Escherichia coli (STEC) causes diarrhea and hemorrhagic colitis with life-threatening complications, such as hemolytic uremic syndrome. The aim of this study was to assess the molecular epidemiologic features of non-O157 STEC strains from different resources in China and illustrate the role of animal reservoirs or animal-derived foodstuffs in human STEC infections. A collection of 301 non-O157 STEC isolates from domestic and wild animals , raw meats , diarrheal patients, and healthy carriers in different regions of China were selected in this study. Of the 301 analyzed STEC isolates, 67 serogroups, and 118 serotypes were identified; this included some predominant serogroups associated with human disease, such as O26, O45, O103, O111, and O121. Eighteen different combinations of stx subtypes were found. Eleven isolates carried the intimin gene eae, 93 isolates contained ehxA, and 73 isolates carried astA. The prevalence of other putative adhesion genes saa, paa, efa1, and toxB was 28.90% (87), 6.98% (21), 2.31% (7), and 1% (3), respectively. The phylogenetic distribution of isolates was analyzed by multilocus sequence typing (MLST). Ninety-four sequence types were assigned across the 301 isolates. A subset of isolates recovered from yak and pika residing in the similar wild environments, Qinghai-Tibetan plateau, showed similar genetic profiles and more tendencies to cluster together. Isolates from goat and mutton exhibited close genetic relatedness with those from human-derived isolates, providing evidence that transmission may have occurred locally within intraspecies or interspecies, and importantly, from animal reservoirs, or raw meats to humans. Comparing isolates in this study with highly virulent strains by MLST, along with serotyping and virulence profiles, it is conceivable that some of isolates from goat, yak, or raw meats may have potential to cause human diseases.Shiga toxin-producing  Escherichia coli (STEC) refers to an E. coli pathotype capable of producing either Shiga toxin 1 (Stx1), Shiga toxin 2 (Stx2), or both toxins. STEC has emerged as an important enteric foodborne zoonotic pathogen causing human gastrointestinal disease and has been implicated in sporadic cases and outbreaks of diarrhea, hemorrhagic colitis (HC), and hemolytic uremic syndrome (HUS) worldwide  have been identified , can lead to the formation of attaching and effacing (A/E) lesions  heat-stable enterotoxin, which is encoded by the astA gene  and screened for stx genes by a duplex PCR assay; the primers used for detecting stx1 and stx2 genes are listed in Table stx1 and/or stx2 genes were plated on CHROMagar™ ECC agar , CHROMagar™ STEC agar , and MacConkey agar  and incubated overnight at 37°C. The following colonies were picked and screened for the presence of stx1 and/or stx2 genes by single colony duplex PCR assay: blue or colorless round moist E. coli-like colonies on ECC agar; mauve colonies on STEC agar; and pink or red colonies on MacConkey agar. Each stx-positive isolate was confirmed to be E. coli by biochemical tests using the commercially available API 20E system . The prevalence of non-O157 STEC in different sources is shown in Table In total, 301 non-O157 STEC isolates were collected from various sources in China; of these, 54, 22, 93, and 63 were isolated from the yak, pika, pig, and raw meats sources reported in our previous studies  were used to confirm the PCR results. The isolates were referred as O-untypable if they did not react with any O antisera. The entire coding sequence of fliC was amplified by PCR using the primers: F-FLIC1 (5′-ATGGCACAAGTCATTAATACCCAAC-3′) and R-FLIC2 (5′-CTAACCCTGCAGCAGAGACA-3′)  to determine the H type of each isolate  were amplified and sequenced according to the E. coli MLST website (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli). Sequences types (STs) for each isolate were assigned based on the allelic profile of the seven housekeeping genes. A neighbor-joining tree was constructed by MEGA 6 based on the concatenated sequences of the seven housekeeping genes, and was used to analyze the phylogenetic relationships among strains. STs of isolates from this study were then compared with those from highly pathogenic STEC strains, including the STs of the HUS-associated enterohemorrhagic E. coli (HUSEC) collection (www.ehec.org) . A minimum spanning tree (MST) based on these STs was generated using the BioNumerics software.Multilocus sequence typing (MLST) was used to characterize phylogenetic relationships and assess the potential risks for human infection. Defined fragments of the seven housekeeping genes  and five isolates were H-untypable (HNT). The predominant were serotypes ONT:H30 and O20:H30, which accounted for 30 and 21 isolates, respectively; other prevalent serotypes were: O2:H32 (10 isolates), ONT:H21 (9 isolates), O21:H25 (9 isolates), O128:H2 (8 isolates), O103:H8 (8 isolates), O2:H45 (7 isolates), O130:H8 (6 isolates), O117:H21 (6 isolates), O8:H16 (5 isolates), and O176:H4 (5 isolates). Nine serotypes  contained four isolates each; 11 serotypes  contained three isolates each; 22 serotypes contained two isolates each; while 64 serotypes contained only one isolate each Table .The majority of non-O157 STEC strains that are a threat to human health are associated with six specific serogroups . In this study, four isolates from diarrheal patients were assigned to these serogroups/serotypes (three were O26:H11 and one was O111:H8). The serotypes O103:H8 and O45:H2 were detected in eight and two goat isolates, respectively; two beef isolates were assigned to serotype O103:H25, and serotype O121:H10 was found in one pork isolate. None of the STEC isolates from antelopes, pikas, yaks, pigs, cattle, or healthy carriers belonged to the six top serogroups Table .stx1 positive only, 192 (63.79%) were stx2 positive only, and 30 (9.97%) were both stx1 and stx2 positive. Overall, three stx1 subtypes  and six stx2 subtypes  were detected; this resulted in a total of 18 different stx1 and stx2 subtype combinations. Among these subtype combinations, stx2e was most prevalent (121 isolates), followed by stx1a (43), stx1c (35), stx2b (26), stx2d (20), stx1a, and stx2d (10), and stx2g(10); this accounted for a total of 265 strains (88.04%)  were stx subtypes and combinations. All of the 107 pig-derived isolates  possessed only stx2e. Two isolates from antelope were only stx1a. The majority of cattle isolates  harbored only stx2; of these, stx2d and stx2g were the dominant subtypes. The isolates from diarrheal patients were largely dominated by only stx1 ; of these, 10 were the stx1a subtype and 11 were the stx1c subtype . ehxA was detected in 93 isolates covering all reservoirs investigated in this study, and the 11 eae positive isolates were all positive for ehxA. Seventy-three isolates contained astA; the majority  of these were pig-derived. The prevalence of the adhesion-associated genes saa, paa, and efa1 was 28.90% (87), 6.98% (21), and 2.31% (7), respectively , beef (3), cattle (1), yak (1), and mutton (1) were ly Table . Notablyaa Table .Ninety-four sequence types (STs) were obtained from the 301 non-O157 STEC isolates. ST710 (27 isolates) and ST993 (19 isolates) were most common, followed by ST155 (15 isolates), ST906 (12 isolates), ST13 (10 isolates), ST540 (10 isolates), and ST3628 (9 isolates). Three STs  contained eight isolates each; ST56, ST297, and ST3692 contained seven isolates each; ST446 and ST953 contained six isolates each. Further, six STs  were detected in five isolates each; five STs  contained four isolates each; six STs contained three isolates each; while 12 STs contained two isolates each and 50 STs were found only once , and virulence gene composition  within a cluster contained isolates from cattle feces and beef. The 24 isolates from diarrheal patients assigned to 13 STs were scattered throughout the phylogenetic tree. Isolate STEC408 from a diarrheal patient sampled in Shanghai city shared the same sequence type (ST155), serotype (O21:H25), stx subtype (stx1a only) and virulence profiles with four isolates from goats sampled in Henan province . Similarly, isolate STEC409 from a diarrheal patient in Shanghai city had the same sequence type (ST993), serotype (ONT:H30), and stx subtype (stx2e only) as 13 pig-derived isolates sampled in Beijing city and Chongqing city, all of which possessed none of the virulence genes tested  in Shenzhen city had the same serotype and stx subtype as a mutton isolate (STEC317) sampled in Beijing city  that shared same STs . Three subsets of STs  from both yak and pika were grouped on similar branches. Notably, four isolates of ST3692, three from yaks and one from pika, shared the same serotype (O2:H45), n Figure . ST88, Sd Figure . A subsey Figure .E. coli MLST database to assess the potential risk for human infection  carried stx1a, stx2d, ehxA, and saa; the isolate from mutton (STEC317) harbored stx1c and saa; while none of the three isolates  possessed any of the virulence genes tested in this study except for stx , goat (STEC007 and STEC009), healthy carrier (STEC435), and raw mutton (STEC362) had the same ST (ST33) as human STECs; four of these isolates were the serotype O91:H14, which is one of the most common serotypes in human pathogenic eae-negative STEC strains; while one isolate from antelope (STEC477) was assigned to serotype O50:H14  living on plateaus with elevations between 3500 and 5500 m, a low partial pressure of oxygen, and high ultraviolet radiation levels, and follows up on our previous studies of STEC in yak and pika residing in the similar extremely harsh wild environments  and raw meats. The prevalence of STEC in yak, pika, pig, cattle, and raw meats samples was 17.58, 1.99, 9.27, 2.73, and 7.4%, respectively  of the isolates, which may represent emerging serogroups or serotypes; however, the potential for causing human infection remains unknown. Even though hundreds of non-O157 serotypes have been described, serotypes O26, O45, O103, O111, O121, and O145 have been suggested to be the most important for human health  that were linked to severe human illness. Almost all animal isolates, with the exception of pig-derived isolates, support our hypothesis that pig-derived STEC isolates have a low potential for causing human disease , O111:H8, O5:HNT, O5:H9, and O78:HNT. This founding supports the notion that the putative adhesion genes toxB and efa1 are present mainly in isolates