Prion disease is neurodegenerative disease that is typically fatal within months of first symptoms. Clinical trials in this rapidly declining symptomatic patient population have proven challenging. Individuals at high lifetime risk for genetic prion disease can be identified decades before symptom onset and provide an opportunity for early therapeutic intervention. However, randomizing pre-symptomatic carriers to a clinical endpoint is not numerically feasible. We therefore launched a cohort study in pre-symptomatic genetic prion disease mutation carriers and controls with the goal of evaluating biomarker endpoints that may enable informative trials in this population.PRNP) mutations (N = 27) and matched controls (N = 16), in a cohort study at Massachusetts General Hospital. We quantified total prion protein (PrP) and real-time quaking-induced conversion (RT-QuIC) prion seeding activity in CSF and neuronal damage markers total tau (T-tau) and neurofilament light chain (NfL) in CSF and plasma. We compared these markers cross-sectionally, evaluated short-term test-retest reliability over 2–4 months, and conducted a pilot longitudinal study over 10–20 months.We collected cerebrospinal fluid (CSF) and blood from pre-symptomatic individuals with prion protein gene (N = 29 participants and over 10–20 months in N = 10 participants. RT-QuIC was negative in 22/23 mutation carriers. The sole individual with positive RT-QuIC seeding activity at two study visits had steady CSF PrP levels and slightly increased tau and NfL concentrations compared with the others, though still within the normal range, and remained asymptomatic 1 year later. T-tau and NfL showed no significant differences between mutation carriers and controls in either CSF or plasma.CSF PrP levels were stable on test-retest with a mean coefficient of variation of 7% for both over 2–4 months in CSF PrP will be interpretable as a pharmacodynamic readout for PrP-lowering therapeutics in pre-symptomatic individuals and may serve as an informative surrogate biomarker in this population. In contrast, markers of prion seeding activity and neuronal damage do not reliably cross-sectionally distinguish mutation carriers from controls. Thus, as PrP-lowering therapeutics for prion disease advance, “secondary prevention” based on prodromal pathology may prove challenging; instead, “primary prevention” trials appear to offer a tractable paradigm for trials in pre-symptomatic individuals. PRNP) gene, many of which are highly penetrant [The well-defined pathobiology of prion disease, with prion protein (PrP) as the sole causal agent , has spuenetrant , may be enetrant , creatinenetrant , pre-symenetrant .“Secondary prevention” trials might recruit pre-symptomatic individuals with prodromal biomarker evidence of disease and aim to stabilize or reverse progression of those markers . While d“Primary prevention” trials might recruit pre-symptomatic individuals at known high genetic risk without requirement of prodromal pathology and treat toward a pharmacodynamic biomarker deemed reasonably likely to predict clinical benefit: CSF PrP levels . For redHere, we recruited and characterized a cohort of pre-symptomatic individuals at risk for genetic prion disease and controls, in order to assess candidate biomarkers for primary and secondary prevention paradigms.http://broad.io/mghprionstudy) primary goal of evaluating the test-retest stability of CSF PrP concentration in individuals at risk for genetic prion disease. Participants were recruited through IRB-approved advertisements shared by word of mouth and social media by Massachusetts General Hospital, Prion Alliance, and CJD Foundation. Participants included known mutation carriers, individuals at risk , and controls including genetic prion disease family members who had already tested negative for a mutation, spouses, and unrelated but demographically matched local controls. Participation required two study visits to Boston and absence of contraindication to lumbar puncture (LP). All participants were cognitively sound and provided written informed consent at the time of study enrollment. This study did not provide predictive genetic testing for genetic prion disease; the research team performed PRNP genotyping on de-identified samples for research purposes only. Participant details are provided in Supplementary Methods.The study, approved by the Partners Institutional Review Board in April 2017 (protocol #2017P000214), was conceived and designed with the pre-specified, publicly announced (The lumbar puncture (LP) for CSF collection was performed using a standardized protocol with a 24-G atraumatic Sprotte needle. The time of day for LP was kept consistent across subjects, and 20 mL CSF was collected per subject where possible. Following collection, CSF was handled uniformly according to an established protocol designed to minimize PrP loss to plastic through measures including (i) highly controlled and minimized plastic exposure, (ii) uniform storage in aliquots no smaller than 40 μL, and (iii) addition of 0.03% CHAPS detergent to a subset of CSF to maintain PrP solubility . SamplesThe study was originally designed to recruit 10 mutation carriers and 10 controls, a number expected to be sufficient to characterize test-retest reliability of CSF PrP as a descriptive statistic; enrollment was subsequently expanded as funding allowed. Additional file N = 26 anonymized pre-mortem cerebrospinal fluid samples from symptomatic sporadic (N = 22) and genetic  prion disease cases collected between 2001 and 2017 were shared by the Australian National Creutzfeldt-Jakob Disease Registry (ANCJDR). All cases were autopsy-confirmed as prion disease, except for N = 2 genetic cases, which due to the presence of the mutation are highly likely to have been prion disease. Samples received in ~ 0.5 mL aliquots were thawed upon receipt, aliquoted to 100 μL volume, refrozen at − 80 °C, and re-thawed only immediately before analysis. Due to sample volume limitations, not all positive controls were utilized in all assays.At each study visit, participants completed a battery of cognitive tests and standardized assessments of mood, neuropsychiatric symptoms, motor function, and activities of daily living. The cognitive battery consisted of standard paper and pencil neuropsychological measures including the Montreal Cognitive Assessment (MoCA) , Verbal Following each LP, participants completed a brief survey that we designed to assess the experience, either on paper or via iPad. They were asked whether they had previously had an LP, and if so, how many. Participants were then asked to mark an X on a 14-cm Likert-type scale to indicate (1) their level of anxiety before the LP procedure and (2) their current feelings at the prospect of a future LP. In both cases, the response was marked on a continuous spectrum bounded by the two extremes of “not anxious at all” and “extremely anxious.” Responses were normalized to the full length of the scale.PRNP single nucleotide variants were identified at the Broad Institute Genomics Platform using a custom targeted capture platform developed by Twist Bioscience combined with deep Illumina sequencing. These genotypes were then confirmed, and the presence or absence of octapeptide repeat insertions determined, using a previously described sequencing and gel analysis protocol [Blood was collected in EDTA tubes, then centrifuged at 1000 rpm to separate plasma for aliquoting into 0.5 mL aliquots. DNA was extracted from whole blood collected on the first study visit. All samples were codified for analysis. Genotypes were used for research purposes only. protocol  implemenPrP levels were quantified at the Broad Institute using the BetaPrion Human ELISA assay , accordiCSF T-tau was measured using the INNOTEST hTau Ag ELISA kit  according to the manufacturer’s instructions. Study samples were diluted 1:4; positive control symptomatic prion disease samples were diluted 1:10. All samples were assayed in duplicate with samples from the same individual co-located on the same plate to facilitate comparison. Following termination of the colorimetric development reaction, absorbance per well was measured at 450 nm as well as at 620 nm for background subtraction using a FLUOStar Optima absorbance plate reader, then fit to an internal standard curve. The operator was blinded to mutation status.CSF NfL was measured using the NF-light RUO ELISA  according to the manufacturer’s instructions. Study samples were diluted 1:2; positive control symptomatic prion disease samples were diluted 1:5. All samples were assayed in duplicate with samples from the same individual co-located on the same plate to facilitate comparison. Following termination of the colorimetric development reaction, absorbance per well was measured at 450 nm as well as at 620 nm for background subtraction using a FLUOStar Optima absorbance plate reader, then fit to an internal standard curve. The operator was blinded to mutation status.Following CSF collection and processing as described above, uniformly handled 0.5 mL CSF aliquots with no additive were stored at − 80 °C until shipment on dry ice to the University of Gothenburg for analysis. CSF neurofilament light (NfL) was measured using an in-house developed ELISA as previously described .Following blood processing as described above, 0.5 mL plasma aliquots were stored at − 80 °C until shipment on dry ice to the University of Gothenburg for analysis. Plasma NfL and total tau levels were measured using the single molecule array (Simoa) HD-1 Analyzer . For T-tau, the commercially available Tau 2.0 kit was used according to the manufacturer’s instructions (Quanterix). For NfL, a previously described in-house Simoa assay was used . CalibraE. coli according to established protocols [g for 5 min at 4 C in a PALL 