Blood biomarkers that can be used for preclinical Alzheimer’s disease (AD) diagnosis would enable trial enrollment at a time when the disease is potentially reversible. Here, we investigated plasma neuronal-derived extracellular vesicle (nEV) cargo in patients along the Alzheimer’s continuum, focusing on cognitively normal controls (NCs) with high brain β-amyloid (Aβ) loads (Aβ+).The study was based on the Sino Longitudinal Study on Cognitive Decline project. We enrolled 246 participants, including 156 NCs, 45 amnestic mild cognitive impairment (aMCI) patients, and 45 AD dementia (ADD) patients. Brain Aβ loads were determined using positron emission tomography. NCs were classified into 84 Aβ− NCs and 72 Aβ+ NCs. Baseline plasma nEVs were isolated by immunoprecipitation with an anti-CD171 antibody. After verification, their cargos, including Aβ, tau phosphorylated at threonine 181, and neurofilament light, were quantified using a single-molecule array. Concentrations of these cargos were compared among the groups, and their receiver operating characteristic (ROC) curves were constructed. A subset of participants underwent follow-up cognitive assessment and magnetic resonance imaging. The relationships of nEV cargo levels with amyloid deposition, longitudinal changes in cognition, and brain regional volume were explored using correlation analysis. Additionally, 458 subjects in the project had previously undergone plasma Aβ quantification.42 in the current study. After normalization of nEVs, the levels of Aβ42 were found to increase gradually across the cognitive continuum, with the lowest in the Aβ− NC group, an increase in the Aβ+ NC group, a further increase in the aMCI group, and the highest in the ADD group, contributing to their diagnoses . Furthermore, nEV Aβ42 was significantly correlated with amyloid deposition, as well as longitudinal changes in cognition and entorhinal volume. There were no differences in plasma Aβ levels among NCs, aMCI, and ADD individuals.Only nEV Aβ was included in the subsequent analysis. We focused on Aβ42 levels in diagnosing AD-induced cognitive impairment and Aβ+ NCs. This biomarker reflects cortical amyloid deposition and predicts cognitive decline and entorhinal atrophy.Our findings suggest the potential use of plasma nEV AβThe online version contains supplementary material available at 10.1186/s13195-022-01010-x. Currently, Alzheimer’s disease (AD) remains the only leading cause of death without an available disease-modifying therapy. It is characterized by the co-existence of aberrantly accumulated amyloid-β (Aβ) and hyperphosphorylated tau . Accordi42, tau phosphorylated at threonine 181 (p-tau181), and t-tau levels in nEVs are closely related to those in the CSF ). We acquired the global and regional AV45 SUVR of each participant using the same methods as in our previous studies , 21, 23.neration ), as welDetails regarding the imaging acquisition protocol and processing steps are provided in Additional file The participants provided blood samples at the time of clinical evaluation. Blood samples were collected in EDTA polypropylene tubes in the morning after an overnight fast. After centrifugation , the supernatant plasma was aliquoted and stored at − 80 °C in the clinical sample center of Xuanwu Hospital. Each sample had undergone 1–2 freeze–thaw cycles before use. We precipitated EVs using Exoquick® and further enriched nEVs using an anti-CD171 antibody. The isolation process has been described previously , 12, witWe performed transmission electron microscopy (TEM) to characterize the morphology of single nEVs, nanoparticle tracking analysis (NTA) to calculate EV concentration and average diameter, and western blotting to verify the nature CD63, TSG101), purity , and neuronal origin  of EVs, following the guidance of MISEV2018 [01, puritPreviously, 458 subjects in the SILCODE project underwent plasma Aβ quantification; their clinical characteristics are displayed in Additional file 1–42 and Aβ1–40 [40 and Aβ42 results were included in the subsequent analysis. We did not analyze glial fibrillary acidic protein, as this is an astrocytic marker.Our pre-experiment results suggested that the electrochemiluminescence method was not sufficiently sensitive to detect Aβ and t-tau in nEVs . Therefore, we used two single-molecule array kits : Neurology 4-Plex E and pTau-181 V2, to measure nEV proteins. Notably, compared to previous single-factor or tri-factor kits, the former was newly developed for highly specific and sensitive measurement of the concentrations of full-length Aβd Aβ1–40 . All asst-tests were used for STT-A/B, AFT, and BNT scales. Kruskal–Wallis H tests followed by multiple post hoc comparisons were used for other continuous variables.In Table H tests followed by multiple post hoc comparisons. Differences in nEV Aβ levels between the two NC groups were further verified after correcting for confounding factors, including age, sex, and APOE ε4 status. The area under the receiver operating characteristic (ROC) curve (AUC) values, with 95% confidence intervals (CIs), were used to evaluate the ability of the indicators to distinguish Aβ− NCs or NCs from other groups.For group comparisons of NTA results  and Aβ concentrations, we performed Kruskal–Wallis 40 and Aβ42, Aβ and cognition, and Aβ and imaging markers, including global brain Aβ deposition and brain regional volumes, Spearman correlation coefficients were calculated. Linear regression models were used to evaluate the above associations further with adjustment for confounding factors (see legends for details). In addition, we used partial correlation analyses to evaluate the relationships between nEV Aβ levels and regional Aβ deposition after correcting for age, sex, and APOE ε4 status. Here, cognition was represented by the MMSE and MoCA-B scales, and the regional volume was expressed as the ratio to the TIV. However, in the regression models, the TIV was used as a covariate. Notably, we also evaluated the associations between baseline Aβ concentrations and longitudinal changes in cognition or regional volumes in a subset of participants. The processing and analysis of longitudinal data were based on previous studies [To explore whether correlations existed between the levels of Aβ studies , 29. Brip < 0.05, and the above analyses were performed using SPSS v24  or R software, version 4.0.1.The significance threshold was set at APOE ε4 allele. Amyloid-PET was available for all NCs and for some patients with aMCI (24.4%) or ADD (26.7%). The aMCI and ADD groups both showed higher AV45 SUVR than did Aβ− NCs. Baseline sMRI was available for 89.3% of the Aβ− NCs, 86.1% of the Aβ+ NCs, 60.0% of the subjects with aMCI, and 62.2% of the subjects with ADD. Compared to NCs, patients with cognitive impairment demonstrated more atrophy in the hippocampus and entorhinal cortex. In contrast, there was no obvious atrophy in the PCC and precuneus, and the apparent upward trends were caused by a significant decrease in the TIV.Two hundred forty-six participants were included in this study: 84 Aβ− NCs, 72 Aβ+ NCs, 45 aMCI, and 45 ADD. Table We followed MISEV2018 requirements to verify the extracted nEVs . More sp40 and Aβ42 results, respectively, after correcting for the particle number to 3 × 108. More specifically, the nEV Aβ42 levels gradually increased across the cognitive continuum, with the lowest in the Aβ− NC group (1.592 ± 0.852 pg/ml), an increase in the Aβ+ NC group , a further increase in the aMCI group , and reaching the highest in the ADD group . Importantly, the differences between the NC groups remained after adjusting for age, sex, and APOE genotype , irrespective of whether outliers were included or excluded , whereas adding demographic characteristics, with or without clinical scales, performed poorly (AUCs of 0.569 and 0.623). The use of sMRI indicators was not helpful for identification (AUCs < 0.60). Compared to the dementia stage, the aMCI stage also has therapeutic potential. As shown in Table 42 levels to discriminate aMCI from NCs was not markedly weakened with the inclusion of Aβ+ NCs (AUC 0.791) and was higher than that of the “demographic model” (AUC 0.657). Similar results were obtained for distinguishing ADD individuals from NCs.As shown in Fig. 42 and Aβ40 levels and their ratios exhibited no differences among clinically diagnosed NCs, aMCI, and ADD individuals ; however, huge overlaps were observed among these groups  and the subgroups . Additionally, there was no interaction between Aβ42 and APOE genotype  had a follow-up cognitive assessment after 14.58 ± 6.37 months. As shown, nEV Aβ42 levels correlated with both baseline scores (p < 0.001) and longitudinal worsening (p < 0.001) in MMSE  had a follow-up sMRI after 13.26 ± 4.7 months. The nEV Aβ42 levels correlated with longitudinal changes in the volumes of the entorhinal cortex and GM, but not that of other regions , but not in Aβ− NCs or in cognitively impaired patients . In contrast, the plasma Aβ concentrations did not change among the groups. More specifically, our study verified previous findings that the nEV Aβ42 assay indeed provides high diagnostic accuracy in identifying patients with cognitive impairment [42 is already increased in Aβ+ NCs, although its diagnostic efficacy was not marked (AUC with APOE genotype = 0.705). Furthermore, nEV Aβ42 levels were strongly associated with global and regional AV45 SUVR, suggesting that they reflect brain Aβ deposition. In addition, baseline nEV Aβ42 levels predicted longitudinal changes in cognition and entorhinal volume.Using a Chinese community-based population, we analyzed the AD-related cargos of EVs and found gradually increasing concentrations of Aβpairment , 13. Morpairment  and provThe specific enrichment of nEVs using immunoprecipitation is a pioneering discovery , opening42 can distinguish cognitively impaired patients from NCs. However, we admit that its ability to recognize Aβ+ NCs is not outstanding. According to our recent study on the discrimination of Aβ+ NCs [42 can predict MCI conversion [40 and Aβ42 were closely correlated, which was contrary to a previous study on nEV Aβ40 [The ability of plasma Aβ to assist in AD diagnoses has been questioned over the years , 34. The Aβ+ NCs , we beli Aβ+ NCs . Our stunversion  and thatnversion . Howevernversion . We founnEV Aβ40  or partinEV Aβ40 . Recent nEV Aβ40 ; therefo42 and AV45 SUVR indicated that nEVs have the potential to reflect brain pathological changes and are emerging as liquid biopsy tools. Furthermore, nEV Aβ42 was also associated with cognitive deterioration, as well as with entorhinal atrophy, suggesting that the blood assay could also serve as a predictor of disease progression, and thus could be used to select individuals most likely to progress during typically short clinical trial periods. We analyzed multiple brain regions; however, only the entorhinal cortex remained significant after correcting for confounding factors. The entorhinal cortex region is characteristically affected by tau pathology at an early stage in AD [42 and entorhinal atrophy only existed in Aβ+ NCs, but not in Aβ− NCs, indicating that the more severe the pathological damage, the more severe the atrophy. However, elevated brain Aβ deposition alone is probably insufficient to produce neuronal damage and cognitive changes [The strong correlations between baseline nEV Aβge in AD . Comparege in AD , 43–45.  changes , and the changes , 48. Nev changes , 49, Aβ  changes , and Aβ  changes .This study has several limitations. First, the small sample size limited the statistical power of our data, and not all participants underwent amyloid-PET. Second, CD171 is not absolutely expressed in the brain, but also in other tissues, and CD171+ EVs are thus not absolutely of neuronal origin only . Third, 42 contributes to the diagnosis of AD-induced cognitive impairment and also discriminates Aβ+ NCs from Aβ− NCs. This blood biomarker reflects cortical amyloid deposition and predicts cognitive decline and entorhinal atrophy. Our findings highlight the clinical utility of nEV Aβ42 in identifying at-risk individuals and conducting disease-modifying trials.In conclusion, our findings suggest that plasma nEV AβAdditional file 1. Supplementary material. Supplementary Table 1. Summary of previous studies on Aβ, tau, and p-tau (detected in the plasma nEV) as biomarkers of AD. Supplementary Table 2. Quality control of the nEV protein. Supplementary Table 3. Baseline characteristics of all subjects with plasma Aβ levels by clinical diagnosis. Supplementary Table 4. Comparisons between participants with and without sMRI or scales data. Supplementary Table 5. Relationships between nEV Aβ42 and age, sex, group, and APOE ε4 status. Supplementary Table 6. Relationship between nEV Aβ42 and AV45 SUVR. Supplementary Table 7. Relationship between baseline nEV Aβ42 and cognitive scales. Supplementary Table 8. Relationship between baseline nEV Aβ42 and brain regional volume. Supplementary Figure 1. Typical TEM images of nEV. Supplementary Figure 2. Typical NTA results. Supplementary Figure 3. NTA results of enrolled subjects. Supplementary Figure 4. Western blot characterization of nEVs. Supplementary Figure 5. nEV Aβ concentrations in different diagnostic groups and the ROC curves. Supplementary Figure 6. Plasma Aβ concentrations in different diagnostic groups.Supplementary Figure 7. Association between nEV Aβ42 concentrations and MoCA-B scale. Supplementary Figure 8. Association between nEV Aβ42 concentrations and baseline brain regional volume. Supplementary Figure 9. Association between nEV Aβ42 concentrations and longitudinal changes in brain regional volume.