of highly prevalent and pathogenic serotypes that are eae positive (Toma et al., Shiga toxin subtypes have been implicated in severe outcome of STEC infection. Stx2 is more frequently associated with disease than Stx1, while Stx2a, Stx2c, and Stx2d are more commonly associated with HC and HUS. The remaining subtypes are only found in patients with uncomplicated diarrhea (Fuller et al., stx subtype, the strains did cluster based on geographic location or animal species. We observed that a subset of isolates recovered from yak and pika in the same unique ecosystem, the Qinghai-Tibetan plateau, shared same STs and were more likely to cluster together. Thus, it is possible that some isolates persist in specific environments and are transmitted locally within intraspecies and interspecies. A similar correlation was also observed in isolates derived from the same animal species regardless of whether they arise from feces or animal meat, e.g., feces of pig and pork or feces of cattle and beef, indicating that most STEC infections due to animal-derived foodstuffs might be traced back to the food producing animals as a specific source, instead of an entry from environmental or human sources along the food chain. Notably, isolates obtained from diarrheal patients did not belong to a specific phylogenetic cluster and were scattered throughout the phylogenetic tree. Furthermore, isolates from goats, pigs, and raw meats were genetically similar to those from human-derived isolates based on MLST, serotypes and virulence profiles, regardless of geographic locations. This also supports the notion of STEC transmission from livestock or raw meats to humans; nevertheless, the transmission route is still unclear and warrants further investigation.MLST has been proposed as an adequate tool for producing genetic profiles for a vast number of isolates, especially under non-epidemic circumstances (Ferdous et al., stx subtypes and virulence gene panels. These data suggest that diarrheal patients, goats, yaks, and raw meats are the important reservoirs of pathogenic STEC strains; while, isolates from antelopes, pikas, pigs, cattle, and healthy carriers likely pose low risks to human health. Given that non-O157 STEC strains are oral-fecal organisms, it is urgent to monitor the local human population for STEC infections, which may be acquired by direct contact with infected patients, wild, or domestic animals as well as consumption of contaminated animal-derived foodstuffs.An analysis of non-O157 STEC isolates in this study against the HUSEC collection, and human STECs of O157 and the top six serogroups revealed that some STEC isolates from diarrheal patients, healthy carriers, goats, pikas, yaks, and raw meats were closely related to pathogenic STEC isolates. Moreover, when combined with serotyping and virulence analysis, it is clear that isolates from diarrheal patients, goats, yaks, and raw meats are more likely to possess highly pathogenic serotypes that have been associated with severe diseases including O26:H11, O111:H8, O45:H2, O91:H14, and O8:H19 Hussein, , as wellstx subtyping, and virulence gene profiling suggest that STEC from different resources across China are highly genetically diverse, that transmission may occur within intraspecies or interspecies, and there is potential for human infections to originate from animal reservoirs or animal-derived foodstuffs. The pathogenicity varied across isolates from different sources. Further work should involve investigation of the epidemiological role of animal reservoirs and animal-derived foodstuffs in the maintenance of STEC, and the genetic markers that related to pathogenicity to determine the direction of transmission and differentiate low- and high-risk non-O157 STEC infections.To the best of our knowledge, this is the first and largest survey to systematically analyze the molecular and phylogenetic features of non-O157 STEC strains in China. Our survey indicates that non-O157 STEC are widely distributed across multiple sources and regions in China. MLST analysis, serotyping, XB, SL and YX designed the project; XB, YX, YJ, HW, QG, BH, SL, and XX carried out the sampling work; XB, BH, YXu, AZ, HS, PB, SF, and RF carried out the experiments and generated data; XB, SL, and YX analyzed data and drafted the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


AbstractMicrocharon Karaman, 1934 (Asellota: Lepidocharontidae) is described from Miho-Uchihama beach, Shizuoka, Japan. Microcharontanakaisp. n. differs from its congeners by having nine simple, five penicillate setae on antennal article 6; one simple distal seta on article 1 of the mandibular palp and having the apical lobe of male pleopod 1 convex, rounded, armed with seven setae. A key to Asian species of the genus and 16S rRNA of the new species are provided.A new species of  Microcharon Karaman, 1934, are free-living interstitial isopods. Being highly adapted to the narrow spaces of the interstitial environment, the species possesses an elongated body without visual organs or pigmentation , a small number of psammolittoral isopods were collected together with other marine interstitial fauna such as, harpacticoid copepods, nematodes and ostracods. The isopod specimens had a typical National Institute of Biological Resources (NIBR), Korea. Two female and one male were transferred to isoamyl acetate for 20 minutes and dried in a critical-point dryer Hitachi E-1010. Dried specimens were mounted onto a SEM stub and coated with gold using a sputter coater to a thickness of 15-30 nm. Coated specimens were examined and photographed with a Hitachi S-3400 scanning electron microscope at Chungang University (Seoul). Measurements were done following the method of Seven female and two male specimens were collected from coarse sand from Miho- Uchihama beach, Shizuoka City, Shizuoka Prefecture, Japan on 7 February 2015  using PCR premix (BIONEER.Co) in TaKaRa PCR thermal cycler . The primers used were 16sar-L (5`-CWAAYCATAAAGAYATTGGNAC-3`) and 16sar-H (5`-ACTTCAGERTGNCCAAARAAYCA-3`)  following the guidelines provided with the kit. DNA was sequenced on an ABI automatic capillary sequencer  using the same set of primers. All obtained sequences were visualized using Finch TV version1.4.0 (http://www.geospiza.com/Products/finchtv.shtml). Each sequence was checked for the quality of signal and sites with possible low resolution, and corrected by comparing forward and reverse strands.Two females from the type locality sample were identified without dissection under an Olympus SZX 12 dissecting microscope. Before amplification, specimens were transferred into distilled water for 20 minutes to remove ethanol and then macerated with a small glass rod. Whole specimens were used to isolate genomic DNA with the aid of the LaboPassTM Kit  following the manufacturer’s protocols. 16S rRNA was amplified with polymerase chain reaction (AYCA-3`) . The ampTaxon classificationAnimaliaIsopodaLepidocharontidaeGenusKaraman, 1934Microcharonstygius  completely dissected and mounted in lactophenol on four slides; paratype 1 female (NIBRIV0000787790) dissected on three slides, paratype 2 female (NIBRIV0000787791) dissected on one slide; adult male pleotelson dissected on three slides; 2 females and 1 male used for SEM.PageBreakPageBreaklateral lobe of maxillula with eleven robust setae; distal apex of male pleopod 1 convex, round, with three apical, four subapical setae; protopod of male pleopod 2.9 times longer than wide; protopod of uropod 3.1 times longer than wide, with fifteen setae.Antennular article 6 smallest, with one aesthetasc, one penicillate, three simple setae distally; article 1 of mandibular palp with one distal simple seta; Body ; propodus, pereopod 5 with two bifid, two simple, one penicillate setae , with sclerite on distal margin, covering proximal part of dactylus ; separated in half by medial sperm tube running from triangular opening on proximal part of medial groove, distolateral edge of hyaline lamella straight, without ornamentations; parallel to lateral margin of pleopod, in lateral view distal end of lobes convex, rounded at distal apex, each with three simple setae distally, four subapical setae on ventral margin.d 1 Figs , 10M: elPleopod 2  and BLAST  was the most similar sequence to M.tanakai resulted by Megablast optimization with the 74% identity, 1e-36 E-value, 92% of query cover, 165 of total and max score. On the other hand, when Blastn optimization was chosen, Betamorphafusiformis  was the most similar sequence to that of our new species with 73% identity, 4e-67 E-value, 100% of query cover, and 266 of total and max score.Partial 16S rRNA sequence was obtained only from a single female. The final length of trimmed sequence was 493 base pairs  body slender, elongate and all somites subequal in width, 2) pereopods 1-4 inserted anterolaterally, pereopods 5-7 inserted posterolaterally 3) coxal plates indiscernible in dorsal view, 4) pleotelson longer than wide, longer than any pereonite, 5) antennal article three with scale laterally 6) antennal flagellum longer than podomeres 7) maxillipedal palps composed of five articles, which are broader than the endite, 8) pereopod 1 leg-like, 9) uropods with long, broad protopod, both endopod and expod shorter than protopod in all other marine species of Microcharon and exopod inserted subapically ; and the endopod of the pleopod 3 with three penicillate setae . The other Asian species, M.kirghisicus and M.raffaellae described from Kyrgyzstan (M.tanakai by the number of articles on the antennula (5 vs 6); both the carpus and propodus of the pereopod with only one simple seta along the medial margin and a difference in the setal formula of the endopod of pleopod 3 (naked vs with three penicillate setae).apically . The newmenistan , it showrgyzstan  and Iranrgyzstan  can be eM.tanakai. To date, 228 16S rRNA sequences of the suborder Janiroidea G.O. Sars, 1897 have been deposited on GenBank belonging to the following 7 families: Desmosomatidae G.O. Sars, 1897, Macrostylidae Hansen, 1916, Joeropsididae Nordenstam, 1933, Janiridae G.O. Sars, 1897, Munnopsidae Lilljeborg, 1864, Haploniscidae Hansen, 1916 and Acanthaspidiidae Menzies, 1962. Although this molecular marker has not been commonly used for barcoding (Serolidae Dana, 1852, Chaetiliidae Dana, 1849 and Munnopsidae Lilljeborg, 1864 (We were able to amplify 16S rRNA partial sequence of arcoding , the 16Srg, 1864 .


