Thermoflexus hugenholtzii JAD2T, the only cultured representative of the Chloroflexota order Thermoflexales, is abundant in Great Boiling Spring (GBS), NV, United States, and close relatives inhabit geothermal systems globally. However, no defined medium exists for T. hugenholtzii JAD2T and no single carbon source is known to support its growth, leaving key knowledge gaps in its metabolism and nutritional needs. Here, we report comparative genomic analysis of the draft genome of T. hugenholtzii JAD2T and eight closely related metagenome-assembled genomes (MAGs) from geothermal sites in China, Japan, and the United States, representing “Candidatus Thermoflexus japonica,” “Candidatus Thermoflexus tengchongensis,” and “Candidatus Thermoflexus sinensis.” Genomics was integrated with targeted exometabolomics and 13C metabolic probing of T. hugenholtzii. The Thermoflexus genomes each code for complete central carbon metabolic pathways and an unusually high abundance and diversity of peptidases, particularly Metallo- and Serine peptidase families, along with ABC transporters for peptides and some amino acids. The T. hugenholtzii JAD2T exometabolome provided evidence of extracellular proteolytic activity based on the accumulation of free amino acids. However, several neutral and polar amino acids appear not to be utilized, based on their accumulation in the medium and the lack of annotated transporters. Adenine and adenosine were scavenged, and thymine and nicotinic acid were released, suggesting interdependency with other organisms in situ. Metabolic probing of T. hugenholtzii JAD2T using 13C-labeled compounds provided evidence of oxidation of glucose, pyruvate, cysteine, and citrate, and functioning glycolytic, tricarboxylic acid (TCA), and oxidative pentose-phosphate pathways (PPPs). However, differential use of position-specific 13C-labeled compounds showed that glycolysis and the TCA cycle were uncoupled. Thus, despite the high abundance of Thermoflexus in sediments of some geothermal systems, they appear to be highly focused on chemoorganotrophy, particularly protein degradation, and may interact extensively with other microorganisms in situ. Chloroflexota (synonym Chloroflexi) continues to be expanded, revealing a global distribution containing broad phylogenetic and physiological diversity. Currently, isolates capable of anoxygenic photosynthesis, obligate organohalide respiration, autotrophy, chemolithotrophy, carboxydotrophy, and fermentation have been described  sediments in Great Boiling Spring (GBS), Nevada, United States, where it can be one of the most abundant organisms (estimated 3.2–60% relative abundance) , filamentous morphology (up to ∼500 μm long), lack of a defined medium, and dependence on complex organic extracts from GBS water for optimal growth. Furthermore, in culture, T. hugenholtzii may have the narrowest growth temperature range of any bacterium or archaeon known (67.5–75°C) (undance) . Similarundance) , rangingundance) ]. The ab.5–75°C) .13C-labeled compounds  provides even more information, including activities of specific enzymes and rates of different metabolic pathways  with a study of the exometabolome and stable isotope probing of T. hugenholtzii to better understand its metabolism and potential ecological role and help inform environmental studies in systems where Thermoflexus species are abundant.Here, we combined analysis of the draft genome of T. hugenholtzii JAD2T for genome sequencing was described in T was created in the Genomes OnLine Database . Scaffolds with a length > 2.5 kbp were assigned to genomic bins using MetaBAT, which is based on read abundance and tetranucleotide word frequency  lineage workflow . RibosomThermoflexus genomes were identified and aligned using the Genome Taxonomy Database Toolkit (GTDB-tk)   from GTDB-tk) . These s1.6.7.a)  was used1.6.7.a)  and SH-l1.6.7.a)  values aT. hugenholtzii genome. Protein sequences were obtained from IMG (IMG Taxon ID: 2140918011) or from NCBI for MAGs and were submitted to MAPLE   is capable of functioning2.The IMG/M system , in comb v2.3.1)  to deter v2.3.1) , using tlone see , 2016. ET. hugenholtzii protein sequences also were analyzed using the MEROPS database (v12) using BLASTP (NCBI BLAST v2.5.0+) and the merops_scan.lib library for comparison between MAGs and T. hugenholtzii. An E-value cutoff of 1E-10 was used and the maximum target sequences matched was set to one.Genes coding for peptidases and peptidase inhibitors and peptidase genomic abundance comparison to other organisms was done using the MEROPS database batch Blast (v10) , 2018. MThermoflexus hugenholtzii proteins were also submitted to BlastKOALA (v2.1) (A (v2.1)  to populA (v2.1)  and SecrA (v2.1)  for idennosDYLZ) and aerobic carbon monoxide dehydrogenase systems (coxMSLF) in MAGs, BLAST+  as the query. Individual amino acid databases for protein-coding genes for MAGs were created and the T. hugenholtzii JAD2T sequences were queried using Megablast . The inoculated replicates (n = 5) and another set of sterile controls (n = 5) were incubated in the dark with no shaking at ∼74 (±2)°C for 7 days.After 7 days of incubation, all samples were placed on ice and then centrifuged at 18,514 rcf for 30 min at 4°C in sterile 50 mL Falcon tubes. The supernatant was decanted and filter-sterilized using 0.2 μm PES filters (VWR) into sterile 50 mL Falcon tubes. Filters were rinsed by the passage of 20 mL of sterile nanopure water prior to filtering the supernatant to remove potential chemical contaminants. Supernatants were stored at −80°C and shipped to Joint Genome Institute (JGI) on dry ice for high-performance liquid chromatography tandem mass spectrometry (HPLC–MS/MS) analysis. All samples were processed together.Cell density was determined by concentrating 1.0 mL of culture from each replicate for 10 min at 22,442 rcf at 4°C. The supernatant was decanted, and the cell pellet was re-suspended in 200 μL of nanopure water. 5.0 μL of the concentrated subsample was loaded onto a Petroff-Hausser counter  and photographed using an Olympus BX51 phase-contrast microscope fitted with a V-TV1x-2 camera (Olympus). Measurements of filament length and density were used to determine cell numbers using an average individual cell length of 4.0 μM .T. hugenholtzii growth were desalted and extracted using solid-phase extraction cartridges . Each cartridge was pre-equilibrated with 1 mL methanol (MeOH) (3×), then 1 mL H2O (5x), then all water expelled with air. Each sample was then acidified with HCl by adding 20 μL of 6 M HCl to 1 mL media, briefly vortexing, then flowing through the PPL cartridge. Each cartridge was then rinsed with 1 mL of 0.01 M HCl (2×) and air-dried. Eluent was collected following rinses of each cartridge with 1 mL MeOH (2×) and 1 mL acetonitrile (2×) into a 5 mL Eppendorf tube. Eluent extracts of the desalted media were then dried in a SpeedVac  and stored at −80°C.HPLC–MS/MS was used to identify metabolites. 1 mL media samples with or without –1, #R435902; d4-lysine, 10 μg mL–1, #61619210; d5-benzoic acid, 10 μg mL–1, #217158 – Sigma), centrifuge-filtered through a 0.22 μm hydrophilic PVDF membrane , and placed into HPLC vials. HPLC-MS/MS was performed on extracts using an Agilent 1290 LC stack, with MS and MS/MS data collected using a Q Exactive Orbitrap MS  in centroid format in both positive and negative ion mode. Full MS spectra were collected from m/z 70–1,050 at 70,000 resolution, with MS/MS fragmentation data acquired using stepped 10, 20, and 30 eV collision energies at 17,500 resolution. Source settings of the mass spectrometer included a sheath gas flow rate of 55 (au), auxiliary gas flow of 20 (au), sweep gas flow of 2 (au), spray voltage of 3 kV and capillary temperature of 400°C. Between each sample injection, a blank was run consisting of 100% methanol. Normal-phase chromatography was performed using a ZIC-pHILIC column , at 40°C, at a flow rate of 0.25 mL−1 with a 2 μL injection volume for each sample. The HILIC column was equilibrated with 100% buffer B (90:10 ACN:H2O w/5 mM ammonium acetate) for 1.5 min, diluting buffer B down to 50% with buffer A (H2O w/5 mM ammonium acetate) for 23.5 min, down to 40% B over 3.2 min, to 0% B over 6.8 min, and followed by isocratic elution in 100% buffer A for 3 min. Metabolites were identified based on exact mass and retention time and comparing MS/MS fragmentation spectra to purchased standards. Raw data files can be obtained through the JGI genome portal under project name “Hedlund 2017 exometabolomics of Thermoflexus hugenholtzii JAD2,” Project ID: 11963743.In preparation for HPLC-MS/MS analysis, dried extracts (eluents) were resuspended in 110 μL MeOH with internal standards  hierarchical classification system (p > 0.05); (ii) either of the comparisons between the non-incubated treatment and the incubated control treatment or the