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Abstract Background Reproducibility is a fundamental requirement in scientific experiments. Some recent publications have claimed that microarrays are unreliable because lists of differentially expressed genes (DEGs) are not reproducible in similar experiments. Meanwhile, new statistical methods for identifying DEGs continue to appear in the scientific literature. The resultant variety of existing and emerging methods exacerbates confusion and continuing debate in the microarray community on the appropriate choice of methods for identifying reliable DEG lists. Results Using the data sets generated by the MicroArray Quality Control (MAQC) project, we investigated the impact on the reproducibility of DEG lists of a few widely used gene selection procedures. We present comprehensive results from inter-site comparisons using the same microarray platform, cross-platform comparisons using multiple microarray platforms, and comparisons between microarray results and those from TaqMan – the widely regarded "standard" gene expression platform. Our results demonstrate that (1) previously reported discordance between DEG lists could simply result from ranking and selecting DEGs solely by statistical significance (P) derived from widely used simple t-tests; (2) when fold change (FC) is used as the ranking criterion with a non-stringent P-value cutoff filtering, the DEG lists become much more reproducible, especially when fewer genes are selected as differentially expressed, as is the case in most microarray studies; and (3) the instability of short DEG lists solely based on P-value ranking is an expected mathematical consequence of the high variability of the t-values; the more stringent the P-value threshold, the less reproducible the DEG list is. These observations are also consistent with results from extensive simulation calculations. Conclusion We recommend the use of FC-ranking plus a non-stringent P cutoff as a straightforward and baseline practice in order to generate more reproducible DEG lists. Specifically, the P-value cutoff should not be stringent (too small) and FC should be as large as possible. Our results provide practical guidance to choose the appropriate FC and P-value cutoffs when selecting a given number of DEGs. The FC criterion enhances reproducibility, whereas the P criterion balances sensitivity and specificity.

Abstract Background Analyses of Expressed Sequence Tags (ESTs) databases suggest that most human genes have multiple alternative splice variants. The alternative splicing of pre-mRNA is tightly regulated during development and in different tissue types. Changes in splicing patterns have been described in disease states. Recently, we used whole-transcriptome shotgun pryrosequencing to characterize 4 malignant pleural mesothelioma (MPM) tumors, 1 lung adenocarcinoma and 1 normal lung. We hypothesized that alternative splicing profiles might be detected in the sequencing data for the expressed genes in these samples. Methods We developed a software pipeline to map the transcriptome read sequences of the 4 MPM samples and 1 normal lung sample onto known exon junction sequences in the comprehensive AceView database of expressed sequences and to count how many reads map to each junction. 13,274,187 transcriptome reads generated by the Roche/454 sequencing platform for 5 samples were compared with 151,486 exon junctions from the AceView database. The exon junction expression index (EJEI) was calculated for each exon junction in each sample to measure the differential expression of alternative splicing events. Top ten exon junctions with the largest EJEI difference between the 4 mesothelioma and the normal lung sample were then examined for differential expression using Quantitative Real Time PCR (qRT-PCR) in the 5 sequenced samples. Two of the differentially expressed exon junctions (ACTG2.aAug05 and CDK4.aAug05) were further examined with qRT-PCR in additional 18 MPM and 18 normal lung specimens. Results We found 70,953 exon junctions covered by at least one sequence read in at least one of the 5 samples. All 10 identified most differentially expressed exon junctions were validated as present by RT-PCR, and 8 were differentially expressed exactly as predicted by the sequence analysis. The differential expression of the AceView exon junctions for the ACTG2 and CDK4 genes were also observed to be statistically significant in an additional 18 MPM and 18 normal lung samples examined using qRT-PCR. The differential expression of these two junctions was shown to successfully classify these mesothelioma and normal lung specimens with high sensitivity (89% and 78%, respectively). Conclusion Whole-transcriptome shotgun sequencing, combined with a downstream bioinformatics pipeline, provides powerful tools for the identification of differentially expressed exon junctions resulting from alternative splice variants. The alternatively spliced genes discovered in the study could serve as useful diagnostic markers as well as potential therapeutic targets for MPM.

Abstract Background Transcriptome sequencing using next-generation sequencing platforms will soon be competing with DNA microarray technologies for global gene expression analysis. As a preliminary evaluation of these promising technologies, we performed deep sequencing of cDNA synthesized from the Microarray Quality Control (MAQC) reference RNA samples using Roche's 454 Genome Sequencer FLX. Results We generated more that 3.6 million sequence reads of average length 250 bp for the MAQC A and B samples and introduced a data analysis pipeline for translating cDNA read counts into gene expression levels. Using BLAST, 90% of the reads mapped to the human genome and 64% of the reads mapped to the RefSeq database of well annotated genes with e-values ≤ 10-20. We measured gene expression levels in the A and B samples by counting the numbers of reads that mapped to individual RefSeq genes in multiple sequencing runs to evaluate the MAQC quality metrics for reproducibility, sensitivity, specificity, and accuracy and compared the results with DNA microarrays and Quantitative RT-PCR (QRTPCR) from the MAQC studies. In addition, 88% of the reads were successfully aligned directly to the human genome using the AceView alignment programs with an average 90% sequence similarity to identify 137,899 unique exon junctions, including 22,193 new exon junctions not yet contained in the RefSeq database. Conclusion Using the MAQC metrics for evaluating the performance of gene expression platforms, the ExpressSeq results for gene expression levels showed excellent reproducibility, sensitivity, and specificity that improved systematically with increasing shotgun sequencing depth, and quantitative accuracy that was comparable to DNA microarrays and QRTPCR. In addition, a careful mapping of the reads to the genome using the AceView alignment programs shed new light on the complexity of the human transcriptome including the discovery of thousands of new splice variants.

