Agrocybe aegerita (AaeUPO), and recombinant galactose oxidase (GAO). The effect of pH on different reaction steps was evaluated and apparent kinetic data  were calculated for different enzyme-substrate ratios and enzyme combinations. Finally, the target product, 2,5-furandicarboxylic acid (FDCA), was prepared in a multi-enzyme cascade reaction combining three fungal oxidoreductases at micro-scale. Furthermore, an oxidase-like reaction is proposed for heme-containing peroxidases, such as UPO, horseradish peroxidase, or catalase, causing the conversion of 5-formyl-2-furancarboxylic acid into FDCA in the absence of exogenous hydrogen peroxide.Enzymatic oxidation of 5-hydroxymethylfurfural (HMF) and its oxidized derivatives was studied using three fungal enzymes: wild-type aryl alcohol oxidase (AAO) from three fungal species, wild-type peroxygenase from  Plastics such as polyethylene terephthalates (PET) have become an integral part of human life, and the world’s annual consumption of such plastics has grown to about 40 million tons in 2014 and is forecasted to increase to over 70 million tons in 2020 . An incrIn addition to bacterial whole-cell conversions ,7, in thAspergillus oryzae were provided by Novozymes AS . Wild-type AaeUPO was produced and purified, as described previously [AaeUPO preparation had a specific activity of 82 U mg−1 measured with veratryl alcohol as substrate according to Ullrich et al. [Recombinant galactose oxidase (GAO) and catalase (Cat) expressed in eviously . The finh et al. . SuperoxPleurotus eryngii (PeryAAO) and Bjerkandera adusta (BaduAAO) were produced in 10-L stirred-tank bioreactors  containing 8 L of the culture media described below (the time courses of enzyme production are given in Pleurotus ostreatus (PostAAO) was cultivated in a 30-L stirred-tank bioreactor using 20 L culture medium , AAO production was stimulated by adding sterile-filtered veratryl alcohol , and afterwards, cultivation was continued over seven days. For both Pleurotus species, the following medium was used to produce AAO: 10.0 g L−1 glucose, 1.0 g L−1 Na acetate, 2.0 g L−1 yeast extract, 5.0 g L−1 peptone (from soybeans), 2.0 g L−1 KH2PO4, 0.5 g L−1 MgSO4·7 H2O, 0.1 g L−1 CaCl2, 0.01 g L−1 FeSO4·7 H2O; pH was adjusted to 5.2 with HCl. Veratryl alcohol was not supplemented and cultivation was carried out over seven days.Wild-type AAOs from dium see . The med® (26 mm × 200 mm) and Mono Q® columns . Eventually, size exclusion chromatography (SEC) was applied using a Sephadex® 75 column . More detailed information, including FPLC elution profiles and an exemplary purification table, are given in the BaduAAO, PeryAAO, and PostAAO were 28.1 U mg−1, 39.0 U mg−1 and 80.6 U mg−1, respectively.AAO-containing culture liquids (crude extracts) were filtered through cotton fabric and then, the filtrates were frozen and thawed to remove dissolved oligosaccharides by precipitation. Afterwards, the liquid was centrifuged  and filtered through glass microfiber filters . The particle-free filtrates were concentrated with an ultra-tangential filtration system using a 10-kDa cut-off membrane . Partial AAO purification was achieved by two steps of ion exchange chromatography steps using Q-sepharoseHydroxymethylfurfural (HMF), 2,5-diformylfuran (DFF), 5-formyl-2-furancarboxylic acid (FFCA), 5-hydroxymethyl-2-furancarboxylic acid (HMFCA), and 2,5-furandicarboxylic acid (FDCA) were purchased from Sigma-Aldrich  with the highest purity grade available. All of the other chemicals used (including culture media components) were obtained from VWR International .+ 8%; Phenomenex, Aschaffenburg, Germany) at 50 °C and the liquid phase was 0.05 N H2SO4 . An exemplary HPLC elution profile is shown in HMF and its oxidation products were analyzed using an Agilent 1200 series HPLC system  equipped with a diode array detector operating between 210 and 500 nm, as well as at specific wavelengths  for calibration. Separation of analytes occurred on a Resex column  or constantly shaken (oxidases) for two hours (or for 15 min during HMF and DFF conversion by AaeUPO) and stopped by adding 50 µL trichloroacetic acid (50%) or by heating (95 °C) for 3 min in the case of FFCA samples.The reaction setup to evaluate pH dependencies of oxidoreductases in the oxidation of HMF and its derivatives was set as follows: 2 mM substrate, 50 mM phosphate buffer of varying pH (3.0 to 9.0), instant one-time-addition of 1 mM Hi, pH 6.0), 0.5 to 140 mM substrate (HMF or DFF), as well as 4 µM, 2 µM, and 0.111 µM of GAO, AAOs, and AaeUPO, respectively. Reactions were stopped with 50 µL sodium azide (10 mM).The reaction setup for the determination of apparent kinetic constants (at varying substrate concentrations) contained in a total volume of 500 µL:50 mM potassium phosphate buffer , 0.02 mg mL−1 AAO , as well as optionally 2.2 µg mL−1 catalase (Cat). They were shaken at 175 rpm for up to 76 h at room temperature. Reactions with AaeUPO were analogously carried out  and contained diverse combinations of AaeUPO (0.04 mg mL−1), Cat (2.2 µg mL−1), SOD (10 µg mL−1), HRP (1 mg mL−1) and H2O2 (5 mM). Furthermore, FFCA (2.5 mM) was treated with AaeUPO (0.04 mg mL−1) in combination with GOD (0.1 mg mL−1), glucose (50 mM), and Cat (2.2 µg mL−1); the latter reaction was also performed in the absence of AaeUPO. Further controls of the aforementioned reaction setups contained: 2.5 mM FFCA in 50 mM KPi (pH 7.25) and optionally 10 mM H2O2, as well as heat-inactivated AaeUPO  or 50 mg mL−1 of porcine hemoglobin or crude hemin (5 mg mL−1) or 1 mM sodium azide.The oxidation of FFCA by selected enzymes was studied in more detail in separate experiments. Reaction mixtures with AAOs contained in a final volume of 0.5 mL following components: 2.5 mM FFCA, 50 mM KPi (pH 6.0 or 7.5), 6 µg mL−1 of BaduAAO, PostAAO or PeryAAO, and 5 mM FFCA. To evaluate whether FFCA oxidation catalyzed by AAOs can be inhibited by H2O2, FFCA was incubated for 24 h  with BaduAAO, PostAAO, and PeryAAO in the presence of different concentrations of H2O2 (0 to 20 mM).FFCA oxidation by AAOs was followed at three pH values and the respective reaction mixtures comprised of 50 mM KP−1), PeryAAO (0.32 mg mL−1), and AaeUPO (0.098 mg mL−1) was realized in a larger reaction volume (10 mL) containing 10 mM HMF as substrate . Samples of 50 µL were taken every 40 min from this reaction solution and analyzed by HPLC. Exogenous H2O2 was not added and AaeUPO was fully supplied with peroxide through the oxidase reactions. All of the enzymatic measurements were carried out in triplicate; if not otherwise indicated, standard deviations were <5%.Eventually, a model cascade reaction combining GAO  and did not form HMFCA. BaduAAO was not efficient in oxidizing HMF and formed only trace amounts of FFCA (<0.1 mM). On the other hand, it was active in a broad pH range . GAO worked best at pH 6.5, but did not produce FFCA. It was the only of the tested enzymes, which was capable of oxidizing HMFCA to FFCA. Interestingly, the sum of HMF and its oxidation products in the reaction mixture dropped to about 1 mM  proceeds roughly 500 times slower than the oxidation of the AAO assay substrate veratryl alcohol. Interestingly, the specific constants  of AaeUPO for HMF and DFF were almost identical (36.6 × 103 and 35.6 × 103 M−1 s−1), although the respective turnover numbers  and Michaelis-Menten constants  differed by an order of magnitude.Apparent catalytic constants for enzymatic HMF and DFF oxidation are given in AaeUPO and the corresponding residual enzymatic activity after the reaction . Interestingly, most FDCA was formed below pH 6.0 where a substantial loss of UPO activity was observed . The resulting total turnover number (ttn) was 170, which is rather unfavorable for an enzymatic conversion. In contrast, UPO activity above pH 6.0 (pH 6.5–8.5) was almost completely preserved, albeit the amounts of FDCA formed were more than five times lower when compared to acidic conditions. Consequentially, pH 6.0 turned out to be the most suitable pH with regard both to the amount of FDCA formed and UPO’s process stability.AaeUPO-containing samples (and controls) revealed a continued FDCA production, although H2O2 had already depleted (data not shown). This phenomenon needed closer inspection and AaeUPO in the absence of exogenous H2O2 (or a peroxide-generating enzyme) and the addition of H2O2 increased the amount to just 0.4 mM. H2O2 alone oxidized FFCA merely to a negligible extent. When AaeUPO was combined with Cat , FDCA formation (1.9 mM) was still intensified, while SOD did neither stimulate nor affect the reaction. Interestingly, the heme enzymes Cat and HRP caused substantial FFCA oxidation  orders of magnitude)). In contrast, the flavin enzyme GOD  inhibited FFCA oxidation by Cat, as well as by AaeUPO and Cat  of  and Cat . Control2O2 were supplemented to the reaction mixtures, FDCA formation decreased . In contrast, PeryAAO produced from the outset twice as much FDCA (0.5 mM), and this was not affected by moderate amounts of H2O2 , and PeryAAO may efficiently oxidize HMF to DFF, along with substantial H2O2 production (to be used by UPO). Under such conditions, more than 95% of the applied HMF (9.7 mM) was already converted after 45 min (first sampling) and after 75 min, it was fully consumed. Concomitantly, DFF and HMFCA appeared in the reaction mixture reaching their maximum concentrations of 2.4 mM and 2.7 mM, respectively, already after 45 min. DFF rapidly disappeared within the next 40 min, whereas HMFCA was rather slowly converted (about 50% within the next 75 min). In both cases, further oxidation yielded FFCA that reached its maximum of 6.3 mM after 75 min (which corresponds to 70% conversion of the applied HMF). Final conversion of FFCA to FDCA turned out to be the bottleneck of the overall oxidation and lasted for the complete remaining reaction time. Nevertheless, FDCA constantly increased and finally reached a concentration of 7.9 mM, which corresponds to an 80% yield related to HMF applied . Furthermore, after 24 h reaction time, the total amount of HMF oxidation products was 9.5 mM, which means an almost complete mass balance.AaeUPO) for their ability to oxidize HMF and its oxidized derivatives. A summarizing formula scheme illustrating the possible reactions is given in Pleurotus ostreatus was reported to detoxify HMF  by means of AAO and it was proposed to integrate the respective gene into fermentation organisms, such as Saccharomyces cerevisiae [We have tested five different fungal oxidoreductases , their individual pH dependencies were notwithstanding fairly different. Carro et al. [−1) and the fact that neither PeryAAO nor PostAAO developed a distinct pH optimum for direct DFF oxidation. However, in the course of our pH dependency studies with HMF, both Pleurotus AAOs formed reasonable amounts of free DFF (about 1 mM at pH 5.5) that obviously had left the active site to be further oxidized after re-entering the active site (compare PeryAAO’s and PostAAO’s active sites—despite high sequence homology (95%)—differ substantially [PeryAAO, on the other hand, is seemingly lacking such a pH-stabilizing mechanism, and hence more affected by the pH of the reaction solvent. These assumptions could be assessed in future studies by comparing the enzymes’ crystal structures or reliable homology models [All of the tested oxidases exhibited pH optima for HMF oxidation around pH 6.0. This corresponds well with earlier findings using recombinant AAO and a mutant GAO ,20. Altho et al.  proposed compare . Maybe, antially ,22. The y models . PeryAAO; PeryAAO, noticeable differences in kcat and KM become evident. The kcat value for HMF of the wild-type enzyme we used is about ten times higher and the KM value 20 times lower than the values of recombinant AAO [E. coli) may have affected the enzymatic performance [AaeUPO exhibited the highest specific constants (kcat/KM) both for HMF (3.66 × 104 M−1 s−1) and DFF (3.56 × 104 M−1 s−1), indicating a certain potential of this enzyme type for further catalytic improvement. Interstingly, AaeUPO’s