The presence of telocytes (TCs) as distinct interstitial cells was previously documented in human dermis. TCs are interstitial cells completely different than dermal fibroblasts. TCs are interconnected in normal dermis in a 3D network and may be involved in skin homeostasis, remodelling, regeneration and repair. The number, distribution and ultrastructure of TCs were recently shown to be affected in systemic scleroderma. Psoriasis is a common inflammatory skin condition (estimated to affect about 0.1–11.8% of population), a keratinization disorder on a genetic background. In psoriasis, the dermis contribution to pathogenesis is frequently eclipsed by remarkable epidermal phenomena. Because of the particular distribution of TCs around blood vessels, we have investigated TCs in the dermis of patients with psoriasis vulgaris using immunohistochemistry (IHC), immunofluorescence (IF), and transmission electron microscopy (TEM). IHC and IF revealed that CD34/PDGFRα-positive TCs are present in human papillary dermis. More TCs were present in the dermis of uninvolved skin and treated skin than in psoriatic dermis. In uninvolved skin, TEM revealed TCs with typical ultrastructural features being involved in a 3D interstitial network in close vicinity to blood vessels in contact with immunoreactive cells in normal and treated skin. In contrast, the number of TCs was significantly decreased in psoriatic plaque. The remaining TCs demonstrated multiple degenerative features: apoptosis, membrane disintegration, cytoplasm fragmentation and nuclear extrusion. We also found changes in the phenotype of vascular smooth muscle cells in small blood vessels that lost the protective envelope formed by TCs. Therefore, impaired TCs could be a ‘missed’ trigger for the characteristic vascular pathology in psoriasis. Our data explain the mechanism of Auspitz’s sign, the most pathognomonic clinical sign of psoriasis vulgaris. This study offers new insights on the cellularity of psoriatic lesions and we suggest that TCs should be considered new cellular targets in forthcoming therapies. We also assessed whether psoriatic skin TCs exhibit (ultra)structural changes. TCs distribution and the pattern of cellular interaction in psoriasis patients could offer new insights into the pathogenesis and progression of this disease.n = 8) or metabolic (n = 2). The skin samples were biopsied three times: from the mature psoriatic lesion, non-lesional skin (40 cm distance from any lesion), and after the clearance induced by local treatment. The treatment followed by the patients was entirely topical: keratolytic  and cytoreductor , followed by calcipotriol and steroid ointments. This study was approved by the Bioethics Committee of the ‘Carol Davila’ University of Medicine and Pharmacy, Bucharest, according to generally accepted international standards. All subjects provided signed informed consent.We studied skin samples from 10 patients  with fully developed (mature) plaques of psoriasis vulgaris. Three of the patients were diagnosed with psoriasis vulgaris type II, and seven were diagnosed with psoriasis vulgaris type I. The triggers of psoriasis were psychological  using a 1 ml syringe until a bleb approximately 5 mm diameter formed under the skin. After testing for numbness, the biopsy was performed with a sterile 6 mm skin punch. After the skin was cored and excess blood cleared the fragment of skin, the biopsy was removed using a scalpel and forceps. The post-biopsy lesion was ligated with two stitches of Ethicon Polyglactin 910 . To prevent infection, the wound was dressed with bacitracin Zn and neomycin sulphate powder  and carefully bandaged.Each of the skin biopsies were divided into two equal fragments, each fragment following the protocol for paraffin embedding for histology, immunohistochemistry (IHC) and immunofluorescence (IF) or Epon embedding for transmission electron microscopy (TEM).Histology and IHC were performed on formalin-fixed, paraffin-embedded, 3-μm-thick tissue sections made from the tissue samples collected from all patients. For histology, standard haematoxylin and eosin staining was performed. For IHC, samples were incubated with primary antibodies Table overnighFor IF we used formalin-fixed paraffin-embedded 3-μm-thick tissue sections. After deparaffinization, the samples were buffered at 97°C with Epitope Retrieval Solution  at pH 6 for PDGFRα and pH 9 for CD34. The samples were washed in PBS with glycine (2 mg/ml) and then blocked with 2% BSA for 1 hr. Samples were incubated with primary antibodies overnight at room temperature with a cocktail consisting of mouse monoclonal CD34  and rabbit polyclonal PDGFRα . After washing three times in EnVisionTM Flex Wash Buffer (Dako) the sections were incubated with anti-mouse Alexa Fluor 488  or anti-rabbit rhodamine  secondary antibodies for another 2 hrs. The nuclei were stained with 4′,6-diamidino-2-phenylindole . Immunofluorescence studies were performed with a Zeiss Axio Imager Z1 microscope (Carl Zeiss MicroImaging GmbH) using 10×, 20×, 40×, and 63× objectives with the appropriate fluorescence filters. Digital images were acquired with a CV-M4+CL CCD camera  linked to a computer running the Isis software .2. The summation of the surfaces of known magnification images was used to obtain this surface area. Data were processed and statistically analysed using Microsoft Excel software.Cell counting was performed on calibrated IF images of known magnification using the NIH ImageJ software 3) tissue fragments processed according to a routine Epon embedding procedure as described previously Transmission electron microscopy was performed on small  had decreased inflammatory infiltrate, and the mild oedema was slightly persistent. Eosinophils and neutrophils were absent from the inflammatory infiltrate. In contrast, the distant uninvolved skin and treated lesion had fewer normally maturated keratinocyte layers and similar rete ridge lengths.CD31 expressed Fig. in the hEndothelial cells were also CD34-positive  in small blood vessels in psoriatic skin Fig.. In addis in smalThe pathogenesis of psoriasis is not fully understood despite efforts to focus on cellular types and signalling molecules We found a decreased number of TCs in psoriatic papillary dermis and observed their recovery after local corticoid therapy. Immunofluorescence showed that the density of TCs identified as CD34/PDGFRα-positive cells was comparable in the dermis of uninvolved and treated skin but decreased in the lesional papillary dermis. Electron microscopy showed that TCs undergo apoptosis and dystrophic changes in psoriatic plaques. Also, IHC showed that CD34-positive cells are not present near dilated capillaries in the dermis of psoriatic plaques. Notably, the interstitial expression of CD34 was slightly increased in the proximity of blood vessels in treated lesional skin. These data suggest TC recovery after topic corticoid therapy despite the residual inflammatory microenvironment We found that TCs usually surround blood vessels in normal skin The 3D reconstruction of dermal TCs by FIB-SEM tomography revealed various conformations of Tps: long, flattened irregular veils (ribbon-like segments) and tubular structures (podomers) with uneven calibre because of irregular dilations (podoms) Vasodilatation tests indicated that arterioles in psoriatic plaques are not normally maximally dilated but have a basal constrictor tone We previously showed that, within the dermis, TCs form an interstitial network based on homocellular (TC-TC junctions) and heterocellular  interactions, suggesting an essential role of TCs in intercellular signalling required for skin homeostasis In conclusion, damaged TCs could be an important step in the pathophysiology of psoriasis. This study offers new insights into the cellularity of psoriatic lesions and we suggest considering TCs as new cellular targets for forthcoming therapies. Because pustular psoriasis is not characterized by Auspitz’s sign, clarifying the involvement of TCs in other forms of psoriasis would be attractive. In the future, we propose extending the research to the pustular and erythrodermic forms of psoriasis.


