Cervical cancer is the most common cancer among women in Sub-Saharan Africa. Cervical cancer is treatable if detected timeously, yet only 20% of South African women have ever been for a Pap smear in their lifetime due to limited access to screening, transport or child care responsibilities.To evaluate the acceptability of self-collection for cervical cancer screening. We aimed to identify which self-collection device women prefer and if they would consider using them for routine cervical cancer screening.HIV-positive women (>18 years) from urban and rural HIV clinics were interviewed following an education session on HIV, human papillomavirus (HPV) and cervical cancer. Participants were shown three self-collection devices; (i) an Evalyn cervical brush, (ii) a Delphilavager and (iii) a tampon-like plastic wand before completing a short questionnaire.A total of 106 women from the urban (n = 52) and rural (n = 54) clinic were interviewed. Overall 51% of women preferred the cervical brush, while fewer women preferred the tampon-like plastic wand (31%) or lavage sampler (18%). More than 75% of women from the rural site preferred the cervical brush, compared to 22% from the urban site (p < 0.001). Women from the urban clinic preferred the tampon-like plastic wand (45%) and then the lavage sampler (33%), as compared to women from the rural clinic .Women from urban or rural settings had different preferences for the various self-collection devices. Patient self-collection with HPV testing may be an acceptable way to improve coverage to cervical cancer screening in high risk HIV-seropositive women. Cervical cancer is the most prevalent cancer in HIV-infected women in sub-Saharan Africa and the leading cancer-related cause of death among women . ApproxiOncogenic types of human papillomavirus (HPV) are the central cause of cervical cancer and its precursor lesions. HPV16, 1HPV infections are more common in HIV-positive women and the prevalence of single and multiple HPV infections is higher among HIV-positive women than HIV-negative women. In HIV-iWomen living in resource-limited countries are especially at risk due to poor access to cervical cancer screening and treatment . InvasivPrecursor lesions of cervical cancer are highly amenable to treatment and therefore identifying effective screening methods allows for the prevention of invasive cervical cancer and associated morbidity and mortality. Screening for cervical cancer in women 30 years and older is well established in resourced countries where the Pap test with the HPV test forms the hallmark of screening. In manyHPV screening through patient self-collection has been shown to have a high sensitivity and specificity for the detection of high risk HPV types which can lead to cervical cancer. Sensitivity and specificity for detection of cervical intraepithelial neoplasia grade 2 or worse (CIN2+)was 87.5% and 77.2% for self-sampling using a cervical brush compared to 96.8% and 79.7% for the direct test (p < 0.001), respectively. HPV testStudies have assessed the acceptability and usability of self-collected sampling for HPV testing in resource-developed settings and some resource-limited settings such as Mexico and China. however there is limited data on the acceptability of HPV self-collection in South African HIV-infected women, a population that are most likely to benefit if implemented –15. We tQuestionnaires assessing attitudes and preferences were conducted among HIV positive patients attending two HIV treatment clinics; ThembaLethu Clinic, Johannesburg, South Africa (urban) and Topsy Clinic, Mpumalanga, South Africa . ThembaLethu Clinic is one of the largest clinics providing ART in South Africa. It is an urban clinic, located at the Helen Joseph Hospital in Johannesburg. The clinic is an accredited public sector Comprehensive Care, Management and Treatment (CCMT) site for HIV-positive patients. Firnhaber and colleagues reported that 95% of women tested at this clinic (n = 148) had HPV DNA with 83% having 1 or more HPV oncogenic types .The Topsy Clinic is located in rural Southern Mpumalanga. This province has one of the higher provincial prevalence rates of HIV, being 15.4% in 2008, and with pregnant women at 32% in 2007. The Topsy Foundation partners with rural communities, empowering people infected with, and affected by HIV and AIDS, through medical care, social support and skills development.HIV-positive adult (>18 years of age) women, willing to participate in the study and provide written informed consent, were enrolled consecutively in the study. Participants were English speaking, between 20 and 65 years of age and were not pregnant.Ethics approval was obtained from the University of the Witwatersrand (protocol number HREC M121051).After obtaining informed consent, women were offered an educational session on HIV, HPV and cervical cancer, presented in English, by the attending clinician. This was followed by a self-administered questionnaire comprising of 15 questions. Participants were shown three different self-collection devices .Three devices were used, each with an acceptable sensitivity (Se) and specificity (Sp) for the detection of high-grade cervical pre-cancer (CIN2+) using physician-collected cervical samples and cytology as the gold standard  or for the detection of oncogenic HPV  –19 Figu. The EvaThe clinician briefly explained each device and how they work. Women could see and touch each device with the opportunity to ask questions before completing the questionnaire. The devices were presented in a different order to distribute the potential order effects equally, as described by Richman et al, 2011. The ordParticipants were asked to complete an anonymous self-administered questionnaire in English. Questions included participant age and self-reported history of Pap-smear (ever and how recent). The questionnaire also included close-ended questions including if they would prefer self- collection devices over conventional Pap-smear and their preference and willingness to use the devices they were shown. In addition, participants were asked if they thought self-testing could be valuable over Pap-smear and if it could prevent cervical cancer.Participant questionnaires were captured in Excel and exported into SAS version 9.3 . Patients were stratified into two groups: urban and rural. Groups were compared using Student t test  or Kruskal-Wallis  for continuous variables and Chi-square (χ2) test or Fisher exact test for proportions, where appropriate. A p value < 0.05 was considered significant. Patient responses were summarized in A total of 106 HIV-positive women were recruited from the waiting rooms of two adult HIV government outpatient clinics: 52 from the urban clinic and 54 from the rural clinic. All women that were approached were willing to participate. Women were of similar age in both groups . More than 30% (n = 36/106) of women had never had a Pap smear, while 51% (n = 54/106) had one in the last 2 years. These numbers were similar in both groups  Overall 51% of women preferred the cervical brush, while fewer women indicated that they would rather use the tampon-like plastic wand (31%) or lavager (18%). There was a strong correlation between liking a device and being willing to use it . All of the women indicated that they would be willing to perform a self-test for cervical cancer screening.There were differences in the acceptability of different self-collection devices among women from urban or rural areas. More than 75% of women from the rural site preferred the cervical brush, compared to 22% from the urban site (p < 0.001). Women from the urban HIV clinic preferred the tampon-like plastic wand (45%) and then lavager (33%) compared to only a few women from the rural clinic  In this cohort of rural and urban HIV-positive women there was a universal acceptance of self-collection as a potential way to improve coverage to cervical cancer screening in high risk HIV-seropositive women and complement conventional Pap smear testing. Urban women seemed to prefer the tampon-like plastic wand whereas rural women preferred the cervical brush. These results may have significant implications for future screening programs for cervical cancer in women with HIV.Results from our study support those from other studies. In a study conducted in Mexico to assess the acceptability of HPV self-sampler, most patients reported being comfortable when using the self-test . We asseStudies have shown that high-risk HPV testing on self-samples appears to be at least as, if not more, sensitive for CIN2+ as cytology on clinician-obtained cervical samples, though often less specific. Variations in clinical performance likely reflect the use of different combinations of collection devices and HPV tests . HoweverThis study did not access barriers or factors that influenced women's perceptions of the different devices. Others have identified the most influential barriers were fear, lack of signs or symptoms of illness, husbands′ influence, cost, lack of time, being unable to read, and lack of trust in the medical community . In a stStudies have shown that HPV self-collected testing significantly improved the participation of women who did not routinely attend cervical cancer screening programs . HoweverBecause it is known that under-screened women are at increased risk of cervical cancer, targeting non-attendees of the screening program could improve the effectiveness of cervical screening , 32. NonWith the recent availability of commercial home-based self-collection tests in South Africa it will be important to assess the acceptability, accuracy and cost-effectiveness of self-sampling compared to cytology based screening in South Africa. It may also be interesting to assess the acceptability and uptake of these tests in urban and rural settings in South Africa in light of our study findings which show that urban women prefer the tampon-like plastic wand whereas rural women preferred the cervical brush.Our findings have implications not only for HPV DNA testing but also for other self-testing methods and suggestthat interventions may need to be context specific in order for them to be effective. Few studies have assessed the acceptability of self-testing for HPV in HIV-positive women, a high-risk population that are most likely to benefit from early detection and improved participation in routine cervical cancer screening. Future studies are needed to assess the acceptability, accuracy and cost-effectiveness of self-collection for HPV, Chlamydia, Gonorrhoea or STD′s in HIV-positive women.Cervical cancer is a preventable cancer and a variety of screening mechanisms need to be evaluated to improve access in resource-limited areas and in areas of HIV prevalence such as Sub-Saharan Africa to reduce the disease burden and mortality of cervical cancer. Patient self-collection with HPV testing may be a way to improve coverage to cervical cancer screening in high risk HIV–seropositive women.


