This research investigates the accelerated hydrolytic degradation process of both anatomically designed bone scaffolds with a pore size gradient and a rectangular shape . The effect of material composition is investigated considering poly-ε-caprolactone (PCL) as the main scaffold material, reinforced with ceramics such as hydroxyapatite (HA), β-tricalcium phosphate (TCP) and bioglass at a concentration of 20 wt%. In the case of rectangular scaffolds, the effect of pore size  is also investigated. The degradation process (accelerated degradation) was investigated during a period of 5 days in a sodium hydroxide (NaOH) medium. Degraded bone bricks and rectangular scaffolds were measured each day to evaluate the weight loss of the samples, which were also morphologically, thermally, chemically and mechanically assessed. The results show that the PCL/bioglass bone brick scaffolds exhibited faster degradation kinetics in comparison with the PCL, PCL/HA and PCL/TCP bone bricks. Furthermore, the degradation kinetics of rectangular scaffolds increased by increasing the pore size from 500 μm to 200 μm. The results also indicate that, for the same material composition, bone bricks degrade slower compared with rectangular scaffolds. The scanning electron microscopy (SEM) images show that the degradation process was faster on the external regions of the bone brick scaffolds (600 μm pore size) compared with the internal regions (200 μm pore size). The thermal gravimetric analysis (TGA) results show that the ceramic concentration remained constant throughout the degradation process, while differential scanning calorimetry (DSC) results show that all scaffolds exhibited a reduction in crystallinity (Xc), enthalpy (Δm) and melting temperature (Tm) throughout the degradation process, while the glass transition temperature (Tg) slightly increased. Finally, the compression results show that the mechanical properties decreased during the degradation process, with PCL/bioglass bone bricks and rectangular scaffolds presenting higher mechanical properties with the same design in comparison with the other materials. A major concern related to the design of polymer-based bone tissue engineering scaffolds is related to the degradation process and degradation kinetics of these constructs as well as the rate of tissue regeneration. After generating an in vitro cell culture of a scaffold/tissue system, the degree of remodelling and replacement of the biological implant by the native tissue needs to be considered ,4,5,6,7.In the case of synthetic polymers such as poly-ε-caprolactone (PCL), hydrolysis is considered the most important degradation mechanism, which strongly depends on the type of chemical bonds, copolymer composition and wettability properties ,23,24,25Despite the significant advances in the field of bone tissue engineering, one of the biggest challenges from a scaffold design point of view is the difficulty in producing structures capable of simultaneously presenting (1) high mechanical strength and (2) proper degradation kinetics.Through the design of anatomical design scaffolds with gradient pore sizes mimicking the structure of bone (bone bricks), it was possible to achieve mechanical properties suitable for bone engineering applications, which represents a significant step forward in the field ,49. This2O5, 45 wt% SiO2, 24.5 wt% Na2O and 24.5 wt% CaO; CeraDynamics Ltd., James Kent Group, Stoke, UK) in powder shape (<10 μm particles size). A melt blending process was used to prepare the blends.PCL  was used as received in the form of pellets. Composite blends were prepared using HA  in nanopowder shape (<20 nm particle size), TCP  in powder shape (ranging between 20 μm and 30 μm) and bioglass 45S5 , were fabricated using the screw-assisted additive manufacturing 3D Discovery  and a continuous path algorithm based on 38 zig-zag double filaments and 14 spiral filaments . Similar3 (20 °C), a solubility of 20 °C and a pH of 14 . The degradation period took place for 5 days. On each day, 5 samples were used from each considered case and measured using a high-precision balance. At each time point, the samples were removed from the NaOH and washed three times with the use of deionised water and left to dry overnight. Once completely dry, the samples were measured to determine the weight reduction. The amount of NaOH used for 5 rectangular scaffolds was 15 mL and for the 5 anatomically designed bone bricks, the amount was 50 mL (due to their size). The pH was monitored throughout the experimental work, and no changes were observed (pH of 14).Accelerated degradation studies were conducted using sodium hydroxide (NaOH) of 5 mol/L (5 N) in aqueous solution  with a density of 1.185 gr/cmScanning electron microscopy (SEM) was used to investigate the morphological characteristics of the samples and to determine pore sizes . Scaffolds were coated (platinum coating) with the use of an EMITECH K550X sputter coater  before imaging. The SEM images were analysed using ImageJ 1.x  (10 measurements per sample).pV is the pore volume, V is the total bulk volume and tP is the porosity.The porosity of the scaffolds was calculated with the use of the following equation:ρ is the density of the unprocessed material, m is the mass measured and V is the volume measured. The calculated density of PCL was 1.124 ± 0.003 g/cm3.The density of PCL used in these experiments was calculated as follows:Thermal gravimetric analysis, using a TGA Q500 , was used to investigate the thermal degradation and to calculate the ceramic content on the printed scaffolds. Experiments were repeated 4 times per considered scaffold (n = 4). The tests were conducted in air atmosphere (50 mL/min) with a temperature ranging from 25 °C to 1000 °C at a rate of 10 °C/min. The weight of each sample was 200 mg, and each test was conducted twice.Differential scanning calorimetry (DSC) tests were performed to determine the melting temperature (Tm), the enthalpy (ΔHm), the crystallinity and the glass transition temperature (Tg) (n = 4). Tests were conducted using a TA Q100  under a nitrogen/air atmosphere (50 mL/min). The heating cycle was as follows: heating from −90 °C to 100 °C at a rate of 10 °C/min and then keeping stable for 2 min. The weight of each considered sample was 20 mg.Compression tests were performed using an INSTRON 3344  (n = 4), at different degradation time points, according to the ASTM D695-15. Force versus displacement curves obtained using the Bluehill Universal Software  were stress–strain curves. The compressive modulus was determined based on the slope of the elastic region of the stress–strain curves.Statistical analysis was performed using a one-way analysis of variance (one-way ANOVA) with Tukey’s post hoc test . Weight loss, TGA and DSC data were analysed using Origin 2021  and are presented as average values of the obtained results.The effects of the scaffold architecture and material composition on the degradation kinetics can also be observed in The thermal gravimetric analysis (TGA) results show that all considered samples exhibit degradation temperatures between 304.12 °C and 437.11 °C, after which only the inorganic materials remain . MoreoveAs samples were printed at 90 °C, the results show that the printing conditions do not induce any material degradation. Additionally, the levels of HA, TCP and bioglass determined with TGA  suggest DSC was used to investigate the crystallinity (Xc), melting temperature (Tm), enthalpy (Δm) and glass transition temperature (Tg) of bone bricks and rectangular scaffolds during the degradation process . The resThe results show that the melting temperature reduced for all the material compositions from day 0 to day 5, with the highest reduction observed for the bioglass scaffolds (there were no significant differences between PCL/bioglass and PCL/HA) and the lowest for TCP scaffolds. This can be explained by the decrease in crystallinity (with the highest reduction in the case of the PCL/HA and PCL/bioglass samples), and the melting temperature can be interpreted as the energy required by a system to destroy ordered regions. The results also show that the degradation occurs mainly through the destruction of the crystalline regions, which is aligned with the variations in both enthalpy and glass transition temperature that decrease and increase during the degradation time, respectively.The compressive modulus results for all considered samples at different degradation time points are indicated in The degradation kinetics of anatomically designed scaffolds with pore size gradients (bone bricks) and rectangular scaffolds with different pore sizes, considering a range of material compositions, was investigated, taking into consideration weight loss and morphological, thermal, chemical and mechanical changes. Due to the long degradation time of PCL, accelerated degradation, using NaOH, was considered. The results show that bone bricks present faster and more controlled degradation in comparison with rectangular scaffolds. The fastest degradation (weight loss) was observed for PCL/bioglass bone bricks and rectangular scaffolds. Moreover, for the same material composition, the degradation is faster in scaffolds presenting large pore sizes. The TGA results show that, in all considered cases, the concentration of the inorganic material remained the same during the degradation process. The DSC results indicate that the crystallinity of all samples at different degradation times decreased, suggesting a faster destruction of the crystalline regions than the amorphous ones. This observation is contrary to other studies suggesting that the degradation of PCL occurs through an initial mass loss that occurs due to the random hydrolytic split of polymeric chains in the amorphous regions, followed by a gradual degradation in the crystalline regions ,64,65,66


Here, we address whether it is possible for protocells to evolve greater metabolic complexity, through positive feedbacks involving nucleotide catalysis. Using mathematical simulations to model metabolic heredity in protocells, based on branch points in protometabolic flux, we show that nucleotide catalysis can indeed promote protocell growth. This outcome only occurs when nucleotides directly catalyse CO2 fixation. Strong nucleotide catalysis of other pathways (e.g. fatty acids and amino acids) generally unbalances metabolism and slows down protocell growth, and when there is competition between catalytic functions cell growth collapses. Autocatalysis of nucleotide synthesis can promote growth but only if nucleotides also catalyse CO2 fixation; autocatalysis alone leads to the accumulation of nucleotides at the expense of CO2 fixation and protocell growth rate. Our findings offer a new framework for the emergence of greater metabolic complexity, in which nucleotides catalyse broad-spectrum processes such as CO2 fixation, hydrogenation and phosphorylation important to the emergence of genetic heredity at the origin of life.The universal core of metabolism could have emerged from thermodynamically favoured prebiotic pathways at the origin of life. Starting with H The qpathways . These apathways –15. Expe archaea –18. Stri archaea , large p archaea ,22–24, g archaea  and gluc archaea , the pen archaea ,25 and s archaea  do occur archaea –31 and c archaea ,33 have  archaea –37.in situ by a geologically sustained disequilibrium, primarily between H2 and CO2 [The advantage of this hypothesis is that it readily explains how various components that makeup protocells could come together to form a living system: the chemistry is driven  and CO2 ,38–41. B and CO2 ,42,43. Y2 fixation [The simplest resolution to the nucleotide-amplification problem is that nucleotides could catalyse their own synthesis, either directly or indirectly. Nucleotide-derived cofactors include coenzyme A, NADH, FADH, ATP, and the pterins and folates involved in COfixation –46. ‘Nakfixation –49. It i2 fixation [2 fixation and specific protometabolic pathways [Theoretical models allow a quantitative analysis of what is possible, given the architecture of the system. We have previously shown that positive feedbacks involving FeS clusters chelated by amino acids can drive the growth of simple protocells composed of fatty acids and amino acids through COfixation . Experimfixation ,52, and fixation . Here, wfixation –57. The fixation . But incpathways ,40,59–61. 23+ and S2– are chelated by monomeric amino acids to form FeS clusters within protocells. These clusters associate with the membrane, where they draw on geochemically sustained proton gradients to facilitate CO2 fixation [a). The first steps of CO2 fixation form a two-carbon prebiotic equivalent to acetyl CoA  hC2 being converted into amino acids (AA), fatty acids (FA), sugars (S) and a primitive energy currency (E) (e.g. acetyl phosphate [b). We do not model individual steps but assume that whole pathways are thermodynamically and kinetically favoured [C2, but these are not considered further as they are irrelevant to turnover. The production of two types of amino acids is considered. The first set, AA1, chelates FeS clusters, which partition to the membrane and catalyse CO2 fixation. The second set, AA2, combines with sugars and energy currency to produce nucleotides (b). We arbitrarily assigned half of all amino acids to each set. The proportion of C2 channelled into the synthesis of each species is inferred from thermodynamic data under alkaline hydrothermal conditions  corresponding to low availability of inorganic phosphate [The model is developed by introducing several metabolic branch points that result in hosphate ; figure favoured . Other oleotides b. We arbditions , three additional catalytic positive feedbacks are considered: (i) catalysis of CO2 fixation by nucleotides (b), (ii) catalysis of individual protometabolic pathways alongside CO2 fixation (c) and (iii) nucleotide autocatalysis alongside CO2 fixation (d). A full description of the model is given in the electronic supplementary material.The dynamics of protocell metabolism are modelled by a system of ordinary differential equations describing the change in the number of molecules of each species over time. These changes are determined by the number of stoichiometric reagents and catalysts present in the system. Besides amino acid catalysis of COll model a, three leotides b, (ii) cfixation c and (iifixation d. A full. 3 (a)2 fixation produces fatty acids (FA), which directly contribute to membrane growth, and amino acids, some of which chelate FeS clusters (AA1), providing a positive feedback loop that enhances CO2 fixation (a). Additional branch points generate further species , which contribute to nucleotide production (b). In the absence of nucleotide production (a) or nucleotide catalysis (i.e. b), this has no effect on cell growth. However, the additional metabolic branch points can be beneficial when nucleotides catalyse CO2 fixation (i.e. b). Nucleotide catalysis of CO2 fixation : for increasing concentrations of all species in the cell  , and for changes in the initial concentration of amino acids and thus the initial ratio of concentrations in the cell . In both analyses, cells with lower concentrations  take longer to reach steady state, but the equilibrium concentrations are the same. We also repeatedly perturbed the concentrations of each species in the cell by adding stochastic segregation of molecules at cell division in a Moran model . At increasing levels of stochasticity , the stable-state division rate remained unchanged  as did the equilibrium concentrations of all species . Our analysis showed no bifurcation points or other unexpected dynamical features. (b)2 fixation as the base-case for comparison (b), we consider the consequences of nucleotide catalysis of individual branch points, enhancing the production of particular species in protocells (c). These consequences are observed through changes to the rate of protocell division . This is because increased amino acid synthesis (AA1) forms more FeS clusters, which promote CO2 fixation and so protocell growth through the positive feedback loop on amino acid synthesis. This positive feedback loop also generates more of the second set of amino acids (AA2), which contribute to nucleotide production, potentially enhancing the catalytic feedback of nucleotides on CO2 fixation. But that effect is self-limiting, as it does not overcome the disadvantage of channelling carbon away from other species. This disadvantage is clearly seen when catalysis increases the rate of the production of only one type of amino acid, leaving the other type unchanged . For these reasons, protocell growth and division collapses with strong catalysis of amino acids (a). If fixed carbon is excessively channelled into amino acid synthesis .Nucleotide catalysis of amino acid synthesis . Even though fatty acids directly contribute to protocell growth (by increasing membrane surface area), there is no positive feedback loop associated with fatty acid synthesis. As a result, the advantage to protocell growth of channelling C2 into the production of fatty acids does not overcome the disadvantage of channelling C2 away from other products. Again, these results are independent of the catalytic strength of nucleotides on CO2 fixation, but are more dramatic with higher b).By contrast, if nucleotides catalyse fatty acid synthesis . The proportion of C2 used in the production of energy and sugars is too low to channel much fixed carbon away from the production of amino acids and fatty acids, even with strong catalysis of these pathways. But if nucleotide catalysis strongly favours CO2 fixation, there is a benefit to channelling more resources towards an energy currency, which becomes more apparent as b). This difference reflects the assumption that less fixed carbon is channelled towards an energy currency than to sugar synthesis (C2) . In this case, the availability of sugars begins to limit nucleotide synthesis, so strong catalysis of sugar production now increases protocell growth.Catalysis of the pathways that synthesize an energy currency (y weakly a. The prncreases b. This d (c)a). That contrasts with catalysis of sugar production (a). The difference reflects the thermodynamic assumption that sugars are formed in excess of the requirements for nucleotide synthesis, so increasing their production does not substantially influence the concentration of nucleotides in the protocell.As well as altering the rate of protocell division , nucleotrotocell a. That cntration a. The diC2 towards amino acids retards the synthesis of sugars and an energy currency, whose production is essential to nucleotide generation (a). In addition, because weak or moderate amino acid catalysis of CO2 fixation drives protocell growth and division (a), nucleotide concentration is halved more regularly with each cell division, lowering their steady-state concentration. By contrast, strong catalysis of amino acid synthesis favours higher concentrations of nucleotides (a) because this collapses protocell growth (AA2) (a). This latter point explains the difference with fatty acid synthesis. Nucleotide catalysis of fatty acid synthesis (a). That is because channelling C2 away from the precursors of nucleotide synthesis  leads to a progressive decrease in the concentration of nucleotides.Nucleotide catalysis of amino acid synthesis  a. This lotocells a. That i2 fixation (2 fixation still generates similar levels of nucleotides in protocells (b). That is because fast catalysis of CO2 fixation . In this case, the energy currency drives nucleotides synthesis, which produces a stronger feedback on cell growth and division that in turn depletes nucleotide concentrations.These qualitative results are largely independent of the catalytic effect of nucleotides on COfixation . Faster otocells b. That i (d)2 fixation through the introduction of additional catalysis of individual pathways (α = 0). This lack of cost is consistent with one type of nucleotide catalysing CO2 fixation and different types catalysing specific pathways. An alternative modelling possibility is that increased catalysis of specific pathways decreases the catalysis of CO2 fixation. This would be the case if the same type of nucleotide performed both catalytic functions (α > 0). In this case, catalysis of individual pathways leads to a decrease in the rate of CO2 fixation, which feeds through to a decrease in the rate of protocell division (c). This effect applies whichever metabolic branch point is catalysed: the stronger the competitive catalysis of specific pathways, the greater the negative effect on CO2 fixation and the corollary decline in protocell growth rate (c).For the results described above, there is no cost to COdivision c. This ewth rate c. (e)d). This can be advantageous for protocell growth when autocatalysis of nucleotides and catalysis of CO2 fixation are coupled and both a). However, this combination does not generate high concentrations of nucleotides in the protocell (b) for similar reasons to those noted above. Although nucleotides are produced at a faster rate, they do not accumulate because with a high rate of CO2 fixation their production is coupled to an increased rate of protocell growth and division. Nucleotide accumulation is greatest when autocatalysis (2 fixation (b), which undermines the rate of protocell division (a) allowing nucleotide accumulation (b). A negative correlation between nucleotide autocatalysis .A final possibility analysed is direct nucleotide catalysis of their own synthesis, i.e. direct rather than network autocatalysis d. This care high a. Howeverotocell b for sim) is low b, which division a allowinmulation b. A negace versa a,b.Figu. 42 fixation alongside a variety of branch points leading to the formation of particular molecular species  as well as their own synthesis either in a direct  or indirect manner (as a system outcome) [Prebiotic nucleotide synthesis is a necessary precursor for any form of RNA world. The model developed here explores how the catalytic activities of nucleotide monomers (naked cofactors) could contribute to the autotrophic growth of protocells and the concentration of nucleotides inside them. Assuming that the first protometabolic systems could, at best, generate trace amounts of nucleotides, nucleotide synthesis would need to be amplified through some form of positive feedback. We have analysed a range of different types of positive feedback, in which nucleotides catalyse COoutcome) ,68,69. T2 fixation, meaning the conversion of CO2 and H2 into the reactive precursors of protometabolism (assumed to be prebiotic thioesters [c). Catalysis of specific pathways does not directly change total flux through the system, but instead diverts flux down one pathway at the expense of others. This adjusts the ratio of products formed away from the thermodynamically assigned proportions. The only useful targets of downstream nucleotide catalysis are therefore amino acid synthesis  and energy currency, albeit in the latter case only when nucleotides strongly catalyse carbon fixation (b). Because both these processes promote CO2 fixation, either directly or indirectly, their catalysis ultimately enhances metabolic flux through the entire network.We find that the most important way in which nucleotides can promote protocell growth is by steepening the driving force that pushes protometabolic flux through the entire network. This is primarily achieved by nucleotide catalysis of COioesters ,71; figuynthesis a,b and efixation b. BecausHowever, there is a critical proviso when considering the catalysis of specific pathways. Strong catalysis of amino acid synthesis unbalances protometabolism, as it shifts too much carbon away from the fatty acids needed for the expansion of membrane surface area, collapsing protocell growth. This finding stresses the primacy of balanced flux for protocell growth: strong catalysis of specific pathways tends to unbalance protometabolism and collapse growth. Conversely, non-specific catalysis of general processes that apply across the network—promiscuous catalysis—limits this problem. In fact, most universally conserved nucleotide cofactors are promiscuous, including NADH, FADH, acetyl CoA and ATP ,55,57,722 fixation, driving balanced flux through the whole network. Similar processes to those performed by nucleotide cofactors occur with FeS clusters, metal ions or minerals, giving a seamless transition from geochemistry to biochemistry [2, CO2, NH3 and HS− are needed, along with metal ions and protons, all of which readily cross leaky fatty acid membranes [Because cofactors can catalyse these reactions without their enzymes –49, the hemistry . This alhemistry ,76. For 2 fixation is fast (b), as the increased number of nucleotides feeds back on protocell growth, speeding cell division and so halving nucleotide concentration more frequently. This important relationship between catalyst concentration and growth rate is emphasized by nucleotide autocatalysis. The prime example of this is purine synthesis, which relies on ATP, meaning that ATP is needed to make ATP [2 fixation weakly, then autocatalysis contributes little to protocell growth, whatever the strength of autocatalysis . Instead, higher concentrations of nucleotides speed up protocell growth, lowering the nucleotide content as protocell division occurs more rapidly . This is an important general point: nucleotides will not accumulate to high concentrations in protocells if they have catalytic functions that speed up protocell growth and division. Thus, the premise for our model—protometabolic nucleotide synthesis requires catalytic positive feedbacks—precludes their continuous accumulation in actively growing protocells.Nucleotide catalysis of specific pathways can affect nucleotide concentration within protocells. Accelerated synthesis of nucleotide precursors  results in varying levels of nucleotide accumulation. Nucleotide concentration only increases sharply when the energy currency pathway is favoured , as ener is fast b, as themake ATP . If nuclWe have deliberately adopted a simple algorithm for cell divisions, which only takes into account the total number of fatty acids (used as a proxy for protocell surface area and volume). More sophisticated models of protocell division dynamics take into account factors such as osmotic pressure  and therOur focus on autotrophic metabolic heredity also sets our work apart from the rich literature of protocell models dealing with the origin of heredity, much of which considers protocells in the context of an RNA world. This literature inspired our theoretical framework, notably Szathmary's stochastic corrector model , for its2 by H2 [2 concentration , the dynamics are not strongly influenced by such factors. The baseline proportion of carbon channelled into each metabolic branch is likewise based on thermodynamic estimates [2 fixation [Is the assumption of autotrophic protometabolism valid? The relative values of the reaction rate constants used here, as well as specific concentrations and other parameters, allow us to capture the topological network constraints on growth, but omit many details. The reactions and catalytic steps assumed in the model link directly with phylogenetic reconstructions of early cells as obligate chemiosmotic autotrophs that grew from the reduction of CO2 by H2 . While tstimates ,15, and fixation ,15 appea2 and CO2 has garnered strength in recent years. Reactive equivalents to the C2 precursor modelled here have been synthesized experimentally under relevant prebiotic conditions [Experimental evidence for a protometabolism deriving from Hnditions ,21. Likenditions ,15 but hnditions ,27–31. Wnditions ,33, thernditions ,15, so inditions –61,91,92nditions –67, and nditions .x(OH)y and Ni3Fe [While there is no definitive evidence that this full set of protometabolic reactions can take place in a single setting, such as protocells, most experimental investigations share commonalities. In particular, in all the examples mentioned above, the chemistry takes place in an aqueous system. pH can vary, with some syntheses taking place in mildly acidic (approx. 4–5) ,23,61 ornd Ni3Fe ,27,94. Pnd Ni3Fe ,97. Othend Ni3Fe , equivalnd Ni3Fe ,61,93–95nd Ni3Fe ,61,93–95nd Ni3Fe  which rend Ni3Fe . OverallAssuming that such an autotrophic protometabolism is indeed feasible, the model developed here shows that there are constraints on how flux must operate in protocells. Catalysts must facilitate balanced flux through protometabolism to promote fast protocell growth via positive feedbacks. Selection for fast growth is likely to oppose the accumulation of catalytic nucleotides within protocells, albeit positive feedbacks favouring the synthesis of an energy currency can increase nucleotide concentrations somewhat. Most importantly, the model gives a new context for the emergence of the genetic code in autotrophic protocells . Whateve


We aimed to investigate the prognostic value of standard EEG  and poor functional outcome, defined by a score on the modified Rankin Scale (mRS) of 3 to 6 (moderate to severe disability or death) at 90 days, were investigated.We performed a retrospective analysis of consecutive ICU patients with severe herpes simplex encephalitis in 38 French centers between 2006 and 2016. Patients with at least one stdEEG study. The first stdEEG was performed after a median time of one (interquartile range (IQR) 0 to 2) day from ICU admission. At the time of recording, 138 (64.5%) patients were under invasive mechanical ventilation. Lateralized periodic discharges were recorded in 91 (42.5%) patients, seizures in 21 (9.8%) and status epilepticus in 16 (7.5%). In the whole population, reactivity to auditory/noxious stimuli was tested in 140/214 (65.4%) patients and was absent in 71/140 (33.2%) cases. In mechanically ventilated patients, stdEEG reactivity was tested in 91/138 (65.9%) subjects, and was absent in 53/91 (58.2%) cases. Absence of reactivity was the only independent stdEEG finding associated with poor functional outcome in the whole population  and in the subgroup of mechanically ventilated patients . Adjusted analyses for common clinical predictors of outcome and sedation at time of stdEEG revealed similar findings in the whole population  and in mechanically ventilated patients .We included 214 patients with at least one available Absence of EEG reactivity to auditory/noxious stimuli is an independent marker of poor functional outcome in severe herpes simplex encephalitis.The online version contains supplementary material available at 10.1186/s13613-023-01110-3. It is astdEEG) .stdEEG changes in HSE has been little studied. In a single-center retrospective study conducted on 103 patients hospitalized for all-cause encephalitis, including 12 HSE, a normal stdEEG was independently associated with a lower relative risk of death [stdEEG abnormalities were not associated with functional outcome [Standard EEG is often considered as an easily accessible, non-specific tool in the intensive care unit (ICU). It is recommended for initial evaluation of patients with suspected encephalitis to detect focal or diffuse changes suggestive of encephalitis and to rule out non-convulsive seizures . Standar outcome . A retro outcome . HoweverstdEEG findings observed in severe adult cases of HSE encephalitis requiring care in the ICU.In the present study, we aimed to investigate the prognostic value of common stdEEG recording performed at admission or during ICU stay. Exclusion criteria were: (a) missing data on clinical outcome at 90 days; (b) missing or incomplete stdEEG data. Standard EEG data was reviewed independently by 2 investigators, including a neurologist (LJ) and a neurophysiologist (GV).We conducted a retrospective analysis of patients from the multicenter HERPETICS database, which included adult patients with HSV encephalitis between January 2006 and December 2016 in 38 ICUs in France . The ethWe used the data previously collected for the HERPETICS database, including patients’ history, neurological symptoms and reason for ICU admission, clinical, biological and imaging data, and therapeutics .stdEEG reports: date of study, indication, and ongoing sedative and/or antiseizure medication at the time of the recording. We followed the American Clinical Neurophysiology Society’s guidelines to describe findings collected from stdEEG reports [stdEEG voltage and/or frequency immediately after an auditory or noxious stimulus. Reactivity on stdEEG recordings was categorized as “present”, “absent”, or “not tested” (when no auditory/noxious stimulus was applied during the recording) [We collected the following data from  reports : backgro reports . Status  reports . Reactivcording) .stdEEG study was available, we collected data from the first and second recordings. We studied evolution of reactivity between subsequent stdEEG studies and classified patients as “preserved reactivity” when both stdEEG were reactive to external stimuli, “absent reactivity” when none of the two stdEEG was reactive, and “variable reactivity” in patients with discordant reactivity between stdEEG studies .If more than one The primary outcome was a poor functional outcome at 90 days, defined by a score on the modified Rankin Scale (mRS) of 3 to 6, indicating moderate to severe disability or death.stdEEG findings with poor functional outcome in the whole cohort by means of univariable and multivariable logistical regression analyses. First, variables associated with outcome in univariable analysis  were entered in the multivariable model. Second, we applied a backward elimination to select variables for the final model. Secondary analyses included a subgroup analysis in patients under mechanical ventilation and adjusted analyses for common clinical predictors of outcome  and sedation at the time of EEG recording. Missing data were treated with multiple imputations using PROC MI and PROC MIANALYZE (SAS Software). All tests were two-sided and a p-value < 0.05 was considered statistically significant. All analyses were performed using SAS software, version 9.4  and R statistical software version 3.5.2 .Patients’ characteristics are expressed as counts and frequencies for categorical variables and medians  for quantitative variables. Comparisons were achieved using the Wilcoxon rank sum test for continuous variables and Fisher’s test for categorical variables. We investigated the association of the first stdEEG, and those of excluded patients who did not have a stdEEG recording or had missing stdEEG data  had two stdEEGs, 14 (6.5%) had 3 and 18 (8.4%) had more than 3 stdEEGs. Data from the first available stdEEG is detailed in Table stdEEG and between acyclovir administration and the first stdEEG were 1 (IQR 0–2) day, and 1 (IQR 1–3) day, respectively. Twenty-seven patients had their first stdEEG before admission to the ICU (with a median delay of 2 days (IQR 1–5) for these patients). Out of 192 stdEEG studies, 105 (54.7%) were performed for suspicion of encephalitis, 79/192 (41.1%) to rule out seizures and status epilepticus and 16/192 (8.3%) for both of these motives. Thirty-two patients out of 209 (15.3%) stdEEG recordings presented with a suppressed or low voltage, 25/213 (11.7%) were asymmetric in voltage and 65 (30.5%) were asymmetric in frequency. Thirteen out of 169 (7.7%) recordings had a maximal background rhythm frequency in the delta range. Lateralized periodic discharges (LPDs) were recorded in 91 (42.5%) patients. Sixty (28%) patients had interictal epileptic activities with spikes or sharp waves in 46 (21.5%) patients and spike-and-waves in 14 (6.5%) patients. Seizures were recorded in 21 (9.8%) patients and 16 (7.5%) presented with status epilepticus at the time of the first stdEEG. Reactivity was tested in 140/214 (65.4%) patients and was present in 69/140 (49.3%) recordings. No differences were found between the characteristics of patients for whom reactivity was tested, and those for whom it was not studied  subjects had one stdEEG included in the multivariable model were maximal background frequency, and stdEEG reactivity. A stdEEG with “absent reactivity” was found to be the only variable independently associated with poor functional outcome . A maximal background frequency recorded in the alpha range was found not to be independently associated with functional outcome (p = 0.187) in the multivariable analysis  patients had a poor functional outcome at 90 days, including 38 (17.8%) deaths. Results of the multivariable analyses of factors associated with poor functional outcome are presented in Table stdEEG reactivity was significantly associated with poor functional outcome in this population . Secondary adjusted analyses for common clinical predictors of outcome and sedation at time of EEG revealed similar findings in the whole population  and in patients under mechanical ventilation .These analyses are presented in Table A secondary subgroup analysis performed in patients under mechanical ventilation (Table stdEEG recording after a median time of 5 (IQR 2–12) days from the ICU admission. As for the first recording, the subsequent stdEEG were performed for seizures in 37/71 (52.1%) patients, for encephalitis suspicion in 14/71 (19.7%) patients, for both of these motives in 1/71 (1.4%) patients or to control the abnormalities shown on the first stdEEG in 16/71 (22.5%) patients. Sixteen (22%) patients had a first stdEEG with “present reactivity” and eight (50%) of them presented with preserved reactivity on the second stdEEG  had poor functional outcome at day 90. On the contrary, 58 (78%) patients had a first stdEEG with absence of reactivity, and among them 37 (50%) had a second stdEEG where reactivity was still absent. A persistent stdEEG with absent reactivity was associated with a poor functional outcome in 31/37 (84%) of these patients. Twenty-nine patients, classified as “variable reactivity”, had discordant reactivity between stdEEGs, and 21/29 (72%) patients had a poor functional outcome.Seventy-four (35%) patients had at least a second of 5 IQR –12 days stdEEG data and assessment of poor functional outcome  at day 90. Our findings suggest that the lack of EEG reactivity to external stimuli in critically ill patients with HSE is independently associated with poor functional outcome. This result was confirmed in a subgroup analysis of mechanically ventilated patients, who can be considered as the most severely affected patients with HSE encephalitis. Moreover, the prognostic value of absent EEG reactivity was independent of common prognostic factors identified in this population  [stdEEG patterns associated with poor neurological prognosis.This retrospective study assembled 214 critically ill patients with HSE from 38 centers, with available mission)  and was stdEEG with a background frequency greater than 4 Hz was the only variable independently associated with a reduced risk of death [The results from our study add to the recent body of evidence highlighting the prognostic role of EEG reactivity in critically ill patients with primary or secondary brain injury –19. Thesof death . Anotherof death . In our stdEEG in the positive diagnosis of HSE, especially in complex cases [stdEEG recordings performed in 25 patients, found stdEEG patterns (i.e. Synek III to V) to be associated with poor clinical outcome at 6 months [stdEEG data only included Synek grade and the presence of LPDs, and reactivity to external stimuli was not reported.Standard EEG is of crucial importance for the positive diagnosis of herpes simplex encephalitis and its complications, notably seizures, status epilepticus and periodic discharges that can indicate parenchymal necrosis. Previous studies have mainly investigated the role of ex cases , and lit6 months . HoweverstdEEG recordings, however, has been studied in brain injuries of other kinds. In a large registry of cardiac arrest patients, absence of EEG reactivity was predictive of unfavorable outcome [stdEEG background pattern [stdEEG reactivity and favorable clinical outcomes in consciousness impairments of various etiologies, but highlights the need for homogenous and consensual methods for reactivity assessment during stdEEG recordings [The prognostic value of  outcome . Preserv pattern . A recencordings .stdEEGs during hospitalization into three groups, we noted a worsening of the neurological prognosis when stdEEGs reactivity remained absent over time. Patients who had reactivity present on both stdEEGs (“preserved reactivity” group) had poor neurological prognosis in 63% of cases. Those who showed reactivity on neither stdEEG (“absent reactivity” group) had a poor prognosis in 84% of cases. The intermediate group (“uncertain reactivity”) with reactivity being present in only one of the two stdEEGs, seemed to evolve towards a poor prognosis in 72% of cases. Although our data was underpowered to allow proper analysis given the small number of patients who benefited from two stdEEGs or more, our study suggests that repeating stdEEG recordings as a follow-up for reactivity could be helpful during the ICU stay. This result requires confirmation by a prospective study with a standardized protocol .Interestingly, the persistence of reactivity over time could be associated with neurological outcome. Indeed, when dividing the 74 patients who had at least two stdEEG monitoring diagnosed with viral or auto-immune encephalitis found 22 (41%) patients who presented seizures [Clinical elements for prognostication in HSE have been identified in previous studies, such as older age, coma, admission body temperature and indirect admission to an intensive care unit . Our stuseizures . PatientstdEEGs recording. Our data was therefore insufficient to determine whether stdEEG could show abnormalities before MRI signs can be detected. A recent multicenter cohort study investigated MRI data associated with poor functional outcome and found that MRI signal abnormalities involving more than 3 cerebral lobes were associated with poor functional prognosis [stdEEG, making the precise localization of lesions and their extension difficult to establish.Since only two patients had a normal MRI at ICU admission in our cohort, MRI lesions were already present in the majority at the time of rognosis . LateralstdEEG reports) and lack of reproducibility between subjects: some patients had a stdEEG upon arrival to the ICU, others were recorded a few days later for complications or delay in the recovery of consciousness. This retrospective design did not allow proper standardization of stdEEG recordings, or a systematic assessment of reactivity with a unique protocol (same stimuli and recording length). Many stdEEG reports reported scarce data, not always in accordance with the American Clinical Neurophysiology Society’s standardized critical care EEG terminology [stdEEG patterns to establish prognosis in adult patients with diffuse anoxic and traumatic encephalopathies [stdEEG tracings by two independent reviewers, instead of stdEEG reports of various origins, could be a way to determine stdEEG patterns in HSE in a more objective way, but such tracings were unfortunately unavailable for retrieval. Moreover, the reports did not mention detailed clinical data at the time of the stdEEG recording, such as Glasgow Coma Scale, FOUR score or response to command, and most of the stdEEG reports had no precisions about the duration of the recording and number of electrodes. For the patients under mechanical ventilation, we could find no precision about the presence and type of sedation at the time of the recording. Secondly, our study lacks continuous stdEEG recordings which could allow a better monitoring of neurophysiological status , as some brief epileptic events might not be recorded with discontinuous short-lasting stdEEGs. This might have underpowered our study in assessing neurological prognosis and mortality in some patients, especially for epileptic complications. Thirdly, our population was limited to patients from intensive care units, which makes it difficult to extrapolate our results to less severely affected populations, such as patients admitted to neurology or infectious diseases departments. Finally, we observed that out of the 286 patients of the Herpetics database, 13 had not benefited from a stdEEG recording, while electroencephalography is recommenced for any suspicion of encephalitis. This could be explained by the large amount of centers in the study, including smaller hospitals, with no on-site Neurophysiology department and a more difficult access to electroencephalography, reflecting real-life access to the usual standard of care.The strength of this study was to assemble a large multicenter cohort of 214 HSE patients recruited from 38 ICUs in reference centers and smaller hospitals, which decreases the risk of center bias. The large number of patients allowed to performed multivariable analyses adjusted for common clinical predictors of outcome in this population. Our study also has limitations: firstly, this study had a retrospective design, which implies missing data  could also be studied, rather than the immediate stdEEG abnormalities in the acute phase. Such a study would allow the assessment of long-term cognitive prognosis, quality of life and epilepsy sequelae. However, HSE cases remain scarce, making the construction of such a protocol difficult.A prospective study with a standardized protocol for the timing of The absence of electroencephalographical reactivity to external stimuli is an independent indicator of poor functional outcome in adult patients with severe herpes simplex encephalitis, notably for mechanically ventilated patients. In addition to its use for the diagnosis of herpes simplex encephalitis and its complications, electroencephalography could help identify prognostic factors in often complex clinical situations.Additional file 1: Figure S1. Patients flow chart. Table S1. Patients’ characteristics, according to the inclusion/exclusion in the study, and according to the testing of reactivity. Table S2. Results of the uni- and multivariable analyses for all patients (n = 214). Table S2bis. Results of the uni- and multivariable analyses for all patients, including clinical data and adjusted for sedation (n = 214). Table S3. Results of the uni- and multivariable analyses for patients under mechanical ventilation (n = 138). Table S3bis. Results of the uni- and multivariable analyses for patients under mechanical ventilation, including clinical data and adjusted for sedation (n = 138). Table S4. Complete case analysis for the whole cohort (n = 194), and among patients under mechanical ventilation (n = 122).


The objectives of this study were to determine the effect of intravenous administration of antimicrobials  on equine fecal bacterial communities over time. Healthy horses were treated for 5 days with enrofloxacin, ceftiofur sodium, oxytetracycline and saline solution, and fecal samples were collected over 30 days. Microbiome analysis was carried out via 16S rRNA gene sequencing. Microbial diversity and abundance were altered using ceftiofur and enrofloxacin. This study showed that antimicrobials alter gut bacterial communities, which could predispose horses to gastrointestinal inflammation, diarrhea and possibly systemic disorders.p < 0.05), but not for other antimicrobials (p > 0.05). Microbial composition was significantly different (p < 0.05) across treatment group and day, but not for interactions between treatment and day, regardless of taxonomic level and beta-diversity distance metric. The most significant antimicrobial effects on relative abundance were noted after intravenous administration of ceftiofur and enrofloxacin. The relative abundance of Fibrobacteres was markedly lower on day 3 compared to other days in the ceftiofur and enrofloxacin treatment groups. There was an increase in Clostridia and Lachnospiraceae from day 0 to days 3 and 5 in ceftiofur and enrofloxacin treated groups. These findings showed the negative effect of antimicrobial drugs on bacterial communities associated with gut health (Fibrobacteres and Lachnospiraceae) and indicate that changes in specific taxa could predispose horses to gastrointestinal inflammation and the development of diarrhea.Alterations in the gastrointestinal microbiota after antimicrobial therapy in horses can result in loss of colonization resistance and changes in bacterial metabolic function. It is hypothesized that these changes facilitate gastrointestinal inflammation, pathogen expansion and the development of diarrhea. The objectives of this study were to determine the effect of intravenous administration of antimicrobial drugs  on equine fecal bacterial communities over time, to investigate whether those changes are detectable after 5 days of treatment and whether they persist over time (30 days). Sixteen horses were randomly assigned into 4 treatment groups: group 1 ; group 2 ; group 3 ; group 4 . Antimicrobial therapy was administered for 5 days. Fecal samples were obtained before (day 0) and at 3, 5 and 30 days of the study period. Bacterial DNA was amplified using specific primers to the hypervariable region V1–V3 of the 16S rRNA gene using a 454 FLX-Titanium pyrosequencer. Antimicrobial therapy failed to cause any changes in physical examination parameters, behavior, appetite or fecal output or consistency throughout the study in any horse. There was a significant effect of treatment on alpha diversity indices (richness) over the treatment interval for ceftiofur on days 0 vs. 3 ( Antimicrobial therapy is implicated in antimicrobial-associated diarrhea (AAD) in horses and humans ,3,4,5,6.Antimicrobial drugs associated with AAD in horses and commonly used in equine practice include tetracyclines, ceftiofur, enrofloxacin and potentiated sulfonamides ,13. ThesThe objectives of this study were to determine how intravenous oxytetracycline, ceftiofur and enrofloxacin modify equine fecal bacterial communities over time, to investigate whether those changes are detectable after 5 days of treatment, and whether they persist over time (30 days). We hypothesized that diversity of the equine fecal microbiome will be reduced by these antimicrobial drugs compared to placebo treatment and that 5 days of treatment will cause significant alterations, some of which will persist over a 30-day period post-treatment.Sixteen healthy horses of the teaching and research herd of The Ohio State University were used for this study with a mean and median age of 13.9 and 14 years old, respectively . Five mares and 11 geldings were used, and breeds represented included 5 Thoroughbreds, 5 Warmbloods, 3 Quarter horses, 2 Standardbreds, 1 Saddlebred and 1 Appaloosa.All horses were housed at the same location under standardized conditions and fed the same diet of grass hay for 3 weeks prior to study inclusion. Horses were considered healthy based upon physical examination, hematology, serum chemistry and fibrinogen concentrations. Horses had no evidence of endoparasitism based on examination of fecal samples. All horses were free of known GI disease, had no history of antimicrobial administration for at least 6 months prior to the study and were up to date on core vaccinations and deworming. The Ohio State University Institutional Animal Care and Use Committee approved this study.Horses were randomly assigned into 4 treatment groups: group 1 ; group 2 ; group 3 ; group 4 . All treatments and sampling were performed during the same time of year under the same conditions in all horses by two investigators .A 14 gauge 5.25 inch (13 cm) polyurethane catheter  was aseptically inserted in the jugular vein of each horse for antibiotic administration. Blood was drawn from each horse for complete blood count and serum chemistry analysis before treatment and at the end of the treatment period. Horses were administered enrofloxacin , ceftiofur sodium , oxytetracycline  and 0.9% sodium chloride solution  for 5 days. Physical examinations  and evaluation of fecal output and consistency were performed twice daily. Antimicrobial drugs were administered after fecal samples were obtained. Fecal samples were collected from the rectum of each horse via a sterile rectal sleeve every morning, frozen in liquid nitrogen and subsequently stored at −80 °C until processing.Samples from baseline , 1, 3, 5 and 30 days post-treatment were analyzed. Bacterial DNA was isolated from fecal samples using a commercial kit . DNA quantity and quality (260/280 ratio) was determined via spectrophotometry .Escherichia coli 16S rRNA gene . The forward primer carried the A pyrosequencing adaptor and a multiplex identifier (MID) sequence, while the reverse primer carried the B pyrosequencing adaptor. The following cycling conditions were used: denaturation at 94 °C for 3 min, followed by 32 cycles of 94 °C for 30 s, annealing at 60 °C for 40 s and 72 °C for 1 min; and a final elongation step at 72 °C for 5 min. A secondary PCR was performed to incorporate linker tags as described for multiplexed 454 FLX amplicon pyrosequencing . Amplified PCR products were purified using Ampure beads .Bacterial DNA was amplified using specific primers to the hypervariable region V1-V3 of the 16S rRNA gene using a 454 FLX-Titanium pyrosequencer  . An apprhttp://www.arb-silva.de, accessed on 15 July 2021) as reference and the RDP database was used for taxonomic classification. All OTUs belonging to the same genus were clustered for further analysis (phylotypes). Random subsampling was performed to avoid bias caused by uneven samples using the smallest number of reads found among all samples.The software Mothur  was usedOperational taxonomic unit counts for each sample were normalized via total sum scaling (relative abundance transformation). Relative abundances were calculated on all samples as the percent of taxonomically classified OTUs at each taxonomic level. Alpha diversity was calculated using Simpson’s (diversity), Chao-1 (richness) and Shannon’s evenness indices (evenness). A generalized linear mixed model was used to investigate differences within and between groups. The dependent variables for time, treatment and their interaction were forced into each model and the model residuals subjectively assessed for normality. To account for repeated sampling of the same horses over time, horse was included as a repeated statement.https://www.r-project.org, accessed on 1 March 2022), and p-values less than 0.05 were considered statistically significant.Principal coordinate analysis (PCoA) and permutational multivariate analysis of variance (PERMANOVA) were performed on beta-diversity metrics (Bray-Curtis and Jaccard distances) at phylum, family and genus level taxonomic grouping to visualize differences in microbial composition among treatment group and day. PERMANOVAs had 9999 permutations stratified by horse to determine significant differences in microbial composition across treatment group, day and treatment-day interactions. Pairwise differences on beta-diversity distances for treatment-day interactions were analyzed using Bray-Curtis distances at genus level taxonomy. Linear mixed models were built with pairwise distance as the outcome, treatment and day with interactions as fixed effects, horse as a random effect and all pairwise p-values being adjusted using Tukey’s HSD. Beta-diversity metrics (Bray-Curtis and Jaccard distances) and PERMANOVAs were calculated using the vegan R package (v. 2.5.7), PCoAs were performed using the ape R package (v. 5.5), and linear mixed models were analyzed using lme4 (v. 1.1.23) and emmeans (1.6.0) R packages. Data analysis was performed using R software v. 3.6.3  and rarefaction curves after subsampling verified adequate coverage of diversity in all samples (data not shown).p < 0.05) on day 0 compared to day 3  across treatment group and day, but not for interactions between treatment and day, regardless of taxonomic level and beta-diversity distance metric  to oxytetracycline and enrofloxacin treated groups compared to ceftiofur (p < 0.05) to oxytetracycline than to ceftiofur treated horses  [Escherichia spp.) eliciting an inflammatory response [The most significant antimicrobial effects were noted after intravenous administration of ceftiofur and enrofloxacin. Only minor changes were seen with oxytetracycline compared to the saline group. All three antimicrobials have broad spectra of activity and have some degree of gastrointestinal excretion; therefore, an impact on gut microbiota was expected. In our study, the relative abundance of Fibrobacteres was markedly lower on day 3 compared to other days in the ceftiofur and enrofloxacin treatment groups. This is of interest because horses with AAD had a reduction in the relative abundance of the Fibrobacteraceae family ,29 in hodiarrhea . The Fibdiarrhea ,31. Furtiraceae) ,32 leadiresponse ,34,35. TLachnospira, Roseburia, Butyrivibrio, Eubacterium, Ruminococcus and Blautia genera) play a crucial role in maintaining homeostasis of the gut [Clostridium perfringens and Clostridiodes difficile [The increase in Clostridia populations and the Lachnospiraceae family from day 0 to days 3 and 5 in ceftiofur and enrofloxacin treated horses is of interest because genera belonging to these taxa  for 5 days orally q12h significantly reduces richness but not diversity, suggesting that antimicrobial drugs affect mainly the low abundance taxa . In our The small number of horses and inclusion of several treatment groups with known interindividual variability of gut microbiota could have increased type II error and decreased statistical power preventing us from identifying further differences between treatment groups ,47. NoneDespite its limitations, studies like this represent an advancement to better understand the effects of exogenous compounds, disease, nutrition, drugs, supplements, prebiotics and probiotics on the equine gastrointestinal microbiome. Information regarding the effects of drugs commonly used in equine practice on the mucosal microbiota is scarce. Implementation of therapies that include the use of nutritional supplements, prebiotics, probiotics and fecal microbial transplantation are controversial because of the paucity of evidence of their benefits. While anecdotal reports and some studies have claimed clinical improvement of various equine gastrointestinal disorders in response to these interventions, the majority have failed to critically demonstrate benefits. These inconsistencies have been the result of inappropriate experimental design, lack of proper controls and inadequate quality and quantity of prebiotics, probiotics and volume of fecal microbial transplants. Additionally, we have learned from numerous microbiome studies in humans that dysbiosis secondary to antibiotic therapy can have long-term detrimental effects. These factors should be considered in the design of future studies as manipulation of the microbiota could potentially improve systemic health and reduce the use of drugs that could be damaging to other body systems.This study showed that antimicrobials commonly used in equine practice  can alter equine gastrointestinal communities, which could lead to gastrointestinal disturbances, promote inflammation and development of diarrhea, further antimicrobial resistance and can influence systemic health. By enhancing our understanding of the effect of antimicrobial drugs on equine gut health, it is a major goal to promote the rational use of these and other drugs, but also implement better antimicrobial stewardship practices.


To investigate the correlation between physical characteristics and disease of cervical lymph node biopsy specimens after contrast-enhanced ultrasound.All patients were biopsied after CEUS, 235 patients were divided into three groups A, B and C according to the physical characteristics of specimens: 92 patients in group A were complete tissue specimens; 113 patients in group B were discontinuous tissue specimens. There were 30 patients in group C, including a small number of tissue and floc, purulent and bloody specimens. Pathological examination, pathogen culture examination and Gene X-Pert MIB examination were completed for all patients in the three groups, and statistical analysis was conducted on the integrity and traits of the specimens.2 value of malignant and benign lymph nodes was 42.401, p = 0.000.Group A included 92 intact tissue specimens, 21 with reactive hyperplasia, 17 with lymphoma, 12 with metastatic carcinoma, 13 with lymphadenopathy, 15 with necrotizing lymphadenitis, and rare lymphadenopathy. In group B, 113 patients were treated with intermittent tissue specimens, including infected lymph nodes, lymphoma in 1 case, metastatic carcinoma in 3 cases and sarcoidosis in 1 case. There were 30 patients in group C, including a small amount of tissue and floc, purulent and bloody specimens, all of which were infected lymph nodes. The χThe physical characteristics of cervical lymph node biopsy specimens after CEUS are correlated with the disease, which has guiding significance for postoperative specimen selection. The common causes of cervical lymph node enlargement are cancer, metastasis, infection, reactive hyperplasia, and so on. Ultrasounds guided needle biopsy is widely used in the diagnosis of lymph node diseases –5. Due tThis study was reviewed and approved by the Medical Ethics Committee of Affiliated Hangzhou Chest Hospital of Zhejiang University, and patients gave informed consent. 235 patients with lymph node diseases treated in our hospital from March 2019 to March 2021 were collected and divided into groups A, B and C according to the specimen characteristics after biopsy. Group A were complete tissue specimens. Group B were discontinuous tissue specimens. Group C consisted of a small number of tissue and floccule, purulent and bloody specimens. The pathological specimens of all patients were biopsied by coarse needle, and the pus and blood specimens were extracted by fine needle. All patients in the three groups completed pathological examination, pathogen culture examination and Gene X-Pert MIB examination, and statistical analysis of specimen integrity and traits was conducted.Inclusion criteria: (1) cervical lymph node enlargement, maximum short diameter of lymph node > 0.5 cm, length to diameter > 1.0 cm; (2) Be above the age of 18; (3) There is no serious cardiopulmonary dysfunction and cannot tolerate needle biopsy; (4) There are visible specimens in the needle groove of puncture biopsy needle: The specimens visible to the naked eye in all the needle groove were group A, Discontinuous specimens or Biopsy needle groove with a sample size greater than 1/3 belonged to group B, The specimens with less than 1/3 of the needle groove or most of them fluid were in group C. Exclusion criteria: (1) In order to reduce allergic reactions and hypersensitivity reactions during the use of SonoVue, patients with a history of allergy to eggs, milk, fish and shrimp were excluded from this study; (2) patients who have taken anticoagulant drugs in the past week; (3) Patients with a history of mental illness who cannot cooperate with complete needle biopsy; (4) Skin damage, skin ulcer and severe psoriasis at the puncture site; (5) Patients whose target lymph nodes are located around blood vessels and cannot be routinely sampled or who have serious bleeding and other complications after sampling.All participants had given written informed consent and were enrolled in institutional protocols approved by our institutional review board.All patients underwent contrast-enhanced ultrasound examination. According to the evaluation of contrast-enhanced ultrasound, the largest lymph node on the affected side or abnormal lymph node with ultrasound was preferentially selected as the target lymph node. Ultrasound-guided puncture biopsy was performed, and all specimens were confirmed by pathology, etiological culture and Gene X-Pert MIB (+),Gene X-PERT MIB is a molecular diagnostic method for tuberculosis in recent years, which can provide diagnostic basis for the diagnosis of lymph node tuberculosis in this study). 94 patients were further confirmed by surgery later.Contrast-enhanced ultrasonography: (1) The largest lymph node on the affected side or lymph node with abnormal ultrasound appearance were selected as the target lymph node. (2) The L9-3 broadband linear array probe with 3.0–9.0 MHz frequency was used in the ultrasound angiography; pulse-inversion harmonic imaging with a low mechanical index (MI) of 0.06 was used in the imaging. The contrast agent was obtained from SonoVue (Bracco), diluted with 5 ml of physiological saline before use and shaken well, then 2.4 ml of the contrast agent was injected into the uperficial elbow vein via bolus injection, followed by 5 ml of physiological saline. (3) Perfusion enhancement of the whole lymph node was observed for 2 min in real time, then the image of the entire imaging process was stored on the instrument hard disk. The contrast-enhanced ultrasound results were categorized into three types of enhancement: homogeneous, heterogeneous and none. Partial enhanced lymph nodes and uniformly enhanced lymph nodes were selected for ultrasound-guided puncture biopsy, with the enhanced biopsy as the main sampling target area.2 test. Diagnostic tests were performed using sensitivity, specificity, positive, and negative predictive values, accuracy, and area under the receiver operating characteristic (ROC) curve. Differences were considered statistically significant at p < 0.05.Data analysis was performed using SPSS 23.0 statistical software. The measurement data are expressed as the mean ± standard deviation (x ± s) and were compared via t-tests. Countable data were expressed as the rate (%), and the two groups were compared using a χThe sex, age and lymph node CEUS mode of patients are shown in Table The comparison of puncture specimen status and disease correlation among patients in the three groups is shown in Table Comparison of physical characteristics of malignant and benign lymph node specimens is shown in Table The causes of superficial lymphadenopathy are various, Table In order to improve the integrity of biopsy specimens, this study used CEUS combined with puncture biopsy. CEUS can accurately display the active components and necrotic areas in lymph nodes. The integrity specimen rate was about 39.1% (group A), and the rate of discontinuous specimen was about 48.1% (group B). The specimen rate of small amount of tissue and floccule, purulent and bloody was 12.8% (group C), and the effective sample rate of selected pathological examination was 87.2% (group A and B), which was consistent with the report [There was a certain correlation between the proportion of enhanced area in lymph nodes and the disease. The integrity of cervical lymph node biopsy specimens was directly related to the proportion of enhanced area in lymph nodes, and homogeneous enhanced lymph nodes had higher specimen integrity. In this study, the specimens integrity of malignant lymph nodes after CEUS combined with puncture biopsy was higher than that of benign lymph nodes, The reason is that tumor cells and lymphoma cells in metastatic lymph nodes are hyperplasia and active, leading to high tension and hard texture in the lymph nodes. After biopsy, the specimens are usually complete strips. When there is necrosis in the lymph nodes, it is usually coagulated necrosis or blood vessel rupture and hemorrhage to form hematoma , showingSome studies have confirmed that lymphoma rarely occurs necrosis, usually caused by trophoblast artery embolization resulting in coagulative necrosis. CEUS can intuitively show the necrotic area in lymphoma , 14–17, There is a great difference in specimen integrity in lymph node infectious diseases Figs. , 3, whicIn this study, necrotizing lymphadenitis, sarcoidosis and other benign lymph node specimens accounted for about 75%, and most cases were located in group A. The integrity of specimens was related to the scope of necrosis. The number of sarcoidosis cases in this study was small, and the integrity of specimens needs to be further studied.For rare lymph node diseases, such as Castleman, Langerhans cell hyperplasia and plasmacytoid blastoid dendritic cell tumor, the puncture specimens in this study are all complete specimens, which are located in group A. Castleman is A hyperplastic disease, and its high integrity is related to its pathological state. The latter two are neoplastic. High integrity may be related to this. Kimura disease is a rich vascular endothelial cell hyperplasia, and a large lymphocyte, eosinophil infiltration of inflammatory lesions , and lymIn conclusion, the integrity of specimens after CEUS combined with biopsy is correlated with diseases to a certain extent. Specimens with high integrity have a high proportion of malignant lymph nodes, which may also be benign diseases such as reactive hyperplasia. CEUS shows homogeneous enhancement. Intermittent and liquid specimens are more likely to be benign lymph nodes. For example, in infectious diseases, specimen submission needs to be diversified, and most of its CEUS manifestations are heterogeneous enhancement and annular enhancement.


The rapid development of biogas plants in China has generated large quantities of digestate. The disparity between the continuity of biogas plant operation and the seasonality of digestate utilization has led to the need to store digestate. Therefore, untargeted profiling of bioactive compounds in the digestate stored under aerobic and anaerobic conditions was performed. The antioxidant and antifungal activity of digestate stored under varying conditions was likewise assessed. The results delineated that digestate storage under varying conditions brought about the degradation of organic acids, alkenes, aldehydes, alcohols, ketones, ethers, amino acids and their derivatives, and esters, leading to the stabilization of digestate components. Together, these new data revealed that digestate storage for up to 20 days under aerobic conditions promotes glycine, serine, and threonine degradation pathways and enhances biotin and vitamins production. In contrast, anaerobic storage enhances the taurine and hypotaurine metabolic pathways and increases the derivation of antimicrobial substances, such as indole alkaloids. Moreover, digestate storage under anaerobic conditions promotes antioxidant and antifungal activity more than storage under aerobic conditions. These findings can contribute to the future development of high-value agricultural products from digestate and the sustainability of biogas plants. Further studies are required for the untargeted metabolomic of digestate under storage to explore the underlying mechanisms of promoting disease resistance by the digestate upon land application. In recent decades, the demand for animal-derived food has risen dramatically due to continuing population growth and a shift in diet toward meat consumption . IncreasDespite its well-known agronomic benefits, the use of digestate as an organic fertilizer or soil amendment in farmlands is dependent on plant and crop seasonality. Besides this, the oversupply and improper distribution of digestate, especially in the regions of intensive biogas plants and its application at an inappropriate time, enhance air and water pollution ,9,10. CoDigestate is usually stored in open and closed reservoirs after solid–liquid separation. Digestate storage in open tanks leads to various problems, such as gaseous and odor emissions, the production of toxic compounds, and pathogen regrowth, thus limiting its potential fertilizing value . Few stuTherefore, to address this knowledge gap, the current study was undertaken for untargeted metabolomics profiling and the meta-analysis of biochemical changes that occur during digestate storage at different temperatures  under aerobic and anaerobic conditions based on the cloud-based mass spectrometric data analysis software (XCMS) integrated with the MetaboAnalyst program. Furthermore, the contents and type of bioactive compounds in the digestate preserved under aerobic and anaerobic conditions were compared. The antifungal and antioxidant activity of the digestate under varying storage conditions was also evaluated.Anaerobic digestate was collected from a continuous;y stirred tank reactor fed with swine manure at Donghuashan Biogas Plant  in Beijing, China. Basic physicochemical characteristics of the digestate are given in To evaluate the variations in untargeted metabolomics profiling of bioactive compounds, about 2 L of digestate was placed in amber glass bottles and incubated for three months at 4 ± 1 °C, 20 ± 1 °C, and 30 ± 1 °C under aerobic and anaerobic conditions. An-4, Ae-4, An-20, Ae-20, An-30, and Ae-30 were the names given to the digestate held under aerobic and anaerobic conditions at the formerly mentioned temperatures. Each treatment was repeated three times, with digestate samples obtained weekly from each treatment.testA). Around 5.006 mg of Trolox was dissolved in distilled water to obtain a 200 μmol Trolox standard stock solution. A total of 1 mL of Trolox standard solution was then mixed with 4.5 mL DPPH solution and used as a control (controlA). A mixture of 1 mL of methanol and 4.5 mL of DPPH solution was used as a blank (blankA) [The antioxidant activity of digestate was determined by the DPPH  method and then converted into the corresponding Trolox equivalents. Firstly, 7.856 mg of DPPH was weighed and dissolved in 200 mL of methanol to make a DPPH solution. After that, 1 mL of the digestate from different storage modes was mixed thoroughly with 4.5 mL of DPPH solution, left in the dark at room temperature for 30 min, and then the absorbance was measured at 517 nm ((Ablank) . The ant−1 was prepared by diluting the Trolox standard stock solution using distilled water. To convert the DPHA radical scavenging rate into Trolox equivalents, a standard curve was drawn, with clearance (x) as the independent variable and Trolox equivalent (y) as the dependent variable.A series of working standards from 20 to 60 μmol Lx is DPPH radical scavenging rate (%), and y is the Trolox equivalent (μmol/L).The regression equation was as follows:Fusarium oxysporum), which was procured from the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China. The fungi were inoculated into a potato dextrose-agar (PDA)  medium on culture plates for 4 to 5 days for rejuvenation and then used as an inoculum. The PDA medium was sterilized by autoclaving at 121 °C for 20 min. When the autoclave temperature was reduced to 40–50 °C, the extracts  from the digestate in different storage modes were added to the melted PDA medium at a concentration of 5%. After thorough mixing, the medium was poured into petri dishes. The rejuvenated fungal colonies were taken out of the incubator for inoculation. Mycelial pads (5 mm diameter) were taken from the leading edge of actively growing colonies using stainless-steel forceps and put upside-down in the middle of the petri dish. The inoculated plates were then incubated at 25 ± 2 °C for one week. In addition, a mixture of pure extractant (Dichloromethane: Methanol = 3:1) and PDA was used as a blank control. The antifungal test was carried out in triplicate. The diameter of the colonies was measured every 24 h for one week using Vernier calipers. The relative inhibition growth (RIG) rate was calculated as described by Tao et al. [D0 = colony diameter mixed with pure extractant (dichloromethane: methanol = 3:1) as the medium (cm), and D = colony diameter as a mixture of different storage modes of methane (cm) as a medium (cm).The antifungal activity of digestate from different storage modes was measured against the pathogenic fungus  at 40 °C and under a vacuum at 96 kPa.The digestate (20 mL) from each storage mode was blended with an equal amount of extractant (dichloromethane: methanol = 3:1) in a covered test tube. After that, 10 g of sodium chloride was added, and the mixture was agitated on an oscillator for one hour. An ultrasonic instrument was used to assist with the extraction process . The samples were first centrifuged for 30 min at 4032× m/z ranging from 35 to 600. The column was programmed so that the temperature increased at a rate of 5 °C min−1, from 60 °C to 180 °C; then, the rate was increased to 10 °C min−1 until the column reached 280 °C. The temperature was then maintained at 280 °C for 10 min. About 1.5 µL of the extract was then injected into the GC–MS using a micro-syringe, and scanning was performed for 50 min. After separation and detection, the compounds were eluted from the column and identified by comparing their mass spectra using the NIST08 data library [Digestate extract analysis was conducted using a GC–MS , equipped with a DB-5MS capillary column  and a quadrupole analyzer, operated in electron impact (70 eV) mode, with an  library .The raw GC–MS data were uploaded to the XCMS online program for peak identification, peak filtering, and peak alignment to obtain a data matrix including the nucleus to mass ratio and retention time and peak area. The processed data were subjected to a multivariate statistical analysis using SIMCA-P . An unsupervised multivariate pattern recognition method, namely a principal component analysis (PCA), was performed to examine the intrinsic variation in the data set. The supervised projection of orthogonal partial least squares discriminant analysis (OPLS-DA)) was performed for class discrimination. The quality of the OPLS-DA model was then assessed using 200 permutation tests. Variables were selected based on their effect on projection (VIP), reflecting the correlation between different storage conditions and the digestate composition.p-values from t-tests. The effect of aerobic or anaerobic storage on the most critical metabolites in the digestate was evaluated under varying storage temperatures, with a similarity index (SI) of >70% for the variables.To confirm the metabolic substances significant for digestate under varying storage conditions, all ions were sorted in descending order according to their VIP values. Potential metabolic markers were selected based on VIP values >1 and critical p) > 2 (p < 0.01) were considered to be the most significant derivative pathways for the generation of metabolites.To discover the differential metabolites that significantly changed in the experimental groups, a pathway analysis was performed using MetaboAnalyst 4.0, a web-based metabolomics data analysis software. An enrichment analysis was performed based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database among these differential metabolites. Pathways with impact values > 0 and –log  were exported from the raw GC–MS data  models were constructed on datasets after preliminary processing to get the intuitive metabolic distribution in the digestate under different storage modes . The PCAN-(2-phenoxy-ethyl)), and esters  were observed in digestate stored at 4 °C under aerobic and anaerobic conditions , the Euclidean distance matrix of the differential metabolites was calculated. Moreover, the top 40 differential metabolites were clustered in an ultimately linked fashion and displayed on a heat map a–c. Signnditions a.N-Acetyltranylcypromine were observed for digestate stored at 20 °C under aerobic and anaerobic conditions, respectively  into MetaboAnalyst 5.0 ) [To compare the metabolic changes in the digestate under aerobic and anaerobic storage conditions, an SMPDB enrichment analysis was performed by importing exclusive metabolites (h 2022)) . As showSome of the metabolic pathways in the digestate were disturbed by different storage modes, leading to an impact on the active components therein, for example, a decrease in the metabolism of vitamin substances and an increase in the purine substance-derived pathway under aerobic storage conditions. An increase in indole acetic acid production by tryptophan metabolism occurred mainly in anaerobic storage.Biotin is a water-soluble vitamin with antioxidant properties derived from the breakdown of volatile fatty acids, promoting plant root growth . Biotin Digestate storage under aerobic conditions promoted the metabolism of glycine, serine, threonine, and purine metabolism. The transamination reaction between serine and glycine to form hydroxyl pyruvate and glycine is catalyzed by glyoxylate aminotransferase and requires oxygen’s involvement b. As theThe metabolism of tryptophan and lysine during anaerobic storage of the digestate resulted in the production of castanospermine and tryptamine e,f. It mTaurine and hypotaurine metabolism pathways were detected in the digestate stored under anaerobic conditions, with the maximum relative abundance observed at 20 °C storage b. TaurinTo identify the effect of different storage modes on the antioxidant properties, digestate’s ability of digestate under varying storage conditions to scavenge DPPH radicals was determined . The antFusarium oxysporum was observed for digestate stored at 20 °C under aerobic and anaerobic conditions   b. ConverDurio zibethinus peel and seed for their antioxidant and antimicrobial activity [The increased antioxidant and antifungal activity in the digestate stored under anaerobic conditions was because of the accumulation of indolizidine alkaloids from ricin and tryptamine. The accumulation of cysteamine and taurine in the digestate stored under anaerobic conditions further promoted antioxidant and antifungal activity . Conversactivity . They reUntargeted metabolomics profiling in digestate stored at varying temperatures  and time (0 to 80 days) under aerobic and anaerobic conditions was performed. The antioxidant and antifungal activity of digestate stored under varying conditions was also evaluated. The results showed that the degradation rate increased with an increase in storage duration. The maximum number of metabolites was detected after 80 days of storage, irrespective of the temperature and mode (aerobic and anaerobic). Moreover, around 49 metabolic pathways of metabolite generation were observed for digestate stored under anaerobic conditions, while 40 metabolic pathways were detected in digestate stored under aerobic conditions. The maximum antioxidant and antifungal activity  were recorded for digestate stored at 20 °C for 20 days under aerobic conditions. This study also showed that metabolomics can be an innovative and promising approach to elucidate the derivation pathways of metabolites and provide new perspectives for a further analysis of digestate under varying storage conditions. More studies are needed for exploring untargeted metabolomic profiling and the antifungal activity of digestate under varying storage conditions to evaluate its disease resistance potential upon land application.