Cardiac development is a complex morphogenetic process initiated as bilateral cardiogenic mesoderm is specified at both sides of the gastrulating embryo. Soon thereafter, these cardiogenic cells fuse at the embryonic midline configuring a symmetrical linear cardiac tube. Left/right bilateral asymmetry is first detected in the forming heart as the cardiac tube bends to the right, and subsequently, atrial and ventricular chambers develop. Molecular signals emanating from the node confer distinct left/right signalling pathways that ultimately lead to activation of the homeobox transcription factor Pitx2 in the left side of distinct embryonic organ anlagen, including the developing heart. Asymmetric expression of Pitx2 has therefore been reported during different cardiac developmental stages, and genetic deletion of Pitx2 provided evidence of key regulatory roles of this transcription factor during cardiogenesis and thus congenital heart diseases. More recently, impaired Pitx2 function has also been linked to arrhythmogenic processes, providing novel roles in the adult heart. In this manuscript, we provide a state-of-the-art review of the fundamental roles of Pitx2 during cardiogenesis, arrhythmogenesis and its contribution to congenital heart diseases. Cardiac development is an intricate morphogenetic process involving distinct cell lineages. The first steps of cardiogenic formation are initiated as bilateral precardiogenic mesoderm becomes specified . Soon thWhile it is rather simple to ascribe embryonic right and left components to the initial symmetrical cardiac tube , the conOver the last decade, our understanding of the molecular regulatory mechanisms underlying cardiovascular development has greatly advanced. Seminal papers reported key roles of distinct growth factors ,15,16 anA particular case is the homeobox transcription factor Pitx2. Pitx2 expression is already present in the developing embryo soon after gastrulation ,31,32,33Homeobox genes encode transcription factors that bind to cognate DNA sequences through their conserved homeodomain regions . Pitx1, The Pitx family is crucial for the formation of several tissues. Pitx1 is involved in development and specification of a number of structures including first branchial arch derivatives, body wall and hind limb musculature ,38,39,40Pitx2 gene, which is located on chromosome 3  in the mouse [Pitx2a and Pitx2b located upstream of exon 1, whereas Pitx2c uses an alternative promoter located upstream of exon 4 and a left side-specific enhancer (ASE) upstream of exon 6 [Pitx2b specific, while exon 4 is Pitx2c specific. Different laboratories assessed by genetic and/or epigenetic studies and Pitx2 knockout mice that this gene is required for the proper development of several organs and structures, such as the pituitary gland, craniofacial region, eyes, heart, abdominal viscera and limbs [PITX2 was identified by positional cloning of the 4q25 locus in patients with Axenfeld-Rieger syndrome [PITX2D) in humans generated by the PITX2C alternative promoter and differential splicing, acting as a dominant negative isoform, specifically against PITX2A and PITX2C , Tetralogy of Fallot , ventricular septal defects (HAND2), double outlet right ventricle (HAND1), either syndromic  or isolated  is the most prevalent developmental abnormality in humans. CHD is indeed the most common non-infectious cause of infant morbidity and mortality. Increasing evidence has demonstrated a crucial contribution of genetic defects in the pathogenesis of CHD. Mutations in several transcription factors such as  reviews ,111). HoPitx2 expression [The adult heart is an asymmetric organ. It is topologically located outside the body midline with the apex oriented to the left. Morphologically, the atrial chambers are rather similar, but conspicuous differences, such as the extent of the smooth region and the abundance of the pectinate muscles, provide hallmarks for distinguishing right and left atrial appendages . Such lepression ,7,113.situs inversus, or not, a condition dubbed situs ambiguus. Several lines of experimental evidence demonstrated that impaired left/right signalling underlies RAI and LAI.Several cardiac abnormalities have been classically reported as defects of the establishment of the left/right body axis . Right atrial isomerism (RAI) is the most common defect in which the heart is formed by isomeric right atria and impaired venous return connections, displaying in most cases the absence of direct pulmonary vein drainage to the atria and bilateral caval veins connections. In this context, both atria display a right-specific pattern; i.e., morphologically right atrial appendages. Left atrial isomerism (LAI) is rather less frequent, presenting isomeric left atria with the absence of direct caval vein drainage to the atria and bilateral pulmonary vein connections, i.e., right pulmonary vein to the right atrium and left pulmonary veins to the left atrium. Similarly LAI also displays a left-specific pattern in both atria; i.e., with morphologically left atrial appendages. These cardiac alterations can occur with an impaired left/right topology of all other body organs, a condition dubbed Pitx2, on the genesis of these cardiac congenital defects.The plausible contribution of left/right signalling to other congenital heart defects such as double outlet right ventricle (DORV), double inlet left ventricle (DILV), atrial or atrioventricular septal defects (ASD and AVSD) remains enigmatic, as well as in syndromic congenital heart defects such as Tetralogy of Fallot. Thus, an important question is when is cardiac asymmetry established during embryonic development and which is the particular contribution of left/right cardiac embryonic tissues to the adult heart. We will provide herein state-of-the-art evidence on the plausible role of left/right signaling, with particular emphasis on the homeobox transcription factor Pitx2 in experimental models revealed complex embryonic defects. Abnormal sidedness was observed in distinct developing organs such as the lungs, gut and heart in mice. Embryonic development was compromised at Embryonic Days (ED) 13–14. Within the cardiovascular system, Pitx2 null mutants displayed right atrial isomerism with impaired venous return [Pitx2c in mice demonstrated that it was mainly this isoform that was responsible for all of the cardiovascular embryonic defects [Pitx2 driving cardiac left-right asymmetry from the secondary heart field [Pitx2 in distinct cardiac compartments. Tessari et al. [Pitx2 function in the early embryonic cardiomyocytes, and surprisingly, right/left cardiac asymmetry was unaltered. Atrial and ventricular-specific deletion in mouse embryos [Pitx2 deletion was carried out in the nascent cardiomyocytes, right atrial isomerism was observed [Pitx2 as a candidate gene for cardiac congenital heart diseases , i.e., DORV and VSD. In all cases, functional analyses of their corresponding PITX2 mutation leads to decreased transcriptional activity and reduced synergistic activation with NKX2.5 [CITED2, identified in a Tetralogy of Fallot patient with aortic stenosis, impaired PITX2 and VEGF expression [PITX2 mutations in isolated CHD, a loss-of-function PITX2 mutation has been identified in an Axenfeld-Rieger syndrome patient carrying cardiac endocardial cushion defects. Such a PITX2 mutation provides no transcriptional and synergistic activity with Nkx2.5 [CHARGE syndrome is characterized by ocular ing gene . Subsequutations . More rediseases , such asdiseases , TGA [12diseases , ventricdiseases ,126 and diseases . A PITX2h NKX2.5 ,126,127 pression , furtherh Nkx2.5 . OverallAtrial fibrillation (AF) is the most common cause of arrhythmogenesis, and it has been related to several risk factors, such as advanced age, male gender, hypertension, obesity, ischaemic heart disease, myocardial infarction, valvular diseases and hyperthyroidism ,135,136.CAV1, HCN4, SYNE2, SYNPO2L, PRRX1 and WNT8A [PITX2 locus [Several genome-wide association studies (GWAS) have been published reporting chromosomal loci in association with atrial fibrillation (AF). Four different genetic loci on chromosomes 1q21-KCNN3, 4q25-Pitx2, 16q22-ZFHX3 and 16q13-IL6R have been associated with this arrhythmia ,140,141.nd WNT8A ,143, butX2 locus ,146,147.X2 locus , identifKCNN3 [TBX5 [HCN4 [KCNJ2 [CAV-1 [KCNQ1 [ZFHX3; SYNE2; Il6R [Tbx5 directly activated Pitx2, and Tbx5 and Pitx2 antagonistically regulated membrane effector genes Scn5a, Gja1, Ryr2, Dsp and Atp2a2 [Distinct laboratories, including ours, have demonstrated that PITX2 potentially regulates AF through modulation of multiple genes implicated in AF; KCNN3 ,145,147;N3 [TBX5 ,149,150;X5 [HCN4 ; KCNJ2 [4 [KCNJ2 ,152; CAV1 [KCNQ1 ,153; ZFHE2; Il6R . In addiPitx2 regulation in a tissue- and isoform-specific fashion [Pitx2 are susceptible to AF during programmed stimulation [Pitx2 haploinsufficiency predisposes to AF in electrically-stimulated adult mice, provoked by ectopic Shox2 expression in the foetal left atrium (LA), which in turn deregulated other pivotal sino-atrial node genes such as Hcn4 and Tbx3 [Genomic regions containing the risk variants have been reported to play a role in  fashion ,155, and fashion ,152,156.mulation . Pitx2 hand Tbx3 .Pitx2 impairment in atrium-specific conditional Pitx2 mouse mutants [Pitx2 dosage has been reported in adult Pitx2 heterozygous mutant mice [Pitx2c heterozygous mutant mice, the expression of TWIK-related acid-sensitive K+ channel (TASK-2) and TASK-like background currents are reduced, although IK1 is not altered [Pitx2 mutant mice has been reported [Pitx2 controls Gja5 expression [PITX2C, contributing to IKs increase and ICa,L reduction [Pitx2 (upregulation or downregulation) is critical for cardiac electrophysiology and, thus, tightly linked to the onset of cardiac arrhythmias. Other substrates for AF are left-sided electrophysiological defects generated by  mutants . In this mutants . Moreoveant mice . In Pitx altered . Impairereported , being ipression ,152 and pression ). Surprieduction . While tPitx2 insufficiency also regulates WNT8 expression, which modulates a complex gene regulatory network, including multiple microRNAs, with a large impact on calcium homeostasis control and pro-arrhythmogenic events [Pitx2 and involved in AF. ChiP-sequencing studies have reported that Pitx2 positively regulates miR-17-92 and its related homologue miR-106b-25. Deficient mice in these miRNAs clusters show arrhythmia susceptibility and dysregulation of Shox2 and Tbx3, similar characteristics to Pitx2-deficient mice [K1 channel expression is modulated by miR-1 [Pitx2 atrial-deficient mice [Pitx2 and Wnt signalling, such as miR-1, miR-26b, miR-29a, miR-106a/b and miR-133 [Our laboratory recently reported that c events . Multiplent mice . IK1 chaby miR-1 , which ient mice . A signi miR-133 ,161.Pitx2 function already alters cardiac function during embryogenesis, as suggested by Wang et al. [However, a question that remains unsolved is whether impaired g et al. , or whetg et al. . We demog et al.  or by prg et al. . Furtherg et al. .Pitx2 to congenital heart diseases. Pitx2 participates in early embryonic left/right signalling and cardiac embryogenesis, and it remains to be expressed in the adult heart. Therefore, the contribution of Pitx2 to CHDs might involve distinct roles at different developmental stages. In this line of thinking, Fakhro et al. [Pitx2 expression in Xenopus experimental models, providing functional links between cilia, left/right, Pitx2 and heterotaxia. Distinct genetic Pitx2 manipulations have also reported distinct congenital heart diseases, establishing temporal and tissue-specific contributions [Pitx2 and atrial fibrillation has been reported [Pitx2 insufficiency are already present at foetal stages, suggesting therefore a congenital substrate on the future onset of atrial arrhythmogenesis. Thus, whether AF might be considered as a developmental disease should be debated. Congenital heart diseases are multifactorial. Over the last few decades, we have witnessed a large advance in the understanding of the genetics of congenital heart diseases. Mutations in different transcription factors have been identified in familial and sporadic cases of distinct congenital heart diseases. Genetic engineering in mice has further provided additional evidence of the functional role of these transcription factors (and their corresponding mutations) in CHDs. Herein, we have provided a state-of-the-art review of the contribution of the homeobox transcription factor o et al.  reportedibutions ,118,120.reported ,165,166.Pitx2 in CHDs, emerging evidence also identified point mutations associated with distinct CHDs, ranging from isolated atrial septal defects to complex cases of Tetralogy of Fallot. At present, evidence of the functional role of these point mutations in these CHDs is rudimentary. CRISPR/Cas9 gene editing of these mutations might in future shed light on the specific contribution of Pitx2 mutations to CHDs.In addition to experimental models demonstrating the functional role of


