The current protein-based CAT or luciferase reporter systems can measure the polyadenylation efficiency of a single pA site or candidate cis element but not the choice of two APA sites. To address this issue, we developed a set of four new bicistronic reporter vectors that harbor either two luciferase or fluorescence protein open reading frames connected with one Internal Ribosome Entry Site (IRES). Transfection of single or dual insertion constructs of these vectors into mammalian cells demonstrated that they could be utilized not only to quantify the strength of a single candidate pA site or cis element, but also to accurately measure the relative usage of two APA sites at both the mRNA (qRT-PCR) and protein levels. This represents the first reporter system that can study polyadenylation efficiency of a single pA site or element and regulation of two APA sites at both the mRNA and protein levels.The link between polyadenylation (pA) and various biological, behavioral, and pathological events of eukaryotes underlines the need to develop in vivo polyadenylation assay methods for characterization of the  Thi 13–25%) ,9,10,11.cis elements in addition to their core cis elements, which have been studied only in a small number of mRNAs [cis elements are the upstream U-rich elements [cis elements facilitate polyadenylation by serving as additional anchors for the polyadenylation machinery, or by recruiting specific trans-acting protein factors [cis elements are yet to be identified.The three types of pA sites, particularly the weaker non-canonical and AAUAAA-like hexamer-independent pA sites, often also possess up- and/or down-stream auxiliary of mRNAs . Among telements , UGUA elelements ,15, and elements  as well elements  and DS1Aelements . These a factors ,17,18,19cis-acting elements defining each APA site and their positions, the isoform and abundance or activity of the core 3’-processing protein factors, and of other protein factors such as transcription factors, splicing factors, RNA-binding proteins that enhance or repress assembly of the core 3’-processing factors to each APA site, and variations in nucleosome organization and epigenetic marks around each APA site [cis elements, exactly how and which trans-acting factors and environmental stimuli impact polyadenylation efficiency and selective/relative usage of APA sites are yet to be elucidated.Due to the presence of two or more alternative pA sites, a large proportion of eukaryotic genes  undergoes alternative polyadenylation (APA), producing several mRNA isoforms with variable length of 3′ coding sequence (coding sequence APA) and/or 3′ untranslated regions  ,21,22,23APA site ,25. SeleAPA site ,27,28,29cis elements defining the AAUAAA-like hexamer-independent pA sites as well as of the additional auxiliary cis elements, trans-acting protein factors, and extracellular stimuli that affect the strength of pA sites and the choice of APA sites necessitates development of in vivo polyadenylation assay methods for quantifying polyadenylation efficiency and relative usage of APA sites. The protein-based CAT [cis elements of the pA site) [Rluc and Fluc), it has only one multiple cloning site and thus still cannot be used to study the relative usage of two APA sites. The RNA-based in vivo tandem polyadenylation assay relying on PhosphorImager quantification of RNase protected RNA bands extracted from the cells transfected with the pCßS-proximal pA-distal pA-BGH pA vector construct [cis element and the relative usage of two APA sites, but has to generate and use α-32P-radiolabeled pA site-specific antisense RNA probe.Characterization of the core ased CAT ,31 or luased CAT ,33,34,35pA site) ,31,32 orpA site) , but is onstruct ,36 can mcis element and the relative usage of two APA sites in insect cell line such as Drosophila melanogaster S2 cells, Helicoverpa zea fatbody cells and Bombyx mori BM-N cells. Our test experiments with the recombinant constructs containing a single pA site, a cis elements or two pA sites demonstrated that these bicistronic reporter vectors could be readily used to characterize cis elements, trans-acting factors, and regulation of APA.In this study, we developed four new bicistronic reporter vectors that harbor either two fluorescence protein ORFs (pCMV-DsRed-MCS-IRES-EGFP-SV40 pA and pTK-DsRed-MCS-IRES-EGFP-SV40 pA) or two luciferase ORFs (pSV40-hRluc-MCS-IRES-hluc-SV40 pA and pTK-hRluc-MCS-IRES-hluc-SV40 pA) connected with a multiple cloning site plus the Internal Ribosome Entry Site (IRES). The two vectors that use the CMV promoter (pCMV) and SV40 promoter (pSV40) are used to determine the strength of a pA site or candidate element and the relative usage of two APA sites in mammalian cells such as human HEK293 and Hela cells, whereas the two vectors that use TK promoter (pTK) are utilized to measure the polyadenylation efficiency of a single pA site or candidate renilla (hRluc) luciferase) and promoters (pCMV or pSV40 vs. pHSV-TK) that drive the transcription of the reporter genes. The two vectors with a pCMV (pCMV-DsRed-MCS-IRES-EGFP-SV40 pA) or pSV40 (pSV40-hRluc-MCS-IRES-hluc-SV40 pA) promoter are compatible with mammalian cell lines such as human HEK293 and Hela cells, whereas the two vectors with a pHSV-TK promoter are used to drive the expression of the reporter genes in insect cell lines such as Drosophila melanogaster S2 cells, Spodoptera frugiperda Sf9, Helicoverpa zea fatbody cells, and Bombyx mori BM-N cells. The fluorescence proteins expressed from the two dual fluorescence vectors can be intuitively and quantitatively detected by a fluorescence microscope or cell flow cytometry, whereas the luciferase expressed from the two dual luciferase vectors can be quantitatively measured by a luminometer.Sequencing of the positive clones verified that we successfully generated a bicistronic reporter system composed of two dual fluorescence (pCMV-DsRed-MCS-IRES-EGFP-SV40 pA and pTK-DsRed-MCS-IRES-EGFP-SV40 pA) A and twoRegardless of the differences in their reporter proteins and promoters, the four vectors share a common structure framework consisting of one promoter, follow by 1st ORF, two restriction enzyme sites, one IRES element, 2nd ORF, one restriction enzyme site, one pA site (SV40 pA), and another restriction enzyme site A. TheoreEcoRI and BamHI sites in the two dual fluorescence vectors or XhoI and EcoRI sites in the two dual luciferase vectors, producing a recombinant construct   of 49 bpnscripts E. By connscripts D,E. Likenscripts F,G. Bothcis-acting elements, we ficrst annealed and extended one common reverse oligo (Red-GFP-PAS-R) with five forward oligos that contain AATAAA (Red-GFP-PAS-wild-F), ANTAAA (Red-GFP-PAS-mut1-F), NANAAN (Red-GFP-PAS-mut3-F), NNNNNN (Red-GFP-PAS-rad-F), or no hexamer (Red-GFP-PAS-del-F) , 2-base , 3-base (TACAAC from Red-GFP-PAS-rad-F), or 4-base (GCTAGC from Red-GFP-PAS-rad-F) variants of the canonical AAUAAA hexamer (In order to test if the bicistronic reporter system can be used to identity core or accessary S-del-F)  to form HI sites A yielded hexamer A.TACAAC, GCTAGC) or 2 A or T to G changes (GAGAAA) exhibited the similar high mRNA and protein ratios of EGFP/DsRed with the wildtype vector and the SPA construct with no hexamer. The other six SPA constructs with an AAUAAA variant of 1-base  or 2-base  substitutions displayed a ratio of between the above two opposite ends  and substitution at the other positions   .cis-acting elements, we annealed and extended one common reverse oligo (PSI-R) with five forward oligos that contain AATAAA (PSI-wild-F), ANTAAA (PSI-mut1-F), NANAAN (PSI-mut3-F), NNNNNN (PSI-rad-F), or no hexamer (PSI-del-F) , two 3-base variant constructs (GAGAAC and CAGAAC from PSI-mut3-F), two 4-base variant constructs (ACGTAG and ATTTTG from PSI-rad-F) and one 5-base variant construct (GCCATT from PSI-rad-F). Dual luciferase reporter assay and qRT-PCR analysis showed that the AAUAAA SPA construct had near-zero mRNA and protein ratios of hluc/hRluc, whereas the wildtype vector and no hexamer SPA construct produced the highest mRNA and protein ratios of hluc/hRluc   to form RI sites A. Sequenuc/hRluc B,C. The uc/hRluc B,C.To test whether the bicistronic reporter system can be utilized to study the relative usage of two APA sites from the same gene, we generated four fluorescence CD47 pA site constructs and four luciferase CD47 pA site constructs by inserting its proximal (pA1), distal (pA2), or both pA sites  and another restriction enzyme site  and protein levels. By contrast, although the luciferase reporter plasmid pPASPORT used by Yao et al. [Rluc and Fluc) connected with one IRES element, it cannot be used to study the relative usage of two APA sites because it does not enable simultaneous insertion of two candidate pA sites. Transfection of any one of the four wildtype vectors we developed here into mammalian or insect cells is expected to produce a bicistronic mRNA with an equal molar ratio of DsRed (or hRluc) and EGFP (or hluc) ORFs. Consistent with this expectation, we observed an EGFP/DsRed or hluc/hRluc mRNA ratio of 1–1.5   demonstr studies ,6,7,10 tnylation  ranked tour data . Fourth,CD47 gene was regulated by 3’ UTR APA [cis-acing elements defining the two APA sits, and the endogenous usage frequency of the two APA sites, validates the utility of the reporter system for study of APA regulation. Specifically, this system can be utilized to identify trans-acting proteins or noncoding RNAs and environmental stimuli that affect/regulate the relative usage of two APA sites.Previous reports have demonstrated that the human  UTR APA ,42, yiel UTR APA . The rel UTR APA ). Such aAs shown in E. coli competent cells and the resulted positive clones were verified by sequencing at Beijing Genomic Institute (BGI) .PCR amplifications of the four inserts  and reverse PCR linearization of the 2 plasmids (pIRES2-EGFP and psiCHECK-2) and 2 bicistronic reporter vectors (pCMV-DsRed-MCS-IRES-EGFP-SV40 pA and pSV40-hRluc-MCS-IRES-hluc-SV40 pA) mentioned above were conducted individually in a 50-μL reaction containing 10 μL 5× PrimeSTAR GXL Buffer, 1 μL PrimeSTAR GXL DNA Polymerase , 5 μL dNTP Mixture (2.5 mM each), 2 μL forward primer, 2 μL reverse primer, 1 μL plasmid DNA template (10 ng), and 39 μL ddH2O. These PCR or reverse PCR reactions were initiated at 95 °C for 5 min, followed by 30 cycles of 98 °C for 10 s, 60 °C for 15 s (4 insert PCRs) or for 1 min per kb (4 linearization PCRs), and a final extension of 10 min at 68 °C. The four bicistronic reporter vectors were then constructed by assembling 1 μL (50 ng) of each linearized plasmid or vector together with 1 μL (25 ng) of the corresponding insert at 50 °C for 15 min in a tube containing 5 μL 2× Seamless Master mix (Taihe Biotechnology) and 3 μL ddH2O. Five μL of each assembling reactions were transformed into DH5α EcoRI and BamHI sites  with one of the 5 forward oligos that contain AATAAA (Red-GFP-PAS-wild-F), ANTAAA (Red-GFP-PAS-mut1-F), NANAAN (Red-GFP-PAS-mut3-F), NNNNNN (Red-GFP-PAS-rad-F), or no hexamer (Red-GFP-PAS-del-F)  to form HI sites A. LikewiHI sites  and insenstructs A. All thCD47 gene  and distal (pA2) pA sites (200 bp up- and down-stream of the corresponding cleavage site) of the human D47 gene A with thD47 gene B. The pr-R-BamHI  were useCD47 pA1 B. LikewiXhoI and CD47PA1-R-EcoRI  high-glucose medium  supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin . For transient transfections, cells seeded onto a 6-well (for the fluorescence vector and constructs) or 12-well (for the luciferase vector or constructs) plate (1 × 106 cells/mL) for 24 h were transfected with a wildtype bicistronic vector or its construct (1 μg/well) using X-tremeGENE HP DNA Transfection Reagent , following the manufacturer’s instructions. Twelve hours post-transfection, we replaced the old culture medium with fresh medium and kept the plates in a CO2 incubator for another 48 h. At least three independent transfections  were conducted for each wildtype vector or construct.Both HEK293 cells and Hela cells were routinely cultured at 37 °C with 5% COn = 3 × 3 = 9). The DsRed and EGFP fluorescence intensities of each construct were normalized by dividing them by the average DsRed and EGFP fluorescence intensities of the wildtype vector, respectively. The average normalized EGFP/DsRed intensity ratio of at least 3 independent transfections per construct or wildtype vector was calculated to represent the relative expression of EGFP and DsRed at the protein level.After Hela  60 h post-transfection. The suspended cells were then subjected to flow cytometry analysis using the MoFlo XDP cell sorter . The cells were excited by a 488 nm laser beam and the emitted EGFP and DsRed fluorescence signals were captured at 532 nm in the detector FL1 and at 561 nm in the detector FL2, respectively. We used the HEK293 cells co-transfected with pIRES2-EGFP and pDsRed-N1 to adjust the green or red fluorescence signals in the FL1 and FL2 channels, respectively. The slopes of the resulted signal plots of the two channels were used to indicate the relative expression of EGFP and DsRed at the protein level.HEK293 cells seeded onto 6-well plates at 1 × 10Hela  for the fluorescence vector and constructs as well as hluc, hRluc, and Amp resistance gene  for the luciferase vector or constructs. The primer pairs for qRT-PCR analyses of the 4 target  and 2 reference genes (Kana and Amp resistance genes) and their amplification efficiencies are summarized in Total RNAs were extracted from HEK293 or Hela cells transfected with the fluorescence or luciferase vector/construct with Trizol reagent  and then treated with DNase I (Promega) and RNase inhibitor (Thermo Fisher Scientific) for 40 min to remove potential genomic DNA (gDNA). One µg of each total RNA sample was reverse transcribed into cDNA at 42 °C for 0.5 h in a 20 µL reaction with 2 µL 6-mer random primer, 2 µL dNTP , 1 µL M-MuLV reverse transcriptase , and 1 µL RNase inhibitor (Thermo Fisher Scientific). The cDNAs were diluted 10-fold and used as the templates for qRT-PCR analyses of DsRed, EGFP, hluc, hRluc, Kana and Amp were individually performed in a 20 µL reaction containing 1 µL 10 fold-diluted cDNA, 0.5 µL of each gene-specific forward and reverse primers (x-axis)-Ct value (y-axis) line, using the formula E = 10−1/slope − 1 [Ct−ΔΔ. We further divided the normalized expression of EGFP or hluc by that of DsRed or hRluc to indicate the relative expression of the two fluorescence or luciferase reporter genes at the mRNA level.qPCRs of lope − 1 . The nort-tests to compare the significance of differences between the two treatments. All of the statistical tests were completed by SPSS version 19.0 software  and GraphPad Prism 5.0 software .One-way analysis of variance (ANOVA) followed by Tukey’s HSD tests were performed to test the significance of differences in the relative expression levels of EGFP and DsRed or hluc and hRluc at the protein (fluorescence intensity or luciferase activity ratios) and mRNA (qRT-PCR data) levels among different fluorescence or luciferase constructs. When there were only two treatments (e.g., wildtype vector vs. synthetic pA site construct in


Acetabular fracture surgery is directed toward anatomical reduction and stable fixation to allow for the early functional rehabilitation of an injured hip joint. Recent biomechanical investigations have shown the superiority of using an additional screw in the infraacetabular (IA) region, thereby transfixing the separated columns to strengthen the construct by closing the periacetabular fixation frame. However, the inter-individual existence and variance concerning secure IA screw corridors are poorly understood.This computer-aided 3-D radiomorphometric study examined 124 CT Digital Imaging and Communications in Medicine (DICOM) datasets of intact human pelves (248 acetabula) to visualize the spatial IA corridors as the sum of all intraosseous screw positions. DICOM files were pre-processed using the Amira® 4.2 visualization software. Final corridor computation was accomplished using a custom-made software algorithm. The volumetric measurement data of each corridor were calculated for further statistical analyses. Correlations between the volumetric values and the biometric data were investigated. Furthermore, the influence of hip dysplasia on the IA corridor configuration was analyzed.3 in males and 9.2 cm3 in female pelves. Corridor volumes were significantly positively correlated with body height and weight and with the diameter of Köhler’s teardrop on standard AP pelvic X-rays. No correlation was observed between hip dysplasia and the IA corridor extent.The IA corridors consistently showed a double-cone shape with the isthmus located at the acetabular fovea. In 97% of male and 91% of female acetabula, a corridor for a 3.5-mm screw could be found. The number of IA corridors was significantly lower in females for screw diameters ≥ 4.5 mm. The mean 3.5-mm screw corridor volume was 16 cmIA corridors are consistently smaller in females. However, 3.5-mm small fragment screws may still be used as the standard implant because sex-specific differences are significant only with screw diameters ≥ 4.5 mm. Congenital hip dysplasia does not affect secure IA screw insertion. The described method allows 3-D shape analyses with highly reliable results. The visualization of secure IA corridors may support the spatial awareness of surgeons. Volumetric data allow the reliable assessment of individual IA corridors using standard AP X-ray views, which aids preoperative planning. Since the prodigious surgical oeuvre of Letournel and Judet in the 1960s, the surgical fixation principles for the treatment of displaced acetabular fractures associated with instability or incongruency of the hip joint have remained mainly unchanged . TherebyDue to demographic changes, the incidence of acetabular fractures in older adults has steadily increased in recent decades, a trend that will inevitably continue in the coming years –12. OperTo address this lack of fixation strength, an additional construct is desirable that would transfix the anterior and posterior columns beneath the acetabular fossa to close the periacetabular fixation frame  Fig. 1)1). EmileFurthermore, the proposed spatial shape of such a screw corridor and its structural anatomical limitations are lacking, which explains why the IA screw has not gained wide acceptance in the acetabular trauma surgeon community. Reports have rarely described the routine application of an IA screw for conventional osteosynthesis of the impaired column(s) .However, recent biomechanical investigations have clearly demonstrated the superiority of a preferably inferiorly positioned interconnecting screw in the IA region beneath the acetabular fossa , 16. GraIn our study, a computer-assisted 3-D radiomorphometric analysis of the IA region was used to assess the presence, true shape, and extent of the intraosseous secure screw corridor. This analysis was performed based on conventional CT Digital Imaging and Communications in Medicine (DICOM) datasets using custom-made software algorithms and differs from Letournel’s original description of the possible screw positions along the quadrilateral wall by allowing the partial penetration of the acetabular fossa without altering the cartilage of the femoral head .Furthermore, a possible relationship between the IA corridor dimensions and the presence of dysplastic acetabular shape variants warrants investigation. In this regard, some authors have reported the widening of the quadrilateral plate due to a traumatic lesion of the triradiate cartilage in infantile acetabula, resulting in posttraumatic dysplasia , 19. ConFor this study, 124 CT datasets of unimpaired human pelves from a Caucasian cohort provided the basis for the virtual analysis of the existence, shape, and variability of the IA corridor. The datasets were primarily prepared by the diagnostic radiology department of the Martin Luther University of Halle-Wittenberg, Germany, for the diagnosis of individual diseases from September 2008 to August 2010. All patients declared their consent for further scientific use of their anonymized imaging data. The CT scans were generated by a SOMATOM Sensation 64 Multislice CT scanner . The image resolution was 512 × 512 pixels, the slice distance was 0.4 mm, and the slice thickness was 0.6 mm.The generated DICOM files were pre-processed semiautomatically by a custom-made C++ software algorithm implemented in the scientific visualization software Amira® . The first preparatory steps included the anonymization, cropping, labeling, and segmentation of the datasets. Next, gray-scale values of the voxels were labeled with a value of “1” if representing bone. Voxels on the exterior of the pelvic bone were labeled with a value of “0”, and the voxels comprised an STL data file. This process represented a key step in transforming the 3-D anatomical shape conditions of the pelvis into a valuable matrix for a mathematical software algorithm. Details of this standardized workflow were described in previously published studies by our working group –25.Twelve anatomical landmarks were set using a mouse-click on the bone surface of each 3-D pelvic model to allow spatial alignment in the standard Matta “inlet” and “outlet” views. To compute the IA corridor, the establishment of three supplemental landmarks was required. For this purpose, the corridor was adjusted in an orthograde manner in a semi-transparent display mode to set the first landmark (P0) on the bony surface in the center of Köhler’s teardrop in the region of the iliopubic eminence. The second landmark (P1) was set on the projection in the region of the ischial tuberosity. The conjunction of these entry and exit points represented the basic valid vector (BVV) for an IA screw track entirely situated in the compact bone, with a voxel transition sequence of “0-1-0” signifying “no bone–bone–no bone”.A rectangular start and a target area were generated at a distance of 50 voxels above P0 and 15 voxels beneath P1 using another custom-made software module. The intersecting BVV corresponded to the center point of each area. Subsequently, the transition sequences from every raster point of the start area and ending on the given raster points of a target area were registered by the script based on the Bresenham algorithm  Fig. 2)2).Fig. 2F) in the acetabular fossa as the cut-off  represented breaks beyond the bone during the course through the IA region and were therefore excluded by the script. Because they were not surgically pertinent, potential entry points medial to the pelvic brim with screw tracks passing through the IA bone region were avoided. For this reason, 5 to 10 landmarks were set by mouse-clicks along the terminal line for each investigated acetabulum. Bresenham lines that passed the bone medial to these landmarks were excluded automatically. Furthermore, screw tracks along the quadrilateral bone stock beyond the fovea acetabuli throughout the posterior column were avoided by an iterative search operation of the program script that determined the thinnest point , Lequesne’s acetabular index (AI), and the Heyman and Herndon (DWI) depth-to-width index were assessed in the AP planar projection of each pelvis bilaterally . Additiop < 0.05). The chi-square test was used to compare the gender-related corridor prevalence to the different screw diameters. The independent samples