This study evaluated the influence of cyclic loading on zirconia bar-shaped specimens after being subjected to three different surface treatments: particle abrasion with either 50 μm or 110 μm alumina and grinding with diamond points, while polished specimens served as a control. Statistical analysis revealed significant reduction (38-67%) in flexure strength (P < 0.001) after three million cycles of dynamic loading for all surface treatments. Scanning electron imaging revealed grain boundary thickening, grain pull-out, and micro-cracking as the main structural defects. The results suggest that various surface treatments of zirconia based dental ceramics may significantly influence their long term fatigue resistance in the oral environment. In the dental field, there is a trend to shift towards metal free restorations due to concerns about possible biological side effects of metallic alloys. Glass ceramic restorations became an interesting alternative because of their well known biocompatibility and superior esthetics, but the low mechanical properties and brittleness of glass ceramic materials represent the main drawback that has faced their widespread use, especially in high load bearing areas (the posterior region of the mouth). Few years ago, the application of glass ceramic materials was limited to small-sized restorations which are not subjected to high stresses, impact shock, or lateral forces since otherwise the restorations had a high risk of fracture during function . PolycryThe introduction of zirconia to the dental field opened the design and application limits of all-ceramic restorations. Thanks to its superior flexure strength and high fracture toughness, long span (multi-unit large restorations) and complex zirconia frameworks are now possible with high reliability and clinically proven success rate ,5,6. TheIn the biomedical field zirconia is used as hip prosthesis replacing femur heads and as dental implants used to replace missing teeth. There are currently lots of concerns about some drawbacks of zirconia as there are several reports indicating unexpected incidences of fracture of zirconia femur heads  which weTo understand the failure mechanisms of zirconia based materials, it is necessary to understand both its fabrication technique and its internal structure. Zirconia is a glass free ceramic material that possesses high hardness and mechanical properties in its fully dense state. The material is usually fabricated by different shaping and milling procedures in its partially dense state (green body) that has mechanical properties almost similar to gypsum products allowing easier and much faster fabrication procedures . AfterwaAt medium temperatures , zirconia is composed of a tetragonal crystal structure that transforms into the more stable monoclinic form upon cooling to room temperature. This self-transformation is accompanied by a volumetric expansion leading to micro-cracking of the sintered structures. To prevent this spontaneous transformation, a secondary phase known as the dopant phase is added to the material to stabilize the tetragonal phase at room temperature . DiffereAddition of 3% mol yttrium dioxide partially stabilizes the tetragonal phase allowing stress induced tetragonal to monoclinic transformation upon exposure to mechanical stimulation. The accompanying volumetric expansion exerts compressive forces upon the propagating crack tip leading to increase in the fracture toughness of the material, a property known as transformation toughening . IncreasOn a microscopic level, zirconia is composed of grains which meet each other at grain boundary regions. The size of the grain structure also influences the mechanical properties and the fatigue behavior of the material. Submicroscopic grains (0.3-0.8 µm) are usually used for biomedical applications. Heating zirconia for prolonged periods above 1,000 °C leads to grain growth and to formation to what is known as the cubic form, which can also utterly influence the long term performance of zirconia based materials . The cubRecent studies reported fracture of zirconia dental restorations at unexpectedly short service periods and under low loading forces. This failure was related to the amount of surface damage introduced during different laboratory and clinical procedures. Increasing the surface roughness of zirconia frameworks using particle abrasion is a common routine used to enhance bonding of resin adhesives to zirconia ,22. A stAt the moment there are no clear guidelines about the ideal surface treatment method of zirconia restorations and the associated influence on their mechanical properties. While some manufacturers recommend rough particle abrasion protocols  other advice against such procedures. Additionally, the influence of cyclic loading and chemically assisted crack propagation on the long term performance of zirconia restorations and their interaction with surface damage remain unclear. These parameters were addressed in the present study.P < 0.001). Subjecting the specimens to dynamic fatigue under water resulted in significant reduction in flexure strength with polished specimens demonstrating the least reduction (38%). Particle abrasion with coarse alumina particles at high pressure and grinding with diamond points resulted in the highest reduction in flexure strength (67%). Previous data are summarized in Statistical analysis revealed significant differences in the flexure strength of the tested specimens before and after cyclic loading  was located at the tensile surface. Airborne particle abrasion resulted in creation of sharp scratches and grooves, while grinding produced parallel lines on the ground surface. Cyclic loading resulted in grain boundary thickening  and micrSecondary electron imaging revealed thickening of the grain boundary region which suffered from micro-cracking and pull-out of some surface grains. The grain boundary regions of zirconia based materials play an important role affecting slow crack growth, low temperature aging, and diffusion of different dopant materials ,34,35. TAn interesting observation was that neither of the tested specimens failed during cyclic loading even with the observed grain boundary thickening and micro-cracking which reflects the capacity of the used zirconia in resisting crack propagation and in accommodating structural damage. The reported reduction in flexure strength of zirconia subjected to cyclic loading was similarly observed in previous studies ,31,33,37The data presented in this study indicate that there is a strong correlation between surface damage and fatigue resistance of the material. Highly polished specimens demonstrated the least reduction in flexure strength after cyclic loading, which could be considered as the theoretical long term performance of the material. Where suitable, a 30% safety factor could be used to predict deterioration in mechanical properties of polished zirconia subjected to cyclic loading. On the contrary, a 60% safety factor could be used when the material is subjected to surface alteration during different phases of the clinical and laboratory procedures. Such precautions will be most relative in thin sections of any zirconia structure subjected to dynamic loading . In a pret al., who reported significant reduction in initial flexure strength for surface damaged zirconia restorations. Survival statistics reported that inducing surface damage could lead to shifting of the theoretical failure stress (60% of the stress of polished specimens) to values as low as 40% of the theoretical strength of surface damaged material which could simply explain the reported fractures after short service time [Previous studies reported that particle abrasion could lead to the formation of deep surface cracks that could extend up to 50 μm from the surface. Such deep cracks would be favorable sites for the initiation of critical cracks especially in the presence of water at the crack tip. The effect of surface damage on the mechanical properties of zirconia was recently evaluated by Wang ice time ,41.In the biomedical field, zirconia implants and prosthetic devices receive different surface treatments and sometimes in combination with different surface coatings to enhance osseo-integration with bone cells. These methods could induce different types of surface damage which could explain unexpected fractures as previously reported ,42. ThusEighty bar-shaped zirconia specimens (2 × 3 × 25 mm) were prepared by cutting partially sintered zirconia blocks  using a precision cutting instrument  and a diamond coated disc . The bars were gently polished on 800 and 1,200 grit silicon carbide paper using a custom made rotating metallographic polishing device. The specimens were finally sintered according to manufacturer’s instructions .1)Particle abrasion with 110 μm aluminum oxide particle at 0.3 MPa pressure and 10 mm distance .2)Particle abrasion with 50 μm aluminum oxide particle at 0.15 MPa pressure and 10 mm distance (P-G 400).3)Grinding with diamond coated points.4)Polishing using 0.5 μm diamond paste. This group was used a control to measure the theoretical strength of the used zirconia.The bars were divided into four groups which received one of the following surface treatments which represent different surface roughening methods produced by different clinical and laboratory procedures (n = 20):All specimens were subjected to three million dynamic loading cycles in a four point flexure test setup (20 × 10 mm span) under water using a custom made pneumatic loading device. The applied load shifted between a maximum and a minimum load which resulted in alternating flexure stress at the tensile surface of the specimens between 50-200 MPa. The stress at the tensile surface was calculated using 4-point flexure strength using the equation mentioned further in the text.The selected stress range represent less than 20% of the theoretical flexure strength of the zirconia used in this study. Such low stress cyclic loading program was selected to study long term fatigue behavior and slow crack growth under water .A 0.5 mm thick silicon sheet was placed between the loading point and the surface of the specimens to prevent generation of contact surface damage which could lead to premature failure. The sheet was replaced every 250,000 cycles. The test was conducted under a water bath at 37 °C at rate of 1 cycle/3 sec.Before and after completion of the three million cycles, the specimens were subjected to one cycle load to failure program performed in the same four point flexure test setup in a universal testing machine  at 0.5 mm/min crosshead speed. The peak load was extracted from computer generated files and the flexure stress at the tensile surface of the bars was calculated using the following formula:The fracture surface was examined using scanning electron imaging  at different magnifications. Secondary electron imaging was conducted by preparing the fracture surface for high magnification imaging. Specimens were ultrasonically cleaned and gold sputter coated . Electro-conductive material was placed on the carrying stub to prevent electro-charging during examination. The spot size (2 μm), kilo voltage (Kv 15), and distance from electron length (20 mm) were adjusted to provide clear image of the examination field under complete vacuum.post hoc tests  were used for statistical analysis .Two way analysis of variance (ANOVA) and Bonferroni in vitro study, it can be concluded that cyclic loading in water combined with different surface treatments of zirconia based dental ceramics significantly reduced their mechanical properties and therefore may affect their long term performance.Within the imitations of this


This data aimed to audit the articles retracted from PubMed indexed dental journals from India. The PubMed indexed journals considered are Indian Journal of Dental research, Journal of Indian Society of Periodontology, Contemporary Clinical Dentistry, Journal of Indian Society of Pedodontics and Preventive Dentistry, Journal of Oral and Maxillofacial Pathology and Journal of Indian Prosthodontic Society for type of article, name of dental specialty, topic of individual dental specialties, causes for retraction of article and authorship trend of retracted articles using web-based search. Among PubMed indexed Indian dental journals,the number of retracted articles were as follows: Indian Journal of Dental research (4) followed by Journal of Indian Society of Periodontology (3), Contemporary Clinical Dentistry (3), Journal of Indian Society of Pedodontics and Preventive Dentistry (2), Journal of Oral and Maxillofacial Pathology (2) and Journal of Indian Prosthodontic Society (1). Out of 15 retracted articles from PubMed indexed Indian dental journals, case reports (7) form a major share followed by original articles(6) and review articles (2). Among the dental specialties of retracted articles, oral pathology and microbiology (5) constitute the major share followed by periodontics (4), pedodontics (4), oral medicine and radiology (1) and prosthodontics (1). Duplicate publication (7), plagiarism (5) and authorship dispute (3) are the causes for the retraction of article. More recently, detailed data of retracted articles in dentistry is published by Nogueira et al. The data was analyzed using descriptive statistics.