Thousands of long non-coding RNAs (lncRNAs) have been identified in mammalian cells. Some have important functions and their dysregulation can contribute to a variety of disease states. However, most lncRNAs have not been functionally characterized. Complicating their study, lncRNAs have widely varying subcellular distributions: some reside predominantly in the nucleus, the cytoplasm or in both compartments. One method to query function is to suppress expression and examine the resulting phenotype. Methods to suppress expression of mRNAs include antisense oligonucleotides (ASOs) and RNA interference (RNAi). Antisense and RNAi-based gene-knockdown methods vary in efficacy between different cellular compartments. It is not known if this affects their ability to suppress lncRNAs. To address whether localization of the lncRNA influences susceptibility to degradation by either ASOs or RNAi, nuclear lncRNAs (MALAT1 and NEAT1), cytoplasmic lncRNAs (DANCR and OIP5-AS1) and dual-localized lncRNAs  were compared for knockdown efficiency. We found that nuclear lncRNAs were more effectively suppressed using ASOs, cytoplasmic lncRNAs were more effectively suppressed using RNAi and dual-localized lncRNAs were suppressed using both methods. A mixed-modality approach combining ASOs and RNAi reagents improved knockdown efficacy, particularly for those lncRNAs that localize to both nuclear and cytoplasmic compartments. Advancements in genomics technologies over the last decade have led to the identification of tens of thousands of previously unsuspected long non-coding RNAs (lncRNAs), revolutionizing the way we view the complex eukaryotic transcriptome ,2. WhileLncRNAs comprise a remarkably diverse population of cellular RNAs. By definition, these are non-protein coding RNAs longer than 200 nt. Length varies from the small 7Sk species (∼330 nt) to the very large Airn (∼118 kb). LncRNAs can be transcribed from intergenic, intragenic or intronic regions. Further, these species can be spliced or remain unspliced and are often 5′-capped and 3′-polyadenylated (reviewed in ). ThrougMethods to degrade cellular RNAs using synthetic oligonucleotides have been used for many years as an approach to study gene function. The present study employs both ‘antisense’ and RNA interference (RNAi) approaches. ‘Antisense methods’ were first used to suppress gene expression over 35 years ago –19 and eTm) of the oligonucleotide, which can lower potency. A chimeric ASO design was developed that employs Tm-enhancing modifications at the ends to increase binding affinity to the target, retaining a central DNA core to form a substrate for RNase H when duplexed with the RNA target (termed a ‘gapmer’)  RNA and locked nucleic acids (LNA)  ,31. The ds (LNA) . ASOs cods (LNA) ,33,34.In recent years, RNA interference (RNAi) has gained favor in the research community as a primary tool to reduce mRNA levels. RNAi is a natural process to regulate gene expression that exists in most eukaryotic cells. RNAi employs small, double-stranded RNA molecules that associate with multiple protein factors to form the RNA-Induced Silencing Complex (RISC), leading to either suppression of translation or degradation of a target mRNA. Natural RNAi effector molecules either arise from processing of endogenous hairpin transcripts by the nucleases Drosha and Dicer (microRNAs or miRNAs) or from long double-stranded RNAs (dsRNAs) processed by Dicer alone  . SynthetAlthough both RNAi and antisense methods have already been successfully used to knockdown specific lncRNAs –45, the We investigated if the intracellular localization of individual lncRNAs influences which knockdown method is optimal to employ. We also compared different ASO and RNAi reagent designs and chemical modification strategies to determine if a specific reagent showed any particular advantage in this application. A set of seven lncRNAs with various cellular distribution patterns was selected from the literature to perform a comparative survey Table . MALAT1 ® Select siRNAs) were designed using the Ambion site selection algorithm .Three design variants were tested for both the ASO and RNAi arms of the study Table . Reagent® Select siRNAs were purchased from Thermo Fisher Scientific  and LNA™ longRNA GapmeRs were purchased from Exiqon . All oligonucleotide sequences are shown in Supplementary Table S1 in the online Supplemental Data.Antisense oligonucleotides (DNA-PS and 2′ OMe-PS gapmers), primers and probes were chemically synthesized using standard phosphoramidite chemistry. RNA oligonucleotides (21mer unmodified siRNAs and 27mer DsiRNAs) were chemically synthesized using t-Butyl-dimethylsilyl (TBDMS) chemistry (Integrated DNA Technologies). Probes for qPCR were purified using reversed phase high performance liquid chromatography (RP-HPLC) (Integrated DNA Technologies) while all other oligonucleotides were prepared as sodium salts. All oligonucleotides were analyzed by electrospray-ionization mass spectrometry (ESI-MS) and were within ±0.02% predicted mass. Oligonucleotide concentrations were calculated using modification-specific extinction coefficients based on measured ultraviolet (UV) absorbance at 260 nm. RNA duplexes were annealed in IDT duplex buffer  (Integrated DNA Technologies). Silencer® 2000 at 10, 3 and 1 nM doses. HeLa cells were cultured in ATCC recommended media. For transfections (optimized for this system), oligonucleotides were reverse transfected in 96-well plates by complexing the various oligonucleotide doses with 0.5 μl Lipofectamine® 2000 (Thermo Fisher Scientific) in OptiMEM® I (Thermo Fisher Scientific) for a total volume of 50 μl in each well. HeLa cells (20 000) were suspended in 100 μl Dulbecco's Modified Essential Medium (DMEM) containing 10% fetal bovine serum (Thermo Fisher Scientific), added to the lipid-oligonucleotide complexes, then incubated for 24 h at 37°C and 5% CO2. For HOTAIR, transfections were performed in 48-well plates with 40 000 HeLa cells per well, with doubling of all reagent volumes. Each 96-well transfection plate included a positive transfection control  and two or three compound-specific negative transfection controls. All transfections were performed in biological triplicate and each experiment was performed at least twice. The results of all transfections performed were averaged.To compare the knockdown efficiency between reagents, oligonucleotides were transfected in biological triplicate into HeLa cells with Lipofectamine® II Reverse Transcriptase (Thermo Fisher Scientific) per the manufacturer's instructions. qPCR reactions were performed using ∼10 ng cDNA with Immolase DNA polymerase , 500 nM of each primer and 250 nM probe in 10 μl reactions in 384-well plate format. Amplification reactions were run on an Applied Biosystems 7900HT (Thermo Fisher Scientific) with cycling conditions consisting of enzyme activation at 95°C for 10 min, followed by 40 cycles of 2-step PCR . All qPCR reactions were performed in triplicate and averaged. Linearized cloned amplicons were used as copy number standards to establish absolute quantitative measurements for each assay. LncRNA expression levels were quantified by multiplexing two 5′-nuclease assays per target (one assay located toward the 5′ end and one located toward the 3′ end of the transcript) and normalized against both HPRT (NM_000194) and SFRS9 (NM_003769) as internal reference controls. LncRNA knockdown levels were calculated by comparing cells transfected with ASO or RNAi reagents to cells transfected with appropriate chemical modification-specific negative control oligonucleotides. Data are plotted graphically with error bars representing standard error of the mean (SEM).LncRNA knockdown was measured by reverse transcription quantitative PCR (RT-qPCR), comparing each knockdown reagent with the appropriate chemically modified negative control oligonucleotide at the same dose and calculated from an average of two or three separate experiments. RNA was isolated 24 h after transfection with the SV 96 Total RNA Isolation Kit  with DNase1 treatment. cDNA was synthesized from ∼200 ng total RNA with anchored oligonucleotide dT and random hexamer primers (Integrated DNA Technologies) using SuperScript® 6.0 software using the non-linear regression formula: log (inhibitor) versus normalized response—variable slope .HeLa and HCT116 cells were transfected in biological triplicates with ASOs or siRNAs as previously described. A range of nine doses for each knockdown reagent was selected based on empirically determined oligonucleotide activity and tested in both cell lines. RNA extraction, cDNA synthesis and qPCR reactions were performed as previously described. IC50 values were calculated with GraphPad PrismO-methoxyethyl phosphorothioate gapmer ASO (ISIS395254) (MALAT1 (metastasis associated in lung adenocarcinoma transcript 1) is a highly abundant ∼8.7 kb lncRNA that localizes to nuclear speckles and plays a role in both regulating alternative splicing and cellular proliferation –67. MALAS395254) .Results are shown for MALAT1 and NEAT1 Figure  and B wiFor the two nuclear lncRNAs studied, the ASOs were consistently more effective at suppressing RNA levels than were the RNAi reagents. For MALAT1 Figure , severalTo verify that the RNAi reagents and ASOs did not cause non-specific inhibition of nuclear lncRNAs, all of the MALAT1 or NEAT reagents were individually transfected into HeLa cells as previously described and RNA levels for both MALAT1 and NEAT1 RNA were measured. None of the MALAT1 knockdown reagents affected NEAT1 levels , and none of the NEAT1 knockdown reagents affected MALAT1 levels , demonstrating target specificity.DANCR (Differentiation Antagonizing Non-Protein Coding RNA) is a small ∼0.8 kb lncRNA involved in maintaining the undifferentiated state of certain cell types in the epidermis and regulating osteoblast differentiation; earlier reports indicated that this target can be suppressed using either shRNAs or siRNAs ,80–82. RThe cytoplasmic lncRNA DANCR was effectively suppressed by all 28 RNAi reagents tested, of which 17 out of 28 showed 80% or higher reduction in DANCR RNA levels at the 1 nM dose Figure . While sin vitro and in vivo, and Bhan et al. inhibited HOTAIR with a DNA-PS ASO , a ∼7.5 kb lncRNA necessary for retinal development and involved in the upregulation of growth-control genes ,16,84–86A-PS ASO ,91,93–98Both ASO and RNAi reagents were able to reduce TUG1 expression Figure , althougA summary compilation of the knockdown studies performed on all seven lncRNAs  at the 10 nM dose is shown in Figure Many lncRNAs are not restricted to either the nuclear or cytoplasmic compartment but exist in variable concentrations between the different subcellular locations . SimultaIt is desirable to employ knockdown reagents which are potent and have minimal toxicity. SiRNAs have been reported to be ≥100-fold more potent than ‘first generation’ DNA-PS ASOs for RNA knockdown, as evidenced by IC50 values in comparative studies at the same target sites –103. ‘Se® Select siRNAs). Oftentimes, potent siRNA sites do not correlate with potent DsiRNA sites and viceversa . However, general knockdown trends were the same between the two classes of ASOs, with good DNA-PS ASO sites also serving as good 2′ OMe-PS ASO sites, which may allow for the less expensive DNA-PS ASOs to be employed for screening ‘walks’ to identify good sites in high value targets with later conversion to a more active 2′ OMe-PS gapmer design.Antisense and RNAi are both widely used techniques for sequence-specific reduction of cellular RNA expression. They employ distinct mechanisms of action with different protein co-factors and/or effector molecules that have different intracellular distribution patterns. While neither method is restricted to a single subcellular compartment, relative activity can vary between compartments and can affect the ease with which that method can be used to knockdown an RNA target. RNase H-mediated ‘degradative’ antisense relies upon hybridization of an ASO to the RNA target to form an RNA:DNA heteroduplex substrate for RNase H1, which is largely (but not exclusively) found in the nucleus –24. The Targeting mRNAs is somewhat simplified by the single purpose of protein coding transcripts, which are made in the nucleus and translated to protein in the cytoplasm. Hence, RNase H active-ASOs can be used to trigger degradation of nascent pre-mRNA transcripts in the nucleus and siRNAs can be used to trigger degradation of mature mRNAs in the cytoplasm; use of either method serves to prevent translation of the targeted mRNA. Although antisense methods were the first gene knockdown technology developed, RNAi has become the dominant technology employed in research applications, largely due to its ease of use. Efforts to develop algorithms to predict effective ASO sites have not had the same success as for RNAi, probably due to their different mechanisms of action. ASO activity relies upon hybridization of the single-stranded oligonucleotide to the target RNA, which is highly dependent upon both the folded structure of the RNA and the presence of RNA-binding proteins that may limit access. Tertiary structure and protein binding are difficult to predict, particularly for longer RNAs of the type studied herein. ASO site selection was done by combining RNA folding prediction, analysis of oligonucleotide sequence properties and a cross-reactivity search to help ensure specificity (for the DNA-PS and 2′ OMe-PS sites) or by Exiqon using proprietary methods (for the LNA-PS sites). RNAi, on the other hand, relies upon a protein effector complex (RISC) that evolved to mediate gene regulation by endogenous dsRNAs and seems to facilitate interaction between the siRNA and the RNA target. Algorithm development for siRNA site selection has been very successful , reducinTargeting lncRNAs is complicated by their wide range of functions and subcellular distribution patterns. LncRNAs play many different roles in the cell, including, for example, association with chromatin or with mRNA, or binding miRNAs or proteins (‘sponge’ effects). Some lncRNAs reside mainly in the nucleus, some reside primarily in the cytoplasm, others are present in both compartments, while yet others concentrate in specific subcellular organelles. It is, therefore, not surprising that lncRNAs might be more difficult to suppress than mRNAs or that greater challenges would be encountered when trying to use the different gene knockdown techniques available.The present study observed that achieving high levels of knockdown of nuclear lncRNAs was easier using antisense methods while knockdown of cytoplasmic lncRNAs was easier using RNAi methods. Consistent with this observation, antisense methods are thought to be particularly effective in suppressing nuclear-retained RNAs, including lncRNAs such as MALAT1 ,114. IntIt is interesting that the cytoplasmic targets DANCR and OIP5-AS1 both associate with 40S/60S ribosomal subunits, placing them in proximity to known sites for high RNAi activity ,51. ThisThe results reported and discussed herein employed measurements of RNA levels taken 24 h post-transfection and, therefore, assess the immediate effects seen on degradation of target molecules present in accessible compartments. Similar results were observed if measurements were taken 48 h post-transfection (data not shown); however, longer incubation periods were not studied. Since transcription occurs in the nucleus, antisense methods, which utilize nuclear RNase H1, should eventually be effective at suppressing all lncRNAs, regardless of where the mature lncRNA product later accumulates in the cell. The time course of this knockdown will vary with the turnover rates for each lncRNA species.The present study employed two RT-qPCR assays to measure levels of each lncRNA. One assay was located toward the 5′-end and the other toward the 3′-end of the target. Using two (or more) assays is needed to help rule out assay-related artifacts, such as false positive results due to interference of the oligonucleotide-based knockdown reagents with reverse transcription or qPCR . Also, tDifferent methods can be used to suppress expression of lncRNAs. Due to their varied function and subcellular distribution, antisense methods (for nuclear species) or RNAi methods (for cytoplasmic species) may show better performance. When the distribution of a lncRNA is unknown, antisense methods may be more reliable for achieving knockdown if only one approach is employed. Combined use of antisense with RNAi methods can lead to better knockdown than use of either method alone, especially for lncRNA targets that are distributed throughout the cell.


Endurance capacity, assessed by 1000-meter (1000 m) run of male university students, is an indicator of cardiovascular fitness in Chinese students physical fitness surveillance. Although cardiovascular fitness is related to endothelial function closely in patients with cardiovascular diseases, it remains unclear whether endurance capacity correlates with endothelial function, especially with circulating endothelial microparticles (EMPs), a new sensitive marker of endothelial dysfunction in young students. The present study aimed to investigate the relationship between endurance capacity and endothelial function in male university students.Forty-seven healthy male university students  were recruited in this study. The measurement procedure of 1000 m run time was followed to Chinese national students Constitutional Health Criterion. Endothelium function was assessed by flow-mediated vasodilation (FMD) in the brachial artery measured by ultrasonic imaging, and the level of circulating EMPs was measured by flow cytometry. Cardiovascular fitness indicator - maximal oxygen uptake (VO2 max) - was also measured on a cycle ergometer using a portable gas analyzer.2max . However, there were no correlations between VO2max and FMD or levels of circulating CD31+/CD42- microparticles. Similarly, no correlations were found between 1000 m run time and FMD, and levels of circulating CD31+/CD42- microparticles in these male university students (p>0.05).1000 m run time was correlated with VOThe correlations between endurance capacity or cardiovascular fitness and endothelial function were not found in healthy Chinese male university students. These results suggest that endurance capacity may not reflect endothelial function in healthy young adults with well preserved FMD and low level of circulating CD31+/CD42-EMPs. The Chinese National Survey in 2010 on Students' Constitution and Health revealed a continuous decline of university students' physical endurance over the past 20 years, supported by declining results in 1000-meter (1000 m) run time 2max), a good indicator of cardiovascular fitness, and endothelial function measured as flow-meditated vasodilation (FMD) in brachial artery of healthy male adults 2max Endothelial dysfunction has been suggested as the initial step to the pathogenesis of cardiovascular disease (CVD) Recently, it has been reported that alteration of circulating EMPs, mostly defined as CD31+/CD42- microparticles, emerged as a new marker for endothelial injury in response to various stimuli Regular aerobic exercise improves physical fitness, especially improve endurance capacity. And it was demonstrated that acute endurance exercise or long-term aerobic exercise lowered the levels of circulating EMPs This cross-sectional study was approved by the Ethics Committee of Zhongshan School of Medicine of Sun Yat-Sen University. Each subject was aware of the investigative nature of the study and gave written informed consent. Forty-seven apparently healthy male student subjects were recruited from grade 2 of the Zhongshan School of Medicine, Sun Yat-Sen University. We recruited subjects with relatively stable endurance capacity, which means 1000 m run time in three consecutive semesters of those subjects, including the latest test two weeks before the lab tests were relatively stable (ranged between mean run time ± 10%). Subjects with clinical history of cardiovascular diseases such as hypertension, hyperlipidemia, diabetes mellitus, ischemic heart diseases, and acute pathologies were excluded. Subjects with familial history of cardiovascular disease were also excluded. All subjects declared no smoking, no alcohol and no medicines at least two weeks before the study.PE-conjugated monoclonal antibody against PECAM-1(PE-CD31) and FITC-conjugated monoclonal antibody against Platelet Glycoprotein Ib (FITC-CD42b) were used to identify EMPs. All the flow cytometry labeling reagents including PE- and FITC-conjugated monoclonal IgGs were obtained from Immunotech (France). Flowcount fluorospherical beads used to determine microparticles' absolute values and 1-µm calibrant beads used to define the upper size limit of the microparticles were from Beckman Coulter (USA).The 1000 m run time was measured on the running track in a stadium of Zhongshan School of Medicine. Following the procedures of Chinese National Students Constitutional Health Criterion, the test was conducted from 16∶30 to 17∶30 at an outdoor temperature of 24°C −27°C. Furthermore,all subjects were clearly instructed to maintain their lifestyle, especially keep their physical exercise habits during the whole study.2max, ventilation volume (VE) and heart rate (HR). Routine laboratory tests including serum total cholesterol, total triglyceride, low density lipoprotein-cholesterol, high density lipoprotein-cholesterol and fasting plasma glucose were measured by standard laboratory methods.The subjects refrained from strenuous physical exercise and consuming caffeine for at least 24 hours before cardiopulmonary test and other measurements. Two weeks after the last 1000 m run test, the venous blood sample for circulating EMPs and routine laboratory tests were drawn from the forearm at early morning in fasting status. After at least 15 minutes resting in a supine position, FMD test was carried out in a controlled environment maintained at 23°C. Then in a laboratory maintained at 25°C∼27°C and relatively humidity at 50%∼70%, the exercise cardiopulmonary test was performed 2∼3 hours after meal to measure VOCirculating CD31+/CD42- microparticles were isolated from 