Yarrowia lipolytica recombinant strains able to grow on raw starch of different plant origin. In the present study, we used the most efficient amylolytic strain as a microbial cell factory of raw-starch-digesting (RSD) amylolytic preparation composed of two enzymes. The RSD-preparation was produced in fed-batch bioreactor cultures. Concentrated and partly purified preparation was then tested in simultaneous saccharification and fermentation (SSF) processes with thermotolerant Kluyveromyces marxianus for ethanol production and Lactobacillus plantarum for production of lactic acid. These processes were conducted as a proof-of-concept that application of the novel RSD-preparation supports sufficient starch hydrolysis enabling microbial growth and production of targeted molecules, as the selected strains were confirmed to lack amylolytic activity. Doses of the preparation and thermal conditions were individually adjusted for the two processes. Additionally, ethanol production was tested under different aeration strategies; and lactic acid production process was tested in thermally pre-treated substrate, as well. Conducted studies demonstrated that the novel RSD-preparation provides satisfactory starch hydrolyzing activity for ethanol and lactic acid production from starch by non-amylolytic microorganisms.Sustainable economy drives increasing demand for raw biomass-decomposing enzymes. Microbial expression platforms exploited as cellular factories of such biocatalysts meet requirements of large-volume production. Previously, we developed  Starch is one of the most abundant carbohydrates, second only to cellulose, found in higher plant biomass. This homopolysaccharide is composed solely of glucose, and as such can be completely hydrolyzed to fermentable sugars without toxic by-products formation. Owing to its abundance, the desirable composition and high carbon content, starch is commonly used as a feedstock in biotechnological production of valued biomolecules, such as ethanol or lactic acid ,2,3. ValTypical starch-based bioprocesses consist of two main steps: (i) starch liquefaction and saccharification, followed by (ii) conversion of glucose released from biopolymer to a target product. At first, the starch-containing feedstock is gelatinized by thermal treatment and then liquefied by thermostable α-amylase at 105 °C, and after cooling down to 60 °C, liquefied starch is saccharified to fermentable sugars by glucoamylase ,8. It haThe application of raw starch degrading enzymes (RSDE) able to hydrolyze granular non-pre-treated starch below the liquefaction temperature can bring significant reduction in the energy consumption compared with the conventional processes ,11. As eAspergillus sp., Penicillium sp. and Rhizopus sp. [Typical RSDE preparations are composed of α-amylase and glucoamylase working synergistically to rapidly and completely hydrolyze raw starch into the fermentable sugars. The amylases having RSD activity can be found in many organisms, including plants, animals and microorganisms, however only the latter can be reasonably exploited in RSDE production ,15. Curropus sp. . As the Komagataella phaffi (Pichia pastoris) is the most popular yeast workhorse in this regard, increasing evidence shows that Yarrowia lipolytica is a highly attractive alternative. Suitability of Y. lipolytica to be an industrial workhorse is repeatedly proved [Y. lipolytica outperforms P. pastoris in terms of heterologous secretory protein production [Y. lipolytica high performance in the heterologous secretory protein production.Apart from the native, fungal producers mentioned above, efforts are being pursued towards development of microbial cell factories being able to produce RSDE of desired characteristics ,18,19. Hy proved ,21,22. Roduction . Its abioduction , resistaoduction , and higoduction  account Y. lipolytica expression platform for heterologous production of amylolytic preparation composed of two enzymes produced simultaneously. To this end, we cloned and expressed RSD α-amylase from rice weevil (Sitophilus oryzae) (SoAMY) and glucoamylase from fungi Thermomyces lanuginosus (TlGAMY) in Y. lipolytica fed-batch bioreactor cultivations. A simple procedure was used to formulate ready-to-use amylolytic preparation, and its yield was analyzed. Finally, the obtained preparation was tested in two proof-of-concept SSF processes: (i) production of ethanol by thermotolerant yeast Kluyveromyces marxianus and (ii) synthesis of lactic acid by Lactobacillus plantarum. Both these strains lack native ability to hydrolyze starch, which was used as the main carbon source; so, the processes completely relied on action of the amylolytic preparation. The target molecules and proof-of-concept processes were selected based on high market demand for the two compounds. Bioethanol is commonly used as an alternative fuel and is thus in high continuous demand. Lactic acid is mainly applied in cosmetics industry and as a platform for the production of green solvents, fuel precursors and biodegradable plastic PLA (polylactic acid) [Therefore, in this study we exploited ic acid) ,27.Y. lipolytica Po1h strain , according to previously described methodology [TlGAMY gene in the first transcription unit and SoAMY gene in the second transcription unit. Then, both genes were transcriptionally fused to a signal peptide of YALI0E22374g [The genes encoding heterologous alpha-amylase and glucoamylase were cloned in hodology . Both gehodology  with TlGKluyveromyces marxianus DSMZ 5422 was purchased from DSMZ (German Collection of Microorganisms and Cell Cultures). The strain was used in a proof-of-concept process of ethanol production from raw starch. The strain was confirmed to be unable to decompose starch through iodine drop test : rich broth, 15 , agar, 20, starch, 10), as described previously [eviously . The strLactobacillus plantarum strain is an own isolate, deposited in the Collection of Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, Poland. The strain was used in a proof-of-concept process of lactic acid production from starch. The strain was confirmed to be unable to decompose starch through iodine drop test, as described above for K. marxianus. The strain was maintained as glycerol stock in 15% glycerol at −80 °C. Prior to each culture run, the strain was streaked on a MRS agar plate ((g/L): ready mix, 62; Oxoid) and incubated at 30 °C for 48 h.Y. lipolytica GGY215 was transferred from fresh YPD agar plate into 200 mL of YPG20 medium ((g/L): YE, 10, Pepton, 20, glycerol (POCh), 20) and cultured over 22 h at 30 °C with shaking 250 rpm . Such a pre-culture was used for inoculation of 1.8 L YPG100 production medium ((g/L): YE, 10, Pepton, 20, glycerol, 100) in Biostat B plus bioreactor , with a total volume of 5 L and a working volume of 2 L (after inoculation)–2.5 L (after feeding). The bioreactor was equipped with two Rushton turbines attached to a stirrer shaft, and the pH, temperature and dissolved oxygen probes. Temperature, pH, vvm and pO2 were maintained through the culturing time at 28 °C, 5.5, 2, 20%, respectively. pO2 was stabilized by activating cascade pO2-stirring and manual control of air flow. When the initial portion of glycerol was consumed (at ~ 24 h), 0.5 L of 5-fold concentrated YPG100 medium was fed into the culture. The cultures were continued for 72 h. Samples were collected in time intervals, centrifuged for 5 min at 12,045× g , and biomass and supernatant were stored separately at −20 °C, until analyzed. Samples were analyzed for concentration of dry biomass (gravimetric method), substrate and metabolites (HPLC) and amylolytic activity in the supernatant (microSIT). The production cultures were run in 4 independent batches.The production strain g in +4 °C . The biomass was discarded, and the supernatant was subjected to protein precipitation with ammonium sulfate. Ammonium sulfate (POCh) was added in portions to reach the final saturation of 80%. The solution was slowly mixed overnight at +10 °C. Then, the solution was centrifuged for 2 h at 5063× g in +4 °C . Samples of the supernatant were stored at −20 °C for analysis. The deposit was resuspended in sterile 0.1 M acetate buffer pH 5.5 and filtered through 0.45 µm low protein retention filter . Subsequent batches of such SoAMY-TlGAMY preparation were standardized based on determination of amylolytic activity through microSIT assay. Such crude preparation was used in the proof-of-concept processes.The total post-culturing suspension (approx. 2.5 L) was centrifuged for 1 h at 5063× To analyze the process of SoAMY-TlGAMY preparation formulation in terms of the enzymes yield and losses, the heterologous enzymes were purified from a control culture to apparent homogeneity, using affinity chromatography, according to a procedure described previously . BrieflyK. marxianus DSMZ 5422 was transferred from a fresh YPD agar plate into 25 mL of premedium ((g/L): YE, 3, malt extract , 3, Peptone, 5, glucose, 10) and cultured over 22 h at 30 °C with shaking 250 rpm. In total, 40 mL of production medium in 100 mL SIMAX bottles ((g/L): native raw starch, 40, YE, 3, ME, 3, KH2PO4 (POCH), 1, CaCl2 (POCH), 0.26, chloramphenicol, 35 µg; pH 5.5) were inoculated at 10% with the preculture. Native rice starch was added to sterilized medium, directly before inoculation, to avoid starch gelatinization. The crude preparation was added into the medium in two dose variants: at 20 and 25 AU per gram of starch, at the moment of inoculation. The SSF process was conducted in a rotary shaker-incubator with shaking at 250 rpm and temperature of 32, 36 and 40 °C for 54 h. Control runs with native starch and the enzymatic preparation, but without the strain, were conducted under corresponding conditions (temperature and doses) in biological triplicate. Samples were collected in time intervals and stored at −20 °C. Ethanol and residual glucose concentrations was monitored throughout the experiment through the HPLC technique. The cultures were carried out in three biological replicates.The SSF cultures were conducted in Minifors 2 bioreactors  with a total volume of 2.6 L and a working volume of 0.5 L. The bioreactor was equipped with two Rushton turbines attached to a stirrer shaft, pH, temperature and dissolved oxygen probes. Temperature, pH and stirring were maintained through the culturing time at 36 °C, 5.5 and 200 rpm respectively. Three different aeration strategies were tested; IS—air provided through a sparger immersed in the culture, air flow of 0.04 vvm throughout the culture, HS–air provided from a headspace, air flow of 0.04 vvm throughout the culture, IS21—air provided through a sparger immersed in the culture, air flow of 0.5 vvm only for the first 21 h of culturing. Raw starch was added to the medium after bioreactor sterilization to avoid gelatinization. The crude amylolytic preparation was added at the amount of 20 AU per gram of starch at the moment of inoculation (at 5% with preculture developed as indicated in 2.5.1).To assess efficiency of SoAMY-TlGAMY crude preparations, three control cultures were conducted: (i) with glucose instead of starch as a substrate, (ii) with application