Chlamydomonas reinhardtii high-starch mutants. Three C. reinhardtii mutants showing high-starch content were generated using gamma irradiation. When grown under nitrogen-deficient conditions, these mutants had more than twice as much starch than a wild-type control. The mechanism of starch over-accumulation in these mutants was studied with comparative transcriptome analysis. In all mutants, induction of phosphoglucomutase 1 (PGM1) expression was detected; PGM1 catalyzes the inter-conversion of glucose 1-phosphate and glucose 6-phosphate in both starch biosynthetic and glycolytic pathway. Interestingly, transcript levels of phosphoglucose isomerase 1 (PGI1), fructose 1,6-bisphosphate aldolase 1 and 2 (FBA1 and FBA2) were down-regulated in all mutants; PGI1, FBA1, and FBA2 act on downstream of glucose 6-phosphate conversion in glycolytic pathway. Therefore, down-regulations of PGI1, FBA1, and FBA2 may lead to accumulation of upstream metabolites, notably glucose 6-phosphate, resulting in induction of PGM1 expression through feed-forward regulation and that PGM1 overexpression caused starch over-accumulation in mutants. These results suggest that PGI1, FBA1, FBA2, and PGM1 correlate with each other in terms of coordinated transcriptional regulation and play central roles for starch over-accumulation in C. reinhardtii.The focus of this study was the mechanism of starch accumulation in  The energy demands of the world are large and increasing and, for many reasons, cannot be met by traditional energy sources alone. Bioenergy is a promising alternative source of energy, particularly appealing as it is renewable. Biofuels are mostly obtained from food crops . HoweverChlamydomonas reinhardtii  mutant showed hyper-accumulation of both starch and TAG under photoautotrophic condition under nutrient deprivation. C. reinhardtii produces starch by photosynthetic carbon fixation under phototrophic condition and also uses carbon-reduced compounds such as acetate under heterotrophic and mixotrophic condition  only involves in the one step conversion, or both acetate kinase (ACK) and phosphate acetyltransferase (PAT) participate in the two step conversion  were used in this study, which were generated from C. reinhardtii strain cc124 (wild-type) by gamma irradiation. For generation of high-starch mutants, C. reinhardtii wild-type (strain cc124) cells grown in liquid Tris-acetate-phosphate (TAP) medium (4Cl was replaced with KCl) media and incubated for 5 days to induce starch accumulation. To select high-starch mutant candidates, iodine vapor staining for colonies was performed. For selected mutant candidates, starch contents were analyzed using starch assay kit as mentioned below. For liquid culture used in this study, approximately 1 × 105 cells were inoculated to 50 ml TAP medium. Culture was shaken continuously at 150 rpm at 25°C under constant white light (40 μmol m-2 s-1). For treatment of nitrogen starvation, 5-day-old sample grown in TAP medium was transferred to TAP-N medium and then cultured for 7 days under same condition as mentioned above. C. reinhardtii mutants.High-starch ) medium  were treThree-day-old cells grown in nitrogen-deficient TAP media were washed with 250 mM phosphate buffer (pH 7.0). Fixation was carried out using 1% (v/v) osmium tetroxide and 2.5% (v/v) glutaraldehyde. Graded ethanol series (50–100%) were utilized for dehydration. After dehydration, samples were embedded in Spurr’s resin and thin sections prepared using an ultramicrotome . Specimens were observed using a transmission electron microscope .5 cells were inoculated in 50 ml of TAP medium and incubated under the same condition as mentioned above. Optical density (OD) at 750 nm was measured every 24 h using a spectrophotometer .Samples grown in nitrogen-deficient TAP media for 3 days were used. Starch content was evaluated using Starch assay kit  according to manufacturer’s instruction. For measurement of growth rate, approximately 1 × 107 cells were used for RNA extraction for each sample. RNA was extracted using TRIzol reagent  according to the manufacturer’s instructions. To remove genomic DNA contamination, one unit of RNase-free DNase  was treated to the total RNA for 30 min. RNA samples were purified according to the manufacturer’s instructions.For RNA extraction, samples were taken at days 3 and 7 after transfer samples to nitrogen-deficient TAP media. Approximately 1 × 10HiSeq2000 . To generate unigene sequences, total RNA (10 μg) from each sample was prepared for creating a normalized cDNA and then amplified according to the Illumina RNA-seq protocol. Sequencing was carried out using Illumina HiSeq2000. Sequence data  were selected using SolexaQA. All sequence reads from different samples were utilized to optimize the de novo assembly using the software Velvet  using BLASTx . For short read mapping, reads were mapped to the assembled unigenes using the bowtie software  was applied to identify the threshold of the p-value in multiple tests using DESeq. Correlation and hierarchical clustering analysis were carried out via AMAP library in R. Functional enrichment analysis for Gene Ontology (GO) and KEGG pathway were conducted using Gene Ontology Database and DAVID2, respectively  according to the manufacturer’s instruction. The constructed library was quantified using the KAPA library quantification kit  following the manufacturer’s protocol. cDNA libraries were sequenced with Illumina e Velvet  and Oasee Velvet . Transcrsoftware . The numsoftware . Identifectively .®III reverse transcriptase , according to manufacturer’s instruction. For quantitative RT-PCR, cDNA was amplified by SYBER Premix Ex TaqTMII  using CFXTM Real-Time System . Reactions were carried out with the following program: 40 cycles at 92°C for 20 s, at 55–60°C for 20 s and at 72°C for 20 s. To confirm maximum efficiency of PCR, standard curve analysis was conducted using serial dilutions of primer pairs. Primer sequences are as follows: PGM1, 5′-GAT TAC GAG GAG TGC GCC AG-3′ and 5′-GGG AGC CAT CTG TGA ACA CG-3′; ACS2, 5′-TGC TGC TGG GCT GTG CAT GA-3′ and 5′-ACG CAG ACA CGA GCG GCT CT-3′; ACS3, 5′-TGA TAA ATT GTC ACG TCG TT-3′ and 5′-GCC TAA ACG GGC CTA GTC-3′; FBA1, 5′-ATT TTG GGA GAG AGC GTT GAG-3′ and 5′-ACA ACA CCA GCA CAA AGC AC-3′; FBA2, 5′-CAG AGC AAC TGC AAC GAG AG-3′ and 5′-GCA CTT CAT CCC TGC TCT TC-3′; STA1, 5′-AAC GCC GAC ATC ACC ATC-3′ and 5′- TCT TGA ACA CGT AGA TGC CC-3′; STA3, 5′-TTC ATC GAG CCC AAG AAC G-3′ and 5′-AGT TGA GGT TGT GGA TGG TG-3′; STA6, 5′-CGT CTG TAT CCT CTG ACC AAG-3′ and 5′-TGG GTG AGG CAG TAA ATC TTG-3′; TubA, 5′-CTC GCT TCG CTT TGA CGG TG-3′ and 5′-CGT GGT ACG CCT TCT CGG C-3′. Specificity of PCR products was tested by melting curve analysis and gel electrophoresis. TubA was served as an internal control. Normalization and quantification were performed using Bio-Rad CFX manager 3.1 program .Reverse transcription (RT) were performed using 5 μg total RNA with 200 units of superscript3).Statistical analyses for quantitative RT-PCR and growth measurement were performed with one-way analysis (ANOVA) using R program (version 3.25Chlamydomonas reinhardtii mutants showing high-starch contents were generated from C. reinhardtii strain cc124 (wild-type) after 100 Gy gamma irradiation (data not shown). Each mutant contained specific mutations compared to cc124 control, which was analyzed using transcriptome in/del analysis . All mutants also showed higher starch contents than cc124 wild-type grown in nitrogen-replete condition (TAP media) for 5 days (data not shown). To determine growth rates of these mutants in TAP media, ODs were measured every 24 h for 7 days. Sm162 and Sm181 displayed similar growth rate with cc124 control until day 2, whereas Sm162 showed lower OD value than cc124 control until day 4 . In the stationary phase, all mutants had greater OD values than a wild-type control. To identify morphological variation on starch accumulation in high-starch mutants, TEM analysis was also conducted . All mutants grown in nitrogen-deficient TAP media for 3 days accumulated large number of starch granules compared to cc124 control.Table 1), which provide at least 10× transcriptome coverage. Trimmed reads from each sample were mapped to the C. reinhardtii genome database (version 5.5) in Phytozome. All samples had over 95% mapped rates (Table 1). Three biological replicates were hired for reducing errors of sample preparation and sequencing. For comparison of gene expression between high-starch mutants and cc124 control, analysis of DEGs was performed . Sm142, Sm 162, and Sm 181 displayed 883, 571, and 518 up-regulated DEGs at day 3 after treatment of nitrogen starvation in comparison with cc124 control, respectively . Up- and down-regulated DEGs from samples at day 3 were used for GO enrichment analysis using GO Database. Large numbers of DEGs at day 3 were classified as nitrogen compound metabolic process, cellular metabolic process, and transferase activity of GO Terms  encoding starch biosynthetic enzyme was found in all mutants at days 3 and 7 after treatment of nitrogen starvation . Whereas, transcript level of phosphoglucose isomerase 1 (PGI1) also decreased in all mutants at days 3 and 7. Surprisingly, key genes encoding starch biosynthetic enzymes, such as ADP-glucose pyrophosphorylase large subunit (STA1), soluble starch synthase III (STA3), and ADP-glucose pyrophosphorylase small subunit (STA6), were significantly down-regulated in all mutants . Quantitative RT-PCR analysis was performed to confirm expression levels of these genes. All mutants displayed down-regulations of STA1, STA3, and STA6 expression at day 3 after treatment of nitrogen starvation . Figure 4 also displays reductions of alpha- and beta-amylase  transcript in mutant at days 3 and 7.To understand mechanism of starch over-accumulation in mutants, expression patterns of genes on starch biosynthesis were investigated by KEGG pathway mapping using transcriptome data  expression were significantly reduced at day 3 in all mutants in comparison with cc124 control . FBA2 transcript was also down-regulated at day 7, whereas no consistent pattern of FBA1 expression was observed between mutants. Expression levels of phosphoenolpyruvate carboxykinase 1 (PCK1), mitochondrial pyruvate dehydrogenase complex 1 (PDC1), and acetyl-CoA synthetases 1 and 2 (ACS2 and ACS3) were down-regulated at days 3 and 7 in all mutants . To confirm expression levels of these genes, quantitative RT-PCR analysis was carried out. Figure 7 shows expression patterns of PGM1, FBA1, FBA2, ACS2, and ACS3 at day 3 after treatment of nitrogen starvation in each mutant. PGM1 expression were highly up-regulated over fourfold in each mutant compared to a wild-type control. Transcript levels of FBA1, FBA2, and ACS2 were significantly reduced. Down-regulations of ACS3 transcripts were found in Sm162 mutant, but Sm142 and Sm181 showed slight reduction of ACS3 expression. Expression patterns of these genes confirmed by quantitative RT-PCR analysis were similar with these by the transcriptome analysis.Expression levels of 48 genes which participate in glycolytic/gluconeogenic pathway including post-glycolytic process were determined between mutants by heatmap analysis . PGM1 catalyzes the inter-conversion between glucose 1-phosphate and glucose 6-phosphate in starch biosynthetic pathway . STA6 catalyzes the conversion of ATP and glucose 1-phosphate to ADP-glucose  transcripts were