Optimizing ventilator strategies may reduce respiratory morbidities in preterm infants. Permissive hypercapnia has been suggested to attenuate lung injury. We aimed to determine if a higher PCO2 target or a control group. The high target group aimed at PCO2 values of 55–65, 60–70, and 65–75 mmHg and the control group at PCO2 values of 40–50, 45–55 and 50–60 mmHg on postnatal days 1–3, 4–6, and 7–14, respectively. TA was analyzed for pro-inflammatory cytokines from postnatal day 2–21. BPD was determined at a postmenstrual age of 36 weeks ± 2 days.During a multicenter trial of permissive hypercapnia in extremely low birthweight infants (PHELBI), preterm infants  requiring mechanical ventilation within 24 h of birth were randomly assigned to a high PCOp = 0.14), IL-8 (p = 0.43), IL-10 (p = 0.24), IL-1β (p = 0.11), macrophage inflammatory protein 1α (p = 0.44), albumin (p = 0.41), neuropeptide Y (p = 0.52), leukotriene B4 (p = 0.11), transforming growth factor-β1 (p = 0.68), nitrite (p = 0.15), and ASM (p = 0.94). Furthermore, most inflammatory mediators were strongly affected by the age of the infants and increased from postnatal day 2 to 21. BPD or death was observed in 14 out of 62 infants, who were distributed evenly between both groups.Levels of inflammatory cytokines and ASM were similar in both groups: interleukin (IL)-6 (2 target levels did not result in lower pulmonary inflammatory activity and thus reflect clinical results. This indicates that high PCO2 target ranges are not effective in reducing ventilator-induced lung injury in preterm infants, as compared to control targets.The results suggest that high PCOISRCTN56143743. Bronchopulmonary dysplasia (BPD), a form of chronic lung disease, is frequently observed in preterm infants. BPD development is associated with long-term oxygen supplementation , 2, and 2). Possible benefits of permissive hypercapnia such as diminished lung injury and pulmonary inflammation  were detected within the first 10 days of life in the bronchoalveolar lavage fluid of preterm infants who subsequently developed BPD compared to infants who did not  , two dif2. The high target group aimed at PCO2 values of 55–65 mmHg on postnatal days (PD) 1–3, followed by 60–70 mmHg on PD 4–6, and 65–75 mmHg on PD 7–14. The control target group aimed at PCO2 values of 40–50 mmHg on PD 1–3, followed by 45–55 mmHg on PD 4–6, and 50–60 mmHg on PD 7–14  was favored over high tidal volumes in both groups. Initial ventilator settings comprised a rate of 60–80/min or greater, inspiratory time of 0.25–0.35 s, positive end-expiratory pressure 3.6 mbar, and a peak inspiratory pressure (PIP) resulting in minimal to moderate chest rise. The rate was allowed to decrease only if the PIP was 14 mbar or lower. Synchronized ventilation or forms of volume control were applied at the discretion of the clinicians in charge of patient care . Thereafter, suctioning was performed and TA collected. Afterward, the suctioning catheter was flushed with 0.5 ml normal saline. TA were transferred to an appropriate tube and immediately centrifuged at 140 × 1 , albumin , and nitrotyrosine . In addition, competitive binding assays were used for neuropeptide Y (NPY)  and LTB4 (R&D Systems GmbH). Nitrite TA levels were determined by a Griess reaction assay (R&D Systems GmbH). All assays were performed according to the manufacturer’s recommendations. For ASM, a proprietary assay was used as described before  was used . Enzyme-d before . To incrd before , 46, we 2 (BPD or death)  , 47.U-test or Fisher’s exact test as appropriate. TA concentrations were compared by mixed model two-way (factors being time and target group) analyses of variance (ANOVA) with a heterogeneous unstructured covariance structure SAS software 9.4 . In the figures, the effect of postnatal age (time) is denoted below the x-axis and the target group effects on each single day at the respective time points.Demographic and clinical outcome data were compared between the high target and the control target group by Mann–Whitney 2 target and the control target group. Demographic data were similar between both groups with respect to the number of patients, gestational age, gender, birthweight, prenatal steroids, and infant age at intubation . Nitrite TA levels were similar between infants assigned to the high target and control target groups from PD 2 to PD 14 . Furthermore, nitrite TA levels markedly decreased in both groups over the study period from PD 2 to PD 21 (p < 0.001).Mixed model ANOVA of target group and postnatal age yielded the p < 0.05). The different PCO2 target levels, however, did not affect IL-6 TA levels from PD 2 to PD 21. Similarly, IL-1β TA levels were not affected by the PCO2 target group . Furthermore, TGF-β1 TA levels increased with advancing postnatal age , but no difference in TGF-β1 TA levels was observed between the high target group and the control target group. IL-10 TA levels were neither affected by the PCO2 target group nor postnatal age, as no significant differences were detected during the study period .Postnatal age also affected IL-6 TA concentration since levels of both, the high target group and the control target group, significantly increased over the study period from PD 2 to PD 21 . However, the subsequent measurement on PD 21 showed no differences of IL-8 TA levels between both target groups. In addition, postnatal age did not affect IL-8 TA levels. In contrast to IL-8, postnatal age strongly affected MIP-1α TA levels, which significantly increased from PD 2 to PD 21 in both target groups . The PCO2 target group did not significantly alter MIP-1α TA levels, as no difference was observed between the groups. Similarly, LTB4 TA levels were not affected by PCO2 target levels from PD 2 to PD 21 .No differences in IL-8 TA levels between the target groups were observed on PD 2 to PD 7 Figure A, but on1 Figure C. Howeve2 target levels did not alter the ASM TA levels, as no significant difference was observed between the high target group and the control target group from PD2 to PD21 . Albumin TA levels were not affected by postnatal age or the target group from PD 2 to PD 21  and pH (high PCO2 target group: 7.23 ± 0.06 and control target group 7.25 ± 0.04) differed significantly between study groups, as intended in the study protocol , suggesting increased weaning efforts in the high target group , which acts by activating the cyclooxygenase pathway and ASM. More recently, ceramide and ASM, the enzyme synthesizing ceramide, were shown to be involved in lung edema formation induced by PAF   . Thus, ithe lung . More rethe lung –66. Hypethe lung , which ithe lung . Moreovethe lung . Anothere of BPD , further1, MIP-1α, NPY, LTB4, and ASM levels. The inflammatory mediators IL-6, IL1β, TGF-β1, MIP-1α, LTB4, and ASM increased with advancing postnatal age, while nitrite and NPY levels declined during the study period.Postnatal age of the infants affected almost every analyzed factor demonstrated by significant changes of TA nitrite, IL-6, IL1β, TGF-βSmall differences in TA cytokine levels may be missed in our study because of the small sample size. However, we limited the collection of TA to the largest center of all participating centers of the PHELBI trial to counteract inter-center variations such as differences of tracheal suctioning procedures and clinical care. Therefore, even a multicenter TA sampling study may not yield more precise results and more statistically significant differences. Furthermore, if there are clinically important differences in a larger sample size, unequivocal trends should already be observed in analyses of inflammatory mediators alike. Thus, we assume that pulmonary inflammatory activity was indeed similar in both study groups and important differences would not have become detectable with a larger sample size. Moreover, the biochemical results go along with the clinical outcome in this study, which indicates that high target levels were as beneficial as control target levels in terms of ventilator-induced lung injury, lung inflammation, and the development of chronic lung disease . In addiAccording to the definition, 12 of 62 infants suffered from BPD in our study, which were distributed statistical comparably in both treatment groups. Thus, similar cytokine levels in both groups were followed by a similar clinical outcome in the patients studied here as well as in the main multicenter trial , which mA limitation of this study is the declining number of infants supplying TA with advancing postnatal age, because infants are extubated as soon as clinically possible to limit potential lung damage from mechanical ventilation. Therefore, infants, who were more prone to develop BPD, remained longer in the study and supply more TA due to continuing invasive mechanical ventilation. Thus, after 14 or 21 days of life, the acquired TA may not be completely representative for the whole group, since TA was only available from infants who still received invasive mechanical ventilation at that time. Furthermore, normalization of TA to correct for dilution is still controversial. Different techniques have been proposed, including albumin content, urea, or secretory immunoglobulin A concentrations. However, no uniformly accepted correction factor is currently available. We did not correct our results for dilution and expressed the data per milliliter of TA, as recommended by the European Respiratory Task Force on Bronchoalveolar Lavage in children , 46.2 targets. In addition, there were no differences in the clinical outcome, which was in accordance with the results of the main multicenter PHELBI trial. We assume that higher PCO2 target ranges are as beneficial as the control target range. Indeed, high target levels may reduce mechanical stress for the pulmonary parenchyma, but possible suboptimal pH-values might impede enzymes, which may be further inhibited directly by high PCO2 concentrations. Overall, the positive effects of high target levels such as less bronchoalveolar damage may be abrogated by the negative effects. This corresponds to the results of the TA measurements.No differences in the levels of most cytokines were found when comparing infants with control or high PCOThis study was carried out in accordance with the recommendations of the International Conference of Harmonization Good Clinical Practice Guidelines and the protocol approved by the institutional review board of the university of Ulm medical faculty. All subjects gave written informed consent in accordance with the Declaration of Helsinki.SG: substantial contribution to acquisition of data, analysis and interpretation of data, as well as drafting the article and giving final approval to be submitted. ML: participated in revising the article critically for important intellectual content and has given final approval of the version to be submitted. UU, YY, and SU: substantial contribution to analysis of tracheal aspirates. HF and HH: made substantial contribution to conception and design of the trial. JD: substantial contribution to statistical analysis of data. UT: substantial contribution to acquisition of data, analysis and interpretation of data, as well as revising the article critically for important intellectual content and giving final approval to be submitted.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.