4 ml of venous citrated blood drawn from forearm as described in our previous reports FMD measurement in the brachial artery was performed with subjects in the supine position for the evaluation of endothelial function. All FMD imaging tests were performed by a highly skilled sonographer who was unaware of the study design. As we described previously 2max was performed by a light-weight breath-by-breath portable gas analyzer  and progressive maximal cycle ergometer . Validity and reliability of the K4b2 portable gas analyzer have been reported elsewhere 2)/oxygen consumption (VO2) >1.1, heart rate (HR) >90% of the age-predicted maximum, and a leveling off of VO2 despite the increment in the workload. Respiratory frequency, HR, VE, VO2, and VCO2 were monitored continuously at all time during the exercise cardiopulmonary test by telemetry. VO2max corresponded to the plateau (average of 30s) in oxygen consumption, despite the increment in the workload.The measurement of VOData of this study are expressed as mean±SD except for circulating CD31+/CD42- microparticles as mean±SEM. Statistical analysis was performed with SPSS 13.0 software for Windows . Pearson's correlation test was used to test the associations among variables in the whole subjects. A two-tailed test (P <0.05) was considered significant.2max  and VO2max/kg . And there was significant correlation between 1000 m run time and oxygen pulse The physiological characteristics of the recruited healthy subjects were shown in 2max and FMD . Coincidently VO2max was not correlated with levels of circulating CD31+/CD42- microparticles  in the all subjects.There was no correlation between VOp>0.05). Neither 1000 m run time correlated with levels of circulating CD31+/CD42- microparticles in the all subjects  endurance run time was unrelated to FMD or the level of circulating EMPs in apparently healthy male university students; 3) endurance run time was significantly correlated with VO2max. The present study is to investigate the relationship between endurance capacity and endothelial function in male university students. It was demonstrated that FMD was in normal range and the level of circulating EMPs was low in the subjects of the study, and there was no relationship between endurance capacity and endothelial function, suggesting that basic endurance capacity can't reflect endothelial function superficially in male university students, hence basic endurance capacity might not serve as a useful predictive marker of cardiovascular disease in apparently healthy young adults.The major findings of the present study are: 1) VO2max. The authors reckoned the improvement of endothelial function implicated the beneficial therapeutic property of statin in increment of VO2max 2max in healthy adults and the elderly Endothelial dysfunction plays an important role in the pathogenesis of cardiovascular disease (CVD) 2max. The possible reason was the study subjects, who were male university students in the same grade, had the similar FMD in normal range. We assumed that FMD of the study population in their university age period might be mainly determined by their original physical status such as heredity background, and physical training might affect FMD mildly in individuals in this age. Actually, our results were in agreement with Ragnom's and Moe's studies 2max but similar FMD, when compared with baseline levels or after a single bout of acute exercise. The authors considered higher cardiovascular function status due to long-term aerobic training didn't improve endothelial function any further in young healthy subjects with well preserved endothelial function. However, in those studies which showed the positive relationship between FMD and VO2max, different age groups or subjects within big age span had been compared. Age heterogeneity of the subjects might be the reason why positive results were found in these studies 2max coincidently.However, in our study we didn't find the relationship between FMD in brachial artery and VO2max is the reference indicator of cardiovascular fitness 2max, which suggested basic endurance capacity represented cardiovascular fitness in our subjects. Considering the possible relationship between VO2max and FMD, it's possible to excavate a more convenient surrogate measurement to assess endothelial function in young adults. Furthermore, does continuous decline of 1000 m run time suggest endothelial dysfunction in university students? Other more sensitive markers except FMD should be introduced to the study to assess the potential endothelial dysfunction.Although the more meticulous experimental parameter-VOThe circulating microparticles of endothelial origin are considered as a sensitive marker for endothelial injury Taken by surprise, relationship between endurance run time and levels of circulating EMPs in male university students had not been demonstrated in this study. It has been reported that the levels of circulating CD62E+EMPs in Africa Americans with inflammatory endothelial dysfunction was significantly decreased after 6 months aerobic endurance physical training It should be pointed out that there were some limitations in the studies. First, this was a cross-sectional study, which suggestive correlations and not casual relationships were observed. Hence, it's impossible to conclude whether declining endurance capacity affected endothelial function or not. Second, the subjects in the current study were in their university age period, whether our results could be extrapolated to other populations with different characteristics remains to be studied. Third, this study selected a narrow sample of subjects, and it just observed the relationship between endurance capacity and endothelial function in the baseline physical condition. Whether there will be any change after an acute exercise or long-term exercise has not been investigated. Further studies with large sample or subjects with different exercise background should be designed to answer these questions.In summary, this study doesn't demonstrate correlation between endurance capacity and endothelial function, which suggests that endurance capacity might not superficially reflect endothelial function in apparently healthy male university students. The result also implies the disability of endurance capacity for predicting cardiovascular disease in young people with well-preserved FMD and low level of circulating CD31+/CD42-EMPs. Further studies are necessary to follow up changes of endurance capacity and level of circulating EMPs in this study population with aging and investigate the relationship between each other in different pathological conditions.


The only enzyme known to reduce N2O is the nitrous oxide reductase, encoded by the nosZ gene, which is present among bacteria and archaea capable of either complete denitrification or only N2O reduction to di-nitrogen gas. To determine whether the occurrence of nosZ, being a proxy for the trait N2O reduction, differed among taxonomic groups, preferred habitats or organisms having either NirK or NirS nitrite reductases encoded by the nirK and nirS genes, respectively, 652 microbial genomes across 18 phyla were compared. Furthermore, the association of different co-occurrence patterns with enzymes reducing nitric oxide to N2O encoded by nor genes was examined. We observed that co-occurrence patterns of denitrification genes were not randomly distributed across taxa, as specific patterns were found to be more dominant or absent than expected within different taxonomic groups. The nosZ gene had a significantly higher frequency of co-occurrence with nirS than with nirK and the presence or absence of a nor gene largely explained this pattern, as nirS almost always co-occurred with nor. This suggests that nirS type denitrifiers are more likely to be capable of complete denitrification and thus contribute less to N2O emissions than nirK type denitrifiers under favorable environmental conditions. Comparative phylogenetic analysis indicated a greater degree of shared evolutionary history between nosZ and nirS. However 30% of the organisms with nosZ did not possess either nir gene, with several of these also lacking nor, suggesting a potentially important role in N2O reduction. Co-occurrence patterns were also non-randomly distributed amongst preferred habitat categories, with several habitats showing significant differences in the frequencies of nirS and nirK type denitrifiers. These results demonstrate that the denitrification pathway is highly modular, thus underpinning the importance of community structure for N2O emissions.Nitrous oxide (N This unique enzyme is found among a wide range of bacterial and archaeal taxa capable of complete denitrification, an anaerobic respiratory process in which soluble nitrate (NO3−) or nitrite (NO2−) is reduced to the gases nitric oxide (NO), N2O and N2 via a series of enzymatic steps. It has been suggested that denitrification is a modular pathway, in that an organism may not always possess the full set of enzymes and thus perform only a subset of steps within the pathway nosZ genes that reduce N2O to N2 yet lack the other denitrification genes have been described 2O produced by denitrification or other N-cycling processes nosZ gene 2O as a terminal product. It was recently shown that manipulation of the proportion of denitrifiers with the nosZ gene in soil microcosms significantly affected the ratio of N2O:N2 production, establishing a causal link between denitrifier community composition and potential N2O emissions 2O emissions ultimately has a genetic basis.The only known sink for N2O is more conserved among specific microbial taxa or functional groups of denitrifiers implies that the composition of the microbial community is not trivial from an ecosystem perspective, however this is not known. Denitrifiers can be divided into two functional groups based on whether they possess the copper binding dissimilatory nitrite reductase (NirK) or the cytochrome cd1 variant (NirS) that catalyze the reduction of NO2− to NO; the key reaction that defines denitrification nosZ co-occurrence patterns in relation to denitrifying organisms with one or the other type of nitrite reductase can provide additional insight on the importance of denitrifier community composition for N2O reduction in different ecosystems.Whether the genetic potential to produce or reduce N2− reduction is performed by NirK or NirS denitrifiers, the link to the enzymatic step reducing NO to N2O needs to be considered. This reaction may be performed by a number of different enzymes that vary in structure and evolutionary relationships, including periplasmic associated flavodiiron, flavorubredoxin, and cytochrome-c type proteins, as well as several variants of the membrane-bound heme-copper oxidases 2O in the denitrification pathway is performed by two variants of the heme-copper oxidase type NO reductases in bacterial and archaeal denitrifiers, encoded by the cnorB or qnorB genes qnorB gene have also been observed in non-denitrifying pathogenic species as well as anaerobic denitrifying methanotrophs, and are