100-kDa filter tube. Eighty microliters of reaction mix and 20 μL of CSF were combined in each well of a black 96-well plate with a clear bottom (Nunc) with final concentrations as follows: 300 mM NaCl, 10 mM phosphate buffer, 1 mM EDTA, 10 uM thioflavin T, 0.002% SDS, and 1 mg/mL SHaPrP. All samples were loaded in quadruplicate with each plate containing negative control CSF  and positive control CSF (symptomatic prion disease patients). After sealing , plates were incubated in a BMG FLUOstar Optima plate reader at 55 °C for 40 h with continuous cycles of 60 s shaking  and 60 s rest and ThT fluorescence measurements every 45 min . Following termination of the experiment, fluorescence readings were merged per well to generate kinetic curves, and the threshold for a positive well was set as the mean value of all negative wells plus 10 standard deviations. A sample was considered overall positive if at least two of four replicates crossed this threshold. The operator was blinded to mutation status. For the bank vole prion protein (BvPrP23-230) alternative protocol, RT-QuIC was performed according to the protocol described by Orru et al. [The assay was performed according to an established protocol for the detection of prion seeds in CSF  that is rotocols , then fru et al. , with thN = 24 participants for whom this was the first LP generally reported lower anxiety about undergoing future LPs than they had felt about the first LP , driven by lower PrP in D178N mutation carriers , replicating previous reports [CSF PrP levels were stable over a 2–4-month interval and similar between carriers (mean CV = 6.8%) and controls (mean CV = 7.5%) Fig. a. CSF Prers Fig. ” section reports , 47.Figp = 0.46, two-sided Kolmogorov-Smirnov test; Fig. p = 0.91, linear regression, Fig. In plasma, T-tau showed high variability (mean CV = 38%) over 2–4 months, limiting interpretability , while CSF NfL was indistinguishable between the two groups . CSF T-tau and NfL were independently measured by ELISA at a second site, and values showed good correlation with those values initially obtained , with no difference between carriers and controls , levels of both proteins remained low with no significant change over time within individuals  in the central nervous system, over time frames likely to be of relevance to dose-finding and biomarker-based trials. In a phase I/II trial of the huntingtin-lowering ASO tominersen, mutant huntingtin protein was reduced by a mean of 40% in CSF in the two highest dose cohorts [PRNP mutations. While our study is biased toward highly motivated carriers willing to participate in research, this same bias will likely apply to trial recruitment, supporting the relevance of these data.Broadly, these findings suggest that CSF PrP levels are stable enough in any one individual, regardless of  cohorts ; our datAcross the candidate disease markers measured, most carriers could not be distinguished from non-carrier controls. Put differently, the present data do not support analogies between the disease state of most adult carriers and the clinically silent incubation phase in prion-infected animals –59. PrevTo our knowledge, ours is the first report of RT-QuIC seeding activity in the absence of prion disease symptom onset. These data may suggest that for individuals harboring the E200K mutation, RT-QuIC seeding activity may offer an early sign of pathological change, before behavioral or cognitive scales can detect changes in performance. But this finding should be interpreted with caution. One previous report found that an E200K individual converted from RT-QuIC negative to positive between 2 and 4 months after symptom onset . A reporOur study has several limitations. As discussed above, orthogonal lines of evidence lend support to the biological validity of our observation that D178N mutation carriers have lower groupwise CSF PrP levels compared to other participants in our study. However, based on our data alone, we cannot exclude the possibility that the ELISA we used to measure CSF PrP may be differentially reactive to some mutant forms of the protein. Our study relied on positive control samples from symptomatic CJD patients collected at various clinical centers. As we have previously shown that heterogenous CSF sample handling precludes reliable quantification of CSF PrP , we wereOur study is relatively young, and our analyses to date provide only short- to medium-term and cross-sectional findings. Moving forward, participants will be seen at annual intervals where feasible, with an eye to enhancing the longitudinal analysis of CSF PrP and enabling longitudinal tracking of pathological biomarkers. Complementary perspectives on the above may be provided by two other efforts, as yet unpublished, to systematically characterize healthy individuals carrying genetic prion disease predisposing mutations: the UK National Prion Monitoring Cohort and UCSF Early Diagnosis of Human Prion Disease studies , 71. AgeA strength of our study is that the highly penetrant prion disease-causing mutations most common in the general population  are alsoThe above characterization of a cohort of pre-symptomatic genetic prion disease mutation carriers and controls suggests that CSF PrP levels are stable in the carrier population and may therefore serve as a meaningful biomarker for PrP-lowering therapeutics. In the meantime, at present, our findings regarding disease stage biomarkers suggest that a “secondary prevention” strategy may not be feasible in genetic prion disease: any prodromal period may be too subtle, too brief, or present in too few individuals at any given time to enable recruitment of a large enough prodromal cohort to enable trials. It remains possible that a fluid biomarker that reliably presages symptom onset further in advance could emerge from further study, particularly in more slowly progressive genetic prion disease variants, allowing subclinical pathology to be tracked in a small cohort of carriers and leveraged toward a secondary prevention trial design. However, our present findings may reflect where the field is likely to stand as therapeutics presently in development approach clinical trials. In this context, pre-symptomatic trials in genetic prion disease may be better served by a primary prevention model based on genetic risk.Additional file 1: Figure S1. Overview of study participant inclusion and exclusion. Figure S2. Pre- and post-procedure anxiety in participants experiencing their first lumbar puncture. Figure S3. Short-term test-retest stability of markers of neuronal damage in carrier and control plasma. Figure S4. Additional measurements and statistics on markers of neuronal damage in carrier and control CSF. Figure S5. RT-QuIC results with recombinant bank vole PrP. Table S1. Measures of cognitive, psychiatric, motor and daily functioning in all MGH study participants. Table S2. CSF PrP levels by mutation status. Table S3. Additional measures of cognitive, psychiatric, motor and daily functioning for one RT-QuIC positive MGH study participant.


One set of heterochiral (PO)3 transitions was assigned, but no structures generated by one of the latest, advanced conformational search codes could account for them. With the aid of a Python program, the carbon atom backbone and then the heterochiral (PO)3 structure were generated using 13C isotopic data. Excellent agreement between theoretical and experimental rotational constants and relative dipole moment components of all three conformers was achieved, especially after applying vibrational corrections to the rotational constants.The chirality controlled conformational landscape of the trimer of propylene oxide (PO), a prototypical chiral molecule, was investigated using rotational spectroscopy and a range of theoretical tools for conformational searches and for evaluating vibrational contributions to effective structures. Two sets of homochiral (PO) 9 initial candidates.What can be done do when theory fails to provide a structural candidate? Using experimental isotopic data and a Python program, we establish a carbon backbone structure and eventually identify the elusive heterochiral propylene oxide trimer shown from 8 By utilizing the classical isotopic substitution approach, we were able to establish the experimental heavy atom backbone of the heterochiral (PO)3, finally leading to the theoretical identification of its structure. Excellent agreement was achieved between experimental and predicted rotational constants using a general hybrid B2/B3So far, no high‐resolution spectroscopic study of PO aggregates beyond the dimer has been undertaken. Here we report the detection of multiple homo‐/heterochiral PO trimers using CP‐FTMW spectroscopy and point out unsolved problems in capturing the structural diversity of (PO)A pair of PO enantiomers, R and S, gives rise to two indistinguishable rotational spectra, whereas different homo‐/heterochiral diastereomeric PO aggregates can be formed 3, are summarized in Tables S1,S2, SI. The large number of possible conformers within such a small energy window highlights the difficulties in identifying them experimentally.For (PO)3 were collected using a 2–6 GHz CP‐FTMW spectrometer.3 conformers were assigned. A racemic PO spectrum is shown in Figure 3 are clearly visible, as well as a new set of strong transitions. This new set was tentatively assigned to a heterochiral (PO)3 since the transitions are only visible with the racemic but not the enantiopure sample. The experimental rotational constants and relative magnitude of dipole moment components are compared with the DFT predictions in Table 3 match those of the two most stable DFT homochiral (PO)3, HOMO1 and HOMO2, quite well, confirming their identification. No reasonable match to a DFT structure, however, could be made for the experimental heterochiral (PO)3 from the list of 53 heterochiral (PO)3 candidates (Table S2). For convenience, we name this experimental structure HETERO1.Broadband spectra of homo‐/heterochiral (PO)3 in all details in our theoretical conformational search, the subtly different binding topologies can be easily discriminated by the CP‐FTMW method with its very high spectral resolution. We therefore resorted to use isotopic substitution, a hallmark of rotational spectroscopy, to further explore the structural details of (PO)3 experimentally.The failure to produce a possible HETERO1 structure theoretically is a significant issue since conformational diversity is important in a wide range of chemistry and biochemistry subfields. While we could not capture the structural diversity of (PO)3, it is ideal to have a single substitution at each heavy atom. Since the enriched 13C and 18O isotopologues of PO are prohibitively expensive, we optimized the experimental conditions and utilized the sensitivity achieved to measure and assign all 18 13C isotopologues of HOMO1 and HETERO1 in their natural abundances. Figures 13C isotopologues of HOMO1 and HETERO1; the experimental transition frequencies and spectroscopic constants are in Tables S7–S10, SI. The 13C isotopologues of HOMO2 could not be detected because of the too low abundance 13C atoms are summarized in Tables S11 and S12 of the SI for HOMO1 and HETERO1, respectively. Since Kraitchman's analyses provide only absolute values of the C atom coordinates, there are 89 possible C‐backbone configurations without considering any chemical constraints because each of the nine carbon atoms has three Cartesian coordinates, leading to a total of (23)9=89 possibilities. For HOMO1, we used the DFT geometry as a guide to determine the signs. The experimental substitution structure of the carbon backbone frame of HOMO1 is compared with the final DFT one (vide infra) in Figure The Kraitchman coordinates for singly substituted 9 possibilities: by utilizing the known PO structure and setting limits for the intramolecular C−C distances, a few hundred possible C‐backbone candidates were obtained initially. The O and H atoms were then put back in, based on the PO monomer geometry. Configurations in which the PO subunits are too close or too far away were then removed, leading to just one unique (PO)3 structure. We tested this procedure first with HOMO1, and then applied it to HETERO1. Details of the search are given in Point S2, SI. The derived HETERO1 C‐backbone is shown in Figure For HETERO1, a Python program was written to extract one unique structural candidate out of the 8−1 less stable than HOMO1 (Table S2).The experimental relative abundance ratio of HETERO1:HOMO1:HOMO2 is estimated to be 3.3:1.0:0.2 for the racemic sample. If their stabilities were the same, one would expect the HETERO1 versus HOMO1 ratio to be 3, based on statistical arguments (Scheme B0) are compared with the equilibrium values (Be). For fully quantitative purposes, the difference between them cannot be neglected. Here we applied a general hybrid approach, referred to as B2/B3, which is based on harmonic/anharmonic force fields computed with the B2PLYP/B3LYP‐D3BJ functionals in conjunction with jun‐cc‐pVTZ and jun‐cc‐pVDZ basis sets, respectively. The B2/B3 approach has been validated for both covalent interactions and non‐covalently bound complexes.In the above discussion, experimental vibrational ground‐state rotational constants B0 are comBe values, relative energies, dipole moment components at the B2 and rPP levels, and their vibrational corrections are summarized in Tables S13‐S18, SI. The results for all singly substituted 13C isotopologues of HOMO1, HOMO2, and HETERO1 are in Tables S19–S30, SI. It is noteworthy that at the rPP level, HETERO1 becomes the global minimum, thus restoring full agreement with the experimental energy ordering. The maximum difference between experimental rotational constants and those from the B2/B3 hybrid Scheme is reduced to 1.2 % in comparison to 3.7 % with just the equilibrium calculations, and the new hybrid rPP/B3 model further reduces the difference to below 0.3 % for all (PO)3 observed (Table 13C isotopologues) of HOMO1 and HETERO1, which were used to refine their theoretical equilibrium structures while fixing the monomer geometries and some intermolecular structural parameters. The refined results are given in Tables S31–34, SI, with some key parameters shown in Figure The six lowest homo‐/heterochiral structures, including HETERO1, were re‐optimized at the B2 and rPP levels and anharmonic vibrational corrections were computed at the B3 level. A full account of the computational details is given in Point S3, SI. The resulting 3, a ternary chiral system of fundamental interest, was investigated in detail using CP‐FTMW spectroscopy and a range of theoretical treatments including the CREST, B3LYP, B2/B3, and rPP/B3 calculations. The high sensitivity/resolution capability of the CP‐FTMW instrument allowed us to uncover the elusive DFT HETERO1 structure based on experimental 13C isotopic data. The study further reveals a slight chirality controlled conformational preference for the heterochiral trimer, continuing the same preference shown in (PO)2. The highly sophisticated semiempirical conformational search program CREST was not able to find the most stable ternary heterochiral aggregate, and the current work may provide benchmark data for further improvements.In conclusion, the conformational panorama of (PO)The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Methods: Short-term colonic fermentations (48 h) of AADE, fructo-oligosaccharides (FOS), and a blank were performed. Microbial metabolites were assessed at 0, 6, 24, and 48 h of colonic incubation via measuring pH, gas pressure, lactate, ammonium, and short-chain fatty acids (SCFAs) levels. Community composition was assessed via targeted qPCRs. Results: After 24 and 48 h of incubation, bifidobacteria levels increased 25-fold with AADE (p < 0.05) and >100-fold with FOS (p < 0.05) compared to blank. Lactobacillus spp. levels only tended to increase with AADE, whereas they increased 10-fold with FOS. At 6 h, pH decreased with AADE and FOS and remained stable until 48 h; however, gas pressure increased significantly till the end of study. Acetate, propionate, and total SCFA production increased significantly with both at all time-points. Lactate levels initially increased but branched SCFA and ammonium levels remained low till 48 h. Conclusion: AADE displayed prebiotic potential by exerting bifidogenic effects that stimulated production of health-related microbial metabolites, which is potentially due to inulin in AADE.Background: Prebiotics used as a dietary supplement, stimulate health-related gut microbiota . This study evaluated potential prebiotic effects of an artichoke aqueous dry extract (AADE) using in vitro gut model based on the Simulator of Human Intestinal Microbial Ecosystem (SHIME Lactobacillus spp.) and Bacteroidetes that are susceptible to alterations. Other phyla are Actinobacteria (including Bifidobacterium spp.), Proteobacteria, Fusobacteria, and Verrucomicrobia. Spatial and temporal discrepancies in gut microbial distribution contribute toward specific metabolic, immunological, and gut-protective functions throughout an individual’s life span [Human gut microbiota consist of over 35,000 bacterial strains, encompassing beneficial and pathogenic species; however, the predominance of positively affecting microbes ensure our well-being . Human gife span ,3. Charaife span .Prebiotics were first defined as, “Nondigestible food ingredients that beneficially affect a host by selectively stimulating growth and/or activity of one or a limited number of bacteria in the colon that are recognized to improve host health” . DysbiosCynara scolymus L.), chicory (Cichorium intybus) roots, and garlic (Allium sativum) are rich in inulin and dietary fibers. Inulin from artichoke is recognized to have the highest degree of polymerization known in plants. Degree of polymerization directly contributes to prebiotic effects and persistence in the colon [Bifidobacterium spp. in the gut [There is a constant need for new prebiotics that can target specific bacterial species and most approaches have focused on non-digestible oligosaccharides, such as galacto-oligosaccharides, soya-oligosaccharides, isomaltooligosaccharides, gluco-oligosaccharides, xylo-oligosaccharides, lacto-sucrose, and inulin-type fructans. However, they are known to have varied prebiotic potential. Inulin has been demonstrated to positively alter gut microbiota in a dose range of 4 to 40 g/d ,14,15,16he colon . Inulin he colon . Additiohe colon . Prebiot the gut ,21. In a the gut ,23.