incubated control and the culture treatment showed significant differences when subjected to a Tukey’s HSD test (p < 0.05); and (iii) at least one treatment had a mean peak height intensity (au) > 105. If samples did not match m/z and retention times for standards, then they were removed from analysis. If significant compounds were found to have a peak height below 105 in all treatments, then they were removed from analysis. If significant compounds were found to have a peak height at or below 105 in some but not all treatments, then individual chromatograms were manually inspected. Compounds were excluded from analysis if satisfactory peak shape was not found upon manual inspection. Shapiro-Wilk and Tukey’s HSD tests were performed using R version 3.4.3.HPLC-MS/MS peak-height values for compounds identified in each treatment were compared to determine biological activity, thermal degradation, or thermal production. The treatment with n system , to simp13C-labeled metabolic probing was conducted with both position-specific and uniformly labeled substrates, and oxidation of the labeled carbon was assessed by using an isotope spectrometer. The cultivation medium used was based on 2 was added to provide enough CO2  for subsequent 13C-CO2 analysis (see below), and then cultures were incubated at 75°C for the duration of the experiment.13C-labeled substrates or uniformly 13C tricarboxylic acid (TCA) metabolites or amino acids were administered to cultures in the peptone-based medium described above. Each 13C treatment was performed in triplicate. 13C position-specific substrate additions consisted of filter-sterilized solutions (21.4 μmol substrate-C mL–1) of sodium pyruvate , sodium acetate (1-13C and 2-13C), and glucose (1-13C and uniformly (U) 13C-labeled) . Uniformly 13C-labeled substrate additions consisted of citrate, L-serine, L-cysteine, L-alanine, and succinate  at a final concentration of 4.0 μg mL–1. 13C-CO2 production rate controls were given natural abundance  pyruvate, acetate, and glucose (as described above for the 13C-labeled compounds). A time 0-h headspace sample (10 mL) was taken immediately prior to 13C-labeled substrate additions, and 1–2 headspace samples (10 mL each) were taken per 24-h period for the next ∼180 h. Cooling was minimized during sampling by placing bottles in a pre-heated (75°C) water bath.At 98.75 h of growth , position-specific 2-free air after injecting samples. This was done to facilitate a sample run time of ∼10 min. on a Picarro 2101-i CO2 and CH4 isotope spectrometer . Picarro data were recorded as 30-s averages of δ13CO2 over a period of near-constant delta readings.The 10 mL headspace samples were injected into a Tedlar air-sample bag  and increased in volume by diluting with CO2 production were set up as described above but without 13C-labeled compounds. Headspace samples (10 mL) were taken over the duration of the experiment and run on a LICOR 6262  to determine headspace CO2 concentrations.Cultures for monitoring the rate of CO2 were added. These controls were incubated for ∼180 h at 75°C to mimic the conditions of the 13C-labeled compound additions in the larger Wheaton bottle cultures , with a G + C content of 67.34%. The genome encodes 2,997 predicted genes, of which 2,944 are protein-coding (89.39%). Also annotated are 48 tRNA-encoding genes, a single copy of 5S and 16S rRNA genes, and a fragmented 23S rRNA gene. A total of 1,928 genes (64.33%) and 1,141 genes (38.07%) were assigned to COGs and KO groups, respectively  were identified manually, the MAPLE analysis provided no support for any complete nitrogen or sulfur metabolism modules, including nitrogen fixation (M00175), ammonia oxidation (M00528), complete denitrification (M00529), dissimilatory nitrate reduction to ammonium (M00530), assimilatory nitrate/nitrite reduction (M00531), complete nitrification , assimilatory sulfate reduction (M00176), dissimilatory sulfate reduction (M00596), or respiratory thiosulfate oxidation  (coxMSLF was identified (2143740265-68 and 2143742206-09)  . The ure M00595)  appears  M00595) . No comp M00595) . A full 2206-09) . Genes c2206-09) , which ioflexota . Componeoflexota  were detThermoflexus hugenholtzii JAD2T contains an unusually high abundance and diversity of annotated peptidases, with 133 genes coding for peptidases and five peptidase inhibitors (T. hugenholtzii JAD2T in the top 3.6% of Bacteria and Archaea for the percentage of genes belonging to a MEROPS protein family4. 17 of the annotated endo- and exopeptidases are predicted to be membrane-bound or extracellular (hibitors . 4.4% ofcellular . Secreticellular . The mosPredicted family M01 (2143742583) and M28F (2143740571), both aminopeptidases, have lipoprotein signal peptides . M28F haNine genes belong to the S1 family of Serine proteases, including seven members of the S1C family. This family of endopeptidases resides in the periplasm of Gram-negative bacteria and can also serve as a general molecular chaperone  Supplem. Five ofSeveral ABC transporters might enable the transport of oligopeptides, free amino acids, or other protein degradation products. Genes for ABC transporters for branched-chain amino acids (LivKHGFM), oligopeptides (OppABDCF), and spermidine/putrescine (PolDGBA) were present . A partiIn contrast, no ABC transporters for general L-amino acids (AapJQMP), cysteine (TcyABC and TcyJKLMN), lysine (LysX1X2Y), histidine (HisJMQP), glutamine (GlnHPQ), arginine (ArtJIMQP), hydroxyproline (LhpPMNO), D-methionine (MetQIN) arginine/ornithine (AotJMQP), glutamate/aspartate (GltIKJL), arginine/lysine/histidine/glutamate (BgtBA), arginine/lysine/histidine (ArtPQR), lysine/arginine/ornithine/histidine/octopine (PA5152-55), neutral amino acids/histidine (NatBCDAE), dipeptide/heme/ δ-aminolevulinic acid (DppABCDF), or dipeptide (DppEBCD) transport were found .Thermoflexus. For example, homoserine, threonine, and glycine could potentially be converted into pyruvate from serine (EC4.3.1.19) and aspartate could be degraded to fumarate , feeding central carbon metabolism  and four medium-quality MAGs   . All bin JZ2_71) . Some rR6.5–7.75 ]. The re unknown . Additio unknown .Chloroflexota, with T. hugenholtzii JAD2T being the only cultured representative  or gluconeogenesis (M00003) modules. The non-oxidative PPP (M00007) was not feasible in “Candidatus Thermoflexus japonica HR22T.” The glyoxylate cycle (M00012) was only feasible in the T. hugenholtzii group and “Candidatus Thermoflexus tengchongensis QQ20T.” All MAGs lacked the delta subunit of the F-type ATPase. The aerobic type-I coxMSLF was conserved across all MAGs. However, Ca. T. sinensis GXS_4 and “Candidatus T. japonica HR22T” had notably lower sequence similarity for coxMLF, and GBS60_20 had lower sequence similarity for coxM, with respect to T. hugenholtzii JAD2T  was found in all MAGs except for Ca. T. sinensis JZ2_71, which was missing nosL, and GBS60_20, which was missing nosYL and had a notably lower sequence similarity for nosZD, with respect to T. hugenholtzii JAD2T  unique to T. hugenholtzii JAD2T, one family (M14B) unique to “Candidatus T. sinensis QQ28T,” and two families  unique to “Candidatus T. japonica HR22T”  was not feasible in any of the “Candidatus T. sinensis” MAGs; an inositol-phosphate transport system (M00599) was feasible in “Candidatus T. japonica HR22T” and “Candidatus T. tengchongensis GXS_4” and “QQ28T,” but not in any others or T. hugenholtzii JAD2T. A PTS transporter for ascorbate was found in all MAGs and T. hugenholtzii JAD2T.With a few exceptions, nucleotide and amino acid metabolism modules were similar across all MAGs and mirrored ii JAD2T . Howeverl others . Similarii JAD2T . A molybThermoflexus hugenholtzii JAD2T grew well (0.26–4.6 × 107 cells/mL) in cultures for exometabolomics, resulting in an average cell yield of 1.1 × 107 cells/mL (T. hugenholtzii growth to have much higher variability than the sterile medium and sterile incubated controls (T. hugenholtzii JAD2T and were represented by the HMDB classes Imidazopyrimidines (adenine) and Purine nucleosides (adenosine). Compounds produced due to biological activity were largely represented by the HMDB class Carboxylic acids and derivatives (6), with one representative of Pyridines and derivatives (nicotinic acid), and one representative of Organoheterocyclic compounds (thymine). All compounds of the Carboxylic acids and derivatives class belonged to the sub-class Amino acids, peptides, and analogs, with direct parent compounds of alpha-amino acids (glycine) or L-alpha amino acids . L-homoserine and L-threonine were not distinguishable with HPLC–MS/MS. In addition, many di- and tri-peptides were identified as possible biological products but were not confirmed with purified standards .cells/mL . NMDS plcontrols , demonstcontrols . Only twOrganic acids and derivatives and Pteridines and derivatives, respectively, were