In addition, numerous new candidate genes were also identified. For example, GRMZM2G154278 in HZS, which belongs to the cell cycle control family, was closely linked to a QTN of the ear height/plant height (EH/PH) trait; GRMZM2G051943 in 207, which encodes an endochitinase precursor (EP) chitinase, was closely linked to a QTN for kernel density; and GRMZM2G170586 in Mo17 was closely linked to a QTN for ear diameter. Complex correlations among these genes were also found. Many IBD segments and genes were included in the formation of FP lines, and complex regulatory networks exist among them. These results provide new insights on the genetic basis of complex traits and provide new candidate IBD regions or genes for the improvement of special traits in maize production.Maize foundation parents (FPs) play no-alternative roles in hybrid breeding because they were widely used in the development of new lines and hybrids. The combination of different identity-by-descent (IBD) segments and genes could account for the formation patterns of different FPs, and knowledge of these IBD regions would provide an extensive foundation for the development of new candidate FP lines in future maize breeding. In this paper, a panel of 304 elite lines derived from FPs, i.e., B73, 207, Mo17, and Huangzaosi (HZS), was collected and analyzed using 43,252 single nucleotide polymorphism (SNP) markers. Most IBD segments specific to particular FP groups were identified, including 116 IBD segments in B73, 105 in Mo17, 111 in 207, and 190 in HZS. In these regions, 423 quantitative trait nucleotides (QTNs) associated with 15 agronomic traits and 804 candidate genes were identified. Some known adaptation-related genes, e.g.,  Zea mays L.) hybrids are important for providing sufficient food, feed and energy for the increasing needs of modern society. Maize hybrid breeding has undergone three stages: (1) double-cross hybrids using inbred lines derived from open-pollinated varieties, including landraces in the 1930s; (2) more productive single-cross hybrids in the late 1950s after the appearance of some heterotic groups, such as Iowa Stiff Stalk Synthetic (SS), Non-Stiff Stalk (NSS) and Iodent (IDT); and (3) superior commercial hybrids for breeding, which became increasingly privatized after the pre-1980s [Excellent maize  markers. We performed a comprehensive analysis of IBD segments transmitted from these FPs to their descendants. Further, 180 representative lines of the 304 lines were selected to evaluate phenotypes based on the results of genotyping analysis. In total, 15 agronomic traits were investigated across multiple environments in three years, and a GWAS was conducted. The consistency between the GWAS results and quantitative trait loci (QTLs) mapping results was investigated to deduce the putative functions for each IBD segment. In addition, 9 representative lines with high membership (more than 0.75) and known pedigrees in each FP group were selected to extract RNA-seq data. Some candidate genes located within IBD segments were identified through genomic, transcriptomic and phenotypic variation analysis. The objectives of this study were to 1) identify specific phenotypes and IBD segments from a given FP and its descendants, 2) uncover the functions of IBD segments, 3) identify major genes involved in the formation of maize FP lines, 4) explore genomic patterns of maize FP formation, and 5) provide new insight for dissecting the genetic basis of complex traits.The four FP lines B73, Mo17, 207, and HZS and their relevant descendants were collected, including 304 lines with co-ancestor membership of greater than 0.5. These lines were divided into four FP groups according to the results of population structure analysis and pedigree information . In totahttp://support.illumina.com/array/array_kits/maizesnp50_dna_analysis_kit.html). When the maize seedlings were one month old, the leaves of five plants were sampled in bulk to extract genomic DNA according to the modified CTAB procedure [http://qteller.com/qteller3/rna_data_sources.php). In addition, 36 inbred lines from a particular group of B73, Mo17, 207 and HZS were sampled according to the top ancestor memberships and known pedigree information, and the transcriptomic expression data of the genes in kernels at 15 days after pollination were extracted from the datasets published previously [The newly collected 180 inbred lines were genotyped using the Maize SNP50 bead chip including 56,110 SNPs .According to the results of the population genetic structure analysis, one group containing a classical FP line and its descendants was denoted as the relevant FP group, which was named the representative FP line. For each group, genetic diversity (GD) was evaluated using 43,252 SNPs in PowerMarker V3.25  and was By combining the results of principal component analysis (PCA), a total of 180 lines covering more than 99.72% of the genetic diversity of the original panel were sampled . This paANOVA was performed using the PROC GLM model. Pearson correlation analysis of each trait across different environments was calculated using the PROC CORR model. Then, the best linear unbiased predictor (BLUP) calculation for each trait was implemented using a PROC MIXED model, with genotype, location, genotype by location, and replications as random effects . All anap values of the SNPs were less than the cutoff value.The BLUP values of all agronomic traits for each line across different environments and 43,252 SNPs were used to perform a phenotype-genotype GWAS, which was implemented in the R package GAPIT  using thhttp://www.maizegdb.org/). The physical positions of each IBD segment, QTL, and QTN were queried against the B73 reference genome (RefGen_v2). On this basis, consistency analysis was conducted by projecting IBD segments, QTLs, and QTNs onto the identical genetic frame diagram. IBD segments containing both QTLs and QTNs were treated as important candidate genetic regions and were analyzed in depth.QTLs associated with the 15 agronomic traits were collected from public data and the literature (2(FPKM) [For each important IBD region, genes predicted by the Filtered Gene Set model  and loca2(FPKM) , ANOVA b2(FPKM) . IntegraThe 304 lines were clustered into four groups  correspoBased on the data of the 15 main agronomic traits in the 180 lines, specific variation characteristics were found in different FP groups . For exavgt1 on chromosome 8 and was significantly associated with DTP, DTS and DTT.The values of BLUPs for the 15 agronomic traits of the 180 lines across 15 environments were used in phenotype-genotype association mapping. The major results are shown in vs. B73, 207 vs. HZS and 207 vs. Mo17, respectively; 168 and 137 segments were common in B73 vs. HZS and B73 vs. Mo17, respectively; and 183 segments were common in HZS vs. Mo17. Triple comparison showed that 133 segments were common among B73, 207 and HZS; 130 among B73, 207 and Mo17; 135 among HZS, 207 and Mo17; and 132 among B73, HZS and Mo17. Quadruple comparison showed that 128 segments were common among these four groups. By contrast, 116 IBD segments were specific to the B73 group, 111 to 207, 190 to HZS, and 105 to Mo17  varying from -3.45 to 5.39, where a minus sign indicates a downward change , ear diameter (ED), ear row number (ER), and kernel number per row (KNPR). “b” shows flowering time-related traits, including DTP, DTS, DTT, and ASI. “c” shows kernel shape-related traits, including 10-kernel length (10KL), 10-kernel width (10KW), 10KL/10KW, and 10-kernel thickness (10KT). “d” shows plant architecture-related traits, including plant height (PH), ear height (EH), and EH/PH. “e” shows kernel weight-related traits, including 100-kernel weight (KWE), 100-kernel volume (KV), and kernel density (KD).(TIF)Click here for additional data file.S2 FigRed and gray lines are defined as 0.05 and 0.01 divided by the SNP number of 43,252, respectively. “a” shows ear-related traits, including ear length (EL), ear diameter (ED), ear row number (ER), and kernel number per row (KNPR). “b” shows flowering time-related traits, including DTP, DTS, DTT, and ASI. “c” shows yield-related traits, including 10-kernel length (10KL), 10-kernel width (10KW), kernel density (KD) and 100-kernel volume (KV).(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 TableEH, PH, EL, ED, KD, KNPR, DTT, DTP, DTS, ASI, KV, 10KW, 10KL, and ER are the abbreviations for ear height, plant height, ear length, ear diameter, kernel density, kernel number per row, days to tasseling, days to pollination, days to silking, interval between silking and anthesis, 100-kernel volume, 10-kernel width, 10-kernel length and ear row number, respectively.(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 TableFPKM is the abbreviation for fragments per kilobase of exon per million fragments mapped.(XLSX)Click here for additional data file.


Epinephelus akaara, in a range of different experimental conditions. Using the software packages geNorm, NormFinder, BestKeeper and refFinder, three traditionally used reference genes, β-actin (β-ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-2-microglobulin (B2M), were identified as not suitable for E. akaara gene expression studies, whereas two newly identified reference genes, conserved oligomeric Golgi complex subunit 5 (Cog5) and brefeldin a-inhibited guanine nucleotide-exchange protein 1 (ARFGEF1), could be universally applied under all the tested conditions. These data provide the foundation for more precise results in RT-qPCR studies of gene expression in E. akaara and other Epinephelus species.Groupers are an economically important fish species in world fishery markets. Because many studies using RT-qPCR have addressed gene expression in groupers, appropriate reference genes are required to obtain reliable and accurate results. In this study, the most suitable reference genes were identified from eleven candidate genes of one of the most valuable species,  However, many studies show that the transcript levels of these genes vary considerably across cellular conditions [Currently, real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) is a ubiquitous mainstay technology for quantifying gene expression because of its advantages, which include accuracy, specificity, sensitivity, reproducibility and convenience , 2. Despnditions –9, whichnditions , buckwhenditions , crazywenditions , deer [1nditions , rice [1nditions , and oilnditions , and thenditions . TherefoVibrio alginolyticus, a causative agent of outbreaks of vibriosis, is one of the most serious diseases and a common problem during various stages of grouper culture that causes fish mortality and serious economic losses [V. alginolyticus in fish includes symptoms of septicaemia, haemorrhage, dark skin, and ulcers on the skin surface in some cases [Epinephelus akaara, we found variable expression levels of some commonly used reference genes, such as β-ACT. Therefore, these genes may not be suitable as reference genes for analyses of gene expression in grouper and could lead to the erroneous normalization of RT-qPCR data. Thus, the identification of suitable reference genes in grouper studies is crucial for the accurate profiling of gene expression.Grouper (Perciformes: Epinephelidae) have high economic value