kcat/KM values for HMF and DFF are in the same range as that of the model substrate veratryl alcohol (3.58 × 104 M−1 s−1) [AaeUPO has significantly contributed to the HMF and DFF oxidation in the cascade experiment and worked jointly with AAOs and GAO.When comparing the catalytic constants of wild-type AAOs  . Hence, −1 s−1). At first glance, this appears astonishing when considering that GAO effectively oxidizes diverse monosaccharides such as galactose and glucose and hence, should be able to oxidize sugar derivatives like HMF as well [n + 2’ system). Considering that GAO also oxidized HMFCA, resulting in FFCA and H2O2 formation, this nevertheless makes the enzyme an interesting candidate for HMF cascade reactions. Thus, HMFCA produced by UPO (that is not a substrate for the other cascade enzymes) can be further used and stoichiometrically returned into the reaction. Moreover, it might be advantageous that HMFCA acts as an ‘H2O2-sink’ to keep the stationary H2O2 concentration in the reaction mixture at a moderate level and prevent UPO from inactivation [GAO turned out to have an exceptional low specific constant for HMF oxidation  [AaeUPO was added to a reaction mixture, which contained afore-produced H2O2 (6 mM by AAO) and residual FFCA (3 mM) that was oxidized to FDCA over 120 h without additional supplementation of H2O2 [AaeUPO is capable of forming FDCA from FFCA in the absence of its natural co-substrate H2O2. The same phenomenon was observed for Cat  was not relevant for the reaction catalyzed by UPO (nor UPO/Cat) [AaeUPO and/or Cat) led us conclude that dioxygen (O2) may be involved in the reaction.Production of FDCA was also achieved with  compare . AaeUPO’us study . In contctivity) . This fi of H2O2 . Closer ses H2O2 ; compareses H2O2 , was notUPO/Cat) ,29. Howe2O2-independent formation of FDCA catalyzed by AaeUPO, Cat or HRP must be the result of a true enzymatic oxidation process, since it was azide-sensitive and was not brought about by plain hemin or hemoglobin. FDCA formation by AaeUPO or Cat was fully inhibited when sodium azide (NaN3) was added, which is known to inactivate heme proteins via catalytic formation of meso-azidoprotoporphyrin IX and/or oxidation of the apoprotein [AaeUPO  possess an oxidase-like activity towards FFCA. This activity was most pronounced in AaeUPO that converted FFCA at much lower concentration (~100-fold) than Cat or HRP. Oxidase-like activities have already been reported for some other heme-containing peroxidases, including HRP (well-studied oxidation of indole acetic acid) and a β,β-carotene-cleaving fungal peroxidase of the DyP-type [AaeUPO’s “usual” peroxygenating activity, but to a putative oxidase-like activity. Another FDCA-producing cascade reaction described in the literature combined a periplasmatic bacterial aldehyde oxidase, HRP, GAO, and Cat [−1 and 0.2 mg mL−1, respectively) and it was not mentioned whether controls solely with catalase were run and if so, whether they produced FDCA. Anyway, the Hoprotein . When coDyP-type ,31,32,33tration ~0-fold th and Cat ,34. Howe2O2 concentration at levels that do not damage UPO [AaeUPO activity was found to decrease mainly in the beginning of the reaction when HMF and DFF (as suitable oxidase substrates) are being converted. With a delay of about 60 min, the decrease of AaeUPO activity slowed down, precisely at that time when only HMFCA, FFCA, and FDCA were left in the reaction mixture. Eventually, it should be mentioned that such an enzyme-based process development will also face various technical challenges to be met in order to achieve a reliable performance. For example, the activity of oxidases is boosted, if pure dioxygen will be used instead of air or if the solubility of dioxygen in the liquid phase will be increased by applying elevated pressure [Overall, our results demonstrate that it is possible to establish enzyme cascade reactions that combine fungal UPO with different peroxide-generating fungal oxidases, such as AAO and GAO, which were never used in combination in previous studies, to produce substantial amounts of FDCA starting from HMF compare . Howevermage UPO . Furtherpressure .


N-methyl-d-aspartate receptor (NMDAR)-dependent neuronal excitoxicity. Here, we have exploited the hypothesis that ZIKV-induced neurodegeneration can be rescued by blocking NMDA overstimulation with memantine. Our results show that ZIKV actively replicates in primary neurons and that virus replication is directly associated with massive neuronal cell death. Interestingly, treatment with memantine or other NMDAR blockers, including dizocilpine (MK-801), agmatine sulfate, or ifenprodil, prevents neuronal death without interfering with the ability of ZIKV to replicate in these cells. Moreover, in vivo experiments demonstrate that therapeutic memantine treatment prevents the increase of intraocular pressure (IOP) induced by infection and massively reduces neurodegeneration and microgliosis in the brain of infected mice. Our results indicate that the blockade of NMDARs by memantine provides potent neuroprotective effects against ZIKV-induced neuronal damage, suggesting it could be a viable treatment for patients at risk for ZIKV infection-induced neurodegeneration.Zika virus (ZIKV) infection is a global health emergency that causes significant neurodegeneration. Neurodegenerative processes may be exacerbated by  in vitro and in vivo. These results suggest that overactivation of NMDARs contributes significantly to the neuronal damage induced by ZIKV infection, and this is amenable to inhibition by drug treatment.Zika virus (ZIKV) infection is a global health emergency associated with serious neurological complications, including microcephaly and Guillain-Barré syndrome. Infection of experimental animals with ZIKV causes significant neuronal damage and microgliosis. Treatment with drugs that block NMDARs prevented neuronal damage both  Zika virus (ZIKV) is a mosquito-borne pathogen that poses significant public health concerns due to the recent rapidly expanding outbreak. It is an emerging pathogen that belongs to the Flavivirus genus and Flaviviridae family, along with other clinically important arboviruses, including Dengue virus (DENV), Chikungunya virus (CHIKV), and West Nile virus  and causes significant neurodegeneration, especially of neural progenitor cells –22. Thes–N-methyl-d-aspartate receptor (NMDAR) blockade by memantine can avoid the death of nearby neurons and decrease neurodegeneration and neuroinflammation associated with ZIKV infection. For instance, the NMDAR antagonist memantine has been approved by international regulatory agencies for the treatment of Alzheimer’s disease (AD)  . In AIDSase (AD) –33.in vitro and in vivo settings, we investigated whether NMDAR blockade could prevent neurodegeneration induced by ZIKV infection. Our data show that blocking NMDARs prevents neuronal death induced by ZIKV, suggesting that memantine may be a useful therapy to prevent neurological disorders in ZIKV-infected patients.Here, using relevant −/−) mice, and type II interferon receptor-deficient (IFN-γR−/−) mice. In all experiments, uninfected control (mock-infected) mice were inoculated with the supernatant of a cell suspension from the mosquito C6/36 Aedes albopictus culture medium, which caused no clinical or biochemical alterations in comparison to uninoculated mice (data not shown). Systemic infection of IFN-α/βR−/− mice with 4 × 105 PFU of ZIKV induced lethality that was observed around the 6th and 7th days after ZIKV inoculation  responses, both type I and type II IFN responses, or other components of the innate immune system –36. Here–culation . Diseaseculation , startin disease . Infectiethality  or body ethality , indicat10.1128/mBio.00350-17.1FIG S1 −/−, and IFN-γR−/− mice (n = 8 mice per group) were inoculated with 4 × 105 PFU of a Brazilian ZIKV strain (HS-2015-BA-01), and lethality rates were analyzed every 12 h for 14 days. Results are shown as percentage of survival. (B) IFN-γR−/− mice (n = 6 mice per group) were inoculated with 1 × 106, 1 × 107, or 1 × 108 PFU of a Brazilian ZIKV strain (HS-2015-BA-01). Change in body weight was analyzed daily for 11 days. Results are expressed as percentage of initial weight loss. p.i., postinfection. Download FIG S1, TIF file, 0.3 MB.Type I interferons are essential to control ZIKV infection, and IFN-γ signaling is not important. (A) WT, IFN-α/βRCopyright © 2017 Costa et al.2017Costa et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the −/− mice. No significant changes in platelet levels , nerve growth factor (NGF), IL-10, or IL-6  after ZIKV infection in WT and IFN-α/βRt levels , hematoct levels , or totat levels  were dett levels , or prodt levels , CCL5  IFN-α/βR−/− mice (n = 5 to 7 mice per group) were inoculated i.v. with 4 × 105 PFU of a Brazilian ZIKV strain (HS-2015-BA-01). Concentrations of brain-derived neurotrophic factor (BDNF [A]), nerve growth factor (NGF [B]), IL-10 (C), and IL-6 (D) in the brain of mice were quantified by enzyme-linked immunosorbent assay (ELISA). Results are shown as picograms per 100 mg of brain tissue. MOCK, uninfected. All results are expressed as mean ± SEM and are representative of at least two experiments. Download FIG S2, TIF file, 0.5 MB.Concentration of inflammatory mediators in the brain of WT and IFN-α/βRCopyright © 2017 Costa et al.2017Costa et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the −/− mice, even 28 days after infection  of mice was evaluated. Results revealed significant increase in IOP on days 3 and 6 after ZIKV infection in both IFN-α/βR−/− and WT mice in comparison to mock-infected matched controls , loss of hippocampal neurons, perivascular cuffs, gliosis, and focal areas with necrotic and apoptotic cells  WT and IFN-α/βR−/− mice (n = 5 to 7 mice per group) were inoculated i.v. with 4 × 105 PFU of a Brazilian ZIKV strain (HS-2015-BA-01). At day 6 of infection, mice were culled, and brain tissue was dissected and sliced (100-µm slices). Staining with fluoro-jade C (FJC) (neurodegeneration) and IBA-I (microgliosis) was performed in cortical and striatal slices, followed by analysis. (A and B) The left panels show the number of neurons positive for fluoro-jade C (indicative of neurodegeneration). The right panels show representative pictures from cortical and striatal slices. (C and D) The left panels show the number of microglia positive for IBA-1 (microgliosis). The right panels show representative pictures from cortical and striatal slices. MOCK, uninfected. Original magnification, ×200. Scale bar, 50 µm. All results are expressed as mean ± SEM and are representative of at least two independent experiments. Download FIG S3, TIF file, 0.7 MB.ZIKV infection induces neurodegeneration and microgliosis in the prefrontal cortex and striatum of IFN-α/βRCopyright © 2017 Costa et al.2017Costa et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the in vitro) at early time points (12 h), reaching a peak of replication at 48 and 72 h after ZIKV infection (multiplicity of infection [MOI] of 1)  . Of noteI] of 1)  and B. NI] of 1) . Figure in vitro  –40. In t–in vitro . To furtarginine . Agmatinarginine . Figure arginine .in vitro mostly express GluN2B-containing NMDARs  after 64 h at an MOI of 0.04. Download FIG S4, TIF file, 0.4 MB.ZIKV infection induces the expression of GRIN2B and FoxO1 genes in human cortical neural progenitor cells (hNPC). RNA-seq-normalization of data and differential analysis between infected and mock-infected conditions were performed by F. Zhang et al. . Black bCopyright © 2017 Costa et al.2017Costa et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the in vivo, we conducted a series of experiments in IFN-α/βR−/− mice infected by ZIKV. Mice were treated with 30 mg/kg memantine, based on previous reports indicating that this dose was sufficient to improve a number of alterations exhibited by AD and stroke mouse models  IFN-α/βR−/− mice (n = 5 to 7 mice per group) were inoculated i.v. with 4 × 105 PFU of a Brazilian ZIKV strain (HS-2015-BA-01). Memantine (MEM) treatment from days 2 to 6 of infection was performed orally b.i.d. (30 mg/kg per mouse). (A) Change in body weight was expressed as percentage of initial weight loss. (B to F) At the peak of infection, day 6 after ZIKV inoculation, mice were culled and blood and tissues collected for the following analysis: (B) total and differential cell counts on blood, represented as the number of differential cell counts  normalized by percentage of total cell counts; (C) neutrophil influx into the brain, assessed by measurement of myeloperoxidase activity; (D to G) concentrations of CXCL-1, CCL5, IL-1β, and TNF-α in mouse brain, quantified by ELISA. Results are shown as picograms per 100 mg of brain tissue. All results are expressed as mean ± SEM and are representative of two independent experiments. *, P < 0.05 compared to control uninfected mice (MOCK); #, P < 0.05 compared to ZIKV-infected mice. VEIC, vehicle. Download FIG S5, TIF file, 0.8 MB.Disease parameters in IFN-α/βRCopyright © 2017 Costa et al.2017Costa et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the in vitro virological data (−/− mice). Overall, our results show that memantine treatment prevents neuronal cell death induced by ZIKV infection without interfering with the ability of the virus to replicate in the host.Corroborating the cal data , viral lcal data  and B. Ical data  without cal data  and F. Mcal data  and F. Ical data  and D in10.1128/mBio.00350-17.6FIG S6 −/− mice. (A) IFN-α/βR−/− mice (n = 5 to 7 mice per group) were inoculated i.v. with 4 × 105 PFU of a Brazilian ZIKV strain (HS-2015-BA-01). Memantine (MEM) treatment from days 2 to 6 of infection was performed orally b.i.d. (30 mg/kg per mouse). At day 6 of infection, mice were culled, and brain tissue was dissected and sliced (100-µm slices). (A and B) Staining with fluoro-jade C (neurodegeneration) and (C and D) IBA-I (microgliosis) was performed in cortical and striatal slices, followed by analysis. (A and B) The left panels show the number of neurons positive for fluoro-jade C (indicative of neurodegeneration). The right panels show representative images from cortical and striatal slices. (C and D) The left panels show the number of microglia positive for IBA-1 (microgliosis). The right panels show representative images from cortical and striatal slices. MOCK, uninfected. *, P < 0.05 compared to the uninfected group; #, P < 0.05 compared to the ZIKV-infected group. Original magnification, ×200. Scale bar, 50 µm. All results are expressed as mean ± SEM and are representative of at least two independent experiments. Download FIG S6, TIF file, 0.7 MB.Memantine prevents neurodegeneration and microgliosis induced by ZIKV infection in the prefrontal cortex and striatum of IFN-α/βRCopyright © 2017 Costa et al.2017Costa et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the in vitro and in vivo. The major findings of this study can be summarized as follows. (i) ZIKV is able to infect and replicate in primary undifferentiated neurons and subsequently induce massive neuronal death. (ii) ZIKV induces neurodegeneration, microgliosis, and ophthalmologic disorders, such as increased intraocular pressure (IOP), in vivo. (iii) NMDAR blockade, by memantine, MK-801, agmatine sulfate, or ifenprodil, prevents neuronal death induced by ZIKV in vitro without affecting the ability of the virus to replicate in those cells. (iv) Neurodegeneration, microgliosis, and overall brain damage were ameliorated in ZIKV-infected mice, and increase in IOP levels was prevented by memantine treatment. These data, therefore, indicate an important neuroprotective effect of NMDAR blockade during ZIKV infection in vitro and in vivo.The rapidly expanding outbreaks of ZIKV and in particular its introduction in the Americas, including its unexpected association with neurological disorders, such as microcephaly and GBS, made this infection a matter of utmost public health concern, with an urgent need for therapies. In the present study, we evaluated the neuroprotective effect of the blockade of NMDAR by using an FDA-approved noncompetitive NMDAR antagonist drug (memantine) and other NMDAR blockers against ZIKV-associated neurodegeneration −/− mice are susceptible to ZIKV infection  from an outbreak that occurred in Brazil in 2015 in wild-type mice or type I or II IFN receptor-deficient mice. Our results are in accordance with the recent literature and show that IFN-α/βRnfection . As expenfection . The finnfection , 49. We  by ZIKV , 51. The by ZIKV , and elein vivo. After infection, a significant increase in intraocular pressure (IOP) was observed in both WT and IFN-α/βR−/− mice, despite the presence of ZIKV only in IFN-α/βR−/− mice. However, no difference was found in RGC counts between the evaluated groups. The latter results may be explained by the short period of ZIKV infection. Our data suggest that infection by itself may be sufficient to elicit the increase of IOP levels in those mice. Corroborating our findings, Foureaux et al. demonstrated that elevated IOP levels found during the early phases of glaucoma induction in Wistar rats were independent of the reduction in the numbers of RGC, which occurred only after 15 days of glaucoma induction . The experimental protocol was approved by the Committee on Animal Ethics of the Universidade Federal de Minas Gerais . All surgeries were performed under ketamine/xylazine anesthesia, and all efforts were made to minimize animal suffering. Studies with ZIKV were conducted under biocontainment level 2 (BCL2) conditions at the Immunopharmacology Lab, Instituto de Ciencias Biológicas (ICB), Federal University of Minas Gerais.KX520666. Virus stocks were propagated in C6/36 Aedes albopictus cells and titrated as described in reference A low-passage-number clinical isolate of ZIKV (HS-2015-BA-01), isolated from a viremic patient with symptomatic infection in Bahia State, Brazil, in 2015, was used. The complete genome of the virus is available in GenBank under accession no. in vivo experiments, wild-type (WT) mice and mice deficient in type I interferon receptor (IFN-α/βR−/−) or type II interferon receptor (IFN-γR−/−), all on the SV129 background, were used. IFN-α/βR−/− mice and their congenic WT controls (SV129/Ev) were originally purchased from B & K Universal Limited (United Kingdom). The IFN-γR−/− strain was originally from Jackson Laboratories (reference no. 002702). All strains were obtained from Bioterio de Matrizes da Universidade de Sao Paulo—USP. Adult mice (7 to 9 weeks old) were kept under specific-pathogen-free conditions. Mice were inoculated with 4 × 105 PFU ZIKV by the intravenous (i.v.) route (tail vein). Disease signs  were monitored daily. Moribund mice with 20% or more body weight loss were euthanized. This time point occurred generally between days 6 and 7 after ZIKV inoculation (the peak of ZIKV infection). In some experiments, therapeutic memantine treatment was performed orally (p.o.) at a dose of 30 mg/kg of memantine per day b.i.d. (from days 3 to 6 after ZIKV infection). For primary culture experiments , Bioterio Central (UFMG) provided wild-type mice on the C57BL/6j (WT) genetic background.For Blood was obtained from the cava vein in heparin-containing syringes at the indicated times after ZIKV infection. Platelets and the hematocrit index were analyzed as described in reference The extent of neutrophil accumulation in the brain of control and ZIKV-infected mice was measured by assaying myeloperoxidase activity, as previously described .The concentration of cytokines , chemokines (CXCL1 and CCL5), and growth factors (NGF and BDNF) in the brain of control and ZIKV-infected mice was measured using commercially available antibodies and according to the procedures supplied by the manufacturer .Brains from control and ZIKV-infected mice, treated or not treated with memantine, were collected and processed for hematoxylin and eosin (H&E) staining as described in reference Histopathological analysis of the eyes was performed as previously described . The retSections from hippocampus, striatum, and prefrontal and motor cortex of mice were assessed for microgliosis (IBA-1 staining) according to the procedures supplied by the manufacturer (Vector Elite kit) and neurodegeneration (fluoro-Jade C staining) as described in reference IOP measurements were performed on days 0, 3, and 5 after ZIKV infection using a Tono-Pen Vet applanation tonometer  as described in reference l-ornithine-coated dishes in neurobasal medium supplemented with N2 and B27 supplements, 2 mM GlutaMAX, 50 μg/ml penicillin, and 50 μg/ml streptomycin, incubated at 37°C and 5% CO2 in a humidified incubator, and cultured for 5 days in vitro. Glial cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 1% streptomycin, and 0.3% amphotericin B and placed in 75-cm2 disposable culture bottles, prepared by coating with 2% gelatin, followed for 14 days of incubation at 37°C and 5% CO2 in a humidified incubator. Culture medium was replenishment every 4 days, and glial cells were plated onto poly-l-ornithine plates. Infection was performed with ZIKV (MOI of 1), followed by an adsorption period of 1 h. Residual virus was removed and titrated (represented by a dashed line on graphs). Wells were then washed twice with incomplete medium. Each well was replaced by a final volume of complete neurobasal medium in the presence or absence of the NMDAR antagonists. Titers are representative of virus accumulation over the whole period of infection, collected at different time points. Beside the kinetic experiments, all experiments evaluating the effects of NMDAR blockers on primary neurons were performed after 72 h of ZIKV infection. In some experiments, neuronal cultures were treated with 30 μM memantine every 24 h.Neuronal cultures were prepared from the cortex and striatal regions of embryonic day 15 (E15) C57BL6j wild-type mouse embryo brains, and glial cells were obtained from the whole brain of newborn C57BL6j wild-type mice (1 to 3 days). After dissection, the brain tissue was submitted to trypsin digestion followed by cell dissociation using a fire-polished Pasteur pipette. Neuronal cells were plated onto poly-in vitro blockade of NMDARs on primary cultured neurons.The commercially available drugs memantine (Eurofarma), MK-801 , agmatine sulfate , and ifenprodil (Tocris) were used for the Neuronal cell death was determined using a LIVE/DEAD assay kit. Briefly, neurons were stained with 2.0 µM calcein acetoxymethyl ester (AM) and 2.0 µM ethidium homodimer 1 for 15 min and the fractions of live  and dead (ethidium homodimer 1-positive) cells were determined. Neurons were visualized by fluorescence microscopy. N1E-115 cell viability was analyzed as described previously .Virus loads in culture supernatant and mouse tissues (optic nerve and brain) were assessed by plaque assay in Vero cells as described in reference post hoc analysis. All analyses were performed using the GraphPad PRISM software 5.0 . Results with a P value of <0.05 were considered significant.Results are shown as the mean ± standard error (SEM), except for viral loads, which were expressed as the median. Body weight was converted to a percentage, and weight loss/gain was calculated by subtracting the basal levels (obtained prior to infection) from those of control and infected mice. Differences were compared using analysis of variance (ANOVA) followed by Student-Newman-Keuls