Candidatus Synechococcus spongiarum” of the marine sponge Aplysina aerophoba, which were collected from two nearby locations in the northern Adriatic Sea. The sequences provide the basis for within-clade comparisons between members of this widespread group of cyanobacterial sponge symbionts.We report here four draft genome sequences belonging to clade F of the cyanobacterium “ Synechococcus/Prochlorococcus clade represents the most dominant cyanobacterial clade in the world’s oceans, as it is responsible for ~20 to 40% of marine chlorophyll biomass and carbon fixation (Porifera) are important components of marine environments due to their immense filter-feeding capacities and consequent impacts upon coastal food webs and biogeochemical cycles. Their associated dense and diverse microbial communities have received considerable research attention, yet much remains unknown about their in situ ecological function(s) (Candidatus Synechococcus spongiarum” are widely distributed and highly abundant in sponges around the world and show diversity on the clade level correlating to host sponge phylogeny (The fixation . Spongesction(s) . The cyahylogeny . This muCa. Synechococcus spongiarum” from different sponge species and geographic locations were compared to each other and to free-living relatives, providing insight into the adaptations of the symbionts to their specific sponge host and to sponges in general  on 7 May 2013, and two were from specimens collected near Rovinj, Croatia  on 6 May 2013, from a water depth of ~5 to 7 m. As described for 15L, “Ca. Synechococcus spongiarum” cells were sorted from freshly prepared sponge-associated prokaryotes by fluorescence-activated cell sorting targeting their phycoerythrin and chlorophyll a autofluorescence (http://jgi.doe.gov), resulting in assemblies of about 0.5 Mb (10D and 10E) and 1.5 Mb (15M and 15N). Estimated genome completeness ranged from 21% (10E) to 80% (15M) according to JGI’s completeness estimation.Here, we present four draft genomes that were sequenced in parallel with the previously published “ clade F . Two of rescence  and autoCa. Synechococcus spongiarum” clade F members of the microbial symbiont consortium of A. aerophoba sampled at two geographic locations at the same time, providing insight into similarities and differences at the subclade level.This data set enables the within-clade comparison of “Ca. Synechococcus spongiarum” strains 10D, 10E, 15M, and 15N were deposited in DDBJ/ENA/GenBank under the accession numbers MWLG00000000, MWLF00000000, MWLD00000000, and MWLE00000000, respectively. The versions described in this paper are the first versions MWLG01000000, MWLF01000000, MWLD01000000, and MWLE01000000.The whole-genome shotgun sequences of “


Rosa hybrida commercial cultivars. In the second trial, aliquots of dried pollen grains of six R. hybrida cultivar were stored under two different temperatures (freezer at T = −20 °C and deep freezer at T = −80 °C) and after a wide range of conservation period, their viability was measured. In the third trial, the effective fertilization capacity of frozen pollen of 19 pollen donor cultivars was evaluated during 2015 crossing breeding plan, performing 44 hybridizations and correlating the number of seeds and the ratio seeds/crossing, obtained by each cultivar, with in vitro pollen germination ability.In the cut flower market, traditional breeding is still the best way to achieve new rose cultivars. The geographical delocalization of cultivar constitution  and plant cultivation (large areas in Africa and South America) represents a limit point for crossing and selection. Rose breeders often need to overcome geographical distances, resulting in asynchrony in flowering among crossing parents, by storing and sending pollen. Hence, a key aspect in breeding programs is linked to pollen availability and conservation, jointly with the identification of parameters related to pollen fertility. In this study we present the results of three different trials. In the first, pollen diameter and pollen viability were chosen as fertility predictors of 10  In the cut flower market, many modern rose cultivars are tetraploid 2n = 4x = 28) and they are produced by traditional genetic improvement methods . Fertility varies considerably among rose cultivars and has led breeders to greatly prefer the more fertile genotypes as parents . Rose brx = 28 anRosa hybrida L. commercial cultivars. Furthermore, an efficient protocol for pollen storage under low temperatures was applied to six cultivars in order to establish pollen preservation procedures useful for rose breeding and germplasm resource research. The effective fertilization capacity of the preserved pollen was studied in 2015 breeding plan of the company NIRP International  and the correlation between pollen viability and fertility was investigated.In the present study, pollen diameter and viability were related to fertility in Rosa hybrida “Alba”, “Aubade”, “Clipper”, “Encanto”, “Green Fashion”, “Golden Fashion”, “Marvelle”, “Peach Aubade”, “Variance”, and “Whisper” plants cultivated in the NIRP International greenhouse . Anthers were air dried for 24 h at standard conditions (T = 24 °C) to favour release and drying of the pollen [w/v, 0.01% H3BO3, 0.01% CaCl2, 0.02% MgSO4·7H2O, 0.01% KH3PO4 and 0.01% chloramphenicol was added to the glass slide and new measurements were performed after 5 min of hydration. Pollen diameter was measured digitally using software LAS software (Leica Application Suite). Since the abnormal pollen maintains irregular shape after hydration [Anthers were collected from e pollen . A totalRosa hybrida “Alba”, “Anastasia”, “Encanto”, “Marvelle”, “Swan” and “Touch of Class” were collected at bud starting blooming stage, from plants cultivated in the NIRP greenhouses. The pollen grains were obtained from a bulk of twelve flowers by gathering the anthers during November 2012. Anthers were air dried for 24 h at room temperature (T = 24 °C 50% humidity) to favour release and drying of the pollen, then they were weighted in aliquots, of 100 mg and stored in polyethylene tubes at T = −20 °C in the freezer and at T = −80 °C in the deep freezer  were stored in plastic boxes in the freezer (T = −20 °C) for a year. Pollen viability was tested in April 2015 by measuring the in vitro germination percentages of 160 samples for each cultivar on the artificial germination medium described above. Aliquots of the same pollen were used as donor in 44 hybridizations listed in Flowers of 19 p ≤ 0.05). Pearson correlation index was calculated between pollen viability (in vitro germination %), the number of seeds and the ratio seeds/crossing. All analyses were performed with SPSS 21.0 Inc. software .Arcsine transformation was performed on all percent incidence data before statistical analysis in order to improve homogeneity of variance. Effects of genotype and storage duration on the analysed traits were evaluated by one-way ANOVA using Ryan-Einot-Gabriel-Welsch’s multiple stepdown F (REGW-F) test  and globular with a diameter larger than 30 µm  . The freρ = 0.343 and ρ = 0.304) between stored pollen in vitro germination and in vivo fertility of the same pollen . A cultivar-dependent behaviour in fresh pollen germination efficiency was observed. The cultivars “Anastasia” and “Marvelle” showed very low in vitro germination rates , soon after flower collection (Day 0). Their pollen grains were not further used in the conservation trials. The best performing cultivars at Day 0 were “Alba” and “Encanto” with 57.1 ± 3.04 and 55.1 ± 1.34% pollen germination, respectively. After 44 days of storage at low temperatures, pollen germination was reduced in the cultivars “Swan” and “Touch of Class”, while no significant differences were observed in “Encanto”. After 134 days of storage pollen germination capacity was also reduced in the cultivar “Alba”. The cultivar “Encanto” with the highest frequency of normal pollen, increased pollen germination after 134 and 190 days of storage both in the freezer and in the deep freezer . Pollen e pollen .The performance of rose pollen has been studied by several authors ,4,12,13.Rosa hybrida cultivars showed diameter lower than 30 μm (“Clipper”) and low fertility could be associated also to low viability and germinability percentages.In Hybrid roses, a few reliable models for fertility prediction have been published. Pipino et al. 2011) reported on the correlation which associates pollen morphology with the number of seeds produced per hybridization. A diameter value of 30 μm was identified by the authors as the threshold between high and low pollen fertility 1 reporte. In thisAccording to our previous observations, the viable level of fresh rose pollen is cultivar dependent and related to the temperature of conservation . At roomOur in vivo hybridization results endorsed the positive correlation between stored pollen in vitro germination and in vivo fertility.Rosa hybrida commercial cultivars.The morphology (mean dry pollen diameter) and the frequency of normal pollen (30 µm) are effective as fertility predictors in Confirming the pioneer experiments of Calvino Mameli E. (1951) , rose po


The epithelial-mesenchymal transition (EMT) is a cellular reprogramming mechanism that is an underlying cause of cancer metastasis. Recent investigations have uncovered an intricate network of regulation involving the TGFβ Wnt, and Notch signaling pathways and small regulatory RNA species called microRNAs (miRNAs). The activity of a transcription factor vital to the maintenance of epithelial stemness, ?Np63a, has been shown to modulate the activity of these EMT pathways to either repress or promote EMT. Furthermore, ?Np63a is a known regulator of miRNA, including those directly involved in EMT. This review discusses the evidence of ?Np63a as a master regulator of EMT components and miRNA, highlighting the need for a deeper understanding of its role in EMT. This expanded knowledge may provide a basis for new developments in the diagnosis and treatment of metastatic cancer. Cancer cell metastasis is the leading cause of death from cancer, however, the cellular mechanisms of metastasis remain incompletely characterized . The proMany epithelial markers, including tight junction proteins such as zona occludens-1 (ZO-1), occludins, and the claudins, as well as the adherens junction protein epithelial cadherin (E-cadherin), are repressed during EMT . The mosAn additional transcription factor recently shown to inhibit EMT is p63, a member of the p53 family. The p63 gene encodes six primary isoforms through differential promoter usage and C-terminal splicing . The altΔNp63α has a vital role in the inhibition of EMT and promotion of the epithelial phenotype. ΔNp63α downregulates mesenchymal genes and simultaneously upregulates epithelial genes, particularly those involved in cell adhesion, such as Claudin1, and integrins involved with cellular adhesion to the extracellular matrix –22. ΔNp6Multiple signaling pathways, including Transforming Growth Factor β (TGFβ), Wnt, and Notch, are involved in the cellular regulation of TWIST, SNAIL, SLUG, and ZEB. The representative signaling cascades are shown in Figure The TGFβ pathway regulates multiple cellular processes, such as proliferation, differentiation, cytoskeletal rearrangements, metastasis, and apoptosis . SignaliSMAD proteins alone bind DNA weakly and, as a functional necessity, associate with other transcriptional factors to increase their binding affinity, including ΔNp63α Figure . SignaliExtracellular matrix-associated Wnt ligands bind to Frizzled and low-density lipoprotein receptor-related protein (LRP) receptors to initiate Wnt signaling. In the absence of Wnt ligand activation, β-catenin is phosphorylated by glycogen synthase kinase-3β (GSK3β), which is in complex with AXIN and adenomatosis polyposis coli (APC). This phosphorylation ultimately targets β-catenin for proteasomal degradation. Induction of Wnt signaling results in activation of Dishevelled (DVL), which recruits GSK3β and Axin to the plasma membrane and preventing GSK3β from phosphorylating β-catenin . This leThere are contradictory observations regarding the role of ΔNp63α within the Wnt pathway Figure . It has The Notch pathway is involved in cellular proliferation, differentiation, apoptosis, and survival . Notch sCells expressing ΔNp63α can induce Notch signaling on adjacent cells while simultaneously repressing this pathway within themselves. This is due