®) model [Cynara scolymus L.); cultivated in Spain, and extracted by the Pure-Hydro Process™ using only water (instead of organic solvents), AADE can be safely used in foods and food supplements.In the present study, we aimed to evaluate the prebiotic effects of artichoke aqueous dry extract (AADE) through an in vitro approach of highly controlled conditions using short-term colonic incubations based on the Simulator of Human Intestinal Microbial Ecosystem (SHIME®) model . In vitrCynara scolymus L.) cultivated in Mediterranean regions of Spain. The AADE was prepared in accordance with the European Pharmacopoeia monograph extracts (Extracta) (No. 0765) using a proprietary water-based extraction process [All chemicals were obtained from Sigma-Aldrich , unless stated otherwise. The test product AADE, also known as Cynamed™, was provided by Euromed S.A. . It is derived from the edible part of the artichoke plant , 2.0 g/L Yeast Extract, 2.0 g/L pepton , 1.0 g/L mucin , 0.5 g/L L-cystein, and 2.0 mL/L Tween80  was added to incubation reactors (90 vol%), already containing the correct amount of the test products for obtaining a final concentration of 0 g/L (Blank) or 5 g/L (for both AADE and FOS), respectively. The reactors were sealed and anaerobiosis was obtained by flushing with N2. Subsequently, fresh fecal material of a healthy human donor (no history of antibiotic use in the six months preceding the study) was collected . After preparation of an anaerobic fecal slurry, this was inoculated at 10 vol% in the aforementioned medium. All incubations were performed in biological triplicate for 48 h at 37 °C under anaerobic conditions with continuous shaking (90 rpm).Short-term colonic fermentations were performed as described recently . BrieflyMicrobial metabolic analyses were performed on samples collected at 0, 6, 24, and 48 h of colonic incubation and levels of pH , gas pressure , lactate, ammonium, and short-chain fatty acids (SCFAs) were measured. Acetate, propionate, butyrate, and branched CFAs (BCFAs)  were quantified as described by De Weirdt et al.  via GC-FLactobacillus spp. (Firmicutes phylum), Bifidobacterium spp. (Actinobacteria phylum), and Akkermansia muciniphila (Verrucomicrobia phylum) were performed using a StepOnePlus™ real-time PCR system . Each sample was analyzed in triplicate. Standard curves for all the different runs had efficiencies between 90–105%. All protocols were initiated for 10 min at 95 °C and terminated with a melting curve from 60 °C to 95 °C. Cycling programs included 40 cycles with a denaturation step of 15 s at 95 °C, an annealing step of 30 s at 60 °C, and an elongation step of 30 s at 72 °C in each cycle. Descriptions of primers used are presented in At the start of colonic incubation and after 24 and 48 h, samples were collected for microbial community analysis. DNA was isolated using the protocol as described by Vilchez-Vargas et al. , startinBesides presenting the absolute levels of the different groups, the ratio between the obtained levels at 24 h and 48 h versus 0 h were calculated for the blank, AADE, and FOS-treated microbiota.p < 0.05. Statistical analysis was performed with the GraphPad Prism software .To evaluate differences in microbial metabolites and microbial community composition between blank and treatment incubations at the different time points, a two-way ANOVA with Tukey multiple comparisons test was performed. Differences were found significant if Bifidobacterium spp., Lactobacillus spp., Bacteroidetes, Firmicutes, and Akkermansia muciniphila) at each of the three time points (0/6/48 h) are presented in Akkermansia muciniphila). First, both at 24 h and 48 h, bifidobacteria levels were significantly increased versus the blank for AADE but especially for FOS . The pH continued to decrease until 24 h and remained stable thereafter, indicating continued microbial fermentation , which reflected enhanced microbial metabolic activity upon AADE/FOS administration. However, the overall increase in total SCFAs was higher with FOS compared with AADE  lower for both AADE (3.42 ± 0.02) and especially FOS (0.34 ± 0.26) when compared with the blank (4.01 ± 0.13). The results for ammonium, another marker for protein fermentation, were similar to those for BCFAs, indicating reduced protein fermentation upon AADE and especially FOS administration. Lactate levels were high (p < 0.05) with AADE and even further increased for FOS compared with blank after the initial 6 h of incubation. Thereafter, lactate levels decreased indicating lactate consumption.As shown in Lactobacillus spp. levels.In the present study, although the effects of AADE on microbial activity and composition were milder as compared to the “gold standard” prebiotic FOS, AADE demonstrated marked prebiotic potential. First, AADE significantly decreased pH and increased gas production, which indicated overall increased microbial activity upon administration of the test product. Saccharolytic metabolites such as acetate and propionate, and thus also total SCFAs, increased, while levels of proteolytic metabolites, BCFAs, and ammonium, significantly decreased upon AADE administration at 48 h. A key finding of this study was the growth-promoting action of AADE, mostly on bifidobacteria which are regarded as health-related members of the intestinal microbiome. Further, AADE also affected Bacteroidetes, Firmicutes, and Lactobacillus spp. and bifidobacteria, and prevent growth of pathogenic bacteria [Based on results of this study, bifidogenic effects of AADE were milder, yet in the same order of magnitude as those of FOS. These findings were similar to those of a previous in vitro study conducted by Barszcz M et al. . Bifidogbacteria ,31.Akkermansia muciniphila. This taxon was not increased for either AADE or FOS and even decreased upon FOS administration. This was likely due to the fact that FOS more strongly decreased the pH (to 5.66 within 24 h), which is a pH at which Akkermansia muciniphila is unable to grow [Akkermansia muciniphila in the distal colon, as shown for inulin in humanized rats [Moreover, in our study, lactate was produced during the initial 6 h of incubation. Subsequently, lactate was consumed and coincided with an increase in propionate levels for both AADE and FOS, with most marked stimulations being noted for FOS. Butyrate was not stimulated by AADE, suggesting that the majority of lactate (that can be used as a substrate for both propionate and butyrate), was cross-fed to propionate upon AADE supplementation. Some Negativicutes  are shown to form propionate  and coul to grow . In vivozed rats .Similarly, acetate and lactate can be cross-fed to butyrate by members of the Ruminococcaceae, Lachnospiraceae, Clostridiaceae, Eubacteriaceae, all members of the Firmicutes phylum ,38. ButyFurthermore, propionate and acetate have been shown to stimulate release of peptide hormones leading to short-term signaling of satiation and satiety to appetite centers in the brain, resulting in reduced food intake by the host ,40,41. SAmmonia and BCFAs are toxic metabolites produced from protein fermentation . In this® model indicate the prebiotic potential of AADE. These findings could be further validated using different models, such as M-SHIME®  which focuses not only on luminal but also mucosal gut-colonizing microbes [Short-term colonic incubations have often been used to gather information on the prebiotic potential of novel ingredients. Results of the present study using an incubation strategy based on the SHIMEmicrobes . Moreove® model, demonstrated that AADE has promising prebiotic potential. Incubation with AADE resulted in an increase of beneficial microbes, which was correlated with their metabolite profile. The promising results of this study justify future investigations using multiple doses in upgraded models to further validate these findings.The present preliminary evaluation, conducted using the SHIME


Grocott-Gomori's-methenamine-silver staining, India ink staining,and calcofluor white staining of CSF demonstrated the existence of unusual morphologicalfeatures like pseudohypha formation, chains of budding yeasts, and structures resemblinggerm tubes  revealed signssuggestive of acute disseminated encephalomyelitis. Cerebrospinal fluid (CSF) showedglucose levels of 64.8 mg/dL and protein levels of 65.6 mg/dL. CSF microscopy showed 42cells/mmrm tubes . Most ofCryptococcus can deviate from its characteristic morphology asencapsulated budding yeast cells, presenting as pseudohyphae or germ tube-likestructures


Agreement analysis showed that bias between TT vs. observers’ mean was lower than between any observers’ mean against each other in the ONL (0.77 ± 0.34 µm vs 3.25 ± 0.33 µm) and INL (1.59 ± 0.28 µm vs 2.82 ± 0.36 µm). Validation dataset showed that TT can detect significant and true ONL thinning (p = 0.006), more sensitive than manual measurement capabilities (p = 0.069). ThicknessTool can measure retina nuclear layers thickness in a fast, accurate, and precise manner with multi-platform capabilities. In addition, the TT can be customized to user preferences and is freely available to download.To develop an automated retina layer thickness measurement tool for the ImageJ platform, to quantitate nuclear layers following the retina contour. We developed the ThicknessTool (TT), an automated thickness measurement plugin for the ImageJ platform. To calibrate TT, we created a calibration dataset of mock binary skeletonized mask images with increasing thickness masks and different rotations. Following, we created a training dataset and performed an agreement analysis of thickness measurements between TT and two masked manual observers. Finally, we tested the performance of TT measurements in a validation dataset of retinal detachment images. In the calibration dataset, there were no differences in layer thickness between measured and known thickness masks, with an overall coefficient of variation of 0.00%. Training dataset measurements of immunofluorescence retina nuclear layers disclosed no significant differences between TT and any observer’s average outer nuclear layer (ONL) ( Among these methods, photoreceptor cell death assays4, outer nuclear layer cell counting5, and outer nuclear layer thickness7, have been used in animal models to quantitate photoreceptor degeneration. Given the technical and time constraints related to cell death assays and manual counting; to date, outer nuclear layer (ONL) thickness has been widely used as a practical proxy method to estimate the depth of photoreceptor cell death8.Progressive photoreceptor cell death is a significant culprit in retinal degenerative diseases. Inasmuch, quantitation of this cell loss has been addressed by a myriad of approaches, predominantly in experimental models of retinal diseases, such as retinal detachment9, to the best of our knowledge, there is currently no freely available tool for the ImageJ platform