In contrast to the cyanobacterial ancestor, chloroplast gene expression is predominantly governed on the post-transcriptional level such as modifications of the RNA sequence, decay rates, exo- and endonucleolytic processing as well as translational events. The concerted function of numerous chloroplast RNA-binding proteins plays a fundamental and often essential role in all these processes but our understanding of their impact in regulation of RNA degradation is only at the beginning. Moreover, metabolic processes and post-translational modifications are thought to affect the function of RNA protectors. These protectors contain a variety of different RNA-recognition motifs, which often appear as multiple repeats. They are required for normal plant growth and development as well as diverse stress responses and acclimation processes. Interestingly, most of the protectors are plant specific which reflects a fast-evolving RNA metabolism in chloroplasts congruent with the diverging RNA targets. Here, we mainly focused on the characteristics of known chloroplast RNA-binding proteins that protect exonuclease-sensitive sites in chloroplasts of vascular plants. Arabidopsis thaliana contains a total of 87 genes encoding mostly ribosomal and photosynthetic subunits, 4 rRNA and 37 tRNA genes [The plastid is the result of an endosymbiotic event that occurred billion years ago and comprised the incorporation of a cyanobacterium into a eukaryotic, mitochondria-possessing cell. The main benefit of the cyanobacterium for the host cell was the supply of additional energy and photoautotrophic growth, granting an enormous selective advantage. The genome of the endosymbiont underwent dramatic transformations, whereby the majority of the cyanobacterial genes were transferred into the nucleus or lost . MeanwhiNA genes .plastid-encoded bacterial type RNA polymerase (PEP) and at least one phage-type nuclear-encoded RNA polymerase (NEP). The PEP is composed of four basic plastid-encoded core subunits (rpo genes) and requires additional nucleus-encoded sigma factors (six in Arabidopsis) for promoter recognition and transcription initiation as well as further factors, which associate to the core during chloroplast development. In contrast, NEP is a one-subunit-enzyme which seems to require additional accessory factors as well. It was assumed that photosynthetic genes are predominantly transcribed by the PEP, while genes for the translational machinery and for PEP subunits are targets of the NEP [rpo genes and genes for the translational machinery, transcripts preferentially generated by the NEP predominate [Similar to the operons of the cyanobacterial ancestor, the chloroplast genes are primarily organized in newly composed transcriptional units and often contain genes that do not belong to the same functional cluster. These units are transcribed as polycistronic pre-mRNAs by two types of RNA polymerases: the  the NEP . However the NEP , whose r the NEP . In mostdominate .psbD–psbC promoter switch in response to blue light in barley, gene-specific transcriptional regulation seems to be an exception rather than the rule [Even though there are examples for gene-specific transcriptional regulation, such as the the rule ,12. Unlithe rule . Most obthe rule . These nthe rule . The prothe rule ,16–18.Arabidopsis, 17 gene products assigned to exhibit ribonucleolytic activity or to contain domains characteristic for ribonucleases were predicted to be localized in the chloroplast [Arabidopsis chloroplasts, RNR1 (RNase R) and PNPase with 3′→5′ specificity, and RNase J with 5′→3′ activity. As in other organisms, plant ribonucleases are thought to be rather unspecific and certain substrate preferences might be determined by the RNA structure or chemical features and less by the RNA sequence [The conversion of polycistronic transcripts into oligocistronic or monocistronic isoforms, the post-transcriptional generation of 5′ and 3′ transcript termini as well as RNA decay in general is accomplished by plastid ribonucleases . In chlooroplast . Among toroplast ,17, whiloroplast . CSP41 that cleave the RNA in unstructured, unprotected regions . AlthougMost of the plastid RNA protectors present in vascular plants are not found in green algae, such as Chlamydomonas, and vice versa, reflecting the diverged genome–plastome interaction and the tight coevolution of the cellular genetic compartments in the respective lineages during endosymbiosis. Comparably dissimilar are post-transcriptional processes in plastids of land plants and green algae. For example, RNA editing requires a plethora of trans-acting factors and is widespread in vascular plants but entirely absent in green algae . Often, pentatricopeptide repeat (PPR) proteins, (2) chloroplast ribonucleoproteins (cpRNPs), and 3) additional RNA protectors, which do not belong to a larger protein family. Our review article mainly focuses on vascular plant chloroplasts as gene expression in Chlamydomonas reinhardtii differs in many respects from vascular plants and extensive reports deserve an independent comprehensive review article. The reader is referred to recent articles reporting on plastid mRNA stability in this unicellular algae  ae e.g. ,33–41 ). .pentatrThe family of plant PPR proteins presumably evolved from tetratricopeptide repeat (TPR) proteins and consists of more than 450 up to approximately 1000 conserved members, shared among the eukaryotic kingdom ,43. PPR atpH and rpl33 as well as the 3′-UTR of atpI and psaJ, whose intergenic regions (atpI–aptH and psaJ–rpl33) share consensus sequences to which PPR10 specifically binds [psaJ, offering new RNA-binding regulation perspectives via different dimerization states [atpH by restructuring its 5′ region and exposing its otherwise masked ribosome-binding site [The best understood PPR protein is PPR10 in maize. PPR10 is essential for the stabilization of two different RNA termini: the 5′-UTR of ly binds   mutants were screened [hcf152 mutant showed defects in the processing and accumulation of the psbB–psbT–psbH–petB–petD transcript cluster accompanied by severe defects in PSII and cytochrome b6f complex formation [psbH–petB intergenic region in monocots and dicots [petB and psbH RNAs respectively, as revealed by precise transcript mapping [In vitro RNA-binding experiments validated that this sRNA represents the direct target of HCF152, suggesting that HCF152 protects the cleaved 5′ petB and 3′ psbH RNAs with identical sequences in their termini against exonucleolytic degradation, which resembles the function of PPR10 [In a first step to identify novel factors in screened . The hcformation ,56. Furtormation . A screed dicots ,50,58, s mapping . In vitrof PPR10 ,24.crp1 (chloroplast RNA processing 1) was identified in a screen for mutants with distinct defects in the expression of plastid-encoded genes. It was shown to lack the cytochrome b6f complex, which could be traced back to defects in the accumulation of monocistronic petB and petD transcripts [petA, petD, and psaC mRNAs [In vivo RIP-chip experiments with the 5′-untranslated regions of psaC and petA mRNAs further contributed to the understanding of CRP1 function as a translational regulator [petA 5′-UTR [Arabidopsis homolog AtCRP1 revealed an association with the 5′-UTR of psaC, the petB–petD intergenic region and to a much weaker extent the petA 5′-UTR [Arabidopsis and maize the overall analyses argue for similar functions of both CRP1 proteins in stabilization of petD 5′ and petB 3′ RNA termini and in translational regulation of psaC and petA mRNAs.The maize mutant nscripts . In addiaC mRNAs . In vivoegulator . ElectroA 5′-UTR . FurtherA 5′-UTR . This isA 5′-UTR . Taken tZea mays mutant ppr53 revealed that 5′ transcript ends of rrn23 and ndhA RNAs were absent. PPR53 was able to bind to the 5′ end of the rrn23 RNA, which was also predicted to be the PPR53 target site, but failed to do so in case of the 5′ end of ndhA RNA. Thus, the authors concluded that PPR53 stabilizes the 23S rRNA by binding to its 5′ region and stimulates the translation of ndhA via another yet unknown factor(s) [Arabidopsis ortholog SOT1 suggest that the binding of SOT1 to the 5′ end of the dicistronic 23S-4.5S rRNA prevents its exonucleolytic degradation [Analysis of the actor(s) . Experimradation . Moreoveradation .pgr3 alleles revealed defects in cytochrome b6f and NDH complex accumulation. These defects were traced back to impaired stabilization of the tricistronic petL–petG–psaJ RNA precursor and putative defects in the translation of petL and presumably a member of the ndh transcripts [in vitro studies revealed an association of recombinant PGR3 with the 5′-UTRs of petL and ndhA, arguing for a role of PGR3 in the stabilization and translational activation of these two RNA targets [petL and ndhA [Arabidopsis pgr3 mutants revealed that the defects in petL stabilization and translation are conserved. However, as pgr3 mutants in maize showed more dramatic effects than the Arabidopsis ones, it was concluded that ZmPGR3 has acquired additional functions compared with the Arabidopsis ortholog [Proton gradient regulation3 (PGR3) is an example for another PPR protein that harbors two functions: transcript stabilization and translation initiation. Initial experiments including different nscripts . Further targets . Finallyand ndhA . Compariortholog .trnG–UCC precursor [In vitro binding assays with recombinant ZmPPR5 could narrow down its binding site to the central region of the trnG–UCC intron, suggesting that PPR5 protects an endonuclease-sensitive site through its binding and might even facilitate the splicing of this group II intron [The stabilizing function of PPR proteins is not restricted to protein coding mRNAs and rRNAs but extends to tRNAs, as demonstrated for the maize PPR5 protein that associates with the recursor . In vitrI intron   mutant showed that rRNA processing and accumulation are affected causing a prevalent reduction of plastid proteins [atpA and atpB translation [atpF–atpA and psaJ–rpl33 intergenic regions, thus acting on similar RNA targets as PPR10. Differences in the mutant phenotype in monocot and dicot species argue for evolutionary diverged functions of ATP4 and SVR7 [Analysis of proteins . A laternslation . Additioand SVR7 .rps12–rps7–ndhB precursor RNA is likely to be processed at not less than two sites in the rps7–ndhB intercistronic region. RNase protection assays revealed that the PPR protein CRR2 is required for intergenic processing of the site close to ndhB [ndhB transcripts by binding to this region, thus hindering 5′-3′ exonucleolytic cleavage. This is reflected by the lack of 5′ processed ndhB transcripts whereas the precursor RNA accumulates at normal levels in crr2 mutants in Arabidopsis, which are unable to accumulate stable and functional NADH dehydrogenase-like complexes mediating cyclic electron flow around photosystem I [in vitro even if the domain was considered to be related to RNA editing [ndhB 5′-UTR has never been investigated, its potential binding site is presumably represented by an abundant RNA footprint downstream of the 5′ end of the monocistronic ndhB mRNA implying that CRR2 could also function in the stabilization of processed RNAs; however, future research is needed to confirm this interaction [The  to ndhB . Additiosystem I . A furth editing . Interes editing . Althougeraction ,50.Physcomitrella patens, later renamed to PpPPR_38, was initially shown to be involved in splicing of clpP and processing of the clpP–rps12 intergenic region resulting in elevated levels of precursor mRNAs [clpP mRNA and to the clpP–rps12 intergenic region in vitro. Studies with isolated chloroplast lysates suggested that binding of PpPPR_38 to the clpP–rps12 intergenic region stabilizes a predicted stem–loop structure that first provides a correct intergenic endonucleolytic processing site and then protects the cleaved clpP mRNA against 3′→5′ exoribonuclease activity [The PPR531-11 protein from or mRNAs . In a foactivity .RNA-Recognition Motifs (RRM)-containing, abundant stroma-localized proteins that associate with various ribosome-free mRNAs and pre-tRNAs [psbA, rbcL, petD, and rps14 as revealed by in vitro studies [Arabidopsis has a more specific function and was shown to be important for editing of different sites and stabilization of several but mainly ndhF mRNAs, whereas CP31B was assumed to support editing of specific CP31A-dependent sites [psbB, psbD, psaA/B, atpB, and ndhB to protect them from degradation under cold stress conditions [Arabidopsis CP33A protein is another example for the global role of cpRNPs in processing and stabilizing of multiple chloroplast RNAs [In general cpRNPs are divided into three different groups according to predicted structural features and phylogenetic considerations: I (cp28 and cp31), II (cp29A and cp29B), and III (cp33) . CpRNPs re-tRNAs ,83. Intere-tRNAs . Meanwhi studies . In addi studies . In cont studies . CP31A int sites . Furthernditions . The Araast RNAs .hcf145 mutant was initially found to specifically lack PSI [psaA–psaB–rps14 transcript encoding the two photosystem I core proteins PsaA and PsaB. Binding of HCF145 to the psaA 5′-UTR probably protects it from exoribonucleotytic attack [transcript-binding motif repeat (TMR) domains of approximately 70 amino acids which are responsible for specific binding to the psaA 5′-UTR. The TMR motifs are present rarely as single domains but often as multiple (up to ten) repeats in quite diverse proteins of unknown function in photosynthetic organisms including moss, red and green algae, and cyanobacteria [The seedling-lethal lack PSI . This coc attack . The HCFbacteria . The preArabidopsis proteins with unknown function and localization [Two additional N-terminal repeated regions of unknown function were found in the HCF145 protein that showed homology to the ubiquitously distributed SRPBCC  binding domains superfamily consisting of 11 Pfam families, including START domains, phosphatidylinositol transfer proteins, Bet_v_1, CalC related, CoxG, and polyketide cyclase related families . The strlization .psaA mRNA, although they do not contribute to the binding on their own. This points to a regulatory role of the SRPBCC domains in adjusting PSI levels [psaA RNA. Further studies will show whether a metabolism-dependent regulation of psaA–psaB–rps14 mRNA levels is important during development, greening and/or acclimation processes.Interestingly, the SRPBCC-related ligand-binding motifs support association to the 5′ I levels  and provprfB mutants described so far, in Arabidopsis lines lacking PrfB1 most plastid UGA stop codon-containing transcripts are unstable [C. reinhardtii, a PrfB-related gene is lacking in the nuclear genome and accordingly TGA stop codons are absent in their plastid genomes [prfB1 mutants. In either case, a hitherto unrecognized surveillance system efficiently removes all these nonfunctional transcripts. This demonstrates an advanced and more sophisticated regulatory level of plastid gene expression in comparison with known eubacterial systems.Different from eukaryotes harboring only one release factor (eRF) for ribosomal release at all three stop codons, eubacteria and organelles contain two release factors, PrfA (RF1) and PrfB (RF2), for termination of translation at UAG and UGA stop codons respectively. The UAA codon is recognized by both factors. PrfB1 and PrfB2 encode the only functional plastid and mitochondrial ribosomal RF2-related proteins in land plants respectively . In contunstable ,95. The  genomes . It is lInterestingly, a possible regulatory function of translational termination at UGA stop codons is reflected by a significant increase in the TGA content in land plant plastid genomes in comparison with those in algae . PresumaArabidopsis are able to terminate translation at UGA stop codons but are specifically affected in the accumulation of the cytochrome b6f complex due to the lack of petB transcripts encoding cytochrome b6. The petB gene is part of the primary pentacistronic psbB–psbT–psbH–petB–petD transcript encoding three proteins of PSII  and two proteins of the cytochrome b6f complex (PetB and PetD). Similar to most plastid polycistronic transcripts, numerous endonucleolytic processing events generate a wealth of overlapping RNA species, which are subjected to individual life times and translational control. Data revealed that petB transcripts that are cleaved at the 3′ end are efficiently generated but rapidly degraded in prfB3 mutants indicating that PrfB3 protects these RNAs from 3′→5′ exonucleolytic attacks. This is in agreement with preferential binding of PrfB3 to the petB 3′-UTR with respect to control transcripts [petB-containing precursor transcripts accumulate in prfB3 but petB is not translated. This indicates that binding of PrfB3 to the 3′ end could have an impact on elongation and/or initiation of translation at the 5′ end or that only processed monocistronic petB transcript are translational competent. Alternatively, a potential weak binding of PrfB3 also to the petB 5′-UTR is required for efficient initiation of translation [Land plant genomes surprisingly encode an additional eubacterial RF2-related protein, named PrfB3, which lost its two most important “SPF” and “GGQ” motifs of a release factor and thus the capability to terminate translation at UGA and UAA stop codons . Accordinscripts . Surprisnslation .petB mRNA levels [b6f amounts. Therefore, PrfB3 could well serve a regulatory function in vivo during acclimation processes by rate limiting levels of petB transcripts when changes in the amount of the cytochrome b6f complex are required. A similar regulatory role was assigned to the plastid PPR protein MCA1 in Chlamydomonas in determining levels of petA mRNA and accordingly its product cytochrome f [A drop in the expression of PrfB3 in partially complemented mutant lines correlated with a decrease in A levels . Most imchrome f .Arabidopsis mutation, which was characterized by a lack of some but not all oligo- and monocistronic psbH transcripts generated by processing of the pentacistronic psbB–psbT–psbH–petB–petD precursor message [psbH messages rather than processing is affected. PsbH encodes an essential 8 kDa phosphoprotein of PSII whose absence leads to a pronounced deficiency of PSII activity and protein levels in the hcf107 mutant [psbH in the nuclear genome, which targets the protein back to the chloroplast, revealed that HCF107 is exclusively required for the expression of psbH [C. reinhardtii that affects expression of psbB [The HCF107 gene was initially identified by high resolution mapping of the corresponding  message ,56. Dete7 mutant ,56. Ecto of psbH . This is of psbB .psbH start codon in agreement with the one repeat—one nucleotide model of PPR proteins [psbH transcripts lacking in hcf107. Expectedly, binding to its target protects the psbH RNA from 5′→3′ exonucleolytic attack in vitro [psbH 5′-UTR remodels the RNA structure, which may also allow ribosome binding and subsequent enhancement of translation, hinting to a dual role of the HCF107 protein. Obviously, PPRs and HATs can act in a similar and presumably analogous way to the recently identified family containing predicted superhelical and repeated RNA-binding TMR motifs in photosynthetic organisms [HCF107 encodes a half-a-tetratricopeptide (HAT) protein containing 11 repeats and no additional domains . It is mproteins  and accoin vitro . RNA strrganisms .Here, we summarized the characteristics of plastid RNA-binding proteins with a protective role in vascular plants. Whether the regulation of endonucleolytic activities, which were assumed to initiate degradation, is rate-limiting for mRNA decay still remains elusive. The RNA-binding proteins addressed here evolved mostly to hinder exonucleolytic attacks of tRNAs and translationally competent mRNAs, some of which were also shown to impact additional processing events and/or translation. Ribosome profiling was shown to partially overcome the challenge to investigate the role of the protectors in translation in particular using genetic approaches with low abundant RNA targets in the respective mutants ,100. NevpetB in Arabidopsis [b6f complex during stress and/or acclimation processes still needs to be shown.Differential expression of RNA protector genes under changing environmental conditions and the effects on their downstream targets speaks in favor of the idea that RNA degradation is mostly regulated at the RNA extremities. One example is provided by a comparable differential expression of AtprfB3 and its target bidopsis . WhetherpsaA stability and thus photosystem I amounts [The progression in mass spectrometric methods will prompt future studies on the metabolic impact and post-translational modifications of RNA protectors on the regulation of their activity, interaction with other hitherto unidentified association partners, and/or RNA affinity in a quantitative manner. Generally, these analyses remained poor, nevertheless the potential integrator HCF145 seems to be a promising candidate for such experiments. The two SRPBCC ligand binding domains of HCF145 offer an excellent platform for their metabolic control by the integration of external stimuli to finally fine-tune photosynthesis by regulating the effect of HCF145 on  amounts . Also, c amounts . Studies amounts ,4.Most protectors are plant specific congruent with the fast divergence of their targets at the 5′- and 3′-UTRs of plastid transcripts . Even ifChloroplast gene expression is predominantly governed on the level of post-transcriptional processes including RNA editing, methylation, polyadenylation, splicing, endo- and exonucleolytic digestion as well as translation.Generally, newly evolved and vascular plant-specific RNA-binding factors encoded in the nuclear genome are required for protecting specific chloroplast RNAs at exo- and endoribonuclease-sensitive sites reflecting a fast-evolving RNA metabolism congruent with the diverging RNA targets.Chloroplast RNA-binding protectors, usually found in high molecular weight complexes with largely unknown components, can act as RNA chaperones, occasionally affect other post-transcriptional processes, such as splicing, editing and translation, and contain a variety of different RNA recognition motifs, which often appear as multiple repeats.Interactions with other proteins, post-translational modifications of protectors, metabolic pathways, and presumably noncoding RNAs may have an important impact on the characteristics of RNA protectors, which are required for normal plant growth and development as well as diverse stress responses and acclimation processes.


Thin boron doped diamond (BDD) film is deposited from trimethyl borate/acetone/hydrogen mixture on Co-cemented tungsten carbide (WC-Co) micro drills by using the hot filament chemical vapor deposition (HFCVD) technique. The boron peak on Raman spectrum confirms the boron incorporation in diamond film. This film is used as an interlayer for subsequent CVD of micro-crystalline diamond (MCD) film. The Rockwell indentation test shows that boron doping could effectively improve the adhesive strength on substrate of as deposited thin diamond films. Dry drilling of graphite is chosen to check the multilayer (BDD + MCD) film performance. For the sake of comparison, machining tests are also carried out under identical conditions using BDD and MCD coated micro drills with no interlayer. The wear mechanism of the tools has been identified and correlated with the criterion used to evaluate the tool life. The results show that the multilayer (BDD + MCD) coated micro drill exhibits the longest tool life. Therefore, thin BDD interlayer is proved to be a new viable alternative and a suitable option for adherent diamond coatings on micro cutting tools. Graphite is the most widely used material for electrical discharge machining (EDM) electrodes due to its low electrode abrasion, light weight, low cost and especially excellent high temperature endurance in the spark arc when compared with cooper ,2. HowevWhen using CVD diamond films in cutting tool applications, cemented carbides (WC-Co) are commonly used as tool material. The major difficulty in depositing diamond on WC-Co tools arises from the binder Co, which induces graphitization during HFCVD procedure. This would result in poor adhesive strength between substrates and diamond coating and further up restrict the application of diamond coated cutting tools. 2B during deposition of BDD film 6 + 10 g KOH + 100 mL H2O) in an ultrasonic vessel for 5 min; (2) etching substrates with Caro’s acid (30 mL H2SO4:70 mL H2O2) for 30 s; (3) ultrasonic treatment in the compound solution of alcohol and diamond powders; (4) ultrasonic washing by distilled water; (5) ultrasonic washing by acetone.The substrates used in this work are commercial tungsten carbide two-flute micro drills (φ = 0.25 mm) which contain 6 wt % cobalt binder, as shown in 3)3) and acetone are used as boron and carbon source, respectively. Trimethyl borate is dissolved in acetone according to a preset boron-substituted carbon (B/C) ratio to obtain boron doped diamond films. The mixed solution is then displaced into a bubbler immersed in glacio-aqueous mix solution to control the flux of carbon and boron source. Ultrahigh purity H2 is used as a carrier gas which flows through the mixed solution and is poured into the vacuum reaction chamber. Two triple-twisted tantalum wires with a spacing of 40 mm are used as hot filaments and are set 12 mm above the top of micro drills. The hot filaments are dragged into a straight position by heat-resisting springs.After pretreatment, un-doped and boron doped diamond films are deposited on micro drills using bias voltage enhanced HFCVD apparatuses. Trimethyl borate . The ingredients and purity of MCD, BDD, BDD interlayer and BDD + MCD films are examined by Raman spectroscopy (SPEC14-03). Rockwell indentation tests are conducted on WC-Co samples to evaluate the adhesion of these coatings by a Hoytom indentation instrument. The conical Rockwell intender is forced into the coating with a load of about 1500 N.3. Suction tubes are applied during machining to sweep away graphite powders. The spindle speed, feed rate and depth of drilled holes are 15,000 r/min, 200 mm/min and 1.3 mm, respectively. After drilling a certain amount of holes, main cutting edge of micro drills are photographed by FE-SEM equipped with measuring system. For each tool, 10 locations of wear land on its flank face are measured and the averaged value is regarded as its flank wear (VB).Cutting experiments are conducted for as-fabricated diamond coated micro drills on a CNC carving machine. The workpiece is cuboid shaped high quality EDM graphite of grade ISO-68  with density of 1.88 g/cmThe surface morphologies of as-fabricated diamond coated micro drills are investigated using FE-SEM. −1, characteristic of high purity sp3 C–C bonding. The broad peak at 1400–1600 cm−1 represents that there are non-diamond phases, including graphite or amorphous carbon, in the film. However, no other peak is visible besides the 1332 cm−1 diamond peak on Raman spectrum of BDD + MCD composite film, which indicates the high purity sp3 C–C bonding in the outer MCD layer. The different amount of graphite or amorphous carbon in these two MCD layer may be that during diamond film deposition, boron dopant could effectively stop binder Co diffusing from tool interior to the surface at high temperature, thus suppress the graphitization of MCD layer on BDD interlayer [The quality of as-deposited diamond coatings is examined by Raman spectroscopy, using a He–Ne laser with an excitation wavelength of 632.8 nm. Raman spectra of these thin films are plotted in terlayer ,20.