The concept of hybrid coronary revascularization (HCR) combines the left internal mammary artery (LIMA)–left anterior descending (LAD) graft and percutaneous coronary intervention (PCI) to non-LAD vessels. Multiple comparative studies have evaluated the safety and feasibility of HCR and coronary artery bypass grafting (CABG) for multivessel coronary artery disease (MCAD). However, the sample size of each study was small, and evidences based on single-institutional experience. The purpose of this meta-analysis was to compare the short-term outcomes of HCR with those of CABG for MCAD.PubMed, EMBASE and Cochrane Library databases, as well as conference proceedings, were searched for eligible studies published up to March 2014. We calculated summary odds ratios (OR) for primary endpoints ; target vessel revascularization (TVR); major adverse cardiac or cerebrovascular events (MACCEs)) and secondary endpoints ; renal failure; length of stay in the intensive care unit (LoS in ICU); length of stay in hospital ; red blood cell (RBC) transfusion). Data from 6176 participants were derived from ten cohort studies.HCR was non-inferior to CABG in terms of MACCEs during hospitalization (odds ratio (OR), 0.68, 95% confidence interval (CI), 0.34–1.33)and at one-year follow-up , and no significant difference was found between HCR and CABG groups in in-hospital and one-year follow-up outcomes of death, MI, stroke, the prevalence of AF and renal failure, whereas HCR was associated with a lower requirement of RBC transfusion and shorter LoS in ICU and LoS in hospital than CABG (weighted mean difference (WMD) –1.25, 95% CI, –1.62 to –0.88; –17.47, –31.01 to –3.93; –1.77, –3.07 to –0.46; respectively).Our meta-analysis indicates that HCR is feasible, safe and effective for the treatment of MCAD, with similar in-hospital and one-year follow-up outcome, significantly lower requirement of RBC transfusion, and faster recovery compared with CABG. Coronary artery bypass grafting  offers superior long-term advantages owing largely to the left internal mammary artery (LIMA) to the left anterior descending (LAD) artery graft ,2. Conve patency ,7. In co patency ,9. HowevThe concept of hybrid coronary revascularization (HCR), which combines the LIMA–LAD graft and percutaneous coronary intervention (PCI) to non-LAD vessels, was first introduced by Angelini and colleagues in 1996 . IntroduTwo independent reviewers  searched PubMed, Embase, Web of Science, and the Cochrane Library for randomized controlled trials (RCTs) and non-RCTs up to 1 March 2014 and compared HCR with CABG for MCAD without language or publication restrictions. The following medical subject heading terms and their variants were used in database searches: hybrid coronary revascularization; coronary artery bypass grafting (on-pump or off-pump); multivessel coronary artery disease. Reference lists within selected studies and abstracts published at major international conferences were also searched.The safety endpoints of this meta-analysis were death, stroke, myocardial infarction (MI) and major adverse cardiac or cerebrovascular events (MACCEs). The efficacy endpoint was revascularization. All the primary endpoints were measured in hospital and one year of follow-up. Death was defined as death from any cause. MI was diagnosed by symptoms, electrocardiography and changes in serum levels of cardiac biomarkers. Target vessel revascularization (TVR) was the need for repeated CABG or percutaneous coronary intervention (PCI). MACCEs were defined as a composite of death, MI, stroke or revascularization.Secondary outcomes were atrial fibrillation (AF), renal failure , length of stay in the intensive care unit (LoS in ICU), length of stay in hospital , and transfusion of red blood cells (RBCs).Five main criteria were used: (i) comparison of HCR with CABG for MCAD; (ii) studies reporting at least one of the outcomes mentioned above; (iii) studies documenting surgical procedures such as one-stop HCR or staged HCR, on-pump or off-pump CABG, and documenting surgical methods such as HCR or CABG; (iv) follow-up duration ≥30 days; (v) non-RCTs with a score >5 as assessed by the Newcastle–Ottawa Scale (NOS) -24.http://www.ohri.ca/programs/clinical_epidemiology/oxford.asp), which addressed three items: patient selection, comparability of groups, and outcome assessment  assessed the methodological quality of the included studies, and disagreement was resolved by consensus and discussion. Quality of non-RCTs was evaluated with the modified NOS . Continuous and dichotomous variables were assessed by weighted mean differences (WMDs) and odds ratios (OR), respectively. A 95% confidence interval (CI) was recorded. Heterogeneity among studies was quantified using the I2 statistic. According to Higgins’ method, IWe identified ten studies eligible for inclusion: nine non-randomized and one randomized Figure , All of P = 0.64; Figure Five studies reported on in-hospital mortality in 5770 patients. Pooled results showed no significant difference in mortality between the HCR group and CABG group <1 month .Patients treated with HCR did not display a significant reduction in risk for MI as compared with those who received CABG within hospital .Stroke was assessed in five studies reporting on 5793 patients. The prevalence of stroke was not significantly different between groups. .MACCEs occurred in 2.5% (10/408) of patients after HCR and 3.6% (181/5092) of patients with CABG.Six studies (5500 patients) provided data on the prevalence of MACCEs. Pooling of the outcomes of these studies revealed no significant differences in the prevalence of MACCEs between patients treated by HCR and those treated by CABG  or the prevalence of renal failure .There was no significant difference in the prevalence of AF between the two groups , LoS in hospital , and fewer instances of RBC transfusion   of patients after HCR and 11.8% (18/152) of patients with CABG at one year of follow-up. The ORs for MACCEs were not significantly different at one year of follow-up  or LoS in hospital  between the two groups.Inclusion of studies with ≥80 patients in each group did not substantially change the pooled point estimate except for LoS in ICU and LoS in hospital. Pooling the outcomes of these studies revealed no significant difference in the LoS in ICU  whereas HCR was associated with a reduced need for RBC transfusion as well as shorter LoS in ICU and LoS in hospital than CABG.We revealed no significant differences in the prevalence of in-hospital and one-year follow-up mortality, MI, stroke, TVR, MACCEs between the two groups, findings which were consistent with the early results of an ongoing RCT . In thatvs 9.6%) after a mean follow-up of 18 months. Shen et al. [P = 0.030).The prevalence of in-hospital one-year follow-up mortality, MI, stroke and MACCEs were similar between the two groups, however, we hypothesized that HCR may be superior to CABG in terms of long-term MACCEs. Hu et al.  reportedn et al.  also repAs duration of follow-up extends, HCR may be superior to CABG in terms of long-term MACCEs.Several reasons support this findings mentioned above. Firstly, avoiding aortic clamping is one of the unique advantages of the hybrid procedure. Aortic manipulation  is necessary during on-pump CABG . SecondlCardiac surgery and administration of contrast dyes during PCI tends to increase the risk of renal failure. Hence, we suggest that the prevalence of renal dysfunction might be higher in patients treated with HCR compared with those undergoing CABG alone. In this meta-analysis, however, no significant difference in the prevalence of renal failure was found between the two groups. The more stable hemodynamics and better urine output in the hybrid group may have resulted in a similar prevalence of renal failure in the present study ,32.In this analysis, the significantly lower requirement of RBC transfusion in the HCR group was attributed to the less invasive nature of minimally invasive direct coronary artery bypass grafting in the hybrid procedure despite continuous perioperative use of aspirin and perioperative administration of clopidogerl ,14,31.LoS in ICU and LoS in hospital were significantly shorter in the HCR group in this analysis, so the hybrid group had a shorter recovery with a substantial reduction in utilization of hospital resources . This phHeterogeneity among studies was observed for several continuous variables, including LoS in ICU and LoS in hospital. This heterogeneity may have resulted from variations in the surgeon’s caseload, the learning-curve effect, the HCR procedure, perioperative management, and standards regarding hospital discharge among the included studies.http://www.clinicaltrials.gov/ct2/show/NCT01699048 and http://www.clinicaltrials.gov/ct2/show/NCT01035567). Ideally, a meta-analysis should include RCTs only, but inclusion of high-quality non-RCTs can improve the statistical power while maintaining an acceptable level of evidence. Abrahama et al. [This meta-analysis had several limitations. The main limitation of our meta-analysis was the retrospective nature of the available data. No RCT has been published but two protocols have been registered (available at a et al.  reportedSecondly, the duration of clinical follow-up was limited to one year in most studies, whereas a meta-analysis of long-term outcomes was not possible due to insufficient data ,17,18,20Thirdly, the definition of endpoints such as MI, MACCEs, and renal failure in different studies varied to some degree, which may have weakened the evidences in our analysis.Finally, obvious heterogeneity was observed for several continuous variables. Therefore, the random-effects model was used.This meta-analysis suggested that HCR is feasible, safe and effective for treatment of MCAD, with similar in-hospital and one-year follow-up outcomes, significantly lower requirement for RBC transfusion, and faster recovery compared with CABG. It may provide a safe and effective alternative for treating selected patients with MCAD. However, to validate the long-term results of HCR for MCAD, more large-scale, multicenter, prospective RCTs are warranted.