t test was performed to analyze sex- and laterality-related differences of the IA corridors. To allow predictive statements, multivariate regression analysis was performed to determine the influence of individual variables on the corridor extent. The descriptive statistics presented in this study include the mean and the first standard deviation.Statistical analysis was completed using SPSS 21® . A confidence interval of 95% was assumed  in total) included 75 males and 49 females. The epidemiological data are shown in Table All IA corridor volumes consistently showed a double-cone shape with the isthmus located in the region of the acetabular fovea as the limiting anatomical structure. The screw entry areas were projected to the iliopectineal eminence. The smaller the screw diameter, the more the shape of the entry area conformed to a teardrop. With increasing screw diameters, the shape thus became more spherical, and the corridor volumes decreased to a tubular shape. The exit areas were consistently localized to the sciatic protuberance. The surface of both areas mutually decreased with higher screw diameters Fig. .Fig. 4Mop = 0.3). However, a safe IA bone path existed for a 4.5-mm screw in 139 male HP (93%), whereas only 75 female HP (77%) displayed sufficient bone space. This difference was highly significant (p = 0.003). This trend continued for screw diameters up to 7.5 mm, with significantly fewer IA corridors present in the female pelves than in the male pelves (p < 0.0001)  and 89 of the 98 female HP (91%), the analysis revealed an existing corridor for a 3.5-mm screw. The chi-square test did not reveal any gender-related differences   notably showed significant sex-specific differences Table . With re01) Fig. .Table 2Vr = 0.3), body height (r = 0.4), and the diameter of Köhler’s teardrop in the AP X-ray view (r = 0.5) showed significant positive correlations with the corridor volume . Therefore, Köhler’s teardrop diameter represented a strong predictive parameter for corridor existence in 100% of our cases, with a cut-off value of ≥ 4 mm . Therefore, our hypothesis of increasing corridor volumes in cases of dysplastic hip variations could not be confirmed.According to our hypothesis of a lateralized rotational center and a lesser roofing of the femoral head leading to a wider bone stock of the quadrilateral plate at the level of the acetabular fovea in dysplastic hip shape variants, the following results were found. In the 248 hemipelves, we observed dysplastic malformations by measuring Wiberg’s lateral center-edge angle in 3 (2.4%), Lequesne’s acetabular index in 37 (14.9%), and the depth-to-width index according to Heyman and Herndon in 13 acetabula (5.2%) . With increasing screw diameters of 4.5 mm or greater, however, the prevalence of a sufficient corridor significantly decreased in females (p < 0.05). Based on these findings, 3.5-mm small fragment screws should be used as the standard implant to transfix fracture separation of the anterior and posterior columns via the IA corridor.Our data showed that the shape, extent, and presence of a safe IA screw corridor differ according to certain individual variables. In both genders, an IA corridor for a 3.5-mm screw existed in more than 90% of all our cases. In females, however, the bone stock of the medial acetabular margin was consistently smaller , which corresponded to the findings of Gras et al. . NotwithFurthermore, the individual epidemiological variables of body height and weight were shown to strongly influence the existence and extent of the IA screw track. Both parameters showed significant positive correlations with increasing corridor volumes. Additionally, linear multivariate regression analyses of the volumetric corridor data revealed a significant correlation between the transverse diameter of Köhler’s teardrop on the standard AP pelvic X-ray view and a boundary value of ≥ 4 mm, allowing the secure insertion of a 3.5-mm screw. Therefore, we achieved the successful transfer of knowledge from virtual volumetric research to an easily measurable value that may serve as an important predictor in preoperative planning.With regard to congenital hip dysplasia, our research did not focus on the joint pathology itself. In fact, it is important to ascertain whether hip dysplasia, which is easily identified on standard AP X-rays, may be used as a helpful indicator of the presence of a capacious IA bone corridor. However, our results disprove the hypothesis of a more capacious quadrilateral bone stock in hip dysplasia that would provide greater space for an IA screw. In contrast to observations in posttraumatic hip dysplasia after juvenile acetabular injury , 19, in Our results provide a comprehensive understanding of the unique anatomic details of the quadrangular bone stock with regard to secure IA screw insertion. The described methods allow the computation of 3-D corridor volumes from conventional pelvic CT data implemented in the C++ computer language as a user-specific module of Amira® software . The worThe primary aim of this study was the determination of consistent anatomical landmarks that could regularly be seen in planar X-rays to allow the prediction of the existence of a safe IA bone corridor for a 3.5-mm screw. As a result, the transverse diameter of Köhler’s teardrop in the standard AP pelvic X-ray turned out as the most predictive value. In our population, the secure IA-bone corridor was found in 100% of the pelves showing a teardrop diameter of ≥ 4 mm. Hence, the teardrop diameter can be considered a safety indicator to support the surgeon’s decision to apply a 3.5-mm screw without preoperative CT.However, an accurate preoperative analysis using conventional X-rays and CT scans remains mandatory for planning the entire surgical intervention.Some limitations of our study must be noted. Semiautomatic CT-based segmentation and computing the IA corridor volume are time-consuming and require expensive software applications. Therefore, the presented methodological workflow is not yet feasible for applications in daily routine clinical practice. The data presented reflect only the physiological situation of the uninjured acetabulum. Any residual dislocation associated with incomplete fracture reduction would thus strongly influence all the volumetric parameters of the IA corridor and would likely lead to an insufficient screw path. Currently, the computed scripts allow only intact pelves to be analyzed. In our study population, the differences between the left and the right acetabulum were not significant; thus, measuring the contralateral acetabulum may be indicative of the corridor existence on the fractured side. Prospectively, advanced software adaptations might enable corridor computation for arbitrary patterns of acetabular fractures and a simulation of residual malreduction. Such innovations are the focus of our future research.


In previous work, we identified 145 active compounds from Bufei Jianpi formula (BJF) by system pharmacology and found that BJF showed short-term effect on chronic obstructive pulmonary disease (COPD) rats. Here, we applied the transcriptomic, proteomic, and metabolomics approaches to illustrate the long-term anti-COPD action and its system mechanism of BJF. BJF has obvious anti-COPD effect through decreasing inflammatory cytokines level, preventing protease-antiprotease imbalance and collagen deposition on week 32 by continuous oral administration to rats from weeks 9 to 20. Subsequently, applying the transcriptomic, proteomic, and metabolomics techniques, we detected a number of regulated genes, proteins, and metabolites, mainly related to antioxidant activity, focal adhesion, or lipid metabolism, in lung tissues of COPD and BJF-treated rats. Afterwards, we integrated system pharmacology target, transcript, protein, and metabolite data sets and found that many genes, proteins, and metabolites in rats BJF-treated group and the target proteins of BJF were mainly attributed to lipid metabolism, inflammatory response, oxidative stress, and focal adhesion. Taken together, BJF displays long-term anti-COPD effect probably by system regulation of the lipid metabolism, inflammatory response pathways oxidative stress, and focal adhesion. Chronic obstructive pulmonary disease (COPD) is a major cause of death with increasing prevalence worldwide, and currently available treatments are largely ineffective , 2. TradPresently, new approaches allow a complete differential profile including transcriptomic, proteomic, and metabolomic analysis to provide a system level method to investigate the therapeutic mechanisms of TCM formula –7. TransHere, we treated COPD rats with BJF during weeks 9 to 20 and evaluated the long-term anti-COPD effect of BJF on week 32. We then performed transcriptome, proteome, and metabolome analyses to identify differentially expressed genes, proteins, and metabolites. Finally, we integrated our transcriptomic, proteomic, metabolic, and system pharmacology data to uncover significantly perturbed pathways at both the transcript, protein, and metabolite levels and to dissect the long-term anti-COPD mechanism of BJF at system level. Klebsiella pneumoniae, tobacco, aminophylline, antibodies, and rats, were prepared as described previously [Chemicals, such aseviously . The aniCOPD rats and BJF formula were prepared as described previously . Normal β, tumor necrosis factor- (TNF-) α, soluble TNF-α receptor 2 (sTNFR2), collagen I, collagen III, collagen IV, matrix metalloproteinase- (MMP-) 2, MMP-9, and tissue inhibitor of MMP (TIMP-) 1 followed the methods described previously. The levels of IL-1β, IL-6, TNF-α, and sTNFR2 in sera were analyzed using ELISA kits.We detected the pulmonary function every four weeks during weeks 0 to 32. Lung tissues were harvested for histological and immunohistochemical examination. The specimens were fixed, embedded, cut into sections, and then stained with hematoxylin and eosin. Immunostaining for interleukin- (IL-) 6, IL-1t-test screening, p < 0.05) and differential expression versus COPD model rats .Total RNA was isolated, purified, amplified, and labeled. Finally, the slides were analyzed. Data were collected with Agilent GeneSpring GX software version 11.0 and filtered for significant detection .The lung tissues were lysed and homogenized. The lysates were digested by trypsin solution. Tryptic peptides were labeled. Peptides were fractionated using strong cation exchange fractionation and then used for LC-MS analysis. Fold changes are higher than 1.0 for upregulation or lower than 1.0 for downregulation. Statistical significance was evaluated using one sample t-test .Lung tissue was prepared as previously . The tisp was < 0.05). Integrated pathway analysis of gene, protein, and metabolomics data was analyzed by Metscape [Cytoscape v3.1.1 plugins, such as BiNGO and CluGO, were used to analyze the molecular function of transcripts and proteins , 14. PatMetscape . The mosMetscape .One-way analysis of variance with the SPSS 19.0 software package  was applied to evaluate the statistical differences. Values are expressed as mean ± standard error of mean.To investigate the long-term anti-COPD action of BJF, we administrated BJF to COPD rats during weeks 9 to 20 and tested the effect of BJF on the functional and morphological changes of lung tissues. In β, IL-6, TNF-α, and sTNFR2 in lung tissues on week 32 . We found these genes were related to many biological functions, such as oxidoreductase, ion channel, or metalloendopeptidase activity  and BJF treatment rats (versus COPD model), respectively . These proteins were mainly involved in these biological functions, such as oxidoreductase activity, peroxiredoxin activity, and nitric-oxide