In eukaryotes, trans-splicing is a process of nuclear pre-mRNA maturation where two different RNA molecules are joined together by the spliceosomal machinery utilizing mechanisms similar to cis-splicing. In diverse taxa oflower eukaryotes, spliced leader (SL) trans-splicing is the most frequent type of trans-splicing, when the same sequencederived from short small nuclear RNA molecules, called SL RNAs, is attached to the 5’ ends of different non-processedpre-mRNAs. One of the functions of SL trans-splicing is processing polycistronic pre-mRNA molecules transcribed fromoperons, when several genes are transcribed as one pre-mRNA molecule. However, only a fraction of trans-splicedgenes reside in operons, suggesting that SL trans-splicing must also have some other, less understood functions. Regenerative flatworms are informative model organisms which hold the keys to understand the mechanism of stemcell regulation and specialization during regeneration and homeostasis. Their ability to regenerate is fueled by thedivision and differentiation of the adult somatic stem cell population called neoblasts. Macrostomum lignano is a flatworm model organism where substantial technological advances have been achieved in recent years, including thedevelopment of transgenesis. Although a large fraction of genes in M. lignano were estimated to be SL trans-spliced,SL trans-splicing was not studied in detail in M. lignano before. Here, we performed the first comprehensive study ofSL trans-splicing in M. lignano. By reanalyzing the existing genome and transcriptome data of M. lignano, we estimatethat 30 % of its genes are SL trans-spliced, 15 % are organized in operons, and almost 40 % are both SL trans-splicedand in operons. We annotated and characterized the sequence of SL RNA and characterized conserved cis- and SL transsplicing motifs. Finally, we found that a majority of SL trans-spliced genes are evolutionarily conserved and significantlyover-represented in neoblast-specific genes. Our findings suggest an important role of SL trans-splicing in the regulation and maintenance of neoblasts in M. lignano. Before being used as templates for protein production, majority of RNA molecules transcribed in the nucleus (pre-mRNA)undergo three major modifications to become mature andfully functional mRNA. This is called RNA processing and itinvolves capping of the 5′ end, polyadenylation of the 3′ end,and splicing. Two types of splicing are distinguished – cis- andtrans-splicing. During cis-splicing all the processing happenswith the same pre-mRNA molecule, resulting in the removalof introns and merging of its exons. During trans-splicing, onthe other hand, two different pre-mRNA molecules expressedfrom distinct genomic loci are joint into a new chimeric transspliced mRNA .Trans-splicing was originally discovered in trypanosomes(Euglenozoa), where it was found that a short 39 bp leadersequence was post-transcriptionally attached to the 5′ ends ofvariant surface glycoproteins pre-mRNA . Later, 5′ end addition of a 22 bp spliced leader (SL)was also observed in Caenorhabitis elegans mRNA of actingene and some other genes . Now thisprocess is well known as SL trans-splicing. A distinct featureof SL trans-splicing is that all such processed transcripts havethe same short SL sequence, or its variant, at their 5′ ends. TheSL sequence is derived from an exon of a non-coding smallnuclear RNA molecule called SL RNA, which is ~100 nt inlength and has 2,2,7-trimethylguanosine cap at its 5′ end instead of 7-methylguanosine cap, which is found in non-transspliced mRNAs . SL RNAs have a splicing donor site at the exon 3′ end,while SL trans-spliced pre-mRNAs have a splicing acceptorsite at the 5′ end of their first exon. SL trans-splicing resultsin removal of the 5′ non-exon pre-mRNA part called outron. It is experimentally shown thatthe only requirement for a gene to be predominantly SLtrans-spliced is an acceptor splicing site close to the 5′ endof the first exon that is not complemented by a donor splicing site upstream in cis . Thus comesanother important feature of SL trans-splicing, namely that itallows formation and resolving of operons – adjacent genestranscribed as a single pre-mRNA from the same promoterregion . However, apart froma clear function in polycistronic transcripts resolution, thefunction of SL trans-splicing for monocystronic transcriptsis still in debate . It is hypothesized thatthe function may be in equalization of 5′ UTRs in length andtheir clearance from out-of-frame AUG start codons, while atthe same time allowing less restricted evolution of 5′ upstreamregulatory sequences, and in additional control of translation. So far, SL transsplicing was found in several clades of eukaryotes: dinoflagellates, euglenozoans, cnidarians, flatworms, nematodes,and ascidians . SL trans-splicing is mostprominent in trypanosomes (100 % genes are trans-spliced)and in nematodes (70 % genes are trans-spliced) . Regenerative flatworms are informative models to understand the mechanism of stem cell regulation and specializationduring regeneration and homeostasis. Their ability to regenerate is driven by the division and differentiation of the adultsomatic stem cell population called neoblasts . Macrostomum lignano is the onlyflatworm species for which a method for stable transgenesisis available so far. The worm also has a number of featuresallowing for efficient cell lineage tracing and phenotypescreening, which makes M. lignano an attractive model tostudy a wide range of biological processes . Well-annotatedM. lignano genome and transcriptome assemblies were recently published . It was estimated that almost 21 % of its genes are SLtrans-spliced to the same 35 bp SL sequence . However, trans-splicing was not studied in details in M. lignano, and its impact on the genome functioningand maintenance is still unknown. Here, we present the firstcomprehensive study of SL trans-splicing in M. lignano andshow that it is strongly connected with genes specific for theneoblasts of the worm.Data. The published M. lignano genome Mlig_3_7  and transcriptome Mlig_RNA_3_7_DV1_v3 assemblies and the correspondingannotation tracks were obtained from (http://gb.macgenome.org/downloads/Mlig_3_7/).Genome deduplication. Mlig_3_7 genome assemblywas deduplicated using purge_dups software (v1.0.1) withdefault settings  and utilizing publishedPacBio genome sequencing data  for thecalculation of contig coverages. Contig names from the deduplicated genome assembly were used to extract respectivegene annotations from the full Mlig_3_7 genome annotation.Motif discovery and SL RNA annotation. Presence ofthe SL sequence at the 5′ end of the M. lignano transcriptswas established in the previous studies . For the annotation of trans-splicedgenes, SL-containing RNA-seq reads were mapped to the Mlig_3_7 genome assembly and the presence of such readsat the beginning of transcripts was used as an evidence ofSL trans-splicing . Therefore, all the SL trans-spliced transcripts have thecorresponding annotation in the Mlig_3_7 genome assembly,and the sequences upstream of their first exon were consideredas outrons. Using the deduplicated annotation track of genecoordinates, we retrieved nucleotide sequences of genomicregions corresponding to exon-intron and exon-outron (forthe trans-spliced genes) junctions with 50 bp flanks in bothdirections. All the sequences were converted to forward orientation and split into three groups corresponding to cis-donor,cis-acceptor, and trans-spliced acceptor sites. The sequencesthen were analysed for the presence of enriched motif usinga stand-alone version of the DREME tool . To determine the SL RNA gene sequence in the genomeassembly, we used the 35 bp M. lignano SL sequence (CGGTCTCTTACTGCGAAGACTCAATTTA TTGCATG) as aseed for a BLASTn  search requiringonly 100 % matching hits. Next, we manually investigatedgenomic sequences surrounding the BLAST hits by matchingthe SL sequence track in the genome browser to the expectedsize of SL RNA (~100 bp). The corresponding sequences werethen checked for folding into secondary structure canonicalfor SL RNA folding using Mfold web server ,and conserved motifs were then manually identified.Prediction of operons. Intergenic distances were retrievedfrom the deduplicated genome annotation file. We only considered distances between immediately adjacent transcriptswith the same transcriptional orientation and not interruptedby transcripts in opposite orientation. Distances were splitinto three categories: between SL trans-spliced genes, between a non-SL trans-spliced gene and an SL trans-splicedgene, and between non-SL trans-spliced genes. To adjust forrepetitive element insertions, we retrieved the correspondingcoordinates from the genome browser RepeatMasker and TRFtracks (http://gb.macgenome.org/) and subtracted them fromthe previously identified intergenic distances. Distribution ofthe distances was visualized as density plots using ggplot2library in R.After the analysis of the graphical data of the distancesdistributions, we selected the threshold value of 1000 bp, below which a pair of adjacent and SL trans-spliced genes wereconsidered as belonging to the same operon. The same appliesif the first gene is non-trans-spliced, but the second is SL transspliced. The distributions of lengths of operons of varioussizes was visualized as violin plots using ggplot2 library in R.Estimation of gene conservation. Gene annotation anddata classifying genes as being specific to neoblasts or germline were retrieved from the previous study . A gene was considered to be conserved if it has anopen reading frame with a detectable homology to a humangene, which is indicated in its annotation, and non-conservedif lacking the homology to human, but has a predicted openreading frame with homology to proteins in other organisms.Otherwise, a gene was considered non-coding. Deduplication of genome assembly. The published Mlig_3_7genome assembly is based on the sequencing data from DV1 M. lignano line. This line has a 2n = 10 karyotype  and was demonstrated to haveundergone a duplication of its large chromosome , while the karyotype of the basal wild typepopulation is 2n = 8 . The size of Mlig_3_7 assembly is764 Mb, which corresponds to the experimental measurementof the genome size in the DV1 line ,and the assembly contains the duplicated large chromosomesequences. To avoid gene overcounting due to the presenceof these duplicated sequences in the Mlig_3_7 assembly,we removed the most redundant scaffolds by deduplicatingMlig_3_7 assembly using purge_dups software . This resulted in approximately 46 % drop in the numberof scaffolds (from 5270 to 2841) and decreased the genomesize to 580 Mb, which is close to the genome size measurements for the NL10 line of M. lignano, which does not havethe chromosomal duplication . Next,we removed the records from transcriptome annotation whichcorresponded to the redundant scaffolds.Motif discovery and SL RNA gene mapping. Investigation of the deduplicated part of the transcriptome shows thata significant fraction of genes, 21 754 out of 71 499 (30 %),are SL trans-spliced in M. lignano. This means that they allhave the same 35 bp SL sequence (CGGTCTCTTACTGCGAAGACTCAATTTA TTGCATG) at the 5′ end of their processed transcripts . Despite this, SL trans-splicing was not characterizedin more detail in M. lignano. First, we retrieved genomicDNA sequences near the cis-splicing and SL trans-splicingexon-intron/exon-outron junction sites and