Trichoderma reesei (TrAA9B) and the thermophilic Thermoascus aurantiacus (TaAA9B) microorganisms in tobacco by plastid transformation with the aim to test this technology as cheap and sustainable manufacture platform. In order to optimize recombinant protein accumulation, two different N-terminal regulatory sequences were used: 5′ untranslated region (5′-UTR) from T7g10 gene (DC41 and DC51 plants), and 5′ translation control region (5′-TCR), containing the 5′-UTR and the first 14 amino acids  of the plastid atpB gene (DC40 and DC50 plants). Protein yields ranged between 0.5 and 5% of total soluble proteins (TSP). The phenotype was unaltered in all transplastomic plants, except for the DC50 line accumulating AA9 LPMO at the highest level, that showed retarded growth and a mild pale green phenotype. Oxidase activity was spectrophotometrically assayed and resulted higher for the recombinant proteins without the N-terminal fusion (DC41 and DC51), with a 3.9- and 3.4-fold increase compared to the fused proteins.Plant biomass is the most abundant renewable resource in nature. In a circular economy perspective, the implementation of its bioconversion into fermentable sugars is of great relevance. Lytic Polysaccharide MonoOxygenases (LPMOs) are accessory enzymes able to break recalcitrant polysaccharides, boosting biomass conversion and subsequently reducing costs. Among them, auxiliary activity of family 9 (AA9) acts on cellulose in synergism with traditional cellulolytic enzymes. Here, we report for the first time, the production of the AA9 LPMOs from the mesophilic  Escherichia coli, Pichia pastoris and fungal strains [In recent years, the circular economy model based on biorefinery platforms that endeavor to reduce waste and increase productivity has had a huge boost. In this new model, plant biomasses are highly significant as a source of energy and high value products. It has been estimated that agricultural activities produce several billion tons of lignocellulosic residues. Although lignocellulosic plant biomasses consist mainly of three types of polymers , the proportion of each polymer varies between residues of diverse origin. Despite their wide application, it is well known that plant biomass breakdown requires a complex enzymatic machinery composed of different classes of cell wall degrading enzymes ,2. Recen strains ,9.Over the past decades, it has been demonstrated that plants represent a promising system to produce recombinant proteins due to low production costs and availability of several strategies to optimize yield and overcome limitations . Among tTrichoderma reseei (termed herein TrAA9B) and the thermophilic Thermoascus auranticus (termed herein TaAA9B) fungi. Indeed, the availability of a green, low-cost and sustainable manufacture platform will allow to overcome one of the major limitations in application of enzymes in biomass conversion reducing production and processing costs. Different expression levels and protein stability were observed for the two LPMO enzymes. Further, the enzymatic assay results demonstrated oxidative activities for the plastid-based LPMO enzymes.The aim of this study was to explore, for first time, the feasibility of plants and, in particular, chloroplast transformation technology as a heterologous platform to produce in tobacco two synthetic Lytic Polysaccharide MonoOxygenases, belonging to AA9 family and derived from the mesophilic TrAA9B) and thermophilic (TaAA9B) LPMOs were constructed. For each transgene, different 5′ regulatory sequences were used (rrn) and the 3′ untranslated region (3′-UTR) of the plastid rbcL gene (TrbcL).To transform tobacco plastid genomes, four vectors containing the mesophilic (ere used . In part10 of E. coli phage T7 (T7g10) in pDC41 and pDC51 vectors or with a translational control region (5′-TCR) containing the 5′-UTR from atpB and 42 N-terminal nucleotides  of atpB coding sequence in pDC40 and pDC50 vectors. A Flag tag was added at C-terminus of all coding regions for protein detection. The expression cassettes target exogenous DNA to the trnV-rps12/7 region of the tobacco plastid genome and contain aminoglycoside 3′ adenylyltransferase (aadA) gene as a selectable marker  from the gene e marker . The biohe probe a. Fragmehe probe b.Transplastomic plants grown in soil under greenhouse conditions generally showed a phenotype similar to that observed in wild type (Nt) and transformed with empty vector (PRV) control plants . Only DCExpression analyses of TaAA9B and TrAA9B transgenes were carried out on T0, T1 and T2 transplastomic plants at both transcription and protein levels. Total RNA was extracted from leaves of plants before flowering stage. Semi-quantitative (27 cycles) RT-PCR amplification of either TaAA9B or TrAA9B genes showed the expected 0.5 kb PCR product . For botE. coli bacterial alkaline phosphatase (FlagBAP) as reference revealed different accumulation levels for the recombinant proteins  into coerulignone . In partt 469 nm ,18.−1 of total soluble protein, respectively, indicating a low oxidase activity. Interestingly, the recombinant AA9 LPMO without the N-terminal modification showed higher specific activity values and in particular, DC41 and DC51 revealed 0.64 ± 0.02 U·g−1 and 0.26 ± 0.01 U·g−1, resulting 5.8 and 2.6-fold higher than those detected for their respective counterparts. Moreover, on the basis of the different expression levels for plastid-based AA9 LPMOs, evaluated by Western blot analysis, for DC40 and DC50 can be estimated specific activities of 16.5 ± 1.0 and 15.1 ± 1.5 U·g−1 AA9 LPMO. Interestingly, the recombinant proteins without the N-terminal fusion, DC41 and DC51, revealed values of 64.6 ± 2.7 U·g−1, and 51.2 ± 1.8 U·g−1, resulting 3.9 and 3.4-fold higher than those of their respective counterparts. Further, the specific activity of TaAA9B in DC41 was 1.3-fold higher than the specific activity of TrAA9B in DC51 protein extracts.The enzymatic assays using crude extracts of DC40, DC41, DC50 and DC51 transplastomic, wild-type (Nt) and PRV control plants, obtained with the acetone precipitation procedure, demonstrated LPMO activity. For all tested samples, except those corresponding to the control plants, an increase in activity (mU/mL), in a dose-dependent manner, was observed . In termLignocellulosic biomass is the most abundant renewable resource in nature suitable for biofuel production. However, the poor efficiency of enzymatic hydrolysis of some recalcitrant lignocellulosic materials remains a key limiting step in their processing . In receT. reesei was chosen as this filamentous fungus is one of the best-studied organisms used in biomass conversion; in fact, it produces a multitude of (hemi)cellulases and is the best known and commercially used cellulolytic system [T. aurantiacus, instead, was selected for its putative thermophilic properties. Neither TrAA9B nor TaAA9B were previously expressed in planta. It is well known that many factors may affect protein yield and stability in plastids. Among them, the choice of regulatory elements at N-terminal of transgene may have a strong impact on accumulation of recombinant proteins [TrAA9B  and TaAA9B  genes: a 5′ untranslated region from T7g10 in DC41 and DC51 plants, and a 5′ translation control region (5′-TCR), containing the 5′-UTR and the first 14 amino acids (DB) of the plastid atpB gene, in DC40 and DC50 plants. For T. reesei AA9 LPMO, the highest protein yield (5% TSP) was obtained in DC50 transplastomic plants, containing the AtpB-TrAA9B fusion protein, while the use of T7g10 5′-UTR in DC51 plants produced a protein accumulation level of 0.5% TSP. The highest expression detected in DC50 transplastomic plants was linked to a mild mutant phenotype mainly due to a growth retardation, since no alteration was observed in all other transplastomic plants expressing AA9 LPMO at lower level. This effect should be ascribed to intrinsic properties of TrAA9B, because when the same protein was accumulated at a lower level (DC51 plants), no mutant phenotype was observed. Phenotypic effects are often reflected in a reduction of the accumulation of Rubisco protein, visible in the SDS-PAGE profile, compared to controls. DC50 plant protein extracts, as well as those of the other transplastomic plants, did not show any alteration in their profiles compared to PRV control plants (T7g10 5′-UTR and atpB 5′-TCR regulatory sequences yielded similar accumulation levels in DC41 (1% TSP) and DC40 (0.7% TSP) transplastomic plants. Likewise, the use of three different DB regions  produced variable expression levels for an endoglucanase (cel6A) and a β-glucosidase (bglC) from Thermobifida fusca [tetC gene was the best choice, while nptII DB yielded the highest accumulation level for bglC expression. However, the variable expression level detected in our transplastomic plants expressing AA9 LPMO enzymes could not be due solely to the regulatory sequences used, but it may also be influenced by the intrinsic properties and/or the microbial origin of the recombinant proteins. For example, Kolotilin et al. [Clostridium cellulovorans gave contrasting results for the accumulation of two fungal xylanases, Xyn10A and Xyn11B, from Aspergillus niger [atpB yielded 0.4% TSP for the endoglucanase from Sulfolobus solfataricus, and a ≥75% TSP accumulation of the β-glucosidase from Pyrococcus furiosus [In the present study, we explored the feasibility of the production of two AA9 LPMO proteins by tobacco plastid transformation, since, previously, this approach enabled very encouraging results in terms of protein yield, hydrolytic activity, and stability of recombinant cellulolytic enzymes . TrAA9B c system ; TaAA9B proteins ,22. Thusl plants . Differeda fusca ,24. Partn et al.  demonstrus niger . In a sigag proteins [T7g10 coding sequence in DC41 and DC51 transplastomic plants detected the recombinant proteins in mature leaves (fifth and seventh), suggesting that either translation or protein stability can limit foreign protein accumulation in the transplastomic lines as already found for some viral antigens [The use of 5′-TCR of the plastidial AtpB protein in DC50 transplastomic plants not only increased the overall protein accumulation but appeared to also protect the recombinant protein from degradation in older leaves of mature plants. Differently, the same regulatory sequence in DC40 plants had neither improved recombinant protein yield nor its in planta stability since no accumulation was detected in older leaves suggesting that TaAA9B was susceptible to proteolytic degradation, as already observed for rotavirus VP6 and HIV1-Pr55proteins ,27. The antigens ,29.