Bacillus thuringiensis serovar kurstaki strain BP865, an isolate from the South Korean phylloplane, and strain HD-133, a reference strain of B. thuringiensis serovar aizawai.We report the draft genome sequences of two insecticidal strains against lepidopteran pests,  Bacillus thuringiensis is a ubiquitous soil bacterium that produces insecticidal crystal proteins, also known as δ-endotoxins (Cry and Cyt toxins), during the sporulation stage , an isolate from the phylloplane in South Korea, was shown to exhibit both insecticidal and fungicidal activities . The biochemical and growth characteristics indicate that BP865 is indistinguishable from HD-1, which is the most widely used bioinsecticide and is recognized as the reference standard in the United States . Strainsera spp. –7.de novo genome assembly was applied using the A5-miseq pipeline  according to the manufacturer’s instructions. Library construction (average insert size of ~400 bp) and genome sequencing were carried out using an Illumina HiSeq 2000 platform at the National Instrumentation Center for Environmental Management . Sequence totals of 1.59 Gb and 1.62 Gb (2 × 101 cycles) were produced from BP865 and HD-133, respectively. A B. thuringiensis genomes from RefSeq (as of 27 October 2016) indicated that BP865 was closest to serovar kurstaki HD-1 , whereas HD-133 was closest to serovar galleriae HD-29 , followed by the two serovar aizawai strains Leapio01  and Hu4-2 . Scaffolds with ≥95% BLAST+ query coverage against complete plasmid sequences for B. thuringiensis available from the RefSeq plasmids were placed to putative plasmids. Through this approach, 87 scaffolds from BP865 (910 kb) and 77 scaffolds from HD-133 (687 kb) could be tentatively assigned to the plasmids (http://genoglobe.kr/kribb/two_Bt_strains_2016).Average nucleotide identity analysis using all 78 available B. thuringiensis strains can be extended beyond the biological control of insect pests.BP865 encodes δ-endotoxins genes for Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ia, and Cry2Ab, whereas HD-133 encodes for Cry1Aa, Cry1Ab, Cry1Ca, Cry1Da, Cry1Ia, Cry2Ab, and Cry9Ea, thereby suggesting that the latter has a wider insecticidal spectrum. However, these include partial or apparently fragmented genes, which necessitates further validation of the complete gene structure. We also identified at least two nonribosomal peptide synthetase (NRPS) genes and two NRPS/polyketide synthetase hybrid genes from both genome assemblies, implying that the potential use of MOXR00000000 (BP865) and MOXQ00000000 (HD-133). The versions described in this paper are the first versions, MOXR01000000 and MOXQ01000000.These whole-genome shotgun projects have been deposited in DDBJ/ENA/GenBank under the accession numbers


Cladosporium in cold ecosystems has been evidenced long before, and most of the knowledge about nutrient utilization of this genus is sporadic. An alpine soil isolate C. neopsychrotolerans SL-16, showing great cold tolerance and significant lignocellulose-degrading capability, was sequenced to form a 35.9 Mb genome that contains 13,456 predicted genes. Functional annotation on predicted genes revealed a wide array of proteins involved in the transport and metabolism of carbohydrate, protein and lipid. Large numbers of transmembrane proteins (967) and CAZymes (571) were identified, and those related to hemicellulose degradation was the most abundant. To undermine the hemicellulose  utilization mechanism of SL-16, the mRNA levels of 23 xylanolytic enzymes were quantified, and representatives of three glycoside hydrolase families were functionally characterized. The enzymes showed similar neutral, cold active and thermolabile properties and synergistic action on xylan degradation (the synergy degree up to 15.32). Kinetic analysis and sequence and structure comparison with mesophilic and thermophilic homologues indicated that these cold-active enzymes employed different cold adaptation strategies to function well in cold environment. These similar and complementary advantages in cold adaptation and catalysis might explain the high efficiency of lignocellulose conversion observed in SL-16 under low temperatures.The occurrence of  Although such habitats experience extreme conditions that are challenging to most life forms, various microorganisms that colonize these cold environments have adapted to low temperatures. Based on their temperature tolerance, Morita2 has grouped these microorganisms into psychrophilic or psychrotolerant: psychrophiles can grow at or below 0 °C and have optimum growth temperatures of ≤15 °C and maximum cardinal temperatures of ≤20 °C, while psychrotolerants have optimum and maximum growth temperatures of >15 °C and >20 °C. However, when extending to higher organisms, such as algae, plants, insects, fish and invertebrates, these definitions are ambiguous and inappropriate, thus Cavicchioli3 introduced ecological terms stenopsychrophile (psychrophile) and eurypsychrophile (psychrotolerant) to differ organisms with narrow and wide growth temperature ranges, respectively.The large fraction of the Earth’s surface, up to 80%, is occupied by permanently cold ecosystems (below 5 °C), which include deep sea as well as polar (Arctic and Antarctic) and alpine regions6. And the strategies adopted by cold-adapted enzymes have been proposed, including but not limited to increased flexibility at the active site8, fewer interactions like ion pairs, aromatic interactions, hydrogen bonds and salt bridges9, larger and more accessible catalytic cavity10, alternations of amino acid composition and increased solvent-accessible area11, and oligomerization12. Unfortunately, how these cold-adapted enzymes acted altogether on biomass degradation and conversion to release fermentable sugars in cold habitats is rarely studied.To survive in extremely cold environments, microorganisms display some common molecular and physiological characteristics, including increased fluidity of cellular membranes, ability to accumulate compatible solutes, expression of cold shock, antifreeze and ice-nucleating proteins, production of cold-active enzymes, and rapid horizontal gene transfer exerted by plasmidsCladosporium as the eurypsychrophilic fungus has been discovered in various cold environments, i.e. the oligotrophic soil15, air16, benthic mats17, marine sponges19 and sea water20 of the Antarctic, the alpine soil of Tibetan plateau21 and Changbai22 and Tianshan23 Mountains, the deep sea of Pacific Ocean24 and South China25, and bat26. Currently 205 species are accepted in Cladosporium, and only a few temperature studies were undertaken in eurypsychrophilic Cladosporium species30. Cladosporium is normally associated with a necrotrophic or hemibiotrophic lifestyle31 and has great capability of degrading lignocellulose36 and polycyclic aromatic hydrocarbons like phenanthrene and anthracene38. However, so for only a few Carbohydrate Active enZymes (CAZymes), including glucoamylase39, xylanase41 and α-galactosidase42, have been functionally characterized. This sporadic knowledge provides some clues to but still far away from the underlying mechanisms of lignocellulose utilization of Cladosporium, not to speak of eurypsychrophilic Cladosporium.C. neopsychrotolerans SL-16 was isolated from the alpine soil of Tianshan Mountain23. To undermine its mechanism in cold adaptation and nutrient utilization, the SL-16 genome was sequenced, and the genes related to plant cell wall degradation were predicted. The genes related to hemicellulose degradation under low temperatures were diverse and abundant, and a xylanolytic enzyme system was selected for qPCR and functional analysis. To our best knowledge, it is the first report on comprehensive genomic characterization of eurypsychrophilic Cladosporium species. We suggest that the synergistic actions on xylan degradation and complementary advantages in cold adaptation of xylanolytic enzymes are important traits of C. neopsychrotolerans for hemicellulose utilization under low temperature conditions.In our previous study, a eurypsychrophilic strain C. neopsychrotolerans SL-16 was initially isolated at 4 °C, and showed optimal growth at 15 °C23. To induce the production of lignocellulose-degrading enzymes, aliquots of spore suspension (1 × 107 CFU) were inoculated into a wheat bran medium43. After 7-day-growth at 4–30 °C, the cultures showed significant cellulase (CMCase and glucanase) and hemicellulase (xylanase and mannanase) activities  was generated in the present study. After assembly the sequence data, the draft genomic DNA sequence of strain SL-16 was 35,929,705 bp in length with the GC content of 52.64%  CAZymes involved in the degradation of hemicellulose (92), pectin (71), the side chains of hemicellulose and pectin (55), cellulose (8), and 9 carbohydrate binding domain (CBM) , confirming the preference of Cladosporium for soft plant tissues as a carbon source36. The CAZymes of SL-16 were also compared with those of the two Cladosporium strains , 83 glycoside transferases (GT), 73 auxiliary activities (AA), 64 carbohydrate-binding modules (CBM), 34 carbohydrate esterases and 19 polysaccharide lyases (PL)  were then selected for functional characterization.Strain SL-16 was distinct due to the high number of putative hemicellulases  competent cells for heterologous expression. After 12-h induction with isopropyl β-D-1-thiogalactopyranoside (IPTG) at 15 °C, the E. coli cells harboring recombinant plasmids pET-xyn10A, pET-xyn11A or pET-xyl43A were disrupted and showed xylanase or xylosidase activities of 1.87 ± 0.06 U/mL, 1.54 ± 0.04 U/mL and 0.87 ± 0.05 U/mL  with beechwood xylan or p-nitrophenyl-β-d-xylopyranoside (pNPX) as the substrate, respectively. The crude enzymes were purified to electrophoretic homogeneity by affinity chromatography with a Ni2+-NTA chelating agarose column. Each recombinant protein migrated a single band of 23–38 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis   Fig. . In comp°C) Fig. . In cont°C) Fig. .Figure 32+, Pb2+, Ag+, SDS and EDTA, and their activities were enhanced by Co2+, Ni2+ or β-mercaptoethanol (>15%). In contrast, the Xyl43A was sensitive to Fe3+, Mn2+, Cu2+, Co2+, Ni2+, Zn2+, Pb2+, Ag+, SDS, and EDTA, losing 46–100% activity.The three xylanolytic enzymes were also tested for tolerance against 15 metal ions and chemical reagents at the concentration of 5 mM  activities. These results indicated that the xylanolytic enzyme system of strain SL-16 is functional against a broad substrate spectrum not limiting to xylan and xylooligosaccharides, which may maximize the utilization of available carbon sources.The substrate spectrum of each enzyme was determined to analyze their degrading capabilities of different carbohydrates Table . Xyn10A d-xylose on the xylosidase activity of Xyl43A was tested under the standard conditions in the presence of 0 to 200 mM xylose. The Ki value of xylose was determined to be 43.3 mM 52, Xyl43A with much higher xylose tolerance is particularly interesting for efficient saccharification of xylan.During xylan biodegradation, xylooligosaccharides of various lengths are produced, which would be hydrolyzed into xylose by xylosidase. The inhibitory effect of Vmax and kcat values but had increased Km value at 20 °C, while Xyn11A and Xyl43A had lower Vmax, kcat, and Km values , the kcat/Km value of Xyl43A was comparable to that of optimal conditions, while Xyn11A showed great improvement in catalytic efficiency at lower temperature. It suggested that the three xylanolytic enzymes employed different mechanisms to function at low temperatures.The kinetics of Xyn10A, Xyn11A and Xyl43A were determined at 20 °C and their temperature optima, respectively. In comparison with the kinetics at optimal temperature, Xyn10A retained high es Table . Xyn10A Tm) values of Xyn10A, Xyn11A and Xyl43A were 56.89 °C, 44.03 °C, and 52.55 °C  instrument. The melting temperature (Hydrolysis products of beechwood xylan and xylooligosaccharides (xylose to xylohextaose) by Xyn10A, Xyn11A and Xyl43A were analyzed by using the high performance anion-exchange chromatography (HPAEC). When using beechwood xylan as the substrate, Xyn10A mainly released xylotriose (67%) and xylobiose (26%), while Xyn11A produced xylotriose (44%), xylotetraose (25%), xylopentaose (13%), and xylobiose (12%). Analysis of the hydrolysis products of xylooligosaccharides confirmed distinctive action modes of Xyn10A and Xyn11A. Xyn10A had no activity against xylobiose and xylotriose, acted weakly on xylotetraose, moderately on xylopentaose, and efficiently on xylohexaose. In contrast, Xyn11A had no capacity of degrading xylobiose, xylotriose and xylotetraose, even lengthening the incubation duration from 