In recent years multiple novel influenza A strains have emerged in humans. We reviewed publically available data to summarise epidemiological characteristics of distinct avian influenza viruses known to cause human infection and describe changes over time. Most recently identified zoonotic strains have emerged in China  – these strains have occurred mostly in association with visiting a live bird market. Most zoonotic AIVs and swine influenza variants typically cause mild infections in humans however severe illness and fatalities are associated with zoonotic H5N6, H10N8, H7N9 and H5N1 serotypes, and the H1N1 1918 Spanish Influenza. The changing landscape of avian influenza globally indicates a need to reassess the risk of a pandemic influenza outbreak of zoonotic origin.The online version of this article (doi:10.1186/s13690-017-0182-z) contains supplementary material, which is available to authorized users. Following the emergence of the zoonotic influenza A H5N1 in humans in 1997, Hong Kong, there was global concern that the virus would evolve to become capable of human to human transmission causing a pandemic similar to the 1918 Spanish flu pandemic which killed over 50 million people  pall influenza A serotypes known to cause human infections. The aim of this report is to identify trends or changes in the epidemiology and characteristics of zoonotic influenza A emergence in humans over time.We previously compared the novel H7N9 which emerged in humans in Shanghai, 2013 to the more extensively studied H5N1 and highlighted some puzzling differences in the epidemiology [For all zoonotic avian influenza virus AIV serotypes except H5N1 and H7N9 , clinical signs, mortalities, details of animal exposure and pathogenic classification of virus. Highly pathogenic (HPAI) outbreaks in poultry typically cause acute, severe mortalities and have significant economic implications due to the severe control measures  imposed following outbreaks. Low pathogenic (LPAI) outbreaks in poultry occur more commonly, have less clinical significance, and are harder to detect. Extracted information is provided in Additional file Details of individual outbreaks and isolated cases were not extracted for non-zoonotic influenza A strains which have caused human infections , as it is outside the scope of this study to record details on the enormous amount of human influenza cases, and their epidemiology have been detailed extensively in previous review articles , 4. SimiWe summarised features of all human and zoonotic serotypes of influenza A which have been documented to cause human infections Table . The emeGenetically distinct influenza A reassortants have emerged in humans on a total of 19 separate occasions since 1918. Of these, 6 strains are able to be efficiently transmitted from human-to-human, 10 are predominantly zoonotic AIVs, and 3 are predominantly zoonotic swine influenza variants.The rate of novel strains emerging in humans has increased in recent years Fig. . In the n = 3), Australia (n = 1) and Hong Kong (n = 2) and from 2013, all novel AIVs have emerged in different geographic regions in China (n = 3) and Taiwan (n = 1).Swine influenza variant viruses  have all emerged in humans in the United States (US) in July 2011, December 2011 and June 2012 respectively , whilst infections caused by exposure to pets, wild animals, or laboratory animals have rarely been documented. In developed countries , human cases were linked to poultry farms  rather than close contact with poultry . HoweverAll zoonotic influenza infections have typically occurred infrequently, in a sporadic pattern typical of animal to human transmission without sustained human to human transmission ensuing. However there are few incidences of larger scale outbreaks which have presented with more cases than would be expected in sporadic transmission: (i) large numbers of H7N9 human cases have occurred each year since it’s emergence in 2013  thMost zoonotic AIVs 6 of 10) and all 3 swine influenza variants typically cause mild infections in humans  improvements in zoonotic AIV case ascertainment, and (ii) a “true” increase in AIV emergence, which could be explained by an increase in AIV circulation and diversity in poultry populations, growths in the poultry industry, and increased human urbanisation.Advances in influenza diagnostics and surveillance capabilities, as well as increased clinician awareness , have likely contributed to better ascertainment of influenza A in humans in China. In the past, AIV reporting have likely been hindered industry and government pressure – in 1994 for example, after H9N2 was discovered in humans in China, further investigations were discouraged by Chinese government officials . In contThis study find there have not been any reports of AIV emergence in humans in low-income, developing countries – this may also be due to case ascertainment bias. Developing countries are both unable to support high levels of active AIV surveillance , and highly regulated agricultural systems with the ability to enforce disease control regulations. Further, poor biosecurity measures at the human-animal interface are common in these countries, which allow for virus to more easily transmit to humans. In recent years, several countries in west and central Africa experienced a resurgence of H5N1 poultry outbreaks: Cameroon, Burkina Faso, Niger, Cote d’Ivoire, Ghana, Nigeria and Nigeria (in these countries reports of H5N1 outbreaks had last occurred in 2006 – 2009) . There iThe increase in AIV emergence in humans may also be a reflection of increased AIV persistence and diversity in poultry. Following the expansion of the poultry industry from the mid-1980s (particularly in China and some other Asian countries) a broad range of AIV lineages have evolved to circulate among domestic poultry species . Prior tThe extent of global AIV persistence and diversity may also be reflected by recently described uncharacteristic AIV outbreaks in animals. For example in high-income countries HPAI incursions have traditionally been quickly contained and eliminated through standard disease elimination protocols, however in 2014 and 2015 HPAI caused unprecedented numbers of outbreaks in commercial poultry farms in the US, and similar outbreaks have occurred in Europe in the past year . AIV traIncreasing urban and agricultural encroachment into previously uninhabited areas, particularly in developing countries, may also play a role in promoting AIV transmission to humans and domestic animals from wild bird reservoir hosts. Wild migratory birds  are thought to be the traditional hosts of AIVs and require use of wetlands and lakes for breeding and wintering. Reduction of these natural habitats for wild birds and can result in birds using agricultural or urbanised areas, promoting interaction with high density farming systems (such as poultry and swine), and also humans. Such close interactions increase the risk of AIV introduction, and zoonotic disease transmission – we recommend AIV control programmes in focus on preventing such interactions particularly in African and Asian countries which are urbanizing rapidly.Zoonotic AIV infections have been sporadically reported in developed regions such as North America, Europe and Australia, with the most recent report occurring in 2013 in Italy. All were caused by subtypes other than HPAI H5N1. Previously it was thought that only H5 and H7 HA types posed a significant pandemic risk, however strains of other HA types  have been able to infect humans and have pandemic potential. The inability to predict which AIV types can infect humans, combined with sheer variety of AIVs circulating in poultry, makes it challenging to focus pandemic preparedness measures to specific strains. A good example is the efforts in pandemic planning globally around H5N1 from 2005 to 2009, which assumed the next pandemic would be H5 related. However, the pandemic which emerged in 2009 was a completely different virus, unrelated to H5N1. Pandemic planning should instead incorporate interventions to prevent the species jump and emergence of a human pandemic strain of influenza.We find there have been recent increases in the number of reports of AIVs infecting humans, predominantly from mainland China. We recommend pandemic preparedness measures focus on preventing zoonotic disease emergence, specifically the strengthening of control efforts to reduce (i) potential introductions of AIVs into poultry populations, (ii) subsequent spread within the poultry sector, and (iii) virus transmission at the human-animal interface (particularly within LBMs). Regional disparities should also be considered. Low-income countries which are undergoing rapid commercialisation of their poultry sector are at highest risk of harbouring AIVs, and are unlikely to detect nor adequately control AIV spread.Additional file 1: Table S1.Epidemiological features of reported outbreaks or isolate cases of distinct avian influenza serotypes. This is a table showing the information we have collected on reported outbreaks or isolate cases of distinct avian influenza strains. (DOCX 70 kb)Additional file 2: Text 1.The following is the reference list for Table


Eating habits, lifestyles, and exposure to specific environmental factors can greatly impact the risk of developing type 2 diabetes (T2D), influence the genome epigenetically, and affect the expression of genes, including genes related to glycemic control, at any stage of life. The epigenetic mechanism underlying obesity and T2D pathogenesis remains poorly understood. Conventional strategies for the treatment of obesity and its comorbidities often have poor long-term adherence, and pharmacological interventions are limited. Bariatric surgery is the most effective current option to treat severe obesity, and Roux-en-Y gastric bypass (RYGB) is the most applied technique worldwide. Epigenetic changes differ depending on the approach used to treat obesity and its associated comorbidities . Compared to primary clinical care, bariatric surgery leads to much greater loss of body weight and higher remission rates of T2D and metabolic syndrome, with methylation profiles in promoter regions of genes in obese individuals becoming similar to those of normal-weight individuals. Bariatric surgery can influence DNA methylation in parallel with changes in gene expression pattern. Changes in clinical biomarkers that reflect improvements in glucose and lipid metabolism after RYGB often occur before major weight loss and are coordinated by surgery-induced changes in intestinal hormones. Therefore, the intestine methylation profile would assist in understanding the mechanisms involved in improved glycemic control after bariatric surgery. The main objectives in this area for the future are to identify epigenetic marks that could be used as early indicators of metabolic risk, and to develop treatments able to delay or even reverse these epigenetic changes. Studies that provide the “human epigenetic profile” will be of considerable value to identify tissue-specific epigenetic signatures and their role in the development of chronic diseases. Further studies should apply methods based on global analysis of the genome to identify methylated sites associated with disease and epigenetic marks associated with the remodeling response to bariatric surgery. This review describes the main epigenetic alterations associated with obesity and T2D and the potential role of RYGB in remodeling these changes. Epigenetics concerns mechanisms for modifying gene expression at the transcriptional level, by chemical modifications of DNA and RNA chromatin. These modifications are heritable by meiotic or mitotic processes, impact gene function, and cannot be explained by changes in the DNA sequence . EnvironIn contrast to gene changes, epigenetic