observed in mutant at days 3 and 7 . Alpha-amylase is a key enzyme that involves in degradation of storage starch granule during seed germination in higher plants , because glycolytic pathway is working in conjunction with starch breakdown. Starch biosynthetic and glycolytic reaction are catalyzed by some of the same enzymes . Possible explanation of starch over-accumulation in mutants is that the down-regulation of PGI1 may reduce conversion of glucose-6 phosphate to fructose-6 phosphate in mutant cells, which could cause accumulation of glucose 6-phosphate. Accumulation of glucose 6-phosphate may have caused the up-regulation of PGM1 that we observed , through feed-forward regulation. This, in turn, could lead to starch over-accumulation in mutants.Down-regulations of amylase r plants . Beta-amrk cycle . Further1 mutant . Therefo enzymes . PGI1 ar enzymes . Arabidoin leave , which iFBA1 and FBA2 expression were determined at day 3 in all mutants compared to a wild-type control . Furthermore, FBA2 transcript was slightly down-regulated at day 7. FBA catalyzes conversion of fructose 1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate in glycolysis . In Arabidopsis, the FBA gene family is classified into plastidic and cytosolic type and expression patterns of these genes varied with developmental stages and stresses  were significantly down-regulated, such as PCK1, ACS2, and ACS3 . Gluconeogenic enzyme PCK1 catalyzes oxaloacetate to phosphoenolpyruvate and ACS converts acetate to acetyl-CoA in acetate metabolism (PCK1 and ACS3 transcript in C. reinhardtii starchless mutant sta6 under nitrogen-deficient condition. Thus, the reason of reductions of PCK1, ACS2, and ACS3 transcript in high-starch mutants may be due to inverse expression against starch content.We did not detect any consistent patterns in the expression of stresses . Recentltabolism . SimilarC. reinhardtii mutants in Figure 8. In short, aberrant expression of PGM1, PGI1, FBA1, and FBA2, encoding starch biosynthetic and glycolytic enzymes, were detected in high-starch mutants. Reductions of PGI1, FBA1, and FBA2 expression caused down-regulation of glycolytic pathway, which may consequently result in accumulation of glucose 6-phosphate. Induction of PGM1 expression is likely due to feed-forward regulation by high level of glucose 6-phosphate content, which enhances starch over-accumulation. Therefore, we propose that PGM1, PGI1, FBA1, and FBA2 correlate each other and play key roles in starch accumulation in C. reinhardtii. Furthermore, PCK1, ACS2, and ACS3 seem to affect starch over-accumulation. This study provides fundamental information of glycolysis/gluconeogenesis and starch biosynthesis in green algae.Based on our results, we propose the scheme of starch over-accumulation in J-WA and W-JJ designed experiments and wrote the manuscript. KK, SJ, and BL performed experiments. J-BK, YJ, H-IC, S-YK, and G-HC commented on the manuscript and analyzed data. All authors contributed to writing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Vigna subterranean) (BG) flour, and its native and hydrothermally modified starches were investigated. The isolated native starch (BNS) was modified by annealing at 50°C for 48 hr (BAS), and heat‐moisture treated at 100°C for 16 hr at 25% moisture level (BHS). The crude protein of BG flour was 16.88%. The amylose content of the flour and native starch was 6.10% and 27.70%, respectively. Hydrothermal modification increased the gelatinization profile of the starch but reduces its pasting properties. Swelling and solubility of the flour and starches increased with increase in temperature. X‐ray diffraction revealed BNS and BAS exhibited “Type C” crystallinity pattern while BHS was “Type A.” The BNS granule was oval, its diameter between 22 and 30 μm, with no significant change in the morphology of BAS and BHS. The BG flour had 33% resistant starch and 11.63% digestible starch. Heat‐moisture treatment enhanced the resistant starch content of the native starch significantly.In‐vitro digestibility and functional properties of Bambara groundnut ( BG bean was milled to produce the Bambara groundnut flour (BGF) used in the study. Its starch was isolated as described by Afolabi (BG ( Afolabi .2.2The native starch of BG starch (BNS) was hydrothermally modified by heat‐moisture treatment at 100°C for 16 hr at 25% moisture level (BHS), while annealing was carried out at 50°C for 48 hr (BAS) following the method of Adebowale, Afolabi, and Olu‐Owolabi . The pur2.3The AOAC International  method w2.4The effect of temperature and pH on solubility and swelling power of the starch samples were investigated following the methods of Afolabi et al. .2.5A Rapid Visco‐Analyser  was used to determine the pasting properties of the starch using AACC  method.2.6The gelatinization parameter of the native and modified BG starch was studied with differential scanning calorimeter (DSC) using the procedure of Afolabi et al. .2.7The crystallinity of the native and modified starch was determined with a Rigaku D‐Max‐ 2200 X‐ray diffractometer . The scanning region of the diffraction angle was from 3 to 40°, with target voltage 40 KV, target current, 100 mA, and aging time 5 min. The relative crystallinity of the starches was determined as enumerated by Afolabi .2.8Granule morphology of the starch was studied by scanning electron microscope, SEM (Hitachi TM‐1000 Table‐top Scanning electron microscope) at 500 magnification.2.9Aspergillus niger for α – amylase production, while spores of Rhizopus oligoshporus were used for glucoamylase synthesis. Both media were incubated at 30°C for 72 hr.Alpha‐amylase and glucoamylase were produced on a solid state fermentation medium containing rice bran, soyabean flour and cassava starch mixed in ratio (10:3:1 w/w) according to Akpan and Adelaja . The mixCrude enzyme extracts were recovered by mixing moldy bran with 0.2 mol/L acetate buffer  in the ratio 1:4 (w/v) in conical flasks. The mixtures were then shaken on an orbital shaker at 150 rpm at 28°C for 1 hr. The extracts were then filtered using muslin cloth. The filtrates were partially purified using 70% ammonium sulfate and kept at 4°C for further use.2.10g for 30 min. The residue was washed twice with 80% ethanol and dried at 60°C. Water (1.56 ml) and 4.0 mol/L KOH (1.5 ml) was added to the dried residue and mixed for 30 min at room temperature. To 1.5 ml of the dispersion; 12 ml of water, 0.65 ml of 2 mol/L acetic acid, and 0.1 ml of amyloglucosidase was added and shaken with the aid of a thermostated shaker  for 90 min at 65°C.The in vitro determination of resistant starch content of the BG flour and starches were analyzed by previously described methods , measuring the absorbance wavelength  at 505 nm. The resistant starch content was calculated as 2.11g, 15 min. Glucose oxidase‐peroxidase kit  was used to measure the glucose content of the supernatant, and the total starch content was calculated as mg of glucose × 0.9.The method of Goni, Alonso, and Saura‐ Calixto  was emplThe digestible starch was determined by calculating the difference between total starch and resistant starch of the sample on dry weight basis.2.12Shapiro‐Wilk test of normality and the Levene's tests of homogenous variance were carried out to assess the assumptions of ANOVA in order to validate the results.All determinations were carried out in triplicate and result reported as the mean ± standard deviation. The swelling and solubility profile were subjected to one‐way analysis of variance (ANOVA) using SPSS statistical software (version 20) to investigate the effect of pH and temperature on starch samples. The 33.1The starch yield of BG on flour basis was 41% Table , but higThe 16.88% protein content of BGF Table , but hig3.2The effect of temperature on swelling power revealed that as the temperature increased, the swelling power of the flour and starch increased Figure . The incThe solubility of BG flour and starches increased as the temperature increased Figure . The obsWilk Statistic for the BNS, BAS, and BHS starch samples are 0.825, 0.815 and 0.837, respectively and their significance were all greater than 0.05. The Levene test has a value of 2.169 with a significance value of 1.20 for swelling index and 2.885 with a significance value of 1.662 for solubility. With respect to swelling index, there was a statistically significant difference between the three starch samples as indicated by one‐way ANOVA  = 36.828, p = .000. A Tukey post hoc test revealed that the mean of the BNS starch sample is significantly higher than the other 2 starches – BAS and BHS and there is a statistically significant difference between the starch samples at 80°C and 90°C only, while there was no statistical difference between the three starch sample's solubility at a different temperature.The statistical analysis of variance (ANOVA) between BNS, BAS, and BHS at different temperature and pH levels showed that the 3.3The pasting and thermal properties of the BG starches are presented in Table Breakdown viscosity which is the difference between the peak and trough viscosity was significantly reduced after hydrothermal modifications from 4252 RVU in BNS to 3925 RVU and 226RVU in BAS and BHS, respectively Table . The pasTo), peak temperature (Tp), and enthalpy change (ΔH) of the BG starch. These increase showed that hydrothermal modification leads to the elevated thermal transition temperature, which is indicative of rearrangement of starch molecular chains to form molecular orders  with enhanced thermal stability  of the starches was 27.60–34.50°C, this is similar to the 22.50–25.40°C reported for runner bean  after hydrothermal treatment from 57.50°C (BNS) to 66.10 and 61.70°C  could be attributed to the increased interaction between amylose/amylose and amylose/amylopectin , with a decrease in gelatinization temperature range (Tc–To)  of BAS could be attributed to the greater influence annealing has on To which represent the melting of the weakest crystallite. ΔH value of the starch decreased after heat‐moisture treatment from 5.57 J/g (BNS) to 4.97 J/g (BHS), this decrease could either be as a result of disturbance of the double helices present in the crystalline and non‐ crystalline regions of the granules , onset and peak temperature  but with the lowest gelatinization range of 27.60°C. Other reports also showed that annealing of starch leads to increase in ΔH and gelatinization temperatures , heat moisture treated (BHS), and annealed (BAS) starches was presented in Figure The resistant starch content of the native, heat moisture treated, and annealed BG starch increased in the following order: BHS > BNS > BAS Figure . TherefoThe digestible starch content of the BG starch increased in the following order: BAS > BNS > BHS Figure . The abo4BG flour and starch is an excellent source of resistant starch, and the versatility of its starch can be enhanced by heat‐moisture treatment and annealing. The use of BG flour and heat‐moisture treated starch in food and food products may be an excellent way to alleviate obesity and diabetes, due to its high resistant starch content. Heat‐moisture treatment also reduced the amylose content of the starch with a consequential decrease in its swelling power, this is a desirable property in the application of the modified starch in food and allied industries.The authors hereby state that there is no conflict of interest to declare on this research.