Abstract Correction to Bemmo A, Benovoy D, Kwan T, Gaffney DJ, Jensen RV, Majewski J: Gene expression and isoform variation analysis using Affymetrix Exon Arrays. BMC Genomics 2008, 9: 529.

Abstract Background Alternative splicing and isoform level expression profiling is an emerging field of interest within genomics. Splicing sensitive microarrays, with probes targeted to individual exons or exon-junctions, are becoming increasingly popular as a tool capable of both expression profiling and finer scale isoform detection. Despite their intuitive appeal, relatively little is known about the performance of such tools, particularly in comparison with more traditional 3' targeted microarrays. Here, we use the well studied Microarray Quality Control (MAQC) dataset to benchmark the Affymetrix Exon Array, and compare it to two other popular platforms: Illumina, and Affymetrix U133. Results We show that at the gene expression level, the Exon Array performs comparably with the two 3' targeted platforms. However, the interplatform correlation of the results is slightly lower than between the two 3' arrays. We show that some of the discrepancies stem from the RNA amplification protocols, e.g. the Exon Array is able to detect expression of non-polyadenylated transcripts. More importantly, we show that many other differences result from the ability of the Exon Array to monitor more detailed isoform-level changes; several examples illustrate that changes detected by the 3' platforms are actually isoform variations, and that the nature of these variations can be resolved using Exon Array data. Finally, we show how the Exon Array can be used to detect alternative isoform differences, such as alternative splicing, transcript termination, and alternative promoter usage. We discuss the possible pitfalls and false positives resulting from isoform-level analysis. Conclusion The Exon Array is a valuable tool that can be used to profile gene expression while providing important additional information regarding the types of gene isoforms that are expressed and variable. However, analysis of alternative splicing requires much more hands on effort and visualization of results in order to correctly interpret the data, and generally results in considerably higher false positive rates than expression analysis. One of the main sources of error in the MAQC dataset is variation in amplification efficiency across transcripts, most likely caused by joint effects of elevated GC content in the 5' ends of genes and reduced likelihood of random-primed first strand synthesis in the 3' ends of genes. These effects are currently not adequately corrected using existing statistical methods. We outline approaches to reduce such errors by filtering out potentially problematic data.

Abstract Background Sinorhizobium meliloti is an agriculturally important model symbiont. There is an ongoing need to update and improve its genome annotation. In this study, we used a high-throughput pyrosequencing approach to sequence the transcriptome of S. meliloti, and search for new bacterial genes missed in the previous genome annotation. This is the first report of sequencing a bacterial transcriptome using the pyrosequencing technology. Results Our pilot sequencing run generated 19,005 reads with an average length of 136 nucleotides per read. From these data, we identified 20 new genes. These new gene transcripts were confirmed by RT-PCR and their possible functions were analyzed. Conclusion Our results indicate that high-throughput sequence analysis of bacterial transcriptomes is feasible and next-generation sequencing technologies will greatly facilitate the discovery of new genes and improve genome annotation.

Abstract Background Lung function at the end of life depends on its peak and subsequent decline. Because obesity is epidemic in young adulthood, we quantified age-related changes in lung function relative to body mass index (BMI). Methods The Coronary Artery Risk Development in Young Adults (CARDIA) study in 1985–86 (year 0) recruited 5,115 black and white men and women, aged 18–30. Spirometry testing was conducted at years 0, 2, 5 and 10. We estimated 10 year change in FVC, FEV1 and FEV1/FVC according to baseline BMI and change in BMI within birth cohorts with initial average ages 20, 24, and 28 years, controlling for race, sex, smoking, asthma, physical activity, and alcohol consumption. Measurements and Main Results Participants with baseline BMI < 21.3 kg/m2 experienced 10 year increases of 71 ml in FVC and 60 ml in FEV1 and neither measure declined through age 38. In contrast, participants with baseline BMI ≥ 26.4 kg/m2 experienced 10 year decreases of 185 ml in FVC and 64 ml in FEV1. FEV1/FVC increased with increasing BMI. Weight gain was also associated with lung function. Those who gained the most weight over 10 years had the largest decrease in FVC, but FVC increased with weight gain in those initially thinnest. In contrast, FEV1 decreased with increasing weight gain in all participants, with maximum decline in obese individuals who gained the most weight during the study. Conclusion Among healthy young adults, increasing BMI in the initially thin participants was associated with increasing then stable lung function through age 38, but there were substantial lung function losses with higher and increasing fatness. These results suggest that the obesity epidemic threatens the lung health of the general population.