observed.In addition, thermal degradation of ketoleucine  and riboflavin, belonging to the HMDB classes T. hugenholtzii JAD2T with 13C-labeled compounds demonstrated heterotrophic activity on a variety of organic substrates, including glucose, organic acids, amino acids, and TCA-cycle intermediates . This may be indicative of a mechanism for balancing intracellular metabolite pools to facilitate the reactions of central carbon metabolic pathways when feeding on proteins . In suppT. hugenholtzii JAD2T metabolize amino acid-derived heat-labile triosephosphates into heat-stable fructose 6-phosphate, rendering metabolite pools stable, and allow metabolic flexibility free from transcriptional regulation  enabling the urea cycle to function  provide gulation . Malate T. hugenholtzii JAD2T growth suggests this pathway may be active despite a gene coding for an arginine deiminase not being identified.Ornithine and ketoleucine accumulated in the medium. Ornithine is a by-product of the urea cycle and ketoleucine can be formed from the incomplete decomposition of branched-chain amino acids, both of which were predicted from the genome . In addition, genes for parts of the arginine deiminase pathway, a pathway for the catabolism of arginine, were identified; however, an arginine deiminase (EC3.5.3.6) was not identified . This paThermoflexus appears to rely on a significant complement of amino acids, which is consistent with an obligately proteolytic lifestyle. For example, many amino acids appear to be metabolic dead ends, meaning that they do not feed into central carbon metabolic pathways, and others have no recognizable de novo biosynthetic clusters. For example, isoleucine, leucine, valine, methionine, phenylalanine, and tryptophan could be transported but are possible metabolic dead ends. The de novo biosynthesis of several amino acids did not appear possible due to the absence of single genes . Most of these absences are supported by comparative genomics with Thermoflexus MAGs, which suggests these are true absences and not artefacts from incomplete genome assembly. This could mean that these amino acids must be scavenged or interconverted, or alternatively that Thermoflexus harbors undefined genes capable of carrying out the missing reactions. In cases where a vast majority of genes are present for a pathway, the presence of undefined genes seems likely . Attempts to design a defined medium using a diversity of amino acid mixtures and CCMP metabolites did not support growth. A defined medium would allow further exploration of the capacity for de novo amino acid biosynthesis by T. hugenholtzii JAD2T and the identification of novel enzymes and metabolic pathways.13C metabolic probing experiments demonstrated broad heterotrophic activity of T. hugenholtzii JAD2T, despite the challenge of growing it in pure culture. This result is generally consistent with genomic predictions, and broad heterotrophic activity demonstrated in GBS sediments where Thermoflexus is abundant  or pyruvate dehydrogenase (2143740152 and 2143740153). If the resulting acetyl group were transferred to oxaloacetate by citrate synthase (2143741275), then C2 and C3 of pyruvate would be oxidized over multiple cycles of the TCA cycle, which was not observed . C. aurantiacus also assimilates acetate through the glyoxylate cycle when growing mixotrophically with H2 and CO2  indicates that glycolysis is highly active relative to the oxidative PPP, which would decarboxylate the C1 position via 6-phosphogluconate dehydrogenase (2143742524). Interpretation of the 13C-glucose and 13C-pyruvate data together suggest a ten-fold higher rate of glycolysis relative to the oxidative PPP, since the only 13CO2 production from universally labeled glucose would occur for C3 and C4 due to pyruvate-ferredoxin oxidoreductase or pyruvate dehydrogenase. This result might not be surprising given the presence of nucleotides and nucleosides in yeast extract coupled with predicted nucleoside transporters, the demonstrated uptake of adenine and adenosine  and to a slightly lesser degree at a high-temperature shelf (∼82°C), with minimal flux observed at low-temperature sites (∼65°C) . Similarly, no stimulation of growth under anaerobic conditions has been observed in the presence of nitrite (2 mM)  . N2O relGBS60_20 , other oe (2 mM) , so the coxMSLF are conserved across all T. hugenholtzii MAGs, but carboxydotrophy has also not been observed for T. hugenholtzii JAD2T. It has been suggested that this system may provide a means for Chloroflexota to persist in times of low nutrient availability and situations requiring dormancy by providing an alternative energy source  was observed when T. hugenholtzii was grown in the presence of O2 (1% of headspace) or anaerobically with nitrite (2 mM) or nitrous oxide (5% of headspace) (data not shown).Genes coding for a type-I y source . This syT. hugenholtzii JAD2T seems to be an obligate chemoheterotroph that depends on proteins and amino acids in light of the high abundance of this organism and close relatives in some hot spring sediments and non-photosynthetic mats (T. hugenholtzii is an abundant member of the sediment community around 80°C (3.2–60% estimated relative abundance), several meters away from photosynthetic mats, which are well-formed in GBS sediments below ∼70°C    and perhme e.g., . Future analyses . Alterna sources . In the Chloroflexota, including both photosynthetic and non-photosynthetic taxa that are abundant and common in circumneutral to alkaline pH geothermal features in Yellowstone National Park. For example, the genera Roseiflexus and Chloroflexus, both within the Chloroflexaceae, are highly abundant in phototrophic mats in the outflow channels of the Octopus Spring and Mushroom Spring in the Lower Geyser Basin, where in situ metabolism has been studied in some detail  Latin neut. adj. Sinae, Chinese; the Chinese Thermoflexus. The nomenclatural type is the metagenomic bin QQ_bins28 (JAEVEY000000000).“Thermoflexus. Predicted to be proteolytic, based an abundance of proteases, and facultatively anaerobic, based on cytochrome c oxidase, nitrous oxide reductase, and a dissimilatory nitrite reduction to ammonium system. Possibly carboxydotrophic, based on a type I carbon monoxide dehydrogenase system. In addition to the nomenclatural type, also includes “Candidatus T. sinensis” GXS_4 (JAFLMU000000000) and “Candidatus T. sinensis” GZ2_71 (JAEVEZ000000000).Currently known only from metagenomic sequence data from circumneutral pH geothermal springs in Tengchong, China. Habitat and genomic features suggest a phenotype conforming to the description of the genus Candidatus Thermoflexus tengchongensis” (teng.chong.en’sis) originating from Tenghchong, a region of Yunnan Province, China; the Thermoflexus from Tengchong. The nomenclatural type is the metagenomic bin QQ_bins20 (NCBI ID JAEVEX000000000).“Thermoflexus. Predicted to be proteolytic, based an abundance of proteases, and facultatively anaerobic, based on cytochrome c oxidase, nitrous oxide reductase, and a dissimilatory nitrite reduction to ammonium system. Possibly carboxydotrophic, based on a type I carbon monoxide dehydrogenase system.Currently known only from metagenomic sequence data from circumneutral pH geothermal springs in Tengchong, China. Habitat and genomic features suggest a phenotype conforming to the description of the genus Candidatus Thermoflexus japonica” (ja.pon’i.ca) Latin neut. adj. Japonicus, Japanese; the Japanese Thermoflexus. The nomenclatural type is the metagenomic bin HR22 (BEHY00000000.1).“Thermoflexus. Predicted to be proteolytic, based an abundance of proteases, and facultatively anaerobic, based on cytochrome c oxidase, nitrous oxide reductase, and a dissimilatory nitrite reduction to ammonium system.Currently known only from metagenomic sequence data from an enrichment culture derived from a subsurface gold mine in Japan. Habitat and genomic features suggest a phenotype conforming to the description of the genus The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/T. hugenholtzii. J-YJ and W-JL assembled and provided the QQ20, QQ28, and JZ2_71 MAGs. ST, CS, SM, TH, DL, and JD completed the bioinformatic analysis. ST, KT, DP, BH, and PD contributed to the experimental design for 13C work. ST, KT, and PD completed the analysis of 13C work. ST, KT, and DP carried out all the culture work. ST, KT, BH, LS, BB, and TN contributed to the experimental design for exometabolomic work. RL, LS, BB, KL, and TN performed the exometabolomic analysis. ST, CS, and SM completed the statistical analysis of exometabolomic data. ST interpreted, compiled, and wrote the manuscript with input from all authors.MH, AC, AS, MP, KP, NV, NM, I-MC, DS, TR, RO’M, CD, NS, NI, NK, TW, and EE-F sequenced and assembled the genome of 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.