checked if they areenriched for some motifs using DREME  . In total, we obtained 187 627 regions around 5′ donorand 3′ acceptor cis-splicing sites and 21 754 regions aroundSL trans-splicing sites. The first most enriched motifs nearcis-splicing 5′ donor and 3′ acceptor sites were GT[G/A]AG and CAG, respectively,corresponding to canonical cis-splicing motifs. A motif [T/C]TNCAG  was thetop enriched motif near SL trans-splicing 3′ acceptor sites.All the motifs were positioned right at the exon-intron/exonoutron junctions of the corresponding sites Next, to confirm the presence of the SL RNA gene in thegenome assembly, we analysed the secondary structure ofthe previously published sequence of M. lignano SL RNAfrom the ML2 version of the genome .However, we found that the reported sequence was erroneously assigned as SL RNA, since it clearly maps to the 5′ endof an SL trans-spliced protein-coding gene  in the Mlig_3_7 assembly, and alsodoes not fold into canonical structure with three hairpin loops(data not shown) . Therefore, we decided toidentify the actual SL RNA gene in the newer Mlig_3_7 assembly. Using SL sequence as a seed for the genomic BLASTnsearch, we mapped a 109 bp sequence, which is repeatedeight times in the deduplicated genome and has the canonicalSL RNA secondary structure predicted by Mfold web server . Subsequent sequence analysisshowed clear signatures of an SL RNA: the SL sequence is at the 5′ end of the gene and forms the first hairpin loop,immediately after the SL sequence there is a clear 5′ donorsplicing site (GTAAG), and between two other hairpin loopsthere is a motif similar to the binding site of Sm spliceosomalprotein  .Operon analysis. The important feature of SL transsplicing is that it allows for processing of long polycistronicpre-mRNA molecules expressed from a single promoter region in a way similar to prokaryote operons. In principle, genome-guided transcriptome assembly using RNA-seq dataallows identification of such operons and their correspondingpre-mRNA sequences, which we previously annotated as transcriptional units . However, it is not always possible to fully reconstructan operon from RNA-seq data alone, since transcriptional unitspredicted from RNA-seq data tend to split in the repeat-richintergenic regions of operons, where read coverage dependson both operon expression level and the frequency of repeatsin the genome. Instead, to estimate what fraction of M. lignano genes are organized in operons based on their genomicorganization, we first explored how intergenic distances between trans- and non-trans-spliced genes are distributed inM. lignano genome . We found that distributionof distances between trans-spliced genes has multimodaldistribution, while it is unimodal distribution for non-transspliced/trans-spliced and non-trans-spliced/non-trans-splicedintergenic distances . SL trans-splicing is anancient evolutionary mechanism , which ismostly abundant in the genomes of simply organized organisms, which have low repetitive content and relatively smallgenomes . We hypothesized that neutralaccumulation of repeats could have influenced the distancesbetween genes in the operons. Interestingly, after we adjustedthe intergenic distances by subtracting the fraction occupiedby repetitive sequences (simple repeats and transposableelements), it had the most impact on the distances betweentrans-spliced genes, revealing a clear bimodal distributionwith the most prominent peak at around 100 bp .This observation indicates that repeats have a substantialcontribution to intergenic distances in operons. To classifygenes as belonging to the same operon, we decided to usethe repeat-adjusted distances with a threshold value of 1 Kb,which separates the two modes of the intergenic distancesbetween trans-spliced genes .Using these criteria for defining operons, we found that10 458 genes  can be assigned to operons, of which1854 (18 %) start from a non-trans-spliced gene . The vast majority of them are comprised of two andthree genes (75 and 18 %), with the maximum operon sizereaching nine genes (two operons) . An exampleof an operon defined in this way is provided in Fig. 2, c.SL trans-splicing is enriched in evolutionary conservedand stem cell genes. We know from a previous study  that evolutionary conserved proteincoding genes, which still have detectable homology betweenM. lignano and human, are enriched in somatic stem cells –neoblasts  . Onthe contrary, only 38 % of germline-specific genes in M. lignano are conserved in human, suggesting their relativelyrecent appearance in evolution of flatworms. We investigatedwhether there is a correlation between gene conservation and SL trans-splicing in M. lignano. We found that 69.9 % of theSL trans-spliced genes are conserved between M. lignanoand human, while 24.3 % are not conserved and 5.8 % arenon-coding . Trans-spliced genes that are locatedin operons have a very similar distribution of conserved,non-conserved and non-coding genes . In contrast,among non-trans-spliced genes only 36.9 % are conserved inhuman, while 42.9 % are non-conserved and 20.2 % are noncoding . Thus, SL trans-spliced genes are stronglyenriched for conserved genes but there is no dependence onwhether these genes are in operons or not.Next, we calculated the fraction of SL trans-spliced genesamong genes enriched in neoblasts (stem cells) and germline – the only proliferation capable cell types in the worm. Intriguingly, 85 % of the stem cellgenes (746) are SL trans-spliced, and almost 86 % (752) areconserved in human (see the Table), and 728 genes are bothconserved in human and SL trans-spliced, which is 96.8 % ofall the conserved genes in neoblasts. Given that out of 33 525conserved genes present in the Mlig_3_7 genome annotation15 201 (45.3 %) are trans-spliced , this represents a 2.13-fold enrichment for conserved SL trans-spliced genesamong neoblast genes relative to the expected from the random distribution . On thecontrary, only 18 % of the germline genes are SL trans-splicedand 37 % are conserved in human. Taken all together, thissuggests that SL trans-splicing plays an important role in stemcell regulation in M. lignano.SL trans-splicing is widespread in diverse flatworm taxa,including both parasitic and free-living species . However, most of the studies of SL trans-splicingwere focused on nematodes and trypanosomes . Here, we performed the firststudy which focuses on SL trans-splicing in the free-livingregenerative flatworm model M. lignano. By reanalyzing theavailable genome and transcriptome data, we found that 30 %of the worm genes are SL trans-spliced, and 15 % are estimatedto be organized in operons . For a comparison, inC. elegans 70 % of genes are SL trans-spliced and 17 % arein operons, in ascidian chordate Ciona intestinalis it is 58 and20 %, respectively, and in the parasitic liver fluke Schistosomamansoni 11 % are SL trans-spliced with a few genes in operons. Among free-living flatworms,trans-splicing was studied before , but there is no firm estimation of its abundance andprevalence of genes in operons. The size of operons in M. lignano also varies similarly to C. elegans, where it ranges fromtwo to eight genes, with the most frequent intergenic distancearound 100 bp, and the majority of operons comprised of twogenes  . The most striking finding of our study is that most ofM. lignano SL trans-spliced genes are evolutionary conserved and, most importantly, that overwhelming majority of neoblast-specific genes (85 %) are SL trans-spliced(see the Table). Interestingly, 39 % of neoblast genes arealso clustered in operons (see the Table), suggesting theirearly evolutionary origin and the necessity for synchronizedexpression and similar transcriptional regulation. Neoblastsare the key players of outstanding regeneration capacity infree-living flatworms, and thus they are the primary subjectof the studies on flatworm regeneration. All the tissue renewaland growth in adult flatworms is due to neoblast proliferationand differentiation . Our data clearly indicates importanceof SL trans-splicing for the gene regulation of neoblasts inM. lignano and lay ground for further studies of how exactlySL trans-splicing machinery contributes to different stages ofneoblast activitySpliced leader trans-splicing affects a substantial fractionof M. lignano genes. We annotated and characterized thesequence of SL RNA, identified the conserved motifs at theexon-intron/exon-outron junction sites in cis- and SL transspliced genes, and provided the first comprehensive analysisof genes comprising operons in M. lignano. Most importantly,we found that the SL trans-spliced fraction is over-representedby evolutionary conserved protein-coding genes, in contrastto the non-trans-spliced part of the genome, and that the stemcell-specific genes are predominantly SL trans-spliced. Ourfindings suggest an important and evolutionary conservedrole of SL trans-splicing in regulation and maintenance ofneoblasts in M. lignano. Thus, a thorough investigation ofthe molecular mechanism of SL trans-splicing is required tofully understand the regulation of regeneration and stem celldifferentiation in flatworms.The authors declare no conflict of interest.Allen M.A., Hillier L.W., Waterston R.H., Blumenthal T. A global analysis of C. elegans trans-splicing. 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To evaluate whether C reactive protein point-of-care testing (CRP POCT) safely reduces antibiotic prescribing for lower respiratory tract infections in nursing home residents.Pragmatic, cluster randomised controlled trial.The UPCARE study included 11 nursing home organisations in the Netherlands.84 physicians from 11 nursing home organisations included 241 participants with suspected lower respiratory tract infections from September 2018 to the end of March 2020.Nursing homes allocated to the intervention group had access to CRP POCT. The control group provided usual care without CRP POCT for patients with suspected lower respiratory tract infections.The primary outcome measure was antibiotic prescribing at initial consultation. Secondary outcome measures were full recovery at three weeks, changes in antibiotic management and additional diagnostics during follow-up at one week and three weeks, and hospital admission and all cause mortality at any point .v 90.8%), 2.2% in all cause mortality rates (3.5% v 1.3%), and 0.7% in hospital admission rates (7.2% v 6.5%). The odds of full recovery at three weeks, and the odds of mortality and hospital admission at any point did not significantly differ between groups.Antibiotics were prescribed at initial consultation for 84 (53.5%) patients in the intervention group and 65 (82.3%) in the control group. Patients in the intervention group had 4.93 higher odds  of not being prescribed antibiotics at initial consultation compared with the control group, irrespective of treating physician and baseline characteristics. The between group difference in antibiotic prescribing at any point from initial consultation to follow-up was 23.6%. Differences in secondary outcomes between the intervention and control groups were 4.4% in full recovery rates at three weeks (86.4% CRP POCT for suspected lower respiratory tract infection safely reduced antibiotic prescribing compared with usual care in nursing home residents. The findings suggest that implementing CRP POCT in nursing homes might contribute to reduced antibiotic use in this setting and help to combat antibiotic resistance.Netherlands Trial Register