Pleurotus ostreatus, we obtained two catalase genes from a draft genome sequence of P. ostreatus, and cloned and characterized them (Po-cat1 and Po-cat2). Po-cat1 (group II) and Po-cat2 (group III) encoded putative peptides of 745 and 528 amino acids, respectively. Furthermore, the gene structures were variant between Po-cat1 and Po-cat2. Further research revealed that these two catalase genes have divergent expression patterns during different developmental stages. Po-cat1/Po-cat1 was at a barely detectable level in mycelia, accumulated gradually during reproductive growth, and was maximal in separated spores. But no catalase activity of Po-cat1 was detected by native-PAGE during any part of the developmental stages. In contrast, high Po-cat2/Po-cat2 expression and Po-cat2 activity found in mycelia were gradually lost during reproductive growth, and at a minimal level in separated spores. In addition, these two genes responded differentially under 32 °C and 40 °C heat stresses. Po-cat1 was up-regulated under both temperature conditions, while Po-cat2 was up-regulated at 32 °C but down-regulated at 40 °C. The accumulation of catalase proteins correlated with gene expression. These results indicate that the two divergent catalases in P. ostreatus may play different roles during development and under heat stress. Catalases are ubiquitous hydrogen peroxide-detoxifying enzymes. They participate in fungal growth and development, such as mycelial growth and cellular differentiation, and in protecting fungi from oxidative damage under stressful conditions. To investigate the potential functions of catalases in  The higive-PAGE . C. neofnditions . In thisnidulans , Paracoc. crassa . All thestreatus .Saccharomyces cerevisiae [Rhodotorula mucilaginosa [A. nidulans [Penicillium marneffei [P. brasiliensis [Po-cat1/Po-cat1 was significantly induced during heat stress, especially under 40 °C, but its high accumulation was not associated with any catalase activity. Po-cat2/Po-cat2 was induced under 32 °C heat stress, and the increased Po-cat2 activity could enhance the resistance of mycelia to adverse external environmental conditions. But Po-cat2 activity decreased under 40 °C heat stress. This is interesting, because one would expect that under this more stressful condition, the mycelia would synthesize more enzymes required for the decomposition of H2O2. In fact, decreased catalase activity under abiotic stresses has been found in many plants and fungi, such as Pisum sativum L. [Brassica napus [Triticum aestivum L. [Candida species [2O2 detoxification pathways function in this situation [Several researchers have reported the induction of catalases during heat stress for multiple species, such as revisiae , Rhodotolaginosa , A. nidunidulans , Penicilarneffei , and P. iliensis . In thistivum L. , Brassicca napus , Triticutivum L. , and som species . One pos species . Anotherituation .P. ostreatus genes, Po-cat1 and Po-cat2, which show sequence similarity to catalase genes. We analyzed their expression patterns during several growth stages and under heat stress. These two genes are diverse in structure and have diametrically opposed expression patterns during different developmental stages, and they also respond differentially under 32 °C and 40 °C heat stress conditions. Only Po-cat2 plays a catalytic function in decomposing H2O2 during any part of the life cycle of P. ostreatus, while the function of Po-cat1 is unknown. Further studies are required to reveal the specific function of Po-cat1 which has high expression during some stages but no detectable catalase activity. These results indicate that the two genes may play different roles during development and under heat stress. This study provides a greater understanding of the biological functions of catalases in edible mushrooms. This study also provides a reference for the elucidation of the mechanisms involved in mushroom development and stress resistance.In this study, we cloned and characterized the


Background: Adenosine can be produced in the heart and acts on cardiac adenosine receptors. One of these receptors is the A2A-adenosine receptor (A2A-AR).Methods and Results: To better understand its role in cardiac function, we generated and characterized mice (A2A-TG) which overexpress the human A2A-AR in cardiomyocytes. In isolated atrial preparations from A2A-TG but not from WT, CGS 21680, an A2A-AR agonist, exerted positive inotropic and chronotropic effects. In ventricular preparations from A2A-TG but not WT, CGS 21680 increased the cAMP content and the phosphorylation state of phospholamban and of the inhibitory subunit of troponin in A2A-TG but not WT. Protein expression of phospholamban, SERCA, triadin, and junctin was unchanged in A2A-TG compared to WT. Protein expression of the α-subunit of the stimulatory G-protein was lower in A2A-TG than in WT but expression of the α-subunit of the inhibitory G-protein was higher in A2A-TG than in WT. While basal hemodynamic parameters like left intraventricular pressure and echocardiographic parameters like the systolic diameter of the interventricular septum were higher in A2A-TG than in WT, after β-adrenergic stimulation these differences disappeared. Interestingly, A2A-TG hearts sustained global ischemia better than WT.Conclusion: We have successfully generated transgenic mice with cardiospecific overexpression of a functional A2A-AR. This receptor is able to increase cardiac function per se and after receptor stimulation. It is speculated that this receptor may be useful to sustain contractility in failing human hearts and upon ischemia and reperfusion. Typically, A1-AR inhibits and A2-AR stimulates adenylyl cyclase activities  in the mammalian heart: some reported a lack of effect  whereasect .2A-receptors in human hearts have been detected on the protein level (2A-KO (constitutive deletion) mouse hearts clearly established that CGS 21680 a classical A2A-receptor agonist was selective: only in WT hearts CGS 21680 (up to 1 μM) increased contractility but not in hearts from A2A-KO mice  mice was performed by standard procedures (mouse strain: FVB/N). One transgenic line overexpressing the A2A-AR under the control of the α-myosin heavy chain (MHC) promoter was established, which was investigated in the present study. Genotypes were identified by PCR analyses of tail-tip DNA using the following primers 5′-acaaagcaggcgatgaag-3′ and 5′-acccttaccccacatagacc-3′. For the reverse transcription 4 μg RNA and random primers were used . The PCR reaction was performed using the Ampliqon Taq DNA polymerase  according to the manufacturer’s instructions. For all experiments, 12–30 weeks old A2A-TG mice and WT littermates of both sexes were used.The PCR generated human A3 and 100 μl 20% SDS. Crude extracts were incubated at 25°C for 30 min before centrifugation to remove debris and thereafter, the supernatants (= homogenates) were separated and stored at -20°C until further use. Western blot analysis was performed as previously described . Following primary antibodies were used in this study: polyclonal rabbit anti calsequestrin, monoclonal mouse anti SERCA2a, polyclonal rabbit anti triadin, and polyclonal rabbit anti junctin , monoclonal mouse anti PLB , polyclonal rabbit anti phospho-PLB . The characteristics and use of these antibodies has been reported repeatedly by our group  for 1 h at room temperature and washing slides were incubated with mouse primary monoclonal antibody to A2A-AR . For detection a goat-anti-mouse antibody conjugated to Alexa Fluor 594  was used. Finally, samples were counterstained for approximately 15 s with DAPI and mounted with Vectashield . For imaging a Zeiss Axiophot2 microscope was used, separate color images  were merged by AxioVision multichannel image processing . Images shown are representative of at least five independent experiments, which gave similar results  in supine position. Additional doses of avertin  were applied during experiments if appropriate to maintain depth of anesthesia. A miniature pressure-volume catheter  was inserted via the right carotid artery and placed in the left ventricle. Increasing doses of dobutamine were administered into the cannulated left external jugular vein using an automated syringe pump . Data were recorded using the MPVS-400 system (Millar Instruments) and Chart5 software . At the end of experiments, animals were euthanized by avertin overdose, and hearts were excised, weighed, and stored at -80°C until further examination. Hemodynamic data were analyzed using Chart5 software (ADInstruments) and PVAN software (Millar Instruments).Mice were anesthetized by i.p. injection of avertin  in 2% solution at a dose of 400 mg kg-1 with a Ca2+-free solution (solution A) composed of (in mM) 140 NaCl, 5.8 KCl, 0.5 KH2PO4, 0.4 NaH2PO4, 0.9 MgSO4, 10 HEPES, 11.1 glucose (pH 7.1). Thereafter, hearts were perfused for 30 min with solution A supplemented with 0.2 mg ml-1 collagenase  and the Ca2+ concentration was gradually increased during digestion. After enzymatic digestion, the hearts were perfused for 10 min with solution A and ventricles were cut into several pieces before myocytes were separated by filtration through a nylon mesh.Ventricular cardiomyocytes were isolated using a published protocol . In brie2A-AR we used the following highly selective antagonists for pre-incubation: 1 μM DPCPX (A1-AR antagonist) or 1 μM ZM 241385 (A2A-AR antagonist). For subsequent stimulation activity 1 μM CGS 21680 [A2A-AR agonist  was present under all experimental conditions. In order to get an insight into the signaling via A agonist ] or isop-1) and hearts were excised. Right and left atria were dissected from isolated A2A-AR transgenic and wild type (WT) mice hearts and mounted in an organ bath. Left atrial preparations were continuously electrically stimulated (field stimulation) with each impulse consisting of 1 Hz, with a voltage of 10–15% above threshold and 5 ms duration. Right atrial preparations were allowed to contract spontaneously. The bathing solution contained (in mM) NaCl 119.8, KCl 5.4, CaCl2 1.8, MgCl2 1.05, NaH2PO4 0.42, NaHCO3 22.6, Na2EDTA 0.05, ascorbic acid 0.28 and glucose 5.0, continuously gassed with 95% O2 and 5% CO2 and maintained at 35°C resulting in a pH of 7.4. Signals detected via an isometric force transducer were amplified and continuously recorded. CGS 21680, ZM 241385or isoproterenol (1 μM each) were cumulatively applied with approximately 20 min for each compound. Contraction experiments were performed after addition of adenosine