in Asian fishery markets, and grouper mariculture has rapidly developed due to the insufficient supply of wild caught fish, which does not satisfy the high demand of markets . Howeverc losses , 23. Theme cases . In receE. akaara and identify the most suitable reference genes for different experimental conditions. To the best of our knowledge, this report is the first assessment of valid reference genes for RT-qPCR studies on groupers. Because the Epinephelus species has the characteristics of a relatively short evolutionary time and closely related phylogeny [E. akaara but also may be applicable to other commercially important Epinephelus species, such as E. coioides, E. lanceolatus, Cromileptes altivelis and Anyperodon leucogrammicus.The objective of this study was to evaluate the appropriateness of traditional reference genes and new candidate reference genes derived from our previous transcriptome sequencing data of hylogeny , the ideE. akaara used in this study were obtained from a local commercial aquatic farm in Fujian Province, China. This study was conducted in strict accordance with the guidelines for the Care and Use of Laboratory Animals. The Institutional Animal Care and Use Committee of Xiamen University approved the protocol. All fish surgery procedures were conducted under MS-222 (Tricaine Methanesulfonate) to induce sedation and anaesthesia. All eggs, larvae, and tissue samples were incubated in an RNA fixer  and stored at -80°C for RNA isolation.All All experiments were performed according to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines . A detaiE. akaara: undifferentiated-phase (UN), developing female (DF), developing male (DM), mature female (MF) and mature male (MM) [For the selection of new candidate reference genes, we analysed our previous gonadal transcriptome sequencing data PRJNA277894) from the following five different gonadal development phases of 7894 fromale (MM) ; and (4)brefeldin a-inhibited guanine nucleotide-exchange protein 1 (ARFGEF1), conserved oligomeric Golgi complex subunit 5 (Cog5), putative ATP-dependent RNA helicase DHX30 (DHX30), neuron navigator 3 (Nav3), homeodomain-interacting protein kinase 3 (Hipk3), probable E3 ubiquitin-protein ligase MYCBP2 (Mycbp2), E3 ubiquitin-protein ligase MGRN1 (Mgrn1), and 60S ribosomal protein L17 (RPL17), and two were traditionally used reference genes, GAPDH and beta-2-microglobulin (B2M). The most commonly used reference gene, β-ACT, in grouper was also evaluated in our study.Based on these selection procedures for the transcriptome sequencing data, 10 genes that had a minor variation in expression were selected. Of these genes, 8 were newly identified candidate reference genes, including We used full-length unigene sequences from gonadal transcriptome data to design specific primers for RT-qPCR. The RT-qPCR products ranged from 86 to 243 bp. The specificities of all the primers were demonstrated by the single bands of expected size in agarose gel electrophoresis and by the single-peak melting curves of the RT-qPCR products . The SanCq = Log (2)/Log (E), where E is the efficiency of the amplification of each primer pair. Then, for both geNorm and NormFinder, the Cq values were transformed to relative quantities using the delta-Cq formula Q = 2delta-Cq, where delta-Cq is the difference between the sample with the lowest Cq and the Cq value of the sample in question. The geNorm program calculates gene expression stability , which is based on the pairwise variation of a single reference candidate gene relative to all other tested control genes. Genes with an M-value below the threshold of 1.5 were those stably expressed and lower M-values indicated greater stability of a gene. The geNorm program also estimates the pairwise variation (Vn/Vn+1) between two sequential normalization factors, NFn and NFn+1, to determine the optimal number of reference genes required. The optimal number of reference genes is necessary to satisfy Vn/Vn+1 below the cut-off value of 0.15. NormFinder uses an ANOVA-based model to identify the most stable reference genes. NormFinder calculates intra- and inter-group variations, and genes with the least variations are those that the most stable. BestKeeper calculates the SD and CV based on the Cq values of each reference gene. Genes with an SD value of < 1.0 are stable, and the gene with the lowest SD and CV values was identified as the most stable. Additionally, refFinder, a user-friendly comprehensive tool, was used to integrate the results from the three programs and select the best reference gene.The PCR data were analysed using three approaches, geNorm , NormFingrowth and differentiation factor-8 (MSTN), was used to validate the expression levels of the most stable, the universal, and the least stable reference genes for E. akaara during gonad development and early ontogenetic development phases. An important immune responsive cytokine, interleukin (IL)-1β, was used to validate the expression levels of the most stable, the universal, and the least stable reference genes for salinity treatment and V. alginolyticus challenged E. akaara. Relative quantification of these two genes in different samples was performed using the 2-ΔΔCt method [To validate the stability of the identified reference genes, the expression levels of two target genes were analysed in different experimental conditions. A non-mammalian myostatin, t method .E. akaara gonad development ranged between 16.0 and 30.5. RPL17 was the most highly expressed gene, with Cq values ranging between 16.0 and 17.7, followed by β-ACT and GAPDH, which exhibited Cq values from 17.9 to 21.7 and from 17.1 to 21.8, respectively. The remaining genes had Cq values greater than 22, with the highest Cq value (30.5) exhibited by Hipk3. For expression stability analysis, the results from the geNorm, NormFinder, and BestKeeper programs were combined, and seven genes, Hipk3, ARFGEF1, B2M, Mycbp2, RPL17, Nav3, and Cog5, were stably expressed during different gonad development phases of E. akaara  and weakly expressed Hipk3 (Cq ranging from 29.4 to 35.4), the other candidate reference genes were expressed at moderate levels. Combining the results from geNorm, NormFinder, and BestKeeper programs, Nav3 and Mgrn1 were stably expressed genes during the entire early ontogenetic development stage of E. akaara ) was considered, the Cq values of all eleven genes ranged from 18.2 to 36.4 . Unlike . akaara . The com17>GAPDH . Howevertermined . TherefoNav3, Mgrn1, Cog5, Mycbp2, β-ACT and ARFGEF1 were stably expressed during the embryonic ontogenesis stage, whereas Cog5, RPL17, Nav3, B2M, GAPDH, β-ACT, ARFGEF1, DHX30 and Mgrn1 were stably expressed during the larval ontogenesis stage  and larval ontogenesis stage (from first feeding larvae to 32 dah), consistent with other reports , better is stage . Accordiis stage . For theectively . TherefoRPL17 and β-ACT were the most highly expressed genes, with Cq values ranging from 17.3 to 18.6 and 18.3 to 19.1, respectively, whereas DHX30 and Hipk3 were the least expressed, with Cq values ranging from 30.8 to 33.2 and 29.1 to 33.4, respectively , hence its limitation as a reference gene in such studies [In previous studies on gene expression using the RT-qPCR method, including investigations on grouper functional genes,  grouper –47, thishallenge , but thehallenge , during hallenge , and undsavignyi . The difmotility . Althoug halibut , embryon halibut , virus-i halibut , and dif halibut . The peractivity , making activity . B2M wasla tarda , 43, 49. studies . In the Cog5 and ARFGEF1 satisfied the stability threshold for all the experimental conditions. Although Cog5 was a newly identified reference gene in fish, previous studies of human non-small cell lung cancer and thyroid cancer used this gene as an internal control [Pacific oyster microarray, Cog5 was also identified as a candidate reference gene because its mRNA level appeared stable across all analysed tissues [Cog5 was closely tied to its function, which is based on the conserved oligomeric Golgi (COG) complex subunit that is involved in the Golgi-associated membrane trafficking and glycoconjugate synthesis [ARFGEF1, is ubiquitous in eukaryotic cells and functions as a regulator of vesicular traffic and actin remodelling [ARFGEF1 is one of the most stable reference genes in plants and animals, including wheat, barley, rye, citrus, and beetle [Cog5 and ARFGEF1 showed similar normalization results with the most stable genes when the relative expression of MSTN and IL-1β was analysed. Therefore, we recommend that Cog5 and ARFGEF1 be used as reference genes for E. akaara under all tested conditions. However, for a specific experimental condition, the most suitable reference genes are strongly recommended because of the better stability and applicability.Clearly, a universal reference gene would be more acceptable to researchers because of its convenience. Of the eleven candidate reference genes, based on the integration of the stability results across all tested conditions, only the stability parameters of  control –57. Furt tissues . The staynthesis . The othodelling . Previoud beetle –64. Moreβ-ACT, GAPDH and B2M, were not applicable for E. akaara gene expression studies in most situations; (2) the two newly identified reference genes, Cog5 and ARFGEF1, could be universally applied in E. akaara under all tested conditions; and (3) the most suitable reference genes were identified for each specific experimental conditions of E. akaara. Additionally, our results might be applicable to other groupers.In summary, the primary conclusions of this study were as follows: (1) the three traditionally used reference genes, S1 Fig(TIF)Click here for additional data file.S2 FigLow M values indicate more stable expression. The least stable genes are on the left, and the most stable genes are on the right. The red line indicates the geNorm cut-off value of 1.5. Ranking of the gene expression stability was performed for the (A) liver, (B) intestine, (C) stomach, (D) spleen, (E) heart, (F) head kidney, (G) gills, and (H) white muscle.(TIF)Click here for additional data file.S3 FigLow V values indicate more stable expression. The least stable genes are on the left, and the most stable genes are on the right. Ranking of the gene expression stability was performed for the (A) liver, (B) intestine, (C) stomach, (D) spleen, (E) heart, (F) head kidney, (G) gills, and (H) white muscle.(TIF)Click here for additional data file.S4 FigLow SD values indicate more stable expression. The least stable genes are on the left, and the most stable genes are on the right. The red line indicates the BestKeeper cut-off value of 1.0. Ranking of the gene expression stability was performed for the (A) liver, (B) intestine, (C) stomach, (D) spleen, (E) heart, (F) head kidney, (G) gills, and (H) white muscle.