to automatedly quantitate multiple layer thicknesses in large images and in different imaging modalities.The utility of retina ONL thickness quantitation to infer photoreceptor degeneration relies on manual calliper measurements. However, the advantage of this approach can be compromised as callipers are manually drawn and measured by an observer. In addition, given the curvature of the retina, manually positioning callipers perpendicular to the layer contour along the long axis can be difficult. Furthermore, given the high magnification of microscope images, a small area of interest is often analysed to expedite the analysis, which can lead to bias. As new imaging modalities also become available, a versatile method that can adapt to multiple imaging modalities is ideal. Therefore, a tool that can adapt to layer architecture and contour to measure thickness in a broad segment, in multiple layers, in either single images or large tiles, and in multiple platforms, is very compelling. Despite pioneering work done in this areaThe purpose of this work was to develop an automated retinal layer thickness measurement tool for the ImageJ platform, which can quantitate nuclear layers following the retina contour, with callipers as close to 1-pixel to each other. For this purpose, we developed the ThicknessTool (TT) and validated its accuracy by objective calibration and agreement analysis with two masked observers. We found that this measurement tool can provide accurate and precise thickness measurements. In addition, TT can process images from multiple imaging modalities.10. C57BL/6J mice were purchased from The Jackson Laboratories and maintained in a standard 12-h light/dark cycle.All animals used in experiments and breeding adhered to the statement of the Association for Research in Vision and Ophthalmology (ARVO). Animal protocols were reviewed and approved by the Animal Care Committee of the Massachusetts Eye and Ear Infirmary. To validate the ThicknessTool in experimental model, a retinal detachment was induced in eight-week-old C57BL/6J mice, as previously describedWe created an image dataset of fifty 8-bit mock images of 1344 × 1024 pixels containing masks of increasing thickness, from 10 to 500-pixel thickness with 10-pixel increments. Following, we created a second dataset of images with a known mask area of 1344 × 200 pixels with 10° rotation intervals, plus 45° and 135° rotations for a total of 21 images. Finally, to emulate layer thinning and thickening, we created a single mock image containing a mask with abrupt thickness changes, with segments of 200, 150, and 250 pixels. All images were stored in TIFF format, and the measured thickness was compared to the known standard.We defined the overall mean thickness limit of agreement to 1 pixel, and the following criteria for the ThicknessTool script calibration: (1) overall mean thickness equal to 200 ± 1 pixel; (2) index of dispersion equal to 0, as the given area is a rectangle and all callipers should be parallel to each other with equal thickness; (3) single calliper measurement at 0°, 45°, 90°, 135°, and 180° rotations equal to 200 pixels; and (4) single calliper measurement equal to 200 pixels ± 2 pixels at the remaining rotations. This difference corresponds to the aliasing effect on area edges at rotations other than the specified on (3). The occurrence of uneven jagged edges requires a broader margin of error (2 pixels) than images with straight  or even (45° and 135°) edges. For comparisons, the measured mean was tested against a hypothesized mean of 200 pixels. An equivalence test with a two one-sided tests (TOST) approach was used to assess minimum and maximum single calliper measurements, and the threshold difference considered equivalent to no difference was defined as 1 for images with even edges and 2 for images with uneven edges.Immunofluorescence digital images of 4′,6-diamidino-2-phenylindole (DAPI)-stained retinal cross-sections were obtained from the Angiogenesis Laboratory fluorescence microscopy database. We selected images from a murine retinal detachment model in order to evaluate TT performance with different ONL thicknesses and various retinal distortions. A total of 64 randomly selected images were included in this dataset and stored in TIFF format for further processing. Training dataset images were measured by an inexperienced observer who never performed the given task before, and by an experienced observer who had performed these measurements before. Images of murine retina displaying the outer (ONL) and inner nuclear layer (INL) were masked and given to observers. Each image was measured twice in a masked manner. Observers measured each layer with six individual callipers, placed across the retinal layer at their discretion. Single callipers were used to calculate mean, minimum, and maximum layer thickness.We randomly selected 16 images from 8 eyes of an experimental murine retinal detachment model. The layer thickness was manually assessed by an observer, who measured ONL and INL in the detached and attached retina, with six individual callipers for each layer.https://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA). To facilitate the use of the script, we designed a graphical user interface dialog to tailor performance parameters to users’ preferences, including image scale, number of callipers, skeleton width, among others  Centroids from these segmented skeletons were isolated as single points and defined by the coordinates ; (3) The skeleton segment angle was calculated by an user-defined grid which evaluated the angle of the segmented mask skeleton in a given area; (4) From each centroid , a positive final vector and a negative initial vector were created perpendicularly to the segmented skeleton. The initial and final vectors were composed by a corresponding horizontal component vector (Xstep), and a vertical component vector (Ystep); (5) The Xstep and Ystep components were created for the initial and final resolved vector at a 1-pixel distance from the centroid in horizontal and vertical directions, respectively. The preliminary initial  and final  coordinates were created from the centroid as described in Eq.  and final  coordinates. If the pixel value returned zero, this was interpreted as the retinal layer mask, and the Xstep and Ystep components were increased by 1-pixel, to analyse the contiguous pixel; (7) This process was iterated until the pixel value was > 0, and this point was assumed as the initial  and final  vector endpoint; (8) Thickness was calculated as the rounded Euclidean distance between initial  and final , as seen in Eq.  and statistical significance. Parallelism plots were constructed fitting a cubic spline with a default lambda of 0.05. Agreement analysis was performed by Bland–Altman plot method13, and as previously described4. Coefficient of variation (CoV) was calculated as the difference between measurements over their mean [(A – B)/(A + B)/2] and CoV% as CoV*100. The index of dispersion was calculated as variance-to-mean ratio. Results are expressed as mean ± standard deviation (SD). A p value of < 0.050 was considered statistically significant.Statistical analysis was performed with SAS software . Normality was assessed with Shapiro–Wilk test. Statistical significance for differences between groups was determined with t-test for matched pairs or Wilcoxon signed-rank test for matched pairs, t-test for independent samples, and one-way ANOVA with Tukey post hoc correction for multiple comparisons. For correlation analysis, values were expressed as Pearson correlation coefficient (p < 0.001). Following, we evaluated the performance of the algorithm at different layer rotations with 200-pixel masks at different angles. We predefined the known or theoretical mean, minimum, and maximum thickness to 200 pixels, a coefficient of variation (CoV) of 0.00, and a margin of error of 1-pixel. As seen in Supplementary Table p = 0.06). Mean thickness for images with even and jagged edges fulfilled the calibration criteria as previously defined, with CoV of 0.00 , indicating that the observed difference does not exceed the defined threshold. Finally, we tested TT capability to detect true thinning and thickening in a mock image with decreasing and increasing thickness, respectively. TT accurately measured varying thickness , with a bias of 0.00 ± 0.00 and no significant difference between known vs. measured markers (p = 1.000) . These results indicate that the ThicknessTool can automatedly measure the different thicknesses and at different rotations in an accurate and precise manner.To calibrate the ThicknessTool algorithm, we created three mock image datasets with masks of known area thickness and rotation. First, the TT was able to accurately measure areas of increasing thickness, from 10 to 500 pixels, with a bias of 0.00 ± 0.00  analysis between TT and observers’ measurements were > 0.88 (p < 0.001) for the ONL and > 0.84 (p < 0.001) for the INL , and no significant differences between TT and any observer average INL measurements. The average for the inexperienced vs. experienced was not significantly different, in either the ONL (p = 0.219) and INL (p = 0.097), with overlapping confidence intervals. Moreover, there were no significant differences between TT and any observer or average ONL/INL ratio measurements, with overlapping 95% confidence intervals. Altogether, these results suggest that no significant difference was evidenced regarding the observers’ prior experience. However, TT ONL and INL measurements stand more closely to experienced observer’s results. Moreover, TT measurements are not significantly different from any observers’ ONL and INL average.The mean ONL thickness was 65.26 ± 7.78 µm, 62.00 ± 7.14 µm, and 62.85 ± 7.33 µm for the inexperienced, experienced, and