of commercial RSDE preparation STARGEN 002 at the same dose of 20 AU per gram of starch, (iii) SHF (separate hydrolysis and fermentation) process: starch hydrolysis for 6 h at 40 °C preceding the inoculation. The control processes were conducted under HS aeration strategy. All the processes were carried out for 72 h. Samples were collected in time intervals and stored at −20 °C. Ethanol and residual glucose concentration was monitored throughout the experiment through the HPLC technique. Microbial growth was assessed through determination of viable counts.Lb. plantarum precultures were developed from glycerol stocks by twice subculturing the strain and increasing volume of MRS liquid medium , 52 (g/L)) for 18–20 h at 30 °C without shaking. The flask production cultures were conducted in 40 mL of the production medium in 100 mL SIMAX bottles ((g/L): native raw starch, 20, YE, 10, ammonium citrate , 2, K2HPO4 (POCH), 2, sodium acetate , 5, MgSO4 × 7 H2O, 0.2, MnSO4 × 4 H2O , 0.05). The starch was added either prior to sterilization (cooked starch variants) or prior to inoculation (raw starch variants). The media with cooked starch were mixed directly after sterilization on a magnetic stirrer to avoid solidification of medium for at least 1 h. The production cultures were inoculated with 2% of the preculture. The SoAMY-TlGAMY crude preparation (60 AU per gram of starch) was added into the medium at the moment of inoculation. The SSF processes were carried out for 72 h at 30 °C without shaking. Control runs with raw starch and the enzymatic preparation, but without the strain were conducted under corresponding conditions (temperature and dose) in biological triplicate. Samples were collected in time intervals and stored at −20 °C. Lactic acid and residual glucose concentration was monitored throughout the experiment through the HPLC technique. The cultures were carried out in three biological replicates.The SSF cultures were conducted in Minifors 2 bioreactors with a total volume of 2.6 L and a working volume of 0.5 L. The bioreactor was equipped with two Rushton turbines attached to a stirrer shaft, pH and temperature probes. Temperature, pH and stirring were maintained through the culturing time at 30 °C, 6.5 and 50 rpm respectively. pH level was established, based on previous literature reports ,36. The Amylolytic activity of the SoAMY-TlGAMY preparations was analyzed using microSIT procedure, described earlier . Rice stConcentration of total proteins contained in the samples was measured using BCA protein assay kit , in a miniaturized form. Standard curve was prepared with a standard BSA solution, ranging from 400 to 1000 µg per mL. All the analyzed samples were first subjected to buffer exchange (20 mM phosphate buffer), to remove interference with incompatible compounds, and diluted to fit within the standard curve range. The reactions were conducted according to the manufacturer’s specifications in technical triplicate.g), 5x diluted in distilled water and passed through a 0.45 µm filter . An Agilent Technologies 1200 series chromatograph, equipped with Rezex ROA 300 × 7.80 mm (Phenomenex), autosampler (G1329B), double pump (G1312B), refractic index detector (G1362A) and diode array detector (G1315C), was used for the analyses. Also, 0.005N H2SO4 was used as the eluent at 0.6 mL/min, under isocratic conditions. The analysis was conducted at 40 °C. In total, 10 µL of the samples were loaded onto the column. Quantitative and qualitative identification of the compounds was carried out using external standards and the peak height .Concentration of glycerol, erythritol, mannitol, citric acid, alpha-ketoglutarate, ethanol and lactic acid was determined using the following HPLC methodology. Samples withdrawn from the cultures were centrifuged  and passed through a 0.45 µm filter . Agilent Technologies 1200 series chromatograph, equipped with Rezex RSO-Oligosaccharide Ag + 200 × 10 mm (Phenomenex) column, autosampler (G1329B), double pump (G1312B), and refractic index detector (G1362A) was used for the analyses. H2O was used as the eluent at 0.3 mL/min, under isocratic conditions. The analysis was conducted at 80 °C. Then, 10 µL of the samples was loaded onto the column. Quantitative and qualitative identification of the compounds was carried out using external standards and the peak height .Concentration of saccharides of polymerization degree from dp1 to dp7 was determined using the following HPLC methodology. Samples withdrawn from the cultures were centrifuged .Where indicated, the biomass concentration in the culture samples was determined using spectrophotometric measurements of absorbance at 600 nm wavelength. The biomass samples were prepared as described in point K. marxianus or Lb. plantarum, respectively), and the plates were incubated at 30 °C or 37 °C for 24 or 48 h. Viable counts were expressed as cfu/mL of the culture sample.Where indicated, microbial growth was analyzed through determination of viable counts per culture volume unit. This technique was used for starch-containing samples, where neither dry cellular weight assessment nor spectrophotometric measurement were useful due to the substrate properties. Upon sampling, culture samples were immediately subjected to decimal dilutions in sterile saline solution. Samples were then surface plated on an agar-solidified medium . Graphical presentation of the obtained data was done using Microsoft Excel 2013 software. Due to difficulties in determination of residual raw starch concentration in the samples from K. marxianus and Lb. plantarum cultivations, yield of ethanol and lactic acid were expressed as gram of the target molecule per gram of the substrate provided at the beginning of the culture, according to methodology and equation 3.69.a.b, page 102 provided in [Obtained results were expressed as mean ± standard deviation (±SD) of the replicates described above and indicated in figure captions. Statistical importance of the differences between compared sets of data was analyzed using one-way analysis of variance (ANOVA) and Tukey’s multiple comparison tests . The levels of significance were set at vided in .Y. lipolytica GGY215 recombinant strain. The strain expresses the two heterologous genes under control of a strong promoter p4UASpTEF. Under the adopted experimental conditions, accumulation of SoAMY-TlGAMY-derived amylolytic activity in the culture supernatant reached 0.327 ± 0.025 AU/mL (Y. lipolytica cultures under adopted conditions. Erythritol (ERY) and citric acid (CA) were the two dominant metabolites, reaching peak concentration of 31.68 ± 7.51 and 32.46 ± 1.78 g/L, respectively. The following decrease in ERY concentration is typical under carbon source limitation for Y. lipolytica cultures. Production of the SoAMY-TlGAMY enzymatic preparation was conducted in fed-batch bioreactor cultures of the 25 AU/mL . ProductTo formulate the crude SoAMY-TlGAMY preparation, the proteins contained in the post-culturing medium were subjected to the ammonium sulfate precipitation. To assess efficiency of such approach and to evaluate the degree of potential losses in SoAMY-TlGAMY, the consecutive protein fractions were analyzed for total protein concentration (microBCA), the protein profile through SDS-PAGE, and for total amylolytic activity , Table 1According to data presented in K. marxianus strain. Initial SSF cultures were conducted in flasks at 32, 36 and 40 °C, with the preparation doses of 20 and 25 AU per gram of starch, to select optimal conditions. Additionally, the profile and time-course of (oligo)saccharides released from raw starch under these conditions was analyzed, within the range dp1-dp7 (The SoAMY-TlGAMY preparation was tested in a proof-of-concept process of bioethanol production from raw starch by  dp1-dp7 A. Actionp < 0.001). The difference in monosugar release between 36 °C and 40 °C was not statistically significant (at p < 0.05). Based on the data presented in K. marxianus, as no residual glucose was detected. Decrease in ethanol concentration in terminal time-points depicted in K. marxianus as the ethanol producer, as it limits the microbe growth.As demonstrated in K. marxianus were focused on adjustment of the aeration strategy. To this end, the air was provided either through (i) a sparger immersed in the culture medium (IS), (ii) through its provision into head-space of the culture (HS), or (iii) through an immersed sparger, but only for the first 21 h of culturing . Conducted statistical analysis confirmed that aeration strategy had a significant impact on ethanol production (p < 0.001). The ethanol yield  ranged from 0.144 to 0.192 g/g (for IS and HS respectively), depending on aeration strategy (p < 0.001). Only at the terminal time-points, the IS21 process turned out to be more efficient . On the other hand, IS process was less efficient (p < 0.001) in terms of ethanol production than HS and IS21 in earlier (48 h) and terminal (72 h) time-points, reaching maximally 7.35 ± 0.07 g/L. The most rapid growth of the cells was observed for IS21 cultures, equal to 107 cfu/mL at 24 h, which was reached in IS processes at 48 h. The viable counts decreased in the following time-points, together with increasing ethanol concentration and limited oxygen provision in all the culturing variants.The initial bioreactor processes of ethanol production from raw starch in the SSF with the SoAMY-TlGAMY and the strategy . As showK. marxianus, raw rice starch, SoAMY-TlGAMY; individually exchanged where indicated), but differing in: (i) the substrate—glucose instead of starch (“control G”), (ii) the type of preparation—commercial RSDE instead of SoAMY-TlGAMY (“STARGEN”), (iii) the mode of operation—SHF instead of SSF (SHF) (p < 0.001). When glucose was used as the sole carbon source, the peak ethanol concentration reached 13.59 ± 0.27 vs. 7.68 ± 0.05 for control G and SSF SoAMY-TlGAMY variants, respectively, at 22 h, but the final ethanol concentration at 72 h was not statistically different between these two variants (at p < 0.05). The process with the SoAMY-TlGAMY preparation was importantly less efficient in terms of ethanol production than the process conducted with commercial enzymatic preparation STARGEN (p < 0.001). The use of commercial RSDE brought the highest ethanol concentration of all tested variants (21.22 ± 1.95 g/L). Concomitantly, the initial glucose concentration released from starch by STARGEN preparation was 7-fold higher than by SoAMY-TlGAMY (35.07 ± 1.47 vs. 5.28 ± 0.28 g/L); and in this regard, was similar to control G variant; which was also reflected by ethanol yields reaching 0.515, 0.340 and 0.173 g/g ; but the final ethanol titer was not significantly higher in this culturing variant .To assess competitiveness of a process with the SoAMY-TlGAMY preparation, comparative cultures were conducted using the same major operation conditions  B. ImportLb. plantarum own isolate strain. Prior to the SSF processes, the optimal temperature for Lb. plantarum growth and lactic acid production was determined. The results presented in p < 0.05), with its top at 30 °C. Correspondingly, the most rapid lactic acid production was