(TIF)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Molecular modeling of Cx39-based channels suggest that a constriction present at the intracellular portion of the para helix region co-localizes with an electronegative patch, imposing an energetic and steric barrier, which in the case of GJCs may hinder channel function. Results reported here demonstrate that Cx39 form HCs and add to our understanding of the functional roles of Cx39 HCs under physiological and pathological conditions in cells that express them.Although connexins (Cxs) are broadly expressed by cells of mammalian organisms, Cx39 has a very restricted pattern of expression and the biophysical properties of Cx39-based channels [hemichannels (HCs) and gap junction channels (GJCs)] remain largely unknown. Here, we used HeLa cells transfected with Cx39 (HeLa-Cx39 cells) in which intercellular electrical coupling was not detected, indicating the absence of GJCs. However, functional HCs were found on the surface of cells exposed to conditions known to increase the open probability of other Cx HCs , extracellular alkaline pH, mechanical stimulus and depolarization to positive membrane potentials). Cx39 HCs were blocked by some traditional Cx HC blockers, but not by others or a pannexin1 channel blocker. HeLa-Cx39 cells showed similar resting membrane potentials (RMPs) to those of parental cells, and exposure to DCFS reduced RMPs in Cx39 transfectants, but not in parental cells. Under these conditions, unitary events of ~75 pS were frequent in HeLa-Cx39 cells and absent in parental cells. Real-time cellular uptake experiments of dyes with different physicochemical features, as well as the application of a machine-learning approach revealed that Cx39 HCs are preferentially permeable to molecules characterized by six categories of descriptors, namely: (1) electronegativity, (2) ionization potential, (3) polarizability, (4) size and geometry, (5) topological flexibility and (6) valence. However, Cx39 HCs opened by mechanical stimulation or alkaline pH were impermeable to Ca Connexins (Cxs) are integral membrane proteins expressed in most tissues of vertebrate animals, and are coded in humans by 21 different genes , 18-β-glycyrrhetinic acid (BGA) and octanol among others , 4′,6-diamidino-2-phenylindole (DAPI+2) and Lucifer yellow (LY−2), among others . We found that HeLa-Cx39 cells do not form functional GJCs since cells remain electrically uncoupled consistent with structural limitations identified by 3, Etd+, procion orange MX2R, brilliant blue G (BBG), Evans blue, propidium iodide, 4-Bromo calcium ionophore A23187 were purchased from Sigma-Aldrich . The 9-amino-6-Chloro-2-Methoxyacridine (ACMA), DAPI+2, hexidium iodide, nuclear yellow, BOBO 1-iodide, 7-aminoactinomycin D (7-ADD), YOYO 1-iodide, TOTO 1-iodide and ethidium homodimer–2–bromide obtained from Invitrogen , indocyanine green from Pulsion medical systems,  methylene blue from Euromed .Octanol, heptanol, carbenoxolone, oleamide, LaCl2/95% air atmosphere. HeLa-Cx39 and HeLa Cx43-EGFP transfected cells were selected with 0.5 mg/mL of puromycin and 0.3 mg of geneticin , respectively, as described previously  overnight. Then, cells were incubated with rabbit anti-Cx39 antibody  for 6 h at room temperature, washed with PBS and incubated 30 min with secondary rabbit anti-IgGs  conjugated to Cy2 or Cy3. Preparations were subsequently washed with PBS, and coverslips were mounted with fluoromont G .The Cx39 transfection was confirmed by immunofluorescence. In brief, cells on glass coverslips, were washed with PBS, and fixed with formaldehyde 4% and incubated with blocking solution : 130 CsCl, 10 AspNa, 0.26 CaCl2, 1 MgCl2, 2 EGTA, 7 TEA-Cl, and 5 HEPES at pH 7.2. The pipette resistance in the bath was 10–15 MΩ. All recordings were performed at room temperature (21–23°C). The microscopic and macroscopic currents were obtained with voltage steps (+60 mV for 40 s) and ramp protocols .Functional HCs were evaluated in whole-cell voltage clamp experiments as previously described  measurements were performed under a whole-cell current clamp configuration at 37°C in individual cells. The pipette solution contained 3 M KCl and the bath solution contained: (in mM) 145 NaCl, 5 KCl, 3 CaCl2, 1.2 CaCl2 and 5 HEPES at pH 7.4. The pipette solution was the same used in HC experiments plus 18 μM DAPI+2 in one pipette to identify the cell in indirect immunofluorescence for Cx39. The junctional current was recorded in an adjacent cell using voltage steps increasing in 20 mV from −100 to 0 mV and a duration of 200 ms each. The holding potential of non-stepped cells was −60 mV and the gap junction conductance (gj) was calculated from gj = −Ib/(Va − Vb), as described previously  were bathed with a solution containing: (in mM) 150 NaCl, 4 KCl, 1 MgCl2 and MgCl2 [divalent cation free solution (DCFS)] to promote HC opening, as shown for other Cx HCs , was recorded as previously described  and images were performed in a Nikon Eclipse Ti inverted microscope and imaged with NIS Elements software .The intracellular free-calcium signal  crystal structure as a template  and GraphPad Prism 6 software .All results are presented as mean ± SE and significant differences between groups were determined using a one-way ANOVA followed by a Tukey's multiple comparisons +2, propidium iodide, Etd+ or neurobiotin , which was not detected between HeLa-Cx39 cells (n = 9)  from Ij recorded at different membrane voltages in Cx43-EGFP yielded similar values within the voltage range illustrated (−40 mV to +40 mV), indicating the absence of voltage dependence  in HeLa-Cx43-EGFP cells generated evident transjunctional current (I) Figure ; values e Figure  right. Ce Figure . On the o Figure , revealiXenopus oocytes. However, positive dye coupling with Alexa488 has been observed in fresh preparations of diaphragm-striated muscle of neonatal mice, composed of myofibers that express only Cx39    , suggesting that inhibition of a few open Cx39 HCs significantly contributed to increasing the electrochemical gradient across the cell membrane. Moreover, the RMPs of HeLa-Cx39 cells bathed in DCFS were significantly lower compared with cells immersed with normal saline solution , indicating that Cx39 HCs are inhibited by a divalent cation likely to be Ca2+, as demonstrated for other HCs , the RMPs of HeLa-Cx39 cells bathed in DCFS were close to those recorded under control conditions  . Accordingly, HCs formed by other Cxs have been shown to be permeable to Na+ .The low open probability of Cx43 HCs might explain the lack of influence on the RMP in HeLa-Cx43 cells  Figure , consist) Figure . Moreove) Figure , and the) Figure . In cont) Figure , suggest+/K+ ATPase pump contributes to the maintenance of RMPs, and its inhibition causes depolarization , which is a Na+/K+ ATPase pump inhibitor  and HeLa-P cells   Figure . In addi) Figure , suggestp > 0.05 n = 10). However, the membrane current of HeLa-Cx39 cells significantly increased upon exposure to extracellular DCFS, particularly at positive membrane potentials (> +25 mV) and drastically decreased upon 200 μM La3+ treatment . It should be noted that the I/V curve recorded in the presence of extracellular divalent cations (control conditions) was comparable to that recorded in DCFS plus La3+  were evident after holding the membrane voltage at +60 mV for about 20 s. However, in cells exposed to DCFS, discrete unitary current events showed a higher open probability (Po = 0.9), and were completely blocked by 200 μM La3+  either in control or DCFS solution    Figure , but bot) Figure .+2, Etd+ and propidium) have been used before to observe changes in membrane permeability due to the presence of Cx HCs , Evans blue (−4), hexidium iodide (+1), methylene blue (+1) and procion orange MX2R (−3), and was canceled out by La3+ , whereas the uptake rate of ethidium homodimer (+4) and BBG (−1) was not significantly affected either by DCFS or La3+ .HeLa-P cells did not show changes in ethidium uptake upon exposure to DCFS Figure , inset. + Figure , inset. ns blue −, hexidiuns blue −, hexidiuThe permeability of Cx39 HCs to molecules varied between large molecules with high net charge, like TOTO MW: 1,300, +4), YOYO  or Evans blue , to smaller molecules with lower net charges, which are not able to pass through HCs-Cx39, such as ACMA dye  or with high net charge, such as ethidium homodimer . Because permeable dyes differ in terms of their molecular nature  have the highest values for the RotBtFrac descriptors . To overcome this limitation, we used a machine-learning approach to explore the physicochemical properties of the different permeability probes tested in the present work. We identified 11 out of 1,000 molecular descriptors Table  that cor2+ uptake, it is necessary to use a stimulus that opens Cx HCs in the presence of extracellular Ca2+, as it occurs upon exposure to alkaline pH   Figure , but Ca2d Figure , inset.  Figures .2+, we induced HC opening by applying mechanical stress, a stimulus that induces opening of HCs constituted of other Cxs and in other cell types  is known to block Cx and Panx1 HCs  electronegativity, (2) ionization potential, (3) polarizability, (4) size and geometry, (5) topological flexibility and (6) valence. Unlike other Cx HCs, Cx39 HCs are not permeable to Ca2+ and show different in sensitivity to classical HC and GJC blockers.Results reported here demonstrate that Cx39 forms functional HCs with distinct unitary conductance (75 ± 5 pS) and inhibited by extracellular Cain silico model and wrote the paper, TPA designed bioinformatics experiments and wrote the paper, OS and CFL wrote the paper, and AAV designed and performed electrophysiological and permeability experiments and wrote the paper and JCS designed research and wrote the paper.BAC performed permeability experiments, FS performed electrophysiological experiments, CU and LAC performed permeability experiments, AHV designed and performed electrophysiological experiments. SEGM, AJMM analyzed bioinformatics data of dyes and wrote the paper, CPB, YE performed The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancer cases, and for the most cancer-related deaths. The survival pathway of Akt, its downstream effectors, the mammalian target of rapamycin (mTOR) and ribosomal protein S6 kinase (p70 S6K), and the Ras-extracellular signal-regulated kinase (Erk1/2) pathways are activated in cancer leading to cell survival and growth. Thus, approaches that inhibit these signaling molecules may prove useful in the fight against lung cancer. Exercise is associated with health benefits and a limited number of studies indicate that serum from physically active individuals inhibit mammary and prostate cancer cell growth. In this study, we examined the effects of post exercise serum on proliferation, survival, and signaling cascades of human NSCLC cells. Blood was collected from male subjects prior to, 5 min, 1 h, and 24 h after a single bout of high intensity interval exercise on a cycle ergometer. Exposure of NSCLC cells to post exercise serum resulted in the inhibition of cell proliferation and survival, as well as significant reduction of phosphorylated/activated Akt, mTOR, p70 S6K, and Erk1/2 levels compared to cells treated with serum taken pre-exercise. Our data suggest that post exercise serum has anti-cancer properties in lung cancer and deserves further systematic investigation in animal models. Lung cancer accounts for the most cancer-related deaths globally and Non-Small Cell Lung Cancer (NSCLC) represents 85% of all lung cancer cases . NSCLC iIncreased proliferation, reduced apoptosis, and increased survival are the main characteristics of cancer cells, which are achieved by modulation of key signaling molecules/pathways . The phoRegular exercise is associated with overall health benefits and reduced risk of many cancers , includiIn the present study, we investigated the effect of serum collected pre-exercise or post high intensity interval exercise (HIE) on human NSCLC cell proliferation, survival, and the effects on Akt, mTOR, p70 S6K, and Erk1/2 phosphorylation/activation and expression.p < 0.05,  residues. Incubation of A549 cells with 10% human serum taken 5 min, 1 h, or 24 h post-exercise resulted in Erk1/2 phosphorylation levels of 60.0 ± 14.2%, 60.0 ± 14.2%, and 52.9 ± 11.2%, respectively, compared to the cells treated with pre-exercise serum A. Total Insulin is a strong growth factor; it directly activates the Akt-mTOR and the Ras-Erk signaling pathways and therefore we measured serum insulin levels to examine if the observed changes could potentially be explained by the changes in serum insulin. Serum insulin levels were not significantly changed  .In this study, we investigated whether serum collected from humans after an acute bout of high intensity exercise could lead to anti-cancer effects in vitro, and what signaling pathways might be affected. Our data are summarized in Apart from the effects of serum collected following an acute bout of exercise, there are studies indicating that serum from individuals participating in exercise programs regularly has anticancer effects in cancer cells in vitro, compared to serum from sedentary controls. The inhibition of prostate ,32,33,34Taken together, the above studies demonstrate that acute exercise as well as exercise training (chronic exercise) induce changes in serum factors that can lead to significant inhibition of cancer cell growth in vitro. Unfortunately, the exact serum factors contributing to the anticancer