in part to crosstalk between NOTCH and the PI3K-AKT pathway Figure . As mentIn conclusion, ΔNp63α is involved with components of signaling pathways found to induce EMT. Therefore, there is a clear connection established between ΔNp63α and the cellular regulation of EMT.miRNAs are small non-coding RNAs shown to target EMT transcription factors, as shown in Table ΔNp63α has been implicated in the direct transcriptional control of miRNA responsible for regulating many cellular processes, including EMT Tables  and 3 5. For exaTGFβ signaling is targeted by additional miRNAs, such as the Let-7 miRNA family in human pancreatic cells, and by miR-365 in lung adenocarcinoma , 65. TheSupporting a role for miRNA in epithelial integrity, expression levels of E-cadherin are positively correlated with those of miR-205 and the miR-200 miRNA family, which also target ZEB1/2 . The miRThe miR-34 family (henceforth referred to as miR-34) also represses EMT through inhibition of SNAIL1 and SLUG expression . FurtherExpression of several other miRNAs have also been found to correlate with ΔNp63α expression, and in silico analysis of these miRNAs identified p63 response elements in their promoters. These miRNAs include Let-7, miR-23, miR-29, miR-134, miR-145, miR-192, and miR-215 . In addiCertain miRNAs target transcripts of ZEB repressors, stabilizing ZEB expression. For example, miR-221 and miR-222 target the ZEB repressor TRPS1, resulting in E-cadherin downregulation to promote EMT . AdditioWith miRNAs clearly involved in dual regulation of EMT, miRNAs regulated by ΔNp63α also share this dichotomy. Interestingly, ΔNp63α has been found to directly downregulate miRNA involved in the suppression of EMT, such as miR-130b and miR-138. . The TGFAs there are a large number of transcripts involved in EMT regulated by miRNA, regulation of microprocessors such as DICER and DROSHA is integral to the procession of EMT. DICER may be a transcriptional target of p63, as expression of DICER has been found to be influenced by the expression of TAp63. The promoters of several microprocessor components, DROSHA, DGCR8, DICER Dicer, and TARBP2, have also been found through computational analysis to have multiple p63 response element sites, with DGCR8 experimentally confirmed to be induced by ΔNp63α , 97.ΔNp63α itself is a target of multiple miRNAs. miR-203 targets the 3’-UTR of ΔNp63α to decrease cellular proliferation, resulting in repression of epithelial stemness . The miRConversely, other miRNAs have either been demonstrated or have the potential to increase ΔNp63α expression. miR-145 is known to increase the expression of p63 through a mechanism yet to be identified . miRNAs Due to the role of EMT in the development of key characteristics of metastatic cancer, including increased motility and resistance to apoptosis, there is therapeutic value in researching regulators of EMT. In cancer, there is often a decrease in expression levels of epithelial biomarkers which lead to increased tumor invasiveness and metastasis. For example, a model of pancreatic β-cell carcinogenesis reveals a link with the loss of E-cadherin and a switch from noninvasive adenoma to invasive carcinoma . AdditiomiRNAs are increasingly being investigated as biomarkers for premalignant lesions . This isΔNp63α and ΔNp63α-regulated miRNAs have utility as predictive biomarkers that can help guide personalized treatment plans to aid in the prevention of metastatic cancer. miRNA expression profiles and miRNA biomarkers that correlate with cancerous and normal phenotypes are now routinely identified using Next Generation Sequencing. miRNA that regulate ΔNp63α in EMT may therefore serve as novel therapeutic targets for inhibiting metastasis, either via blockade of metastatic-inducing miRNAs through antagonistic mRNA-mimic complementary base pairing or through exogenous introduction of miRNAs that induce tumor-suppressing effects . FinallyThere is growing evidence for the involvement of in EMT in the development of metastatic cancers, with implications in breast, lung, prostate, bladder, and gastric cancers. Identifying the mechanisms by which EMT induces a motile and invasive cell phenotype is therefore valuable in combating the various types of malignant carcinoma.This review presents information from multiple studies detailing the diverse involvement of miRNAs and ΔNp63α in the development of EMT-mediated metastatic cancer. miRNAs have emerged as regulators of key EMT transcripts, whose expression patterns could indicate the genomic balance indicative of EMT. The transcription factor ΔNp63α has been shown to regulate multiple miRNAs in addition to cell signaling pathways, including TGFβ, Wnt, and Notch, involved in EMT, as described throughout this review. Since ΔNp63α expression and activity are altered by these signaling pathways to either promote or antagonize EMT, ΔNp63α-regulated miRNAs will also be altered in a similar manner. Understanding ΔNp63α-regulated miRNA profiles could therefore provide clues as to whether specific cancer types will become migratory and invasive. This highlights the importance of uncovering the regulatory network of miRNAs by ΔNp63α within the context of EMT.Principally, identification of ΔNp63α-regulated miRNAs as novel biomarkers may provide a powerful tool for the prediction of metastatic potential. Further, Next Generation Sequencing may facilitate generation of a miRNA profile of cells undergoing EMT. With further study, such profiling has the potential to create a more personalized patient prognosis and therapeutic development for treatment of high-risk invasive tumors.


The influence of genetic variation on these CSF biomarkers has been investigated in candidate or genome-wide association studies (GWAS). However, the investigation of statistically modest associations in GWAS in the context of biological networks is still an under-explored topic in AD studies. The main objective of this study is to gain further biological insights via the integration of statistical gene associations in AD with physical protein interaction networks.The cerebrospinal fluid (CSF) levels of total tau (t-tau) and Aβ1–42 ratio using quality controlled genotype data, including 563,980 single nucleotide polymorphisms (SNPs), with age, sex and diagnosis as covariates. Gene-level p-values were obtained by VEGAS2. Genes with p-value ≤ 0.05 were mapped on to a protein-protein interaction (PPI) network . We integrated a consensus model strategy into the iPINBPA network analysis framework, and named it as CM-iPINBPA. Four consensus modules (CMs) were discovered by CM-iPINBPA, and were functionally annotated using the pathway analysis tool Enrichr. The intersection of four CMs forms a common subnetwork of 29 genes, including those related to tau phosphorylation , amyloid beta production , and AD .The CSF and genotyping data of 843 study subjects  from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) were analyzed. PLINK was used to perform GWAS on the t-tau/AβThis study coupled a consensus module (CM) strategy with the iPINBPA network analysis framework, and applied it to the GWAS of CSF t-tau/Aβ1-42 ratio in an AD study. The genome-wide network analysis yielded 4 enriched CMs that share not only genes related to tau phosphorylation or amyloid beta production but also multiple genes enriching several KEGG pathways such as Alzheimer’s disease, colorectal cancer, gliomas, renal cell carcinoma, Huntington’s disease, and others. This study demonstrated that integration of gene-level associations with CMs could yield statistically significant findings to offer valuable biological insights  and suggest high confidence candidates for subsequent analyses. In addition, the total tau (t-tau) protein level has been shown significantly elevated in the CSF of AD patients. As a result, the CSF t-tau/Aβ1–42 ratio has also been studied as a biomarker for differentiating AD from normal older adults ) 1–42 ratio as the test phenotype or quantitative trait (QT), downloaded the PPI network from the Human Protein Reference Database (HPRD) (http://www.hprd.org/), and applied the iPINBPA analysis to the GWAS findings of the CSF t-tau/Aβ1–42 ratio. Our goal was to search for subnetworks or network modules enriched by the CSF GWAS findings, which may offer valuable biological insights and suggest high confidence candidates for subsequent analyses.With these observations, in this work, we performed a genome-wide network-based pathway analysis for CSF studies in AD. We analyzed an AD cohort from Alzheimer’s Disease Neuroimaging Initiative (ADNI), used the CSF biomarker t-tau/Aβp-values, which were then assigned to 22,179 genes. The gene assignment and gene-based p-values were calculated using the VEGAS2 software  for 1000 times was applied in iPINBPA [A is defined as:A random sampling of gene sets of size  iPINBPA  to deterA is the network score, and μk and σk are respectively the mean and the standard deviation of the background distribution of the network scores at size k.where Zp-values and high weights. Details about the algorithm is available in Wang et al. [In Step 3, a greedy algorithm was developed to search for modules with high network scores, i.e., those enriched in genes with low g et al. .In this work, the parameters were set in iPINBPA as follows: r = 0.5, T = 5, NetScore > 3.0, NetSize ≥ 5, and MaxNetSize ≤ 300. Given the stochastic nature of the iPINBPA algorithm, we ran iPINBPA ten times, respectively by setting the random seed value from 1 to 10. Consequently, we obtained ten groups of enriched subnetworks (GNs) identified by iPINBPA. Below, we couple a consensus module (CM) strategy with iPINBPA (named as CM-iPINBPA) to generate consensus findings from these analyses.m and n, we use Dice’s coefficient DC to measure their similarity:Given two subnetworks TNa be the top subnetwork identified in Run a ∈ {1, 2, …, 10}. We first find SNb(TNa), which is the most similar subnetwork to TNa in Run b ∈ {1, 2, …, 10}\{a}. Clearly we haveIn this work, we only focused on analyzing the top subnetwork (TN) in each iPINBPA run. Let sn is any subnetwork enriched in Run b. After that, we define the consensus module (CM) based on Run a as follows:where CMa = TNa ∩ ).CMa is the intersection of TNa and its most similar subnetworks identified in all the other runs.Namely, http://amp.pharm.mssm.edu/Enrichr/) and the Kyoto Encyclopedia of Genes and Genomes database  [Cytoscape 3.2  was usedp/kegg/)  was applp = 0.764) was not significantly different across the five diagnostic groups; however, gender demonstrated a significant difference (p = 0.002). Furthermore, as expected, age, APOE e4 status, clinical dementia rating–sum of boxes (CDR-SOB), mini mental status examination (MMSE), logical memory immediate recall, and logical memory delayed recall exhibited significant differences across the five groups (p < 0.001). Also as expected, the phenotype t-tau/Aβ1–42 ratio significantly differed across the diagnostic groups (p < 0.001).The demographic and clinical characteristics for the 843 ADNI subjects in this study are presented in Table APOC1 gene, p = 2.576E-28), which was previously reported by Lars Bertram et al. [APOE gene on chromosome 19 was also significant with p = 4.98E-23, and was previously reported by Soerensen et al. [TOMM40 SNP rs2075650  secretion in fibroblasts from AD patients [TP53 was a novel gene as a biomarker of AD and was related to neurodegenerative processes [Consensus modules (CMs) were identified by CM-iPINBPA network analysis strategy. Subnetwork search was conducted on the GWAS findings using iPINBPA ten times by varying the seed value of random number generator, which ranged from 1 to 10. Table patients , 44. Culrocesses .Table 3R2) is 0.96335. Thanks to the overlapping subnetworks, only four unique CMs were identified  shown in Fig. GSK3B, MAPK1, PSEN2, CALM1, CALM2 and CASP8. CM2  shown in Fig. BACE1, GSK3B, MAPK1, PSEN2, CALM1, CALM2 and CASP8. CM3  shown in Fig. GSK3B, CALM1 and CASP8. CM4  shown in Fig. BACE1, GSK3B, MAPK1, PSEN2, CALM1, CALM2 and CASP8.Table ed Table . These fCALM1, CASP8, and GSK3B, and 26 other genes.In addition, the intersection of the four CMs was extracted and named as the common subnetwork. The common subnetwork is shown in Fig. p-value ≤ 0.05. Then, these identified pathways of the four CMs were plotted as a heat map shown in Fig. To test the hypothesis that CMs enriched by the GWAS findings might be significantly overrepresented in AD and other relevant