modifications are reversible. This characteristic may allow for the identification of novel therapeutics based on functional gene restoration. Therefore, an understanding of epigenetic mechanisms can be clinically relevant for the treatment of some chronic diseases , 4. ThisAll of the cells in a given organism’s tissues have identical DNA. This genetic material contains key information to perform any function, although not all genes are expressed at once. Moreover, DNA does not determine all of the cells’ features. Gene regulation, through epigenetic marks in the DNA or cell nucleus proteins, allow each cell to acquire its specific function . Environ3) to a cytosine base in DNA, converting cytosine into 5-methylcytosine  sequences, termed dinucleotides or CpG islands. There are approximately 29,000 CpG islands in the human genome . CpG islThe DNA methylation pattern is not uniform among eukaryotes or between species . MethylaThe influence of DNA methylation on gene expression appears to depend on where methylation takes place in the gene sequence . MethylaA key discovery in epigenetics science was that DNA 5mC may be oxidized by a family of α-ketoglutarate–dependent oxygenases known as ten-eleven translocation (TET) proteins. These proteins originate 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5FC), and 5-carboxylcytosine (5caC). Thymine DNA glycosylase (TDG) can further process these 5mC derivatives, which are submitted to base excision repair or replication-dependent dilution, thereby leading to DNA demethylation . The 5mCIn short, epigenetic marks, which can be reversible, may change the way that gene transcription is controlled in the cell. Recent discoveries have increased interest in the role of epigenetic changes in the development of chronic diseases, as well as in potential therapeutics that can reverse these changes .Different forms of genetic variability, particularly single nucleotide polymorphisms (SNPs), have been associated with the current obesity and T2D epidemic . Some poDNA methylation varies among healthy individuals due to a combination of genetic and environmental influences and stochastic events, such as surgery for weight loss . StudiesSeveral studies have assessed the relation between site-specific DNA methylation and obesity. These studies focused on potential gene targets for obesity, appetite control, and insulin signaling, some of which were selected based on previous findings of altered gene expression in the same subject. Most of these studies had a cross-sectional design , preventing researchers from being able to establish whether DNA methylation is a cause or a consequence of the obesity phenotype .TNF) and leptin (LEP) genes and high levels of methylation in proopiomelanocortin (POMC) and aryl hydrocarbon receptor nuclear translocator-1 (BMAL1) genes were identified in whole blood samples or peripheral blood leukocytes of obese subjects , as well as altered methylation and gene expression levels for 102 genes, including cyclin dependent kinase inhibitor 1A (CDKN1A), phosphodiesterase 7B (PDE7B), septin 9 (SEPT9), and exocyst complex component 3 like 2 (EXOC3L2). Importantly, methylation of these genes can affect pancreatic cell function , ACLY, and phospholipase C gamma 1 [PLCG1]) and insulin signaling  were associated with NAFLD. When the analysis was replicated in 39 independent liver samples from NAFLD obese patients, high transcription levels in CpG regions of zinc finger protein 274 (ZNF274), PGC1A, and sterol regulatory element binding transcription factor 2 (SREBP2) were associated with liver disease [n = 23) underwent bariatric surgery and were reevaluated 5–9 months after this procedure. Despite the expected improvement of liver histology, the intraindividual comparison of liver biopsies before and after bariatric surgery highlighted methylation changes associated with liver disease, which appeared to be partially reversible by the surgery. An inverse correlation of the NASH phenotype and bariatric surgery was observed, indicating that methylation changes associated with NASH can be reversed after surgery. Specific methylation signatures of NAFLD-related genes  were clearly distinct after the bariatric procedure, both in gene ontology and transcription factor binding site analyses. The receptor protein tyrosine phosphatase epsilon (PTPRE)-encoding gene exhibited altered expression and methylation patterns before and after surgery. Hypermethylation and low expression of PTPRE are negative regulators of insulin signaling in skeletal muscle, suggesting that changes in its expression and methylation after bariatric surgery may be involved in the hepatic insulin sensitivity restoration observed after the procedure [Organ-specific epigenetic signatures after bariatric surgery have been identified in organs involved in obesity and T2D pathogenesis. Ahrens et al. obtained liver biopsies from 63 patients, classified histologically as normal controls (enotypes . Altered disease . Some ofrocedure .(HDAC4), solute carrier family 37 member 3 (SLC37A3), and DENN domain containing 1 (DENND1C), also known as FAM31C, in subcutaneous adipose tissue, correlated with fasting glycemia levels. Significantly different mRNA levels of all studied genes presenting with methylation in their promoter regions were found when comparing before and after RYGB. These findings suggest that altered DNA methylation of adipose tissue may have a role in obesity development and can be changed by RYGB, potentially contributing to glucose homeostasis [Adipose tissue plays a key role in the metabolic disorder related to obesity. Another study on organ-specific epigenetic signatures after bariatric surgery assessed DNA methylation profiles in 15 obese women before and a mean of 17.5 months after RYGB, when patients had achieved up to 27% of body weight loss . After Reostasis .Despite the benefits associated with metabolic and weight loss responses to bariatric surgeries, patients who undergo these procedures still exhibit a higher risk of chronic diseases (including T2D relapse) and mortality compared to the general population . Postobe2) and from 14 never-obese women (BMI 25 ± 2 kg/m2), both presenting stable weight for the previous 6 months. Gene expression was assessed in the adipose tissues of 9 women from each group. Compared to the never-obese group, the bariatric surgery group showed significant global DNA hypomethylation and a greater number of differentially methylated sites in adipogenesis genes. In a secondary analysis, the authors assessed DNA methylation and expression of adipogenesis genes in 15 and 11 obese women, respectively. Differential methylation of adipogenesis genes was accompanied by altered expression levels in obese but not postobese women. The authors concluded that altered methylation of adipogenesis genes in fat cells can contribute to adipose tissue hyperplasia in women after bariatric surgery [A clinical study tested the hypothesis that differentially methylated DNA sites are associated with adipose tissue hyperplasia after bariatric surgery-induced weight loss. Genome-wide DNA methylation was analyzed in abdominal subcutaneous fat cells collected from 16 women 2 years after gastric bypass surgery with significant weight loss  were significantly lower in obese patients presenting relevant weight loss after bariatric surgery. Mechanisms dependent on the inhibitor of plasminogen activator (PAI-1) encoded by SERPINE-1 are involved in the pathogenesis of obesity, IR, and T2D [SERPINE-1 have been associated with obesity and metabolic syndrome [SERPINE-1 gene methylation may be a predictive marker of weight loss response to RYGB [Current findings are aiding in the identification of epigenetic markers with potential predictive value for clinical responses induced by the surgical procedure . For ins and T2D , and metsyndrome . Therefo to RYGB .® emerging as the most widely used platform for this purpose. This method analyzes more than 450,000 methylation regions in CpG sites and other nearby regions, providing reproducible quantitative data that could facilitate comparisons across studies [Thanks to advances in next-generation technologies, the field of epigenetics is growing quickly. Not long ago, researchers believed that DNA methylation represented only a gene-silencing mark, and that its increase in promoter regions was associated with decreased gene expression. Recent studies suggest that DNA methylation also affects noncoding RNA expression, transcriptional elongation, splicing events, and overall genomic stability, depending on the genomic location in CpG regions where methylation occurs . Some of studies .Epigenetic researchers in the areas of obesity and T2D seek to identify biomarkers that can predict the risk of developing these diseases and their associated complications. They aim to understand the environmental factors related to obesity, which can modulate gene expression by affecting epigenetic mechanisms. Such understandings would allow the design of new therapeutic strategies. However, the characterization of all of the factors able to modify epigenetic signatures and their true importance for obesity and T2D are affected by the small and cumulative magnitude of changes produced by dietary and environmental factors, which differ largely between biological samples .Peripheral blood is the DNA source most applied in epigenetic studies because of the ease of analysis . HoweverRecent findings suggest that epigenetic changes in specific tissues of patients with T2D are at least partially co-regulated by variations in DNA sequence. Body weight reduction can have a dynamic effect on the epigenome. Considering the role of obesity in T2D development, the epigenome is an innovative target for pharmacological and environmental therapeutic interventions . The idePotential biomarkers for obesity can be detected early, enabling prediction of disease risk at a young age, before the phenotype develops. This observation opens the promising possibility to design intervention strategies for the prevention of obesity and, consequently, T2D. Bariatric surgery can influence DNA methylation in parallel with changes in gene expression pattern. Weight loss that can be achieved by these procedures is associated with changes in the methylation of CpG sites and between exon regions near the transcription sites. Dynamic changes in DNA methylation might be an early event that drives the transcription of genes involved in the orchestral regulation of insulin sensitivity in human obesity .Epigenetic regulation seems to be tissue specific; therefore, it is important to investigate a homogeneous cell type or tissue from a target organ for development of the studied disease. The pancreatic islet is one of the most thoroughly investigated cell types, and the liver, muscle, and adipose tissues are the main investigated tissues for T2D. Changes in clinical biomarkers that reflect improvements in glucose and lipid metabolism after RYGB often occur before major weight loss and are coordinated by surgery-induced changes in intestinal hormones. Therefore, the intestine has been the subject of major studies on T2D and bariatric surgery , 66. TheIn conclusion, evidence supports a potential role of epigenetic changes in obesity and T2D pathogenesis. Epigenetic profiles seem to be altered by bariatric surgeries towards metabolic homeostasis. The main objectives in this area for the future are to identify epigenetic marks that could be used as early indicators of metabolic risk, and to develop treatments able to delay or even reverse these epigenetic changes. Further studies should apply methods based on global analysis of the genome  to identify methylated sites associated with disease and epigenetic marks associated with the remodeling response to bariatric surgery.