NL5054 Antimicrobial resistance is a growing worldwide public health threat. An important driving force is inappropriate antibiotic use.C reactive protein point-of-care testing (CRP POCT) for suspected lower respiratory tract infections might contribute to prompt and appropriate decisions of whether or not to prescribe antibiotics, or to suggest additional investigations. CRP is an acute phase protein synthesised by the liver as a non-specific response to inflammatory stimuli. CRP levels respond dynamically to the presence (increase within six hours) and relief  of inflammation.Further evidence from primary care has established that CRP POCT is a cost effective tool for reducing antibiotic prescribing for lower respiratory tract infections without negative consequences for clinical recoveryWe performed a pragmatic, open label, cluster randomised controlled trial. The trial was conducted in accordance with a previously published protocol.We recruited 11 nursing homes across the Netherlands that each accommodated 400 residents on average. Physicians in these nursing homes collected data from September 2018 to the end of March 2020. We used a simple randomisation procedure with a 1:1 ratio, which resulted in six intervention group nursing homes that used CRP POCT and five control group nursing homes that provided care as usual. CRP POCT devices  were provided by primary care diagnostic centre Saltro  for the duration of the trial, including the run-in period.All somatic, psychogeriatric, and short stay  nursing home residents received trial information from the researchers shortly before the start of the trial or upon admission of a new resident to the nursing home during the trial. Patients with a suspected lower respiratory tract infection, according to their physician’s assessment, were eligible for participation. This broad inclusion criterion corresponded with the pragmatic nature of the trial . Exclusion criteria were current or recent (in the past week) infection or use of antibiotics, or a recorded statement to withhold antibiotic treatment. A two phase informed consent process was used. The first phase allowed all residents to opt out of participation. In the second phase, physicians asked for written informed consent only from patients who were eligible for participation, during or shortly after initial consultation. The physician asked the patient’s representative for consent if the patient definitely did not have decision making capacity. Deferred consent (informed consent requested after the use of CRP POCT) was obtained when the patient was critically ill or the patient’s representative was not available during the initial consultation.Data on clinical status, additional diagnostics, and management decisions were collected for all participants on initial consultation and one week and three weeks later. During each consultation, treating physicians filled out electronic case report forms that were integrated into the nursing home electronic patient record system. These forms were automatically uploaded  to the secure database portal of the research team.Physicians employed by Dutch nursing homes have their principal site of practice within the nursing home.Intervention group physicians received a medical training session from the research team on the correct use of CRP POCT and its interpretation based on the available evidence and the current Dutch guideline recommendations on lower respiratory tract infections.Main analysis—the primary outcome measure of the main analysis was antibiotic prescribing at initial consultation (yes or no). Secondary outcome measures were the use of additional diagnostics, including repeated CRP tests at one and three weeks (yes or no at each time point); changes in antibiotic treatment policy at one and three weeks (yes or no at each time point); complications (descriptively presented) and safety indicators—full recovery at three weeks according to the physician , hospital admission at baseline, one week, or three weeks (yes or no), and all cause mortality at baseline, one week, or three weeks (yes or no).Secondary analyses—we also explored the extent to which total antibiotic prescribing in nursing homes was influenced by any potential difference in antibiotic prescribing for lower respiratory tract infections. We collected pseudonymised pharmacy dispensing data from participating nursing homes on all systemic, short term antibiotic prescriptions (ATC code J01) for all indications during the trial period and eight months preceding the trial. Finally, we studied the range of CRP values within which physicians decided to prescribe antibiotics. CRP values were shown continuously and categorised per 20 mg/L up to 100 mg/L, and 100-200 mg/L.Main analysis—in our protocol paper we presented the full sample size calculation that adjusted for an intra-cluster correlation coefficient of 0.06 and resulted in a total of 671 participants.Secondary analyses—we plotted categories of CRP values against the decision for antibiotic treatment at initial consultation with a stacked diagram. We aimed to explore descriptively at which point—for which range of CRP values—antibiotics were increasingly prescribed. Also, we plotted physician specific antibiotic prescribing decisions across continuous CRP values. For the analysis of pharmacy dispensing data, we calculated the average number of antibiotic prescriptions per 1000 resident days per month and defined daily dose. We plotted the monthly defined daily dose and average number of prescriptions for the intervention and control groups before and during the trial period to explore potential trend differences.For missing data in the pharmacy dispensing data we imputed the standard dosage, or if this was not possible, we used the average value of other prescriptions for the same drug type. For prescriptions with no chronic or short term description, we excluded prescriptions with treatment durations >70 days. After further inspection of indications, we also excluded prescriptions with treatment durations >42 days.We used SPSS statistical software package version 26 for descriptive statistics. For the logistic generalised estimating equation analysis, Stata statistical software package version 14 was used.No patients were involved in setting the research question or the outcome measures, nor were they involved in developing plans for recruitment, design, or implementation of the study. No patients were asked to advise on interpretation or writing up of results. The nursing home organisations involved their client councils before study commencement, which was either to inform the council about the study participation, or to ask the council for their consent to participate in the study. Some client councils of participating nursing homes reviewed the information letter before study commencement.A total of 242 patients from 11 nursing home organisations were included in the UPCARE study. One participant retrospectively did not fit the inclusion criteria. We did not have any baseline data of three other participants. Baseline characteristics were comparable between the trial groups . Sixty fAntibiotics were prescribed at initial consultation for 84 patients (53.5%) in the intervention group and 65 patients (82.3%) in the control group. The between group difference in initial antibiotic prescribing at any point from initial consultation to follow-up was 23.6% (table S2). The naïve logistic regression analysis  showed tv control group: 6.7% v 2.6%) and a switch in treatment regimen (4.6% v 10.3% at one week and 1.4% v 7.8% at three weeks). Cessation or prolongation of antibiotic treatment during follow-up was uncommon (<4%). Antibiotic treatment changes  occurred less often in the intervention group during follow-up compared with the control group , irrespective of treating physician and baseline characteristics. Table S2 shows that the most pronounced differences in treatment changes between the groups were an initial start of treatment in the first week after initial consultation  was larger than the anticipated 15% clinically relevant difference. During follow-up, this difference was smaller (23.6%). The low occurrence of hospital admission and mortality, and the relatively low between group difference in full recovery at three weeks indicate safe use of CRP POCT.A strength of the study involved the pragmatic design, which increases generalisability of the results.A limitation to the data collection was the absence of a standardised question about changes in diagnosis . Therefore, we could not explore or correct for different diagnoses with regard to full recovery at three weeks. Furthermore, physicians filled out any potential complications in text fields of clinical status or reasons for hospital admission see  footnoteAnother potential limitation was the differential inclusion number between groups. A general concern for cluster randomised trials with inclusion post randomisation is selection bias.In our main analysis we corrected for clustering only at the physician level. We also checked potential clustering at the nursing home level, but the intra-cluster correlation coefficient at the nursing home level was near zero in a mixed model analysis that included both the physician and nursing home levels, while clustering did appear at the physician level (intra-cluster correlation coefficient for physician in three level model 0.175). Finally, we did not achieve the initially anticipated sample size. However, given the observed clustering and magnitude of the effect, the results seem sufficiently powered for the primary outcome measure.v 80% and 95%). Potentially, this relates to generally increased attention to antimicrobial resistance, for instance in the media and with increasing developments in antibiotic stewardship efforts.The findings echo those of large trials in general practice that have shown decreased antibiotic prescribing in CRP guided groups compared with usual care groups.The high full recovery rate in both groups, the low occurrence of hospital admissions and mortality, and the non-significant difference in odds of these secondary outcomes between groups suggest that CRP POCT can be safely used in nursing homes. Hospital admission of patients from Dutch nursing homes is generally uncommon compared with other countries, partly because of the availability of in-house specialised elderly care physicians.Because of the pragmatic trial design, we believe that the results can be generalised to other nursing homes and other countries with similar long term care facilities. Evidence for large scale implementation in nursing homes could be reinforced if our economic evaluation shows cost effectiveness of CRP POCT compared with usual care. We also conducted a process evaluation that aimed to improve implementation knowledge and practical guidance for CRP POCT. Both studies will be published separately.In countries where hospital admission of nursing home residents is more common, new studies could focus on the effect of CRP POCT on improved patient selection for hospital admission.Diagnostic uncertainty about suspected lower respiratory tract infections in nursing home residents contributes to inappropriate antibiotic prescribingC reactive protein point-of-care testing effectively reduces antibiotic prescribing for respiratory tract infections and chronic obstructive pulmonary disease in primary careC reactive protein point-of-care testing (CRP POCT) was found to safely reduce antibiotic prescribing for suspected lower respiratory tract infections in nursing home residents compared with usual care CRP POCT in nursing homes might contribute to reduced antibiotic use in this setting and help to combat antibiotic resistance


