Lactiplantibacillus plantarum WCFS1 possess two metabolicpathways for lactose metabolism, involving β-galactosidase and 6Pβ-galactosidase  activities. As L. plantarum WCFS1 genome lacks a putative 6Pβ-galgene, the 11 GH1 family proteins, in which their 6Pβ-glucosidase(6Pβ-glc) activity was experimentally demonstrated,, were assayedfor 6Pβ-gal activity. Among them, only Lp_3525 (Pbg9) also exhibiteda high 6Pβ-gal activity. The sequence comparison of this dual6Pβ-gal/6Pβ-glc GH1 protein to previously described dualGH1 proteins revealed that L. plantarum WCFS1 Lp_3525 belonged to a new group of dual 6Pβ-gal/6Pβ-glcGH1 proteins, as it possessed conserved residues and structural motifsmainly present in 6Pβ-glc GH1 proteins. Finally, Lp_3525 exhibited,under intestinal conditions, an adequate 6Pβ-gal activity withpossible relevance for lactose maldigestion management.Intestinal lacticacid bacteria can help alleviate lactose maldigestionby promoting lactose hydrolysis in the small intestine. This studyshows that protein extracts from probiotic bacterium  Individualswho suffer from lactose maldigestion must reduce lactose in theirdiet and avoid milk consumption. A conventional strategy for lactosemaldigestion management is to use milk products with reduced lactosecontent or to supplement the diet with exogenous β-gal. However,oral administration of β-gal is inconvenient and ineffectivebecause the exogenous enzyme has normally poor pH stability and israpidly degraded by proteases in the gastrointestinal tract.1 Two different metabolic pathwaysfor the lactose uptake have been reported in lactic acid bacteria.One pathway involves the translocation of intact lactose and furtherhydrolysis by a β-gal. In the other pathway, lactose is transportedinto cells via a lactose-specific phosphoenolpyruvate-dependent-phosphotransferasesystem (PEP–PTS), in which lactose is phosphorylated duringtransport and then hydrolyzed into 6-phospho-β-d-galactoseand d-glucose by 6P-β-galactosidase .4β-gal plays an important role inthe management of lactoseintolerance. Because β-galactosidases are also produced by gutmicrobiota, intestinal lactic acid bacteria can help alleviate lactosemaldigestion. Lactic acid bacteria that can reach and inhabit theintestinal tract may alleviate clinical symptoms brought about byundigested lactose and could promote lactose hydrolysis in the smallintestine.Lactiplantibacillus plantarum isone of the few lactic acid bacteria species that has successfullyadapted to different foods and is also part of the human colonic microbiota.7L. plantarum possess both metabolicpathways for lactose metabolism, as demonstrated by the fact thatcell-free extracts from several strains exhibited β-gal and6Pβ-gal activities.11 Several glycosidases exhibiting β-gal activityhave been described in L. plantarum strains, such as LacA13 and LacLM.17 However, the glycosidase(s) responsible for the 6Pβ-gal activityfound in L. plantarum cell extractsremains unknown.L. plantarum WCFS1 strain becauseit was isolatedfrom human saliva and persists in the digestive tract better thanother Lactobacillus spp. isolated fromthe human intestine.18 In this study, 6Pβ-galactivity was assayed in 11 glycoside hydrolases from L. plantarum WCFS1 possessing 6P-β-glucosidase(6Pβ-glc) activity. One of these proteins exhibited also a high6Pβ-gal activity and it was subsequently characterized to betterelucidate its dual 6Pβ-glc/6Pβ-gal glycosidase activity.This work aimed to study 6Pβ-gal activityin the L. plantarum WCFS1 strain used in this study waskindly provided by Dr. Michiel Kleerebezem . This strain is a colony isolate of L.plantarum NCIMB 8826, which was isolated from humansaliva. L. plantarum WCFS1 was grownin the modified MRS broth  at 30 °C. The MRS mediumwas modified by replacing glucose with galactose to avoid possiblecatabolite repression by glucose.19Escherichiacoli DH10B,used for DNA manipulations, and E. coli BL21(DE3), used for protein expression, were cultured in the Luria–Bertani(LB) medium containing ampicillin (100 μg/mL) at 37 °Cand 140 rpm.The L. plantarum WCFS1 were used to determine β-gal and 6Pβ-gal activitiesby using as substrates synthetic p-nitrophenyl-glucosidederivatives, pNP-β-d-galactopyranoside (Sigma-Aldrich) and pNP-6-phospho-β-d-galactopyranoside  (GoldBio),respectively.Restingcells and cell-free protein extracts from 20 Briefly,for the resting cell assays, L. plantarum WCFS1 cells were cultured in the MRS-Gal medium at 30 °C until0.5 OD600nm was reached. Then, cells were harvested andwashed twice with 0.9% w/v NaCl. After washing, cells were resuspendedin 50 mM MOPS buffer (pH 7.0) containing 20 mM NaCl and 1 mM DTT,and the corresponding p-nitrophenyl-glycoside (10mM) was added. Reactions were incubated at 30 °C for 10 min.Resting cells and cell-free protein extracts wereprepared as previously described.L. plantarum WCFS1 was grown in the MRS-Gal medium at 30 °C, until lateexponential phase. Cells were harvested, washed twice with 50 mM MOPSbuffer, NaCl 20 mM, 1 mM DTT, pH 7.0, and subsequently resuspendedin the same buffer for cell rupture. Bacterial cells were disintegratedby using a French Press and subsequently, cells were additionally broken with glass beadsin FastPrep TM Fp120 equipment (Savant). The disintegrated cell suspensionwas centrifuged at 4 °C in order to remove cell debris. Supernatantscontaining soluble proteins were filtered, and the obtained proteinextracts were incubated in the presence of the corresponding substrate(10 mM) at 30 °C for 10 min.For cell-free protein extracts, 20 The rate of hydrolysis of p-nitrophenyl (pNP) glycosides for 10 minat 30 °C was measured in 50 mM MOPS buffer pH 7.0 containing20 mM NaCl and 1 mM DTT, at 420 nm in a microplate spectrophotometerPowerWave HT . Reactions were stopped by the additionof 1 M sodium carbonate (pH 9.0). Control reactions containing noenzyme were utilized to detect any spontaneous hydrolysis of the substratestested. Enzyme assays were performed in triplicate.β-gal and 6Pβ-galactivities in resting cells and cell-freeprotein extracts were determined by a spectrophotometric method previouslydescribed.L.plantarum WCFS1 were produced as previously described.20 The hydrolytic activity of these 12 glycoside hydrolases was determinedby using a library of 24 pNP-glycoside derivatives.13 The assay was performed in a 96-well flat bottomplate (Sarstedt), where each well contained a different substrate(10 mM). Briefly, the reaction consisted of 4 μg protein in50 mM MOPS buffer pH 7.0 containing 20 mM NaCl and 1 mM DTT. The reactionwas incubated at 30 °C for 10 min and stopped by the additionof 1 M sodium carbonate at pH 9.0. Hydrolysis of each pNP-glycoside derivative was colorimetrically measured by liberationof p-nitrophenolate (pNP) at 420nm in a microplate spectrophotometer PowerWave HT .Controls without the enzyme for evaluating spontaneous hydrolysisof the tested substrates were carried out. Experiments were performedin triplicate, and results were expressed as an average activities± standard deviation.Glycosidehydrolases from the GH1 family  and GH42 family (LacA or Lp_3469) from pNP-6-phospho-β-d-glucopyranoside (pNP-6P-Glc) and pNP-6-phospho-β-d-galactopyranoside  as substrates, for 6Pβ-glc and 6Pβ-gal activities,respectively. The optimal pH was determined by using citrate (pH 3.0),acetic acid-sodium acetate (pH 4.0–6.0), MOPS (pH 6.5 and 7.0),and Tris–HCl (pH 8.0) buffers (50 mM). The optimal temperaturewas assayed by incubation of Lp_3525 in 50 mM MOPS buffer pH 7.0 containing20 mM NaCl and 1 mM DTT at 4, 22, 30, 37, 45, and 65 °C. Assayswere performed in triplicate.The effects of temperature and pH on Lp_3525 weredetermined using L. plantarum OSU in asurvey of lactobacilli for β-galactosidase and phospho-β-galactosidaseactivities using pNP-galactoside derivatives as substrates.8 Later, using the same substrates, both activitieswere also identified in additional L. plantarum strains, extracts,10 or toluene-treatedcell suspensions.11 However, none of theseassays evaluated the activity exhibited by L. plantarum whole cells. When the lactose uptake occurs through the PEP-PTS,whole cells should not exhibit 6Pβ-gal activity because lactoseneeds to be phosphorylated during its transport into the cell. Inorder to know whether β-gal and 6Pβ-gal activities existin the probiotic L. plantarum WCFS1strain, both activities were assayed in resting cells and in cell-freeprotein extracts by using pNP-Gal and pNP-6P-Gal as substrates. As expected, resting cells did not show6Pβ-gal activity, and they exhibited only a low β-galactivity (0.47 ± 0.14 μmol min–1 mg–1). By contrast, both activities were observed at thesame extent in cell-free extracts, β-gal (3.67 ± 0.08 μmolmin–1 mg–1) and 6Pβ-galactivity (3.69 ± 0.06 μmol min–1 mg–1). This is the first report describing similar β-galand 6Pβ-gal activities in L. plantarum cell-free extracts. Previously, higher β-gal than 6Pβ-galactivities were reported in L. plantarum strains,11 except for L. plantarum ATCC 14917 and L. plantarum ATCC 8014 strains, which exhibited higher 6Pβ-gal activity.11 As β-gal activity was generally the predominantactivity in the previous reports, it was speculated that 6Pβ-galcould be a spurious enzymatic activity in L. plantarum.15Twodifferent pathways for lactose hydrolysis have been described in lacticacid bacteria, involving proteins exhibiting β-gal and 6Pβ-galactivities. These two activities were reported in crude cell-freeextracts from L. plantarum WCFS1 cell-free extracts was clearly confirmed, the proteins involvedin both activities should be identified. The available complete genomesequence of L. plantarum WCFS1 revealedthe existence of a high number of proteins annotated as “glycosidehydrolases”. Among the families included in the