deaminase (1 μg ml-1) and DPCPX (1 μM) to avoid interference from endogenous adenosine or A1 adenosine receptor activation.Mice were anesthetized by i.p. injection of pentobarbital sodium  containing 118 mM NaCl, 25 mM NaHCO3, 0.5 mM Na-EDTA, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2.5 mM CaCl2, and 11 mM glucose in an isolated heart system . The heart preparations were allowed to equilibrate for 30 min before measurements. Hearts were stimulated at 8 Hz and heart rate, aortic pressure, and LV pressure were measured and monitored continuously. The first derivative of LV pressure (+dP/dt and -dP/dt) was calculated . To generate global ischemia, the perfusion was stopped for 20 min  and thereafter the hearts were reperfused for 40 min.Heart preparations were utilized as described previously . Mice weMeasurements of intracellular cAMP levels were performed by using the Biotrak direct enzyme immunoassay  according to the manufacturer’s instructions. The cAMP containing supernatant was acetylated , and divided into the donkey anti-rabbit IgG coated wells of a microtiter plate prefilled with anti-cAMP serum. Competition between unlabeled cAMP in the sample and a fixed quantity of peroxidase-labeled cAMP, for a limited number of binding sites on the cAMP specific antibody allowed quantification of intracellular cAMP.g and 4°C . The supernatant was centrifuged at 45,000 × g . The resulting supernatant was stored at -20°C. The pellet was resuspended in buffer B, containing KCl 600 mM, histidine 30 nM (pH = 7.0) and again centrifuged for 45 min at 64,000 × g. The pellet was resuspended in 250 μl buffer C, containing sucrose 250 mM, histidine 10 mM (pH = 7.0) and stored at -80°C until it was used for ligand binding experiments.Freshly obtained ventricular tissue was manually minced in small slices. Homogenization was performed at 4°C with Polytron  three times for 30 s at the speed of 20,000 rpm in buffer A, containing sucrose 250 mM, histidine 30 mM (pH = 7.4) and thereafter using Virsonic 100  for 2 s. This suspension was centrifuged for 10 min at 1,500 × 2A-AR was determined using [3H]-CGS 21680. Membrane proteins were diluted to the concentration of 2 μg μl-1. The binding assays contained 40 μg of membranes in A2A-TG and 400 μg in WT and in addition 8 nmol of [3H]-CGS 21680 in a final volume of 200 μl (A2A-TG) and 2 ml (WT), which contained also 10 U ml-1 adenosine deaminase. Unspecific binding was determined by addition of 1 μM ZM 241385. Binding assay was performed by incubation at 37°C for 90 min. Initially, the experiments for time dependency of ligand association were performed for 15–120 min. Furthermore, protein linearity was measured in the range of 20–120 μg protein. In order to determine Bmax and KD 16 increasing concentrations of [3H]-CGS 21680 from 0.1 to 20 nM were used. For assessment of the specificity of radioligand binding incubations in the presence of the A1-AR antagonist PSB36 (100 nM) as well as of the A3-AR antagonist MRS1220 were performed. Incubations were terminated by filtration . Radioactivity bound to the filters was determined by adding 5 ml scintillation fluid  in a counter .The density of At-test or using the χ2-test as appropriate. A p-value < 0.05 was considered as significant.Data shown are means ± SE. Statistical significance was estimated by analysis of variance followed by Bonferroni’s All other chemicals were of analytical grade. Deionized water was used throughout the experiments. Stock solutions were freshly prepared daily.2A-AR, we noted that the A2A-AR could be detected on the protein level for the following reasons. First of all, using very sensitive radioligand binding experiments, we could detect binding of the [3H]-labeled A2A-AR-agonist CGS 21680 samples form A2A-TG hearts. This binding of [3H]-labeled A2A-AR-agonist CGS 21680 could be almost completely blocked by addition of the (non-radioactively labeled) A2A-AR-antagonist ZM 241385, whereas (non-radioactively labeled) A1-AR-antagonist PSB36 or (non-radioactively labeled) A3-AR-antagonist MRS 1220 did not affect the binding of [3H]-CGS 21680. Interestingly, under the same experimental conditions used in samples from A2A-TG heart, no specific binding of A2A-AR using the [3H]-labeled A2A-AR-agonist CGS 21680 could be established in samples of the hearts from WT littermates . Secondly, the A2A-AR could be visualized using immunohistology in A2A-TG hearts. We tentatively identified the localization of the receptor in A2A-TG to the vicinity of the sarcolemma . Relative heart weight, an established parameter of cardiac hypertrophy, did not differ in whole hearts, ventricles or right atria between A2A-TG and WT (Table 1). These data exclude a cardiac hypertrophy of A2A-AR hearts at the two time points studied.In transgenic mice which overexpress the A2+ ATPase 2a], and triadin and junctin , were unchanged between WT and A2A-TG. However, PLB (phospholamban) expression was tentatively but not significantly increased in A2A-TG and is currently regarded as unchanged. Interestingly, at weeks 12 and 30 the expression of Gsα was lower in A2A-TG compared to WT ventricles (Table 2). On the opposite note, in ventricle from 30 weeks of A2A-TG mice an increase of Giα proteins was detected, but not at age 12 weeks or in atrial preparations (Table 2).The expression of CSQ , SERCA . At wee2A-TG and WT, invasively left ventricular function was assessed by a catheter while drug was continuously infused with a syringe pump into the jugular vein. The increase of the beating rate to infusion of dobutamine  was similar in A2A-TG and WT . Basal left +dP/dT was higher in A2A-TG compared to WT animals . However, in WT, dobutamine increased +dP/dT to a higher extent than in A2A-TG . In subsequent echocardiographic studies, basal heart rate and interventricular systolic septum thickness were increased in A2A-TG compared to WT. However, the effects of β-adrenoceptor agonist isoproterenol on both parameters were comparable between WT and A2A-TG animals .In Ap < 0.05 vs. WT, n = 5–7). During global ischemia (20 min duration), contractile response (here +dP/dt) rapidly stopped . Protective effects were smaller but also present after 40 and 60 min of global ischemia and reperfusion . Notably, basal rates of pressure development were higher in A2A-TG than in WT . Upon reperfusion, cardiac contractile response gradually resumed and relative similar pressure values were reached as before the ischemia in A2A-TG and WT . It is noteworthy that diastolic function is quite sensitive to ischemia in A2A-TG  and the increase in diastolic pressure and its normalization as well as increase and normalization of rates of pressure development were sensitive to 100 nM SCH 442416, an A2A-AR antagonist . Moreover, CGS 21680 was able to hasten and elevate the percentile restoration of ventricular + dP/dT .Global ischemia was induced in paced (8 Hz) isolated Langendorff-perfused hearts by complete stop of the perfusion. Hearts were paced because differences in heart rate, alone, might influence contractility. Basal heart rates before pacing amounted to 421.5 ± 14.5 bpm for WT and 467.8 ± 11.7 bpm for TG  and/or increased beating rate in isolated right atrial preparation (or in isolated perfused hearts or catheterized hearts of echocardiography). We reported this chain of events before for 5HT4 receptor overexpressing mice .The continuous stimulation of cAMP content by the overexpressed A2A-AR expression  is supported by the fact that an A2A-AR agonist increases force in left atrial preparations and beating rate in isolated right atrial preparations. Consistent with this the inotropic and chronotropic effects could be reduced by an appropriate antagonist.Our interpretation of a functional A1-AR overexpression as well as A3-AR receptor overexpression can protect cardiac contractility against short term ischemia [preconditioning . We extend those data  by describing a reduced expression of Gs in TG, studying isolated atrial preparations  and by studying the signal transduction in more detail. We concur with their data that cAMP (they measured adenylyl cyclase activity) is not increased in TG under basal conditions and therefore basal phosphorylation state of TnI and phospholamban was not enhanced (which they did not measure). We further extend their work by showing that the overexpressed A2A-receptor exhibits the expected pharmacology: namely the A2A-AR agonist CGS 21680 concentration dependently increased cAMP content in TG (but not WT) and likewise, probably by stimulating the activity of PKA, leads to increased phosphorylation state of phospholamban on its PKA sensitive phosphorylation site and in increased phosphorylation state addition of TnI.In contrast to our data they noted cardiac hypertrophy Table 1; . Similarin vivo functional interaction of A1A coexpression with A2A-receptors  not only in cardiomyocytes but also in cardiac endothelial cells and smooth muscle cells. Hence, any effect we observe in perfused hearts (in the absence of drug application) is probably due to the action of endogenously produced adenosine on the human A2A-AR in cardiomyocytes. Another advantage of our approach  but also on endothelial, smooth muscle and fibroblasts . These receptors may therefore be a target for pharmacological treatment of reperfusion injury. However, more detailed studies are called for and are expected with interest.Finally, the data on ischemia may suggest that the A2A-AR increases -physiological relevance. In this regard it might be of interest that during ischemia in isolated perfused hearts the expression of Ancreases . A2A agopatients .1 receptor mediated. We have not been able to perform similar experiments in human ventricular tissue . However, some years ago we reported at least an increase in the mRNA of A2A-AR in failing human ventricular samples compared to non-failing (2A-AR may sustain contractility in end stage human heart failure. Hence, A2A-AR may serve a physiological role in humans and, as shown for adenoviral expression of adenylate cyclase, genetic manipulation of A2A-AR may offer a novel venue for treating human heart failure.Furthermore, the direct comparison with the human situation is not easy. In earlier work, we reported that adenosine can in some atrial samples from patients exert a positive inotropic effect . However-failing . Thus, i2A-AR overexpression to cardiomyocytes using the established alpha myosin heavy chain promoter. Moreover, transgenic A2A-AR is functional after drug-induced stimulation to increase force and beating rate in mouse hearts or mouse heart preparations and the transgenic A2A-AR can protect the mouse heart against ischemia.We describe a transgenic mouse with AKD, JE, SG-W, LF, FS, and UK performed the research. PB, FM, WS, and JN designed the research study. PB, PK, NZ, UK, and JN analyzed the data. PB, UG, and JN wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