Steel plates with open holes are commonly used in structural assemblies. The fatigue properties of such details are influenced by bolted clamp-up and hole fabrication methods. The fracture surface, stiffness degradation and fatigue life of test specimens are investigated in detail and compared with the contemporary test data. The analysis results show that the presence of draglines greatly influences the fatigue crack initiation at the open-hole cut by laser. The bolted clamp-up condition greatly enhances the stiffness and the fatigue life of the open-hole details. A discussion is also made from a comparison with the referred fatigue life of hole fabrication details, such as the influence of plate thickness and plasma cutting, drilling and oxy-fuel gas cutting, with the details studied herein. This work could enhance the understanding of the fatigue property and design of such details. Steel plates with open holes are commonly used in structural components to enable an assembly with bolts. The gusset plates and cross-frames are components which can be regarded as examples of such types. As evidenced from the basic configurations of these components, the weaknesses of the components are determined by fabrication methods as well as clamping conditions ,2, whichFor the sake of evaluating the mechanical characteristics of the components with hole fabrication, tensile tests have been widely used in obtaining the basic mechanical properties of the connections. Punched holes as a fast and cost-effective method of forming holes are widely used for secondary tension members. Also, holes may be formed by full-size drilling and thermal cutting, which a concern of most early studies. From the tests, Chesson and Munse  concludeAlthough the weakness of punched holes may be improved using reamed full-size to remove the damaged zone surrounding the hole, laser cutting as a substitute has shown good applications in engineering ,14. The In this paper, the fatigue properties of open-hole steel plates using laser cutting as a modern and common fabrication method will be studied in terms of failure mode and life results. Also, the fatigue life of the open-hole steel plates with the bolted washer clamp-up condition is also examined. Finally, the effects of clamping and fabrication methods on the fatigue life of such structural details will be discussed further. The results obtained in this study can be taken as a basic reference for further study involved in the fatigue life extension of related structural details.The material used for the test specimens was Grade 345B low alloy steel which conforms to the Chinese national standard GB/T1591. This material has good toughness and ductility. The chemical composition and mechanical properties are listed in The test specimens were designed to consider two configuration details, i.e., the open-hole details and the bolted washer clamp-up details. All test specimens had similar dimensions, as shown in The test specimens were tested using the Shimadzu EHF-UV050k2-020-0A fatigue testing machine of 50 kN capacity. Output and instant information from the testing machine were monitored and recorded by an automatic data acquisition system controlled by Windows Software for 4830 3.40b. Tensile fatigue tests were conducted following the Chinese code GB/T 3075-2008 . ConstanThe fractures of the steel plate exclusively originated at the edge of the hole. The typical crack initiation through the thickness of the plate around the hole appeared to be unsymmetrical on both sides of the hole. The presented fracture surface was taken from the test specimens and was able to demonstrate a typical observation similar to the others. For the test specimen without the bolted clamp-up loaded under the stress range of 220 MPa, for example, the typical fracture surface and its magnification of approximately 30× under the scanning electron microscope  are shown in ith cycle can be obtained as the ratio between the measured force range and the deformation range, which can be given by:i,max and σi,min are the maximum and minimum stress components at the ith cycle; di,max and di,min are the deformation components corresponding to σi,max and σi,min, respectively, which were measured from the testing machine and while eliminating the initial error of deformation arising from the grip assembly; Am is the plate net cross-section area excepting the hole.Stiffness is a well-defined engineering property, easily measured, and not involved in the destruction of the test specimen . During The comparison of the stiffness degradation behaviour of specimens with open-hole details and bolted washer clamp-up details is shown in N, and the applied stress range, ΔS, as:m is the slope of the S-N relation; C is the material constant-related parameter m)(1/. Thus, σmax = 290 MPa was considered for comparison. Using the above-mentioned method, the fatigue life results of the test specimens are compared with the reference regarding other fabrication methods as shown in The fatigue life with a 95% level of reliability and a 95% confidence level for structural fatigue details is calculatedas: ce level . Given tThe fatigue crack of test specimens mostly initiated not only at the edge of the hole but also in the vicinity of the middle thickness of the steel plate with the presence of predominant draglines within the thickness for the laser-cut hole.The open-hole plate using laser cutting exhibits notable stiffness degradation in its appearing cycle and magnitude. This trend seems to be reduced to some extent when the bolted clamp-up details are introduced.The bolted clamp-up details greatly enhance the fatigue life of the open-hole plate from category E to approaching category A as codified in JSSC, from category B’ to approaching category A as codified in AASHTO, and from category 100 to approaching category 160 as codified in Eurocode.The fatigue life of open-hole details with laser cutting is greater than that with punched holes but lower than those with plasma cutting, drilling and oxy-fuel gas cutting. The introduction of bolted clamp-up details can be used to compensate for the laser cutting holes when a lower fatigue detail coefficient is concerned.The fatigue property of open-hole steel plates was investigated in this research with a focus on the influences of the bolted clamp-up condition and hole fabrication methods. Through analyzing the fracture surface, stiffness degradation and fatigue life results, the following conclusions can be drawn:Based on the basic understandings obtained from this study, further research is still needed incorporating a wider range of parameters, such as plate thickness, etc., in a follow-up study.


We modified the conventional aortic arch replacement procedure to avoid circulation arrest and a prolonged extracorporeal circulation time, especially in cases of acute aortic dissection. We herein present our experience with a modified branch-first approach to acute aortic dissection, with anastomosis of the supra aortic vessels prior to commencing cardiopulmonary bypass.Since 2012, 41 patients  have undergone the modified procedure. Procedurally, the implanted graft was used as a landing zone for second-stage endovascular stent-graft deployment intended to manage the residual descending dissection. Antegrade and retrograde systemic perfusion was instituted during cardioplegic arrest. The brain was actively perfused via the graft throughout the procedure.Arch replacement surgery could generally be completed within approximately 4 h. During a 2-year period of aortic dissection or arch aneurysm treatment, only four anastomoses were required during the first stage of operation: two in the aorta, and one each in the innominate and left common carotid arteries. No patient died of surgical causes, and no stent grafts were deployed into the false lumen, a characteristic of procedures using traditionally antegrade deployment.We recommend that our procedure for acute aortic dissection be performed in two stages (graft replacement first and stent graft deployment second), particularly for patients underwent preoperative hypotesion. If malperfusion syndrome still exists after graft replacement, stent graft should be deployed in one stage. The arch aneurysm can be treated in one stage because there is no concern about false lumen deployment.The online version of this article (10.1186/s13019-017-0689-y) contains supplementary material, which is available to authorized users. We modified the conventional aortic arch replacement procedure, especially for acute aortic dissection, to avoid circulation arrest and a prolonged extracorporeal circulation time, which have been identified as independent risk factors for morbidity and mortality , 2.Preparation: We recommend performing this procedure in a hybrid operation room. Bilateral cerebral oxygen saturation and invasive artery pressure in the bilateral upper limbs were monitored continuously during the operation.After performing sternotomy and opening the pericardium, the tissues were dissected free as much as possible to expose the aortic arch and supra-arch branches. The adventitia was reserved as much as possible to facilitate suturing of the fragile arteries in an acute setting. The patient was subsequently heparinised.Additional file 1:Video 1 showed the procedural steps of simplified arch replacement approach. (MP4 286720 kb)We used a fine paediatric vascular clamp, such as a Pilling clamp 354,486, to fully close the left carotid artery (LCA) and thus avoid secondary injury to the intima. We usually sutured the intima to the adventitia, which had been severely torn, using a 7–0 prolene suture and mattress-suturing and added a pericardial strip outside the carotid artery. Next, we completed the end-to-side anastomosis between the branch and LCA using a 6–0 prolene suture.We then measured the length of the second branch of the aortic graft  and clipped it, and clamped and transected the innominate artery and trimmed the end to prepare for anastomosis. We anastomosed the graft branch and innominate artery in an end-to-end manner using a 5–0 prolene suture, and completed the end-to-side anastomosis between the branch and left common carotid artery. These end-to-side and end-to-end anastomoses are key to understanding how to actively perfuse the brain. Specifically, this method does not cause brain circulatory arrest of blood flow from the carotid to the innominate artery during the procedure, yet it allows the aorta to be clamped near the carotid artery. The proximal end of the innominate artery does not require suturing if the segment has been severely dissected; rather, clamping can be performed until the aorta is closed.The left femoral artery was exposed and cannulated using a cannula connected to the cardiopulmonary bypass to yield retrograde artery perfusion. A hybrid operating room can confirm that the cannula has been placed in the true aortic lumen. We inserted a two-stage cannula in the right atrium, and established another artery cannula branched from the artery end of cardiopulmonary bypass using a Y-shaped connector to the perfusion branch of the graft. Accordingly, the aorta was perfused in both an antegrade and retrograde manner, which allowed adequate perfusion of the viscera, even in the presence of preoperative malperfusion syndrome. The cardiopulmonary bypass was established, and the blood was cooled to 28 °C.Once the blood temperature reached 32 °C, the aorta was clamped near the left common carotid