Mixed communities of fungi and bacteria have been shown to be more efficient in degrading wood than fungi alone. Some standardised protocols for quantification of the wood decay ability of fungi have been developed . Here, we describe a step-by-step protocol developed from the official standard DIN V ENV12038 to test combinations of bacteria and fungi for their combined wood degradation ability. Equally sized wood blocks are inoculated with wood decay fungi and bacterial strains. Axenic controls allow the analysis of varying degradation rates via comparison of the wood dry weights at the end of the experiments. This protocol provides new opportunities in exploration of inter- and intra-kingdom interactions in the wood-related environment and forms the basis for microcosm experiments.Key features• Quantification of wood decay ability of mixed cultures.• Allows testing if fungi are more efficient in degrading wood when bacteria are present. In nature, basidiomycetous fungi are associated with other microbes including prokaryotes and eukaryotes. This results in complex competitive and antagonistic interactions as well as commensal and mutualistic behaviour , article in prep.].To date, several protocols and studies exist for the assessment of wood resistance against wood-destroying basidiomycetes Bacterial strains to test 1% agarose gel for gel electrophoresis system Polyethylene glycol 6000 (PEG) 20% 80% EtOH (-20 °C)Nuclease-free waterTryptone soya agar (TSA) plates (see Recipes)TSA soft agar  (see Recipes)Liquid Tryptone soya (TS) medium (see Recipes)Malt extract agar (MEA) (see Recipes)Modified Melin-Norkrans (MMN) (see Recipes)Hutner’s Trace metals (see Recipes)0.85% NaCl solution (see Recipes)Sodium borate buffer  RecipesTryptone soya agar (TSA) platesCasein peptone 1.5% (w/v), 15 gSoy peptone 0.5% (w/v), 5 gNaCl 0.5% (w/v), 5 gAgar 1.8% (w/v), 18 g2O, add up to 1 LdH(pH = 7.3 ± 0.2)Prepare TSA medium, autoclave (120 °C), let it cool down, pour into Petri dishes, and let cool to room temperature (RT). Store at 4 °C.TSA soft agarCasein peptone 1.5% (w/v), 15 gSoy peptone 0.5% (w/v), 5 gNaCl 0.5% (w/v), 5 gAgar 0.7% (w/v), 7 g2O, add up to 1 LdH(pH = 7.3 ± 0.2)Prepare TSA medium and autoclave (120 °C). Store at ~50 °C until usage for plate casting after Koch.Liquid Tryptone soya TS mediumCasein peptone 1.5% (w/v), 15 gSoy peptone 0.5% (w/v), 5 gNaCl 0.5% (w/v), 5 g2O, add up to 1 LdH(pH = 7.3 ± 0.2)Prepare TS liquid medium, autoclave (120 °C), and let it cool down. Store at 4 °C.Malt extract agar (MEA)Malt extract 3% (w/v), 30 gSoy peptone 0.3% (w/v), 3 gAgar 1.8% (w/v), 18 g2O, add up to 1 LdH(pH = 3.5 ± 0.2)Prepare MEA medium, autoclave (120 °C), and cool it down to 50 °C. Pour into Petri dishes and let cool to RT. Store at 4 °C.Modified Melin-Norkrans (MMN) [after Tauber et al. (2016)]Glucose, 5 g4)2HPO4, 0.25 g, 1.2 mLFeClThiamine-HCl (10%), 1 μLHutner’s trace metals (0.01×), 100 μLAgar, 20 g2O, add up to 1 LdH(pH = 5.6)3, Thiamine-HCl (10%), and Hutner’s trace metals (0.01×) (Prepare FeCl (0.01×)  separateHutner’s trace metals2OEDTA disodium salt, 50 g in 250 mL dH4·7H2O, 22 g in 100 mLZnSO3BO3, 11.4 g in 200 mLH2·4H2O, 5.06 g in 50 mLMnCl2·6H2O, 1.61 g in 50 mLCoCl4·5H2O, 1.57 g in 50 mLCuSO4)6Mo7O24·4H2O, 1.10 g in 50 mL4·7H2O should be prepared last to avoid oxidation.For 1 L final mix, dissolve each component in the volume of water indicated. The EDTA should be dissolved in boiling water, and the FeSOMix all solutions except EDTA. Bring to boil, then add the EDTA solution. The colour of the mixture turns to green. When everything is dissolved, let cool to 70 °C. While keeping the temperature at 70 °C, add 85 mL of hot KOH (20%). Cool to RT and fill up to 1 L final volume. Close the flask with a cotton plug  and swirl it once a day while incubating for 1–2 weeks. Usually, the solution will initially be clear green but turns dark red or purple over the next few days, leaving a rust-brown precipitate. If no precipitate forms or the solution remains green, check the pH .Filter through two layers of Whatman #1 filter paper and repeat, if necessary, until the solution is clear. Store refrigerated or frozen in convenient aliquots.0.85% NaCl SolutionSodium chloride 0.85% (w/v), 8.5 g2O, add up to 1 LdHPrepare solution, autoclave (120 °C), and let cool down. Store at RT.Sodium borate buffer Boric acid, 48 g (concentration 1 M)NaOH pellets, 8 g2O, add up to 1 LdH2O in a suitable container and add the boric acid and the sodium hydroxide to the solution. Stir until all solids are dissolved. Adjust pH to 8 by adding boric acid or NaOH. Fill to 1 L with dH2O.Prepare 900 mL of dHDilute the 20× stock solution to 1× before usage for gel electrophoresis (can be used for preparation for agarose gels and in the gel tank).Eppendorf centrifuge 5810 equipped with A-4-81 rotor TM 17 microcentrifuge equipped with 24 × 1.5/2.0 mL rotor with ClickSealTM biocontainment lid Heraeus Fresco2, Assistent, Glaswarenfabrik Karl Hecht, Sondheim vor der Rhön, Germany)Counting chamber Thoma Incubator ® 40/40R)Benchtop orbital incubator shaker TM, Microfil®, Merck Millipore Billerica, USA)Vacuum filter manifold and sterile filtration funnels Overhead shaker model REAX2 Heating oven, 3.8 cu ft, 230 VAC ®, Mettler-Toledo, Vienna, Austria)Balance Carton package (to guarantee incubation in darkness)®, Carl Roth, Karlsruhe, Germany)Sterile glass Petri dishes TweezersPCR cycler TM Electrophoresis Cell, EmBi Tech, San Diego, USA)Horizontal gel electrophoresis system Gel Doc® comfort 5355 ThermomixerMicrosoft Excel version 2209RStudio version 4.0.3 (2020-10-10)Pre-experiments to determine bacterial viability on wood blocksGrow bacterial isolates in 5 mL of TS liquid medium overnight .g for 10 min).Harvest bacteria and wash them with 1 mL of 0.85% NaCl solution .Use a Thoma chamber to adjust the bacterial suspension to 10Immerse the autoclaved wood blocks with bacterial suspension and incubate them in a sterile glass Petri dish overnight at 25 °C.The next day, imprint the wood blocks gently on TSA plates, using a sterilised tweezer. Remove the wood blocks. Incubate at 25 °C overnight to 24 h.If bacteria grow rapidly on TSA plates, they are useable for the main experiment.Preparation of the main experimentS. lacrymans was pre-cultivated on modified Melin-Norkrans (MMN) for 3–4 weeks at 25 °C).Establish fungus on solid agar medium  to vessels filled with 25 mL of MEA medium.Note: Use the peripheral zone of the pre-culture(s). Set aside some vessels, as they serve as control .Incubate in darkness for four weeks (25 °C).In the meantime:Picea abies).Number the wood blocks  and note their weights . Keep them in a sterile environment until use.Put a minimum of four moisture control specimen wood blocks in the oven and dry them at ~50 °C for 72 h.Weigh and record the dry mass of the moisture control wood blocks.Number and weigh glass Petri dishes.Setup of the main experimentStart to prepare bacteria approximately 2–3 days before fungal cultures are ready. Culture them on TSA medium (25 °C).Inoculate 5 mL of liquid TS medium with bacteria .g for 10 min)Harvest bacteria and wash them with 0.85% NaCl solution .Use a Thoma chamber to adjust the bacterial suspension to 10Immerse the numbered, autoclaved, and weighed wood blocks with the bacterial suspension, remove them immediately, drain the excess suspension (sterilised filter paper) and put one (immersed) wood block in each vessel with fungal mycelium. Prepare a minimum of five replicates.Note: Set aside some vessels as they serve as control.For each approach, put wood blocks in vessels filled with MEA but without mycelium, as these are additional controls  (minimum triplicates per bacterium).Treat a minimum of 14 wood blocks solely with 0.85% NaCl solution and place them on top of the mycelium .Incubate the vessels at 25 °C in darkness  for eight weeks .HarvestingRemove superficial mycelium from the wood blocks with sterilised tweezers . CollectImprint the wood blocks on TSA plates and incubate them at 25 °C.Put each wood in one numbered and pre-weighed glass Petri dish; the numeration should match.Weigh the wood blocks + Petri dish.Dry the wood blocks at approximately 50 °C.Weigh the mycelium collected from the wood.Dilute the mycelium sample 1:10 with NaCl solution (0.85%).Shake the mycelium solution for 30 min on a rotary shaker.Plate 100 μL of the solution on TSA agar plates and incubate at 25 °C.Use the residual solution.For plate casting after Koch (if less than 1 mL):i. Mix residual solution with 3 mL of TSA soft agar.ii. Vortex gently.iii. Pour evenly on TSA plates.For concentration on a nitrocellulose filter with vacuum (if more than 1 mL):i. Clean the filter unit with 96% EtOH and sterilise the tweezers.ii. Install the sterile filtration funnel and the filter.iii. Pour the solution on the filter.iv. Apply vacuum.v. Put the filter on TSA medium.Incubate all agar plates at 25 °C and check daily for growth .EvaluationWeigh dried wood blocks.The wood blocks can stay in the glass Petri dish, as the weight was determined at the start of the experiment.Weigh them daily until they are dry (should take no longer than 2–3 days).Check TSA plates for growth, documenting on which plates growth occurs.Use single bacteria colonies for establishment of pure cultures .Perform colony PCR and check if bacteria match with applied species by sequencing the 16S rRNA gene .i. Prepare PCR reaction: mix 12.5 μL of Red Taq DNA Polymerase Master Mix (2×) with 0.625 mL of each of the primers 27F and 1492R (10 μmol), 10.75 μL of distilled water, and 0.5 μL of BSA (2%) .ii. Pick a single bacterial colony not older than three days with a sterile toothpick and add it to the PCR mixture.iii. Transfer the reactions to the PCR cycler: PCR conditions are 95 °C for 10 min, 30 cycles of 95 °C for 30 s, 53 °C for 30 s, 72 °C for 45 s, and a final elongation step at 72 °C for 10 min.iv. Check the PCR products by agarose gel electrophoresis  to confirm their correct size .v. Purify the PCR reaction(s) (PEG protocol by Travis Glenn).1) Transfer 18 μL of the respective PCR reaction(s) into a 1.5 mL tube and mix with 16 μL of PEG (20%) by constantly pipetting the solution up and down.2) Put it on the thermo shaker  for 15 min.g for 15 min at RT (21 °C).3) Centrifuge at 15,000× 4) Discharge the supernatant .5) Wash the pellet with 50 μL of ice-cold 80% ethanol (-20 °C).g for 2 min at RT (21 °C).6) Centrifuge at 15,000× 7) Remove the supernatant.8) Repeat the centrifugation step and remove the supernatant completely.9) Incubate the tubes on the thermo shaker (37 °C and 800 rpm) with lid open until no trace (visible drops as well as smell) of ethanol is left.10) Add 18 μL of nuclease-free water.11) Pipette the nuclease-free water up and down several times (this ensures that the DNA is fully resuspended in the water).vi. Check the purified PCR products again with agarose gel electrophoresis  .vii. Send for sequencing .Wood decay