synthase regulator activity Figures , and attFurthermore, we observed the COPD model group (191 proteins) shared 127 proteins of the BJF-treated-group (168 proteins). Expression changes of 82 proteins (among the 127 proteins) in COPD model rats were suppressed by BJF (Supplementary Table  A5). The 82 proteins were mainly related to these biological functions, including oxidoreductase, glutathione transferase, NAD or NADH binding, and eukaryotic cell surface binding activity , which wAfter metabolomics analysis, 41 and 68 differentially regulated metabolites were identified in model (versus control) and BJF-treated (versus model) rats, respectively (Supplementary Tables  A6 and A7). MetaboAnalyst was applied to analyze metabolic pathway Tables  and 6. WHere, many significantly regulated genes, proteins, and metabolites, related to the therapeutic effect of BJF, were identified. For further holistic analysis, the genes, proteins, and metabolites were integrated to provide a systems biological interpretation.We firstly built the gene-metabolite networks based on the transcriptomics and metabolomics data of model and BJF-treated rats. We observed these genes and metabolites were primarily involved in lipid, purine, or energy metabolism Figures . SimilarWe previously identified 175 targets of the BJF using system pharmacology methods (Supplementary Table  A8) and examined the short-term anti-COPD of BJF. Now, the above data indicated that BJF has long-term efficiency for the treatment of COPD rats. Next, we sought to illustrate the system mechanism of the long-term anti-COPD effect of BJF by combining the transcriptomics, proteomics, and metabolomics and system pharmacology data.Initially we explored the biological functions of the overlapping proteins between potential targets of BJF and transcripts in BJF-treated rats. The result showed that 11 overlapping proteins  were related to many different biological functions including oxidoreductase activity, MAP kinase activity, and NF-kappa-B binding . Next, 9Finally, we constructed a system picture of the molecular mechanisms of long-term anti-COPD effect of BJF based on above comprehensive data. As shown in α. We also found these metabolites, including arachidonate, PGE2, and LTB4, elevated in the airways of COPD patients [κB were the predicted targets of BJF. We also found that BJF treatment could sustainably decrease the level of IL-1β, IL-6 TNF-α, and sTNFR2 in lung tissues and serum of COPD rats.In lipid metabolism analysis, BJF decreased the levels of metabolites, such as lecithin, arachidonate, PGE2, LTB4, and 11-epi-PGF2patients –25. In aThe oxidative stress continuously generates reactive oxygen species, causes lipid peroxidation, and further leads to sustained inflammatory responses, which is a major contributing factor to obstructive disorders in lungs –28. GlutIn summary, these data suggested that BJF showed the long-term anti-COPD therapeutic effect on rats through modulating system biological functions, including lipid metabolism, inflammatory response, oxidative stress, and focal adhesion pathway.Combining transcriptomics, proteomics, and metabolomics approaches is becoming more important in investigating the mechanisms of the therapeutic effect of Chinese herbal medicine and will most likely change the way we approach investigations of complex disease development. However, attempts thus far to integrate 3-omics data have been met with limited success. In this study, a system level approach based on integrating transcriptomics, proteomics, metabolomics, and system pharmacology data analysis was applied to clarify the long-term anti-COPD effect and its system mechanism of BJF. We administrated COPD rats with BJF during weeks 9 to 20 and then found BJF had long-term anti-COPD effect and could effectively inhibit the inflammatory cytokine expression, protease-antiprotease imbalance, and the collagen deposition on week 32. The transcriptomics, proteomics, and metabolomics profiles model and BJF-treated rats were further characterized, which could help us to dissect the long-term therapeutic mechanism of BJF at system level. The results showed that the regulated transcripts, proteins, and metabolites were related to many different biological functions, including oxidoreductase activity, antioxidant activity, and lipid metabolism. Furthermore, the comprehensive analysis results suggested that BJF showed its long-term anti-COPD effect probably through modulating lipid metabolism, inflammatory response, oxidative stress, and focal adhesion pathways. Taken together, an integrated transcriptomics, proteomics, and metabolomics approach combined with system pharmacology has the potential to considerably advance our understanding of the system mechanism of long-term therapeutic effect of traditional Chinese medicine. However, the main limitation of this work is its direct demand for further experimental validation such as the significant regulated genes, proteins, and metabolites in COPD and BJF-treated rats, which will be resolved in the follow-up works.Related transcriptomics parameters of lung tissue of COPD and BJF-treated rats were showed in supplementary material.


BTK) is an emerging therapeutic target in IgE receptor (IgER)‐cross‐linked basophils.Recent data suggest that Bruton's tyrosine kinase  on IgE‐dependent activation and histamine release in blood basophils obtained from allergic patients (n=11) and nonallergic donors (n=5). In addition, we examined the effects of these drugs on the growth of the human basophil cell line KU812 and the human mast cell line HMC‐1.We examined the effects of four BTK blockers were found to inhibit anti‐IgE‐induced histamine release from basophils in nonallergic subjects and allergen‐induced histamine liberation from basophils in allergic donors. Drug effects on allergen‐induced histamine release were dose dependent, with IC50 values ranging between 0.001 and 0.5 μmol/L, and the following rank order of potency: ibrutinib>AVL‐292>dasatinib>CNX‐774. The basophil‐targeting effect of ibrutinib was confirmed by demonstrating that IgE‐dependent histamine release in ex vivo blood basophils is largely suppressed in a leukemia patient treated with ibrutinib. Dasatinib and ibrutinib were also found to counteract anti‐IgE‐induced and allergen‐induced upregulation of CD13, CD63, CD164, and CD203c on basophils, whereas AVL‐292 and CNX‐774 showed no significant effects. Whereas dasatinib and CNX‐774 were found to inhibit the growth of HMC‐1 cells and KU812 cells, no substantial effects were seen with ibrutinib or AVL‐292.All four BTK‐targeting drugs are potent inhibitors of IgE‐dependent histamine release in human basophils. The clinical value of BTK inhibition in the context of allergic diseases remains to be determined. In addition, we examined the effects of these drugs on growth of BA and MC. The results of our study show that BTK inhibition by ibrutinib is a potent approach to suppress allergen‐induced histamine release and activation in human BA.22.1The anti‐IgE mAb E124.2.8 (Dε2), the fluorescein isothiocyanate (FITC)‐labeled mAb CLB‐gran12 (CD63), and phycoerythrin (PE)‐conjugated mAb 97A6 (CD203c) were purchased from Immunotech , and human IgE from Merck Millipore . A detailed description of mAb is listed in Table 2.2Peripheral blood was obtained from 5 healthy individuals and 11 patients allergic to Der p 2 and/or Phl p 5. Patients were diagnosed according to standard diagnostic procedures and their molecular IgE reactivity profiles were determined by ISAC  technology.2.3The human MC line HMC‐1 was kindly provided by Dr. J.H. Butterfield . Two subclones were used, HMC‐1.1 expressing KIT V560G and HMC‐1.2 expressing KIT V560G and KIT D816V.2.4ex vivo obtained BA were incubated in HRB in the absence or presence of anti‐IgE antibody E‐124.2.8 (0.001‐10 μg/mL) at 37°C for 30 minutes. Then, histamine release was measured as described above.The histamine release assay was performed on dextran‐enriched BA  essentially as described.2.5stim) and unstimulated (MFIcontrol) cells, and expressed as stimulation index, SI (MFIstim:MFIcontrol).+ BA was quantified by multicolor flow cytometry on a FACSCalibur as reported.Whole‐blood cells were incubated with various tyrosine kinase inhibitors  (0.001‐10 μmol/L) at 37°C for 30 minutes. Then, cells were washed and incubated with anti‐IgE mAb E124.2.8 (1 μg/mL) or allergens (1 μg/mL) together with fluorochrome‐labeled mAb against CD13, CD63, CD164, or CD203c for 15 minutes. Thereafter, cells were subjected to erythrocyte lysis and analyzed by multicolor flow cytometry on a FACSCalibur as described.2.63H‐thymidine was added . Cells were then harvested on filter membranes in a Filtermate 196 harvester . Filters were air‐dried, and the bound radioactivity was counted in a β‐counter . All experiments were performed in triplicates.HMC‐1 cells and KU812 cells were incubated in control medium or in various concentrations of ibrutinib, AVL‐292, CNX‐774, or P505‐15 (range: 0.001‐10 μmol/L) or dasatinib (0.000001‐10 μmol/L) at 37°C for 48 hours. Thereafter, 0.5 μCi 2.7t test was applied. In case of multiple comparisons, the Bonferroni correction was performed. A P value of <0.05 was considered to indicate statistical significance.To determine the level of significance in drug incubation experiments, histamine release experiments and surface staining experiments in BA and human cell lines, the paired Student's 33.1To study drug effects on BTK activation and to explore the specificity of these effects, we examined the phosphorylation status of various IgER downstream signaling molecules in drug‐exposed and IgER‐cross‐linked BA as well as in untreated or drug‐exposed cell lines . We found that dasatinib, ibrutinib, AVL‐292, and CNX‐774 counteract anti‐IgE‐induced expression of pBTK in BA Figure . Of all 3.250 values ranging between 0.003 and 0.03 μmol/L (0.01±0.01 μmol/L) in healthy donors   Figure B,C. Dasa3.53H‐thymidine uptake, ibrutinib, AVL‐292, and P505‐15 showed no significant effects on proliferation of HMC‐1.1, HMC‐1.2, and KU812 cells unless high concentrations (≥1 μmol/L) were applied   and HMC‐1.1 cells were lower compared to that obtained with HMC‐1.2 cells (1.45 μmol/L)  and allergen‐exposed BA obtained from patients allergic to Der p 2 and Phl p 5 were comparable and were found to be within a pharmacologically meaningful range, suggesting that the drug may indeed be able to block histamine secretion in patients with IgE‐dependent allergies. This hypothesis was confirmed by studies on ex vivo BA obtained from a CLL patient receiving ibrutinib. In this experiment, IgE‐dependent histamine release in ex vivo obtained BA was almost completely suppressed during therapy compared to pretreatment results.Recent data suggest that ibrutinib blocks IgE‐dependent activation and histamine release in normal blood BA.A number of previous and more recent data suggest that BTK is an essential IgER downstream regulator of activation and mediator secretion in BA and MC.50 values. In addition, we found that ibrutinib inhibits IgE‐dependent upregulation of CD13 and CD164 on BA. Unexpectedly, however, the other BTK inhibitors tested did not counteract upregulation of CD13, CD63, CD164, or CD203c on BA. From these data, one could speculate that the effects of ibrutinib on IgE‐dependent upregulation of these activation antigens were mediated by other  drug targets in BA. Alternatively, the inhibitory effects of the other drugs on BTK activation were too weak to result in downregulation of these CD molecules.Recent data suggest that ibrutinib inhibits IgE‐dependent upregulation of CD63 and CD203c on normal BA.The severity of an allergic reaction may not only depend on the type of allergen, IgE levels, cytokine exposure, and the microenvironment but also on the total number of effector cells involved in the reaction.50 values. Moreover, we found that other BTK blockers also counteract mediator secretion in BA. These effects may have clinical implications and may provide a basis for the development of new treatment concepts in IgE‐dependent allergies.In summary, our data show that ibrutinib is a most potent inhibitor of allergen‐induced (IgE‐dependent) activation and histamine release in human BA and MC. The effects of the drug were seen in normal BA as well as in allergen‐exposed BA obtained from patients allergic to Der p 2 or Phl p 5, with meaningful ICD.S. performed proliferation and apoptosis assays, flow cytometry experiments, and cell activation experiments, and wrote parts of the manuscript; K.B. performed flow cytometry experiments and wrote parts of the manuscript; G.S. performed cell activation experiments and histamine measurements; Y.D., M.F.