artery. The ascending aorta was opened, and cardioplegia fluid was perfused into the left and right coronary arteries to induce cardiac arrest. We trimmed the proximal end of the aorta and reinforced the aortic wall with two Dacron strip patches using the sandwich technique. The distal end of the aorta was treated in the same manner. The distal anastomosis of the aorta and graft was achieved with a 3–0 prolene suture, after which we released the clamp distal to the graft to determine whether any bleeding would occur. We found that it was safer and more efficient to wrap the anastomosis with a strip patch than to use sutures. The proximal anastomosis of the aorta with the graft was performed in the same manner.Finally, the patient was rewarmed and the heart beat recovered. The root of the left common carotid artery was closed using a 5–0 prolene suture to prevent retrograde blood flow into the false lumen. From that point, the aortic dissection was changed from type A to type B. Extracorporeal circulation was restored in a stepwise manner to complete the procedure. Angiography revealed the remaining distal dissection. Endovascular repair was not immediately required except in cases of low perfusion syndrome or a threatened rupture.Two weeks later, the patient returned to the operating room, where a femoral approach to expose the femoral artery was performed under local infiltration anaesthesia. Once the catheter reached the ascending aorta, a superstiff guidewire  was introduced for stent graft deployment. Finally, a 20-cm-long stent graft  was deployed near the second graft branch orifice.Morbidity and mortality can be improved by reducing blood perfusion; the durations of general anaesthesia, cardiopulmonary bypass, and abnormal hemodynamics; and carefully protecting the myocardium and brain. Since 2012, 41 patients  have undergone a procedure that was gradually modified, including the brain perfusion manner, branch priority, retrograde stent graft deployment via the femoral approach, a two-stage concept involving two-branch anastomosis. Arch replacement surgery could be completed within approximately 4 h, and during 2 years of treating aortic dissection or arch aneurysm, only four anastomoses were required during the first stage of surgery: two in the aorta and one each in the innominate and left common carotid arteries. No patient died of surgical causes, and no stent grafts deployed into the false lumen, which tends to occur with traditionally antegrade deployment.The left subclavian artery remained perfused by the aorta from the true or false lumen after graft replacement after the first stage. It could be covered by the stent graft or reserved for the second stage. We were also able to reconstruct blood flow in all three supra-arch branches. For example, if a patient had an advantage of the left vertebral artery, we might consider an end-to-end anastomosis between the left subclavian artery and third graft branch during the rewarming period. The left carotid artery could be transected at the root and pushed aside to facilitate dissection of the subclavian artery see Fig. .Fig. 1ShPerfusion via the graft helps to avoid the deployment of other types of perfusion, e.g. auxiliary artery retrograde perfusion and antegrade cerebral perfusion via a catheter. Anastomosis of the supra-arch branches is performed before initiating CPB, and can thus be performed easily to ensure quality. The body temperature is lowered only to 28–32 °C during cardiac arrest, thus reducing the rewarming period. Altogether, these modifications save approximately 3 h.If the left common carotid artery is occluded by a torn intima, the first anastomosis can be performed between the left subclavian artery and graft branch .The very stiff guidewire and stent profile, which are curved prior to surgery, help to deliver the stent graft into the distal aortic graft.For type A dissection, in order to shorten surgery time as possible, we recommend that our procedure be performed in two stages, especially for patients with the most severe conditions . However, one-stage treatment is required if intraoperative angiography (contract agent injected through the graft branch) confirms malperfusion of the viscera or lower extremities. The retrograde manner of stent deployment avoids plunging the stent graft into the false lumen through the torn intima (compare Figs. An arch aneurysm can be treated in a single stage, as false lumen deployment is not a concern. This is very similar to hemi-arch replacement with a stented graft/elephant trunk into the proximal descending aorta. In the former, we have more choosable specifications and models of the interventional stent grafts than the graft of the latter in China. That is important for protecting the descending aorta containing stent graft. The best stent graft we think is as soft as it can be to fit the diameter and shape of descending aorta. Further more, although aneurysm surgery is easier than dissection, we still suggest avoiding cardiac arrest if possible, unless the femoral approach of stent graft delivery is not permitted.Although simplified, this procedure remains a major surgery for patients experiencing great distress, malperfusion, and abnormal hemodynamics resulting from an aortic dissection type A. If a sufficient segment of normal ascending aorta remains, a TEVAR procedure, which reserves the supra-arch branches, or a debranch procedure is recommended for simplicity and safety . There r


Graft hyperperfusion demonstrated by increased hepatic vascular velocities and a small-sized graft in the early postoperative period contributes to hepatic regeneration 2 weeks after LDLT.Hepatic regeneration is essential to meet the metabolic demands of partial liver grafts following living donor liver transplantation (LDLT). Hepatic regeneration is promoted by portal hyperperfusion of partial grafts, which produces shear stress on the sinusoidal endothelium. Hepatic regeneration is difficult to assess within the first 2 weeks after LDLT as the size of liver graft could be overestimated in the presence of postsurgical graft edema. In this study, we evaluated the effects of graft hyperperfusion on the rate of hepatic regeneration 2 weeks after LDLT by measuring hepatic hemodynamic parameters. Thirty-six patients undergoing LDLT were enrolled in this study. Hepatic hemodynamic parameters including peak portal venous flow velocity (PVV) were measured using spectral Doppler ultrasonography on postoperative day 1. Subsequently, we calculated the ratio of each velocity to 100 g of the initial graft weight (GW) obtained immediately after graft retrieval on the day of LDLT. Ratios of GW to recipient weight (GRWR) and to standard liver volume (GW/SLV) were also obtained. The hepatic regeneration rate was defined as the ratio of the regenerated volume measured using computed tomographic volumetry at postoperative week 2 to the initial GW. Correlations of the hemodynamic parameters, GRWR, and GW/SLV with the hepatic regeneration rate were assessed using a linear regression analysis. The liver grafts regenerated to approximately 1.7 times their initial GW (1.7 ± 0.3 [mean ± standard deviation]). PVV/100 g of GW ( Although a substantial amount of liver regeneration occurs within the first 2 postoperative weeks followed by a plateau within 1 to 2 months after LDLT,,3 a large amount of graft edema also occurs secondary to portal hyperperfusion of partial grafts and thus, precludes an accurate assessment of hepatic regeneration within 2 weeks after LDLT.Living donor liver transplantation (LDLT) is a well-established treatment for end-stage liver disease, especially in Asian countries with a shortage of cadaveric donors. Unlike the whole liver allografts which are transplanted from deceased donors, LDLT uses a smaller partial liver graft and therefore, it is important to balance the requirements for maintaining adequate remnant liver volume in the donor with those for ensuring appropriate graft volume in the recipient, in order to meet the metabolic demands of the recipient and ensure the safety of both patients.Therefore, we investigated the effects of graft hyperperfusion during the early postoperative period on hepatic regeneration 2 weeks after LDLT by assessing the correlations of hepatic hemodynamic parameters per initial graft weight (GW), which were measured by spectral Doppler ultrasonography on postoperative day (POD) 1; graft-to-recipient weight ratio (GRWR); and GW to standard liver volume ratio (GW/SLV), which were obtained immediately after the graft retrieval, with the hepatic regeneration rate at 2 weeks after LDLT.22.1After obtaining study approval from Daegu Catholic University Medical Center Institutional Review Board and written informed consent from the patients, this prospective observational cohort study was conducted using 36 consecutive patients undergoing LDLT (31 right-lobe and 5 left-lobe grafts) from March 2013 to January 2014. Patients were included if they had liver cirrhosis with or without concurrent hepatocellular carcinoma. Patients were excluded if they underwent LDLT due to acute liver failure or if they required repeat surgical interventions within the study period; if they failed to be weaned from mechanical ventilation within 24 hours of arrival to the surgical intensive care unit after surgery; if they had vascular complications of the portal vein, hepatic artery, or hepatic vein; or if they had missing data.2.22 mixture with continuous infusion of remifentanil. Desflurane and remifentanil were regulated to maintain a bispectral index value between 40 and 60 along with mean arterial pressure and heart rate within 25% of preinduction values. Rocuronium was continuously infused at a rate of 1 mg/kg/h for neuromuscular blockade during the surgery. All patients were mechanically ventilated without positive end expiratory pressure at a constant tidal volume of 6 to 10 mL/kg. The respiratory rate and fraction of inspired oxygen were adjusted to maintain arterial carbon dioxide and oxygen tensions between 35 to 40 mm Hg and 150 to 200 mm Hg, respectively. Prostaglandin E1 was continuously administered from the beginning of anastomosis of hepatic artery at a rate of 0.01 μg/kg/min. After surgery, all patients were admitted to a surgical intensive care unit and extubated within 24 hours of arrival.Anesthesia was induced using 1.5 to 2 mg/kg of propofol in combination with 0.01 to 0.05 μg/kg/min of remifentanil. Endotracheal intubation was facilitated with 1 mg/kg of rocuronium. Anesthesia was maintained using desflurane in an air/O2.3 Following full mobilization of the native liver and its vascular exclusion, total hepatectomy was performed with preservation of the inferior vena cava by selective clamping of the right hepatic vein and, middle and left hepatic vein trunk without venovenous bypass. One of the orifices of the middle and left hepatic vein trunk and the right hepatic vein was closed according to the type of liver graft and the remaining orifice was anastomosed to the donor hepatic vein under partial clamping of the inferior vena cava. After the end-to-end anastomosis of the portal vein, the liver graft was reperfused. Finally, the hepatic artery and bile duct were reconstructed and the abdominal wall was closed.When the right lobe of a donor was used, a single and large hepatic venous outflow was constructed by one-orifice venoplasty of the graft's right hepatic vein and reconstructed middle hepatic vein.2.4One day after LDLT, peak portal venous flow velocity (PVV), peak systolic (PSV), and end diastolic velocities (EDV) of the hepatic artery, as well as peak hepatic venous flow velocity (HVV) were measured under spectral Doppler ultrasonography using a 1 to 5 MHz curved array transducer  equipped to the diagnostic ultrasound system   = 706.2 × body surface area (m2) + 2.4, where body surface area (m2) = body weight (kg)0.425 × body height (cm)0.725 × 0.007184.Using the initial GW measured immediately after retrieval of the graft from donors, GRWR and GW/SLV were obtained. GRWR was calculated by dividing the GW by recipients’ body weight. As an index of expected size of the native liver in the recipient,,16 In this study, the hepatic regeneration rate was defined as the ratio of regenerated volume measured using computed tomographic volumetry at postoperative week 2 to the initial GW.To assess the postoperative regeneration of liver grafts, the regenerated liver volume was calculated by computed tomographic volumetry of multiple computed tomographic images obtained with a slice thickness of 3 mm at postoperative week 2  and monitored for the development of small-for-size syndrome. This syndrome is defined as delayed functional hyperbilirubinemia: total serum bilirubin > 20 mg/dL for >7 consecutive days occurring after the 7th POD, excluding technical, immunological, and hepatitis factors.2.7The primary end point of this study was hepatic regeneration rate at postoperative week 2. A total of 40 patients were required to achieve a statistical power of 80% with significance level of 0.05 (2-tailed) and drop-out rate of 10%, assuming that the alternative hypothesis of a 0.2 coefficient of determination between peak PVV/100 g of the initial GW (PVV/GW) on POD 1 and hepatic regeneration rate at postoperative week 2 is true.2.8P < 0.05 was considered to be statistically significant.The data are expressed as the mean ± standard deviation for normally distributed data and number of patients (percentage) for categorical data. The Kolmogorov-Smirnov and Shapiro-Wilk tests were used to assess the normality of distribution of the data. If at least one of the null hypotheses of the tests was not rejected, the assumption of normality was determined to be met. Simple linear regression analysis was used to investigate the relationship between hepatic hemodynamic parameter values and their values per 100 g of the initial GW on POD 1 and hepatic regeneration rate at postoperative week 2. All statistical analyses were performed using IBM SPSS Statistics software, version 19.0.0 . A 2-tailed 3 and 35% for GRWR and GW/SLV, respectively), no patient developed small-for-size syndrome. The liver grafts regenerated to an average 170% of the initial GW (1123.3 ± 195.6 g), at postoperative week 2.Four patients were excluded from this study because they underwent reinterventions during the study period including exploratory laparotomy for postoperative bleeding on POD 3 n = 1) and spectral Doppler ultrasonography examinations more than 1 day after LDLT (n = 3). The mean initial GW was 681.7 g   and between GW/SLV and hepatic regeneration rate  , EDV/100 g of the initial GW (EDV/GW), and HVV/100 g of the initial GW (HVV/GW) Table  became p4–21 For these reasons, we preferred measuring the blood flow velocities of vessels to calculating their blood flow volume in order to assess the graft hyperperfusion. Therefore, higher velocities of inflow to the liver  represent more severe graft hyperperfusion. If inflow to the liver is constant, a small liver graft (with low GRWR or GW/SLV) would be hyperperfused. Based on the above concepts, positive correlations of PVV/GW, PSV/GW, and EDV/GW as well as negative correlations of GRWR and GW/SLV, with the hepatic regeneration rate indicate the contribution of hyperperfusion of liver graft to hepatic regeneration in the present study.In this study, the extent of graft hyperperfusion was evaluated by measuring the hepatic hemodynamic parameters per 100 g of the initial GW on POD 1, as well as the GRWR, and GW/SLV. We believe that the blood flow velocities of vessels in a liver graft represent their blood flow volume in the absence of vascular complications that significantly affect the patency of the blood flow. In addition, the blood flow volume per minute, which is calculated by multiplying the time-velocity integral per heart beat by heart rate and cross-sectional area of a vessel, is not accurate due to inter- and intraobserver variation producing high standard deviations. On the contrary, other studies found significant correlations between PVVs and various other indices representing hepatic regeneration obtained at different postoperative time points.,9 However, these studies did not analyze data with PVV/GW. At this point, it is difficult to explain discrepancies in results between the studies because of differences in study designs.Our results showed a significant correlation between the PVV/GW on POD 1 and hepatic regeneration rate at postoperative week 2. However, there was no correlation between the PVV (not standardized by dividing with the GW) and hepatic regeneration rate. Likewise, a previous study reported a significant correlation between the PVV/GW on POD 1 and hepatic regeneration rate at postoperative week 1, but no correlation between PVV and hepatic regeneration rate. However, during the first 2 postoperative weeks, augmentation of graft volume might occur due to graft edema which prevents appropriate evaluation of hepatic regeneration. Therefore, the assessment of hepatic regeneration at postoperative week 2 seems reasonable to determine the extent of postoperative hepatic regeneration as early as possible in the absence of confounding effects caused by graft edema. Consequently, we evaluated hepatic regeneration at postoperative week 2. In contrast, the previous studies did not measure regenerated graft volume at postoperative week 2, preventing the possibility of a fair comparison.The liver graft transplanted to a cirrhotic patient undergoes rapid hepatic regeneration that plateaus at GW/SLV of 100% at 2 weeks after LDLT, following which regeneration almost completely ceases. several correlations of mean PVVs measured on POD 1, 7, or 28 with GW/SLV at postoperative week 1 or 2 and postoperative month 1 or 3 were found. However, the number of patients (n = 15) was too small to achieve sufficient statistical power. Furthermore, the etiology of liver disease was not consistent (4 patients with fulminant hepatic failure and 9 patients with liver cirrhosis). If fulminant hepatic failure develops in a patient with previously normal hepatic physiology, the amount of PVF to a liver graft would be significantly lower than that of a liver graft transplanted to a cirrhotic patient. Hence, the analysis of a patient group with mixed etiology is inappropriate. In addition, the slice width of their computed tomographic scan (7 mm) for the volumetric study was twice more than ours (3 mm). Another study, which showed the correlation of difference in PVF per 100 g of GW (PVF/GW) between recipient and donor with percent change in GW between time of graft harvest and postoperative month 2, enrolled 22 patients which is still smaller than number of patients (n = 36) in the present study. In addition, the accuracy of volumetric assessment could not be determined due to the absence of description of the slice width in magnetic resonance imaging. A more recent study reported the correlations of PVV/GW on POD 1 and PVF/GW on POD 1 and 5 or 6 with the hepatic regeneration rate at postoperative week 7. However, this study also included 3 patients with fulminant hepatic failure and assessed the hepatic regeneration rate earlier than 2 weeks after LDLT, when the effects of early graft edema on the liver graft are still prevalent. Lastly, Jiang et al's study, which showed the correlations of PVV and PVF on POD 1 and 3 with percent change in GW between preoperative period and POD 30, included only 18 patients. All the above studies standardized only the surgical technique without considering standardization of anesthetic management. To improve the design of our own study, we considered the limitations of previous studies, and estimated the sample size required to achieve a sufficient statistical power, tried to enroll patients with homogeneous etiology, used a uniform surgical and anesthesia protocol, and enhanced the accuracy of the computed tomographic volumetry with thinner slice width.Although several studies have demonstrated the beneficial effect of portal hyperperfusion on hepatic regeneration in LDLT recipients, these studies have certain limitations. In the study by Eguchi et al, we did not expect that hepatic arterial blood flow would contribute to hepatic regeneration. However, we observed significant correlations between PSV/GW and EDV/GW with the hepatic regeneration rate. This unexpected effect of hepatic arterial blood flow might be attributed to the hepatic arterial buffer response blunted by the administration of prostaglandin E1 before the reperfusion of the hepatic artery.Due to the effects of hepatic arterial buffer response, which represents a reciprocal decrease in hepatic arterial blood flow in response to an increase in PVF on the liver graft, Thus, the significant correlation between HVV/GW and hepatic regeneration in the present study strongly suggests that potent hepatic venous outflow that effectively delivers blood flow from the portal vein and hepatic artery can also contribute to hepatic regeneration. Therefore, to facilitate hepatic venous outflow, we performed a simplified one-orifice technique in the present study. However, until further evidence on the contribution of HVV to hepatic regeneration becomes available, this parameter should be used only as a reference and other factors which contribute to hepatic regeneration should be prioritized.The relationship between hepatic venous flow or HVV and hepatic regeneration has not been extensively investigated. Although the grafts can regenerate successfully despite partial deprivation of venous drainage, hepatic venous outflow obstruction can cause graft congestion resulting in inappropriate regeneration of the affected area. we cannot entirely exclude the possibility that graft edema persists after this time period and could have influenced our results. Third, despite continuous regeneration between the reperfusion of graft and POD 1, the initial GW was used to calculate the velocities per GW due to unavailability of the liver volume data on POD 1. Fourth, this study did not evaluate the relationship between hepatic blood flow and regeneration due to poor reproducibility of blood flow estimation using time-velocity integral and diameter of blood vessels. Lastly, multicollinearity caused by the high intercorrelations between the velocities, GRWR, and GW/SLV did not allow multivariate analysis in the present study.This study has several limitations as well. First, patients were followed up for only 2 weeks which prevented the assessment of long-term outcomes. 2nd, although it is known that graft edema resolves during the 2nd postoperative week,In conclusion, we demonstrated positive correlations of the velocities of hepatic vessels per 100 g of GW measured on POD 1 with the hepatic regeneration rate measured 2 weeks after LDLT and negative correlations of GRWR and GW/SLV with the hepatic regeneration rate. In conclusion, graft hyperperfusion contributes to hepatic regeneration in the early postoperative period.


I want to report that the launch of our double‐blind procedure for manuscript review has been successful, thanks to the diligence and discretion of our Associate Editors and Reviewers. Our goal was to publish four issues in the calendar year 2004. We are going to meet this goal: the Fall issue is scheduled for publication in December. I wish to offer many thanks to all of you. You have all shown faith in the JACMP and supported it.I also want to recognize the many others who make the JACMP possible. The JACMP survives on volunteer efforts. Everyone associated with the JACMP volunteers his or her time and efforts. These individuals, like you, are deeply committed to and firmly believe in the need for a clinically oriented journal. Below you will see listed the names of many of these volunteers. When you see them in person, I would greatly appreciate it if you let them know how much you appreciate their efforts on behalf of the JACMP.In addition, and as mentioned in the previous issue, we owe a tremendous debt of gratitude to our commercial sponsors. You can show your gratitude in a number of ways. For instance, please keep in mind we are counting every use of our banner ads. Every time you visit a vendor through the JACMP, you help the JACMP. Using the banner ads demonstrates to our sponsors the importance of the journal to you and the effectiveness of our journal as an avenue to reach them. Another way to show your thanks is to consider our sponsors whenever you need a product or service or want to visit a vendor's web site: do so using the banner ads. Use the list of our sponsors. Mention the journal in your communications with them. Ultimately, our existence depends on you in so many ways, reading our articles, recognizing our volunteers, and supporting our sponsors. We hope you continue to make the JACMP and its support an easy habit.Michael D. MillsEditor‐in‐Chief


To elucidate whether any differences are present in the stiffness map obtained with a multiscale direct inversion algorithm (MSDI) vs that with a multimodel direct inversion algorithm (MMDI), both qualitatively and quantitatively. The MR elastography (MRE) data of 37 consecutive patients who underwent liver MR elastography between September and October 2014 were retrospectively analyzed by using both MSDI and MMDI. Two radiologists qualitatively assessed the stiffness maps for the image quality in consensus, and the measured liver stiffness and measurable areas were quantitatively compared between MSDI and MMDI.2) was larger than that by MSDI (37.5 ± 14.7 cm2) (P < 0.05). Liver stiffness measured by MMDI (4.51 ± 2.32 kPa) was slightly (7%), but significantly less than that by MSDI (4.86 ± 2.44 kPa) (P < 0.05).MMDI provided a stiffness map of better image quality, with comparable or slightly less artifacts. Measurable areas by MMDI (43.7 ± 17.8 cmMMDI can provide stiffness map of better image quality, and slightly lower stiffness values as compared to MSDI at 3T MRE, which radiologists should be aware of. Regarding the quantitative consistency among different inversion algorithms, little clinical data has been reported so far. To our knowledge, there has been only one report comparing the results between MSDI and MMDI, in which little difference (9%) in the stiffness values of the liver measured by either method was reported using 1.5T MRE system.16 MMDI is a relatively new inversion algorithm, and has been reported to have several improvements over MSDI, including superior resolution, less noise, and shorter processing time.16In the management of patients with chronic liver diseases and cirrhosis, the assessment of the degree of liver fibrosis is of great importance, because progressive liver fibrosis can result in portal hypertension, liver cancer, and finally patient death.The purpose of this study is to elucidate whether liver stiffness values calculated with MSDI and MMDI are comparable at 3T MRE, and also whether any differences are present in the stiffness map (elastogram) obtained using the two algorithms.Out institutional review board approved this study and waivered to obtain informed consent because of its retrospective nature.1-weighted in- and out-of-phase images, diffusion-weighted images, dynamic-enhanced studies, T2-weighed images, and hepatobiliary phase images. There were 22 men and 15 women, with age ranging from 31 to 85 years old (mean 62). Among these, 8 and 17 had hepatitis B and C hepatitis/cirrhosis, and three had alcoholic liver diseases, and underwent MR imaging for the assessment of hepatocellular carcinoma; remaining nine patients had no chronic liver disease and underwent MR imaging to rule out liver focal lesions suspected on ultrasonography.Between 2014 September 1 and October 30, 37 consecutive patients underwent gadoxetate  enhanced liver MR imaging including MRE in our institute. The protocol included precontrast T18 Four slices were obtained including the level of the hepatic hilum under 16-s breath-holding. Wave images and elastogram images (stiffness map) with cross-hatching marks were automatically generated on the operating console. Cross-hatching marks represent the 95% confidence threshold mask, indicating areas unsuitable for stiffness measurement.18 Inversion algorithm used for stiffness map calculation at that time was MSDI. Liver stiffness was measured by one experienced radiologist (KY) using a copy-and-paste method,20 by placing as large free-hand region-of-interests (ROI) as possible on the stiffness map, mainly in the right hepatic lobe, avoiding apparent pathologies, large vessels, areas with inadequate wave propagation (hot spot or dark spot artifacts), and cross hatching marks.20 An average of the four slices was used to represent the liver stiffness of each patient. Measured stiffness and size of ROI (mm2) for each slice was recorded in a separate laptop computer at the time of routine clinical practice and stiffness measurement was not repeated especially for this study.The MR equipment used was a 3T clinical unit  along with a 32-element phased-array coil, and MRE was obtained before contrast enhancement. A 19-cm-diameter passive pneumatic driver was positioned over the center of the right rib cage at the level of the xiphoid process and attached to an acoustic waveform generator. A 60-Hz acoustic wave was propagated to the driver. A 2D spin-echo echo-planar MRE sequence  acquired magnitude and phase difference wave images using 42 cm field-of-view.In June 2015, a new inversion algorithm software MMDI was installed, and the digital imaging and communication in medicine (DICOM) data of MRE of the above mentioned patients were additionally analyzed, and stiffness map or elastogram with cross-hatching marks were newly generated with MMDI.20 Overall image quality was determined based on the homogeneity of the whole image. Hot spots and dark spots were defined as areas showing focally elevated or decreased stiffness within areas without cross-hatching marks, respectively, showing no focal abnormality in any other sequences of the protocol, possibly attributable to wave overlap or interference.20Two radiologists (KY and YS) reviewed the two stiffness maps originated from MSDI and MMDI, referring to the wave images, anatomical images, and other relevant images, including diffusion-weighted images and hepatobiliary phase images for the presence of any focal lesions, and qualitative assessment was performed in consensus, and determined which stiffness map was better than the other; namely, MSDI is worse, similar, or better, in quality, as compared to MMDI. The items assessed were overall image quality of the stiffness map, and the prominence of hot spots or dark spots artifacts within the areas without cross-hatching marks, namely measurable areas.2, and the stiffness values. Measurable areas were compared between the two systems at slice basis (n = 148). Measured stiffness in kPa was compared at patient basis (n = 37).Measurement of the liver stiffness was then repeated on the MMDI stiffness map by the same radiologist (KY) and in the same manner, as the MSDI stiffness map measurement, and quantitative assessment was performed. The items for quantitative assessment were the measurable areas, which is represented by the size of ROI in cm21 .For comparison between two parametric variables, paired There were 148 images in total for review (four slices per patient in 37 patients) and stiffness measurement was feasible in all slices. The measured stiffness per patient ranged from 1.9 to 15.1 kPa as measured with MSDI, and from 1.8 to 13.6 kPa as measured by MMDI, which would correspond to a full range of fibrosis grades .As for the overall image quality of stiffness maps, MSDI was worse in 103 (70%), similar in 39 (26%), and better in 6 (4%), out of 148 slices, as compared to MMDI.As for hot spots/dark spots artifacts, MSDI had more prominent ones in 22 (15%), similar ones in 114 (77%), and less prominent artifacts in 12 (8%), out of 148 slices, as compared to MMDI.2 for MSDI, and 43.7 ± 17.8 cm2 for MMDI, showing statistically significant difference (P < 0.001).Measurable areas, or the area of ROI, was 37.5 ± 14.7 cmP < 0.001), and the relationship between the two are shown in Bland-Altman analysis suggests the difference in the liver stiffness between MSDI and MMDI becomes larger as the stiffness value increases  assessed, however, the number of slices (images) were relatively large (n = 148), and the measured stiffness ranged widely enough to cover the whole fibrosis grades . We therIn conclusion, MMDI can provide elastograms or stiffness maps of better image quality as compared to MSDI at 3T MRE, and the stiffness values obtained by MMDI is slightly (7%) lower than those obtained by MSDI. Radiologists should be aware of this issue, particularly when patients are assessed longitudinally using these two different inversion algorithms.