Cancer is a complicated disease that has a significant characteristic of evading cell death (apoptosis). The induction of apoptosis in cancerous cells seems a promising procedure to use as a cancer treatment. In the family of the tumor necrosis factor (TNF) proteins, there are some ligands with the capability to induce apoptosis. Several recombinant TNF apoptosis-inducing ligands have been designed over the years, and their characteristics have been improved. This review provides an overview of the studies done in different stages of the TNF apoptosis-inducing ligands as cancer treatments and the strategies to surpass their natural limitations to improve their effectiveness.Cancer is a complex disease with apoptosis evasion as one of its hallmarks; therefore, apoptosis induction in transformed cells seems a promising approach as a cancer treatment. TNF apoptosis-inducing ligands, which are naturally present in the body and possess tumoricidal activity, are attractive candidates. The most studied proteins are TNF-α, FasL, and TNF-related apoptosis-inducing ligand (TRAIL). Over the years, different recombinant TNF family-derived apoptosis-inducing ligands and agonists have been designed. Their stability, specificity, and half-life have been improved because most of the TNF ligands have the disadvantages of having a short half-life and affinity to more than one receptor. Here, we review the outlook on apoptosis-inducing ligands as cancer treatments in diverse preclinical and clinical stages and summarize strategies of overcoming their natural limitations to improve their effectiveness. Cancer is one of the leading causes of death globally; it is a complex disease that involves bypassing growth suppressors, angiogenesis, metastasis, and apoptosis evasion . The mosApoptosis evasion is one of the main hallmarks of cancer; its dysregulation is related to overexpression of antiapoptotic genes or survival signals and downregulation/mutation of proapoptotic genes . TherefoThis review provides an overview of the TNF family of receptors and their apoptosis-inducing ligands as cancer treatments. Additionally, we describe several engineered death receptor agonists and how gene therapy may improve the apoptosis-inducing ligands’ efficacy.Apoptosis is a regulated cell death process that keeps the cell population balanced in a living organism . It is aThe intrinsic pathway is activated when the outer membrane of the mitochondria is permeabilized, releasing cytochrome c. Then, it binds to the apoptotic protease activating factor 1 (APAF1), which leads to the formation of the apoptosome. The apoptosome recruits caspase-9, which activates caspases-3 and -7, leading to cell death. Besides cytochrome c, the mitochondria also release the second mitochondria-derived activator of caspase (SMAC/DIABLO) and the serine protease HtrA2/Omi. These factors impede the inhibitor of apoptosis proteins (IAPs), boosting the induction of apoptosis ,9. The mThe extrinsic pathway’s activation requires the stimulation of death receptors (DRs); these are transmembrane receptors that belong to the TNF receptor superfamily . They haIndependently of which pathway is activated, both lead to the activation of caspases-3, -6, and -7, which induce apoptosis . SeveralEven though the DRs are well known for their apoptosis induction characteristics, they may also trigger necroptosis. A regulated necrosis process mediated by the RIP kinase family members can be induced, in some cell lines, under certain circumstances, such as the use of caspase inhibitors , downregIn recent years, members of the TNF superfamily of cytokines, TNF-α, Fas ligand (FasL), and TNF-related apoptosis-inducing ligand (TRAIL), have been studied for their apoptosis induction capability when bound to their receptors that contain a DD . The mosThe death receptors are type I transmembrane proteins with cysteine-rich extracellular domains. The DRs have been detected in a wide variety of healthy ,18 Figu and cancThe analysis of Fas expression revealed its downregulation in some colon carcinomas , lung caInterestingly, the TNF apoptosis-inducing ligands also bind to decoy receptors (DcRs). These receptors are not able to trigger the apoptosis cascade, but they can activate survival and migration signaling ,30; the TNF-α, FasL, and TRAIL are expressed mainly in immune cells, including granulocytes, monocytes, T cells, B cells, dendritic cells, and NK cells . Most apTNF-α is a type II transmembrane protein with different biological roles: mediation of the inflammatory response, regulation of immune cells, and cytotoxicity. TNF-α binds to tumor necrosis factor receptor 1 (TNF-R1), also known as death receptor 1 (DR1), and tumor necrosis factor receptor 2 (TNF-R2); both receptors are involved in prosurvival signaling and proliferation by activating the NF-kB pathway. However, only TNF-R1 has a death domain that can trigger apoptosis through caspase cascade activation under certain conditions, such as the absence of the IAPs ,36. TNF-TNF-R1, TNF- R2, and TNF-α can be cleaved by the metalloprotease TNF-α converting enzyme (TACE or ADAM-17), resulting in the soluble forms of TNF-α (sTNF-α), sTNR-R1, and sTNR-R2 ,40,41,42Recombinant TNF-α has been studied as a cancer treatment. Unfortunately, in clinical trials, it showed systemic toxicity when administrated intravenously. Therefore, treatment directed to a specific organ or an area of the body seems to be a better option .FasL (CD95L) is a type II transmembrane protein expressed in diverse immune cells, like B, T, and NK cells . It can Under normal circumstances, DcR3 is expressed in the brain, gastrointestinal tract, ovary, kidney, and urinary bladder, but it is difficult to detect it in serum ,49. HoweA soluble form of FasL (sFasL) can be produced either by proteolytic cleavage or alternative splicing . HoweverSo far, recombinant FasL (rFasL) has not reached clinical trials because of the liver toxicity induced by the systemic administration of rFasL in mice ,55.TRAIL is a homotrimeric transmembrane protein type II. TRAIL is expressed on the surface of T cells, macrophages, and NK cells; its principal role is to modulate the immune response . TRAIL bTRAIL is a homotrimeric protein; a zinc ion stabilizes the trimeric conformation in the Cys230 residue . This coDulanermin is a recombinant human soluble TRAIL  protein that was tested in several clinical trials alone or in combination with chemotherapy. In phase I, it was tested as a treatment for advanced cancer  and metaA circular recombinant mutant of human TRAIL known as circularly permuted TRAIL (CPT) was developed; it consists of the N-terminal amino acids (121–135) fused with a flexible linker to the C-terminal amino acids (135–281) of TRAIL. It has a longer half-life compared to dulanermin. CPT has been tested in patients (phases Ib and II) with relapsed or refractory multiple myeloma (RRMM), where in general it was well tolerated, albeit elevation of liver enzymes was reported ,75. In RAs mentioned before, the apoptosis-inducing ligands from the TNF superfamily interact with more than one receptor. This promiscuous interaction, in some cases, can block the activation of the apoptotic pathway , as in tThe study of antibody-mediated therapies to treat cancer, where monoclonal antibodies (mAbs) are designed to target a specific antigen , such asAlthough antibody targeting of TNF-R1 as cancer therapy seems promising due to its proinflammatory signaling , diverseA monoclonal antibody against murine Fas developed in the early 1990s showed liver toxicity in mice after intraperitoneal administration . Later, Many DR4- and DR5-targeting antibodies have been designed for cancer treatment. Mapatumumab (HGS-ETR1) is a fully human agonist monoclonal antibody (mAb) that binds with high affinity to DR4 . This moOf several monoclonal antibodies designed for targeting DR5, only five reached clinical trials: conatumumab ,112,113,As mentioned previously, the TNF apoptosis-inducing ligands are present as trimeric transmembrane proteins that can be cleaved, resulting in soluble proteins . These pThe activation of Fas by sFasL requires the oligomerization of sFasL ; with thIn the case of TRAIL, different approaches have been followed to help stabilize the trimer conformation, from using a fused leucine zipper motif, the N-terminal of sTRAIL (LZ-TRAIL) , to usinFusion proteins have been used to improve the activity of the soluble form of the apoptosis-inducing ligands. For years, it has been known that soluble ligands have a binding activity differing from that of membrane-bound ligands . As descH) and light (VL) of the immunoglobulin; these two regions are bonded together by a small and flexible linker [Fusion proteins are molecules with multifunctional properties depending on the moieties that conform to them . A commoe linker . This ene linker .Several fusion proteins containing TNF-α have been designed as a cancer treatment . A fusioAnother TNF-α fusion protein is the IL-12-L19-TNFα a triple fusion protein containing interleukin-12 (IL-12), an antibody fragment specific to fibronectin (L19) extra-domain B, and TNF-α. It was tested in vitro, where it showed cytotoxic activity in murine L-M fibroblast . HoweverThe fusion protein L19-TNFα was designed for localized administration and reached clinical trials. L19 has an affinity for the EB-D domain of fibronectin, which is considered a marker of angiogenesis in cancer patients . It was Although membrane-bound FasL can induce apoptosis, sFasL lacks the apoptotic activity, which can be regained by fusing sFasL with an scFv. Different FasL fusion proteins have been designed to improve FasL apoptotic activity. Those proteins  have bee+ xenograft mouse models at doses in the fmol/kg range [The use of engineered antibodies in the format of an scFv is an explored approach to create TRAIL fusion proteins with high stability and the capacity to activate the apoptotic pathway . The fuskg range . The TRAAnother TRAIL fusion protein in clinical trials is ABBV-621. It is now in phase I study as a therapy for patients with previously treated solid tumors and hematologic malignancies (NCT03082209). ABBV-621 is a fusion protein that contains an immunoglobulin G1 (IgG1)-fragment crystallizable region (Fc) portion fused to a single chain trimer of TRAIL subunits. This fusion protein was designed to maximize the clustering of TRAIL receptors ,166.Gene therapy may deliver proapoptotic genes to express these proteins to induce cell apoptosis ,168. TheMany viral vectors have been designed to deliver apoptosis-inducing ligands throughout the years, but only a few reached clinical trials, such as the adenoviral vectors TNFerade ,174 and TNFerade is a second-generation replication-defective adenovirus armed with human TNF-α cDNA. The vector includes the radiation-inducible promoter early growth response (Egf-1) upstream of the transcriptional start site of human TNF-α . The EgfTNFerade was used in clinical trial phases I and II to treat different types of cancers, where the patients tolerated the treatment well. However, in phase III, TNFerade was used to treat advanced pancreatic cancer but was not effective ,177,179.VB-111 (ofranergene obadenovec) is a replication-defective adenovirus serotype 5 vector armed with a modified murine pre-proendothelin promoter (PPE-1) and human Fas-chimera transgene. The modified PPE-1 promoter has a hypoxia-responsive factor and three copies of the