B lymphocytes are thought to play a relevant role in multiple sclerosis (MS) pathology. The in vivo analysis of intrathecally produced B cell-related cytokines may help to clarify the mechanisms of B cell recruitment and immunoglobulin production within the central nervous system (CNS) in MS.Paired cerebrospinal fluid (CSF) and serum specimens from 40 clinically isolated syndrome suggestive of MS or early-onset relapsing-remitting MS patients (CIS/eRRMS) and 17 healthy controls (HC) were analyzed for the intrathecal synthesis of IgG , CXCL13, BAFF, and IL-21. 3D-FLAIR, 3D-DIR, and 3D-T1 MRI sequences were applied to evaluate white matter (WM) and gray matter (GM) lesions and global cortical thickness (gCTh).p < 0.01), lower values of BAFF Index , and higher CSF CXCL13 levels . In these patients, BAFF Index but not CSF CXCL13 levels inversely correlated with the intrathecal IgG synthesis . CSF leukocyte counts were significantly higher in IgGOB+ compared to IgGOB− (p < 0.05) and HC (p < 0.01), and correlated to CSF CXCL13 concentrations .Compared to HC, CIS/eRRMS having IgGOB  had higher intrathecal IgG indexes (p < 0.05), while no difference in MRI parameters of WM and GM pathology was observed between IgGOB+ and IgGOB−.The gCTh was significantly lower in patients with higher CSF CXCL13 levels . In this study, CXCL13 identifies a subgroup of MS patients characterized by higher leukocyte counts in the CSF and early evidence of cortical thinning, further suggesting a role for this chemokine as a possible marker of disease severity. Several lines of evidence suggest that B cells play a central role in the pathogenesis and/or the clinical evolution of multiple sclerosis (MS). First, it is well known that the great majority of MS patients have quantitative (increased indexes) and/or qualitative  immunoglobulin changes in the cerebrospinal fluid (CSF), indicating an intrathecal Ig production , 2. SecoThe role of B cells in MS pathology may be multifaceted . Beside The presence of meningeal follicle-like structures (FLS) has stimulated the hypothesis of a “germinal center reaction” within the CNS of MS. Briefly, naïve B cells recruited in the CNS  may locally meet and present CNS antigens to T helper lymphocytes. Both B and T cells actively collaborate  to establish a germinal center reaction that leads B cell maturation to IgG-secreting PCs. PCs will survive within the CNS thanks to intrathecally produced B cell survival factors, such as B cell-activating factor (BAFF), as suggested by a recent observation [We studied serum and CSF levels of soluble factors involved in naïve B cell recruitment (CXCL13), in follicular reaction (IL-21) and intrathecal B cell survival (BAFF) in MS patients at clinical onset and correlated their intrathecal synthesis with local Ig production and with magnetic resonance imaging (MRI) parameters of white and gray matter damage. The aim of the study was to explore whether, at clinical onset, B cell-related cytokines could serve as immunological markers of MS severity/prognosis.Forty consecutive patients with a diagnosis of clinically isolated syndrome suggestive of MS (CIS) or early-onset relapsing-remitting MS (eRRMS), whose diagnosis was achieved in agreement with the most recent diagnostic criteria , were enSeventeen healthy controls (HC) were also enrolled in the study. These subjects complained tension headache, subjective transient sensory symptoms, psychosomatic disorders, etc. that underwent lumbar puncture for diagnostic purposes having unexpected, subtle, but unspecific white matter lesions at MRI. Immunological and coagulation screenings, microbiological and standard CSF analysis were normal; also upper aortic artery and Transcranic ultrasound EcoColorDoppler and visual evoked potentials were normal.No differences for age and gender were observed between eRRMS and HC Table . The stuIgG), CSF/serum albumin ratio (QAlb) to estimate the integrity of the blood-brain barrier (BBB), calculation of intrathecal IgG synthesis by means of quantitative formulae (IgG Index) [Alb was higher than the normal value for patient’s age . After cell centrifugation, both CSF and serum were stored at −80 °C until cytokine analysis.CSF was collected by non-traumatic lumbar puncture between 8:00 and 9:00 am. Routine examination on paired CSF and serum specimens included cell count and differentiation, CSF/serum IgG ratio (QG Index)  IgG ReibG Index)  and LocaG Index) , and demBAFF, QCXCL13, and QIL-21) and Index  were calculated.CSF and serum levels of BAFF , IL-21 (Bender MedSystems Cat. Numb. BMS2043TEN), and CXCL13  were measured by a highly sensitive immune-enzymatic assay ELISA according to the manufacturer’s instruction. BAFF concentrations (pg/mL) were measured on 1:2 diluted sera and on native CSF. IL-21 and CXCL13 concentrations (pg/mL) were measured on native sera and CSF. Cytokine ratio (CSF/serum cytokine: Q3. This sequence was acquired before and after gadolinium administration. (b) 3D-fluid attenuated inversion recovery (FLAIR): RT 4800 ms; ET 310 ms; inversion time (IT) 1650 ms; 365 contiguous axial slices with thickness of 1.0 mm; matrix size 256 × 256; and FOV = 256 × 256 × 182 mm3; (c) 3D-double inversion recovery (DIR): RT 13,000 ms, ET 10 ms, IT 3400/325 ms; 40 contiguous axial slices, resolution 1 × 1 × 3 mm, FOV 230 × 200 mm, time 3.5 min. Two experienced observers, blinded to the patient’s identity, assessed all images. The global cortical thickness (gCTh) was analyzed by means of Freesurfer on 3D-T1 sequences. WM lesions were identified on FLAIR sequences, while cortical lesions (CL) were identified on DIR scans by two blinded evaluators  using published consensus recommendations.Images were acquired using a 3T scanner  with 33 mT/m power gradient and a 32-channel head coil. No major hardware upgrades occurred during the study, and bimonthly quality-assurance sessions assured measurement stability. The following images were acquired for each subject: (a) three-dimensional (3D) turbo field echo : repetition time (RT) 7.8 ms; echo time (ET) 3.6 ms; 180 contiguous axial slices with the off-center positioned on zero with thickness of 1.0 mm; flip angle = 8°; matrix size = 220 × 220; FOV = 220 × 220 × 180 mmT test (when two groups were compared) or the ANOVA (for more than two group comparisons) were performed applying the Bonferroni correction. For ordinal categorical variables, the Mann-Whitney U test was performed, while for no ordinal variables, Pearson’s chi-square test was used. Linear correlation between variables was tested using Pearson’s single or multiple linear model when all variables were normally distributed. The significance level was set at p < 0.05.For normally distributed variables, the IgG, IgG Index, IgGLoc, and IgGIF) were significantly higher in CIS/eRRMS compared to HC. Higher QAlb values were observed in CIS/eRRMS (5.3 ± 2.3 × 10−3) than in HC , but only six CIS/eRRMS patients  had BBB damage.Table CXCL13 and QAlb , even when only CXCL13-positive CSF were included in the analysis . Since these data indicated that CXCL13 was intrathecally produced, rather than derived from passive filtration from blood, only the CSF concentrations of this cytokine were considered in further analyses. CXCL13 was detected more frequently  and at higher concentration  in the CSF of CIS/eRRMS than HC.In HC, CXCL13 was found in all sera and, at very low levels (0.9 ± 1.5 pg/mL), in 8/17 (47%) CSF. In these subjects, no correlation was observed between Qp = 0.3).In HC, IL-21 was detectable in all CSF (28.1 ± 29.1 pg/mL) but only in one serum, thus suggesting that CSF IL-21 was intrathecally produced. No difference in CSF IL-21 levels was observed between HC (28.1 ± 29.1 pg/mL) and CIS/eRRMS , indicating that CSF BAFF may result from both choroid plexus filtration and active production within the CNS. The BAFF Index was lower in CIS/eRRMS (12.4 ± 5.5 pg/mL) than in HC .BAFF was detected in all the sera and CSF of HC and CIS/eRRMS. In HC Qp < 0.01) and IgGOB+ (p < 0.05).Based on the presence of IgGOB in the CSF, CIS/eRRMS patients were divided in IgGOB positive  and in IgGOB negative  Table . Furtherp < 0.05) and HC (p < 0.005) .CSF CXCL13 concentrations were higher in IgGOB+ compared to both IgGOB− (05) Fig. . A higheNo difference in CSF IL-21 levels was observed between HC, IgGOB−, and IgGOB+ Fig. .p < 0.05, and 11.9 ± 6.1, both p < 0.005, respectively) but not in IgGOB−  compared to HC , MS patients , IgGOB− , and IgGOB+ .Finally, BAFF Index did not correlate to CSF CXCL13 concentrations in HC (p < 0.05). CSF CXCL13 concentrations, but not BAFF Index, significantly correlated to leukocyte counts  compared to HC  and HC (p < 0.01). In IgGOB+, CSF CXCL13 concentrations, but not BAFF Index values, significantly correlated to leukocyte number  than IgGOB−  compared to HC (2.34–2.58 mm). In order to find associations between CSF  and MRI parameters, we performed a subgroup analysis.μ + 4δ) that allowed the identification of two groups of CIS/eRRMS patients having CSF CXCL13 values below  or above  this limit. All HC had CXCL13 values below this limit.On the base CXCL13 concentrations in the CSF of HC, we found a cutoff value of 6.8 pg/mL .p < 0.05; Fig. μ + 3δ, i.e., 5.2 pg/mL) was applied.Interestingly, no difference in any MRI parameter was observed between IgGOB+ and IgGOB− or between IgGLoc− and IgGLoc+. CXCL13+ patients had a significant thinning of gCTh (2.42 ± 0.09 mm) compared to CXCL13− , that is correlated to disability progression in MS.Our findings are in line with the previous reports on the association of CXCL13 to leukocyte CSF counts , higher Other cytokines were found to increase in MS CSF . Among tOur findings indicate that intrathecally synthesized IgG are produced by PCs that enter the CNS from the peripheral blood at a given time and are locally retained as long-term PCs. A role for FLS as source of intrathecal IgG in early disease phases is not supported by our observations on CXCL13.In our study, CXCL13 identifies a subgroup of MS patients characterized by the early evidence of cortical thinning, further suggesting a role for this chemokine as marker of disease severity.