‐T., W.R.S., and R.V. contributed patient material and vital reagents; C.S., W.R.S., and U.J. contributed patient material and parts of the study design; and P.V. contributed the study conception and design, patient material, and wrote parts of the manuscript.The authors declare that they have no conflict of interest in the current study. Conflict of interest unrelated to the present study: U.J. received honoraria from Janssen‐Cilag. R.V. received research grants from Biomay Ag, Vienna, Austria; Thermo Fisher, Uppsala, Sweden; and Fresenius Medical Care, Bad Homburg, Germany; and serves as a consultant for these companies. W.R.S. received a research grant from Lipomed, Arlesheim, Switzerland. P.V. received research grants from Novartis, Deciphera, and Blueprint, and honoraria from Novartis, Ariad, Pfizer, BMS, Deciphera, and Celgene. Click here for additional data file.


Rhodotorula mucilaginosa (NCYC 65) was discovered in which sugar substrate is cleaved but the products are not metabolised. We propose that engineering this and some of the other properties discovered in this study and transferring such properties to conventional industrial yeast strains could greatly expand their biotechnological utility.In addition to ethanol, yeasts have the potential to produce many other industrially-relevant chemicals from numerous different carbon sources. However there remains a paucity of information about overall capability across the yeast family tree. Here, 11 diverse species of yeasts with genetic backgrounds representative of different branches of the family tree were investigated. They were compared for their abilities to grow on a range of sugar carbon sources, to produce potential platform chemicals from such substrates and to ferment hydrothermally pretreated rice straw under simultaneous saccharification and fermentation conditions. The yeasts differed considerably in their metabolic capabilities and production of ethanol. A number could produce significant amounts of ethyl acetate, arabinitol, glycerol and acetate in addition to ethanol, including from hitherto unreported carbon sources. They also demonstrated widely differing efficiencies in the fermentation of sugars derived from pre-treated rice straw biomass and differential sensitivities to fermentation inhibitors. A new catabolic property of  Alternative energy sources are therefore of considerable interest and have been widely investigated, including sources such as water, solar, wind, nuclear fission/fusion and biomass4. As a possible alternative technology to petroleum refining, bio-refining shows many advantages with the potential of improving the quality of soil, water and air5. Ideally, bio-refining processes are needed to convert different feedstocks including biomass from municipal waste, agricultural waste, plant residues and industrial wastes to fuels or chemicals6. A priority list of chemicals which might be derived from biomass has been compiled by the US Department of Energy (DOE), including key renewable chemical targets such as ethanol, arabinitol, succinic acid, lactic acid and levulinic acid7.Large amounts of fossil-derived energy and chemicals are consumed globally each year. Unfortunately, the fossil resources, which are responsible for about 75% of the anthropogenic emission of CO10. Saccharomyces cerevisiae has been considered as the preferred yeast for ethanol fermentation, and can grow on simple sugars such as glucose, and on the disaccharide sucrose. However, more species of yeasts have been reported as being able to produce not only ethanol but also some highly-sought-after chemicals11. Therefore, diverse yeasts might have the potential to produce a range of bio-products via fermentation of different substrates.It is the microorganism employed which determines the category of products produced from such substrates. Yeasts are fungi capable of converting sugars to a range of metabolites. They are widely used in the food industry for producing bread and wineIn this study, to further explore the untapped potential of yeasts, and to inform future yeast screening studies, we have investigated and compared a specially selected set of genetically highly diverse yeast strains for their natural comparative abilities to ferment a range of carbon sources and to produce metabolites of potential interest in the renewable chemicals industry. Moreover, we have assessed how those selected yeast strains react to complex conditions when converting glucose from pretreated raw material to elucidate the potential of such yeast strains in industrial bio-refining.The 11 yeast strains selected for this study Fig.  were choInitially, aerobic growth of the 11 diverse yeasts on 13 different sugars (including pentose (C5), hexose (C6) and disaccharides (DIS) derived from plant tissues and microbial fermentation products) were tracked for 72 hours by recording turbidity every 30 mins. Subsequently, the lag phase (LP), doubling time (DT) and efficiency (ΔOD) were calculated by using PRECOG (see  Methodology). Figure Final fermentation liquors of the (anaerobic) yeast fermentations were analysed for the levels of unmetabolized sugar substrates Fig.  and ethaInterestingly, NCYC 16 consumed many of the sugars to a large degree but it failed to produce the expected stoichiometric levels of ethanol. Whilst NCYC 16 consumed all the glucose, it produced only half the amount of ethanol produced by NCYC 568. Surprisingly, NCYC 65 consumed virtually all sucrose whilst producing almost no ethanol. This wide range of fermentation behaviours indicated that metabolic products other than ethanol might be produced.To assess the production of other metabolites which could be of industrial significance, fermented liquors were analysed by a new high throughput nuclear magnetic resonance (NMR) method. This enabled the identification and quantification of 16 significant metabolites. Data was collected and is presented in Fig. Of the 16 chemicals quantified, 2,3-butanediol, acetic acid, arabinitol, citric acid, ethanol, glycerol, lactic acid, pyruvic acid, succinic acid, ethyl acetate and malic acid were produced in the highest quantities. The other metabolites were produced in trace quantities. Ethanol was the most common product detected after fermentation of all sugars as well as acetic acid. Notwithstanding differences between the yeasts, arabinitol, glycerol, succinic acid and ethyl acetate were produced from 8 sugars but not from lactose. Although the yield of lactic acid was generally low; it was still produced from all sugars. Citric acid was produced widely but negligibly from most of sugars but significantly from galactose. Like citric acid, 2,3-butanediol was significantly produced by only maltose and galactose. Higher yields of pyruvic acid were detected from glucose, fructose, mannose and sucrose compared with the other sugars. Interestingly, malic acid was only produced by NCYC 16 and then only from fructose. Ethyl acetate was significantly produced from 8 of 9 sugars except galactose by several strains. NCYC 16 produced highest amount of ethyl acetate from those sugars. Arabinitol was also significantly produced from 8 of the 9 sugars (excluding lactose) by several strains especially by NCYC 10.Multiple additional products were also significantly detected by NCYC 568 and NCYC 2577 fermentation from several fermented sugars. For example, NCYC 568 could produce glycerol and ethyl acetate in addition to ethanol from several sugars. Similarly, 3 different readily quantifiable chemicals could be produced by NCYC 2577 - in addition to ethanol: acetic acid was produced from glucose, fructose, mannose, sucrose and galactose; glycerol was produced from glucose, fructose, mannose and galactose. Interestingly, ethanol was insignificantly produced from cellobiose by all strains except NCYC 31.Basidiomycetous yeast, consumed nearly 100% of sucrose without producing a reasonable amount of any product . At an initial substrate loading of 5% w/w , the maximum theoretical ethanol yield is 9.89 mg/ml  = 2CH3CH2OH (ethanol) + 2CO2, maximum ethanol theoretically produced should be no more than 9.885 mg/ml). Initially, SSF was carried out using the complete pre-treated slurry. The results , 5-hydroxymethylfurfural (5-HMF), formic acid and levulinic acid.The fermentation conditions created using pre-treated raw materials are much more complex than those involving purified sugars. To evaluate the ability of the different yeast strains to utilise real world biomass substrates to produce ethanol, rice straw material characterised previously by Wood, lts Fig.  show thaTherefore, pre-treated rice straw samples were washed with distilled water 3 times prior to resuspension and evaluation for SSF. The results are shown in Fig. 13 since xylose is the second most abundant fermentable sugar in lignocellulosic materials. Naturally-occurring pentose-fermenting yeasts are limited, and include Pichia (Scheffersomyces stipites)14; Candida shehatae15. Hence there has been significant effort to genetically modify yeasts to enhance this property whilst improving hydrolysate inhibitor tolerance16. Pentoses and lactose can be fermented by bacteria, fungi and certain yeast strains19.In this study, the paucity of xylose and arabinose fermenters is notable. There is considerable interest in biofuel production from lignocellulosic hemicelluloses20, however recent production of it was only 1.7 million tons mainly produced via chemical processes21. In this study, ethyl acetate was produced in largest quantities by NCYC 16  from a range of sugar substrates, extending the findings of Walker22 who identified Pichia anomala  as a high ethyl acetate producer and compared it with S. cerevisiae, and Kurita23 and Rojas, et al.24 who had carried out fermentation of P. anomala on malt agar medium and glucose respectively. The present study has further demonstrated that the yield of ethyl acetate varies significantly when fermented in different sugars . For example, ethyl acetate was produced more from fructose than from glucose by NCYC 16. This may be due to the efficiency of conversion via different metabolic pathways of P. anomala. Glucose is firstly metabolised via multiple pathways - to glucose-6-phosphate (glucose-6-P) and then portioned for growing biomass (cells) or converted to fructose-6-P (fructose-6-phosphate)26. In contrast, fructose is directly metabolised to fructose-6-P and then further irreversibly phosphorylated to fructose-1, 6-bisphosphate27. Yields may also be affected by levels of oxygen which can inhibit or enhance ethyl acetate production29. Further research will be required to optimise the conditions for enhancing ethyl acetate production by NCYC 16 and to compare its phenotype to additional closely related NCYC strains. Preliminary results have strongly-indicated that ethyl acetate production may be significantly enhanced by intermediate levels of air or oxygen (results not shown).Ethyl acetate is an important platform chemical that can be used as a microbiologically-degradable and environmentally friendly solvent in the manufacture of food, glues, inks and perfumes. The world’s annual demand of ethyl acetate was 2.5 million tons32. This study also observed, we believe for the first time, that high arabinitol yields could be produced by NCYC 568 (Zygosaccharomyces rouxii) and NCYC 2577 (Kazachstania servazzii) from a range of sugars (both hexoses and pentoses), and in NCYC49  from mannose. NCYC 10 (Debaryomyces hansenii) was also found to produce considerable quantities of arabinitol at levels higher even than ethanol. This confirms and extends previous studies that reported accumulation of arabinitol from a range of carbon sources34.Arabinitol is currently synthesised via a chemical reaction requiring catalysis at high temperature, and it is an interesting platform chemical that has the potential to be used as a non-nutritive sweetener and as a feedstock in producing ethylene glycol, propylene, enantiopure compounds, arabinoic and xylonic acids35 and may provide useful genetic information for bioethanol production. Similarly, NCYC 65 may be a potential enzyme producer which can simply cleave sucrose to glucose and fructose monomers, presumably by invertase activity. However, how NCYC 65 obtains energy is not clear – a careful mass balance will need to be carried out to clarify whether any of the glucose or fructose are metabolised. It is interesting that NCYC 65 grows more rapidly in sucrose than in glucose, although we did not assess growth of the yeast in both fructose and glucose mixtures. An understanding of the genetics and biochemistry in comparison with other strains might be used as a first step to improve conversion of sucrose to glucose and fructose. NCYC 65 was unable to ferment any of the 13 sugars significantly. However, Lee et al. reported that a strain of R. mucilaginosa could produce acetylxylan esterase36. Such an esterase could remove certain substituents of hemicelluloses such as acetyl groups, which could potentially enhance the activity of xylanases38. Moreover, a high fatty acids accumulation strain of R. mucilaginosa was reported in the research of Li, et al.39. They mentioned those fatty acids could be used for biodiesel.NCYC 31 was the only strain in the set which could convert cellobiose largely into ethanol due to its ability to produce β-glucosidase40. There are some ways to reduce the effects of inhibitors and enhance the yield of products. For example, detoxification of hydrolysates and the use of fully or partially inhibitor resistant yeast strains43. It may be possible to improve the resistance of yeasts to inhibitors by genetic improvement40. Additional fine-tuning of the pre-treatment process may also be helpful44.The fermentation conditions created using pre-treated raw materials will be much more complex than environments created by purified sugars. In this study, each strain’s ability to produce ethanol was assessed and compared between enzymatic hydrolysates of rice straw and fermentation on purified sugars. Results were very consistent between the two substrates for all strains except NCYC 2577. NCYC 2577 could ferment purified glucose but was not very effective at fermenting the glucose from rice straw. It is possible that NCYC 2577 is particularly sensitive to inhibitors at very low levels (that might remain even after washing). Inhibitors of yeast fermentation are produced during unavoidable pre-treatments and yeasts can demonstrate considerable variation in their response to such chemicalsIn conclusion, this study presented information on yeast behaviours when grown on a range of pure carbon sources, identifying the key metabolites produced by some of strains across the different sugar substrates. It has demonstrated that the chosen phylogenetic diversity of the strain set was matched by phenotype diversity, highlighting the importance of screening widely across the vast yeast taxonomy for key bio-industrial traits. Furthermore, by evaluating the ability of yeast strains to ferment rice straw hydrolysates and comparing those data with fermentation of purified sugars, this study has highlighted the challenges that need to be addressed when attempting to exploit yeasts industrially. Nevertheless, we have identified significant gaps in knowledge that would be worthy of further research, including the possibility of creating ethyl acetate or D-arabinitol from raw materials, and the exploitation of NCYC 65 for sucrose hydrolysis.http://opendata.ifr.ac.uk/NCYC/)45. Draft genome assemblies were obtained from the raw reads for each of the 94 strains using the A546 assembly pipeline (20130326 version). A phylogenetic tree was estimated from the resulting 94 genome contig files using the FFP software47 with the RY alphabet and a word size of k = 14. The resulting Newick tree file was input to the Core Collection of Diverse taxa (CCD) software48 and the 10 maximally diverse yeast strains were selected under the Phylogenetic Diversity (PD) principle  +1% glucose  at 25 °C for 72 hours before use. The 13 laboratory-purified sugars were obtained from . 500 ml of a 10 mg/ml solution of each sugar in YNB was prepared and autoclaved.The genomes of a set of 94 diverse yeast strains selected from the National Collection of Yeast Cultures (NCYC) were paired-end sequenced at the Earlham Institute, Norwich  using an Illumina HiSeq. 2000 sequencer and TruSeq libraries, with 2 × 100 bp reads derived from inserts of size 500 bp. Raw reads for a subset of these strains may be found at the NCYC OpenData website  which contained 180 µl of each sugar (10 mg/ml). The reader plate was placed in VersaMax ELISA Microplate Reader  and incubated at 25 °C for 72 hours by which time growth had ceased. Growth of yeasts was recorded every 30 min during the 72-hour incubation and then further processed by using PRECOG2SO4 mobile phase at a flow rate of 0.6 ml/min, column temperature 65 °C. Injection volume was 25 µl.Small-scale fermentation was carried out in triplicate in 96 deep well (2 ml) plates . 980 µl of sugar solution (10 mg/ml) was added in to a set of 11 wells and then one of the 11 yeasts (20 µl) was added to each well. This was repeated for all sugars. Plates were sealed with a clear polypropylene PCR seal . They were then, incubated on an orbital shaker (135 rpm) in a 25 °C incubation room for 72 hours. The fermentation was terminated by heating at 100 °C for 10 mins. After cooling on ice, the supernatants were filtered  filters) and analysed for residual sugars and ethanol by High Performance Liquid Chromatography (HPLC) using a Series 200 LC instrument  equipped with both a refractive index detector and photodiode array detector. Separations were performed on a BIO-RAD Aminex® HPX-87H organic acid analysis column , protected by a matching guard column, eluting with 0.004 mol/l H1H NMR was used to identify and quantify key metabolites in the triplicate fermentation liquors. Samples were prepared by mixing 300 µl of liquor with 300 µl of NMR phosphate buffer -propionate-d4 (TSP) as a chemical shift and concentration reference, 40 mg sodium azide in 200 ml deuterium oxide, pH 6.4). 500 µl aliquots of the mixture were transferred into 5-mm NMR tubes for spectral acquisition. The 1H NMR spectra were recorded at 600 MHz on a Bruker Avance spectrometer  running Topspin 3.2 software and fitted with a cryoprobe. Each 1H NMR spectrum was acquired with 64 scans, a spectral width of 12300 Hz, an acquisition time of 2.7 s, and a relaxation delay of 3.0 s. The “noesygppr1d” pre-saturation sequence was used to suppress the residual water signal with a low-power selective irradiation at the water frequency during the recycle delay and a mixing time of 10 ms. Spectra were transformed with a 0.3-Hz line broadening, manually phased, baseline corrected, and referenced by setting the TSP methyl signal to 0 ppm. The metabolites were quantified using the software Chenomx NMR suite 7.6™. Metabolites were identified using information found in the literature, on the web , in the Chenomx standards library, or by use of the 2D-NMR methods, correlation spectroscopy (COSY), heteronuclear single quantum coherence (HSQC), and heteronuclear multiple bond coherence (HMBC). Some additional spectra of standards were run in-house to supplement those available in the Chenomx library. Heat-maps as presented were edited by using R , hence yellow and light orange represented compounds were produced as trace quantities. (https://www.r-project.org/).12 rice straw was grown in a rice paddy field at the Ba Vi National Park, Hanoi, Vietnam. After mature straws were harvested in spring 2012, the biomass was further fumigated and air-dried under ambient conditions  at the Agricultural Genetics Institute, Hanoi, Vietnam12. Cell wall polysaccharides were previously identified as containing rhamnose, fucose, arabinose, xylose, mannose, galactose and glucose. Rice straw used in this study contains 39% ± 0.65% of glucose, 23% ± 0.31% of xylose, 4% ± 0.14% of arabinose and 1.4% ± 0.04% of galactose. Only minor concentration of rhamnose, mannose and fucose were present.As described previouslyRice straw (<2 cm) was milled in a 6700EFM Freezer/Mill . Samples were pre-frozen in liquid nitrogen for 10 mins. Then, freeze milled for five mins. These samples were then pre-treated for SSF (explained below).et al.50 as those severities were selected from different thermal degradation zones. Tubes were cooled with compressed air to ambient, and stored at −20 °C before further experimentation.Milled rice straw was pretreated by using a BIOTAGE® Initiator + reactor . 4 samples (each 0.75 mg) had been weighed into 20 ml microwave pressure tubes. Water (14.25 ml) was then added into each of those tubes to give a 5% (w/w) suspension. Capped tubes were treated at severities 1.57, 3.65, 5.15, 5.45 . Experiments on each pre-treatment severity regime were performed in duplicate. The pre-treated samples were defrosted and then each agitated to provide a uniform suspension. Whilst mixing with small magnetic stirrer bars, 937.5 µl slurry was transferred into each Matrix tube. Cellic® CTec2   and one of the pre-grown yeast strains (each 50 µl) were then added. The tubes were sealed by screw caps and set into 96-tube Matrix plates were then incubated on shaker (135 rpm) at 25 °C for 72 hours. After fermentation, those tubes were heated at 100 °C for 10 min, then cooled on ice and centrifuged (3000 rpm) for 10 min. 400 µl supernatants of each sample were filtered using a Pall filter plate 0.2 µm  then multi-pipetted into a 96 well deep-well plate for HPLC analysis. Ethanol standards were made for quantifying ethanol produced by yeast fermentation.SSF of pretreated and washed rice straw was carried out in the same way. The washing involved centrifugation to sediment the pretreated residue, removal of the supernatant by decanting, followed by resuspension of the pellet in distilled water to 15 ml. This was carried out 3 times.All data generated or analysed during this study are included in this published article.Supplementary Information