30 min to 12 h, and was active against xylopentaose and xylohexaose. The results indicated that Xyn11A requires at least five subsites for binding and cleaves substrate randomly, while Xyn10A requires at least four subsites for binding and cleaves substrate in a relatively strict mode. This difference may also account for the broad functional spectra of Xyn10A, which is more flexible in substrate binding. Xyl43A was capable of degrading all tested xylooligosaccharides and attacking xylan to produce xylose slowly.How the three xylanolytic components, xylan-specific Xyn11A, multifunctional Xyn10A, and bifunctional xylosidase-arabinofurasidase Xyl43A, cooperate in conversion of xylan substrate to xylose (pentose) is much intriguing. When using xylans from beechwood, birchwood, and wheat as substrates, the amounts of released reducing sugars  by individual or combined enzyme(s), each at the dosage of 20 U/g dry matter, were determined. As shown in Table Thermoascus aurantiacus (TaXyn10)54 and GH11 xylanase of Thermomyces lanuginosus (TlXyn11)55 are thermophilic, while the GH43 xylosidase-arabinofuranosidase from a compost metagenome (CoXyl43)57 is mesophilic. Their structure elements, protein interaction and accessible surface area were compared to reveal the cold-adapted strategies. In comparison to the mesophilic and thermophilic counterparts, the three enzymes have less hydrophobic residues in common. Moreover, Xyn10A has more Gly and higher Gly/Pro ratio, Xyn11A has more Arg and higher Gly/Pro ratio, and Xyl43A has more Gly. Protein interactions, including hydrogen bonds, salt bridge, disulfide bonds, hydrophobic bonds, aromatic-aromatic interactions, and cation-Pi interactions, are also negatively correlated with enzyme flexibility. Relative to the counterparts, Xyn10A has less hydrogen and hydrophobic bonds and no disulfide bond, Xyn11A has less salt bridges and cation-Pi interactions and no disulfide bond, and the Xyl43A has less protein interactions checked in this study. Another structure feature of enzyme flexibility is increased interactions with the solvent. Xyn10A and Xyn11A have larger accessible surface area (ASA) but vary in the amounts of exposed nonpolar, polar and charged ASA, while Xyl43A has less ASA. It suggested that the three xylanolytic enzymes employ different strategies to function at low temperatures.The parameters related to structure flexibility of Xyn10A, Xyn11A and Xyl43A and their structure-resolved counterparts were analyzed and shown in Table Cladosporium is one of the largest and most heterogeneous genera of hyphomycetes that are widely distributed in all types of environments59. It has been attracting much research interest in recent years because of the broad physiological capabilities 61 and pathogenicity in plants, animals and humans63. Two genomes of Cladosporium have been sequenced and functionally annotated, and their nutritional modes may account for the evolution of functional genes. The tomato leaf mold pathogen C. fulvum has a genome of 61.1 Mb that contains 14,127 predicted genes31, while the human allergen and occasional pathogen C. sphaerospermum has a genome of 26.9 Mb that contains 9,652 predicted genes64. Both genomes encode wide arrays of CAZymes (445 for C. fulvum and 605 for C. sphaerospermum), which are related to their nutritional strategy and host specificity65. In comparison to the biotrophic C. fulvum and saprophytic C. sphaerospermum, the hemibiotrophic C. neopsychrotolerans SL-16 isolated from the rhizosphere soil of snow lotus has a moderate-size genome (35.9 Mb) that contains 13,456 predicted genes and encodes 571 CAZymes. Gene classification analysis indicated that most of the predicted genes of Cladosporium are related to the transport and metabolism of carbohydrate, protein and lipid. In comparison to other known biomass-degrading fungal strains, all the three Cladosporium species tend to have more CAZymes (58–92 vs. 31–53) to degrade hemicellulose , the 23 xylanolytic enzymes showed high expression levels  and low temperature (35–40 °C) and inactivation by the application of moderate heat (>40 °C). In comparison to the two cold-active fungal xylanases from psychrotrophic Penicillium chrysogenum and Bispora antennata69 that have temperature optima at 25 °C and 35 °C, the xylanolytic enzymes of eurypsychrophilic C. neopsychrotolerans showed similar or higher temperature optima and retained less or comparable activity at 0 °C (10.1–21.2% vs. 50% and 20%). Further kinetic analysis of these enzymes revealed different cold-adaptive catalytic mechanisms: under low temperature (20 °C), Xyn10A retained high enzyme velocity and turnover number, while Xyn11A and Xyl43A showed increased substrate affinity , Xyn11A has psychrophilic nature while Xyn10A and Xyl43A are psychrotolerant. Moreover, these enzymes also varied in pH adaptability and stability and chemical resistance. In comparison with Xyn10A and Xyn11A, Xyl43A had adaptability and stability over a narrower pH range and was sensitive to most tested metal ions and chemical reagents. This divergence might be ascribed to their evolution of different functional strategies: Xyn10A and Xyn11A are extracellular and more adaptive to harsh conditions, while intracellular Xyl43A is relatively demanding and lacks adaptation to environmental stress. The similar and complementary properties of these xylanolytic enzymes may contribute to the efficient hemicellulose utilization of C. neopsychrotolerans SL-16 over a broad temperature range (0–60 °C).erature 1 °C, the 71. The functionally characterized xylanolytic enzymes of strain SL-16 showed the same substrate spectra as described above. Xyn10A had broad substrate specificity including xylan, cellulose and glucan, Xyn11A was specific for xylan substrates, and Xyl43A had both xylosidase and arabinofuranosidase activities. Combination of these enzymes would broaden the substrate spectrum and act synergistically on complex biomass by using different cleavage modes72. In this study, the hydrolysis capacities of the xylanolytic enzyme system against three xylan substrates were tested. Of them, hardwood xylan (beechwood and birchwood) contains approximately 90% xylose that are linked by β-1,4-glycosidic bonds as the main chain, while wheat arabinoxylan is featured with relatively complex composition (xylose/glucose/arabinose of ~75:15:10)74. Although all enzyme combinations had no significant synergy (0.74–1.04-fold) on the release of reducing sugars, more reducing sugars (up to 1.27-fold) were detected in the sequential rather than simultaneous reactions with hardwood xylan as the substrate 23. To test the ability of SL-16 to grow at different temperatures, it was grown in wheat bran medium43 at 4–30 °C for 7 days. The lignocellulose-degrading capability of the fungus were assayed at pH 6.0 and 30 °C for 10 with 1% (w/v) beechwood xylan, barley β-glucan, and carboxymethyl cellulose (CMC) as substrates as described below.C. neopsychrotolerans SL-16 was extracted using the CTAB method. The 500-bp insert library was sequenced using the Illumina PE250 system. The clean reads were then assembled by SOAPdenovo 77 for data processing. Genes were subsequently predicted using Augustus 3.2.178 and GeneMark-ES 4.2179 with default parameters, which were further integrated by the GLEAN (https://sourceforge.net/projects/glean-gene/). All predicted gene models were functionally annotated against InterProScan80, SwissProt81, KEGG82, and KOG83. Interpro and SwissProt hits were used to map GO terms (www.geneontology.org), and KEGG hits were used to assign EC numbers (www.expasy.org/enzyme). Genes associated with carbohydrate utilization were identified by performing a local BLASTp against the CAZy database (http://www.cazy.org) using the criteria of e-value threshold ≤1e−5, identity exceeding 50% and subject coverage exceeding 70%. SignalP84 and TMHMM85 were employed to predict the presence of signal peptide and transmembrane α-helix structures. The protein sequences of eight biomass-degrading fungal representatives were downloaded from different databases for orthologous gene comparison (Table The genomic DNA of 260/A280 and A260/A230. cDNA was synthesized according to the protocol of ReverTra Ace-α-TM kit (TOYOBO). The gene fragments coding for mature xylan-main-chain degrading enzymes (xylanase and xylosidase) with >50% identity to known proteins were amplified by PCR and ligated into the pEasy-T3 vector (Tiangen) for Sanger sequencing.The mycelia of strain SL-16 were collected after 2-day-growth in wheat bran medium at 15 °C and immediately ground to a fine powder in liquid nitrogen. Total RNA was extracted using the SV Total RNA Isolation System (Promega) according to the manufacturer’s protocol. The quantity and purity of RNA was determined using an Ultrospec 2100 pro UV/visible spectrophotometer (Amersham Biosciences) based on the absorbance ratios of A7 copies of each gene. All qPCR amplifications followed the thermal program consisting of the following cycles: 95 °C for 5 min, 40 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s. Fluorescence data were collected at the last step of each cycle. The relative fold change of each gene was calculated from the Ct values after normalization against β-tubulin. All operations for qPCR followed the MIQE86.qPCR was performed using the QuantStudio 6 Flex real-time qPCR system (Thermo Fisher Scientific) with the SYBR Green SuperReal PreMix (TianGen), SL-16 cDNA template, and specific primers of each gene  (Novagen) were digested by restriction enzymes , purified, and connected by the T4 DNA ligase (New England Biolab) to construct recombinant plasmids, which were further transformed into E. coli BL21(DE3) competent cells. Enzyme induction with IPTG (0.6–0.8 mM) at 15 °C and purification through nickel affinity chromatography followed the previous protocols87. Xylanolytic activities of the sonication-disrupted cell pellets and purified recombinant enzymes were assayed as described below. The molecular mass and purity of each purified recombinant enzymes were evaluated by the SDS-PAGE. The protein concentrations were determined using bovine serum albumin as the standard88.The gene fragments of 89 with 1% (w/v) beechwood xylan, locust bean gum, CMC or barley β-glucan (Sigma-Aldrich) as the substrate. One unit (U) of xylanase/mannanase/CMCase/glucanase activity was defined by the amount of enzyme releasing 1 μmol of reducing sugar per min at given assay conditions. The xylosidase activity towards pNPX (1 mM) was assayed as described by Shao et al.90. One unit of xylosidase activity was defined as the amount of enzyme that released 1 μmol of p-nitrophenol per min.Xylanase, mannanase, CMCase or glucanase activity was assayed using 3,5-dinitrosalicylic acid (DNS) methodThe pH properties of Xyn10A, Xyn11A and Xyl43A were determined in the 100 mM of McIlvaine buffer (pH 3.0–8.0), Tris-HCl (pH 8.0–9.0) and glycine-NaOH (pH 9.0–12.0). The pH-activity profiles were examined at 30 °C over the pH range of 3.0–9.0 for 10 min. The temperature optima were examined at each optimal pH over the temperature range of 0 to 60 °C. For pH stability assays, all enzymes were pre-incubated at pH 3.0–11.0, 30 °C for 1 h without the substrate, and their residual enzyme activities were determined under optimal conditions. The thermostability assays were performed by incubating the enzymes at optimal pH and at 30 °C or 40 °C without substrate for 0–60 min, and then measuring the residual enzyme activities under the optimal assay conditions.The effects of different metal ions, chemical reagents and detergents on enzyme activities were determined under optimal conditions and compared to the blank controls.pNP derivatives including pNPX, pNPAf, pNPG, pNPGal, pNPAb, and pNPC. All substrates were purchased from Sigma-Aldrich or Megazyme.The substrate specificities of Xyn10A, Xyn11A and Xyl43A were tested by measuring their enzyme activities against 1% (w/v) of polysaccharides  or 1 mM of Ki) of d-xylose for Xyl43A was determined by fitting to the Dixon plot91. Reaction systems (500 μL) containing 125 μL of 1.0 or 1.25 mM pNPX, 275 μL of d-xylose solution  and 100 μL of suitably diluted enzyme were incubated at room temperature for 10 min. Enzyme activities were determined as described above.The inhibition constant  was used for data analysis using the Michaelis-Menten model. DSC was used to assay the thermodynamics of Xyn10A, Xyn11A and Xyl43A on a MicroCal™ VP-Capillary DSC  as described previously92. Aliquots of proteins (200 μg) were dissolved in 1 mL of 20 mM McIlvaine buffer (pH 6.5) and loaded into the capillary automatically. The Tm value corresponded to the maximum of the transition peak over 20–90 °C within a heating and scanning rate of 2 °C/min. The same McIlvaine buffer was used as the blank control.The 93.Reaction systems (1 mL) containing 0.5 U of Xyn10A, Xyn11A or Xyl43A and 1% (w/v) substrate  in McIlvaine buffer (pH 6.5) were incubated at room temperature for 12 h and treated as baselines. The simultaneous reactions of Xyn10A, Xyn11A and Xyl43A contained 0.5 U of each enzyme. For sequential reactions, first reactions containing either Xyn10A or Xyn11A (0.5 U of each) were incubated at room temperature for 12 h, boiled in water bath for 10 min to inactivate the enzyme(s), followed by secondary and tertiary incubation(s) with the other xylanase and xylosidase for another 12 h. The released reducing sugars in the supernatants were measured as xylose equivalents by using the DNS method, and the appropriately diluted hydrolysates (50–200 times) were analyzed by the HPAEC  equipped with a pulsed amperometric detector (PAD) and xylooligosaccharides (Sigma-Aldrich) as standards. The XCR was defined as the percentage of xylose amount against the total amount of reducing sugars  released. The degree of synergy was defined as the ratio of xylose released when enzymes were incubated simultaneously or sequentially to the sum of the xylose released by each enzyme aloneT. aurantiacus 53, GH11 xylanase of T. lanuginosus 55, and GH43 xylosidase-arabinofuranosidase of a metagenome 57, were selected for the analysis of cold-adapted strategies. Homology modeling of Xyn10A, Xyn11A and Xyl43A was conducted by using the Swiss-modeler (https://swissmodel.expasy.org/interactive). The amino acid composition was analyzed by the Vector NTI Advance v10.0 software. PIC (http://pic.mbu.iisc.ernet.in/) and VADAR v1.8 (http://vadar.wishartlab.com/) were used to calculate the protein interactions and ASA.Three structure-resolved thermophilic or mesophilic homologues, the GH10 xylanase of Supplementary materials


The results indicated that the maternal LED markedly decreased the BW, small intestinal weight, and the ratio of jejunum and ileum villus height to crypt depth in the offspring. Moreover, the activities of lactase and sucrase in newborn piglets’ intestine, and sucrase and maltase in weaning piglet intestine were markedly decreased by the maternal LED. In addition, maternal LED significantly increased the mRNA relative expression of ileal IL-6 and TNF-α in newborn piglets. Plasma IL-1β concentration and colonic Escherichia coli amount were affected by maternal diet (p < 0.05) and LPS challenge (p < 0.001). Maternal LED significant increased the mRNA relative expression of ileal TLR-4, IL-1β and NF-κB as well as decreased ZO-1 in weaning pigs after LPS challenge (p < 0.05). In conclusion, decreasing energy intake could suppress the offspring’s intestinal digestion and absorption function, and increase the susceptibility of weaning piglets to LPS challenge.Maternal nutrition during gestation is involved in the offspring’s intestinal development and immunity. The aim of this study was to (1) determine the effects of maternal energy on intestinal digestion and absorption function in offspring, using pigs as a model; and (2) to evaluate the potential effect and mechanisms of maternal energy in modulating immune responses of lipopolysaccharide (LPS)-challenged piglets. After mating, thirty-six nine-parity sows (Landrace × Yorkshire), body weight (BW)  were allocated to two dietary treatment groups; a control diet (CON) group and a low-energy diet (LED) group. The nutrient levels of the CON were based on the nutrient recommendations by the National Research Council , and contained 3.40 MCal digestible energy (DE)/kg diet and 7.3% crude protein; while the LED contained 3.00 MCal DE/kg diet. The dietary treatments were introduced from day 1 of gestation to farrowing. Intestine samples were collected from the pigs’ offspring at birth, and at weaning (day 28 post-birth). At weaning, male pigs from control and LED groups were intraperitoneally injected with LPS (50 μg/kg body weight) or saline ( Maternal malnutrition in pregnancy can cause maternal endocrine and metabolic disorders, further damaging the growth of the placenta, thereby affecting the mother-to-fetus nutrient transport and weakening the growth and development of the fetus . PregnanThe study protocol was reviewed and approved by the Care and Use Committee of Sichuan Agricultural University, and followed the current laws of animal protection .ad libitum. Throughout the experiment, all animals had free access to water. The ambient temperature for sows was maintained at 20–25 °C. Heating light and pads were provided for suckling piglets, and the temperature was maintained at 26–32 °C, which was gradually decreased with increase in age. All sows were housed in individual feed stalls during gestation and, during lactation, were housed in farrowing pens.A total of thirty-six nine-parity sows (Landrace × Yorkshire), with initial body weights (BW) of 233.56 ± 2.77 kg and backfat thickness (BF) <14 mm at day 0 of gestation were used in this study. Sows were artificially inseminated with pooled semen obtained from a littermate boar (housed on the research farm) on the day of estrus, and then 12 and 24 h later. After artificial insemination, sows were randomly assigned to one of two diets: the control (CON) diet was designed as per the recommendations of the National Research Council  for gestating sows, and contained 3.40 MCal digestible energy (DE) per kilogram, and 7.3% crude protein, together with 5% crude fiber; while the low-energy diet (LED) contained 3.00 MCal DE/kg, with the same crude protein and crude fiber as CON. Sows were fed 2.2 kg/day during early pregnancy (days 1–30 of gestation), 2.4 kg/day mid-pregnancy (days 31–90), and 2.8 kg/day late pregnancy (days 91 to parturition). After farrowing, sows were fed a lactation diet in accordance with NRC 2012, and were offered the diet three times per day , starting at 2.0 kg/day, then gradually increasing by 1 kg/day until the sixth day, after which the sows could feed Escherichia coli L2880, Sigma-Aldrich, Los Angeles, CA, USA) was dissolved in sterile saline (9 g/L) to make the LPS solution . The dosage of the LPS injection and the time to sacrifice piglets were described previously [At birth, six piglets (one piglet from each of six litters), each having approximately the same body weight (±10%) as the mean body weight of the litter, were selected from each group and slaughtered before suckling. At the day of weaning (day 28 post-birth), twelve healthy male piglets were selected from each of the two treatments. Six piglets per treatment were intraperitoneally injected with LPS solution to administer LPS at 50 µg/kg body weight, in order to make a model of immunological challenge, and another six piglets in each treatment were intraperitoneally injected with an equivalent amount of sterile saline. Feed was removed 4 h before the injection. The rectal temperature of each piglet was measured every 2 h. LPS  and stored at −20 °C until analysis. Immediately after collection of blood samples at 4 h, piglets were slaughtered by jugular puncture, and the entire intestine was removed as quickly as possible. The small intestine (SI) was quickly measured for weight and length. The middle portion (approximately 4 cm) of each segment of the small intestine  was rapidly obtained and placed in liquid nitrogen, and then stored at −80 °C for subsequent analysis, as previously described [The pigs were anesthetized with an intravenous injection of pentobarbital sodium (50 mg/kg BW) before blood sample collection, and then slaughtered. From each group, six newborn piglets without eating colostrum  were slaughtered, as well as 12 weaning piglets deprived of breast nursing from sows for 4 h before the LPS injection . On the day of birth and at 0 and 4 h after injection of LPS or normal saline, blood samples were collected from the anterior vena cava and placed into heparinized tubes, centrifuged , and then embedded in transparent paraffin and sliced . All tisIntestinal enzyme activity was measured as previously described . After t®Premix Ex Taq II (Tli RNaseH Plus) and 0.2 μL ROX Reference Dye II (50×), 0.8 μL of the primers, 1 μL of RT products and 3 μL diethylpyrocarbonate-treated water. The PCR protocol was used as follows: 1 cycle (95 °C for 30 s), 40 cycles  and 1 cycle . The relative mRNA abundance of the analyzed genes was calculated using the 2−ΔΔCt method, as described previously by Mou et al. [Gene expression levels were detected by using a reverse transcription polymerase chain reaction (RT-PCR) similar to the procedure previously described . Brieflyu et al. . The mosPlasma TNF-α, IL-1β, IL-10, IgA and GLP-2 concentrations were measured by using ELISA kits suitable for pigs  according to the manufacturer’s protocol.Routine analysis of blood samples in heparinized-plasma venipuncture vials were performed by automatic blood analyzer .g, 4 °C) for 10 min. The supernatant (1.2 mL) was removed by aspiration into another 5-mL centrifuge tube, added to 0.24 mL of 25% metaphosphate, vortex mixed for 30 s, and then left to stand for 30 min at 4 °C. Next, the contents were centrifuged  for 10 min, and then 1.2 mL of the supernatant was removed by aspiration, added to 23.3 μL of 210 mmol/L cortonic acid, and vortex mixed for 30 s. Then, 0.3 mL of the mixed solution was removed and placed into another 2-mL tube, added to 0.9 mL Carbinol, and vortex mixed for 30 s for subsequent gas chromatography analysis. The samples were analyzed by CP-3800 gas chromatography  equipped with a micro-injector (10 μL), a flame ionization detector and a capillary chromatographic column . The injector temperature was 220 °C, the detector temperature was 250 °C, hydrogen flux was 40 mL/min, and air flux was 450 mL/min. The temperature program was as follows: 100 °C, hold 1 min, increase to 190 °C at 20 °C/min, increase to 190 °C, hold 3 min. Peaks were identified by comparing their retention times with individual reference standard fatty acids.SCFAs, including acetate, propionate and butyrate in digesta and mucosa samples, were analyzed with a modification of the previous method . Briefly® Green PCR reagents , whereas quantitative RT-PCR for Bifidobacterium, Lactobacillus, and Escherichia coli were performed with Taq primers and fluorescent oligonucleotide probes that had been commercially synthesized , as listed in Bifidobacterium, Lactobacillus, and Escherichia coli were set up in a total volume of 10 μL, and contained 0.15 μL of probe, 0.5 μL of each primer, 0.5 μL of probe enhancer solution, 4 μL of Real Master Mix, 0.5 μL of genomic DNA, and 3.85 μL of sterile deionized water. Amplification was performed using a CFX 96 System . As for total bacteria, the amplification program consisted of one cycle of 95 °C for 30 s and then 40 cycles of 95 °C for 5 s, 60 °C for 34 s, and one cycle of 95 °C for 15 s, 60 °C for 1 min, 95 °C for 15 s. For Bifidobacterium, Lactobacillus, and Escherichia coli, the amplification program consisted of one cycle of 50 °C for 2 min, 95 °C for 10 min and then 40 cycles of 95 °C for 15 s and 55.6–59.5 °C for 1 min. Melting curve analysis and size determination of PCR fragment on agarose gels verified amplification of the target fragments. Standard curves were generated as previously described [Quantitative RT-PCR of total bacteria was performed with SYBRescribed .t-test was used