Allobates femoralis, a poison frog with predominantly male parental care, to investigate whether tadpole transport can be induced in both sexes by transferring unrelated tadpoles to the backs of adults in the field. Specifically, we asked whether the presence of tadpoles on an adult's back can override the decision-making rules preceding tadpole pick-up and induce the recall of spatial memory necessary for finding tadpole deposition sites. We used telemetry to facilitate accurate tracking of individual frogs and spatial analysis to compare movement trajectories. All tested individuals transported their foster-tadpoles to water pools outside their home area. Contrary to our expectation, we found no sex difference in the likelihood to transport or in the spatial accuracy of finding tadpole deposition sites. We reveal that a stereotypical cascade of parental behaviours that naturally involves sex-specific offspring recognition strategies and the use of spatial memory can be manipulated by experimental placement of unrelated tadpoles on adult frogs. As individuals remained inside their home area when only the jelly from tadpole-containing clutches was brushed on the back, we speculate that tactile rather than chemical stimuli trigger these parental behaviours.Understanding the external stimuli and natural contexts that elicit complex behaviours, such as parental care, is key in linking behavioural mechanisms to their real-life function. Poison frogs provide obligate parental care by shuttling their tadpoles from terrestrial clutches to aquatic nurseries, but little is known about the proximate mechanisms that control these behaviours. In this study, we used  Summary: Placement of unrelated tadpoles on adult poison frogs triggers a cascade of parental behaviours involving tadpole transport and spatial memory use in both sexes, despite the asymmetric parental sex roles. Studying the external stimuli and contexts that induce and modulate complex behaviours, such as parental care, is key to linking the proximate mechanisms to the function of behaviour . UnderstAllobates femoralis have revealed a surprising degree of flexibility in the parental care strategies of this species. In A. femoralis tadpole transport is primarily performed by males (Poison frogs (Dendrobatoidea) are a well-studied group of Neotropical frogs with complex life histories that involve parental care, including egg attendance and larval transport from terrestrial clutches to aquatic deposition sites . The adaby males . While mby males , males bby males , thus adby males , and wilby males .A. femoralis follows a sequence of stereotypical behaviours (Movie 1). First, to pick up the tadpoles, the parent lowers its body posture and rotates in the clutch, waiting for the tadpoles to wriggle onto its back. The tadpole carrier will then transport the offspring to, and sometimes distribute them between, small terrestrial pools, tens to hundreds of metres away from the territory . The frogs in the study area rely primarily on an array of 13 artificial plastic pools  for tadpole deposition.The study was carried out from January to March 2016 and 2017 in an experimental population established on a 5 ha river island  near theAllobates femoralis (Boulenger 1884) is a small diurnal frog common throughout the Amazon basin and the Guiana Shield .To obtain tadpoles for the experiments, we searched through the leaf-litter around the males' calling sites. In ge 24–25 ; usuallyA. femoralis usually initiate tadpole transport . We transferred 8–12 tadpoles, which corresponds to the average number of tadpoles transported naturally. We waited for a few minutes until the tadpoles settled on the back, then released the frogs as gently as possible to prevent the larvae from falling off. Occasionally, the frog lost a few tadpoles with a sudden escape movement during release. We never observed the frog losing additional tadpoles after the initial release. We continuously observed the frog for 5–15 min immediately after release, during which time most frogs remained immobile. During this period, we often observed the tadpoles wiggling on the frog and sometimes repositioning themselves on its back. To minimize the chances of testing a male with his own tadpoles, we only used tadpoles collected outside the respective male's territory and its immediate neighbourhood . To minimize the chances of testing a female with her own tadpoles, we used tadpoles collected at least 50 m away from the capture site of each tested female, as females preferentially mate with males within an approximately 20 m radius . We used a commercially available transceiver  for tracking. Following release, all frogs were located every 15–30 min during their daylight activity hours (∼07:30 h to 18:30 h). In each case, we carefully approached the location of the signal and attempted to detect the frog visually. In cases of poor visibility or if an individual was hiding, we narrowed the signal to less than 1 m. Every position fix was recorded on the background GIS map as described above. Whenever the frog was visible, we also recorded the number of tadpoles present and the current behaviour . In 2016 (tadpole group), we followed the frogs until all tadpoles were deposited. Most frogs completed tadpole transport and deposition within 1 day (mean±s.d. duration 4.47±2.48 h), but two individuals were tracked for 2 consecutive days (Table S1). All males were untagged immediately after tadpole deposition and were recaptured back in their territory over the following days. As females do not show conspicuous behaviour such as calling or territorial aggression, it was not always possible to confirm female homing by recapture. Thus, we tracked three females that had moved the longest distance after deposition to confirm that females also return to their home areas after tadpole transport. All three females tracked showed homing behaviour and moved back to their original home areas after tadpole deposition. In 2017 (jelly group), the frogs were tracked for 1 day between 6.7 h and 9.4 h (mean duration 7.92 h) and untagged in the evening between 17:30 h and 18:45 h.We used the harmonic direction-finding telemetry technique to track individuals and followed a protocol that has been successfully used in ransport ,b. The sA. femoralis do not move at night, for the two frogs that were tracked overnight, we subtracted 12 h for the calculation of movement speed. We compared the total duration of tadpole transport between males and females with and without subtracting the 12 h.Visualization and extraction of coordinates were done in the GIS software ArcGIS™ 10 (ESRI). To describe the spatio-temporal characteristics of frog movement to the pools, we quantified the latency to move, the speed of movement and the distance travelled to the first deposition site, and the total duration until all tadpoles were deposited. The latency to move was defined as the time taken to leave a 5 m radius area from the capture/release site. This cut-off was chosen based on the distance to the closest deposition site across all frogs and it allowed us to separate various local movements around the release site from the directional movement towards the deposition site. As U-test for sex differences in temporal and spatial parameters. We also used the Mann–Whitney U-test to compare the speed of movement and SC of induced male tadpole transport with natural male tadpole transport (see http://www.R-project.org/).To estimate orientation accuracy, we calculated a straightness coefficient (SC), defined as the ratio between the straight-line distance and the actual path distance between the starting point and the first deposition site (such that SC=1 would indicate a perfectly straight path). SC is a simple but robust estimator of the efficiency of a goal-oriented path . For theport see . No tracOur study was approved by the Animal Welfare Board of the University of Vienna  and by the scientific committee of the research station where fieldwork was conducted. All necessary permits were provided by the local authorities (DEAL: ARRETE no. 2011-44/DEAL/SMNBSP/BSP). All sampling was conducted in strict accordance with current French and EU law and followed the ‘Guidelines for use of live amphibians and reptiles in the field and laboratory research’ by the Herpetological Animal Care and Use Committee (HACC) of the American Society of Ichthyologists and Herpetologists.female=35±31 m, pathmale=38±20 m) at an average speed of 10 m h−1 . The average latency to leave the 5 m-radius area around the capture site was 1.92 h  and the total duration until deposition was on average 6.47 h . The average orientation accuracy (SC) was 0.87 . We did not find any significant differences between the sexes in any temporal and spatial parameters measured . We also found no significant difference between movement speed and orientation accuracy of males during induced and natural tadpole transport .All 20 individuals from the tadpole group transported and deposited the cross-fostered larvae. Seventeen frogs deposited in artificial pools and three frogs used natural pools A. Two fr−1 and only three frogs  left the 5 m-radius area around the capture sites.Only one frog  out of 20 moved to a pool after being swabbed with jelly from tadpole-containing clutches. During the entire tracking period, jelly group frogs moved on average 8.16±6.45 m (mean±s.d.) at an average speed of 1.1±0.9 m hA. femoralis females rarely and selectively transport tadpoles, both sexes were equally likely to transport cross-fostered tadpoles. We also found no difference in the accuracy or speed of finding deposition sites between males and females.We successfully triggered tadpole transport and deposition in all tested frogs by transferring unrelated tadpoles onto their backs. In contrast, all frogs except one subjected to the same handling procedure and brushed with jelly from tadpole-containing clutches remained in their home areas. Even though A. femoralis do not use direct cues for offspring recognition (Oophaga pumilio (Our findings show that the decision-making processes and motor actions involved in tadpole pick-up do not need to precede successful tadpole transport B. They aognition , similar pumilio . Our abi pumilio  and the  pumilio . It suggOur study provides limited information about the exact cues that trigger tadpole transport, but we speculate that tactile stimuli may play a predominant role. Dorsal swabbing with jelly from clutches that contained ready-for-transport tadpoles did not trigger the parental behaviour, suggesting that chemical cues passively released by tadpoles are not sufficient as a trigger. Tadpoles wriggling up and onto the back of a parent could provide a variety of tactile cues to initiate tadpole transport. Tadpoles of several tropical anurans, including dendrobatid frogs, are known to use tactile and vibrational cues for parent–offspring communication in the context of begging behaviour . The tacA. femoralis, females can produce clutches on average every 8 days and males commonly have several clutches simultaneously inside their territory (Allobates femoralis males appear to be an exception to this pattern as they maintain high levels of aggression and territoriality while being in a state of parental readiness. Neuroendocrine control of parental care in poison frogs has recently attracted some attention (Even though tadpole transport was triggered in all tested individuals, there was a marked and highly variable latency between tadpole transfer and the initiation of movement towards a deposition site. Several factors such as the nature of the trigger or the spatio-cognitive processes preceding long-distance movement may influence this latency, but we speculate that the motivational/hormonal state of the individual is likely to be the key element. Complex cycles of neuroendocrine parental care regulation in mammals, birds and fish are often preceded by reproductive triggers such as mating . In A. ferritory . As a reerritory , includierritory . Allobatttention , but to Ammophila spp.; Philanthus spp.; A. femoralis, the presence of tadpoles on the back induces a stereotypical sequence of orientation, fast directional movement, pool inspection, tadpole deposition and homing – behaviours dependent on the recall of long-term spatial memory (To sum up, our results echo the findings of classical ethological studies on parental spatial behaviour of digger wasps (l memory , 2016. T