believed to be involved in detoxification 2 and O2nor), however this enzyme is not considered to be involved in energy conservation 2O production from a given ecosystem could be considered a sum of the activities of each N2O-genic NO reductase, although the contribution of qnorB and cnorB likely outweighs that of other Nor-types due to their prominent role in anaerobic respiration To understand the ecological implications of whether NOnosZ, being a proxy for the trait N2O reduction, differed among nirK and nirS type denitrifiers, how this relates to taxonomic affiliation, and whether these patterns could be explained by the presence of a nor gene encoding one of the canonical NO reductases or the p450nor variant for fungal denitrifiers. This was investigated by examining the distribution of the genes nirK, nirS, nor and nosZ in 652 publicly available microbial genomes across 18 phyla. The increasing number of genomes from organisms found in a diverse range of environments 2O reduction is from an evolutionary perspective.The aim of our study was to determine whether patterns of co-occurrence of www.ncbi.nlm.nih.gov). To ensure that homology searches were as comprehensive as possible, a two-step procedure was performed for each gene. First, an initial TBLASTN search www.ncbi.nlm.nih.gov/sutils/genom_table.cgi) was performed using translated nirS, nirK, and nosZ gene sequences from either Paracoccus denitrificans PD1222 or Bradyrhizobium japonicum USDA110 as queries. Resulting hits were then translated to amino acid sequences and aligned using SATÉ v2.2.3 nirK, nirS and nosZ, with 477, 150 and 282 sequences, respectively, were then used to create Position Specific Score Matrices (PSSM) nirK, nirS and nosZ amino acid sequences. Strains with identical sequences were kept when a unique co-occurrence pattern of denitrification genes was observed, or when the sequence of another denitrification gene was not identical, resulting in a dataset of 652 organisms  to allow for an equal representation of both variants within the initial PSSM. For the eukaryotic species, the amino acid sequence for the P450nor from Fusarium oxysporumnor based on previously described conserved amino acid positions A local database was constructed by downloading all 4135 draft and completed microbial genome nucleotide sequences available (November 2012) at the National Center for Biology Information  Small subunit (SSU) rRNA gene sequences corresponding to the organisms were retrieved from the local genome database using Infernal nirK, nirS, norB, nosZ, and P450nor were created using the MUSCLE alignment algorithm nirK and nosZ, LG+Γ+F for norB and nirSnor. Subsequently, new alignments were inferred using SATÉ, and after manual inspection of the resulting alignments maximum likelihood (ML) phylogenetic trees were inferred using 30 RAxML tree searches and selecting the best likelihood topology, followed by bootstrap analysis with 250 replicates. Small subunit rRNA sequences were aligned using SINA Preliminary amino acid alignments of full-length gene sequences of 2O reduction between denitrifiers with nirK and nirS, the nosZ gene was used as a proxy for the capacity of an organism to reduce N2O and mapped onto the corresponding taxa within the nirK and nirS phylogenies. Quantification of the phylogenetic patterns of nosZ distribution for both genes was performed using Fritz and Purvis' D), implemented in the ‘caper’ package within the R statistical programming environment . This statistic measures whether a trait is highly clumped (D<0), displays clumping patterns due to Brownian evolutionary processes (D∼0) or random distribution patterns (D = 1), or is highly over-dispersed (D>1) across a given phylogeny. In order to also determine the degree to which the ability to reduce N2O is conserved amongst nirK and nirS denitrifiers, we used the phylogenetic metric ConsenTRAIT D) of nosZ as a trait on nirK and nirS gene trees in the R environment. Trait depth is a measure of the average distance between organisms exhibiting the trait and their last common ancestor, such that increasing values of τD indicate more conserved traits.To compare the phylogenetic pattern of NnirK, nirS, nor and nosZ were investigated in relation to taxonomic affiliation based on the 16S rRNA gene phylogeny at different taxonomic ranks. In order to determine whether the co-occurrence patterns of nir and nos genes were randomly distributed among taxa, contingency tables of co-occurrence patterns by taxonomic affiliation at the phylum, class, order and family was performed, followed by Chi-squared goodness of fit tests in the R environment. Lower taxonomic ranks were not examined due to low frequencies (<5) in the majority of cells. In addition, the standardized residuals of the resulting cell values were examined to distinguish taxa that differed significantly from the expected values for each pattern of co-occurrence, and presented as a mosaic plot generated in the R environment using the ‘vcd’ package. For each possible combination of nosZ and nir genes, the fraction harboring a nor gene was determined.The genomic co-occurrence patterns of nirK, nirS and nosZ were also correlated with available information on the organism's lifestyle as determined by habitat preference and for each possible combination, the fraction harboring a nor gene was determined. Information on habitat preference could be retrieved for 626 organisms that were grouped into eight general habitat categories: wastewater treatment plants, extremophilic, marine systems, fresh water systems, soil, plant, animal, and food associated organisms, and those found in multiple habitats. Here, extremophilic represents habitats for thermo-, psychro-, halo-, acido- and alkalophiles as well as oil degrading, radiation resistant and arsenic resistant organisms. The plant and animal associated organisms are specified as being directly associated to a plant or animal host, whereas food associated are those used in food production. Organisms isolated from multiple sources without any specific preferences were lumped into the ‘multiple habitats’ category. The different categories of lifestyle versus the gene occurrence patterns were tested in the same way as for taxa affiliation.The co-occurrence patterns of nirK or nirS homolog, respectively, including ten genomes that harbored both nir genes. As stated earlier, we limited our search of NO-reductases to the cnorB and qnorB variants encoding NorB found in the canonical denitrification pathway, as well as the p450nor variant for fungal denitrifiers. This resulted in a total of 431 organisms with a nor gene, whereas 314 genomes were found to possess a nosZ gene. Interestingly, a large part of the nirK organisms (35%) had a truncated pathway lacking a canonical nor and a majority (70%) did not have nosZ. By contrast, only 3.6% of the nirS-type denitrifiers lacked nor and 20% did not have a nosZ gene. Of all organisms harboring nosZ, 30% did not possess any nir genes and about 24% did not have nor. In general, genomes containing both nosZ and nor also had a nir gene, whereas organisms with nosZ that did not have a nor gene also lacked a nir gene  of 0.335, which indicates a moderately clumped distribution that was significantly different from both random (p<0.001) and Brownian distributions (p = 0.002) of phylogenetic trait patterns.Phylogenetic analysis of amino acid alignments resulted in well-supported tree topologies, with 48%, 63%, 62% and 66% of nodes having bootstrap support greater than 70% for ectively –S5. Simihylogeny  could benorB phylogeny could be clearly divided into cnorB and qnorB variants, with a diverse range of bacterial and archaeal phyla found within each clade  that was also significantly non-random (p<0.001) and did not correspond to a Brownian process (p = 0.02), yet was slightly less clumped than the pattern of nosZ distribution observed for the nirK phylogeny. Additional analysis using the ConsenTRAIT metric (τD), performed using nosZ occurrence as a proxy for the trait N2O reduction on both the nirS and nirK phylogenies, indicated a higher degree of evolutionary conservation of nosZ with nirS (τD = 0.089) than nirK (τD = 0.043).In contrast to logenies  and S3 wnir, nor and nos genes (nirK only (K-type), nirK+nosZ (KZ-type), nirK+nirS (KS-type), nirK+nirS+nosZ (KSZ-type), nirS only (S-type), nirS+nosZ (SZ-type) and only nosZ (Z-type) were indeed not randomly distributed amongst groupings at either the phylum, class, or order levels , but also within the Deltaproteobacteria (71.4%), Firmicutes (27.5%), and Euryarchaeota (35.7%), with the frequencies of Z-type organisms being significantly higher than expected within these groups  was significantly higher than expected, as well as organisms with a KSZ pattern. The frequency of organisms with only nosZ was overrepresented in marine ecosystems, yet lower than expected in the category of plant host-associated lifestyles. The Z-type organisms exhibited a low occurrence of nor in marine (12%) and host associated environments, whereas the opposite was observed in fresh water, soil and wastewater.When comparing data relating to lifestyle in terms of habitat preference with nirK and a nirS gene in the same genome. Although that is still rare, it contradicts the previous assumption that the two nitrite reductases are mutually exclusive nir genes was previously reported in the genome of a Methylomonas strain nir genes in co-existing subpopulations affiliated to Pseudovibrio detected in the metagenome of a marine denitrifying community. Nevertheless, it has not yet been demonstrated if the two types of nitrite reductases are functional when present in the same genome, although it is not unlikely considering that two divergent copies of nirS in a Thauera sp. isolate were expressed under different conditions The genome data-set analyzed in this study demonstrated that the denitrification pathway is modular with no less than seven different co-occurrence types of the genes coding for the reductases NirK, NirS and NosZ. Interestingly, we found ten organisms that possess both a nosZ gene was used as a proxy for the capacity of an organism to reduce N2O and we found that this gene occurred much more frequently in the genomes of organisms with nirS than in those with nirK. This was previously suggested nor gene partly explained this pattern, as nirS almost always co-occurred with nor, although there were exceptions. The substantial difference in co-occurrence of nosZ with nirS vs. nirK in the genome data-set is likely governed by an underlying ecological or evolutionary mechanism that constrains the loss of nor and nosZ in organisms with nirS. This is also supported by the higher degree of phylogenetic conservation observed for nosZ presence when mapped onto the nirS phylogeny than onto that of nirK. Previous studies have shown that loss of the nos regulon may occur through either short-term adaptive processes, such as phase variation in nirK Azospirillum soil isolates nirK Neisseria species nirS, nor and nosZ co-occurrence to regulatory mechanisms that may be shared between the three enzyme