The effects of age, height, and gender on magnetic central and peripheral motor conduction times  were analyzed using a multiple regression model.Motor evoked potentials were recorded in 91 healthy volunteers. Magnetic stimulation was performed over the primary motor cortex  and over the cervical and lumbar spines . The spinal latency was taken as an estimate of PMCT and was subtracted from cortical latency to yield CMCT.Lower limb CMCT correlated significantly with height only; there were no significant predictors for upper limb CMCT. Upper and lower limb PMCT correlated with both age and height.Muscle Nerve51:706–712, 2015This is among the largest studies of CMCT in normal subjects. The multiple regression model unifies previously reported simple regression analyses, reconciles past discrepancies, and allows normal ranges to be individualized.  AHabductor hallucisAPBabductor pollicis brevisCMCTcentral motor conduction timeCSTcorticospinal tractEDBextensor digitorum brevisEDCextensor digitorum communisEMGelectromyogramFDIfirst dorsal interosseousFDSflexor digitorum superficialisGCgastrocnemiusM1primary motor cortexMATSmagnetic augmented translumbosacral stimulationMEPmotor evoked potentialPMCTperipheral motor conduction timeSEPsomatosensory evoked potentialTAtibialis anteriorThe function of the corticospinal tract (CST) can be assessed non‐invasively using magnetic stimulation. Central motor conduction time (CMCT) has emerged as the most reliable parameter,Normal ranges of CMCT have been described in several reports . The set of muscles examined was usually small and varied between studies, and results are only in partial agreement where comparisons are possible. This is illustrated by the large spread of mean CMCT to distal muscles: 5.2–8 ms  in the upper limb and 13.4–18.2 ms  in the lower limb. Contributing factors may include small study populations and methodological discrepancies, particularly with regard to the type of stimulator and coil used. In addition, the lower limb representation of M1 was stimulated with a circular coil, whereas a double cone coil is probably better suited to this task, especially for distal muscles.,Previous studies have also considered the effect of age, height, and gender on CMCT. Statistical methods ranged from comparisons between discrete groupsIn this study we sought to clarify the effects of age, height, and gender on CMCT using a stepwise multiple regression model in a large study population stratified by age. PMCT data were modeled similarly. We employed modern equipment and routine clinical methods, including use of standard circular and double cone coils to stimulate the upper and lower limb representations of M1, respectively.At least 15 volunteers were recruited for each decade of age between 20 and 80 years (50 men and 41 women). Age ranged from 22 to 77 years, and height averaged 171.0 ± 9.6 cm . Eighty‐two subjects were right‐handed, and 9 were left‐handed, based on self‐report. None had a history of neurological disorders or diabetes mellitus, contraindications to magnetic stimulation, or used neurotropic medications. All subjects provided written informed consent. The study was approved by the research ethics committee of the Newcastle University Medical Faculty, and conformed to the Declaration of Helsinki.z to 2 kHz; Model D360; Digitimer, Welwyn Garden City, UK) and digitized at 5 kHz .Every effort was made to maintain subjects at a constant level of alertness, and all assessments were carried out on the dominant side. Subjects were seated in a comfortable chair with the arm resting on a cushion. Surface electromyography (EMG) was recorded from abductor pollicis brevis (APB), first dorsal interosseous (FDI), flexor digitorum superficialis (FDS), and extensor digitorum communis (EDC) in the upper limb, and from the extensor digitorum brevis (EDB), abductor hallucis (AH), tibialis anterior (TA), and gastrocnemius (GC) in the lower limb. Adhesive electrodes  were placed in a belly‐tendon montage over the intrinsic muscles of the hand or foot. For the long muscles of the forearm or calf, a belly‐tendon montage would have resulted in large interelectrode distances, thus increasing cross‐talk from neighboring muscles. Hence, electrodes were placed 4 cm apart, one‐third of the distance along the long muscle from its proximal origin. Signals were amplified, band‐pass filtered , a circular coil (13 cm outer diameter) was held over the vertex with its orientation optimized for stimulation of the dominant hemisphere . For lower limb cortical MEPs, a double cone coil was used in an analogous manner . Stimulation intensity was set at 10% of maximum stimulator output above the resting motor threshold as defined by the Rossini‐Rothwell method.Magnetic stimulation was delivered using a stimulator device  at a frequency of 0.2 HAnalysis was performed in MATLAB  using custom scripts. The shortest onset latency for each set of 10 MEPs was assigned interactively. In the presence of a background contraction, the earliest deflection of the MEP with the shortest latency was often ambiguous on superimposed raw traces because of background EMG activity but could be identified easily on averages of rectified MEPs . Each step involved evaluating the residuals of the model and the associated probability for each predictor and moving a single predictor into or out of the model as recommended by the interactive tool. Significance thresholds were set at ≤0.05 for a predictor to enter the model and at ≥0.10 for it to be removed. The model was considered complete when no further movement of predictors was recommended.One subject did not tolerate lower limb cortical MEPs, but all remaining subjects completed all parts of the protocol.Means, standard deviations, and regression models are listed numerically in Tables Upper limb CMCT was not significantly related to any of the potential predictors Table A–C.r2; Table Lower limb CMCT showed a significant positive relationship with height; there was no significant relationship with age or gender. The regression models accounted for approximately 5–12% of the variance observed  coil, Matsumoto and colleagues selectively stimulated either the lumbosacral nerve roots near the exit foramina, akin to conventional magnetic root stimulation, or the conus medullaris within the spinal canal.1; CMCT = 0.083 × height − 0.47, P < 0.000114).Our finding of a significant relationship between lower limb CMCT‐M and height is in agreement with several previous studies describing similar results in someIt has been suggested that this relationship may be attributable to the peripheral component of CMCT‐M. If so, no such relationship should exist for CMCT‐F. Although several studies have reported both CMCT‐M and CMCT‐F, few have considered how both types of CMCT may differ in their relationship to height. One report showed that CMCT‐M was related to height in 1 of 3 muscles, but CMCT‐F was not,There is consensus that upper limb CMCT does not correlate with height.,The effect of age on CMCT is controversial. We observed no correlation between age and CMCT‐M. This concurs with 6 earlier reports measuring both CMCT‐M and CMCT‐F and finding that neither of them were significantly related to age.Similar to our findings, previous investigations showed CMCT to be unaffected by gender,2) was greater for PMCT than for CMCT. We are not aware of any previous multiple regression models for PMCT, but the proportion of variance explained by our model for PMCT is in broad agreement with values reported for multiple regression models of related peripheral conduction parameters.It is well known that age and height correlate negatively with peripheral nerve conduction velocities and correlate positively with distal motor and F‐wave latencies, whereas gender is generally not considered a significant predictor.The relationship between PMCT and height is explained readily by the strong correlation between height and limb length and thus the length of the peripheral nerves.CMCT and PMCT showed greater spread in the lower limb than in the upper limb Tables  and 2. Tq1 − α/2 standard deviations, where α is the desired significance level and q is the inverse of the normal cumulative distribution function. For example, a 95% prediction interval has α = 0.05 and q1 − α/2 = q0.975 = 1.960; this upper bound would be exceeded by α/2 = 0.025 = 2.5% of normal readings.In clinical practice, numerical results are typically compared with normal ranges or cut‐off values, which constitutes fixed‐level testing at a predetermined significance level. Here, an appropriate cut‐off would be the upper bound of a chosen prediction interval. The bound can be approximated by evaluating the regression model with the parameters of the patient and adding Z‐score is calculated as Z =  / standard deviation of residuals. The corresponding probability is then computed as ϕ(−|Z|), where ϕ is the cumulative normal distribution.Alternatively, we can evaluate the probability of observing a latency at least as high as that of the patient under the null hypothesis that the latency of the patient is normal. The regression model is evaluated with the parameters of the patient, and the The data provided allow either approach to be implemented easily; we have deliberately not provided prescribed cut‐off values, as they would force the reader into fixed‐level testing with a chosen significance level.In conclusion, this is among the largest studies of CMCT‐M in normal subjects and, to our knowledge, the only study to employ multiple regression modeling. Such an approach was applied to SEPs more than 3 decades ago, and its application to MEP data has helped to reconcile controversies surrounding the effects of age and height on CMCT. In addition, the model accounts for 5–12% (CMCT) or 19–53% (PMCT) of variance. Paired with side‐to‐side comparisons within a given subject, this should boost the diagnostic accuracy and precision of CMCT‐M.Supplementary InformationClick here for additional data file.