effects of exercise are not known. Exercise affects the levels of circulating hormones, growth factors, and cytokines, and in recent years contracting/exercising skeletal muscle has been recognized as an important tissue producing and releasing into the bloodstream myokines and other substances that affect the functionality of other tissues, resulting in health benefits . The effMedia from electrically-stimulated myotubes inhibited MCF-7 mammary cancer cell proliferation and induced apoptosis, an effect that was similar to the effects of post-exercise serum . These bInterestingly, serum collected 24 h post-exercise had a similar inhibitory effect on cancer cell survival, Akt, mTOR, p70S6K, and Erk phosphorylation, as serum collected 5 min and 1 h post-exercise. These data indicate that changes in serum factors, induced by exercise and responsible for its anticancer effects, persist 24 h post-exercise. We measured insulin levels and found no significant changes. Additionally, it should be noted that the serum used in the present study was assessed for cytokine levels previously . IL-1α, Our study showed that post-exercise serum was able to inhibit the activation of Erk1/2 in A549 cells, suggesting that this significant inhibition of Erk1/2 contributes to the inhibition in cell proliferation and survival that was seen. One previous study has also shown that serum taken from men following 3 weeks of low-fat diet and moderate aerobic exercise intervention was associated with a significant reduction of phosphorylated and total levels of Erk1/2 compared to pre-intervention serum in HepG-2 liver cells .A549 cells harbour a mutation in the Ras GTPase protein (KRAS), which leads to constitutive activation of the Ras-PI3K-Akt signaling cascade and enhanced Ras-MAPK (Erk1/2) signaling. This cell line has high basal levels of Akt and Erk1/2 activation  and reprFuture studies should explore the effects of post-exercise serum on other signaling molecules, such as AMPK, p53, and caspases. Studies using animal models of cancer should be employed to investigate the potential risks/benefits of acute versus chronic exercise as well as which modes of exercise  may be more beneficial than others. Furthermore, because exercise is already considered an important component of maintaining general health, clinical trials with cancer patients have recently begun, although their clinical benefits remain unclear ,50, thus3VO2, glycerol, and dimethyl sulfoxide (DMSO) were purchased from Sigma . Clarity Western enhanced chemiluminescence substrate (ECL), sodium dodecyl sulfate (SDS), 30% acrylamide/bis solution 37 (5:1), ammonium persulfate (APS), Tween 20, tetramethylethylenediamine (TEMED), bromophenol blue, 2-mercaptoethanol, Biorad protein assay reagent, polyvinylidene difluoride (PVDF) membranes, molecular weight protein standards, and electrophoresis reagents were from BioRad . Glycine was purchased from BioShop . Tris-HCl was purchased from Fisher chemical . Skim milk was purchased from Great Value . Triton X-100, EGTA, and sodium pyrophosphate were purchased form EMD . NaCl was purchased from ACP . EDTA was purchased from Caledon . Bovine serum albumin was from Calbiochem .Human A549, H460, and H1299 NSCLC cells and MRC5 normal lung fibroblasts were obtained from the American Type Culture Collection (ATCC). Cell culture (RPMI and DMEM) media, fetal bovine serum (FBS), trypsin, and antibiotic-antimycotic were from GIBCO . Total and phospho-specific antibodies Akt (S473), mTOR (S2448), p70 S6K (T389), Erk1/2 , β-actin, and secondary anti-rabbit IgG HRP-linked antibodies were from Cell Signaling Technology via New England Biolabs . Bovine serum albumin (BSA), leupeptin, PMSF, Nag and 4 °C for 15 min. Serum was aliquoted into polypropylene microfuge tubes. The serum collected from the study participants was not pooled. Serum collected from the different subjects was stored at −80 °C until use.Recreationally active male University students  were recruited to participate in a high intensity exercise (HIE) study previously described . Particiv/v) FBS, and 1% (v/v) antibiotic-antimycotic solution in a humidified atmosphere of 5% CO2 at 37 °C. Cells were serum deprived for 3 h prior to immunoblotting to synchronize cells by arresting them in the G0/G1 phase before treatment with 10% of either FBS or serum taken pre-exercise, 5 min, 1 h, or 24 h post HIE. Serum from the different subjects was used to treat cells in triplicates.A549, H460, and H1299 cells were grown in RPMI media and MRC5 cells were grown in DMEM media, both supplemented with 10% , allowed to adhere overnight, and incubated with media containing either 10% FBS, pre-exercise, 5 min, 1 h, or 24 h post exercise serum for 30 min. Following the treatment, cell lysates were prepared, protein content was measured using the Biorad protein assay reagent, and samples (20 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein was then transferred to a polyvinylidene fluoride (PVDF) membrane for 1.5–3 h, blocked for 1 h with 5% skim milk, and incubated overnight with the indicated primary antibodies. The following day the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h. Protein was visualized using a LI-COR C-Digit blot scanner (LI-COR Biosciences) and corresponding software. Ponceau S (Biorad) was used to confirm the total protein content and transfer efficacy. Densitometric analysis was performed using ImageJ software.Serum insulin was determined using a commercial quantitative sandwich enzyme immunoassay, with a detection range from 15.6 to 500 pmol/L and a sensitivity of 0.3 pmol/L. The intra assay coefficient of variation (CV) was 8.5%.p < 0.05 using Graphpad Prism 5 software.The results are expressed as the mean ± SEM of the indicated number of independent experiments. Repeated measures analysis of variance (ANOVA) with Tukey post-hoc tests were performed and statistical significance was assumed at In summary, we were able to show that serum collected from young men after acute exercise resulted in significant inhibition of lung cancer cell survival and proliferation in vitro which was associated with significant inhibition of phosphorylation/activation of Akt, mTOR, p70 S6K, and Erk1/2 levels compared to cells treated with serum taken pre-exercise. Our data suggest that post-exercise serum has anti-cancer properties in lung cancer and deserves further systematic investigation in animal models of cancer and in humans.


Treatment with VPA increased the number of megakaryocyte/erythroid progenitors (MEP), accompanied by inhibition of megakaryocyte differentiation, whereas treatment with NAM accelerated megakaryocyte development, and stimulated polyploidisation. Treatment with both KDACi resulted in no significant effects on erythrocyte differentiation, suggesting that the effects of KDACi primarily affect megakaryocyte lineage development. H3K27Ac ChIP-sequencing analysis revealed that genes involved in myeloid development, as well as megakaryocyte/erythroid (ME)-lineage differentiation are uniquely modulated by specific KDACi treatment. Taken together, our data reveal distinct effects of specific KDACi on megakaryocyte development, and ME-lineage decisions, which can be partially explained by direct effects on promoter acetylation of genes involved in myeloid differentiation.Treatment with lysine deacetylase inhibitors (KDACi) for haematological malignancies, is accompanied by haematological side effects including thrombocytopenia, suggesting that modulation of protein acetylation affects normal myeloid development, and specifically megakaryocyte development. In the current study, utilising ex- Transcriptional regulation of the megakaryocyte/erythroid (ME) lineage is regulated by a relatively small number of master regulators. Differentiation of the common myeloid progenitor (CMP) towards the ME-lineage in humans is mainly regulated by GATA-1, the key-regulator of both megakaryocyte, and erythrocyte development . ProgresIn addition to transcriptional regulation of normal and aberrant myeloid differentiation, the role of epigenetic regulatory networks, and post-translational modifications has been identified in recent years –16. ThisResults of phase I/II clinical trials with KDACi suggest no unfavorable effects on the normal haematopoietic progenitor cell (HPC) compartment, however both hypergranulocytosis, and prolonged thrombocytopenia have been described , 28–31. In this study, we compared the effects of VPA treatment with NAM treatment on human ME-lineage development, and further progression into the megakaryocytic lineage. Our data demonstrate for the first time that KDAC and SIRT inhibition differentially modulates the expansion and differentiation of MEP. Treatment with VPA increases the MEP compartment, yet inhibits megakaryocyte development, while erythroid development is normal. NAM treatment stimulates megakaryocyte differentiation at the expense of proliferation, while the effects on erythroid development resemble the effects of VPA treatment. Utilising a histone 3 lysine 27 acetylation (H3K27ac) chromatin immunoprecipitation- (ChIP) sequencing approach, we identified key regulatory genes implicated in myeloid progenitor function, and ME-lineage differentiation, directly regulated by VPA and NAM treatment. Taken together, our study provides novel insights into the effects of KDACi on ME-lineage development, and increases our knowledge of the role of HDAC and sirtuins in normal human haematopoiesis.Data were obtained from the Utrecht Patient Oriented Database (UPOD). The content of UPOD and its setting have been described in detail elsewhere , 45. Fro5) cells were cultured in Stemspan Serum Free Expansion Medium (SFEM) . Cells were differentiated towards megakaryocytes for 11 days upon addition of stem cell factor (SCF) (50 ng/mL), thrombopoietin (TPO) (20 ng/mL), and interleukin 6 (IL-6) (10 ng/mL). Cells were differentiated towards erythrocytes for 11 days upon addition of SCF (50 ng/mL) interleukin-3 (IL-3) (0.1 nmol/L), and eryhtropoietin (EPO) (5IE/mL). Every 3 days, cells were counted with trypan blue, and fresh medium was added to a density of 5.0 x 105 cells/mL for megakaryocytes, and 2.0 x 105 for erythrocytes. After 7 days of differentiation only TPO and IL-6 were added to the megakaryocyte cultures. The KDAC inhibitors trichostatin A (TSA), sodium butyrate (SB) valproic acid (VPA)  and SIRT inhibitor nicotinamide  were added to the fresh medium every 3 days. Umbilical cord blood was collected after written informed consent was provided according to the Declaration of Helsinki. The use of umbilical cord blood for this study was approved by the ethics committee of the University Medical Center Utrecht.CD34+ cells were isolated from human umbilical cord blood as previously described (32). CD34+ , and glycoprotein 1B (CD42b), and by polyploidy (DNA content >4N). Mature erythrocytes can be characterized by transferrin receptor protein 1 (CD71), and glycophorin A (CD235a) expression. Megakaryocyte precursor cells were isolated at day 7, and 11 of differentiation, washed with PBS, followed by incubation phyoerythrin (PE)-labeled anti-CD61 antibody, and a fluorescein isothiocyanate (FITC)-labeled anti-CD42b antibody  in PBS/5% FCS on ice, for 30 minutes in the dark. Next, cells were washed in PBS, resuspended in PBS/5% FCS, and analysed by FACS. Erythrocyte precursor cells were isolated after 11 days, and incubated with PE-labeled anti-CD71 antibody, and FITC-labeled anti-CD235a antibody, and prepared for FACS analysis as described above. Isotype antibody staining was used for gating. Analysis of polyploidy was performed after 7, and 11 days of differentiation. Cells were stained with FITC-labeled conjugated CD42b antibody as described before. After that, cells were fixed and permeabilised in ice-cold ethanol for 30 minutes on ice. Cells were washed with PBS, resuspended in PBS/5mM EDTA and incubated with 40μg/mL RNAse for 30 minutes at RT. Subsequently the cellular DNA content was stained with 0.1 mg/mL propidium iodide  for 5 minutes and analysed in the CD42b-positive cell population by FACS . Due to normal variation between donors, we expressed the effects of VPA and NAM