rates0), dry four moisture control specimen wood blocks at ~50 °C for 72 h after autoclaving and weighing (point B5). This can be done in parallel to the assay. Our specimen lost on average 0.6905 g (≙8.62%), hence 91.38% of the mass remained unchanged. Calculate dry m0 as: dry m0 = m d0 × 0.9138, with m d0 being the initial weight of the wood block after autoclaving (before exposure to microbes). Calculate mass loss (ML) difference as: mT (g) = (dry m0 - m1), where mT is the difference of ML after exposure to microorganisms, and m1 and dry m0 are the dry masses after and before degradation, respectively. The ML in % is the measure for the extent of fungal degradation [mT (%) = (dry m0 ÷ m1) × 100]. Prepare a minimum of five replicates for each approach and of three replicates per control approach. Calculate average wood weight loss in % and standard deviation for each approach and illustrate e.g., in a boxplot. Conduct statistical analysis with pairwise.t.test function for pairwise comparison using t-tests with pooled SD  in the psych package v2.2.5 of the R programming language (RStudio). Additionally, calculate single-factor variance analysis .To ensure that the initial moisture content was uniform throughout all wood blocks and to calculate the estimated dry weight on day zero  and optimise for highly similar sequences (megablast). According to the results of the Blast search, assign the bacteria to genera and compare with the bacterial strains applied at the beginning. This allows to estimate if the bacteria survived the procedure.Check sequencing results for quality by evaluating the corresponding chromatograms. If 16S rRNA gene sequences of the applied microbes are available for comparison, compare initial sequences with the sequences of the re-isolated strains by aligning them. Alternatively, blast sequences of the 16S rRNA gene with the Note: Some mould spores may have survived and developed; we recommend recording affected plates/tins. We excluded these specimens from the calculations.Prepare enough tins with pre-cultured fungus, as contaminations occur easily since some steps cannot be performed entirely sterile. Before starting the experiment, we recommend careful planning and listing of what should be prepared.Mind the controls:S. lacrymans with wood that is immersed with 0.85% NaCl solution.Axenic fungal control: Wood sterile control: wood that is immersed with 0.85% NaCl solution, without microbes.8 CFU/mL without fungus.Axenic bacterial control: wood that is immersed with bacterial suspension that was adjusted to 100).Minimum four moisture control specimen wood blocks (without them it is not possible to estimate the initial dry weight dry mIt is also possible to sterilise the wood blocks via UV light, as this would preserve the initial state of the wood better than autoclaving. The disadvantage is that more moulds and other contaminants survive this procedure, which might cause problems in the experiment.


Klebsiella pneumoniae harboring rmpA and molecular characteristics of the bacteria. We retrospectively investigated adult patients with K. pneumoniae BSI from January 2010 to March 2021 at Nagasaki University Hospital. A matched case–control study in a 1:3 ratio was conducted to clarify the clinical and bacterial characteristics of BSI caused by rmpA-positive K. pneumoniae compared with those caused by rmpA-negative isolates. Antimicrobial susceptibility testing and multilocus sequence typing (MLST) were performed for rmpA-positive isolates. The rmpA was detected in 36 (13.4%) of the 268 isolates. Of these 36 isolates, 31 (86.1%) harbored iucA and 35 (97.2%) each possessed peg-344 and iroB; capsular types were identified as K1 in 9 (25.0%) and K2 in 10 isolates (27.8%). Contrarily, of the 108 rmpA-negative isolates, which were matched for case–control studies, 5 isolates (4.6%) harbored iucA and 1 (0.9%) each possessed peg-344 and iroB; 2 (1.9%) and 3 isolates (2.8%) had K1 and K2 capsular types, respectively. Among the rmpA-positive isolates, ST23/K1 (eight isolates) was the most frequent, followed by ST412/non-K1/K2 (seven isolates), ST86/K2 (five isolates), and ST268/non-K1/K2 (four isolates). In a multivariate analysis using clinical factors, liver abscess positively correlated with rmpA-positive isolates, whereas biliary tract infection and use of anticancer drugs negatively correlated with rmpA-positive isolates in patients with K. pneumoniae BSI. Considering the correlation between rmpA-positive isolates and clinical features, rmpA can be used as a marker for understanding the pathophysiology of K. pneumoniae BSI.We investigated the clinical features of bloodstream infections (BSIs) caused by  Klebsiella pneumoniae causes various infections, such as pneumonia, urinary tract infections, and bacteremia, commonly in hosts with comorbidities2. However, in recent decades, there have been several reports, primarily from Taiwan, of cases of community-acquired bloodstream infections (BSIs) with liver abscess caused by hypervirulent K. pneumoniae (hvKp) in healthy individuals3, and the spread of the bacterium is a major concern worldwide. Unlike classical K. pneumoniae, hvKp causes infections, including meningitis, necrotizing fasciitis, endophthalmitis, and metastatic infections in multiple organs4.Classical K. pneumoniae  have been reported5. Capsules protect the bacterial cells from phagocytosis and antimicrobial peptides and suppress host immunological responses9. HvKp produces a hypercapsule, which consists of a mucoviscous extracellular polysaccharide; it envelopes the bacterial surface more robustly than a typical capsule. Specific capsular types such as K1 and K2 are associated with increased hvKp pathogenicity5.Four major virulence factors of rmpA) is a transcriptional regulator and enhances capsular polysaccharide synthesis and capsule production11. Previous studies have shown that the deletion of rmpA reduces colony mucoviscosity11, virulence in mice, and resistance to human serum10. Additionally, plasmid-borne rmpA has been found to be an accurate marker of hvKp with high sensitivity (0.98) and specificity (0.93)4. Therefore, in this study, we focused on K. pneumoniae harboring plasmid-borne rmpA and investigated the molecular epidemiology and clinical features of BSI caused by the bacterium in our university hospital located in western Japan.Plasmid-borne regulator of mucoid phenotype A  and sex-matched patients to each case were listed among patients from whom rmpA-negative K. pneumoniae was isolated, and three patients per case were randomly selected as controls using Microsoft Excel (Microsoft Corporation). The clinical characteristics of the patients with BSI caused by K. pneumoniae were compared between the rmpA-positive and rmpA-negative groups. We evaluated the infection sites with bloodstream infection, such as pneumonia, biliary tract infection, and urinary tract infection, from which K. pneumoniae was isolated from each site. Other infection sites, including liver abscess, endophthalmitis, meningitis, and purulent spondylitis, were evaluated regardless of K. pneumoniae isolation from each site. The severity of BSI was assessed using the Pitt bacteremia score14. The study was performed in accordance with tenets of the Declaration of Helsinki and the Ethical Guidelines for Medical and Biological Research Involving Human Subjects. The study protocol including the waiver of consent was approved by the Institutional Review Board of Nagasaki University Hospital .We retrospectively investigated 15. Bacterial DNA was extracted using the boiling method previously described12, with minor modifications. Three to five colonies were mixed with 100 µL Tris–EDTA buffer containing 250 U/mL achromopeptidase . After incubation at 40 °C for 15 min, 250 µL of 10% Chelex 100 Resin (Bio-Rad) was added, and the mixture was boiled at 99 °C for 5 min, cooled on ice for 1 min, and centrifuged at 12,000 rpm for 1 min. The supernatant was used for the subsequent analyses.Hypermucoviscosity was assessed using the string test, which was considered positive if the viscous string was greater than 5 mm in length when the colony was stretched using a loop on an agar platermpA, iucA, peg-344, and iroB, which have been reported to be accurate makers of hvKp4, as well as capsular types, including K1 (magA), K2, and K5, were evaluated using PCR. The PCR primers used were as follows: rmpA forward, 5′-ACTGGGCTACCTCTGCTTCA-3′; rmpA reverse, 5′-CTTGCATGAGCCATCTTTCA-3′17; iucA forward, 5′-AATCAATGGCTATTCCCGCTG-3′; iucA reverse, 5′-CGCTTCACTTCTTTCACTGACAGG-3′18; K1 (magA) forward, 5′-GGTGCTCTTTACATCATTGC-3′; K1 (magA) reverse, 5′-GCAATGGCCATTTGCGTTAG-3′15; K2 forward, 5′-GACCCGATATTCATACTTGACAGAG-3′; K2 reverse, 5′-CCTGAAGTAAAATCGTAAATAGATGGC-3′19. peg-344 forward, 5′-CTTGAAACTATCCCTCCAGTC-3′; peg-344 reverse, 5′-CCAGCGAAAGAATAACCCC-3′4; iroB forward, 5′-ATCTCATCATCTACCCTCCGCTC-3′; iroB reverse, 5′-GGTTCGCCGTCGTTTTCAA-3′4; K5 forward, 5′-TGGTAGTGATGCTCGCGA-3′; K5 reverse, 5′-CCTGAACCCACCCCAATC-3′19.In this study, plasmid-borne rmpA, 50 °C for iucA, K1 (magA), and K2], and 1 min at 72 °C, and 7 min at 72 °C for the final extension; 10 min at 95 °C, 35 cycles of 30 s at 95 °C, 30 s at the annealing temperature (53 °C for peg-344 and 59 °C for iroB), and 40 s for peg-344 and 30 s for iroB at 72 °C, and 7 min at 72 °C for the final extension; for K5, 1 min at 94 °C, 30 cycles of 30 s at 94 °C, 45 s at 59 °C, and 90 s at 72 °C, and 6 min at 72 °C for the final extension.DNA was amplified under the following conditions: 5 min at 94 °C, 35 cycles of 30 s at 94 °C, 30 s at the annealing temperature [46 °C for Antimicrobial susceptibility was examined using BD Phoenix M50 (Becton Dickinson), according to the manufacturer’s instructions, and determined according to the Clinical and Laboratory Standards Institute (CLSI) M100-Ed33.rmpA-positive isolates, based on the sequences of seven housekeeping genes . The primers used have been described in the Klebsiella pneumoniae MLST database (https://bigsdb.pasteur.fr/klebsiella/primers-used/). Direct sequencing was performed as follows. DNA was amplified using primers for each housekeeping gene under the following conditions: 2 min at 94 °C, 35 cycles of 30 s at 94 °C, 1 min at 50 °C, and 30 s at 72 °C, and 5 min at 72 °C for the final extension. The products were purified using a QIA quick PCR purification kit (QIAGEN) or ExoSAP-IT (Applied Biosystems). Fluorescence-based cycle sequencing reactions were performed using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). After purification using the BigDye Xterminator Purification Kit (Applied Biosystems), the products were analyzed using the SeqStudio Genetic Analyzer (Applied Biosystems). Allele sequences and STs were determined according to the Klebsiella pneumoniae MLST database (https://bigsdb.pasteur.fr/klebsiella/).Multilocus sequence typing (MLST) was carried out for Numerical variables are expressed as median (interquartile range) and compared using Wilcoxon rank-sum test between groups. Categorical variables were compared using Fisher’s exact test. In the multivariate analysis, variables with P < 0.2 in the univariate analysis were selected and adjusted using the conditional logistic regression model. Data were analyzed using JMP v16 (SAS Institute Inc.), and results with P < 0.05 were considered statistically significant.K. pneumoniae isolated from the blood of individual patients, 268 were available. Of these 268 isolates, rmpA was detected in 36 isolates (13.4%). Of the remaining 232 isolates without rmpA, 108 were matched as rmpA-negative controls based on the age (± 5 years) and sex of the patients  harbored iucA and 35 (97.2%) each possessed peg-344 and iroB. Capsular types were identified as K1 in 9 (25.0%), K2 in 10 (27.8%), and K5 in 1 (2.8%) isolates, respectively. Among the 108 rmpA-negative isolates, 5 (4.6%) harbored iucA and 1 (0.9%) each possessed peg-344 and iroB; 2 (1.9%), 3 (2.8%), and 1 (0.9%) isolates had K1, K2, and K5 capsular types, respectively. Hyperviscosity was found in 30 rmpA-positive isolates (83.3%), which was higher than that in rmpA-negative isolates .Table rmpA-positive isolates. No carbapenem-resistant isolates and three (8.3%) extended-spectrum β-lactamase (ESBL) producers were identified. Two isolates (5.6%) were resistant to ciprofloxacin.Table rmpA-positive isolates. ST23/K1 (eight isolates) was the most frequent ST/capsular type, followed by ST412/non-K1/K2 (seven isolates), ST86/K2 (five isolates), and ST268/non-K1/K2 (four isolates).Table rmpA-positive K. pneumoniae, compared with those caused by rmpA-negative isolates . The use of anticancer drugs was significantly higher in the rmpA-negative group than in the rmpA-positive group. Similarly, the presence of malignancy tended to be higher in the rmpA-negative group than in the rmpA-positive group, but the difference was not significant. Other comorbidities and use of medical devices did not differ between the groups.We investigated the baseline characteristics and clinical features of BSI caused by es Table . Of the rmpA-negative group, and the rate was higher than that in the rmpA-positive group . Conversely, liver abscess was a more frequent infection in the rmpA-positive group  than in the rmpA-negative group . Disease severity assessed using the Pitt bacteremia score was similar, and the mortality rates did not show significant differences between the groups.The biliary tract was the most frequent infection site with BSI  in the rmpA-positive isolates and the clinical factors of patients with K. pneumoniae BSI. Variables with P < 0.2 in the univariate analysis , the positive rates of iucA, peg-344, and iroB were remarkably higher in the rmpA-positive group  than in the rmpA-negative group . In addition to rmpA, iucA, peg-344, and iroB have been reported to be accurate markers of hvKp4. The high detection rates of these markers in rmpA-positive isolates support that rmpA is a useful marker of hvKp. Furthermore, K1 and K2 capsular types were identified in 25.0% and 27.8% of the isolates, respectively, in the rmpA-positive group, which were clearly higher than those in the rmpA-negative group . The STs of K. pneumoniae from patients with BSI vary geographically20. Our results showed that ST23/K1 was the most prevalent (eight of 36 isolates) in rmpA-positive K. pneumoniae causing BSI, which is supported by the findings of a previous study on hvKp from Japan21. Additionally, we identified ST65/K2 and ST86/K2, similar to that in a previous study in Japan21.Our study demonstrated the molecular epidemiology of K. pneumoniae harboring rmpA. Liver abscess was recorded in 22.2% of the patients with BSI in the rmpA-positive group, three times more frequently than that in the rmpA-negative group (7.4%). The multivariate analysis showed that liver abscess significantly correlated with rmpA-positive isolates . In addition, all eight rmpA-positive K. pneumoniae isolates causing liver abscess showed hyperviscosity and carried iucA. These results are supported by a recent report that rmpA, positive string test, and aerobactin are associated with K. pneumoniae causing liver abscess in patients with community-acquired BSI22. Furthermore, ST23/K1 (three isolates) and ST65/K2 (two isolates) were identified in five (62.5%) of the eight rmpA-positive K. pneumoniae isolates that caused liver abscess in our study, which is consistent with previous reports that they are the common ST/capsular types associated with liver abscess in East Asian countries25. The remaining types that caused liver abscess were ST412/non-K1/K2 (two isolates) and ST268/non-K1/K2 (one isolate).This study showed the clinical characteristics of BSI caused by rmpA-positive isolates. Classical K. pneumoniae is known to cause bacteremia especially in immunocompromised patients2. Therefore, the use of anticancer drugs may reflect the immunocompromised condition of the host. Additionally, biliary tract was a frequent infection site in the rmpA-negative group (27.8%) compared with that in the rmpA-positive group (8.3%) in this study. A recent study reported a similar result that biliary tract infection was observed more frequently in classical K. pneumoniae BSI26.Meanwhile, the use of anticancer drugs and the presence of biliary tract infection negatively correlated with rmpA-positive isolates in this study, we could not analyze the microbiological characteristics of rmpA-negative isolates in detail.This study has a few limitations. First, as this was a retrospective study, some variables of clinical factors might not have been recorded by attending physicians. Second, the sample size was limited because this study was conducted in a single center, and some isolates were unavailable during the study period. Finally, because we focused on K. pneumoniae harboring rmpA, isolated from patients with BSI in our hospital. The presence of rmpA correlated with the clinical characteristics of K. pneumoniae BSI and can be used as a marker for understanding the pathophysiology of K. pneumoniae BSI.In conclusion, our study revealed the molecular epidemiology of Supplementary Table 1.


Aquilaria crassna (AC) is a beneficial plant widely used to alleviate various health ailments. Nevertheless, the neuroprotection, antiaging, and xenobiotic detoxification against high benzo[a]pyrene induction have not been investigated. This study aimed to investigate the effects of ethanolic extract of AC leaves (ACEE) in vitro using SH-SY5Y cells and in vivo using Caenorhabditis elegans (C. elegans). Neuroprotective activities and cell cycle progression were studied using SH-SY5Y cells. Additionally, C. elegans was used to determine longevity, health span, and transcriptional analysis. Furthermore, ACEE possible active compounds were analyzed by gas chromatograph–mass spectrometry (GC-MS) analysis and the possible active compounds were evaluated using a molecular docking study. First, ACEE possessed neuroprotective effects by normalizing cell cycle progression via the regulation of AhR/CYP1A1/cyclin D1 pathway. Next, ACEE played a role in xenobiotic detoxification in high B[a]P-induced C. elegans by the amelioration of lifespan reduction, and body length and size decrease through the reduction in gene expression in hexokinase (hxk) and CYP35 pathway. Finally, phytochemicals of ACEE were identified and we uncovered that clionasterol was the possible active constituent in powerfully inhibiting both CYP1A1 and hexokinase II receptor. Essentially, ACEE was recognized as a potential alternative medicine to defend against high B[a]P effects on neurotoxicity and xenobiotic detoxification. Aquilaria crassna Pierre ex Lecomte (AC) is known by the name of agarwood or eagle wood. It is a highly precious fragrant plant and widely used as an ingredient in the traditional Chinese and Korean medicines. Likewise, it has different pharmacological activities, including the attenuation of cigarette smoke-induced inflammation and oxidative stress in airway epithelial cells P is regarded as a dangerous environmental pollutant that can cause serious health ailments, particularly brain tissue and neuronal damage. Based on previous studies, AC possesses multipotent pharmacological properties for human health. Nonetheless, the protective effect of AC on B[a]P-induced toxicity has not been clarified. Consequently, we have been studying the protective activity and underlying mechanisms of AC extract against B[a]P-induced neuronal cell damage and xenobiotic processing in human neuroblastoma SH-SY5Y cells and nematode Aquilaria crassna Pierre ex Lecomte (AC) were collected from HRH Princess Maha Chakri Sirindhorn Herbal Garden, Rayong Province, Thailand. The plant was botanically identified and deposited at the herbarium of Kasin Suvatabhandhu, Department of Botany, Faculty of Science, Chulalongkorn University (Voucher specimen: A17634(BCU)). Plant material was extracted by the maceration method P of 0–40 µM and ACEE of 0–100 µg/mL were used to determine both the toxic concentration of B[a]P and nontoxic concentration of ACEE with SH-SY5Y cells. Then, to find out the protective dose of ACEE against B[a]P-induced cytotoxicity, the cells were cotreated with a toxic concentration (40 µM) of B[a]P and ACEE of 0–25 µg/mL for 48 h. Afterwards, 5 mg/mL of MTT reagent  was added to each well and incubated at 37 °C for 4 h to induce formazan product formation. Next, 10% SDS in 0.01 N HCl was added and the cells were incubated at 37 °C overnight. The amount of formazan is proportional to the number of alive cells [Benzo[a]pyrene or B[a]P  [Cell cycle was analyzed by the assessment of DNA content distribution. Having been treated with either B[a]P or ACEE and incubated for 48 h, fixed cells were prepared by incubating with 70% ethanol at −20 °C for 2 h. Then, the cells were washed 3 times and stained with propidium iodide containing RNase A for 15 min in the dark. The percentages of cell population were analyzed using a BD FACSCalibur flow cytometer and BD CellQuestCA, USA) .w/v) nonfat milk and 0.05% Tween-20. Western blotting was permitted to stay overnight at 4 °C with primary antibodies including AH Receptor , cyclin D1 , CYP1A1 , and β-actin . Either rabbit  or mouse IgG  horseradish peroxidase-conjugated antibodies and chemiluminescence detection reagent were used to allow the visualization of bands. These protein bands were obtained using ImageJ software version 1.53 P, or cotreatment of 40 µM B[a]P with 5 µg/mL ACEE. At the L4 stage, new NGM was prepared and L4 worms were transferred to separate them from their progeny and to avoid starvation. The number of dead and live worms was counted and recorded every day until all worms were dead .N2 synchronized L1 larvae were cultured on different NGM coated with B[a]P at 0, 100, 200, and 300 µM, and were allowed to grow into adult day 1. Later, a 10× objective lens of brightfield microscope was