PEBP1)/Raf Kinase inhibitory protein (RKIP) expression can be used to identify a significant proportion of patients who performed poorly after DC vaccination. This result was validated by q-PCR analysis on blood samples from a second cohort of 95 patients treated with DC vaccination in four different centers. We conclude that low PEBP1 expression correlates with poor overall survival after DC vaccination. Intriguingly, this was only the case for expression of PEBP1 after, but not prior to, DC vaccination. Moreover, the change in PEBP1 expression upon vaccination correlated well with survival. Further analyses revealed that PEBP1 expression positively correlated with genes involved in T cell responses but inversely correlated with genes associated with myeloid cells and aberrant inflammation including STAT3, NOTCH1, and MAPK1. Concordantly, PEBP1 inversely correlated with the myeloid/lymphoid-ratio and was suppressed in patients suffering from chronic inflammatory disease.Immunotherapy for metastatic melanoma offers great promise but, to date, only a subset of patients have responded. There is an urgent need to identify ways of allocating patients to the most beneficial therapy, to increase survival and decrease therapy-associated morbidity and costs. Blood-based biomarkers are of particular interest because of their straightforward implementation in routine clinical care. We sought to identify markers for dendritic cell (DC) vaccine-based immunotherapy against metastatic melanoma through gene expression analysis of peripheral blood mononuclear cells. A large-scale microarray analysis of 74 samples from two treatment centers, taken directly after the first round of DC vaccination, was performed. We found that phosphatidylethanolamine binding protein 1 ( These fPEBP1), also known as Raf-1 kinase inhibitor protein (RKIP), as a biomarker that can be used to predict patient survival after DC vaccination. PEBP1 has previously been described to modulate several major cancer and inflammatory signaling pathways . The correlation plot, however, indicated that a survival of 14 months rather than 12 months best delineated the patients with low PEBP1 expression and low survival. Indeed, patient selection was further improved when the maximal survival time for the short survival group was extended to 14 months ) allowed us to identify patients who were surviving less than 14 months after vaccination with a sensitivity of 46% and a specificity of 89%. Using this expression cut-off we were thus able to select a patient population with a significantly poorer clinical outcome after vaccination, that is unlikely to respond to further DC therapy in the applied format and which would be in immediate need of other or additional treatments  is indicative of patient responses to DC vaccination using overall survival as an endpoint, and as such may provide a mechanistic insight into what determines a successful treatment response.We then investigated whether t Figure . The difs Figure . Notablyf Figure . Interesf Figure . A similf Figure . TogethePEBP1 expression may reflect the response to DC vaccination, we interrogated the original MA data to identify genes that that were positively or negatively correlated with PEBP1 to understand more about the molecular context in which this protein may act. First, we analyzed genes ranked on their correlation with PEBP1, for the enrichment of previously described blood transcriptional modules (BTMs) using Gene set enrichment analysis (GSEA) [PEBP1 expression correlated well with genes associated with an adaptive immune response (e.g. T cells and their activation) as well as the regulation of transcription (Table To obtain further insights into how s (GSEA) –37. Inteon Table . HoweverPEBP1 may represent a state of systemic inflammation and stress accompanied by low protein biosynthesis. This systemic state may hamper an effective response to DC vaccination.In the search for more mechanistic insights, we subjected the genes that were positively- or negatively-correlated with PEBP1 to protein-protein-interaction and gene ontology (GO) analyses. We found that genes correlating with PEBP1 expression mapped to extremely well-connected, mostly ribosomal, proteins, suggesting high protein synthesis (ability) in these patient samples. Negatively-correlating genes were clustered around the signaling proteins STAT3, NOTCH1, MAPK1 and UBB Figure . MoreoveTo investigate whether these functional implications of PEBP1 expression may stretch beyond DC vaccination and/or metastatic melanoma, we interrogated a large co-expression database of immune cells (immune-navigator) and found that also in this database PEBP1 expression correlated with adaptive immune cell responses and anti-correlated with monocytes and generic immune activation Figure . MoreovePEBP1 expression after DC vaccination may reflect the ineffective development of a productive, adaptive immune response and instead may be associated with myeloid cells that can negatively regulate immune responses in a state of aberrant/chronic inflammation. Recently, several studies have reported the deleterious effect of myeloid-derived suppressor cells (MDSCs) in melanoma and in response to DC vaccination [PEBP1 expression levels and MDSC frequencies in the Nijmegen samples. However, this relationship could not be confirmed for samples of the other centers, possibly due to the non-optimal conditions induced by sample freezing and differences in the MDSC identification strategy .We here describe PEBP1 levels after DC vaccination, but not before, are associated with a poor survival. Interestingly, we found that in patients who responded poorly, PEBP1 levels decreased even further, whereas patients who did well demonstrated a rise in PEBP1 levels upon vaccination. Based on the molecular context in which we find PEBP1 to be expressed, we postulate that a decrease in PEBP1 may be indicative of a skewing of the (DC-vaccine-triggered) immune response towards chronic inflammation/myeloid immune suppression rather than towards an effective anti-tumor response. Such a scenario would be in line with both the requirement for a robust adaptive response to combat the tumor, and the reported high prevalence of inflammation and/or tumor-induced MDSCs in patients with a poor prognosis [PEBP1 levels were negatively correlated with monocytic MDSC numbers. These experiments that aimed to determine the relationship between PEBP1 and MDSC are however by no means conclusive, and were hampered by great variability in MDSCs numbers between centers. The use of frozen material and the non-standardized MDSC identification methods used limited these analyses [We tested and validated the association of PEBP1 with survival on independent samples from different treatment centers. Our data indicate that low rognosis , 38, 39.analyses . Going fPEBP1 is directly driving the skewing of T cell-mediated immunity towards robust anti-tumor responses, or is merely a bystander gene reflecting the state of the immune system. PEBP1, also known as RKIP, is a mostly-cytoplasmic protein that is widely expressed and has diverse functions affecting several important signaling cascades including NFκB, MAPK/ERK and GSK3β signaling, to regulate cell proliferation, migration, and activation  (by PPI). Possibly the discriminative power of PEBP1 demonstrated here may relate to the signaling pathways associated with these molecules. PEBP1 expression has been reported to dampen the inflammatory response, acting on both ERK/MAPK and NFκB signaling to reduce pro-inflammatory cytokine release [PEBP1 expression has also recently been shown to attenuate STAT3 signaling, which in turn has been shown to be a key factor for the suppressive effect of MDSCs [PEBP1 acts on these signaling pathways to prevent/dampen excessive systemic inflammation that may hamper an effective adaptive immune response following DC vaccination. This hypothesis is supported by our finding that PEBP1 levels are also diminished in patient suffering from other diseases associated with chronic inflammation.It remains to be determined whether iewed by , 41). Inreported , 32. Alt release , 42, 43.of MDSCs –46. MDSCof MDSCs , 47, 48.of MDSCs , 50. ConPEBP1 may be more sophisticated. In CD8+ T cells PEBP1 expression is associated with enhanced IFN-γ signaling downstream of the TCR [PEBP1 may thus have dual functions in inhibiting deleterious systemic inflammation that drives immunosuppression, whilst at the same time promoting adaptive responses. In line with this view, we find in our gene expression data an inverse correlation of PEBP1 with genes connected to innate and inflammatory responses, while genes associated with T cell responses positively correlate with PEBP1 expression. Furthermore, we observed that PEBP1 is also highly correlated with the expression of ribosomal subunits suggesting PEBP1 expression to be associated with high protein biosynthesis capacity. This may facilitate an effective adaptive immune response which is highly dependent on the production of large amounts of inflammatory mediators  and immune cell expansion. Further research on the expression and function of PEBP1 in different immune cells is required to shed further light on how exactly PEBP1 relates to the skewing of immune responses following DC vaccination.Besides regulating inflammation in general, the action of  the TCR . PEBP1 mPEBP1 expression may all represent contraindications for further DC vaccination in its current form. More studies are required to obtain detailed mechanistic insight into the interaction of these factors. Recent literature suggests that lifting this state of aberrant inflammation and concordant suppression of immune responses in cancer patients, by e.g. interfering with STAT signaling or by certain regiments of chemotherapy could increase the effectiveness of various types of immunotherapy [PEBP1 could represent a facile and effective blood-based biomarker to monitor whether the fitness of the immune system is adequate for successful immunotherapy.Our study once more indicates that the presence of excessive/chronic inflammation and /or a lack of “fitness” of the immune system may be a severe obstacle to the success of DC vaccination. Suppressive and exhausted immune cells may hamper the effectiveness of treatment modalities that are aimed at inducing or boosting adaptive immune responses. Together, the data presented here and from other studies, suggest that the presence of chronic inflammation, MDSCs and now also low otherapy , 52, 53.www.clinicaltrials.gov NCT00053391 and [Clinical data and biological samples from melanoma patients undergoing DCs vaccination were obtained from the University of Erlangen, Erlangen, Germany , fro3391 and , 60) andex vivo from autologous monocytes by culture with GM-CSF and IL-4 and subsequently matured with a cytokine cocktail consisting of IL-1 beta, IL-6, TNF-alpha and PGE2 ;in vitro transcribed to synthesize biotin-labeled complementary RNA (cRNA) using the Illumina TotalPrep-96 RNA Amplification Kit . A total of 750ng of biotin-labeled cRNA was then hybridized to Illumina HumanHT-12 V3.0 expression BeadChips at 55C for 18 h. The hybridized BeadChip was washed and stained with streptavidin-Cy3 according to the manufacturers protocols using Illumina whole-genome gene expression direct hybridization assay . GenomeStudio Data Analysis Software used to visualize and analyse images generated. This software provides data in standard file formats that can be readily processed with most commercial and/or public accessible gene expression analysis software programs to identify significantly differentially expressed genes with pathways and networks involved. Raw data from BeadStudio were exported as text files and processed using the Bioconductor R package Lumi [RNA was extracted from biobanked PBMCs and moDCs using the Ambion RNA mirvana™ extraction kit. RNA quality and abundance were determined using an Agilent 2100 Bioanalyzer and Nanodrop ND-1000 spectrophotometer, respectively. 500ng of total RNAs were reverse transcribed to synthesize first- and second- strand complementary DNA (cDNA), purified and age Lumi . Qualityth percentile FDR = 0 to exclude the presence of false positive survival associated probes. The resulting probes were then associated with a fold change by dividing their mean expression values in long survivors by their mean expression value in short survivors. A fold-change cut-off of 2 was then applied to restrict the number of probes.The significance analysis of microarrays (SAM) as implemented in the Bioconductor R package Rsam  includesGene expression was also investigated through the multivariate methods implemented in the R package mixOmics . In partin-vitro matured DCs, we performed a paired PBMC vs matured DC two-class analysis. Only significant probes were analyzed in the second pass, consisting in an unpaired two-class (long vs short survivors) SAM. Also, in this case, we selected probes at 90th percentile FDR = 0 to statistically exclude the detection of false positive results. A 2 fold change cut-off was used for further selection.For this analysis two consecutive two-class SAMs were drawn: in the first pass, we searched for a “vaccination signature” by testing for differential expression in PBMCs obtained from donors after vaccination and the mature DCs included in the vaccine. Since each patient received autologous Established housekeeping genes for qPCR normalization of PBMCs samples are not consistently reported in literature. Genes that are routinely used as reference genes such as GAPDH do not perform well on whole blood or PBMC samples , 66. We PBMCs were obtained from each of the Center's Biobanks. RNA was isolated using the RNeasy mini kit  or Trizol  following manufacturers protocol. RNA quantity was determined on NanoDrop 2000c (Thermo Scientific) and RNA quality was determined via agarose gel electrophoresis. 2 ug of RNA was DNAse I treated to remove residual genomic DNA and reverse transcribed into cDNA by M-MLV reverse transcriptase (Life Technologies) to obtain 25ul of cDNA. cDNA was diluted 25x in nuclease free water. For each reaction, 4ul diluted cDNA, 300nM primers, 10ul SYBR Green (Roche) and water were added to a final volume of 20ul. Each sample was amplified using a CFX96 sequence detection system (BioRad). The following qPCR cycling conditions were used: 50°C/2min, 95°C/10min, 40cycles of 95°C/15s; 60°C/1min, melt analysis 60°C - 95°C with increment 0.5°C/5s. The gene-specific oligonucleotide primers used to determine the expression of the genes of interest are listed in http://sysimm.ifrec.osaka-u.ac.jp/immuno-navigator/) was used as input. For GSEA standard operating setting was applied taking into account only the highest ranking gene sets with a minimum 10 genes.Using the full microarray dataset, the Pearson correlation and p-value of all probes in relation to PEBP1 expression on the array was determined in the R programming environment. Genes were ranked based on their log-transformed Pearson correlation coefficient, and this ranked list was used as input for Gene Set Enrichment Analysis (GSEA) against the blood transcriptional modules described by LI at al. –37. Altehttp://string-db.org) to find any direct  or indirect  associations [3D for visualization (http://www.biolayout.org/) [To build PPI networks the top 500 of genes, correlating or anti-correlating with PEBP1 expression were loaded into STRING database (ciations . Using tut.org/) .