−1 in order to get more information of the boron doped diamond films. It can be seen that the symmetric Lorentzain peak at ~1332 cm−1 characteristic of undoped diamond changes to an asymmetric Fano-like lineshape because of a quantum mechanical interference between the zone-center Raman-active optical mode and the continuum of electronic states induced by the high concentration of dopant [−1 for BDD films and BDD interlayer), accompanied by two broad bands around 500 cm−1 and 1220 cm−1 [−1 is related to the vibrational modes of boron pairs due to the distortion in the diamond lattice caused by these isolated defects [In the case of BDD film and BDD interlayer, the as deposited diamond films are examined by Raman spectroscopy in the region of 300–1800 cmf dopant . Another220 cm−1 ,22,23,24220 cm−1  and the  defects .2B in BDD layer. Meanwhile, these compounds could effectively stop cobalt from moving outside the substrate during the deposition procedure.The Rockwell indentation tests are conducted on the MCD, BDD, BDD interlayer and BDD + MCD coated tungsten carbide samples to evaluate their respective interfacial coating–substrate adhesion strength. It can be seen in The effectiveness of BDD interlayer on cutting performance of diamond coated micro drills is investigated in dry drilling of high quality EDM graphite. For the sake of comparison, pure MCD and BDD coated micro drills are involved. In graphite machining, brittle fracture of polycrystalline aggregates takes place instead of plastic deformation in metal machining. Thus, the tool failure mode in graphite machining is mainly abrasive wear of graphite powders. As abrasive wear has the largest influence on flank wear , the maiAs exhibited in To assess the wear mechanism of these coated micro drills, FE-SEM images of main cutting edges are captured after drilling a certain amount of holes, as shown in It is noted that much less flank wear could be seen on BDD + MCD c2 coatedSequentially, only BDD + MCD coated micro drill is used to drill graphite, its flank wear begins to rise up and diamond films are found peeling off at some locations after drilling 3600 holes, as can be seen in −1 and 500 cm−1, which are the feature of boron incorporation in the diamond lattice, are visible in Raman spectrum of BDD coating and BDD interlayer on micro drills. Results from Raman spectra show that the purity of MCD on the BDD interlayer is higher than that of MCD without the BDD interlayer. Detached coatings around the indentation can be seen on MCD film, while only micro cracks are observable on BDD coating and BDD interlayer after indentation test. That is to say, boron doping could effectively improve adhesion between diamond film and substrate. In dry machining of graphite, the tool life of BDD + MCD coated micro drill is about 2–3 times than that of MCD or BDD coated micro drill. BDD interlayer before MCD deposition could effectively enhance cutting performance of diamond coated micro drills. The main wear type of BDD + MCD coated micro drill is flank wear. However, chipping and coating delamination could also be observed in addition to flank wear on BDD and MCD coated micro drills. The difference of these wear types is attributed to relatively good adhesive strength between substrate but low wear resistance of BDD layer and relatively poor adhesive strength between substrate but high wear resistance of MCD layer. MCD, BDD and BDD + MCD composite films with thickness of about 2 μm are uniformly coated on cutting part of the micro drills. The broad peaks around 1220 cm


Animal’s efficiency in converting feed into lean gain is a critical issue for the profitability of meat industries. This study aimed to describe shared and specific molecular responses in different tissues of pigs divergently selected over eight generations for residual feed intake (RFI).longissimus muscle, liver and two adipose tissues using a porcine microarray and analyzed for the line effect (n = 24 pigs per line). The most apparent effect of the line was seen in muscle, whereas subcutaneous adipose tissue was the less affected tissue. Molecular data were analyzed by bioinformatics and subjected to multidimensional statistics to identify common biological processes across tissues and key genes participating to differences in the genetics of feed efficiency. Immune response, response to oxidative stress and protein metabolism were the main biological pathways shared by the four tissues that distinguished pigs from the low or high RFI lines. Many immune genes were under-expressed in the four tissues of the most efficient pigs. The main genes contributing to difference between pigs from the low vs high RFI lines were CD40, CTSC and NTN1. Different genes associated with energy use were modulated in a tissue-specific manner between the two lines. The gene expression program related to glycogen utilization was specifically up-regulated in muscle of pigs from the low RFI line (more efficient). Genes involved in fatty acid oxidation were down-regulated in muscle but were promoted in adipose tissues of the same pigs when compared with pigs from the high RFI line (less efficient). This underlined opposite line-associated strategies for energy use in skeletal muscle and adipose tissue. Genes related to cholesterol synthesis and efflux in liver and perirenal fat were also differentially regulated in pigs from the low vs high RFI lines.Pigs from the low RFI line had an improved gain-to-feed ratio during the test period and displayed higher leanness but similar adiposity when compared with pigs from the high RFI line at 132 days of age. Transcriptomics data were generated from Non-productive functions such as immunity, defense against pathogens and oxidative stress contribute likely to inter-individual variations in feed efficiency.The online version of this article (doi:10.1186/s12864-017-3639-0) contains supplementary material, which is available to authorized users. Feed cost is the major component (two-third or more) of the total cost of pig meat production , so thatTo date, some DNA variants playing a role in feed efficiency traits have been proposed from analyses of SNP locations by whole-genome association in pigs , 5 and blongissimus muscle, liver and two adipose tissues obtained in pigs from two lines divergently selected for RFI. Pigs from the low RFI line had an improved G:F ratio and they consistently demonstrated greater loin muscle size when compared with pigs from the high RFI line [vs. specific molecular pathways in different tissues which might be important in an efficiency phenotype.This study aimed to analyze the transcriptomic profiles of RFI line , 18. Thin = 24 per line) that were divergently selected over eight generations for RFI were compared at the same age (132 days). Pigs from the low RFI line grew faster during the test period (P < 0.001), ate somewhat less feed (P < 0.10) and were heavier at the same age than pigs from the high RFI line (Table P < 0.001) in pigs from the low RFI line. The longissimus muscle, the major fast-twitch muscle in the loin part of the body, had a greater mass (relative to BW) in pigs from the low RFI line, but liver was lighter in those pigs as compared with pigs from the high RFI line. The relative proportions of dorsal (backfat) subcutaneous (SCAT) and perirenal  adipose tissues, which are used as surrogates of carcass adiposity, were almost similar in both lines.Forty-eight pigs from two genetic lines . By using the same cut-offs for the four tissues examined, the global effects of the line on the numbers of differentially expressed probes (DEP) and their corresponding genes (DEG) were found much more pronounced in muscle than in the other examined tissues, with a total of 2 494 genes being differentially-expressed in muscle  were over-expressed in pigs from the low RFI line when compared with pigs from the high RFI line  were affected by the line across the four tissues. Intracellular (protein) transport and immune response, which includes antigen processing and presentation, were the top-enriched biological processes. In addition, membrane lipid metabolic process, macromolecule (protein) catabolic process, translational elongation, phospholipid metabolic process, the response to oxidative stress, and an heterogeneous cluster related to cell death, cytokine production and protein kinase activity were also listed as important biological processes modulated due to line. These eight pathways can be thus considered as the main biological responses shared by different tissues and participating in difference in feed efficiency.The 147 DEG were submitted to an enrichment test to explore the biological processes which were common to the four tissues. As indicated in Table SOD2 and PON1 (involved in the response to oxidative stress), IL1 and CTSC (acting in immunity) and NTN1 (regulating apoptosis). Two additional networks were also related to immunity  were involved in a network of genes including the phosphoinositide protein kinase (PDK1), an enzyme participating in lymphocyte homeostasis and function [CD40, TRAF6) were associated with nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) and C-jun N-terminal kinase (JNK) signaling pathways in another small network. Another significant network was clearly dedicated to protein metabolism , protein catabolism , and protein transport (YWHAZ).In parallel, highly-interconnected non-redundant small networks were released using Ingenuity Pathway Analysis® (IPA) tool, with particular genes (out of the 147 DEG) represented as nodes and relationships between them or with neighboring genes represented as edges. This analysis allowed pointing key genes in different functional networks which were mainly related to response to oxidative stress, apoptosis, immunity and(or) protein metabolism. Through low density lipoproteins (LDL), the first network Fig.  describefunction ; two genism Fig.  with difCREB1 (cAMP responsive element binding protein 1), a gene known to induce transcription of genes involved in fatty acid metabolism. This network also included YME1L1 (YME1 like 1 ATPase), a gene involved in mitochondrial protein metabolism, and PSEN1 (prenesilin-1), a gene encoding a protein that has been proposed to be the catalytic component of the gamma-secretase protein complex involved in development and cell regulatory processes.A last informative network , which means that part of the molecular mechanisms summarized by Dim1 contributed to inter-individual difference in muscularity. The second dimension (Dim2) summarized only 6% of the variability of transcriptomic changes and this did not allow any distinction between pigs from the low or high RFI lines. Therefore, only Dim1 was further considered as a relevant summary of the tissue molecular changes due to line.In addition to bioinformatics analyses, a Multiple Factor Analysis (MFA) was performed to aggregate the whole transcriptomic datasets. This data integration mathematical procedure ensures that each tissue influence was balanced in the joint analysis when latent synthetic variables were calculated as linear combinations of the most highly correlated DEP. The first dimension (Dim1) of the MFA summarized 24% of the whole molecular variability. As shown in Fig. P < 0.001) and commonly found in the four tissues are provided in Table The first latent variables in muscle (Dim1_muscle), liver (Dim1_Liver), PRAT (Dim1_PRAT) and SCAT (Dim1_SCAT) had a contribution near |1| to Dim1 in the MFA diagnostic plot Fig. : this mePSEN1 (presenilin-1) was again identified; it had the strongest positive correlation with Dim1. Moreover, USP33 (ubiquitin specific peptidase 33) and IPP  related to protein ubiquitination, RPL6 and RPL14 acting in translation, and PLA2G12A and PIGL involved in phospholipid metabolic process, also had positive contributions to Dim1. On the opposite, four genes involved in immune response  were identified as negative contributors to Dim1. Netrin (NTN1) and two other genes participating to oxido-reduction process  were also among the top negative contributors to Dim1. Finally, PHYH (phytanoyl-CoA 2-hydroxylase) was represented by different DEPs being either positively or negatively associated to Dim1. This was due to an opposite regulation of PHYH expression in adipose tissues (up-regulated in PRAT and SCAT in pigs from low vs high RFI lines) and lean tissues (down-regulated in muscle and liver in pigs from low vs high RFI lines).Among the top-ranked common genes, In a second step of analysis, genes that were differentially expressed only in one tissue were submitted to an enrichment test to reveal tissue specificities. Functional clusters were listed, tissue by tissue, in Tables INSR, INS-IGF2 and IRS2), coded for various enzymes acting in glycogen metabolic process (~8 genes) and other glucose metabolic processes such as glycolysis, tricarboxylic acid cycle, and finally the respiratory chain  to achieve the oxidative process of energy production from glucose  and insulin-growth factor-1  families , APOA1 (apolipoprotein-A1) coding for a major component of high-density lipoproteins, and LCAT (lecithin–cholesterol acyltransferase) which is responsible for the formation of plasma cholesteryl esters from free cholesterol  produced by lymphocytes and its receptor (IL10RB), which is required for IL10-induced signal transduction  or no (PRAT) significantly enriched pathways. Only 37% of the DEG in PRAT was also reported in SCAT , was identified as another enriched biological process in SCAT of pigs from the low RFI line when compared with pigs from the high RFI line. Conversely, defense response was the main enriched pathway of under-expressed genes in SCAT in pigs from the low RFI line: this concerned chemokines  and attractin (ATRN), a secreted protein regulating the chemotactic activity.In SCAT, the predominant biological pathway shared by over-expressed adipose genes in pigs from the low RFI line concerned the generation of precursor metabolites and energy. Notably, glucose metabolic process (5 genes) and lipid catabolic process 6 genes) were up-regulated processes in those pigs when compared with pigs from the high RFI line (Table  genes weSREBF2 (sterol regulatory element binding transcription factor 2) acting as a key transcription factor to control cholesterol homeostasis , regulation of apoptosis and cell death , and cholesterol and steroid metabolic processes  thus emerged again as modulated pathways due to line. Importantly, fatty acid catabolic process was represented by genes that were under-expressed in muscle of pigs from the low RFI line and by genes that were up-regulated in adipose tissues of the same pigs when compared with pigs from the high RFI line.P < 0.10) than what was expected based on data acquired in sib mates raised in the selection farm  and reported in former generations raised in experimental farms under conventional diets . Pigs of the low RFI line also had greater muscle mass when compared with pigs from the high RFI line, which agrees with previous data [This study considered pigs from two genetic lines divergently selected for RFI, a biological estimate of the efficiency of feed utilization. Phenotypically, pigs from the low RFI line had an improved gain-to-feed ratio during the test period, the difference between lines being commensurate to our previous observations in a former generation of selection . In this. Pious data , multivaCD40 (TNF receptor superfamily member 5) in the four examined tissues was notably proposed as one of the hallmarks of pigs from the high RFI line. This may be due to the regulatory role of this gene (which is expressed by T and B lymphocytes) in a wide spectrum of molecular processes during the initiation and progression of adaptive immunity [CTSC (cathepsin-C). This gene codes for a lysosomal cysteine proteinase playing a central role in bacterial killing and immune regulation in T lymphocytes [NTN1 (netrin-1) as another key gene for which a differential expression in the four tissues brought an important contribution to genetics difference for RFI, may be related to the role of this laminin-related secreted protein in directing the retention of macrophages in adipose tissue [TLR2 and TLR4), which are known to be expressed by both myoblasts and myotubes [Immune response appears to be the most important biological pathway of clustered under-expressed genes in the four tissues examined in pigs from the low RFI line (more efficient) when compared with pigs from the high RFI line (less efficient) at 132 days of age. In agreement with our results, levels of serum inflammatory markers such as the acute phase protein haptoglobin have been reported to be lower in Yorkshire pigs selected for low RFI . In cattimmunity , 26. Thiimmunity . Anotherphocytes . Identife tissue . In accomyotubes ; genes imyotubes . From thmyotubes . Moreovemyotubes  and aftemyotubes . These dPON1) had a higher expression level in the four tissues of pigs from the low RFI line when compared with pigs from the high RFI line. This glycoprotein is transferred from liver to prevent the accumulation of lipid peroxides and reduce macrophage inflammatory response in tissues [UQCRB, a gene coding for the ubiquinol-cytochrome c reductase binding protein which plays roles in oxygen sensing and ROS-mediated signaling [CAT, SOD1, DUOX2) when pigs from the low RFI line were compared with pigs from the high RFI line. This suggests that feed efficiency might be associated with different ROS production between adipose and lean tissues, and(or) that these tissues dealt differently with ROS detoxification.The present study identifies response to oxidative stress as another biological process shared by the same genes which were differentially-regulated in the four tissues depending of the line. A small network of co-expressed genes also linked response to oxidative stress with immunity and apoptosis. In agreement with these findings, it is generally admitted that immune system activation releases highly reactive species to kill pathogens, which can cause oxidative damage to tissues. Especially, paraoxonase-1  and USP33 (ubiquitin specific peptidase 33) regulating ubiquitin-dependent protein catabolic process, were among the top molecular contributors in the differences between low and high RFI lines. Many ubiquitin-conjugating enzymes were also specifically over-expressed in muscle of the most efficient pigs. Both protein targeting and the ubiquitin system are known to regulate virtually all aspects of cellular functions [Finally, the largest number of genes differentially regulated in the four tissues between pigs from the low or high RFI lines belonged to protein metabolic processes. Similarly, candidate genes in pathways related to protein metabolism have been suggested in genomic regions accounting for feed conversion ratio in cattle . Ribosomunctions . Therefounctions , up-regulongissimus muscle, the gene expression program related to glycogen utilization was up-regulated in pigs from the low RFI line. This is in agreement with our previous findings of higher level of glucose issued from glycogen hydrolysis and elevated glycolytic potential in pigs selected for low RFI [PHYH. This gene codes for the phytanoyl-CoA 2-hydroxylase, an enzyme involved in metabolizing phytanic acid, a metabolite of phytol regulating the alpha-oxidation process in the peroxisome. Examples for such an opposite regulation of PHYH gene expression between liver and adipose tissue have been provided in rodents fed high-fat diets [Tight regulation of glucose homeostasis is essential for fast-twitch muscle where glucose metabolites are the major energy sources. In  low RFI . Moreove low RFI . In the  low RFI . Altoget low RFI , furtherat diets , 46. To at diets . The preCYP) superfamily of enzymes that catalyze reactions in the synthesis of cholesterol and steroids were down-regulated in liver and PRAT of pigs from the low RFI line (more efficient). Similarly to our results, other CYP genes were found as down-regulated genes in liver of low RFI cattle [LCAT (lecithin cholesterol-acyltransferase) responsible for the formation of most plasma cholesteryl esters, and its cofactor APOA1 (apolipoprotein A1) which promotes cholesterol efflux, were up-regulated in liver of pigs from the low RFI line. Taken together, these changes suggest that cholesterol level in liver may be reduced  in more efficient pigs. Interestingly, positive relationships between cholesterol plasma level and RFI values have been previously reported in pigs [Finally, members of the cytochrome P450  have been investigated [IGF1 expression in pig muscle decreased with advancing age [Among the four examined tissues, the ent pigs . On the stigated . Considecing age , this sucing age . AltogetThis study presents novel findings on the transcriptomic modulations related to feed efficiency considering four tissues involved in energy generation and use in pigs. Immune response, protein metabolism and response to oxidative stress were identified as the main molecular pathways that were associated with a genetic difference for RFI and a general difference for efficiency phenotypes in pigs at 132 days of age. Therefore, non-productive functions in metabolic tissues are likely important processes for feed efficiency. It may be further suggested that dietary compounds with both anti-inflammatory and anti-oxidant activities can improve feed efficiency in growing pigs.http://ethique.ipbs.fr/sdv/charteexpeanimale.pdf). All pigs were reared in compliance with national regulations and according to procedures approved by the French Veterinary Services at INRA experimental facilities.The care and use of pigs were performed following the guidelines edited by the French Ministries of High Education and Research, and of Agriculture and Fisheries (Purebred Large White pigs were considered in the present study. These pigs originated from two lines divergently selected over eight generations for RFI. Principles of selection were described by Gilbert and colleagues . Brieflyn = 24 per line) originated from the selection herd  were reared at INRA Pegase . From 74 d of age onwards, pigs were randomized by line in two dietary groups (n = 12 per diet and per line), a high-fat high-fiber diet or a low-fat low-fiber diet. Detailed information about diets can be found in our associated publication [longissimus muscle just below SCAT sampling in the right carcass side. After opening the abdominal cavity, the liver and the entire perirenal adipose tissue (PRAT) were collected and weighed. Tissue samples were immediately cut, frozen in liquid nitrogen, and stored at −75 °C until analyses. The day after slaughtering, the entire dorsal SCAT (backfat) and the longissimus muscle were removed from the left side of the chilled carcasses and weighed.In this experiment, 48 barrows  were thus generated for further analyses.Total RNA was extracted from each tissue by homogenization in Trizol reagent  and purified using a silica-membrane technology under vacuum . The method included a DNase digestion step to remove contaminating DNA. The quantification of RNA was performed by using a NanoDrop® ND-1000 spectrophotometer . Ratios of A260/280 and A260/230 were greater than 1.7 in all samples, denoting good purity. The RNA integrity was assessed using the Agilent RNA 6000 Nano kit and an Agilent 2100 Bioanalyzer . Average RNA integrity numbers were 8.4 ± 0.4 (mean ± SD). Altogether, 192 RNA samples . Each of the 192 RNA samples was labeled with Cy3 dye using the Low Input Quick Amp Labeling kit (Agilent Technologies) following the manufacturer’s instructions. Briefly, a two-step procedure generates fluorescent complementary RNA (cRNA) by using T7 RNA polymerase, which simultaneously amplifies target and incorporates cyanine-labeled CTP. Samples were then purified with an RNeasy Mini kit . Microarray hybridizations were carried out in Agilent’s hybridization chambers containing 600 ng of Cy3-labeled cRNA sample per hybridization. Hybridization reactions were performed at 65 °C for 17 h using Agilent’s Gene Expression Hybridization kit. After washing, microarrays were scanned at 3 μm/pixel resolution using the Agilent DNA Microarray Scanner, and images were analyzed with Agilent Feature Extraction Software .Analyses were performed using the R software version 2.10.0. Raw spots intensities were first submitted to quality filtration based on four criteria: intensity, uniformity, saturation and outliers detection. Intensities of filtered spots were transformed into log2(Cy3), and data were normalized by median centering, i.e. subtracting the median value across all probes from all raw values for each sample.http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=) GSE84092 for longissimus muscle, GSE84093 for liver, GSE70837 for PRAT and GSE70836 for SCAT. First, the microarray data obtained in each tissue were analyzed by a two-way analysis of variance (ANOVA) with RFI line, diet and the interaction between line and diet as the main effects. Because there is no significant interaction between line and diet (cut-off P > 0.05), the line and diet effects can be separately inferred. In this study, only the line effect on transcriptomes was analyzed and discussed. For the diet effect on adipose tissue transcriptomes, one may refer to our associated publication [P-value < 0.01 and fold-change between conditions > |1.1|) was considered as a heuristic way [P-values for a control of the FDR were also calculated in the four tissues  and are accessible through GEO subseries accession numbers  were projected as supplementary variables in the principal MFA plane . Calculating the correlations between each DEP and the first two MFA dimensions allows deciphering the DEP which were mainly responsible for variability in overall molecular responses. In the present study, the retained correlation threshold was r > |0.70| (P < 0.001).Complementary to this descriptive analysis, the data integration method MFA was used to highlight the most important contributors to RFI difference. Details for this mathematical method that analyses the relationships between tissue-wise probe sets can be read elsewhere . Its abihttp://david.abcc.ncifcrf.gov/ [P-value were obtained. In this study, E ≥ 1 and P-value ≤ 0.05 were retained to list the groups of genes. Plausible regulatory networks that included expression changes were then visualized using QIAGEN’s Ingenuity® Pathway Analysis , with the main goals to generate functional hypotheses and identify central molecules in small sub-networks.The gene ontology terms for biological processes (GOBP) were automatically searched within lists of DEG, using the Database for Annotation, Visualization and Integrated Discovery (DAVID) bioinformatics resource database (crf.gov/ ). This acrf.gov/ . The resn = 48 pigs) in the four tissues, by using High Capacity RNA to cDNA Kit . Primers were designed from porcine sequences available in Ensembl or NCBI databases using Primer Express® v3.0 software (Applied Biosystems). Detailed information for primer sequences (forward and reverse) is provided in Additional file TM real-time PCR system (Applied Biosystems). As stated by the GenNorm algorithm among three other tested reference genes, TOP2B gene was identified as the most stable housekeeping gene and was used for normalization. Using R software, the ANOVA model used for microarray analyses was then applied to determine significant effect of line on gene expression levels.Technical validation of microarrays data was performed by examining expression levels of target genes by qPCR. First-strand cDNA synthesis was performed with 1 μg of total RNA used for microarray analysis Additional file 2:Lists of genes differentially expressed between pigs from the low or high RFI lines in muscle, liver and two adipose tissues. (XLSX 366 kb)Additional file 3:Comparison between microarrays and qPCR for target genes. (DOCX 26 kb)Additional file 4:Differentially expressed genes shared by muscle, liver and two adipose tissues between pigs from the low or high RFI lines. (XLSX 31 kb)Additional file 5:Networks of genes involved in immune response (DOCX 658 kb)Additional file 6:Network of genes encompassing intracellular signaling pathways (DOCX 346 kb)Additional file 7:Top molecular contributors to RFI difference across tissues as indicated a multiple factor analysis (MFA). (DOCX 28 kb)Additional file 8:Genes participating to relevant GO biological processes specifically in longissimus muscle. (DOCX 28 kb)Additional file 9:Genes participating to relevant GO biological processes specifically in liver. (DOCX 22 kb)Additional file 10:Genes participating to relevant GO biological processes specifically in adipose tissues. (DOCX 22 kb)


And the expression level of ERCC1 was significantly higher in CAD cases than controls. Multiplicative interactions among SNP rs11615, alcohol drinking, history of T2DM, and history of hyperlipidemia could increase 5.06-fold risk of CAD (P = 1.59 × 10−9). No significant association of rs2298881 and rs3212986 with CAD risk was identified. Taken together, SNP rs11615 in ERCC1 gene might confer susceptibility to CAD and severity of coronary atherosclerosis in a Chinese Han population.Excision repair cross-complementing 1 (ERCC1) gene encodes ERCC1 protein, which is mainly responsible for the repair of DNA damage in different diseases including coronary artery atherosclerosis by acting as a rate-limiting element in nucleotide excision repair (NER). Using a three-stage case-control study with 3037 coronary artery disease (CAD) patients and 3002 controls, we investigated associations of three single nucleotide polymorphisms (SNPs) with CAD risk and severity of coronary artery atherosclerosis in Chinese Han population. In the discovery set, the variant allele T of rs11615 was significantly associated with higher CAD risk  and severity of coronary artery atherosclerosis . These associations were more remarkable in the merged set (adjusted OR = 1.23, P = 8 × 10 The main pathogenesis of CAD is atherosclerosis, a form of chronic inflammation characterized by accumulated deposition of lipoproteins, migration of monocyte and macrophage, and eventually atheroma formation and rupture3. Recent publications report that CAD is etiologically related to DNA damages caused by reactive oxygen species (ROS)4, lipoprotein oxidation and peroxidation5. DNA damage associating with atherosclerosis occurs in circulating cells, vascular smooth muscle cells (VSMCs) and endothelial cells of the vessel wall. Unrepaired DNA damage promotes endothelial cell dysfunction, monocyte migration, macrophage and foam cell death, VSMC senescence, and eventually contributes to plague rupture via inducing expression of adhesion molecules and release of inflammatory cytokines8. One of the most important DNA repair systems is nucleotide excision repair (NER), which is highly versatile and involved in repairing a wide variety of sophisticated DNA damage, such as helix-distorting DNA lesions and DNA adducts, induced by endogenous and exogenous stimuli10.Coronary artery disease (CAD) is the most common heart disease, which is caused by stenosis in coronary arteries and its branches due to genetic and environmental factors11. ERCC1 is mainly responsible for making incision at the 5′ terminus of DNA lesions by forming a structure-specific endonuclease complex with xeroderma pigmentosum complementation group F (XPF) and may contribute to positioning the nuclease complex at the lesion site by interacting with Xeroderma pigmentosum complementation group A (XPA)9. Besides evidence of molecular studies, in animal model studies, the knockdown and overexpression of ERCC1 was related highly to the increase and decrease of infarct volume in the ischemic brain respectively12. Mice with ERCC1 deficiency showed vascular dysfunctions, including elevated blood pressure and increased vascular stiffness13 and also led to elevated serum cholesterol level and the expression change of genes involved in metabolism of cholesterol, which have been considered as traditional risk factors for CAD15. Based on the function of ERCC1 and the effect of ERCC1 deficiency on brain ischemia, vascular dysfunction, and cholesterol metabolism, we guessed that the SNPs related to ERCC1 expression might also contribute to CAD risk since CAD and stroke are both belong to atherosclerosis cardiovascular diseases due to high cholesterol levels. Then, we chose three SNPs in ERCC1 for further study. One is synonymous C19007T (rs11615) polymorphism locating at exon IV, which has been reported to be associated with differential ERCC1 mRNA and protein levels16. The other is C8092A (rs3212986) in 3′-untranslated region (UTR) and may regulate ERCC1 expression by affecting ERCC1 mRNA stability17. The last is rs2298881 locating in intron I, which has been reported to alter ERCC1 expression by affecting ERCC1 promoter activity18.Excision repair cross-complementing 1 (ERCC1) is the rate-limiting element in NER systemTherefore, in the present study, we performed a three-stage case-control study to investigate the association of these three SNPs with CAD risk and severity of coronary artery atherosclerosis in a Chinese Han population.Baseline and clinical characteristics of study populations were shown in Table −6  after adjusting for age, sex, BMI, smoking, alcohol drinking, hypertension, T2DM and hyperlipidemia (Supplementary Table −4 in the dominant model)  , T2DM (Pinter = 0.013) and hyperlipidemia (Pinter = 0.018) to increase CAD risk  of SNP rs11615 with increased CAD risk in almost all subgroups, expect for participants without alcohol drinking habit Table . After t−9) increased risk of CAD in the participants with variant genotypes (CT + TT) of SNP rs11615, alcohol drinking and history of T2DM and hyperlipidemia compared to the reference group, which may be an evidence of genetic and environmental interactions  analyses based on the result of multiplicative interaction analyses. Hyperlipidemia had the greatest influence on CAD risk among the factors included, which consisted with it being the initial split node in CART analyses. Subsequent inspection of the CART tree revealed a 5.06-fold (P = 1.59 × 10−5)  , validation set P = 0.004), replication set (P = 0.015) and merged set  and plasma ERCC1 level in 78 subjects (39 CAD patients vs 39 controls), all of whom were randomly selected from the validation and replication sets. After adjusting for covariates, ANCOVA models showed that ERCC1 mRNA expression  were higher in CAD patients than controls and then a significant association was found between variant genotypes (CT + TT) and decreased ERCC1 mRNA expression in CAD patients (P = 0.033) but not in controls (P = 0.488)  and participants with alcohol drinking, T2DM and hyperlipidemia. And CART analyses shown that variant genotypes (CT + TT) of SNP rs11615, combined with the above three CAD risk factors, could increase CAD risk up to 5.06 times. Furthermore, variant genotypes (CT+TT) of SNP rs11615 contributed to the severity of coronary artery atherosclerosis. In addition, it presented that CAD patients had a significantly higher ERCC1 mRNA expression levels than controls, however we found the statistical association between the CT + TT genotype and a lower ERCC1 expression level only in CAD patients.19. In this study, we found a higher frequency of variant genotypes (CT + TT) in CAD patients. And the patients with variant genotypes (CT + TT) had a decreased level of ERCC1 expression. Besides, a previous study in cell lines got coincident results that ERCC1 levels in human ovarian cancer cell (MCAS) with T allele of SNP rs11615 reduced almost 60% of that in human ovarian cancer cell (A2781/CP70) with wild-type ERCC1 sequence20. So we inferred that the T allele of SNP rs11615 was associated with increased risk and severity of CAD in Chinese Han population, probably by reducing the expression level of ERCC1 compared to the C allele. However, potential mechanism needs to be further studied.Actually previous studies have reported that mice with ERCC1 deficiency showed a higher mutation frequency, increased genomic instability, the dramatic accumulation of unrepaired lesions and then the development of cardiovascular disease like CAD22. Subsequent bioinformatics using ENCODE at UCSC (http://genome.ucsc.edu/ENCODE/) further revealed that variant allele of SNP rs11615 changed the binding site of ZNF263 transcription factors, which might have the ability to influence the expression of the target gene23. Moreover, Epigenome Browser (http://epigenomegateway.wustl.edu/) also showed that the wild type of SNP rs11615 located in a region having potential enhancer activity and the variant allele of this locus had the possibility to relatively down-regulate ERCC1 expression  at codon 118 of the ERCC1 gene, converting a high-usage codon (AAC) to a relatively infrequent one (AAT), could affect ERCC1 translation and the level of ERCC1 protein and thereby impair repair activity on account of codon usage being decreased by almost half24. It is common knowledge that differential extent of DNA damage accompanied with varying degree of expression of DNA repair-related genes. Consistent with this, immunoreactivity for 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dG), an oxidative DNA damage marker, was detected in atherosclerotic plaque VSMCs, macrophages and endothelial cells, but not in VSMCs of adjacent normal media25. Therefore, ERCC1 expression levels in peripheral blood, only relatively reflect the repair situation of coronary artery in CAD patients to a certain extent, and future studies are needed to detect ERCC1 expression in atherosclerotic plaque.In the present study, we just found the association of variant genotypes of SNP rs11615 with ERCC1 mRNA expression levels in CAD patients, but not in controls. These results could arise from the restriction of small sample study, or dominants of DNA damage in patients, or because peripheral blood cells are not in step with plaque tissue. DNA damage occurred mainly in atherosclerotic plaque, ranging from deletions of parts of chromosomes to DNA strand breaks and DNA adducts and that was more serious than DNA damage in peripheral white blood cells26. Accumulation of the unrepaired DNA damage which resulted directly from alcohol drinking or indirectly from DNA repair enzyme (ERCC1) deficiency, contributed to the progression of CAD by activating the release of inflammatory cytokines13. And it was reported that interaction of SNP rs11615 with alcohol drinking could increase the risk of laryngeal cancer27. For history of T2DM, recent studies have shown that abnormal glucose metabolism could enhance ERCC1 expression and protein levels by activating the release of insulin and that mice with ERCC1 deficiency showed a progeroid phenotype with disturbance of glucose metabolism29. As for hyperlipidemia, lipid oxidation and peroxidation was common reason for DNA damage and then aggravated the development of atherosclerosis. By the way, ERCC1 deficiency could affect lipid metabolism by up-regulating genes related to extracellular efflux of cholesterol15. All the above evidence, combined with the proved effect of SNP rs11615 on ERCC1 expressions, support that development of CAD could be further aggravated by interaction between SNP rs11615 and traditional CAD factors . However, further functional studies are needed to explore the potential mechanism of this interaction.To explore potential genetic and environmental interaction effect on CAD risk, we performed CART analyses and found that participants with variant genotypes (CT + TT) of SNP rs11615, alcohol drinking and history of T2DM and hyperlipidemia tended to have increased CAD risk. Heavy alcohol drinking always generated a great deal of reactive oxygen species (ROS), which subsequently induced a variety of DNA damage as well as low density lipoprotein (LDL) oxidation12 and the report that ERCC1-deficient mice showed increased vascular stiffness and vascular dysfunction13. Therefore, combined with the association of SNP rs11615 with ERCC1 expression, we speculated that SNP rs11615 might accelerate the progress of coronary artery atherosclerosis.The present study evaluated severity of coronary artery atherosclerosis by calculating Gensini score and revealed that variant genotype (CT + TT) of SNP rs11615 was associated significantly with higher Gensini scores. This result was supported by a recent study that knockdown of ERCC1 expression exhibited an increasing infarct volume in the ischemic rat brainSome limitations should be taken into consideration. First, we just genotyped three most common SNPs in ERCC1 and failed to explore the effect of other ERCC1 genetic variants on CAD risk. Second, although we have adjusted for common CAD risk factors, other genetic and environmental factors could also involve the development of CAD. Finally, ERCC1 expression and protein levels in plaques were not detected in our study due to restriction of samples.In conclusion, this three-stage case-control study of 3037 cases and 3002 controls, for the first time, suggested significant association of SNP rs11615 with CAD risk as well as possible interactions among SNP rs11615, alcohol drinking and history of T2DM and hyperlipidemia. Functional studies are required to validate our findings and illuminate the potential mechanism.This three-stage case-control study, involving 3037 CAD patients and 3002 controls, was selected from three sets: the discovery set (study 1) with 806 CAD cases and 816 controls from Zhongnan Hospital of Wuhan University between January 2011 and December 2012; the validation set (study 2) with 1124 CAD cases and 1118 controls from Wuhan Asia Heart Hospital between March 2013 and October 2014; and the replication set (study 3) with 1107 CAD cases and 1068 controls from the above two centers between March 2015 and May 2016. All study participants were Han nationality by self-description.31 including cigarette smoking, alcohol drinking and histories of hypertension, type 2 diabetes mellitus (T2DM) and hyperlipidemia  and clinical data, such as blood pressure, body mass index (BMI), fasting plasma glucose (FPG) and lipid levels. This study and informed consent were approved by Medical Institutional Review Board of Zhongnan Hospital of Wuhan University and conformed to guidelines of the Declaration of Helsinki.The diagnosis criteria of CAD was stenosis of more than 50%, confirmed by coronary angiography, in at least one segment of main coronary artery or their main branches. Patients with the following diseases were excluded: cardiac diseases including acute heart failure, congenital heart disease, myocardial bridge or cardiomyopathy and coronary artery spasm, as well as systemic disease such as malignancy, autoimmune disease, severe liver or renal disease and immunosuppressive drug use. Controls were age- and gender-matched participants without detectible luminal stenosis identified by coronary angiography (1054 controls) and healthy individuals without above-mentioned cardiac or systemic diseases discovered by physical examination (1948 controls). The following data were extracted, traditional CAD risk factorshttp://www.1000genomes.org/)32, three most common SNPs, which were rs11615 in exon IV, rs3212986 in 3′-untranslated region (UTR), and rs2298881 in intron I, were selected (Supplementary Table A large number of studies investigated the association of single nucleotide polymorphisms (SNPs) in ERCC1 gene with cancer. Based on the 1000 Genome Database   programs. CART adopted Gini index as splitting criterion to build a hierarchical classification tree for finding an optimal combination of genetic and environmental factors, which could predict CAD risk more forcefully38. Logistic regression analyses were used to assess the association of each terminal node (TN) with CAD risk.To evaluate the potential high-order genetic and environmental interactions in CAD risk, we carried out classification and regression tree (CART)−ΔΔCq method was used to calculate the relative expression of ERCC1 by normalizing to the internal reference gene (GAPDH).After extracting from human peripheral blood leukocytes using Trizol reagent , total RNA was prepared to remove DNA contamination using the RNase-Free gDNA eraser and then conduct reverse transcription using a reverse transcriptase kit . The cDNA product was used to determine ERCC1 mRNA expression using real-time quantitative PCR (RT-qPCR) analysis with the SYBR-Green kit on a CFX96 Touch system . The 2Plasma samples were isolated from the whole blood by centrifugation and stored at −80 °C immediately until use. According to the manufacturer’s instructions, we measured plasma concentration of ERCC1 by an enzyme-linked immunosorbent assay (ELISA)  and then quantified it using a standard curve with the detection range of 30–1200 pg/ml.The differences in clinical characteristics between cases and controls were analyzed by the Pearson chi-square test  and the Student’s t-test (continuous variables). The Pearson chi-square test was also used to assess Hardy-Weinberg equilibrium (HWE) of each SNP in three independent study populations. Odds ratio (OR) and their 95% confidence intervals (CIs) were calculated by logistic regression analyses to estimate the association of ERCC1 SNPs with CAD risk before or after adjusting for age, sex, BMI, smoking, alcohol drinking, hypertension, T2DM and hyperlipidemia. Genetic and environmental interactions of ERCC1 SNPs and selected stratification variables on CAD risk were evaluated by the multiplicative likelihood ratio test. When assessing severity of coronary artery atherosclerosis by Gensini score, Mann-Whitney U test and logistic regression analyses were used to analyze the association of ERCC1 SNPs with Gensini score. All the analyses above were carried out using SPSS 22.0  and P values were considered statistically significant below the cut-off value of 0.05. Also, Bonferroni correction was performed for multiple comparisons in the entire analyses. Moreover, power analyses were performed using Power and Sample Size program (PS) 3.0 .All data generated or analyzed during this study are included in this published article (and its Supplementary Information files).Supplementary Information


Supplemental Digital Content is available in the text. The latter procedures are performed using our original variation of the Lejour breast reduction technique,2 whose main complications include moderate wound dehiscence and limited fat necrosis with an overall rate of 10%. Other complications include postoperative hematoma requiring surgical revision (4%), temporary nipple sensation loss (2%), and major fat necrosis with wide wound dehiscence (2%), the latter being reported only in patients with large adipose breasts.In our laboratories, we are carrying out investigations on human fibroblasts under different experimental conditions.3 The patient’s body mass index at the time of the operation was 34; her medical history included long-standing hypothyroidism successfully treated with thyroid hormone replacement therapy and an anxious syndrome treated with citalopram, a selective serotonin reuptake inhibitor. Due to a relevant breast asymmetry, the reduction accounted for 1,169.5 g in the right breast and 674 g in the left one, and the reduction of the distance between the clavicular notch and the nipple was 17 cm on the right and 13 cm on the left.A few months ago, we performed a breast reduction in a formerly obese nonsmoker patient, in apparently healthy conditions, complaining of chronic cervical and dorsal pain related to her breast hypertrophy.Equisetum arvense- and collagen-based ointment that eventually allowed a complete wound healing in 10 weeks.A slow onset venous congestion in the nipple-areola complex yielding to a bilateral partial nipple-areola complex necrosis and a major wound dehiscence with fat necrosis in both breasts complicated the postoperative course. The patient underwent surgical debridement and a combination of local treatments including negative pressure therapy, hyaluronan felt application, iodine powder–based advanced dressing, and an 4 displayed an unusually torpid fibroblast growth trend in comparison with the cultures derived from other patients . The viable cell counts in the fibroblast culture from this patient at 24, 48, 72, 96, and 120 hours reported markedly lower values than the counts at the same times in a standard control culture.Interestingly, the skin samples harvested from this patient and cultured in Dulbecco’s Modified Eagle Medium (DMEM) enriched with fetal bovine serum, penicillin, streptomycin, gentamicin, and L-glutamineThe latter evidence raises the question whether the unpredictable complications reported in elective plastic surgery procedures that are not attributable to surgical misconduct or patient’s comorbidities and/or risk factors might be related to an unexplored intrinsic cell torpidity that would be worth investigating with genetic and/or metabolic tests. Indeed, although a preliminary preoperative in vitro screening is not feasible at the moment in all the patients undergoing elective plastic surgery, an ex post study investigating such a peculiar cellular behavior might be proposed in selected cases to properly address or rebalance the respective patient/physician liabilities within the context of medico-legal litigation.