used to image adult day 1 worms in each group. Later, the software Motic Image Plus 3.0. was used to analyze the body length and size of the adult worms .C. elegans, L1 larvae stage worms were treated with 300 µM B[a]P and 5 µg/mL ACEE for 72 h into adult day 1 to investigate the pathway by which B[a]P stimulated toxicity and metabolism in C. elegans. Additionally, RNA from both SH-SY5Y and the worms was extracted by GENEzolTM reagent  following RNA extraction protocol procedure. Reverse transcription and the qPCR were accomplished in the recommended protocol of iTAQ universal SYBR green supermix  and CFX Real time PCR, respectively. The measured fluorescent signals revealed the PCR results. All primer sequences are shown in This assay was performed to understand the underlying signaling pathways that drive the biological effects of B[a]P and ACEE. For cell line, to identify the pathway by which ACEE ameliorates the effect of B[a]P on the induction of cell cycle arrest in neuronal cells, SH-SY5Y cells were treated with 40 µM B[a]P and 5 µg/mL ACEE and cultured for 48 h. As well, for The X-ray crystallographic structures of cytochrome P450 1A1 (PDB ID: 4I8V)  and hexop-value ≥ 0.05). In case the assumptions had been violated, the data were then analyzed using a Kruskal–Wallis test followed by a post hoc Mann–Whitney test. The p-value < 0.05 was considered statistically significant.All data were displayed as the mean ± standard error of the mean (SEM). Means were from at least 3 independent experiments. Prior to conducting the multiple comparison tests, the data were run to check for the normality and the homogeneity of variance. A one-way analysis of variance (ANOVA) followed by a post hoc Tukey test was performed for the data that met the assumptions of both the normal distribution and the homogeneity of variance , friedelan-3-one (12.76%), vitamin E (10.00%), neophytadiene (8.53%), lupenone (6.6%), and acetyleburicoic acid (6.05%). The components which corresponded to the peaks are shown in w/w). Interestingly, ACEE contained 61.4 ± 0.90 mg GAE/g dry weight for total phenolic content and 20.61 ± 2.79 mg QE/g dry weight for total flavonoid content. ACEE had antioxidant capacities of 62.16 ± 2.39 mg of vitamin C equivalent antioxidant capacity (VCEAC)/g dry weight in DPPH and 81.97 ± 0.90 mg VCEAC/g dry weight in ABTS. Furthermore, ACEE at the concentration of 5 µg/mL possessed the free radical scavenging capacities of 49.09 ± 2.24% and 94.91 ± 0.95% against DPPH and ABTS radicals, respectively.The ACEE extraction yield was 8.50%  and 100 µg/mL  compared to the 0.1% DMSO control group. On the contrary, we found that B[a]P at the concentration of 40 µM could significantly decrease cell viability  . Therefore, 40 µM B[a]P and 5 µg/mL ACEE were used for the further experiment.The nontoxic concentrations on cell viability of ACEE were at 5–25 µg/mL a. Cell v= 0.000) b. Intere= 0.000) c. The efp value = 0.015). After treatment combined with 40 µM B[a]P and 5 µg/mL ACEE, the percentage of cells in the G0/G1 phase was significantly higher . The flow cytometer results are shown in To further understand the protection of ACEE extract on B[a]P-induced cell cycle disturbance, a flow cytometer generated the data that showed the percentage of cells in the G0/G1 phase when treated with 40 µM B[a]P was significantly lower than in the 0.1% DMSO control group , 5.75 ± 1.63-fold change , and 1.51 ± 0.08-fold change , respectively) in the B[a]P-treated SH-SY5Y cells compared with the 0.1% DMSO control group. After co-treatment with 40 µM B[a]P and 5 µg/mL ACEE, ACEE extract could attenuate the B[a]P effect and normalize the cell cycle progression. Hence, the relative mRNA expression of AHR, CYP1A1 and cyclin D1 in the ACEE-treated group was significantly decreased , and the relative mRNA expression of AHR, CYP1A1 and cyclin D1 was 1.03 ± 0.17-fold change , 1.08 ± 0.19-fold change , and 0.94 ± 0.09-fold change  compared to the B[a]P-exposed group, respectively. The results in p < 0.05 vs. control). The relative protein expression of CYP1A1 and cyclin D1 compared to the control group was 1.13 ± 0.01-fold change  and 1.94 ± 0.02-fold change , respectively. When treatment was combined with 40 µM B[a]P and 5 µg/mL ACEE, both CYP1A1 and cyclin D1 proteins were significantly decreased but only the AHR expression was not changed in response to the cotreatment with ACEE . The relative protein expression of CYP1A1 and cyclin D1 compared to the 40 µM B[a]P-treated group was 0.79 ± 0.03-fold change  and 1.45 ± 0.18-fold change , respectively. Moreover, Our study indicates that high B[a]P-fed worms were manifested by shortened lifespan. The results in p = 0.000 for 100 B[a]P-treated group; 0.74 ± 0.01-fold change, p = 0.000 for 200 B[a]P-treated group; 0.77 ± 0.01-fold change, p = 0.000 for 300 B[a]P-treated group; 0.75 ± 0.01-fold change, p = 0.000 for 400 B[a]P-treated group). The toxicity of B[a]P was determined by body length assay. We found that all B[a]P at the concentrations of 100–400 µM could significantly reduce the body length compared to the 0.1% DMSO control group . In contrast, ACEE activity on the body length improvement could not be detected in the 400 B[a]P-treated group.After the cotreatment of B[a]P and 5 µg/mL ACEE, ACEE could attenuate the toxic effect of B[a]P and cause the improvement in the body length  and 0.69 ± 0.03-fold change , respectively, * p < 0.05 vs. control). After cotreatment 300 B[a]P and 5 µg/mL ACEE, ACEE could attenuate the toxic effect of B[a]P and cause the improvement in the body length ; p value = 0.000); #p < 0.05 vs. 300 µM B[a]P alone).Not only body length assay, but as seen in cyp-35A1, cyp-35A2, cyp-35A3, cyp-35A4, cyp-35A5, cyp-35B2, cyp-35B3 and cyp-35C1) was studied.To identify the protective potential of ACEE on xenobiotic processing, mRNA expression of eight representative genes of the CYP35 family . As the treatment combined B[a]P with ACEE, the ACEE could significantly reduce mRNA expression compared with B[a]P-fed worms. mRNA expression was 0.80 ± 0.30-fold change, p value = 0.034 for cyp-35A1; 0.91 ± 0.21-fold change, p value = 0.021 for cyp-35A2; 1.42 ± 0.11-fold change, p value = 0.034 for cyp-35A3; 1.74 ± 0.65-fold change, p value = 0.043 for cyp-35A4; 1.68 ± 0.23-fold change, p value = 0.034 for cyp-35A5; 0.79 ± 0.13-fold change, p value = 0.021 for cyp-35B2; 5.94 ± 0.81-fold change, p value = 0.034 for cyp-35B3; 3.08 ± 0.46-fold change, p value = 0.034 for cyp-35C1 (#p < 0.05 vs. 300 µM B[a]P-fed worms). We found that 300 µM B[a]P could significantly increase the mRNA expression of all ed group , mRNA exhxk genes , 2.89 ± 0.4-fold change , and 2.46 ± 0.25-fold change , respectively (* p < 0.05 vs. control). mRNA levels of hxk-1, hxk-2, and hxk-3 in the ACEE extract-treated group were lower than those in the B[a]P-treated group. The fold change of mRNA levels was 0.55 ± 0.1-fold change  for hxk-1, 0.98 ± 0.49-fold change  for hxk-2, and 0.86 ± 0.36-fold change  for hxk-3 (#p < 0.05 vs. 300 µM B[a]P-fed worms). In this study, we aimed to investigate the effect of B[a]P and ACEE on mRNA expression of xk genes . We founTo further identify the competence of ACEE on CYP1A1 and hexokinase II, we used alizarin and purpurin as positive control for CYP1A1 , and metBased on the docking results in Additionally, as shown in We found that the other three phytochemicals, including β-amyrin , lupenone , and clionasterol , showed strong inhibition against hexokinase II with the higher binding energy .Aquilaria crassna (ACEE) exerted the neuroprotective effect against the increase in cyclin D1 in B[a]P-induced neurotoxicity through an AHR-mediated CYP1A1 pathway. Interestingly, cyclin D1 is an influential protein of cell cycle activator that directs the progression of cell cycle and is upregulated in neurotrauma or in traumatic brain injury [Benzo[a]pyrene or B[a]P is a carcinogen and mostly discovered in cigarette smoke, vehicle exhausts, crude oil, coal tar, tobacco, and cooked food, especially grilled meats . B[a]P mn injury . C. elegans were used as in vivo model. CYP35 family plays a crucial role in the xenobiotic processing in C. elegans lacking classical CYP1A1 enzyme P including lipid metabolism and alteration in longevity [C. elegans worms lacking either cyp-35A1 or cyp-35A3 were characterized by an absence of a B[a]P-induced increase in toxicity and lipid metabolism P exposure P-fed worms.To further understand the effect of B[a]P and ACEE on organism survival, 1 enzyme . In thisongevity . C. elegtabolism ,44,45. Mexposure . In addiexposure . Finallyexposure . These rhxk-1, hxk-2, and hxk-3 were identified in C. elegans exposed to 3-Bromopyruvate (3-BrPA). The high expression of these genes might be responding to the large ATP consumption during 3-BrPA metabolism P and ACEE in both neuronal cell line and animal models. Further analysis of the dose-response relationship could be performed using the linear-quadratic dose-response model in order to better understand and predict the cellular response to B[a]P or ACEE exposure. Collectively, ACEE might be an effective neuroprotectant and detoxification agent against B[a]P exposure with a possible active constituent including clionasterol.Overall, the findings from this present study support that ACEE could provide a neuroprotective effect against high B[a]P-induced neurotoxicity in human neuronal SH-SY5Y cells by the induction of cell cycle progression. Likewise, ACEE could attenuate the effect of B[a]P on shortening cyp-35 and hxk pathway. Additionally, clionasterol might be the prominent phytochemical that inhibits CYP-induced B[a]P’s metabolism into toxic metabolites. Essentially, ACEE could be advanced as an agent for the protection of B[a]P-induced xenobiotic toxicity in neuronal cells and C. elegans.In summary, the AC leaf is composed of rich bioactive compounds and antioxidant properties. ACEE could represent neuroprotectant from B[a]P-induced neurotoxicity. ACEE can protect neuronal cells from B[a]P-induced cell damage, including the induction of cell cycle normalization through the AHR/CYP1A1/CCND1 signaling pathway. ACEE also utilizes lifespan extension and xenobiotic detoxification via the