to compare the differences between the dietary groups. CON and the LED results after LPS challenge were performed by two-way ANOVA; data was tested for normality and homogeneity of variances  and, when necessary, data was normalized  to achieve ANOVA assumptions. Additionally, Duncan’s multiple range tests were conducted to determine the difference among the treatments. All values are means with their standard errors (SE), and differences between treatments were considered significant when p < 0.05.SPSS 21.0  was used for all statistical analyses. Newborn piglets’ results were tested for variance using Levene’s test; a two-tailed p < 0.01). Maternal LED piglets had a higher ratio of SI length to BW than CON piglets (p < 0.01). We also found the maternal low-energy diet significantly reduced the ratio of SI weight to length in newborn piglets (p < 0.05). However, maternal diet did not affect the weight of the small intestine (SI), the length of the SI, or the ratio of SI weight to BW in the newborn piglets (p > 0.05).As shown in p < 0.001, p < 0.001, p < 0.05 and p < 0.001, respectively) (p = 0.001).In weaning piglets, maternal diet significantly lowered BW, SI weight, SI length, and SI weight/length (ctively) . SI weigctively) . Comparep < 0.05, The villus height of jejunum and ileum, and the VCR (ratio of villus height to crypt depth) of the duodenum and ileum were lower in LED newborn piglets than in CON piglets , but not by LPS challenge, the duodenum villus height (p < 0.05) was also significantly influenced by Diet × LPS (p < 0.05). The duodenum crypt depth in −LPS LED piglets was lower than CON piglets (p < 0.05), whereas there was no difference in +LPS LED piglets.Maternal diet significantly affected duodenum villus height and crypt depth (CD), jejunum villus height and ileum VCR of the weaning piglets (et × LPS . Comparep < 0.05) in the LED group in newborn piglets (The activity of lactase in jejunum and sucrase in ileum markedly decreased ( piglets .p < 0.01), it is not affected by the LPS challenge and Diet × LPS (p > 0.05), the sucrase in the LED group was lower than in the CON group , but was significantly influenced by maternal diet . The ileal IL-10 mRNA abundance in the +LPS LED group was higher than −LPS CON and −LPS LED groups (p < 0.05). Compared with +LPS CON group, the mRNA relative expression of ileal ZO-1 was significantly decreased in the +LPS LED group  to decrease the mRNA relative expression of the ileal GLP-2R in the newborn piglets (The plasma GLP-2 concentration was lower in LED newborn piglets than in CON piglets ( piglets B.p < 0.01) .Plasma GLP-2 concentration was significantly affected by maternal diet in the weaning piglets ( < 0.01) A, whereaET × LPS B. The plp < 0.001) rectal temperature at 4 h  and LPS challenge (p < 0.05), while plasma IgM concentration was affected by maternal diet (p < 0.01) and Diet × LPS interaction (p < 0.01), and a tendency (p < 0.10) towards decrease was also observed following LPS challenge (p < 0.05) in the LED group compared with −LPS CON group. Compared with −LPS CON group, the plasma IgM concentration significantly decreased in the remaining groups at weaning (p < 0.05).Plasma IgA concentration was affected by maternal diet , LPS challenge (p < 0.001), and Diet × LPS interaction (p < 0.05) (p < 0.01) and Diet × LPS interaction (p < 0.05) (p < 0.05) in the +LPS LED than in the remaining groups. In addition, plasma IL-1β concentration was higher (p < 0.05) in the +LPS CON group than in the −LPS CON group.Plasma IL-1β concentration was significantly affected by maternal diet ( < 0.05) A, while  < 0.05) B. Plasmap < 0.05) (p < 0.05) in the +LPS treatments than in the −LPS CON treatment, whereas propionic acid concentration in caecal content was lower (p < 0.05) in the +LPS LED group than in the −LPS CON group.Acetate and propionic acid concentrations in caecal content were significantly decreased by LPS challenge ( < 0.05) . AcetateEscherichia coli in colonic content (p < 0.05)  .In the embryonic period, the gastrointestinal system is one of the earliest systems of polarization, and the last few weeks before the birth of the fetus are a very important period for intestinal potential and digestive function maturity . Some stIntegrity of the intestinal structure is required for the maintenance of intestinal nutrition . FetusesTNF-α and IL-6, were significantly up-regulated by maternal LED in newborn piglets, and plasma IgA and IgM were significantly reduced by the maternal dietary treatment in weaning piglets. Meanwhile, it was observed that LPS challenge resulted in increased plasma IL-1β concentration, which was consistent with gene expression results. Meanwhile, LPS challenge and low-energy diets increased TLR-4 and NF-kB gene expression in weaning pigs’ ileum, which corresponded positively with inflammatory response. Previous studies also found maternal restraint stress during gestation in pigs enhanced the magnitude of the TNF-α and IL-6 responses to LPS in the offspring [Nutritional and other molecular events in fetal and neonatal life lay the foundation for future health, which includes effects on immune system function. The second aim of this study was to determine the potential effect of maternal LED on the immune responses of LPS-challenged piglets. The increased body temperature, plasma TNF-α, and IL-1β concentrations indicated the successful establishment of the immune model following LPS challenge. It has been demonstrated that the TLR-4–Myd88–NF-kB signal pathway is involved in inflammation . Moreoveffspring . TherefoZO-1 gene in weaning piglets. At the same time, maternal LED significantly reduced the GLP-2 levels in newborn and weaning piglet plasma. GLP-2 increases the rate of crypt cell proliferation and villus elongation, and reduces apoptosis, leading to improved barrier function [The intestinal epithelial barrier includes the presence of a single epithelial cell (EC) layer, tight junctions (TJs) between ECs, and the presence of associated immune cells . ZO-1, Ofunction . Meanwhifunction . In the function . The decfunction . These rIn conclusion, our study demonstrates that decreasing energy intake during gestation suppressed the offspring’s intestinal digestion and absorption function and increased susceptibility of the weaning piglets to LPS challenge.


In total, 2385 patients with Graves’ hyperthyroidism  were involved in our study. Of these, 1552 cases with hepatic dysfunction received 131I treatment. All clinical data were retrospectively reviewed to explore the risk factors associated with hepatic dysfunction using logistic regression analysis. Furthermore, we observed thyroid and liver function indices for the 1552 subjects at 3, 6 and 12 months after 131I treatment, in order to evaluate efficacy. Overall, 65% patients were affected by hepatic dysfunction. The most common abnormality was elevated alkaline phosphatase (ALP), of which the prevalence was 52.3%. The percentages of hepatocellular injury type, bile stasis, and mixed type were 45.8%, 32.4%, and 21.8%, respectively. Both univariate and multivariate analyses demonstrated that age, duration of Graves hyperthyroidism, free triiodothyronine (FT3)level, and thyrotrophin receptor antibody (TRAb) concentration were the most significant risk factors predicting hepatic dysfunction. Additionally, the patients with mild hepatic dysfunction, or hepatocellular injury type were more likely to attain normal liver function after 131I treatment. Furthermore, after 131I treatment, liver function was more likely to return to normal in the cured group of patients compared with the uncured group. Older patients and cases with a longer history of Graves’ hyperthyroidism, higher FT3 or TRAb concentration were more likely to be associated with hepatic dysfunction, and the prognosis of hepatic dysfunction was closely associated with the outcomes of Graves’ hyperthyroidism after 131I treatment.Hepatic dysfunction is often observed in patients with Graves’ hyperthyroidism. The aims of this study were to investigate the risk factors for hepatic dysfunction and to analyze the efficacy of  In clinical practice, hepatic dysfunction in patients with Graves hyperthyroidism is common. This has proven to be a relatively safe procedure with a high cost-effective ratio in retrospective studies. The normal interaction between the thyroid and liver is critical for maintaining homeostasis. However, to date, reports demonstrating predictive factors related to hepatic dysfunction in Graves’ hyperthyroidism are both scarce and controversial, and studies exploring RIT and liver function are rare. Therefore, identification of the factors resulting in hepatic dysfunction is crucial.There are 3 main factors that contribute to hepatic dysfunction in the context of hyperthyroidism, including the independent effects of excessive thyroid hormones, antithyroid drug (ATD)-related liver injury, and the presence of concomitant liver disease. Historically, the control of hyperthyroidism has required 3 options including ATD, surgery, and radioiodine-131 therapy (RIT). In China, the current treatment guideline for Graves hyperthyroidism states that RIT should be the first choice of treatment in patients with hepatic dysfunction.131I treatment.In the present study, we retrospectively collected large-scale clinical data on 2385 patients with Graves hyperthyroidism to investigate the risk factors for hepatic dysfunction in patients with Graves hyperthyroidism and further analyzed the therapeutic effect of 22.1This study is a retrospective clinical study. It is the summary and analysis of a large number of clinical data. The ethics committee of Tianjin Medical University General Hospital waived the need to obtain written informed consent from all patients. All clinical data used in this study were analyzed anonymously.2.2 Of these, 1073 patients had been treated with ATD before, and 1552 cases were associated with hepatic dysfunction and received RIT. We used hepatitis virus markers, abdominal ultrasonography, echocardiography, and determination of auto-antibodies and immunoglobulin subtypes for patients with hepatic dysfunction, in order to exclude other apparent causes of liver damage. Other causes included viral hepatitis, liver cirrhosis or biliary tract disease, chronic cardiac dysfunction or autoimmune liver disease.A total of 2385 patients who were diagnosed with Graves hyperthyroidism  were retrospectively reviewed. Graves hyperthyroidism was diagnosed on the basis of thyrotoxicosis, increased thyroid hormones and decreased thyroid-stimulating hormone (TSH), elevated thyroid radioiodine uptake, and diffuse goiter, or exophthalmos, or pretibial myxedema, or positive TRAb.2.33) (X6), free thyroxine (FT4) (X7), TSH (X8), TRAb (X9), anti-thyroid peroxidase antibody (TPOAb) (X10) and anti-thyroglobulin antibody (TgAb) (X11), RAIUmax (maximum radioiodine uptake) (X12), and thyroid weight (X13). Meanwhile, liver function tests including serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), ALP, gammaglutamyl transpeptidase (GGT), total bilirubin (TBIL), and direct bilirubin (DBIL) were also collected.Data were recorded, including patient age (named X1), gender (X2), duration of Graves hyperthyroidism (X3), the course of ATD treatment (X4), heart rate (X5), serum free triiodothyronine , ALP < 2 ULN and/or TBIL, DBIL < 2.5 ULN was defined as mild hepatic dysfunction; 3 ULN≤ ALT or AST < 20 ULN, 3 ULN≤ GGT < 10 ULN, 2 ULN≤ ALP < 5 ULN and/or 2.5 ULN≤ TBIL, DBIL < 5 ULN as moderate; ALT or AST ≥20 ULN, GGT ≥10 ULN, ALP ≥5 ULN and/or TBIL, DBIL≥5 ULN as severe.The diagnosis of hepatic dysfunction was based on the following criteria:According to the different liver function indices, hepatic dysfunction was further classified as hepatocellular injury type, bile stasis type, or a mixed type.2.4Thyroid function tests were measured by chemiluminescence immunoassays (ADVIA CENTAUR XP SIEMENS AG). TPOAb and TgAb were detected by immulite (Immulite 2000 SIEMENS AG), and TRAb was detected by ELISA (enzyme-linked immunosorbent