observed at 30°C reaching 13.31 g/L in 24 h . Within this temperature range, residual glucose level was comparable, without significant differences (at p < 0.05).The second exemplary process implementing the SoAMY-TlGAMY preparation was oriented on lactic acid production by Lb. plantarum, three doses of the preparation were tested  at 30 °C. As presented in (p < 0.001), reaching 5.15 ± 0.089 g/L at 24 h of hydrolysis for 60 AU per gram of starch.To determine the amount of assimilable sugar released by the SoAMY-TlGAMY crude preparation from the native rice starch under thermal conditions facilitating growth and lactic acid production by ented in A, at thiK. marxianus). Therefore, apart from the typical SSF conducted without thermal pre-treatment of starch, a completely sterilized medium was included in these experiments (“cooked starch” variants). Comparison of the SSF processes conducted with raw starch and cooked starch with Lb. plantarum and SoAMY-TlGAMY preparation (60 AU per gram of starch) is presented in p < 0.01), which resulted from better accessibility of the cooked biopolymer for SoAMY-TlGAMY enzymes and higher provision of the assimilable carbon (peak 9.54 ± 0.65 vs. 1.43 ± 0.58 g/L at 4 h). Lactic acid production from cooked starch was the most rapid during approximately the first 32 h of culturing, reaching a value of 15.32 ± 0.79 g/L and high lactic acid yield 0.654 g/g at 24 h  and Lb. plantarum are shown in While the trials of lactic acid production by 9 cfu/mL) and ceased in the following time-points. Glucose consumption rate was higher than its release from starch, as its level was maintained close to zero throughout the process, while starch concentration decreased.The lactic acid production rapidly rose during the first 24 h of culturing up to 9.30 ± 0.27 g/L and yield of 0.465 g/g , reachinY. lipolytica [Y. lipolytica, synthetic fusions with different signal peptides (SP) were constructed [Y. lipolytica strains expressing the two heterologous genes encoding complementary amylolytic activities alpha-amylase SoAMY and glucoamylase TlGAMY from filamentous fungi [In our previous studies, after cloning and expression of an insect-derived gene encoding alpha-amylase in polytica ,32, we epolytica  and the polytica . To furtstructed ; the besus fungi . In thatIn the present study, we used the most efficient amylolytic strain from that previous set of the SoAMY-TlGAMY-producing strains, with the aim to use it as a microbial cell factory of RSD amylolytic preparation composed of two enzymes. The strain GGY215 (SP3-G1TG2S) was cultivated in fed-batch bioreactor cultivations in rich medium, to promote production of the enzymes. The cultivation protocol largely relied on our previous experience with single protein-producing strains ,38 in teK. marxianus DSMZ 5422 strain for the production of ethanol, and with the Lb. plantarum own isolate for the production of lactic acid. These processes were conducted as a proof-of-concept that application of the SoAMY-TlGAMY crude preparation supports sufficient starch hydrolysis enabling the microbial growth and production of the targeted molecules as the selected strains were confirmed to lack amylolytic activity. The crude preparation was also tested for the profile of oligosaccharides released from starch after treatment. Notably, saccharides from dp1 to dp7 were tested, however, only dp1 (glucose)  was present at detectable level in the post-reaction mixtures. Such an outcome was expected, as previously determined dp profile for SoAMY amylase solely was also dominated by glucose [The concentrated and partly purified preparation SoAMY-TlGAMY was then tested in the SSF processes carried out in the media with starch as the main carbon source with the thermotolerant  glucose . It cannY. lipolytica-produced TlGAMY [K. marxianus, the cultivation temperatures  were chosen based on literature data, suggesting a range between 30 and 40 °C [Lb. plantarum, the reported optimal temperatures are highly variable, starting from 30 °C [Lb. plantarum strain. As shown in Compromising thermal optima for enzymatic and microbial catalysts action is the key, initial challenge in developing a new SSF process. As previously shown, the SoAMY alpha-amylase contained in the preparation exhibits maximum activity at 40 °C . The glud TlGAMY  indicatend 40 °C , or 40 °nd 40 °C ,44,45. Hnd 40 °C  to 42 °Cnd 40 °C ,48. Thisom 30 °C , throughom 30 °C  up to 45om 30 °C . Here, oK. marxianus can grow at higher temperatures, close to the optimum for the enzymes (40 °C), lower doses were tested in this temperature range (20 and 25 AU per gram of starch). Due to growth limitation of Lb. plantarum at >32 °C, the cultivations had to be conducted at 30 °C, and thus higher doses of the preparation were tested in these temperatures . Expectedly, for the K. marxianus processes, higher doses of the enzymatic preparation gave higher concentration of produced ethanol by generating higher levels of fermentable sugars . Since in this process further manipulation with cultivation temperature, enabling lowering the preparation doses, was not possible, a higher dose of the preparation was chosen for Lb. plantarum cultivations on starch.Simultaneously with the investigation into the optimal growth temperature for the two strains to be used in the proof-of-concept processes, different doses of the crude SoAMY-TlGAMY preparation were tested. Starting doses were established based on previous reports on the SoAMY and TlGAMY ,41 compae sugars A, but thLb. plantarum SSF process development, recurring contaminations with undesired microbiota from raw-starch, and inability to provide antibiotics protection, made it reasonable to conduct trials with sterilized substrate. Improved accessibility of autoclaved starch to the amylase’s activity significantly improved efficiency of the process (15.58 ± 0.50 vs. 5.59 ± 0.10 g/L lactic acid) and prevented contaminations (routinely checked by microscopic observations). Notably, studies on SSF with Lactobacilli spp. are conducted on heat-pre-treated (autoclaved) soluble starch rather than raw substrate e.g. [Lb. plantarum in the presence of the SoAMY-TlGAMY was close to the results reported for Lb. plantarum grown on cassava starch  [Lactobacilli spp. exploitation in the SSF processes were conducted at higher substrate load, up to 150 g/L of starch [During the ate e.g. ,51,52,53f starch , which gK. marxianus and the SoAMY-TlGAMY preparation  could be conducted with raw, non-thermally pre-treated starch, and thus—fully exploit potential of the SoAMY-TlGAMY RSD preparation. Upon developing the proof-of-concept process we much focused on establishing appropriate aeration strategy. It is known that under aerobic conditions, ethanol synthesis is blocked, but also K. marxianus synthesizes large amounts of volatile compounds, namely ethyl acetate, resulting in losses of ethanol yield [K. marxianus under three aeration condition  and showed that maximum ethanol yield was obtained under anoxia, followed by hypoxia. In contrast, Kuloyo et al. [K. marxianus strain. The maximum ethanol concentration in the K. marxianus SSF cultures on raw starch reached 12.11 ± 0.26 g/L (SSF processes with the ol yield ,56. In tol yield  analyzedo et al.  showed t0.26 g/L A. When cK. marxianus SSF process in raw starch with the SoAMY-TlGAMY preparation was evaluated in comparison to three reference cultures (Finally, efficiency of the cultures B. When cY. lipolytica as a heterologous protein expression platform to produce the RSD-amylolytic preparation composed of two enzymatic activities: alpha-amylase SoAMY and glucoamylase TlGAMY. The partly purified preparation was then used in the two proof-of-concept processes operated according to the SSF strategy, exploiting microbial biocatalysts devoid of amylolytic activities in their native form. The thermotolerant K. marxianus was used for the production of ethanol, while the Lb. plantarum strain was used for the lactic acid formation. Depending on the used microorganisms, the two SSF processes were subjected to different limitations and bottlenecks, and thus the optimization studies were focused on different aspects. The principal limitation encountered with Lb. plantarum was inability to operate with raw, non-sterilized substrate, which caused recurring contaminations. Additionally, the thermal growth optima for the LAB strain was low in relation to the thermal optimum of the RSD-preparation activity. Altogether, these factors forced application of large quantities of the preparation and operation in thermally pre-treated starch, which was not desired. On the other hand, the thermotolerance of K. marxianus and possibility of implementing antibiotic protection, to eliminate undesired microbiota, allowed to fully exploit the RSD potential of the new preparation in the ethanol production process. After insight into oxygen control strategy, the process with the novel RSD-preparation was compared with the process conducted with commercial preparation, full availability of assimilable sugars and the SHF process. Comparisons with the SHF process and the control run conducted on glucose showed that the SSF with SoAMY-TlGAMY preparation on raw starch holds promise of efficient process, after further optimization. Nevertheless, as evidenced by a process conducted with the commercial preparation, further insightful studies are needed.In summary, this study falls into a general trend of exploiting microorganisms as cellular factories providing the market with desired biomolecules. Here we used


Animal Microbiome. We launch today with ‘Distinct microbiotas of anatomical gut regions display idiosyncratic seasonal variation in an avian folivore [I am proud to announce the official launch of folivore ’ and havMicrobiome was launched in 2013. This journal has gone from strength to strength with the current impact factor being 9.133. Indeed, for scientists focussed on microbiomes, this journal has become the go-to place to publish due to its reputation. This is excellent, but has led to a strain on capacity to deal with a high influx of submissions. The journal was also originally set up to focus on clinical and environmental microbiomes, but in recent years there has been a continual increase in the number of animal microbiome related publications. As a Senior Editor of Microbiome in the animal section, I can say that we are inundated with excellent submissions but often they may not have quite the impact required for Microbiome. So where do you submit to if your paper is rejected from Microbiome? There is, of course, an array of journal options, but there is a gap for an impactful journal focusing solely on animal microbiome studies, and this is the main impetus for launching the journal Animal Microbiome.So you may ask why do we need yet another journal with a focus on microbiomes? When I was approached regarding my thoughts on the need for an animal microbiome focussed journal, I asked myself the same question: is it really needed? The rapid rise in microbiome research .Being a sister journal to Animal Microbiome will address all aspects of non-human animal-associated microbiomes, including (but not limited to): domestic, wild and livestock animals. We will also welcome studies encompassing (but not limited to): marker gene surveys; ‘-omics’ surveys , bioinformatic and other analytical tools, which have an underlying strong hypothesis for using such techniques. The use of meta-omic approaches is strongly encouraged in order to substantially advance scientific understanding. Animal microbiome operates under the same principles and policies as Microbiome. Once accepted, all articles published in Animal Microbiome will be freely available through the journal web portal and within major research publication databases such as PubMed Central in due course. Also, because authors, not the publisher, will hold the copyright to their work, they will be free to distribute published articles. Open access science is crucial for ensuring the most impact from published research.Animal Microbiome, my strategy is for the journal to become a platform for understanding animal-associated microbiomes and allow scientific exchange of ideas to increase the impact of research in this field. We hope that you will support this journal in order to advance fundamental understanding of animal associated microbial communities globally.The number of submissions we have already had bodes well for the need for this journal and the future of the journal. As the Editor-in-Chief for