pathways, the Enrichr method was performed for pathway analysis. For the genes in each CM, a pathway enrichment analysis was conducted, and the nominally enriched pathways were identified with adjusted p-values were determined and shown in Fig. 1–42 ratio as a quantitative trait (QT) in this study enabled us to examine the effect of genes previously known to be associated with the QT as well as to identify novel genes. Table p-values. Given the Bonferroni-corrected threshold of p < 2.25 × 10−6 , we found five significant genes. As expected, significant associations were identified between loci on chromosome 19 and the CSF t-tau/Aβ1–42 ratio  genes code for proteins related to the clearance of Aβ and mitochondrial functions [PVRL2 gene was previously reported as related to risk factors that contribute to AD pathogenesis [PDK2 (p = 5 × 10−6) shows a trend towards the significance, and the overexpression of this gene may be related to cancer and diabetes[CCL7 mRNA is highly increased by Aβ1–42 stimulation [CCL7 gene was previously reported as related to the chemotaxis of pro-inflammatory cells to the inflamed location [PTGER3 gene was previously reported as being related to the inflammatory response [In the GWAS analysis, the gene-level unctions ; and theogenesis . PDK2  gene. GABRB1 codes for the β1 subunit of gamma-aminobutyric acid A (GABAA) receptors [GABRB1 gene has been demonstrated to be involved in the thalamus structure and its interactive effects on intelligence [GABRB1 gene had also been associated with many neuropsychological diseases, such as schizophrenia, major depression, bipolar disorder, and Alzheimer’s disease [The genes in the CMs might not show a direct statistical significance but could interact with some genes identified in our GWAS. These genes can demonstrate indirect association with the studied QT, and may potentially provide valuable biological interpretation. For example, Consensus Module 1 contains the protein gamma-aminobutyric acid (GABA) A receptor  were involved in the top twenty pathways. The gene PDGFRB has been confirmed as causative of primary familial brain calcifications (PFBC) [PIK3R1 has been shown to be involved in Alzheimer’s disease [CALM1 encodes for tau protein and regulates the subcellular localization and function of calmodulin in neurons [CFLAR suppresses death receptor-induced apoptosis and TCR activation, which induces cell death by inhibiting caspase-8 activation [RHOA was implicated in Aβ neurotoxicity, and the activation generates cytoskeletal changes [NGFR ligands play an important role in preventing fundamental tau-related pathologic mechanisms in Alzheimer’s disease [Table s (PFBC) . The gen disease . CALM1 e neurons . CFLAR stivation . RHOA wa changes . NGFR li disease . These pDue to the limited number of subjects available to us, we were only able to perform a discovery study in this work. When more data become available in the future, replication studies in independent cohorts are required to evaluate and validate the identified network modules in our study. In addition, in this work, we reported the results using the default parameter setting provided by the software tool and according to Lili et al. , except 1–42 ratio was performed, using a sample of 843 subjects from the ADNI database. To our knowledge, this is the first genome-wide network-based pathway study on the CSF biomarker t-tau/Aβ1–42 ratio in Alzheimer’s disease. Due to the stochastic nature of the iPINBPA method, we employed a consensus module (CM) strategy to run iPINBPA multiple times and aimed to identify CMs from these different runs. We identified 4 CMs. These CMs contain not only genes from KEGG AD pathways, including BACE1, GSK3B, MAPK1, PSEN2, CALM1, CALM2, CASP8, and SK3B; but also interesting genes with relevant biological functions such as GABRB1, MMP2, CDK17, and IGFBP3. In sum, besides confirming previous findings , this study has also suggested new susceptible genes, CMs and pathways underlying Alzheimer’s disease.Network-based methods form a new generation of enrichment analysis strategy, and they can overcome the limitation of traditional enrichment analysis where only a fixed set of pre-defined pathways are examined. In this study, a genome-wide network-based pathway analysis of the CSF biomarker of the t-tau/Aβ


Streptomyces is one of the largest resources for bioactive metabolites, particularly antibiotics. Antibiotic production and other metabolic processes are tightly regulated at the transcriptional level. Sigma (σ) factors are components of bacterial RNA polymerases that determine promoter specificity. In Streptomyces, σ factors also play essential roles in signal transduction and in regulatory networks, thereby assisting in their survival in complex environments. However, our current understanding of σ factors in Streptomyces is still limited. In this mini-review, we demonstrate the roles of Streptomyces σ factors, illustrating that these serve as linkers of different metabolic pathways. Further investigations on σ factors may improve our knowledge of Streptomyces physiology and benefit exploitation of Streptomyces resources.The gram-positive filamentous bacterium  Streptomyces is the largest genus of Actinobacteria and is characterized by complex morphological differentiation and the ability to produce valuable secondary metabolites  based on differential physical functions, phylogenic relationships, and possession of conserved domains σ1 (region 1.1), σ2 , σ3 (region 3.0–3.2), and σ4 (region 4.1–4.2)   of variable length located between regions 1.2 and 2.1 . NCR is involved in core interactions and promoter escape  is not only required for essential gene expression, but is also involved in morphological differentiation and secondary metabolism  and undecylprodigiosin (RED) by initiating the transcription of the pathway-specific regulatory genes actII-ORF4 and redD  is greater than other commonly used promoters in Streptomyces, the native promoters of the antibiotic biosynthesis genes or pathway-specific regulatory genes are replaced with hrdBp to improve antibiotic yield. For example, nikkomycin yield is significantly enhanced by introducing hrdBp-driven sanG, which encodes the pathway-specific activator of nikkomycin biosynthesis in S. ansochromogenes TH322 (Because σover 50% . In the es TH322 .Table 1)  . The Strnd σHrdD . The majHrdD is transcribed during growth, and the σHrdD-RNAP holoenzyme can initiate transcription of genes that are involved in antibiotic biosynthesis  and differentiation  in vitro, hrdD mutants do not exhibit changes in secondary metabolism and differentiation , which are referred to as alternative σ factors that are used to direct RNAP to genes involved in adaptive responses  and scnD (encodes a P450 monooxygenase). The deletion of scnC or scnD results in the accumulation of two natamycin analogs, 4,5-deepoxy-natamycinolide and 4,5-deepoxy-natamycin, respectively, but abolishes natamycin biosynthesis (whiG expression is regulated by bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP), an important second messenger that imparts global effects in Streptomyces (reviewed by whiG transcription in a (c-di-GMP)-dependent manner. At the vegetative growth stage, high concentrations of c-di-GMP lead to the formation of BldD-(c-di-GMP) complexes that bind to whiG promoter regions, repressing its transcription. When morphological differentiation starts, c-di-GMP concentrations decline, causing the release of BldD from the target DNA, thereby relieving transcriptional suppression of whiG , all belonging to the σB-like σ factors, which are homologs of Bacillus subtilis σB  and antibiotic biosynthetic genes   , 2011. Fynthesis . Guanosiynthesis . In S. cgulators . σB inflene relA .B responds to environmental stress signals and activates the stress-related regulon. First, σB controls osmotic defensive responses by initiating the transcription of genes that are involved in the synthesis and uptake of osmotic compatible solutes  biosynthesis and the expression of catalase  and σ4 domains . As group 4 σ factors usually correlate with extracytoplasmic stimuli, these are also called extracytoplasmic function (ECF) σ factors .Group 4 σ factors are the most divergent members of the σStreptomyces spp. One of the most extensively studied stress-responsive ECF σ factor is S. coelicolor σR. It is induced by oxidants and translation-inhibiting antibiotics and subsequently activates the oxidative damage reparatory system, ribosome-associated products, and transcriptional regulators  , translational (repressed by translation initiation factor IF3), and post-translational (degraded by Clp proteases) levels  . Additiog stress . These fregulons . The exp) levels . Taken tBldN, is required for aerial mycelium formation . The LuxR-family regulator MibR is responsible for the activation of mibABCDTUV, and the ECF σ factor σMibX initiates the transcription of mibJYZO, mibQ, mibR, mibXW, and mibEFHSN. Under normal conditions, the transcriptional activity of σMibX is sequestered by its cognate anti-σ factor MibW, abolishing microbisporicin production. Upon nutrient limitation, cellular ppGpp concentrations increase and subsequently activate mibR transcription in a σMibX-independent manner, followed by the expression of the mibABCDTUV operon, thereby inducing the biosynthesis of immature microbisporicin. Subsequently, the immature antibiotic inactivates MibW, leading to the release of σMibX, which in turn upregulates the expression of mature microbisporicin (Regulation of secondary metabolism by ECF σ factors is commonly identified among actinomycetes, and some pathway-specific ECF σ factors are located within the antibiotic biosynthesis gene cluster. σsporicin . Microbisporicin . This moStreptomyces, the only identified cluster-situated ECF σ factor is σAntA, which has been reported in S. albus. Unlike σMibX, no anti-σ factor regulating the transcriptional activity of σAntA has been reported to date (AntA expression and antimycin production diminish upon differentiation (AntA may be “switched off” by some specific regulatory mechanism that is coupled with differentiation, yet needs further investigation.In  to date . Interesntiation , suggestStreptomyces metabolism. However, compared to various other ECF σ factors in this particular bacterial species, researches investigations on ECF σ factors and the mechanism underlying their response to signals are limited.In summary, ECF σ factors play essential roles in responding to signals and modulate Streptomyces regulatory network.Further investigations on ECF σ factors, the corresponding signals, and the relationship between these signals and biological processes are necessary to enrich our understanding of the 70 family in Streptomyces, illustrating their function and underlying mechanisms.Transcriptional regulation is a crucial mechanism in modulating gene expression, and RNAP is the key element in transcription. Sigma factors are the only variable elements of holo-RNAP, indicating that the selection of a particular target gene is mainly dependent on σ factors. In most cases, unlike other transcriptional factors that upregulate or downregulate gene expression, σ factors act as “switches” that turn expression on or off. In this mini-review, we focused on the members of the σStreptomyces species undergo complex morphological differentiation and synthesize various secondary metabolites, which are governed by a complex regulatory network. As a crucial component of the transcriptional regulatory system, σ factors fine-tune gene expression and regulate metabolic fluxes. Thus, engineering of specific σ factors is a feasible method of generating high-producing antibiotic strains (Streptomyces σ factors should be conducted. strains , which rStreptomyces has a subtle signal sensing and transduction network that responds to extracellular and intracellular signals. Recent studies have revealed that the majority of antibiotic biosynthesis gene clusters in Streptomyces are not expressed, and these clusters are described as “silent.” These silent metabolites are not produced under normal conditions, suggesting that special signals are needed to activate them (reviewed by Streptomyces signal transduction system, thereby promoting antibiotic production and the discovery of novel secondary metabolites.DS, CL, and JZ conducted the literature study and wrote the draft manuscript. WL edited and revised 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.


Vicia faba genotypes, Aguadulce (AD), Luz d’Otonio (LO) and Reina Mora (RM) to water deficit. Multiple physiological and biochemical parameters were used to analyse the response of the three genotypes to two soil water contents (80% and 40% of field capacity). A significant lower decrease in shoot, root and nodule dry weight was observed for AD compared to LO and RM. The better growth performance of AD was correlated to higher carbon and nitrogen content than in LO and RM under water deficit. Leaf parameters such as relative water content, mass area, efficiency of photosystem II and chlorophyll and carotenoid content were significantly less affected in AD than in LO and RM. Significantly higher accumulation of proline was correlated to the higher performance of AD compared to LO and RM. Additionally, the better growth of AD genotype was related to an important mobilisation of antioxidant enzyme activities such as ascorbate peroxidase and catalase. Taken together, these results allow us to suggest that AD is a water deficit tolerant genotype compared to LO and RM. Our multiple physiological and biochemical analyses show that nitrogen content, leaf proline accumulation, reduced leaf hydrogen peroxide accumulation and leaf antioxidant enzymatic activities  are potential biological markers useful to screen for water deficit resistant Vicia faba genotypes.Climate change is increasingly impacting the water deficit over the world. Because of drought and the high pressure of the rising human population, water is becoming a scarce and expensive commodity, especially in developing countries. The identification of crops presenting a higher acclimation to drought stress is thus an important objective in agriculture. The present investigation aimed to assess the adaptation of three  Due to global warming and the rising human population, water availability is becoming an increasing issue for agriculture. One of the main challenges of modern agriculture is to maintain growth and crop productivity under adverse environmental stress conditions such as water deficit. Water deficit impairs plant development leading to consequent loss of crop yield. It has been reported that more than 50% of the average yield of most major crops is lost due to drought stress . Under t2 assimilation rates due to reduced stomatal conductance [When plants grow in a water-limited environment, they undergo physiological, and biochemical modifications considered as water deficiency adaptation mechanisms . Growth ductance . Total cductance ,7. Also,ductance ,10.Plants have evolved many mechanisms for drought stress tolerance including a number of physiological and biochemical processes such as maintenance of water-use efficiency, osmotic adjustment and protection of the cellular machinery ,12,13. OVicia faba L.) is considered as one of the most extensively grown grain legumes cultivated in diverse environments [Faba bean  suspension at 108 bacteria per mL. The experiments were conducted in a greenhouse (approximate temperature of 26/20°C (day/night), 50–80% of relative humidity and 16 h photoperiod) at the Faculty of Sciences and Techniques, Marrakesh . Water deficit was initiated when seedlings were almost 20 day-old, attaining 2–3 true leaves and it was applied by maintaining the soil moisture at 40% of substrate field capacity (FC) for the stressed plants, and 80% FC for the well-watered plants. The experimental plant pots were arranged in a simple randomized design with twelve replicates per treatment per genotype. The nutrient solution applied once a week was composed as follows: MgSO4 (100 μmol.L-1), K2SO4 (750 μmol.L-1), KH2PO4 (125 μmol.L-1), CaCl2 (1650 μmol.L-1), Sequestrene (16 μmol.L-1), MnSO4 (6 μmol.L-1), H3BO3 (4 μmol.L-1), ZnSO4 (1 μmol.L-1), NaMoO4 (0.1 μmol.L-1), and CuSO4 (1 μmol.L-1). At plant flowering stage, about 50–60 day-old plants were harvested for growth assessment, and physiological and biochemical analyses.Three faba bean genotypes, Aguadulce (AD), Luz d’Otono (LO) and Reina Mora (RM), frequently grown at the Haouz region of Morocco, were used in this study. Before sowing, seeds of all genotypes were surface sterilized with 2.5% sodium hypochlorite for 20 min, then were washed with sterile deionized water and left to germination in sterile sand at 23°C for five days. Seedlings with homogenous stages of development were selected and put in pots filled with a mixture of sterilised sand and peat (4:1). Then, they were inoculated with a Shoot dry weight (SDW), root dry weight (RDW) and nodule dry weight (NDW) were measured for the three faba bean genotypes. Once harvested, shoots, roots and detached nodules were washed and dried at 70°C for 48 h. Three replicates (six plants for each treatment per genotype) were performed. For nitrogen (N) and carbon (C) analyses, samples  from six plants were considered as three replicates and were analysed using a CHN Elemental Analyser .The relative water content (RWC) was determined on leaf discs of 1 cm diameter from fully expanded third leaves and calculated as:http://rsb.info.nih.gov/ij/index.html). Three replicates (six plants for each treatment per genotype) were performed.Where FW is the fresh weight, TW is the turgid weight after water saturation of the discs at 4°C and DW is the dry weight after samples drying at 75°C until obtaining a constant weight . The leaMaximum quantum efficiency of Photosystem II (PSII) (Fv/Fm) in individual mature leaves was analysed using a fluorescence meter  according to the method of Jifon and Syvertsen . After 3-1 g-1 DW. Total chlorophyll was determined using the following formula:Photosynthetic pigments were measured as described by Takele . ChloropFour replicates per treatment were performed.-1 FW. Three replicates per treatment were performed.After 35 days of water stress, plants were harvested and samples from fully expanded third leaves, roots and nodules were collected for osmoprotectant content measurements. Free proline was extracted from 0.2 g of fresh plant material samples in aqueous sulphosalicylic acid  and estimated by using ninhydrin reagent according to the method of Bates et al. . The abs2SO4. Five hundred microliter were collected and cooled in ice for 1 h, before adding 200 μL of cold KI-I2 reagent. The tubes were stored at 4°C for 16 h and then centrifuged at 10 000 rpm for 15 min at 4°C. The supernatant was removed. Periodite crystals were dissolved in 4 mL of 1,2-dichloroethane. After 2 h, the absorbance was measured at 365 nm. GB concentration was determined using calibration curves and expressed as nmol glycine betaine g-1 DW. Three replicates per treatment per genotype were performed.Glycine betaine (GB) was determined by the method described previously by Grieve and Grattan  with som-1 cm-1. Three replicates per treatment per genotype were performed.After 35 days of water stress, plants were harvested and samples from fully expanded third leaves, roots and nodules were collected for oxidative stress indicator measurements. Lipid peroxidation was determined by estimating the malonyldialdehyde (MDA) content in 0.2 g leaf fresh weight according to Madhava Rao and Sresty . MDA is 2O2) was measured as described by Velikova et al. [2O2 content was calculated by comparison with a standard calibration curve plotted using known concentrations of H2O2. Each treatment included three replicates.Hydrogen peroxide  using a chilled mortar and pestle. The homogenate was then centrifuged at 12 000xg for 20 min at 4°C. Supernatant was used for enzyme activity and protein content assays. All extracts were handled at 4°C. Total protein content of the enzyme extracts was determined following Bradford [2O2 using enzyme reaction solution containing 25 mM phosphate buffer (pH 7.0), 9 mM guaiacol, 10 mM H2O2 and enzyme extract. The increase of absorbance due to formation of tetraguaiacol was assessed at 470 nm [2O2 and 0.5 mM sodium ascorbate were used in the reaction solution. Catalase (EC 1.11.1.6) activity was assayed according to Aebi [2O2 consumption [2O2 were used in the reaction solution. Polyphenol oxidase (EC 1.14.18.1) activity was determined according to the protocol described by [Peroxidase (EC 1.11.1.7) activity was determined according to Fielding and Hall  by the ot 470 nm . The actt 470 nm , by meas to Aebi . The decsumption . In thisribed by . The reaThe statistical analysis was performed using SPSS Ver.10 software  and a Two-way analysis of variance (ANOVA II). The data were expressed as the mean ± standard error. Means were statistically compared using Student-Newman-Keuls’s multiple-range test at the level of p < 0.05.Growth performances of three different faba bean genotypes were analysed under water deficit. The most common symptom of dehydration injury is the inhibition of growth, which is reflected in a reduction in the dry matter yield ,43. The The carbon-to-nitrogen ratio (C:N ratio) is an important parameter associated with optimal growth and plant development ,45,46. WTaken together, these results showed that the C:N ratios were significantly reduced in all the considered organs. However, the increased N content in roots and shoots of “AD” under water deficit is a striking difference with “LO” and “RM” genotypes.The effect of water deficit on faba bean was analysed based on multiple leaf physiological markers . RelativProline and glycine betaine (GB) are known osmolytes produced by plants under drought stress. We measured the content of these two molecules in leaves, roots and nodules . Proline2O2). We measured the content of MDA and H2O2 in leaves, roots and nodules  and ROS production such as hydrogen peroxide , guaiacol peroxidase (GaPX), catalases (CAT) and polyphenol oxidases (PPO) were measured as parameters of antioxidant defence. APX and GaPX activities were significantly higher in roots and nodules than in leaves of the three genotypes under control conditions Figs  and 7. BIn contrast to APX and GaPX activities, CAT and PPO activities did not show a clear general induction pattern associated to the better growth performance of “AD” Figs  and 9. N2O2 content observed in leaves and nodules of “AD” compared to the two other genotypes is correlated to the increased antioxidant activities detected in the “AD” genotype.All results considered, the low increase of MDA and HV. faba genotypes under mild water deficit conditions and to identify reliable biological markers correlating plant adaptation to water deficit. Our results showed that water deficit significantly decreased the plant growth of the three V. faba genotypes. However, “AD” presented a significantly higher leaf and root DW than “LO” and “RM”, showing that amongst the most common genotypes used in Morocco, “AD” is the one that presents the better growth under water deficit conditions  showed that the “AD” genotype presented the lowest alteration of these five parameters compared to “LO” and “RM”. Thus, these results support the idea that “AD” is less affected than “LO” and “RM” under water deficit. Amongst these markers, LMA which is not significantly different in control and stressed “AD”, and total chlorophyll which is not highly reduced under water deficit in “AD” compared to “LO” and “RM” appear to be two robust markers of V. faba, the four-fold higher accumulation of proline in “AD” compared to the other genotypes indicate that proline content is a good marker for plant resistance to water deficit. A similar conclusion was suggested from a recent study in field experiments [Proline and glycine betaine (GB) are potent osmoprotectants accumulated under water deficit . Prolineeriments .2O2 under water deficit. However, significant lower accumulation of H2O2 in all the organs and of MDA in shoots was detected in “AD” compared to “LO” and “RM”. These results suggest that oxidative stress is less marked in “AD” than in the two other genotypes. Interestingly, H2O2 accumulation was similar in leaves of “AD” in control and water limited conditions (2O2 accumulation in leaves should allow resistant genotypes to be differentiated from sensitive ones.Analysis of the different organs showed accumulation of MDA and Hnditions . In the 2O2 correlated with the better water deficit adaptation suggests that detoxification mechanisms could be more efficient in the “AD” genotype than in the two other genotypes. The induction of the cellular antioxidant machinery plays a crucial role for protection against drought-induced oxidative stress. In our experiments, APX activity increased significantly in leaves, roots and nodules of “AD” under water deficit. This increase was not observed in the two other genotypes which grew less well under water deficiency (V. faba (The lower level of Hficiency . In a re(V. faba . The betMedicago truncatula plants [Nitrogen content was more important in “AD” than in “LO” and “RM” suggesting that the biological nitrogen fixation is more efficient in “AD” than in “LO” and “RM”. Analyses of osmolyte accumulation showed that both proline and glycine betaine accumulate more in AD nodules than in “LO” and “RM” nodules. Increased osmolyte accumulation is correlated to an enhanced tolerance to drought stress ,58 and ba plants . In our a plants ,56, evena plants . In addia plants 2O2 content. The higher catalase activity in root nodules may be associated to both symbiotic partners and it may play a significant role in nitrogen fixation efficiency [V. faba genotypes tolerant to mild water stress conditions.A common feature of nodule drought stress is a decreased catalase activity in nodule ,61,62,63ficiency . Thus, nV. faba genotypes in future breeding programs.Based on this research, we suggest that the “AD” genotype presents a higher tolerance to water deficiency than “LO” and “RM”. Thereby, the “AD” genotype may be considered as an interesting candidate to be used in crosses for tolerance breeding. In parallel, our study shows that various parameters are differentially altered in the genotypes depending on their tolerance levels. Both “LO” and “RM”, two genotypes with a lower resistance to water deficit, show a similar behavior which is different from that of “AD”. The different biological markers that show the most differentiated profiles will be helpful to screen for water deficit tolerant


Previous studies have addressed the involvement of phosphoinositide-specifc phospholipase γ1 (PLCγ1) and protein kinase B (PKB/Akt) in osteoarthritis (OA) pathogenesis, but it is not ascertained the possibility of them to be potential targets for OA therapy. Here, through local intra-articular injection of PLCγ or Akt inhibitor in a rat OA model induced by anterior cruciate ligament transaction plus medial meniscus resection, the architecture of chondrocyte and matrix organization of articular cartilage were observed using histopathological assays and Aggrecan, Col2, PLCγ1, and Akt levels were detected using immunohistochemistry assays. By treatment of Akt or PLCγ inhibitor and transfection of different PLCγ1- or Akt-expressing vectors in rat OA model chondrocytes, Aggrecan, Col2, PLCγ1, p-PLCγ1, Akt, and p-Akt levels were detected using western blotting analysis. The binding between PLCγ1 and Akt was assessed with co-immunoprecipitation assays in human OA chondrocytes. These results showed that PLCγ inhibition protected chondrocytes against OA, but Akt inhibition did not dramatically aggravate OA progression. There were mutual antagonism and binding between PLCγ1 and Akt that could be regulated by their phosphorylation levels. Consequently, the data reveal that the inhibition of PLCγ1 may provide an attractive therapeutic target for OA therapy, implicating its binding to Akt. Osteoarthritis (OA) is a chronic and degenerative disease of articular cartilage, mainly characterized by chondrocyte death and articular cartilage matrix degradation in OA pathogenesis. Thus, either suppressing matrix degradation or promoting matrix synthesis could mitigate or prevent OA degeneration. Some signal molecules, such as p38, JNK and ERK MAP kinases, and PI-3 kinase/Akt, could regulate the expression levels of Aggrecan(a proteoglycan) and type II collagen Col 2), which are main components in articular cartilage matrix, to suppress or promote matrix synthesis . Recent , which aPhosphoinositide-specifc phospholipase γ (PLCγ), including PLCγ1 and PLCγ2, could be activated by many extracellular factors to induce hydrolysis of phosphatidylinositol 4,5-bisphosphateP2) to form the second messages diacylglycerol(DAG) and inositol 1,4,5-trisphosphate(IP3). Both DAG and IP3 in turn activate a number of signaling pathways to regulate cell metabolism in mammalian cells . PLCγ1 iAlthough most signal molecules have been examined in isolation, it is becoming clear that there is a significant crosstalk among them and the overall effects on OA pathogenesis depend on the balance in activity of them . InvestiIn this study, it is investigated the role of PLCγ and Akt and the relationship between them in OA chondrocytes. We showed that local intra-articular injection of PLCγ inhibitor could protect chondrocytes against OA in a rat OA model, whereas Akt inhibitor could not dramatically aggravate OA progression. There were mutual antagonism and binding between PLCγ1 and Akt that could be regulated by their phosphorylation levels. Therefore, the inhibition of PLCγ1 may serve as a better solution for OA therapy than Akt activation, partially, implicating its binding to Akt.*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, vs NS group). Nevertheless, the architecture of chondrocyte and matrix of articular cartilage in TCN-treated groups at different concentrations were similar to that in NS group along with the decreased thickness of articular cartilage and increased OARSI scores . Although TCN (10 μg/kg) caused a significant decrease at the grade of OARSI scoring system compared with NS group , the other two concentration of TCN did not change the thickness of articular cartilage and OARSI scores compared with NS group. Taken the data together, U73122 treatment for either 1 or 2 month had a significant chondroprotective effect on articular cartilage in a rat OA model, but TCN had not any intensifying effect on OA cartilage degeneration.Given the number of signal cascades triggered by activated Akt, which might make it difficult to understand its regulatory mechanism, the effect of Akt on articular cartilage was detected with the treatment of its pharmaceutical inhibitor Triciribine (TCN) in this study. Using a rat OA model induced by ACLT+MMx, we investigated the effect of PLCγ1 and Akt inhibition on articular cartilage with local intra-articular injection of its pharmaceutical inhibitor U73122 (U) and TCN. Rats were killed after 1 and 2 month-injection with U73122 or TCN, respectively, the specimens were then fixed, sectioned into 3 μm thicknesses, and stained with H.E. and Safranin O –Fast green stainings. The results after injection for 1 and 2 month were shown in Figures ***P < 0.001). In 2 month-treated groups, the expression level of Aggrecan in U73122-treated groups at different concentrations was close to that in normal group . In 2 month-treated groups, the expression level of Col 2 in U73122 (10 μg/kg)-treated group was close to that in normal group, whereas Col 2 expressions in the other two groups of U73122 were less than that in normal group, indicating that the efficacy of U73122 (10 μg/kg) treatment was superior to that of the two other groups . Therefore, treatment of both U73122  and U73122  could enhance the expression of Col 2 reaching normal level, and that the efficacy of U73122 may partially depend on the time and concentration of its treatment. Consequently, definite concentration PLCγ1 inhibitors promoted matrix synthesis through increasing Aggrecan and Col 2 expression levels in a rat OA model.Aggrecan and Collagen Type 2(Col 2) are main biomarkers of cartilage matrix synthesis, the effect of PLCγ1 inhibition on them was then assessed with immunohistochemistry assay in a rat OA model. Considering the heavy cartilage degeneration in NS group, the difference between U73122-treated and normal groups was analysed. In 1 month-treated group, Figure p Figure . These r***P < 0.001, ****P < 0.0001). Meanwhile, the treatment U73122 at different concentrations for either 1 or 2 month enhanced Akt expression level, close to that in normal group, indicating that U73122 also promoted Akt expression reaching normal level. Therefore, there was a reciprocal modulation between PLCγ1 and Akt, a possible mutual antagonism in chondrocytes of a rat OA model. Moreover, compared with Figures To ascertain whether there is a crosstalk between PLCγ1 and Akt in OA pathogenesis, the expression levels of PLCγ1 or Akt were assessed with immunohistochemistry assay in PLCγ1 and Akt inhibitor-treated groups, respectively. Either 1 or 2 month-treated groups, PLCγ1 had significantly higher expression in different concentration TCN-treated groups than that in normal group, indicating that TCN treatment enhanced PLCγ1 expression level  treatment. Given that both PLCγ1 and Akt, their phosphorylations are attributed to their regulatory effects on cell processes, the phosphorylation levels of PLCγ1 at Y783 site (p- PLCγ1) and Akt at S473 site (p-Akt) were measured with western blotting analysis. Figure Given that targeting molecule therapy is thought to be an effective solution for OA therapy –4, and tPLCγ1 has been detected at elevated level in human OA cartilage, implicated as a possible contributor to cartilage degeneration , 14. Theet al. study that Akt binds to and phosphorylates PLCγ1 in response to epidermal growth factor [In agreement with Wang Y h factor , there wIn conclusions, PLCγ inhibition by its pharmaceutical inhibitor or PLCγ1/Y783A mutant could protect chondrocytes from OA degeneration in both a rat OA model and human OA chondrocytes, and that there is a mutual antagonism between PLCγ1 and Akt in a rat OA model , Akt, p-Akt (S473), Aggrecan, Col 2, and β-actin were purchased from Cell Signalling Technology, Inc.  and Santa Cruz Biotechnology , respectively. Unless otherwise specified, inhibitors U73122 (U), Triciribine (TCN), and the rest of the reagents were purchased from Sigma-Aldrich .n = 4), such as normal, OA, NS, U73122 (U) , and Triciribine (TCN) . The rat experimental model of OA was induced with anterior cruciate ligament transection in combination with resection of medial menisci (ACLT + MMx) as previously described [The protocol was approved by the Committee on the Ethics of Animal Experiments of the University of Xiamen (ID no.20140306). 9-week-old male Sprague–Dawley rats (250–300 g) were acclimatized to the laboratory environment for 1 week before the experiments and were randomly divided into two parts, 1 and 2 months. Each part included nine groups , the sections were incubated overnight at 4°C with primary antibody: Col II (1: 1000), Aggrecan (1:800), PLCγ1 (1:200), and Akt (1:200) dilutions, respectively, prior to incubation with secondary antibodies. Diaminobenzidine (DAB) was then used to visualize the immunohistochemical reaction followed by being counterstained with haematoxylin. Finally, dark brown cells were considered to be positive. Photomicrographs were taken with OLYMPUS BX41 microscope equipped with a digital camera. The percentage of positive chondrocytes was measured and analyzed using Image-Pro Plus 6.0 Software and GraphPad Prism version 5, representing relative protein expression level , 23.Neonatal male Sprague–Dawley rats (within 24-72 hrs after birth) were killed after approval of the ethical Committee of Medical School, Xiamen University (ID No. 20110920). Articular cartilages of rat hint limbs were removed under sterile conditions and chondrocytes were then cultured as previously described , 18, 26.In addition, after receiving all patient consent and in accordance with the hospital ethical guidelines approved by the Ethics Committee of Zhongshan Hospital, Xiamen University ID No. 20100426), China, human OA cartilage was obtained from 79 patients  with advanced OA who were undergoing total knee replacement surgery  and transferred to PVDF membranes  for western blotting analysis as described in previous studies , 29. TheAs described previously , 30, prot-test with GraphPad Prism version 5 . A value of P < 0.05 was considered as being significant.All data were expressed as the means ± S.E.M. for three or five independent experiments for each group. The differences between the groups were examined for statistical significance using the unpaired


A copula is a function which joins (or ‘couples’) a bivariate distribution function to its marginal  distribution functions. In this paper, we obtain Chebyshev type inequalities by utilising copulas. C is a function  Property (C2) is referred to as the 2-increasing property, or moderate growth [C: (C4)C is nondecreasing in each variable;(C5)C satisfies the Lipschitz condition: for all  For further reading on copulas, we refer the readers to [A copula is a function which joins (or ‘couples’) a bivariate distribution function to its marginal  distribution functions. Mathematically defined, a copula mentclass2pt{minimaders to .t-norms join membership functions of fuzzy sets, and hence combining probabilistic information and combining fuzzy information are not so different [t-norm T is a function Commutativity: Associativity: Monotonicity: Boundary condition: While copulas join probability distributions, ifferent . Mathemaoperties : Commutat-norm if and only if it is associative; conversely, a t-norm is a copula if and only if it is 1-Lipschitz [t-norms, namely the minimum operator Iff, gare synchronous onandwherethenFis Chebyshevian onConsequently, the following Chebyshev type inequalities can be stated Letbe integrable and nonnegative onLetIfFis Chebyshevian onthenfor allLetbe integrable onIffandgare synchronous onthen we have Chebyshev’s inequalityfor allf defined on a measure space ω on Ω with If mentclass2pt{minimThe 2-increasing property of copulas gives us the following result.Letbe a copula andbe an integrable function. ThenCis Chebyshevian onIfpis nonnegative, thenProof follows by  and PropNow we state a more general form of this inequality. We start with the following lemma.Letbe two synchronous functions andCbe a copula. Thendefined byis Chebyshevian onf and g are synchronous, they have the same monotonicity on A be the collection of subsets of f and g are both nondecreasing. Suppose that f and g are nondecreasing. Therefore, the 2-increasing property of C givesf and g are decreasing. Therefore, the 2-increasing property of C givesF is Chebyshevian in both cases. □Since Lemma LetCbe a copula, be two synchronous functions, be an integrable function. Ifpis nonnegative, thenW copula) and the rest follows from the Fréchet-Hoeffding bound (M copula), and the rest follows from the Fréchet-Hoeffding bounds (In this example, we obtain some Chebyshev type inequalities by choosing some examples of copulas. Let mentclass2pt{minimmentclass2pt{minimng bound . Similarmentclass2pt{minimg bounds .In what follows, we generalise Theorem Letbe a measure space, be a measurable function, andCbe a copula. Thendefined byis Chebyshevian onWe also have, for a nonnegative integrable functionF follows from the 2-increasing property of copulas. Therefore, we have The Chebyshevian property of In this example, we obtain some Chebyshev type inequalities by choosing some examples of copulas. Let We also have the following result.Letbe a measure space, be a measurable function. Letωbe a positive weight on Ω withLetbe a copula. We have the following inequalities:The 2-increasing property of copulas gives usIn the next section, we provide further inequalities of this type.μ-integrable with μ-measurable and We denote the following:f defined on a measure space ω on Ω with We denote by Letbe a measure space, be measurable functions. Letωbe a positive weight on Ω withLetbe a copula. We have the following inequalities:Firstly, we haveLetbe a measure space, be measurable functions. Letωbe a positive weight on Ω withLetbe a copula. Iffandgsatisfythen we have the inequalitiesf with the property that The proof follows from Theorem Recall the notationLetbeμ-integrable withLetbeμ-measurable andIfis a copula, thenIn particular, we haveWe also haveIn particular,μ-ω-integrable functions k and l, we haveWe know that for any g bounds , we obtas proves . We obtas proves  by settit}f≡g in .From , we also0}dμ. By . Finally0}dμ. By  by settit}f≡g in . □Ifis a copula, then we havefor anyUsing the Fréchet-Hoeffding bounds  and the Consider the quantitiesBy Schwarz’s inequality, we also haveWe have the following result.Letbeμ-integrable withLetbeμ-measurable and such thatIfis a copula, then (with the notation in Theoremwe haveIn particular, we haveWe also haveIn particular, we haveFrom Lemma We denote the following quantities:Let  Then by  we get\Take Some particular instances of interest:


I , doublets (gene pairs) and MIC based on the maximal information coefficient. It is unclear whether dendrogram-based I and doublets can capture synergies efficiently. Although MIC can capture a wide range of interaction, it has a high computational cost triggered by its 3-D search. In this paper, we developed a simple and fast approach based on abs conversion type (i.e. Z = |X1 − X2|) and t-test, to detect interactions in simulation and real-world datasets. Our results showed that dendrogram-based I and doublets are helpless for discovering pair-wise gene interactions, our approach can discover typical pair-wise synergic genes efficiently. These synergic genes can reach comparable accuracy to the individually discriminant genes using the same number of genes. Classifier cannot learn well if synergic genes have not been converted properly. Combining individually discriminant and synergic genes can improve the prediction performance.Selecting informative genes, including individually discriminant genes and synergic genes, from expression data has been useful for medical diagnosis and prognosis. Detecting synergic genes is more difficult than selecting individually discriminant genes. Several efforts have recently been made to detect gene-gene synergies, such as dendrogram-based  However, they cannot provide insights into the interaction of these genes. According to information theory, the pair-wise interactions I 3 is defined asI is the symbol for mutual information, I  is the individual effect of gene X1 relative to phenotype Y, I  is the individual effect of gene X2 relative to Y, and I  is the joint effect of X1 and X2 relative to Y. A positive value of I  indicates synergy, while a negative value of I  indicates redundancy.Selection of informative genes, including individually discriminant genes and synergic genes, from expression data has been useful for medical diagnosis and prognosis. Individual gene ranking techniques such as et al.4 4 or uses the maximal information coefficient directly to measure MIC5. The way to discretize continuous variable is the key to estimate the value of mutual information. Binarization, such as the dendrogram-based4 technique, simplifies the estimation, and provides simple logical functions in the connection of the genes. However, it may result in information loss and estimation error. Although MIC5 can capture a wide range of interactions, it has a high computational cost triggered by its 3-D search. The other is the conversion strategy, such as doublets6 and top scoring pair (TSP)7. They employ a new variable Z derived from the combinations between X1 and X2  to measure I  instead of I. This strategy is low computational cost, due to the search space reduced from 3-D to 2-D. However, it is unclear whether this conversion strategy can capture synergies8 efficiently.Detecting synergic genes is more difficult than selecting individually discriminant genes. Several efforts have recently been made to detect gene–gene synergies. These efforts often fall into one of the two strategies. One is the non-conversion strategy, which uses formula (1) directly to measure Y = |X1 − X2|. The only difference between them is the value ranges of independent variables. Although Doublets6 included sum, diff, mul and sign conversion types (TSP is similar to sign), it, unfortunately, ignored abs conversion type.Inspecting Fig. abs conversion type and t-test, to discover pair-wise synergic genes that are related to cancer. Furthermore, we validated these synergic genes by using classification performance with simulation and real-world datasets. Our results show that these synergic genes can enhance the individually discriminant model and improve the prediction performance. We also demonstrated that these synergic genes should be converted into new variables (Z) prior to be used as input features for classifiers, especially for many pairs of synergistic genes.In this work, we developed a simple and fast approach based on 9.Four binary class datasets are involved in this work. The reference, sample size, number of genes in each dataset, and the number of samples in each class are summarized in Table n samples and m genes, and can be denoted as {Yi, Xij}, i = 1,2,…,n; j = 1,2,…,m. Xij represents the expression value of the jth gene (Gj) in the ith sample; and Yi represents the class label of ith sample. Yi ∈ {0, 1}, 0 denotes cancerous and 1 denotes normal tissue samples. Rank-based methods7 are robust to quantization effects and to overcome background differences between gene pairs. Therefore, let Rij denote the rank of the ith sample in the jth gene, we replace the expression values Xij by their ranks Rij and get a new data matrix {Yi, Rij}.Suppose that a dataset has Gp and Gq, Doublets6 lists four conversion types.For two genes i = 1,2,…,n; p = 1,2,…, m; q = 1,2,…, m; p ≠ q; s = 1,2,…, m(m−1)/2. Again, we get a new data matrix {Yi, Zis}. For each converted feature Zs, we use the t-score, instead of I , to rank the association between Z and Y, since Y ∈ {0, 1}.We add a new conversion type:t- score.The individually discriminant genes are also ranked by t- score based on all samples. The Top N gene pairs and/or the Top N individually discriminant genes are selected as input features. Support Vector Machine (SVM) Classifier is available at http://www.csie.ntu.edu.tw/~cjlin/libsvm/10. We simply use the average accuracy of five-fold cross-validation (CV) to evaluate the classifier performance as the datasets involved in this paper have balanced numbers of positive and negative samples.Each gene pairs and each individually discriminant genes are ranked by abs conversion type and six reference methods in Prostate1 dataset11. In Fig. abs conversion type and MIC are different, they share the same pattern: each individual gene is unrelated to cancer by individual gene evaluation, but the pair-wise genes are sufficient to distinguish the cancer from normal samples. Figure sum, diff, mul, sign and dendrogram-based I methods. As an example  are related with cancer directly. However, they are individually discriminant rather than synergic genes. In a word, only abs conversion type and MIC can capture typical pair-wise synergies, dendrogram-based I and doublets are helpless for discovering pair-wise gene interactions.Figure ple Fig. , the higInd, Sum, Diff, Mul, Sign and Abs methods  selected by ype Fig.  and diffype Fig.  can distabs conversion type have any biological relevance to cancer, we further validated the top10 gene pairs (16 genes) according to UniHI12 database (http://www.unihi.org/) and PubMed (Table PARP1-HMGB1 and CCHCR1-GRAP) that are associated with interaction in UniHI. The interaction between PARP1 and HMGB1 has been verified by Dara et al. (2007)13, the activation of PARP1 induces release of the pro-inflammatory mediator HMGB1 from the nucleus15. Of the 16 genes, 15 of them have been reported to relate to cancer. Four of them have been reported to relate to prostate cancer directly. Although LINC01278 has not yet been reported to relate to cancer, abs conversion type suggests that it is an important informative gene. LINC01278 occurred three times in the top 10 gene pairs  cannot distinguish cancer from normal samples. It also indicates that the input features for classifiers should be conversion feature Z  (Table e.g dataset9 and dataset10) (Table Z) prior to be used as input features for classifiers, especially for many pairs of synergistic genes.For the less input features (2) Table , all of 0) Table , althougY is determined by individually discriminant genes X1 and X2, and pair-wise synergic genes X3–X4 and X5–X6. In other words, the true genetic model is X1, X2, X3, X4, X5, X6}, X7–X1000 are genes unrelated to Y. Now we get the dataset {Y, X1, X2,…, X1000} and want to construct a genomic prediction model16 based on machine learning, but don’t know the true genetic model. Even the individual discriminant genes X1 and X2 can be highlighted by t-test, and the synergic genes X3, X4, X5 and X6 can be highlighted by Abs conversion type or MIC, classifier cannot learn well when the input features space is {X1, X2, X3, X4, X5, X6}. It means that learning machine can never tell us the true optimal subset, if synergic genes have not been converted properly. This indicates the complexity of genomic prediction, also provides a new explain for “missing heritability” in GWAS study.This is a surprising and important discovery. Suppose phenotype abs conversion type, we also evaluated the prediction performance of individually discriminant and synergic genes with three more recent and larger publicly available datasets   When the input features involves 20 genes, the average accuracies for Top20_Ind, Top10_Syn and Top10_Ind + Top5_Syn are 78.74%, 81.67%, and 83.74%, respectively. When the input features involves 40 genes, the average accuracies for Top40_Ind, Top20_Syn and Top20_Ind + Top10_Syn are 80.36%, 84.63%, and 85.75%, respectively. These indicate that combining individually discriminant and synergic genes, rather than only using the individually discriminant genes or the synergic genes, can receive better prediction accuracies. 3) The classification performances of the label randomization tests drop to random, it validate the reliability of synergic genes selected by abs conversion type.Table The minimum number of individually discriminant and synergic genes required in the optimal subset remains to be determined by the further research.Abs conversion type have more powerful ability to improve prediction performance for the individually discriminant model than the genes selected by the other conversion types.We also compared the prediction performance of the 5 conversion types Table . The resabs conversion type and t-test, to detect gene–gene synergy. We find that dendrogram-based I and doublets are helpless for discovering pair-wise gene interactions, and the synergic genes selected by our method and the MIC method are consistent with the typical pair-wise synergy. However, MIC has a higher computational cost. For example, the running time of the entire process on Prostate1 dataset  by MIC method is approximately 20 hours (Intel Core i5-4590@3.3 GHz), whereas it is only 47 minutes by our method. Experiments on simulated and real-world data showed that combining the individually discriminant genes selected by t-test and the synergic genes selected by our methods can improve prediction performance. These synergic genes should be converted into new variables (Z) prior to be used as input features for classifiers.In this paper, we propose a fast approach based on the combination of


These results open up the potential for multi-directional in situ alignment of carbon nanotubes providing one viable route to the fabrication of many novel optoelectronic devices.A plasma-enhanced chemical vapour deposition reactor has been developed to synthesis horizontally aligned carbon nanotubes. The width of the aligning sheath was modelled based on a collisionless, quasi-neutral, Child’s law ion sheath where these estimates were empirically validated by direct Langmuir probe measurements, thereby confirming the proposed reactors ability to extend the existing sheath fields by up to 7 mm. A 7 mbar growth atmosphere combined with a 25 W plasma permitted the concurrent growth and alignment of carbon nanotubes with electric fields of the order of 0.04 V μm Most are based on either graphoepitaxy [Various reported oepitaxy ,23,24,25oepitaxy , magnetioepitaxy , gas flooepitaxy ,29,30,31oepitaxy ,19,21. Goepitaxy . Despiteoepitaxy . Here thet al. [et al. [−1 whereas Law et al. [et al. [−5 nN, a force approximately four orders of magnitude greater than the weight of the catalyst particle, whilst Hertel et al. [et al. [et al. [et al. [et al. [in situ horizontal alignment of CNTs.Zhang et al.  and Ural [et al.  reportedw et al.  determin [et al.  estimatel et al.  showed t [et al.  proposed [et al.  presente [et al.  postulat [et al.  suggeste [et al. ,39 where−1 was applied post-growth. The CNTs strongly interacted with the electric field and become increasingly laterally dispersed by up to 20 µm in some cases, though little alignment was evident. The similarly wide distribution of catalyst particles would also suggest that the catalyst also interacts strongly with the field and that tip growth is ultimately preferential, supporting the posits of Chen et al. [−1). We believe the variations in alignment to be the product of disparities in the electrode fabrication though alignment can certainly be attributed to the electric field given the extremely linear growth. Other than the low-linear densities, a major limitation to this local electric-field alignment is the requirement for microelectrodes patterned directly on the substrate. These structures interfere with post-growth processing and device fabrication. Substrate independent electrodes obviate these limitations. The electric field can then be applied across entire samples. To the best of the author’s knowledge, no one has hitherto demonstrated such global alignment horizontally. A new horizontal nanotube PE-CVD reactor was modelled, designed and fabricated.Catalyst activity was initially validated using samples grown by traditional thermal-CVD (T-CVD), at 700 °C for 900 s, and vertical PE-CVD, as per our earlier work . No horin et al. . T-CVD seT) and glow potential (pV), the relation between the sheath width (S), charge carrier density (n) and cathode potential (oV) is given by [o is the permittivity of free space, k is Boltzmann’s constant and e is the electronic charge. Here the Debye length (Dλ) is given by;en is the electron density. In the presented reactor the Debye length is ~22 µm which is significantly smaller than the characteristic dimensions of the reactor (~1 mm). This validates our initial assumption of stable and largely collisionless plasma. Combining Equations (1) and (2), and assuming quasi-neutrality (n ~ en), gives;k = (6.12 × 104) m5/2V−3/4K1/4 and F denotes the pressure and plasma power dependence of the electron temperature and the electron density. Note that the plasma potential pV << 0V, where pV ~5 V and 0V ≥ 500 V. Typical growth profiles (Vo ~ 600 V and en ~ 1017 m−3) produce a 1.6 mm sheath for eT = 1.5 × 104 K [It has been widely postulated that CNT alignment is mediated by anisotropic torque induction within a narrow plasma sheath . This shgiven by ,42;(1) × 104 K .0V = 600 V) and electron density (en = 8.2 × 1016 m−3) have been indicated (black dot) for clarity. Our estimates suggest that a ten-fold increase in sheath width is possible by tuning the cathode bias, electron temperature and electron density.Equation (3) suggests that the sheath width is strongly dependent on the electron density and weakly dependent on the cathode bias. The derived dependencies are illustrated in en ≈ n+ = n) the corresponding currents are;C is the mean charge carrier velocity, given by;em, m+) are known. The measured current is the sum of the electron and ion currents (|I|=|eI| = |I+|). However, eT >> T+ (∴ Ce >> C+) , hence |I|≈|eI|. The electrons exhibit a Maxwellian energy distribution which modulates the current accordingly to give [V (=pV − V) is the potential difference between the plasma and probe. The electron density can now be found from Equation (4). The parameters pV, eI and eT are empirically accessible through the Langmuir probe I–V data. We find that for typical process conditions (3.2 mbar/ 50 W/ 550 V), en = 2.5 × 1017 m−3 and eT = 0.7 eV; values which show excellent agreement with those reported by Blaek et al. [We directly controlled the cathode bias and indirectly controlled the electron temperature and electron density by controlling the plasma power and reactor pressure. The measured variation in sheath width with plasma power and reactor pressure is shown in  to give ;(6)whek et al. .et al. [−1, with increasing reactor pressure (en). However, the sheath width also increases at low pressures (to increase eT). Consequently, a more detailed functional dependence on en and eT must be evaluated. Equations (7) and (8) show the functional fits, where θ denotes the plasma power, and where separation of variables has been assumed for en.k' represents an averaging term which negates the scaling effects introduced by the assumed separation of variables. Equations (3), (7) and (8) can be combined to give a sheath width of the form S = S. Hence,The electron density linearly increased with plasma power a, a simiet al. , and quapressure d. SheathUL / υ where U is the free-stream flow velocity, υ = μ / ρ is the kinematic viscosity (the ratio of the dynamic viscosity to density) and L is a characteristic dimension of the flow-perturbing feature. For an ideal gas a Reynolds number of ~100 is estimated, based on the data of Sun et al. [3 flow at 3.5 mbar (250 sccm)/700 °C, which indicates a predominately laminar flow (turbulent flow → Re > 2300). Thus, in principle, limited alignment degradation should occur as a result of the feedstock/passivation gases. Moreover, low gas flows (<300 sccm ~1.7 cm/s) were employed throughout to avoid turbulent interactions and to ensure the alignment was solely attributed to the electric field. Indeed, Xin et al. [Catalyst samples were mounted with the gas flow parallel to the substrates surface. It is possible that this may enhance, or even account for any observed alignment, as per the findings of ,30,31. Fn et al.  on the d4 cm−1, consistent with the linear packing densities achieved by local electric field experiments reported elsewhere . 104 cm−1 ,20,21.et al. [et al. [Consider the two extreme cases of sheath extension as illustrated in et al.  reported [et al.  found an3:C2H2). W electrodes were patterned on Si/SiO2 (200 nm) substrates, separated by a 50 µm gap by standard photolithography. Ni catalyst strips and 80 nm catalyst dots (Al2Ox/Fe–10/1 nm) were patterned between the electrodes using poly(methylmethacrylate) mediated electron beam lithography. W (25 nm) supported Fe (5 nm) samples were photolithographically defined and prepared by magnetron sputtering. Metallic supports were intentionally used to minimise the interaction between the growing nanotubes and the Si/SiO2 substrate. Catalyst samples were loaded into the reactor following an acetone and IPA degreasing step and nanotubes were grown at 700–800 °C.Nanotubes were grown by thermal chemical vapour deposition using an etchant-to-feedstock gas ratio of 200:50 sccm  at 700 °C. The probes tip was 2–5 mm from the cathode centre in the glow discharge region. Following 300 s of plasma stabilisation the reactor pressure was reduced by either throttling the attached roughing pump or varying the input flow rates, followed by a second plasma stabilisation period of 300 s. The reactor pressure was varied from 2.5 to 6 mbar and the I–V characteristics were recorded using a Velleman DVM890. The probe bias was incrementally increased from 0 to 25 V using a Manson EP-613 source.Plasma characteristics were measured using a cylindrical Langmuir probe of length 102H2:NH3 growth atmosphere with a plasma power of 25 W. The presented reactor permits the concurrent growth and alignment of carbon nanotubes with electric fields of the order of 0.04 V μm−1 with linear packing densities of up to ~5 × 104 cm−1. These results open up the potential for multi-directional in situ alignment of carbon nanotubes providing one viable route to the fabrication of many novel optoelectronic devices.A new plasma-enhanced chemical vapour deposition reactor is reported offering a new route to synthesis horizontally aligned carbon nanotubes. The reactor does not require time consuming and costly substrate preparatory processes and can synthesis CNTs with an alignment factor of Str37 = 0.5. A model based on a collisionless, quasi-neutral ion sheath is presented and has been validated by direct Langmuir probe measurements confirming the proposed reactors ability to extend the existing sheath fields by up to 7 mm using a 7 mbar, C