−1 AA9 LPMO, respectively, whereas a 3.9- and 3.4-fold lower activity was estimated for DC40 and DC50 protein extracts . Structural and characterization analyses revealed that AA9 auxiliary family are copper-dependent enzymes and that the Cu2+ ion is coordinated by a motif denoted as histidine brace (His-brace) formed by the highly conserved N-terminal histidine and a second histidine later in the sequence [Neurospora crassa NcLPMO9C and NcLMPO9F activities to be 32.2 and 2.2 U·g−1, respectively, while Myriococcum thermophilum was 30.9 U·g−1. Similarly, de Gouvêa et al. [−1 for the AfAA9_B LPMO of Aspergillus fumigatus, and 17 U·g−1 for the LPMO from Myceliophthora thermophila C1 (MtC1LPMO), respectively.Despite the absence of a specific assay to test the enzymatic activity of the LPMO family, a spectrophotometric method described by Breslmayr, et al.  was usedsequence . The Hissequence ,32 and isequence ,33,34,35sequence  found tha et al.  and Guo a et al.  reportedOur results demonstrated the suitability of plastid transformation as a platform for heterologous production of AA9 LPMO proteins and indicate that to retain their oxidative activity, these auxiliary enzymes need to be expressed without any N-terminal fusion. Despite the stable production of plastid-based AA9 family proteins being demonstrated in all the plant generations tested, further investigation is necessary to increase the protein yield obtained in this work and to carry out an in-depth evaluation of their booster activity in biomass digestion. To this aim, better results could be achieved by using alternative 5′-UTRs and/or bacterial LPMOs coding sequences, since it has been largely proved that chloroplasts, due to their endosymbiotic origin, can successfully produce recombinant proteins from bacterial sources ,39,40,41An improved production of plastid-based auxiliary enzymes, in terms of protein yields and specific activity, still represents an important chance to promote a sustainable and optimized biomass hydrolysis. Indeed, one major limitation of enzyme application is due to their high costs of production, that could be strongly reduced using chloroplast transformation technology as a green and renewable platform.Nicotiana tabacum L. cv. Petite Havana for plastid transformation were grown in sterile conditions on hormone-free medium containing MS salts and B5 vitamins , 30 g·L−1 sucrose and 8 g·L−1 agar, pH 5.6, at 24 °C with a 16 h photoperiod of 40 μmol photons m−2·s−1.Plants of −1 sucrose, 0.1 mg·L−1 NAA, 80 g·L−1 agar and with 500 mg·L−1 of spectinomycin under controlled conditions, and subsequently transferred to soil in a growth chamber  for seeds production.Homoplasmic transplastomic lines were rooted and propagated on medium containing MS salts with B5 vitamins, 30 g·LTaAA9B or TrAA9B genes (DC series) or with the empty control vector (PRV) were sown in vitro under controlled conditions (16 h light 40 μmol photons m−2 s−1 and 8 h dark at 24 °C) on Murashige and Skoog (MS) medium with B5 vitamins (Duchefa), solidified with 0.8% (w/v) agar, with 20 g·L−1 sucrose and 500 mg·L−1 of spectinomycin, or they were sown in soil in a growth chamber .Seeds derived from transplastomic plants transformed with either TrAA9B and TaAA9B genes from Trichoderma reesei (GenBank accession number AY281372.1) and Thermoascus auranticus (GenBank accession number KF170230.1), respectively, were synthetized by GeneArt .The TaAA9B and TrAA9B genes were digested with NheI and XbaI restriction enzymes and cloned into pHK30 and pHK40 plasmids [neo coding gene to develop pDC40/41 and pDC50/51 vectors, respectively.The synthetic plasmids ,43 replarrn promoter, the 5′-UTR of E. coli phage T7 gene 10 and the plastid rbcL gene 3′-UTR, whereas in pDC40 and pDC50 constructs the rrn promoter was fused with 5′ translation control region (5′-TCR) that includes the 5′-UTR and 42 N-terminal nucleotides (DB) of the plastid atpB gene  and used for delivery in tobacco leaf tissue. Biolistic experiments, in vitro regeneration and a selection of shoots were carried out according to the protocol described in Scotti and Cardi .® Plant Mini kit (Qiagen). cDNA was synthetized using the RevertAid RT Reverse Transcription kit  following the manufacturer’s instructions. Gene specific RT-PCR primers  was performed to verify the expression of recombinant proteins. Total RNA from the leaf of transplastomic and control was extracted with the RNeasy primers  were desv/v) SDS, 0.2% (v/v) Triton X100, 2% (v/v) 2-mercaptoethanol, 1 mM PMSF, 1× protease inhibitor cocktail  (leaves (g): buffer A (mL) ratio 1:3). The homogenate was centrifuged at 16,000× g and 4 °C for 20 min and supernatant recovered. Samples were electrophoresed either in a 10% or 12% SDS-PAGE and blotted onto Hybond ECL nitrocellulose membrane . Recombinant FlagBAP protein (Sigma) was used as control. Membranes were incubated with a 1:4000 dilution of monoclonal anti-Flag M2 antisera (Sigma) and subsequently with an HRP-conjugated anti-mouse antibody . Chemiluminescent signal was measured using a ChemiDocTM XRS+ and images were analysed using the Image LabTM Software .Leaves from transplastomic and control plants were grinded in liquid nitrogen and total proteins were extracted by homogenization in buffer A , incubated on ice for 15 min, stirred for 1 h at 4 °C and centrifuged at 20,000× g for 15 min. Pellet was then washed with solution C and then resuspended in buffer A (pellet (g): buffer A (mL) ratio 1:10). Following incubation on ice for 15 min, stirring for 1 h at 4 °C and centrifugation, supernatant was recovered and dialyzed three times against 20 vol 20 mM Na phosphate buffer pH 7.5.Crude protein samples for enzymatic assays were extracted using acetonic precipitation. Briefly, leaves from transplastomic and control plants grinded in liquid nitrogen were homogenated in 80.8% acetone containing 0.2% 2-mercaptoethanol (solution B) (leaves (g):solution B (mL) ratio 1:10), incubated on ice for 15 min, stirred for 1 h at 4 °C and centrifuged at 20,000× 2O2, as described by Breslmayr, et al. [469 = 53,200 M−1 cm−1) per minute under the described conditions.The crude extracts of DC40, DC41, DC50, DC51 transplastomic plants, and PRV and wild-type (Nt) plants used as controls, obtained with the acetonic precipitation, were used for the enzymatic assay. The LPMO activity was measured using 2,6-dimethoxyphenol  and H, et al. . The rea


Voxel-by-voxel correlation analysis was done for both groups in all networks. Results: In HiAmy, IFC was reduced in all networks except SN. A negative correlation in DMN, CEN, SRN and SMN suggests high Aβ related to IFC reduction; However, a positive correlation in SN suggests high Aβ related to an increase in IFC. In LowAmy, IFC increased in CEN, SMN, SN and SRN. Positive correlation in all major brain networks. Conclusion: The level of Aβ accumulated demonstrated differential effects on IFC in various brain networks. As the treatment to reduce Aβ plaque deposition is available in the market, it may be an option for the HiAmy group to improve their IFC in major brain networks.Introduction: Amyloid-β protein (Aβ) is one of the biomarkers for Alzheimer’s disease (AD). The recent application of interhemispheric functional connectivity (IFC) in resting-state fMRI has been used as a non-invasive diagnostic tool for early dementia. In this study, we focused on the level of Aβ accumulated and its effects on the major functional networks, including default mode network (DMN), central executive network (CEN), salience network (SN), self-referential network (SRN) and sensory motor network (SMN). Methods: 58 participants (27 Hi Aβ (HiAmy) and 31 low Aβ (LowAmy)) and 25 healthy controls (HC) were recruited. [ All imaging scans were conducted within one week. A group of HC was recruited from the university hospital, with age-matched with all recruited participants (diseased group). HC underwent clinical evaluation, neuropsychological test and MRI only, due to the restricted application of radiopharmaceuticals to HC.All participants underwent clinical evaluation, neuropsychological test  , [18F]fl18F]flutemetamol PET/CT scan), clinical (baseline and follow-up) and neuropsychological (HK-MoCA test) findings. The clinical diagnostic criteria for AD and MCI patients were in reference to the McKhann et al., 2011 study [18F]flutemetamol PET/CT scan result. For VD patients, the diagnosis was made with reference to the clinical criteria described in the Roman 1993 study [18F]flutemetamol PET/CT scan, as well as macro-vascular MRA and microvascular MRI abnormalities. The MR ASL perfusion scan provided additional information related to the perfusion patterns when it was necessary.The final diagnosis of each participant was determined by a multi-disciplinary panel, which consisted of two geriatricians (SYF and CPKC) and one neuroradiologist (MHKF) based on the imaging  of 5 mm was applied. Scanning time was limited to 30 min.The Aβ protein accumulation was assessed by  = 6.75 ms, echo time [TE] = 3.163 ms, inversion time [TI] = 844 ms, flip angle = 8°, slice thickness = 1.2 mm, 256 sagittal slices; acquisition matrix = 256 × 256, field of view = 256 × 256 mm2, acquisition matrix = 72 × 72, 36 slices, pixel dimension: 144 × 144 mm2, voxel size = 1.6 × 1.6 × 4 mm3).Each participant was asked to relax and remain quiet during the scan, with their eyes kept closed but not fall asleep. Resting state functional MRI (rs-fMRI) images were obtained using multi-echo echo planar imaging (EPI) sequence, with following parameters: 180 time points; TR = 2000 ms, TE = 30 ms, Flip angle = 90°, slice thickness = 4 mm, FOV = 230 × 230 mmhttp://www.rfmri.org/dparsf, accessed on 22 June 2022); it is a software built on the MATLAB platform 2018a , with Statistical Parametric Mapping software . Pre-processing was performed with standard processing steps as follows flutemetamol uptake in white matter was non-specific, i.e., the uptake in the white matter region was not specifically related to Aβ. Therefore, a white matter mask was created using WFU Pickatlas version 3.0.5 [18F]flutemetamol.The mean SUVr map of each group was obtained. The flutemetamol uptake maps. The maps for HiAmy and LowAmy groups were displayed in The Aβ protein accumulation was assessed using flutemetamol PET/CT scan was offered to diseased groups only, healthy controls did not undergo PET scan due to radiation consideration. The final diagnosis of each participant was not confirmed by histopathology as gold standard. Furthermore, the tau PET imaging, vascular comorbidity, white matter hyper-intensity were not included in this study; these techniques were commonly used for characterizing AD and its phenotypes were considered as a substitute of histopathology recently, and might be more commonly used in the future [This study was conducted in a real-life clinical setting and the participant recruitment was consecutive, while the sample was limited. [e future ,65. Furte future .The level of amyloid-β accumulated demonstrated differential effects on IFC in different brain networks. As the treatment to reduce amyloid-β plaques deposition is available in the market, it may be an option for HiAmy group to improve their IFC in major brain networks.