treatment on MK differentiation related to MK differentiation in the controls.4 differentiating megakaryocytes and were fixed in methanol for 3 minutes. After fixation, cytospins were stained in a 50% eosin methylene blue solution according to May-Grunwald  for 15 minutes, rinsed in water for 5 seconds, and nuclei were counterstained with 10% Giemsa solution  for 20 minutes. Megakaryocyte maturation can be characterised by polyploidisation. Erythrocyte differentiation can be characterised by enucleation to produce reticulocytes. Micrographs were acquired with an Axiostar plus microscope  fitted with an 100x/1.3 NA EC Plan Neofluor oil objective using Immersol 518F oil (Carl Zeiss), a Canon Powershot G5 camera , and Canon Zoombrowser EX image acquisition software. Photoshop CS5 was used for image processing .May-Grunwald Giemsa staining was used to analyse megakaryocyte differentiation, and erythrocyte differentiation. Cytospins were prepared from 5.0 x 10et al. [- cells are common myeloid progenitors (CMP), whereas Lin-, CD34+, CD38+, CD123+, and CD45RA+ cells are granulocyte-macrophage progenitors (GMP). The Lin- CD34+, CD38+ CD123- and CD45RA- cell population contains the megakaryocyte-erythroid progenitors (MEP). Cell populations containing HSC, CMP, GMP and MEP were characterized by FACS analysis . Isotype antibody staining was used to ensure gating of the correct population.Haematopoietic progenitor populations were characterised as described by Manz et al. . BrieflyCD34+ cells were differentiated towards megakaryocytes for 4 days, followed by overnight treatment with VPA, or NAM. Lysates were prepared, and ChIP was performed as described previously  utilisin− ΔΔCt and ß2 microglobulin was used as reference gene. The sequences of the primers are listed in CD34+ cells were differentiated towards megakaryocytes for 4 days, followed by overnight treatment with VPA, or NAM. mRNA was extracted using the RNeasy Isolation Kit . According to the manufactures protocol for single-stranded cDNA synthesis, 500 ng of total RNA was reverse transcribed using iScript cDNA synthesis kit . cDNA samples were amplified using SYBR green supermix (BIO-Rad), in a MyiQ single-color real time PCR detection system (BIO-Rad) according to the manufacturers’ protocol. To quantify the data, the comparative Ct method was used. Relative quantity was defined as 2The correlation between VPA plasma concentration and the absolute number of platelets was evaluated using linear regression analysis. Data analysis was performed using SPSS 17.0 . For analysis of the other experiments, a one-way ANOVA analysis was followed by a Dunnett multiple comparison test to compare the differences between the control and KDACi-treated cells, or a Bonferroni multiple comparison test to compare all treatment conditions. A p-value of 0.05 or less was considered significant: * p<0.05, ** p<0.01.The unique process of megakaryocyte development is characterised by lineage progression from a bipotent progenitor (MEP), followed by endomitosis, resulting in highly specialised, polyploid cells . The effin vitro, we differentiated human umbilical cord blood (UCB)-derived CD34+ hematopoietic progenitor cells towards megakaryocytes in the absence or presence of the pan KDACi TSA, two different class I/II KDACi (VPA or SB), and compared this with treatment with the class III (sirtuin) inhibitor nicotinamide (NAM). Megakaryocyte differentiation was determined based on integrin β3 (CD61), and glycoprotein 1B (CD42b) expression, and cytospin analysis of megakaryocyte morphology. While we observed no clear effects of TSA on megakaryocyte differentiation, VPA and SB treatment resulted in a significantly decreased CD61/CD42b expression  progenitor expansion and survival. Upon treatment of VPA, and NAM, a concentration dependent inhibition of megakaryocyte progenitor expansion was observed as determined by counting trypan blue-negative cells during differentiation . Next, tet al. [We have previously demonstrated that VPA treatment of CD34+ myeloid progenitors, differentiated towards the GM-lineage, resulted in an increased percentage of MEP . To inveet al. . CompareIn addition to increased expression, and activity of megakaryocyte lineage-specific genes, megakaryocyte development is also dependent on downregulation of erythroid lineage specific genes ,53. SincWhile VPA and NAM have both been described to affect lysine deacetylase activity, treatment of CD34+ cells resulted in differential effects on MEP expansion and differentiation towards the megakaryocyte differentiation, but the effects on erythroid differentiation were similar. Therefore, we next investigated the potential underlying molecular mechanisms involved in these effects, by differentiating UCB-derived CD34+ cells to megakaryocytes for 4 days, followed by overnight treatment with VPA or NAM, before performing H3K27ac chromatin immunoprecipitation (ChIP). H3K27 acetylation is a histone modification implicated in chromatin remodelling, and characterizes active enhancers and promoters . H3K27acTreatment with VPA or NAM resulted in 1011 VPA-specific, and 580 NAM-specific genes with > 2-fold increased acetylation, and an overlap of 482 genes. Likewise, we found 1497 VPA-specific, and 614 NAM-specific genes with > 2-fold decreased acetylation, and an overlap of 612 genes . As expeNext, utilizing DAVID functional annotation software, gene-ontology analysis was performed. While no specific regulatory network involved in megakaryocyte development was identified, both treatment with VPA and NAM was linked to haematopoiesis and myelopoiesis, apoptosis, and gene-specific transcription . Based oTaken together these data demonstrate that VPA and NAM treatment differentially modulates histone acetylation marks, associated with active transcription at genes involved in cell fate decisions during myeloid development, and more specifically ME-lineage differentiation.Finally, to investigate whether the changes in H3K27 promotor acetylation of ME-lineage specific genes, indeed lead to increased or decreased mRNA expression, we evaluated the mRNA level of several ME-lineage specific genes, by differentiating UCB-derived CD34+ cells to megakaryocytes for 4 days, followed by overnight treatment with VPA or NAM, before performing real time qRT-PCR. Treatment with VPA resulted in increased mRNA expression of LMO2 and RUNX2, and decreased mRNA expression of GATA1 compared to the control . Upon trex-vivo human CD34+ differentiation system, we investigated the effects of VPA, a widely used class I, and to a lesser extent, class IIa KDACi [et al. demonstrated that the combined conditional deletion of HDAC1 and HDAC2, both class I HDAC, in human haematopoietic cells, resulted in anaemia and thrombocytopenia, accompanied by apoptosis of megakaryocytes, while single deletions resulted in no haematological phenotype. In addition it was suggested that high HDAC1 expression levels are required for ME-lineage development, while HDAC1 overexpression has been demonstrated in AML cell lines, and primary AML cells [For the treatment of haematological malignancies, KDACi, in combination with conventional chemotherapy, or other epigenetic agents, have shown to improve haematological outcome –27. SincIa KDACi , 59, andML cells , 61. StuML cells , 59. TakML cells . Taken iML cells , our datML cells , 65.ChIP-sequencing analysis demonstrated that VPA treatment of megakaryocyte progenitors was associated with increased acetylation of H3K27 at the promoters of genes involved in HSC differentiation, and ME-lineage development . H3K27acTreatment with the SIRT1/2 inhibitor NAM resulted in accelerated differentiation, and increased polyploidisation of megakaryocyte precursors at the expense of proliferation Figs . Little Possibly, patients with thrombocytopenia of unknown origin could benefit from NAM treatment, as it might increase megakaryocyte differentiation and therefore peripheral blood platelet counts. On the opposite, due to possible skewing of ME-lineage differentiation toward the erythroid lineage, in patients with (non-haemolytic) anaemia, VPA might increase the red blood cell count and haemoglobin levels.In conclusion, we have demonstrated that VPA and NAM, two functionally distinct KDACi, differentially regulate cell fate decisions during megakaryocyte, and erythroid development. Our data suggest that these effects can at least be partially explained by direct effects on H3K27-promoter acetylation, and subsequent transcription of genes involved in HSC and haematopoietic progenitor differentiation, and more specifically in ME-lineage development. Taken together our data increase the understanding of the effects of KDACi on normal haematopoiesis, and the role of HDAC and sirtuins in the regulation of normal myeloid development and megakaryocyte development in particular.S1 FigHSC differentiate into the bipotent MEP, followed by megakaryocyte lineage progression, characterized by polyploidization, and cytoplasmic maturation, accompanied by sequential increased surface expression of integrin ß3 (CD61), and glycoprotein Ib (CD42b), and activity of the transcription factors GATA1, RUNX1, GATA2, LMO2, FOG1, NF-E2, FLI1 and TAL1.(EPS)Click here for additional data file.S2 FigUCB-derived CD34+ cells were differentiated to erythrocytes during 11 days in the absence or presence of 100–200μM, or 1-5mM NAM. (A) At all culture time points, trypan blue negative cells were counted. Data represent the fold expansion of erythroid precursors during development. (B) At day 11 of differentiation, surface marker expression of CD71, and CD235a was analysed by FACS. Data represent the percentage of double positive cells, compared to the control. (C) Cytospin analysis of erythroid precursor cells. Arrows indicate enucleation. Data are representative for 3 (VPA), or 2 (NAM) independent experiments. Error bars represent SEM, * p<0.05, ** p< 0.01.(EPS)Click here for additional data file.S3 FigUCB-derived CD34+ cells were differentiated towards megakaryocytes. At day 4 of differentiation, cells were treated overnight with 200μM, or 5mM NAM. Next, lysates were prepared, followed by ChIP-sequencing see . Data re(EPS)Click here for additional data file.S4 FigUCB-derived CD34+ cells were differentiated towards megakaryocytes. At day 4 of differentiation, cells were treated overnight with 200μM VPA, or 5mM NAM. Next, lysates were prepared, followed by ChIP-sequencing see . Plots r(EPS)Click here for additional data file.


Acinetobacter baumannii strains. These samples were obtained from patients at the University of Colorado Hospital in 2007 and 2013 and encode an estimated 20 and 13 resistance genes, respectively.We report here the draft genome sequences of two clinically isolated  Acinetobacter have been named as a serious threat to public health . Antibiotic resistance genes were identified with ARG-ANNOT . The Nextera XT DNA library kit was used to build paired-end 250-bp libraries, which were sequenced on an Illumina MiSeq, generating 1,644,910 and 1,081,729 reads for CU060707 and CU032113, respectively. FASTQ files were filtered using the sliding window mode in Trimmomatic  in 240 contigs with an N50 of 150,723 bp. The genome contains 56 tRNAs, seven rRNAs, one CRISPR, and 3,831 coding sequences. The 20 resistance genes detected include six aminoglycosides , nine β-lactams , two macrolide-lincosamide-streptogramins , one chloramphenicol (catB8), one sulfonamide (sulI), and one tetracycline (tetB). Most resistance genes are encoded on the genome, though OXA-235 appears to be on a plasmid, similar to GenBank CP015486.1.The draft assembly of A. baumannii CU032113 is 4,182,411 bp in length  in 322 contigs with an N50 of 59,362 bp. There are 70 tRNAs, nine rRNAs, two CRISPRs, and 3,882 coding sequences. The 13 resistance genes found include two aminoglycosides (strA and strB), eight β-lactams , two chloramphenicols (catA1 and cmlB1), and one sulfonamide (sulII). OXA-24 is the only gene not on the genome; it appears to be on a plasmid, similar to GenBank JN207494.1  and LXYW00000000 (for CU032113).This whole-genome shotgun sequencing project has been deposited in GenBank under the accession numbers