In recent years, biomaterials have advanced into a burgeoning interdisciplinary research field combining traditional material science, engineering, biology and medicine. Over the last half-century, many types of lifeless materials such as metals, ceramics and polymers have become ‘bio’-materials, making their way into the fabrication of medical devices for the repair or replacement of damaged or deteriorating human tissues and organs. As we proceed into this new century, this process has accelerated. In the last decade alone, the integration of advances in materials and life sciences with cutting-edge medical sciences and nanotechnologies has driven biomaterial research into an explosive phase. In our pursuit of happiness, longevity and quality of life, we aspire to acquire abilities not only to repair damaged tissues and organs, but also to regenerate them. Driven by this desire and the advances in stem cell and developmental biology, biomaterials science has entered a ‘regenerative’ era. A new crop of materials has gained regenerative properties in becoming ‘smart biomaterials’ that can not only provide mechanical support, but also function as sophisticated regulators of biological activities by activating stem cells and triggering the body’s own regenerative processes to restore functions of damaged tissues and organs.Regenerative Biomaterials’, a Chinese Society for Biomaterials (CSBM) journal and a platform devoted to disseminating the latest research advances in regenerative biomaterials as well as traditional biomaterials and medical devices.The potential of regenerative biomaterials is limitless. Even traditionally considered non-regenerative tissues such as nerves and teeth can now regenerate with the help of advanced biomaterials and stem cells. Materials with regenerative properties have laid the foundation upon which regenerative medicine and stem cell therapies can be built, and spawned a new research field of ‘regenerative biomaterials’, which encompasses a variety of research areas including stem cell therapy, tissue engineering and biomedical engineering. Many scientists from these diverse disciplines have been attracted to this rapidly expanding field, bringing in new ideas, new approaches and a new flood of research articles on this red-hot topic. Against this backdrop, we are launching ‘Journal of Biomedical Materials Research , the official journal of the Society for Biomaterials (USA), the Japanese Society for Biomaterials, the Australasian Society for Biomaterials and the Korean Society for Biomaterials; the Journal of Materials Science: Materials in Medicine (published by Springer), the official journal of the European Society for Biomaterials; and Biomaterials Research , the official journal of the Korean Society for Biomaterials. Therefore, it is quite necessary for the CSBM to have its own official journal for nurturing the development of Chinese biomaterial research and cultivating interaction with scientists from other parts of the world. In addition, the ever-growing number of high-quality manuscripts submitted annually from China also propels the need of a new journal and guarantees the standard of our publication. To forge international collaboration, we also accept high-quality manuscripts from scientists worldwide.Founded in April 2012, the CSBM is a non-profit academic organization for Chinese biomaterial researchers inside China and abroad. The CSBM is one of the six founding members of the International Union of Societies for Biomaterials Science and Engineering (IUSBSE). In June 2012, CSBM successfully organized the 9th World Biomaterials Congress in Chengdu, which received great attendance and applause from scientists worldwide. As we all know, almost all influential societies in the field of biomaterials have their official journals, such as the Regenerative Biomaterials will be an international, interdisciplinary, peer-reviewed journal concerning frontiers and advances in the new generation of biomaterials and regenerative medicine. The aim of the journal is to provide a forum for original research papers, reviews, clinical case reports and commentaries on the topics relevant to the development of advanced regenerative biomaterials. Manuscripts using novel regenerative technologies and therapeutic approaches for the regeneration and repair of damaged tissues and organs, and manuscripts addressing interactions between biomaterials and cells or tissue, especially stem cells, will be considered with high priority. However, studies on cells or stem cells in regenerative medicine but without materials will not be included. The journal will be a platform for communication among various disciplines concerning biomaterials; these include nanotechnologies, stem cell biology, regenerative medicine, and clinical medicine. We hope that the new journal will contribute to the world community of biomaterials with rapid publication of high-level, creative research.


Degradation of cellular proteins by the proteasome is critical for the generation of MHC-associated peptides. The constitutive proteasome and the IFNγ-induced immunoproteasome (IP) differ in the use of three catalytic β subunits, which alters production of MHC class I epitopes. The potential for a disparate repertoire of epitopes produced between inflammatory/non-inflammatory tumours therefore arises.We have extensively investigated this phenomenon both broadly, in terms of the cancer cell as a whole, and specifically, by evaluating presentation of three NY-ESO-1 HLA-Cw3 restricted epitopes.We profiled the immunopeptidome of a melanoma cell line under steady state or IFNγ-treated conditions; by mass spectrometry analysis of HLA Class I bound peptides. Profound changes in the overall epitope profile presented on HLA Class I molecules, leading to extensive alteration in the overall immunogenicity of the cell, were observed.We studied processing and presentation of three NY-ESO-1 HLA-Cw3 restricted epitopes by melanoma cell lines. Using specific T-lymphocyte clones, we examined presentation of each epitope following processing through proteasome subtypes. We further investigated processing of these epitopes by selective inhibition of the IP catalytic subunit LMP7 by siRNA knockdown, or small molecule inhibition.In vivo, we demonstrate that melanoma cells can be induced to switch proteasome subtype following treatments which induce IFNγ at the tumour site.Our data demonstrate broad changes in epitopes presented by melanoma cells under inflammatory versus non-inflammatory conditions. These results illustrate a little-studied mechanism of immune escape by tumour cells. Awareness of how individual cancer epitopes are processed by melanoma cells is therefore critical to inform development of future therapies which involve cancer vaccination, adoptive T-lymphocyte transfer, or combination treatments including these.


C. elegans strains originated from various geographic areas. We found a negative correlation between lifespan and developmental rate. In contrast, we did not find such negative correlation between longevity and developmental rate among the individuals of C. elegans strains. These data imply that polymorphic genetic variants among wild isolates determine resource allocation to longevity and developmental rate.Genetic studies using model organisms have shown that many long-lived mutants display impaired fitness, such as reduced fecundity and delayed development. However, in several wild animals, the association between longevity and fitness does not seem to be inevitable. Thus, the relationship between longevity and fitness in wild organisms remains inconclusive. Here, we determined the correlation between lifespan and fitness, developmental rate and brood size, by using 16 wild-derived  Despite extensive research efforts during the past several decades, aging remains as a mysterious process that almost all organisms experience. Many theories have been proposed to explain the causes of aging. The antagonistic pleiotropy theory suggests that genes that cause aging confer beneficial effects in early life but have harmful effects in the later life stages of an organism . The disCaenorhabditis elegans is one of the best-established model organisms for aging research. C. elegans is relatively short-lived, genetically tractable, and amenable to track life-historical traits . Stiewed in , 12).C. elegans genetics research. Many other wild C. elegans strains from various geographic areas have also been isolated and characterized. Large-scale analysis of single-nucleotide polymorphism (SNP) patterns, phylogeny, and transcriptomic data indicates genetic variations among the wild isolates of C. elegans . Worms were washed off replete NGM plates with M9 buffer, and remaining eggs were incubated at 20°C for 1-2 hours for synchronization. Forty newly-hatched L1 progeny were transferred to new OP50-seeded NGM plates and were allowed to develop. Worms that have at least one egg in their bodies were considered as adults. The number of adult worms was counted every 2 hours after 40 hours from the L1 transfer. The developmental time of all 16 wild analysis . Data wehttp://sbi.postech.ac.kr/oasis) was used for statistical analysis for lifespan and developmental time [Adult worms were washed off replete NGM plates with M9 buffer, and remaining eggs were allowed to hatch at 20°C for 1 hour for synchronization. Newly-hatched individual L1 worms were transferred to new individual OP50-seeded NGM plates, and allowed to develop. Worms that have at least one egg in their bodies were considered as adults. The developmental time for each worm was recorded every 2 hours until the worm reached adulthood. The adult worms were then transferred to new individual NGM plates every 12 hours until they did not lay eggs. The brood size of each worm was the total number of hatched progeny during the duration of the assay. Worms that ceased producing eggs were transferred to new individual NGM plates for continued lifespan assays. Worms were considered as alive if they moved when prodded. All assays were conducted at 20°C three times independently. OASIS  for correlations in this study. The obtained p values for several datasets were also manually confirmed as follows. The statistical significance of the Pearson correlation coefficient r between two variables ix and iy  was tested under the null hypothesis that there is no association between ix and iy. We numerically obtained the null distribution of r, when ix's (iy's) were randomly permuted against iy's (ix's). From this null distribution, two-sided p values were computed.A web-based calculator was used to obtain


For this purpose, the HMMER program was used to align 3,016 epitopes reported in the Immune Epitope Database and Analysis Resource (IEDB) and distributed in 34,294 stored sequences in the Pfam database. Eighteen epitopes were identified: 8 in HA, 5 in NA, 3 in NP, and 2 in M2. These epitopes have remained constant since they were first identified (~91 years) and are present in strains that have circulated on 5 continents. These sites could be targets for vaccination design strategies based on epitopes and/or as markers in the implementation of diagnostic techniques.The unpredictable, evolutionary nature of the influenza A virus (IAV) is the primary problem when generating a vaccine and when designing diagnostic strategies; thus, it is necessary to determine the constant regions in viral proteins. In this study, we completed an  The influenza A virus (IAV) appears seasonally, causes annual epidemics, and occasionally presents a new strain with pandemic reach, leading to severe consequences for global health and for the global economy , 2. EverIAV is an enveloped virus and a member of the orthomyxoviridae family; its genome consists of eight segments of simple chain RNA of negative polarity that code for 3 structural proteins , 1 membrane protein (M1), and 6 nonstructural proteins  .IAV is classified according to its two principal membrane antigens: HA 18 subtypes) and NA 11 subtypes) , 7. In h subtypes subtypesThe following three subtypes are responsible for the pandemics of the past century: H1N1 , H2N2 , and H3N2  .The last influenza pandemic occurred in 2009 due to a new pandemic virus A H1N1 (A H1N1pdm 09). This virus was detected in 214 cities on 5 continents and, up to July 30, 2010, had caused 18,389 cases [These pandemic viruses arose due to a combination of aviary, pig, and human influenza viruses –15 becauThese mutation mechanisms confer an unpredictable, evolving character to the influenza viruses, which is the principal difficulty to overcome when designing a vaccine. This difficulty occurs because while vaccination has been an effective method against 60 to 90% of seasonal strains , it has Inactivated trivalent vaccines that contain the hemagglutinin (HA) protein of the influenza A (H1N1) and A (H3N2) viruses and influenza B virus are the only authorized commercial vaccines . Due to Antiviral strategies that are currently in development include neutralizing antibodies , small m in silico identification of highly conserved epitopes with diagnostic and vaccination potential in the HA, NA, MP, and M2 proteins of the influenza virus that have been reported from 1918 to 2014 for the primary strains that have circulated in the world .Therefore, it is of vital importance to know the sites of the proteins or antigenic determinants among the different strains of the influenza virus that historically have been recognized by the immune system. Therefore, the present study completed anhttp://www.iedb.org/), the following inclusion criteria were taken into account: experimentally reported epitopes for HA, NA, NP, and M2 proteins corresponding to the H1N1, H2N2, H3N2, and H5N1 subtypes of influenza A from 1918 up to 2011.To complete the epitope search on the IEDB site .http://pfam.xfam.org/), sequences corresponding to the hemagglutinin (PF00509), neuraminidase (PF00064), nucleoprotein (PF00506), and matrix 2 (PF00599) protein families were downloaded from the Pfam-A entries along with sequences from the NCBI database, representing manually refined, high-quality families.Using the Pfam database , each one of the alignments downloaded from the Pfam database was used in the construction of hidden Markov models. This algorithm generates a file with the extension ∗.hmm that contains the consensus sequence for each family of proteins.Using the hmmalign algorithm was used to align the epitopes with their corresponding hidden Markov models (∗.hmm). This algorithm generates a file with the extension ∗.sto that contains the epitopes aligned with the consensus sequence for each protein.Subsequently, the hmmalign program, the regions that presented the greatest frequency of reports of epitopes, at least 2 standard deviations above the mean, were selected for each protein and strain analyzed. Subsequently, two new alignments were completed with the help of the Megalign program . The first alignment was between the present epitopes in each region to determine the consensus recognition sequence. The second was between this consensus sequence and some current strains that circulated between 2013 and 2014 to determine the similitude percentage with strains that currently circulate.Based on the files generated by the hmmbuild program, mold structures downloaded from the Protein Data Bank (PDB) with similitude percentages ≥70% were utilized. With these structures as a template, a three-dimensional structure of each protein was modeled in the Swiss-Model  virtual platform; the consensus regions of each epitope were highlighted in the model utilizing the PyMOL program .To complete the modeling of the consensus sequences generated using theFor each epitope group, a phylogenetic tree with the complete sequence of the protein to which it belongs was constructed using the MEGA program ; the treTo determine the reach that using these epitopes would have, in terms of vaccination or diagnosis in the world population, an analysis of the strains containing the consensus epitopes determined in this study was completed; for each epitope, data from viruses isolated from cases that were reported were collected.The stages of the entire analysis and the programs used are summarized in Descriptive statistics were used to determine the regions of the proteins that were recognized with the greatest frequency. For this purpose, the protein was divided into groups, each containing 10 amino acids, the frequencies of epitopes present in each group were observed, and the means and standard deviations were calculated. Up to 3 regions with frequencies at least 2 standard deviations above the mean were chosen for each protein.In total, 3,016 epitopes and 34,294 sequences were identified. Of the epitopes and sequences, respectively, 1,352 and 15,102 belonged to H1N1, 91 and 412 belonged to H2N2, 1,006 and 12,186 belonged to H3N2, and, finally, 567 and 6,594 belonged to H5N1. In all cases, HA is the protein for which more epitopes and sequences have been reported . hmmalign program, 25 sites were identified which showed the greatest frequency to which the epitopes aligned. These sites were distributed in the following manner: 8 for H1N1, 3 for H2N2, 6 for H3n2, and 8 for H5N1  or a strain reported in 2013 (in the case of H5N1). The mutation rates for nucleotide substitutions per site per year were 1.2 × 10 strains .The circulation analysis revealed that the epitopes defined in this study have been present in isolates obtained from 5 continents: the H1N1 epitopes have been identified in China, Mongolia, the USA, France, and Puerto Rico; the H2N2 epitopes have only been identified in Japan; the H3N2 epitopes were isolated in Panama, Argentina, Canada, the USA, China, Holland, France, and Australia; and, finally, the H5N1 epitopes were observed in Indonesia, Thailand, Vietnam, China, Japan, France, and Egypt .While the interest in the scientific community is focused on the production of an influenza vaccine that provides cross-protection against multiple subtypes , thus fa in silico analysis using previously reported epitopes and those for which evidence indicates they are capable of inducing an immunological response.This necessity emphasizes the importance of identifying IAV sequence regions that remain constant and that, moreover, have the capacity to induce immunological responses from T and B cells. Therefore, this study completed anAs was to be expected, given that H1N1 was the first IAV subtype that was recognized and has been circulating in the world since 1918, of the epitopes used in this study, those corresponding to H1N1 represented 44.8%, while those corresponding to H2N2 only represented 3%. This difference could, in part, be due to the fact that H2N2 only circulated after the 1957 pandemic up until 1968, when it was displaced by the H3N2 subtype .However, for all of the subtypes, with the exception of H2N2, the protein in which the greatest number of epitopes has been described is HA, with the following percentages: H1N1 50.7%, H3N2 52.2%, and H5N1 67.7%. This finding is similar to observations made by Bulimo and Cols in 2012 . InteresIt is important to note that although the H5N1 subtype has only been detected sporadically in humans, it is generally associated with cases of zoonosis in individuals who have very close contact with poultry or undomesticated birds as a result of work activities or hunting, respectively . The epiStudying the highly recognized zones or those zones with the greatest numbers of epitopes within the IAV proteins over the years is related to site conservation; their remaining constant makes it possible to identify the strains that will circulate in the coming years. Some of the consensus epitopes defined in this study are so constant that they have circulated for 91 years; these epitopes have been described in various studies conducted using sequences belonging to the 1918 strain and reported from 1983 (by Hackett and Cols) until 2012 (by Vergara and Cols). In the case of H2N2, there are epitopes that have been maintained for 44 years and have been described in studies completed from 1983 (by Lamb and Cols) until 2009 (by Babon and Cols). This study is the first to consider epitopes reported in strains that have circulated since 1918 to determine their conservation over such a long period of time.It is not unusual that, for some IAV proteins, almost their entire sequence can be considered an epitope. Nevertheless, not all of these epitopes have the capacity to produce a protective response by inducing neutralizing antibodies, a fact that has been well verified in studies such as Gelder et al. in 1995  and in 1Of the 3,016 epitopes analyzed in this study, 18 consensus epitopes were identified, according to what was reported in the IEDB. Twelve of the epitopes were recognized exclusively by T lymphocytes , 39, 43, in silico manner, the minimum sequence that could be recognized by the immune system ; thus, we can infer that this region in general and, more specifically, the region close to the sialic acid-binding site show a greater degree of conservation than the stem region. These findings are different than those reported by Bulimo et al. in Kenya in 2012  in a stuIn the case of the NA protein, the consensus epitopes were identified far from the zanamivir-binding site, which would be expected, as this site is the site of greater selective pressure, resulting in a tendency for more modification. These findings coincide with those reported for the H1N1 subtype by Moscona in 2009  and by BFor the M2 protein, consensus epitopes were detected in the membrane and transmembrane regions, even though this is the Amantadine-binding site. Although resistance to ion channel blockers is less common for the M2 protein  than theAnother point to consider when searching for constant sites is the mutation rate for IAV proteins, which, contrary to what was expected, are 3-fold lower for the HA proteins of the H1N1 subtype than for HA proteins of the H3 and H5 subtypes. Similarly, the mutation rates are also 3-fold lower than that reported by Klein and colleagues  in 2014.Finally, one aspect to explore is the reach of the vaccination or diagnostic strategies that could develop from these epitopes, as, currently, seasonal influenza vaccine design is based on strain circulation , indepen in silico manner, it is necessary to demonstrate the potential of these findings experimentally in future studies.In conclusion, this study was able to identify 18 epitopes present in the HA, NA, NP, and M2 proteins of IAV that are, in accordance with previous studies, able to induce an immune response via T and B cells. These epitopes have remained constant for up to 91 years and have circulated on various continents. Nevertheless, because the analysis was completed in an


Hereditary Cancer in Clinical Practice would like to thank all our reviewers who have contributed to the journal in 2015.The editors of Hereditary Cancer in Clinical Practice. We look forward to your continued support in 2016.Without the participation of skilful reviewers, no academic journal could succeed, and we are grateful to the committed individuals who have given their time and expertise to the peer review of manuscripts for  Stefan AretzGermanyGabriela BaloghArgentinaElizabeth BancroftUKMorgan ButrickUSABerta CamposSpainJohn V ConaglenNew ZealandCezary CybulskiPolandTadeusz DebniakPolandPawel DomagalaPolandThilo DorkGermanyDagmara DymerskaPolandD Gareth EvansUKThomas HansenDenmarkShirley HodgsonUKFrans HogervorstNetherlandsEvgeny ImyanitovRussiaArvids IrmejsLatviaAnna JakubowskaPolandWojciech KluźniakPolandKristina Lagerstedt RobinsonSwedenMarcin LenerPolandAnnika LindblomSwedenFinlay MacraeAustraliaDiptasri MandalUSAAlexandra MartinsFranceCliff MeldrumAustraliaJanusz MenkiszakPolandCaterina MianItalyPal MollerNorwaySteven NarodCanadaJan OosterwijkNetherlandsCarmen Radecki BreitkopfUSAMalgorzata Stawicka NielacnaPolandBente Talseth-PalmerAustraliaAmanda TolandUSAAnnemieke van der HoutNetherlandsRob van der LuijtNetherlandsMichelle Wong-BrownAustraliaXingnan ZhengUSA


When designing micro-scale tactile probes, a design trade-off must be made between the stiffness and flexibility of the probing element. The probe must be flexible enough to ensure sensitive parts are not damaged during contact, but it must be stiff enough to overcome attractive surface forces, ensure it is not excessively fragile, easily damaged or sensitive to inertial loads. To address the need for a probing element that is both flexible and stiff, a novel micro-scale tactile probe has been designed and tested that makes use of an active suspension structure. The suspension structure is used to modulate the probe stiffness as required to ensure optimal stiffness conditions for each phase of the measurement process. In this paper, a novel control system is presented that monitors and controls stiffness, allowing two probe stiffness values (“stiff” and “flexible”) to be defined and switched between. During switching, the stylus tip undergoes a displacement of approximately 18 µm, however, the control system is able ensure a consistent flexible mode tip deflection to within 12 nm in the vertical axis. The overall uncertainty for three-dimensional displacement measurements using the probing system is estimated to be 58 nm, which demonstrates the potential of this innovative variable stiffness micro-scale probe system. Micro-scale coordinate measuring machines (micro-CMMs) make it possible to measure micro-scale features on parts that are small, intricate and fragile. Micro-CMMs can demonstrate very high accuracies, with volumetric uncertainty levels of a few hundred nanometres or less ,2. MicroTo achieve low contact forces, micro-probes are designed to incorporate highly compliant suspension structures, onto which the stylus system can be mounted . WhIn recent work, a variable stiffness probing system has been proposed to tackle the problem of optimising probe stiffness, and minimise the need for design compromise. The variable stiffness probing system makes it possible to modulate probe