Similar results were observed in the HBV decline period post drug administration to the HBV transgenic mouse model, although these three TFV prodrugs finally achieved the same anti-HBV effect after 42 days treatments. Furthermore, TFV ester prodrugs showed a correcting effect on disordered host hepatic biochemical metabolism, including TCA cycle, glycolysis, pentose phosphate pathway, purine/pyrimidine metabolism, amino acid metabolism, ketone body metabolism and phospholipid metabolism. The callback effects of the three TFV ester prodrugs were ranked as TMF > TAF > TDF. These advantages of TMF were believed to be attributed to its greater bioavailability in preclinical animals  and better target loading, especially in terms of the higher hepatic level of the pharmacologically active metabolite TFV-DP, which was tightly related to anti-HBV efficacy. Further analysis indicated that stability in intestinal fluid determined the actual amount of TFV prodrug at the absorption site, and hepatic/intestinal stability determined the maintenance amount of prodrug in circulation, both of which influenced the oral bioavailability of TFV prodrugs. In conclusion, our research revealed that improved pharmacokinetics of TFV ester prodrugs  strengthened the inhibition of HBV replication and the rebalance of hepatocellular metabolism, which provides new insights and a basis for the design, modification and evaluation of new TFV prodrugs in the future.Tenofovir (TFV) ester prodrugs, a class of nucleotide analogs (NAs), are the first-line clinical anti-hepatitis B virus (HBV) drugs with potent antiviral efficacy, low resistance rate and high safety. In this work, three marketed TFV ester drugs, tenofovir disoproxil fumarate (TDF), tenofovir alafenamide fumarate (TAF) and tenofovir amibufenamide fumarate (TMF), were used as probes to investigate the relationships among prodrug structures, pharmacokinetic characteristics, metabolic activations, pharmacological responses and to reveal the key factors of TFV ester prodrug design. The results indicated that TMF and TAF exhibited significantly stronger inhibition of HBV DNA replication than did TDF in HBV-positive HepG2.2.15 cells. The anti-HBV activity of TMF was slightly stronger than TAF after 9 days of treatment (EC Hepatitis B virus (HBV) infection is a severe and worldwide public health problem that can cause chronic hepatitis and advanced-stage liver diseases, such as fibrosis, cirrhosis and even hepatocellular carcinoma . Accordiin vitro through only two-steps phosphorylation. It bypasses the first phosphorylation step due to its already having one phosphate group , tenofovir alafenamide fumarate (TAF) and tenofovir amibufenamide fumarate (TMF). TDF , the firTMF, TAF, TDF, and TFV (purity >98%) were purchased from MedChemExpress . Tenofovir diphosphate (TFV-DP) (purity >98%) was purchased from Mason Chem, Inc. . Lamivudine (purity >99%) was purchased from Tokyo Chemical Company . HPLC-grade acetonitrile and methanol were purchased from Merck . Deionized water was purified by a Milli-Q system . All other chemicals were purchased from commercial sources and were of analytical grade.−1 penicillin and streptomycin  at 37°C with 5% CO2. The cells in this study were used between passages 10 and 20 and were negative for mycoplasma infection. Primary rat hepatocytes were isolated from rat liver by a modified two-step collagenase perfusion method and treated and cultured as we published previously  at 4°C for 2 min. After aspirating the supernatant, the cells were re-suspended in DMEM with equal volume of percoll solution and centrifuged (50 g) at 4°C for 2 min. Next, the cells were washed and cell viability was determined. Lastly, the cells were seeded in rat tail collagen-coated 24-well cell culture plates for experiments.HBV-positive HepG2.2.15 cells were obtained from the China Center for Type Culture Collection in Wuhan . Cells were grown in DMEM supplemented with 10% fetal bovine serum and 100 U·mleviously . In brie4 cells/well, and incubated with cell culture medium containing TMF/TAF/TDF at various concentrations (ranging from 0.5 nM to 20 μM). On the 3rd, 6th, and 9th days post-drug incubation, the cell culture medium was harvested for HBV DNA analysis and replaced with a freshly prepared drug-containing medium. The EC50 was calculated with GraphPad Prism.HepG2.2.15 cells were seeded in 96-well plates at 4 × 10Six-week-old male HBV transgenic mice and wild-type C57BL/6 mice (18 ± 2 g) were purchased from Beijing Vitalstar Biotechnology Co., Ltd. . Sprague-Dawley rats (200 ± 20 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. . Beagle dogs (8.8 ± 1.0 kg) were provided by the Yadong Laboratory Animal Research Center . All the animals were housed under controlled conditions with a 12/12 h light/dark cycle and allowed free access to food and water. HBV transgenic mice were housed in Beijing Vitalstar Biotechnology Co., Ltd. . Animal studies were carried out in accordance with the Institutional Animal Care and Use Committee of Beijing Vitalstar Biotechnology Co., Ltd. , and the Guidelines for Animal Experimentation of China Pharmaceutical University .7 copies/mL). These HBV transgenic mice were randomly divided into four groups with 8 mice each. Three groups were orally administered 60.7 μmol/kg TMF, TDF or TAF for 84 days, respectively, and the last group was treated with an equal volume of normal saline once daily as a positive control . Another 8 wild-type C57BL/6 mice served as a negative control. Blood samples were collected every week to monitor serum HBV DNA levels. All the samples were assayed by the laboratory department of Jiangsu Province Hospital of Chinese Medicine.The HBV transgenic mouse model was built by pronuclear microinjecting a linearized DNA fragment, 1.28-fold the length of the HBV genome , into the fertilized egg of a C57BL/6NCrl mouse and stably transmitted HBV to the fifth generation with high titers of HBV DNA in serum .HepG2.2.15 cells were treated with 5 μM TMF/TAF/TDF for 3, 6, and 9 days, and then cells were collected for untargeted metabolomics analysis and targeted lipidomics analysis with metabolite identification based on LC-Q/TOF-MS as we described previously . The com2 and seeded in 24-well cell culture plates to approximately 70%–80% confluence. Then, the cells were incubated with TMF, TAF or TDF (5 μM) for 2, 4, 8, 12, and 24 h and collected for LC-MS/MS analysis. Similar experiments were also performed in primary rat hepatocytes.HepG2.2.15 cells were cultured in fresh DMEM (10% FBS) at 37°C with 5% COn = 6, male and female for half), C57BL/6 mice  and beagle dogs . Blood samples were collected at 0.083, 0.25, 0.5, 1, 2, 6, 12, and 24 h postdose from rats (via jugular vein) and dogs (via forelimb vein), or at 0.083, 0.25, 1, 4, 10, and 24 h postdose from mice (two time points per mouse) into EDTA-containing tubes for LC-MS/MS analysis. Pharmacokinetic parameters were calculated by a noncompartmental model using WinNonlin Pro 6.4 .Considering the features of ester prodrugs, three common animal models (including rodent and non-rodent) were used to compare the differences of the pharmacokinetic behaviors of TFV prodrugs comprehensively. The plasma pharmacokinetics of different TFV prodrugs were investigated in SD rats  were orally dosed with 30 μmol/kg TMF, TAF or TDF. Tissues including heart, liver, spleen, lung, kidney, thymus, stomach and small intestines were harvested into 50% acetonitrile immediately and homogenized separately. Tissue homogenates (W/V: 0.1 g/ml) were analyzed by LC-MS/MS.To explore the biodistribution of different prodrugs, SD rats (Simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were prepared according to the Chinese Pharmacopoeia . Rat blood and tissue homogenates (intestine and liver) were freshly collected and prepared. TMF, TAF, or TDF (5 μM) was incubated with each at 37°C for 0, 5, 15, 30, 60, 90, 120 min, and then the reaction was stopped and followed by LC-MS/MS analysis.in situ single-pass perfusion experiment was carried out as we reported previously (eff) was calculated as in is the flow rate (mL/min); A is the surface area of the intestinal segment (cm2); Cin is the initial donor concentration (5 μM); Cout(corr) is the corrected concentration of perfusate samples after applying the weight correction factor: To explore the intestinal permeability of different TFV prodrugs, an eviously . Brieflyn = 6) were anesthetized, fixed and administered 30 μmol/kg TMF, TAF or TDF orally. Blood (200 μl) was collected at 5, 30 min and 1 h post-dose from the rat hepatic portal vein (HPV) and inferior vena cava (IVC) for LC-MS/MS analysis.18 male SD rats  was 2.34 times and 1.67 times smaller than that of TDF (17.09 ± 2.45 nM) and TAF (12.17 ± 0.56 nM), respectively..The HBV-positive hepatic cell line HepG2.2.15, which stably secretes HBV DNA into the supernatant medium, was used to compare the anti-HBV activities of different nucleotide analog(s) 0 values . TMF and10 (copies/mL) difference (p < 0.05) during the HBV DNA decline period, which was approximately 16% of the maximal anti-HBV effect in this transgenic mouse model. Furthermore, when each prodrug was withdrawn (day 84), the rebound of serum HBV DNA in the TMF-treated group was the slowest.In HBV transgenic mice, all the drugs exhibited significant anti-HBV effects, as there was a sharp reduction in HBV DNA copies in serum to the same level after the first 42 days post-drug administration. The antiviral response of each prodrug was sustained until dosing discontinuance on day 84 due to the persistence of genomic HBV . In partAlong with the inhibition of HBV DNA replication, host intracellular biochemical metabolism was altered by TFV ester prodrugs. PCA analysis indicated that the metabolomic profiles of HepG2.2.15 cells were markedly shifted from the bottom-right direction to the upper-left direction along with the continuous replication of HBV DNA from 3–9 days. These shifts were corrected to different extents by TFV ester prodrugs, of which TMF was the most potent, while TDF was the weakest . Next, bFurthermore, the callback efficacy of TFV ester prodrugs in lipids was investigated by lipidomics. A total of 680 lipids, including 283 triacylglycerols (TAGs), 39 diacylglycerols (DAGs), 3 monoglycerides (MAGs), 350 lipoids and 5 free fatty acids (FFAs), were detected in our method, and PCA analysis indicated that these prodrugs were more potently rebalanced in lipoids than in total lipids, so the subsequent analysis focused on lipoids . From thAs shown in As shown in max and t1/2 of TFV for each prodrug were similar regardless of the dose  were so rapidly hydrolyzed into TFV that the prodrug form could not be detected in plasma. The TFV levels and the exposure of TFV in plasma for each dosage group of the same prodrug were presented in a dose dependent manner. Furthermore, the values of tthe dose . The chathe dose .max of 0.25 h, a t1/2 of 1.73 h and a bioavailability of 9.70%, while neither TAF nor TDF itself could be detected in plasma. Meanwhile, the AUC of TFV in plasma showed a striking difference among the three TFV ester prodrugs, in which the AUC of the TMF group (16.26 μM h) was 3.01 or 1.75 times that of the TDF (5.39 μM h) and TAF (9.30 μM h) groups. Therefore, the total bioavailability of these prodrugs was TMF (47.26%) > TAF (22.59%) > TDF (13.55%), of which the total bioavailability included both ester and TFV forms for TMF but only TFV forms for TAF and TDF  and exposed higher (AUC0-t 3.68 ± 2.20 μM h vs. 2.35 ± 1.20 μM h) in the ester form in plasma than did TAF, and thus led to the bioavailability difference between TMF and TAF (53.49% ± 11.34% vs. 32.96% ± 6.46%)  . For the± 6.46%) . Hence, To explain the absorption differences among TMF, TDF, and TAF, potential steps involved in prodrug absorption, including gastrointestinal fluid stability, intestinal permeability and first pass effect, were evaluated.1/2 of 13.7 min; TAF showed higher stability in SIF with an elimination t1/2 of 48.4 min but a higher degradation in SGF than TDF (the degradation t1/2 of TAF in SGF was 71.3 min). Comparatively, TMF was extremely stable in SIF with a t1/2 over 120 min and relatively stable in SGF.As shown in in situ single-pass perfusion experiment on rat jejunum (eff 53.7 × 10−5 cm/s) than TAF (Peff 8.1 × 10−5 cm/s) or TMF (Peff 5.6 × 10−5 cm/s). The absorption rate constants (ka) of TMF, TAF and TDF were 0.675 × 10−3/h, 0.807 × 10−3/h and 2.095 × 10−3/h, respectively.An  jejunum  revealed1/2 of 3.58 min in rat blood, and approximately 0.3 min in rat intestine and in liver homogenates (0.32 and 0.36 min). TMF was more stable than TAF in rat intestine and in liver homogenate , despite a higher degradation in blood (t1/2 6.97 min vs. 9.31 min). As mentioned above, the absorption of the TFV ester prodrug was related to many factors; therefore, the normalized weighting coefficient, defined as the ratio of the AUC or Peff of each prodrug to the sum of the AUC or Peff of the three prodrugs, was calculated to evaluate the absorption of the TFV ester prodrug from an overall perspective. in vivo. Furthermore, among different factors, intestinal stability, including the intestinal lumen and intestinal epithelium, was the main factor affecting the bioavailability of the TFV ester prodrug in vivo.For the first-pass effect, the prodrug stabilities in intestine/liver homogenate and blood were assessed. As shown in Current treatment of HBV infection relies on NAs and IFNs, and NAs are superior due to their effective viral suppression, good-tolerance and favorable pharmacokinetics . TFV is in vitro based on HBV-positive HepG2.2.15 cells, which can secrete HBV DNA particles. TMF displayed 2.3-fold higher antiviral activity than TDF and 1.6-fold better efficacy than TAF after 9 days of treatment  from the baseline (10 (copies/mL) on the 28th day and achieved 3.1 Δlog10 (copies/mL) for a 1–2 weeks delay. Such a 0.5 Δlog10 (copies/mL) lower difference  between TMF and TAF/TDF is analogous to the improvement from entecavir to TDF with 0.2 Δlog10 (copies/mL) (in vitro results (TMF > TAF > TDF). This might be related to species differences, because in vitro experiments were carried out with human derived cells, while in vivo experiments were carried out on mice. Meanwhile, the in vivo anti-HBV effects of the three TFV prodrugs in mice were quite correlated with their in vivo exposure in mice, as the AUC of TDF was close to that of TAF in mice, and both of them were much smaller than that of TMF in mice  , which s in mice .Idh3a, Dlst, Suclg2) were downregulated in HBV-transgenic mice , glycolysis, nucleotide metabolism and ketone body metabolism were upregulated, while pentose phosphate pathway, TCA cycle and amino acid metabolism were downregulated . Wan, Q nic mice . Taken tnic mice . HBV hasnic mice , and thenic mice . On thisSince the regulatory effect of TMF on HBV positive hepatic metabolism was considered to be mediated by HBV inhibition through TFV-DP, the superiority of TMF in liver enrichment and metabolic activation was elucidated. The tissue distribution of each TFV prodrug was determined and compared in rats. TMF, TAF, and TDF were detected only in the stomach and the intestine, and their released TFV was distributed widely after being absorbed into circulation and mainly accumulated in the liver and kidney . The greBioavailability improvement might be an important assurance of liver enrichment. Hence, the bioavailabilities of TMF, TAF and TDF were compared in various preclinical animals, including SD rats, C57BL/6 mice and beagle dogs. All the three prodrugs effectively delivered TFV to the system circulation , and TMFeff values of all three prodrugs were at the level of 10−5 cm/s, which indicated high permeability (in vitro stability in intestinal homogenate (Subsequently, the possible mechanisms that influence the bioavailability of TFV ester prodrugs were analyzed. The main factors for oral drug absorption into systemic circulation include stomach fluid, intestinal fluid, intestinal barrier, liver and blood. Therefore, gastrointestinal fluid stability was analyzed first. Although the three prodrugs are similar in structure, different moieties contribute to the distinct stability behaviors of TFV prodrugs. TDF was the most stable in SGF but the most unstable in SIF; moreover, TAF was more stable in SIF than in SGF . TMF waseability  (Figure eability . Therefoeability . The oramogenate . When thmogenate . After fmogenate . Considein vitro and in vivo, while TMF was slightly superior to TAF. These different pharmacological activities of TFV prodrugs are believed to be attributed to their differences in pharmacokinetic characteristics: the greater intestinal stability and bioavailability of TFV prodrug in preclinical animals provide better target loading, especially higher hepatocytes level of pharmacologically active TFV-DP, which is tightly related with the efficacy of such prodrug (In our study, the antiviral activity, hepatic biochemical metabolism regulating effect, targeted distribution and metabolic activation, bioavailability, gastrointestinal fluid stability, intestinal permeability and first-pass effect of three currently marketed TFV ester prodrugs were evaluated and compared. TMF and TAF exhibited significantly stronger anti-HBV efficacy and correcting effects for disordered hepatic biochemical metabolism than TDF  prodrug . Therefo


Antimicrobial use (AMU) is a key driver of antimicrobial resistance (AMR). There are few data on AMU, to inform optimizing antibiotic stewardship, in the Lao PDR (Laos).Point prevalence surveys (PPS) of AMU were conducted at four-month intervals in six general hospitals across Laos from 2017 to 2020, using modified Global-PPS data collection tools. The surveys focused on AMU amongst hospitalized inpatients.The overall prevalence of inpatient AMU was 71% , varying by hospital and survey round from 50·4% (135/268) to 88·4% (61/69). Of 4,377 patients, 44% received >one antimicrobial. The total number of prescriptions assessed was 6,555. Ceftriaxone was the most commonly used (39·6%) antimicrobial, followed by metronidazole (17%) and gentamicin (10%). Pneumonia was the most common diagnosis among those prescribed antimicrobials in both children aged ≤5 years (29% among aged ≤1 year and 27% among aged >1 to ≤5years) and adults aged ≥15 years at 9%. The percentage of antimicrobial use compliant with local treatment guidelines was 26%; inappropriate use was mainly found for surgical prophylaxis (99%). Adult patients received ACCESS group antimicrobials less commonly than children . Most WATCH group prescriptions (99%) were without a microbiological indication.AMU among hospitalized patients in Laos is high with frequent inappropriate use of antimicrobials, especially as surgical prophylaxis. Continued monitoring and enhanced antimicrobial stewardship interventions are needed in Lao hospitals.The Wellcome Trust [Grant numbers 220211/Z/20/Z and 214207/Z/18/Z] and bioMérieux. Overall use of ACCESS group antimicrobials was 50% in Laos which was less than the WHO target of 60% and higher in children than adults.•Pneumonia was the number one indication for antimicrobial prescription in both children and adults.•Appropriate use of antimicrobials in hospitals in Laos according to prescribing indication was only 26%. Inappropriate use of antimicrobials was mainly explained by use for prophylaxis.•These data suggest targets for appropriate solutions to improve antimicrobial use to control AMR in Laos.•Appropriate antimicrobial stewardship programs, including comprehensive antimicrobial use guidelines and regular monitoring of AMU should be applied to the whole country.Alt-text: Unlabelled box,https://livedataoxford.shinyapps.io/amulaos/). The aim of this study was to describe the patterns of hospital antimicrobial use in six general hospitals across Laos over a 4-year period, from 2017 to 2020, as a basis for informing AMR policy, action and implementation in Laos.Acquired antimicrobial resistance (AMR) emerges quickly in some pathogens soon after the deployment of many antimicrobials, reducing their efficacyPoint prevalence surveys (PPS) of antimicrobial use were repeatedly conducted in inpatients in six hospitals: two referral  and four provincial hospitals (60–100 beds), including Luang Namtha, Xiengkhuang, Vientiane and Salavan Provincial Hospitals. Mahosot, Xiengkhuang, Luang Namtha and Salavan participated in the surveys from mid-2017, with Vientiane Provincial Hospital joining in 2018 and Savannakhet in 2019. These six hospitals were included in the survey because they were participating in aetiology of fever studies in Laos and because they were geographically dispersed, with two hospitals located in the north, two in the centre, and two in the south of Laos. These six hospitals in one prefecture and five out of 17 provinces were considered representative of different regions in Laos .Data collection was performed using an adapted version of the Global-PPS protocol (2019)PPS were conducted three times a year from July 2017 to December 2020. The survey rounds were based on Lao seasons with the wet season from May to October and dry season from November to April.et al.,Spectrum (broad or narrow) of antibiotics was classified based on Hagedoorn Descriptive analysis was performed for patients’ characteristics, the trend of antimicrobial prescriptions through seasons, survey round, and antimicrobial prescriptions by medical specialty and organ involved. Frequency, mean ), and median with interquartile range (IQR) were used to describe data. Antimicrobial prescriptions were described based on the prescribing indications, WHO AWaRe classification Monitoring AMU was introduced as a routine surveillance activity in Laos and no patient identifiable data were collected. Surveys were conducted with the permission of the participating hospitals and the Lao Ministry of Health and were not subject to ethical review.This work was funded by the Wellcome Trust [Grant numbers 220211/Z/20/Z and 214207/Z/18/Z] and bioMérieux – Singapore who provided a startup grant for the project. The funders of this study had no role in the study design, data collection, analysis, interpretation or writing the report. For the purpose of open access, the author has applied a CC BY public copyright licence to any Author Accepted manuscript version arising from this submission.Between July 2017 and December 2020 (41 months), ten rounds of antimicrobial use PPS were conducted in six hospitals across Laos . Over these 41 months, records of 6,188 inpatients were screened for antimicrobial use; revealing that 4,377 ) inpatients received antimicrobial treatment, with 6,555 antimicrobial prescriptions in total. The use of antimicrobials varied from the lowest prevalence of 50·4% (135/268) in one survey round in Savannakhet Provincial Hospital to the highest of 88·4% (61/69) in Salavan Provincial Hospital .Figure 1p = 0·03; Vientiane Province: p = 0·009; Xiengkhuang: p = 0·002; Luang Namtha: p = 0·04 and Savannakhet: p < 0·0001, except Salavan: p = 0·93). Season had no discernable effect on antimicrobial use at Mahosot Hospital (p = 0·45), Xiengkhuang (p = 0·10) and Salavan (p = 0·96). However, the use of antimicrobials varied by seasons in Vientiane Province, Luang Namtha and Savannakhet (p = 0·03 (Supplementary Table 3). After controlling for seasonality, there was no trend in the proportion of patients prescribed antimicrobials over the survey period in each survey hospital ; except Savannakhet: p = 0·0005  was found at Salavan Provincial Hospital at 82·3% (80·2%-84·4%), followed by Luang Namtha at 74·8% (68·8%-80·9%), Vientiane Province at 72·5% (66·4%-78·6%), Xiengkhuang at 70% (65·2%-74·8%), Mahosot at 68·6% (65·4%-71·8%-77·4%). There were significant differences in the proportion of patients prescribed antimicrobials in each survey round by individual hospital , anti-tuberculosis drugs for 49/6,555 (0·8%), antifungals for 47/6,555 (0·7%), antivirals  for 13/6,555 (0·2%), anti-helminthics for 12 (0·2%) and an antimalarial for 1 (0·02%). Of all prescriptions, the cephalosporins class was the most prescribed at 2,919/6,555 (44·5%), followed by penicillins at 1,116/6,555 (17%), then nitroimidazoles at 1,104/6,555 (16·8%), aminoglycosides at 679/6,555 (10·4%) and macrolides at 213/6,555 (3·2%) (Supplementary Table 4).Of the cephalosporins, ceftriaxone accounted for 2,595/9,19 (89%) prescriptions. Cefalexin was the only first-generation cephalosporin used, accounting for 154/2,919 (5%) prescriptions and 4/2,919 (0·1%) patients were prescribed second generation cephalosporins (cefuroxime). Ampicillin was the most prescribed of the penicillin class at 41%  prescriptions, followed by amoxicillin at 35%  and cloxacillin at 19% . The majority of beta lactam/beta-lactamase inhibitors (BL/BLIs) and carbapenem use was in Mahosot Hospital at 67/78 (86%) and 56/57 (98%) prescriptions, respectively. Of BL/BLIs, amoxicillin/clavulanic acid was the most prescribed at 45/78 (58%), followed by ceftriaxone/sulbactam at 30/37 (38%) and cefoperazone/sulbactam at 3/78 (3%) (Supplementary Table 4).The majority of prescribed antibiotics were broad spectrump < 0·0001). Overall, cephalosporins, penicillins and aminoglycosides were prescribed more frequently in children aged >5 to ≤14 years at 2,590/5,410 (48%), than children aged ≤5 years at 329/1,145 (29%), p < 0·0001. Cephalosporin prescriptions were predominantly in older children (>5 to ≤15 years) and adults (≥15 years), accounting for 2,590/5,410 (48%) prescriptions, compared with 329/1,145 (29%) prescriptions in children ≤5 years (p < 0·0001). Antimalarials, antivirals and lincosamides were used in adults only .The proportion of patients prescribed antimicrobials varied by age group. Penicillins tended to be used more frequently in infants (aged ≤1 year) at (306/754 (41%)), compared to 109/391 (28%) children aged >1 to ≤5 years and only 55/317 (17%) children aged >5 to ≤14 years (p < 0·0001). Aminoglycosides and penicillins were prescribed more in children than adults (p < 0·0001) for pneumonia. A similar pattern was observed in patients with sepsis or skin and soft tissue infection  at 254 (55%) than children aged ≤28 days, >28 days to ≤1 year, aged >1 to ≤5 years and >5 to ≤14 years at 5 (15%), 49 (27%), 51 (49%) and 10 (50%), respectively , followed by Escherichia coli infection for 8/58 (14%), and Staphylococcus aureus infection for 7/58 (12%)). Of 75 patients with confirmed infection, tuberculosis was diagnosed in 17 (23%) patients by either microscopy or GeneXpert (Supplementary Table 5). Among survey hospitals, confirmed diagnoses based on laboratory results were mostly (52/75 (69%) from Mahosot Hospital patients.Just over half ) of all prescriptions were for treating infections, while 2,310/6,555 (35%) were for either medical prophylaxis ) or surgical prophylaxis ) . During p < 0·0001). ACCESS group use was highest in infants aged ≤1 year at 77% (578/754) and 52% (199/384) in children aged between >1 and ≤5 years, 46% (141/309) in children aged between >5 and ≤14 years, and 47%  in adults . Use of ACCESS group antibiotics varied by hospitals in each survey and ranged from 44%-59% prescriptions. Use of WHO AWaRE ‘Not recommended’ antibiotics was only found in Mahosot Hospital in 33/2,029 (2%) prescriptions  patients  prescriptions) who received antibiotics, the use of ACCESS antibiotics over these four years made up 3,244/6,433 (50%) prescriptions, while WATCH antibiotics accounted for 3,156/6,433 (49%). No RESERVE group antibiotic prescription was captured in this study. However, antibiotics from the ‘Not recommended” group of WHO AWaRe were prescribed to 33/4,255 (0·8%) patients, including ceftriaxone-sulbactam in 30/33 (91%) and cefoperazone-sulbactam in 3/33 (9%). Among the WATCH group used, 44/3,156 (1%) were based on microbiology results while no prescriptions of antibiotics in the ‘Not recommended’ group were suggested by microbiology results. ACCESS antibiotics were more commonly used in children (63%) than adults (47%)  could be assessed as to whether the prescriptions adhered to the pre-2021 guidelines, whilst, 2,158 (33%) were unable to be assessed because there was no guidelines or prescribing indications were unknown/unclear. During this 4-year monitoring period, 26%  adhered to pre-2021 guidelines and nearly three-quarters of prescriptions ) did not adhere. Retrospective analysis of the hypothetical compliance with the new 2021 antimicrobial prescribing guidelines (2021-guidelines)Based on pre-2021 guidelines, adherence to the guidelines was more common among children aged ≤5 years at 44%  than for older children aged >5 to ≤14 years and adults at 20% . Prescriptions not adherent to the guidelines were found most commonly in obstetric/gynaecology departments at 98% (637/648), followed by surgery departments at 81% . The five most common indications for prescriptions without guidelines to assess against were eye, cardiovascular system, bone and joints, male genital conditions, and skin and soft tissue problems at 97% (33/34), 94% (33/35), 90% (162/180), 89% (81/91) and 76% (407/535), respectively. The top five indications for prescriptions without guidelines to assess, based on 2021-guidelines, were eye, male genital conditions, cardiovascular system, central nervous system and skin and soft tissue at 94% (32/34), 81% (81/91), 51% (18/35), 46% (80/174) and 38% (202/535), respectively.p < 0·0001). The proportions of prescriptions adhering to the pre-2021 guidelines were 51% (157/304) for sepsis, 69% (48/70) for meningoencephalitis, 64% (35/55) for suspected typhoid fever, 86% (67/78) for suspected rickettsial infections, and 78% (52/67) for melioidosis (both suspected and confirmed). Of 29 patients  with microbiologically confirmed melioidosis, antimicrobial prescriptions for two (7%) patients did not adhere to the guidelines.Pneumonia was the most common indication identified for antimicrobial prescription in both children and adults; the proportion of pre-2021 guideline adherence in children for which treatment guidelines were available was high, at 77% (166/216) in those aged ≤1 year, 72% (76/105) in children aged >1 to ≤5 years and 56% (10/18) in children aged >5 to ≤14 years but it was much lower in adults at 6% (26/465) , penicillins (17%), nitroimidazoles (16·8%), aminoglycosides (10·4%) and macrolides (3·2%) were the most commonly used antimicrobials. AMU prevalence varied across hospitals over time, and was as high as 88·4% in some hospitals on occasions. This is an alarming message for Laos. This prevalence is higher than reported from other regions of the globe. Our results show higher AMU than the overall proportion from the Global-PPS of 303 hospitals in 53 countries at 34%, in which the highest proportion was from African countries at 50%. The proportion of antimicrobial use in six East and South Asian countries  was 37% and 42% in west and central Asian countries .,,,,rd or 4th generation cephalosporins, fluoroquinolones or glycopeptides.,Escherichia coli among cultured isolates of E.coli is increasing year on yearE. coli in Laos. With rising levels of antibiotic resistance in Laos for some critical pathogens,Choice of antimicrobial use varies by country. The prescription patterns in Laos resemble other low- and middle-income countries,,,,,https://clinicaltrials.gov/ct2/show/NCT04914793), will hopefully lead to increased appropriate use of antimicrobials in Lao hospitals over time. Existing evidence has suggested that guidelines can improve the quality of antimicrobial prescribing. However other factors such as education, policy and public engagement also play important roles in sustainably improving the appropriateness of antimicrobial use.33The proportion of antimicrobial use adherent to the local pre-2021 treatment guidelines in this study was 26%, while 33% of prescriptions could not be evaluated because of missing guidelines or unknown/unclear indications. Comparison with adherence to 2021-guidelines found an increase in guidelines adherence to 35% and the proportion of use which could not be assessed reduced from 25% to 14%. This is much lower than estimates of appropriate antimicrobial use from other Asian countries (Vietnam (69·2%) in 2008, Myanmar 31·9%) in 2019, Indonesia (52·2%) in 2019 and India (77·4%) in 2017).·9% in 20Inappropriate surgical prophylaxis (99%) accounted for most inappropriate use. The latest local surgical prophylaxis guidelines were released in 1996 and have not been updated. Antibiotics of choice in the 1996 guidelines were mainly ampicillin (intravenous), cefalotin (intravenous), ± gentamicin or metronidazole; while our current study showed that the majority of the antimicrobial prescriptions for surgical prophylaxis were for ceftriaxone at 50%, whilst ampicillin accounted for 1% of prescriptions and no cefalotin prescriptions were captured. Cefazolin was commonly used in Oceania, North America, Western Europe for surgical prophylaxis, whilst ceftriaxone was used more often in Eastern and Southern Europe, Africa,,,,The WHO AWaRe list is being used increasingly in many countries to monitor the rational use of antibiotics.This study has key limitations. The total duration of therapy was not captured because it was a point prevalence survey. There were no Lao guidelines for treatment or prevention of some infections meaning appropriateness could not be assessed in every patient. The assessment of appropriateness of antimicrobial use in this study was solely based on choice of antimicrobial(s), regardless of the dose, duration and route of administration; this could lead to an overestimate of the appropriateness of use. Data indicating antimicrobial use by hospital-acquired or community-acquired infections were not available. As we did not survey outpatient prescriptions, we do not have the complete picture of hospital antimicrobial use in the country.These results from repeated surveys over 41 months suggest that Laos has a consistently high prevalence of antimicrobial use in hospitals. Inappropriate use of antibiotics was highest by surgical and obstetrics/gynecology specialties. The choice of available antibiotics to prescribe in Laos is limited. Some useful antibiotics  are available but rarely used. Access to microbiology diagnosis is also limited outside Vientiane City but has been expanding to many provincial hospitals recently with the support of the Lao government and international funders. Antimicrobial stewardship activities to promote the rational use of antimicrobials in hospitals were sporadic over this survey period. As in many low- and middle-income countries,VC, TR, MM, EA and PN conceptualized and designed the study, SK, PP, VP, CP, SS, KD and SB provided data and facilitated data collection, VC, AD and DC collected data, VC and TR performed data curation, VC analysed data and wrote the original draft. All authors reviewed and approved final version of the manuscript. VC is the guarantor.Data from this study are a part of national antimicrobial surveillance. De-identified data are available on reasonable request to Lao Ministry of Health.The Lancet Group takes a neutral position with respect to territorial claims in published maps and institutional affiliations.H Rogier van Doorn is board member of SEDRIC (Surveillance and Epidemiology of Drug Resistant Infections Consortium). All other authors declare no completing interests. The study received a grant from bioMérieux Singapore to start the project. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