Over the past two decades the primary driver of mass loss from the West Antarctic Ice Sheet (WAIS) has been warm ocean water underneath coastal ice shelves, not a warmer atmosphere. Yet, surface melt occurs sporadically over low-lying areas of the WAIS and is not fully understood. Here we report on an episode of extensive and prolonged surface melting observed in the Ross Sea sector of the WAIS in January 2016. A comprehensive cloud and radiation experiment at the WAIS ice divide, downwind of the melt region, provided detailed insight into the physical processes at play during the event. The unusual extent and duration of the melting are linked to strong and sustained advection of warm marine air toward the area, likely favoured by the concurrent strong El Niño event. The increase in the number of extreme El Niño events projected for the twenty-first century could expose the WAIS to more frequent major melt events. in situ observations and model simulations to highlight the role of the strong El Niño event.Sporadic surface melt over the West Antarctic Ice Sheet is not fully understood. Here, the authors report on an extensive melting episode in the Ross Ice Shelf area in 2016 and use comprehensive  Episodes of widespread summer melt have been sporadic in West Antarctica since the phenomenon started being monitored from space in the late 1970s refs . Their i267893Here we document a prominent surface melt event that occurred in January 2016 and affected a large portion of the Ross Ice Shelf. This event happened while an important field campaign, the Atmospheric Radiation Measurement West Antarctic Radiation Experiment (AWARE), was ongoing in central West Antarctica. The observations collected during this campaign provided unique insight into some of the physical mechanisms governing surface melting in this otherwise data-sparse region. In particular, these observations highlighted the presence of low-level liquid-water clouds, which may have aided the radiative heating of the snow surface. Furthermore, we explore the large-scale atmospheric factors behind the melt event, namely the role played by the strong 2015–2016 El Niño event and the positive SAM. Building on existing literature and new idealized climate model simulations, we show that the El Niño event is likely responsible for setting up the atmospheric circulation pattern that steered warm air towards the Ross Ice Shelf. We also show that the positive SAM counteracted to some extent the El Niño influence and thus likely mitigated the overall magnitude of the melt event.Passive microwave satellite observations  indicateThe satellite observations were corroborated on the ground by a number of automatic weather stations (AWSs) that recorded near-surface temperatures near or above 0 °C for several consecutive days during 10–21 January . The onsThe January 2016 melt event also coincided with the AWARE field campaignTs) up to the melting point (through exchange of sensible heat), especially at low elevations. However, Ts is ultimately controlled by the full surface energy budget (SEB), being the net of radiative (short- and longwave) and turbulent (sensible and latent) heat fluxes. Clouds exert an important influence on the SEB by modulating the radiative fluxes121315During the short Antarctic summer, strong onshore winds may by themselves raise the ice sheet’s surface temperature  mission for 12 January 2016  indicate−2, that is, the range where the cloud radiative enhancement effect previously observed over Greenland−2 simulated by ERA-Interim during the same period (−2 (−2), mainly attributable to enhanced downwelling longwave radiation  over the 90–120°W sector of the Southern Ocean . By creaOn a broader scale, the melt event occurred during one of the strongest El Niño events on record  and 6b. 620The SAM, which characterizes the strength of the westerly winds around Antarctica, is an important modulator of the tropical influence in the South Pacific sector of the Southern Ocean8Properly understanding the mechanisms responsible for the January 2016 melt event requires investigating the potential roles of El Niño and the SAM. This in turn can provide insight into the recurrence of such event in the future (see Discussion). These relationships are by no means simple. For example, not all major El Niño events are accompanied by widespread surface melt in West Antarctica ; not all prominent West Antarctic melt events coincide with strong El Niño events ; and the magnitude of West Antarctic melt does not scale with the intensity of El Niño events. Accordingly, it is not possible to establish with certainty whether the 2015–2016 El Niño caused (in a deterministic sense) the January 2016 melt event, a problem inherent to weather and climate phenomenaWe used the Community Atmosphere Model (CAM) version 4 to conduct a set of four 15-year idealized simulations (see Methods for details). Three simulations were forced with annually repeating SSTs characteristic of past strong El Niño events, representing a total of 45 El Niño events. The fourth simulation was forced with annually repeating climatological SSTs to serve as control run. A model SAM index was calculated for each simulation based on principal component analysis of Southern Hemisphere monthly 500 hPa geopotential height anomalies. Here again, we considered the average SAM Index for November–January.Estimating surface melt occurrence in the CAM model can be problematic as the process is affected by model grid resolution, model temperature biases and model deficiencies in the placement of key atmospheric features252627P<0.01, meaning that the type of event is significantly dependent on the combined states of El Niño and SAM. Such dependence confirms findings from previous literature729Based on the model SAM Index and series of warm and cold events, we generated a contingency table  tallyingExpanding on the previous analysis, we also find that 9 out of the top 10 warmest events occur during a negative or neutral SAM phase, while 9 out of the top 10 coldest events occur during a positive or neutral SAM phase. This demonstrates that the Ross sector is much more likely to experience conditions favourable to surface melting during El Niño events when a negative or neutral SAM is present. Given that the SAM was in a predominantly positive phase before and during the January 2016 melt event, our model results suggest that the state of the SAM likely mitigated the magnitude  of surface melt in West Antarctica during the austral summer of 2015–2016. In other words, the 2015–2016 melt season would likely have been more prominent had the SAM been in a negative or neutral phase, more commonly associated with El Niño events.Further research is needed to better understand the various mechanisms behind major West Antarctic melt events and to accurately predict their future occurrence. Accurate prediction is contingent on the ability of climate models to resolve the broad range of factors responsible for these events, from the large-scale climate drivers to the regional atmospheric circulation to the microphysical and radiative processes. Among them, the simulation of ENSO and its teleconnections, and the representation of high-latitude mixed-phase clouds (such as those observed at WAIS Divide in January 2016) are two key areas in need of improvement in climate models1531Future changes in the intensity of ENSO events are currently estimated with greater confidence than ENSO-related long-distance climate changes, owing in large part to model difficulties in simulating the present-day mean state climate32327810Finally, the January 2016 melt event demonstrates that the present-day climate of West Antarctica already allows for extensive surface melt to occur occasionally. In this regard, two recent modelling studies37Surface melt over ice sheets can be easily detected from space as the appearance of liquid water in the snowpack causes a sharp increase in microwave brightness temperature12melt) defined as Tbmelt=Tbref + ΔT, where ΔT=30 K and Tbref is a reference temperature. Tbref was calculated as the 12-month average from 1 April–31 March after filtering out all melt days as in ref. ref could not be calculated , we used the Tbref value from the previous or following year (whichever matched the sensor/satellite). This overall algorithm was shown to be particularly well suited for detecting melt in dry-snow areasFor a given grid cell and a given day, we determined that melt was occurring as soon as one of the two daily Tb observations exceeded a threshold value  was computed as follows:The 2015–2016 AWARE field campaign ran from 4 December 2015 through 18 January 2016, and deployed ARM Mobile Facility instruments464846Sdir is the direct solar beam from the normal incidence pyrheliometer, θz is the solar zenith angle and Sdiff is the diffuse flux from a shaded pyranometer. Surface latent and sensible heat fluxes were derived using the algorithm of ref. Here, A basic description of our modelling experiment is already provided in the main text for clarity. A few additional details are given here. We conducted four 15-year simulations using the CAM version 4 ref.  with seahttps://eosweb.larc.nasa.gov/clouds). Details about the cloud phase discrimination algorithm are given in ref. The vertical profiles of cloud particle phase shown in ftp://amrc.ssec.wisc.edu/pub/aws/10min/rdr/). On these stations, the temperature sensor is at a height of three meters above the surface. The CLWP data used in http://apps.ecmwf.int/datasets/). Note that CLWP corresponds to ERA-Interim total cloud liquid water field. The SST data used in https://www.ncdc.noaa.gov/oisst). The SAM Indexhttps://legacy.bas.ac.uk/met/gjma/sam.html). The Equatorial SOI used in http://www.cpc.ncep.noaa.gov/data/indices/).The AWS temperature estimates used in http://www.cesm.ucar.edu/models/ccsm4.0/cam/).The source code of the CAM4.0 global atmospheric model can be obtained free of charge through the Community Earth System webpage ( http://www.archive.arm.gov/discovery/). For quick access, users can enter ‘AWARE’ in the search box (top-left corner of the page) and click on AWR—AWARE .Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Tables and Supplementary References


Surgical site marking is one important cornerstone for the principles of safe surgery suggested by the WHO. Generally it is recommended that the attending surgeon performs the surgical site marking. Particularly in the case of same day surgery, this recommendation is almost not feasible. Therefore we systematically monitored, whether surgical site marking can be performed by trained nursing staff.The aim of the study was to find out whether surgical site marking can be carried out reliably and correctly by nurses.The prospective non-controlled interventional study took place in a single primary care hospital of Uster in Switzerland. During a pilot phase of 3 months (starting October 2012) the nursing staff of a single ward was trained and applied the surgical site marking on behalf of the responsible surgeon. After this initial phase the new concept was introduced in the entire surgical department. 12 months after the introduction of the new concept an interim evaluation was performed asking whether the new process facilitates daily routine and surgical site marking was performed correctly. 22 months after the introduction a prospective data collection monitored for one month whether the nursing staff carried out surgical site marking independently and correctly. Data were collected by a patient-accompanying checklist that was completed by the nursing staff, the staff in the operating room and the responsible surgeons.n = 115/128) of the surgical site markings were correctly performed. For the remaining 10% either a surgical site marking was not necessary or the nursing staff asked a surgeon to mark the correct surgical site. During the whole study time of almost 3 years, no wrong-site surgery occurred.The stepwise implementation of the new concept of surgical site marking was well accepted by the entire staff. 150 patient-accompanying checklists were analyzed. 22 data sheets were excluded from the analysis. 90% (Surgical site marking can be performed by trained nurses. However, the attending surgeon remains fully responsible of the correct operation on the correct patient. Wrong-site surgery (WSS) is a complication with potentially devastating effects , 2. The There is a large variance in the adherence to the principals of safe surgery and the use of checklists , 10, 11.Therefore we asked whether surgical site marking could be assigned by trained nursing staff at the admission without affecting patient’s safety.The prospective non-controlled interventional study took place from 2012 until 2015 at the clinic for general and orthopedic surgery in the primary care hospital of Uster in Switzerland. The hospital of Uster is a public teaching hospital with a catchment area of app. 200.000 inhabitants with 5.000 in-patients and 19.000 out-patients per year in all surgical unities. The different surgical specialties include orthopaedic surgery, general and trauma surgery, abdominal surgery, hand surgery and urology. The hospital follows the guidelines of the WHO surgical safety checklist. Before 2012 the attending surgeon or the surgical resident assigned surgical site marking. A timeline of the study is given in Fig. During 3 months (August 2012–October 2012) the nursing staff on a single surgical ward applied the pre-operative site marking and completed a patient-accompanying checklist regarding the upcoming intervention. All participating nurses were trained regarding the correct surgical site marking.Quality control: 22 months after implementation an additional questionnaire determined whether the surgical site marking was attached independently, whether uncertainties existed regarding the marking, or whether a surgeon was called to label the surgical site correctly. This questionnaire was filled in simultaneously by the surgeon in charge, the nurses applying the surgical site mark, and the staff of the OR. Data were collected from October 15th until November 15th 2014. During the team time-out shortly before surgery, the attending surgeon recorded on the checklist whether the site marking was done properly. All surgical site markings were recorded prospectively with a patient-accompanying checklist and questionnaire. Emergencies were excluded from data collection.After the pilot phase, the process was established on all surgical wards of the clinic in January 2013. Twelve months after implementation a questionnaire-based first evaluation of the new process was done asking nursing staff and physicians about feasibility and satisfaction about the new process. After closing the pilot phase, a written evaluation filled in by 13 participating nurses showed a positive attitude towards the new process and a significant benefit in the daily business Fig. . ThereafTwelve months after implementation 50 members of the nursing staff were asked whether they felt confident to place the surgical site marking and whether the process facilitated daily routine. 40 surgeons were asked whether surgical site marking was performed correctly and reliably by the nursing staff Fig. .Fig. 3EvEighty-seven percent of the nursing staff felt confident to place the surgical site marking and 89% mentioned that the process facilitated daily routine. Almost all surgeons (97%) trusted the surgical site marking done by the nursing staff and 86% of the surgeons noticed facilitation. Both surgeons and nursing staff felt a positive side effect in inter-professional collaboration Fig. .n = 150/353). Twenty two data sheets were incomplete and therefore were excluded from the analysis. One hundred twenty-eight data sheets were available for analysis  surgical site markings were correctly performed. In only two cases  the nursing staff were not able to perform the site marking and asked a surgeon to mark the patient. In 10% (n = 13/128) surgical site marking was performed although it was not necessary . Analysis of the team-time out before surgery indicated that surgical site marking was done correctly in all of the 353 patients and no WSS occurred during the study period.Twenty two months after implementation a prospective data collection took place. During one month 353 patients for elective surgery were recorded and surgical site marking was performed by the nursing staff. The return rate of the patient-accompanying checklist and questionnaires was 42%  Swiss Foundation for Patient Safety b) Swiss Professional Association of Care and c) Cantonal Medical Service of Zurich. All three lawyers agreed that surgical site marking can be delegated to the nursing staff. However, the surgeon remains fully responsible for the correct intervention.The results of the study also suggest that the implementation of the principles of safe surgery including safety checklists and surgical site marking are more important than the question of who should perform surgical site marking. Although the surgeon is responsible for his intervention, all staff has to carry out the principles of safe surgery to avoid mistakes , 19.Based on this study we have introduced surgical site marking during the admission process as a task of the nursing staff and hereby facilitated the daily routine of surgeons and nurses, especially in the setting of same day surgery. However, as wrong site surgery is a very rare surgical event, future large-scale studies are necessary to strengthen the results of our study. It should be emphasized that every practising surgeon remains fully responsible of performing the correct operation on the correct patient.


We investigate the gas flow behavior of unidirectional porous ceramics processed by ice-templating. The pore volume ranged between 54% and 72% and pore size between 2.9   Porous ceramics are used in a variety of applications where high permeability is required, such as filtration of gases and liquids or catalytic supports. The mainIn general, ceramics processed by the replica method exhibit accurate porosity control and the pore size is relatively large 200–3000 000 μm, ao et al.  conductestrength.The idea of using ice crystals to template porosity in materials and in ceramics in particular have been known for a very long time –8, and tIn ice-templated materials, Fukasawa et al.  studied Fukushima et al.  studied ry model. AssumingPekor et al.  studied While there are several studies that discuss permeability in ice-templated ceramics, there is no single study that systematically explores the effects of pore volume, pore size, and pore morphology on permeability. Further, no article connects the mechanical properties of the samples with their permeability, and establishing this link is critical to the industrial application of ice-templated materials.In this work, we aim to provide a deep understanding of permeability in ice-templated materials across different pore volumes, sizes, and morphologies. We systematically explore the permeability of ice-templated samples with different pore morphologies over the 54–72% porosity range with controlled pore sizes ranging from 2.9 to 19.1 ous work  in order2. 2.1. Porous samples with unidirectional, aligned porosity were prepared by ice-templating. Suspensions were prepared by mixing distilled water with 0.75 wt% of dispersant , 3 wt% of PVA  as a binder, and 3 mol% yttria-stabilized zirconia  at different solids loading ranging from 50 wt% (14.7 vol.%) to 65 wt% (24.2 vol.%). In some suspensions, zirconium acetate  placed on top of a copper plate. The top of the mold was exposed to air and kept at room temperature. Samples were unidirectionally frozen from the bottom to the top circulating silicone oil by the freezing plate. Temperature of the oil was regulated by a cryothermostat . The cooling rate was set at 2 porosity  and furtTo evaluate the effect of pore directionality, samples with randomly oriented porosity were prepared using pore formers. The same zirconia powder was mixed with commercially available pore former polypropylene with a mean particle size (2.2. m) and volume (V) of the samples with respect to that of fully dense TZ-3YS  was calculated based on the mass . Some samples were sectioned parallel to the freezing direction to evaluate the directionality of the pores. All the images were obtained using a scanning electron microscope . Furthermore, pore size results were confirmed by mercury intrusion porosimetry  with an applied pressure up to 0.31 bar.L) of 12–15 mm and a diameter 15 mm. In the case of ice-templated specimens, pores were always aligned parallel to the flow during the test.Gas permeability was measured using a custom built equipment and is shown in Figure Permeability was evaluated using the Forchheimer’s equation adapted for compressible fluids, Equation :(3) ΔPL=where L is the thickness of the sample , 3. 3.1. Figure o 65 wt% 4.2 vol.%Table Figure Since air flow in porous materials is a volumetric phenomenon and is highly sensitive to pore size, a complementary evaluation to image analysis was performed by mercury porosimetry. This technique probes the entire volume of the sample in contrast to the local measurements obtained by image analysis.Figure Results obtained by both techniques, image analysis and mercury intrusion porosimetry, are in excellent agreement, as shown Table 3.2. Figure All samples followed a linear relationship between Equation ). The fiEquation  considerhe fluid. The exceion μνs/k in EquatNevertheless, careful considerations should be made if behavior.Figure The values obtained in this work are in agreement with those found in the literature for ice-templated samples with similar microstructure. Pekor et al.  reporteda et al.  obtainedComparing the permeability exhibited by ice-templated materials with other macroporous materials obtained by different techniques, we can see in Figure Additionally, we evaluated the effect of pore directionality  k1However, this approach is hard to apply to cellular materials due to the difficulties in extracting the characteristic parameter that could replace the particle size (ize (dc)  and thenThe experimental values of Equation . Since tThe main factor that explains the deviation between experimental and calculated values of Equation  was origEquation ) to reprEquation  describeEquation  for sampThe microstructure of ice-templated materials better resemble a capillary system where pores are aligned parallel to the gas flow than a porous granular media. The maximum permeability in an ideal capillary model is described by Equation :15](9) (9) 15](9where The capillary permeability (Equation  and usinEquation  where τ=ncreases. Unlike i4. The air permeability of ice-templated materials was evaluated as a function of different morphological parameters such as pore volume, size, and morphology. The results showed that pore size is the main parameter controlling the pressure drop and is therefore of capital importance to increase the permeability of porous materials without a detrimental effect on the mechanical stability. Based on this knowledge we produced ice-templated samples with a 53% pore volume and a permeability equivalent to 72% pore volume through a pore shape modification. This reduction on total pore volume allowed samples to be obtained with a high resistance coupled with a high permeability, two properties seemingly inversely related.Microstructure control using ice-templating allows us to tailor the mechanical and gas flow properties almost independently and consequently could be of potential application in products such as filters or catalytic supports. Using a pressure drop test we evaluated the tortuosity of ice-templated samples and we obtained a value remarkably close to the ideal case (