Previous studies examining the association between kidney stone disease (KSD) and arterial stiffness have been limited. Both age and gender have been found to have an impact on KSD, but their influence on the relationship between KSD and increased arterial stiffness is unclear. This study included 6694 subjects from October 2006 to August 2009. The diagnosis of kidney stone was based on the results of ultrasonographic examination. Increased arterial stiffness was defined as right-sided brachial-ankle pulse wave velocity (baPWV) ≥ 14 m/s. Associations between KSD and increased arterial stiffness were analyzed using multiple logistic regression models. KSD was positively related to increased arterial stiffness in both male and female groups  after adjusting for confounding factors. Subgroup analysis by age group (<50 and ≥50 years) showed a significant positive relationship only in the groups ≥ 50 years for both genders , but not in the groups < 50 years. In conclusion, KSD is associated with a higher risk of increased arterial stiffness in individuals aged ≥ 50 years, but not in those aged < 50 years for both genders. Increased arterial stiffness is a marker for increased risk of cardiovascular diseases arising from atherosclerosis, including coronary heart disease, heart failure, and stroke . The cliKidney stone disease (KSD) has traditionally been viewed as an isolated urinary system disorder. However, in recent decades, KSD has been found to be related to several systemic disorders, including obesity, metabolic syndrome, hypertension, diabetes, chronic kidney disease, and cardiovascular diseases ,10,11,12Currently, there have been only two studies on the association between KSD and arterial stiffness ,14. One n = 25) or heart disease (n = 152), either side of ankle-brachial index < 0.9 or > 1.3 (n = 177), abdominal sonography showing gouty nephropathy or uric acid deposition in the kidney (n = 43), and missing data (n = 467), a total of 6694 subjects were finally enrolled in this study for data analysis. This study was approved by the Ethical Committee for Human Research at the National Cheng Kung University Hospital in Taiwan (IRB number: B-ER-106-064). Data in this study were analyzed anonymously; thus, the informed consent of the subjects was not needed.This was a cross-sectional study for which the study population was drawn from subjects who had a health check-up at the Health Examination Center of the National Cheng Kung University Hospital from October 2006 to August 2009. They received different health check-up packages depending on their needs. Initially, we enrolled 7503 subjects aged ≥ 20 years who had undergone an abdominal sonography examination and a measurement of arterial stiffness. After excluding subjects with a history of stroke  was calculated as weight in kilograms divided by height in meters squared. Brachial systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured with an automatic blood pressure monitor, with the subjects in a supine position after resting for at least five minutes. Hypertension was diagnosed when subjects had a history of hypertension or SBP/DBP ≥ 140/90 mmHg. Blood samples were collected from all subjects after at least an 8 h overnight fast for measurement of fasting plasma glucose (FPG), glycated hemoglobin (HbA1c), total cholesterol, triglyceride, high-density lipoprotein cholesterol (HDL-C), creatinine, uric acid, calcium, and C-reactive protein (CRP) values. Except for subjects who were pregnant and those with a history of diabetes, the two-hour post-load glucose (2-h PG) level was checked after a 75-g oral glucose tolerance test. Diabetes mellitus was diagnosed when subjects had a history of diabetes, FPG ≥ 126 mg/dL, 2h-PG ≥ 200 mg/dL, or HbA1c ≥ 6.5%. The estimated glomerular filtration rate (eGFR) was calculated using the Modification of Diet in Renal Disease formula .All subjects received an abdominal ultrasound examination performed by experienced radiologists using a convex-type real-time electronic scanner  after at least an 8 h overnight fast. The diagnosis of kidney stone was based on ultrasonographic results, which showed echogenic foci with or without acoustic shadowing  in the renal pelvis or calices .We used the baPWV to evaluate arterial stiffness. The baPWV value was calculated as the pulse wave transmission distance divided by transmission time between the brachial and tibial arteries. It was measured automatically using a non-invasive vascular screening device  by wrapping pressure cuffs around each of the four extremities to measure the blood pressure and pulse waves of the brachial arteries of both arms and tibial arteries of both legs simultaneously after five minutes of bed rest. Increased arterial stiffness was defined as right-sided baPWV ≥ 14 m/s .t-test was used to compare continuous variables, and the Chi-square test was applied to compare categorical variables between subjects with and without increased arterial stiffness. The prevalence of increased arterial stiffness and KSD by age group in total population, males and females were compared using the Mantel–Haenszel Chi-square test for trend. In the multivariate analyses, the adjusted odds ratio (OR) and 95% confidence interval (CI) were calculated with binary logistic regression models to determine the relationship of KSD and other clinical variables with increased arterial stiffness. A p value < 0.05 was regarded as statistically significant.The 17th version of SPSS software  was used to perform the statistical analyses. Continuous variables were expressed as mean ± standard deviation, and categorical variables were presented as a number (percentage). In the univariate analyses, the Student’s Among the 6694 subjects, 2280 (34.1%) had increased arterial stiffness. Compared with subjects with normal arterial stiffness, subjects with increased arterial stiffness were older and male predominant, and they also had higher BMI, SBP, DBP, HbA1c, FPG, 2-h PG, total cholesterol, triglyceride, creatinine, uric acid, and calcium levels, but had lower HDL-C and eGFR values . In addiThe prevalence of both increased arterial stiffness and KSD showed increasing trends with increasing age in total population. The similar trends were also observed in both male and female groups. The prevalence of KSD peaked in the age group of 60–69 years in males. Males also had significantly higher prevalence of increased arterial stiffness and KSD compared to females .After adjusting for age, gender, BMI, hypertension, diabetes, total cholesterol/HDL-C, triglyceride, eGFR, uric acid, calcium, highest quartile of CRP, smoking, alcohol consumption, and regular exercise, KSD was positively related to increased arterial stiffness  . The posn = 84) [This is the first study to investigate the impact of gender and age on the association between KSD and increased arterial stiffness. To the best of our knowledge, there have been only two studies examining the relationship between KSD and increased arterial stiffness, and gender and age were not examined as a factor in either study. These previous two studies demonstrated a positive relation between KSD and increased arterial stiffness ,14. Howen = 84) . Their sn = 84) . As for n = 84) , a baPWVn = 84) . In addin = 84) ,14. In oThe exact mechanism regarding the association between KSD and increased arterial stiffness is still unknown. First, although they share common risk factors such as aging, obesity, hypertension, diabetes, and dyslipidemia, the positive relationship between KSD and increased arterial stiffness remains the same after adjusting for the above risk factors. Second, chronic systemic inflammation may play a role in the common pathogenesis of KSD  and incrHowever, we found different results for the relationship between KSD and increased arterial stiffness in people aged ≥ 50 and <50 years. The aged artery is characterized by endothelial dysfunction, chronic inflammation, migration and proliferation of vascular smooth muscle cells, elastin fragmentation, extracellular matrix deposition, and matrix calcification/amyloidosis/glycation ,26. LeveUric acid was positively associated with increased arterial stiffness in the female group but not in the male group in this study. This result was similar to that of a Korean study, which found that elevated serum uric acid was independently associated with increased baPWV in healthy Korean women, but not in men . Serum uIt is well known that aging and hypertension are factors that are strongly related to increased arterial stiffness, whereas the correlation between increased arterial stiffness and other established cardiovascular risk factors such as diabetes mellitus, dyslipidemia, renal dysfunction, BMI, and smoking have been shown to be less strong or insignificant ,42,43. TThere are some limitations in this study. First, due to the cross-sectional design, we could not elucidate the causal relationship between KSD and arterial stiffness. Second, this study was based on a Taiwanese population; thus, caution should be taken while applying the results to other ethnic groups. Third, the composition of kidney stones is unknown; hence we could not further specify the association between different types of kidney stones and arterial stiffness. Fourth, bilateral or multiple stones were related to higher rates of metabolic abnormalities ,53, but In conclusion, this study demonstrated that KSD is associated with a higher risk of increased arterial stiffness in individuals aged ≥ 50 years, but not in those < 50 years for both genders. Risk of cardiovascular diseases may be evaluated in people aged ≥ 50 years with KSD. Further studies are warranted to investigate the underlying mechanism linking KSD and increased arterial stiffness by age group.