Streptomyces being the most common genus. Three of the target strains were chosen for scale-up fermentation after preliminary screening of antibacterial activities and the metabolomics investigation using LC-MS. These strains were Penicillium sp. NX-S-6, Streptomyces sp. YHLB-L-2 and Streptomyces sp. ZLBB-S-6. Twenty-three secondary metabolites (1–23), including a new sorbicillin analogue (1), were produced as a result of antibacterial activity-guided isolation. Through spectroscopic analysis using MS and NMR, the structures of yield compounds were clarified. According to antibacterial data, S. aureus or B. subtilis were inhibited to varying degrees by sorrentanone (3), emodic acid (8), GKK1032 B (10), linoleic acid (14), toyocamycin (17) and quinomycin A (21). The most effective antimicrobial agent against S. aureus, B. subtilis, E. coli and A. baumannii was quinomycin A (21). In addition, quinomycin A showed strong antifungal activity against Aspergillus fumigatus, Cryptococcus neoformans, and two clinical isolated strains Aspergillus fumigatus #176 and #339, with MIC as 16, 4, 16 and 16 µg/mL, respectively. This is the first time that bioprospecting of actinobacteria and their secondary metabolites from medicinal plants in Fenghuang Mountain was reported. The finding demonstrates the potential of endophytic microbes in medical plants to produce a variety of natural products. Endophytic microbes will be an important source for new antibiotics.One of the largest concerns with world health today is still antibiotic resistance, which is making it imperative to find efficient alternatives as soon as possible. It has been demonstrated that microbes are reliable sources for the creation of therapeutic antibiotics. This research intends to investigate the endophytic microorganisms from several medicinal plants in Fenghuang Mountain  and to discover new antibiotics from their secondary metabolites. A total of 269 endophytic strains were isolated from nine distinct medicinal plants. Taxonomic analysis revealed that there were 20 distinct species among these endophytes, with  Currently, antibiotic resistance is a significant issue that needs to be tackled globally . Researc1–23), including a new sorbicillin analogue, were produced after a bioassay-guided isolation, and they were then identified by using spectroscopic data analysis. We tested each isolated compound for the presence of antibacterial or antifungal properties  is a treasure trove of diverse natural medicines . Due to operties .Achyranthes bidentata Blume, Ainsliaea fragrans Champ., Artemisia capillaris Thunb., Daucus carota L., Leonurus japonicus Houtt., Perilla frutescens (L.) Britt., Prunella vulgris L., Salvia miltiorrhiza Bge. and Stemona sessilifolia (Miq.) Miq.] were gathered for endophyte cultivation. After plant tissues were surface sterilized, 269 endophytic microbes were gradually isolated and identified. With a few bacteria and one fungus (Penicillium sp. NX-S-6 from Achyranthes bidentata), the endophytes were primarily actinomycetes. Twenty genera, including Streptomyces, Bacillus, Nocardia, Micromonospora, and others, were represented by all of the strains, etc. Seventy-five percent of the total was Streptomyces  in some way (see Achyranthes bidentata (Ainsliaea fragrans (E. coli and S. aureus. More strains exhibited inhibitory effects on S. aureus compared to E. coli. Penicillium sp. NX-S-6 (Streptomyces sp. YHLB-L-2 (Streptomyces sp. ZLBB-S-6 (The majority of the isolated endophytic strains (1 mg/mL) showed inhibitory activity against the tested pathogenic bacteria , followed by extraction, fractionation and chromatographic purification (1), along with thirteen previously identified compounds: oxosorbicillinol (2) –; (+)-ESI-MS: m/z 264.2 [M + H]+; (+)-HR-ESI-MS m/z 264.1231 [M + H]+ .3, 8, 10, 14, 17 and 21 against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii varied in intensity (21) [B. subtilis. In order to further assess the antifungal efficacy of quinomycin A, standard and isolated clinical strains were used. With MIC values of 16, 4, 16 and 16 µg/mL, respectively, this natural product demonstrated potent inhibition against Aspergillus fumigatus, Cryptococcus neoformans, Aspergillus fumigatus #176 and Aspergillus fumigatus #339  , was evetus #339 .1–23) were isolated by bioassay-guided purification from three of the most active endophytes. Along with 2–6, compound 1 was designed as a new compound that belongs to the sorbicillin family. The unusual hybrid sorbicillinoids known as bisorbicillchaetones A and B  were recently identified as coming from the deep-sea derived fungus Penicillium sp. SCSIO06868 with anti-inflammatory activities [7–9 by an endophyte has been demonstrated to occur often in secondary metabolites from medicinal plants [10) was previously produced from Penicillium sp. GKK1032. It was confirmed further by the isolation of the triterpenoid molecule soyasapogenol B (11) from an endophytic fungus in Achyranthes bidentata that plant endophytes might interact with their hosts’ metabolic process and generate analogs [12) is frequently present in plants [18, 19) are polyene macrolides with remarkable antifungal activities against Corynespora cassiicola [Fusarium oxysporum f. sp. cucumerinum [21) was the most effective antimicrobial compound against both Gram-positive and negative bacteria, as well as the standard and clinical isolated fungal strains, out of the 23 isolated compounds. Even better than ampicillin and polymyxin, quinomycin A’s MIC against B. subtilis was less than 0.125 µg/mL. Quinomycin A, also known as echinomycin, has previously been recognized as a possible anticancer drug that has a great affinity for double-stranded DNA [One of the biggest concerns with human health worldwide continues to be antibiotic resistance. To address this issue, a thorough search for new and better antibiotics is currently in progress. A total of 269 endophytic strains were isolated and identified from the nine medicinal plants that were collected in Fenghuang Mountain. Twenty-three secondary metabolites . Nuclear magnetic resonance spectra were measured with a Bruker Advance AV500 spectrometer . LC-MS was performed with an Agilent 1290 system equipped with a 6120 Quadrupole MSD mass spectrometer . HRESIMS spectra were recorded with a AB Sciex TripleTOF 5600 mass spectrometer . HPLC analyses were conducted on an Agilent 1260 system with a PDA detector  and a Waters 2695 system with a 2998 PDA detector . Preparative HPLC separation was conducted on a Waters 1525EF LC system (Waters). MCI Gel high-porous polymer (75–150 μm) was obtained from Mitsubishi Chemical Corporation . Sephadex LH-20 resin (25–100 μm) was obtained from GE Healthcare Company . Amberlite XAD16N resin was purchased from Taitan Company . Chemicals were purchased from Aldrich or Juyou Company .Oatmeal agar: oatmeal agar 65.0 g/L.ISP4 agar: ISP4 37.0 g/L and agar 15.0 g/L.3 0.02 g/L, NaCl 0.2 g/L, MgSO4·7H2O 0.05 g/L, K2HPO4 2.0 g/L, FeSO4·7H2O 0.01 g/L and agar 18.0 g/L.LNMS agar: soluble starch 0.1 g/L, yeast extract 0.1 g/L, CaCO2HPO4 1.5 g/L, MgSO4·7H2O 0.05 g/L and agar 15.0 g/L, pH was 7.2.KMB agar: glycerin 10.0 g/L, peptone 20.0 g/L, KModified Gaoshi No.2 agar: glucose 1.0 g/L, peptone 0.5 g/L, tryptone 0.3 g/L, NaCl 0.5 g/L, multivitamins  and agar 20.0 g/L, pH was 7.2.M2 agar: malt extract 10.0 g/L, glucose 10.0 g/L, yeast extract 4.0 g/L and agar 15.0 g/L.CMA agar: corn flour 40.0 g/L and agar, 15.0 g/L.YG agar: glucose 5.0 g/L, yeast extract 3.0 g/L, tryptone 5.0 g/L and agar 15.0 g/L.2HPO4·3H2O 0.5 g/L, (NH4)2SO4 1.0 g/L and CaCO3 2.0 g/L.Seed medium: glucose 10.0 g/L, corn flour 40.0 g/L, gluten powder 5.0 g/L, bran 10.0 g/L, KSDB medium: peptone, 10.0 g/L and glucose, 40.0 g/L.4·7H2O 0.5 g/L, NaCl 4.0 g/L, K2PO4 0.5 g/L and CaCO3 2.0 g/L.Medium A: glucose 10.0 g/L, soluble starch 20.0 g/L, yeast extract 5.0 g/L, peptone 5.0 g/L, MgSOAchyranthes bidentata, Ainsliaea fragrans, Artemisia capillaris, Daucus carota, Leonurus japonicus, Perilla frutescens, Prunella vulgris, Salvia miltiorrhiza, and Stemona sessilifolia were gathered. All the plants mentioned above were identified by co-author Dr. Yang Hu. The voucher specimens (No. 2020005–2020013) were deposited in our Jiangsu Key Laboratory for Functional Substances of Chinese Medicine. Plant parts  were first cleaned with water before being submerged in 75% ethanol for 5 min and 2% sodium hypochlorite for 5 min, respectively. Tissue samples were washed three times in sterile water following each treatment. The surface-sterilized samples were divided into small pieces aseptically and 0.2 g of each was suspended in sterile water (1.0 mL) and heated at for one minute at 75 °C to kill nonsporulating bacteria [From Fenghuang Mountain , fresh samples of bacteria . ISP4 agbacteria .https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 20 February 2023). The accession numbers for each of the retrieved 16S rRNA sequences were deposited in the NCBI nucleotide database.The target 16S rRNA gene was amplified using universal primers  after bacteria were seeded in 4 mL of TSB broth for three days at 28 °C . The amphttps://blast.ncbi.nlm.nih.gov/Blast.cgi) for searching the sequence that was the closest match. The ITS sequence was deposited in the NCBI nucleotide database with accession number OQ438028.From a fresh strain agar plate, 100 mg of fruiting bodies or mycelia were taken and ground with liquid nitrogen. The Fungal Genomic DNA Rapid Extraction Kit  was used to extract the DNA. The DNA samples were dissolved in 100 µL of TE Buffer and kept at −20 °C for storage. Using the universal primers: ITS1 5′-TCCGTAGGTGAACCTGCGG-3′; ITS4 5′-TCCTCCGCT TATTGATATGC-3′, the partial ITS gene fragment was amplified . The ampUsing 250 mL baffled conical flasks, individual microbial colonies were cultivated for 7 days at 28 °C in seed medium (50 mL). Then, prior to harvesting, 1 g of XAD-16 resin was applied. The culture of each strain was centrifuged in a falcon tube for 15 min at 5000 rpm. The supernatant was discarded and 30 mL of distilled water was used to wash the mycelia and XAD resin component twice before vortexing and centrifuging. The crude extract was then obtained by centrifuging and evaporating the supernatant after adding 15 mL of methanol and extracting by sonication for 30 min.2O with 0.05% FA (B), and the gradient program was 5–100% A in 0–5 min, 100% B in 5–7 min, 100–5% B in 7–8 min, 5% B in 8–10 min. The flow rate was 0.3 mL/min. The spectrum of wavelengths was 200–600 nm. 120 eV for API-MS. Prioritization would begin with strains that produced an abundance of products with comparable UV absorbances , mobile phases were acetonitrile (A) and Horbances .Staphylococcus aureus (No. ATCC 29213) and Escherichia coli (No. ATCC 25922) were acquired from the China Center of Industrial Culture Collection. All of the examined microorganisms were incubated in LB broth media overnight at 37 °C. The cultures were quantified using a spectrophotometer and diluted to A = 0.02 (OD600) before being distributed to 96-well plates (100 µL/well). The crude extracts of endophytes (1.0 mg/mL) and positive control  were subsequently added to the 96-well plates . The plates were examined for absorbance at 600 nm using a microplate reader to determine the inhibitory rate following a 16 h incubation at 37 °C.Two different media were used to ferment the strain twice .w/v) and stirred for six hours. Following filtration, the resin was three times (500 mL each) washed with water, and three times (500 mL each) extracted with methanol. HPLC results showed that the metabolite profiles of the mycelial and supernatant fractions were comparable; thus, they