Abstract Background Breast cancer is a heterogeneous disease, presenting with a wide range of histologic, clinical, and genetic features. Microarray technology has shown promise in predicting outcome in these patients. Methods We profiled 162 breast tumors using expression microarrays to stratify tumors based on gene expression. A subset of 55 tumors with extensive follow-up was used to identify gene sets that predicted outcome. The predictive gene set was further tested in previously published data sets. Results We used different statistical methods to identify three gene sets associated with disease free survival. A fourth gene set, consisting of 21 genes in common to all three sets, also had the ability to predict patient outcome. To validate the predictive utility of this derived gene set, it was tested in two published data sets from other groups. This gene set resulted in significant separation of patients on the basis of survival in these data sets, correctly predicting outcome in 62–65% of patients. By comparing outcome prediction within subgroups based on ER status, grade, and nodal status, we found that our gene set was most effective in predicting outcome in ER positive and node negative tumors. Conclusion This robust gene selection with extensive validation has identified a predictive gene set that may have clinical utility for outcome prediction in breast cancer patients.

Abstract Background The TyrA protein family includes members that catalyze two dehydrogenase reactions in distinct pathways leading to L-tyrosine and a third reaction that is not part of tyrosine biosynthesis. Family members share a catalytic core region of about 30 kDa, where inhibitors operate competitively by acting as substrate mimics. This protein family typifies many that are challenging for bioinformatic analysis because of relatively modest sequence conservation and small size. Results Phylogenetic relationships of TyrA domains were evaluated in the context of combinatorial patterns of specificity for the two substrates, as well as the presence or absence of a variety of fusions. An interactive tool is provided for prediction of substrate specificity. Interactive alignments for a suite of catalytic-core TyrA domains of differing specificity are also provided to facilitate phylogenetic analysis. tyrA membership in apparent operons (or supraoperons) was examined, and patterns of conserved synteny in relationship to organismal positions on the 16S rRNA tree were ascertained for members of the domain Bacteria. A number of aromatic-pathway genes (hisHb, aroF, aroQ) have fused with tyrA, and it must be more than coincidental that the free-standing counterparts of all of the latter fused genes exhibit a distinct trace of syntenic association. Conclusion We propose that the ancestral TyrA dehydrogenase had broad specificity for both the cyclohexadienyl and pyridine nucleotide substrates. Indeed, TyrA proteins of this type persist today, but it is also common to find instances of narrowed substrate specificities, as well as of acquisition via gene fusion of additional catalytic domains or regulatory domains. In some clades a qualitative change associated with either narrowed substrate specificity or gene fusion has produced an evolutionary "jump" in the vertical genealogy of TyrA homologs. The evolutionary history of gene organizations that include tyrA can be deduced in genome assemblages of sufficiently close relatives, the most fruitful opportunities currently being in the Proteobacteria. The evolution of TyrA proteins within the broader context of how their regulation evolved and to what extent TyrA co-evolved with other genes as common members of aromatic-pathway regulons is now feasible as an emerging topic of ongoing inquiry.

Abstract Background The growing conviction that lateral gene transfer plays a significant role in prokaryote genealogy opens up a need for comprehensive evaluations of gene-enzyme systems on a case-by-case basis. Genes of tryptophan biosynthesis are frequently organized as whole-pathway operons, an attribute that is expected to facilitate multi-gene transfer in a single step. We have asked whether events of lateral gene transfer are sufficient to have obscured our ability to track the vertical genealogy that underpins tryptophan biosynthesis. Results In 47 complete-genome Bacteria, the genes encoding the seven catalytic domains that participate in primary tryptophan biosynthesis were distinguished from any paralogs or xenologs engaged in other specialized functions. A reliable list of orthologs with carefully ascertained functional roles has thus been assembled and should be valuable as an annotation resource. The protein domains associated with primary tryptophan biosynthesis were then concatenated, yielding single amino-acid sequence strings that represent the entire tryptophan pathway. Lateral gene transfer of several whole-pathway trp operons was demonstrated by use of phylogenetic analysis. Lateral gene transfer of partial-pathway trp operons was also shown, with newly recruited genes functioning either in primary biosynthesis (rarely) or specialized metabolism (more frequently). Conclusions (i) Concatenated tryptophan protein trees are congruent with 16S rRNA subtrees provided that the genomes represented are of sufficiently close phylogenetic spacing. There are currently seven tryptophan congruency groups in the Bacteria. Recognition of a succession of others can be expected in the near future, but ultimately these should coalesce to a single grouping that parallels the 16S rRNA tree (except for cases of lateral gene transfer). (ii) The vertical trace of evolution for tryptophan biosynthesis can be deduced. The daunting complexities engendered by paralogy, xenology, and idiosyncrasies of nomenclature at this point in time have necessitated an expert-assisted manual effort to achieve a correct analysis. Once recognized and sorted out, paralogy and xenology can be viewed as features that enrich evolutionary histories.