Delirium is characterized by disturbances in circadian rhythm. Melatonin regulates our circadian rhythm. Our aim was to compare preoperative cerebrospinal fluid (CSF) melatonin levels in patients with and without postoperative delirium.Prospective cohort study with hip fracture patients ≥ 65 years who were acutely admitted to the hospital for surgical treatment and received spinal anaesthesia. CSF was collected after cannulation, before administering anaesthetics. Melatonin was measured by radioimmunoassay (RIA). Data on delirium was obtained from medical and nursing records. Nurses screened every shift for delirium using the Delirium Observation Screening Scale (DOSS). If the DOSS was ≥3, a psychiatrist was consulted to diagnose possible delirium using the DSM-IV criteria. At admission, demographic data, medical history, and information on functional and cognitive status was obtained.p-value 0.47. No differences between patients with and without delirium were found in mean melatonin levels in analyses stratified for cognitive impairment or age.Seventy-six patients met the inclusion criteria. Sixty patients were included in the analysis. Main reasons for exclusion were technical difficulties, insufficient CSF or exogenous melatonin use. Thirteen patients (21.7%) experienced delirium during hospitalisation. Baseline characteristics did not differ between patients with and without postoperative delirium. In patients with and without postoperative delirium melatonin levels were 12.88 pg/ml (SD 6.3) and 11.72 pg/ml (SD 4.5) respectively, Preoperative CSF melatonin levels did not differ between patients with and without postoperative delirium. This suggests that, if disturbances in melatonin secretion occur, these might occur after surgery due to postoperative inflammation. Delirium is a neuropsychiatric syndrome that is highly prevalent in older persons admitted to the hospital. PatientsRecent studies found that plasma melatonin levels differed in patients with dementia, after surgery and with ageing in general.–14 In olThe majority of studies have measured melatonin in blood. Because of the proximity to the pineal gland and the brain itself, cerebrospinal fluid (CSF) measurements are an interesting target for research and could perhaps better reflect cerebral processes. As melatonin is released directly into CSF, this could provide more accurate information on melatonin production and secretion. Presently, little is known about human CSF melatonin levels. Research in CSF and brain tissue of diseased patients showed that patients with Alzheimer’s disease had lower levels of melatonin in CSF and in brain tissue compared to healthy controls.–21 Also,Elderly patients with a hip fracture are prone to develop delirium after surgery and commonly receive spinal anesthesia whereby CSF can be obtained without an additional invasive procedure. If alterations in CSF melatonin levels were associated with higher delirium incidence, this would provide more insight into delirium pathophysiology. No CSF melatonin concentrations are known in patients with delirium. Therefore, the aim of this study was to compare preoperative CSF melatonin levels in elderly patients with and without subsequent (postoperative) delirium.We conducted a prospective cohort study. From March 2012 to April 2014, individuals aged 65 years and older who were acutely admitted for surgical repair of a hip fracture to the Sint Lucas Andreas Hospital in Amsterdam were recruited. Written informed consent was obtained from the patient or legal representative in case of incompetence. Patients who did not receive spinal anesthesia, were unable to speak or understand Dutch or English, or left the surgical ward within 48 hours after admission, were excluded. The choice for general or spinal anesthesia was at the doctors’ and patients’ discretion and based upon the hospital’s protocol. The Medical Ethics Committee of the Academic Medical Center Amsterdam approved the study protocol. The study was conducted according to the Good Clinical Practice guidelines and in compliance with the Declaration of Helsinki.Demographic data, medication use at home and medical history were collected at admission. Functional decline was measured by the 6-item KATZ Index of Activities of Daily Living in which the current status was compared to the situation two weeks prior to admission with a maximal score of 6.The primaNurses screened all patients 3 times a day (once every shift) for delirium using the Delirium Observation Screening Scale (DOSS) seven days a week. In the Netherlands, delirium screening with the DOSS is recommended in the Dutch delirium guideline and is part of daily clinical practice in most hospitals., 29 A cuSurgery was performed daily and was not limited to office hours. CSF was collected after cannulation for the introduction of spinal anesthesia, prior to administering anesthetics. CSF samples were immediately brought to the laboratory. After centrifugation, the samples were alliquoted and stored at -80°C until melatonin measurement. Melatonin was measured in duplicate by a direct radioimmunoassay . If the two measurements differed < 12.5% from each other (the coefficient of variation of the RIA), we took the mean of the measurements. If the measurements differed > 12.5%, we did not use these and performed a new measurement in duplicate. The lower detection limit of the assay was 3 pg/ml.2 test, Fisher’s exact test or Mann-Whitney U test, as appropriate. Differences in melatonin level between groups were assessed with an unpaired T- test or with an one-way ANOVA with Bonferroni post-hoc tests. To assess the correlation between age and CSF melatonin levels, a Spearman’s rank correlation test was used to avoid normal distribution assumptions of the data. We determined the season when the sample was collected to control for their influence as melatonin levels might be lower in winter compared to summer. We defined season by the month of surgery. Summer was defined as June, July or August, autumn as September, October or November, winter as December, January or February and spring as March, April or May.All analyses were performed using SPSS version 22. DescriptIn total, 250 consecutive patients underwent hip surgery from March 2012 to April 2014 of which the vast majority were over the age of 65 years old. One hundred and twenty-four patients were enrolled in our study. Of these enrolled patients, 76 persons received spinal anesthesia and provided CSF. Sixty patients were included in the analysis. Main reasons for exclusion were that samples could not be identified, exogenous melatonin use or insufficient CSF for melatonin measurement.  Of thesep-value 0.47. (p = 0.13). No differences in mean melatonin levels were found between patients who experienced postoperative delirium and those who did not in analyses stratified for cognitive impairment.In all samples, the melatonin concentrations were within the detection limit of the assay. Mean preoperative CSF melatonin level was 11.87 pg/ml (SD 4.9 pg/ml). In patients with postoperative delirium, melatonin levels were 12.88 pg/ml (SD 6.3 pg/ml) and in patients without postoperative delirium, melatonin levels were 11.72 pg/ml (SD 4.5 pg/ml), ue 0.47.  One patiTo the best of our knowledge, this is the first study that measured melatonin in CSF in older hip fracture patients and evaluated the association of CSF melatonin levels with delirium. We could not demonstrate a difference in preoperative CSF melatonin levels in older hip fracture patients that experienced postoperative delirium compared to older hip fracture patients who did not. This suggests that the melatonin level is not different for patients at risk for delirium.Alterations in melatonin secretion or secretion pattern may cause older persons to develop delirium or possibly even induce delirium. Cronin aMean CSF melatonin levels in our study were 11.87 pg/ml. Previous studies reported CSF concentrations from 1.47 pg/ml up to 542 pg/ml in physiological conditions., 33–37 TDue to the preliminary nature of this study and small number of patients with delirium, no multivariate analysis was performed. However, analyses stratified for the most important confounders, age and cognitive impairment did not show differences in CSF melatonin levels. In our population, baseline characteristics that were associated with a higher risk of developing delirium, like higher age and cognitive impairment, did not differ between patients who experienced delirium and patients who did not. This could indicate that the number of patients experiencing delirium in our study was too small.CSF samples were obtained under bright light conditions and under stressful circumstances for the patient. Although the time to surgery was short, hospital stay and experiencing pain could already have induced sleep disturbances to some extent in any of our patients. These factors could have confounded our findings and endorse the need for CSF measurements in non-surgical populations and for determination of physiological CSF melatonin levels in elderly. More research is required to further explore our preliminary findings.In this study, a CSF sample was obtained before surgery and delirium usually was diagnosed within 48 hours after surgery. Therefore, differences in CSF melatonin concentrations may have been missed due to the time between sampling and the occurrence of delirium. No information about the secretion pattern of melatonin could be obtained because we were only able to obtain one CSF sample due to practical and ethical reasons. As circadian rhythm disturbances frequently occur in delirium, alterations in melatonin concentrations could happen later on in the disease process and preoperative sampling may be premature to see meaningful change. No conclusions about CSF melatonin levels during delirium could be drawn as only one person was delirious at the time of CSF sampling. Timing of sampling appears very important and could have been a cause for not finding an association between CSF melatonin levels and postoperative delirium in this study.Overall, the incidence of delirium was 21.7% in this study which is line with previous studies describing delirium incidence in postoperative older people. The timeFuture research should focus on melatonin levels in the perioperative period, and repeated sampling before, during and after delirium. Also the relationship between CSF melatonin levels and blood melatonin levels must be further explored to see if blood levels correlate with CSF melatonin concentrations. To further explore our preliminary findings, future research should include a larger sample size to exclude a type 2 error, diagnose delirium according to the DSM criteria, and include measurements in the non-surgical population.S1 File(SAV)Click here for additional data file.


