary gene flow between various BWM populations and a sympatric congener, the Javan Myna (Acridotheres javanicus). This assessment allows us to provide recommendations for immediate and urgent conservation action.In this study, we use thousands of genome-wide markers from 85 captive individuals across the morphological spectrum in the BWM complex to evaluate whether the phenotypic differences between the two geographically terminal taxa available in melanopterus, tertius or hybrids based on plumage and studbook records. Our phenotypic score range extended from 0 to 24 along a cline of increasing melanism , with two in particular exhibiting traits typically diagnostic of tricolor (grey back and white rump)  and six individuals had a score of 22–24 . Birds that were not scored were classified as ‘Unknown’ in our subsequent genomic analysis—however, it should be noted that these ‘Unknown’ individuals were all listed as ‘melanopterus’ by their breeding facilities, and are thus assumed to fall within the pure melanopterus score range of 0–6.We scored melanistic characteristics of a subset of BWMs, sourced from two conservation breeding facilities, that were classified as either ity see “”. HoweveAcridotheres javanicus) and Common Mynas (Acridotheres tristis) , from our double digest Restriction-Site Associated DNA sequencing (ddRADSeq) data. These datasets included: (1) all 85 individuals, (2) individuals with no first order kinship, (3) founder individuals identified by pedigree information from Cikananga Conservation Breeding Centre and any individuals with a kinship coefficient less than 0.25, and (4) a genus-level subset for introgression testing, which included congeneric Javan Mynas , and a second exclusively containing Balinese tertius individuals  plots, our sampled BWMs are roughly divided into two clusters separated along principal component PC1: one cluster containing west Javan als Fig. . While t47   Fig. .ND2 gene sequences of BWMs from GenBank, including one from the rare East Javan taxon tricolor, which is not represented in any modern conservation-breeding facilities and therefore almost impossible to obtain. We compared these sequences with a subset of ND2 sequences generated from our dataset. Our phylogenetic analysis revealed limited differentiation among all three subspecies  internal clade to the exclusion of all melanopterus samples, the single tricolor sample, and phenotypic hybrids. However, raw pairwise ND2 divergence was very low among all samples (< 1.5%), especially when compared to mitochondrial divergences typically associated with sister species level in the bird barcoding literature (~ 2–3%)50.We were able to source multiple formerly published mitochondrial MC1R gene across the subset of individuals we sequenced from our fresh dataset   in eight of 28 tertius-melanopterus pair combinations tested in the arrangement depicted in Fig. melanopterus. We estimated an admixture fraction fG of 1.16% for the population level test, indicating that the latter percentage of the tertius genome is estimated to be secondarily derived from the Javan Myna through introgression.Population-based testing for secondary gene flow on dataset 4 Table  revealedmelanopterus and tertius), fineRADstructure indicated higher overall allelic sharing with tertius than with melanopterus  is practically extinct in the wild except for a small free-roaming flock inside a Javan wildlife park, while the other two subspecies each number < 200 individuals in the wild. Of equal worry, only one subspecies (melanopterus) was covered by an active conservation-breeding program numbering a few dozen individuals at the time of writing25, while the other two subspecies were either held in numbers too low for a viable breeding program (tertius) or were completely unrepresented in any of the world’s recognized conservation-breeding facilities and zoos (tricolor)31. All three subspecies of Black-winged Myna have been protected under the Indonesian Ministry of Environment & Forestry’s regulations since 2018.The Black-winged Myna is representative of a growing global panel of terminally endangered species whose fate and continued survival crucially hinges on ex-situ programs are uncertain about best practices regarding the breeding of individuals bearing traits that reflect minor gene flow between subspecies. Specifically, the breeding program of melanopterus has been beset by a surprising incidence of individuals with isolated grey mantle feathers on otherwise white upperparts  into the more melanistic tertius subspecies of BWM . When investigating allele sharing between Javan Mynas and the two geographically terminal BWM subspecies (melanopterus and tertius), fineRADstructure indicated higher overall allelic sharing with tertius than with melanopterus : in certain areas of southeastern North America, Coyotes co-occur and hybridize with slightly larger-sized canids that have long been mistaken for an independent, highly endangered species, the ‘Red Wolf’ (Canis rufus)66. Recently, the Red Wolf was shown to be extremely similar to Coyotes genomically, albeit with a signature of excess allelic sharing with Grey Wolves (Canis lupus)67, suggesting that Red Wolves may not have existed in North America in pre-human times, but instead emerged as the result of a locally dying Grey Wolf population being assimilated with Coyotes. Regardless of ongoing disputes about the perceived conservation value of Red Wolves68, knowledge of the true evolutionary history of an introgressed lineage carries immense implications for conservation.Extreme phenotypes that have been inherited through small-scale introgression can be easily mistaken as signals of species-level differentiation even if actual genomic differentiation between ‘pure’ and ‘introgressed’ populations is low. In a conservation context, such misdiagnoses can lead to poor practice. One of the most illustrative examples is the Coyote  whereas the other two urgently need to be established. The aim of this recommendation is to sample and preserve the whole range of morphological and genetic diversity that used to be found naturally in the range of this species, while retaining the option of cross-breeding subspecies. While we show that melanism is a poor indicator of overall genomic divergence in BWM, it is likely linked to local adaptations in each population that generate apparent phylogeographic breaks and resultant phenotypic clustering, as evidenced empirically in Western Barn Owls  across Europe, as well as with simulations70. Melanistic phenotypes therefore likely provide good visual indicators of local adaptations, which may be crucial for the success of any possible wild reintroductions in the future72, while effectively hedging each captive population as insurance against the detrimental effects of genetic diversity loss over time, since individuals from each program can be drawn as donors for the genetic rescue of another73.In the present environmental and funding environment, we recommend aiming for three separate breeding sub-programs under the umbrella of a single species-wide program without a strict separation. One of these sub-programs already exists  and Bali Bird Park   Table . Samplesmelanopterus; colour score range 0–6) to the darkest bird  secondary coverts , (ii) scapulars, (iii) mantle, (iv) mid back, (v) rump, and (vi) tail coverts. Scores across all six features were then added up to produce a single aggregate colour score for each individual. All individuals were scored by a single researcher to preclude observer bias.75 was then conducted using the original data representing the