A novel method was conducted to synthesize conductive polyaniline (PANI) doped with dodecyl benzene sulfonic acid (DBSA) (PANDB) in xylene by using chemical oxidative polymerization at 25 °C. Meanwhile, the synthesis process was photographed. Results showed as the reaction time was increased, and the color of the product was gradually turned into dark green. The influence of different synthesis time on properties of synthesized PANDB was then examined by a Fourier transform infrared (FTIR) spectrometer, an ultraviolet-visible spectrophotometer (UV-vis), a four-point measurement method, and a Field-emittance scanning electron microscope (FE-SEM). The result indicated that the optimum reaction time was 24 h with conductivity at around 2.03 S/cm. FE-SEM images and the conductivity testing showed that the more needle-like shapes in resulted PANDB, the higher the conductivity. The synthesized PANDB solution was blended with UV curable coating firstly and then coated on polyethylene terephthalate (PET) sheet. The UV coating/PANDB conductive composite films displayed an impressive translucency along with an adequate flexibility at room temperature. The UV coating/PANDB conductive composite film on PET sheet was flexible, transparent, and with antistatic function. Inherently conductive polymers (ICPs) are a specific category of synthetic polymers with distinctive electro-optic properties . These p12H25). It was favorable to dissolve PANDB in common organic solvents. Thus, the PANDB-based materials have appropriate applications such as antistatic materials [However, if the reaction time was not long enough, the PANI synthesis process might produce toxic by-products (benzidine and trans-azobenzene) . Meanwhiaterials ,11,12,13aterials ,15,16, baterials , sensorsaterials , etc.Wang et al. prepared the conductive PANDB/PI microfiber membranes by polymerized PANDB on the surface of PI nanofiber membrane. The PANI/PI microfiber membranes had potential use in novel functional fiber and fabric applications, including antistatic and electromagnetic shielding fabric, electrical resistive heating fabric and intellectual fabric . Chu et However, the PANDB synthesized in aqueous solution or in emulsion polymerization could not be properly dissolved in organic solvents. Thus, in order to improve the solubility of PANDB in xylene, a novel polymerization method was developed to synthesize PANDB in xylene directly in this study. The influence of synthesis time on properties of synthesized PANDB was examined by a Fourier transform infrared (FTIR) spectrometer, an ultraviolet-visible spectrophotometer (UV-vis), a four-point measurement method, and a Field-emittance scanning electron microscope (FE-SEM). The synthesized PANDB was blended with UV curable coating firstly and then coated on polyethylene terephthalate (PET) sheet. The translucency and antistatic function of UV coating/PANDB conductive composite film were also examined.Aniline (AN) and ammonium persulfate (APS) were purchased from Merck . Benzoyl peroxide (BPO) and xylene were purchased from Shimakyu Chemical Co. . Dodecylbenzene sulfonic acid (DBSA) was purchased from Tokyo Kasei . UV curable coatings were provided by Eternal Materials Co., Ltd. .PANDB synthesized in xylene was illustrated as below. Firstly, 6.2 g of BPO (0.026 mol) was added into a beaker containing 100 mL of xylene under constant stirring until completely dissolved (solution A). Then, 3.0 g of AN (0.032 mol) and 8.4 g of DBSA (0.026 mol) were added in another beaker containing 100 mL of xylene under constant stirring until completely dissolved (solution B). After that, pouring the solution A into the solution B for chemical oxidative polymerization of AN at 25 °C. The reaction time was controlled at 6 h, 12 h, 24 h, 36 h, and 48 h, respectively. When the reaction time was reached, reverse osmosis (RO) water was poured into the reaction solution and then the excess DBSA was filtered out. The resulted product was dark green emeraldine salt (ES) type conductive PANI doped with DBSA (PANDB) and it could be well dispersed in xylene. In order to obtain dried PANDB for characterization, the product solution was dried in an oven at 80 °C.In order to compare the dispersibility and solubility of PANDB synthesized in xylene and in aqueous solution, we followed the previous experimental procedure to synthesize PANDB in aqueous solution . In thatThe synthesized PANDB (5 wt% and 10 wt%) with the optimized conductivity was added into a fixed proportion of UV curable coating. The UV coating/PANDB conductive composite solution was subjected to ultrasonic treatment for 10 min and then a spin coater  was used to coat UV curable coating, UV coating/PANDB conductive composite solution on a flexible and translucency PET sheet. At room temperature, 1 mL UV curable coating, UV coating/5 wt% PANDB, and UV coating/10 wt% PANDB conductive composite solutions were dropped on the surface of the PET sheet, respectively. Then, the rotation speed of the spin coater was set at two stages. The first stage was set at 500 rpm for 5 s. The second stage was set at 1200 rpm for 10 s. The coated UV coating and UV coating/PANDB conductive composite films were put into an oven for drying at 80 °C for 10 min in order to volatilize the solvent. After that, the dried UV coating and UV coating/PANDB conductive composite film were irradiated with a wavelength of 365 nm UV lamp for 30 min to complete the curing process of UV curable coating.−1, and the scanning rate was 64/s.The chemical structures of samples synthesized in reactions with differing durations were identified using a Fourier transform infrared (FTIR) spectrometer . The samples and dehydrated potassium bromide (KBr) (weight ratio of sample/KBr = 1/99) were grounded together into fine powders and then the homogeneous mixtures were pressed to form pellets for analysis. The wavenumber was ranged from 600–4000 cmThe samples were well dispersed in xylene at room temperature. Then, optical absorbance of the PANDB solution in the wavelength range of 250–900 nm was determined using an ultraviolet-visible spectrophotometer . The UV–vis spectrophotometer was also used to examine the degree of transparence of the UV coating/PANDB conductive composite film coated on the surface of PET sheet.5 psi for 2 min at room temperature. The electrical conductivity σ (S/cm) of the sample was examined by a four-point probe meter  at room temperature. Each synthesized PANDB was firstly compressed into a sample with a diameter of 10 mm. Then, 10 different locations on the sample surface were measured. The obtained results were averaged and reported. The surface resistance (Ω/sq.) of PET sheet, pure UV-coating film, and UV coating/PANDB conductive composite films were also examined and reported at room temperature by the same instrument.0.1 g PANDB was weighed and then pressed under 3.0 × 10The surface morphology of PANDB synthesized at different times was investigated by a Field-emittance scanning electron microscope (FE-SEM) at 10 kV with a JEOL  JSM-840A scanning microscope. All specimens were coated with a conductive layer of sputtered platinum.The process of synthesized conductive PANDB with the molar ratio of AN:BPO:DBSA equal to 1.0:0.8:0.8 was shown in 3)1/2) was significantly higher than that in xylene 1/2). As a result, the PANDB synthesized in aqueous solution may be arranged tightly among the molecules. Therefore, PANDB synthesized in aqueous solution cannot be uniformly nano-dispersed and dissolved in xylene.If PANDB synthesized in xylene and in aqueous solution were respectively dissolved in xylene, it can be found that most of the PANDB synthesized in xylene can be uniformly nano-dispersed and a small part of PANDB can be even dissolved in xylene  and benzenoid ring (N–B–N), respectively. The characteristic peak appearing at around 1300 cm−1 was attributed to C–N stretching vibration of the secondary amine in the PANDB backbone. The characteristic peak appearing at around 1030 cm−1 was the absorption peak of the stretching vibration of the sulfonic acid group (-SO3H), which was part of the DBSA molecular structure. Additionally, the characteristic peak appearing at 829 cm−1 was due to aromatic C–H bending vibration band due to the 1, 4-disubstituted benzene ring.−1, the ratio of quinoid (Q) to benzenoid (B) structure present in the PANDB, which indicated the oxidation level of synthesized PANDB, can be estimated [From the ratio of peak areas corresponding to the 1590 and 1490 cmstimated . Figure π–π* transition of the benzenoid rings on the PANDB backbone. The peaks appearing at around 450 and 800 nm were attributed to polaron–π* transition and excition transition of quinoid rings, respectively. The second band at 450 nm was attributed to the polaron–π* transition, or quinoid rings. The broad band corresponds to the π–polaron transition, or formation of the exciton. The bands at 450 and 730~800 nm both involved formation of the polaron and were greatly affected by the level of doping of the polymer. These peaks were shown to be characteristic of doping with DBSA, although these peaks can shift slightly according to the degree of doping, conjugation length and solvent used to synthesize PANDB [ze PANDB .Appropriate reaction time resulted in the increasing of needle-like particles in products and also increasing the inner contact surface area of products, which might be the reason for the higher conductivity. Therefore, the optimum reaction time was set as 24 h in this synthesis system. According to our previous research results, different synthesis conditions can result in different surface morphology of the product . This wa12, 1.1 × 1012, 1.7 × 1010, and 1.5 × 109 Ω/sq., respectively. Results implied the surface resistance could be decreased with increasing PANDB amount in UV coating. The surface resistances of PET sheet, pure UV coating film, UV coating/5 wt% PANDB and UV coating/10 wt% PANDB conductive composite films were around 7.0 × 10The conductive PANDB was synthesized directly in xylene by using a novel chemical oxidative polymerization with different reaction time at 25 °C. The results showed that most of the PANDB synthesized in xylene could be uniformly dispersed in xylene, and a small part of the PANDB could even be dissolved in xylene without forming any precipitation. On the contrary, for the PANDB synthesized in aqueous solution, it could not be uniformly dispersed in xylene, and part of PANDB could even be precipitated in xylene. The influences of synthesis time on properties of PANDB synthesized in xylene were also examined. The results illustrated that if the reaction time was too short, the chemical oxidation reaction was insufficient, and if the reaction time was too long, it resulted in an excessive oxidation and other side reactions. Both phenomena can affect the conductivity of the product. In this synthesis system, the optimum reaction time was found and then set as 24 h. Its conductivity at this optimized condition was around 2.03 S/cm. FE-SEM images showed that different synthesis conditions resulted in different surface morphology of PANDB. The more needle-like particles founded in the synthesized PANDB, the greater conductivity the product will have. The synthesized PANDB was blended with UV curable coating firstly and then coated on PET sheet. The UV coating/5 wt% PANDB and UV coating/10 wt% PANDB conductive composite films displayed an impressive translucency (around 92.0 and 89.1% at 550 nm) along with an adequate flexibility at room temperature. Furthermore, the functional UV coating/PANDB conductive composite film can be applied as antistatic products and biosensors.


Virtual Communities of Practice (vCoP) is a nascent approach to professional development for simulation educators :124-30). vCoPs overcome geographic barriers to accessing expertise and professional networks and may promote ‘democratisation’ of voices in the simulation community. However, the optimal process for creating, nurturing and joining vCoPs in healthcare simulation is not well understood.Simulcast (www.simulationpodcast.com)—utilising the conceptual framework of Wenger’s three dimensions of Communities of Practice. In exploring these dimensions—joint enterprise, mutual engagement and shared repertoire—we hope to contextualise vCoP within professional development approaches for simulation faculty and invite readers to engage with our existing community.We report on the establishment of our healthcare simulation hybrid podcast/blog— Thom context .Not every collection of individuals engaged in learning is necessarily a community of practice , and WenWhat it is about: its joint enterprise as understood and continually renegotiated by its members.How it functions: the relationships of mutual engagement that bind members together into a social entity.What capability it has produced: the shared repertoire of communal resources  that members have developed over time [ver time .Connecting simulation practitioners with resources, experts, academics and each other remain persistently challenging. VCoP has been promoted as potential lifelines of professional development within the simulation education community, in part based on the success of parallel communities in emergency medicine –8 and heemphasis on active apprenticeship and legitimate peripheral participation as a model for ‘becoming’ a central member of an established community [Wenger’s theories have an ommunity , while oommunity . Within Simulcast was established in July 2016, as a monthly podcast focused on professional development for simulation educators. Early podcast episodes focused on topics such as simulation debriefing, psychological safety and scenario design, with a format of host interviewing an academic or expert in the field. In parallel, we established a monthly journal club, with a format based on the Academic Life in Emergency Medicine (ALiEM) ‘MeDIC’ series [’ series —a writteSimulcast and the open access journal Advances in Simulation have collaborated to produce bimonthly podcasts discussing an article nominated by the journal Editor in Chief, including a simulcast producer, article author and additional discussant with expertise in the relevant area.Since 2018, Simulcast has also partnered with conferences relevant to the simulation community—Australasian Simulation Congress (ASC), International Meeting for Simulation in Healthcare (IMSH) and Society in Europe Applied to Simulation in Medicine (SESAM)—to ‘cover’ the events, similar to a mainstream media approach.Simulcast production are managed solely by the producers. The website is a WordPress (https://wordpress.com/) blog-based platform, hosted on a VPN by Bluehost (https://www.bluehost.com/). When guests and hosts are connecting remotely, the podcast audio is recorded using Zencastr (https://zencastr.com/), then edited using either GarageBand (https://www.apple.com/au/ios/garageband/) or Hindenburg (https://hindenburg.com/) and optimised using Auphonic (https://auphonic.com/). The podcasts are then published to the hosting platform Podbean (https://www.podbean.com/) and distributed to iTunes Podcasts and Spotify (https://www.spotify.com/au/) via RSS feed. There are many alternatives for website build, hosting, recording editing and distribution, and these mentioned are solely for demonstration and transparency of our process.The technical elements of Our resources and podcasts are free, and financial and other costs are borne by the producers.At the time of writing, the site hosts an archive of over 80 podcasts and more than 30 online journal club discussions, with over 80k podcast downloads and 100k site hits.Simulcast home page—‘to bridge the gap between the simulation researcher, working in the academic centres, and the clinician delivering education at the point-of-care.’ This has provided an anchoring set of values for decisions regarding production and style or ‘brand’, relationships and subject focus.Our mission is articulated on the The production and style have been shaped by the feedback from the community—suggesting guests, topics and technical enhancements and also reflecting on how changes have affected their contribution to the blog. Production quality is a priority but within the time and financial constraints of being impartial and free. Relationships have been considered in terms of shared mission, values and brand associations, as opposed to commercial partnerships. Profiling technology and industry advances without the encumbrance of financial relationships allows credible critique of new products and techniques.The original mission was born of desire for translation from academic to practical, a ‘bridge’, with a flow of ideas into the community for reflection and discussion. With active participation of readers and listeners, this bridge has become increasingly bidirectional—with space created for discussion between researchers and authors and health professional educators at the clinical coalface. As described by Wenger, a community of practice continually renegotiates its joint enterprise.The CoP starts with the Simulcast producers. Vic is an emergency physician, academic and educator whose simulation experience includes undergraduate education, translational simulation, high performance team training and faculty development in health professional education. Her interest in social media was encouraged by participation in the emergency medicine vCoP. Ben is a paediatric emergency physician and simulation consultant with experience delivering translational and educational simulation in regional Queensland and a degree in visual arts in animation. He had previously established an international simulation journal club distributed via email. Jesse is an intensive care nurse with experience in nursing, medical and corporate health professional education and translational and educational simulation in settings throughout hospitals, public spaces and primary healthcare facilities. Jesse also had prior interest and experience in blog and podcast production and research interest in vCoP in health education .don’t confuse traffic with audience, and audience with community. In aspiring to the description of a vCoP, we consider our engagement as a network—with academics, journals, book editors, faculty development providers, events, industry and ‘coalface’ simulation educators—rather than a repository of information to be disseminated. This has required strategies to cater for network members with a broad range of motivations, learning needs and social media proficiency.Although seeking broad connection and reach, our strategy has been Through interviews, book reviews, infographics and journal clubs featuring new publications, we have worked with academics to translate their knowledge to the broader simulation community. Fulfilling the promise of democratisation, our vCoP sees lead authors contribute to journal club to thank and reflect on the comments of those ‘legitimate peripheral participants’.Advances in Simulation has connected our social media reach with the journal’s academic credibility and reputation. We have drawn on our own professional networks for guest appearances, advice and inspiration and added to those networks during interviews and through developing content for Simulcast. Our existing collaborations are not exclusive, and we aspire to more.Collaboration with Simulcast to be more than a broadcast, we have intentional social media strategies to encourage bidirectional engagement and ‘deliberate group cultivation’ [Simulcast team also contribute to ‘outposts’—other blogs and podcasts in which we are guests, but also ambassadors for Simulcast, in a strategy inspired by ALiEM [In considering ivation’ . Applyinivation’ , the Simby ALiEM .Although rising in popularity, social media-based resources for health professional education require new (and old) processes to ensure quality and credibility , while rSimulcast originated as a podcast, as part of a global trend in popularity of this media format. There is broad appeal in the convenience of ‘short form’ media accessed on a phone, consumed in a drive or train ride, combined with the neurochemical triggers of powerful audio storytelling [ytelling . Podcastytelling  and espeytelling , 7. Our Simulcast Journal Club is the primary pathway for bidirectional connection between our community and hosts via social media, blog comments or real-life interactions. In many ways, the journal club demonstrates key features of legitimate peripheral participation, with simulation enthusiasts and researchers having the opportunity to contribute to discussion between prominent world experts and relative novices in a social, relaxed format. Participants have the option to titrate their interaction based on comfort level and their perception of ‘membership’, moving from