Microbes able to convert gaseous one-carbon (C1) waste feedstocks are increasingly important to transition to the sustainable production of renewable chemicals and fuels. Acetogens are interesting biocatalysts since gas fermentation using Clostridium autoethanogenum has been commercialised. However, most acetogen strains need complex nutrients, display slow growth, and are not robust for bioreactor fermentations. In this work, we used three different and independent adaptive laboratory evolution (ALE) strategies to evolve the wild-type C. autoethanogenum to grow faster, without yeast extract and to be robust in operating continuous bioreactor cultures. Multiple evolved strains with improved phenotypes were isolated on minimal media with one strain, named "LAbrini", exhibiting superior performance regarding the maximum specific growth rate, product profile, and robustness in continuous cultures. Whole-genome sequencing of the evolved strains identified 25 mutations. Of particular interest are two genes that acquired seven different mutations across the three ALE strategies, potentially as a result of convergent evolution. Reverse genetic engineering of mutations in potentially sporulation-related genes CLAU&#95;3129 (spo0A) and CLAU&#95;1957 recovered all three superior features of our ALE strains through triggering significant proteomic rearrangements. This work provides a robust C. autoethanogenum strain "LAbrini" to accelerate phenotyping and genetic engineering and to better understand acetogen metabolism., Competing Interests: Declaration of Competing Interest We declare that LanzaTech has interest in commercial gas fermentation with C. autoethanogenum and Audrey Harris, Steven D. Brown, Grant Hawkins, Jim Daleiden, Loan Tran, Hunter Zeleznik, Rasmus O. Jensen, Vinicio Reynoso, Heidi Schindel, and Michael Köpke are employees of LanzaTech., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The genus Clostridium is a large and diverse group within the Bacillota (formerly Firmicutes), whose members can encode useful complex traits such as solvent production, gas-fermentation, and lignocellulose breakdown. We describe 270 genome sequences of solventogenic clostridia from a comprehensive industrial strain collection assembled by Professor David Jones that includes 194 C. beijerinckii, 57 C. saccharobutylicum, 4 C. saccharoperbutylacetonicum, 5 C. butyricum, 7 C. acetobutylicum, and 3 C. tetanomorphum genomes. We report methods, analyses and characterization for phylogeny, key attributes, core biosynthetic genes, secondary metabolites, plasmids, prophage/CRISPR diversity, cellulosomes and quorum sensing for the 6 species. The expanded genomic data described here will facilitate engineering of solvent-producing clostridia as well as non-model microorganisms with innately desirable traits. Sequences could be applied in conventional platform biocatalysts such as yeast or Escherichia coli for enhanced chemical production. Recently, gene sequences from this collection were used to engineer Clostridium autoethanogenum, a gas-fermenting autotrophic acetogen, for continuous acetone or isopropanol production, as well as butanol, butanoic acid, hexanol and hexanoic acid production., (© 2024. The Author(s).)