percentage scores for each individual to determine if the analysed birds fall into distinct morphological clusters, and elucidate the optimum number of clusters suitable to describe the data.A K means cluster analysis using the R package ClusterND2)76, including three melanopterus, one tertius and three presumed morphological hybrids, whilst a similarly diverse subset of five were selected for Melanocortin-1 receptor (MC1R)77 sequencing. The subsets of samples chosen for ND2 and MC1R sequencing were selected to represent the entire breadth of taxonomic and melanistic variation within our larger dataset, respectively. Polymerase chain reaction (PCR) was carried out in a C1000 Thermal Cycler. We performed PCR amplifications in individual 25 μl reaction volumes, which comprised 2.5 μl DreamTaq buffer, 0.5 μl dNTP mix (working concentration 10 mM), 0.5 μl of each primer (working concentration 10 μm), 0.125 μl DreamTaq polymerase, 2 μl mtDNA template and 18.8 μl molecular grade water. PCR product clean-up was carried out using ExoSAP-IT, and the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems Inc.) was used to cycle-sequence the samples. Sequences were obtained by capillary electrophoresis using an Applied Biosystems 3130xl Genetic Analyzer.DNA extractions were carried out using the Qiagen DNEasy Blood & Tissue Kit following the manufacturer’s recommended protocol. A Qubit 2.0 Fluorometer was used to quantify DNA concentrations of the extracts. A subset of seven samples were selected for sequencing of the mitochondrial gene NADH dehydrogenase subunit 2  were used to double digest the samples for 3.5hrs at 37 °C, followed by clean-up using a 1.1X ratio of Sera-Mag SpeedBead Carboxylate-Modified Magnetic Particles (Thermo Scientific). Samples were re-quantified and then ligated with unique barcodes (PIE adaptors) by T4 DNA Ligase (New England Biolabs Inc.) at 16 °C for 16 h.Double-digest restriction enzyme associated DNA sequencing (ddRADSeq) was performed as per Tang et al.For subsequent library preparation, the samples were split into eight pools for distribution across two Illumina HiSeq 4000 lanes (4 pools/lane) according to their post-restriction concentrations. Samples similar in concentration were pooled to avoid either over- or under-representation of any one sample. Library clean ups were performed for each of the eight pools with AMPure XP beads (Agencourt) using a 1.5X bead ratio. Size selection was carried out for these eight pools using a Pippin Prep Gel Electrophoresis system (Sage Science) to isolate fragments for a sample peak of 420 bp in length, followed by another AMPure XP clean up. PCR amplification of size-selected fragments was run for 12 cycles, followed by a final AMPure XP clean up step. Pools were screened for quality control on a Fragment Analyzer  and quantified on a Qubit 2.0 Fluorometer before being pooled in equimolar proportions to form two final libraries. Final libraries were spiked with 30% PhiX and transferred to the Genome Institute of Singapore for Illumina sequencing, achieving a mean sequencing depth of 143X.melanopterus, tertius and morphological hybrids, were additionally sequenced on a separate Illumina HiSeq 4000 lane to a mean sequencing depth of 359X to obtain more SNPs for introgression testing. We also sequenced three samples of Common Myna (Acridotheres tristis) and six samples of Javan Myna (Acridotheres javanicus) in the same way for introgression testing (Table A subset of eleven Black-winged Myna (BWM) samples, including representatives of 79. Samples with less than one million reads were removed from subsequent analysis. The remaining sequence reads were then aligned against the Javan Myna (Acridotheres javanicus) reference genome80 using BWA-MEM81.We used FastQC (Babraham Bioinformatics) to analyze sequence quality across all base positions. Demultiplexing was then performed using the “process-radtags” command in Stacks v1.3479 was used to call SNPs. We employed a minimum stack depth of 10 in our analyses. We only retained loci that were found in more than 90% of individuals, using options available in the populations module of Stacks. As a first step to reduce the effects of linkage disequilibrium in subsequent analyses, we only accepted one SNP from each locus using the –write_single_snp option provided, leading to a set of 7,592 SNPs.The pipeline ref_map.pl in Stacks v1.3482 was run to determine the amount of missing data present across the 85 retained samples and filter out any individuals with more than 30% missing data, and filter SNPs under linkage disequilibrium (r2 > 0.9) using a 25-SNP window sliding 10 SNPs at a time.PLINK 2.083 to estimate pairwise relatedness by means of maximum likelihood estimation. We then removed a single individual from each pair which displayed a kinship coefficient above 0.25 (first order kinship). We generated a total of four datasets using the methods described above, with the exception of employing a stack depth of 5 rather than 10 in the ref_map.pl pipeline for dataset (4) in order to retain more loci  were run using both datasets 2 and 3 for comparison. Dataset 4 was used for introgression testing and fineRADstructure, and all datasets were used for comparative STRUCTURE analyses.84 for all datasets. STRUCTURE was run from K = 2 to K = 10 with ten iterations per K and without a priori hypotheses of cluster membership. For each iteration we implemented a burn-in of 50 000 generations and Markov chain Monte Carlo iterations (MCMC) for 250,000 generations. We used STRUCTURE Harvester Web v0.6.9485 and the Evanno method47 to determine the statistically most significant K value. Results were averaged across replicates by evaluating individual ancestry coefficients  with CLUMPP v1.1.286 using the Greedy option provided. Results for dataset 2 . We used a missing data cut-off of 10%, and utilized RADpainter to calculate the coancestry matrix followed by assigning individuals to populations at default parameters, including a burn-in period of 100 000 and 100 000 MCMC iterations.We investigated individual-based pairwise coancestry using the MCMC coalescence algorithm as implemented in fineRADstructure v0.3.154 using SNP dataset 4. A simple version of this test uses the relative frequencies of two discordant SNP distributions (ABBA vs BABA) in a four-taxon pectinate phylogenetic topology  into tertius (P2) relative to melanopterus (P1), and vice versa, using Common Mynas as the outgroup  length. We ran a maximum likelihood tree using RAxML GUI 1.590 under a GTR + Gamma model of sequence evolution and a rapid bootstrap algorithm with 10 000 replicates. A trimmed alignment of 902 bp, which excluded any ambiguous positions or gaps, was used to generate a median-joining haplotype network using Network version 1091.DNA sequences from Sanger sequencing were vetted using CodonCode Aligner version 8.0.1 (CodonCode Corporation). We supplemented our ND2 dataset with additional Black-winged Myna sequences from GenBank, including two MC1R sequences from six individuals were assembled as described above in order to generate an 835 base pair alignment. Nucleotide sequences were translated into amino acids using MEGA version 7 and compared to check for non-silent mutations using Geneious 11.1.5 (https://www.geneious.com).All experimental protocols were approved and conducted in accordance with regulations outlined by the National University of Singapore’s Office of Safety, Health, and Environment. Animal sampling was approved by the Institutional Animal Care and Use Committee (IACUC) for the National University of Singapore.Supplementary Information.


No changes in TUG or HR-QoL were observed at POST. Concurrent NMES exercise may be an effective exercise intervention for augmenting physical function in participants with cancer and moderate and poor functional status. Implications for cancer survivors: By allowing participants to achieve therapeutic levels of exercise, concurrent NMES may be an effective supportive intervention in cancer rehabilitation.The primary aim of this study was to investigate the functional, physiological and subjective responses to NMES exercise in cancer patients. Participants with a cancer diagnosis, currently undergoing treatment, and an had an Eastern Cooperative Oncology Group (ECOG) performance status (ECOG) of 1 and 2 were recommended to participate by their oncologist. Following a 2-week, no-NMES control period, each participant was asked to undertake a concurrent NMES exercise intervention over a 4-week period. Functional muscle strength [30 s sit-to-stand (30STS)], mobility [timed up and go (TUG)], exercise capacity [6-min walk test (6MWT)] and health related quality of life (HR-QoL) were assessed at baseline 1 (BL1), 2-week post control (BL2) and post 4-week NMES exercise intervention (POST). Physiological and subjective responses to LF-NMES were assessed during a 10-stage incremental session, recorded at BL2 and POST. Fourteen participants [mean age: 62 years (10)] completed the intervention. No adverse events were reported. 30STS (+ 2.4 reps,  Although poorly understood, the mechanisms behind cancer related fatigue have been linked to the role of skeletal muscle3. Common treatments such as chemotherapy and hormone therapy can have catastrophic effects on skeletal muscle structure and function, with an accelerated aging phenotype observed 4. These losses are associated with impaired muscle strength in common cancers 6. Chemotherapy can also impair peak oxygen capacity (VO2peak), leading to poorer exercise tolerance7. These treatment complications can increase sedentary behaviour, impair physical function and compromise independence.Both cancer pathology and associated life extending treatments are linked to complications which can impair physical function and quality of life. Some complications are acute in nature, persisting across a treatment cycle and normalising during treatment recovery. However, common chronic complications following cancer treatment can persist for years. For example, fatigue, which is seen in almost 100% of patients, may persist for up to 10-years post treatment cessation8. However, despite its recognised benefits, muscle strengthening, and aerobic exercise can be challenging in this population. Many individuals with impaired functional status while undergoing treatment experience exercise limiting symptoms. This translates to as few as 5% of individuals who are undergoing treatment being physically active9. Therefore, alternative exercise methods which can be undertaken during cancer treatment to minimise the loss of or augment functional outcomes and accelerate return to conventional exercise are warranted.Conventional exercise (i.e. aerobic and resistance exercise) is now recognised as a primary adjunct therapy to help offset disease/treatment complications. There is strong evidence to support prescription of conventional exercise to improve cancer related health outcomes including fatigue, quality of life, and physical function across the cancer trajectory10. High frequency tetanic NMES (HF-NMES) has a proven muscle strengthening effect using frequencies ≥ 20 Hz in healthy and patient populations11. Emerging evidence suggests that the application of sub-tetanic low frequency NMES  can elicit a comfortable and sustainable aerobic exercise response, and enhance aerobic exercise capacity and exercise endurance in healthy and patient populations13. Recently, a concurrent model of NMES exercise delivery (both LF and HF-NMES phases) has been evaluated in patients with mixed cancer diagnoses15. This early work involving a case series and a case report has demonstrated safety, feasibility and patterns of improvement in functional and quality of life outcomes.Neuromuscular electrical stimulation (NMES) is an emerging field in oncology rehabilitation15. This is a common finding in the general NMES literature18, and is a pragmatic consideration given the exercise limiting symptoms experienced by those with advanced disease stages. However, adherence to concurrent NMES exercise for extremely deconditioned individuals can be affected by the ability to apply NMES garments unsupervised19. This is unsurprising given that up to one third of those with cancer experience difficulty in basic activities of daily living including getting dressed20. Therefore, this may have important implications for long-term compliance to home-based unsupervised NMES exercise. In addition, no studies have investigated the functional response to concurrent NMES exercise in a controlled setting. Furthermore, although the physiological response to LF-NMES has been investigated in healthy participants21, the physiological and subjective response in an cancer population is currently unknown.Preliminary, exploratory work in an oncology population suggests that concurrent NMES exercise may be best implemented in more deconditioned individuals to enhance functional outcomesTherefore, the primary aim of this prospective controlled study was to assess the effects of concurrent NMES exercise in comparison to no NMES exercise intervention on functional and quality of life outcomes for participants undergoing active treatment who were unable to exercise independently in cancer rehabilitation. A secondary aim was to investigate the physiological response to LF-NMES was examined using a series of physiological and subjective dependent variables.22, baseline 6-min walk test (6MWT) distance was also used to categorise participants according to functional status, . 6-min walk test scores of < 350 m are classified as poor in comparator clinical populations23 and have been shown to correlate with performance status in patients with recurrent glioma24. Participants were excluded if they had limitations which affected their ability to complete functional exercise tests, a serious, uncontrolled cardiac arrhythmia, any cognitive impairment which may affect their ability to apply NMES safely without direct supervision, deep vein thrombosis within the last 6 months, metastatic lesions of the femur, or a cardiac pacemaker. Prior to study entry, participants provided written informed consent.Participants were volunteers by self-selection who had a cancer diagnosis, were currently undergoing treatment (chemotherapy or hormone therapy) and had an ECOG performance status of 1 or 2. They were recommended to participate by their oncologist where physical limitations due to muscle weakness and asthenia which limited voluntary exercise participation were identified. Due to the subjective nature of the ECOG scale25. In addition, this experimental design offset the need for a no/sham NMES control group and was selected due to practical reasons (high attrition rates associated with exercise interventions in cancer populations). To help control for cancer treatment effects, participants entered the study at least 1 week after commencing a treatment cycle, with testing sessions at BL2 and POST occurring at least 1 week before/after a treatment cycle.This was a one group pre-test post-test prospective cohort study design comprising a 2-week control period  and a 4-week intervention period (Wk 3–6). Functional outcomes , participant reported outcomes  and physical activity levels (PA) were recorded at three separate time points: start of control period [Baseline 1 (BL1)], post 2-week control period (BL2), and post 4-week NMES exercise intervention (POST). All participants were required to attend the university laboratory at each time point for assessment. The two-week control period was designed to show relative stability of the outcomes without intervention or possible decline as supported by the literature in this area2), heart rate (HR)] and subjective (rate of perceived exertion (RPE) and discomfort) outcome measures were recorded during two supervised 10-stage incremental NMES sessions to assess change in these outcomes selected as indices of improved cardiorespiratory fitness and intervention tolerability. These incremental sessions occurred at BL2 and POST. A schematic of the study design is depicted in Fig. Physiological  and 6MWT  = 5.385, p = 0.022] performance differed significantly between time-points. Post hoc tests using Fisher’s LSD revealed that STS performance improved by an average of 2.4 reps (2.8) (p = 0.007) and 6MWT performance improved by an average of 44.3 m (67.2) (p = 0.028) after the 4-week intervention (POST) from BL2  at each study phase BL1, BL2 and POST) are summarised in Table  and POSTQoL data were not interval and were not normally distributed during normality tests. Group median results for all QoL functional scales and the fatigue symptom scale are presented at the three time points where differences across study time points were noted Table .Table 3Gp < 0.001). Peak HR and discomfort values were also higher at the second incremental session; however, these values were not significantly different  vs 56.6 mA 14.6), .6, p < 0This is the first study to investigate the functional, physiological and subjective effects of a home-based concurrent NMES exercise intervention in participants with a cancer diagnosis, who were concurrently undergoing treatment and were deemed to have moderate or poor functional status. All participants had been identified by their oncologist to have limitations in exercise participation. The main finding of this study was that a concurrent NMES exercise intervention resulted in significant and clinically meaningful improvements in lower limb functional muscle strength and functional exercise capacity as measured by 30STS and 6MWT. Higher maximum LF-NMES intensities were achieved at a similar level of subjective discomfort during the second incremental NMES session suggesting early habituation to the intervention.15; however, these studies were designed to provide preliminary data on safety and feasibility and the study design employed was uncontrolled in nature. Although randomised controlled trials (RCT’s) are the gold standard, these designs are not always pragmatic and may be undermined by issues including intervention delivery and participant recruitment and retention35. As such the current study used a two-week control period to address the difficulties described and the need for a no/sham NMES control group, with stability in this population across this control period. In addition to this, we attempted to control for residual treatment effects by timing assessments to treatment administration (at least 1-week post treatment infusion).The primary reason for conducting this study was to assess the effects of a relatively modest dose of concurrent NMES exercise  on functional outcomes in participants with cancer. Previous work in oncology using this concurrent progressive approach has demonstrated a trend for improvement in lower limb strength and functional exercise capacity following NMES exercise application8, many patients undergoing active treatment experience exercise limiting complications. Exercise which uses concurrent NMES exercise has capacity to provide a pragmatic alternative to conventional exercise. In the current study, significant improvements in the primary outcome, 30STS performance were observed in favour of the concurrent NMES stimulation phase. Improvements in this outcome were reported in 9 of 14 participants, with eight participants achieving the MCID. Potential mechanisms behind muscle strength gains include greater motor unit recruitment which has been seen after 4 weeks of HF-NMES36; however, in the absence of supporting data this is speculative. It should be noted that although the improvement was significant, the increase was modest . In addition, when comparing 30STS change by functional group, no significant difference was found. These results are encouraging, meet the MCID reported in comparator clinical populations and highlights the potential use of concurrent NMES to elicit a strengthening effect in cancer patients unable to partake in conventional exercise.Although recent physical activity guidelines from the American College of Sports Medicine (ACSM) for individuals with cancer provide strong evidence for the participation in concurrent exercise (aerobic and strengthening) for improving physical function20. We observed significant improvements in 6MWT distance, with a mean increase of 44 m, (+ 14%) after the concurrent NMES exercise intervention. Improvements were reported in 11 of the 14 participants, with eight participants exceeding the MCID. Potential mechanisms behind these improvements include an increase in muscle oxidative capacity which is shown to occur following protracted exposure to LF-NMES37; however, again, without supporting data this is speculative. Of note, our results are greater than those reported by Banerjee et al.13, who noted a 7.5% (~ 39 m) increase in 6MWT distance following 6 weeks of LF-NMES in patients with chronic heart failure (CHF). Discrepancies between study results could be linked to the more overall deconditioned state of the participants in the current study (baseline 6MWT: 308 m vs 415 m). However, the additional stimulus of concurrent NMES exercise should also be considered. For example, reduced lower limb muscle strength is associated with impaired walking performance39. Therefore, the ability of concurrent NMES exercise to potentially target multiple body systems may well have provided greater benefits than either NMES exercise modality alone.Disabilities related to basic activities of daily living (ADL) such as walking are prevalent in > 50% of patients with cancer8. In addition, exercise interventions exceeding 12 weeks are recommended to see improvements in this health outcome8.We observed no significant changes in any QoL categories. This is likely due to the study being underpowered to detect a change in these outcomes. However, it is also important to highlight that clinically meaningful changes in Global QoL, Physical QoL subscale and Fatigue symptom subscale were noted. Global QoL deteriorated by 12 points from BL2 to POST whilst both Physical QoL and Fatigue improved by 20 and 16 points respectively over the same time period. The deterioration in Global QoL is unsurprising despite clinically meaningful improvements in Physical and Fatigue subscales given that this health outcome is known to deteriorate across a patient’s treatment cycle, and encompasses a range of factors that may have a more varied response in individuals2max), translating into an increase in aerobic capacity and exercise performance21. This training was also accompanied by a significant increase in maximum heart rate (HRmax) at the second incremental NMES session21. We attempted to investigate the aerobic exercise response to LF-NMES in oncology. Unfortunately, complications with gas analyser equipment meant VO2 data was not possible. Therefore, we reported HRmax achieved during the first and second incremental NMES sessions. When compared with the HRmax response reported by Crognale et al.21, who targeted the same muscle groups in a healthy active group, HRmax achieved in the current study was lower at both the first [92.0 beats.min−1 (21.0) vs 114.1 beats.min−1 (20.7)] and second [100.0 beats.min−1 (18.9) vs 134.8 beats.min−1 (21.9)] incremental sessions. Differences may be explained by a lower absolute maximum NMES intensity [77.6 (28.6) mA vs 103.8 (18.0) mA], and leg fat free mass which is correlated with tolerance in more severe disease40.LF-NMES in healthy participants has been shown to elicit a comfortable and sustained therapeutic aerobic exercise response  to LF-NMES. Indeed, LF-NMES has been shown to enhance citrate synthase (CS) activity by 9 to 31% in healthy and patient populations when used `3x/week, for 4 to 10 weeksWhen identifying suitable outcome measures for future work in this population, the limitations of this cohort must be considered. Although the response to TUG was not significant, individuals with advanced cancer may struggle with prolonged periods of walking. As such, the 30STS or TUG tests may be the most appropriate outcome measures for focus in future work. An adapted Short Physical Performance Battery (SPPB), which includes the 30STS in favor of the 5STS (due to the floor effect) may be an appropriate battery of tests to assess the effectiveness of concurrent NMES exercise in cancer rehabilitation. Furthermore, working towards establishing a minimum data set for future work should consider the 30STS and TUG.A strength of our study design was the inclusion of a control period in which we demonstrated stability in the participants included. In addition, we controlled for residual treatment complications by assessing participants at least 1-week pre/post treatment However, our study has several limitations. A parallel ‘standard care’ control group would have strengthened our results. Future work should consider an RCT design. Although we were successful in recruiting the minimum number of participants required to find significant change in our primary outcome, our sample size was still small and our study population is heterogeneous, likely leading to a lack of statistical power for some outcomes, and making it hard to draw definitive conclusions regarding the effects of concurrent NMES on functional and QoL outcomes. In addition, because of our small sample, we used Fisher LSD during our analysis when comparing groups across time periods. Fishers LSD is less conservative than Bonferroni correction, therefore increasing Type 1 error rate. As such, future studies with a larger sample are needed to confirm our results. Finally, we acknowledge that our sample was relatively young and may not be representative of those in an older and frailer oncology population.In conclusion, concurrent NMES exercise may be effective for enhancing functional muscle strength and exercise capacity in participants undergoing treatment for cancer. This study does not demonstrate that LF-NMES can elicit an aerobic exercise response in cancer patients. However, peripheral muscle adaptations following LF-NMES, combined with the muscle strengthening effects of HF-NMES may have contributed to functional improvements noted. Future work is required to elaborate on the mechanisms behind concurrent NMES exercise adaptations. A need remains to identify the most appropriate candidates for concurrent NMES exercise. Future large trials are required to expand on the findings of this study.


Chronic migraine is a condition with gradually increasing prevalence among adolescents which causes severe headaches resulting in functionality loss. Factors contributing to migraine becoming chronic and negatively affecting quality of life in adolescence are still unclear. Parallel with these, we aimed to examine the effect of psychiatric symptoms on headache severity and functionality loss among adolescents with chronic migraine.We evaluated features of 50 adolescents who were diagnosed with chronic migraine according to International Classification of Headache Disorders-3 for the first time in their lives by an experienced neurologist. Sociodemographic and clinical data were collected and Pediatric Migraine Disability Assessment Score, Visual Analogue Score and DSM-5 Level 1 Cross-Cutting Symptom Measure Scores (CCSM-5) were evaluated. Semi-structured psychiatric interviews were done to those who scored higher than cut-off scores on CCSM-5. Healthy control group was constituted of cases which had similar age and sex distribution to case group.Majority of the case group was female (%78). There was a positive correlation between headache severity and computerized tomography history in emergency department. All of the psychiatric symptom scores were significantly higher in case group except for psychotic symptoms; but attention problems and manic symptoms clusters did not have significant difference according to the thresholds of CCSM-5. Receiving a psychiatric diagnosis did not affect frequency, severity or duration of headaches. There were also no relationship between depression/anxiety diagnosis and severity of headache/functionality loss.Findings suggest that; more rational treatment methods with lesser functionality loss should be developed by adopting multidisciplinary and prospective approach via psychiatric screening for adolescents with chronic migraine. Headache is the most frequent somatic symptom and major cause of chronic moderately severe pains during adolescence –3. PrevaChronic pains have negative effects on cognitive functions  as well as “state” emotional  and “trait” emotional  features . HeadachBiological, cognitive, emotional and behavioral factors are known to play major roles in occurrence of headaches  during adolescence; thus evaluation of comorbid psychiatric conditions are suggested in order to provide more comprehensive and effective treatment , 16. In 2(m2)] and classified as normal or over-weight/obese according to Turkish standart percentiles : This is a semi-structured psychiatric interview which was developed by Kaufman et al. in order to diagnose psychiatric disorders amon children and adolescent and was updated according to DSM-5 diagnostic criteria in 2016 , 27. Unap < 0.05 was accepted as statistically significant.Data was analysed using the Social Sciences software version 21.0 . The Kolmogorov-Smirnov test was used to check whether the data were normally distributed. In order to compare data between groups; Independent T-Test and ANOVA were used for parametric data and Kruskal-Wallis (KW) and Mann-Whitney U (MWU) tests were used for non-parametric data. Categorical data were analysed with Chi-Square test. Mean [standart deviation (±SD)] values were given for parametric data, median [inter-quartile range (IQR)] values were given for non-parametric data and frequencies (percentage) were given for categorical data. Correlations between continuous data were analysed with Pearson Correlation test; where as relationship between categorical data was analysed with Spearmen Correlatino test. The value of p = 0.011, Table Majority of the patients were female (%78) and had mean age of 15.5 (±1.45) years. Time between emergence of headache symptoms and clinical referral was 18.8 ±21.3) months and they had mean head ache duration of 20.7 (±6.78) days per month. Median value of VAS was 7 (2) and median value of PEDMIDAS was 2 (3). Analgesic drug over-use was present among 14% of the patients and ages of analgesic over-users ranged between 16 and 18 years. Over-weight/obese patients constituted only %26 of case group. Rate of at least one other chronic illness among adolescents with CM was 16% . Majority of the referrals were during autumn (16%) and winter (20%). During the emergency department referrals; 46% of patients under-went sinus x-ray and 18% of patients under-wnt CT  and PEDMIDAS  scores. In addition there was a positive correlation between somtatic symptom scores on CCSM-5 and PEDMIDAS  scores (Tablo 5). Patients who received MDD and/or GAD diagnosis did not differ from patient who did not receive these diagnoses regarding VAS  and PEDMIDAS .Total of 14 CM patients (28%) had a history of at least one previous psychiatric referral, total of 29 CM patients (58%) received a new psychiatric diagnosis according to K-SADS-PL-DSM-5 during the time period of the study and the most common (22%) psychiatric diagnosis was the comorbidity of Major Depressive Disorder (MDD) and Generalised Anxiety Disorder (GAD). Among adolescents with CM; total of 8 patients MDD (16%) and 6 patients received GAD (12%) diagnosis. Other Psychiatric diagnoses included; bipolar disorder (2%), obsessive-compulsive disorder (OCD) (2%), OCD and MDD comorbidity (2%) and attention deficit and hyperactivity disorder (ADHD) (2%). All of the psychiatric symptom scores in CCSM-5 were significantly higher in case group except for psychotic symptoms; but attention problems and manic symptoms clusters did not have significant difference according to the thresholds of CCSM-5  did not stop attending their educations which may reflect some social and cultural differences can play important roles on the outcomes of CM , 31. PEDSomatic, anxious, aggressive, attention deficit, irritable and depressive symptoms in CCSM-5 were significantly higer in adolescents with CM compared to healthy controls. CCSM-5 has questions about headache, nausea and vomiting in the section of somatic symptomatology and this might explain the higher somatic symptom scores seen on case group. In addition, pain is known to be a complex and multi-dimensional phenomenon which constitutes of sensorial, emotional/ motivational and cognitive components . PsitiveInvolvement of psychiatric point of view in headache field extends a long history and as a consequence, comprehensive investigation and wholesome definition of this complex phenomenon have accelerated . RelatioAdditionally problems caused by headaches which these patients experience during their daily lives  may contribute to the development of psychiatric problems. As previously mentioned, headache symptoms limit and negatively affect daily activities of these patients; thus result in psychiatric disorders and decreased quality of life. In the literature, most of the studies which used Pediatric Quality of Life Inventory (PedsQL) report that children and adolescents who are diagnosed with migraine have worse quality of life compared to healthy controls –46. Howep = 0.005, Pearson correlation)/ PEDMIDAS  scores  have found that adolescents with migraine have higher scores not only in internalizing symptoms (depression and anxiety) but also in externalizing symptoms and total problem scores which suggest that externalizing problems also play critical roles on migraine symptomatology . Even thThis study only included adolescents CM patients; thus it was conducted on a fairly homogenous clinical headache sample which can be counted as its methodological strength. Also it is a rather detailed clinical research in which numerous individual and environmental risk factors and their effects on functionality, headache severity and psychiatric symptomatology were examined by both a neurologist and a child and adolescent psychiatrist. Furthermore, studies on adolescent migraine patients which used DSM-5 criteria are limited; implementation of ICHD-3 beta version and DSM-5 criteria in order to examine the association between CM and psychopathology in adolescent age group is the major strength of our research. Despite its important findings and strenghts, our study also has some limitations. Firstly, we did not examine clinical and environmental risk factors for migraine in control group; so it was not the main of this study, it could not be determined if these risk factors really effect pathophysiology of migraine or not. Secondly, psychiatric screening tool that we used (CCSM-5) only evaluates the symptoms during last 2 weeks and instruments which examine psychosymptomatology in a longer time period may better explain the psychopathological processes underlying CM. Thirdly, cross-sectional design and relatively small sample size of our study might make it hard to fully clarify a cause and effect relationship between clinical conditions and generalize its results to a larger population. In addition we would like to add that, even though our sample size is sufficient according to the power analysis; further studies with larger sample sizes are needed in order to portray a more defined framework about the relationship between CM and psychological well-being.In conclusion, migraine is a debilitating neurological condition which disturbs daily activites, quality of life and psychological well-being of adolescents. In order to fully understand the mechanisms leading to this rather enigmatic association between migraine and psychiatric problems, futher studies with larger sample size and longitudinal design which use DSM-5 criteria are needed. We believe that revealing this relationship will enhance a multidisciplinary treatment approach which will cover somatic, cognitive and psychiatric aspects of the clinical complaints in adolescents with CM. Thereby, management of these conditions and pain will become easier and rational medication modalities will have positive effects on both individual and population-wide economies. Another point which should be emphasized is the need of detailed anamnesis for CM patients who were referred to emergency departments. Acquiring comprehensive psychological and neurological background of CM patients in emergency departments will not only guide effective treatment modalities; but also prevent negative radiation exposure and economical effects of unnecessary imaging techniques like x-rays or CTs.The study was conducted in accordance with the ethical guidelines, including the World Medical Association Declaration of Helsinki 2008, and the legal requirements of the Ethics Committee of the institution it was conducted in . All methods were carried out in accordance with relevant guidelines and regulations. Informed consent was obtained from from a parent. All experimental protocols were approved by a named institutional committee.


Hypertension is a major modifiable risk factor for arteriosclerosis that can lead to target organ damage (TOD) of heart, kidneys, and peripheral arteries. A recent epigenome-wide association study for blood pressure (BP) identified 13 CpG sites, but it is not known whether DNA methylation at these sites is also associated with TOD.In 1218 African Americans from the Genetic Epidemiology Network of Arteriopathy (GENOA) study, a cohort of hypertensive sibships, we evaluated the associations between methylation at these 13 CpG sites measured in peripheral blood leukocytes and five TOD traits assessed approximately 5 years later.P < 0.05). Mendelian randomization analysis suggests that methylation at two sites (cg16246545 and cg14476101) in PHGDH may causally influence UACR.Ten significant associations were found after adjustment for age, sex, blood cell counts, time difference between CpG and TOD measurement, and 10 genetic principal components (FDR q < 0.1): two with estimated glomerular filtration rate , six with urinary albumin-to-creatinine ratio , and two with left ventricular mass indexed to height . All associations with eGFR and four associations with UACR remained significant after further adjustment for body mass index (BMI), smoking status, and diabetes. We also found significant interactions between cg06690548 and BMI on UACR, and between 3 CpG sites  and diabetes on UACR (FDR q < 0.1). Mediation analysis showed that 4.7% to 38.1% of the relationship between two CpG sites (cg19693031 and cg00574958) and two TOD measures (UACR and LVMI) was mediated by blood pressure (Bonferroni-corrected In conclusion, we found compelling evidence for associations between arteriosclerotic traits of kidney and heart and previously identified blood pressure-associated DNA methylation sites. This study may lend insight into the role of DNA methylation in pathological mechanisms underlying target organ damage from hypertension. More than 100 million Americans have high blood pressure, which costs more than 50 billion dollars a year , 3. TOD N = 17,010) identified 13 significant CpG sites [Genome-wide association studies have identified genetic variation that influences TOD traits –13. EpigpG sites . In a crpG sites . HoweverHere, we investigate the association between the 13 DNA methylation sites associated with blood pressure and TOD of the heart, kidneys, and peripheral arteries in a cohort of largely hypertensive African Americans, and investigate whether these effects are modified by other risk factors for arteriosclerosis including body mass index (BMI), smoking status, and diabetes or are mediated by blood pressure.The Genetic Epidemiology Network of Arteriopathy (GENOA) is a community-based study designed to identify genes influencing blood pressure . In the N = 316 from 450 K and N = 1100 from EPIC). The Minfi R package [2 ratio of the intensity of the methylated probe versus the unmethylated probe [ComBat in R [DNA methylation was assessed, and bisulfite converted from genomic DNA extracted from stored peripheral blood leukocytes collected at GENOA Phase 1 using Illumina Infinium HumanMethylation BeadChips  less than 0.01. Imputation was performed separately by chip (Affymetrix or Illumina) using the 1000G Phase3 v5 reference panel, and post-imputation comparison between the two groups revealed no substantial differences in genotype and allele frequencies. SNPs with imputation quality less than 0.8 were removed before MR analysis.2.7, LVMI), and a measure of the peripheral arteries . TOD measurement methods are described in detail elsewhere [Measures of systolic blood pressure (SBP) and diastolic blood pressure (DBP) were the average of the last two of three sequential readings taken in a sitting position for at least 5 min at Phase 1 . SBP andlsewhere . BMI wasOutliers greater than four standard deviations from the mean of each TOD outcome and covariate were removed prior to analysis, and UACR was natural log transformed as ln (UACR+ 1). Ten genotype principal components (PCs) were included in all models to adjust for population stratification. Analyses were conducted in R (Version 3.4.1)  and SAS N = 422) were included. To examine whether the blood pressure-associated CpG sites from Richard et al. (2017) were also associated with SBP and DBP in the full GENOA sample with methylation data and TOD traits (N = 1212), we used a linear mixed effects model while accounting for family structure. Covariates included age at Phase 1, sex, BMI, smoking status, and 10 PCs.In the Richard et al. (2017) EWAS, a subset of GENOA African Americans with 450 K methylation data , we tested for interaction between the CpG site and each of the three risk factors  on the TOD measure. For each significant interaction, we plotted an interaction using a scatter plot by risk factor status (e.g. diabetes VS non-diabetes). The y-axis of each plot was a predicted value of TOD measure estimated from each linear regression model.mediation R package to examine whether the effects of the CpG sites on TOD measures are mediated through blood pressure [For significant associations between CpG sites and TOD measures prior to adding arteriosclerosis risk factors (FDR q < 0.1), formal mediation analyses were conducted using the pressure . We usedP < 0.1) both with a CpG site and a TOD measure, we performed a formal mediation analysis. We used a linear mixed effects model adjusted for family structure with covariates including sex, age when gene expression was measured, and 10 PCs. When DNA methylation M-value or gene expression level was the independent variable, it was pre-adjusted for sex and age before conducting the association analysis. In formal mediation analysis, we tested the indirect association between a CpG site and a TOD measure through changes in proximal gene expression levels using the bootstrapping method in the mediation R package [To examine whether the associations between CpG sites and TOD measures prior to adding arteriosclerosis risk factors (FDR q < 0.1) are mediated by gene expression levels, we evaluated the associations between 1) CpG sites and gene expression levels from genes that were located within ±1 Mb of the corresponding CpG sites, and 2) between the gene expression levels and TOD traits, separately. For gene expression levels that had at least a marginal association ( package .N = 1151 and FDR q < 0.05) [2 = 0.1 and physical distance threshold = 100 kb). The associations between IVs with CpGs and TOD measures were examined to obtain the beta and standard error estimates in GENOA African Americans. Inverse-variance weighting (IVW) was used to evaluate causation for each CpG on TOD measures. If the IV is comprised of only one variant, the Wald-ratio MR method was used to examine the causal effect [TwoSampleMR R package [P-value to be significent (P < 0.005). For each significant MR result, we created a scatter plot to depict the relationship of the SNP effects on the exposure against the SNP effects on the outcome using the same package. A line was drawn in each plot and the slope corresponds to the estimated causal effect from the IVW method. We used the MR Egger test to examine pleiotropy because IVW MR is invalid in the presence of horizontal pleiotropic effects of IVs [To investigate whether the previously identified CpG sites (FDR q < 0.1 prior to adding arteriosclerosis risk factors) might contribute causally to TOD measures, we performed Mendelian randomization (MR) analysis using 1000G imputed SNPs in GENOA African Americans. Since public mQTL databases and TOD GWAS data are limited in African Americans, we performed one-sample MR analysis using data from GENOA African Americans. Instrumental variables (IVs) for DNA methylation were drawn from cis-mQTL analysis in GENOA using SNPs within ±1 Mb of each corresponding CpG site using RVTEST ( < 0.05)  and cluml effect . Causal  package . We conss of IVs .2 (SD: 6.7), 12% of participants were current smokers, and 31% had diabetes.Descriptive statistics at Phase 1 (SBP and DBP) and Phase 2 (other variables) are shown in Table P < 0.05) with SBP and DBP, respectively, after adjustment for age, sex, BMI, and smoking status: cg19693031 and cg00574958 with both SBP and DBP; cg18120259 with SBP only (Table P < 0.0038) . Effect directions for nine CpG sites for SBP and 12 CpG sites for DBP were negative in our analysis, while the effect directions for all of the 13 CpG sites were negative in Richard et al. (2017).Three and two out of the 13 evaluated CpG sites had at a least marginal association  on TOD measures, we added interaction terms in the baseline association models. We found four significant interactions on UACR: cg06690548-BMI, cg14476101-diabetes, cg19693031-diabetes, and cg06690548-diabetes  through changes in blood pressure. Mediation analysis showed that seven associations between CpG sites and two TOD measures (UACR and LVMI) were significantly mediated by blood pressure  with both the corresponding CpG site and the TOD trait . We then performed a formal mediation analysis for these five associations but found no significant (P < 0.05) mediating effect of gene expression on the associations.Using the 10 significant CpG site-TOD measure associations prior to adding arteriosclerosis risk factors (FDR q < 0.1), we conducted a 2-step association analysis to identify potential mediating effects of gene expression level: an association between each CpG site and expression of proximal genes, and an association between gene expression and the TOD measure. A total of five genes with expression data that were located within 1 Mb of a corresponding CpG site were at least marginally associated  and cg14476101  tests to examine forward causality of DNA methylation on TOD. The results of MR analysis of CpG-TOD association are shown in the Table Scis gene expression mediating the associations between CpG sites and TOD measures. MR analysis provided supporting evidence of the forward direction of association for 2 CpG sites located near PHGDH with UACR.We identified ten significant associations between previously identified CpG sites associated with blood pressure and TOD measures including eGFR, UACR, and/or LVMI in a sample of 1218 African Americans. Half of these associations remained significant even after further adjustment for other TOD risk factors including BMI, smoking status, and diabetes. Among the CpG sites from the ten significant associations, one significant interaction with BMI and three significant interactions with diabetes on UACR were identified in an interaction analysis. Seven associations between CpG sites and two TOD measures were partially mediated by blood pressure (4.7 to 38.1%), but there was no significant Only a small portion of previously identified CpG sites from Richard et al. (2017) were also significantly associated with blood pressure in GENOA after multiple testing correction (three out of 13 CpG sites). Syme et al. (2019) found three out of the 13 CpG sites had significant associations with blood pressure in 135 adolescents and 110 middle-aged adults . Most ofResults from the mediation analysis suggests the significant associations between the CpG sites and TOD are not always mediated through blood pressure. Since the GENOA participants were selected based on their own or their sibling’s hypertension status, the average blood pressure in GENOA was elevated compared to the general population. Some of these CpG sites from Richard et al. (2017) may operate at a lower blood pressure level, but may have weaker effects at higher blood pressures, so associations may not be as pronounced in GENOA. Despite the lower rate of replication with blood pressure, we found 10 significant associations with TOD measures in the kidney and heart .SLC7A11), cg19693031 (TXNIP), and cg00574958 (CPT1A) and TOD in the kidney even further after adjustment for BMI, smoking status, and diabetes. cg06690548 (SLC7A11), cg19693031 (TXNIP) and cg00574958 (CPT1A) have previously been associated with triglyceride levels, in addition to blood pressure, in EWAS studies [PHGDH that may causally influence affect UACR. PHGDH encodes an enzyme which facilitates amino acid L-serine biosynthesis. Disturbances in serine metabolism are associated with endothelial dysfunction, inflammation, and atherosclerosis in the kidney, and there is evidence that the mechanism of these effects may be through the 1-carbon (DNA methylation) metabolism pathway [We identified significant associations between cg10601624, cg06690548  or those who were obese (cg06690548 in SLC7A11). PHGDH is previously reported to be involved in the progression of glucose intolerance through serine biosynthesis in mice adipose tissues [TXNIP is increased in the presence of high glucose, which promotes oxidative stress and β-cell apoptosis [The inverse association between DNA methylation and UACR was stronger in those with diabetes , we ascertained the relationships between TOD measures and previously identified DNA methylation levels associated with blood pressure using state-of-the-art analytic methods in a population with a high burden of hypertension. In GENOA, TOD traits were measured approximately 5 years later than methylation, thus helping to establish the temporal relationship between changes in methylation and TOD. We were also able to characterize the direction of association using MR techniques. However, for two CpG sites (cg06690548 and cg10601624), we were not able to examine the causal direction of the association because of the lack of appropriate instruments. Also, despite of the five-year lag between measurements, the level of TOD trait could have been changed before methylation level changed. In addition, we lacked the ability to account for the duration of hypertension, which preceded DNA methylation for over two thirds of the study population. When we adjusted for hypertension in the baseline association models, the significance remained for eGFR and UACR, but attenuated in LVMI. Studies with longitudinal measures of DNA methylation at multiple timepoints may help better elucidate these temporal relationships.PHGDH in the kidney, potentially through endothelial dysfunction, inflammation, and atherosclerosis. Future research should further confirm these initial findings in populations of other ancestries. This study may lend insight into the role of DNA methylation in complex pathological mechanisms underlying target organ damage from hypertension and provide potential clinical targets for treatment and prevention.In conclusion, we found compelling evidence for associations between arteriosclerotic traits of kidney and heart and previously identified blood pressure-associated DNA methylation sites in a cohort of older African Americans. For the identified associations, we additionally examined the interactions between CpGs and other risk factors, potential mediation by blood pressure, and the causal relationship connecting CpG sites and TOD measures. We found evidence supporting the causal relationship of epigenetic regulation of Additional file 1: Table S1. Interactions between CpG sites and target organ damage risk factors including BMI, current smoking, and diabetes on target organ damage phenotypes in GENOA African ancestry population. Table S2. Results of mediation analysis of CpG sites-systolic blood pressure-target organ damage phenotypes in GENOA African ancestry population. Table S3. Results of mediation analysis of CpG sites-systolic blood pressure-target organ damage phenotypes adjusted for BMI, current smoking, and diabetes in GENOA African ancestry population. Table S4. Mendelian randomization results showing the inverse-variance weighted effects of multiple cis-SNP used as instrumental variables in the association of DNA methylation and target organ damage measures in GENOA African ancestry population. Figure S1. Interaction plots between a CpG site and the traditional risk factors for arteriosclerosis (BMI and diabetes) on urine albumin-to-creatinine ratio (UACR). Figure S2. Mendelian randomization (MR) scatterplot of urine albumin-to-creatinine ratio vs. CpG with estimates from inverse-variance weighted (IVW) MR using cis-SNPs within ±1 Mb of corresponding CpG site in GENOA African Americans.


Routine clinical and pathological examinations usually cannot fully conclusively determine the relationship between different lesions of lung cancer. Detailed genetic analysis of tumor samples may supply important additional information and identify second primary lung cancers.EML4–ALK rearrangement of the squamous cell carcinoma.In the present study, we report a case of synchronous multiple primary lung cancer (MPLC) composed of two distinct pathological subtypes with epidermal growth factor receptor (EGFR) gene mutations L858R of the acinar adenocarcinoma subtype and The present report highlights the clinical importance of molecular cancer biomarkers detection to guide management decisions in MPLC cases. EML4–ALK) rearrangement.Multiple primary lung cancer (MPLC) is defined as two or more primary lung cancers occurring in the same patient and can be classified into synchronous or metachronous multiple primary lung cancer based on the time of occurrence . NumerouA 76-year-old male smoker with no family history was admitted to our hospital because of cough. Chest computed topography (CT) showed a 6 cm well-defined mass in the right lower lung, while another faint nodular lesion with a ground glass pattern and cavity was detected in the right middle lobe, together with emphysema and interstitial changes Fig. . A positALK-EML4 rearrangement in the lung adenocarcinoma and squamous cell carcinoma encouraged us to explore the tumor origin, and both tumors presented as treatment-naïve mutants occurring synchronously.Despite the low incidence of MPLC in non-small cell lung cancer (NSCLC) , 5, earlALK rearrangement is one of the crucial molecular alterations in NSCLC, especially in adenocarcinoma with the incidence between 2 and 13% . HoweverEML-4ALK rearrangement in squamous cell carcinoma. Our case was confirmed as stage I for adenocarcinoma (T1aN0M0), and thus the EGFR-TKI was not indicated. There are two main findings. First, the ground glass opacity in the middle lobe was not metastasized from the huge mass in the right lower lobe. Treatment naïve adenocarcinoma and squamous cell carcinoma occurred together in the same patient was rare, highlighting the importance of radical surgical intervention to improve the prognosis. Second, to the best of our knowledge, ALK fusion and EGFR L858R mutations in separate squamous cell carcinoma and adenocarcinoma diagnosed at the same time has not been reported. It showed that various kinds of driver gene mutations can present in different lobes simultaneously even from the very beginning [EGFR mutations have also frequently been reported in MPLC patients, whereas complicated mutations  were only found in female patients . Our maleginning . Complex


Previous studies have shown that the Vav1/Rac1 pathway affects the macrophage cytoskeleton and cell migration, but its role in the pathogenesis of diabetic atherosclerosis is unknown.  Macrophage-derived foam cells play a central role in atherosclerosis, and their ultimate fate includes apoptosis, promotion of vascular inflammation, or migration to other tissues. N−/− mouse model and primary peritoneal macrophages, respectively. The results showed that the atherosclerotic plaques of diabetic foot amputation patients and diabetic ApoE−/− mice were larger than those of the control group. Inhibition of the Vav1/Rac1 pathway reduced vascular plaques and promoted the migration of macrophages to lymph nodes. Transwell and wound healing assays showed that the migratory ability of macrophage-derived foam cells was inhibited by CML. Cytoskeletal staining showed that advanced glycation end products inhibited the formation of lamellipodia in foam cells, and inhibition of the Vav1/Rac1 pathway restored the formation of lamellipodia.  In this study, we used anterior tibiofibular vascular samples from diabetic foot amputation patients and accident amputation patients, and histological and cytological tests were performed using a diabetic ApoE CML inhibits the migration of foam cells from blood vessels via the Vav1/Rac1 pathway, and this process affects the formation of lamellipodia. The recruitment of macrophages to the intima and phagocytosis of lipids to form foam cells is an essential step in the progression of atherosclerosis , 2. Regaε-Carboxymethyl-lysine (CML) is a major active ingredient in advanced glycation end products. Studies have shown that Nε-carboxymethyl-lysine can promote foam cell formation, inhibit foam cell migration, and accelerate the progression of atherosclerosis, but the specific mechanism is not yet clear [Net clear , 6. Vav1et clear . Rahamanet clear , 9; in vet clear , but wheMembers of the Rho GTPase family play an important role in intracellular signaling pathways. Previous studies have shown that the Rho GTPase family can participate in intercellular function changes, including cell adhesion, contraction, proliferation, and migration, by regulating the polymerization state of the cytoskeleton . During n = 4) and from normal amputees (n = 4) were obtained from the Affiliated Hospital of Jiangsu University  from February 2015 to June 2017. This study was approved by the Chinese Clinical Trial Registry and conducted in agreement with the institutional guidelines. Written informed consent was obtained from all patients. Ethical review number is ChiECRCT20190206.Anterior tibial arteries from human diabetic amputees (-\- mice with a C57BL/6J background were obtained from the Jackson Laboratory (USA). The diabetic ApoE-\- model was established as described previously. In brief, at 6 weeks of age, the mice were rendered diabetic through intraperitoneal injection for five consecutive days of 40 mg/kg STZ dissolved in 100 mM citrate buffer (pH 4.5). Mice with blood glucose levels of ≥300 mg/dL after 2 weeks of STZ administration were considered diabetic.The experimental protocols were approved by the Institutional Animal Care and Use Committee of Jiangsu University  and performed in accordance with the Guidelines for Animal Experimentation of the National Institutes of Health. Male ApoEφ) were harvested from C57BL/6 mice 4 days after thioglycollate injection by peritoneal lavage as described. Isolated PMφ were plated on glass coverslips. After 1 hour of incubation at 37°C, nonadherent cells were removed by gentle washing, and the remaining cells were cultured overnight in DMEM containing 10% FBS before use in the phagocytosis assay.Peritoneal macrophages .Atherosclerotic lesions in the aorta or para-aortic lymph nodes of ApoEn = 4/group) were injected intraperitoneally with 3% thioglycollate to trigger sterile peritonitis. After 4 days, peritoneal macrophages were labeled by injection of 1 μm Fluoresbrite green fluorescent plain microspheres (Polysciences). On the next day, the mice were injected with an inflammatory stimulus (400 ng LPS) to induce efflux of macrophages from the peritoneum to the draining lymph nodes. Following LPS injection (3 hours), the percentage of macrophages in the lavage fluid was quantified by flow cytometry using the PE/Cy7-conjugated anti-mouse F4/80 antibodies (BioLegend).The mice  containing 1 mmol/L PMSF protease inhibitor  and phosphatase inhibitors  on ice and then centrifuged at 12,000 g and 4°C for 15 min. Protein concentrations were determined using a micro BCA protein assay kit . Protein samples were loaded onto a polyacrylamide gel and transferred onto PVDF membranes by using a semidry method. The membranes were blocked in 5% milk in TBST, followed by overnight incubation with primary antibodies. The membranes were subsequently washed with TBST and then incubated with HRP-conjugated secondary antibodies. Immunoreactive bands were visualized by chemiluminescence (ECL Amersham Pharmacia). Protein expression levels were quantified by densitometric analysis by using LANE 1D software.t-test. Comparisons among multiple treatment groups were assessed by one-way ANOVA, followed by a post hoc LSD test. P < 0.05 was considered statistically significant.Data are presented as the mean ± S.D., and SPSS 17.0 software was used to analyze the data. Comparisons between two variables were analyzed using the unpaired Student's Anterior tibial arteries from diabetic and control amputees (accident amputation) were obtained from the Affiliated Hospital of Jiangsu University. Masson's staining revealed that the fiber cap of atherosclerosis lesion in diabetic amputees was thinner than that in control amputees . We perfThe Vav family acts as important guanine exchange factors (GEFs) that regulate the activity of small GTPases. By regulating the binding and dissociation of small GTPases and GTP in atherosclerosis, we detected intravascular small GTPase activity in patients with diabetic foot amputation and accident amputation. The results showed that enzymatic activity of the small GTPase family members Rac1 and RhoA (with GTP-binding state) increased, but the expression of another small GTPase, Cdc42, did not change significantly .−/− mouse model based on previous methods in our group and administered Vav1 antibody to ApoE−/− mice. Oil Red O staining and CD68 immunogold staining showed increased lipid accumulation in the plaques of high-fat diet-fed ApoE−/− mice, increased foam cells in plaques, vascular plaque area in diabetic ApoE−/− mice, and lipid accumulation in foam cells. The ApoE−/− mice fed with a high-fat diet had further elevation of these factors, and the antibody interfered with the expression of Vav1  are formed. CDC42 was later shown to promote and activate Rac, and there is considerable evidence that the activity of Rac maintains the directional front end of protruding lamellipodia . In contOur data provide additional research support for the final fate and specific matrix of foam cells in diabetes atherosclerosis and validate the role of peritoneal macrophage efflux experiments in foam cell migration studies in diabetic mice, while the role of Vav1/Rac1 in advanced glycation end products inhibiting foam cells needs to be further elucidated. The Vav1/Rac1 pathway inhibits foam cell migration by inhibiting the formation of lamellipodia in macrophages, and blocking each loop of this pathway inhibits the effect of advanced glycation end products. These findings provide further understanding of new ways to promote the regression of atherosclerotic plaques.