An innovative method for prestressing structural elements through the use of shape memory alloys (SMAs) is gaining increasing attention in research as this method does not require the use of mechanical anchorages for tendons. The activation of the memory effect by means of temperature variations (Joule effect) in effect produces high stresses in SMA components attached to concrete components as reported in the literature. This paper presents the work performed for the purpose of prestressing concrete hollow cylinders with the use of nickel–titanium (Ni–Ti) SMA wires. In the tests, a variety of hollow cylinders were made using the same concrete mix and with the same wall thickness (20 mm), but with different external diameters . Their prestressing was achieved by the means of Ni-Ti SMA wires of different diameters  wrapped around the cylinders. Longitudinal and circumferential strain during the thermal activation of the SMA wires by Joule heating was measured using gauges located on the internal surface of the hollow cylinders. The experimental protocol, recorded observations, and discussion of the effectiveness of the prestressing of concrete elements as a function of the test parameters are included in the text in detail. Comments on the conditions for effective prestressing of concrete cylinders with SMA wires are proposed in the conclusions of the paper. Technology relating to the prestressing of structures is widely known in civil engineering and is used mainly in the construction of bridges, tanks, industrial structures, and public buildings. At the current level of development of this method, prestressing techniques with conventional steel tendons  and tendons formed from composites developed in the last 30 years ,2,3 are The use of shape memory alloys (SMAs) as another innovative technology used in the field of structural strengthening is an increasingly popular subject for considerations and developments. SMAs belong to the group of smart (intelligent) materials. Martensitic transformations triggered by temperature and stress occur in these metallic alloys. At zero stress, an SMA is in the austenite (A) phase at “high” temperatures, and it is in the martensite (M) phase at “low” temperatures. The A→M and M→A transformations also occur at constant “high” ambient temperatures upon mechanical loading and unloading, respectively. For various use of the SMA materials, as for example microactuators, a good understanding of the phase transformations and of the role of influencing parameters is important . WhateveNickel–titanium (Ni-Ti) and, more generally, Ni–Ti-based alloys are the most frequently used SMAs in engineering applications, especially in the form of wires. In , for insThe confinement of plain concrete cylinders using wound SMA wires is one of the proposed applications in civil engineering to create a state of compression; see, for example, ,19,20,2155.44Ti (wt.%) for the 1 mm wire and Ni55.84Ti (wt.%) for the other two diameters of the wires. s), austenite-finish (Af), martensite-start (Ms), and martensite-finish (Mf). Values were obtained by means of dilatometric testing. Detailed information on the property testing of SMA wires can be found in ;c,SMA—circumferential compression stress in concrete at the residual state [MPa];σres—stress in shape memory wire at the residual state [MPa];σSMA—force in SMA wire remaining after prestress execution [N].FThe following parameters were used for the calculations of stress and force in the SMA wires: Ec, SMA) and mean values of the residual stress in the prestressing wire σres calculated on the basis of measurements are included in The mean values of the circumferential stress in concrete caused by the prestressing wire action , the force in SMA wire was much lower than for the remaining specimens of the same type . Such lower effectiveness was mainly related to the difficulty in blocking the wire ends at the beginning of the test. An additional influence may be related to degradation of the SMA wire by the flow of electrical current. The value of the electric current  used in these tests was much greater than the value influencing the properties of SMA wires, as reported, for example, in . A summaFor the investigation of the relationship between the value of the initial predeformation of the wire and the level of prestressing obtained in the concrete hollow cylinders, four specimens with external diameters of 250 mm prestressed with Ni–Ti wire with a diameter of 2 mm were tested. The initial predeformation was at two different values (6% and 3%); there were two cylinders for each predeformation value. Tests I and II refer to the initial predeformation of 6%, while tests III and IV refer to the 3% predeformation.After the initial predeformation, the wire was wound on the hollow cylinder and was connected to the electrical current. A diagram of wire temperature variations over time during the heating and cooling periods is presented in Variations of the circumferential concrete strain as a function of SMA wire heating time for the four tests are shown in Variations of the concrete strain in the circumferential direction related to temperature for the four tests described above are presented in Test I , −545 μm/m;Test II , −547 μm/m;Test III , −428 μm/m;Test IV , −305 μm/m.SMA wire predeformed to a higher initial strain value needed a longer duration of electrical current flow to reach the expected temperature. In the case of wire with a diameter of 2 mm predeformed to 6%, the time needed to reach a temperature of 45 °C was three times longer than the time needed to heat the same wire predeformed to 3%.As a consequence of the longer heating time required for the wire predeformed to 6%, the concrete hollow cylinder was also heated more and this provoked an additional thermal deformation of the concrete.At a wire temperature of 45 °C, the circumferential strain of the concrete in the hollow cylinder prestressed with the wire predeformed to 6% was around 50% higher than the strain in the concrete hollow cylinder with the wire predeformed to 3%; the measured concrete strain included the influence of prestressing with the SMA wire and the thermal deformation of the concrete.res) in this wire was higher than in the case of a wire predeformed to 3%.It can be seen in The results collected during the tests performed allowed the following conclusions to be formulated:res in the wound wire. A constant pitch of the wound wires of 4 mm was successfully achieved in order to exclude any risk of electrical short cirquit. Nevertheless, the following potential imperfections of the tests were observed:Limited space between the wires eliminated the possibility to install temperature sensors on the external surface of the hollow cylinders. For the needs of analysis, it was assumed that the external surface temperature was equal to the temperature of the wound wire—such an assumption is rather rough, as contact between the wires and the concrete is not uniform.As a consequence of the above point, the external concrete surface was not heated evenly.During the execution of the prestressing of the hollow cylinders, their position was vertical and this could have inhibited the ventilation and cooling of the internal surface.During the tests, attention was paid to the proper registration of the concrete strain and the temperature of the internal surface of the hollow cylinders, as well as that of the wire. The results were used for the determination of the final stress in the concrete hollow cylinders in the circumferential direction and the residual stress σThe speed of increase in the wire temperature under the influence of current flow depended on the type of wire, as well as its diameter. Together with the temperature of the wires, the temperature of the concrete cylinders also increased. The progress of these variations depended on the current parameters, the wire impedance, the wire and cylinder thermal capacity, and the ambient temperature. Diagrams of the temperature increase of the wire and concrete above the initial level registered during prestressing of the hollow cylinders are presented in The temperature variation of the Ni–Ti wire with a diameter of 1 mm was the most rapid of all wire diameters. The maximal value of the wire temperature was reached after 6 to 10 mins of heating. At the same time, the temperature of the internal surface of the concrete remained low (10 °C to 30 °C); thus, the temperature gradient between the SMA wire and the internal concrete surface could reach around 75 °C after 3 mins of heating (as was the case for the hollow cylinder with a diameter of 250 mm). After 5 mins, this gradient dropped to 30 °C. For all specimens, the temperature of the wire decreased quickly after the end of heating, while the temperature of concrete after reaching its maximum decreased less quickly. For the hollow cylinders with a diameter of 200 mm, the temperature of the concrete 7 mins after the test was higher than the temperature of the wire. This may be explained by the lowest thermal capacity of the 200 mm concrete cylinder from all specimens. For hollow cylinders of 250 mm and 300 mm prestressed with the 1 mm diameter wire, the temperature of the wire and of the concrete became equal after 20 mins, while, for the 200 mm cylinder, the concrete temperature was higher for a much longer period of time.Prestressing of the concrete hollow cylinders with SMA wires with diameters of 2 mm and 3 mm required a substantially longer heating time, as, under the effect of lower electrical impedance of the material, less heat was released. A heating time of 20 to 30 mins also resulted in a systematic increase in temperature of the internal surface of the hollow cylinder. The temperature gradient during the heating time did not exceed 25 °C b,c. At tDuring the execution of the entire testing program, two failures of concrete hollow cylinders occurred, as is typical for a new direction of research; two hollow cylinders (R1 and R2), each with an external diameter of 300 mm, broke during prestressing. In the first case, hollow cylinder R1  was subjThe wire temperature increase in relation to time and the variations of concrete strain on the internal surface of the hollow cylinder in the longitudinal and circumferential directions are shown in During further execution of the test program, concrete strains recorded with the strain gauges installed in the circumferential direction linearly increased to 250 s. After this moment, a significant increase in the strains registered by gauges G1 and G3 was observed. Destruction of all strain gauges in the circumferential direction and in one longitudinal direction (G6) occurred simultaneously at 275 s. The upper part of the hollow cylinder separated from the lower part with a bang and was thrown to a height of around 1.5 m. Loosened coils of the Ni–Ti wire absorbed its kinetic energy, and the separated part fell to the floor. The Ni–Ti wire was neither broken nor damaged b.In the other case, the hollow cylinder R2 was destroyed during prestressing with the same diameter of Ni–Ti wire (3 mm). The specimen was prepared according to the procedure described in Analysis of the presented diagrams led to the