endothelium-specific element that increase the specificity for angiogenic vessels—the vector was designed to target Fas-chimera transgene expression in angiogenic blood vessels to induce apoptosis . In a phLater, VB-111 was tested in combination with bevacizumab in a phase III study (NTC02511405), where it did not show efficacy. It is essential to consider that the treatment setting was different from the previous phase I/II study. In this trial, the patients had a combinational treatment of VB-111 and bevacizumab. Coughesy et al. thought that the lack of effectiveness in this trial was because bevacizumab antagonized VB-111 . VB-111 The adenovirus vectors can also be engineered as oncolytic viruses; they can replicate selectively in cancer cells and kill them, releasing new viral particles that can infect neighboring and distant cancer cells . An examAnother example of an oncolytic vector is the NDV/Anh-TRAIL . This veIn the case of TRAIL, no gene therapy has reached clinical trials yet. However, several viral vectors have been designed to increase TRAIL effectiveness and avoid repeated administration ; those v10 viral particles (vp), the tumor size decreased by around 90%. The fusion protein was detected in plasma 60 days after infection with a concentration of around 200 μg/mL. It did not show liver toxicity, thus proving to be safe for systemic administration [Additionally, Ad-scFv425:sTRAIL was tested in an established renal carcinoma xenograft in nude mice. After an intraocular administration of 10stration .As a cancer treatment, induction of cell death by apoptosis appears an interesting pathway to follow. In search of apoptosis-inducing ligands, targeting the death receptors (DRs) from the TNF superfamily seems the right approach. The cytokines from the TNF superfamily, TNF-α, FasL, and TRAIL, can induce apoptosis by activating the extrinsic pathway when they bind to DR1, DR2, DR4, or DR5 .Although TNF-α was identified as promising cancer therapeutic and several agonists were developed for that purpose, TNF-α antagonists have a greater value in treating inflammatory and autoimmune diseases (rheumatoid arthritis) ,217. RecIn clinical trials, some of the recombinant death ligands and agonists developed as cancer treatments showed limited activity and, in some cases, systemic toxicity ,70. TherThe use of viral vectors can improve the apoptosis-inducing ligands’ effectiveness; using tumor-specific promoters can give a localized effect, thus averting systemic toxicity. The transgene’s continuous production can help overcome the short half-life of the ligands and avoid the need for multiple administrations. For instance, the use of Fas agonists in a systemic administration can lead to liver toxicity, which may be prevented by localized administration with adenoviral vectors and tumor-specific promoters. The drawback of TRAIL’s short half-life can be overcome using an adenoviral vector, as Bremer et al. demonstrated .Although TNF apoptosis-inducing ligands have been designed as cancer therapies and analyzed in preclinical studies showing promising results, only a few have reached clinical trials. The majority of them faltered in the first stages of the studies, showing a lack of effectiveness. Therefore, the use of gene therapy as a tool to improve the pharmacokinetics and meet the need for better biomarkers of resistance, which help screen and select the target population to design tailored anticancer approaches from single to combination agents, may help overcome the lack of effectiveness during the clinical trials. Additionally, the TNF apoptosis-inducing ligands’ effectiveness may also be boosted by using them in combination with other anticancer therapeutics.Considering that the gene therapy techniques are being developed and improved to such an extent that vaccines against SARS-CoV-2 were developed in record time using Ad5  and lipi


The accelerated SARS-CoV-2 evolution under selective pressure by massive deployment of neutralizing antibody-based therapeutics is a concern with potentially severe implications for public health. We review here reports of documented immune escape after treatment with monoclonal antibodies and COVID-19-convalescent plasma (CCP). While the former is mainly associated with specific single amino acid mutations at residues within the receptor-binding domain , a few cases of immune evasion after CCP were associated with recurrent deletions within the N-terminal domain of the spike protein . The continuous genomic monitoring of non-responders is needed to better understand immune escape frequencies and the fitness of emerging variants. The SARS-CoV-2 spike protein is the target of neutralizing antibody (nAb)-based therapeutics. Control of the COVID-19 pandemic is being hampered by continued evolution of SARS-CoV-2, which includes mutations in the spike protein that can affect immunogenicity and antibody-mediated neutralization. Evolutionary modeling suggests that SARS-CoV-2 strains harboring 1–2 deleterious mutations naturally exist, and their frequency increases steeply under positive selection by monoclonal antibodies (mAb) and vaccines  or molnuhttps://jbloomlab.github.io/SARS-CoV-2-RBD_MAP_clinical_Abs/) [Mutations that confer in vitro resistance to therapeutic anti-spike mAbs have been characterized with various methods and are informative about treatment-emergent immune escape. Deep mutational scanning (DMS) predicts protein expression, ACE2 binding, and mAb binding . The metal_Abs/)  and finaal_Abs/) . nAb binal_Abs/) ,10.4 led to ΔF140 spike mutation at day 45, followed by E484K at day 73, and an insertion in the N-terminal domain (NTD): these accumulating mutations led to complete immune escape [Although DMS was also applied to polyclonal antibodies in COVID-19-convalescent plasma (CCP) , the proe escape . Similare escape . AlthougIn vivo, while intrahost-SARS-CoV-2 mutation development is typically very low , faster We mined PubMed  for keywords related to COVID-19 , immune escape  and nAb-based therapeutics  both in vitro and in vivo. Clinical cases were annotated for eventual underlying immune deficiency, concurrent treatments and outcome. The 3D structural coordinates of the full spike protein   and the Our literature search revealed 32 papers that were then manually inspected to determine whether they included relevant information that was then retrieved, evaluated and organized into Tables.Escape from nAb-based therapeutics provides a crucial demonstration that these immune therapies target protective antigens, which the pathogen actively evades. Hence, the emergence of neutralizing-resistant variants in individuals receiving mAb and CCP provides powerful evidence for their antiviral activity. This evidence is independent of reduction in viral load, which has been reported with mAbs given early in disease but have been an inconsistent finding in randomized controlled trials (RCT) of CCP for COVID-19 .Obtaining the frequencies for this phenomenon from case series is not possible due to the high risk of selection biases, which would yield unrealistically high frequencies. In contrast, RCTs with their control groups are the suggested reference. With bamlanivimab, resistance was reported in 7% of patients, regardless of dosage (700/2800/7000 mg) versus <1% in patients treated with placebo ,29. AparIn contrast to mAb therapeutics, immune escape under CCP has not been investigated in RCTs. Hence, evidence exclusively stems from case series and case reports  and is fDeletions of amino acids from a protein structure generally result in greater structural changes than single amino acid changes, since these reduce the size of the protein and can trigger changes that propagate through the whole structure. Furthermore, the mechanism for the emergence of deletion variants appears to be very different from the single amino acid changes that are frequent from error-prone RNA replication and could involve deletions from RNA editing. Since CCP targets a large number of epitopes in the spike protein while mAbs target a single epitope, these molecular differences parallel what is expected from their respective selection pressures in the sense that escape from polyclonal preparations requires larger antigenic structural changes than escape from mAbs. In contrast to escape mutations selected for by mAb therapy, CCP selection yields point mutations throughout the spike protein. This reflects the vast antigenic surface area covered by the polyclonal antibodies within CCP. Escape mutations would be theoretically selected for on the basis of the most potent antibodies present in a particular CCP unit, which may vary markedly from donor to donor, which could explain the generally divergent evolution of SARS-CoV-2 in the presence of CCP. However, residues 141–144 and 243–244 are the sites of mutations or deletions in several cases, indicating these sites may offer effective escape from CCP derived from many donors, possibly by triggering a large-scale conformational rearrangement, as discussed above. As RBD binding antibodies are often neutralizing via ACE2 receptor occlusion, it is interesting that only 23% of CCP case studies identified the escape mutations within the RBD . This suNothing can be inferred about the fitness of an emerging mutant in the absence of selective pressure, but it is of interest that one variant with the E484K mutant that emerged after bamlanivimab therapy was able to infect multiple household contacts . In vitrWithin-host variation  is a natural phenomenon which has been reported for SARS-CoV-2 in immunocompetent patients and ultimately facilitates the persistence of infection. Among 33 patients having positive NPS PCR for an average of 18 days, Voloch et al., observed a distinguishing pattern of mutations over the course of the infection mainly driven by increasing A→U and decreasing G→A signatures, including spike mutations . G→A mutations are driven by the RNA-editing enzyme activities typical of innate immunity . NeverthImmunosuppression has been postulated to be an accelerator for viral evolution. Actually, On the other hand, co-administered small chemical antivirals can be mutagenic per se. Remdesivir has both amino and imino tautomers when pairing with RNA bases . Both amIn summary, our survey of the available mutational data show that escape variants associated with mAb and CCP therapy manifest different type of mutations. For mAbs, most mutations are single amino acid replacements in the RBD domain, while most variants eliciited in patients treated with CCP exhibited amino acid deletions. In fact, it is noteworthy that RBD mutations were relatively rare in CCP escape variants. Although the numbers are relatively small, which suggests caution in making generalizations, this dichotomy in geography of mAb and CCP mutations could reflect the fact that mAbs target a single epitope where the mAb–antigen interaction can be significantly altered by single amino acid changes while CCP targets many epitopes and has several mechanisms of action, such that evading polyclonal antibody immunity is likely to require much larger spike protein structural changes. Despite the relatively small set of variants for which there is molecular data available, the large variation of molecular solutions that allow SARS-CoV-2 to escape antibody-mediated protection is striking and suggest the need for continued vigilance in genomic surveillance, especially in cases refractory to therapy.


Interprofessional practice (IPP) has been shown to contribute to quality service provision and improved health outcomes. This knowledge has led to the integration of interprofessional education into course curricula for many health-care disciplines. Offering interprofessional education in rural areas to students undertaking work integrated learning placements is challenging particularly because of the diversity of students and placement dates combined with the student focus on the assessable placement curriculum.This research investigated and evaluated the utility of an escape room as an educational modality that facilitates learning whilst providing a supportive and motivating learning environment. Our project focused on the acquisition of interprofessional practice knowledge and experience by a health professional student cohort.This study used the novel intervention of an escape room combined with an interactive teaching session to test student engagement and learning about interprofessional practice and teamwork. The research used a mixed methods single group pre-post design.Fifty students  from seventeen universities and seven professions participated in teams of three to six members. Most participants (66%) had not previously completed an escape room. The results showed that the intervention provided effective and engaging learning and was intrinsically appealing to students despite its non-assessable nature. Individual student reflection on their participation showed developing insight into the critical importance of clear communication and intentional team member collaboration in the provision of effective interprofessional practice.The escape room intervention added value to the placement curriculum and proved flexible for a heterogeneous student cohort.The online version contains supplementary material available at 10.1186/s12909-021-02666-z. Collaborative or interprofessional practice (IPP) involves multiple health workers from different professional backgrounds working together with patients, their families, carers and communities to deliver health care , 2. IPP In Australia, regional, rural and remote areas have a shortage of health workforce, in part because university-based health professional training programs tend to be located in major cities and rural training exposure (e.g. placements) tend to be of short duration . One strRecognising the importance of interprofessional practice, our organisation is working to embed interprofessional learning into placement offerings for all students . This isEscape rooms are a relatively recent international entertainment phenomenon and are immersive “team-based games where players discover clues, solve puzzles, and accomplish tasks in one or more rooms in order to accomplish a specific goal … in a limited amount of time” . Escape The literature on escape rooms as educational environments, and the benefits of gamification where game elements are used in health education  is growiAs a result of the body of available evidence and our requirements to provide an educational session that both demonstrated team functioning and provided a social activity for students, a purpose-designed educational session incorporating an escape room was developed. This was to be used as a novel mechanism for interprofessional learning for placement students in our region and a means of contributing to the growing body of literature around escape rooms. With the assistance of a professional escape room designer, an escape room with a scenario which authentically highlighted the unique aspects of working as a health professional in our isolated tropical context, Nana’s Nightmare, was designed and piloted . FollowiTo investigate the utility of an escape room coupled with a debriefing workshop as an effective and engaging interprofessional learning activityTo evaluate the impact of the escape room on participant knowledge about interprofessional practice and teamworkTo evaluate the impact of the escape room through participant reflection on their personal contributions to the teamThis paper describes our research evaluating the effectiveness of using the format of the escape room to deliver an educational activity. The overall research question is whether an educational session incorporating an escape room can facilitate the acquisition of interprofessional practice knowledge and competencies in health professional students. Specific evaluation objectives were:The research used a mixed methods single group pre-post design  to evaluParticipants, health professional students, were recruited from the University database using email, via social media, posters in student common areas and workplaces, and indirectly through clinical supervisors and course coordinators. Snowball recruitment  occurredThe intervention was a session comprised of two components. It commenced with the escape room and was followed by an educational workshop focused on teamwork and interprofessional practice. Development and delivery of our portable, educational escape room for this project was completed with a budget of $3000 AUD. This included initial consultation fees for a professional escape room designer. The ‘room’ consisted of nine health-themed puzzles , props , and lesson materials that could be packed into a large suitcase and set up in most lecture or meeting-type rooms.The escape room started with an announcement to participants that the region was experiencing a cyclone and as a team of health professionals they had been requested to assist a residential aged care facility. Participants completed a locum staff registration form and then as a team undertook a check of the facility to ensure that all residents were safe. The team was provided with an emergency kit to assist residents as needed. During their room check, the team ‘discovered’ a resident, Nana, fallen out of bed and unconscious. By solving a series of puzzles that highlighted various health professional roles and Nana’s healthcare issues, the final puzzle required the team to call an ambulance for Nana and thus ‘escape the room’. After their escape, the team worked together to develop a health care plan for Nana and then participated in the workshop component. Table the characteristics of an effective teamthe definition, features and benefits of interprofessional practicethe barriers to, and enablers of, interprofessional practiceThe simulated, locum staff registration form completed by participants at the commencement of the escape room component collected participant demographic characteristics and their individual pre-intervention definition of interprofessional practice. The educational session was facilitated by one or two of the researchers and included didactic content and group-work (based on information obtained during the escape room) to present, or elicit:During the educational session participants individually assessed the team’s effectiveness on each of 11 team characteristics using an adaptation of Sharif and Nahas’ questionnaire –36. The Quantitative data was analysed descriptively and paired-samples t-tests used to compare pre and post session self-rated changes in knowledge. Qualitative data was synthesised using an inductive, descriptive, thematic analysis method based on processes described by Braun and Clarke , 38. Codn = 33, 66%) had not previously participated in an escape room of educational or entertainment design, some  had participated in one room, and one had participated in ten.The session was run eleven times with a total of fifty participants. All participants completed all parts of the session and evaluation. The number of participants in each session ranged from three to six. Most (98%) of participants were aged 19–35 years mean = 25.5). There were more females (78%) than males (22%). Participants were enrolled in seventeen universities, both Australian and international, with the most students from our own university; and came from years 2–6 of courses from seven different disciplines . When asked to rank a range of learning formats , 90% of participants ranked the escape room session as their preferred format to learn about teamwork and interprofessional practice.‘great chance to … learn more about interdisciplinary teamwork and … how I can improve as an effective team player’ (P29). ‘it was very educational’ (P43), ‘have gained insight into the need for IPP and will strive to implement to (sic) future care’ (P2), ‘I learnt a lot especially about interprofessional patient care’ (P35). They also mentioned the authentic nature of the scenario, ‘very hands on/visual, allows teamwork to solve realistic puzzles, very close to real life situations’ (P28), ‘liked the health/medical content’ (P13).Analysis of participant comments on the reason for their rating showed two main themes: the educational value (48% of participants) and the learning novelty (over 50% of participants). Concerning educational value, participants commented on their knowledge acquisition: Over 50% of participants mentioned the novel nature of the learning in the escape room. They used words such as fun, interesting, enjoyable, entertaining and engaging and specifically valued the organised interactive challenge that the escape room format provided. ‘I enjoyed how the escape room was an entertaining experience and how the debrief session was able to link the activity to interprofessional practice. It made the concept much easier to understand’ (P1).Three members of one team who were unsuccessful with ‘escaping’ the room rated the value of the experience 5 or less. One criticism was that ‘There didn’t seem to be a clear start with so many clues to work through. It was too manic to learn’ (P4). Interestingly, one member of this team provided two ratings indicating they differentiated between success in the game: ‘a one for ‘not making it out’, and the interactive game format: [a nine for] ‘presenting IPP in a fun interesting engaging manner’ (P48).Participants were asked to recall as many as possible of the eleven characteristics of an effective team which were presented to them during the 60-min educational session. Most participants recalled at least five of these characteristics. The top four characteristics recalled were ‘Communication’ (88%), ‘Clear purpose’ (54%), ‘Appropriate Culture’ (52%) and ‘Suitable leadership’ (52%). ‘Distinct roles’ were recalled by a third (32%) of participants. The least recalled characteristics were ‘Cohesion, trust and social relationship’ (22%), Commitment (14%), Decision-making and Conflict management (14%), ‘Relevant members’ (12%), flexibility (12%) and ‘Adequate resources’ (8%).interprofessional practice and multi-professional practice. Most frequently, participant’s post-session definitions included the concept of ‘working together to achieve a common goal for patient care’.Participant pre-session definitions of interprofessional practice ranged from ‘I don’t know’ (P20), through to ‘Interprofessional practice refers to using a multi-disciplinary approach to achieve the best outcome. In allied health this could refer to best patient care and outcome’ (P15). After the session, participants reflected on their original definitions and changed them if desired to reflect their newly acquired knowledge. Thirty-seven (74%) of participants adapted their definition. Overall the changes made indicated the participants had learned of the difference between Paired-samples t-test showed a statistically significant increase in knowledge for all five learning objectives. This is shown in Table Analysis of the free-text responses demonstrated participant’s increased understanding of interprofessional practice as a result of the session. The most frequently mentioned learning was the importance of different professions and skills contributing to a team (52%). Other frequently reported learnings were the characteristics of effective teams (28%), the barriers, enablers and benefits of interprofessional practice (26%) and the difference between interprofessional and multidisciplinary practice (22%).Participants reflected on their individual contributions as a team member during the escape room component. Secondarily, they reflected on whether the workshop component had triggered ideas to increase their effectiveness as an interprofessional team member. There were three main themes in the reflections.Improving communication (57%) was the most common theme. Participants noted that better communication required listening and recognising the ideas of others: ‘(be) more patient with other’s ideas. Sometimes if I thought someone wasn’t on the right track, they did actually end up being correct’ (P37). Improved communication also required increased discussion and a willingness to contribute your ideas and plans ‘I would make more of an effort to speak out and think out loud so other members of the team could feed off from my ideas’ (P22).The second theme related to taking a pro-active role (41%) in team relationships and leadership. Contributing more actively is illustrated by the participant who said they would: ‘try to play a more active role in the team’ (P45). Taking a leadership role encompassed understanding individual strengths and contributions as exemplified by: ‘have more clear roles and designated leadership possibly. In terms of my contribution perhaps do more to ensure everyone continues to work collaboratively’ (P24).The other common theme in responses (35%) focused on planning, increased organisation as a team and working together to solve problems: ‘stop and discuss what our plan is as a team and delegate roles and plan of attack, to avoid confusion and be time efficient’ (P4). This theme involved a recognition that teamwork required intentional effort and time invested by team members in order to get the best outcome: ‘define roles from the beginning’ (P10).The results appear to support the use of an escape room to engage a cohort of multidisciplinary, multi-university health professional students in learning about interprofessional practice and teamwork. High ratings of the session, combined with participant preference for the escape room over more traditional learning modes such as lectures, aligns with other evidence  that escOur result of lower enjoyment ratings from participants who failed to ‘escape’ also aligns with the literature. Hermanns et al. , who hadThe literature shows that learners value authentic over theoretical approaches to learning, and that appropriately contextualised learning supports optimal outcomes in interprofessional education . The gamIt has been argued that game-based learning impacts cognitive development, motivation, and decision making, because it triggers emotion . HeighteThis study demonstrated increased knowledge about interprofessional practice through the pre-post definitions provided by participants, and about teamwork and health professional roles by participant recall of team characteristics. This result combined with the participant reflection regarding their personal practices in the escape room team was interesting because it highlighted the new insights gained and the positive changes they would consider when contributing in future teams. Most participants recalled ‘communication’ as an important characteristic of teams and additionally highlighted the importance of improving communication to achieve better team outcomes. Our scenario encouraged communication between participants in the team and required participants to actively contribute to collaborative decision making to solve the problem. Interprofessional practice in healthcare provision requires collaboration and communication , 4, 41 bThe importance of more actively contributing, stepping up to leadership, is an attribute observed in well-functioning teams  that wasOur study, while comprising a reasonable size sample, was limited by the size of each professional group in the sample which was not reflective of the health workforce profile in our region. In addition, having the knowledge gain measured immediately post-workshop does not indicate the effectiveness of long-term learning. Longer term retention and application of knowledge could be a useful next step in our body of research on escape room learning. In addition, it is acknowledged that participant insights gained from the escape room experience may not directly transfer into workplace team practices, however our results suggest that the experience was beneficial for learning.As a standalone session, the escape room format with a post-game debrief and educational workshop appears to promote learning and reflective insights in a diverse range of student professionals. As an innately fun and social activity, it offered a motivating and novel learning environment compared with more traditional methods. Participants were able to participate at their own level of comfort and knowledge and were then scaffolded to reflect on and apply their experience to better understand interprofessional practice and effective team functioning. The benefit of offering the educational activity in this format was that it added value to the placement curriculum, was sufficiently flexible for a heterogeneous student cohort and was intrinsically engaging despite its non-assessable nature. By providing a setting for social learning and motivating students to learn, escape rooms can provide a useful adjunct to traditional classroom learning methods.Additional file 1.


Effective information management in the emergency department (ED) can improve the control and management of ED processes. Dashboards, known as data management tools, efficiently provide information and contribute greatly to control and management of ED. This study aimed to identify performance indicators quality dashboard functionalities, and analyze the challenges associated with dashboard implementation in the ED. This systematic review began with a search in four databases  from 2000 to May 30, 2020, when the final search for papers was conducted. The data were collected using a data extraction form and the contents of the extracted papers were analyzed through ED performance indicators, dashboard functionalities, and implementation challenges. Performance indicators reported in the reviewed papers were classified as the quality of care, patient flow, timeliness, costs, and resources. The main dashboard functionalities noted in the papers included reporting, customization, alert creation, resource management, and real-time information display. The dashboard implementation challenges included data sources, data quality, integration with other systems, adaptability of dashboard functionalities to user needs, and selection of appropriate performance indicators. Quality dashboards facilitate processes, communication, and situation awareness in the ED; hence, they can improve care provision in this department. To enhance the effectiveness and efficiency of ED dashboards, officials should set performance indicators and consider the conformity of dashboard functionalities with user needs. They should also integrate dashboards with other relevant systems at the departmental and hospital levels. Since the emergency department (ED) provides complex services and hosts patients in critical physical conditions, timely and precise service provision in ED is considered a challenge. MoreoveIt is important to employ quality dashboards to obtain timely information for effective control and management of ED processes. For effective information management and communications in the ED, key performance indicators and functionalities of the quality dashboards should also be determined in terms of user needs , 19. Thi2.1. Data sources and search strategyData search and extraction phases were performed based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist . The seaThe final search for papers was conducted on May 30, 2020. Moreover, this study spanned from June 1 to December 30. One researcher (SA) independently searched and retrieved the papers, whereas uncertainties were discussed with the other two authors (RR and HM). The relevant papers were also retrieved through a search in Google Scholar and Google. The search for papers was finalized with a bibliographic check of the designated papers. 2.2. Inclusion and exclusion criteriaThe inclusion criteria were: 1) papers written in English; 2) papers on ED quality dashboards; and 3) papers on performance indicators and functionalities of ED quality dashboards. Moreover, the exclusion criteria were: 1) non-English papers; 2) papers merely designing a clinical dashboard for the ED; or 3) papers examining the dashboards of other hospital wards.2.3. Paper selection, paper evaluation, and data extractionIn the screening step, three authors  checked the titles and abstracts of the papers and irrelevant papers were removed. In the eligibility step, the papers were independently checked by the same noted authors. The bibliography check was then conducted by one of the authors (SA). The quality assessment of the papers based on Cochrane Effective Practice and Organization of Care (EPOC) guideline  was indeFor data extraction, the performance indicators of the ED were first extracted from the papers. The performance indicators used by the ED quality dashboards mentioned in the reviewed papers were classified as patient flow, timeliness, quality of care, costs, and resources  22). Th. Th22). A total of 1275 papers were retrieved through the search in databases. Additionally, four papers were retrieved from a search attempt on Google Scholar and Google. After removing the duplicates using endnote software, 484 papers remained. The titles and abstracts of the papers were then reviewed. As a result, 423 papers were removed and 61 remained. In the next step, the full texts of papers were checked and this resulted in the removal of 42 papers. Ultimately, 18 papers remained for analysis. One more paper was also retrieved when performing a bibliographic check of the designated papers. No paper was removed after quality assessment, and all the papers entered the final analysis phase. Finally, 19 papers were reviewed in this study. 3.1. Quality assessmentAccording to the quality assessment of papers, three studies , 23, 24 3.2. ED quality dashboard performance indicatorsThe performance indicators  were reported in five main categories . The majority of performance indicators were related to patient flow (8 indicators), timeliness (7 indicators), quality of care (4 indicators), and costs (2 indicators), respectively. The following indicators belonged to the patient flow category: the number of patients discharged and the type of discharge  (in 11 papers), the total number of people visiting the ED (n=10), the number of patients in the ED  (n=8), the number of patients admitted per triage level (n=6), how the patients visited the ED , and the number of patients for whom a decision was made in six hours (n=4). The timeliness category consisted of the following performance indicators: the patients’ mean length of stay (n=16), the time of triaging (per each triage level) (n=4), and the mean time elapsed since the patients’ arrival in the ED until the onset of triaging (n=4). Only three out of the 19 papers had mentioned the quality of care performance indicators, which included patient revisits (before 72 hours) (n=3) and the percentage of mortality among the patients admitted to the ED (in=2). The cost performance indicators included the number of tests ordered by the doctors (n=8) and the number of consultations given (n=3). Finally, resource performance indicators were the number of beds  (n=7) and the number of personnel in the ED (divided by discipline and gender) (n=6).3.3. ED quality dashboard functionalitiesAll the 19 papers discussed the reporting functionality. This was followed by customization (n=12), alerting (n=10), 32-35, 3.4. Challenges to use of quality dashboards in EDThe challenges associated with the use of quality dashboards in the ED included a lack of integration with other hospital systems and inputting the data manually, 32, lacThis study aimed to identify the performance indicators, functionalities, and challenges of quality dashboards in the ED. Some studies had examined the effects of clinical/quality dashboards on patient care improvement or analyzed the functionalities and positive and negative effects of using dashboards in hospital, 41. DetThere are limitations with the current study that could be addressed in future research. First of all, the current study reviewed studies published in English and there might be useful relevant studies that were excluded. In addition, although we systematically studied the key performance indicators of quality dashboard in the emergency department, we did not include studies that addressed clinical dashboards of this department due to aim of the study. In the analysis step, an attempt was made to avoid or minimize the risk of bias, as any possible discrepancy experienced by either of the first three authors were discussed in group. However, there may have been issues that did not catch our attention. The findings of the current study indicated that applying key performance indicators for ED quality dashboard, including quality of care, patient flow, timeliness, costs, and resources, could improve utilization management and quality of care. Moreover, a quality dashboard should support functions such as customization, reporting, real-time information display, resource management, alert creation, and automated data collection. In addition, the use of drill-up, drill-down, and drill-through features could help improve efficiency and effectiveness of ED quality dashboard. With respect to the challenges noted, it is suggested that in further developments of ED dashboard, attention should be paid to data sources, the quality of data, and integration with other systems both in the ED and in other hospital departments. None.Concept and study design: SA, RR, HM, MVA; literature search, study selection and data collection: SA; analysis and interpretation: SA, RR, HM; Writing the article: SA; critical revision of the article: RR, HM; final approval of the article: SA, RR, HM, MVA.This systematic review did not receive any funding. This review was part of a larger study, approved by the Ethics Committee of Shahid Beheshti University of Medical Sciences, Iran (IR.SBMU.RETECH.REC.1399.565). The authors declare that they have no competing interests.


Tumor necrosis factor alpha (TNF-α) plays important roles in processes such as immunomodulation, fever, inflammatory response, inhibition of tumor formation, and inhibition of viral replication. TNF-α and its receptors are ubiquitously expressed in developing organs and they regulate the survival, proliferation, and apoptosis of embryonic stem cells (ESCs) and progenitor cells. TNF-α is an important inflammatory factor that also regulates the inflammatory response during organogenesis, and its cytotoxic effects can interfere with normal developmental processes, even leading to the onset of diseases. This review summarizes the various roles of TNF-α in organogenesis in terms of its secreting pattern, concentration-dependent activities, and interactions with other signaling pathways. We also explored new potential functions of TNF-α. Tumor necrosis factor alpha (TNF-α) belongs to the TNF superfamily  of proteThis review summarizes the roles of TNF-α in the development of various organs and associated diseases. We also highlight the mechanisms of TNF-α from multiple aspects, including its effects on stem cells/progenitor cells, secreted forms, concentration-dependent activities, and interactions with other signaling pathways.The effects of TNF-α can be traced back to gastrulation, during which it promotes embryonic differentiation and cell apoptosis . Organogin vivo and in vitro , progenitor cells, and differentiated cells. It not only inhibits the self-renewal of mouse ESCs but also induces their apoptosis and inhibits their differentiation into embryos . TNF-α pin vitro .Tumor necrosis factor alpha is also involved in regulating the proliferation, apoptosis, and differentiation of progenitor cells. It promotes the proliferation and differentiation of neuroblasts into astrocytes in the human fetal cortex , inhibitAlthough transmembrane TNF-α (pro-TNF) is biologically active, TNF-α primarily exerts autocrine and paracrine functions in a soluble, trimeric form during developmental processes. TNF-α acts as an autocrine and paracrine growth factor that stimulates the proliferation of hematopoietic cells and B cells . AutocriLow concentrations of TNF-α tend to promote cell proliferation, whereas high concentrations tend to inhibit cell proliferation and even induce the apoptosis of neural stem cells/progenitor cells in the subventricular zone of neonatal mice, and of osteoclast progenitor cells and intestinal stem cells in fetal mice . The bioTumor necrosis factor alpha interacts extensively with fibroblast growth factor (FGF) family, Wnt family, and TGF-β superfamily members to co-regulate developmental processes. It promotes angiogenesis by inducing basic FGF and FGF-1 expression in endothelial cells .Tumor necrosis factor alpha inhibition promotes the functional recovery of nerves by activating the Wnt3a signaling pathway in BMSCs . TNF-α iTumor necrosis factor alpha regulates the differentiation of osteoblasts by affecting the bone morphogenetic proteins (BMP) signaling pathway . It alsoTumor necrosis factor alpha induces the expression of hepatocyte growth factor in human bone marrow- or adipose-derived progenitor cells and in MSCs to promote tissue growth and repair . TNF-α hin vivo and in vitro and negatively affects brain development in vivo  or surrounding cells . TNF-α tends to promote cell proliferation at low concentrations and inhibit cell proliferation and induce apoptosis at high concentrations. TNF-α interacts extensively with the FGF, Wnt, and TGF-β signaling pathways to co-regulate organogenesis. It is also an important inflammatory factor that regulates the inflammatory response and exerts cytotoxic effects. The overwhelming cytotoxic effect of TNF-α during organogenesis due to inflammation interferes with normal developmental processes and can trigger disease onset. Despite considerable knowledge about TNF-α and its functions in organogenesis, several questions remain. For example, how TNF-α prevents the activation of undesirable immune responses during developmental processes remains obscure. Low concentrations of TNF-α promote the development of intestinal epithelium without triggering inflammatory response , but wheKY was in charge of acquisition of reference articles and critical revision of the manuscript for important intellectual content. HG and ZY were in charge of critical revision of the manuscript for important intellectual content. XX was in charge of drafting of the manuscript and supervision. All authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Tumour necrosis factor (TNF)-α plays an important role in inflammatory, infectious, and tumor processes, and it is closely related to the early stages of gastric cancer. It is a pro-inflammatory cytokine, produced not only by macrophages and monocytes but also by the lymphocytes, mast cells, neutrophils, keratinocytes, smooth muscle cells, and some tumor cell lines. Large amounts of TNF are released upon contact of macrophages, CD4 + T lymphocytes, and natural killer (NK) cells with lipopolysaccharides, bacterial products, and interleukin 1. TNF-alpha inducing protein (Tipa) is a unique carcinogenic factor of Helicobacter pylori, secreted into culture broth. The biological activities of TNF alpha and deletion mutant were studied. TNF alpha protein specifically binds to cell-surface nucleolin and then enters the gastric cancer cells, where TNF-a and chemokine gene expressions are induced by NF-jB activation. Nucleolin localizes on the surface of gastric cancer cells, and interaction between TNF alpha and cell-surface nucleolin causes a cancer-oriented microenvironment that increases the risk of gastric cancer. This paper discusses a mechanism of gastric cancer development and the clinical significance of tumor necrosis-alpha and carcinoembryonic antigen in gastric cancer. The carcinoembryonic antigen (CEA) is a glycoprotein with a molecular weight of 200 kDa, containing approximately 40% proteins and 60% carbohydrates. CEA is one of the oncofetal antigens produced during embryonic life and fetal development. From the immunohistological perspective, CEA may be determined in high concentrations in the fetal gastrointestinal tract and pancreas, colorectal adenocarcinomas, and low concentrations in normal tissue and the intestinal mucosa, exocrine pancreas, and liver , 2. ArouSerological tumor markers are widely used for the diagnosis, the effect of the treatment, and gastric cancer surveillance. Numerous studies have assessed and analyzed the plasma values of tumor markers for gastric cancer, but no correlation has been made between TNF alpha, the carcinoembryonic antigen, CA19-9, and CA 72-4 . A studyInflammation is considered the most important factor in the pathogenesis of cancer, so this is why the polymorphism of genes involved in inflammation has been carefully studied in recent years. TNF alpha is the most studied marker in gastric cancer, and it has been shown that at the cellular level, its polymorphism is of particular importance as the gene is identified at the level of chromosome 6. The biologically active form of TNF-α is the trimer, and, in addition to this soluble form, there is also a membrane-bound form located on the surface of the TNF alpha-producing cells, which serves as a support for the soluble form by proteolytic cleavage. The aim of this review paper is to highlight the clinical significance of these specific markers, i.e., TNF alpha and CEA, for the development of gastric cancer as well as for the risk of other inflammatory conditions and a possible correlation of other biomarkers like CA 72-4 in patients with gastric cancer.We searched the PubMed database for studies related to the 2 proteins  we have chosen in the study. Consequently, we selected the most recent and significant results to illustrate the clinical significance of the two markers and their correlations in patients diagnosed with gastric cancer and intestinal inflammation. We studied articles from the database from 2008 to 2020, respectively 28 articles about TNF alpha and 16 CEA articles related to gastric cancer.Recent studies show that serum concentrations of TNF alpha and CEA may be associated with gastric cancer and the pathogenesis of the gastrointestinal disease. TNF alpha and CEA may be potential predictors of gastric cancer in patients with benign gastric lesions. Serum CEA levels provide additional prognostic information for patients with primary gastric cancer. In particular, elevated serum CEA levels provide additional prognostic information and are useful predictors of curability in patients undergoing gastrectomy. Serum CEA levels are an independent prognostic factor in patients with primary gastric cancer .TNF-alpha clearly plays a major role in establishing a link between inflammation and cancer. Because TNF-alpha is also needed for the proper functioning of the immune system, complete suppression of TNF-alpha over a long period is likely to prove harmful. Any chronic inflammatory condition linked to the majority of the inflammatory diseases could be a potential target for anti-TNF-alpha therapy –17.The tumor necrosis factor TNFαlpha plays an important role in the process of inflammation, infection, and tumors and has central importance in the early stages of gastric cancer. The relationship between TNFα and CEA is well established but contradictory and controversial. A pro-inflammatory cytokine genetic profile increases the risk of non-cardia gastric adenocarcinoma but not other upper gastrointestinal cancers, possibly by inducing a hypochlorhydria and atrophic response to gastric H. pylori infection.This review described the characteristics of gastric cancer, taking into account epidemiology, risk factors, and possible correlations between TNFα and CEA. Geographical variations and gender need to be taken into account. Several risk factors such as family history, diet, alcohol, smoking, Helicobacter pylori, and Epstein-Barr infection are also important. Polymorphisms in the TNF-α promoter affect GC risk. High TNF producer alleles are associated with increased risk. The TNF-α polymorphism is H. It may enhance carcinogenesis through chronic inflammation of Helicobacter pylori. Previous studies have outlined many controversial results based on the correlation between specific and non-specific biomarkers of gastric cancer. Therefore, there is an urgent need to identify other links between biomarkers, immunohistochemical studies, and molecular biology tests.The authors declare no conflict of interest.MCR and BDT designed and conducted the research. SDM and ROP revised the manuscript and made critical changes. PDM and AA collected and analyzed data to reduce bias.


International Journal of Neonatal Screening (IJNS). Here, I will kick this off, with an appraisal of Issue 4 of Volume 6 of IJNS [The Editorial team  is trying something new. We will take turns in helping you to make your choice of the wide variety of papers we offer in each new issue of the  of IJNS .Issue 4 lists 21 papers. Sadly, this issue holds two obituaries of our beloved colleagues Fred Lorey , which yThen, many of these contributions belong to Special Issues; there are five papers in the Special Issue “Neonatal Screening for Mucopolysaccharidoses” , four inIJNS is preparing, and we already have some very exciting contributions to this Special Issue  [The paper of Dr. Mei Baker and co-workers on molecular technologies in newborn screening is a must-read . It is aof 2021) .Of course, we recommend you dive into all the papers of this issue, but my recommendation would be “We All Have a Role to Play: Redressing Inequities for Children Living with CAH and Other Chronic Health Conditions of Childhood in Resource-Poor Settings” by Kate Armstrong et al. . AdmitteI feel that this paper is inspiring, with authors from five countries that together share a success story based on two frameworks. These frameworks can easily be adopted and applied in other regional settings. In the words of the authors: “share the wheel, don’t reinvent it”. This paper is about promoting equity and disseminating knowledge, and that, I repeat, is at the heart of the mission of ISNS.