TT, respectively . In addition, the coefficient of variability between measurements was significantly different between inexperienced and observers’ average measurements for both ONL and INL (p < 0.001) and between the experienced observer and observers’ average measurements for the ONL (p < 0.009) . In contrast, we found the lowest bias when comparing TT vs. observers’ means . For the INL group, we found statistically significant differences when comparing inexperienced mean vs. experienced mean (p < 0.001), TT vs. inexperienced mean (p < 0.001), and TT vs. observers’ mean (p < 0.001). Most importantly, the bias between TT vs. observers’ INL mean (1.59 ± 0.28 µm) was lower than the those between any observers mean against each other (2.82 ± 0.36 µm). In addition, Bland–Altman plots displayed random variability within measurements  to 500 pixels (twice the outer nuclear layer measured in our dataset). In addition, we created a dataset with multiple mask rotations to mimic the random mask orientations. This step is necessary as TT callipers are constructed in a vectorial manner, in number and angles as the user or region of interest requires. Moreover, we tested TT capability to detect true thinning and thickening in a mock image with decreasing and increasing thickness, respectively. We found that the TT can automatedly and accurately measure thickness when challenged with different thicknesses and rotations.In the development of new measurement tools, calibration images are mandatory to benchmark and finetune the performance of image processing algorithms to a standard16. Such errors are not only misleading to the scientific community but also high priced.Accurate assessment of photoreceptor cell death currently relies on manual counting by masked observers. Ideally, two masked two observers quantitate retina thickness, and their average is used to determine the overall outcome. We found that single observers’ measurements were prone to over and underestimation, regardless of their previous experience. In addition, observers draw incorrectly angled callipers, which can indirectly contribute to the abovementioned bias. Moreover, callipers were not located in corresponding locations in repeated same-image analysis, which dampens the reproducibility of the measurements. Altogether, this leads to a lack of accurate or reproducible retina thickness quantitation. This matter is of critical importance, as this bias can lead to α or β errors. For example, finding no difference in retinal thickness in cases where a neuroprotective treatment is effective, or even worse, finding a significant difference in retinal thickness in an ineffective treatment17. As authors describe, such agreement must be quantified, and cannot be assessed by mean comparison or hypothesis testing, as it does not exist in a present or absent binary form13. In addition, high correlation does not necessarily imply a high agreement between two methods, as it quantifies the degree to which two methods are related18. Agreement analysis showed lower bias of TT against observers than observers’ average against each other for both ONL and INL. Taken into consideration TT capability to detect true mask thinning and thickening, collectively, these results confer to TT a higher measurement agreement.Regardless of the performance of manual measurements to date, it remains the gold standard. For purposes of method agreement, we refrained from relying on mean comparison or correlation, as Bland and Altman have previously reported in their seminal work9. However, their approach was not validated by thickness agreement analysis with manual observers. In their work, authors also described a method to quantify structural distortions in retinal tissue before and after injury, also known as distortion index. In essence, the authors stated that structural changes in the ONL are directly proportional to the distortion index. In our work, we estimated overall distortion in a similar approach using the index of dispersion (ID), calculated as the variance-to-mean ratio, as previously defined by Cox and Lewis19. Similarly, the index of dispersion allows the assessment of the overall calliper variation, which can provide detailed layer architecture data. Briefly, an ID of 0 represents no dispersion; therefore, we used the ID for calibration purposes, as mock images of known and constant thickness were uniform rectangles (ID = 0). We speculate that the index of dispersion can be equally used for further morphometric assessment.Moreover, the automated thickness analysis by TT yields a series of convenient metrics that remain hidden otherwise in manual measurements. On this note, Byun et al. have developed pioneering methods to estimate the ONL thickness exclusively4. We recommend users to first tailor and validate their segmentation algorithm for optimal performance. Finally, regardless of the potential image modality application, this tool has been validated only for ONL and INL measurements in DAPI nuclei-stained murine retina cryosections. We believe future work can help expand the applicability of this tool for three-dimensional images, including upcoming imaging platforms.Several limitations to this work should be considered. First, despite the calibration analysis performed beforehand, the validation of TT can only be performed against manual measurements. This process is cumbersome, as observers’ measurements carry an intrinsic bias. However, observers showed comparable performance, with overlapping confidence intervals. Second, TT analysis should be performed in areas distant from image edges, as skeleton are trimmed to avoid calliper-to-end image measurements. Despite the fact that TT allows skeleton and edge trimming, users should evaluate processed images for qualitative control. Third, TT measures thickness in binary segmented masks. Therefore, its performance is as strong as the segmentation algorithm used. In our case, we build this tool based on a previously validated algorithm for retina layer segmentationIn summary, we developed a fast, accurate, and precise retinal thickness measurement tool with multi-platform capabilities. In addition, the TT can be customized to user-defined preferences. This includes calliper number with a 1-pixel minimum distance between callipers, skeleton and edge trimming, and automated or manual layer segmentation. In addition, given the potential use with multi-platform capabilities, the source code can be easily modified to fit different applications. Finally, this tool is freely available to download and use. We expect this measurement tool could improve the outcomes and reduce bias in retina research.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.


Klebsiella pneumoniae carbapenemase (KPC)–3–producing multidrug-resistant K. pneumoniae with ceftazidime/avibactam (CAZ-AVI) in a patient who developed postneurosurgical meningitis and bacteremia. Therapeutic drug monitoring of cerebrospinal fluid and blood samples demonstrated CAZ-AVI concentration levels 20-fold greater than the minimum inhibitory concentration in the first 60 minutes postinfusion, providing evidence for the utility of CAZ-AVI in treating KPC–Klebsiella pneumoniae central nervous system infections.This report describes the treatment of  Enterobacteriaceae (CRE) are difficult to treat. Selecting an effective antibiotic regimen to overcome antimicrobial resistance while achieving and maintaining adequate CNS levels is particularly challenging. Moreover, clinical accounts treating CNS infections with newer antimicrobial agents are lacking, and data pertaining to their pharmacokinetic/pharmacodynamic (PK/PD) properties remains scarce. Carbapenem-hydrolyzing β-lactamases such as Klebsiella pneumoniae carbapenemase (KPC) confer resistance to most β-lactams, including carbapenems ) was determined to be 38%, while bacterial loads decreased by a mean of 5.66 log10 CFU as a result of therapy [CAZ-AVI is currently approved for intra-abdominal infections, complicated urinary tract infections, hospital-acquired bacterial pneumonia, and ventilator-associated bacterial pneumonia. The clinical experience with CAZ-AVI in the treatment CNS infections is uncommonly reported, and the capacity of this combination to cross the blood–brain barrier remains unknown. In this setting, effective therapy depends on the ability of CAZ-AVI to maintain elevated CSF concentrations that achieve PK/PD thresholds seen in prior simulation models for CNS infections. The only available experimental data were conducted in an animal model inoculating AmpC-producing t1/2 for CAZ and AVI, concentrations were likely well above the MIC for the entire 8-hour dosing interval in the CSF and plasma. In fact, CAZ concentrations were likely 4–5 times higher than the MIC for the entire dosing interval in both the CSF and plasma [In our patient, TDM of CAZ and AVI was conducted to determine how effectively CAZ and AVI distributed into the CNS. Consistent with the Food and Drug Administration–approved dosing, the patient received 2.5 g of CAZ-AVI administered as a 2-hour infusion every 8 hours . Based on our current understanding of the target PK/PD indices for CAZ-AVI , the mead plasma . Similard plasma , 13 and d plasma , 15. TheKp is unclear. Our patient received an additional 2 weeks of CAZ-AVI and intrathecal amikacin to achieve resolution of CSF neutrophilic pleocytosis and microbiologic cure. Although the objective of measuring CAZ-AVI concentrations in the CSF of a patient with a KPC-Kp-associated CNS infection was achieved, these data should be interpreted with caution as they represent only 2 CSF sample measurements. Furthermore, conclusions regarding CAZ-AVI serum ratios/percentages cannot be generalized using this study design due to the sample size, interpatient variability, and other clinical variables.Only 2 published reports have described CRE-associated CNS infections treated with CAZ-AVI. In both cases, postneurosurgical meningitis was caused by CRKP (KPC production was not confirmed). Samuel et al.  employedAdditional studies that measure CSF concentrations of CAZ-AVI using more samples from multiple patients and throughout the entire dosing interval are required to determine the PK/PD parameters for CNS infections with highly resistant organisms. However, the levels attained and measured in our patient using this dosing regimen appear to have been adequate.KPC-Kp nosocomial meningitis. A novel CAZ-AVI TDM method was successfully applied to demonstrate that CAZ-AVI achieves CSF levels well above the MIC in the first 180 minutes postinfusion. Further clinical studies are required to assess the complete profile of CAZ-AVI in the CSF. Our case provides evidence on the use of this agent in treating KPC-Kp.This report describes the measurement of CAZ-AVI concentration levels in the CSF of a patient suffering from


The study aims to investigate the association between plasma Lp-PLA2 and CSF.Coronary slow flow (CSF) is characterized by delayed opacification of distal epicardial coronary arteries without significant coronary stenosis. In addition, The changes of lipoprotein-associated phospholipase A2  method, CSF was defined as TFC > 27. Serum Lp-PLA2 levels were higher in the CSF group than in the control group  and were significantly correlated with the mean coronary artery thrombolysis in myocardial infarction (TIMI) frame count . Logistic regression analysis showed that high Lp-PLA2 was independently associated with CSF after adjustment for conventional risk factors . Male sex  and hypertension  were also CSF risk factors. Receiver-operating characteristic curve (ROC) analysis showed that Lp-PLA2 levels can predict CSF severity; the predictive power was higher than the other risk factors.Lp-PLA2 than normal controls. After adjustment for potential confounders, increased Lp-PLA2 was independently associated with presence of CSF.Our study demonstrated that patients with CSF had higher circulating levels of Lp-PLA The coronary slow flow (CSF) phenomenon is a special phenomenon involving coronary microcirculation dysfunction, which is characterized by delayed distal vascular opacification without significant epicardial coronary stenosis. Studies have shown that slow blood flow is often associated with adverse cardiovascular events, including angina, myocardial infarction, malignant arrhythmias and even sudden cardiac death, suggesting a poor prognosis . Althoug2), a leukocyte-derived enzyme, is involved in the metabolism of low-density lipoprotein (LDL), propagating atherogenesis, and mediating the inflammatory process of the vascular wall [2 circulates in plasma in its active form with complexes of LDL and high density lipoprotein (HDL) [2 plays an important role in the pathophysiology of atherosclerosis and as a predictive biomarker for predicting future cardiovascular events [2 has been found to be closely associated with inflammation and atherosclerosis, we speculate that Lp-PLA2 may be associated with CSF [Lipoprotein-associated phospholipase A2  . Recent r events . Accordiwith CSF . TherefoThe retrospective study was carried out from January 2017 to September 2019 at Anzhen Hospital. A total of 170 patients who underwent coronary angiography with clinical chest pain and without significant coronary stenosis were consecutively enrolled in the observational study; 78 of the patients had slow coronary flow without any stenosis evidenced by coronary angiography, and 92 patients had normal coronary arteries and normal flow. Patients with prior evidence of coronary artery disease , coronary ectasia, coronary calcification), congenital heart disease, valvular heart disease, cardiomyopathy, ischemic electrocardiogram, hematological system disease, tumors, heart failure, liver dysfunction, or kidney dysfunction or who had recently undergone surgery were excluded from the study. The study was approved by the Beijing An zhen Hospital Ethics Committee of Capital Medical University, and all patients provided informed consent.2 activity was measured using an enzyme-linked immunoassay . Glucose, urea, creatinine, total cholesterol (TC), triglyceride (TG), HDL, and LDL levels were measured using a chemiluminescence method with a Roche Diagnostics Cobas analyzer Cobas 8000, c702 module. High sensitivity C-reactive protein (hs-CRP) levels were also measured by the nephelometric method. All samples were tested in triplicate according to the manufacturer’s protocols.The study measured demographic characteristics, including age, sex, height, weight, body mass index (BMI) and waist circumference. Smoking, hypertension, diabetes mellitus and medication use were collected from electronic medical records. After fasting for at least 8 h, peripheral blood was collected on the morning of the first day of admission and stored at − 70 °C. The plasma Lp-PLA. The diagnostic criteria of diabetes mellitus was according to WHO guidelines or by indication for insulin or anti-diabetic medications [ACS was diagnosed according to European Society of Cardiology guidelines in 2015 . Cardiomications .CSF was defined according to the thrombolysis in myocardial infarction (TIMI) frame count (TFC) method. Coronary angiogram was performed using a digital subtraction angiography system , which used the standard Judkins technique to obtain images at the rate of 30 frames/s. The injection rate was 4–5 ml/s in the left coronary artery and 3–4 ml/s in the right coronary artery (RCA). The left coronary artery was injected with 8-10 ml contrast agent and RCA was injected with 6-8 ml contrast agent. The left anterior descending coronary artery (LAD) was imaged as the right anterior oblique projection with an angle of 20–25°, the left circumflex coronary artery (LCX) was imaged as the right anterior oblique projection with an angle of 20–25°, and the RCA was imaged as the positive projection with an angle of 30°. The first frame was defined when the contrast agent touched two medial walls of the coronary artery and advanced steadily with a diameter of more than 70%, and the last frame was defined when the leading edge of the contrast agent reached the end of the branches of the coronary artery. The frame counts in the LAD were divided by a factor of 1.7 to correct for its longer length. According to Gibson et al., any frame count over 27 is considered abnormal and indicates significant CSF . All pat2 and other risk factors. We used multivariate logistic regression analysis to assess the value of Lp-PLA2 in predicting the presence of CSF with adjustment for risk factors. Additionally, a receiver operating characteristic (ROC) curve was plotted for the plasma Lp-PLA2 level to evaluate the ability of the variable to classify the severity of CSF. The ROC curve were calculated in an area under the curve (AUC) and 95% confidence intervals. A P value< 0.05 (two tailed) was considered significant.Data were analyzed with IBM SPSS software version 23.0. Categorical variables were presented as frequencies and percentages, and continuous variables were expressed as the mean and standard deviation. Continuous variables with a normal distribution were compared using Student’s t-test. Kruskal-Wallis test and one-way ANOVA were used to analyze the differences between groups. The categorical variables were tested by chi-square test. Spearman ρ test correlation analysis was used to examine the correlation between Lp-PLAP = 0.018). However, the other demographic factors, such as age, BMI, waist circumference, prevalence of hypertension, prevalence of diabetes and current smoking, did not differ significantly between the CSF and NCF groups. Except for TGs, TC, and high-density lipoprotein cholesterol (HDL-c), the two groups showed no significant differences in most laboratory tests. The levels of HDL-C and TC were lower in the CSF group than in the NCF group , and the TG level was significantly higher in the CSF group .The baseline clinical characteristics of the study participants are shown in Table 2 levels in the CSF group were significantly higher than those in the control group  , and negatively correlated with HDL-C  and ejection fraction  , male sex  and hypertension  were risk factors for CSF. After adjustment for traditional confounders, we found that the Lp-PLA2 levels remained to be significantly and independently associated with the presence of CSF   was better in predicting CFS than TC , TG , male sexand hypertension . The sum of the sensitivity and specificity for the prediction of the extent of CSF was maximal at a level of Lp-PLA2 ≥ 260.5 ng/ml .The ROC curve analysis Fig.  showed t2 and CSF phenomenon. The major finding was that Lp-PLA2 levels were positively correlated with CSF and significantly elevated in subjects with CSF. In addition, multivariate logistic regression analysis and ROC curves showed that Lp-PLA2 was still independently associated with the presence and severity of CSF. Male sex and hypertension also independently predicted the presence of the CSF phenomenon.As far as we know, the present study demonstrated for the first time that there was an independent relationship between Lp-PLAAlthough CSF has been found to be related to microvascular diseases, vascular endothelial dysfunction, coronary atherosclerosis, oxidative stress, insulin resistance, adipocytokines, and abnormal blood composition, the exact mechanisms are still unknown –20. Tamb2 was shown to be an independent predictor of CSF. Lp-PLA2, known as platelet-activating factor acetylhydrolase, is a calcium-independent serine lipase [2 is synthesized by macrophages and other inflammatory cells and circulates in the human blood mainly with LDL granules (80–85%) and less often with HDL [2 is involved in the oxidative modification of vascular wall LDL to generate oxidized phospholipids and oxidized non-esterified fatty acids, which can promote the development of vascular inflammation and atherosclerotic plaques [2 has been identified as an independent predictor of cardiovascular disease (CVD),and has been recommended as an adjunct to traditional risk assessment in patients with moderate and high 10-year risk of CVD as defined by Framingham risk scores [2 activity was associated with coronary heart disease (CHD) risk over 5.3 years based on Cox proportional hazards regression [2. Additionally multivariate logistic regression analysis showed that Lp-PLA2 could independently predict CSF. However, the mechanisms underlying the relationship between Lp-PLA2 and CSF are not entirely understood. The most reliable hypothesis seems to be that CSF is caused by increased microvascular resistance induced by endothelial dysfunction due to Lp-PLA2 mediated chronic inflammation.The role of chronic inflammation-mediated endothelial injury in CSF has been widely recognized. Muhammed Oylumlu et al. revealed that the platelet-to-lymphocyte ratio could be an important risk factor in patients with CSF . Li et ae lipase . Lp-PLA2with HDL . Lp-PLA2 plaques . Lp-PLA2k scores . In the gression . Our stu2, male gender,c and hypertension are independent risk factor of the presence of CSF phenomena. Tsimikas found in his study that there was a significant correlation between Lp-PLA2 and LDL, non-HDL, and HDL [Numerous studies have attempted to define the demographics, characteristics and independent predictors of patients with CSF. Arbey Y found that current smoking was associated with CSF . Other s and HDL . However2 and CSF. (4)The use of drugs, such as lipid-lowering drugs, may affect the results of experiments. (5)Finally, we determined only the mass of Lp-PLA2 instead of its activity due to our laboratory limitations.Our study has some limitations: (1)This study was limited by a small and nonrandomized sample, which may restrict the generalizability of our results. (2)The study did not examine the ethnic background or socioeconomic status of the study population.(3)This study was also a single center, retrospective study. Multicenter randomized controlled trials must be conducted to explore the relationship between Lp-PLA2 levels were positively correlated with CSF. The multivariate regression results showed that Lp-PLA2 was independently associated with the presence and severity of CSF. Currently, there is still no effective method to treat CSF. This study indicated the association between Lp-PLA2 and CSF, which may provide a new therapeutic target for the treatment of CSF. However, the exact pathophysiological mechanisms of Lp-PLA2 in CSF require further study to elucidate.In summary, the main finding was that Lp-PLA


Listeria monocytogenes (L. monocytogenes) is a facultative intracellular bacterial pathogen which can invade different mammalian cells and reach to the central nervous system (CNS), leading to meningoencephalitis and brain abscesses. In the diagnosis of L. monocytogenes meningoencephalitis (LMM), the traditional test often reports negative owing to the antibiotic treatment or a low number of bacteria in the cerebrospinal fluid. To date, timely diagnosis and accurate treatment remains a challenge for patients with listeria infections.We present the case of a 66-year-old woman whose clinical manifestations were suspected as tuberculous meningoencephalitis, but the case was finally properly diagnosed as LMM by next-generation sequencing (NGS). The patient was successfully treated using a combined antibacterial therapy, comprising ampicillin and trimethoprim-sulfamethoxazole.L. monocytogenes. Hence, the clinical utility of this approach can be very helpful since it provides quickly and trust results.To improve the sensitivity of LMM diagnosis, we used NGS for the detection of  Listeria monocytogenes (L. monocytogenes) is a Gram-positive bacillus, its genetic machinery allows bacterial life as a facultative intracellular pathogen. L. monocytogenes widely exists in the environment at high salt concentrations and fairly low moisture content [ content . It is w content . The conL. monocytogenes in the cerebrospinal fluid (CSF) [Invasive forms of listeriosis can lead to infection of CNS, in these cases even though the adequate antibiotic treatment the overall mortality rates remains nearly 30% . Due to id (CSF) . Therefoid (CSF) . Unlike To date, only a few studies have evaluated the use of NGS for the clinical diagnosis of LMM. Herein, We report the use of NGS as a helpful diagnostic method, which enabled the identification of LMM in this case, leading to proper treatment and fully recovery of the patient. To our knowledge, this is the first reported case in which the patient was initially misdiagnosed as tuberculous meningoencephalitis and finally diagnosed as LMM by NGS.2O, with 580 × 106/L white cells . The protein level was significantly elevated (978 mg/L), while the CSF glucose level  and chloride level (110.8 mmol/L) were decreased. Gram stain, acid-fast stain, and ink stain of the CSF were negative. Additionally, CSF and blood cultures were negative. Laboratory examination suggested hyponatremia (129 mmol/L). Immunological screenings, including an evaluation of humoral immunity (immunoglobulin levels and subclass IgG), cellular immunity , and complement (C3 and C4), were normal. The tumor-related screening was normal. Tests for human immunodeficiency virus (HIV) and infectious hepatitis were negative. Brain computed tomography (CT) and magnetic resonance imaging (MRI) indicated no obvious abnormalities. Therefore, the patient was diagnosed with tuberculous meningoencephalitis. Following the administration of anti-tubercular therapy (ATT), the headache and fever were relieved. On reexamination, LP improved but remained abnormal. Three days later, the NGS of CSF was suggestive of Listeria, and we immediately amended the therapeutic regimen to ampicillin plus trimethoprim-sulfamethoxazole (TMP-SMX). After 4 weeks, the headache, and fever resolved, and the LP results were completely normal.A 66-year-old formerly healthy woman was admitted to the hospital with a 4-day history of headache, fever, nausea, and vomiting, followed by changes in consciousness. On admission, she presented with neck stiffness, positive Kernig sign, and a Glasgow Coma Scale (GCS) score of 12. A lumbar puncture (LP) was performed: the CSF was canary yellow, the opening pressure was 250 mmHThe patient originated from China and lived in Changde, Hunan province, since birth. She was a housewife without recent travel experience and denied eating raw or semi-raw food, or unpasteurized dairy products. She had no history of autoimmune disease and did not use tobacco or illicit drugs.Approximately 2–3 mL of CSF was collected and sealed using a sterile technique, shipped on dry ice to Hugobiotech Co., Ltd.  to perform NGS detection. The DNA was extracted and purified from 200 μL of CSF supernatant according to the manufacturer’s instructions for the TIANGEN DNA Mini kit DP316 . The DNA libraries were constructed via end-repaired adaptation and application of polymerase chain reaction (PCR). The concentration and quality of the libraries were evaluated using Qubit 1*dsDNA HS Assay Kit  and agarose gel electrophoresis . Qualified libraries with different barcode labeling were pooled together, and then sequenced on an Illumina NexSeq platform .L. monocytogenes genome. After excluded the reads from human host, L. monocytogenes reads show dominant abundance in all microbial species, with a percentage of 81.27%  using Scalable Nucleotide Alignment Program software . The remaining data were aligned to the microbial genome database using the Burrows-Wheeler Alignment . The database collected microbial genomes from the National Center for Biotechnology Information, and contained more than 20,000 microorganisms, including 11,910 bacteria, 7103 viruses, 1046 fungi, and 305 parasites. The alignment results were used to calculate the coverage and depth of each species calculated. The number of sequencing reads was 5,942,851. Of these, 486 reads uniquely aligned to the Listeria demonstrated younger demographic [China is one of the greatest contributors to the global tuberculosis burden, with a high prevalence rate of smear-positive tuberculosis, approximating 59/100,000 , 8. Accoographic . The cliographic . Previouographic . The CSFThe timely identification of pathogens and the initiation of appropriate antibiotic treatment is the key for a favorable prognosis. Methods for pathogen discovery, such as bacterial culture, have existed for years and remain the gold standard for the diagnosis of listeria infections, however, these methods are time-consuming, cumbersome and lack sensitivity. In addition, the positive rate of CSF culture is low (around 40%) . MoleculL. monocytogenes, which could explain the improvements observed in the patient’s headache and CSF results during ATT [The combination of ampicillin and TMP-SMX has previously been described as an effective treatment for CNS listeriosis , 22. A rring ATT , 24. Furring ATT , 26. Furring ATT .A Danish cohort study reported that patients with listeria encephalitis presented a significantly higher mortality rate than the general population within 5 years of diagnosis, primarily due to cancer . To imprDue to the non-specific CSF findings and low positivity rate of culture, the early diagnosis of LMM remains a clinical challenge. This case highlights the feasibility of using NGS as a early diagnostic assay for CNS listeriosis, and proposes that in patients with clinically suspected tuberculous meningoencephalitis, the possibility of Listeria infection needs to be considered.