Objective: Focused assessment with sonography for trauma (FAST) is a valuable ultrasound procedure in emergency settings, and there is a need for evidence-based education in FAST to ensure competencies. Immersive virtual reality (IVR) is a progressive training modality gaining traction in the field of ultrasound training. IVR holds several economic and practical advantages to the common instructor-led FAST courses using screen-based simulation (SBS)., Methods: This prospective, interventional cohort study investigated whether training FAST using IVR unsupervised and out-of-hospital was non-inferior to a historical control group training at a 90 min SBS course in terms of developing FAST competencies in novices. Competencies were assessed in both groups using the same post-training simulation-based FAST test with validity evidence, and a non-inferiority margin of 2 points was chosen., Results: A total of 27 medical students attended the IVR course, and 27 junior doctors attended the SBS course. The IVR group trained for a median time of 117 min and scored a mean 14.2 ± 2.0 points, compared with a mean 13.7 ± 2.5 points in the SBS group. As the lower bound of the 95% confidence interval at 13.6 was within the range of the non-inferiority margin (11.7-13.7 points), training FAST in IVR for a median of 117 min was found non-inferior to training at a 90 min SBS course. No significant correlation was found between time spent in IVR and test scores., Conclusion: Within the limitations of the use of a historical control group, the results suggest that IVR could be an alternative to SBS FAST training and suitable for unsupervised, out-of-hospital courses in basic FAST competencies., Competing Interests: Conflict of interest K.J., J.D.L., R.O.J., and O.G. were all involved in development of the IVR module. R.O.J. is the chief executive officer and co-founder of VitaSim, which provided the virtual reality software used in this study. J.D.L. was previously employed by VitaSim as a part-time learning consultant. C.F. has voiced a previous VitaSim IVR module. Neither participated in the assessment of the participants’ US skills or data analysis. The remaining authors do not have any conflicts of interest to disclose., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Intestinal resection and a proximal stoma is the preferred surgical approach in patients with severe secondary peritonitis due to perforation of the small intestine. However, proximal stomas may result in significant nutritional problems and long-term parenteral nutrition. This study aimed to assess whether primary anastomosis or suturing of small intestine perforation is feasible and safe using the open abdomen principle with vacuum-assisted abdominal closure (VAC)., Methods: Between January 2005 and June 2018, we performed a retrospective chart review of 20 patients (> 18 years) with diffuse faecal peritonitis caused by small intestinal perforation and treated with primary anastomosis/suturing and subsequent open abdomen with VAC., Results: The median age was 65 years (range: 23-90 years). Twelve patients were female (60%). Simple suturing of the small intestinal perforation was performed in three cases and intestinal resection with primary anastomosis in 17 cases. Four patients (20%) died within 90-days postoperatively. Leakage occurred in five cases (25%), and three patients developed an enteroatmospheric fistula (15%). Thirteen of 16 patients (83%) who survived were discharged without a stoma. The rest had a permanent stoma., Conclusions: Primary suturing or resection with anastomosis and open abdomen with VAC in small intestinal perforation with severe faecal peritonitis is associated with a high rate of leakage and enteroatmospheric fistula formation., Trial Registration: The study was approved by the Danish Patient Safety Authority (case number 3-3013-1555/1) and the Danish Data Protection Agency (file number 18/28,404). No funding was received., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Objectives: Using virtual reality (VR), students of radiography can practice acquisition and positioning of musculoskeletal radiographs and get immediate feedback on their performance within the simulator. The purpose of this study was to assess usability of a newly developed VR simulator and to explore self-perceived clinical readiness (SPCR) of radiography students before and after training acquisition of wrist radiographs in the VR simulator., Material and Methods: A prospective methodology was applied where the students ( n = 10) estimated their own SPCR in regard to acquisition of wrist radiographs pre- and post-VR training. A questionnaire on usability, realism, and educational value of the simulator was answered post-VR training. Usability and SPCR scores were calculated. The student's paired t -test was applied to explore the impact of VR training on SPCR., Results: The students (90%) reported that the simulator was realistic and they thought that it could contribute to learning. The pre- and post-SPCR scores were 75 (95% confidence interval [CI]: 54-96) and 77 (95% CI: 59-95), respectively. There was no significant difference ( P = 0.4574) between the pre- and post-SPCR scores., Conclusion: Results indicated that the concept of training acquisition and positioning of wrist radiographs in a VR simulator is feasible with positive feedback from the students. The SPCR scores improved slightly, although not statistically significant, after completion of the training session., Competing Interests: R.O.J. is a co-founder and co-owner of VitaSim. R.O.J. advised the authors M.G.T. and S.K.K.G. on technical solutions to developing the X-ray training module in virtual reality. R.O.J. had no influence on data collection, data analysis, interpretation, or publishing decisions., (© 2023 Published by Scientific Scholar on behalf of Journal of Clinical Imaging Science.)

Focused lung ultrasound (FLUS) has high diagnostic accuracy in many common conditions seen in a variety of emergency settings. Competencies are essential for diagnostic success and patient safety but can be challenging to acquire in clinical environments. Immersive virtual reality (IVR) offers an interactive risk-free learning environment and is progressing as an educational tool. First, this study explored the educational impact of novice FLUS users participating in a gamified or non-gamified IVR training module in FLUS by comparing test scores using a test with proven validity evidence. Second, the learning effect was assessed by comparing scores of each group with known test scores of novices, intermediates and experienced users in FLUS. A total of 48 participants were included: 24 received gamified and 24 received non-gamified IVR training. No significant difference was found between gamified (mean = 15.5 points) and non-gamified (mean = 15.2 points), indicating that chosen gamification elements for our setup did not affect learning outcome (p = 0.66). The mean scores of both groups did not significantly differ from those of known intermediate users in FLUS (gamified p = 0.63, non-gamified p = 0.24), indicating that both IVR modules could be used as unsupervised out-of-hospital training for novice trainees in FLUS., Competing Interests: Conflict of interest disclosure R.O.J. is a co-founder and co-owner of VitaSim. R.O.J. advised the main author J.D.L. on technical solutions to developing the ultrasound training module in virtual reality. R.O.J. had no influence on data collection, data analysis, interpretation or publishing decisions. The remaining authors declare no conflicts of interest., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Summary Statement: Simulation-based training using virtual reality head-mounted displays (VR-HMD) is increasingly being used within the field of medical education. This article systematically reviews and appraises the quality of the literature on the use of VR-HMDs in medical education. A search in the databases PubMed/MEDLINE, Embase, ERIC, Scopus, Web of Science, Cochrane Library, and PsychINFO was carried out. Studies were screened according to predefined exclusion criteria, and quality was assessed using the Medical Education Research Study Quality Instrument. In total, 41 articles were included and thematically divided into 5 groups: anatomy, procedural skills, surgical procedures, communication skills, and clinical decision making. Participants highly appreciated using VR-HMD and rated it better than most other training methods. Virtual reality head-mounted display outperformed traditional methods of learning surgical procedures. Although VR-HMD showed promising results when learning anatomy, it was not considered better than other available study materials. No conclusive findings could be synthesized regarding the remaining 3 groups., Competing Interests: R.O.J. is employed by and co-owner of VitaSim ApS. The authors declare no conflict of interest., (Copyright © 2022 Society for Simulation in Healthcare.)

This study was aimed at comparing the learning efficacy of a traditional instructor-led lesson with that of a completely virtual, self-directed lesson in immersive virtual reality (IVR) in teaching basic point-of-care ultrasound (PoCUS) skills. We conducted a blinded, non-inferiority, parallel-group, randomized controlled trial in which final-year medical students were randomized to an instructor-led (n = 53) or IVR (n = 51) lesson. Participants' learning efficacy was evaluated by blinded assessors, who rated each participant's performance using the Objective Structured Assessment of Ultrasound Skills (OSAUS) assessment tool.The mean total scores for participants were 11.0 points (95% confidence interval: 9.8-12.2) for the instructor-led lesson and 10.3 points (95% confidence interval: 9.0-11.5) for the IVR lesson. No significant differences were observed between the groups with respect to total score (p = 0.36) or subgroup objectives of the OSAUS score (p = 0.34 for familiarity, p = 0.45 for image optimization, p = 0.96 for systematic approach and p = 0.07 for interpretation). Maintenance costs for both courses were estimated at 400 euros each. Startup costs for the instructor-led course were estimated 16 times higher than those for the IVR course. The learning efficacy of an instructor-led lesson on basic US did not differ significantly from that of a self-directed lesson in IVR, as assessed using the OSAUS. The results suggest that IVR could be an equivalent alternative to instructor-led lessons in future basic US courses, but further research is warranted to clarify the role of IVR in PoCUS courses., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Many industrial chemicals that are produced from fossil resources could be manufactured more sustainably through fermentation. Here we describe the development of a carbon-negative fermentation route to producing the industrially important chemicals acetone and isopropanol from abundant, low-cost waste gas feedstocks, such as industrial emissions and syngas. Using a combinatorial pathway library approach, we first mined a historical industrial strain collection for superior enzymes that we used to engineer the autotrophic acetogen Clostridium autoethanogenum. Next, we used omics analysis, kinetic modeling and cell-free prototyping to optimize flux. Finally, we scaled-up our optimized strains for continuous production at rates of up to ~3 g/L/h and ~90% selectivity. Life cycle analysis confirmed a negative carbon footprint for the products. Unlike traditional production processes, which result in release of greenhouse gases, our process fixes carbon. These results show that engineered acetogens enable sustainable, high-efficiency, high-selectivity chemicals production. We expect that our approach can be readily adapted to a wide range of commodity chemicals., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Abstract: The primary aim was to evaluate the effect of immersive virtual reality learning for training medical students in basic clinical ultrasound. Secondary outcomes were to explore if virtual reality learning had an effect on hand-eye coordination skills and if the medical students wanted more virtual reality learning.This pilot study was a double-blind, parallel-group, block-randomized, controlled trial. Participants (n = 20) were blinded and randomized to virtual reality or e-learning for basic ultrasound education. Medical students with no previous ultrasound education were recruited voluntarily from the University of Southern Denmark. Data were collected during introductory courses on ultrasound from March to May 2019. Participants were assessed with Objective Structured Assessment on Ultrasound Skills. Assessing supervisors were blinded.The virtual reality group (n = 11) scored a significantly higher Objective Structured Assessment on Ultrasound Skills score (143 [95% confidence interval {CI}, 135 to 151]) compared with the e-learning group (n = 9; 126 [95% CI, 113 to 138]; mean difference, 17 points [95% CI, 4 to 30]; P < 0.01). No significant effect on the hand-eye score was found (mean difference, 3 points [95 % CI, -3 to 9]; P = 0.32). Ninety-one percent of the virtual reality group wanted more virtual reality learning.Immersive virtual reality learning improved medical students' ultrasound skills significantly compared with e-learning. The hand-eye score was higher in the virtual reality group, although not at a significant level. Students wanted more virtual reality learning. Further research is needed to clarify immersive virtual reality's educational role in the future., Competing Interests: R.O.J. is a cofounder and co-owner of VitaSim. The remaining authors declare no conflict of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Introduction: Immersive virtual reality (IVR)-based training is gaining ground as an educational tool in healthcare. When combined with well-established educational methods, IVR can potentially increase competency and autonomy in ultrasound (US)-guided peripheral venous cannulation.The aim of this study was to examine the impact of adding IVR training to a course in US-guided peripheral venous cannulation., Methods: Medical students (n = 19) from the University of Southern Denmark with no former standardized US education were recruited to voluntarily participate in a pilot study, designed as a randomized controlled trial. The primary outcome was the proportion of successful peripheral venous cannulations on a phantom. Secondary outcomes included the proportion of surface punctures on the phantom and procedure time. Participants received e-learning on the basic US before randomization to either IVR (n = 10) or no further training (n = 9). The additional IVR training comprised 10 virtual scenarios for US-guided peripheral venous catheter (PVC) placement. Students were subsequently evaluated in peripheral venous cannulation by a blinded assessor., Results: The proportion of successful peripheral venous cannulations was significantly higher in the IVR group (P ≤ .001). The proportions of successful cannulations were significantly higher in the IVR group compared to the control group for the 1st and 2nd PVC (P = .011, P = .023), but not for the 3rd PVC (P = .087). Similar results were found for the proportion of surface punctures (1st: P ≤ .001, 2nd: P = .001, and 3rd: P = .114). No significant differences in procedure times were found between the groups., Conclusion: This pilot study showed that adding an IVR-based training simulation to an existing e-learning curriculum significantly increased the learning efficacy of US-guided PVC placement for medical students., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc.)