conclusion that the wire was quickly heated to a temperature of 38.7 °C, while, on the internal surface of the concrete hollow cylinder, the temperature was nearly stable for a period of 100 s and remained at a level of + 5 °C. After 136 s from the start of the test, the difference between temperatures on the external and internal surfaces of the hollow cylinder was 32 °C. At this time, the hollow cylinder burst was observed 0.21 m from the bottom. The final temperatures of the wire and concrete were 26 °C and 5 °C, respectively.A careful analysis of the strain development in concrete in both measured directions shown in The above-described conditions were characteristic of the moment of creation of the circumferential cracks, perpendicular to the longitudinal direction of the hollow cylinder. The temperature difference (gradient) between the external and internal surfaces of the concrete at 64 s was 16 °C. Considering the value of concrete elasticity modulus at the level of 36 GPa, this gave a tensile stress in concrete of 5.9 MPa. The estimated value was higher than the concrete tensile resistance in the axial load, and it was 4.4 MPa on the basis of laboratory tests.To conclude, it is expressed that hollow cylinder failures were provoked by stress exceeding concrete tensile resistance. Thermal actions generated at the time of cylinders prestressing, together with the load applied to hollow cylinders by means of wound wire with prestressing force activated within it, were the cause of the damage.Cylindrical concrete elements may be effectively prestressed with use of initially predeformed wire formed from shape memory alloy (SMA), in which prestressing is activated in the method of heating the wires with a flow of electrical current.c, as well as values of permanent stress in the SMA wires σres, depend on the type and diameter of the wire, the initial predeformation value, and the hollow cylinder diameter and thickness.Values of the circumferential compressive stress in concrete σc, as well as higher values of permanent stress in the prestressing wire σres, than for the specimens with wire predeformation of 3%.Prestressing of the concrete hollow cylinders with SMA wires with a higher level of initial deformation (6%) is more effective as it brings higher values of circumferential compression stress in concrete σThe performed testing program showed that activation of the shape memory effect in the wires with use of a flow of electrical current is effective in practical usage. The selection of the Ni–Ti wire diameter should relate to the conditions of the prestressing execution. It should be noted that wire predeformed to 6% requires a much longer action of electrical current than wires predeformed to 3%. Longer heating of SMA wires also results in the heating of concrete, and this may lead to thermal deformation of the concrete.Too much heat in the wire may result in a high thermal gradient in the hollow cylinder wall, which may consequently provoke high tensile stress in concrete; such conditions may provoke exceeding of the concrete tensile strength and, ultimately, the failure of the hollow cylinder. Overheating may also damage the SMA wire.As a result of the performed research, the following conclusions can be drawn:


Identifying healthcare workers (HCW) who have less awareness and knowledge on antibiotic use and resistance represents a challenge for public health, since it might help the development of novel educational and training initiatives tailored on specific subgroups of professionals. This work aims to compare knowledge, attitudes and behaviors on antibiotic use and resistance across different groups of Italian HCW.We used data from the multi-country and multi-professional survey launched by the European Centre for Disease Prevention and Control between 28 January to 4 March 2019 to assess knowledge, attitude and behaviors of HCW on antibiotics, antibiotic use and resistance. We distinguished three clusters of HCW using the Two-Step Cluster analysis, based on their personal and professional characteristics .In general, cluster 1 consisted mostly of allied healthcare workers, while clusters 2 and 3 were made up almost completely of pharmacists and medical doctors, respectively. Interestingly, healthcare workers in cluster 3 had the highest knowledge on antibiotic use and resistance. Workers in cluster 1, instead, were those reporting the highest awareness of the importance and role of hand hygiene as an infection prevention and control measure. However, HCW in cluster 2 were those who recognized more their role of advisors on prudent antibiotic use.Italian HCW exhibited different knowledge, attitudes, and behaviors on antibiotic use and resistance. These findings raised the need for educational and training interventions targeting specific professional groups.The online version contains supplementary material available at 10.1186/s13756-021-01002-w. Antibiotic resistance represents one of the major threats for public health worldwide. It has been estimated that every year approximately 700,000 deaths are caused by infections with antibiotic-resistant microorganisms , 2. WithAlthough antibiotic resistance is considered a multifactorial issue, the lack of awareness on this issue itself and on the prudent use of antibiotics remain the leading causes for overuse and misuse of antibiotics and inappropriate infection prevention and control (IPC) practices , 5–7. InIndeed, most of the epidemiological and clinical burden of infections caused by antibiotic-resistant microorganisms is associated with healthcare across the European Union (EU) and European Economic Area (EEA) , 11. HowSurvey of health-care workers’ knowledge, attitudes and behaviors on antibiotics, antibiotic use and antibiotic resistance in the EU/EEA [In this scenario, identifying the HCW and professional figures who have less awareness and knowledge on antibiotic resistance could be a strategy for developing novel educational and training initiatives designed to drive behavior changes on antibiotic use. In 2018, the European Centre for Disease Prevention and Control (ECDC), in the framework of the European Antibiotic Awareness Day (EAAD) initiative, launched the e EU/EEA . This wae EU/EEA . Overalle EU/EEA –17. The e EU/EEA . Specifie EU/EEA .Enterobacterales and Acinetobacter baumannii, and methicillin-resistant Staphylococcus aureus [The situation of antibiotic use and resistance in Italian hospitals and regions poses a major public health threat to the country, due to high level of carbapenem-resistant s aureus . During s aureus . In genes aureus . Thus, wThe ECDC launched the survey in 2019 across thirty EU/EEA countries. The development of the survey was based on a theoretical model (COM-B) to understand the Capabilities, Opportunities and Motivations, which enable prudent Behavior on antibiotic use amongst European HCW. Survey development and study design were fully described elsewhere . In brieWe employed a clustering approach to distinguish different groups of HCW, not only based on their profession but also considering other personal and professional characteristics. A Two-Step Cluster analysis was applied to partition the original dataset into different clusters of participants, with high within-cluster homogeneity and between-cluster variability. This algorithm, based on the Schwarz’s Bayesian Information Criterion (BIC), allowed to handle categorical and continuous variables and to choose the exact number of clusters across different clustering solutions . We chosThe European HCW completed 18,365 questionnaires. Among them, the Italian health professionals provided responses with 2167 questionnaires, exceeding (169%) the calculated target survey sample size for our country.We analyzed data from 1693 Italian HCW who provided a complete assessment of personal and professional characteristics. In particular, the assessment was completed by 832 medical doctors, 555 pharmacists or pharmaceutical technicians, 241 allied HCW (including 85.1% of nurses), and 65 other HCW.The Two-Step Cluster analysis distinguished three different clusters of participants, which were related strictly to the type of profession and to the activities as prescriber and/or as antibiotic use advisor. Indeed, clusters 2 and 3 were made up almost completely of pharmacists and medical doctors : the former were also mostly antibiotic use advisors; the latter were both antibiotic use advisors and prescribers. Cluster 1, instead, represented a heterogeneous group with a majority of allied HCW (53.8%). Table We also noted some differences across clusters in terms of social media used, with cluster 3 showing the lowest proportion of users compared to other clusters. Moreover, we observed that the use of different platforms varied across clusters . In contrast, participants in cluster 2 provided the highest proportion of correct answer compared to the other participants for the question “Unnecessary use of antibiotics makes them become ineffective” (94.2%) Fig. A. Using Figure Next, we assessed the availability of informational material and opportunities relevant for preventing and controlling antibiotic resistance for survey respondents. In particular, the percentage of participants who agreed or strongly agreed that they had easy access to guidelines on managing infections was higher in clusters 3 (70.5%) and 1 (68.5%) than in cluster 2 (48.5%) , than those in cluster 2 and 3 (p < 0.001) , the third one was extremely heterogeneous for personal and professional characteristics. This did not allow us to understand whether the observed differences with the other two clusters were related to the entire cluster or to specific subgroups of HCW. Indeed, previous studies conducted in different countries suggested that knowledge and attitudes on antibiotic use and resistance might differ among HCW, and that this difference could be attributed to their level of education, professional experience, and type of hospital where they work , 17, 30.In conclusion, our study highlights different knowledge, attitudes, and behaviors on antibiotic use and resistance among Italian HCW. Medical doctors were those with the highest level of knowledge, probably for their greater awareness of the relationship between antibiotic use and resistance and their easiest access to guidelines. Although our analysis provides data and information that will be useful to plan future campaigns and initiatives targeting HCW, there is still a need for further studies to explore what other social and professional factors affect the level of knowledge and awareness on these issues.Additional file 1: Figure S1 Proportion of healthcare workers participating in the survey on antibiotic use and antibiotic resistance, stratified by cluster and social media use, Italy, 2019 . ** p-values <0.01 or *** p-values <0.001. Differential use of social media by the HCWs who participated in the ECDC survey on antibiotic use and antibiotic resistance.Additional file 2: Figure S2 Proportion of healthcare workers participating in the survey on antibiotic use and antibiotic resistance, stratified by cluster and awareness of the Italian national Action Plan on antibiotic resistance, the European Awareness Antibiotic Day (EAAD) and Week (WAAW), Italy, 2019 . *** p-values <0.001. Differential awareness of EAAD, WAAW and the national action plan on antibiotic resistance by the HCWs who participated in the ECDC survey on antibiotic use and antibiotic resistance.