assay) (MEDIPAN Germany). Liver function indexes were measured by colorimetry (Hitachi C7600 Japan). The 131I dose (MBq) = estimated thyroid weight (g) × absorption dose (Gy/g) × 0.67/ . Absorption dose = 100 Gy/g and 0.67 is a correction coefficient. The dosage range of 131I was 74–555 MBq (240.5 ± 81.4 MBq).Length, breadth, and depth of the 2 lobes of the thyroid gland were measured by ultrasound using the formula for a prolate ellipsoid.2.5131I therapy to evaluate the treatment efficacy. We defined complete remission (euthyroid) and hypothyroidism as “cure” (cured group), and partial remission, inefficacy, or recurrence as “uncure” (uncured group).We measured serum levels of thyroid and liver function indices on the 1552 patients at 1, 3, 6, and 12 months after 2.6P values presented were 2-tailed, and values < 0.05 were considered to be statistically significant. Statistical analysis was performed using SPSS  for windows, version 12.0 .A chi square test was used to analyze the difference between the ratios. To identify the risk factors for hepatic dysfunction, we used a bivariate logistic regression model  and stepwise logistic regression  with a variable entrance criterion of 0.05 or less. Missing values for TgAb and TPOAb were assigned to the mean level. All 33.1P = 0.005).Overall, 65% (1552/2385) of patients with Graves hyperthyroidism were affected by hepatic dysfunction. Moreover, we found the prevalence of hepatic dysfunction was 68.1% (731/1073) in ATD-treated patients, and prevalence was 62.6% (821/1312) in patients without ATD treatment. The patients who received ATD treatment had a significantly higher prevalence of hepatic dysfunction compared with those who did not receive ATD treatment , 34.9% (542/1552), and 6.6% (102/1552), respectively. The most common abnormality was elevated ALP, with a prevalence of 52.3%. Additionally, the percentages of hepatocellular injury type, bile stasis, and mixed type were 45.8%, 32.4%, and 21.8%, respectively.3.23 level >19.5 pmol/L (3 ULN), TRAb concentration >15 IU/L (10 ULN), and positive TPOAb were more likely to be associated with hepatic dysfunction .Patient characteristics were compared using bivariate logistic regression analysis between the 2 groups Table . The res1 = age/10; X2 = 1 if the patient was male, and X2 = 2 if the patient was female; X3 = 1, 2, 3, 4, 5, if the duration of hyperthyroidism was less than 1 year, 1–2 years, 2–3 years, 3–4 years and longer than 4 years, respectively; X4 = 1, 2, 3, 4, 5, if the patient did not receive ATD treatment, or accepted drug therapy that was shorter than 1 year, 1–2 years, 2–3 years, and longer than 3 years; X5 = 1, 2, 3, 4, if the heart rate was lower than 80, 80–90, 90–100, and exceeded 100bpm; X6 = 1, 2, 3, 4, if FT3 level was lower than twice the upper limit, 2–3, 3–5, and higher than 5 times the upper limit; X7 = 1, 2, 3, 4, if FT4 level was lower than twice, 2–3, 3–5, and higher than 5 times the upper limit; X8 = 1 if TSH was 0.1–0.3 mIU/L, and X8 = 2 if it was lower than 0.1 mIU/L; X9 = 1, 2, 3, 4, if TRAb was normal, 1.5 (upper limit)-15, 15–40 (determination limit), >40 IU/L; X10 = 1, 2, 3, if TgAb was negative, 40 (upper limit)-3000 (determination limit), >3000 IU/mL; X11 = 1, 2, 3, if TPOAb was negative, 35 (upper limit) – 1000 (determination limit), >1000IU/ml; X12 = RAIUmax (%) × 10; X13 = thyroid weight (g)/10. Resultant variable: Y = 1 for the patient with hepatic dysfunction, and Y = 0 for the patient without hepatic dysfunction. Forward stepwise regression analysis rejecting for trend ultimately revealed that age, duration of Graves hyperthyroidism, FT3 level and TRAb concentration were independent factors predicting hepatic dysfunction in patients with Graves hyperthyroidism .Furthermore, we used multivariate logistic regression analysis to screen for relevant factors. In our study, we suggested a new assignment for causal variables: X3.3131I treatment is displayed in Table 131I treatment. Moreover, we found that the remission rates in patients with mild, moderate, and severe hepatic dysfunction were 86.8%, 65.5% and 55.9%, respectively. Additionally, the remission rate of mild hepatic dysfunction was higher than that of moderate (P < 0.001) or severe dysfunction (P < 0.001). Similarly, we found the remission rates with hepatocellular injury type, bile stasis, and mixed type were 91.7%, 59.2%, and 74.0%, respectively, and the remission rate of hepatocellular injury type was also higher than that of bile stasis (P < 0.001) or the mixed type (P < 0.001).The outcome of hepatic dysfunction after 3.4131I treatment to evaluate the therapeutic effect. In the present study, we defined complete remission (euthyroid) and hypothyroidism as cure (cured group), and partial remission, inefficacy or recurrence as uncure (uncured group). The relationship between efficacy of Graves’ hyperthyroidism and outcomes of hepatic dysfunction after 131I treatment are displayed in Table We measured serum levels of thyroid and liver function indices on the 1552 patients with Graves’ hyperthyroidism at 3, 6, and 12 months after 131I therapy. Hypothyroidism occurred in about 34.1% of (530/1552) patients. The liver function tests in 90.8% (1022/1126) of the cured group patients returned to normal, although the remission rate of hepatic dysfunction was only 41.8% (178/426) in the uncured group of patients. The remission rate of hepatic dysfunction in the cured group was higher than that of the uncured group (P < 0.001). Thus, the prognosis for hepatic dysfunction was significantly associated with the outcomes of Graves’ hyperthyroidism.The cure rate of Graves’ hyperthyroidism (including euthyroid and hypothyroidism) was 72.6% (1126/1552) after initial 4–15 (1) long-term excessive thyroid hormones produnction has direct toxicity on the liver; (2) thyrotoxicosis hastens liver glycogen and protein decomposition, which causes liver cell degeneration; (3) A hypermetabolic state leads to an increasement in organic energy consumption and liver burden. However, the blood supply to the liver does not concomitantly increase. This causes hepatocellular anoxia and free radical damage; (4) autoimmune mechanisms might have a role in the process of hepatocyte injury; (5) hyperthyroid heart disease with cardiac insufficiency might cause hepatic ecchymosis and hepatocyte necrosis; 6. ATD has direct toxicity on the liver. It might be related to the individual's heterogeneous reaction.In clinical practice, hepatic dysfunction in Graves’ hyperthyroidism patients is common. Most of these patients have no obvious symptoms except for mild nonspecific discomfort. Only a minority of patients suffer from severe liver damage and develop liver failure. The mechanism may be related to the following factors:Our study demonstrated that 65% of patients with Graves’ hyperthyroidism were associated with different degrees of hepatic dysfunction. Most of these patients had mild or moderate liver injury, and severe hepatic dysfunction occurred in only 6.6% of patients. Additionally, an increase in ALP was found to be the most common abnormal indicator, and the prevalence was 52.3%.3 level >19.5 pmol/L (OR = 2.116), TRAb concentration >15IU/L (OR = 1.857), and positive TPOAb (OR = 1.453) using univariate analysis . Additionally, we found that older subjects, cases with longer duration of Graves’ hyperthyroidism, higher FT3 level or higher TRAb concentrations were more likely to be associated with hepatic dysfunction as patients with Graves’ hyperthyroidism using multivariate logistic regression analysis. The findings are different from previous research data of Li's group. Li's research showed that the influential factors for hepatic function injury were age, hyperthyroidism duration, heart rate, thyroid weight, FT4, RAIU, TgAb, TPOAb, and TRAb. However, their research subjects were patients with hyperthyroid hepatic injury and those patients who had been treated with ATD were excluded from their analysis.To explore the risk factors of hepatic dysfunction for patients with Graves’ hyperthyroidism, we analyzed 13 related factors, such as age and gender, and found that hepatic dysfunction is more likely to occur in patients aged ≥45 (OR = 2.386), a history of Graves’ hyperthyroidism >3 years (OR = 3.985), heart rate >90 bpm (OR = 1.617), FT The liver plays an important role in thyroid hormone metabolism. In those patients with long-term duration of Graves’ hyperthyroidism, excess thyroid hormones increase liver burden, and have direct toxicity on the liver, so the likelihood of hepatic dysfunction rises.,14 Increased TRAb concentration indicates an active autoimmune response, which results in hyperfunction of the thyroid gland, an increased excitability of multiple systems and augmented metabolism.,20 Our study showed that patients with hepatic dysfunction had higher TRAb levels, which corroborates recent reports. FT3 has more tissue-activity than FT4, and studies have confirmed excess T3 induces apoptosis of hepatocytes. Excessive thyroid hormones secretion may cause superfluous oxidative stress, an imbalance between pro-oxidant and antioxidant, which can then lead to hepatocyte damage. In addition, excessive thyroid hormones increase heart rate and cardiac output. However, in the liver, increased tissue oxygen consumption may cause hepatocellular anoxia resulting in liver injury.,25 Thus, higher TRAb or FT3 level can increase the risk of hepatic dysfunction for patients with Graves’ hyperthyroidism.Older patients are more likely to have chronic cardiac insufficiency, hepatic vein congestion, or even liver centrilobular necrosis.131I treatment in 77.3% of patients. Moreover, the remission rate of mild hepatic dysfunction (86.8%) was higher than that of moderate (65.5%) or severe dysfunction (55.9%), and the remission rate of hepatic dysfunction for hepatocellular injury type (91.7%) was higher than that for bile stasis (59.2%) or the mixed type (74.0%). Therefore, the remission rate of hepatic dysfunction is closely related to its severity and types. Additionally, our study revealed that progntosis of hepatic dysfunction was associated with the outcomes of Graves’ hyperthyroidism. The remission rate of hepatic dysfunction in the cured group of patients (90.8%) was higher than that in the uncured group of patients (41.8%). 131I treatment should be the prior choice for those patients with hepatic dysfunction in order to control hyperthyroidism timely and effectively.In total, hepatic function indices returned to normal 6 months after 131I usually does not cause radiation damage to the liver. In our study, hepatic dysfunction mildly deteriorated in the 2 weeks after 131I treatment in a small minority of patients with severe liver damage. This may be related to the early release of thyroid hormones and the transient aggravation of hyperthyroidism. We did not observe an obvious exacerbation of hepatic dysfunction through long-term follow-up.The metabolic process of Our study had some inherent limitations associated with the retrospective analysis. The 1552 patients involved in our study had hepatic dysfunction induced by Graves hyperthyroidism and/or ATD. Those patients with liver diseases (such as autoimmune liver disease) or biliary tract diseases were rejected. However, corresponding testing or examination was not universally performed to exclude these patients. Additionally, we selected the cases with complete data to perform our retrospective analysis. The exclusion of a few patients who were lost to follow-up might result in potential bias. Therefore, rigorous prospective studies will need to be performed to confirm these preliminary findings.53 or TRAb concentrations. Additionally, the prognosis of hepatic dysfunction is closely associated with the outcomes of Graves hyperthyroidism after 131I treatment.Hepatic dysfunction is more likely to occur in older patients, and cases with a longer history of Graves’ hyperthyroidism, and higher FTThe authors thank the native English speaking scientists of Elixigen Company  for editing our manuscript.