were combined and concentrated to afford crude extract (101.63 g). The crude extract underwent further ethyl acetate (EtOAc) extraction to afford EtOAc extract (29.56 g), which was then isolated using a silica gel chromatographic column  with gradient petroleum ether/EtOAc system to produce 13 fractions (A–M). Fraction C (1.13 g) was purified on a Sephadex LH-20 column eluted with DCM: MeOH = 1: 1 to obtain sub-fraction C1 (0.1 g), which was further purified by a preparative HPLC (80% MeCN) to obtain compounds 13 (4.1 mg) and 14 (28.4 mg). Fraction D (5.05 g) was crystallized to obtain compound 12 (182.1 mg). Fraction F (0.56 g) and fraction J (0.95 g) were combined and purified on a Sephadex LH-20 column with DCM: MeOH = 1: 1 to yield compounds 9 (15.7 mg) and 10 (14.1 mg). Fraction I (0.47 g) was purified through a Sephadex LH-20 column (DCM: MeOH = 1: 1) to obtain compound 11 (20.0 mg). Sub-fraction I1 (18.9 mg) was purified by using a preparative HPLC (40% MeCN) to acquire compound 1 (5.6 mg).Firstly, portions of the fully grown strain were transferred from M2 agar plates into ten 250 mL Erlenmeyer flasks with 50 mL of seed medium. The seed cultures (each 1 mL) were moved from the three days of incubation at 28 °C (200 rpm) to the 50 mL seed media . After 7 days of incubation at 28 °C with 200 rpm agitation, the fermentation broth was combined and filtered to afford mycelium and supernatant. The mycelium component was extracted using MeOH (3 × 2.0 L) under sonication. The supernatant portion was then mixed with XAD-16 resin  were transferred to 100 mL of SDB media . The fermentation broth was combined and centrifuged at 4 °C  following two weeks of incubation at 28 °C with 200 rpm agitation. The mycelium component was extracted using MeOH (3 × 2.0 L) with sonication, and 54.46 g of crude extract A was produced after the solvent was evaporated. Crude extract B (17.69 g) was produced after the supernatant part was extracted with EtOAc three times and evaporated. Crude extract A (54.46 g) was isolated by using a silica gel column  and a petroleum ether/EtOAc system to yield 14 fractions (A′–N′). Fraction K′ (0.24 g) was purified on a Sephadex LH-20 column using MeOH to obtain sub-fraction K′1 (0.12 g) and compound 7 (3.1 mg). Sub-fraction K′1 was then purified by using a preparative HPLC (40% aqueous MeCN) to acquire compounds 3 (2.4 mg), 4 (14.6 mg) and 5 (3.9 mg). Compound 6 (10.1 mg) was isolated form fraction M′ (0.11 g) by using preparative HPLC (45% MeCN). Fraction N′ (0.80 g) was purified on a Sephadex LH-20 column using MeOH to obtain sub-fraction N′1 (61 mg) and compound 8 (2.1 mg). Sub-fraction N′1 was then isolated by using a preparative HPLC (30% MeCN) to yield compound 2 (7.1 mg).Secondly, Streptomyces sp. YHLB-L-2 was grown for 7 days at 28 °C on M2 agar plates, as shown in w/v) and agitated for six hours. Following filtration, the resin was rinsed with water three times (500 mL each), and three times extracted with methanol (500 mL each). HPLC results showed that the metabolite profiles of mycelial and supernatant fractions were comparable; thus, they were mixed and concentrated to produce crude extract (56.32 g). The crude extract was firstly subjected to an MCI column  using a gradient MeOH/H2O system to obtain 11 fractions (A–K). Fraction C (1.32 g) was purified with a Sephadex LH-20 column using 80% MeOH as eluent to obtain compound 17 (39.0 mg) and sub-fraction C1. Compound 16 (7.7 mg) was isolated from sub-fraction C1 (142.6 mg) by using a preparative HPLC (23% MeOH). Fraction E (3.02 g) and fraction F (1.93 g) were mixed and purified by using a Sephadex LH-20 column (80% MeOH) to acquire compounds 15 (58.5 mg) and 20 (16.5 mg). Fraction H and fraction J were identified to be pure compounds 19 (0.74 g) and 18 (0.19 g) respectively.Streptomyces sp. ZLBB-S-6 was grown for 7 days at 28 °C on M2 agar plates. In total, ten flasks of 50 mL of seed medium were inoculated with cut pieces of agar containing a fully grown strain. The seed cultures (each 1 mL) were transferred to 100 mL seed medium  after three days of incubation at 28 °C (200 rpm). The fermentation broth was combined after 7 days of incubation at 28 °C with 200 rpm agitation and then filtered to afford the mycelium and supernatant. Under sonication, the mycelium component was extracted with MeOH (3 × 2.0 L). XAD-16 resin (4% w/v) was added to the supernatant portion, and the mixture was agitated for six hours. Following filtration, the resin was three times washed with water (500 mL each), and three times extracted with methanol (500 mL each). The HPLC results showed that the metabolite profiles of mycelial and supernatant fractions were comparable; thus, they were combined and concentrated to produce crude extract (71.08 g). The crude extract was firstly subjected to an MCI column  using a gradient MeOH/H2O system to obtain 9 fractions (A-I). Fraction C (1.61 g) was isolated by using a Sephadex LH-20 column (80% MeOH) to obtain sub-fraction C1 (99.0 mg), which was further purified by preparative HPLC using 5% MeOH to obtain compound 22 (6.6 mg). Compounds 21 (85.0 mg) and 23 (20.4 mg) were isolated from fraction H (3.52 g) by using a flash column silica-cs (50 g) eluted with a gradient of DCM/MeOH.As shown in 13C NMR shielding constants were calculated with the time-dependent density-function theory (TD-DFT) methodology at the B3LYP/6-311+G level in CDCl3 with the IEFPCM solvation model using Gaussian 09. Frequency analysis was performed as well to confirm that the re-optimized conformers were at the energy minima. The spectra were combined after Boltzmann weighting according to their population contributions using SpecDis 1.64 software [13C NMR data of 6S-1 was consistent with experimental data (2) of 0.9976.The Chem3D 15.0 software with PM3 force field was used to establish the initial conformations of target molecules. Then the conformational searches were conducted via random searching in the Schrodinger Maestro 2019 using MMFFs force field with an energy cutoff of 10.0 kcal/mol. Duplicate conformations were identified and removed when the root-mean-square distances (RMSD) were less than 0.5 Å for any two geometry-optimized conformations. Subsequently, the remaining conformations were re-optimized at the B3LYP/6-311G level in vacuo by using the Gaussian 09 program . The 13Csoftware . Utilizisoftware , the comtal data , with a Staphylococcus aureus (ATCC 29213), Bacillus subtilis (A186), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853) and Acinetobacter baumannii (ATCC 19606) were purchased. The bacteria were inoculated in LB Broth media overnight at 37 °C. The cultures were quantified with a spectrophotometer, diluted to A = 0.02 (OD600) and then dispensed to 96-well plates (100 µL/well). All isolated compounds 1–23, positive controls ampicillin and polymyxin (1–128 µM by double dilution method) and DMSO (placebo control) were then added (n = 3). Plates were incubated for 16 h at 37 °C, and the inhibitory rate was calculated by measuring the absorbance at 600 nm. When compared to positive control groups, bacterial growth inhibition greater than 80% was used to determine the MICs.From CICC , standard strains of Aspergillus fumigatus 1160, Candida albicans (ATCC 210231), Cryptococcus neoformans H99 and the clinically isolated strains Aspergillus fumigatus 77, 176, 185, 331, 339 and 353 were used as test strains. On YG agar plates, fungi were grown at a temperature of 37 °C. After two days, spores were collected, and the RPMI-1640 culture was used to modify the suspension concentrations. By using a blood counting chamber, the Aspergillus fumigatus strain final inoculated concentrations were prepared as 0.4 × 104 to 5.0 × 104 CFU/mL. The final inoculated concentrations of Candida albicans and Cryptococcus neoformans strains were prepared with McFar Land (OD530) to be 500~2500 CFU/mL. By using the double dilution procedure, compound 21 (0.125–32 µM) was added to 96-well plates (n = 3) and maintained for 48 h at 37 °C. Itraconazole served as the positive control. The MICs were acquired by ocular inspection without mycelia developing.Standard


Bilaterally absent cortical somatosensory evoked potentials (SSEPs) reliably predict poor outcome in comatose cardiac arrest (CA) patients. Cortical SSEP amplitudes are a recent prognostic extension; however, amplitude thresholds, inter-recording, and inter-rater agreement remain uncertain.In a retrospective multicenter cohort study, we determined cortical SSEP amplitudes of comatose CA patients using a standardized evaluation pathway. We studied inter-recording agreement in repeated SSEPs and inter-rater agreement by four raters independently determining 100 cortical SSEP amplitudes. Primary outcome was assessed using the cerebral performance category (CPC) upon intensive care unit discharge dichotomized into good (CPC 1–3) and poor outcome (CPC 4–5).Of 706 patients with SSEPs with median 3 days after CA, 277 (39.2%) had good and 429 (60.8%) poor outcome. Of patients with bilaterally absent cortical SSEPs, one (0.8%) survived with CPC 3 and 130 (99.2%) had poor outcome. Otherwise, the lowest cortical SSEP amplitude in good outcome patients was 0.5 µV. 184 (42.9%) of 429 poor outcome patients had lower cortical SSEP amplitudes. In 106 repeated SSEPs, there were 6 (5.7%) with prognostication-relevant changes in SSEP categories. Following a standardized evaluation pathway, inter-rater agreement was almost perfect with a Fleiss’ kappa of 0.88.Bilaterally absent and cortical SSEP amplitudes below 0.5 µV predicted poor outcome with high specificity. A standardized evaluation pathway provided high inter-rater and inter-recording agreement. Regain of consciousness in patients with bilaterally absent cortical SSEPs rarely occurs. High-amplitude cortical SSEP amplitudes likely indicate the absence of severe brain injury.The online version contains supplementary material available at 10.1007/s00415-023-11951-4. In multimodal neuroprognostication after cardiac arrest (CA), cortical somatosensory evoked potentials (SSEP) are recommended to predict the severity of hypoxic-ischemic encephalopathy (HIE) , 2. BilaIn few studies, low-amplitude cortical SSEP amplitudes predicted poor outcome, whereas high-amplitude cortical SSEP amplitudes indicated absence of severe HIE –12. The To further investigate the validity of cortical SSEP amplitude as a prognostic test in comatose CA patients, we conducted a large, retrospective, multicenter study with focus on a standardized SSEP evaluation, inter-recording agreement, and inter-rater agreement.Local ethics committees fully approved this study and waived the need for patient consent (EA4/004/14). The study was conducted according to the Declaration of Helsinki.We retrospectively enrolled adult comatose CA patient investigated with median nerve SSEP in four academic centers . Local postresuscitation care protocols adhered to international guidelines , 19. Thimax and Pmin within 5–10 ms after stimulus. Only when noise level was below 0.25 µV in all cortical recordings and cortical potentials were not reproducible, SSEPs were classified as bilaterally absent. For reproducible cortical potentials (at least 4.5 ms after the N13), we determined cortical amplitudes of baseline–N1, N1–P1, baseline–P1, and Nmax–Pmin in atypical SSEP morphology. The cortical SSEP amplitude was defined as the highest amplitude of all recordings. Amplitudes were rounded to two decimal places.We placed electrodes at CP3/CP4, C7  and over the supraclavicular fossa (NErb) following the international 10–20 system and used a midfrontal (Fz) cortical reference electrode , 23. To In patients with repeated SSEPs, we evaluated the second cortical SSEP amplitude blinded for the results of the first SSEP. Based on previous own studies , 24, 25,Furthermore, we studied inter-rater agreement on determination of cortical SSEP amplitudes. Three raters with different neurophysiological expertise  underwent a training session with supervisor C.E.  on 20 patients with typical SSEP morphologies  following the standardized evaluation pathway . Subsequently, each rater independently evaluated the last 100 SSEPs not included in the training session, blinded to clinical data and the results of the other raters.We assessed clinical outcome as primary outcome using the Cerebral Performance Category (CPC) scale upon intensive care unit (ICU) discharge, and classified CPC 1–3 as good and CPC 4–5 as poor outcome. To investigate potential confounders of coma, center specifics in order of prognostic testing, and ICU duration, we reviewed patients with CPC 4 and cortical SSEP amplitudes above 2.5 µV , 24, 25.Baseline demographics are presented as medians with inter-quartile range (IQR) or absolute numbers with percentage as appropriate. To illustrate the association between cortical SSEP amplitude and investigated parameters, we used scatter plots, and calculated median and IQRs. We used the Kolmogorov–Smirnov test to check for normal distribution and a two-sided Wilcoxon rank-sum test to compare groups as appropriate. Sensitivity and specificity were calculated for outcome prediction. A heatmap illustrates the inter-recording agreement. To analyze inter-rater agreement, SSEPs were categorized into excluded, bilaterally absent, up to 0.5 µV, larger than 0.5–2.5 µV, and above 2.5 µV; and agreement between the four raters was described numerically. Fleiss’s kappa was calculated according to previous conventions  as folloAnonymized data are available on reasonable request to the corresponding author.We enrolled 816 patients of whom 9 (1.1%) had incomplete recordings, 48 (5.9%) bilaterally non-reproducible cervical potentials with low-amplitude cortical SSEP amplitudes, and 53 (6.5%) patients had too high noise levels. Supplementary Table 2 and Supplementary Table 3 shows the patient flow for the standardized evaluation pathway stratified by each center. Table Figure n = 16) or CPC 5 (n = 114) yielding a sensitivity of 30.3% to predict poor outcome. Presence of cortical SSEPs yielded a positive predictive value of 48.0% (276/575) to predict good outcome. The lowest cortical SSEP amplitude of CPC 1 patients was 0.50 µV and 0.63 µV in CPC 2 patients. Of 429 patients with CPC 4–5, 184 (42.9%) had cortical SSEP amplitudes below 0.50 µV.Of 277 patients with good outcome, one patient with CPC 3 had bilaterally absent cortical SSEP in a first SSEP and 0.51 µV in a repeated SSEP. This was an 18-year-old male who regained consciousness, but suffered from a severe spastic tetraparesis, dysphagia, anarthria, generalized dystonia, and severe cognitive deficits. He did not improve in a 13 months follow-up. Except for this one patient, all other 130 (99.2%) patients with bilaterally absent cortical SSEP had CPC 4 , range of median cortical SSEP amplitude were 2.38–5.59 µV in CPC 1–3, 0.87–3.11 µV in CPC 4, and 0.25–1.05 µV in CPC 5 patients, respectively. Among CPC 1–3 patients, the lowest cortical SSEP amplitudes was 1.02 µV in center 2, 1.25 µV in center 3, 0.78 µV in center 4, and bilaterally absent and 0.50 µV, respectively, in center 1.p < 0.001) in 400 (56.7%) patients without regain of consciousness. In center 4, 86 patients without regain of consciousness had higher cortical SSEP amplitudes compared to other centers . 58 patients, who regained consciousness in center 4, had significantly higher cortical SSEP amplitudes  compared to 219 patients of center 1 .In 306 (43.3%) patients who regained consciousness, median cortical SSEP amplitude was 2.8 µV (1.8–5.4) compared to 0.7 µV  with regain of consciousness had cortical SSEP amplitudes below 1 µV, 176 (57.5%) above 2.5 µV, and 85 (27.8%) above 5 µV. The positive predictive value for a threshold of 2.5 and 5 µV to predict regain of consciousness was 64.0% (176/275) and 68.6% (85/124), respectively.Figure The positive predictive value to predict good outcome using cortical SSEP amplitudes above 2.5 µV was 57.8% (159/275). 42 (40.4%) of 104 CPC 4 patients had cortical SSEP amplitudes above 2.5 µV of whom 21 patients improved neurologically or died early after ICU discharge with potential death causes other than HIE. Considering improvement after discharge and death causes from other than HIE, the proportion of CPC 4 patients with cortical SSEP above 2.5 µV without identifiable confounders ranged between 0 and 23.6% in the four centers. 22.8% (74/325) of CPC 5 patients had cortical SSEP amplitudes above 2.5 µV.The lower cortical SSEP amplitude threshold to predict poor outcome did not relevantly change during the first days after CA . Except for one patient with CPC 3 and bilaterally absent SSEPs, the lowest cortical SSEP amplitude of good outcome patients was 0.76 µV, 0.50 µV, 0.63 µV, 0.59 µV, and 0.73 µV, on days 1, 2, 3, 4, and 5, respectively.106 patients had a repeated SSEP at a median of 6 days (4–7) after CA. Figure 6 (5.7%) patients had prognostication-relevant changes in SSEP categories. In 3 patients with SSEP amplitude decrease, a second CA, refractory cardiogenic shock, and progressive global brain edema were likely reasons. In 3 patients with SSEP amplitude increase, noise levels might have contributed to different cortical SSEP amplitudes between recordings in one patient, apart from the CPC 3 patient with bilaterally absent cortical SSEPs and cortical amplitudes of 0.51 µV in the repeated SSEP.Supplementary Table 4 shows the patient flow of the cortical SSEP amplitude stratification by the four raters. Fleiss’ kappa was 0.88 indicating almost perfect inter-rater agreement. In 85 of 100 SSEPs, all four raters agreed on the cortical SSEP category, and at least three of four raters agreed in 96 of 100 SSEPs  bilaterally absent and cortical SSEP amplitudes below 0.5 µV predicted poor outcome with high specificity. (2) We found one patient among 131 patients with bilaterally absent cortical SSEPs, who regained consciousness, but had severe long-term neurological deficits. (3) High-amplitude cortical SSEP amplitudes argued against, but did not exclude severe HIE. (4) Repeated SSEP recordings yielded mostly identical cortical SSEP amplitude categories within the first days after CA. (5) Following a standardized evaluation pathway, inter-rater agreement on cortical SSEP amplitudes was high.Cortical SSEP amplitude are generated by the summation of postsynaptic potentials in the somatosensory cortex , 27–29; In our study, bilaterally absent cortical SSEP predicted poor outcome with high specificity of 99.2% corroborating previous studies , 30–32. In our current study, one 18-year-old patient with initially bilaterally absent cortical SSEPs regained consciousness, but suffered from severe long-term neurological deficits. This illustrates that bilaterally absent SSEPs do not preclude recovery of consciousness with absolute certainty –38. RareExcept for this case, the lowest cortical SSEP amplitude in good outcome was 0.5 µV, and patients without regain of consciousness had a median of 0.7 µV. This is in line with several studies , which fWe assessed clinical outcome upon ICU discharge. Hence, we cannot exclude that CPC 4 patients with bilaterally absent or low-amplitude cortical SSEP amplitudes improved neurologically in long-term. The median duration of the ICU stay of CPC 4 patients was 23 and 29 days in center 1 and 2. We have previously shown that long-term improvement in CPC 4 patients beyond this observation period on the ICU is rare and was not observed among patients with poor early prognostic testing such as bilaterally absent SSEP . Other sImportantly, adherence to international guidelines , 23 and Finding prognostic tests to predict the absence of severe HIE is difficult. We previously found that high cortical SSEP amplitudes indicated absence of severe HIE , 25 at aTwo previous studies found moderate to substantial inter-rater agreement on classifying cortical SSEPs as bilaterally present or absent with noise as main confounder of disagreement , 16. InsIn our study, four raters with different expertise achieved almost perfect inter-rater agreement following a standardized SSEP evaluation pathway with a tolerable noise level of 0.25 µV. Our Fleiss’ kappa of 0.88 was higher compared to 0.34–0.75 in previous studies , 16, whiPrognostic results from repeated SSEPs were shown to be stable between recordings within the first days after CA , 46. CorOur study has strengths and limitations. Strengths include the large sample size, four participating centers with different recording devices, recording parameters, and cohorts, blinded SSEP amplitude assessment using a standardized SSEP evaluation pathway, blinded evaluation of inter-rater agreement, and a large number of repeated SSEPs. Altogether, these features support generalizability of our results.Due to the retrospective and multicenter design, our study cohort was heterogenous. However, the main results remained unchanged in the subgroup analyses, and we could study the effect of patient selection on cortical SSEP amplitudes. We assessed clinical outcome upon ICU discharge and, due to legal restrictions, could not routinely obtain long-term follow-up. Importantly, CA patients may substantially recovery during rehabilitation –49. TherIn summary, in this retrospective multicenter study of 706 comatose patients with SSEPs after CA, bilaterally absent and cortical SSEP amplitudes below 0.5 µV predicted poor outcome with high specificity. One young patient with bilaterally absent cortical SSEPs regained consciousness with severe long-term neurological deficits. High-amplitude cortical SSEP amplitudes likely indicate absence of severe HIE. Using a standardized evaluation pathway, inter-recording agreement and inter-rater agreement were reliable to routinely use cortical SSEP amplitudes in multimodal neuroprognostication.Supplementary file1 (PDF 5318 KB)Below is the link to the electronic supplementary material.