anonymous readers to enthusiastic contributors at a pace that suits their learning needs. The journal club enhances appeal through combining the monthly podcast, frequent infographic summaries of literature, case vignettes that form a serial, satirical melodrama when read in sequence and downloadable Ebook collections. Over 3 years, there has been a global reach—with contributions from South Africa, China, the UK, New Zealand, Australia, Canada and the USA. There have been over 700 blog post comments on the website with over 650 of these being active discussant contributions in journal clubs.The the impact of Simulcast. Outcomes we expected have been consumption by individual podcast listeners, increased Altmetrics for journal articles featured on Simulcast and many new friends and professional colleagues. Less expected outcomes have included the use of Simulcast content in short courses and fellowship programs and integrated into academic curricula. Our online journal club has been embraced by IRL  journal clubs for local institutions/simulation programs.We have numeric analytics for downloads and website hits, but we have not performed any formal evaluation of scope has evolved—our ability to deliver focused, in depth content is in tension with the diversity of practitioners we aim to include in the Simulcast vCoP. We recognise a bias toward debriefing and educational aspects of simulation in our content and know that we cannot provide a comprehensive scope that is fully reflective of the diversity within healthcare simulation.Our quality—technical and educational—is a critical element in an era of overwhelming online possibilities. Our posts and podcast content are not peer reviewed, but we have appreciated some frank feedback from colleagues. Although technology has dramatically lowered the barriers to entry for podcasters—inexpensive hardware and intuitive software for recording and editing and a plethora of online advice [Podcast e advice —high quaEstablishing a safe container for online learning has proved challenging. Despite promoting reflection and shared vulnerability, simulation educators remain as reticent as other learners when it comes to intellectual candour [ candour . Delibersustainability is supported by a producer trio with different perspectives and skill sets, allowing workload sharing and diverse outputs. We note cautions from others who are active in promoting vCoPs in over-extending our capacity [Our capacity . Our hosSimulcast.We hope this review assists others considering establishing or joining a virtual community of practice and invite readers to participate as listeners, contributors or colleagues with Additional file 1. Simulcast Infographic. An infographic depicting Simulcast through the lens of Wenger’s pillars of communities of practice.


For the first set of phantoms, 10 measurements for each of the eight inclusions were performed by each observer, whereas the second set of phantoms was used to evaluate the influence of depth on the stiffness measurements. Variability of the stiffness measurements was evaluated in vivo on 10 healthy livers, with 10 measurements for each hepatic segment. Texture analyses were performed in B-mode, obtaining elastography images for every hepatic segment. Results: Stiffness measurements were influenced by depth, particularly when exceeding 7 cm. In vivo measurements demonstrated that measurements of segments I, VII, and VIII were less reliable, mainly due to their deeper locations. The protocols used were more flexible in terms of acquisition setup and probe placement than those currently used with Fibroscan®. For texture analysis on the B-mode images, 12 features showed low variability regardless of the evaluated hepatic segment. On elastogram, only two features showed low variability, but not in every segment. Conclusion: We demonstrated the robustness of two methodologies for the quantification of liver stiffness and heterogeneity. Further clinical studies should evaluate whether these techniques can assess tumor responses to treatment and, therefore, have the potential to be used as imaging biomarkers.Aims and objectives: In order to evaluate the responses of hepatic lesions to treatment in terms of tissue stiffness and heterogeneity, this work investigated the robustness of 2D shear-wave elastography (2D SWE) stiffness measurements and texture analyses in vitro and in vivo in terms of repeatability and variability. Methods and materials: A multioperator ( Numerous new anticancer molecules recently emerged, such as antiangiogenic therapies, targeted therapies, and immunotherapies. Each molecule has its own impact on tumoral tissue. In addition to a possible decrease in size, which is well described in the literature with cytotoxic therapies, numerous changes in tumor characteristics are possible, such as partial or total angiogenesis decrease, necrosis, or lymphocyte influx into the tumor. These different outcomes make evaluation of tumor responses difficult. The evaluation of treatment response was initially based on tumor size evaluation . These criteria, which are still used as clinical references, may not reflect accurate treatment responses since, in many situations, favorable responses to treatment were observed without decreases in size ,3,4. UndSeveral new criteria were proposed to assess tumor responses in situations where Response evaluation criteria in solid tumors (RECIST) is faulty, such as the Choi criteria developed for gastro-intestinal stromal tumor (GIST), in particular, based not only on size decrease but also on density changes of the lesion. In the same field, studies conducted on the use of dynamic contrast- enhanced ultrasound (DCE-US) in the evaluation of tumor responses validated a new response biomarker that is particularly useful in the evaluation of antiangiogenic treatments .Ultrasound-based elastography (UBE) methods are now widely available at low cost and have the advantage of no radiation. They are currently of great interest with a growing community, receiving attention in recent years for noninvasive assessment of tissue mechanical properties . DiffereAccording to the World Federation for Ultrasound in Medicine and Biology (WFSUMB)  recommenIn recent years, several teams investigated the possibility of characterizing liver lesions on the basis of their stiffness, therefore enabling them to distinguish benign tumors from cancers ,10 and tUsing ultrasound-based elastography and texture analysis to assess possible modifications of tumor stiffness with treatment is an interesting approach. However, this approach only makes sense if stiffness measurements are reliable over the whole liver and not restricted to the right lobe, as reported in the literature, and if texture analysis on B-mode and SWI images of the liver is reliable regarding finding potential imaging biomarkers. As for SWI exploration, these features must be reliable over the different hepatic segments. To our knowledge, there are no studies or recommendations regarding the use of ultrasound-based elastography or texture analysis to evaluate the therapeutic responses of liver lesions in oncology via exploration of all hepatic segments. Thus, the purpose of our work was to evaluate the robustness of UBE and texture analyses in this field through the assessment of reproducibility, repeatability, and variability in vitro on phantoms and in vivo on healthy livers. p-value threshold of 0.05 was considered statistically significant.Stiffness measurements based on high-frequency shear-wave elastography were performed using the ultrasonography machine Aplio i900 and the convex probe PVI-475BX, . The main acquisition parameters were as follows: Frame rate = 1; precision = 4; aplipure = 1; Dynamic Range = 70 dB; general frequency = 4 MHz. Stiffness measurements were directly reported and acquired images were saved in Digital Imaging and Communication in Medicine (DICOM) format for texture analysis processing. Stiffness measurements were characterized in terms of reproducibility with a coefficient of variation (CV) defined as CV = σ/μ, where σ is the standard deviation and μ is the mean of the corresponding parameters. All statistical analyses were performed using RStudio  software. A Two sets of phantoms were used to assess both interoperator variability on stiffness measurements and the impact of depth on these measurements. The first phantom, model 049 “Elasticity Phantom—Spherical target map”  provided a set of reference standards to perform stiffness measurements. As illustrated in The first phantom was used to assess interoperator variability based on the operators’ experience with ultrasound. Five operators had various experience with the use of classical ultrasound probe, whereas none of them had prior experience with stiffness measurements using UBE. Each operator performed 10 measurements per inclusion, starting with the biggest and the stiffest inclusion, for a total of 80 measurements. Measurements of inclusion size were performed using region of interest (ROI) located at the center of each inclusion. The influence of the size of the inclusions on the measured stiffness was evaluated prior to evaluation of interoperator variability. The robustness of UBE was first evaluated through the assessment of reproducibility, repeatability, and variability regarding the influence of the size of the inclusion on the measured stiffness, then according to inclusion type. The influence of the size of the inclusion was assessed by comparing the measured stiffness of the inclusions of 10 and 20 mm for each calibrated type. For every inclusion, the percent bias, i.e., the difference between measured and expected calibrated stiffness divided by the expected stiffness, was computed to evaluate the quality of the measured stiffness. Then, the influence of the operator on the measured stiffness was assessed by computing the coefficient of variation (CV), i.e., the ratio of the standard deviation on the mean of the measured stiffness, between operators. The second set of phantoms was used to evaluate the influence of depth on the measured stiffness value. For each of the four phantoms, 10 measurements were made at 3, 5, 7, and 9 cm depth. The phantom was then modified, adding a 2-cm gel pad, to perform 10 measurements at a depth called “9 cm modified” b. This eTen healthy volunteer livers were assessed by two radiologists who performed 10 