stiffness, so the optimum stiffness can be selected as required. Stiffness modulation is achieved through the use of a novel suspension structure that is combined with a set of piezoelectric actuators. The actuators are configured to apply compressive forces to the suspension structure, resulting in a reduction of stiffness. Controlling the magnitude of the compressive force, therefore, allows the probe stiffness to be controlled. A full description of the design and working principle behind the variable stiffness probing system is presented elsewhere ,12.The previous work illustrates the concept of variable stiffness, and demonstrates the potential performance in terms of stiffness modulation. In this paper, the results for a performance evaluation are presented for this new form of variable stiffness probing system. A brief description of the probing system and the associated fixtures is first presented. Then, to set the context for the experimental evaluation, the intended measurement strategy for the probe is defined. In addition, the novel control system used to set, monitor and control probe stiffness is discussed. The method used to complete a three-dimensional (3D) characterisation of the probe is then presented. Based on the characterisation results, a number of probing system errors are evaluated, including linearity, repeatability and long-term stability. To investigate the capability to modify stiffness during use, the probe is assessed under different compliance conditions, and following repeated switching between these states.A schema of the variable stiffness micro-scale probing system is shown in The stiffness of the probing system can be modulated in two operating modes: “stiff” and “flexible”, which can be selected by switching the actuator force, supplied by the three actuators, between set-point values. To identify the expected vibration modes of the probing system, modes representative of vertical and lateral stiffness were estimated using finite element analysis of the full structure including the stylus system, and the selected mode shapes can be seen in −1 in the vertical direction, and around 610 Nm−1 in the lateral direction. For the flexible mode, the displacement is set at 2.2 µm and the frequency is 600 Hz; the vertical stiffness is 914 Nm−1 and lateral stiffness is 410 Nm−1, as illustrated in To investigate the relationship between applied actuator load and probe stiffness, a finite element model (FEM) of the probe was developed. In the FEM, for simplicity, the effect of the actuator was modelled as a static displacement. For a range of applied actuator displacements, the stiffness and first two resonant modes of the probe were evaluated using a previously validated modelling process . Figure The sequence of operation for a variable stiffness probing system consists of four steps. The first step is to drive the probing system to a position where the stylus tip is close to the measurement surface. During the first step, it is advantageous for the micro-CMM to move the probing system as fast as possible towards the target surface. Therefore, the probe will be in the stiff mode to prevent inertial loads, generated as the probing system is moved by the micro-CMM, from causing misleading deflections of the probe tip. The second step is to switch the probe to be in a flexible mode, so it is ready to make contact with the target surface. During this step, a potential difference is applied across the piezo-electric actuators to achieve the desired reduction in stiffness. To ensure that a repeatable stiffness value is reached, a feedback control loop is used see . The thiThe open-loop control capability of piezoelectric stack actuators is significantly limited due to hysteresis and creep effects ,14. TherTo operate the flexible probing system in the two modes requires the use of two PID control loops. The two loops were implemented in LabVIEW software from National Instruments; To provide a stable environment for testing the capability of the variable stiffness probing system, the experimental work was completed in a temperature controlled laboratory (20 ± 0.5 °C). An overview of the experimental setup is shown in To simulate the measurement process, a nano-positioning stage  was used to displace the probe tip while the probe fixture remained static. The nano-positioning stage had a 100 µm maximum displacement, nonlinearity error of 0.1% and a repeatability of less than 10 nm . In thisTo provide additional environmental stability, the actuators and probing system were housed within a foam enclosure. This enclosure provided an insulating barrier to reduce thermal fluctuations, and reduce the impact of acoustic vibrations and air turbulence. Two thermocouples were used see  to measuSignal synchronisation and excitation were achieved using National Instruments hardware (NI cRIO-9022) and LabVIEW FPGA (field programmable gate array). A lookup table was used to store waveforms for the linear chirp signal in the FPGA. A control system was designed to operate the probe using LabVIEW Real-Time. Coordination of the FPGA and Real-Time controller was achieved using Direct Memory Access, and the resulting control loop had a cycle time of approximately 51 ms. The maximum measuring rate of the capacitive sensor was 10 kHz, which is well above the maximum expected resonant frequency for the probing system (approximately 1 kHz).Two kinds of experiments have been designed to investigate the performance of the micro-probing system. First, the behaviour of the probing system in the stiff mode, where the system can work as a normal micro-scale probing system, is considered. This initial investigation determines long-term stability, sensitivity of the probe in 3D and repeatability. Second, the probing system is investigated in the flexible mode, and the ability of the probe to switch between stiff and flexible modes is examined. For example, is the probing system able to repeatedly switch from stiff to flexible mode with no resultant off-set errors, and will the sensitivity of the probing system be changed as a result of switching between modes?The stability of the probing system in the lab environment was assessed over a continuous eighty hour period. During this time, the probing system output, and the temperature and humidity in the isolation enclosure see  were reci.e., displacement along the x, y and z axes, as shown in 1, δ2, δ3 and δ4 displacement vectors described below, and then relate these to motion in a Cartesian frame . To calibrate the probing system in the stiff mode, the stylus tip was displaced over an incremental range using the nano-positioning stage. To minimise the effect of humidity-related drift, the calibration was conducted over a one minute period for each direction. To correctly align the nano-positioning stage for the three lateral displacements, the triangular body of the probe fixture was used to provide datum faces  relate the displacement of the stylus tip in the four directions described see  to the tIn Equations (1) and (2), the first subscript denotes the Cartesian axis they relate to, and the second subscript denotes the sensor. Finally, Equation (3) relates the sensor voltage directly to the displacement of the stylus tip with respect to x, y and z axesIt is important for accurate probe measurements that the stylus tip has a well-known and repeatable position before and after being displaced by contact. Therefore, the repeatability of the stylus tip initial position was evaluated for both lateral (y) and vertical (z) displacements . For each displacement direction, the repeatable return to a datum position, henceforth called the “zero offset condition” was recorded for three nominal displacements of 4 µm, 8 µm and 14 µm. For each of the nominal displacements, the probe was displaced and then returned to the zero offset condition, with ten repeats being made at each displacement. Data is presented for both the return to zero offset, and the deviation between readings for the nominal displacements. As described in Using the transfer function described in Equation (4) (see below), the impact of actuator creep on the stylus tip displacement was calculated. From In order to investigate the sensitivity of the probing system in the flexible mode, it was calibrated using the same procedure as for the stiff mode. The position of the nano-positioning stage was maintained between the calibration of the stiff and flexible mode in each direction to minimise error. The calibration was completed within one minute, reasonably limiting drift to less than 6 nm, as described above. The transfer function resulting from the calibration of the four directions is given in Equation (4).In order to illustrate the linearity of the probe’s response to displacement, the residual is plotted in To demonstrate that changing between modes does not significantly affect the calibration, a calibration was first performed in flexible mode, and then the probing system was set to stiff mode before returning to flexible mode for a repeat calibration. The coefficients generated from each calibration cycle are presented in in vice versa.As well as determining the effect that switching from stiff to flexible mode has on sensitivity, it was also necessary to determine any unwanted stylus tip position offset error. This was determined with the knowledge that the load applied to the beams will result in a deflection of the stylus tip, which is most dominant in the z direction . If the To overcome this issue, the frequency-based probe stiffness control system , was use−1. The control system is able to tune the frequency within a range of 0.075 Hz . This validates the feasibility of variable stiffness probing systems; showing the ability to switch between stiff and flexible modes with minimal impact on probing system performance.Combining stage errors with the probing system linearity error, the stylus tip zero offset position error and the probing system measurement repeatability, gives an estimate of a combined uncertainty for the probing system or of 29 nm. The dominant uncertainty comes from the linearity error of the probing system. The expanded uncertainty is 58 nm (While changing stiffness causes minor repeatability errors, it must be noted that as a result of switching, the stylus tip undergoes a displacement of approximately 18 um, which may cause issues for future application of the probing system. This displacement results from the tendency of the suspension structure to bend under the influence of uneven compressive load. The displacement at the probe tip is equal to −13.78 µm, 4.36 µm and 12.51 µm in x, y and z direction. The deflection of the stylus tip by this amount could cause large contact forces to occur when the probe is changed from flexible to stiff mode. To accommodate this displacement, three displacement sensors could be integrated to make sure three displacements are equal from each actuator, which would avoid the lateral deflection of probe tip during the translation between modes. Then to account for the motion in the vertical direction, the probe tip could be manipulated by the CMM in synchronisation with switching of the mode. This would limit the contact force generated by the probing system. Even if the displacement sensors were not applied, and a complex displacement in three directions was observed, the CMM could still be controlled in synchronisation with the probing system such that as the stiffness is changed the CMM retracts the stylus tip to compensate for any resulting deflection. This compensation procedure would be learned as part of the calibration routine for the probe, by slowly switching stiffness and monitoring the stylus tip deflection as a function of actuator voltage; the learned compensation displacement path would be applied by the CMM during stiffness mode transitions.In this paper, the performance of the first variable stiffness micro-CMM probing system has been characterised. The evaluation was based on a large body of experimental data, that the interested reader is able to access via an online database . Using tA control system, based on measuring resonant frequency, has been developed and its ability to tune the stiffness in both stiff and flexible modes was demonstrated. When actuated from one mode to another, the suspension geometry is altered, and the stylus tip moves, but the rest position for both modes is shown to be predictable and repeatable to within 12 nm between the stiff and flexible mode in the z direction.A practical limitation of the probing system is that it takes approximately twenty seconds to reach a steady state when the controller switches to flexible mode, as illustrated in The optimum stiffness for the probe would be zero stiffness for contacting and infinite for removal. In the paper we present a probe that has the ability to modulate frequency, and prove that it is possible to dynamically modulate stiffness during probing. However, more work is now needed to further increase the size of the stiffness change that is possible.


Journal of Venomous Animals and Toxins including Tropical Diseases (JVATiTD – www.jvat.org) [The year 2015 marks 350 years since the appearance of the world’s first scientific publication and 20 years since the publication of the vat.org) .Journal des Sçavans and Philosophical Transactions. Since then, the structure of a scientific work has respected the basic paradigms “introduction, methods, results and discussion” – one of humanity’s oldest formats.The printing-press revolution, one of the most important breakthroughs for humanity, began about 1465 when Johannes Gutenberg invented a moveable mechanical type for printing. In 1665, the first scientific journals appeared in France and England, called respectively the In 1900, 9,000 scientific articles were published per year; by 2000, this number had grown to 900,000. In 2010, PubMed Central, which does not index the majority of the world’s journals, received 1,100,000 papers for indexing, i.e., approximately 3,000 per day . These dhttp://arxiv.org/), which was considered the first electronic scientific publication [In August of 1991, Paul Ginsparg, a physicist at Cornell University (USA), founded the Los Alamos e-print archives  circulated until 2002. Between 1995 and 1999, it was distributed on floppy disks, between 2000 and 2002 on CD-ROM, and only from 2003 on by the Internet, initially via the website http://www.jvat.org.br. In the same year, the publication was broadened to include tropical diseases, and was then called the Journal of Venomous Animals and Toxins including Tropical Diseases (JVATiTD) [In 1995, there were about 100 electronic journals in the world, and the commercial Internet was starting in Brazil. In this context, the first Brazilian electronic scientific journal was launched in Botucatu, covering the area of Toxinology. This new journal started one hundred years after Vital Brazil lived in Botucatu. It is a homage we paid to this brilliant Brazilian scientist . The JouJVATiTD) .In 2006, the journal was selected to be included in the Science Citation Index Expanded (ISI – Web of Science™ – Thomson Reuters) and in 2007, received its first impact factor, 0.43. Since between 2007 and 2012 the impact factor remained steady between 0.3 and 0.5, the Editorial Board decided to migrate to a renowned international publisher that would maintain the open access policy and contribute to improving the quality of the publication. It was decided at the time by BioMed Central, Springer. After initiating the partnership, the following goals were established: to index immediately in PubMed Central; to maintain the policy that APCs (article-processing charges) would be free to UNESP researchers; to restructure the editorial board by inviting new researchers, preferentially foreigners; and to increase the impact factor to 2.0 within five years.In January of 2013 there was a migration to BioMed Central and indexing in the following databases: PubMed, PubMed Central, PubMed Central Canada, UK PubMed Central, Depot , SpringerLink, Springer Images/Author Mapper, CrossRef, Elsevier/ELSAMS, Quertle, Scirus, EPO (European Patent Office), OhioLINK, SIMID, CISTI (Canada Institute for STI), Proquest, GALE and WebCite/Webcitation. Five associate editors were invited and the editorial board than comprised 15 Brazilian members and 13 foreigners . In 2015, the impact factor was 0.8, demonstrating the growth of the publication.A study conducted by Eysenbach  showed tBarraviera  lists thThe creation of an electronic scientific journal 20 years ago, when the commercial Internet was not available in Brazil, was a truly daring act! This paradigm shift could come true because it was developed step by step, i.e., first the floppy disk attached inside the cover with the possibility of printing, next the CD-ROM enabling reading on a computer screen, and then the availability of content and submission of papers only online. The electronic format itself became a reality in 2013, when the journal moved to the continuous publication model. Our story clearly shows that some paradigm shifts must occur gradually so that success can be achieved.


Nonsurgical embryo transfer (NSET) of blastocysts to pseudopregnant female recipients provides many benefits over surgical implantation with less distress for the mice, no anesthesia or analgesia required and a considerable reduction in implantation time per mouse. Although a disposable device to perform NSET is on the market since 2009, it is not generally used in transgenic facilities, most likely because surgical implantation is efficient and inexpensive. Here, we report that with several refinements to the original protocol, the NSET method becomes very attractive and outperforms the traditional surgical transfer on basis of pregnancy rate, birth rate and implantation-related discomfort. Furthermore, repeated use of the same NSET device on several recipient females reduces the costs to a reasonable level. The data presented covers all embryo transfers over the last 5 years at the transgenic facility of the Netherlands Cancer Institute, of which the last 2 years were performed exclusively with NSET.The online version of this article (doi:10.1007/s11248-014-9802-3) contains supplementary material, which is available to authorized users. Generating genetically engineered mice requires a diversity of skills and a considerable investment in time. Typically, embryonic stem cells (ESCs) are modified in vitro to introduce specific genetic alterations, after which the ESCs are injected into blastocysts. Next, the ESC-injected blastocysts are transferred in pseudopregnant female recipients in order to obtain chimeric mice with contribution of the modified ESCs to all tissues in order to achieve transmission of the genetic modifications to the next generation. All steps are crucial and need to be optimized. Recently, we have reported our efforts to optimize both the ESC culture conditions and the ESC microinjection method  Avoid contamination with mineral oil. After microinjection, we pool the embryos in a drop of KSOM. This drop is covered with mineral oil to prevent evaporation. Before the transfer, the embryos are washed and transferred to separate drops of KSOM, with 15 blastocysts per drop  Transfer 15 injected blastocysts per recipient female mouse. The protocol provided with the NSET device indicates a range of 12–20 embryos per recipient. Typically, we implant 15 embryos. We have transferred higher numbers, but then encountered more non-pregnant mice as well as fosters having difficulties giving natural birth. In cases where the mice did not deliver on due date, a caesarian section was performed a day later. Here, we often observed few, but large pups and embryo resorption in the uterus.(III) Use the B6CBAF1/OlaHsd (Harlan) strain for the pseudopregnant recipients. Initially, we used the CD-1 strain (Charles River) as a recipient as suggested by ParaTechs, however we had difficulties to achieve successful entry into the uterine horn. We therefore, returned to using the strain we routinely used for our embryo transfer, i.e. the B6CBAF1/OlaHsd. The females on this background produce decent numbers of offspring and are caring mothers. Furthermore, the B6CBAF1/OlaHsd mice are generally leaner and calmer as opposed to CD-1. We start using the recipients when they reach 8 weeks of age and weigh between 20 and 35 g. Once the mice weight passes 35 g we no longer use them regardless of age.(IV) Carefully select recipient female mice. One of the downsides of the NSET approach is the inability to evaluate whether a recipient female is stimulated or primed for implantation by observing the ovaries and uterus as can be done during surgical implantation. Transfer of embryos in unstimulated females will generally not yield successful pregnancies. We follow a set of simple rules for selecting appropriate recipient female mice suitable for the NSET method. First, only recipient female mice in their pro-estrous or estrous cycle are mated with vasectomised males as assessed by appearance of the vagina  Position recipient females at the correct angle to allow undisturbed insertion of the NSET catheter tip. Select a recipient mouse in a calm state and let her grab the wire grid with the forelimbs and raise the back to insert the small speculum Fig. d. Upon i(VI) Only use the small speculum. The NSET protocol suggests to use first a small speculum to open the vagina followed by a larger speculum. We find that only using the small speculum to insert the catheter tip works equally well. Using sense of touch, the catheter tip is inserted through the small speculum and once it touches the edge of the speculum the tip has entered either one of the uterine horns Fig. g. It is (VII) The same NSET device can be used for multiple transfers in 1 day. One of drawbacks for the NSET device is the cost involved. A box comes with ten devices, with a list price of $200–250 depending on the order size. ParaTechs strongly advices single use, which makes $20–25 per transfer. We routinely re-use our NSET devices for multiple transfers in 1 day. Some caution has to be taken. Avoid possible cross-contamination; only re-use the NSET device when the same type of ESC-injected blastocysts are to be implanted in multiple recipients. Between transfers, carefully clean the catheter tip by flushing with KSOM medium  Practice makes perfect. Although the NSET method is straightforward and easy to learn, especially in comparison to the surgical implantation technique, still it requires experience to achieve the optimal results. In our first year of using the NSET method we observed reduced pregnancy rates and a strong drop in the number of live-born pups compared to the surgical transfer Table . The refIn conclusion, a range of relatively minor adaptations to the original NSET protocol improve the overall success rate of embryo transfer, even surpassing the generally used surgical embryo transfer. The adapted protocol is as simple as the original protocol and can easily be implemented in any transgenic facility. Our production data represent transfer of all our ESC-injected blastocysts over the last 5 years and do not include specific experiments to test the contribution of each individual adjustment to the protocol. We are therefore unable to state which adjustments provides most gain. In light of the 3R’s in animal research, i.e. replacement, reduction and refinement, the NSET method is a valuable “refinement” step as it causes less discomfort to the mice Below is the link to the electronic supplementary material.


A study was undertaken to evaluate the effect of artificial aging through steam and thermal treatment as influencing the shear bond strength between three different commercially available zirconia core materials, namely, Upcera, Ziecon, and Cercon, layered with VITA VM9 veneering ceramic using Universal Testing Machine. The mode of failure between zirconia and ceramic was further analyzed as adhesive, cohesive, or mixed using stereomicroscope. X-ray diffraction and SEM (scanning electron microscope) analysis were done to estimate the phase transformation (m-phase fraction) and surface grain size of zirconia particles, respectively. The purpose of this study was to simulate the clinical environment by artificial aging through steam and thermal treatment so as the clinical function and nature of the bond between zirconia and veneering material as in a clinical trial of 15 years could be evaluated. The most recent introduction to the dental ceramics family is zirconia, which in its pure form is a polymorphic material. Fixed partial denture and crowns are available options for replacing lost tooth structure. Fabrication of fixed prosthesis involves layering of metallic substructure or all ceramic core structure with an aesthetic ceramic veneering material. The underlying substructure provides the required strength and the overlying ceramic gives the required esthetics .Yttrium oxide partially stabilized tetragonal zirconia polycrystal (Y-TZP) was introduced in dentistry as a core material in the 1990s. The name of zirconium is said to be derived from the Persian word “Zar”-“gun” meaning golden in color .Many studies on the use of zirconia have been conducted, but some features still remain unclear. There is a lack of information on how temperature change and water treatments affect the material properties and clinical durability of veneered zirconia cores. Unlike other prostheses in the body, oral prosthesis is constantly subjected to moisture with fluctuating pH and temperature. Recent studies have shown an indication that water molecules have an influence on the bond between veneered and veneering material . Water mThe substructure (zirconia core) of the restoration is covered by veneering ceramic. Studies have shown the bond between the zirconium core and the veneering ceramic to be influenced by water molecules which eventually affects the clinical durability of the restoration in function , 9.μm) using X-ray diffractometer and SEM (scanning electron microscope), respectively, between aged and nonaged specimens. The objective of this study was to simulate the clinical environment so the durability, clinical function, and nature of the bond between zirconia and veneering material, as in a clinical trial of 15 years, can be evaluated. The null hypothesis tested was that the bond strength between different zirconia framework and veneering ceramic will not be affected by artificial aging.Aging of zirconia may have detrimental effects on its bonding with veneering ceramics; mechanical stresses and wetness exposure accelerate this process . PresentThree types of zirconia were selected for this study: Cercon , Ziecon , and Upcera  . A totalThough thermocycling has been advocated in the literature as a conventional aging test, autoclaving induced low-temperature degradation is an established method for accelerated aging of Y-TZP materials. Autoclaving at 134°C for 5 hours is the standard aging protocol according to ISO 13356 valid for Y-TZP implants for surgery. 20 samples from each group were autoclaved at 134°C for 5 hrs to simulate oral conditions for 15 years [The prepared samples were steam cleaned and taken up for ceramic layering application. Ninety samples of different zirconia core material were layered using VITA VM9  veneering ceramic material . First, A metal jig was used for holding the samples to determine the shear bond strength. The samples were held in such a manner that the junction of zirconia substructure/veneering ceramic interface was faced towards the chisel load applicator. Universal Testing Machine  with a 10 kN load cell and crosshead speed of 0.5 mm/min was used. A chisel load applicator was used to direct a parallel shearing force to the substructure/veneer ceramic interface. The fracture load was obtained with the help of the graph on the digital monitor attached to the machine. The drop in the graph determined the point of debonding (fracture load) of each sample. Fracture load (kg) was converted to the shear bond strength (MPa) by use of the cross-sectional area of disc. The following formula was used to calculate the shear bond strength (MPa): After shear bond strength analysis, all the debonded samples were analysed under a stereomicroscope (20x) to evaluate the nature of bond failure. Each specimen was placed on carbon coated flat platform in such a manner that the surface between zirconia substructure and veneering ceramic faces the pointer of stereomicroscope, so as to identify whether there was cohesive failure, adhesive failure, or a combination of both, that is, mixed failure.Xm) was calculated by equation given by Garvie and Nicholson. The m-phase fraction (Xm) was calculated by the following equation [Im(111) is intensity of the peak (areas under the 31.5° peak) that represents the m-phase, It(111) is intensity of the peak (areas under the 30.3° peak) that represents the t-phase.The crystalline phases on the surfaces of the specimens were analysed by a Philips X'Pert 1 X-ray diffractometer. Scans were performed in the 2Θ range of 25° to 35° with a step size of 0.01° and 0.5 s/step interval. The m-phase fraction (equation :(2)Xm=ImAfter phase analysis, to observe the surface microstructure for the surface particle size evaluation, specimens were sectioned and polished with a diamond wheel and grinding machine and coated with gold using a sputter-coating technique. The specimens were observed by field emission scanning electron microscopy. Electron imaging was performed at an accelerating voltage of 15 kV to investigate surface geometry with Zeiss EVO 40 scanning electron microscope at the magnification of 30,000x. 10 areas were randomly selected and analysed for each specimen Figures .t-test and one-way analysis of variance (ANOVA) followed by post hoc comparison by Bonferroni method. For each group and subgroup p < 0.05 was considered significant.The data was entered into MS Excel spreadsheet and analysed, using SPSS version 11 statistical software. Descriptive statistics were calculated for each variable for the three groups. Techniques applied were Student's p = 0.66). Among the test groups  Cercon showed the highest mean value (24.20 ± 8.87 MPa) followed by Upcera (24.0 ± 7.53 MPa) and the lowest mean value was shown by Ziecon (23.9 ± 8.03 MPa). The statistical difference was found to be nonsignificant among the subgroups (p = 0.99).p = 0.00). Among the test groups  Cercon showed the highest mean value (14.3 ± 0.70%) followed by Upcera (12.4 ± 1.9%) and the lowest mean value was shown by Ziecon (11.0 ± 0.18%). The statistical difference was found to be significant among the subgroups (p = 0.04).μm) followed by Ziecon (0.41 ± 0.064 μm) and the lowest mean value was shown by Cercon (0.39 ± 0.060 μm). The statistical difference was found to be nonsignificant among the subgroups (p = 0.532). Among the test groups  Upcera showed the highest mean value (0.42 ± 0.034 μm) followed by Ziecon (0.40 ± 0.05 μm) and the lowest mean value was shown by Cercon (0.40 ± 0.032 μm). The statistical difference was found to be nonsignificant among the subgroups (p = 0.781).Based on the results obtained, the proposed hypothesis was accepted. There is no significant difference in the mean values of shear bond strength among all samples for both aging and nonaging groups. Hence the proposed hypothesis was accepted and probably multicentric trials could be taken to get the explicable results. Zirconia is classified as a high strength ceramic material; it was introduced for use in dentistry as a biomaterial with unsurpassed mechanical properties. Its clinical application expanded from single crowns, short-span fixed partial denture, and multiunit full-arch zirconia frameworks to implant abutments and complex implant superstructures –15. TherThe results showed that there is no significant difference in the mean values of shear bond strength among all samples . SimilarAnother factor that may affect the shear bond strength is the multiple firing of veneer ceramic over zirconia core; due to multiple firing there is relaxation of residual stresses and phase changes from tetragonal to monoclinic transformation . This noZirconia veneered samples of all groups (aged as well as nonaged) after SBS test showed combined and cohesive (within veneer) bond failure as seen under stereomicroscope, with the predominance of cohesive failure in the veneer layer with no adhesive failure . SimilarIn the present study different analytical methods are applied, namely, XRD and SEM, to analyse the aging characteristics of the zirconia ceramics.ISO Standards allow a maximum of 25% of m-phase to be present , post acThe present study on simulated aging has shown that LTD is observed to be insignificant over a simulated period of 15 years of clinical usage.The literature is well supplemented with research on phase transformation and its effect on physical properties such as flexural strength and fracture toughness. However, very few studies have evaluated the quantitative phase transformation within zirconia frameworks due to LTD and the correlation of phase transformation with the bond strength between zirconia substructure and veneering ceramic.In a study Xiao et al. observed the change in the monoclinic phase and compared the low-temperature resistance aging performances of the three clinical frequently used zirconia core materials Lava Frame, Cercon Smart, and Upcera before and after aging. XRD analysis showed that the m-phase contents of the three zirconia materials increased by prolonging the aging time, where in Upcera zirconia was the most sensitive to the aging treatment .The differences in LTD observed in the three different brands of yttria-stabilized zirconia ceramics may be attributed to differences in grain sizes, distributions of the grains, and additives  . Other fControl of grain size in zirconia ceramics is important to maximize the mechanical properties and minimize the possibility of LTD. Grain size may also affect the bond strength; a SEM analysis among zirconia groups of Upcera, Ziecon, and Cercon revealed that, after postaging treatment, the grain sizes in all the three zirconia ceramics were found to be insignificantly the same as that of samples without aging . The resThe strength and other physical properties of zirconia are related to the quality of the zirconia block, that is, its composition, method of compaction and converting into a blank, and homogeneity within the blank. Sintering mechanism may have a determinant influence on the phase transformation, grain size, and the physical and mechanical properties. A wide scope of research is open to investigate further the challenges in improving the properties of zirconia restorations. The study is in vitro, where clinical situations have been simulated. Therefore, the results of this study may not directly transpolate into serviceability of the prosthesis in clinical setting. However, the results do indicate the acceptable values of bond strength upon aging of all commercially available zirconia used in the study as compared to samples without any aging procedure.From the present study it can be concluded that 15 yrs of clinical usage as induced by artificial aging has no effect on the bond strength of zirconia and the veneering ceramic. The effect of stress generated by clinical usage such as masticatory forces, surface cracks, premature contact, or any other mechanism leads to stress formation within prosthesis which could not be simulated in the study samples. This concentration of stress with the passage of time (aging) could affect the performance of zirconia based restorations clinically.(1)Bond strength between zirconia and veneering ceramic in all samples was comparatively same (24.43 MPa to 27.9 MPa) and aging of 15 years had no effect on bond strength (23.9 MPa to 24.20 MPa).(2)The zirconia veneer interface came out to be stronger than the strength of the veneering ceramic; on average 70% of the samples have shown combined failure; hence to improve the longevity of zirconia restorations the strength of veneering ceramic has to be improved.(3)Bonding of zirconia veneered restoration is not influenced if the phase transformation (% of monoclinic content) is up to 14%. ISO Standards allow a maximum of 25 wt% of m-phase to be present, post accelerated aging test conducted at 134°C with 2-bar pressure for 5 hrs for any commercial zirconia ceramics to be used in dentistry.(4)μm–0.42 ± 0.034 μm) may not be affected by the phase transformation (% of monoclinic content) if the zirconia veneered restorations are placed in oral cavity for 15 years .Surface grain size (0.40 ± 0.032 Within the limitations of the study, following conclusions were drawn:


Starch retrogradation is a term used to define the process in which gelatinized starch undergoes a disorder-to-order transition. A thorough understanding of starch retrogradation behavior plays an important role in maintaining the quality of starchy foods during storage. By means of DSC, we have demonstrated for the first time that at low water contents, the enthalpy change of retrograded starch is higher than that of native starch. In terms of FTIR and Raman spectroscopic results, we showed that the molecular order of reheated retrograded starch samples is lower than that of DSC gelatinized starch. These findings have led us to conclude that enthalpy change of retrograded starch at low water contents involves the melting of recrystallized starch during storage and residual starch crystallites after DSC gelatinization, and that the endothermic transition of retrograded starch gels at low water contents does not fully represent the retrogradation behavior of starch. Very low or high water contents do not favor the occurrence of starch retrogradation. Rice and wheat are two of the most economically important crops, contributing food for more than 90% of the world’s populationGelatinization of starch causes loss of molecular order in both the amylose and amylopectin components. Subsequent retrogradation is the process in which the disaggregated starch chains reassociate into an ordered structure, which is different from the initial state37789Starch retrogradation may be characterized by determining the enthalpy change of retrograded starch gels after storage at 4 °C. The degree of retrogradation (DR) is often described by a parameter obtained by dividing the gelatinization enthalpy change of starch granules by the enthalpy change of reheated retrograded starch gels11Oryza sativa) sample was purchased from a supermarket in Tianjin, China. Amylose (A0512) and amylopectin (A8515) from potato starch were purchased from Sigma Chemical Co. . Other chemical reagents were of all analytical grade.The wheat (Zhoumai 18) flour was kindly provided by the Institute of Crop Science, Chinese Academy of Agricultural Science. The rice (Rice starch (RS) was isolated according to the method of Spigno and De Faveri8The amount of damaged starch in the isolated starches was determined using the Megazyme Starch Damage Kit . Amylose content was determined by iodine binding according to ChrastilG) and enthalpy change on reheating of retrograded starch gels (ΔHR) were obtained using data recording software. All measurements were performed in triplicate. Degree of retrogradation (%DR) was calculated according to the formula:Differential scanning calorimetry (DSC) measurements were performed using a Differential Scanning Calorimeter  equipped with a thermal analysis data station. Starch (approximately 3 mg wet weight) was weighed accurately into an aluminum sample pan. Distilled water was added with a pipette to obtain starch:water ratios of 1:0.5, 1:0.75, 1:1, 1:1.5, 1:2.5 and 1:4 (w/v) in the DSC pans, corresponding to the water content of 33, 43, 50, 60, 71 and 80%, respectively. The pans were sealed and allowed to stand overnight at room temperature before analysis. The pans were heated from 20 to 100 °C at a rate of 10 °C/min. An empty aluminum pan was used as the reference. Starch retrogradation was determined on the same gelatinized samples after storage at 4 °C for 7 days. The retrograded starch samples were re-scanned using the heating profiles described for starch gelatinization. Gelatinization enthalpy change of native starch  and Raman spectroscopic analysis.To obtain sufficient material to gain a better understanding of starch retrogradation behavior, retrograded starches were also prepared by simulating the DSC heating conditions without stirring using the RVA to prepare starch gels followed by storage at 4 °C for 7 days. The retrograded starch samples prepared in this way were freeze-dried, ground using a mortar and pestle, and passed through a 100 μm sieve. The resulting retrograded starch powders were mixed with water in ratios of 1:0.5, 1:0.75, 1:1, and 1:1.5 (w/v) in the DSC pans and analyzed using the same procedures for DSC measurements as described above.−1 was used to estimate the short-range molecular order of starch.The residual molecular order of native starch after DSC heating and of retrograded starch gels after DSC reheating was determined directly using a Thermo Scientific Nicolet IS50 spectrometer . The ratio of absorbance at 1047/1022 cm−1, with a resolution of approximately 7 cm−1. The full width at half height (FWHH) of the band at 480 cm−1, which can be used to characterize the molecular order of starchThe molecular order of native starch after DSC heating and of retrograded starch gels after DSC reheating was also determined by using a Renishaw Invia Raman microscope system , which was equipped with a Leica microscope  and a 785 nm green diode laser source. Spectra were collected on at least five different spots of gelatinized and retrograded starch samples in the range of 3200–100 cmResults are reported as the mean values and standard deviations of at least duplicate measurements. Analyses of variance (ANOVA) by Duncan’s test (p < 0.05) were conducted using the SPSS 17.0 Statistical Software Program .Amylose contents of the rice and wheat starches were 11.5% and 25.0%, respectively . The amy1617The DSC curves of starch gelatinization and from reheated retrograded starch gels are presented in o, Tp and Tc of rice starch were in the range of 61.2–62.1 °C, 66.9–68.3 °C and 71.0–73.9°, respectively. The transition temperature range (Tc–To) was from 9.8 to 11.7 °C. The To, Tp and Tc of wheat starch ranged from 55.1, 60.8 and 64.6 °C to 56.8, 62.1 and 67.4 °C, respectively. The thermal transition broadened with Tc–To ranging from 9.5 to 11.3 °C. These observations are consistent with previous results, which showed that the conclusion temperature of pea and wheat starches increased gradually with increasing water content7Thermal transition temperatures of native starch and retrograded starch gels at different water contents are listed in o and Tp, were much lower than those of native starch granules , νs (C1-O-C4), νs (C1-O-C5), skeletal (C-C-O), and ν (C-H) modes, respectively2930−1 are often used to characterize the molecular order of native starch granules or the changes in molecular order of starch samples during gelatinization or retrogradation6293233−1 is often used to characterize the relative crystallinity of starch samples; this parameter is most responsive to changes in crystallinityR were higher than those for FWHMG over the whole range of water content , indicating that the relative crystallinity of retrograded starch gels after DSC reheating was lower than that of gelatinized starch samples, consistent with the ATR-FTIR results.As the LCM-Raman spectra of starch samples were similar, only those of wheat starch are presented . SeveralThe maximum enthalpy change of retrograded starch gels was observed at water content around 50%, which does not correspond to the maximum degree of starch retrogradation. The crystallites formed during retrogradation are less ordered and less stable than crystallites in native starch granules. The observed higher enthalpy change of retrograded starch gels at low water content indicated that the enthalpy change of retrograded starch gels involves not only the melting of new starch crystallites formed on storage, but also further melting of starch crystallites remaining after gelatinization. This conclusion was supported by the ATR-FTIR and Raman spectroscopy analysis of molecular order of starch after gelatinization and after reheating of retrograded starch gels. Several studies investigated the effect of water content on retrogradation of starch21222335362122p and Tc of retrograded starch gels with increasing water content. Two hypotheses, not mutually exclusive, are proposed to interpret this observation. The crystallites formed during retrogradation could become progressively less stable with increasing water content, leading to lower Tp and Tc of retrograded starch gels. Another explanation is that the higher Tp and Tc of retrograded starch gels at low water content is due to further melting of residual starch crystallites that remained in retrograded starch gels. The melting of starch crystallites is not complete at the end of DSC heating, especially at low water content38c of retrograded rice and wheat starch gels was slightly lower than those of native starch granules at low water content, and that the transition temperature range of retrograded starch gels decreased with increasing water content .