Purpose: This systematic review investigates whether infrared thermography (IRT) can measure systemic vasoconstriction and addresses the value of IRT in assessing circulatory deficiency and prognoses., Methods: Design was based on the PRISMA criteria and a systematic search of 6 databases was performed., Results: Of 3,198 records, five articles were included. Three clinical studies were identified; two found significant correlations between IRT obtained temperatures and mortality. An experimental study found an association between peripheral temperature and stroke volume. An animal study found that central-peripheral temperature differences correlated with shock index, mean arterial pressure, and disease progression., Conclusions: Data from the most valid study suggests that central-peripheral temperature differences should be investigated further, both on its own, and integrated with other variables.

The design and optimization of biosynthetic pathways for industrially relevant, non-model organisms is challenging due to transformation idiosyncrasies, reduced numbers of validated genetic parts and a lack of high-throughput workflows. Here we describe a platform for in vitro prototyping and rapid optimization of biosynthetic enzymes (iPROBE) to accelerate this process. In iPROBE, cell lysates are enriched with biosynthetic enzymes by cell-free protein synthesis and then metabolic pathways are assembled in a mix-and-match fashion to assess pathway performance. We demonstrate iPROBE by screening 54 different cell-free pathways for 3-hydroxybutyrate production and optimizing a six-step butanol pathway across 205 permutations using data-driven design. Observing a strong correlation (r = 0.79) between cell-free and cellular performance, we then scaled up our highest-performing pathway, which improved in vivo 3-HB production in Clostridium by 20-fold to 14.63 ± 0.48 g l -1 . We expect iPROBE to accelerate design-build-test cycles for industrial biotechnology.

Background: Prognostic uncertainty inhibits clinicians from initiating timely end-of-life discussions and advance care planning. This study evaluates the efficacy of the CriSTAL (Criteria for Screening and Triaging to Appropriate aLternative care) checklist in emergency departments., Methods: Prospective cohort study of patients aged ≥65 years with any diagnosis admitted via emergency departments in ten hospitals in Australia, Denmark and Ireland. Electronic and paper clinical records will be used to extract risk factors such as nursing home residency, physiological deterioration warranting a rapid response call, personal history of active chronic disease, history of hospitalisations or intensive care unit admission in the past year, evidence of proteinuria or ECG abnormalities, and evidence of frailty to be concurrently measured with Fried Score and Clinical Frailty Scale. Patients or their informal caregivers will be contacted by telephone around three months after initial assessment to ascertain survival, self-reported health, post-discharge frailty and health service utilisation since discharge. Logistic regression and bootstrapping techniques and AUROC curves will be used to test the predictive accuracy of CriSTAL for death within 90 days of admission and in-hospital death., Discussion: The CriSTAL checklist is an objective and practical tool for use in emergency departments among older patients to determine individual probability of death in the short-term. Its validation in this cohort is expected to reduce clinicians' prognostic uncertainty on the time to patients' death and encourage timely end-of-life conversations to support clinical decisions with older frail patients and their families about their imminent or future care choices., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Abstract: To determine the validity of the Australian clinical prediction tool Criteria for Screening and Triaging to Appropriate aLternative care (CRISTAL) based on objective clinical criteria to accurately identify risk of death within 3 months of admission among older patients., Methods: Prospective study of ≥ 65 year-olds presenting at emergency departments in five Australian (Aus) and four Danish (DK) hospitals. Logistic regression analysis was used to model factors for death prediction; Sensitivity, specificity, area under the ROC curve and calibration with bootstrapping techniques were used to describe predictive accuracy., Results: 2493 patients, with median age 78-80 years (DK-Aus). The deceased had significantly higher mean CriSTAL with Australian mean of 8.1 (95% CI 7.7-8.6 vs. 5.8 95% CI 5.6-5.9) and Danish mean 7.1 (95% CI 6.6-7.5 vs. 5.5 95% CI 5.4-5.6). The model with Fried Frailty score was optimal for the Australian cohort but prediction with the Clinical Frailty Scale (CFS) was also good (AUROC 0.825 and 0.81, respectively). Values for the Danish cohort were AUROC 0.764 with Fried and 0.794 using CFS. The most significant independent predictors of short-term death in both cohorts were advanced malignancy, frailty, male gender and advanced age. CriSTAL's accuracy was only modest for in-hospital death prediction in either setting., Conclusions: The modified CriSTAL tool (with CFS instead of Fried's frailty instrument) has good discriminant power to improve prognostic certainty of short-term mortality for ED physicians in both health systems. This shows promise in enhancing clinician's confidence in initiating earlier end-of-life discussions., Competing Interests: MC and KH developed the risk prediction tool in 2015 and have tested it retrospectively in several hospitals. This might be perceived by readers as non-financial conflict of interest given their knowledge of the subject matter or materials discussed in this manuscript. However, they have no affiliations with or involvement in any organisation or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; or expert testimony or patent-licensing arrangements), that may influence the validity of the study. All other authors certify that they have NO conflict of interest to report. The analyses were led by two co-authors (RMT and FG) who were not involved in the development of the tool, data collection or outcome ascertainment for this validation study.The study was approved in Australia by the hospital management teams and by the South Eastern Sydney Local Health District Ethics Committee [#15/026 HREC/15/POW/55 and data was stored in the UNSW secure server. The need for approval in Denmark was waivered by the Regional Ethics Committee of Southern Denmark, as this project was considered to be a quality improvement initiative without an intervention [42].Verbal information on the study preceded the request for written informed consent which was obtained from each individual if cognitively competent or their surrogate at the time of recruitment. Participants consented to being interviewed, giving access to hospital records during admission, and being contacted for follow-up outcome ascertainment. The original consent remained with the researchers and a copy went to the patient or their family.

Background: Vacuum-assisted closure (VAC) has, in many instances, become the treatment of choice in patients with abdominal catastrophes. This study describes the use and outcome of ABThera KCI® VAC in the Region Southern Denmark covering a population of approximately 1.202 mill inhabitants., Method: A prospective multicenter study including all patients treated with VAC during an eleven-month period., Results: A total of 74 consecutive patients were included. Median age was 64.4 (9-89) years, 64% were men, and median body mass index was 25 (17-42). Duration of VAC treatment was median 4.5 (0-39) days with median 1 (0-16) dressing changes. Seventy per cent of the patients attended the intensive care unit. The 90-day mortality was 15%. A secondary closure of the fascia was obtained in 84% of the surviving patients. Only one patient developed an enteroatmospheric fistula. Patients with secondary closure were less likely to develop large hernias and had better self-evaluated physical health score ( p < 0,05). No difference in mental health was found., Conclusion: The abdominal VAC treatment in patients with abdominal catastrophes is safe and with a relative low complication rate. Whether it might be superior to conventional treatment with primary closure when possible has yet to be proven in a randomized study.

Technological solutions to reduce greenhouse gas (GHG) emissions from anthropogenic sources are required. Heavy industrial processes, such as steel making, contribute considerably to GHG emissions. Fermentation of carbon monoxide (CO)-rich off gases with wild-type acetogenic bacteria can be used to produce ethanol, acetate, and 2,3-butanediol, thereby, reducing the carbon footprint of heavy industries. Here, the processes for the production of ethanol from CO-rich off gases are discussed and a perspective on further routes towards an integrated biorefinery at a steel mill is given. Recent achievements in genetic engineering as well as integration of other biotechnology platforms to increase the product portfolio are summarized. Already, yields have been increased and the portfolio of products broadened. To develop a commercially viable process, however, the extraction from dilute product streams is a critical step and alternatives to distillation are discussed. Finally, another critical step is waste(water) treatment with the possibility to recover resources., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Virulence in Staphylococcus aureus is regulated via agr-dependent quorum sensing in which an autoinducing peptide (AIP) activates AgrC, a histidine protein kinase. AIPs are usually thiolactones containing seven to nine amino acid residues in which the thiol of the central cysteine is linked to the alpha-carboxyl of the C-terminal amino acid residue. The staphylococcal agr locus has diverged such that the AIPs of the four different S. aureus agr groups self-activate but cross-inhibit. Consequently, although the agr system is conserved among the staphylococci, it has undergone significant evolutionary divergence whereby to retain functionality, any changes in the AIP-encoding gene (agrD) that modifies AIP structure must be accompanied by corresponding changes in the AgrC receptor. Since AIP-1 and AIP-4 only differ by a single amino acid, we compared the transmembrane topology of AgrC1 and AgrC4 to identify amino acid residues involved in AIP recognition. As only two of the three predicted extracellular loops exhibited amino acid differences, site-specific mutagenesis was used to exchange the key AgrC1 and AgrC4 amino acid residues in each loop either singly or in combination. A novel lux-based agrP3 reporter gene fusion was constructed to evaluate the response of the mutated AgrC receptors. The data obtained revealed that while differential recognition of AIP-1 and AIP-4 depends primarily on three amino acid residues in loop 2, loop 1 is essential for receptor activation by the cognate AIP. Furthermore, a single mutation in the AgrC1 loop 2 resulted in conversion of (Ala5)AIP-1 from a potent antagonist to an activator, essentially resulting in the forced evolution of a new AIP group. Taken together, our data indicate that loop 2 constitutes the predicted hydrophobic pocket that binds the AIP thiolactone ring while the exocyclic amino acid tail interacts with loop 1 to facilitate receptor activation.