carbohydrate-activeenzyme (CAZy) database that contain proteins possessing β-galactivity, only GH1, GH2, and GH42 families are present in L. plantarum WCFS1. The presence of proteins annotatedas β-gal belonging to the families GH1 (Lp_3629), GH2 (Lp_3483and Lp_3484), and GH42 (Lp_3469) are found in the genome of L. plantarum WCFS1. L. plantarum β-gal from GH2 (LacLM) and GH42 (LacA) families have beenpreviously recombinantly produced and biochemically characterized.16 Both β-gal possess high transgalactosylation activity forthe synthesis of prebiotic galacto-oligosaccharides or other galactosylatedderivatives.16 In relation to GH1 family β-gal, it was experimentally proventhat Lp_3629 possesses β-gal activity, and not β-glc activityas it was originally annotated.21 Whenimmobilized, this β-gal successfully catalyzed the hydrolysisof lactose and lactulose, and the formation of different oligosaccharidesfrom galactose and lactulose was detected.22Once the presence of β-galand 6Pβ-gal activities in L. plantarum WCFS1 possesses11 genes encoding for GH1 family proteins that are annotated as 6PB-glc;however, there is no protein annotated as putative 6Pβ-gal.These 11 GH1 proteins have been recently characterized. All of thempresented 6Pβ-glc activity, and in addition, 8 proteins exhibited6-phospho-β-d-thioglucosidase activity.20 Therefore, it is possible that some of theseGH1 proteins could also possess 6Pβ-gal activity, being responsiblefor the 6Pβ-gal activity exhibited by L. plantarum WCFS1 cell-free extracts.According to the CAZy database, 6Pβ-gal is categorizedasa GH1 family enzyme. A total of 21 different enzymatic activitiesare currently classified in the GH1 family. Some enzymes, however,have broad substrate specificity, meaning that they can utilize morethan one substrate and catalyze more than one reaction. The completegenome of 20 The hydrolyticactivity of these enzymes was assayed by using pNP-β-d-galactopyranoside  and pNP-6-phospho-β-d-galactopyranoside  as substrates, for β-gal and 6Pβ-gal activities,respectively. LacA GH42 proteins were used as the control for β-galactivity.13 In addition, the catalyticactivity of these proteins toward 24 pNP glucosidederivatives was also assayed. Despite all the GH1 proteins exhibiting6Pβ-glc activity,20 only one of them,Lp_3525 (Pbg9), effectively hydrolyzed pNP-6P-Galand showed high 6Pβ-gal activity .The 11 GH1 6Pβ-glc proteinsand LacA β-gal were recombinantlyproduced as previously described.activity 1. Low 6Pactivity 1.13 NoneThe obtained results indicatedthat Lp_3525 has broad specificityas it could hydrolyze more than one phosphorylated substrate.23 Galacto- and gluco-derived substrates are very similar and haveidentical structures, differing only in the position of their O4 hydroxylgroup, which is an axial position in the galacto-epimer versus anequatorial position in the gluco-epimer. It was hypothesized thatboth enzymes evolved from a common ancestor, but at some point, 6Pβ-galevolved independently, so that a tryptophan residue was acquired inorder to accommodate galactose-6′P (rather than glucose 6-phosphate)at the active site of subsite −1.23Comparativestructural analysis suggests that a tryptophan instead of a methionineor alanine residue at subsite −1 may contribute to the catalyticand substrate selectivity to structurally similar 6Pβ-gal and6Pβ-glc assigned to the GH1 family.Geobacillus stearothermophilus is known.25 Gan1D has been shown to exhibitbifunctional activity possessing both 6Pβ-gal and 6Pβ-glcactivities. The different ligands trapped in the active site adoptdifferent binding modes to the protein, providing a structural basisfor the dual gal/glc activity observed for Gan1D enzymes. In thisprotein, specific mutations were performed on one of the active siteresidues (Trp-433), shifting the enzyme specificity from dual activityto a significant preference toward 6Pβ-glc activity from Lactococcus lactis subsp. lactis, and 4GPN (Bgl) from Streptococcusmutans as protein models for each specific activity,respectively (BlBglC numbering)involved in the hydrogen bond are not conserved in Lp_3525. Instead,Gly-426 and Val-431 residues (Lp_3525 numbering) are found. Theseresidues are conserved in the 6Pβ-glc 4GPN from S. mutans  or in the previously described dual 6Pβ-gal/6Pβ-glcprotein (Trp-433 in BlBglC) suggested that Lp_3525belongs to a different dual 6Pβ-gal/6Pβ-glc protein group, the residues involved in glyconbinding (−1 subsite), and those involved in aglycon binding(+1 subsite) are conserved.20 Moreover,the Lp_3525 sequence presents a long extra C-terminal segment uniqueto 6Pβ-glc, which varies in length and sequence. This is a typical6Pβ-glc motif that is absent in the 6Pβ-gal hydrolases.29 In addition, Lp_3525 lacks the lid motif presentin 6Pβ-gal that blocks the entrance to the active site instead of the asparagine and tryptophan present in the 6Pβ-gal(Asn-297 and Trp-429 in 4PBG)  thanto 4PBG 6Pβ-gal (35.76% identity). All these data seem to indicatethat Lp_3525 belongs to a dual 6Pβ-gal/6Pβ-glc enzymegroup but more closely resembling the structural features of 6Pβ-glcproteins.The sequence of Lp_3525 has typicalresidues conserved in 6Pβ-glc.ive site 1.29 Lp_3in 4PBG) 1.30 All BlBglC, GK3214, and Gan1Dproteins havebeen previously described as “dual 6Pβ-gal/6Pβ-glc”,28 despite presenting a similarsequence identity to 4GPN and 4PBG .Moreover, they possess residues and motifs specific for 6Pβ-gal,such as the asparagine and tryptophan residues (Asn-297 and Trp-429in 4PBG),30 and the lid motif that blocks the entranceto the active site.29 Additionally, thelong extra C-terminal segment typical of 6Pβ-glc is absent inBlBglC, GK3214, and Gan1D proteins. These data indicated, that contrarilyto Lp_3525, these dual 6Pβ-gal/6Pβ-glc are more similarto 6Pβ-gal enzymes.However, Lactobacillus gasseri ATCC33323, four enzymes showing6Pβ-gal activity  were described.Phylogenetic analysis showed that LacG1 and LacG2 belongs to the 6Pβ-galcluster and Pbg1 and Pbg2 belongs to the 6Pβ-glc cluster, asthey showed closer similarity to 6Pβ-glc (about 50%) than to6Pβ-gal (30–35%).31 The four L. gasseri GH1 glycosidases exhibited dual 6Pβ-gal/6Pβ-glcactivity, as they were able to hydrolyze oNP-6P-Galand oNP-6P-Gal.32 As inthe case of Lp_3525, Pbg1 and Pbg2  showed all the residues and motifs conservedin 6Pβ-glc .28 However, gathered evidenceindicates that there is a different group of GH1 glycosidases possessingdual 6Pβ-gal/6Pβ-glc activity whose the structural basisfor their enzyme bifunctionality remains unknown.Therefore, so far, the differences in gluco and galactosubstrate-bindinginteractions in dual 6Pβ-gal/6Pβ-glc GH1 glycosidaseshave only been analyzed in proteins sharing sequence and structuralfeatures with 6Pβ-gal  assayed with pNP-6P-Glc  and LacG2 (40 °C) from L. gasseri for both substrates.32 In addition, Lp_3525 possessed a different optimal pH foreach enzymatic activities, being more acidic for 6Pβ-gal (pH4.0) than for 6Pβ-glc (pH 6) activity 34 to 50 °C (LacG from S. cremoris and Pbg1 from L. gasseri JCM1032).35 Most of these 6Pβ-gal were inactivated above 60 °C, asit happened in Lp_3525 whose optimal temperature was 45 °C 36 or 2% at60 °C (CelD)37 whereas Lp_3525 retainedat 65 °C more than 95% of its maximal activity 36 or 25% at pH 7.5 (CelD).37When Lp_3525 6Pβ-glcactivity was compared to other 6Pβ-glcpreviously described, it was observed that Lp_3525 possessed a broadrange of optimal temperature and pH 2. Lp_352activity 2b. As meL. gasseri ATCC 33323), Lp_3525, which belongs tothe 6Pβ-glc group, possessed different biochemical propertiesconcerning the hydrolyzed substrate, pNP-6P-Gal or pNP-6P-Glc. Moreover, Lp_3525 exhibited a broad range oftemperatures and pH for its maximal 6Pβ-glc activity, althoughit displayed more constricting conditions for 6Pβ-gal activity.The obtained data confirmed that similarlyto dual 6Pβ-gal/6Pβ-glcbelonging to the 6Pβ-gal group  butalso 6Pβ-glc. This strain contained seven different genes associatedwith 6Pβ-glycosidases, at least four of them encoded dual 6Pβ-gal/6Pβ-glcproteins. Pbg1 and Pbg2 belong to the 6Pβ-glc group describedin this study for the first time, whereas the primary enzymes forlactose utilization, LacG1 and LacG2, belong to the 6Pβ-galgroup previously described.32 In L.plantarum WCFS1, among the 11 proteins having 6Pβ-glcactivity,20 only Lp_3525 possessed high6Pβ-gal activity. Although it appears to be a waste of energythat several enzymes possess apparently the same function, resultspreviously obtained by using 6Pβ-glc mutants,43 and those described in this work confirmed that not allthe proteins are functionally complementary. It has been describedthat the gene encoding Lp_3525, the L. plantarum WCFS1 dual 6Pβ-gal/6Pβ-glc protein, is controlled bycarbon catabolite repression,44 but furtherstudies are needed in order to know the regulation and expressionof Lp_3525 in the presence of lactose and different carbon sources,and its possible relevance for lactose maldigestion management.45 Asa consequence, these enzymes could be useful in bioprocessing approachesfor the valorization of some cereal byproducts following their inclusionin lactic acid bacteria starters with targeted metabolic activities.46In addition to lactose metabolism, in this work, we have demonstratedthat Lp_3525 could be able to hydrolyze phospho-β-galacto/gluco-derivedsubstrates over a broad temperature and pH ranges, especially forthe 6Pβ-glc activity. Although the capability of phospho-β-glycosidaseactivities is still rather unexplored, this type of enzymes may catalyzethe degradation of phosphorylated glucosides, glycosylated lignin,and fiber-related disaccharides during plant-based fermentations and contribute to the release ofa wide range of phenolic compounds.