The possibility ofobtaining cocrystals of kojic acidwith organiccoformers has been investigated by both computational and experimentalapproaches. Cocrystallization attempts have been carried out withabout 50 coformers, in different stoichiometric ratios, by solution,slurry, and mechanochemical methods. Cocrystals were obtained with3-hydroxybenzoic acid, imidazole, 4-pyridone, DABCO, and urotropine,while piperazine yielded a salt with the kojiate anion; cocrystallizationwith theophylline and 4-aminopyridine resulted in stoichiometric crystallinecomplexes that could not be described with certainty as cocrystalsor salts. In the cases of panthenol, nicotinamide, urea, and salicylicacid the eutectic systems with kojic acid were investigated via differentialscanning calorimetry. In all other preparations the resulting materialswere constituted of a mixture of the reactants. All compounds wereinvestigated by powder X-ray diffraction; the five cocrystals andthe salt were fully characterized via single crystal X-ray diffraction.The stability of the cocrystals and the intermolecular interactionsin all characterized compounds have been investigated by computationalmethods based on the electronic structure and pairwise energy calculations,respectively. Cocrystals of kojic acid, not easilyprone to cocrystalformation, and salt(s) were obtained by slurry, mechanochemistry,and solution crystallization; eutectic mixtures were also explored.Structures and stabilities were investigated by combined computational,diffraction, and calorimetric methods. Inthe pharmaceutical field, in particular, cocrystallization with pharmaceuticallyacceptable coformers is extensively explored to improve fundamentalproperties of the active pharmaceutical ingredients (API) such asthe solubility and dissolution rate, but also thermal stability andprocessability. In addition to this, the API can also be designedas a codrug by cocrystallization with another active ingredient. Then,not only do the solid-state physicochemical properties of APIs changerelative to those of parent crystals, but biological activity mayalso yield significantly different results.Cocrystallization is one of the most fruitfulapplications of thecrystal engineering idea,7  is a naturalfungal metabolite,first isolatedfrom 12Indeed, the motivation for this studystems from the need to fightthe emerging threat of antimicrobial resistance (AMR). AMR is becomingone of the major medical challenges in most healthcare systems, andrefers to the ability of microorganisms to develop resistance to theaction of originally efficacious antibiotics; that is, the sensitivityto drugs is reduced.13 piperazine,14 3-hydroxybenzoic acid,15 and urotropine.16 Furthermore, it isknown that 4-pyridine and 1,4-diazabicyclo[2.2.2]octane (DABCO) areeffective antioxidants. 4-Pyridine is also an important raw materialfor the synthesis of antibacterial active pyridine quaternary ammoniumsalts,17 and the DABCO bis-quaternary saltsalso exhibit antibacterial activity.18 Theophyllinehas also been shown to possess antiaging and anticytotoxic effectsin human skin.19 Caffeine is highly biologicallyactive, has the ability to penetrate the skin barrier, and also preventsexcessive accumulation of fat in skin cells.20 Theobromine can scavenge reactive oxygen species (ROS) producedin the skin after UV exposure.22 Nicotinamide is alsowidely used as a cosmetic skin care ingredient for its whitening,antiaging, and skin barrier building properties.24 The list was further extended to comprise 4-aminopyridine, whichis also known to enhance the release of presynaptic acetylcholineand increase the force of muscle contractions, thus behaving as anantagonist of the neuromuscular blockade effects of antibiotics.26In this work we explorethe possibility of preparing a series ofcocrystals of kojic acid with a number of selected coformers to enrichthe library of novel materials with potential antibacterial properties.In order to select adequate coformers, we are guided by the informationavailable in the literature on the antibacterial properties of moleculessuch as imidazoles,27 and their stability evaluatedby available computational methods.Beside organic compounds already known to possess mild antimicrobialactivity, a number of other cocrystallization partners have been selectedby using data mining tools available within the Cambridge StructuralDatabase,The coformers that haveyielded novel materials by solution, slurry,and mechanochemical methods, whether as cocrystals, salts, or eutecticcompositions by cocrystallization with kojic acid, are listed in Kojic acid was purchased from TCI. 3-Hydroxybenzoicacid (3HBA), imidazole, 4-pyridone, DABCO, urotropine, theophylline,piperazine (PIP), panthenol, nicotinamide, urea, salicylic acid (SA),4-aminopyridine, and solvents were purchased from Sigma-Aldrich. Distilledwater was used. All compounds were used without further purification.2PIP][KA]2·2H2O was obtained inthe same ball milling conditions as HKA·imidazole and HKA·4-pyridone,but two drops of ethanol had to be added to the reacting mixture.The complex (HKA)4·(theophylline)3 canbe produced by ball milling in the presence of two drops of methanol,ethanol, acetone, ethyl acetate, or acetonitrile. HKA·DABCO wasobtained by manual grinding for 10 min, with an agate mortar and pestle,and equimolar amounts of HKA (0.5 mmol) and DABCO (0.5 mmol), eitherin the absence of solvent or in the presence of two drops of water,ethanol, or methanol.The cocrystals HKA·imidazole,HKA·4-pyridone, and the complex HKA·4-aminopyridine wereobtained by ball milling equimolar quantities of HKA (0.5 mmol) andcoformer (0.5 mmol), in a 5 mL agate jar in the presence of two drops(100 μL) of water, and two 3 mm agate balls, for 60 min in aRetsch MM200 ball miller, operated at a frequency of 20 Hz. [KA]2·2H2O were recoveredby slow evaporation at ambient conditions of a methanol:ethanol 1:1solution containing equimolar amounts of HKA (0.5 mmol) and piperazine(0.5 mmol).Single crystals of (HKA)Table S1 reports datacollection and refinement details. Structural solution and refinementby least-squares methods against F2 were carried out usingSHELXT28 and SHELXL29 implemented in the Olex230 software.Non-hydrogen atoms were refined anisotropically. Highest peaks ofresidual electron density from Fourier maps indicated, for all structures,the position of all the hydrogen atoms belonging to -NH and -OH groups,thus confirming the cocrystal/salt attribution; for sake of betterrefinement, these hydrogens were added in calculated positions andrefined riding on their respective N or O atoms. The program Mercury31 was used for graphic representations and forsimulation of X-ray powder patterns on the basis of single crystaldata, either retrieved from the Cambridge Structural Database (CSD)or collected in this work. The program PLATON was used for calculationof intermolecular hydrogen bonds.32Single crystal X-raydiffraction data were collected at room temperature with an OxfordDiffraction X’Calibur equipped with a graphite monochromatorand a CCD detector. Mo–Kα radiation (λ = 0.71073Å) was used. The powder X-ray diffraction(PXRD) patterns were collected on a PANalytical X’Pert Proautomated diffractometer equipped with an X’Celerator detectorin Bragg–Brentano geometry, using Cu–Kα radiation(λ = 1.5418 Å), without a monochromator, in the 2θrange 5°–40° .X-ray powderdiffractograms in the 5–40° 2θ range were collectedon a PANalytical X’Pert PRO automated diffractometer equippedwith an X’Celerator detector and an Anton Paar TTK 450 systemfor measurements at controlled temperature. The data were collectedin open air in Bragg–Brentano geometry using CuKα radiationwithout a monochromator.2 atmosphere in the35–200 °C temperature range, at the heating rate of 10.00/5.00/2.00°C min–1.DSC traceswere recorded using a PerkinElmer Diamond differential scanning calorimeter.All samples (ca. 5–10 mg) were placed in open Al-pans. Allmeasurements were conducted under N4·(theophylline)3 and HKA·4-aminopyridine were performed with a PerkinElmerTGA7 in the temperature range 40–300/500 °C under N2 gas flow at a heating rate of 5.00 °C min–1.TGA measurements forthe crystalline complexes (HKA)solidus line and the eutectic temperature,while peak temperatures were used to draw the liquidus lines and determine the eutectic composition.33HKA:Urea, HKA:Salicylicacid, HKA:Panthenol, and HKA:Nicotinamide mixtures with HKA mole fractionsranging from 0 to 1 were thoroughly ground with an agate mortar andpestle at ambient temperature for 10 min; the microcrystalline powdersthus obtained were analyzed via powder X-ray diffraction and differentialscanning calorimetry (DSC). DSC measurements were performed from roomtemperature to a maximum temperature exceeding the one observed forthe highest melting component, at heating rates of 10/5/2 K/min see ; as for –5 Ha peratom, atomic displacements to 5 × 10–3 Å,and maximum forces to 2 × 10–2 Ha Å–1. The lattice parameters were kept fixed during theminimizations.The experimentalstructures2 were op41 using the Perdew–Burke–Ernzerhofgeneralized gradient approximation exchange-correlation density functional42 and ultrasoft pseudopotentials43 with the addition of the MBD* dispersion correction.44 The number of k-points were chosen to providea maximum spacing of 2π·0.04 Å–1, and a basis set cutoff of 1180 eV was applied. Optimizations wereconsidered complete when energies converged to better than 2 ×10–5 eV per atom, atomic displacements to 1 ×10–3 Å, maximum forces to 5 × 10–2 eV Å–1, and maximum stresses to 0.1 GPa.These computational settings were found to yield sufficiently accurateenergies and converged minimizations. Atomic positions and latticeparameters were optimized. In the case of the DABCO cocrystal, thedisorder was resolved by calculating the energy for the two potentiallyorder structures, and for all calculations the weighted average ofthe two ordered structures was used.Furthermore, the experimental structures 1 were opEcocrystalstab) was calculated basedon Ecocrystaltot, Ecoformertot, and EHKAtot correspond to the PBE-MBD* energies of thecocrystal, coformer and HKA, respectively, and n tothe stoichiometric ratio.The cocrystal stability .The intermolecular energies were calculated between all unique nearestneighbors. The model (CE-B3LYP) uses B3LYP/6-31G molecular wavefunctions, calculated by applying the molecular geometries extractedfrom the crystal structures. This approach uses electron densitiesof unperturbed monomers to obtain four separate energy