Capecitabine has been commonly used in recurrent or metastatic nasopharyngeal carcinoma. However, radiation recall after capecitabine for nasopharyngeal carcinoma has not been reported.We report the case of a 64-year-old Chinese woman with locoregionally advanced nasopharyngeal carcinoma previously treated with induction chemotherapy followed by concurrent chemoradiation 6 years ago. She developed cervical, mediastinal, and abdominal nodal relapses 14 months later. She then received capecitabine with initial excellent tumor response for 1 year but disease recurrence was noticed at the peripancreatic nodal region, which was successfully treated with concurrent chemoradiation with capecitabine. Unfortunately, she developed progressive erythema of the face and neck region at exactly the previous irradiation site for her initial nasopharyngeal carcinoma, 2 months after taking capecitabine. She initially ignored it, but it became more confluent and serious. Eventually, a facial skin biopsy was performed showing nonspecific chronic inflammation only. The diagnosis was most likely radiation recall phenomenon since capecitabine was the only drug she received before development of this dermatological manifestation on her previously irradiated face and neck. Treatment was conservative and supportive albeit with no significant clinical improvement.Radiation oncologists should be aware of this potential risk of capecitabine, especially when it is administered for a long period of time. Radiation recall is a rare and acute dermatological condition typically occurring at the site of previous radiation therapy, after administration of a certain type of oral or intravenous medication particularly chemotherapeutic agent(s) or, less likely, targeted therapies –4. Skin Capecitabine, a prodrug of 5-fluorouracil, has been commonly employed in the treatment of breast cancer, upper and lower gastrointestinal cancer, and pancreatic cancers and thus all patients who were previously reported to suffer from radiation recall after capecitabine had their primary tumors in these tracts –11. Cape2 twice daily from day 1 to 14 and every 3 weeks afterward and titrated up to 1250 mg/m2 twice daily from day to 1 to 14 every 3 weeks from cycle 3 onward with a significant drop of plasma EBV DNA to 0 copies/mL after taking capecitabine for 7 months. She continued capecitabine up to 1 year until further disease progression with increasing in size of the peripancreatic lymph node metastasis with plasma EBV DNA elevation to 64 copies/mL. In view of the solitary peripancreatic nodal disease, she received conformal radiotherapy (50 Gy in 25 fractions over 5 weeks) to the peripancreatic lymph node concurrent with capecitabine 825 mg/m2 twice daily and 5 days a week on radiotherapy days only. Plasma EBV DNA dropped to 0 copies/mL and her PET-CT scan 9 months later confirmed complete metabolic response of the peripancreatic lymph node. Capecitabine was then stopped after the complete response demonstrated in this latest PET-CT scan. Unfortunately, she noticed that her face gradually developed progressive erythema and increasing warmth with relative sparing of the nostrils and perinasal region 2 months after taking capecitabine. It was initially ignored by our patient until the skin erythema became more confluent and prominent at the irradiation site of the previous IMRT for her initial NPC  6 years ago. Pretreatment plasma Epstein-Barr virus (EBV) deoxyribonucleic acid (DNA) was 827 copies/mL. She received induction chemotherapy consisting of one cycle of cisplatin and 5-fluorouracil and two more cycles of carboplatin  plus 5-fluorouracil in view of her deteriorating renal function, followed by radical chemoradiation using carboplatin concurrent with intensity-modulated radiation therapy (IMRT) with 70 Gy in 33 fractions to the gross primary tumor and neck nodes and 66 Gy in 33 fractions to the high-risk primary and neck nodal regions, all over 6.5 weeks delivered by Simultaneous Accelerated Radiation Therapy (SMART) technique completed 5 months later. Posttreatment plasma Epstein-Barr virus deoxyribonucleic acid (EBV DNA) became undetectable at 8 weeks after IMRT. Unfortunately, her disease relapsed 14 months later with lymph node metastases to the left supraclavicular fossa and the right superior mediastinal region, together with a distant lymph node metastasis at the peripancreatic region revealed by [18 F]fluorodeoxyglucose positron emission tomography with integrated computed tomography (PET-CT) (Fig. NPC Fig. . It did NPC Fig. . Unfortuet al. revealed that external radiotherapy induces thymidine phosphorylase and it enhanced the efficacy of capecitabine in human cancer xenografts [Capecitabine is rarely used as part of systemic chemotherapy in head and neck squamous cell carcinoma but it is more commonly used in breast, esophageal, gastric, colorectal, and pancreatic cancers –11. On tnografts . Thymidinografts , 25. UprIn summary, this is the first case of radiation recall related to capecitabine in a patient with recurrent NPC. Radiation oncologists should be vigilant of this potential complication associated with capecitabine especially when it is used for a long period of time. Treatment is mainly supportive though significant clinical improvement is not expected.


Calcification of the ligamentum flavum (CLF) can cause myelopathy due to spinal cord compression. Only several cases in Caucasian patients have been described. Neurological deterioration can only be stopped by surgical decompression. We report a 63-year-old Caucasian woman presenting with progressive pins-and-needles sensations in both hands, worsened by painful paresthesia in both lower extremities. MRI showed a dorsal compressive mass extending from C2 to Th3 vertebrae with myelopathy at the level of C6. A laminectomy was performed, which improved clinical symptoms. Histological examination showed CLF. Early recognition of CLF and early spinal cord decompression are needed to improve neurological outcome. Calcification of the ligamentum flavum (CLF) is a rare disease mainly occurring in the cervical spine (Miyasaka et al. A 63-year-old female presented with progressive pins-and-needles sensations in both hands over the past 3 years, worsened by tingling and burning pain in both lower extremities since 4 weeks. Physical examination revealed bilateral hypesthesia of all digits of the hands and loss of vibration sense up to the clavicle following the dermatomes C5–C8. The biceps, triceps and brachioradialis reflexes were all slightly increased. For lower extremities physical examination showed changing hypesthesia on the lateral sides of both legs and feet with a loss in vibration up to the knee. The knee and ankle jerk reflexes were slightly increased with an indifferent plantar reflex bilaterally. There were no changes in muscle tone and Lhermitte’s sign was positive. MRI showed an intraspinal extradural compressive mass extending from C2 to Th3 posterior of the spinal cord. Spinal cord was compressed at the level of C5/6 and C6/7 Fig. . The lesHistology showed ligamentous and cartilage tissue containing extensive dystrophic calcifications intertwined with ligamentum flavum without traces of old blood or mature bone formation Fig. . HistoloOne-month MRI follow-up showed no progression of the lesion and a clear decompression of the spinal cord at the operated level Fig. . Eight mDorsal spinal cord compression can be caused by different pathological conditions. MRI imaging is important to differentiate from neoplasm, meningioma, lymphoma and chronic calcified epidural hematoma (Boutarbouch et al. The only treatment for CLF is surgical decompression of the spinal cord (Ahn et al. Although rare, CLF should be considered as a possible diagnosis of extensive dorsal spinal cord compression. It is important to recognize and treat CLF at an early stage, since timely decompression results in better neurologic outcome.


Atherosclerosis is a chronic inflammatory process that, in the presence of hyperlipidaemia, promotes the formation of atheromatous plaques in large vessels of the cardiovascular system. It also affects peripheral arteries with major implications for a number of other non-vascular tissues such as the skeletal muscle, the liver and the kidney. The aim of this review is to critically discuss and assimilate current knowledge on the impact of peripheral atherosclerosis and its implications on skeletal muscle homeostasis. Accumulating data suggests that manifestations of peripheral atherosclerosis in skeletal muscle originates in a combination of increased i)-oxidative stress, ii)-inflammation, iii)-mitochondrial deficits, iv)-altered myofibre morphology and fibrosis, v)-chronic ischemia followed by impaired oxygen supply, vi)-reduced capillary density, vii)- proteolysis and viii)-apoptosis. These structural, biochemical and pathophysiological alterations impact on skeletal muscle metabolic and physiologic homeostasis and its capacity to generate force, which further affects the individual’s quality of life. Particular emphasis is given on two major areas representing basic and applied science respectively: a)-the abundant evidence from a well-recognised atherogenic model; the Apolipoprotein E deficient mouse and the role of a western-type diet and b)-on skeletal myopathy and oxidative stress-induced myofibre damage from human studies on peripheral arterial disease. A significant source of reactive oxygen species production and oxidative stress in cardiovascular disease is the family of NADPH oxidases that contribute to several pathologies. Finally, strategies targeting NADPH oxidases in skeletal muscle in an attempt to attenuate cellular oxidative stress are highlighted, providing a better understanding of the crossroads between peripheral atherosclerosis and skeletal muscle pathophysiology. Hyperlipidaemia – defined as increased levels of lipids and/or lipoproteins in blood – is a major risk factor of atherosclerosis and cardiovascular disease as well as an independent risk factor for peripheral arterial disease (PAD) , 2. AtheAPOB) or E (APOE) lead to familial hypercholesterolaemia or dysbetalipoproteinaemia; diseases that are associated with premature atherosclerosis [The transport of triglycerides, chylomicrons, chylomicron remnants and other lipids through the lymphatic and circulatory system is regulated by apolipoproteins. These proteins bind with lipids and phospholipids to form lipoprotein complexes in a variety of sizes according to the relative amounts of lipids and proteins they incorporate . Lipoproclerosis .Apolipoprotein E (APOE) regulates blood lipid and lipoprotein levels in multiple ways, including acting as a ligand for low-density lipoprotein receptor (LDLR), the transfer of lipoproteins to the liver, the formation of chylomicrons and very low-density lipoproteins (VLDL) as well as affecting the activities of hepatic and lipoprotein lipases , 21. HowThe aim of this review is to critically discuss and assimilate current knowledge on the impact of peripheral atherosclerosis and its implications on skeletal muscle homeostasis. Accumulating data suggest that manifestations of peripheral atherosclerosis in skeletal muscle originate in a combination of increased i) oxidative stress, ii) inflammation, iii) mitochondriopathy, iv) altered myofibre morphology and fibrosis, v) chronic ischemia followed by impaired oxygen supply, vi) reduced capillary density and vii) apoptosis , 9, 27. APOE gene is located on chromosome 19 and is comprised of four exons and three introns [APOE gene has two polymorphisms at a single locus that lead to three APOE alleles, ɛ2 (APOE2), ɛ3 (APOE3) and ɛ4 (APOE4) [Apolipoprotein E is synthesized by several tissues, primarily the liver, brain, adrenal glands, adipose tissue, kidney and macrophages –30. APOE introns . APOE is introns , 34, 36. (APOE4) . APOE3 i (APOE4) . APOE2,  (APOE4) . APOE4 o (APOE4) –40. The  (APOE4) , 42.APOE has a major anti-atherogenic function, as it regulates plasma homeostasis by promoting the clearance of lipoproteins, chylomicrons and their remnants, induces VLDL secretion from the liver as well as the reverse cholesterol efflux of VLDL and chylomicron remnants from the liver and macrophages , 37, 42.-/-) leads to hyperlipidaemia and spontaneous development of atherosclerotic lesions from the age of 10 weeks, designating the ApoE-/- mouse model as one of the best established experimental tools to study atherosclerosis [-/- mice has addressed the impact of the initiation and progression of atherogenesis on the vascular system [APOE deficiency in humans is a rare phenomenon but has a broad range of phenotypes spanning from mild hypercholesterolaemia to severe xanthomatosis, hyperlipoproteinaemia and development of premature atherosclerosis , 44, 45.clerosis , 47. Conr system –51.