This study aimed to investigate an unsettled issue that whether T4 esophageal cancer could benefit from surgery.Patients with T4N0-3M0 esophageal cancer from 2004 to 2015 from the Surveillance, Epidemiology, and End Results (SEER) database were included in this study. Kaplan–Meier method, Cox proportional hazard regression, and propensity score matching (PSM) were used to compare overall survival (OS) between the surgery and no-surgery group.P < 0.001) in T4N0-3M0 cohort, 0.471 (0.354–0.627) (P < 0.001) in T4aN0-3M0 cohort, and 0.480 (0.335–0.689) (P < 0.001) in T4bN0-3M0 cohort. The HR (95% CI) for neoadjuvant therapy plus surgery vs. no surgery and surgery without neoadjuvant therapy vs. no surgery were 0.548 (0.461–0.650) (P < 0.001) and 0.464 (0.375–0.574) (P < 0.001), respectively. No significant OS difference was observed between neoadjuvant therapy plus surgery and surgery without neoadjuvant therapy: 0.966 (0.686–1.360) (P = 0.843). Subgroup analyses and PSM-adjusted analyses showed consistent results.A total of 1822 patients were analyzed. The multivariable Cox regression showed the HR (95% CI) for surgery vs. no surgery was 0.492 (0.427–0.567) (Surgery might bring OS improvement for T4N0-3M0 esophageal cancer patients, no matter in T4a disease or in T4b disease. Surgery with and without neoadjuvant therapy might both achieve better OS than no surgery.The online version contains supplementary material available at 10.1186/s12957-023-03239-8. Esophageal cancer accounts for 3.1% of all newly diagnosed cancers and the 5-year overall survival (OS) is approximately 30% . As an aThe milestone randomized controlled trials (RCTs), RTOG 85–01 trial and CROSS trial validated the efficacy of chemoradiotherapy alone and chemoradiotherapy plus surgery for locally advanced esophageal cancer, respectively , 7. HoweAs the role of surgery in T4 esophageal cancer is still unclear, we downloaded data of patients with T4N0-3M0 (8th version) esophageal cancer and conducted this multicenter study to compare the OS outcome between the surgery group and the surgery group.https://seer.cancer.gov/) and contained information on cancer of about 28% of the population of the USA. A subset of data submitted by 17 cancer registries was used in the extraction. After transforming the 6th or 7th version American Joint Committee on Cancer (AJCC) TNM staging system into the 8th version, patients diagnosed with AJCC 8th T4N0-3M0 esophageal cancer (only one primary) from January 2004 to December 2015 were included for screening. The exclusion criteria are as follows: patients who were aged < 18 years, diagnosed with autopsy/death certificate without pathological confirmation, whether received surgery or not was not known. The esophageal cancer was recognized using the “Site recode the 3rd edition of International Classification of Disease for Oncology (ICD-O-3)” of “Esophagus”. The histological types were classified based on ICD-O-3 classification: adenocarcinoma , squamous cell carcinoma , and other histological types  of the surgery group was 7 (6–7) months, and the corresponding data of surgery group was 19 (17–23) months. The 1-, 3-, and 5-year OS rate (95% CI) of the no surgery group was 29.0% (26.8%-31.4%), 9.5% (8.1–11.1%), and 5.4% (4.3–6.7%), respectively, and the corresponding rates of the surgery group was 66.3% (61.5–71.5%), 33.9% (29.3–39.4%), and 26.1% (21.8%-31.3%), respectively. The Kaplan–Meier curves in Fig. P < 0.001).In the study cohort, 1412 of the no-surgery group (P < 0.1) for OS of T4N0-3M0 esophageal cancer patients. After multivariable Cox analysis, all these variables remained as the independent prognostic factors except for age. In Tables P < 0.001) and 0.493 (0.417–0.582) (P < 0.001).In Table The forest plots of subgroup analyses showing HR (95%CI) for surgery vs. no surgery in T4N0-3M0, T4aN0-3M0, and T4bN0-3M0 cohort were presented in Fig. P < 0.001 ] or PSM-adjusted Cox analysis [0.892 (0.58–1.372) (P = 0.603)]  P = 0.03]  conducted by Fujita et al. comparing esophagectomy after chemoradiotherapy and chemoradiotherapy alone in AJCC 6th T4N0-1M0 squamous cell carcinoma in thoracic esophagus suggested that surgery did not decrease the mortality risk for responders to chemoradiotherapy, but the nonresponders showed a tendency to benefit from esophagectomy without statistical significance [P < 0.001. These different results might be caused by a different study cohort of only squamous cell carcinoma in the thoracic esophagus and a very small sample size in Hiromasa et al.’s study. Two retrospective studies directly compared surgery with no surgery in T4 esophageal cancer, reporting unfavorable results for surgery [n = 58) with induction chemoradiotherapy plus surgery (n = 13), and no significant OS difference was observed [Only a few studies exploring the role of surgery in T4N0-3M0 esophageal cancer were reported. A prospective study  of the whole study cohort (n = 1822) underwent surgery, indicating a small proportion of T4N0-3M0 esophageal cancers were resectable probably. For some unresectable T4 esophageal cancer patients, resectability could be achieved after chemoradiotherapy [As shown in Table otherapy , 20. Shiotherapy . Therefootherapy . Taken trt n = 18 underwenotherapy .Some limitations of the present study have to be admitted. First, there may be some bias that cannot be adjusted because of the study’s retrospective nature. Second, a Mixture of no and unknown radiotherapy/chemotherapy in the SEER database, and some factors like exposure to smoking/alcohol, multiple advanced underlying diseases performance status, regimens of chemotherapy, dose of radiation, R0/1/2 resection can influence the survival outcome but were not recorded in SEER database. Hence, RCTs with a large sample size are needed to validate the benefit of surgery.Surgery might bring OS improvement for T4N0-3M0 esophageal cancer patients, no matter in T4a disease or T4b disease. Surgery with and without neoadjuvant therapy might both achieve better OS than no surgery.Additional file 1: Supplementary Table S1. Selection procedure of study cohort. Supplementary Table S2. Univariable and multivariable Cox regression analysis comparing no surgery with surgery for the OS of AJCC 8th T4aN0-3M0 EC patients. Supplementary Table S3. Univariable and multivariable Cox regression analysis comparing no surgery with surgery for the OS of AJCC 8th T4bN0-3M0 EC patients. Supplementary Table S4. Univariable and multivariable Cox regression analysis comparing no surgery with neoadjuvant therapy plus surgery for the OS of AJCC 8th T4N0-3M0 EC patients. Supplementary Table S5. Univariable and multivariable Cox regression analysis comparing no surgery with no neoadjuvant therapy plus surgery for the OS of AJCC 8th T4N0-3M0 EC patients. Supplementary Table S6. Univariable and multivariable Cox regression analysis comparing neoadjuvant therapy plus surgery with no neoadjuvant therapy plus surgery for the OS of AJCC 8th T4N0-3M0 EC patients. Supplementary Table S7. Univariable and multivariable Cox regression analysis comparing no surgery with surgery for the OS of AJCC 8th IIIB-IVA esophageal adenocarcinoma patients. Supplementary Table S8. Univariable and multivariable Cox regression analysis comparing no surgery with surgery for the OS of AJCC 8th IIIB-IVA esophageal squamous cell carcinoma patients. Supplementary Figure S1. Baseline standardized mean difference before and after PSM for surgery vs. no surgery in T4N0-3M0 EC (A), surgery vs. no surgery in T4aN0-3M0 EC (B), surgery vs. no surgery in T4bN0-3M0 EC (C), Neo + surgery vs. no surgery in T4N0-3M0 EC (D), surgery without Neo vs. no surgery in T4N0-3M0 EC (E), and Neo + surgery vs. surgery without Neo in T4N0-3M0 EC (F). PSM, propensity score matching; EC, esophageal cancer; Neo, neoadjuvant therapy. Supplementary Figure S2. Subgroup analysis of HR for surgery vs. no surgery in OS of stage T4aN0-3M0 EC. HR, hazard ratio; OS, overall survival; EC, esophageal cancer; CI, confidential interval. Supplementary Figure S3. Subgroup analysis of HR for surgery vs. no surgery in OS of stage T4bN0-3M0 EC. HR, hazard ratio; OS, overall survival; EC, esophageal cancer; CI, confidential interval. Supplementary Figure S4. Baseline standardized mean difference before and after PSM for surgery vs. no surgery in IIIB-IVA esophageal adenocarcinoma patients (A), and IIIB-IVA esophageal squamous cell carcinoma patients (B). PSM, propensity score matching. Supplementary Figure S5. Survival curves of OS for stage IIIB-IVA esophageal adenocarcinoma comparing surgery with no surgery before PSM (A) and after PSM (B), IIIB-IVA esophageal squamous cell carcinoma comparing surgery with no surgery before PSM (C) and after PSM (D). OS, overall survival; PSM, propensity score matching.