Corynebacterium striatum (C. striatum) as causative agent in a 53-year-old woman.We present an extremely rare case of tubo-ovarian abscesses involving Prevotella spp. and C. striatum. We concluded that C. striatum from skin contaminated by psoriasis vulgaris had caused the tubo-ovarian abscesses by way of ascending infection.The patient presented with stomach pain, chills, and nausea. Her medical history included poorly controlled psoriasis vulgaris and diabetes. Laboratory and imaging findings led to diagnosis of septic shock due to tubo-ovarian abscesses. She was treated with antibiotic therapy and surgery to remove the left adnexa. Various cultures detected C. striatum. In patients whose skin has been weakened by psoriasis vulgaris or other infections, Corynebacterium should be considered as causative microorganisms, and antibiotic therapy including vancomycin should be administered.This may be the first known case of tubo-ovarian abscesses due to  Tubo-ovarian abscesses are inflammatory masses involving the fallopian tubes and ovaries as well as adjacent pelvic internal organs, and usually arise as lower genital tract infections that ascend and seed the upper reproductive tract  in women of reproductive age . Tubo-ovCorynebacterium striatum (C. striatum) as causative agent.Here, we present an extremely rare case of tubo-ovarian abscesses involving A 53-year-old woman presented to the emergency department for evaluation of abdominal pain, chills, and nausea. Her medical history was notable for psoriasis vulgaris, diagnosed at age 42, for which she had received intermittent treatment, but had not received any treatment in the preceding 6 months Fig. . She alsThe patient had felt abdominal bloating and pain for 3 days prior to presentation. Two days before presentation, the pain had moved to the lower left part of the abdomen, where it was localized. On the day of presentation, she had felt chills and nausea and requested transportation by ambulance for emergency treatment.2. Her vital signs were as follows: body temperature, 40.1 °C; blood pressure, 98/65 mmHg; pulse, 108 beats/min; respiratory rate, 16 breaths/min; and oxygen saturation, 98 % on room air. On physical examination, she was tender to palpation throughout the left abdomen, but no clear peritoneal irritation symptoms were noted. Poorly controlled psoriasis vulgaris also was found, and because she had not bathed for more than 10 days, the skin was covered by filthy sebum secondary to poor hygiene. Laboratory findings were as follows: leukocyte count, 5480/μL; neutrophils, 92 %; C-reactive protein level, 20.3 mg/dL; procalcitonin level, 2.41 ng/mL; blood urea nitrogen level, 22.2 mg/dL; and creatinine level, 1.99 mg/dL  and addition of a vasopressor became necessary. Urine volume also declined and renal function worsened, such that maintaining blood pressure became increasingly difficult even with a vasopressor. On the second day of hospitalization, given that the patient was demonstrating signs of septic shock, emergency abdominal surgery was performed to control the infection source.An abscess had formed from the left ovary, entangling the fallopian tube; thus, resection of the left adnexa was performed. While no evidence of rupture was found, there were significant adhesions in the large intestine around the left adnexa. Infection lesions were found in the left ovarian parenchyma extending to the surrounding organs, including the fimbriae of the fallopian tube, with diffuse infiltration of inflammatory cells of neutrophil bodies across a wide range, and randomly spaced formation of large and small abscesses. No malignant findings were noted. Gross findings of the excised specimens are shown in Fig. Prevotella spp., ovarian abscess cultures identified Prevotella and C. striatum, and vaginal and perineal skin cultures also revealed C. striatum. C. striatum was identified by using the API Coryne identification panel . Vaginal and ovarian abscess cultures were negative for Chlamydia trachomatis, Neisseria gonorrhoeae, and other microbes. Also, Chlamydia trachomatis and Neisseria gonorrhoeae were negative according to polymerase chain reaction. Based on these culture results, meropenem was changed to ampicillin/sulbactam 3 g every 8 h, and vancomycin and minocycline were continued.Blood cultures detected After surgery, the patient’s hemodynamics slowly stabilized, urine volume gradually increased, and renal function improved. Blood cultures on the eighth day were negative; thus, antibiotic therapy was implemented for 14 days. On the 15th day, she was moved from the intensive care unit to a general ward. A summary of the patient’s clinical course is shown in Fig. C. striatum and Prevotella. Tubo-ovarian abscess due to C. striatum is extremely rare. We concluded that C. striatum from skin contaminated by psoriasis vulgaris had caused the tubo-ovarian abscesses by way of ascending infection. Although vaginal cultures were negative for anaerobic bacteria, we considered that ascending infection also occurred due to Prevotella spp.We presented a case of tubo-ovarian abscess due to Escherichia coli, aerobic streptococci, Bacteroides fragilis, Prevotella, and other anaerobes, such as Peptostreptococcus [Candida [Pasteurella multocida [Salmonella [Streptococcus pneumoniae [Corynebacterium.Infection paths of tubo-ovarian abscesses include ascending infection from the vagina; hematogenous, lymphatic, or other descending infection; or spread of intra-abdominal infection after surgery . Common tococcus , while s[Candida , Pasteurultocida , Salmonelmonella , and Streumoniae . There aeumoniae . To the C. striatum was detected in cultures of the ovarian abscesses, vaginal secretions, and perineal skin. In addition, weakening of the skin barrier due to psoriasis vulgaris, skin contamination secondary to poor hygiene, and contamination around the perineum were strongly evident. Based on these findings, we concluded that C. striatum from skin resident microbiota had caused the tubo-ovarian abscesses by way of ascending infection.In this case, Corynebacterium bacteria are part of the normal flora of the skin and upper respiratory tract, and are usually not identified in specimens from sputum or the nasal cavities. In addition, because Corynebacterium is often regarded as contamination in such specimens, drug sensitivity tests are rarely performed. In addition, even when detected in blood cultures, if the patient’s clinical condition is poor, some physicians might conclude that it is due to contamination when the specimens were drawn. However, C. striatum has attracted attention for its involvement in respiratory infections, wound infections, infectious endocarditis, and urinary tract infections as an opportunistic infection or microbial-substitution disease [ disease , 11.Neisseria gonorrhoeae and C. trachomatis, even though these bacteria are rarely isolated from tubo-ovarian abscesses) is recommended. Specifically, any combined use of cefoxitin and doxycycline; cefotetan and doxycycline; or ampicillin/sulbactam and doxycycline is recommended as a first-line treatment [Commonly, tubo-ovarian abscess is caused by pelvic inflammatory disease, and a treatment regimen covering the above-mentioned high-frequency causal microorganisms and sexually transmitted pathogens (including reatment , 12–14. Prevotella and C. striatum. When the patient’s condition deteriorated due to septic shock, emergency surgery also was performed. The above-mentioned recommended first-line therapy did not successfully treat the C. striatum. Therefore, in cases with psoriasis vulgaris or other skin diseases in which the skin barrier is weakened or skin contamination is strong, we believe that an antibiotic regimen including vancomycin is necessary out of consideration for Corynebacterium or methicillin-resistant Staphylococcus aureus. Finally, appropriate timing of surgery is essential for successful treatment of tubo-ovarian abscesses.In the present case, the diagnosis was tubo-ovarian abscesses and severe sepsis. Given the severity of the disease, broad-spectrum antibiotics were used, including meropenem, minocycline, and vancomycin, though they are not typically first-line treatments. This regimen covered both C. striatum. In patients whose skin has been weakened by psoriasis vulgaris or other infections, Corynebacterium, which is normally considered as a skin contaminant, should be considered as a causative microorganism, and antibiotic therapy including vancomycin should be administered.This may be the first known case of tubo-ovarian abscesses due to


Implementation of evidence-based treatment for pre-hospital status epilepticus can improve outcomes. We hypothesized that publication of a pivotal pre-hospital clinical trial (RAMPART), demonstrating superiority of intramuscular midazolam over intravenous lorazepam, altered the national utilization rates of midazolam for pre-hospital benzodiazepine-treated seizures, while upholding its safety and efficacy outside the trial setting.This is a retrospective, observational cohort study of pre-hospital patient encounters throughout the United States in the National Emergency Medicine Services Information System database, from January 2010 through December 2014. We compared the rates and odds of midazolam use as first-line treatment among all adult and pediatric benzodiazepine-treated seizures before and after RAMPART publication (February 2012). Secondary analyses were conducted for rates of airway interventions and rescue therapy, as proxies for safety and efficacy of seizure termination. 156,539 benzodiazepine-treated seizures were identified. Midazolam use increased from 26.1% in January 2010 to 61.7% in December 2014 . The annual rate of midazolam adoption increased significantly from 5.9% per year to 8.9% per year after the publication of RAMPART . Overall frequency of rescue therapy and airway interventions changed little after the publication of RAMPART.These data are consistent with effective, ongoing, but incomplete clinical translation of the RAMPART results. The effects of the trial, however, cannot be isolated. The study was limited by broad inclusion of all benzodiazepine-treated seizures as well as a lack of information on route of drug of administration. The safety and effectiveness of midazolam for benzodiazepine-treated seizures in prehospital clinical practice appear consistent with trial data, which should encourage continuing increases in utilization. Second stage knowledge translation—the process by which clinical trial findings move into accepted clinical practice—has often been slow and incomplete. Indeed, the Institute of Medicine reported an average of 17 years interval between reports of randomized controlled trials and broad incorporation into practice.The 2012 publication of the results of the Rapid Anticonvulsant Medication Prior to Arrival Trial (RAMPART) providesWe therefore determined the changes in the first-line use of midazolam for pre-hospital benzodiazepine-treated seizures before and after publication of RAMPART. Additionally, we evaluated the generalizability of the RAMPART findings by evaluating the effectiveness of pre-hospital midazolam for the termination of prolonged seizures in routine clinical practice on a national level.This is a phase IV, retrospective, observational cohort study of the impact of the publication of the RAMPART results on the pre-hospital use of midazolam for benzodiazepine-treated seizures. Since the duration between 911 call and EMS arrival is almost universally greater than five minutes in the United States, we used the presence of benzodiazepine treatment for seizures as a proxy for status epilepticus. The RAMPART trial began in June 2009, concluded in January 2011, and the results were published in the New England Journal of Medicine in February 2012. The University of Michigan Institutional Review Board determined this project, which used de-identified data, was not regulated human subjects research.The National Emergency Medical Services Information System (NEMSIS) is a national repository of standardized patient care reports from EMS activations from up to 48 U.S. States and territories depending on year.[Using the NEMSIS dataset, we identified all patient care events involving adult and pediatric patients who were treated with benzodiazepines for seizures and presumed convulsive status epilepticus in the pre-hospital setting, from January 1, 2010, through December 31, 2014. Since the NEMSIS database does not include a diagnostic code to differentiate status epilepticus from other seizures, and duration between 911 call and EMS arrival is almost universally greater than five minutes in the United States, presumed status epilepticus was operationalized as the presence of a seizure and the administration of a benzodiazepine. In an attempt to minimize the over-inclusion of benzodiazepine-treated seizures that would not qualify as status epilepticus we utilized two sampling strategies for benzodiazepine-treated seizures and based the analysis on the narrower strategy. First, using the following Field Codes, events were sampled where seizure was coded in either the chief complaint as reported by dispatch (455), the CMS condition code (8017), or the EMS provider’s primary (1710) or secondary impression (1845). Second, sampling was narrowed to the primary or secondary impression. After limiting the samples to those treated with benzodiazepines, the total eligible population was similar for the two seizure sampling strategies, so the latter, narrow strategy was chosen for a more precise operational definition of presumed status epilepticus.The primary outcome measure is midazolam use as first-line treatment, as a proportion of all benzodiazepine-treated seizures, evaluated monthly. First-line medication was defined as the first medication listed in the “medications given” variable for a patient care event. While RAMPART demonstrated the efficacy specifically of intramuscular midazolam, medication route is not available in the NEMSIS database. Venous access procedures could not be used as a proxy for intravenous (IV) administration of medications, as they were equally as common for those treated with midazolam as for those treated with other, IV-administrable, benzodiazepines .The main predictor is an indicator variable for events occurring after RAMPART publication in February 2012. Age, reported in units of hours, days, months, and years, was converted to years and analyzed continuously and categorically according to the age categories used in RAMPART. Gender was evenly distributed at baseline .Patient race  and ethnicity  were included to look for disparities in rates of knowledge translation. Geographic distribution of events was defined by US Census Regions , and levels of urbanicity . Characteristics of the EMS encounter, including service level (basic life support (BLS), advanced life support (ALS), air, other), primary role of the