-/- mice is distributed in VLDL and intermediate density lipoproteins (IDL)/LDL particles [-/- mice as summarised in Table -/- mice not only have lower levels of HDL cholesterol, but the HDL particles are significantly altered in their composition [The majority of cholesterol in the ApoEarticles , 53. It position .Table 1O-/- mice develop atherosclerotic lesions at a young age. Monocytes begin to adhere to the vascular wall at the age of 6–8 weeks and approximately after 2 weeks; foam cells and lesions are observed leading to atherosclerotic plaques [Due to their hyperlipidaemic profile, ApoE plaques , 59, 60. plaques . At the  plaques . Specifi plaques , 62. Apa plaques –67. None plaques .-/- mice exhibit a 61% occlusion of the lumen and decreased endothelial-dependent relaxation to acetylcholine, an increase in stiffness of conduit arteries leading to elastic laminar fragmentation, while endothelium independent relaxation remains intact [-/- mice appears to be focal rather than systemic [As atherosclerotic lesions advance with age, plasma levels of VLDL and LDL either increase or remain unchanged , 68. Pers intact –74. In csystemic , 75. Addsystemic .-/- mice advances in an age-dependent manner and lesion composition and progression is very much similar in humans [-/- mice and humans, ApoE-/- mice do not develop lesions in the coronary arteries as humans [-/- mice exhibit dilated cardiomyopathy characterised by impaired left ventricular function and increased fibrosis of the myocardium and endocardium [-/- deficiency in combination with ageing and diet seem to impact on cardiac morphology [At 17–18 months of age, lumen occlusion can reach 90% and lesions are found mostly in the aortic arch, aortic root and in the proximal and distal segments of thoracic aorta, while small lesions appear in the central thoracic aorta , 72, 76.n humans , 77. It s humans , 61. Mans humans , 78–80. ocardium . Althougrphology , there irphology , 81, 82,-/- mice of the same age and diet, with male mice displaying higher levels [-/- mice showed that the lesion burden in the aortic sinus and abdominal aorta was greater that in control ApoE-/- mice, while estradiol 2 treatment of gonadectomised male and female ApoE-/- mice led to a 50% decrease of lesion area throughout the aorta [The progression of atherosclerosis may be regulated by sex . Some evr levels , 56, 77.r levels , 84. Furr levels , 85. Stuhe aorta , 87. On he aorta , 89. In -/- mice and abundant evidence suggests that such dietary challenges result in aggravation of the atherosclerotic phenotype [High-fat diets are usually composed of 15–36% w/w fat which corresponds to 34–60% kcal from this source containing mostly saturated fats –95. Howehenotype , 95, 97.-/- mice [-/- mice kept on a chow diet, although absolute levels of total cholesterol vary amongst studies as shown in Table One to eight weeks on a western-type diet increases total plasma cholesterol levels by 3- to 7-fold and atherosclerotic lesions in the aortic sinus are evident from the fourth week in young ApoE-/- mice , 98. WesAlong with increased inflammation, increased oxidative stress is also evident. By 7 weeks of western-type diet, reactive oxygen species production increases by 2-fold in the aorta and after 8 weeks there is a 4-fold increase in the media of the aorta and a 3-fold increase in the perivascular fat of the ascending aorta , 57. Thi-/- mice after 16 weeks of western-type diet when compared to the wild type mice on a chow diet [-/- mice fed a western-type diet for 20 weeks have increased eNOS protein expression in aortas when compared to wild type fed the same diet, potentially as a compensatory mechanism against the increased expression of Nox2 that reduces nitric oxide bioavailability [2+ dependent eNOS activity as judged by decreased basal levels of cyclic guanosine monophosphate [Endothelial dysfunction is evident from the fifth week of western-type diet and in particular, endothelium-dependent relaxation to acetylcholine is impaired in the proximal descending aorta whereas it remains unaltered in the thoracic aorta , 106. Enhow diet . Converslability . Endothehosphate . The walhosphate . In resihosphate , 110. Sihosphate .In addition to endothelial dysfunction, increased inflammation and oxidative stress, there is evidence that ApoE deficiency may impact on cardiac muscle per se. Standard chow diet supplemented with 0.15% w/w cholesterol for 18 months induces cardiac hypertrophy followed by impaired cardiac function which is characterised by a reduced ejection fraction and a reduced maximum rate of change of left ventricular pressure . In turn-/- mice, LDL receptor deficient (Ldlr-/-) mice represent another model of systemic atherosclerosis that shows mechanistic differences in the development of atherosclerosis reviewed elsewhere [-/- mice on a chow diet have also increased total plasma cholesterol but lesion formation is rather observed in aged animals and is limited to the aortic root, whereas in ApoE-/-  mice lesions form at 8–10 weeks of age and subsequently develop throughout the aorta. When ApoE-/- mice are fed a western-type diet total plasma cholesterol is further augmented and lesions form at an earlier age (i.e. 4–6 weeks old) and progress more rapidly [-/- mice fed an atherogenic diet for 12 weeks, develop lesions in the aortic root and increase thereafter robustly extending to the thoracic and abdominal regions of the aorta [-/- mice subjected to either a chow- or an atherogenic- diet carry most of their cholesterol in the apoB48-containing VLDL particles, whereas Ldlr-/- mice on a chow diet have predominantly apoB100-containing LDL particles, a feature that is more relevant to human pathogenesis of atherosclerosis [-/- mice exhibit increased LDL as well as VLDL particles [-/- mice may be attributed not only to the plasma lipid levels but also to the loss of the antiatherogenic functions of ApoE [Apart from the ApoElsewhere , 93. In  rapidly , 116. Cohe aorta , 117. Anclerosis , 93. Howarticles . Further of ApoE .Beyond the well-documented evidence on the detrimental effects of atherogenic diets with regards to the cardiovascular system and progression of atherosclerosis, there has recently been an expansion of interest on new areas that will be discussed below. Emphasis is given on i) the manifestations of atherosclerosis in peripheral tissues such as skeletal muscle and ii) the role of Noxs in vascular disease and the subsequent potential emerging from strategies to attenuate vascular oxidative stress by targeting NADPH oxidases.Skeletal muscle constitutes one of the largest tissues in the human body comprising of almost 40% total body weight . SkeletaAs discussed above, central atherosclerosis is a chronic inflammatory disease that leads to plaque formation and occlusion of major arteries, initiated by increased oxidative stress and pro-inflammatory signalling that lead to endothelial dysfunction , 137. HoIn fact, limb skeletal muscles of PAD patients exhibit increased protein carbonylation and 4-hydroxy-2-nonenal (4-HNE) adducts, which increase further as the disease advances , 9, 141.Apart from increased oxidative damage, inflammation is another major initiator of central atherosclerosis. The majority of studies have focused on identifying markers of systemic inflammation and their potential role in PAD pathophysiology , 142–144MARCKS and phospholipid transfer protein, PLP), proteolysis  and immune function  in intermediate lesions, whereas genes controlling cell metabolism  are downregulated [COL1A1 and Collagen Type III Alpha 1 Chain, COL3A1) and a downregulation of genes involved in cell cycle  apoptosis  and RNA metabolism  is observed [Inflammation in lesions of femoral arteries is increased in patients with CLI as compared with patients with IC, is mainly localised in the intima and is characterised by elevated number of macrophages , 147. Imegulated . In advaobserved . Anotherobserved .Beyond oxidative damage and systemic inflammation, patients with PAD develop mitochondriopathy , 141. MiThe myopathy in PAD is also characterised by the alteration in the myofibre morphology. Several groups have reported that myofibres reduce in size as the disease advances and the loss in cross sectional area (CSA) is correlated with the increase in oxidative damage , 151. HoAtherosclerosis in the arteries of the lower limbs leads to chronic ischemia of the muscle due to reduced oxygen delivery . NonethePAD patients exhibit vascular endothelial dysfunction not only in the large vessels but also in the microvasculature –188. ET-Consequently, peripheral atherosclerosis impacts on skeletal muscle as outlined by increased oxidative damage, inflammation, mitochondriopathy, loss of number and function of myofibres, impaired capillary density and apoptosis. Although ischemia– reperfusion cycles are considered the principal cause of skeletal muscle pathophysiology in PAD, the impact of other comorbidities such as hyperlipidaemia on endothelial dysfunction and skeletal muscle pathophysiology should not be overlooked.-/- and LDL receptor deficient (Ldlr-/-) mice are widely used as models for systemic atherosclerosis, currently there is sparse scientific evidence evaluating the skeletal muscle pathophysiology of these two experimental models of atherosclerosis, as summarised in Table -/- mice on normal chow diet have decreased capillary density in the gastrocnemius from the age of 12 weeks and nitric oxide bioavailability in skeletal muscle arterioles declines from the age of 20 weeks without changes in endothelial function [-/- mice exhibit extensive plaque formation in the aorta and atherosclerotic plaques are also found in the femoral arteries [-/- mice administered a high-fat diet  compared to wild type mice [-/- mice on a different high-fat diet consisting of a higher percentage of fat (60% v/v fat) exhibit elevated inflammation and greater levels of hydrogen peroxide in the muscle as compared with wild type mice [-/- mice at the age of 22 weeks have decreased capillary density in the gastrocnemius, which is less pronounced than in the ApoE-/- and the wall to lumen ratio is elevated in the skeletal muscle arterioles [Although ApoEfunction , 195. Atarteries . Blood parteries , 197. Cuype mice . This stype mice . Ldlr-/-terioles . Taken tThe study of skeletal muscle pathophysiology in PAD has been modelled in animal studies using the “femoral artery ligation” model of hind-limb ischemia, with the majority of data being obtained from young and healthy animals in the absence of any cardiovascular risk factors or comorbidities associated with PAD such as hyperlipidaemia , 196. Th-/- mice as a model for PAD by inducing limb ischemia via femoral artery ligation [-/- mice subjected to ischemia followed by exercise have similar hyperaemic response with exercising PAD patients and exhibit delayed regeneration and increased inflammation [-/- mice on a western-type diet and subsequent induced-ischemia, display impaired ambulation compared to wild type mice due to delayed restoration of blood perfusion [-/- mice exhibit impaired muscle regeneration [-/- mice display impaired myofibre regeneration with increased fat infiltration and calcification. The authors concluded that the delay in muscle healing is due to impaired macrophage phagocytic activity [-/- mice are also used as a model for PAD by ligating the femoral artery and inducing limb ischemia [-/- mice on a western-type diet and subjected to ligation of both femoral and iliac arteries have reduced blood perfusion as compared to wild type mice [-/- mice show complete blood flow recovery at 10 days after injury, whereas wild type mice recover fully after 7 days [In this regard, a few studies have used ApoEligation , 215. Spammation , 215. Inerfusion . In the neration . Similaractivity . Ldlr-/-ischemia –221. Ldlype mice . In a dir 7 days . HoweverReactive oxygen species are a diverse class of reactive chemical molecules that consist of highly reactive oxygen atoms . ROS pla-/-/p47phox-/- double knockout mice at the age of 16 weeks have similar total plasma cholesterol levels and decreased vascular superoxide production but the same lesion area in the aortic sinus with ApoE-/- mice [-/-/p47phox-/- double knockout mice at the age of 30 weeks have reduced lesion area by 75% in the whole aorta and under a high-fat diet the reduction reaches 50% at the age of 18 weeks [-/-/p47phox-/- and ApoE-/- followed by a high-fat diet  leads to decrease of lesion coverage (>50%), smaller lesion size, fewer macrophages and lower endothelial superoxide production in both groups [-/- mice with bone marrow from ApoE-/-/p47phox-/- have lower levels of oxidised LDL and ApoE-/-/p47phox-/- with bone marrow from ApoE-/- have reduced expression of cellular adhesion molecules. These findings suggest that on one hand the NADPH oxidase activity contributes to lesion formation and on the other hand vascular wall cells and bone marrow promote atherogenesis through different processes [Noxs were first identified in phagocytic cells and they are responsible for the respiratory burst that is essential for innate immune response and phagocytosis of pathogens , 222. In-/- mice . Althoug18 weeks . Allogenh groups . ApoE-/-rocesses , 229. Alrocesses .Table 5T-/- mice (Nox1-/y/ApoE-/-) fed a high-fat diet  exhibited reduced superoxide production and a decreased lesion area by 35% in the aortic arch and 28% in the entire aorta. Furthermore, the lesions contained less macrophages [-/y/ApoE-/- mice fed a western-type diet exhibited similar lesion coverage throughout the aorta and the thickening of the intima was larger at the level of the aortic sinus compared to ApoE-/- mice [-/- mice reduced lesion coverage, attenuated macrophage infiltration, expression of pro-inflammatory chemokines and ROS production, while it did not have any effect in ApoE-/- mice [The role of Nox1 in atherosclerosis is not fully elucidated and there are contradictory data from studies on Nox1 depletion. Sheehan et al. reported that Nox1 deficiency in ApoErophages . On the -/- mice . Additio-/- mice . Similar-/- mice . Thus, N-/- mice on a normal chow diet does not seem to impact on lesion area and lipid deposition in the aortic sinus at the age of 24 weeks with male ApoE-/-/gp91-/- mice having decreased HDL levels and increased LDL levels by 60% compared to ApoE-/- whereas these differences in plasma cholesterol were not evident in female double knockout mice [-/- mice overexpressing Nox2 specifically in the endothelial cells (Nox2Tg ApoE-/- mice) on a normal chow diet have elevated vascular superoxide production, but surprisingly, the lesion area is equivalent to ApoE-/- mice with comparable lesion composition and progression [-/- mice [-/y/ApoE-/- male mice administered a western-type diet  either for 7 or 14 weeks have similar plasma cholesterol levels with ApoE-/- and although the lesion area coverage in the aortic sinus is similar, a 50% decrease in lesion area throughout the aorta is evident [-/y/ApoE-/- exhibit reduced vascular superoxide production by 75% and improved nitric oxide bioavailability compared to ApoE-/- [-/- mice on a western-type diet  and treated with a Nox2-specific inhibitor, the Nox2ds-tat peptide, showed decreased lesion coverage throughout the aorta and the carotid arteries [Nox2  deletion in ApoEout mice . ApoE-/-gression . Interes-/- mice . Therefo-/- mice . On the  evident . Further ApoE-/- . Importaarteries . Moreovearteries . The Noxarteries –236. Thiarteries , 94, 225Skeletal muscle expresses two Nox members i.e. Nox2 and Nox4 although in cultured muscle cells apart from Nox2 and 4 expression, Nox1 and Duox 1 and 2 expression has been also reported . Nox2 isThe “metabolic hypothesis” was introduced almost three decades ago to suggest a direct relationship between the local blood flow and the metabolic demand of the muscle , 250. Th+ and Ca+ and iv) dysregulation of myocellular ions, (i.e. K+ and Na+) that further compromise cellular function [Most studies that investigate the impact of atherosclerosis on PAD focus on haemodynamic changes from ischemia-reperfusion cycles , 260–264function . The onsfunction . Higher function . Furtherfunction . This fiThere is currently sparse experimental evidence on the effect of central atherosclerosis on skeletal muscle biology. Hyperlipidaemia–a major risk factor of atherosclerosis and PAD–is considered an important step in atherogenesis , 266–268-/- and ApoE-/- experimental atherosclerotic mouse models with increased plasma concentrations of TNFα, IL1β and MCP1 [Since hyperlipidaemia impacts on the liver and adipose tissue, it can be speculated that skeletal muscle can be affected as well taken into account the molecular crosstalk among these metabolically active tissues , 275–278and MCP1 . Pro-infand MCP1 , 278. Co-/-/ApoE-/- mice that exhibited diminished atherosclerotic burden and vascular ROS production [-/- mice with an AT1 receptor antagonist inhibited progression of atherosclerosis [-/- mice with an AT1 receptor antagonist and simultaneous infusion with angiotensin II inhibited progression of atherosclerosis by decreasing the expression of pro-inflammatory markers and ROS production from the endothelium [-/- mice with a calcium antagonist or a vasodilator did not affect lesion progression [Another factor that may be affecting muscle pathophysiology is the peptide hormone angiotensin II that has been found to contribute to atherogenesis and progression of atherosclerosis . Angioteoduction . Importaclerosis . In a diothelium . The afogression , 282.Angiotensin II might affect skeletal muscle homeostasis in atherosclerosis, not only due to its significant impact on the vasculature, but due to a rather direct effect on skeletal muscle. For instance, angiotensin II in congestive heart failure and chronic kidney disease contributes to muscle wasting , 284. Th-/- mice. Several inhibitors have been developed in an attempt to reduce atherosclerosis, with Nox2ds-tat recently showing that treatment of ApoE-/- with this peptide reduced lesion size in the aorta and carotid arteries [Peripheral atherosclerosis has serious implications for human health and impacts in several tissues including skeletal muscle. Accumulating data over the past decade from human studies with PAD patients and some evidence from rodent studies have improved our understanding about the pathophysiology of skeletal muscle in the context of atherosclerosis. There is a number of structural, functional, biochemical and metabolic deficits taking place in the skeletal muscle that compromise its homeostasis and functional capacity. There are signs of myopathy, fibrosis, mitochondrial de-regulation, oxidative stress, inflammation and apoptosis, followed by impaired capillary density, chronic ischemia and muscle pain. Such manifestations in skeletal muscle may in turn compromise the capacity to generate force or ambulate and impact on the quality of life. By identifying the crossroads between skeletal muscle metabolism, dietary challenges and peripheral atherosclerosis we will be able to better prepare new strategies to cope with such abnormalities and improve muscle physiology and quality of life in patients with cardiovascular complications such as peripheral atherosclerosis. Experimental models of atherosclerosis with a skeletal muscle phenotype may offer an attractive tool to develop new strategies to combat the manifestations of atherosclerosis in peripheral tissues. NADPH oxidases are key components of increased cellular oxidative stress and their role in atherogenesis has received much attention recently. Several studies have shown the beneficial effect of Nox2 deficiency in the initiation and lesion progression in the aorta of the ApoEarteries . The imp


Ostrea lurida) have received considerable attention with regard to restoration, however there is limited information on adaptive population structure. Using oysters raised under common conditions for up to two generations (F1s and F2s), we tested for evidence of divergence in reproduction, larval growth, and juvenile growth among three populations in Puget Sound, Washington. We found that the population with the fastest growth rate also exhibited delayed and reduced reproductive activity, indicating a potential adaptive trade-off. Our results corroborate and extend upon a previous reciprocal transplant study on F1 oysters from the same populations, indicating that variation in growth rate and differences in reproductive timing are consistent across both natural and laboratory environments and have a strongly heritable component that cannot be entirely attributed to plasticity.Adaptive evolution and plasticity are two mechanisms that facilitate phenotypic differences between populations living in different environments. Understanding which mechanism underlies variation in fitness-related traits is a crucial step in designing conservation and restoration management strategies for taxa at risk from anthropogenic stressors. Olympia oysters ( This process of local adaptation can be opposed by homogenizing gene flow from dispersal, a significant factor for marine species with an extended planktonic dispersal phase2. However, if the scale of dispersal extends across strong selective gradients, adaptive population divergence can still occur through phenotype-environment mismatch— where strong purifying selection occurs each generation following dispersal4. Characterizing the spatial scale and magnitude of adaptive divergence is a crucial step in designing conservation and restoration management strategies for taxa at risk from anthropogenic stressors6.Natural environments exhibit spatial heterogeneity in both abiotic and biotic factors, oftentimes driving populations to evolve traits that confer a fitness advantage in their native habitat over foreign genotypes7, which may confound inferences of local adaptation8. Phenotypic plasticity is widespread in marine species11, and for marine invertebrates the most common trigger for plasticity appears to be the abiotic environment11. Organisms can be raised their entire lives in common conditions in order to minimize the effects of phenotypic plasticity. However this approach may fail with strong transgenerational plasticity (TGP) - defined here as when the environment or phenotype of the parent affects the phenotype of the offspring12. The ideal experimental design to distinguish TGP from genetic change involves raising and breeding individuals for at least two generations in a common setting1, although TGP has persisted for more than two generations in some laboratory studies13. Whenever breeding organisms for multiple generations, care should be taken to evaluate and reduce the influences of artificial selection and genetic drift14. In a recent review of experimental evidence for local adaptation in marine invertebrates, only 11 out of 59 studies utilized 2 or more generations15. Distinguishing between plastic responses and adaptive evolution to the environment is key to understanding the potential for marine species to acclimate or adapt to changing environmental conditions16.Conclusively demonstrating adaptive divergence is complicated by phenotypic plasticity, where individuals adjust their phenotype according to the conditions they experience18, resulting in an increasing need to understand the relative importance of adaptive and plastic processes in shaping phenotypic variation. Evidence that TGP might be common for marine molluscs is growing (see ref.19 for a thorough review), however only a handful of studies investigating adaptive differentiation in this clade have compared organisms raised in common conditions for at least 2 generations, and of those most involved Gastropoda (see refs24).Marine molluscs, and bivalves in particular, constitute some of our most economically and ecologically important marine invertebrates. Like many other marine invertebrates, they exhibit complex life cycles which include both planktonic larval stages as well as benthic juvenile and adult stages. The larval stage of many marine molluscs has been shown to be particularly sensitive to risks from ocean acidification and warmingOstrea lurida) is an estuarine species natively distributed from the central coast of Canada to Baja California. As a rhythmic consecutive hermaphrodite, spawning events in O. lurida are thought to be synchronized between males and females based on environmental cues of temperature and seasonality25. Considered ecosystem engineers in estuaries, they provide structured habitat, remove suspended sediments, and limit eutrophication26. Following devastating commercial exploitation in the early 20th century, recovery of Olympia oyster populations has been stifled by other anthropogenic threats 28. There is political and economic pressure to restore abundance and recover ecosystem services offered by this species, which has spurred increasing interest in understanding the genetic, phenotypic, and epigenetic variation at both local and regional scales29.The Olympia oyster  in 2014. Variation in survival, growth rate, and reproductive activity was observed among populations and the four transplant sites. In particular, oysters from Fidalgo Bay had faster growth rates and reduced or delayed reproductive activity at most sites, while oysters from Dabob Bay had better survival yet slower growth rates, indicating potential adaptive trade-offs30.A recent study on Olympia oysters in central California provided evidence for spatial adaptive differentiation through a reciprocal transplant experiment with first-generation laboratory-reared (F1) oysters. The authors also found suggestive, although not statistically significant, evidence of population-level differences in low salinity tolerance in second-generation, laboratory-reared (F2) oysters, and hypothesized adaptive divergence may occur in Olympia oysters over distances as short as 20–100 kmet al.30. Three fitness-related traits were measured across populations- reproductive activity, larval growth rate, and juvenile growth rate. We also estimated the relatedness and genetic diversity of the F1 generation using SNP data in order to demonstrate that the population-specific traits described here are not primarily due to family-level variation from few effective breeders.Although both of these studies controlled for environmental exposure of broodstock for up to 5 months prior to producing F1 oysters and attempted to maximize genetic diversity, they did not sufficiently minimize concerns about TGP or evaluate the relatedness of their laboratory-reared populations. This study aims to mitigate the influence of TGP on inferring adaptive differentiation by testing if phenotypic differences among populations of Olympia oysters are consistent across generations. In the summer of 2015, we conducted a common garden experiment on F1 and F2 oysters derived from the same three Puget Sound populations as Heare p = 0.0421), with F1 oysters from Fidalgo producing the fewest , with Fidalgo oysters exhibiting delayed reproduction compared to the other populations and much higher variance in the date of initial larval release .The onset of larval release also differed significantly among populations . By Day 7, size varied significantly among populations (linear mixed model(LMM), p = 7.939e−5). Fidalgo larvae were 8% larger than Dabob larvae  and 6% larger than Oyster Bay larvae . After 14 days, size still varied significantly among populations , but only the comparison between Fidalgo and Dabob larvae remained significant  . Growth rate between Day 0 and Day 48 diverged among populations . Fidalgo oysters grew 46% faster than Dabob oysters , but all other pairwise comparisons were not significant. Between 48 and 68 days, shell growth continued to differ among populations . Dabob oysters grew slower over this time period than Fidalgo oysters  and tended to grow slower than Oyster Bay oysters  , expected heterozygosity (He) and inbreeding coefficient (Fis) represent values averaged over loci that are polymorphic within the population, while kinship coefficient (ϕ) is averaged over all pairwise comparisons of individuals. Values of ϕ over 0.1768 are considered full-sibs. Observed heterozygosity varied across loci and did not significantly differ from expected heterozygosity except in Dabob oysters . Sibship reconstruction identified families of full-sib pairs within the 50 oysters sampled from each population. Fidalgo oysters comprised of 33 families , none of which included more than 8% of the total individuals. Dabob oysters had 21 full-sib families and Oyster Bay oysters had 17 families, the largest of which contained 16% and 24% of individuals, respectively. Families for all populations were well mixed between spawning groups for production of F2 oysters.Population-level estimates of genetic diversity and relatedness for F1 oysters are shown in Table et al.30 study and showing consistent interpopulation differences between first-generation (F1) and second-generation (F2), commonly reared oysters, this study is the first to demonstrate statistically significant phenotypic population structure extending through the F2 generation in Ostrea lurida, and one of the few to do so in a mollusc. Persistent population differences in growth rate and reproductive timing across several generations have also been documented for selectively-bred strains of the eastern oyster, Crassostrea virginica31, suggesting population divergence at these traits may be general across Ostreidae.The results presented here provide evidence for a strong heritable component underlying phenotypic variation in growth and reproduction among three populations of Olympia oysters in Puget Sound, WA. By building on the Heare et al. By showing consistent reproduction patterns in both variable natural environments and controlled laboratory conditions, we demonstrated that reproductive timing in this species is not exclusively mediated by environmental conditions, but is also under genetic and/or epigenetic control. This result is in concordance with recent studies indicating that nearby populations of Olympia oysters vary in temperature thresholds for reproduction33. The development of asynchronous reproduction on such small spatial scales has major implications for limiting gene flow and contributing to population divergence34.Reproductive activity was characterized for the adult F1 oysters, with Oyster Bay oysters producing the most larvae over the course of 7 weeks and the Fidalgo population demonstrating delayed reproductive activity, in accord with the reciprocal transplant study by Heare et al., indicating a fixed underpinning for growth rate differences that manifests during both the larval and juvenile life stages. Interestingly, Fidalgo oysters also exhibited severely reduced and delayed reproductive activity. One explanation for this is an adaptive trade-off in energy allocation36, where Fidalgo oysters are devoting more energy to shell growth and less to gonad development. Heare et al. also observed a potential adaptive trade-off in their reciprocal transplant study, where the population with slowest growth also had the highest survival across sites. Further investigation is required to fully resolve the link between growth, reproductive activity, and survival.Fidalgo oysters exhibited the fastest growth during both the F2 larval and juvenile stages, while Dabob oysters exhibited the slowest growth, resulting in a significantly smaller size than Fidalgo oysters and smaller  than Oyster Bay oysters. This result is consistent with findings in F1 oysters by Heare Ostrea species38, we estimated genetic diversity and relatedness for a subset of F1 oysters to ensure our approach of separating broodstock into smaller groups of 20–25 oysters produces offspring with many different families represented. Assuming the original wild population had kinship coefficients close to zero, our results do indicate that a reduced number of the original broodstock contributed to the F1 generation. However, by identifying between 17 and 33 different families within 50 sampled individuals, we are confident that the total pool of 94–101 F1 individuals per population represents upwards of 40 wild genotypes and that our spawning method of mixing these families across smaller groups produced a genetically diverse F2 generation.Given the characteristically high variance in reproductive success for 30, supporting the possibility of selection driving the phenotypic variation observed in this study. In particular, up to 10 fold less chlorophyll a has been observed in Fidalgo Bay compared to the other sites. The proposed trade-off in energy allocation for this population could be driven by selection from such environmental differences in the quantity and timing of food supply40. As mentioned previously, selection could act through local adaptation or phenotype-environment mismatch, depending on the spatial scale of dispersal relative to the scale of environmental heterogeneity. Although dispersal information is not currently available for Olympia oyster larvae in Puget Sound, estimates using chemical fingerprinting in Southern California identified considerable larval exchange among estuaries separated by up to 75 km41, which encompasses the distance between the populations used in this study. Further research using both chemical fingerprinting and genetic approaches are required to understand patterns of dispersal within Puget Sound in order to elucidate the mechanisms of adaptive population divergence.Environmental conditions in temperature, freshwater input, primary productivity, and anthropogenic waste effluent are known to vary among these sites27. These efforts have focused on multiple sites in Puget Sound, including those covered in this study. Our results support this practice, with a couple of caveats: (1) If local adaptation is indeed driving the observed phenotypic population structure, populations may be adapted to historical, rather than current, conditions. The convention of replenishing populations with only local broodstock sources may not provide the genetic diversity required to adapt in the face of rapid anthropogenic-induced environmental changes42. (2) Hatchery conditions vary dramatically from natural summer spawning conditions, and therefore artificial selective pressures are likely at play in the production of both the F1 and F2 oysters used in this study14. However, the observed differences in size among populations are corroborated by field observations of wild oysters .Our results have implications for ongoing restoration efforts attempting to rebuild Olympia oyster populations. Current protocols for hatchery-based Olympia oyster restoration in Puget Sound involve using wild broodstock to produce hatchery-raised juveniles for outplanting in the same source population as the broodstockDespite the relatively close proximity of these populations and the potential for high gene flow, we observed significant phenotypic differences in fitness-related traits, even after multiple generations in the same environment. Of note, the population with the fastest growth rate also exhibited delayed and reduced reproductive activity, indicating a potential adaptive trade-off. By mitigating the influence of transgenerational plasticity, our results suggest these trait differences are heritable. Further research is now required to understand the mechanisms of inheritance underlying these observed differences, whether they be genetic or epigenetic.Crassostrea), Olympia oyster females are fertilized with spermatozeugmata (‘sperm packets’) from the water column and brood larvae for approximately 10–12 days. After being released into the water column, larvae are planktonic for approximately two weeks before attaching to a hard substrate (‘settlement’). To ensure genetic diversity, each population was allowed to spawn in 24 separate groups of 20–25 oysters. Larvae produced from each population were reared in tanks based on spawning group, settled on very small pieces of oyster shell, then fed ad libitum. In August 2013, 480 juvenile oysters (5–10 mm) from each source population were outplanted at Clam Bay located in central Puget Sound , a different site than any of the source populations. For the purposes of this study we will refer to the cohort outplanted at Clam Bay as F1s. Reproductive and growth characteristics of F1 oysters at Clam Bay have been described by Heare et al.30. In June 2015, F1 oysters were moved into NOAA’s Kenneth K. Chew Center for Shellfish Research and Restoration in Manchester, WA and maintained in mesh bags suspended in separate 18.9 L buckets with a diet of mixed live algae in flowing seawater.Adult oysters were collected from three locations in Puget Sound, Washington; Fidalgo Bay , Oyster Bay , Dabob Bay  during November and December 2012. Oysters were held for 5 months in common conditions in Port Gamble, Washington and spawned in June 2013. Unlike many other oyster genera that broadcast spawn both eggs and sperm (e.g. μM) and counted with triplicate drop counts. A cohort of these F2 larvae were used in the larval growth trials with the remainder raised in tanks (100 L) for the juvenile growth trial.Spawning of broodstock (F1) was induced by elevating temperature to 18–20 °C in May 2015 and maintaining algae supplementation at 60,000–80,000 cells/mL. To ensure genetic diversity, each population  was divided into 5 groups of 16–21 oysters. In addition to ensuring that multiple females were involved in reproduction, these replicate groups allowed us to statistically test population-level differences in reproductive activity. Larval release was checked and quantified every one to three days with larvae filtered  containing a 50/50 mix of live T. isochrysis and diatom algae . Approximately 20 larvae were sampled haphazardly by pipette from the initial larval pools at Day 0 and each replicate at Day 7 and Day 14 of the experiment, fixed in 10% buffered formalin, and photographed under a microscope for analysis using ImageJ v1.5143 software to determine shell area and length. We report results based on shell length, although shell area gave qualitatively similar results (not shown).To investigate differences in growth rate among populations at the larval stage, we set up a larval growth rate experiment starting on a day when all three populations were producing at least 25,000 total larvae across multiple buckets . Larvae were pooled by population and mixed well in 1 L beakers. The concentration of larvae was estimated using triplicate drop counts, and 900 larvae per population were added to each of three replicate plastic beakers (1 L) in order to account for random effects due to the beaker environment. These beakers were each outfitted with a “silo”, a 7.62 cm (3”) diameter section of conditioned PVC pipe covered in 100 μM  were moved to new tanks (100 L) where air was bubbled to maintain oxygen levels and stimulate water movement. These tanks were lined with PVC tiles (10 × 10 cm) roughed on one side using coarse sandpaper to promote oyster settlement. After four weeks, settled oysters were culled to fewer than 30 oysters/tile to avoid overgrowth interactions . Tiles were randomized and attached to four protected outplanting trays that were suspended from the dock at NOAA Manchester Research Station (depth = 6 m). Photos were taken of oysters on tiles prior to outplanting (Day 0), after 48 days, and after 68 days for analysis using ImageJ software to determine shell area.Larvae from each population greater than 224 44, we sequenced 279 individuals and 18 technical replicates from the F1 generation for a total of 297 samples across 5 lanes of Illumina HiSeq. The technical replicates are necessary for quality assessment and genotyping recalibration of downstream analyses. For each population, we mapped reads from the 50 individuals with the highest read depth as well as 4–5 technical replicates to a draft O. lurida genome of 8,733 scaffolds over 10 kb in length. For each sample in this subset, approximately 30% of reads mapped to the draft genome using Bowtie2. After genotyping single nucleotide polymorphisms (SNPs) using the UnifiedGenotyper tool in GATK v3.645, we filtered SNPs for quality and excess heterozygosity based on Hardy-Weinberg equilibrium within populations using VCFtools46 and custom scripts by Mikhail Matz https://githb.com/z0on/2bRADGATK. For subsequent analyses of relatedness and genetic diversity, we thinned our dataset to one SNP per 2b-RAD tag for 677 SNPs confidently identified in at least 75% of individuals.Using a 2b-RAD reduced-representation sequencing approachTo compare differences in reproductive activity among populations in the F1 generation, the five separate broodstock buckets per population were considered as independent replicates. The number of larvae released in each bucket on each sampling day was normalized by the number of adults in that bucket. To determine if there was a difference in total reproductive output among populations, the cumulative number of larvae produced throughout the 7 weeks for each bucket was analyzed using a one-way analysis of variance  with post hoc Tukey’s tests. In order to determine if populations differed in their timing of reproductive activity, one-way ANOVAs were also conducted on the number of days until the first observed larval release in each bucket after spawning conditions were induced, as well as the number of days until spawning peaked in each bucket.lme447. For the larval growth experiment, randomly selected live larvae were measured on Day 0, Day 7 and Day 14 with 10–12 larvae  larvae per replicate. Dead oysters are easily distinguished from living oysters by having an entirely clear protoshell and no observable tissue. As O. lurida larvae grow prior to maternal release, a one-way ANOVA was used to test if size varied among populations on Day 0 prior to separating out into replicate beakers. For the LMM analysis, population was a fixed effect and replicate beaker was a random effect. Prior to running the LMM, size distributions were tested for normality using the Shapiro-Wilkes test with the stats R package. Significance of the LMM results was established using a Likelihood Ratio Test against a null model based only on random effects. Shell length was compared at each time point using pairwise t-tests with a Bonferroni correction for multiple testing.Linear mixed models (LMMs) were used to measure the effect of source population on oyster growth using the R package PMCMR package)48.For the juvenile experiment, growth was tracked on an individual basis (growth rate = Δarea/# days). All oysters that were alive  and not extending > 50% off the tile at each time point were measured for shell area. For the linear mixed model, population was a fixed effect and tray containing the tile was a random effect. Growth rate was natural log-transformed based on indications of non-normal distributions from the Shapiro-Wilkes test. Pairwise comparisons for populations at each time point were performed with the Nemenyi post hoc test using information from the Kruskal-Wallis test (49 using 677 high-quality SNPs obtained by 2b-RAD sequencing. Mean expected and observed heterozygosity was calculated for each population using hierfstat50 and compared with Bartlett’s test of homogeneity of variances51. Pairwise estimates of relatedness were calculated using the KING algorithm52 as implemented in VCFtools46. Full sib pairs are classified by having a kinship coefficient over 54, with the following parameters: polygamous males and females, long run length, full-likelihood analysis, high-likelihood precision, update allele frequencies during run, no prior information, and allelic dropout rate of 0.001.Estimates of genetic diversity and relatedness in the F1 generation were calculated in R v3.4.1www.github.com/ksil91/PS-Oly-Larvae-Growth.The datasets generated during current study are available on figshare, 10.6084/m9.figshare.5975452. Reproducible R Markdown notebooks detailing the code used for statistical analyses can be found at


We attempted to explore the molecular mechanism underlying PHY906 intervention of colon cancer. The microarray data of tumors treated by PHY906 and PBS alone were downloaded from the public Gene Expression Omnibus database. The dataset was further analyzed for the differentially expressed genes (DEGs) and their related biological functions were analyzed, followed by function and pathways. Protein-protein interaction (PPI) network was constructed and the significant nodes were screened by network centralities and then the significant modules analysis. Besides, they were clustered and transcriptional factors (TFs) were predicted. The gene expression patterns changed induced by PHY906 treatment, including 414 upregulated and 337 downregulated DEGs. The biological process of response to steroid hormone stimulus and regulation of interferon-gamma production were significantly enriched by DEGs. Ezh2 (enhancer of zeste 2) was found to be the key node in PPI network. There are 12 significant TFs predicted for module 1 genes and 3 TFs for module 2 genes.γ level and mediating cancer cell death by activating the response to steroid hormone stimulus and activating the response to steroid hormone stimulus. E2f1, Hsfy2, and Nfyb may be therapeutic targets for colon cancer. PHY906 showed treatment efficacy in modulating cell apoptosis by intervening interferon-gamma production and response to steroid hormone stimulus. Ezh2 and its TFs such as E2f1, Hsfy2, and Nfyb may be the potential therapeutic targets for anticancer agents development. PHY906 treatment may function in protecting the epithelial barrier against tumor cell invasion by modulating IFN- Colon cancer is the common tumor derived from gastrointestinal systems, which has affected 1.4 million new cases in 2012 and results in 694,000 deaths globally . The sur Glycyrrhiza uralensis Fisch. (G), Paeonia lactiflora Pall. (P), Scutellaria baicalensis Georgi (S), and Ziziphus jujuba Mill. (Z). It has been widely used for treating gastrointestinal symptoms in Eastern countries for about 1800 years [PHY906 is a TCM prepared from four medical herbs, such as00 years . In rece00 years . It playCurrently, microarray technology has been widely applied to elucidate the tumor progression in different conditions. Wang et al. developed the microarray data of mice bearing colon tumor following treatment with PHY906 plus CPT-11 and suggested that the combination of PHY906 and CPT-11 created a unique response in colon tumors .In order to explore the potential mechanism of PHY906 in treating colon tumors, we reanalyzed the published microarray data of colon tumor treated with PHY906 and PBS  individually. The differentially expressed genes (DEGs) induced by PHY906 intervention were identified and then the DEGs related function and pathways were analyzed.n = 10), PHY-906 (n = 9), CPT-1 (n = 10), and the combination of PHY-906 and CPT-1 (n = 9). In order to explore the therapeutic effect of PHY-906, we only selected the dataset of 9 samples of BDF-1 mice bearing colon 38 tumors treated with PHY906 and 10 tumor samples of those treated with PBS as controls for further analysis. The raw data were downloaded based on the platform of CCDTM Mm-CCDTM36k (derived from GPL5960).In order to evaluate the effect of PHY906 on colon cancer, we mined the gene expression data from fully public GEO database. The microarray data with the series number GSE25192 were downloaded from GEO database, which was contributed by Wang et al. . The micP values for DEGs were calculated with nonpaired t-test with the application of limma package. For DEGs identification, the cutoff value was set at P < 0.05 and |log2FC(fold change)| ≥ 0.58.The raw file data (in gpr format) were preprocessed in limma package , includiP value < 0.05 were set as the cutoff value.The gene sets that share the common biological function and pathways can be analyzed by DAVID online tool . In the The protein-protein interactions (PPIs) were predicted based on the information deposited in STRING  database . The proThe essential nodes within PPI network were analyzed based on 3 network centralities, such as degree centrality , betweenP < 2.0E − 6 were considered as significant.The gene sets in the modules of network generally share the common biological process. The significant modules in PPI network were analyzed by hierarchical cluster analysis with the application of ClusterONE  and the The TFs that played a regulatory role in module were predicted by iRegulon Cytoscape plugin . The TF-Total 751 DEGs were identified in PHY906 treated colon tumor tissues, including 414 up- and 337 downregulated DEGs. As shown in The DEGs were enriched in 3 GO Categories, such as biological process (BP), cellar component (CC), and molecular function (MF) . The uprBased on the protein interactions corresponding to DEGs, the PPI network was constructed, which was comprised of 365 DEGs and 1005 protein pairs . Then, tAfter cluster analysis, 2 significant modules were screened. Function enrichment analysis showed that the module 1 genes were closely related to sensory perception of smell, sensory perception of chemical stimulus related biological process, olfactory receptor activity related MF, and olfactory transduction pathway. The module 2 genes were significantly enriched in cell cycle and DNA replication related BP and pathways .PHY906, as one of the traditional Chinese herbal formulations, possesses the advantage in decreasing the side effect and complications following chemo- and radiotherapies . PHY906 Our data demonstrated that the PHY906 administration induced significant alterations in gene expression levels of colon tumors comparable to PBS treatment. In the presents study, total 751 DEGs were screened out in PHY906 treated tumors including 414 upregulated DEGs and 337 downregulated ones. Function analysis showed the regulation of interferon-gamma production and response to steroid hormone stimulus were dysregulated in PHY906 treated tumors.γ) production is significantly enriched by downregulated genes. It has been reported that IFN-γ affects the barrier function of epithelial monolayers [γ has been found to affect the actin distribution and tight junction permeability is affected by cytoskeletal rearrangements [γ may decrease the epithelial barrier function by rearranging cytoskeletal actin. It is in line with our finding that the actin cytoskeleton was activated by upregulated genes in PHY906 treated colon tumors. Our work also found that IFN-γ production was decreased by involvement with downregulated genes, which could illustrate that PHY906 treatment protected the epithelial barrier against tumor cell invasion by modulating IFN-γ level.The biological process of regulation of interferon-gamma (IFN-nolayers . The connolayers . IFN-γ hngements . IFN-γ mIn this paper, response to steroid hormone stimulus was significantly enriched by upregulated genes. Steroid hormone is suggested to be a major regulator in cell apoptosis . PreviouThe original paper suggested that PHY906 directly modulated the cancer cell survival in cell cycle and apoptosis in tumor cells, which is similar to our findings. PPI network analysis also showed that the genes in module 2 were closely related to cell cycle and genes in module 1 were significantly enriched in sensory perception of chemical stimulus. It is reported that PHY906 reduces chemotherapy-induced gastrointestinal toxicity in colon cancer treatment . The celMoreover, Ezh2 was found to be the key node in PPI network according to both degree and betweenness. Ezh2 was also clustered in module 2. A line of evidence showed that Ezh2 played key role in stimulating cell growth and proliferation in colon cancer . The expDespite the significant findings in this paper, the microarray data analyzed were derived from murine colon tumor tissues. It is unknown whether the similar findings can be produced over human colon cancer tissues. It is a limitation in the present study and further analyses based on human samples are warranted in the near future.In conclusion, PHY906 showed characteristic in controlling cell apoptosis by intervening interferon-gamma production and response to steroid hormone stimulus. The significant node of Ezh2 and its TFs such as E2f1, Hsfy2, and Nfyb may show the potential therapeutic effect on colon cancer. Our study may provide insights to understand the role of PHY906 in treating colon cancer and provide valuable information for novel anticancer agents development in the future. However, further studies are urgently needed.


Lung cancer is the most common cause of cancer mortality worldwide, with an estimated 1.4 million deaths each year. Here we report whole-exome sequencing of nine tumor/normal tissue pairs from Chinese patients with non-small cell lung carcinoma (NSCLC). This allows us to identify a number of significantly mutated genes in NSCLC, which were highly enriched in DNA damage repair, NF-κB pathway, JAK/STAT signaling and chromatin modification. Notably, we identify a histone-lysine methyltransferase gene, namely, MLL2, as one of the most significantly mutated genes in our screen. In a following validation study, we identify deleterious mutations of MLL2 in 12 out of 105 (11.4%) NSCLC patients. Additionally, reduced or lost expression of MLL2 was commonly observed in tumor tissues as compared with paired adjacent non-tumor tissues regardless of mutation status. Together, our study defines the landscape of somatic mutations in Chinese NSCLC and supports the role of MLL2 mutation in the pathogenesis of the disease. Lung cancer is the most commonly diagnosed cancer as well as the leading cause of cancer deaths worldwide. It has been classified into two major types, including NSCLC and small cell lung cancer (SCLC)1Cancer is a highly heterogeneous disease associated with various environmental exposures and genetic susceptibility which leads to unrestrained cell proliferation and neoplasm formation. The accumulation of somatic DNA alterations is considered as one of the contributing factors in carcinogenesis232567811018567141617919Here we used massively parallel sequencing to sequence the exomes of nine NSCLCs and matched normal adjacent tissue pairs. This allows us to identify a number of known and previously unreported genes that are significantly mutated in these patients. Pathway analysis and network reconstruction revealed several key signaling cascades affected in NSCLC including cell-cycle progressing, JAK/STAT signaling and chromatin remodeling. These results provide new insights into the molecular pathogenesis of NSCLC in Chinese patients.vs 52 ± 16 per nonsmk, p = 0.001, Student's t-test). Consistent with previous studies916In this study, we performed whole-exome sequencing of paired tumour/control DNA from nine Chinese patients with NSCLC, including eight ADCs and one SCC, with a mean depth of 104× and 93.8% of bases covered to at least 20× . Altoget56915We then selected a subset of nonsynonymous mutations from genes that are mutated in multiple patients for further validation. Using Sequenom MassARRAY genotyping, we validated 101 out of the 108 (93.5%) nonsynonymous mutations examined , indicatThis systematic approach allowed us to identify several well-described target genes as well as a number of previously unreported genes that are recurrently mutated in our NSCLC cohort. Notably, we found nonsynonymous mutations in 62 census cancer genes as reported in Catalogue of Somatic Mutations in Cancer (COSMIC) database, indicating that they were highly mutated in NSCLC and causally linked to the pathogenesis of the disease. This included many known genes associated with lung cancer, such as TP53, EGFR, KRAS and ERBB4. Indeed, TP53 was the second most frequently mutated gene identified in this study, with two nonsense and three missense mutations in five tumor samples (56%). A missense mutation and a mutation in 5′-UTR region of EGFR were identified in two different samples. We also found one missense mutation of KRAS and one missense mutation of ERBB4 in different patients. TTN is the most frequently mutated proteins in our study which contains 13 missense mutations in 6 out of 9 samples (67%). This is consistent with results from COSMIC that 244 out of the 467 (52%) lung cancer samples sequenced have somatic mutations in TTN. TTN encodes a very large, high abundant protein of striated muscle. However, considering the large size of the protein and prevalence of TTN mutations identified in other studies unrelated to cancer, it is presently hard to determine whether mutations in TTN act as drivers or are only passengers in NSCLCWe then used MuSiC algorithm221924252622In addition to mutations, we also identified small insertions/deletions (indels) as well as copy number variations (CNVs) in these nine patients using different computational approaches (see methods). All together we identified 93 high confident indels in these nine samples . We founAlthough it seems that the mutations are tremendously diverse and complex, however, they might disturb genes involved in the same signaling pathway or regulatory module6282919242527We then mapped mutant genes into a global signaling network using cytoscape32The frequent mutations of genes involved in chromatin modification and the discovery of MLL2 as one of the most frequently mutated genes in NSCLC lead us to study this gene in greater detail. We then resequenced the coding regions of MLL2 in the discovery cohort as well as an additional 96 NSCLC samples by targeted sequencing. The two cohorts together encompass 105 NSCLC cancers, including 81 ADC and 24 SCC. Specifically, all exons of MLL2 in each tumor tissue were amplified using long-range PCR, followed by barcoded sequencing on the Illumina Genome Analyzer with an average coverage of ~1000× per sample. After removing known mutations in dbSNP and 1000 Genomes, we resequenced all nonsynonmous mutations in tumor/normal pairs to screen for novel somatic mutations using Sequenom followed by Sanger sequencing. All together we identified inactivating mutations or protein deficiency of MLL2 in 12 out of 105 (11.4%) individuals , Table 3−6, Student's t-test), regardless of the mutation status. All together, we found that MLL2 was frequently mutated and repressed in NSCLC, supporting its role as a critical tumor suppressor.We then used quantitative real-time PCR to study MLL2 expression in 23 NSCLC samples with good RNA quality, including the nine samples in the discovery cohort. Somatic mutations of MLL2 were identified only in three tumors in the discovery cohort. Interestingly, we found that MLL2 expression was either silenced or significantly reduced in all tumor tissues compared with adjacent non-tumor lung tissues , purified, and hybridized to the NimbleGen 44 M human exome array for enrichment. Each captured library was then loaded on Hiseq2000 platform, and we performed high-throughput sequencing for each captured library. Each sample was sequenced at the mean depth of 100× to achieve high sensitivity and accuracy for mutations detection. Raw image files were processed by Illumina basecalling Software 1.7 for base-calling with default parameters and the sequences of each individual were generated as 90 bp pair-end reads.http://snp.gs.washington.edu/SeattleSeqAnnotation137/).SOAPalignerWe use MuSic  to identify significantly mutated genes which have a observable higher mutation rate than background mutation rates. Several basic elements were inputted into MuSiC, including the TCGA MAF format files of somatic mutations of 9 lung cancer patients, the BAM files for the 9 lung cancer samples and their matched normal, and the target exons regions coordinated with the MAF files above (hg19). We applied SMG (the significantly mutated gene) test, a module in MuSiC, to compare each category of nonsynonymous mutation rates to the appropriate BMR. The probability that a gene contains significantly more mutations was determined by three independent test including FCPT, LRT and CT integrated as part of the algorithm.Subsets of nonsynonymous mutations from genes that are mutated in multiple patients identified in our initial screen were selected for validation using Sequenom MassARRAY platform. Genotyping of each tumor/normal pair involved an initial locus-specific PCR reaction, followed by single base extension using mass-modified dideoxynucleotide terminators of a primer which anneals immediately upstream of the potential polymorphic site. PCR primers and extension primers were designed by MassARRAY Assay Design 3.1 software with the default parameters. The extension products were nanodispensed to SpectroCHIP II-G384 chips and analyzed by MassARRAY Analyzer and MassARRAY Typer 4.0 software.Ingenuity Pathway Analysis (IPA) was performed to identify signaling pathways that contain higher number of mutant genes than expected by chance. Network reconstruction and Gene ontology based module search was performed with GeneMANIAWe used 15 long-range PCR to amplify the coding regions of the MLL2 gene in 105 NSCLC tumor samples  was used as reference and amplified using G-F: 5′- AAGGTGAAGGTCGGAGTCAA-3′ and G-R: 5′-AATGAAGGGGTCATTGATGG-3′. Student's t-test was used to compare levels of expression between tumor and adjacent non-tumor tissues.Supplementary Information