The properties of cement concrete using waste materials—namely, recycled cement mortar, fly ash–slag, and recycled concrete aggregate—are presented. A treatment process for waste materials is proposed. Two research experiments were conducted. In the first, concretes were made with fly ash–slag mix (FAS) and recycled cement mortar (RCM) as additions. The most favorable content of the concrete additive in the form of RCM and FAS was determined experimentally, and their influence on the physical and mechanical properties of concrete was established. For this purpose, 10 test series were carried out according to the experimental plan. In the second study, concretes containing FAS–RCM and recycled concrete aggregate (RCA) as a 30% replacement of natural aggregate (NA) were prepared. The compressive strength, frost resistance, water absorption, volume density, thermal conductivity, and microstructure were researched. The test results show that the addition of FAS–RCM and RCA can produce composites with better physical and mechanical properties compared with concrete made only of natural raw materials and cement. The detailed results show that FAS–RCM can be a valuable substitute for cement and RCA as a replacement for natural aggregates. Compared with traditional cement concretes, concretes made of FAS, RCM, and RCA are characterized by a higher compressive strength: 7% higher in the case of 30% replacement of NA by RCA with the additional use of the innovative FAS–RCM additive as 30% of the cement mass. The cement industry alone accounts for approximately 4.1% of the EU’s and ~8 to 10% of the world’s anthropogenic COIn 2017, about 45 billion tons of natural aggregates were produced, and it is estimated that the extraction rate will rise to 66 billion tons in 2025 . The con2 emissions [Concrete production requires increasing amounts of raw materials, while industrial by-products can be used to meet this demand ,10,11,12missions . Industrmissions ,24,25,26The global production of coal fly ash is estimated to be 750 million tons per annum. Japan is the leader in the utilization of coal combustion by-products (utilization rate above 90%) . The gloIn light of the data above, the use of waste materials in new concrete as a substitute for natural aggregates or cement is perfectly justified. Therefore, many authors have devoted their research efforts towards the use of recycling aggregates in new concretes, determining, for example, the most advantageous method of obtaining them ,30,31,32Fly ash from biomass has also become the subject of research for many scientists . Biomass2 emissions can also be lowered significantly, as less cement is needed for concrete production. To tackle the problems regarding the waste produced by the concrete industry, a great deal of research has been carried out regarding the use of recycled concrete rubble as a binder and an aggregate.For the above-mentioned reasons, researchers are making efforts to study recycling opportunities in concrete and cement production. After all, recycling has three benefits: it reduces the demand for new resources, it cuts down on production energy costs, and it recycles waste that would otherwise be landfilled . For insIn line with the circular economy principle, huge amounts of concrete waste and the high availability of waste from the energy industry , the solution to environmentally friendly sustainable development is the production of concrete with recycled coarse aggregates (RCA), recycled cement mortar (RCM), and fly ash–slag mixtures (FAS), which is explored in this article. The novelty of the article is to show the concrete properties with obtained secondary materials, received from one technological process and according to the comprehensive patented method .The preparation of cement composites, due to the two types of waste used (fly–ash slag and concrete rubble), was based on two main types of treatment: (1) the process of producing recycled coarse aggregate (RCA) and recycled cement mortar (RCM) according to the thermomechanical treatment of concrete rubble according to PAT.229887  and (2) Stage I: Concrete rubble was crushed in a jaw crusher to a size < 40 mm.Stage II: The rubble obtained in this way was placed in a thermal furnace and annealed with the following parameters according to PAT.229887 (650 °C for 60 min) .Stage III: The debris, after being removed from the furnace, was placed in a Los Angeles drum and subjected to mechanical treatment according to PAT.229887  to finalStage IV: The cooled material was sieved through a 4 mm sieve to separate the fine fraction (<4 mm) from the coarse fraction (≥4 mm). Coarse aggregate (≥4 mm) was additionally divided into 4–8 mm and 8–16 mm fractions ready for use in the new concrete mix.Stage V: Recycled mortar (RCM) was milled again in the LA drum for 20 min.Stage VI: Fly ash–slag (FAS) was milled in the LA drum for 20 min, after previously being dried at 60 °C.The purpose of processing RCM and FAS in the ball mill was to increase the proportion of the finest particles in the material and thus increase their real surface area. RCM, which is thermally activated in the recycling process (PAT.229887) has pozzolanic properties, which were proven by .This article presents the results of research on the physical and mechanical properties, and the microstructure of concretes prepared with FAS–RCM and RCA.Cement Standards—Part 1: Content, Requirements and Compliance Criteria of Commonly Used Cements” was used in this study.Portland cement (CEM I 42.5R) meeting the requirements of the EN 197-1 standard “The concrete rubble was obtained as a result of the thermomechanical treatment of pre-crushed concrete road curbs of the declared strength class C 30/37 received from a Polish industrial company. The rubble was sieved to obtain recycled aggregate separated into the 4–8 mm and 8–16 mm fractions, which were used in the following research. In In accordance with the research plan, the sand fraction of 0–2 mm, the gravel fraction of 2–4 mm, and dolomite fractions of 4–8 mm and 8–16 mm were used as the natural aggregates. The dolomite NAs are presented in In The gradation curves are similar because the recycled aggregate was divided into fractions and used in percentages, similar to the natural aggregate. Standard upper and lower curves are also shown.The results above indicate that the recycled aggregate, obtained as a result of the thermal (650 °C) and mechanical treatment of concrete rubble, has properties similar to those of the natural aggregate. As a result of the treatment according to , the cemAs a result of the thermomechanical treatment of the concrete rubble, as well as the RCA, RCM (<4 mm) was obtained.To develop the higher specific area (as in the case of FAS) of the RCM, the additional process of milling in the LA drum for approximately 20 min was applied. The RCM after the additional milling process is ready to use in further tests.The fly ash–slag mixture is post-production waste that was generated as an unavoidable by-product of energy production in conventional coal-fired power plants. The furnace waste was fly ash from electrostatic precipitators and slag from the slag removers, which, in the case of wet storage, forms ash and slag mixtures. The FAS obtained for the tests was characterized by high humidity (approximately 15%); therefore, before starting the tests, it was dried at 60 °C for approximately 24 h. After that, to activate the material (as in the case of the recycled mortar) and to increase the proportion of the finest fractions, the regrinding process was carried out in the LA drum for about 20 min.In The Master Glenium SKY 638 superplasticizer based on third-generation polycarboxylate ethers was used for the tests. The correct configuration of the polymer chains allows the adsorption processes to be spread over time and avoids admixture build-up of cement hydration products. As a result, the fluidity effects of the concrete mixture are maintained without adversely affecting the early strength of the hardened concrete. The recommended dosage is 0.2–2.0% relative to the cement mass.3 samples. The compressive strength test was carried out on six cubic samples following the EN 12390-3 standard “Testing Hardened Concrete—Part 3: Compressive Strength of Test Samples”. The tests of water absorption were performed according to Polish standard PN-88/B-06250 (Ordinary Concrete) on 6 and 12 samples (depending on the experiment).The physical–mechanical tests of the concrete were conducted on 100 × 100 × 100 mmTesting Hardened Concrete. Density of Hardened Concrete”. The volume was calculated using the proven nominal dimensions of the molds, 100 × 100 × 100 mm3. The samples were weighed in a saturated, surface-dry state and dried to a constant weight after 28 days of maturation. The surface frost resistance test was carried out for Series 3, 9, and 10. This test consists of determining the mass of the exfoliated material from the surface of the sample covered with a 3% NaCl solution after a given number of freezing and thawing cycles. The test procedure according to PKN-CEN/TS 12390-9 corresponds to the description given in the standard for precast roading concrete (EN 1339:2003 “Concrete Paving Flags Requirements and Test Methods”). Thermal conductivity tests were prepared according to the EN 1946-3:2000 standard “Thermal Performance of Building Products and Components—Specific Criteria for the Assessment of Laboratories Measuring Heat Transfer Properties—Part 3: Measurements by Heat Flow Meter Method”.The volume density in the dry and saturated state was tested according to EN 12390-7 standard “−1. The working temperature ranged from ambient temperature to approximately 1000 °C.To determine the chemical composition of the raw materials , X-ray fluorescence (XRF) was performed. ZSX PMIMUS IV  carried out with an instrument with CuKα . Diffraction patterns were measured between 10° and 70° at a rate of 1 2σºmin. The morphology of two samples  were obtained using scanning electron microscopy (SEM) and backscattered electron (BSE) imaging. A JEOL JSM 7800F  was used. Small portions of the samples were cut and then placed in epoxy resin for 48 h. After 48 h, the samples were polished and sputtered with carbon. Furthermore, simultaneous thermogravimetric analysis and differential thermal analysis TGA/DTA were carried out for three hardened samples. TGA/DTA was performed at a heating rate of 5 °C·minThe experiment was carried out in two stages. In the first stage (Experiment I), the most favorable content of the concrete additives in the form of RCM and FAS was determined experimentally, along with their influence on the physical and mechanical properties of concrete. For this purpose, 10 test series were carried out according to the experimental plan described in detail in To better determine the best amount and the impact of FAS–RCM on the concrete’s properties, the experiment was carried out based on two variables:X1: the amount of RCM (0 ≤ X1 ≤ 50 as a percentage of the summary mass of FAS–RCM);X2: the amount of FAS–RCM (0 ≤ X2 ≤ 30 as a percentage of cement mass).The amounts of RCM and FAS resulted from the authors’ previous experiences and the limit of the content of additives as a cement substitute according to the EN-206 standard. The details of the range of variability and the levels of the analyzed factors are presented in Additionally, a control series was made without the addition of FAS–RCM. The test experimental plan is presented in w/s ratio of 0.45 with the following constant aggregate proportions: sand constituted 35%, the 2–4 mm fraction made up 10%, the 4–8 mm fraction made up 25%, and the 8–16 mm fraction made up 30%. The variables were the content of recycled concrete mortar (0–50%) and the content of the FAS–RCM mixture (10–30% w/w).All recipes maintained a constant The additives were introduced into the concrete in the same way as fly ash. It was assumed that the additives would be included in the minimum amount of cement in concrete and the equivalent water–cement coefficient would be determined by the coefficient k, the size of which depends on the class and type of cement. The maximum amount of additive (FAS–RCM) included in the k value was as follows: FAS–RCM/cement (C) ≤ 0.33 (by weight).3.For the strength class of CEM 42.5 cement, the value of k = 0.4 was established. The minimum cement content required in the appropriate exposure class may be reduced by a maximum amount of k × (minimum cement content in a given exposure class—200) kg/m3, meeting the requirements of all exposure classes according to EN 206:2014 and accounting for strong chemical aggression (the concrete was exposed to contact with soil and groundwater). We also assumed a w/s ratio of 0.45, which fell within the permissible range, and the minimum concrete class was C35/45. The final compositions of the concrete mixtures are presented in As a result, 40% of the planned cement mass was replaced with FAS–RCM, and the aggregate made up the remaining part. The starting series for the concrete composition design was the control series (No. 10), which did not include additives. The initial amount of cement was assumed to be 360 kg/m3. First, weighed aggregate, cement, and possibly FAS and RCM, were poured into the mixer drum and mixed until dry. The mixing water was dosed with the superplasticizer and mixed until the material had a homogeneous consistency. For the preparation of samples, in each of the test stages, cube-shaped forms with dimensions of 100 × 100 × 100 mm3 (2). The vibration time was 15–30 s.The concrete mix for the samples in the tests was prepared in a laboratory counter-rotating concrete mixer. Each preparation was about 18 dm 100 mm3 , meetingAfter the samples had been stored, they were stored for a minimum of 16 h in molds covered with glass fleece in order to limit water loss from the surface of the samples. After demolding, the samples were stored in water at a temperature of 20 ± 2 °C until the tests were carried out.Series 1: made solely from sand and dolomite aggregate (control series).Series 2: 30% of the NA (>4 mm) was replaced with recycled aggregate based on dolomite. This is the acceptable percentage of replacement of coarse aggregate with recycled aggregate for exposure classes other than X0 permitted by the EN 206:2014 standard.Series 3: 30% of the NA (>4 mm) was replaced with recycled aggregate based on dolomite, and an additive was introduced in the form of a mix of FAS–RCM, where FAS and RCM each accounted for 50% of the weight of the additive.In order to determine the impact of RCA with the addition of FAS–RCM on the strength properties of concretes, another experiment was prepared, which used RCA derived from waste generated in the production process of elements at Jadar Sp. z o. o. The basis of the concrete rubble was dolomite aggregate. The following series of concrete mixes were prepared:3 of the mixture.2 and CaO values were close, which may indicate that this RCA was obtained from siliceous aggregates. Very similar values were also obtained for RCM. For the fly ash–slag mix, the major compound was SiO2. This result is similar to that of Goncalves et al. [To know the chemical composition of the raw materials, XRF was performed . In the s et al. .Due to the variability in the composition of natural aggregates, recycled concrete aggregate, recycled masonry aggregate, and the fly ash and slag mixture, For recycled concrete aggregate, the phases found were quartz (05-0490), calcite 05-0586), portlandite 44-1481), illite (02-0050), and larnite (09-0351) . The sam481, illi6, portlaThe average test results of the compressive strength for each series after 28 days of curing are shown in x1) and the amount of FAS–RCM (x2) are presented in The changes in the concrete compressive strength depending on the amount of RCM . The test results after 28 cycles of freezing and thawing are summarized in 2. In the presence of 30% FAS (No. 3), the weight of the exfoliated material was reduced by 5% compared with the control series. On the other hand, the presence of 50% cement mortar in the FAS–RCM mixture resulted in a decrease in the mass of exfoliated material by an average of 13% compared with the control series. This proves the beneficial effect of the presence of the FAS–RCM mixture on sealing the concrete structure. All series of concretes can be classified as the same category of surface frost resistance as concrete in the FT1 pavement .As shown by the test results , in all In As shown in In As shown by the results of the tests conducted , the watThe results of the bulk density in the saturated and dry states are presented in As can be seen from To observe their microstructure, the samples with the highest compressive strength (FAS–RCM + RCA) and the reference sample (control) were analyzed. Si is the main element of natural aggregate A, and thConcretes made with FAS, RCM, and RCA were characterized by a higher compressive strength (by about 7%) compared with traditionally produced concrete in the case where 30% of the natural aggregates are replaced by recycled aggregates, with the additional use of the innovative FAS–RCM additive as 30% of the cement mass.The water absorption of concrete with recycled aggregates and the addition of FAS–RCM was very low (not exceeding 3.5%); this value was obtained as a result of the waste FAS–RCM and RCA affecting the sealing of the composite structure. Usually, low water absorption in concrete is obtained as a result of the use of expensive plasticizers or superplasticizers.3 in the case of 30% recycled aggregates).Concretes based on FAS–RCM and RCA were characterized by low dry and saturated bulk density values , while concretes containing 30% RCA and 30% FAS–RCM tested with de-icing salts could be classified as FT1 .Concrete with waste materials had a lower thermal conductivity coefficient than standard composites because of the waste materials (FAS–RCM and RCA).The portlandite and larnite phases (XRD) found in recycled cement mortar and recycled concrete aggregate improved the compressive strengthSEM showed cement hydration products at the interfacial transition zone (ITZ) between the recycled aggregate and the cement paste.


Aconitum alkaloids and explore potential metabolic markers to understand the differences between raw and processed Fuzi with different steaming time points. Moreover, the selected metabolic markers were visualized by DESI-MSI, and six index alkaloids’ contents were determined through HPLC. The results indicated visible differences among raw and processed Fuzi with different steaming times, and 4.0 h is the proper time for toxicity attenuation and efficacy reservation. A total of 42 metabolic markers were identified to discriminate raw Fuzi and those steamed for 4.0 and 8.0 h, which were clearly visualized in DESI-MSI. The transformation from diester-diterpenoid alkaloids to monoester-diterpenoid alkaloids and then to non-esterified diterpene alkaloids through hydrolysis is the major toxicity attenuation process during steaming. DESI-MSI combined with metabolomics provides an efficient method to visualize the changeable rules and screen the metabolic markers of Aconitum alkaloids during steaming. The wide application of this technique could help identify markers and reveal the possible chemical transition mechanism in the “Paozhi” processes of Fuzi. It also provides an efficient and easy way to quality control and ensures the safety of Fuzi and other toxic traditional Chinese medicine.Fuzi is a famous toxic traditional herbal medicine, which has long been used for the treatment of various diseases in China and many other Asian countries because of its extraordinary pharmacological activities and high toxicity. Different processing methods to attenuate the toxicity of Fuzi are important for its safe clinical use. In this study, desorption electrospray ionization mass spectrometry imaging (DESI-MSI) with a metabolomics-combined multivariate statistical analysis approach was applied to investigate a series of  Aconitum carmichaelii Debeaux (Fam. Ranunculaceae), primarily cultivated in Jiangyou and Butuo in Sichuan, China, has been an indispensable herb in traditional Chinese medicine (TCM) for thousands of years , monoester-diterpenoid alkaloids (MDAs), non-esterified diterpene alkaloids (NDAs)  , and lips (NDAs) . Based os (NDAs) . TherefoAconitum alkaloids are also responsible for the pharmacological activity and therapeutic efficacy of Fuzi, and efficacy preservation should be observed during toxicity attenuation. Recent research has indicated that DDAs, MDAs, and water-soluble active constituents have a massive loss after soaking in Danba and subsequent rinsing. Moreover, inorganic impurities are easily introduced to Fuzi  (2020 Edition), soaking in Danba , then continuous soaking with salt, steaming or decocting, long rinsing, and oven drying are the major processing methods for Fuzi . During  to Fuzi . Thus, aAt present, the high-performance liquid chromatography (HPLC) and liquid chromatography coupled with mass spectrometry (LC-MS) are commonly used methods for alkaloid analysis of Fuzi . HoweverMass spectrometry imaging (MSI) integrates sensitivities and high-throughput screening mass spectrometry with spatial and temporal chemical information and enables the visualized distribution of specific metabolites within non-destructive tissues . Matrix- The extensive application of DESI-MSI to determine natural products (primary and secondary metabolites) demonstrated its power and good practicability .In this study, an easy-handling and comprehensive method combining DESI-MSI with metabolomics was applied to investigate the variation of key ester-diterpenoid alkaloids and identify the metabolic markers in raw and processed Fuzi with different steaming time points. In addition, the contents of six ester-type alkaloids were determined through HPLC.40), mesaconitine (22), hypaconitine (36), benzoylaconine (34), benzoylmesaconine (32), and benzoylhypaconine (29) were obtained from Chengdu Herbpurify Co., Ltd. . LC-MS-grade methanol (MeOH), formic acid (FA), acetonitrile, and isopropanol were purchased from Sigma-Aldrich . Ammonium acetate of HPLC grade was obtained from Chengdu Chron Chemical Co., Ltd. . The Elga