components:electrostatic (EE), polarization (EP), dispersion (ED), and exchange-repulsion (ER). Eachenergy term was scaled independently to fit a large training set ofB3LYP-D2/6-31G counterpoise-corrected energies from both organicand inorganic crystals.47The optimizedPBE-TS structures were then used as starting points for pairwise intermolecularenergy calculations,50 that possesses two hydrogenbond donors and four acceptors; it is also a fairly rigid molecule,with only one torsional degree of freedom shown by the hydroxymethylgroup. While some metal complexes with kojic acid are known in theliterature, together with their structures, neither organic cocrystalsnor organic salts have been reported. A search in the Cambridge CrystallographicDatabase27 was thus conducted, via theprogram Mercury,31 to screen for suitablecoformers by Molecular Complementarity52 and multicomponentHydrogen Bond Propensity (HBP)54 methods. The HBP methodwas used to restrict the large number of potential coformers obtainedvia the molecular complementarity method. To this end, a multicomponent(MC) score was calculated: while a positive MC value indicates probablecocrystal formation, if the value is close to zero no conclusion canbe formed. Tables S3 and S4 show the resultsof both methods, with 14 possible coformers (if scores down to zeroare included). The combined use of the two methods, therefore, helpsin narrowing the number of compounds that should be tried as possiblecoformers. It is interesting to note that in the case of piperazineand theophylline, that reacted with HKA to form stoichiometric compounds,the MC method produces “pass” flags, i.e., the predictionis of possible cocrystal formation, while the HBP method assigns tothese coformers a negative value (see Tables S3 and S4). The two methods, therefore, should be tested and usedas an additional tool in crystallization experiments, not as a hard-and-fastrule for molecules selection. The amino acids were also tried, butthey did not give positive results, as the reagents were in the zwitterionicform, which, according to both MC and HBP searches, are not good candidates(see Tables S3 and S4).Kojic acid isa highly solublecompound55 For this reason, we decided to further explorethe possible formation of cocrystals of HKA with molecules that areknown to act as coformers in cocrystals of 3-hydroxybenzoic acid.To this end, a search in the CSD was conducted, and, among other systems,we selected molecular solids possessing interesting properties inthe skin care or in the antibacterial fields, and therefore they couldbe used as enhancers of HKA properties. A second list of possiblecoformers was thus compiled (see Thereaction of HKA with the 3-hydroxybenzoic acid(3-HBA) resultedin a cocrystal with striking structural similarity with the crystalstructures of both HKA and 3-HBA Form II, although the cocrystallizationwas conducted with 3-HBA Form I. Incidentally, the 3-HBA polymorphshave been recently investigated in detail by one of us.iled see 4. SchemeTable S2, pure 3-HBA is knownto exist in three polymorphic modifications.55 Form I is the thermodynamically stable form, while form II and IIIare metastable. The commercial 3-HBA employed in the reaction withHKA was form I. Both ball milling and slurry of the solid mixturewere unsuccessful, while crystallization from a water/ethanol solutionresulted in the formation of HKA·3-HBA cocrystals. The cell parametersobtained from a single crystal, and listed in Table S2 together with those of HKA, are closely related tothe values for 3-HBA Form II, and bear some relationship with thoseof crystalline HKA.Crystallization fromsolution of HKAand 3-HBA resulted in the formation of the cocrystal HKA·3-HBA.As listed in 55 We investigated the difference incocrystal formation enthalpy, i.e. form I → cocrystal and formII → cocrystal. The energy difference between 3-HBA forms Iand II was measured and calculated to be in the range of 0.5 to 0.6kJ mol–1.55 Therefore,the stabilization energy  differsmarginally, by less than 1 kJ mol–1, ifform I or II is used as the starting material.Similarly to the cocrystallization experiments,cooling crystallization of 3-HBA (from ethyl acetate) resulted inform II, whereas grinding experiments favored the formation of formI.OH hydrogen bonds molecules of the same type structures in parts of the cocrystal and in HKA . CO bonds[O(H)···OCO 2.857(7) Å], while HBswith urotropine are external [O(H)···N distances 2.764(6)Å]. In 2·urotropine, shown in b-axis, with hydrogen bonds contributing as an additional“glue” to the cocrystal stability duct see S1. Figurduct see S1. Figurduct see S1. CO boduct see S1. CO bo see the  section.56The compound 4-pyridone is thepredominant, keto-tautomer of 4-hydroxypyridine in liquid media andsolids, as shown in Figure S2). CO and OCO···H–N with 4-pyridone molecules only;the 4-pyridone molecule, in turn, forms hydrogen bonds of the N–H···OCO and OCO···H–O with neighboringHKA molecules only . A view down the crystallographic a-axis . HKA and DABCO are perfectly complementary as far as hydrogen bondingfunctions are involved, and the two opposite OH groups on each HKAinteract with the N atoms of two neighboring DABCO molecules, thusforming an infinite chain of alternating HKA and DABCO extending parallelto the crystallographic [101] direction . The cocrystal can be describedas a layered structure, as shown in hydroxymethyl···OCO [O(H)···O distances2.658(2) and 2.706(2) Å], bridged by imidazole molecules viaN–H···OCO [N(H)···Odistances 2.795 and 2.808(3) Å] and N···H–Ohydroxyl . The stacking of anions is made possible by the highly stabilizingelectrostatic contribution of the interlayers cation···anioninteractions , with theaddition of charge assisted hydrogen bonds of the N–H···(−)O type and bridging water molecules, as shown in (−)O 2.747(2) Å, N(H)···OCO 2.882 (2) Å, N(H)···OW 2.715(2) Å,O(H)W···OCO 2.685(2) Å,and O(H)W···(−)O 2.710(2)Å].ions see S35, with64 We thus prepared via ball-milling  solid-state mixtures ofHKA containing mole fractions from 0 to 1 of the second componentof interest. DSC measurements were then performed, and the resultingthermograms  used for the construction of the binary phase diagrams . Figures S19–S22). In all cases the patterns confirm the presence of physical mixtures,as no peaks are observed in addition to the peaks of the two components.Cocrystalformation could not be observed for the remaining selected coformers,despite the many attempts with different cocrystallization techniques, stoichiometric ratios,solvents, and temperatures. In the end, we decided to investigatethe phase diagrams of a small number of binary systems, to determinetheir eutectic composition and temperature: eutectic compositionsare currently recognized as a possible alternative, when cocrystalformation is not possible, to modify the physical properties of acompound, such as thermal stability and solubility.As it can be observed in The phase diagram of the HKA:nicotinamide binarysystem is shownin Figure S18) show only a widening of the peaks due to melting, andno separation of the solidus/liquidus temperature is possible fora molar fraction of panthenol higher than 0.75.The HKA:panthenol phase diagram is shown in 15).65The chosenoptimization method was successful in reproducing the experimentalstructures 6. The st2·2H2O, thesalt and a hypothetical cocrystal (proton moved) were used as inputfor structure optimization calculations (PBE-MBD*). Proton transferto the salt was observed for the hypothetical cocrystal structure.The experimental salt structure was obtained in both cases after structureoptimization. Thus, the calculations reflect the experimental finding,i.e. salt instead of cocrystal formation.In the case of [H2PIP][KA]Ecocrystalstab is negative for an exothermic reaction, indicating thatthe cocrystal should be stable with respect to its components (ignoringentropic effects). The enthalpy was estimated to be negative for thefive cocrystals, indicating that for all cocrystals are enthalpicallystabilized. The comparison of the Dmol3 calculations (no cell optimization)to the PBE-MBD* optimizations reveals that the cheaper  method already indicates the trend, i.e., cocrystalformation, albeit some of the ΔEcocrystalstab valuesdiffer between the two applied methods.The stabilizationenthalpy was calculated for each of the cocrystal according to Supporting Information (Table S6).The strongestpairwise interactions (hydrogen-bonding and aromatic) observed inthe HKA, coformer, and cocrystal structures are given in Z′ = 2 cocrystal (imidazole HKA) the two independent moleculesform interactions of similar strengths. The strongest interaction,−71.6 kJ/mol  coformers with hydrogen-bonding donor and hydrogen-bondingacceptor capabilities and (2) coformers featuring only hydrogen-bondingacceptor groups. A comparison of the intermolecular interactions seenin HKA, 3-HBA, imidazole, pyridine, and their respective cocrystalreveals that the strongest interaction is always formed in the cocrystal,despite the fact that HKA and coformers can form strong interactionswith the single component states 8. Furthe6 kJ/mol 8, is seeBoth, DABCOand urotropine, do not exhibit strong hydrogen-bondingdonor groups. Therefore, it is not surprising that the pairwise interactionsseen in the cocrystal are of higher strengths than in the coformeralone 9. ThecoIn this paper we have reported our experimentaland computationalresults obtained in the investigation of a family of cocrystals ofkojic acid. The large number of unsuccessful experiments has confirmedthe poor cocrystallization tendency of this important organic compound.Nonetheless, we have been able to identify and structurally characterizefive new cocrystals with the cocoformers 3-hydroxybenzoic acid (3-HBA),imidazole, 4-pyridone, DABCO, and urotropine; in the case of the stoichiometriccompounds of kojic acid with theophylline and 4-aminopyridine no fullstructural characterization was achieved, and therefore the distinctionbetween salt and cocrystal with could not be made with certainty.In the case of panthenol, nicotinamide, urea, and salicylic acid theirmixtures with kojic acid were investigated via differential scanningcalorimetry and powder diffraction, and their eutectic compositionsand temperatures determined. In contrast, proton transfer and saltformation have been observed in the case of piperazine.2PIP][KA]2·2H2O are in agreement with the experimental findings,the formation of a molecular salt instead of a cocrystal.Thestability and intermolecular interactions in all structurallycharacterized compounds have been unraveled based on periodic electronicstructure calculations and pairwise interaction energy estimations.Furthermore, the calculations for [H66 Analogously, we have shown that the antimicrobial compoundproflavine, and recently also kojic acid, can be cocrystallized withmetal salts of silver, zinc, and gallium to increase the minimal inhibitionconcentration of the drug activity toward bacteria.69 The preparationof cocrystals of known drugs, as well as the quest for new combinationsof natural antibacterials, have been shown to be successful in thedevelopment of improved or novel drugs; extensive work of other researchgroups is well documented in a recent review by Nangia.70Aspointed out in the As a logical follow-up of the resultsreported in this study, weare now investigating the antimicrobial and bacteriostatic behaviorof the novel cocrystals discussed above. The results will be reportedin the near future in specialized publications.