Consequently, concrete-related CO2 emissions have tripled between 1990 and 2020, and its contribution to global emissions has risen from 5% to 9%. We propose that the policy agenda should focus more on limiting production growth by changing how concrete structures are designed, constructed, used, and disposed of to address the sand and climate crises.Concrete production poses multiple sustainability challenges, including resource over-exploitation and climate change. Here we show that growing global demand for buildings and infrastructure over the past three decades has quadrupled concrete production, reaching ∼26 Gt/year in 2020. As a result, annual requirements for virgin concrete aggregates (∼20 Gt/year) exceeded the extraction of all fossil fuels (∼15 Gt/year), exacerbating sand scarcity, ecosystem destruction, and social conflict. We also show that despite industry efforts to reduce CO  •Global concrete production quadrupled from 1990 to 2020•Demand for virgin concrete aggregates exceeded the extraction of all fossil fuels•2 emissions rose to 9% in 2020The concrete industry’s share of global CO Environmental science; Global change; Civil engineering Such a lack of systems-based understanding masks the scale of the challenges and the opportunities for future intervention.Addressing these challenges requires a systems-based understanding of the material flows and the implications associated with the entire concrete cycle, from resource extraction to processing, manufacturing, construction, use, and end-of-life management. However, despite its universal use in society, the extent to which concrete has been produced, stockpiled, and disposed of is poorly understood on a global scale. Accordingly, information on resource requirements and CO2 emissions of concrete production.,,,,2 emissions and their driving forces have not been investigated in detail. A key question is, therefore, to what extent has global concrete production contributed to the extraction of scarce resources and the increase in CO2 emissions over time?Several key studies have addressed some of these knowledge gaps by quantifying the resource requirements and CO2 emissions associated with the global concrete cycle. The analysis begins with the collection and reconciliation of fragmented statistical data on material flows. The concrete-related CO2 emissions are then calculated by compiling a dataset documenting the energy consumption in each process and their associated emission factors. The analysis also reveals the drivers of change in CO2 emissions through an index decomposition analysis. Collectively, this study provides a systems-based understanding of the challenges and the opportunities for intervention in the impending sand and climate crises.This study aims to answer this question by calculating and mapping a series of material flows and COBased on our analysis, the global production of concrete and mortar (referred to hereafter as concrete for simplicity) quadrupled between 1990 and 2020, reaching ∼26 Gt/yr in 2020 . AccordiA consequence of such rapid growth in concrete production and limited circularity is a surge in the requirements for virgin aggregates . In 1990, virgin aggregates that were used for concrete manufacturing accounted for only about 60% of all fossil fuel extraction . HoweverReliable data on total aggregate production are scarce, but global aggregate production in 2020 is estimated to be approximately 42 Mt/yr.2 emissions tripled in the last 30 years, reaching ∼3100 Mt-CO2/yr in 2020 (2 emissions are on-site placement (∼6%) and transportation (∼4%). The contributions of aggregate production (∼2%), mixing and batching (∼1%), and admixture preparation (<1%) are relatively small, resulting in cement production-related emissions accounting for 86% of the total emissions from the concrete cycle. In contrast, over the same 30-year period, total energy-related CO2 emissions  increased by a factor of 1.5.2 emissions increased from approximately 5% in 1990 to 9% in 2020.The sustainability issues associated with the increase in concrete production are not only restricted to destructive resource extraction but also extend to climate change. According to our analysis, concrete-related global CO in 2020 A. Emissi2 emissions? We explored the drivers of changes in CO2 emissions associated with cement production using a logarithmic mean Divisia index I approach.2/yr) was attributed to substituting clinker with supplementary cementitious materials . Thermal and electrical energy efficiency improvements resulted in further emission savings of 119 Mt-CO2/yr and 21 Mt-CO2/yr, respectively. Additional emission savings through the use of alternative fuels and electricity decarbonization amounted to 16 Mt-CO2/yr and 25 Mt-CO2/yr, respectively. Collectively, the abatement efforts of the cement industry resulted in emission savings of 414 Mt-CO2/yr in 2020, which is equivalent to 44% of 1990 emissions. In turn, CO2 emissions per unit of cement production were reduced by ∼20% between 1990 and 2020. However, the 4-fold increase in cement production has exceeded these savings, resulting in a nearly 3-fold increase in emissions in 2020 compared to 1990 levels. Namely, the increased contribution of the concrete industry to total emissions is primarily due to the fact that the industry’s efforts to reduce CO2 emissions have been outweighed by the increase in the societal demand for cement and concrete.An important question is which factors have contributed the most to the change in CO2 emissions, the CO2 savings were entirely outweighed by the surge in concrete production in recent decades. These findings demonstrate the difficulties of tackling resource conservation and affecting deep emissions reductions while continuing to expand cement and concrete production. Improvements in production process efficiency have always been offset by production growth. We propose that the policy agenda should be focused more on how to limit the growth of cement and concrete production by changing the way that concrete structures are designed, constructed, used, and disposed of.Ultimately, the findings of this study highlight the growing role that the global concrete cycle plays in the impending sand and climate crises. The need for sand, gravel, and crushed stone for concrete manufacturing already far exceeds the extraction of all fossil fuels, exacerbating the global sand crisis. Clearly, how we produce and use concrete holds the key to addressing the issue. It is also important to note that, despite the considerable efforts by the cement and concrete industry to reduce CO,,,Multiple lines of evidence have shown that those efforts produce significant resource and emission savings throughout the design, construction, use, and end-of-life phases.,Sand, gravel, and crushed stone have been largely ignored in international targets and goals, including the Sustainable Development Goals.With each new planned building or infrastructure development, we have an opportunity to ask: Do we really need to build more? Can we make do with less? Can we adapt what we already have? Asking such questions could offer a unique opportunity to address both sand and climate crises in one stroke. The findings of this study provide a foundation for discussions in these areas and can inform the development of effective policy solutions.It is important to note that all of the estimates in this study are based on disparate and incomplete data, which have a high degree of uncertainty. Monte Carlo simulations attempt to capture such uncertainty, but the uncertainty range of system variables is not based on actual measurements. Therefore, the uncertainty range should not be interpreted as an exact range but only as being sufficient to determine whether the trends in the estimation results are sufficiently robust. Simplification of cement applications, such as excluding cementitious solidifiers from the analysis, is another limitation of the study. According to a study conducted in the UK, approximately 4% of cement is used for applications other than concrete and mortar.watari.takuma@nies.go.jp).Further information and requests for resources should be directed to, and will be fulfilled by, the lead contact, Takuma Watari . These flows are estimated by multiplying the estimated concrete waste by its treatment rate (When the concrete used in buildings and infrastructure reaches the end of its useful life, it is crushed into large pieces. The crushed concrete waste is then either landfilled or sorted and downcycled for use as road-base materials or aggregates in lieu of virgin aggregates sourced from mining. There are also so-called hibernating stocks, which remain ent rate .,,,,,The main sources of data are as follows: cement production,,,The uncertainty of estimates derived using disparate and incomplete data is captured by using Monte Carlo simulations. The uncertain system variables considered are cement production, market share of concrete and mortar, lifetime, concrete mixture, and mortar mixture, with a range of uncertainties based on several literature sources , and S5.2 emissions associated with the concrete cycle are calculated based on a comprehensive dataset documenting the energy consumption in each process and their associated emission factors (CO factors . The emi,2 emission factor for various processes associated with the concrete cycle.,,,The main sources of data are as follows: thermal efficiency in the cement kilnThis study identifies the drivers of changes in carbon emissions associated with cement production using an index decomposition analysis. This is done using a logarithmic mean Divisia index I approach because of its theoretical basis, adaptability, ease of use and interpretation of results, and complete decomposition.


Via radical polymerization, three polyurethane-modified polycarboxylate molecules of various comb topologies were synthesized. This study investigated the effects of varying types and concentrations of supplementary cementitious materials (SCMs) on the surface tension, flowability, and zeta potential of cement. An elevation in the molar ratio between isoamyl alcohol polyoxyethylene (TPEG) and acrylic acid (AA) from 1:1 to 5:1 reduced the surface tension of the polycarboxylate molecule from 47.70 mN/m to 35.53 mN/m and increased flowability from 280 mm to 310 mm, as the results indicated. An increase in the SCM and polycarboxylate dosage proportionally decreased liquid-phase surface tension and increased flowability. A decrease in the water-to-cement (w/c) ratio from 0.5 to 0.3 corresponded to an observed increase in the zeta potential of cement pastes. However, a rise in the quantity of polycarboxylate and SCMs corresponded to a decrease in the zeta potential at a w/c ratio of 0.3. The hydration products of Portland cement allow these groups to attach to the products’ surface, which also breaks down the flocculating structure between silicate particles while forming an adsorption layer. The steric hindrance and electrostatic repulsion of the polycarboxylate superplasticizer, when added, can significantly alter the interaction between cement particles at the interface between water and solid activator; this will improve flowability and other chemical properties once the Portland cement particles are evenly distributed [42− would increase the dissolution concentration of Ca2+ in calcium-silicate-hydrate gel (C–S–H), which increased polycarboxylate superplasticizer aggregates and sharply reduced PCE molecule dispersion in Portland cement. Therefore, it is necessary to strictly control the content of PCE in a concrete system. The research results from Schmid et al. [Polycarboxylate superplasticizer molecules contain many functional groups, including –COOH, –SOtributed ,2,3,4. Ctributed . Differetributed ,7,8, andtributed  believedtributed  thought tributed  found thWen, Xiaodong, et al.  studied From the references above, however, we found that there are few studies on the adsorption and dispersion characteristics of Portland cement and SCMs in combination with different water-reducing agents. In this study, polyurethane-modified polycarboxylate superplasticizer  with the same main chain length and different side chain lengths was synthesized by controlling the molar ratio of large monomers to methacrylic acid. The interaction between MPCE and Portland cement mixed with supplementary cementitious materials was studied. We hope to accurately regulate the molecular structure of polycarboxylic acids based on polyurethane side chain macromers, effectively elucidate the structure–activity relationship, and reveal the interaction mechanism between polyurethane side chain macromers and Portland cement. The relevant research results are expected to provide sufficient theoretical basis for the design, synthesis, structure–activity relationship, and interaction mechanism with Portland cement of polyurethane polycarboxylate.The raw materials included P·I42.5 cement, mineral powder, fly ash, and metakaolin. Their chemical components and particle size distribution are shown in The raw materials were terminal hydroxy siloxane, dimethyl propionic acid, polyether diethyl alcohol, 1,4-butanediol, isoamyl polyoxyethylene ether (TPEG-2400 g/mol), n-methyl pyrrolidone, and isophorone diisocyanate. Acrylic acid, p-toluenesulfonic acid, thioglycolic acid, ascorbic acid (Vc), dibutyltin laurate, polyethylene glycol 200 (PEG), hydroquinone, benzoyl peroxide, hydrogen peroxide, ammonium persulfate, and deionized water were among the pure synthetic materials identified via chemical analysis. Ingredients included sodium gluconate, tartaric acid, citric acid, sodium hexametaphosphate, and white sugar, which were all of industrial grade.First, 22.2 g of isophorone diisocyanate was placed in a three-mouth flask, and then added, drop by drop, into the polyethylene glycol 1000 containing dibutyltin dilaurate using a dropping funnel (the solution of dibutyltin dilaurate and dimethyl propionic acid/N-methyl pyrrolidone was 0.2 g per 25 g polyethylene glycol 1000 and 3.35 g dimethyl propionic acid dissolved in 5 mL N-methyl pyrrolidone). A 50 g quantity of water was added next. The mixture of 1,4-butanediol and terminal hydroxy siloxane was mixed in, 2.5 g of ethylenediamine was added, and then 1.5 g of sodium dodecylbenzene sulfonate was added one at a time until the temperature reached 70 degrees Celsius. After that the solution was stirred, while 16% of the free –NCO weight was incorporated into the mixed solution. The solution was removed from heat, and the polyurethane prepolymer was obtained.Component A: Component B: (a certain amount of Mercaptoacetic acid and ammonium persulfate)Components A and B were dropped into the beaker using a peristaltic pump at a speed of 2 mL/min, and the process lasted about 2.5 h. After heat preservation for about 1 or 2 h (this range obtained after multiple experiments), the modified polycarboxylate mother solution M was obtained by adjusting the pH by using NaOH to 6–7.To create a 1.0 g/L solution, the PCE sample was diluted. The weight-average molecular weight (Mw) and conversion rate of PCE macromonomers were determined using a differential refractive index detector (RID) and gel permeation chromatography (GPC). The mobile phase used for GPC consisted of pure water containing 0.05% sodium azide, with a flow rate of 0.8 mL/min and column temperature maintained at 40 °C.PCE was vibrated in the constant temperature shaker at a set temperature to reach adsorption equilibrium, and it was then pumped and filtered. The obtained filtrate was centrifuged to obtain the supernatant, which was centrifuged twice. The residual concentration of polycarboxylate was measured using a total organic carbon tester (TOC). Then the adsorption capacity of polycarboxylate on the surface of cement particles was calculated based on the following equation.eQ represents the amount of adsorption (mg/g). C0 stands for the concentration before the adsorption of the PCE solution (mg/g). eC means the concentration after the adsorption of the PCE solution (mg/g). V is the volume of the solution (the unit was the number 1); additionally, M stands for the mass of the cement (g).In this equation, The liquor of the water reducer was initially produced in accordance with the planned w/c ratio and PCE percentage. Next, the solution was mixed with gelled materials and stirred via magnetic force for 10 min. The cementitious pastes were separated with a centrifuge for 10 min after being oscillated on the oscillator. The supernatant that had been separated was taken out and put through a 0.45 m nylon microporous filter membrane. In a volumetric flask, the filtrate was transferred and diluted 100 times with deionized water. Liquid TOC from the German company Elementar was used to measure the carbon content of the diluted solution. The concentration of PCE in the solution was determined based on its carbon content, and its adsorption capacity was then determined. The cement content, water-to-cement ratio, and polycarboxylate content were set at 50 g, 0.4, and 0.2%, respectively. The surface tension of the PCE solution at different concentrations was tested by using A-60 automatic surface tensiometer produced by American Cono Industries Co., Ltd, Boston, MA, USA.The test of surface tension of cement pastes between the liquid and the solid phases test: A beaker containing 50 g of Portland cement and a set w/c ratio of 0.4 was used. The polycarboxylate ether (PCE) was mixed with 20 g of water and added into the beaker at varying concentrations . The liquid was first mixed slowly for 2 min, followed by vigorous mixing for an additional minute. The resulting cement mixture was then centrifuged and the surface tension of the supernatant was measured.On the basis of GB/T8077-2012 , the holPolycarboxylate was added to a beaker containing 50 g of gelled material with a water-to-cement ratio of 0.4. The solid concentrations of the PCE mixture were 0, 0.1%, 0.2%, 0.3%, 0.4%, and 0.5% of the mass of the gelled material, respectively. While varying the w/c ratios and polycarboxylate concentrations, we swirled the cement pastes at low speed for two minutes, followed by high speed for one minute. The average of the 5–7 zeta potential measurements performed on each sample over the course of 10 min was recorded.The mortar self-shrinkage of the self-compaction cement mortar was measured by using a corrugated pipe and a non-contact probe. The corrugated pipe had an inner diameter of 20 mm and length of 340 ± 5 mm, and the volume transformation of the fluid mortar could be converted into the length transformation. The water-reducing agent concentration in the cement mortar was 0.3–0.5%, and the w/c ratio was 0.3. Free shrinkage experiment, standard sand, benchmark cement, and a 2:1 sand-binder ratio used throughout. Additionally, above 140 mm, cement paste flowability was maintained. For 72 h after casting, the self-shrinkage variations of cement mortar were measured continuously.TAM Air, an 8-channel standard isothermal calorimeter made in Stockholm, Sweden, was used to quantitatively analyze the total heat release and the heat release rate of the cement pastes with SCMs. A 4 g quantity of gelled material was put into a beaker. A solid content of 0.2% of polycarboxylate was incorporated into the mixture at a water-to-cement ratio of 0.4. The hydration heat of the newly mixed cement samples was tested for 72 h. The temperature in the surrounding area was kept at 20 °C.Compared to other reaction systems, the REDOX system offers several advantages, including lower activation energy and a faster polymerization rate. The lower activation energy required to initiate polymerization means that the reaction can occur at lower temperatures. Among the three types of composite REDOX systems screened, ammonium persulfate at a mass of 1% of the monomer mass was found to be optimal under typical reaction conditions. Three types of multiple REDOX systems were selected and employed at typical reaction temperatures while a constant mass of ammonium persulfate at 1% of the monomer mass, namely hydrogen peroxide + ammonium persulfate-sodium bisulfite, hydrogen peroxide + ammonium persulfate-Vc, and hydrogen peroxide + ammonium persulfate-rongalit, was maintained. To investigate the properties of polycarboxylate in the three different initiation systems, three types of polycarboxylate named M1, M2, and M3 were synthesized while the other factors were kept constant. The presented findings provide evidence that, even when a constant TPEG-to-acrylic acid molar ratio is maintained, variation in the initiator system can significantly impact the flowability of cement pastes. The initial flowability values exhibited significant change, and over time, the difference in flowability values between the pastes increased steadily. In particular, the type M1 polycarboxylate demonstrated the lowest surface tension in the hydrogen peroxide + ammonium persulfate-sodium bisulfite initiation system. Regarding both conversion data and molecular weight distribution, type M2 exhibited the most favorable behavior, as the monomer conversion of the molecule was the highest and the molecular weight distribution was the most concentrated. Via analysis, it was determined that the polycarboxylate synthesized at room temperature using the hydrogen peroxide + ammonium persulfate-VC composite initiation system exhibited the best properties.9(34) was conducted. This experiment examined the law of polycarboxylate produced at ambient temperature and referred to the pertinent literature and experimental experience. The orthogonal table was used to set up the experiment. Numerous experimental settings could be used to identify representative conditions, and fewer trials could be used to infer superior manufacturing conditions. Meanwhile, further statistical analysis could be carried out to obtain more accurate results. Via a single-factor experiment, four primary variables affecting the dispersion characteristics of ethers and polycarboxylates were determined: n(H2O2), n(TGA), n(H2O2):n(APS):n(VC), n(AA):n(TPEG). A three-level, four-factor orthogonal experiment was designed for this study. In order to study polycarboxylate synthesis at room temperature, an orthogonal experiment on four components and three levels LemQ, the two indices were comprehensively treated to obtain the comprehensive indices, as the table illustrates. The specific methods were as follows: First, the maximum and minimum value of the saturated adsorption capacity emQ were 100 and 0, respectively. The maximum and minimum value of the surface tension were also 100 and 0. In this way, a centesimal approach might be additionally used to measured values of the two indices. Since the saturated adsorption amount and the weight of surface tension were equal, a comprehensive index was obtained via a 50% weighted average calculation. The average value of the comprehensive indices of all levels referred to K1–K3, and the range means R. Based on the aforementioned results, the optimum synthesis process was A1, D2, C2, B3, which was the same as n(H2O2) = 0.05 mol, n(TGA) = 0.01 mol, n(AA):n(TPEG) = 2:1, n(H2O2):n(APS):n(VC) = 15:1:1.In order to thoroughly assess how many parameters affect surface tension and saturation adsorption capacity The ideal process conditions described above were used to create the polycarboxylate. The reaction took place at a temperature of 40 °C over 4 h. We tested the dispersivity of the polycarboxylate and named it M4; the results are shown in According to the results in Similar to below, the w/c ratio was 0.4.The addition of various cementitious ingredients led to a decrease in the surface tension of the produced pastes. The surface tension further decreased with increasing admixture content. This can be attributed to the smaller particle size of the mixed mineral powder and fly ash compared to Portland cement. These materials consist of aluminosilicate glass beads and sponges that resemble glass beads, with a small average particle size and internal specific surface area. Aluminosilicate glass beads exhibit advantages such as dense texture, smooth surface, good flowability, and reduced adsorption of free water, thus acting as “ball bearings” in the concrete mixture. These products effectively fill the voids left by Portland cement hydration products and can significantly reduce internal porosity, resulting in refinement of the pore size.2+, Na+, and OH− in the liquid phase decreased due to the lower solubility of the admixture compared to Portland cement particles. During the early hydration of Portland cement, the Si-O bond would break in the alkaline environment, and then combine with hydrated and dissociated ions to form C–S–H gels. This caused a decrease in the concentration of reverse ions on the surface of Portland cement particles, which in turn influenced the liquid-phase surface tension [After the addition of the SCMs, the concentrations of plasma Ca tension ,17,18,19In previous studies, the impact of incorporating various SCMs on rheological properties adsorption that of cement has been tested, and the results indicate that an increase in the SCMs content led to a continuous decrease in the rheological properties of the cement ,21,22,23Mineral powder, being mainly composed of vitreous material, had an irregular shape similar to that of Portland cement particles. Its crystallinity was low, and its particle arrangement was disordered, resulting in a relatively large specific surface area. After adding the admixture, the specific surface area of the cement pastes containing mineral powder increased, leading to more adsorption of polycarboxylate superplasticizers by the mineral powder in the pastes ,25. ConsFly ash is characterized by its spherical shape, loose structure, and microporosity, which make it highly adsorbable. Fly ash added to cement paste effectively adsorbs polycarboxylate molecules, and the spherical particles of fly ash can effectively fill in the gaps between Portland cement particles, leading to a more compact structure and smaller interparticle spacing . ConsequAfter being calcined at high temperatures, the original 1:1 shape of the metakaolin dioctahedral silicate structure is transformed into a debris topography consisting of lamellar octahedral alumina and other components, resulting in a transition phase with relatively poor crystallinity. This process leads to the disappearance of the accumulation phenomenon and an increase in interparticle pore size. The distinctive interlayer structure of metakaolin facilitates the adsorption of water and polycarboxylate molecules ,28, resuThe zeta potential of cement pastes mixed with M4 was investigated with varying levels of SCM content  and water-reducing agent content  while maintaining a constant w/c ratio of 0.4, as presented in 2O molecules in both pure Portland cement pastes and those blended with the three supplementary cementitious materials. The formation of a solvent hydration shell resulted in lubrication and steric hindrance effects [The surface potential of the particles underwent changes during the process of Portland cement hydration, with corresponding changes to the potential of the diffusion layer. The maintenance of electrostatic repulsion was crucial to maintaining zeta potential. The adsorption of polycarboxylate superplasticizers onto cement particle surfaces led to the extension of functional group-polyoxymethylene chain segments into the solution. This resulted in the formation of hydrogen bonds with H effects . These eIn a gel system, zeta potential reflects the potential of double electrode layers; additionally, between molecules, it has a significant correlation with electrostatic repulsion. From the Debye–Huckel formula:FES stands for electrostatic repulsion, ε represents the relative dielectric constant of water, ε0 is the vacuum dielectric constant, ψ represents electrodynamic potential, K stands for the Debye–Huckel parameter, h denotes the distance between two particles, and l indicates the thickness of the adsorption layer. PCE follows Langmuir’s monolayer adsorption law on the outside of particles of Portland cement [l remains unchanged, and the distance between two particles h is decreased. In this way, the electrostatic repulsion force FES is decreased [In this equation, d cement . It can ecreased . The staecreased . +, Ca2+, and Na+ plasma concentrations in its liquid phase [2O molecules in the pastes. The addition of these two SCMs reduces the amount of Portland cement in the mixture, leading to a decrease in the total production of hydration products, especially Ca (OH)2 and hydrated calcium silicate, which play a crucial role in improving zeta potential. As a result, the stability of the pastes decreases, and zeta potential also decreases correspondingly [It is evident that the zeta potential in cement pastes decreased when the admixture content of polycarboxylate superplasticizer was 0–0.2% and the three SCMs, namely fly ash, mineral powder, and metakaolin, were added. This decrease in zeta potential is one of the main reasons for the reduction in flowability and liquid-phase surface tension of the newly mixed cement pastes. The solubility of Portland cement particles is higher than that of mineral powder and fly ash, resulting in an increase in Kid phase . Mineralondingly . −) is removed at the calcination temperature of 600–800 °C, causing the initial 1:1 shape of dioctahedral silicate to break down and degrade into a lamellar octahedral fragment morphology with low crystallinity, producing an Al2O3–SiO2 transition phase. This structural change causes amorphous Al2O3–SiO2 to have very high pozzolanic activity, and metakaolin has particularly strong adsorption of H2O [When metakaolin is added, the hydroxy (OHn of H2O . Consequ2+ and Mg2+ replaced Al3+ on the lattice) resulted in a negative charge at the crystal level of the metakaolin. After coming into contact with water, metakaolin formed a constant charge surface, which was positively charged at the particle edge and negatively charged at the crystal level. The zeta potential values of metakaolin were determined from the presence of ions with opposite charges on the shear plane during solid-liquid sliding. Under alkaline conditions, these ions were negatively charged [3A and C4AF in Portland cement correspondingly increased. As they were positively charged, the zeta potential became positive [Additionally, in the absence of polycarboxylate superplasticizer addition, it was observed that increasing the admixture content of metakaolin from 20% to 30% resulted in an increase in zeta potential from −1.9 mV to 1.0 mV. Furthermore, as the admixture content of polycarboxylate superplasticizer increased, the negative zeta potential value of the combination of metakaolin and cement pastes became positive. This was due to the Al–OH bond breaking and becoming positively charged on the faceted pebble of the metakaolin lattice edge. However, factors such as lattice defects and isomorphic substitution , metakaolin was negatively charged at the crystal level. After contact with water, metakaolin formed a constant charge surface, which was positively charged at the particle edge and negatively charged at the crystal level. The reverse-sign ions on the shear plane of the solid-liquid slide determined the positive and negative zeta potentials of metakaolin. Under alkaline conditions, the reverse-sign ions were negative and negatively charged [3A and C4AF in Portland cement increased correspondingly; as they were positively charged, the zeta potential also became positive [The Al–OH bond broke and became positively charged on the faceted pebble at the metakaolin lattice edge. However, due to factors including lattice defects and isomorphic substitution . The hydrated calcium sulfoaluminate coating the cement particles hinders the interaction of water with cement, and the heat release rate begins to increase during the subsequent process. The coating layer is destroyed under pressure, which increases the reaction rate of water and cement, leading to the maximum heat release. By replacing part of the Portland cement, the amount of clinker in the cement is reduced, resulting in a decrease in the overall heat release. During the induction and acceleration periods of the second and third stages of hydration, the total heat release decreases . C exhibited a compact high monomer conversion rate and molecular weight distribution when compared to other reducing agents such as sodium bisulfite and rongalit. This led to excellent overall performance, including longer side chains and improved steric hindrance and dispersion compared to regular PCE. The dispersibility of the synthesized polycarboxylate was found to be improved with an increase in the molar ratio of AA/TPEG under identical experimental conditions. The optimal synthesis process was determined to be at room temperature using TGA and H2O2 molar masses of 0.01 and 0.05, respectively, with AA and TPEG in a 2:1 molar ratio and an H2O2 to APS to Vc ratio of 15:1:1.The polycarboxylate molecules produced by the REDOX system of hydrogen peroxide+ammonium persulfate-VAs the content of SCMs and polycarboxylate superplasticizer admixture increased, there was a decrease in the surface tension of cement pastes when combined with metakaolin, fly ash, and mineral powder. This decrease in surface tension also resulted in a decrease in the stability of the cement pastes, as well as a decrease in the zeta potential value.The zeta potential of cement pastes increased upon addition of supplementary cementitious materials and reduction in w/c ratio, which resulted in improved steric hindrance effect and system stability when mixed with mineral powder, fly ash, and metakaolin. This effect was accompanied by an increase in the paste consistency and a more noticeable change in zeta potential.As a result of its unique lamellar structure and smaller particle size, metakaolin exhibits a stronger adsorption effect on water and polycarboxylate superplasticizer than Portland cement. Therefore, the inclusion of metakaolin had a greater impact on the surface tension, flowability, and zeta potential of cement pastes.In cement pastes mixed with polycarboxylate superplasticizer (w/c = 0.3), the addition of mineral powder, fly ash, and metakaolin slowed down its self-shrinkage, with the most significant effect observed with fly ash, followed by metakaolin and mineral powder. Additionally, increasing the content of PCE resulted in a further reduction in both overall heat release and heat release rate.


Bloodstream infections by carbapenemase-producing Enterobacterales and other MDRO were higher among the Lebanese than the French group of patients . For the French group, rapid identification of species and mechanisms of resistance significantly shortened turnaround time for definitive laboratory diagnosis and increased antibiotic therapy adequacy. No statistically significant differences were noted in targeted antibiotic therapy between the two groups. This study suggests that, in settings where bacterial resistance is prevalent, rapid microbiological methods have not provided any additional value. The clinical and economic impact of rapid microbiological methods will likely depend on local CPE, VRE, and other MDRO epidemiology and are areas for future research.In recent years, the diagnosis of bloodstream infections has been complemented by rapid microbiological methods, unattainable to most clinical laboratories in resource-limited settings. We evaluated the impact of their shortage on antibiotic therapy adequacy. We conducted a prospective multicenter cohort study including 150 adult Gram-negative bacilli bacteremia episodes, evenly distributed across three university hospitals: one in Lebanon, a resource-limited setting, and two in France, a resource-rich setting. Previous colonization by multidrug-resistant organisms (MDRO) was significantly more prevalent among the Lebanese than the French group of patients (16/50 vs. 5/100;  Clostridioides difficile and to resistant pathogens [Bloodstream infections are severe but treatable conditions requiring prompt microbial identification and susceptibility testing to make the most informed therapeutic decision . In factathogens . Moreoveathogens ,5. In reathogens . Not onlathogens ,8. Direcathogens ,10. Whilathogens . Despiteathogens . In this® BD Diagnostics, Franklin Lakes, NJ, USA, in Hôtel-Dieu de France de Beyrouth and in Avicenne Hospital, and BacT/ALERT® VIRTUO® bioMérieux Marcy l’Etoile, France, in Saint-Joseph Hospital), subcultures onto specific media plates and a Gram stain were performed. Gram stain results were immediately communicated to the treating physician and exported into the electronic information system. Bacterial identification was achieved by macroscopic characteristics of colonies accompanied by biochemical tests using API® bioMérieux systems France, in Beirut, Lebanon or by Bruker® MALDI-TOF MS, NJ, USA, applied directly on positive blood broths or on colonies in Paris and Bobigny, France. Rapid colorimetric methods, such as the BetaLACTA® Test (BLT) Bio-Rad® , which help determine resistance mechanisms were performed exclusively in France, as described [Enterobacterales (ESBL-PE) strains were detected by the double-disk synergy test when bacteria were susceptible to cefoxitin (10 µg) [Enterobacterales (CPE) strains were detected when they showed resistance to ertapenem, in accordance with the CA-SFM/EUCAST algorithm, and confirmed using MASTDISCS® Combi Carba plus UK, in Beirut, Lebanon, or Cepheid Xpert Carba-R® Sunnyvale, CA, USA, assay in Paris and Bobigny, France [Pseudomonas spp., or Fluoroquinolones on Enterobacterales in the absence of an alternative. In the absence of the availability in Lebanon of molecules such as aztreonam, ceftazidime-avibactam, and cefiderocol, colistin was considered the best available therapy for the management of these infections. We have decided to classify the use of colistin monotherapy for the treatment of CPE-related infections as “undetermined”. As for CPE infections resistant to ceftazidime-avibactam and fluoroquinolones, we considered the combination of colistin and aminoglycoside as adequate as they were the only available and microbiologically active agents. We also evaluated antibiotic therapy adequacy according to clinical judgement which takes factors such as the causative agent, route of infection, bacterial epidemiology, type of infection, and colonization into consideration.We conducted a prospective cohort study, from January to June 2022, in three university hospitals: Hôtel-Dieu de France de Beyrouth, a Lebanese 450-bed hospital; Avicenne Hospital, a French 500-bed hospital; and Saint-Joseph Hospital, a French 600-bed hospital. A total of 150 patients were consecutively enrolled, evenly distributed among the three hospitals. All patients were aged 18 and older, hospitalized, and presented a Gram-negative bacilli bacteremia. Immunocompromised patients and patients with known drug allergies were excluded from this study. Once a blood culture was signaled positive for growth by the automated systems (BACTECescribed . Antimic (10 µg) . Carbape, France ,17. We aEnterococcus (VRE), CPE, and/or other multidrug-resistant organisms (MDRO) such as extended-spectrum beta-lactamase-producing Enterobacterales and multidrug-resistant Pseudomonas aeruginosa). Microbiological data such as the causative pathogen, mechanism of resistance, time elapsed to genus and species identification, rapid colorimetric assays for mechanisms of resistance, and definitive antibiotic susceptibility testing were collected. Empirical antibiotic therapy as well as eventual adjustments were noted in order to assess antibiotic therapy adequacy at the three different timepoints previously mentioned. In addition, we recorded patients’ outcome parameters, including eventual complications, length of hospital stays, and 30-day mortality. Eventual complications include tissue damage, organ failure, metabolic acidosis, severe hypoxemia, and septic thrombosis.Demographic data such as age, gender, and ward of hospitalization were collected. Clinically, various data were collected such as route of infection, type of infection , and patient-related factors for antibiotic resistance .One hundred and fifty patients were analyzed. p < 0.01). Six cases of CPE were detected which were all from Lebanon. On the first day of blood culture positivity, identification of the causative pathogens and their respective mechanisms of resistance were unachievable in the Lebanese group, as opposed to the French group. Turnaround time to definitive laboratory diagnosis was significantly faster in the French group.Distribution of the causative agents and their mechanisms of resistance are summarized in p < 0.01). Unlike prior hospitalization, colonization with MDRO in the last 12 months was significantly linked with MDRO bloodstream infections. Empirical antibiotic therapy was significantly more adequate for CPE and other MDRO than non-MDRO. However, no statistically significant differences were found in the adequacy of targeted antibiotic therapy and 30-day mortality . However, based on clinical judgments, no statistically significant differences were noted between the two groups. Concerning optimal antibiotic therapy after identification of species and mechanisms of resistance in France, ineffective antibiotic therapy decreased, while adequate antibiotic usage increased. Antibiotic usage remained unchanged for the Lebanese, where rapid colorimetric or molecular tests were not available. Concerning targeted antibiotic therapy based on antibiotic susceptibility testing, the use of adequate antibiotics was statistically more significant in the Lebanese group. Moreover, the use of unnecessary broad-spectrum antibiotics was statistically more significant in the French group. Otherwise, based on clinical judgments, no statistically significant differences were noted between the two groups in targeted antibiotic therapy. Adequacy of antibiotic therapy remained undetermined for patients who underwent hospital transfers . Furthermore, a statistically significant association was found between mortality rate and the use of antibiotics in the last three months, as well as infections related to CPE . Patients who received initial effective antibiotic therapy exhibited a notable improved survival rate (p = 0.049).A multivariate mortality analysis was performed . InfectiWe evaluated the effect of the lack of rapid microbiological methods including direct bacterial identification and rapid detection of resistance profiles from blood culture broth using MALDI-TOF MS and rapid colorimetric methods, respectively. In the absence of these diagnostic methods, identification of the causative pathogen and mechanism of resistance were unachievable at the first day of positivity. While these rapid methods certainly enhance and facilitate the effective and targeted treatment such as the observed increase from 14% to 32% in adequacy following their use in the French group, their lack was not associated with less antibiotic therapy adequacy in our study and had no effect on patients’ clinical outcomes. In fact, we observed more statistically significant bloodstream infections with ESBL-PE and CPE in the Lebanese group, which could explain the adequacy of broad-spectrum antibiotic use in the absence of a rapid detection of resistance. The lack of certain antibiotics from the Lebanese market led to the use of relatively broader-spectrum agents that otherwise would have been reserved for resistant pathogens. For instance, due to the unavailability of piperacillin, a combination of piperacillin-tazobactam was used for the management of piperacillin-susceptible pseudomonas bloodstream infection. In the Lebanese hospital, the lack of proper stewardship implementation, particularly in the absence of proper guidance by infectious disease specialists, may be associated with the use of unnecessary broad-spectrum antibiotics. For example, in our study, a combination of colistin and meropenem was used to treat an ESBL infection susceptible to piperacillin-tazobactam.Acinetobacter spp. (70.3%), compared to 33.9% in the United States and 33.9% in European Union countries, followed by third-generation cephalosporin–resistant Enterobacterales, methicillin-resistant Staphylococcus aureus, and CPE [Even though comprehensive data remain scarce, previous studies suggest that low-to-middle-income countries (LMICs) including Lebanon are likely to be most affected by the declining effectiveness of antibiotics. LMICs’ higher exposure is by dint of pre-existing developmental and economical challenges and likely deficiencies in the healthcare system . In fact and CPE . High prDespite a shortage in rapid diagnostic methods, adequacy was not affected in our study. Although bloodstream infections to resistant pathogens were more prevalent in the Lebanese cohort of patients, empirical therapy was more appropriate. Meanwhile, in the French cohort of patients, targeted antibiotic therapy was overused. Hence, our study showed that, in settings where the prevalence of bacterial resistance is high, rapid microbiological methods have not provided any additional value. Therefore, epidemiological surveillance is of utmost importance since it influences the choice of empirical antibiotic therapy and provides the necessary input for developing therapy guidelines, antimicrobial stewardship programs, and public health interventions. In fact, the choice of empirical antibiotic therapy can be influenced by epidemiologic risk factors, such as a high prevalence of VRE, CPE, or other MDRO, that can influence the probability of antibiotic resistance and likelihood of successful treatment. Mehl et al. prospectively recorded 1995 episodes of bloodstream infections between 2002 and 2013 in a medium-sized Norwegian hospital where antimicrobial resistance was relatively low. In this cohort, appropriate empirical antibiotic therapy was achieved by replacing second- and third-generation cephalosporins with penicillin and gentamicin . In contOn the other hand, the choice of empirical antibiotic therapy may be determined by assessing clinical scoring systems that predict the likely source of infection. Goodman et al. evaluated a clinical decision algorithm predicting ESBL-PE bloodstream infections in 1288 patients at Johns Hopkins Hospital. Of the 194 patients with ESBL-PE bloodstream infections, 68 (35%) received empirical carbapenem therapy within 6 h after causative pathogen identification. This tool correctly identified one-third of the original 68 patients, as well as an additional 78 cases. Use of the clinical decision algorithm would have increased by 50% ESBL-PE detection during the empirical treatment, justifying the use of agents covering ESBL-PE .Clostridioides difficile infection, time to resolution of fever, time to discharge from hospital, or de-escalation of broad-spectrum therapy between the two groups. While it was shown that microbial identification was quicker in the rapid diagnosis group, time to effective antimicrobial therapy was no shorter [The role of rapid diagnostics in bloodstream infections has been subject to a number of publications. The RAPIDO multicenter randomized control trial, including 8628 patients across seven National Health Service acute hospital trusts in England and Wales, compared the impact of rapid microbial identification and conventional methods on clinical outcomes in bloodstream infection. In this study, conventional methods included performing a Gram stain and microscopy followed by culture to isolate individual species, and biochemical tests to identity and reculture to test antimicrobial susceptibility. In the rapid diagnosis arm, direct MALDI-TOF MS after extraction was used in addition to conventional methods. Findings concerning 30-day mortality and length of hospital stay are in concordance with ours, despite several limitations in our study. No significant differences were observed in total antimicrobial consumption,  shorter . Finding shorter . Indeed, shorter ,28. A 20 shorter .Clostridioides difficile infections [On another note, despite the use of rapid microbiological methods, the overuse of broad-spectrum antibiotics increased from 40% to 49% in the French hospitals. Also, the overuse of broad-spectrum antibiotics in targeted antibiotic therapy, based on clinical judgments, was observed in 19% and 28% of the included patients in the French and Lebanese hospitals, respectively . Inapprofections . Howeverfections ,32. At afections .Our multicenter study has some limitations. Its major limitation is the small number of patients included, particularly in the resource-limited hospital, which may explain the observed absence of difference between the two studied groups. Therefore, our findings should be investigated on a larger sample of patients. Also, this study was conducted in only one university hospital in Lebanon. Even though Hôtel-Dieu de France is a major hospital in Beirut, the findings of our study may not be generalized to other hospitals, especially in the absence of antimicrobial stewardship programs and national recommendations for bloodstream infections. Moreover, differences in laboratory workflow and infectious disease consultations between the French and Lebanese hospitals were not taken into account. Finally, the absence of significant differences in 30-day mortality between the two groups is difficult to defend insofar as we have not taken into account the multiple confounding factors that may be involved.In conclusion, bloodstream infections are life-threatening situations where rapid pathogen identification and antibiotic susceptibility testing enable clinicians to optimize antibiotic therapy, consequently leading to better patient clinical outcomes. Advancements in rapid diagnostics shorten laboratory turnaround time. Co-implementation of rapid microbiological methods and antimicrobial stewardship interventions may significantly enhance patients’ outcomes. In our experience and in situations of a high prevalence of resistance, rapid microbiological methods have not provided any additional value. The clinical and economic impact of rapid microbiological methods will likely depend on local CPE, VRE, and other MDRO epidemiology and are areas for future research.


This paper presents a framework that can transform reconfigurable structures into systems with continuous equilibrium. The method involves adding optimized springs that counteract gravity to achieve a system with a nearly flat potential energy curve. The resulting structures can move or reconfigure effortlessly through their kinematic paths and remain stable in all configurations. Remarkably, our framework can design systems that maintain continuous equilibrium during reorientation, so that a system maintains a nearly flat potential energy curve even when it is rotated with respect to a global reference frame. This ability to reorient while maintaining continuous equilibrium greatly enhances the versatility of deployable and reconfigurable structures by ensuring they remain efficient and stable for use in different scenarios. We apply our framework to several planar four-bar linkages and explore how spring placement, spring types, and system kinematics affect the optimized potential energy curves. Next, we show the generality of our method with more complex linkage systems that carry external masses and with a three-dimensional origami-inspired deployable structure. Finally, we adopt a traditional structural engineering approach to give insight on practical issues related to the stiffness, reduced actuation forces, and locking of continuous equilibrium systems. Physical prototypes support the computational results and demonstrate the effectiveness of our method. The framework introduced in this work enables the stable, and efficient actuation of reconfigurable structures under gravity, regardless of their global orientation. These principles have the potential to revolutionize the design of robotic limbs, retractable roofs, furniture, consumer products, vehicle systems, and more. Examples include robots and robotic arms2, adaptive building facades3, deployable bridges and canopies5, stowable solar arrays6, furniture7, micro-grippers9, and retractable roofs10. For decades, research has focused on the kinematics, mobility, and stress states of reconfigurable structures14. A fundamental challenge that remains is efficiently actuating them while preserving stiffness and stability, especially in applications where gravity has a significant effect. In many designs, reconfiguration requires a large input of energy, resulting in inefficient, over-designed, and costly structures that are impractical to fabricate and operate.Reconfigurable structures are systems with components that move, or 17. Advantages of systems with continuous equilibrium include low energy required for actuation and an inherently stable reconfiguration path that avoids instabilities and dynamic snap-through behaviors.Continuous equilibrium systems are a subset of reconfigurable structures with a kinematic mode that allows them to reconfigure with a negligible input of energy. Continuous equilibrium is also described as neutral stability or zero stiffness, and is characterized by a constant potential energy curve throughout reconfiguration19. Continuous equilibrium is attained when the potential energy contributions of these components offset the potential energy due to gravity. Examples include the Anglepoise desk lamp, where pre-stressed springs allow the lamp to be easily repositioned20, bascule bridges which utilize a counterbalance to open22, and chairs which can be easily adjusted to recline at any angle23.Under gravity, most reconfigurable structures do not have continuous equilibrium with a constant potential energy curve; rather, the potential energy is affected by gravity as the structure moves through its kinematic path. The potential energy curve of a system is also affected by elements such as counterweights, springs, or magnets25, an initial plastic deformation27, a temperature gradient28, thermal residual stresses29, or coupled components with offsetting deformations31. Structures can be designed to match a prescribed energy landscape (including a landscape corresponding to continuous equilibrium) by numerically computing the appropriate spring properties32. Despite these examples, there is currently no comprehensive framework to transform structures into systems with continuous equilibrium while considering gravity. In most previous studies, gravity has been ignored, or systems are either trivial to design or designed by trial and error. Additionally, all previous work has focused on achieving continuous equilibrium in only one specific orientation. If the entire structure is reoriented with respect to the ground , continuous equilibrium is not maintained.Continuous equilibrium has been attained in structures through the addition of zero-free-length springsreconfigure though their kinematic path and are reoriented with respect to a global reference frame. The method involves computing properties of springs that directly offset the potential energy due to gravity. In this paper, we first discuss the optimization setup used to find spring parameters that transform a simple linkage into a continuous equilibrium structure. Next, we explore how continuous equilibrium can be maintained as systems are reoriented. Then we apply the method to other linkages and expand to more complex systems. Finally, we use traditional structural engineering methods to explore the practical considerations for the use of these systems.In this work, we present a framework to design structures that maintain continuous equilibrium as they 33, biomechanics and bio-inspired design36, automotive steering37, surgical instruments38, and many other fields. A planar four-bar linkage consists of four rigid members connected with pinned joints, resulting in a one-DOF mechanism39. The four bars are identified as the input link, output link, coupler (floating) link, and ground (fixed) link, which is an imaginary bar connecting the two support nodes , external extensional spring, with one end attached to the Scissor Mechanism and the other end anchored to an external support  Fig. B. We comath Fig. C. Largerentclass1pt{minimaIn reality, the optimized continuous equilibrium structures will remain flexible (similar to a mechanism) and locking of the system would be necessary to provide stiffness for functional load-bearing applications. The structural stiffness of the Watt’s linkage with internal torsional springs is computed using the stiffness matrix formulation where a unit force is applied in the middle of the linkage. Without locking, the linkage only has high stiffness in the horizontal direction at the center of the kinematic path, where the midpoint of the floating bar traces a vertical path Fig. F. In theMore details on the computational methods and physical experiments are included in the Supplementary Materials.fmincon in MATLAB. The results presented in the ‘Considerations for Practical Implementation’ section are based on a formulation of a structural stiffness matrix where torsional springs are added at one end of classical beam elements.The optimization problems were solved using the function Physical prototypes of the Watt’s linkage and Scissor Mechanism were fabricated as proof-of-concept continuous equilibrium systems. Linkage members were cut from acrylic sheets and have dimensions of 0.3048 m (12″) by 0.0381 m (1.5″) with a thickness of 8.4 mm (0.329″) for the Watt’s linkage and 16.2 mm (0.638″) for the Scissor Mechanism). A low-friction frame was used to support the Scissor Mechanism. Torsional springs were purchased from McMaster-Carr and attached using acrylic pieces. The reconfiguration force was measured using a load cell.In this paper, we introduce a comprehensive method for designing reconfigurable structures that maintain continuous equilibrium. Our method involves optimizing the properties of internal, external, torsional, and extensional springs that counteract gravity to minimize the fluctuation of the potential energy curve throughout the kinematic path. The optimization framework is extended to optimize structures for a range of orientations, leading to one design that has continuous equilibrium properties even as the orientation of the structure changes. Combinations of springs with asymmetric kinematics tend to result in better performance, and external springs are the most effective when considering a structure at multiple orientations. We demonstrated how our design framework can be applied to real-world systems including a linkage with an external mass carried along a linear path, a linkage with a mass carried along a radial path, and a three-dimensional deployable origami arch. Using computational simulations and physical proof-of-concept prototypes, we show that the proposed continuous equilibrium structures enable more efficient actuation. Using optimization to design for continuous equilibrium results in reconfigurable structures that are more stable, efficient, and versatile for any application scenario. The framework presented in this work will expand the ability of designers and engineers to create multi-functional systems to be used in robotics, infrastructure, consumer products, architecture, and more.Supplementary Movie 1.Supplementary Movie 2.Supplementary Movie 3.Supplementary Movie 4.Supplementary Movie 5.Supplementary Movie 6.Supplementary Movie 7.Supplementary Movie 8.Supplementary Information.