Labwater Purelab system  was used to obtain purified water for UPLC analyses. All other reagents were of analytical grade.The reference standards  of aconitine  and authenticated by Professor Yi Zhang from Chengdu University of TCM . The vouCleaned raw Fuzi were randomly divided into 11 groups, one of which was selected as raw Fuzi (without steaming), and the rest were placed into a suitable container for steaming for 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 h. All the samples were directly sliced, and the thickness of cross-sectional slices was approximately 5 mm. Subsequently, the sample was glued onto glass slides using double-sided tape and then subjected to DESI-MSI analysis.2O, 0.2% FA, and 0.1 mM leucine enkephalin (LE) delivered at 2 μl/min; capillary voltage: 4.5 kV; and ionization mode: positive. The pixel size (X and Y pixel size of 200 μm) was determined on the basis of the total scan time of the mass spectra and the x–y scanner speed. A mass range of m/z 100–1,000 and the scan rate of 400 μm/s were used for imaging, and the images were viewed using high-definition image (HDI) v1.5 software . The MS images were created from Raw MS files through HDI with LE as the lockmass (m/z 556.2766) for resolution MS.Imaging experiments were conducted using a Waters Xevo G2-XS QTof mass spectrometer equipped with a DESI source . The following DESI parameters were optimized to obtain good signal intensity: nebulizing gas (dry nitrogen) pressure: 0.45 MPa; spray solvent 70% MeOH, 30% Hm/z and signal intensity. The data matrix was imported into SIMCA-P 13.0  to establish PAC, PLS-DA, and OPLS-DA models for distinguishing classification analysis and selecting crucial variable constituents for Fuzi steaming for different time points. The data were standardized using a Pareto-scaling technique.MS raw data files were imported into HDI for imaging. The regions of interest (ROIs) were selected from root areas. Each sample had six ROIs that were converted into a data matrix of mass v/v) for 30 min . Following extraction, isopropanol–ethyl acetate  was added to compensate for the lost weight during extraction and then filtered. 25 ml of filtrate was accurately measured and evaporated under reduced pressure at 40°C to dryness. Three milliliters of 0.05 M hydrochloric acid–methanol solution was precisely added to dissolve the residue. The supernatants were used as the sample solution after being centrifuged at 15,000 rpm for 30 min. The concentration of aconitine , mesaconitine , hypaconitine , benzoylaconine , benzoylmesaconine , and benzoylhypaconine  in every sample was calculated on the basis of established calibration curves.According to the CP (2020 Edition) , all the18 column  and maintained at 30°C. The mobile phase consisted of 0.04 M of ammonium acetate solution  and acetonitrile (solvent B) using the following gradient: 20%–28% B for 0–10 min, 28%–30% B for 10–20 min, 30%–35% B for 20–50 min, and 35%–38% B for 50–60 min. The flow rate was kept at 1 ml/min, and the injection volume was 10 μl. The UV detection wavelength was set at 235 nm.The filtered sample was analyzed using Agilent Technology 1260 Infinity  equipped with a binary pump, mobile phase degasser, temperature-controlled autosampler, column thermostat, and diode array detector (1260-DAD). HPLC separation was performed on an Agilent Eclipse XDB CHPLC-DAD was validated with regard to linearity, stability, recovery, repeatability, and precision. The storage solutions of six compounds were used in the regression equations between the peak areas and concentrations of six compounds. The limit of detection (LOD) and quantification (LOQ) were determined at signal-to-noise ratios of 3 and 10, respectively.In DESI analysis, the ratio selection of solvent and acid is an important step to achieve sensitive and high MS signal response to target compounds. The solvent compositions are selected based on the analyte of interest and the tissue surface, which play a major role in the elution of each category of the compound . A large22), hypaconitine , and aconitine . In addition, the total amount of MDAs, including benzoylmesaconine , benzoylaconine , and benzoylhypaconine , should not be less than 0.010%.As a highly toxic medicinal material, improper use of Fuzi will seriously affect the safety of people. According to the CP (2020 edition), DDAs shall not exceed 0.020% with regard to the total amount of mesaconitine , aconitine , hypaconitine , benzoylmesaconine , benzoylaconine , and benzoylhypaconine , were visually presented and compared. With the extension of steaming time, the concentrations of three DDAs decreased sharply, whereas the three MDAs increased significantly, among which mesaconitine (22) and hypaconitine (36) decreased faster than aconitine (40) and could not be visualized after 1.0 h, and the intensity of benzoylmesaconine (32) and benzoylhypaconine (29) increased faster than that of benzoylaconine (34). The DESI-MSI results are consistent with those of HPLC experiments, which indicated its reliability.S images , six alkm/z and signal intensity from DESI-MSI was submitted to PCA and PLS-DA to discriminate the variations of Fuzi with different steaming time points, visualize the underlying trend, and understand the metabolic differentiation among them. The PCA score plot (R2X = 0.884 and Q2 = 0.798) showed that all the ingredients could be classified into three major groups  (A data matrix of mass r groups . Raw Fuz= 0.701)  was in ghttps://scifinder.cas.org/), and further confirmation was obtained through comparisons with authentic standards and published literature , benzoylhypaconine , neoline , benzoylmesaconine , mesaconitine , aconitine , and indaconitine , changed after steaming for 4.0 h. Thus, these metabolites were considered as chemical markers to distinguish raw Fuzi and processed Fuzi steamed for 4.0 h. In addition, a total of 20 metabolites, such as neoline , karacoline , isotalatizidine/talatizidine , benzoylhypaconine , songorine , fuzitine , and talatisamine , were set as the markers to distinguish Fuzi steamed for 4.0 and 8.0 h.A total of 42 discriminating metabolites were tentatively identified, and the detailed information is given in 22), aconitine (40), hypaconitine (36), and some of the MDAs decreased dramatically within two steaming hours. The content of napelline (7), denudatine/Guanfu base H (3), karakolidine/Chuanfumine (11), karakanine (9), episcopalisine (24), and aconicarchamine B (25) declined to low levels after steaming for 4.0–5.0 h, but such content increased again when steaming was continued for 10.0 h. Nine metabolites, including dehydrated benzoylhypaconine (26), fuziline (19), senbusine A/B (15), guiwuline (16), karacoline (10), talatisamine (14), dictysine (5), 16-hydroxycardiopetaline (8), and nevadenine (12), showed volatility decreases, and most of them belong to NDAs. As an isoquinoline alkaloid, fuzitine (2) generally exists in the form of salt, which showed a particular changing rule during steaming. The content reached the highest on steaming after 4.0 h and decreased or transformed to other metabolites with the extension of steaming time. The heat map also indicated that some of the metabolites were unstable when steamed for 3.0 h and then tended to be stable. When steamed for 10.0 h, the content of metabolites became similar to samples steamed for 4.0–5.0 h. This phenomenon corresponds to the aforementioned PCA and PLS-DA results (A heat map was used to visualize the changes in the content of the 42 metabolites in raw Fuzi and processed Fuzi for different steaming hours. As shown in  results .As a typical toxic traditional herbal medicine that is known for its extraordinary pharmacological activities and toxicity, Fuzi has long been used as an irreplaceable traditional herbal medicine in TCM, Kampo medicine, and homeopathy for thousands of years .As toxicity and efficacy are interdependent in Fuzi, the toxicities of processed products such as Yanfuzi, Heishunpian, Baifupian, Danfupian, and Paofupian dramatically decreased compared with raw Fuzi , but theAconitum alkaloids. Combining metabolism and multivariate statistical analysis, the metabolites and important markers would be identified, which are essential to assess the quality of steamed Fuzi.Investigations of basal metabolism are important to improve the quality control of TCMs . DDAs, tHPLC was initially applied to determine variation in the content of three MDAs and three DDAs. A total of 10 steaming time points were set, and HPLC data indicated that the content limit of steamed Fuzi met the requirements of the CP after 3.0 h of steaming and became steady after 4.0 h. DESI-MSI was performed to visualize the HPLC results with high resolutions in mass and space: three MDAs were difficult to detect, and three DDAs were highly visible in raw Fuzi. The images showed an increase in MDAs and a decrease in DDAs during steaming. After 4.0 h of steaming, DDAs and MDAs met the standards of CP, and parts of the DDAs still remain. PCA and PLS-DA analyses also illustrated that the samples after steaming for over 4.0 h were grouped closer and located in the coordinate origin region. Consequently, 4.0 h of steaming was recommended for the toxicity attenuation and efficacy reservation of Fuzi.19-ester-diterpenoid alkaloids were proposed (1 and 2 in path C also indicated two important reversible processes, which explained why some of the alkaloids’ content fluctuated during steaming. However, NDAs were the important hydrolysis products after long and continuous steaming, and the pharmaceutical activities will then decrease.Chemometric analysis was combined with a heat map to identify the significant metabolic markers, and 4.0 h was set as the key time node. The DESI-MSI comparison of the 42 metabolites of raw Fuzi and processed Fuzi steamed for 4.0 and 8.0 h showed that the content of highly toxic DDAs decreased dramatically, and a large part of the content of MDAs and NDAs fluctuated during steaming, which indicated a reversible process . DDAs anproposed . Except proposed , esterif19-ester alkaloids were summarized. Therefore, the novel method provides an efficient approach to visualize the changeable rules and screen the metabolic markers of alkaloids during steaming. The wide application of the method could help to identify biomarkers and reveal the possible chemical transition mechanism in the “Paozhi” processes of TCM.In this study, the combination of DESI-MSI and metabolomics for rapid and high-resolution characterization of ester alkaloids in raw and steamed Fuzi was developed. The changes in alkaloids of raw and processed Fuzi with different steaming time periods were observed, and the results of HPLC, DESI-MS coupled with PCA, PLS-DA, and OPLS-DA all indicated that 4.0 h of steaming was appropriate for toxicity attenuation and efficacy reservation of Fuzi. In addition, a total of 37 metabolic markers to distinguish raw and processed Fuzi steamed for 4.0 h and 22 metabolites to distinguish Fuzi steamed for 4.0 and 8.0 h were identified through DESI-MSI-based metabolomics. Moreover, three major chemical transformation pathways of C