complexes, but not with NirK, which is fully functional without accessory proteins. The expression of all three enzymes is controlled by oxygen and NO levels within the cell nirS, nor and nosZ expression has been described to date. Based on the gene co-occurrence patterns, the majority of nirS organisms are more likely to perform complete denitrification to N2 than the nirK types under favorable environmental conditions and we suggest that the presence of nor in the genome of an organism also harboring nir or nosZ serve as an indicator of whether the organism is a denitrifier sensu stricto or merely a nitrite or nitrous oxide reducer.The nosZ, it is interesting that a range of studies have shown evidence of niche differentiation between nirS and nirK denitrifying communities nirK bearing organisms would be more likely to emit more N2O than one dominated by denitrifiers with nirS under the same environmental conditions. Indeed, the ratio of nirS/nirK type denitrifiers was recently demonstrated to have a significant and negative relationship with the capacity of a soil to act as an N2O sink when modeling the relative influence of abiotic and denitrifier community factors on the potential for soil N2O reduction nirK abundance and N2O emissions from arable and woodland soils, and based on qPCR measurements estimated that only 10–30% of nirK type denitrifiers also carry the nosZ gene, depending on soil type. Similarly, Cuhel et al. 2O emissions to total denitrification activity (i.e. N2 + N2O) and the relative abundance of nirS-type denitrifiers in pastureland soils.In the context of different co-occurrence patterns with nosZ gene without having nir or a putatively N2O producing nor gene, and these were predominantly found within nosZ clade II. Several representatives from this clade, including the host-associated bacteria Wolinella succinogenes and Campylobacter fetus, as well as the thermophile Geobacillus thermodenitrificans and the soil bacterium Anaeromyxobacter dehalogenans, have been demonstrated to grow with N2O as the single electron acceptor 2O reduction. Net negative N2O fluxes in field measurements have been reported nosZ clade II largely explained the soil N2O sink capacity 2O reduction were predominant in clade II communities, especially those associated with nosZ II lineages from organisms lacking nir. Because clade II has been shown to be both diverse and abundant in different ecosystems 2O consumers in the environment.A highly truncated version of the denitrification pathway was observed among the substantial fraction of organisms that possessed the nir/nos co-occurrence patterns were not randomly distributed across taxa, from phylum down to order level, with significant patterns related to certain taxonomic groups. The data set was too small to detect any patterns at lower taxonomic ranks. Accordingly, closely related organisms do not necessarily share this trait, and even if they do the denitrification genes may be distantly related or they may not have the same set of genes nosZ gene without possessing any nir gene were mainly found amongst the Bacteroidetes, and Firmicutes belonging to nosZ clade II, and predominantly lacked a nor gene. Thus, environments in which these taxa are highly abundant may have substantially lower N2O emission rates than those dominated by organisms that only have nir and nor genes, such as several representatives among the Actinobacteria and Ascomycota. This coincides with the results of Philippot et al 2O production and denitrification rates on a field that was subjected to different cattle grazing regimes. Interestingly, the percentage of N2O to total denitrification activity (N2 + N2O) was lowest in the region of the field with the highest relative abundance of Bacteroidetes and lowest abundance of Actinobacteria. Furthermore, regions of the field in which nirS and nosZ clade I were most abundant also had the highest relative abundance of Betaproteobacteria, corresponding to our finding that the co-occurrence of nirS and nosZ is the predominant pattern of denitrification genes within this class. Thus, general patterns of gene co-occurrences in conjunction with taxonomic information can potentially aid in our interpretation of experimental data and predictions of genetic N2O emission potential.Although denitrification as a trait is widely distributed across different phyla, the observed K-types over S-types amongst animal host associated organisms, suggesting that nirK might be involved to a greater extent during animal host adaptation and pathogenicity. Previous studies examining the relationship between nitrite reduction and pathogenicity of Neisseria and Brucella species have indicated that nirK is expressed primarily to help the bacterium cope with low oxygen levels within the host nirS has also been shown to be important for virulence in Pseudomonas aeruginosa, albeit by regulation of virulence factors through maintaining a steady state of NO within the host nirK gene alone is sufficient to encode a functional protein while nirS requires several accessory genes nirK rather than nirS. Interestingly, both the KS- and KSZ- types were significantly overrepresented in wastewater. One could speculate that carrying both types of nir genes could imply an adaptive advantage in an environment supporting denitrification activity, provided that both genes are transcribed to functional nitrite reductases under different conditions. The SZ type was more often found among genomes isolated from wastewater and freshwater than expected by chance. In agreement, it has been suggested that nirS denitrifiers are better adapted to stable, high water content conditions where oxygen availability is expected to be consistently low, while nirK communities tend to dominate under more recurrently changing conditions nirS communities are more frequently detected, more diverse and more phylogenetically clustered in marine systems than nirK communities nirS denitrifiers dominate in this habitat and that nirS denitrifying communities are shaped by habitat filtering more so than nirK communities. However, our analysis based on habitat data recovered from the GOLD database does not provide enough data or detail to confirm these hypotheses.Comparisons of gene co-occurrence patterns to preferred habitat categories showed differential associations of denitrifier gene combinations with specific habitats or lifestyles. Most notable was the prevalence of nirK and nirS denitrifiers. Although N2O emissions are subject to environmental factors and inherent cellular regulatory mechanisms, the ultimate control over whether N2O is emitted during denitrification is the presence of nosZ in the genomes of organisms that comprise the denitrifying community. Thus, the substantial difference between taxa as well as nirS, nirK and nor co-occurrence with nosZ likely plays a significant role in determining the genetic N2O emission potential from a given ecosystem, thereby underpinning the significance of microbial community structure for N2O emissions. Further research examining the underlying physiological or evolutionary mechanisms that result in the non-random patterns of nir/nor/nos gene occurrence observed here may provide additional predictive value for future N2O mitigation strategies.In conclusion, our results show that the co-occurrences of denitrification genes are not randomly distributed among taxonomic groups, preferred habitats or Figure S1Maximum likelihood phylogeny of full-length 16S and 18S rRNA sequences from 652 organisms harboring denitrification genes. The inner colored ring represents taxonomic affiliation as indicated by the legend. The four outer bar-chart rings show the presence of nirK (orange), nirS (purple), nor (turquoise) and nosZ (magenta). Bar height represents the number of copies (≤4). Bootstrap values >70% are indicated by grey circles, and the scale bar denotes nucleotide substitution rate (GTR+Γ).(EPS)Click here for additional data file.Figure S2Maximum likelihood phylogeny of 458 full-length nirK amino acid sequences, rooted at midpoint. Co-occurrences of nirS, nor and nosZ genes in the genomes are indicated with purple, turquoise and magenta bars, respectively. Bar height depicts the number of co-occurring gene copies (≤3) and the scale bar denotes nucleotide substitution rate (LG+Γ). Strain names are colored to indicate taxonomic affiliation according to the legend, and bootstrap values >70% are designated by circles.(EPS)Click here for additional data file.Figure S3Maximum likelihood phylogeny of 110 full-length nirS amino acid sequences, rooted at midpoint. Co-occurrences of nirK, nor and nosZ genes in the genomes are indicated with orange, turquoise and magenta bars, respectively. Bar height depicts the number of co-occurring gene copies (≤2) and the scale bar denotes nucleotide substitution rate (LG+Γ+F). Strain names are colored to indicate taxonomic affiliation according to the legend, and bootstrap values >70% are designated by circles.(EPS)Click here for additional data file.Figure S4Maximum likelihood phylogenies of 413 prokaryotic and 18 fungal (inserted graph) full-length nor amino acid sequences. Both phylogenies are rooted at midpoint. Co-occurrences of nirK, nirS and nosZ genes in the genomes are indicated with orange, purple and magenta bars, respectively. Bar height depicts the number of co-occurring gene copies (≤4) and the scale bar denotes nucleotide substitution rate (LG+Γ+F and WAG+Γ+F respectively). cnorB sequences are shaded in orange and qnorB sequences in green. Strain names are colored to indicate taxonomic affiliation according to the legend, and bootstrap values >70% are designated by circles.(EPS)Click here for additional data file.Figure S5Maximum likelihood phylogeny of 314 full-length nosZ amino acid sequences, rooted at midpoint. Co-occurrences of nirK, nirS and nor genes in the genomes are indicated with orange, purple and turquoise bars, respectively. Bar height depicts the number of co-occurring gene copies (≤4) and the scale bar denotes nucleotide substitution rate (LG+Γ). Clade I is shaded in orange, clade II in blue and the halophilic Archaea in turquoise. Strain names are colored to indicate taxonomic affiliation according to the legend, and bootstrap values >70% are designated by circles.(EPS)Click here for additional data file.Table S1Taxon identification number and project name according to NCBI of 652 organisms harboring denitrification genes. Copy numbers of nirK, nirS, nor and nosZ are indicated.(PDF)Click here for additional data file.Table S2Frequency table of gene co-occurrence patterns and taxonomic affiliation at class level. The nirK (K), nirS (S) and nosZ (Z) co-occurrence types and percentage of organisms within each class that also harbor a nor gene are indicated.(PDF)Click here for additional data file.Table S3Frequency table of gene co-occurrence patterns and taxonomic affiliation at order level. The nirK (K), nirS (S) and nosZ (Z) co-occurrence types and percentage of organisms within each order that also harbor a nor gene are indicated.(PDF)Click here for additional data file.


Metabarcoding has become an important tool in the discovery of biodiversity, including fungi, which are the second most speciose group of eukaryotes, with diverse and important ecological roles in terrestrial ecosystems. We have designed and tested new PCR primers that target the D1 variable region of nuclear large subunit (LSU) ribosomal DNA; one set that targets the phylum Ascomycota and another that recovers all other fungal phyla. The primers yield amplicons compatible with the Illumina MiSeq platform, which is cost-effective and has a lower error rate than other high throughput sequencing platforms. The new primer set LSU200A-F/LSU476A-R (Ascomycota) yielded 95–98% of reads of target taxa from environmental samples, and primers LSU200-F/LSU481-R  yielded 72–80% of target reads. Both primer sets have fairly low rates of data loss, and together they cover a wide variety of fungal taxa. We compared our results with these primers by amplifying and sequencing a subset of samples using the previously described ITS3_KYO2/ITS4_KYO3 primers, which amplify the internal transcribed spacer 2 (ITS2) of Ascomycota and Basidiomycota. With approximately equivalent read depth, our LSU primers recovered a greater number and phylogenetic diversity of sequences than the ITS2 primers. For instance, ITS3_KYO2/ITS4_KYO3 primers failed to pick up any members of Eurotiales, Mytilinidiales, Pezizales, Saccharomycetales, or Venturiales within Ascomycota, or members of Exobasidiomycetes, Microbotryomycetes, Pucciniomycetes, or Tremellomycetes within Basidiomycota, which were retrieved in good numbers from the same samples by our LSU primers. Among the OTUs recovered using the LSU primers were 127 genera and 28 species that were not obtained using the ITS2 primers, although the ITS2 primers recovered 10 unique genera and 16 species that were not obtained using either of the LSU primers These features identify the new primer sets developed in this study as useful complements to other universal primers for the study of fungal diversity and community composition. Identification and classification of a wide variety of organisms using DNA sequences has helped overcome many limitations of traditional morphological approaches, including providing faster analyses, resolving convergent morphologies, recognition of closely related or sister species, and the ability to identify cryptic organisms from complex or opaque substrates ,2. This However, the utility of DNA as a tool to catalogue biodiversity, resolve phylogenies, or explore patterns in ecological communities depends strongly on choice or design of primers for selecting the appropriate genetic markers . Primer http://www.illumina.com/systems/sequencing.html). Unfortunately, no primers have been developed to target the D1 region across a wide diversity of fungi with a short amplicon suitable for the Illumina MiSeq platform. Here we introduce and evaluate two primer sets targeting the D1 region of the large subunit (LSU) of ribosomal DNA to evaluate fungal diversity. We demonstrate how these primers can be applied for discovery of major fungal groups to recover a broad range of fungal taxa with confident higher-level placement  and potential species or species-group identification.An ongoing challenge in diversity studies is the pairing of appropriate genetic markers and targeted primer development that works with contemporary technologies. Next Generation Sequencing (NGS) can capture low abundance DNA and provide detailed analyses of relative abundances in microbial communities  at a relSaccharomyces cerevisiae  and anchored with the sequence of  J01355; ). This a J01355; ) was cre J01355;  and the  J01355; . The aliThe region of LSU between bases 150 and 550 from different fungal groups was used to query GenBank using BLAST , separatSaitoella, 209C in Taphrina) or were mismatches at the 3’ end that occurred only rarely . Primer layout was as follows:The reverse primer LSU481-R had two pairs of mismatches to most Ascomycota at bases 468–469 and 474–475, which were corrected, and the revised primer LSU476A-R was moved 5 bases 3’-ward for greater specificity against plants and other non-fungal eukaryotes. Both forward and reverse primers were modified on their 5’ ends to include the following: forward and reverse Illumina MiSeq adaptors to allow sequences to bind to the flow cells, a 4 bp random linker (NNNN) to increase the diversity of the first positions sequenced and so enable the instrument to more easily separate clusters and set appropriate fluorescence levels, and unique 8 nt barcode sequences with an edit distance of at least 4 , and the reverse primer is ITS4_KYO3 (5′– CTB TTV CCK CTT CAC TCG –3′). These primers target diverse taxonomic groups within Ascomycota and Basidiomycota. Primers were ordered with an Illumina Adaptor, linker, and unique barcode as described above.We compared our developed primer sets against another primer set currently used in Illumina sequencing  described by Toju et al. , where tSphagnum-dominated fen at the White River Experimental Research sites near White River, Ontario, Canada , managed by the Ontario Forest Research Institute and the Ontario Ministry of Natural Resources, with permission. Eight large, intact peat cores complete with above-ground herbaceous and shrubby vegetation were collected  Supplem(DOCX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Tablea, 941 098b, 134 391, and 96 830 reads, respectively; n = 3 for all).Relative frequency (%) calculated using total number of reads for each soil type (348 237(DOCX)Click here for additional data file.


Prasugrel is more effective than clopidogrel in reducing platelet aggregation in acute coronary syndromes. Data available on prasugrel reloading in clopidogrel treated patients with high residual platelet reactivity (HRPR) i.e. poor responders, is limited.To determine the effects of prasugrel loading on platelet function in patients on clopidogrel and high platelet reactivity undergoing percutaneous coronary intervention for acute coronary syndrome (ACS).Patients with ACS on clopidogrel who were scheduled for PCI found to have a platelet reactivity ≥40 AUC with the Multiplate Analyzer, i.e. “poor responders” were randomised to prasugrel (60 mg loading and 10 mg maintenance dose) or clopidogrel (600 mg reloading and 150 mg maintenance dose). The primary outcome measure was proportion of patients with platelet reactivity <40 AUC 4 hours after loading with study medication, and also at one hour (secondary outcome). 44 patients were enrolled and the study was terminated early as clopidogrel use decreased sharply due to introduction of newer P2Y12 inhibitors.At 4 hours after study medication 100% of patients treated with prasugrel compared to 91% of those treated with clopidogrel had platelet reactivity <40 AUC (p = 0.49), while at 1 hour the proportions were 95% and 64% respectively (p = 0.02). Mean platelet reactivity at 4 and 1 hours after study medication in prasugrel and clopidogrel groups respectively were 12 versus 22 (p = 0.005) and 19 versus 34 (p = 0.01) respectively.Routine platelet function testing identifies patients with high residual platelet reactivity (“poor responders”) on clopidogrel. A strategy of prasugrel rather than clopidogrel reloading results in earlier and more sustained suppression of platelet reactivity. Future trials need to identify if this translates into clinical benefit.NCT01339026ClinicalTrials.gov  Dual antiplatelet therapy (DAPT) consisting of aspirin and an adenosine diphosphate (ADP) receptor inhibitor has been shown to reduce the risk of subsequent vascular events including myocardial infarction (MI) and stent thrombosis in patients with acute coronary syndromes (ACS). Some of APACS was a randomised, open label study carried out in 1 centre in Germany and 4 centres in the UK comparing prasugrel with clopidogrel reloading in ACS patients pre-treated with clopidogrel who had high residual platelet reactivity (“poor responders”). PCI had to be planned to take place as early as possible and no later than 72 hours from admission.Patients with prior clopidogrel loading within 24h before planned PCI or receiving chronic treatment with clopidogrel  who had high platelet reactivity ≥ 40 AUC were randomised to prasugrel (60 mg loading and 10 mg maintenance dose) or a high dose clopidogrel regimen (600 mg reloading followed by 150 mg maintenance dose). There are no large clinical endpoint studies evaluating cut-off values for platelet reactivity measured with Multiplate Analyzer in the early phase e.g. 4h after loading dose in ACS patients to predict early periprocedural events. 