unit , and type of service requested  were included given our a priori belief of an association with midazolam use. Average annual call volume for represented years was calculated and used as a proxy for EMS agency size.Secondary outcome measures include airway intervention and rescue therapy, as proxies for safety and efficacy of seizure termination, respectively. Airway intervention was defined as any invasive procedure to secure the tracheal airway, including any of the following procedure codes: 31.420 (direct laryngoscopy), 96.042 (rapid sequence intubation), 96.051 (combitube blind insertion airway device), 96.030 , 96.053 (King LT blind insertion airway device), 96.052 , 96.041 , 96.040 , and 31.120 . Rescue therapy, or the administration of a second drug or dose to terminate a seizure, was defined by the presence of a second benzodiazepine listed under “medications given.”Explanatory variables for secondary analysis are identical to the primary analysis with the addition of a binary variable for the use of midazolam versus other benzodiazepines, and its interaction with time.Midazolam use as a proportion of all benzodiazepines was plotted graphically by month, and by levels of each categorical predictor, covering two phases: 1) pre-RAMPART publication phase (January 2010 through January 2012), and 2) post-RAMPART publication phase (February 2012 through December 2014). Rates of midazolam adoption during the two phases were compared using a piecewise linear regression model (spline).A series of nested random-effects hierarchical logistic regression models, using Laplace approximation, were fit with a primary outcome variable of midazolam use versus all other benzodiazepines. A random intercept for EMS agency was applied to adjust for and measure within-agency correlation. Intra-class correlation (ICC) was calculated using the method of Snijders and Bosker (Equation below). The numModel 0 is the intercept-only model, which assesses the proportion of midazolam used by the average EMS agency during the study period. Model 1 assesses the unadjusted impact of RAMPART publication. Model 2 assesses the impact of RAMPART publication, adjusting for secular trends. Model 3 further adjusts for patient demographics. Model 4, the fully adjusted model, further adjusts for characteristics of the EMS call.Three variables  were omitted from the logistic regression models due to high percentages of missing data, which would significantly limit the number of observations in the more fully adjusted models. All other variables had less than 1.5% missing data and were included in the models. In order to reduce any bias caused by diminishing numbers of events in later models that incorporate variables with missing data, all nested models were limited to events with non-missing data for all variables in the final model. Sensitivity analyses were carried out with the inclusion of each of the 3 highly missing variables included in the fully adjusted model. The census region Island Areas was omitted from regression analysis due to low representation.Secondary analyses compared rates of airway interventions and rescue therapy before and after RAMPART publication and between those treated with midazolam and other benzodiazepines. Because midazolam dose and route may have also changed over time or in response to the trial, we also modeled the interaction with time for each agent in a series of nested hierarchical logistic regression models for the prediction of secondary outcomes. Model 0 is the intercept-only model, which assesses the frequency of rescue therapy and airway interventions by the average EMS agency during the study period. Model 1 assesses the unadjusted impact of midazolam use. Model 2 assesses the unadjusted interaction of midazolam use and time. Model 3 assesses the interaction of midazolam use and time, adjusted for demographics. Model 4, the fully adjusted models, further adjust for characteristics of the EMS call.We conducted data manipulation and analysis with SAS .After observing that the rate of midazolam use appeared to increase more rapidly at the end of 2011, prior to the publication of RAMPART, we conducted two post-hoc analyses of potential confounders on the rate of change in midazolam adoption. A spline model of midazolam use among EMS agencies that contributed data to NEMSIS in all 5 study years was used to reduce artifact that could reflect the onboarding of new states and EMS agencies into the dataset at the beginning of each year. Additionally, we examined the effect of the national diazepam shortages on benzodiazepine use patterns with a series of spline models with indicator variables for the months in which national diazepam shortages began, were publically announced, and subsequently resolved. Effects of lorazepam shortages were not investigated as the diversity of manufacturers was felt to likely preclude any perceptible impact.After fitting the hierarchical logistic models of the primary outcome and seeing the high ICC, we fit a multinomial logistic regression model of midazolam use levels per EMS agency-year  in order to determine the effect of agency-level characteristics on the overall odds of midazolam use. We chose multinomial logistic regression despite the ordered nature of the data as we could not dependably assume equal odds associations between midazolam use levels. In addition, the high proportion of agencies using no midazolam or all midazolam made Poisson and linear regression distributionally inappropriate. Predictors included the proportion of each agency’s patient population that is in a particular age bracket , or is female, black, or Hispanic. Additional predictors included agency size , catchment area (number of counties in which agency operates), urbanicity , highest service level , transport capability, and provision of 911 scene response. The model was limited to agency-years for which each of the above variables was non-missing. There were no problems with multicollinearity, with a variance inflation factor threshold of 5.Among the 93.7 million patient care events  in the NEMSIS database from 2010–2014 , 1.9 milThe mean patient age was 39.4 ± 21.8 years . Additional event characteristics for each year of the study are listed in Midazolam use for pre-hospital benzodiazepine-treated seizures increased from 26.1% (326 of 1249) to 61.7% (2412 of 3909) of cases, between January 2010 and December 2014 . This corresponds to a 7.7% increase per year . Midazolam use generally increased over time for all categorical predictors . Spline In the hierarchical logistic regression models, after adjusting for secular trends as well as demographic characteristics , EMS encounter characteristics , and agency size (model 4), the odds of receiving midazolam were 24% higher after RAMPART publication  and were not included in the multivariable models.Of the 2709 EMS agencies that used benzodiazepines to treat a seizure in more than one year, allowing for comparison between years, 1094 (40%) increased their midazolam use, 476 (18%) decreased use, and 1139 (42%) had no change in use. Of the agencies that did not change their midazolam use, 462 (41%) consistently used midazolam and 632 (55%) never used midazolam.The results of the post hoc multinomial logistic regression can be seen in Overall, independent of agent, frequency of rescue therapy and airway interventions changed little before and after publication of RAMPART. Rescue therapy was used 0.5%  more often after publication , and airway interventions were used 0.3%  less often after publication .Those initially treated with midazolam were both more prevalent in post publication period and had a 3.3%  lower rate of use of rescue therapy than those treated with other benzodiazepines . They also had a 1.0%  higher rate of airway interventions .Furthermore, rates of rescue therapy and airway interventions for those treated with midazolam have decreased over time, while the rates associated with treatment with other benzodiazepines have increased or remained constant, respectively . In the In this retrospective, observational, cohort study, we found that midazolam utilization for pre-hospital benzodiazepine-treated seizures increased substantially from 2010–2014, and the rate of adoption increased significantly after the publication of RAMPART. These data are consistent with effective but incomplete clinical translation of the RAMPART results, but the effects of the trial cannot be isolated. The nature of underlying trends is unclear and the potential effects of already changing practices, drug shortages, and early release of trial results to the Federal Drug Administration and the EMS agencies that participated in RAMPART are potentially confounding. Increasing use of midazolam in the pre-publication phase may represent a pre-existing trend that is entirely agnostic to the trial. If so, this trend may or may not have continued in the post-publication period even if there had been no trial data. In this scenario, the trial data may have had little effect on the secular trend, may have been permissive, or may have positively reinforced the trend. It is also possible that the trend in the pre-publication period may be related to the occurrence of the trial, reducing the apparent impact of the published results. The relatively steep pre-publication trend in midazolam use is consistent with this later hypothesis, as a 5.9% per year rate of increase is not likely to have been sustained for more than 4 years prior to the study period. However, the nature of the trend prior to the study period is unknown, as is its association with other publications on midazolam’s efficacy. These oAfter adjusting for secular trends and other factors, the odds of receiving midazolam were 24% higher after the publication of RAMPART, which reflects the significantly increased rate of adoption of midazolam around that time. The increasing use of midazolam could be explained by growing acknowledgment of the benefits of midazolam for the treatment of pre-hospital status epilepticus. Midazolam is not only as effective as alternative intravenous medications , but is The increasing use of midazolam relative to alternative benzodiazepines is consistent with the effects of several alternative or additive events, as described above, including the effective but incomplete clinical translation of the RAMPART results. The problem of knowledge translation has been distilled into two distinct components: “getting the evidence straight” and “getting the evidence used”. There hThere were small but statistically significant differences in rates of rescue therapy and airway interventions before and after RAMPART publication and between seizures treated with midazolam and other benzodiazepines. Given that the magnitude of these differences are small and their directions opposite, the increased use of midazolam was not associated with increased complications overall. Furthermore, the safety and efficacy of midazolam improved over time relative to other benzodiazepines. These results likely reflect changing patterns of midazolam dose, route of administration, or both. If our data reflect the increased adoption of non-IV routes of midazolam administration compared to IV midazolam, our study could indicate that the non-IV route in general is both safer and more efficacious. However, in addition to RAMPART’s demonstration of the efficacy of IM midazolam over intravenous medications, a number of studies have also demonstrated the efficacy of nasal and buccal midazolam, which may be leading many EMS agencies to prefer these routes to IM midazolam for the treatment of pre-hospital status epilepticus.–20 ShoulSeveral limitations of this study should be noted. The study utilized data that were collected by EMS personnel for purposes other than this study and therefore have limited ability to answer all study questions. Additional limitations of the NEMSIS dataset have been described elsewhere . NotablyDue to the limitations of diagnostic codes in NEMSIS and the fact that few patients would have 911 call-to-EMS arrival durations of less than five minutes, we operationalized status epilepticus as any benzodiazepine-treated seizure. It is possible that some patients had cessation of seizures after 911 was called and were treated prematurely for any recurrent seizures, however the administrative database does not provide a level of clinical granularity to discern this. While the incidence of early treatment of seizures prior to the onset of status epilepticus is not known, our selection of roughly 8% of seizures that were treated with benzodiazepines is only modestly higher than other reports of a 5–7% incidence of status epilepticus among acute seizures in emergency department cohorts. This suggests premature benzodiazepine treatment was not a substantial occurrence in our cohort.–24 FurthHowever, while our selection of benzodiazepine-treated seizures was consistent across the duration of the study and not substantially greater than reported incidences of status epilepticus among acute seizures, the intubation rate in our study suggests the possibility of a significant sampling bias. We found that the overall intubation rate for all benzodiazepine-treated seizures was 2.0%, which is far less than other reports , 25, 26.While RAMPART only addressed cases of convulsive status epilepticus and we were unable to make any distinction between any types of seizures, we believe that the incidence of nonconvulsive status epilepticus in our pre-hospital cohort was likely minimal. Paramedic training and protocols in the United States emphasize the diagnosis of seizure by witnessing motor convulsions, making diagnosis and treatment of non-convulsive status epilepticus unusual in the pre-hospital setting. Our focus on benzodiazepine-treated seizures was therefore likely self-limited to cases of convulsive status epilepticus.Lastly, the inclusion of progressively more patients in later years of the analysis—representing the inclusion of additional states and EMS agencies in the NEMSIS database—could have introduced selection bias. However, our post-hoc sensitivity analysis of the rate of midazolam adoption that was limited to EMS agencies for which we have data for all five study years  demonstrOur data are consistent with effective, ongoing, but incomplete clinical translation of the RAMPART results. The effects of the trial, however, could not be isolated. The safety and effectiveness of midazolam for benzodiazepine-treated seizures in the prehospital setting in clinical practice appear consistent with trial data. Along with midazolam’s ease of administration, low cost, long shelf life, and low susceptibility to shortages, this should encourage continuing increases in midazolam utilization. Since the use of midazolam is highly dependent on the responding EMS agency, future research should focus on the decision-making process of EMS medical directors across the country.