Dear Editor,5 These discoveries were paralleled by the description of phylogenetically closely related bovine astrovirus isolates (BoAstV) from the nervous tissue of cattle with encephalitis of unknown etiology in the USA (BoAstV NeuroS1) and by our laboratory in Switzerland (BoAstV-CH13).7 BoAstV-CH13 was retrospectively found in approximately one-quarter (5/22) of the cattle with etiologically unresolved non-suppurative encephalitis in Switzerland; however, most of these cases (17/22) were unrelated to BoAstV-CH13. Here, we report the identification of yet another neuroinvasive astrovirus in this set of cattle with non-suppurative encephalitis, which has less than 65% genetic similarity to currently known astrovirus isolates.Human astroviruses are of particular importance as one of the most common pathogens that cause juvenile gastroenteritis. In recent years, several astrovirus isolates were identified as potential causes of encephalitis in immunocompromised human patients.In 2006, a 4-year-old Braunvieh cow (case ID 42535) was notified as a clinical bovine spongiform encephalopathy (BSE) suspect to the Swiss authorities. Precise information about the clinical presentation was not available, but the spectrum of signs in BSE-suspect animals usually involves changes in behavior and temperament, hyper-reactivity, and incoordination. Post mortem BSE testing was negative, and histopathological examination led to the diagnosis of severe non-suppurative meningo-encephalomyelitis . This inin-silico subtraction of reads that aligned to the bovine reference genome and de novo assembly of the remaining reads, we identified four contiguous sequences (contigs) of 792, 1010, 1103, and 3170 nucleotides that matched with the highest amino acid sequence similarity (64%–83%) to different proteins of a sheep astrovirus isolate entry (accession number NC_002469.1) of the National Center for Biotechnology Information database. Gaps between the contigs were bridged by RT-PCR followed by Sanger sequencing. The 5′ and 3′ ends of the RNA molecule were determined by rapid amplification of cDNA ends (Illumina sequencing of frozen brain tissue RNA extracts (medulla oblongata) from animal 42 535 resulted in 21 443 420 read pairs. After DNA ends . This reDNA ends . RT-PCR DNA ends . Taken t8 The same topology was obtained in a maximum-likelihood tree that was based on the full-length capsid protein amino acid sequences (Full genome phylogenetic comparison placed the BoAstV-CH15 in the same cluster of previously described neurotropic astroviruses (equences . A slidiequences .n = 22) from the retrospective study mentioned above7 using the BoAstV-CH15 RT-PCR protocol. Besides case 42 535, one additional encephalitis case was reactive and showed the specific 388-bp amplicon. This animal was an unrelated neurologically diseased 7-year-old cow that was diagnosed with severe non-suppurative poliomeningoencephalitis and ganglioneuritis in 2007 , these conflicting results could be explained by the presence of either a different type of astrovirus or by coinfection with both viruses in the same animal. An RNA extract from the brain tissue of animal 42 799 was therefore similarly subjected to NGS. Mapping the obtained reads to the BoAstV-CH13 and BoAstV-CH15 genomes identified specific reads for 99% of the BoAstV-CH13 genome and 34% of the BoAstV-CH15 genome  in the brains of two neurologically affected cows. These findings extend previous reports on neurotropic astrovirus isolates in humans, mink, and cattle.5 This notion is supported by the finding that viruses of this clade have also been identified in the feces of a diseased patient10 and that there is evidence for a high prevalence of infection in healthy individuals as well.11In human patients, neurotropic astrovirus isolates revealed ∼95% nucleotide identity with each other, most of them belonged to the HuAstV VA1/HMO-C clade, and they were all identified in immunocompromised patients. It has therefore been postulated that central nervous system invasion occurred via the enteric system as a consequence of immune system impairment.14 and second, there is no evidence for an immunocompromised condition in the animals under investigation. The age of the affected cows ranged from 1.5 to seven years, and, thus, the presence of an underlying inherited primary immunodeficiency is unlikely. On the other hand, dairy cows held under intensive production management are often exposed to stressors, such as crowding, pregnancy, lactation, and metabolic imbalances, which may have immunosuppressive effects.15 However, information on clinical signs and disease history in these animals is sparse, and any conclusion regarding the disease pathogenesis in cattle would be premature. More comprehensive epidemiological investigations on the clinical presentation, prevalence, and diversity of astrovirus infections in cattle are needed.In bovines, the situation may be different. First, the neurotropic isolates have not yet been identified in feces samples of cattle,1 The diversity of the HMO clade suggests that transmission between animal species and between animals and humans occurred at different time points in astrovirus evolution. For instance, our data suggest that BoAstV-CH15 originated from a common ancestor with the enteric ovine isolate rather than with bovine enteric isolates. In this sense, future research into astrovirus infections should include activities in both veterinary and human medicine in terms of a ‘one-health' concept.The close phylogenetic relationship between animal and human feces and encephalitis isolates within the phylogenetic HMO clade raises an important question concerning the role of cross-species transmission events in astrovirus infection and evolution. Apart from one boy with astrovirus encephalitis living in the vicinity of a mink farm, there have been no clear epidemiological links between astrovirus infections in humans and animals.


Enterovirus species C (EV-C) consists of more than 20 types, among which the three serotypes of polioviruses, the etiological agents of poliomyelitis, are included. Biodiversity and evolution of EV-C genomes are shaped by frequent recombination events. Therefore, identification and characterization of circulating EV-C strains require the sequencing of different genomic regions. A simple method was developed to quickly sequence the entire genome of EV-C isolates. Four overlapping fragments were produced separately by RT-PCR performed with generic primers. The four amplicons were then pooled and purified prior to being sequenced by a high-throughput technique. The method was assessed on a panel of EV-Cs belonging to a wide-range of types. It can be used to determine full-length genome sequences through de novo assembly of thousands of reads. It was also able to discriminate reads from closely related viruses in mixtures. By decreasing the workload compared to classical Sanger-based techniques, this method will serve as a precious tool for sequencing large panels of EV-Cs isolated in cell cultures during environmental surveillance or from patients, including vaccine-derived polioviruses.Enteroviruses are among the most common viruses infecting humans and can cause diverse clinical syndromes ranging from minor febrile illness to severe and potentially fatal diseases.  Enterovirus species C (EV-C), genus Enterovirus, family Picornaviridae, are non-enveloped viruses with a single positive strand RNA genome. The virions contain one copy of the genome, which is about 7500 nucleotides in length and consists of two untranslated regions (5′- and 3′-UTR) flanking a unique large open reading frame. The polyprotein encoded by this open reading frame is first cleaved into three precursors (P1–P3) that are subsequently cleaved into functional proteins. P1 gives rise to the four structural capsid proteins (VP1–VP4) while P2 and P3 generate non-structural proteins involved in the viral cycle. Enteroviruses cause a wide spectrum of human diseases, with clinical signs ranging from mild febrile illness, such as the common cold, to severe forms, such as acute haemorrhagic conjunctivitis, myocarditis, encephalitis, and acute flaccid paralysis . Field isolates originated from stool samples collected in Madagascar in 2002  A1 Tompkins and CV-A19 NIH-8663, see below. These latter cannot be propagated in cell lines but can infect suckling mice. RNA of these two viruses was extracted from brains of mice inoculated intracerebrally. These brains were retrieved from the laboratory collection and no mice were used during this study. Brains were crushed in PBS before RNA extraction.EV-C full-length nucleotide sequences retrieved from the GenBank database were aligned using CLC Main Workbench 7.6.4 software (CLC bio). Eight degenerated primers were designed to target conserved genomic regions Table .Viral RNA was extracted from 250 μL of culture supernatants or clarified brain extracts using the High Pure Viral RNA kit , following the manufacturer's instructions.In order to check the RNA extraction step, a one-step real-time RT-PCR assay was performed on extraction products using a pan-enterovirus generic assay previously described . The thermocycler profile was 2 min at 94°C followed by 30 cycles of 30 s at 94°C, 30 s at 55°C, and 3 min at 72°C.The cDNA was then used as a template for amplification in four PCRs carried out in a final volume of 50 μL that included 5 μL of 10X PCR Buffer w/o MgClTen microliters of each PCR product were analyzed on ethidium bromide-stained agarose gels. For each virus, the four PCR products were pooled, purified on silica columns  and eluted in 30 μL of water. No purification of the amplicons was performed by gel-excision, even when additional bands were detected on ethidium bromide-stained agarose gels.The sensitivity of the assay was evaluated using serial three-fold dilutions of a cell culture supernatant infected by CV-A13 isolate 67001 whose titer was determined according to the WHO standard protocol . Libraries were built using 1 ng of DNA with the Nextera XT DNA Library Preparation kit in a SureCycler 8800 thermocycler (Agilent). After purification on AMPure beads (Beckman), the libraries were controlled using the High Sensitivity D1000 assay (Agilent) on a TapeStation 2200. Sizing was achieved by electrophoresis on a PippinPrep System with the PippinPrep kit CDF1510 (Ozyme). Finally, the libraries were quantified using the KAPA Quantification kit on a LightCycler 96 System (Roche). Sequencing was performed on a NextSeq500 with the HighOutput or MidOutput kits. All kits were used following manufacturer's instructions.Reads were demultiplexed followed by the removal of tags and adaptors. After importation in CLC Genomics Workbench 8.5 (CLCbio), reads were trimmed using the following parameters: Trim quality score limit = 0.01; Trim ambiguous nucleotides with Maximum number of ambiguities = 1. To avoid the presence of primer sequences in the reads, the 26 5′- and 3′-terminal nucleotides were removed. After trimming, reads shorter than 50 nucleotides were discarded.For each sample, the trimmed reads were mapped against the reference sequence of the corresponding virus. The sequences used as references were the sequences previously determined by the Sanger method. The entire genome of all field isolates were previously sequenced in our laboratory  that span the entire viral genome. Fragments ranged from ~1200 to ~3000 nucleotides in length. Overlapping regions were approximately 240-nt-long for fragments A and B and fragments C and D , the PCR performed with the four primer pairs produced gel bands at the expected size for all the viruses (data not shown). Some additional bands were also observed for certain RT-PCR products. Among our panel, CV-A19 NIH-8663 RNA gave the weakest bands on ethidium bromide-stained gel after the RT-PCR step. It is not possible to exclude the hypothesis that the four primer pairs had a low efficiency in amplifying the genome of this virus through PCR because of mismatches. Nonetheless, since the RNA of this virus was extracted from a mouse brain collected more than 20 years ago, this result is more likely due to the low amount of full-length virus RNA in this sample.The products of RT-PCR amplification of non-polio EV were sequenced by Illumina technology. Due to French regulations relating to PV containment, the sequencing platform was not allowed to handle the PV RT-PCR products.For each sample, the four RT-PCR fragments were produced separately and then pooled prior to purification. Sequencing resulted in a list of reads of which most were ~100 nucleotide-long after trimming. Because all of the samples were not sequenced during the same run, the number of reads varied from sample to sample. Thus, the number of reads after trimming ranged from 76,802 to 714,322 .de novo assembly . This method can be used to determine the full-length sequence of viruses isolated from clinical or environmental samples.The second method consists in constructing contigs through For each sample, reads were mapped against the corresponding sequence previously determined by the Sanger method Table . For allFor all samples, the reference sequence was fully covered without any gaps (breadth of coverage >99.0%). The breadth of coverage never reached 100.0% because the sequences corresponding to the outer primers C004 and C005 and the few nucleotides located downstream C005 were absent in the final contig.Average depth of coverage, which is the average number of times a base is sequenced, was very high (>980) but varied greatly from sample to sample Table , dependiThe consensus sequences of field strains deduced from mapping were compared to the consensus sequences determined by the Sanger method using RNA extracted from the same cell culture supernatants. For each virus, the two consensus sequences were identical indicating that high-throughput sequencing did not introduced nucleotide changes in the consensus sequences compared to the Sanger method.In order to assess the ability of the method to determine full-length sequences of the original isolates, the trimmed reads of each sample were assembled without using a reference.de novo assembly tool of CLC Genomics Workbench succeeded in assembling the virus genome into a unique contig that covered the entire genome between the outer primers C004 and C005. The number of reads included in the final full-length contigs was virtually similar to the number of reads previously found to map against the reference  by BLAST analysis. These contigs were from non-specific amplification of cellular nucleic acids during the RT-PCR process or from contamination of the viral RNA with cellular DNA during extraction. The number of such contigs varied tremendously from sample to sample, ranging from 0 to 783 , the number of virus reads was too low to reconstitute the full-length genome sequence through de novo assembly. By contrast, de novo assembly succeeded in building the full-length genome for all samples with dilution factor ≤ 39, including the last one in which only 2.5% of the reads were from the virus. For this sample, the coverage depth ranged from 5 to 95 along the contig (average of 28). In spite of this relatively low depth, the contig sequence generated for this sample was identical to the Sanger consensus sequence.After pooling and purification, the RT-PCR products of each dilution were sequenced during the same run. The total number of reads varied from sample to sample, from 88,930 to 316,336 Table . In the These results demonstrated that the method was able to generate full-length sequences of EV-C isolates from a few thousand reads and indicated that sequencing can be attempted even in the absence of detectable bands on agarose gels.Supernatants of cell cultures inoculated with clinical and environmental samples often contain mixtures of enteroviruses. In order to determine whether the method was able to sequence mixtures by assembling properly different viral contigs, four mixtures of viruses were prepared by mixing cell culture supernatants of two viruses, 67900 and 67001. These two viruses belong to the same serotype (CV-A13) and display together a nucleotide identity of 83%. The four mixtures contained the same volume of 67900 supernatant but different volumes of 67001 supernatant Table . Thus, tde novo assembly generated separate contigs for reads from 67900 and from 67001. As expected, the proportion of reads included in the 67900 contigs increased from mixture 1 to mixture 4 while the proportion of reads in the 67001 contigs decreased  as it does on the amount of virus genome copies in the sample, increasing the sequencing depth would overcome the low yield of RT-PCR amplification that could be observed for some samples. Another advantage of high-throughput sequencing compared to Sanger-based technics is that no gel-purification of the RT-PCR products was required, even in the presence of contaminating bands. In case of numerous samples to be analyzed, the RT-PCR products could be purified faster by using ultrafiltration-based or silica-based 96-well plates rather than individual silica-based columns.de novo assembly.For all samples, the coverage depth was heterogeneous along the genome. Heterogeneous coverage depths are often observed after high-throughput sequencing, partly because of the processing of the samples , the Fondation Total, and the US Department of Health and Human Services (grant No. 5 IDSEP140020-02-00). Patsy Polston is granted by a Pasteur Foundation's Gillings Pasteur Fellowship.This work was supported by the 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.


Ethylene is a simple two carbon atom molecule with profound effects on plants. There are quite a few review papers covering all aspects of ethylene biology in plants, including its biosynthesis, signaling and physiology. This is merely a logical consequence of the fascinating and pleiotropic nature of this gaseous plant hormone. Its biochemical precursor, 1-aminocyclopropane-1-carboxylic acid (ACC) is also a fairly simple molecule, but perhaps its role in plant biology is seriously underestimated. This triangularly shaped amino acid has many more features than just being the precursor of the lead-role player ethylene. For example, ACC can be conjugated to three different derivatives, but their biological role remains vague. ACC can also be metabolized by bacteria using ACC-deaminase, favoring plant growth and lowering stress susceptibility. ACC is also subjected to a sophisticated transport mechanism to ensure local and long-distance ethylene responses. Last but not least, there are now a few exciting studies where ACC has been reported to function as a signal itself, independently from ethylene. This review puts ACC in the spotlight, not to give it the lead-role, but to create a picture of the stunning co-production of the hormone and its precursor. Figure 1). Figure 1). 13 years later, 6) dependent enzyme inhibitor, which was later known to inhibit the enzymatic conversion of SAM toward ACC. The identification of ACC as the precursor of ethylene was a major breakthrough in the understanding of the ethylene biosynthesis pathway in plants, and was part of the foundation for many new discoveries in the field of ethylene biology.The discovery of ethylene as a plant growth regulator can be attributed to the work of the Russian scientist Arabidopsis, eight of which encode functional ACC synthases . Expression of different members of the tomato ACO family in Escherichia coli showed that each isoform had a specific in vitro enzyme activity (The second ethylene biosynthesis protein is ACC-oxidase (ACO), which converts ACC to ethylene in the presence of oxygen. It took a long time before ACO activity could be demonstrated ong time , it was ong time . This ca and CN- . The reaoalanine . ACO belctivator . The subctivator , while octivator . Althougactivity .Arabidopsis  by ATP-dependent tonoplast carriers , was discovered by carriers . Nonethecarriers . The abif tomato .in vitro studies, stated that GACC was the most abundant ACC derivative in tomato (in vivo in tomato fruit during climacteric ripening (Arabidopsis genome contains four genes (GGT1-4), of which only GGT1 and GGT2 are catalytically active , which was discovered in crude tomato extracts of ACC-N-malonyltransferase . These cn tomato , anotherripening . The Aray active . Both GGlar GACC . A knocke number . The effArabidopsis tissue  showed that the JA-ACC-induced root inhibition was independent of JA signaling. Furthermore, the ethylene signaling mutant etr1-1 and the double mutant etr1-1 jar1-1 were insensitive to the JA-ACC treatment and displayed no inhibition of root growth, indicating that JA-ACC acts via the ethylene signaling pathway (A third derivative of ACC is jasmonyl-ACC (JA-ACC). This molecule was discovered by screening for amino acid conjugates of JA, using GC-MS . Four ams tissue . Interess leaves . Recombi pathway . Most liThe three above-mentioned derivatives of ACC  are perhaps not the only ones. Future metabolic studies might reveal additional conjugates of ACC. Nonetheless, these three derivatives can potentially play an important role in the regulation of the pool of available ACC, which in turn can affect eventual ethylene production levels, with physiological and developmental consequences. Genetic perturbations of the formation of ACC derivatives could be a useful tool to unravel their exact roles. In addition, a more detailed structural and biochemical characterization of the enzymes involved in the formation of ACC derivatives is essential.Pseudomonas sp. strain ACP , but so do other amino acids like L-alanine, DL-alanine, and DL-valine  promotor of some Rhizobia, linking ACC deaminase with nodule formation . It should be noted that the foliar applied ACC was also rapidly converted into MACC, which was not found to be transported via the phloem (ACS isoforms are expressed (but not exclusively) in the vascular tissue  secured the ability of ACO to produce ethylene in cells with a high ACO expression  during different developmental processes. Tissue specific gene expression profiling of maize root cells showed that there are differences between patterns . ACO wasobserved . Differepothesis . A similpothesis , 1999, wOf course one should take into account that gene expression levels not always reflect actual protein levels, and that post-translational modifications can play an important role in protein stability and activity. A targeted metabolomics and proteomics study by Besides long-distance, short-distance intracellular transport of ACC was also observed in barley and wheat mesophyll cells  and maizClearly, more research is needed to further unravel the exact molecular and biochemical mechanisms which assure intracellular, inter- and intra-tissue and long-distance ACC transport in plants, and their corresponding physiological effects.1-Aminocyclopropane-1-carboxylic acid holds a key position in many physiological processes as it is the direct precursor in the biosynthesis of ethylene. A balanced supply and consumption of ACC is essential to achieve the necessary production level of ethylene within a given spatial and temporal context. As shown above, the pool of ACC is regulated by a complex interaction of production, consumption, modification, and transport. Interestingly, recent findings have suggested a perhaps even more important role for ACC, as a signaling molecule independent from ethylene .fei1 fei2 mutant displays a severe defect in anisotropic root growth due to a reduced cellulose microfiber content in the cell wall at the root tip . The fei1 fei2 phenotype can be reversed by the application of ethylene biosynthesis inhibitors, but not by ethylene signaling inhibitors. The application of both aminooxy-acetic acid (AOA) or α-aminoisobutyric acid (AIB) specifically inhibits ethylene biosynthesis and can reverse the phenotype of the fei1 fei2 mutant . AOA is an inhibitor of PLP-dependent enzymes, and will affect the activity of ACS resulting in a reduced ethylene production. AIB on the other hand is a structural analog of ACC and acts as a competitive inhibitor of ACC preventing ethylene production at the level of ACO. Ethylene signaling inhibitors such as 1-methylcyclopropane (1-MCP) and silver ions, did not affect the fei1 fei2 phenotype . Similarly, genetics showed that the fei1 fei2 mutant crossed with etr1-3 (a mutation in the ethylene receptor causing severe ethylene insensitivity), nor ein2-50  could reverse the phenotype. All together this study showed that the typical fei1 fei2 phenotype was not affected by ethylene signaling, but could be reversed by ethylene biosynthesis inhibitors. This suggests that the signal reversing the fei1 fei2 phenotype originated independent from ethylene signaling, involved ACS and is possibly ACC itself  into the hexuple mutant acs2,4,5,6,7,9 creating an octuple mutant line with complete or severe inhibition of ACS function. The lines that showed a very strong silencing of ACS8 and ACS11, displayed embryo lethality. This suggests that ethylene biosynthesis (or ACC biosynthesis) is essential for Arabidopsis viability, while this is not the case for the single (ctr1 and ein2) and double (ctr1 ein2) ethylene signaling mutants , could shed light on the role of ACC as a signaling molecule. It also still needs to be elucidated whether this unique title of “signaling molecule” is to be awarded to ACC, or rather to one if its (unknown) downstream derivatives.All together these reports suggest a role for ACC as a signaling molecule to regulate plant development and growth, independent from ethylene. The exact molecular mechanism by which ACC signaling operates, and whether or not there is an ACC receptor and downstream signaling components, remains to be investigated. Future biochemical studies with specific ethylene biosynthesis and signaling inhibitors, in combination with genetics to create higher order ethylene biosynthesis/signaling mutants  ACC-deaminase, adding another layer of metabolic complexity to the regulation of ACC levels. It is also well established that ACC can be easily transported over short (intracellular and intra-tissue) and long-distances (via the xylem and phloem), providing the plant with an elaborate system to control local and remote ethylene responses. Last but not least, ACC has been identified as a potential signaling molecule, independent of ethylene. This property of ACC is perhaps the most exciting, opening new avenues in ACC research, with potentially profound effects on plant physiology. The molecular mechanism by which ACC is signaling and the identity of other putative signaling components in such an ‘ACC pathway’ remain to be discovered.Bram Van de Poel and Dominique Van Der Straeten conceived the topic and wrote the manuscriptThe 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.


Much variation in individual-level cognitive function in late life remains unexplained, with little exploration of area-level/contextual factors to date. Income inequality is a contextual factor that may plausibly influence cognitive function.In a nationally-representative cohort of older Americans from the Health and Retirement Study, we examined state- and metropolitan statistical area (MSA)-level income inequality as predictors of individual-level cognitive function measured by the 27-point Telephone Interview for Cognitive Status (TICS-m) scale. We modeled latency periods of 8–20 years, and controlled for state-/metropolitan statistical area (MSA)-level and individual-level factors.Higher MSA-level income inequality predicted lower cognitive function 16–18 years later. Using a 16-year lag, living in a MSA in the highest income inequality quartile predicted a 0.9-point lower TICS-m score , roughly equivalent to the magnitude associated with five years of aging. We observed no associations for state-level income inequality. The findings were robust to sensitivity analyses using propensity score methods.Among older Americans, MSA-level income inequality appears to influence cognitive function nearly two decades later. Policies reducing income inequality levels within cities may help address the growing burden of declining cognitive function among older populations within the United States. Epidemiological studies suggest that cognitive impairment and decline are linked to individual-level characteristics including age , race/etIncome inequality, the degree of unequal distribution of income within populations, is an area-level/contextual factor hypothesized to adversely affect health –16. PropWhile the effects of income inequality on health have been extensively studied, the literature is mixed. In a meta-analysis of multilevel cohort studies, higher within-country income inequality predicted a 1.08 times higher risk of individual-level mortality . Other rThe effects of income inequality on cognitive function plausibly vary by the time period separating the measures of income inequality and cognitive function . For example, as with many chronic diseases, the pathological process of cognitive impairment likely begins years prior to being expressed clinically in late adulthood . The "cognitive reserve" hypothesis posits that exposure to environmental factors that provide more cognitive enrichment such as higher educational attainment may help build a higher cognitive reserve of nervous system functioning and confer a resilience to neuropathological insults , such that a higher neuropathological threshold must be reached before impairment of cognitive function manifests clinically , 23. StuUsing nationally representative data on older Americans from the Health and Retirement Study (HRS), we examined the lagged associations of income inequality measured at both the MSA and state levels with individual-level cognitive function, controlling for state-/MSA-level and individual-level factors.The Health and Retirement Study (HRS) is a nationally-representative prospective cohort study of U.S. adults born in 1947 or earlier, designed to investigate health, social, and economic outcomes in aging Americans. The original HRS study cohort that served as our study population consisted of 10,620 participants who were members of households with at least one person born during the period 1931–1941 i.e., aged 51–61 years in 1992. Addresses were geocoded to 1990 Census boundaries based on the MSA/state of residence in 1992; 134 (1.3%) participants could not be geocoded. Of the remaining cohort, 2,573 (24.2%) participants lived in a non-MSA at baseline and were excluded from the MSA-level analyses . An additional 392 (3.7%) participants were missing baseline Gini coefficient data for the MSA-level analyses and omitted from these analyses. For the state-level analyses, no additional participants had missing Gini coefficient data; 24 residents of the District of Columbia were excluded. Finally, 1,353 (12.7%) participants and 1,841 (17.3%) participants from the MSA- and state-level analyses, respectively, could not be interviewed because they were too impaired to complete the cognitive assessments, resulting in a proxy respondent being interviewed. These individuals were omitted because the cognitive outcome used for primary respondents was not administered to proxy respondents; the exclusion of these participants from our analyses is unfortunate. Study participants with follow-up assessments were retained in the study sample regardless of subsequent moves to a different MSA or state at follow-up than at baseline in 1992. The final baseline sample eligible for the MSA-level analyses was comprised of 5,455 adults in 1992; the final sample eligible for the state-level analyses consisted of 7,568 adults in 1992.We used a modified version of the Telephone Interview for Cognitive Status (TICS-m), a validated measure of global cognitive function, administered at each wave between 1998 and 2010 , 34. TheIncome inequality was modeled as a predictor of cognitive function with the Gini coefficient. Theoretical values for the Gini coefficient range from 0, reflecting perfect equality, to 1, corresponding to perfect inequality . We examAll linear regression models were adjusted for multiple factors at the individual and area levels that could confound the relationship between income inequality and cognitive function. These factors consisted of 1992 baseline age , gender, race/ethnicity , education , and net wealth . Models also controlled for baseline self-reported medical diagnoses of diabetes, hypertension, heart disease, and stroke, and measures of body mass index. The earliest measure of TICS-m in 1995/1996 was further included in all models as a proxy for baseline cognitive function. We also controlled for the 1990 Census-derived percentage Black and median household income measured at the same geographic level as the respective Gini coefficient.We calculated descriptive statistics for all individual- and MSA/state-level characteristics of the baseline analytic samples. We then plotted the marginal means of the TICS-m scores  for those residing within each MSA/state Gini coefficient quartile across biennial years 1998 through 2010.t  was regressed as a continuous variable on the quartile of income inequality of MSA/state residence and the individual and MSA-/state-level covariates. The quartile of lowest MSA-/state-level income inequality served as reference group. All results were weighted using sampling weights at baseline in 1992 to account for the complex multistage survey design, including stratification, clustering, and additional post-stratification.Using multivariate linear regression, we next incorporated varying lag periods between the baseline value of the Gini coefficient and cognitive function measures . The lag period is the number of years between the 1990 income inequality exposure (assuming that the study participant lived in the same MSA/state in 1990 as at the baseline survey in 1992) and the TICS-m score year. In separate models, the TICS-m score measured in year All analyses incorporated attrition weights to adjust for non-response bias . Specifically, we included and tested in each model the significance of the interaction terms for the cross-product of each factor with the income inequality quartiles. We used joint F-tests to assess the statistical significance of each set of interactions, applying a 1% significance level in light of multiple testing.Finally, we performed sensitivity analyses using propensity score methods to reduce potential bias due to non-random selection of individuals into quartiles of income inequality  by observable factors. Using TWANG , GeneralHRS participants provided verbal informed consent. All respondents were read a confidentiality statement when first contacted, and gave oral or implied consent by agreeing to do the interview. Verbal consent was obtained because most interviews were conducted by telephone. HRS staff at the University of Michigan recorded consent in writing. The Institutional Review Board (IRB) at the University of Michigan approved the methods, consent procedure, and data collection in the HRS. The IRB at the RAND (Research ANd Development) Corporation also approved analysis of these data.At baseline in 1992, participants from the original HRS cohort in the MSA-level analytic sample ranged in age from 51–61 years; 55.2% were female, 80.2% were White, and 42.9% had attained high school education . The meaBetween 1998 and 2010, with each higher income inequality quartile, we observed a gradual decrease in the means of the TICS-m scores . For 199Findings from the models that examined MSA-level income inequality are shown in Findings based on the models with the state-level Gini coefficient are displayed in We found no consistent and significant interactions between gender, race/ethnicity, and net wealth with MSA-level income inequality in their estimated effects on cognitive function (data not shown). Furthermore, we did not identify any interactions between gender, net wealth, or living in a MSA (vs. non-MSA) area with state-level income inequality.In sensitivity analyses using propensity scores, we obtained qualitatively similar results to those shown in Tables In a large, nationally representative, prospective cohort of older Americans, we found independent, graded associations of higher MSA-level income inequality with lower cognitive function measured at ages 67–79, using lag periods of 16 to 18 years. Living in a MSA in the quartile of highest income inequality was independently associated with a 0.9-point lower level of cognitive function 16 years later. Based on the marginal TICS-m scores we observed across waves, this effect size roughly corresponds to the magnitude associated with five years of aging.Our study is the first to explore the relation between income inequality and cognitive function; past studies of contextual determinants of cognitive function have been confined to neighborhood SES. Furthermore, these studies have either been cross-sectional in design –13 or noAlthough prior studies of income inequality on mortality and self-rated health have identified stronger effects at the state than MSA or county levels, our findings support associations of income inequality at the MSA but not state level; in analyses of state-level income inequality, we observed no differences in income inequality associations according to residence in MSA vs. non-MSA areas. Given this evidence compatible with a geographic specificity of income inequality effects at the MSA level (vs. the state level), the MSA-level income inequality associations could reflect pathways acting at more local levels. If feelings of relative deprivation are primarily experienced through comparisons with others locally within the same metropolitan area  rather than comparisons with others across one’s state, the negative consequences of relative deprivation on cognition  would be expected to be more salient with higher levels of income inequality at the metropolitan level. Past evidence has linked perceptions of local relative deprivation to mental well-being .Modeling income inequality measured several years prior to cognitive function assessment, we identified a temporal relation in our results. Furthermore, by modeling a range of lag periods, we determined the lag periods with the strongest associations on cognitive function in late life. Additional strengths of our analysis include the use of nationally-representative samples of older adults; re-weighting at the analysis stage for non-response to reduce selection bias; control for multiple area- and individual-level factors including co-morbid conditions and baseline cognitive function to limit residual confounding; exploration of associations at both the MSA and state levels; and sensitivity analyses using propensity score weights to further minimize selection bias.Our significant associations of income inequality with cognitive function corresponded to lag periods of 16 and 18 years. These lagged associations for income inequality measured in mid-life are in keeping with previous work exploring lag periods between area-level income inequality and self-rated health, and support the manifestation of effects of income inequality on cognitive function after more than a decade. This temporal association plausibly reflects the average time that it takes for income inequality to adversely affect individual cognitive function, for example mediated by feelings of relative deprivation and psychosocial factors such as depression, or mediated by the erosion of collective social capital and trust, which in turn may influence behavioral and psychosocial determinants of cognitive function.Our study had several limitations. First, income inequality assigned to study participants was based on the MSA/state of residence at baseline in 1992. Some study participants may have moved into other MSAs/states when they were cognitively assessed between 1998 and 2010. Our sensitivity analysis restricted to non-movers further indicated the robustness of our findings to migration to a different MSA over the study period. Second, both for the lack of observed associations for state-level income inequality and presence of associations for MSA-level income inequality, we cannot rule out residual confounding by income inequality or area-level SES  at smaller geographical scales . Third, although we accounted for non-response through survey weights and conducted sensitivity analyses using propensity score methods  and by restricting to the sample of individuals who were cognitively assessed in every wave from 1998 to 2010 , none of these approaches eliminates selection bias due to unobservable factors. Selection bias could partly explain some of the observed null findings, if attrition/omission due to disability  or deaths removed those most susceptible to cognitive decline from the analytic sample. If the remaining cohort members were characterized by higher cognitive reserve capacities, they may not have been as susceptible to neuropathological insults of income inequality. However, the significant findings of income inequality at the MSA level  coupled with the apparent absence of effect modification by MSA/non-MSA residence in the state-level analyses do not favor selection bias due to attrition as a major explanation of the null state-level findings, and lend support to the presence of true effects of income inequality at the metropolitan level. Finally, while we controlled for multiple baseline co-morbid conditions to reduce confounding, we did not adjust for time-varying health events between the measures of income inequality and cognitive function, which could have led to residual confounding.In summary, this study offers novel evidence on the associations of MSA-level income inequality with cognitive function nearly two decades later in a nationally-representative sample of older Americans. These findings may aid policymakers in identifying vulnerable populations at risk of long-term adverse effects on cognitive function from mid-life exposure to high levels of income inequality. By investigating possible mediating pathways, we may better elucidate how income inequality within cities shapes cognitive function. Policies that reduce levels of income inequality at the metropolitan level may be potential levers to address the growing public health burden of declining cognitive function among older populations within the United States.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.


Breast cancer is one of the most important causes of cancer-related deaths worldwide in women. In addition to gene expression studies, the progressing work in the miRNA area including miRNA microarray studies, brings new aspects to the research on the cancer development and progression. Microarray technology has been widely used to find new biomarkers in research and many transcriptomic microarray studies are available in public databases. In this study, the breast cancer miRNA and mRNA microarray studies were collected according to the availability of their data and clinical information, and combined by a newly developed ranking-based meta-analysis approach to find out candidate miRNA biomarkers (meta-miRNAs) that classify breast cancers according to their grades and explain the relation between miRNAs and mRNAs. This approach provided meta-miRNAs specific to breast cancer grades, pointing out let-7 family members as grade classifiers. The qRT-PCR studies performed with independent breast tumors confirmed the potential biomarker role of let-7 family members (meta-miRNAs). The concordance between the meta-mRNAs and miRNA target genes specific to tumor grade (common genes) supported the idea of mRNAs as miRNA targets. The pathway analysis results showed that most of the let-7 family miRNA targets, and also common genes, were significantly taking part in cancer-related pathways. The qRT-PCR studies, together with bioinformatic analyses, confirmed the results of meta-analysis approach, which is dynamic and allows combining datasets from different platforms. Breast cancer (BC) is the second leading cause of cancer-related deaths worldwide in women, after lung cancer. Despite advances in early detection and treatment, the estimated number of deaths in 2014 due to BC is about 40,000. The chance of a woman having invasive breast cancer during her lifetime is about 1 in 8. The chance of dying from breast cancer is about 1 in 36 [The clinical management of breast cancer depends on the availability of robust clinical and pathological prognostic factors. One of the best-established prognostic factors for BC is histological grade, which is based on the degree of differentiation of the tumor tissue. Recent microarray-based expression profiling studies have provided evidence that tumors of different grades show distinct genomic, transcriptomic and immunohistochemical profiles .Caenorhabditis elegans, these short regulatory RNAs have been found to be an abundant class of RNAs in plants, animals, and DNA viruses. miRNAs induce mRNA degradation (some studies indicate that destabilization of target mRNAs is the predominant reason for reduced protein output), translational repression, or both depending upon the complementarity of the miRNA to its mRNA target [MicroRNAs (miRNAs) are small non-coding RNA molecules, which negatively regulate gene expression at the post-transcriptional level . FollowiA target –8. miRNAA target . Future A target ,5.C. elegans. It is known that mature let-7 family members are highly conserved across animal species and that these miRNAs are key regulators of cell proliferation and differentiation. Let-7 family members are often reported as tumor suppressors in humans. Altered let-7 family expression is likely to contribute to cancer formation and also progression. Accordingly, these miRNAs may have the potential to be used as novel predictive markers in cancer [The let-7 family of miRNAs was initially discovered as important developmental molecules in n cancer ,11.http://www.ncbi.nlm.nih.gov/geo/) and Array Express[http://www.ebi.ac.uk/arrayexpress/) [Microarray technology has been widely used in research to find new biomarkers in the past decade. Many transcriptomic microarrays have been generated and made available in public databases such as the Gene Expression Omnibus (GEO) from NCBy Express from EBIxpress/) ,15.Meta-analysis may briefly defined as combining independent studies using various methods to increase the statistical power by increasing the number of samples analyzed ,17. MetaTo overcome these limitations, we developed a ranking-based meta-analysis approach. It is known that ranking-based meta-analysis approaches are successful in eliminating the differences that originate from platform variability . The metWe explained the relation between miRNAs and mRNAs in addition to the identification of miRNA signatures in this study. Accordingly the study steps were designed for both miRNA and mRNA together .www.ncbi.nlm.nih.gov/geo/) and ArrayExpress [www.ebi.ac.uk/arrayexpress) using the search terms “miRNA” or “mRNA”, “microarray” and “breast cancer” together. With these keywords we found 96 studies in which 52 of the microarray studies were performed with cell lines and 4 of them were formalin-fixed, paraffin-embedded (FFPE) studies. The datasets that were generated by using cell lines and FFPE tissue samples were excluded. The rest of the datasets were evaluated according to the presence of their data and grade information. Only studies with miRNA and mRNA microarray expression data conducted in human breast cancer tissues that had clinical information regarding grade status were included for further analysis.miRNA and mRNA microarray studies related to breast tumors were searched in two microarray databases, Gene Expression Omnibus   among samples with grade I, II and III. For each study, the miRNA and mRNA lists were generated considering their p values, and miRNAs and mRNAs were noted according to their ranks in the list. For each miRNA or mRNA, the mean of its rank in all studies was calculated and this value became its ce value .http://bioinfo.vanderbilt.edu/webgestalt/) pathway analysis tool was used for enrichment analysis of the targets of the let-7 family members and the common targets of the meta-miRNAs and meta-mRNAs. The enrichment results of the let-7 family target genes were visualized by a heatmap . In a reverse-transcription reaction, mature miRNAs were polyadenylated by poly(A) polymerase and subsequently converted into cDNA by reverse transcriptase with oligo-dT priming. The cDNA was then used for qRT-PCR profiling of mature miRNA expression. The cDNA was diluted at a ratio of 1:10 before being used as a PCR template and stored at -20°C until further use.qRT-PCR analysis was performed on the Roche LightCycler 480 by using specific miRNA primers and the miScript SYBR Green PCR Kit . The amplification mixtures contained 1.0 μl 1:10 diluted cDNA, 5.0 μl SYBR Green PCR Master Mix, 1.0 μl miScript Universal Primer , 1.0 μl miScript Primer Assay (miRNA specific forward primer) and 2 μl RNase-free water in a total volume of 10 μl. Cycling conditions were as follows: 95°C for 15 min for initial activation and then 40 cycles of 94°C for 15 s, 55°C for 30 s, and 70°C for 30 s during amplification. The ΔΔCt method was used for the analysis of qRT-PCR results and RNU6-2 was used as a normalization factor.http://www.umm.uni-heidelberg.de/apps/zmf/mirwalk/index.html). miRWalk is a comprehensive database that involves not only in silico predicted but also experimentally validated targets. To compare the meta-miRNA targets with meta-mRNAs, common genes were identified by using Venny [http://bioinfogp.cnb.csic.es/tools/venny/)The validated targets of the grade-specific meta-miRNAs were identified bioinformatically by using miRWalk   . The expression range of each miRNA in each grade  was shown in terms of normalized Ct values according to a normalization factor RNU6 as box-whisker-plots in (p≤0.05) .To validate the reliability of let-7 family members in grade status discrimination, an integrated analysis was designed in which the targets of the let-7 family members were compared with the meta-mRNAs that are supposed to be grade predictors. For this integrated analysis the meta-analysis approach that was applied to miRNA datasets was also applied to the 2 mRNA microarray studies conducted in breast cancer  and metaThe intersection of meta-mRNAs (top 20% of 13355 mRNAs) and let-7-target genes (837 targets without repetition) was found by Venny analysis and the results showed that they shared 116 common genes . The patTo further validate the prediction power of the 116 common genes an independent microarray data from GSE22219 [Meta-analysis is a procedure for statistically combining the results of many different studies. The data obtained by meta-analysis is more generalized and stable since the information is not specific to a case but the combination of many different studies. The common problem of various meta-analysis approaches is the technical differences resulting from the usage of different platforms. This necessitates the development of new methodologies with the potential to eliminate the differences resulting from the different probe design, normalization and preprocessing methods specific to each platform. We combined independent miRNA microarray datasets to identify miRNA signatures that could classify the breast cancer samples according to their histological grade in this study. An ANOVA-dependent ranking-based meta-analysis approach that makes identification of novel candidate miRNAs possible with the potential to eliminate differences emerging from microarray platform variability was developed in MATLAB for this purpose. The code is so dynamic that it allows the addition of newly generated data for meta-analysis. In a ranking-based meta-analysis study, Phan et al. compared their Rank Average Meta-Analysis with 5 other meta-analysis approaches  and found that combining microarray datasets by averaging ranks effectively increases the sample size while enabling robust analysis of heterogeneous data. Another The method enabled us to combine three independent miRNA microarray studies performed with three different platforms and two different mRNA microarray studies performed with the same platform. Since miRNA microarray studies are mostly concentrated on the comparison of tumor and normal tissues, the pathological information (including the grade information) was not available, which limited the number of studies we used for meta-analysis.The meta-analysis study was performed with the tumor samples obtained from independent studies in which the clinical information of the tumors was available. In this study we concentrated on meta-miRNAs that are predictive for histological grade status, one of the important prognostic factors in breast cancer. In the meta-miRNA list obtained from the meta-analysis, 8 of the first 20 miRNAs were found to be let-7 family members; let-7c, let-7a, let-7f, let-7d, let-7e, let-7i, let-7g and let-7b . The enrNext, we investigated the let-7 family targets in silico in order to further analyze their predictive potential regarding histological grade. The validated target gene lists were obtained for each miRNA . Further characterization of the genes was performed by pathway enrichment analysis. The genes were significantly enriched in pathways important for cancer development and progression such as Jak-STAT, MAPK, TGF-beta, mTOR signaling pathways, cell cycle and apoptosis . Since mMany studies have shown the relation of miRNAs with the clinical status of breast cancer and reported their role in breast cancer pathogenesis, development and progression ,41,46–49In conclusion, the meta-miRNAs that were obtained by the meta-analysis developed for this study could be verified experimentally in independent breast tumors and were comparable with previous studies related to breast cancer. We have demonstrated that the meta-analysis approach is reliable and robust and also that the let-7 family members of meta-miRNAs may be alternative and potential biomarkers for grade prediction. Meta-analysis of miRNA microarray datasets also has the potential to lead to more comprehensive measures of the existing differential miRNA expression data and can therefore provide miRNA and miRNA-target gene sets with high prognostic value.S1 PRISMA ChecklistThe page numbers corresponding to the items listed in the checklist were filled out according to the meta-analysis study presented.(DOC)Click here for additional data file.S1 TablemiRNAs were noted according to their ranks in the list. For each miRNA the mean of its rank in all studies was calculated with their p values (the mean of rank column) and this value became its real rank in the list within the real rank column, which was used instead of the significance value.(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


Missed injury is commonly used as a quality indicator in trauma care. The trauma tertiary survey (TTS) has been proposed to reduce missed injuries. However a systematic review assessing the effect of the TTS on missed injury rates in trauma patients found only observational studies, only suggesting a possible increase in early detection and reduction in missed injuries, with significant potential biases. Therefore, more robust methods are necessary to test whether implementation of a formal TTS will increase early in-hospital injury detection, decrease delayed diagnosis and decrease missed injuries after hospital discharge.We propose a cluster-randomised, controlled trial to evaluate trauma care enhanced with a formalised TTS procedure. Currently, 20 to 25% of trauma patients routinely have a TTS performed. We expect this to increase to at least 75%. The design is for 6,380 multi-trauma patients in approximately 16 hospitals recruited over 24 months. In the first 12 months, patients will be randomised  and allocated 1:1 to receive either the intervention (Group 1) or usual care (Group 2). The recruitment for the second 12 months will entail Group 1 hospitals continuing the TTS, and the Group 2 hospitals beginning it to enable estimates of the persistence of the intervention. The intervention is complex: implementation of formal TTS form, small group education, and executive directive to mandate both. Outcome data will be prospectively collected from (electronic) medical records and patient (telephone follow-up) questionnaires. Missed injuries will be adjudicated by a blinded expert panel. The primary outcome is missed injuries after hospital discharge; secondary outcomes are maintenance of the intervention effect, in-hospital missed injuries, tertiary survey performance rate, hospital and ICU bed days, interventions required for missed injuries, advanced diagnostic imaging requirements, readmissions to hospital, days of work and quality of life (EQ-5D-5 L) and mortality.The findings of this study may alter the delivery of international trauma care. If formal TTS is (cost-) effective this intervention should be implemented widely. If not, where already partly implemented, it should be abandoned. Study findings will be disseminated widely to relevant clinicians and health funders.ACTRN12613001218785, prospectively registered, 5 November 2013ANZCTR: The online version of this article (doi:10.1186/s13063-015-0733-y) contains supplementary material, which is available to authorized users. Missed injuries after multi-trauma are a significant health problem, affecting young to middle-aged adults (mainly men). Estimates of incidence are highly variable, but most likely between 1 to 5% of all trauma patients have missed injuries . Missed The standard primary and secondary surveys by ED, intensive care unit (ICU) and surgical teams have been shown to miss injuries ,2,5,6. PThe TTS would be expected to reduce missed injuries and therefore improve trauma care. However, our recent systematic review  found suAmong the deficiencies was the substantial variation in outcome definitions for missed injury, leading to a recommendation for a classification (See Table P < 0.001), this may not have been enough to detect any potential differences in missed injury rates if they exist.A retrospective study  in our Apost hoc, [Missed injuries may be minor or only require conservative treatment. Therefore, the focus ideally should be on clinically significant missed injuries. Few studies have defined ‘clinically significant’ ost hoc, ,11,12 wiost hoc, .early trauma care, where the tertiary survey by can be conducted up to 24 hours after ED assessment.Current international guidelines such as Early Management for Severe Trauma (EMST) do mention consideration of tertiary surveys, but do not mandate or formalise their implementation.  This mayThere are a number of potential concerns regarding performing a formalised TTS on every multi-trauma patient. It will take more medical and nursing staff time to perform, and may lead to over-investigation. It could lead to over-diagnosis by labelling minor self-limiting conditions with subsequent patient concern or overconsumption of medical resources. As such, it may feed the notion that even minor, self-limiting injuries should always be diagnosed and actively managed. Additionally, re-examination by the same clinician or team may miss the same injury twice due to systematic error or persistent cognitive error. Ultimately, the decision to implement a formalised TTS procedure relies on the assessment of the clinical effect, as well as the value of the clinical benefits, compared to the associated change in costs. However, in order to provide an evaluation of the cost-effectiveness of a formalised TTS, reliable estimates of the effectiveness of a formal TTS is a necessary requirement.In summary, questions remain regarding the utility and (cost-) effectiveness of the TTS. Although there is a sound rationale for performing formalised TTS, the current data is inconclusive since the quality of data is low and effect size variable. The health care costs to provide this extra layer of safety-net may not prove cost-effective. This protocol specifies the design for a multicentre clinical trial to assess the effectiveness and feasibility of implementing a formal TSS. The results of the study will provide high level evidence that either a TTS should be mandatory in trauma patients, or that we can save resources if no benefit is found.Our primary aim is to determine whether the implementation of a formalised TTS will decrease missed injuries post hospital discharge (Type III injuries) and, as secondary aim, increase injury detection in hospital (Type I and II injuries combined). We further hypothesise that the intervention effect will be maintained and that the intervention would lead to decreased clinically significant missed injuries as judged by a blinded expert panel, decreased hospital and ICU bed days, decreased readmissions to hospital, improved quality of life and decreased time off work. We expect complications of the injuries to be the same, but complications of care to be less. If no difference between injury rates is found, we hypothesise that both arms will be equivalent in resource utilisation .www.spirit-statement.org). The Gold Coast Hospital and Health Service Human Research and Ethics Committee approved the study including informed consent processes as outlined under approval number HREC/14/QGC/79.This protocol adheres to the SPIRIT statement  practice and standard practice with additional formalised trauma tertiary survey completion. This protocol was developed by a small group of experts in the field of tertiary survey in trauma, (including Emergency Physician and Trauma Surgeon) and methodology .This trial is to be conducted in approximately 16 trauma receiving hospitals across Australia in at least 4 states: Queensland, New South Wales, Victoria and Western Australia. Participating hospitals do not have a dedicated trauma service or formalised process for review of admitted trauma patients.All multi-trauma patients admitted to hospital for at least 24 hours will be prospectively identified using and Emergency Department Information System  and are eligible for recruitment if they meet the inclusion criteria based on previous work ,16.16 years and olderAdmitted for at least 24 hours (since TTS can be performed up to 24 hrs) and any of the following:Injuries in two or more body regionsHigh impact mechanism Chest or abdominal injuriesFractured neck of femur under the age of 65 years.Inclusion criteria as used in previous studies ,16 are aInformed consent for 1, 6 and 12-month telephone follow-up interviews will be obtained from the patient or proxy within 24 hours of enrolment.Participating hospitals (clusters) will be 1:1 randomised to either immediate (Group 1) or delayed (Group 2) introduction of TTS ; however, we will seek permission to provide limited disclosure to the patient. Specifically, we do not plan to advise them of the study while they are in the ED/hospital, for the following reasons:, used in two previous studies [The implementation of a formalised TTS form . ThiAll medical and nursing staff on trauma admitting wards will attend two education sessions on the rationale and use of the TTS form, provided by a chief or associate researcher in small-group work-shop format.Key stake holder engagement  with a clear directive to all levels of medical and nursing staff for compliance with training and education sessions and mandatory TTS form completion as part of routine care within 24 hours of admission.The primary intervention will be at the hospital level and outcomes will be measured at the patient level. The intervention will consist of the following:Standard (existing) care will be at the discretion of the trauma admitting teams of the control hospitals. Standard care is defined as performing a clinical examination based on clinical judgment and without standardised forms. Data collection forms and procedures will be identical for both study arms. A TTS in the intervention phases is defined as completion of the formal TTS form within 24 hours of admission, where during standard care it is defined as a documented tertiary survey as part of a clinical examination within 24 hours of admission. A review of the previously known injuries only is NOT a tertiary survey.Based on our own descriptive data ,16, routAs outlined in Figure Data collection processes are the same for both Groups. The prospectively identified eligible patients will have their primary and secondary survey performed in the ED. All clinical reviews (including ward rounds) in the first 24 hours on the ward after surgical or interventional radiology intervention will be documented using a previously used ,16 data Data related to the inpatient admission will include whether a  TTS is documented during admission and which components of the TTS are performed. A list of injuries, newly detected injuries and changes in management will be collected. A central blinded panel of experts, comprised of at least two emergency physicians and 2 (trauma) surgeons, will class newly detected injuries in-hospital as Type I, Type II or not a new injury  or a complication of care or injury. An injury will only be classified as definite or likely (Type III) ‘missed injury’ if there is no evidence  of the self-reported injury during the hospital stay. The panel will determine the clinical significance of these injuries based on change in management and expected effect on (duration) of morbidity.Patients who reported a missed injury will be offered appropriate pathways for follow-up. Finally, we will undertake a search of the Death Registry to identify the mortality rate at 12 months post hospital discharge and cause of death.Appropriate data on the associated cost-implications will be collected during the trial in order to conduct a cost-effectiveness analysis. Items identified that require appropriate measurement and valuation include: medical and nursing time used to assess patients in the first 24 hours, requested advanced imaging type and frequency, ICU length of stay, hospital length of stay, ICD-10 diagnostic and procedure codes, Australian-Revised Diagnostic-Related Group (AR-DRG) codes, patient time-off work, community medical services provided, and re-admissions to hospital.The primary outcome is Type III missed  injury rate. This study aims to compare the effect of a formalised TTS compared to routine care on missed injury rates after hospital discharge (type III).Maintenance of intervention effect (comparing two 12-month periods in Group 1)During Hospital StayIn-hospital missed injury rate (Type I and II combined)Proportion of TTS performedHospital bed days, ICU bed daysDuring Follow-up Clinically significant missed injuries (as determined by expert panel)Interventions required for delayed diagnosis (Type I and II) or missed injuries (Type III)Diagnostic imaging usage Predefined complications of care and injury (see above)Readmissions to hospitalDays off workQuality of life (EQ-5D-5 L)Days off workMortalitySecondary outcomes are:The primary analysis will be by intention-to-treat (ITT). Per-protocol analyses will also be performed, based on all patients completing 1, 6 and 12 months follow-up. Based on our prospective study , compari.The sample size for a cluster-randomized trial requires adjustment of the number of patients that a corresponding individually randomized trial would have needed. This is due to variation between the clusters, and results in a correction using the intra-cluster correlation coefficient (ICC) . Using a3This calculation is consistent with a previous study specifically examining the ICC in trauma receiving hospitals. ICC varied depending on outcome, but the average ICC for blunt trauma the ICC was 0.01 . AssuminThe study is adequately powered for the primary hypothesis, and for several secondary outcomes, such as pre- and post-comparison in Group 2, but not for the pre- and post- comparison in Group 1, measuring maintenance of the intervention effect.t-test and chi-square test will be used to compare differences in proportions. All results will be presented with 95% confidence intervals.De-identified data will be analysed using SPSS v20.0 software . For continuous variables, we will use an independent P value of 0.05 or less will be deemed statistically significant.Health economic analyses will include a within trial incremental cost-effectiveness evaluation of formal TTS versus standard care. The outcome measure, incremental cost-effectiveness ratio, directly compares the difference in costs between the formal TTS and standard care with the difference in health related quality of life. Health-related quality of life will be measured in quality adjusted life years (QALYs) derived from changes in EQ-5D-5 L measured at multiple time points. A cost-effective threshold of $AUD 50,000 per QALY will be used to determine whether the intervention was cost-effective and a cost-effectiveness acceptability analysis will be conducted using a range of threshold values from $20,000 to $100,000. A 95% confidence interval around the incremental cost-effectiveness ratio will be estimated using bias-adjusted bootstrapping. In addition, sensitivity analyses and sub-group analyses will be conducted to identify the parameters and patient characteristics that affect the likelihood of the intervention being cost-effective. A Blinding the treating staff to the intervention is challenging, but the patient will be blinded since all patients will be receiving a standard care variation  and will not be informed of the specific intervention. However, relatively objective endpoints will be adjudicated by a blinded expert panel and a prospective randomised open-label, blinded end-point (PROBE) methodology can be used  and has Missed injury in trauma is a common condition in young adults, with potential to lead to prolonged morbidity. The findings of this study may alter the delivery of international trauma care. If formal TTS is (cost-) effective this intervention should be implemented widely, inform guidelines and change practice. If not, where already partly implemented, it should be abandoned. Study findings will be disseminated widely to relevant clinicians and health funders.Recruitment has not yet begun because we are awaiting funding.


To date, we have demonstrated that both methods of chemical cross-linking improved the elasticity and tensile strength of the collagen implants. Differential scanning calorimetry and biocompatibility studies indicate comparable, and in some cases, enhanced properties compared to that of the EDC/NHS controls. In vitro studies showed that human corneal epithelial cells and neuronal progenitor cell lines proliferated on these hydrogels. In addition, improvement of cell proliferation on the surfaces of the materials was observed when neurite promoting laminin epitope, IKVAV, and adhesion peptide, YIGSR, were incorporated. However, the elasticity decreased with peptide incorporation and will require further optimization. Nevertheless, we have shown that epoxy cross-linkers should be further explored in the fabrication of collagen-based hydrogels, as alternatives to or in conjunction with carbodiimide cross-linkers.A bi-functional epoxy-based cross-linker, 1,4-Butanediol diglycidyl ether (BDDGE), was investigated in the fabrication of collagen based corneal substitutes. Two synthetic strategies were explored in the preparation of the cross-linked collagen scaffolds. The lysine residues of Type 1 porcine collagen were directly cross-linked using l,4-Butanediol diglycidyl ether (BDDGE) under basic conditions at pH 11. Alternatively, under conventional methodology, using both BDDGE and 1-Ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) as cross-linkers, hydrogels were fabricated under acidic conditions. In this latter strategy, Cu(BF Corneal disease leading to vision loss is a major cause of blindness worldwide and a World Health Organization priority disease, particularly in the developing world . While mWe have now shown that biomimetic collagen-based materials that exhibit characteristics similar to that of the native extracellular matrix (ECM) can be developed as scaffolds to enable regeneration of the human cornea through recruitment of endogenous progenitor cells. Collagen is the most abundant ECM constituent in the body and contains the molecular recognition elements needed to elicit cellular responses and potentially carry out functions normally innate to that of the native tissue ,6,7. In However, these early implants were susceptible to microcrack formation and shearing . Hence, Cross-linking of bi-functional BDDGE is pH dependent and occurs through secondary amine bond formation via epoxide ring opening by amine groups of collagen under basic pH conditions . We examOur earlier clinical trial results demonstrated that stroma in-growth to the central portions of the EDC based cross-linked implants took between 12 and 18 months  to purple formazancrystals. Herein, the amount of formazan crystal formation is directly proportional to the number of live cells. The MTS assay was performed on days 1, 4 and 7 of cell culture. Hydrogels were rinsed three times in PBS and subsequently transferred into new wells prior to MTS assay to avoid the absorbance from any cells that may have been growing on the tissue culture plastic beneath the hydrogels. Subsequently, 20% v/v of MTS reagent in cell culture medium was added and cultures were incubated further for 4 h at 37 °C. Absorbance was measured at 490 nm using a spectrophotometric microplate reader .The biocompatibility of the different hydrogel formulations with HCEC survival was examined by using live/dead staining on day 1 and day 4 of culture. Calceinacetoxymethyl  and ethidium homodimer-1 (EthD-1) (Invitrogen) were used to determine the viability of cells on the hydrogels. The concentration of Calcein AM and EthD-1staining solution were 2 µM and 4 µM respectively. The fluorescence images were visualized under fluorescent confocal microscope . Morphological inspection of the cells for flattening and spread (as opposed to rounded cells) gave an indication of the ability of each hydrogel formulation to support cell adhesion and spread.n = 3). Data obtained from the different experiments are presented as mean values. Statistical significance of differences between two hydrogels cell proliferation was analyzed using single factor analysis of variance (ANOVA).Cell proliferation studies on hydrogels were repeated separately for three times and run in triplicate (The BDDGE hydrogels exhibit high tailorability, given that the mechanical properties could be manipulated depending on the conditions in which the hydrogels were fabricated. Specifically, the BDDGE-EDC/NHS hydrogels, fabricated by BDDGE coupling followed by carbodiimide chemistry resulted in materials with high elasticity. Interestingly, the slow gelation time of the BDDGE cross-linking may allow for drug encapsulation within the collagen matrix upon fabrication for therapeutic use. In addition, the YIGSR and IKVAV peptide-BDDGE-based hydrogels may also serve as ideal candidates for corneal substitutes given their ability to enhance epithelial and nerve cell growth.


The treatment of an occluded saphenous vein graft (SVG) with percutaneous coronary intervention may encounter major adverse cardiac events (MACE). MACE rates have been reduced significantly by using the embolic protection device (EPD).The aim of this study was to clarify the risks and the benefits of embolic protection devices.In a prospective, non-randomized observational study, patients aged 33 to 85 years old who underwent elective percutaneous coronary intervention due to SVG stenosis at our tertiary care center were enrolled between 2009 and 2011. The incidence rates of adverse events, including MACE, were obtained during the patients’ hospitalization and at 30-day and 6-month follow-up. MACE included death, Q-wave and non-Q-wave myocardial infarction, in-stent thrombosis, target lesion revascularization, and target vessel revascularization.From 150 patients enrolled to the study, 128 (85.3%) patients underwent direct stenting and the rest underwent the EPD procedure. In-hospital MACE occurred in 17.2% of the patients in the direct stenting group versus only 9.1% in the EPD group (P = 0.530). MACE incidence was gradually increased at one and 6-month follow-up periods in the direct stenting group , and remained unchanged in the EPD group (9.1% at six-month follow-up). Multivariate logistic regression model showed that the stenting procedure type could not predict early and midterm MACE with the presence of baseline characteristics as cofounders.Despite the considerable lower early and midterm MACE rates, numerically following the EPD procedure compared to direct stenting, the difference in the MACE rates between the two groups was not significant. Saphenous vein graft (SVG) percutaneous coronary intervention (PCI) carries unique technical challenges, requires the use of the embolic protection device (EPD) to reduce the adverse events associated with distal embolization. Distal embolization is a common and almost omnipresent consequence of SVG PCI due to the soft and friable nature of SVG lesions . AdequatThere are reports on the safety and efficacy of direct stenting in SVG lesions in the current study. Therefore, the present study draws a direct comparison between the two techniques. Our objective was to perform a comparative effectiveness study to examine the benefits associated with using EPD to remove thrombi or protect against distal embolization in patients underwent occluded SVG PCI of occluded saphenous vein graft (SVG).Data was collected prospectively on a cohort of patients underwent PCI on the SVG at our tertiary care center from 2009 to 2011. The inclusion criteria was SVG occlusion confirmed by coronary angiography, and the exclusion criteria included refusal to continue the study, occurrence of noncardiac adverse events leading to death within the study protocol, and presentation of acute coronary syndrome. Baseline measurements included demographics, cardiac history, graft age, and stent type. The patients were treated with one of the two methods of direct stenting or EPD. The study was conducted in accordance with the declaration of Helsinki protocol and was approved by the Ethics committee of Tehran University of Medical Sciences. All the patients provided written and informed consent prior to entering the study. Information on adverse events - including MACE - was obtained during the patients’ hospitalization and at 30-day and six-month follow-up periods. MACE included death, Q-wave and non-Q-wave myocardial infarction, in-stent thrombosis, target lesion revascularization, and target vessel revascularization. The follow-up visits were performed either by the referring cardiologist or alternatively via telephone contact.The results were reported as mean ± standard deviation (SD) for the quantitative variables and percentages for the categorical variables. The groups were compared using Student's t-test for the continuous variables and chi-squared test (or Fisher's exact test if required) for the categorical variables. Predictors exhibiting a statistically significant relation with MACE in the two groups in univariate analyses were taken for multivariate logistic regression analysis to investigate their independence as predictors. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. P values ≤ 0.05 were considered statistically significant. All the statistical analyses were performed using SPSS .The study population consisted of 150 patients with a mean age of 63.23 ± 9.53 years. Direct stenting was performed in 128 (85.3%) patients, and 22 (14.7%) of the patients underwent the EPD procedure. Stent type in the direct stenting and EPD groups were respectively 18.8% and 9.1% drug-eluting stents versus 81.3% and 90% bare-metal stents; there was no statistically significant association between the two groups (P = 0.3). The in-hospital mortality rate was two patients at one-month follow-up, and two patients at six-month follow-up in the direct stenting group compared to none in the EPD group at the same time points. In-hospital thrombosis occurred in one case at one-month follow-up and in 3 cases at 6-month follow-up in the direct stenting group compared to none in the EPD group at the same points in time. In the direct stenting group, there was no case of target vessel or target lesion revascularization at one-month follow-up, but there was one case of target vessel revascularization and five cases of target lesion revascularization at six-month follow-up. In the EDP group, there were no cases of target vessel or target lesion revascularization at one and 6-month follow-up periods. No significant difference was found between the two groups regarding gender distribution and mean age (P > 0.05). Except for current smoking which was more prevalent in the EPD group (54.5%) (P = 0.001), the prevalence of coronary disease risk factors including history of hypertension, hyperlipidemia, diabetes mellitus, and family history of coronary artery disease were similar between the two study groups . The meaAssessment of the MACE rates showed a gradual increase in the direct stenting group  and no change in the EPD group at one-month and six-month follow-up . In the Performing PCI on the SVG is associated with a high risk of MACE, mainly periprocedural myocardial infarction resulting predominantly from distal embolization of atherosclerotic plaque and friable debris within the graft and causing microvascular occlusion and no reflow -17. The Since 2002, 4000 patients have been enrolled in studies evaluating the EPD , 22, 23.In-hospital stent thrombosis occurred in 20 patients (15.6%) of the direct stenting group (n = 128), and none of the EPD group patients (P = 0.9). In one study, the mean patient age was 69 years, including 82% male and 41% diabetic subjects; these findings are consistent with our results . We evalDiabetes mellitus is generally associated with a higher risk of adverse events after PCI. In one study, the diabetic patients were significantly younger, had lower SVG degeneration scores, and had smaller estimated plaque volumes by compared to non-diabetic subjects; these differences may account for the lower event rates among the former group in that data set . In our Several risk factors, including thrombus and graft age, have been described for SVG PCI without distal embolic protection . InteresWe finally evaluated the predictive value of baseline indicators in predicting midterm MACE related to PCI on the SVG, and our results confirmed that only 14.7% of the entire study population received EPD stenting, while most of them received direct stents. Although the MACE rate at different time periods was higher in the direct stenting group compared to the EPD group, but this difference was not statistically significant. Although the MACE rate was gradually increased during follow-up in the direct stenting group, it remained unchanged in the EPD group. Since patients’ survival can be manifested by the MACE indicator, it can be concluded that the EPD procedure can be more appropriate than direct stenting to improve patients’ survival and is, thus superior to the latter procedure. Some investigators have emphasized the superiority of direct stenting over the EPD procedure due to its more cost-effectiveness and availability, while some others have reported similar or higher effectiveness in the EPD procedure. These discrepancies can be due to the type of study , sample size or inclusion criteria for patient selection. Accordingly, clinical trials with greater sample sizes are required to reach reliable conclusions and compare direct stenting and EPD regarding reducing MACE following SVG PCI.Identifying the predictors of MACE allows reliable prediction of patient outcomes and confirms consistent treatment benefits by using the EPD across the range of patients tested in randomized trials. Despite considerable lower early and midterm MACE rates following the EPD procedure compared to direct stenting, the difference in the MACE rates between our two study groups was not statistically significant. More studies with greater sample sizes are needed to confirm our results. The number of patients included in the EPD group was relatively low, partly due to the scarcity of such devices. Nevertheless, the results of the present study require further consideration.