measurements of stiffness per hepatic segment. These measurements allowed assessment of operator influence on the measurements and impact of the acquisition protocol on the results. The acquisition protocol was left to the free judgment of the operator and was carried out blindly regarding depth of the measurements, the approach , and ROI positioning. Measurements were performed with ROI ≥ 10 mm and ROI ≤ 20 mm. Depth was defined as the distance between the volunteer’s skin and the ROI’s center. The protocol also allowed volunteers to breathe normally. One parameter that was reported to influence stiffness measurements was the corpulence of the volunteers. The volunteers were then divided into three distinct body mass index (BMI) groups, i.e., Group 1: BMI < 22; Group 2: 22 ≤ BMI ≤ 25; Group 3: BMI > 25.The variability of texture features was assessed using images acquired from one healthy volunteer liver. Each image was comprised of a stiffness map and a B-mode image. For each hepatic segment, 10 images were collected for a total of 80 images. Measurements were performed on ROIs of 20 mm, which were placed manually at the same anatomical location on B-mode images and on stiffness maps, as illustrated in Measurements were performed by five operators using the first phantom to assess the influence of the size of the inclusions, the influence of the calibrated stiffness, and the influence of the operator on the measured stiffness. The influence of the size of the inclusions was assessed for every type of inclusion. As seen in The second set of phantoms was used to evaluate the influence of depth on the stiffness measurements. Qualitative analysis of one phantom was performed in order to compare homogeneity of the stiffness maps. On healthy volunteer livers, stiffness measurements could be made on all segments of the liver; CVs are reported in p ≈ 0.5). The impact of the BMI on measurement depth and variability was also assessed, but no statistically significant results were observed.The interoperator variability was also evaluated. All tests suggested that there was no operator influence on the measurements . These CVs were much higher than those reported by Huang et al. [Some parameters, such as BMI, are known to affect stiffness measurements, which may also interfere with evaluation of the therapeutic response . In healTexture analysis was performed on 20-mm ROIs in every hepatic segment and the robustness of each feature was evaluated. Results showed that, using B-mode or gray-scale images, 12 features presented low variability regardless of the evaluated hepatic segment. In contrast, only two features were stable when extracted from the stiffness maps, and not for every segment. Whereas these features were expected to be more reliable and less variable when evaluated with parametric maps (2D–SWE elastograms), the inhomogeneous nature of the produced maps seemed to be an obstacle for the quantification of reliable texture features. Jian et al.  demonstrTwo quantitative approaches were evaluated regarding their robustness with the aim to further investigate liver lesions.Measurements of liver stiffness using 2D–SWE can be performed in a reliable and reproducible manner on hepatic segments II to V, as long as the measurements are performed at a depth inferior to 7 cm. The values btained in segments I, VII, and VIII were variable according to the depth of measurement. Texture features were extracted to assess their robustness on a healthy liver. A set of features was demonstrated as stable regardless of the considered hepatic segment when evaluated using B-mode images, whereas few features evaluated using stiffness maps were stable only for some hepatic segments. Ultrasound-based elastography and texture analysis of B-mode imaging should be further evaluated to assess tumor responses to treatment.


Acute beer or alcohol ingestion reduces arterial stiffness, but the dose required to reduce arterial stiffness is unclear. Therefore, this study aimed to determine the acute effects of ingesting various amounts of beer on arterial stiffness in healthy men. Nine men (20–22 years) participated, in eight trials in random order on different days. The participants each consumed 25, 50, 100, or 200 mL of alcohol-free beer  or regular beer , and were monitored for 60 min thereafter. Arterial stiffness did not significantly change among all AFB and B25. However, B50, B100, and B200 caused a significant decrease in arterial stiffness for approximately 30–60 min: heart-brachial pulse wave velocity ; brachial-ankle pulse wave velocity ; heart-ankle pulse wave velocity ; and cardio-ankle vascular index . Positive control alcoholic beverages reduced arterial stiffness, and these reductions did not significantly differ regardless of the type of beverage. Our data show that consuming about 50 mL of beer can start to reduce arterial stiffness, and that the reduced arterial stiffness is mainly attributable to the alcohol in beer. Arterial stiffening occurs with advancing age and impairs the ability of arteries to buffer BP pulsation and blood flow . Thus, iEpidemiological studies have identified a J-shaped association between alcohol intake and arterial stiffness, and arterial stiffness is significantly lower in those who consume light-to-moderate amounts of alcohol than in those who consume none . Beer isIn contrast, many studies have indicated that moderate-to-excessive alcohol consumption increases arterial stiffness and the risk of cardiovascular diseases . Low conBased on this background, the present study aimed to determine how much beer should be consumed to elicit reductions in arterial stiffness. Therefore, we investigated the acute effects of ingesting various amounts of beer on arterial stiffness in healthy young men. Considering our previous results , we hypo2, and 21.4 ± 2.0%, respectively. Because elastic properties of arteries fluctuate significantly with the phases of the menstrual cycle in young female , and body fat in nine healthy young males participated in this study were 21.1 ± 0.2 years, 171.0 ± 2.1 cm, 67.3 ± 3.8 kg, 22.9 ± 1.1 kg/mg female , only ma∗Power 3. In accordance with our previous findings , and the mean values were analyzed . The coeWe conducted a positive control study to confirm whether alcohol in beer is the main source of changes in arterial stiffness . SpirytuF-value was significant, the Bonferroni correction was applied for post hoc multiple comparisons. Relationships were assessed using Pearson’s correlation coefficient. All data were statistically analyzed using IBM SPSS Statistics 25J  and Bell Curve for Excel Statistics 3.10 . To quantify the magnitude of the experimental effect between baseline and object value, effect size (ES) was calculated using G∗Power 3. Differences were considered significant at P < 0.05.Results are presented as means ± SEM. Parameters at each baseline were compared using one-way repeated-measures ANOVA. Changes in each parameter were analyzed by two-way  repeated-measures ANOVA. When the P < 0.01). In particular, the BAC at B100 increased until 30 min (P < 0.01) and that of B200 increased throughout the study period (P < 0.01).Two-way repeated-measures ANOVA revealed significant interaction in BAC . BaselinP < 0.05), but not in the B25 trial (ES = 0.39).Two-way repeated-measures ANOVA also revealed significant interactions in hbPWV, baPWV, haPWV, and CAVI . BaselinBaseline HR and BP did not significantly differ. Although HR in B25, B50, and B100 tended to increase, the values did not reach statistical significance. HR in B200 significantly increased at 15 min post-ingestion (ES = 0.76), but not in any AF trial. Systolic BP, diastolic BP, and mean BP did not significantly change throughout the experimental period, but post-ingestion diastolic BP slightly decreased in the B50, B100, and B200 trials .r = −0.47, P < 0.001), baPWV , haPWV , and CAVI . Statistical correlations tended to be stronger between circulating alcohol and hbPWV, haPWV, and CAVI than baPWV. However, although the same volume of beer was consumed, changes in arterial stiffness did not correlate significantly with the volume of beer consumed per body mass .In all pooled data of main experiments in beer trials from 15 min to 60 min, estimated circulating alcohol levels were significantly and negatively correlated with changes in hbPWV  begins to reduce arterial stiffness, and that the reduction in arterial stiffness is mainly attributable to the alcohol in beer. These findings could thus offer new insights into the development of better methods for preventing arterial stiffening, regulating the circulation system, and vascular biology, especially from the viewpoint of blood distribution during physiological tasks or alcohol ingestion.All datasets generated for this study are included in the article/The studies involving human participants were reviewed and approved by the Human Ethics Committee at the Osaka Institute of Technology . The patients/participants provided their written informed consent to participate in this study.MN, TY, and RN conceived, designed, and performed the study and analyzed the data. MN, TY, RN, and NM wrote the manuscript. MN and NM interpreted the data. All authors approved the final version of 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.


Correction to: Journal of Orthopaedic Surgery and Research 15, 19 (2020)https://doi.org/10.1186/s13018-019-1496-zFollowing publication of the original article , we haveCorrect:[25]. Akgül, T., et al., Double intertrochanteric osteotomy for trochanteric overgrowth and a short femoral neck in adolescents. Journal of orthopaedic surgery (Hong Kong), 2016. 24(3): p. 387–391.[26]. Boese, C.K., et al., The Modified Femoral Neck-Shaft Angle: Age- and Sex-Dependent Reference Values and Reliability Analysis. BioMed Research International, 2016. 2016: p. 1–7.[27]. Genest, F. and L. Seefried, Subtrochanteric and diaphyseal femoral fractures in hypophosphatasia—not atypical at all. Osteoporosis International, 2018. 29(8): p. 1815–1825.[28]. Jiang, N., et al., Femoral Version, Neck-Shaft Angle, and Acetabular Anteversion in Chinese Han Population. Medicine, 2015. 94(21): p. e891.[29]. Emrah Kovalaka, C.E.T.A., Management of unstable pertrochanteric fractures with proximal femoral locking compression plates and affect of neck-shaft angle on functional outcomes. Journal of Clinical Orthopaedics and Trauma, 2017(421): p. 1–6.