40 AUC was a prespecified arbitrary cut-off value based on previous observations, showing that a similar degree of platelet reactivity can be achieved at ~4h after loading with newer platelet inhibitors prasugrel and ticagrelor. [Screened patients identified with low platelet reactivity indicating a good response to clopidogrel were entered into a registry. The trial was registered at clinicaltrial.gov (NCT01339026). The APACS study was conducted in compliance with the principles of the Declaration of Helsinki and the International Conference on Harmonization consolidated guidelines and was approved by the NHS Research Ethics Committee and the ethical review committees of Tübingen University. Patients gave written informed consent prior to study participation.Inclusion criteria were: ACS, age: 18–75 years, intention to perform PCI <72 hours from admission, prior or chronic treatment with clopidogrel (defined as prior clopidogrel loading within 24h before planned PCI or chronic >24 hours and at least 7 days of maintenance dose treatment with clopidogrel), high platelet reactivity as defined by ADP induced platelet activation of ≥ 40 AUC by Multiplate analyser with timing of platelet function assay at least 2 hours after pre-PCI loading dose, provision of informed consent by participating patient. Inclusion criteria for the registry were the same as above except for a platelet reactivity of <40 AUC.9/L at the time of screening, International Normalized Ratio (INR)> 1.5 at the time of screening, Hb<10g/dL, intracranial neoplasm, arteriovenous malformation or aneurysm, severe hepatic impairment (Child Pugh class C) or intention to use the following medications: oral anticoagulation, other antiplatelet therapy (including GPIIb/IIIa inhibitors) besides aspirin, non-steroidal anti-inflammatory drugs (NSAIDs) or cyclooxygenase-2 (COX-2) inhibitors, female patients who are pregnant, planning pregnancy, or who are breastfeeding, known allergy, hypersensitivity or other contraindications to prasugrel or clopidogrel or hemodynamically unstable patients in whom study related procedures might cause unnecessary delays to urgent revascularization.Exclusion Criteria were body weight <60 kg, pre-treatment with prasugrel within 7 days of randomisation, history of stroke or transient ischemic attack, patients with increased bleeding risk e.g. recent major trauma or surgery, gastrointestinal bleeding or active peptic ulceration, platelet count < 100 x10The Multiplate analyzer, a whole blood platelet function assay, was used to study the platelet aggregation level. A 600μL blood sample acquired in hirudinized tubes (Sarstedt) was obtained to perform ADP and TRAP tests in each patient. The blood samples were taken before the loading dose, 1h, 4 hours and 24h after the loading dose. The area under the aggregation curve (AUC) was used as a measure of the overall platelet aggregation. Tests were performed 30 minutes to 3 hours after taking blood.Patients were randomised to either open label clopidogrel  or prasugrel . The randomisation algorithm is presented in Randomised patients were followed up to 30 days for clinical status, study medication, blood samples for adenosine diphosphate ADP) and thrombin receptor activating peptide (TRAP) test and serious adverse events (SAEs). Registry patients were followed up to 30 days for survival. Major Adverse Cardiac Events (MACE) were defined as death, stroke, myocardial infarction and repeat revascularization. Bleeding events were defined according to the bleeding academic research consortium criteria (BARC) criteria as previously reported.Primary endpoint was the proportion of patients with improved platelet response i.e. platelet reactivity under the cut-off value of 40 AUC in the prasugrel reloading arm compared to the clopidogrel reloading arm at 4 hours after randomisation in patients with initial high platelet reactivity.The secondary endpoints were ADP induced platelet reactivity at 1 and 24 hours, TRAP induced platelet reactivity at 1, 4 and 24 hours, and MACE including death, stroke, myocardial infarction, repeat revascularization and major bleedings defined according to BARC.For an absolute difference of 20% in conversion of patients with initial high platelet reactivity to a platelet reactivity under the cut-off value of <40 AUC  after reloading with prasugrel compared to clopidogrel the estimated minimum sample size was 62 patients per arm  to detect this difference. Baseline variables were compared with the use of chi-square tests for categorical variables; t-tests were used for normally distributed continuous variables and the Wilcoxon rank-sum for variables with non-Gaussian distributions. Comparison of the primary endpoint and secondary endpoints was carried out using a chi squared or Fishers exact test. The confidence intervals were two-sided with a 95% confidence level, and all hypothesis tests were two-sided carried out at a significance level of 0.05.The study started enrolment in February 2012. During the course of the trial clinicians had started to adopt clinical guidelines recommending prasugrel or ticagrelor over clopidogrel for ACS patients.,10 whichOf the ACS cohort 40.7% was found to have HRPR at the time of PCI despite pre-treatment with clopidogrel. 44 patients fulfilled all eligibility criteria, consented to study participation and were enrolled. Another 64 patients fulfilled eligibility criteria but had normal platelet reactivity (i.e. <40 AUC) and entered the registry . The basWith respect to TRAP induced platelet aggregation there was a significant difference at baseline between both arms. Patients randomised to prasugrel had lower TRAP induced platelet aggregation compared to patients allocated to clopidogrel reloading . The reduced TRAP induced aggregation values in the prasugrel compared to the clopidogrel treated arm persisted over time mean 66.9 AUC  vs. mean 78.9 AUC  at 1 hour, mean 63.0 AUC  vs. mean 67.4  at 24 hours; .Clinical events are shown in The primary endpoint of the proportion of patients with platelet reactivity under the cut-off value of 40 AUC after randomisation was not significantly different in the prasugrel reloading arm compared to the clopidogrel reloading arm at 4 hours although this analysis lacks statistical power due to early termination. In the secondary analysis, reloading with prasugrel provided a better antiplatelet response at one hour than reloading with clopidogrel. The findings can be explained by the greater antiplatelet efficacy and faster onset of prasugrel shown in mechanistic studies and better clinical outcomes as seen in TRITON-TIMI38. However a direct comparison of these two agents in clopidogrel “poor responders” in the setting of ACS and PCI has not been carried out.12 inhibitor naïve patients. To date there is no data from randomised trials comparing the peri-procedural effects of prasugrel versus high dose clopidogrel in thienopyridine pre-treated ACS patients which is a common clinical scenario. In fact, randomised studies investigating pharmacodynamic/pharmacokinetic profile suggest that even with the third generation P2Y12 inhibitors prasugrel and ticagrelor, the platelet inhibitory effect in real world ACS patients is insufficient at the time of the procedure.[rd generation P2Y12 receptor blockers. On the other hand, early and potent platelet inhibition by prasugrel loading in the upstream period has been associated with an excess of procedural related major bleedings without ischemic risk reduction in the ACCOAST trial.[Poor platelet inhibition in the early phase of ACS and PCI is associated with MACE including acute stent thrombosis. In a recrocedure.,16 This ST trial. It is thST trial.The present findings suggest that early prasugrel reloading in clopidogrel pre-treated ACS patients who exhibit high platelet reactivity at the time of PCI may provide a more rapid and complete maximum platelet inhibition compared to reloading with 600mg clopidogrel followed by a high dose clopidogrel maintenance dose regimen. The study further indicates that about 40% of patients have high platelet reactivity at the time of PCI despite adequate clopidogrel pre-treatment. This observation corresponds well with previous reports of up to 50% of NSTE-ACS5 and 64.5% of STEMI patients  expressiWhereas the benefit of platelet function test guided antiplatelet therapy for the post-ACS long-term phase has been debated ,21, therTo the best of our knowledge, there are no data from randomised clinical trials investigating the role of platelet function testing using multiple impedance aggregometry in the very early procedural time window in ACS patients. The present results suggest that preselecting patients might help to improve platelet inhibition in this critical phase of an acute coronary event. In the present study, repeated clopidogrel loading dose regimen was not sufficient to overcome high platelet reactivity in ACS patients. This is in line with previous results from pharmacodynamic studies in stable CAD patients. A high dose clopidogrel regimen was only able to reduce high platelet reactivity in non-carriers of the loss-of-function CYP2C19*2 genotype in contrast to prasugrel.,2612 receptor antagonist in the upstream period of NSTE-ACS, clopidogrel is still widely used in the ambulance setting and the majority of NSTE-ACS patients in Europe and North America are still pre-treated with clopidogrel. This will likely persist due to higher costs for newer antiplatelet agents. We did not evaluate the impact of the loss- or gain of function genotypes that might have influenced metabolism of the P2Y12 receptor antagonists in particular clopidogrel in the acute setting.[This trial has some limitations. First of all it was prematurely terminated due to changing guideline adherence and before the calculated sample size for the primary endpoint was reached. The active comparator of high dose clopidogrel is not favoured by current guidelines. However, due to current treatment patterns and lack of evidence and safety concerns of novel P2Y setting. The conc setting.,29 The s setting.In conclusion our data provides evidence that residual high platelet reactivity can be corrected by prasugrel in patients with ACS undergoing PCI who have been treated with clopidogrel. The role of platelet function testing is evolving and is not currently recommended as a routine test in guidelines ,9 while S1 CONSORT Checklist(PDF)Click here for additional data file.S1 Protocol(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.