The effects of alkaline copper quaternary (ACQ) and zinc borate (ZB) on the resistance of corn stalk fiber (CSF)-reinforced high-density polyethylene (HDPE) composites to biodegradation were examined. Both biocides could inhibit termites, mold fungi, and wood-decay fungi, even at high CSF formulations . Additionally, ACQ enhanced the resistance of the composite materials to certain biotic stresses better than ZB. The CSF/HDPE composites treated with ACQ at the 3.0% level exhibited a superior performance against termites, white rot fungi, and brown rot fungi. ACQ treatment at the 1% level was optimal for inhibiting soft rot fungi. Furthermore, mold growth was not observed on ACQ-treated CSF/HDPE samples. The untreated CSF/HDPE composites were more susceptible to mold infections and decay than the untreated poplar/HDPE composites, likely because of an incomplete removal of the pith. The chemical features of the corn stalk may also have influenced these differences, but this possibility will need to be explored in future investigations. Furthermore, the CSF component of CSF/HDPE composites is highly susceptible to fungal attacks, with the soft rot fungus inducing the largest mass losses, followed by the white rot fungus, and then the brown rot fungus. Corn is an important agricultural crop in northeastern China, and its annual production in the Heilongjiang province of 33.43 million tons accounts for more than 14.5% of the total yield in China . AgriculBecause of a shortage of available wood resources and the detrimental environmental impact of burning agricultural residues, studies have been conducted over the last few decades regarding agricultural fiber-based composites in China ,7. One vAFPCs have prevailed as outdoor building materials, such as dock fences, pavilions, and road signs, which are exposed to cyclical wet and dry outdoor environments. Though HDPE and Maleated polyethylene (MAPE) materials are immune to biological attacks, the AFPCs are susceptible to bio-deterioration under prolonged exposures to high humidity or water, which can severely affect the economic value and utility of the products. In fact, the agricultural fibers in AFPCs are susceptible to wood-decay fungi, termites, and mold fungi ,5. A preACQ is one of the copper amine-based preservatives, which has the advantages of easy treating practices, low volatile organic compound emissions, and improved anti-biological properties. With the proven efficacy of ACQ treatments of wood products, it is hypothesized that using ACQ-treated agricultural fibers as raw materials during the production of AFPCs will decrease the susceptibility of the final products to biological degradation and photo degradation. As one of the preservatives used to minimize the damage caused by insects and microbes, ACQ can extend the service life of decking materials . ZB was The objective of this study was to investigate the feasibility of using ACQ and ZB as preservatives during the production of AFPCs from CSF. The effects of various ACQ and ZB treatments on the performance of composites against termites, mold fungi, and wood-decay fungi were evaluated.3 and flow index: 0.7 × 10−4 kg/min at 190 °C) was used as the matrix . MAPE (Epolene CMG9804 with a grafting rate of 0.9%) was used as the coupling agent . Paraffin was used as lubricant .Corn stalks were collected from farmland in Harbin, China. After removing the inner pith, the corn stalks were granulated using a laboratory mill to the size required to pass through a 60-mesh screen. The average length of CSF was 1 mm, and the average diameter was 0.1 mm. The ground material was dried to a moisture content of about 3% prior to use. We used commercially available ACQ and ZB as preservatives . Preservative-treated CSF was prepared by vacuum-impregnating samples with ACQ or ZB. High-density polyethylene (HDPE) (density: 954 kg/mPopulus sp.) wood, which is susceptible to termites, were used as reference material to be tested under the same conditions. The aboveground subterranean termite field test protocol  used in this study was designed according to Wong  × 100%. The schematic drawing is presented in The test blocks (30 mm × 30 mm × 4 mm) were oven-dried at 40 °C to reach a constant weight (W1), which was the initial weight before the decay test. The test blocks meant for the A scanning electron microscope  was used to obtain microphotographs of CSF/HDPE composites. The samples were coated with a layer of gold-palladium before being observed with SEM at an accelerating voltage of 12.5 kV.Termites are distributed in tropical and subtropical regions and like to nest in areas close to water. The required nutrients of termites are derived from plant cellulose, lignin, and their products. The termite test data are summarized in The average mass losses for the ACQ-treated CSF/HDPE, ZB-treated CSF/HDPE, and untreated CSF/HDPE with different formulations are provided in C. curvignathus. This result indicated that the CSF material in the CSF/HDPE composite was heavily damaged by termites. With increasing ACQ retention levels, the termite resistance initially increased rapidly, but then increased more slowly for both formulations. Termite resistance increased 2.6- and 1.2-fold at an ACQ level of 3% for the 50/50 and 60/40 formulations, respectively. Shang et al. [In general, a higher mass loss was observed with increasing CSF content, likely because the termites were able to feed on more CSF material in this no-choice test. These findings are consistent with the results of a previous study . As expeg et al.  also repg et al. . This isIn the short term, mold fungi may be more important for WPCs than wood-decay fungi because they develop more quickly on the WPCs . Data reThe median mold infection severities for the ZB-treated CSF/HDPE, untreated CSF/HDPE, and untreated poplar/HDPE with different formulations are presented in When the CSF content increased to 60%, the median mold infection severity increased from 12.4% to 24%, which corresponded to a rating increase from 2 to 3, depending on the formulation type. The superior mold resistance of the 50/50 composites is probably because the lower CSF content in the composites ensures that it is well encapsulated and protected by the plastic. These findings are consistent with previous observations, which suggested that there is a linear correlation between wood content and mold infection severity. This may be attributed to an increase of wood not encapsulated by the polymer matrix . Our resThe curvilinear trend of all samples was similar, and the median mold severity was increased with an increasing test time. As can be seen, the mold increment was apparent within the first 20 days, and then gradually became slower. This is because the limited CSF was on the surface of composites, so the mold growth rate decreased with the reduced CSF content.The weight loss results for the CSF/HDPE and control specimens after analyzing the resistance to the white-rot fungus are presented in P. sanguineus was uniformly low for all composites tested. However, the mass loss for the untreated poplar sapwood reference materials was 44.27%, indicating that the CSF/HDPE composites were far more decay-resistant than solid wood, even at high CSF formulations. This is likely because the weight loss in WPCs is only based on the fiber filler, and the WPC fibers are a possible food source for fungi [P. sanguineus induced a significant weight loss in untreated CSF/HDPE composites, but not in poplar/HDPE. The weight loss in untreated CSF/HDPE composites was 4.48% in the 50/50 formulations and 5.46% in the 60/40 formulations. However, the fact that the P. sanguineus induced weight loss was only 0.49% (50/50) and 1.29% (60/40) for poplar/HDPE, suggests that CSF is more susceptible to the white rot fungus than the poplar fibers.In general, the mass loss caused by G. trabeum) was smaller than that caused by the white rot fungus in all untreated CSF/HDPE samples, regardless of the formulation. These results are in agreement with those described by Verhey et al. [G. trabeum was consistently lower for all tested composites than for the poplar control specimens (70.73%), indicating the existence of an invasive decay environment and active fungal viability in poplar. These findings are consistent with the results of a previous study by Simonsen [A summary of the mass losses for the CSF/HDPE composites with different formulations and control specimens is provided in y et al. . This diSimonsen . MeanwhiSimonsen , suggestG. trabeum lost considerably more weight than the samples treated with ACQ or ZB. Additionally, increasing preservative concentrations resulted in a decreasing mass loss for the same formulations (The untreated composite samples exposed to ulations . For exaC. globosum) caused greater mass losses in all untreated CSF/HDPE composites at different formulations. This suggests that CSF/HDPE composites are highly susceptible to soft rot fungal infections. Regarding the influence of CSF content, a lower soft-rot resistance was observed with an increasing CSF content. This was the expected result because CSF can provide an environment conducive for composite decay. Thus, mass losses were higher for the 60/40 CSF formulation than for the 50/50 formulation. These findings imply that the CSF content is an important factor affecting the degradation behavior of CSF/HDPE composites infected by the mixed soft rot fungal inocula. Our data is in agreement with the results described in earlier publications [The average soft rot fungus-induced mass losses in composite materials treated with ACQ or ZB are provided in ications ,24. As eC. globosum/unsterile soil mixture than ZB.The ACQ and ZB-treated composites retained more weight than the untreated samples, indicating that the preservatives may be useful for enhancing soft-rot resistance. A previous study concluded that the incorporation of ZB in composite materials can significantly decrease mass losses, even at high fiber component formulations . With inP. sanguineus can damage the surface of composite materials by degrading the CSF. The scanning electron microscopy results are consistent with the mass loss data presented above.The scanning electron microscopy images of the surface and internal microstructures of samples before and after the decay resistance test are presented in CSF/HDPE containing 3% ACQ is more resistant to termites, white rot fungi, and brown rot fungi than the untreated control composites, even at high CSF formulations . Additionally, ACQ enhances the biotic stress resistance of CSF/HDPE composites more than ZB.Compared with the untreated poplar/HDPE composite, the untreated CSF/HDPE is more susceptible to mold infections and decay.The CSF of the CSF/HDPE composites is susceptible to fungal attacks, with the soft rot decay causing the largest mass losses, followed by the white rot fungus, and then the brown rot fungus.Manufacturing AFPCs using preservative-treated CSF may be feasible, and represents a potentially viable way to convert an agricultural waste product into value-added composites.Our results revealed that the incorporation of ACQ or ZB by CSF/HDPE composites can significantly decrease mass losses and mold growth. This implies that these biocides may be inhibitory toward termites, mold fungi, and wood-decay fungi, even at high CSF formulations . The following are specific conclusions that are supported by the results described herein: