Herein, new polyborosiloxanes(PBSs) were prepared using a straightforwardsynthetic approach to obtain a core–shell structure as a materialwith various features such as better adhesion ability to the appliedsurface and enhanced thermal properties. In this concept, in situcore–shell formation was allowed by sequential addition ofingredients with fixed conversions. First, pre-condensed polysiloxanewas synthesized, with a 60% conversion, as a core by the reactionof phenyltriethoxysilane in the presence of water in an acidic condition.Subsequent addition of boric acid into the pre-condensate and a furthercondensation reaction resulted in the formation of the shell layerthrough the introduction of the −Si–O–B–bonds to the network of the PBS. The resulting resin was used as abinder for heat-resistant paint in combination with an aluminum pigment,and the paint applied on a metal plate was found to be resistant upto 600 °C in terms of adhesion strength. It was also demonstratedthat the incorporation of boron in the core–shell structureshowed better adhesion strength than the one-pot preparation of PBS.Using this method, not only the heat resistance requirement of theindustrial coating was achieved but also the flame-retardant abilitywas introduced. The latter is due to the nature of the −B–O–Si–bond in the network structure, which splits and combines reversiblydue to moisture susceptibility. On the other hand, the high bondingenergies of B–O and Si–O, 537.6 kJ/mol and 460.5 kJ/mol,respectively, boost the thermal properties in combination with theglassy film formation of boron compounds.7 Anotherfactor affecting the thermal stability is the organic substituentof the Si–R bond, where R is usually selected from the methylor phenyl groups due to their long half-life time at 250 °C,which are approximately 14 months and 11 years, respectively.15 As mentioned briefly above, it was the aim toprepare phenyl-containing PBS resin due to its unique properties.Several “one-pot” methods have been described for PBSpreparation in the literature using silanes and various boron sourcessuch as boric acid,10 trialkyl borate,17 and borohydride.18 Linear or partially crosslinkedPBSs were also prepared by reacting polydimethylsiloxane derivativeswith sodium tetraborate12 and/or boricacid.19 Apart from the above-mentioned routes, ourstrategy is to prepare PBS as an outer layer of polysiloxane resin,which has been proposed for the first time, to be used as a heat-resistantbinder.In recent years, boron- and silicon-containingpolymers, namely,polyborosiloxanes (PBSs) have been extensively studied due to theirheat resistant,20Heat-resistant paints mainly composed of methyl/phenylsubstitutedsilicon resin as a binder and thermally stable pigments such as aluminumor iron oxide are used as heat-resistant coatings up to 600–650°C especially for metal surfaces such as stoves, chimneys, andheat exchangers.As an importantnovel method, we have described PBS resins composedof polysiloxane as a core encapsulated by a PBS shell. PBS along withan aluminum pigment was incorporated into a heat-resistant paint formulationand coated on a metal panel with a 35 μm dry film thickness.The results showed that the adhesion strength of the coatings heldup to 600 °C for 2 h, which was quite satisfactory as GT0. Wecharacterized the resins using Fourier transform infrared (FTIR) spectroscopy,thermogravimetric analysis (TGA), NMR, scanning electron microscopy(SEM), transmission electron microscopy (TEM), and X-ray photoelectronspectroscopy (XPS) analysis.A small manipulationof the synthetic processes during the preparation of polymers allowedmany opportunities for designing the polymeric network structure.In our case, addition of the reagent during the condensation reactionin two steps resulted in the formation of a polysiloxane core anda PBS shell layer as a novel strategy, different from the commonlyemployed one-pot route in the literature for randomly distributedPBS. From the view of the structural aspect, this minor change corenetwork was formed through −Si–O–Si– linkages,whereas the shell was formed through −Si–O–B–and −Si–O–Si– linkages 1.2CH3 peakat 960 cm–1 and the −OH vibration signalat 3400 cm–1 disappear with time, which indicatesthat the condensation between alkoxy silane and hydroxy sources takesplaces. Classical Si–O–Si and B–O signals areobserved at around 980–1130 and 1350–1450 cm–1, respectively. The peaks appearing at 670 and 905 cm–1 were associated with Si–O–B bonds, which confirmsthe formation of PBS.9 C=C vibrationpeaks at 1600 cm–1 and C–H vibration peaksat 3020–3070 cm–1 are due to the phenyl groupsof the solvent and silane reagent. Moreover, aromatic C–H bendingvibrations belonging to phenyl groups appeared at 881 cm–1.The FTIR spectrum of the PBS resin is presented in Figure S1.TGA performed under a nitrogen atmosphere showsthat the solventis evaporated at around 120–150 °C 2. A new 11B NMR wasperformed to observe the boron linkage characteristicin PBS-2. There appeared one broad peak at 18.7 ppm, which indicatedthe formation of both B(OSi)3 and B(OSi)2OHstructures . SEM–energy-dispersiveX-ray spectroscopy (EDX) and SEM images of the PBS-1 and PBS-2 resinswere obtained for observing the core–shell structure of theboron-based resins. No meaningful results were obtained .S5. First, the xylene solution containing 65wt % PBS was dropped onto the copper grid, and then, TEM images wereobtained from the dried sample. The images revealed the formationof the core–shell structure via color contrast between thepolysiloxane core and PBS shell. The core–shell structureswere irregular, as expected.The morphology andcore–shell structure of the resultingPBSs (PBS-1 and PBS-2) were investigated via TEM images, as shownin S6). In the spectrum, it wasobserved that the PBS-2 sample surface consists of O, C, B, and Siwith binding energies of O 1s, C 1s, B 1s, Si 2s, and Si 2p at 533.1,284.5, 191.63, 153, and 102 eV, respectively. The B 1s peak regionrevealed the B–O–Si and B–OH bond existence atthe surface of the PBS sample.21 The C 1s peak areaof the PBS-2 sample proved the presence of three chemical states,corresponding to C–Si, C–C/C=C, and C–Obonds. Two binding energies were observed at 532.22 and 533.65 eV,attributed to Si–OH and Si–O–Si bonds, respectively,for the O 1s peak .XPS was used to determine the chemical states of the PBSsamplesPBS-1 and PBS-2 4 and S6.The main conceptof the heat-resistant paint is to be stable up to a required temperaturesuch as 400 °C for decorative paints and 600 °C for industrialpaints. Additional properties such as corrosion resistance or flameretardancy may also be demanded depending on the final application.Our motivation was to invent not only heat-resistant but also flame-retardantresin in one product. The latter is a natural consequence of possessingboron in the structure. To understand in a better way, PBS-2 was driedat room temperature and subjected to a continuous flame, and quenchingof the flame was noted right after the removal of the flame source,while the commercial product keeps burning.In terms of heat-resistantproperties or adhesion strength, it was found that the resin type,boron content, film thickness, and application temperature play importantroles. Adhesion strength is measured by a cross-hatch tape test, asgiven below in It is important to note that core–shell structures weredemonstrated to be resistant up to 600 °C as GT0–GT1 eventhough the boron content was low see 6, while On the other hand, PBS-4 was found to be resistant up to 400 °C,which was attributed to the increased binding sites compared to thatof PBS-3 7.In terInsummary, we have prepared PBS resins composed of polysiloxaneas a core encapsulated by a PBS shell for the first time. PBS as acombined product has not only enhanced the thermal stability but alsoflame-retardant properties. PBS prepared via a core–shell strategywas found to be an excellent platform for heat-resistant coating applicationsup to 600 °C. It was also revealed that increasing the bindingsites increased the adhesion. We believe that our study can open newpaths toward designing innovative coatings. In a future work, theflame retardant property of PBS will be investigated.Phenyltriethoxysilane (PTES), boric acid(Eti Maden), sulfuric acid, xylene, non-leafing aluminum pigment pastewith 65% active content (Eckart), and BENTONE 038 (Elementis) pastewith 12% active content were supplied by Denge Kimya, and all thechemicals were of industrial grade and not less than 97% purity.61.7 g of PTES (256.7 mmol)and 0.1 g of sulfuricacid (1.02 mmol) were mixed at room temperature for 10 min in a 100mL round-bottom flask, and then, 6.4 g of water (355.5 mmol) was addeddropwise. The mixture was stirred overnight, and ethanol was distilledoff as a side product at 95 °C. During distillation, 2 g of boricacid (32.3 mmol) and 8.5 g of xylene were added to the reaction mediumat 57 and 74% yields, respectively. Distillation was completed upto 90% yield at 115 °C under 0.5 bar vacuum. The final productwas diluted with xylene/isobutanol to give a clear solution and 65%solid content, and the catalyst was neutralized with the requiredamount of sodium bicarbonate at room temperature.61.7 g of PTES (256.7 mmol) and 0.1 g of sulfuricacid (1.02 mmol) were mixed at room temperature for 10 min in a 100mL round-bottom flask, and then, 5.37 g of water (298.3 mmol) wasadded dropwise. The mixture was stirred overnight, and ethanol wasdistilled off as a side product at 95 °C. During distillation,3.9 g of boric acid (63.07 mmol) and 8.5 g of xylene were added tothe reaction medium at 46 and 82% yields, respectively. Distillationwas completed up to 92% yield at 115 °C under 0.5 bar vacuum.The final product was diluted with xylene/isobutanol to give a clearsolution and 65% solid content, and the catalyst was neutralized withthe required amount of sodium bicarbonate at room temperature.61.7 g of PTES and 0.1 g of sulfuric acid weremixed at room temperature for 10 min in a 100 mL round-bottom flask,and then, 6.4 g of water (dropwise) and 2 g of boric acid were addedinto the flask. The mixture was stirred overnight, and ethanol wasdistilled off as a side product at 95 °C. In order to preventgelation, 10 and 5 g of xylene were added to the reaction medium at64 and 76% yields, respectively. Distillation was completed up to92% yield at 115 °C under 0.3 bar vacuum. The final product wasdiluted with xylene/isobutanol to give a clear solution and 65% solidcontent, and the catalyst was neutralized with the required amountof sodium bicarbonate at room temperature.61.7 g ofPTES and 0.1 g of sulfuric acid weremixed at room temperature for 10 min in a 100 mL round-bottom flask,and then, 5.37 g of water (dropwise) and 3.9 g of boric acid wereadded into the flask. The mixture was stirred overnight, and ethanolwas distilled off as a side product at 95 °C. In order to preventgelation, 10 and 5 g of xylene were added to the reaction medium at65 and 74% yields, respectively. Distillation was completed up to90% yield at 115 °C under 0.4 bar vacuum. The final product wasdiluted with xylene/isobutanol to give a clear solution and 65% solidcontent, and the catalyst was neutralized with the required amountof sodium bicarbonate at room temperature.61.7 g of PTES and 0.1 g of sulfuricacid were mixed at room temperature for 10 min in a 100 mL round-bottomflask, and then, 5.37 g of water was added dropwise. The mixture wasstirred overnight, and the catalyst was neutralized. 10 g of xylenewas added to the reaction medium when 22 g of ethanol was distilledoff at 95 °C. Distillation was completed up to 90% yield at 115°C under vacuum. The final product was diluted with xylene togive a 65–70% solid content.2 metal plate ,whichwas cleaned with xylene and dried prior to the application.Heat-resistant paint containingPBS was prepared using a guideformulation given in The painted metal platesreached tack-free time in less than anhour at room temperature and then were cured at 200 °C in a mufflefurnace for 30 min. Afterward, the samples were kept at 400 °Cfor 2 h and cooled down to room temperature, and a cross-cut testwas performed to measure the adhesion strength. Likewise, differentplates were prepared at 600 °C, and the same procedure was repeated.11B NMR spectra of the samples were recorded by using CDCl3 as a solvent on a Bruker Biospin AVANCE III-400 MHz spectrometer.An FEI Quanta FEG 250 FE-SEM instrument was used to obtain informationabout the morphology of the PBS. The surface structures of the sampleswere determined using a high-resolution transmission electron microscope(Hitachi 7700) equipped with an acceleration voltage of 120 kV. XPSwas performed using an XPS system  with a monochromaticAl K X-ray source. The range of the binding energy was 0–400eV.FTIR spectra were obtained using aThermo Scientific Nicolet iS50 FTIR Spectrometer. TGA was carriedout under a nitrogen atmosphere using a PerkinElmer Diamond TGA 4000instrument at a heating rate of 20 °C/min till 900 °C.


Riemerella anatipestifer is an important bacterial pathogen associated with epizootic infections in waterfowl and various other birds. The complete genome sequence of Riemerella anatipestifer xi1, isolated in China, was sequenced. The genome consisted of 2,062,911 bp carrying 1,879 predicted genes. Riemerella anatipestifer, a member of the genus Riemerella in the family Flavobacteriaceae, mainly infects domestic ducks, geese, turkeys, and other birds, causing pericarditis, perihepatitis, and meningitis  tablets containing 5% bovine serum and cultured overnight at 37°C in an atmosphere of 5% CO2. Single colonies were selected for further R. anatipestifer identification, including Gram stain, specific PCR amplification, and biochemical identification. The primers used in PCR identification are listed in Taq mix enzyme, 1 μL of each primer, 1 μL of template, and 5 μL of double-distilled water (ddH2O), with the following procedure: 94°C predenaturation for 3 min, 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 1 min, a total of 35 cycles, and then extension at 72°C for 10 min and holding at 4°C. Two specific bands at 644 bp and 194 bp were obtained after amplification. Biochemical testing was carried out with bacterial microbiochemical reaction tubes  following the manufacturer’s instructions, in which the isolated strains could use oxidase, urease, and contact enzyme, could not use carbohydrates, citrate, urea, arginase, and lysine enzyme, and could not produce H2S and indole. The identified strain, named xi1, was cultured in TBS containing bovine serum in an atmosphere of 5% CO2 for 16 h at 37°C for genome extraction. The genome was prepared and sequenced using an Illumina NovaSeq 6000 instrument  in paired-end (2 × 150 bp) format.ningitis . In the mic loss . A totalentified . A limitvailable –7. R. anAfter sequencing, 3,269,733 raw reads were obtained and subjected to extraction of host data. A total of 1,981,433 raw reads were preprocessed for quality checking using FastQC version 0.11.9  and furtR. anatipestifer xi1 will provide the genetic background for understanding the pathogenicity mechanisms of this pathogen using comparative genomics.Overall, the genome sequence of avirulent JAJDFB000000000. The version described in this paper is version JAJDFB010000000. The BioProject number is PRJNA772301, and the SRA accession number is SRR16620534.This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession number


BackgroundAcute appendicitis is one of the events most frequently encountered by general surgeons. Despite the high incidence, serious problems are experienced in the diagnosis and clinical follow-up. In the pathogenesis of the disease, oxidative stress and impaired antioxidant defense mechanisms created in the body by this stress play an important role. As dynamic thiol-disulfide hemostasis is closely related to oxidative stress and is known to have a crucial role in the pathogenesis of oxidative stress, this study aimed to compare its value with other inflammatory markers in the diagnosis and follow-up of acute appendicitis.MethodologyThis study included cases admitted for surgery with a diagnosis of acute abdomen at Keçiören Research and Training Hospital General Surgery Clinic between April 2015 and July 2015 who were intraoperatively diagnosed with acute appendicitis and underwent routine appendectomy. In the preoperative period and after clinical healing before discharge, blood samples were obtained to examine white blood cell (WBC), mean platelet volume (MPV), total bilirubin, C-reactive protein (CRP), and thiol-disulfide balance, and the results were recorded.ResultsA total of 68 cases were operated on for acute appendicitis, and 59 were evaluated comprising 23 (39%) females and 36 (61%) males with a mean age of 35.6 years (range = 19-65 years). The mean duration of hospital stay was two days (range = 1-8 days). The results of the tests performed preoperatively and before discharge and their p-values were as follows: native thiol (-SH) 393.5 ± 9.4 µmol/L and 369.3 ± 9.5 µmol/L (p = 0.04), total thiol 434 ± 9.7 µmol/L and 396.7 ± 10.2 µmol/L (p = 0.03), disulfide (-S-S) 16.8 ± 0.7 µmol/L and 15.7 ± 0.9 µmol/L (p = 0.3), WBC 13.2 ± 0.5 × 10³/mL and 9.2 ± 0.4 × 10³/mL (p = 0.0), CRP 8.17 ± 1.24 mg/L and 7.84 ± 0.82 mg/L (p = 0.17), MPV 7.4 ± 0.37 fL and 7.97 ± 0.19 fL (p = 1.0), and total bilirubin 0.86 ± 0.08 mg/dL and 0.69 ± 0.06 mg/dL (p = 0.08).ConclusionsIn the clinical follow-up of acute appendicitis patients, the decrease in WBC, total thiol, and native thiol values can be helpful to clinicians as markers of clinical healing. However, CRP may not be a useful marker of clinical healing in acute appendicitis patients who are discharged early. Acute appendicitis is one of the most common reasons for acute abdomen diseases, and appendectomy is one of the most frequently performed operations by general surgeons . DespiteThiol components are known to have anti-oxidant effects in the body and are formed of disulfide bonds (RSSR) entering an oxidation reaction against oxidants. Oxidative stress among thiol groups may lead to the formation of a mixed disulfide structure. These disulfides can again be reduced to thiol groups, and thus the dynamic thiol-disulfide hemostasis can be maintained. Oxidative stress is known to play a crucial role in the pathogenesis of dynamic thiol-disulfide hemostasis which has a close relationship with oxidative stress; therefore, it has been considered that it can be critical in the diagnosis and follow-up of acute appendicitis .This study aimed to compare the dynamic thiol-disulfide values of patients admitted to the hospital with acute appendicitis with other laboratory parameters , bilirubin, mean platelet volume (MPV)) which can be helpful in monitoring the inflammatory process, both on admission and before discharge from the hospital.This prospective clinical study included cases admitted for surgery with a diagnosis of acute abdomen at Keçiören Research and Training Hospital General Surgery Clinic between April 2015 and July 2015 who were intraoperatively diagnosed with acute appendicitis and underwent routine appendectomy. The study was approved by the Ethics Committee on Clinical Research of Ankara Atatürk Sanatorium Teaching and Research Hospital (2015-KAEK-B.10.4.İSM.4.06.68.49). The diagnosis of acute appendicitis was made with clinical symptoms and physical examination as well as laboratory tests and imaging techniques such as ultrasonography and computed tomography. The intraoperative appendix perforation status was recorded. Patients were excluded if they were over 65 years of age, if they had a chronic disease , if a procedure apart from routine appendectomy was performed, or if appendicitis was not observed in the pathological examination. During the preoperative period and to monitor the inflammatory process after clinical healing before discharge, blood samples were obtained to examine hemogram , biochemistry , serology (CRP), and thiol-disulfide balance, and the results were recorded. A record was made of the duration of hospital stay and events which prolonged the hospitalization during the clinical follow-up.The basis of thiol-disulfide homeostasis is that dynamic disulfide bonds (-S-S-) are broken down to functional thiol groups (-SH) by sodium borohydride. In the assay, the sodium borohydride remnants were removed with formaldehyde. The total thiol content of the sample was assayed using the modified Ellman reagent. The native thiol content was calculated from the total thiol content and half of the acquired difference provided the disulfide bond amount. The native thiol-disulfide ratio (-S-S-/-SH) was calculated with native thiol (-SH) and the disulfide (-S-S) amount.Statistical evaluation was done using SPSS version 18 software . Conformity to normal distribution of the variables on admittance to the hospital and before discharge was examined using visual and analytical methods. When values showed normal distribution , the comparison was done using the Student’s t-test. Values that did not show normal distribution  were compared using the Wilcoxon test. P-values of <0.05 were accepted as statistically significant.A total of 68 cases were admitted for acute appendicitis operation. A total of nine cases were excluded; in four cases, procedures other than routine appendectomy were performed because of complicated appendicitis, and in five cases, pathologies other than appendicitis were observed. Of the remaining 59 cases for evaluation, 23 (39%) were female and 36 61%) were male, with a mean age of 35.6 years (range = 19-65 years) . The results of the tests performed preoperatively and before discharge and their p-values were as follows: native thiol (SH) 393.5 ± 9.4 µmol/L and 369.3 ± 9.5 µmol/L (p = 0.04), total thiol 434 ± 9.7 µmol/L and 396.7 ± 10.2 µmol/L (p = 0.03), disulfide (-S-S) 16.8 ± 0.7 µmol/L and 15.7 ± 0.9 µmol/L (p = 0.3), WBC 13.2 ± 0.5 × 10³/mL and 9.2 ± 0.4 × 10³/mL (p = 0.0), CRP 8.17 ± 1.24 mg/L and 7.84 ± 0.82 mg/L (p = 0.17), MPV 7.4 ± 0.37 fL and 7.97 ± 0.19 fL (p = 1.0), and total bilirubin 0.86 ± 0.08 mg/dL and 0.69 ± 0.06 mg/dL (p = 0.08)  and total antioxidant status (TAS) were evaluated to define oxidative stress in patients with acute appendicitis. Both the TOS and TAS values were observed to be higher in acute appendicitis patients compared to the control group . SimilarAlthough not statistically significant, there was also an increase in the disulfide values. That the difference in the disulfide values was not significant can be attributed to not having included advanced-stage complicated appendicitis in this study.CRP is an acute-phase reactant used in the diagnosis and follow-up of several diseases, primarily inflammatory diseases. This plasma protein, which is produced in the liver, increases rapidly as a response to wounds, infections, and other inflammatory stimuli in the body ,10. ElevIn the literature, there is no consensus on the use of CRP in the diagnosis of acute appendicitis, but it has been emphasized that CRP can be helpful in the diagnosis of advanced and complicated appendicitis ,12. In tIt has been reported that in the inflammatory process, serum bilirubin levels are increased without any liver or cholestatic disease. The mechanism of the increase has been attributed to cholestasis which forms due to bacterial endotoxins or increased bilirubin expression through the effect on the hepatobiliary system of oxidative stress, which affects many systems in the body. Although several studies in the literature have shown a correlation between elevated bilirubin in acute appendicitis and the severity of the disease, it has been emphasized that bilirubin alone may not be sufficient for the diagnosis of cases in the early stages ,14. In tMPV showing thrombocyte dimensions is routinely used in complete blood count (CBC) analyses but is a test that is generally overlooked by clinicians. It has been shown in the literature that the MPV value is affected in the inflammatory process, just as in many diseases. Bozkurt et al. investigated the diagnostic value of MPV in acute appendicitis. Although no significant difference was observed in the MPV values between the control and appendicitis groups, a lower MPV value was observed in complicated appendicitis patients compared to the control or non-complicated groups . In thisThe inflammatory biomarker most commonly used by clinicians in the diagnosis of acute appendicitis is WBC. However, as reported by Yokoyama et al., some clinicians believe that WBC is not an indicative factor for surgery . Given tIn previous studies concerning acute appendicitis and laboratory tests, the research has generally focused on diagnosing the disease or determining its severity. However, when medical treatment has been debated in the current treatment of the disease, the determination of the clinical course demands as much importance as the determination of the severity of the disease. Okuş et al. evaluated the CRP and WBC values in acute appendicitis patients where medical treatment was not successful. A substantial increase was determined in the CRP values of patients with unsuccessful treatment . HoweverOur study was designed as a prospective study. Therefore, more reliable data have been obtained compared to various retrospective studies. There are several limitations in this study that should be taken into consideration. First is the inclusion of a relatively small number of patients who were admitted to a single center. Therefore, studies with a large number of participants can provide more valuable results. Other diagnostic aids such as procalcitonin and imaging techniques such as ultrasonography and/or computed tomography and Alvarado score were not correlated with thiol/disulfide homeostasis parameters.Although acute appendicitis is frequently encountered by general surgeons, serious problems are experienced in diagnosis and follow-up. This study demonstrated that dynamic thiol/disulfide homeostasis shifted toward disulfide formation as a result of thiol oxidation in patients with acute appendicitis. A thorough clinical examination which is correlated with laboratory tests plays a significant role in the clinical follow-up. In the determination of the status of clinical recovery, reductions in total thiol, native thiol, and WBC values are important. However, the benefit is not provided at a sufficient level from CRP, which is a test with a long half-life, as a significant reduction was not achieved in this process. Prospective and randomized controlled trials are necessary to confirm the pathophysiologic role of thiol/disulfide homeostasis in acute appendicitis. Further studies are required to optimize the use of this novel oxidative stress marker in conjunction with other established approaches.


Sargassum hystrix. The brown seaweed was extracted using 4 methods, namely, A , B , C , and D . The antioxidant activity testing was carried out using the 2,2-diphenyl-1-picrylhydrazyl (DPPH), Ferric-Reducing Antioxidant Power (FRAP), and Hydroxyl Radical Scavenging Activity (HRSA). The yield for methods of A, B, C, and D was 2.46 ± 0.30, 0.68 ± 0.34, 1.18 ± 0.15, and 0.62 ± 0.25%, with fucose content of 39.97 ± 4.82, 26.72 ± 3.38, 41.08 ± 9.49, and 40.62 ± 8.59%, xylose content of 8.07 ± 0.92, 5.63 ± 0.40, 6.80 ± 0.83, and 7.83 ± 1.83%, and the sulfate content of 11.47 ± 2.20, 15.31 ± 2.47, 30.62 ± 2.76, and 27.80 ± 3.59%. The result indicated the occurrence of a sulfate ester group in the functional group analysis with numerous similarities with the commercial fucoidan. The highest antioxidant activity of fucoidan from S. hystrix was found in method C, which was influenced by sulfate levels. Therefore, the extraction method of fucoidan from S. hystrix affects the characteristics and antioxidant activity.Fucoidan is a bioactive compound of brown seaweed with antioxidant characteristics. This study examined the aftermath of the extraction method on the yield, fucose content, xylose content, sulfate content, total sugar, antioxidant activity, and functional groups of fucoidan from  The human body comprises trillions of cells that routinely produce free radicals and reactive oxygen species (ROS), which are part of the metabolism process . OxidatiSargassum sp. of previous studies includes Sargassum crassifolium [Sargassum cristaefolium [Sargassum hystrix [Sargassum muticum [Fucoidan is a sulfated polysaccharide that originates from various brown seaweed species, and it possesses antioxidant biological activities , which hsifolium , Sargassaefolium , Sargass hystrix , and Sar muticum . Accordi muticum  and Sinu muticum , the chaSargassum binderi, using different solvents under varying conditions. However, no study has been carried out to examine the aftermath of the different extraction methods on the characteristics and antioxidant activity of fucoidan from S. hystrix. Therefore, this study examined the aftermath of the extraction methods on the characteristics and antioxidant activity of brown seaweed fucoidan from S. hystrix.Sinurat and Kusumawati  researchS. hystrix was obtained from Teluk Awur, Jepara, Central Java, in November 2019. The seaweed was washed and dried for 24 hours in an oven at 60°C [ at 60°C  before iThe schematic diagram of fucoidan extraction is shown in v/v), which was stirred and left for 2 hours. The mixture was centrifugated at 8,000 rpm for 5 minutes at 4°C. The supernatant was further neutralized using a 0.5 M NaOH. It was then stirred for 30 minutes at room temperature and centrifugated at 8,000 rpm for 15 minutes. Finally, ethanol (1 : 3) was added to the collected supernatant and centrifuged at 8,000 rpm for 15 minutes, thereby leading to the formation of pellets in the form of a paste, which was dried by freezing.This fucoidan extraction method was proposed by Purbomartono et al. . A total2 (1 : 20) (w/v). It was then extracted and stirred with a hot plate stirrer for 4 hours at 85°C. Afterward, it was filtered with a 625-mesh sieve with the filtrate collected and centrifuged at 3,000 rpm for 45 minutes at 5°C. The supernatant was collected in a jar while the sediment was disposed. In addition, ethanol was added to the filtrate at a ratio of 1 : 2, while the precipitate was dissolved in water and centrifuged at 3,000 rpm for 15 minutes. Consequently, the residue obtained was dialyzed with 0.5 M NaCl and distilled water and dried with a freeze dryer.This method was proposed by Sinurat and Kusumawati . The sea2 and left overnight at a temperature of 4°C. The mixture was radiated from a central point at 3,000 rpm for 10 minutes. Then, the supernatant was added with 96% ethanol until the concentration of ethanol in 30% solution and incubated at 4°C for 4 hours. The solution was readded using ethanol until the ethanol concentration was reduced to 70% and incubated at 4°C for 24 hours. A sediment's appearance is followed by filtering the solution using a filter paper to collect the residue, which was dried using a freeze dryer.Palanisamy et al.  modifiedw/v) and stirred in a water bath for 3 hours at 70°C. The solution was chilled at room temperature and filtered using a 625-mesh sieve. Ethanol (1 : 5) was added to the filtrate and then centrifuged at 3,000 rpm for 10 minutes. The filtrate collected was added to 96% ethanol (3 : 5) and centrifuged at 3,000 rpm for 10 minutes. The pellets were then freeze-dried while the sediments were discarded.This method was proposed by Zhao et al. . A totalThe fucoidan yield was collected by dividing the weight of fucoidan by the weight of dry seaweed powder and multiplying it by 100. S. hystrix. The process was conducted using the method proposed by Sinurat and Kusumawati [−1. Subsequently, the commercial fucoidan from M. pyrifera , which was used as the standard, was given a similar treatment as the sample.This analysis was tested with a Fourier Transform Infrared (FT-IR) Spectrophotometer (Perkin-Elmer 577) to examine the form of functional groups in fucoidan from sumawati  with 2 mS. hystrix. The phenol-sulfate test was conducted by adding 2.5 mL of concentrated H2SO4 to each solution and shaken till the mixture became homogeneous. It was then soaked in ice for 20 minutes, after which 0.5 mL (5%) of phenol was added and shaken till a homogeneous mixture was obtained, which was further soaked in ice for 30 minutes. A UV-VIS spectrophotometer was utilized to measure the absorbance of all samples, while the fructose and xylose standards were determined at a wavelength of 490 nm and 480 nm, respectively.The total sugar content was determined using the phenol-sulfuric acid test based on the method proposed by Dubois et al. . The sug2-gelatin method was used in this research to analyze the sulfate content of fucoidan from S. hystrix. A homogenous solution was obtained by dissolving 100 mL of aquabides and 0.5 g gelatin in a hot plate stirrer at temperatures ranging between 60 and 70°C. Furthermore, 0.5 g of BaCl2 was added to the solution and left for 24 hours at a temperature of 4°C. The sample was also processed by dissolving 6 mg of fucoidan from S. hystrix in 2 mL of 3.5 N HCl and stirred till homogeneous. Furthermore, it is centrifugated to ensure that only the supernatant is collected. The commercial fucoidan solution of M. pyrifera was given a similar treatment as the sample. Standard K2SO4 at concentrations of 200, 400, 600, 800, and 1,000 ppm were prepared. Moreover, the sample solution, 3% TCA, and BaCl2-gelatin were mixed in a cuvette at a successive ratio of 200, 600, and 300 μL. The mixture was manually stirred and left for 15 minutes, after which a UV-VIS spectrophotometer was used to measure the absorbance at a wavelength of 360 nm. Furthermore, the standard K2SO4 and the commercial fucoidan from M. pyrifera solutions were given similar treatment.The Dodgson and Price  BaCl2-geS. hystrix and commercial fucoidan from M. pyrifera  was prepared using distilled water and allowed to homogenize. Furthermore, 0.76 mM DPPH solution was prepared by dissolving 3 mg of DPPH powder in 10 mL distilled water and stored at a temperature of 4°C for 24 hours. The solution was further incubated at room temperature for 30 minutes with a UV-VIS spectrophotometer used to analyze the absorbance at a wavelength of 517 nm. In addition, the antioxidant was represented in accordance with the inhibition percentage of IC50, as follows:A is the sample (800 μL sample + 200 μL 0.76 mM DPPH), B is the control sample (800 μL sample + 200 μL distilled water), C is the negative control (800 μL distilled water + 200 μL 0.76 mM DPPH), and D is the blank (800 μL distilled water).The RSA DPPH antioxidant activity test was conducted according to the modified method proposed by Zaranappa et al. . A sampl3+ to Fe2+ using a spectrophotometer at a wavelength of 595 nm. The changes were analyzed to determine the formation of a blue color in the solution. Furthermore, 0.775 g of sodium acetate trihydrate (CH3COON·3H2O) was added to 4 mL of concentrated acetic acid which was dissolved in water to determine the acetate buffer solution with pH 3.6. The buffers were stored as stock solutions at a temperature of 4°C with 0.15 g of TPTZ in 40 mM HCl dissolved in 50 mL of distilled water to obtain 10 mM of 2,4,6-tripyridyl-s-triazine (TPTZ). Furthermore, 0.828 mL of concentrated HCl was dissolved in 250 mL of distilled water and a TPTZ solution stored at 4°C for 24 hours to obtain 40 mM HCl. A solution of 20 mM FeCl3·6H2O was also prepared by dissolving 0.54 g of FeCl3·6H2O in 100 mL of distilled water for 24 hours at a temperature of 4°C before usage. A FRAP reagent was also prepared by mixing 25 mL of acetate buffer, 2.5 mL of TPTZ, FeCl3·6H2O solutions (10 : 1 : 1), and 100 mL of distilled water. Furthermore, the standard FeSO4·7H2O  solutions were produced by dissolving 2.78 g FeSO4·7H2O in 1,000 mL distilled water, before serially diluting to attain 50, 100, 150, 200, 250, and 300 ppm concentrations. This was followed by preparing sample solutions from S. hystrix  and commercial fucoidan . Furthermore, a 20 μL solution was added to 150 μL of FRAP reagent in a 96-well microplate using a UV-VIS spectrophotometer at a wavelength of 595 nm (Multiple Go). The standard FeSO4 solution and commercial fucoidan were also given similar treatment.The FRAP test was conducted using the Clarke et al.  model, w4, 1 mL 0.3% of H2O2, and 0.5 mL 9 mM salicylic acid. The mixture was dissolved in 5 mL of distilled water. It was then incubated at 37°C for 30 minutes. A UV-VIS spectrophotometer with a wavelength of 510 nm was used to determine the absorbance. The percentage of HRSA and IC50 represented the antioxidant activity. The following formula was used to calculate the HRSA percentage. A0 is the absorbance of a sample, A1 is the absorbance of control, and A2 is the absorbance of reagent.The HRSA test was conducted using the Zhao et al.  proposedS. hystrix was mixed with 25 mL water and then produced 3 mL of the reagent . The fucoidan solution and the reagent were mixed till it was homogenized and incubated for 90 minutes at 5°C, after which the mixture was cooled and then vortexed. A spectrophotometer with a wavelength of 695 nm was used to measure the absorbance. Commercial fucoidan from M. pyrifera was given a similar treatment as the sample. Vitamin C was used as a standard curve. Therefore, the total value of antioxidant capacity obtained was denoted in mg of the Ascorbic Acid Equivalent (AAE)/g. The following formula was used to calculate the total antioxidant capacity:The total antioxidant activity test was conducted using the method proposed by Salma et al. . A totala is the concentration of vitamin C in the test sample (mg/L), V is the total volume of test solution (mL), G is the weight of extract (g), and 1000 is the conversion factor to total volume of solution (mL).where n = 3) of all the values was expressed. The data was processed using Excel 2013 and Statistical Package for Social Sciences (SPSS) version 20.0 for Windows . Normality was determined using the Kolmogorov-Smirnov test. However, assuming the data was normally distributed, there is a need to carry out the parametric analysis using the LSD and Duncan tests. When the data are not normally distributed, a nonparametric analysis is determined using the Kruskal-Wallis and Mann-Whitney tests.The standard deviation  which has two kinds of polarities, namely, the OH (polar) and the alkyl (nonpolar) groups, was used. Therefore, there is a possibility that some fucoidan dissolved in the polar group, thereby leading to a low yield. There was an insignificant difference between C and B because the solvents (CaCl2 and ethanol) used tend to remove components that are insoluble in acidic solvents. In addition, CaCl2 specifically deposits alginate, thereby affecting the purity of fucoidan extract, which results in low yields [Fucoidan yield is the ratio of the weight of its extract to the weight of dry seaweed flour expressed in percent (%) . The yiew yields .S. hystrix extracted using methods A and D produced higher yields than others. This is due to the acid solvents (HCl and EDTA), which led to the degradation of the seaweed polymer chains [Fucoidan from r chains . HCl solr chains , fucoidaS. hystrix as shown in S. hystrix extracted using method C yielded higher fucose. This is due to the use of ethanol, which specifically precipitates the fucoidan with the help of distilled water during the extraction process. In addition, fucose is soluble in distilled water. According to Sinurat and Maulida [S. hystrix extracted with ethanol solvent has a greater level of purity than other solvents. Because ethanol acted especially to precipitate fucoidan, the ethanol solvent yields a high fucose content, and the extraction process was aided by the use of distilled water because fucose was very soluble in distilled water. Fucose was the major component of fucoidan, according to Sinurat and Maulida [2 solvent was used. According to Sinurat and Kusumawati [2 precipitates alginate, thereby yielding low fucoidan.The extraction methods used on the fucose content were derived from  Maulida , fucose  Maulida ; hence,  Maulida . Meanwhisumawati , CaCl2 pS. hystrix is shown in S. hystrix fucoidan using methods A, B, C, and D is 8.07 ± 0.92, 5.63 ± 0.40, 6.80 ± 0.83, and 7.83 ± 1.83%, respectively. On the contrary, the xylose contained in the commercial fucoidan from M. pyrifera was 8.07 ± 6.79%. Subsequently, the xylose content of the four fucoidan S. hystrix and commercial fucoidan from M. pyrifera is insignificantly different (p > 0.05).According to Lim et al. , the maiS. hystrix as shown in S. hystrix extracted using method B has the least amount of sugar, 26.72 ± 3.74%, compared to A, C, and D. The total sugar content obtained using method B was significantly different (p < 0.05) from the commercial fucoidan from M. pyrifera. Conversely, fucoidan extracted using methods A, C, and D were insignificantly different (p > 0.05) compared to the total sugar obtained from the commercial fucoidan from M. pyrifera.The extraction methods used on the total sugar content were obtained from the fucoidan from S. hystrix is shown in S. hystrix has a high sugar content, the sulfate content was similarly proportional to the sugar content; the higher the sugar, the more sulfate ions were bound to the sugar. The low sulfate concentration of fucoidan extracted with HCl solvent may be owing to the strong acid solvent destroying sulfate as well as extracting fucoidan. While the sulfate content of fucoidan extracted with EDTA solvent was better than that extracted with HCl solvent, it was still lower than that extracted with ethanol solvent. This could be due to the fact that EDTA solvent was a weak acid, allowing it to extract fucoidan while maintaining its structure [2 solvent has a relatively low sulfate level compared to ethanol and EDTA. This could be due to the ability of CaCl2 to bind sulfate. Ca2+ ions in CaCl2 can attach to sulfate ions (SO42-) to create CaSO4, which causes the sulfate content to be transported away by CaCl2 [The effect of the extraction method on the sulfate content derived from tructure . Fucoidaby CaCl2 .S. hystrix and commercial fucoidan are shown in S. hystrix fucoidan.The functional groups of fucoidan compound extracts were determined using FT-IR analysis. This involves carrying out infrared absorbance testing on a compound to distinguish its functional groups based on the absorbance pattern . The FT-S. hystrix  and commercial fucoidans  have similar wavelengths. These indicate that several functional groups are present in the compounds. This also includes the absorption area, which has a wavelength of 3453.96 cm−1 in commercial fucoidan and 3434.22 cm−1 in fucoidan from S. hystrix thereby showing the hydroxyl (OH) functional group [−1 and 1631.98 cm−1 in the commercial fucoidan and fucoidan from S. hystrix, respectively, shows the presence of a carbonyl group (C = O). On the contrary, an absorption area of approximately 1600 cm−1 indicates the presence of uronic acid in both compounds [Fucoidan from ompounds .−1 indicates the presence of sulfate groups in the compounds, for example, wavelengths of 1029.64 cm−1 and 1260.64 cm−1 in commercial fucoidan and 1038.46 cm−1 and 1252.70 cm−1 in fucoidan from S. hystrix. According to Sinurat and Kusumawati [−1, is a characteristic of fucoidan compounds. This shows that the S. hystrix extract sample is a fucoidan. However, several functional groups in commercial fucoidan are absent in the S. hystrix fucoidan, namely, the absorption area with a wavelength of 1166.97 cm−1 indicates sulfate ester, while a wavelength of 851.44 cm−1 implies sulfated polysaccharides (COS), which strengthens the characteristics of these compounds [−1 signifies the presence of a C-6 group from the paranoid ring [S. hystrix, an absorption area with a wavelength of 1420.39 cm−1 indicates the presence of a methyl (CH3) functional group [The absorption area with a wavelength of relatively 1000 to 1300 cmsumawati , the sulompounds . In addioid ring . In fuco50 of 4.336, 5.409, 5.616, and 2.200 ppm, respectively. Conversely, the commercial fucoidan from M. pyrifera had an IC50 of 1.634 ppm (S. hystrix extracted using method C showed the least activity. This is because it was extracted with ethanol (method C), which has a lesser primary antioxidant activity than the secondary. The RSA DPPH method is used for determining the primary antioxidant activity. Therefore S. hystrix shows low antioxidant activity when it was tested with this method [S. hystrix extracted with ethanol solvent is thought to have formed hydrogen bonds between the hydroxyl groups, which act as electron donors in the primary antioxidant mechanism [The DPPH activity of fucoidan extracted using methods A, B, C, and D had an IC.634 ppm . The DPPs method . Fucoidaechanism . UnfortuS. hystrix on the FRAP value is shown in 2 (method B) solvents, which have a lesser sulfate content. According to Sinurat and Maulida [The effect of the extraction method of fucoidan from  Maulida , the antS. hystrix on HRSA values is shown in S. hystrix extracted using methods A, B, C, and D had IC50 HRSA 2312.51 ± 362.15, 1693.97 ± 515.07, 849.37 ± 67.54, and 2360.07 ± 536.93 ppm, respectively, while the commercial fucoidan IC50 was 1906.39 ± 537.58 ppm. Based on these results, fucoidan extracted using method C had the least IC50 value (highest antioxidant activity). Conversely, method D had the highest IC50 value (least antioxidant activity). This was because method C has higher sulfate content than D. According to Wang et al. [50 value, S. hystrix fucoidan with ethanol solution has an IC50 value of 849.37 ± 67.54 ppm, which is lower than the commercial IC50 of fucoidan value of 1906.39 ± 537.58 ppm. This suggested that fucoidan S. hystrix has a stronger antioxidant activity than commercial fucoidan, despite the fact that statistical testing shows that the IC50 values of the two samples were not substantially different since p > 0.05. Although S. hystrix fucoidan has a lower sulfate level, it has stronger antioxidant activity than commercial fucoidan. It is likely that other components in the fucoidan extract play a role in its bioactivity besides sulfate. This is because the bioactivity of fucoidan compounds is influenced by three factors: chemical structure, phenol content, and sulfate content [S. hystrix and influence the strong antioxidant activity. Fucoidan molecules, on the other hand, have a relatively high bioactivity when compared to other secondary metabolites. This is due to the heterocyclic structure of fucoidan, which is made up of carbon atoms bound to sulfate ions. The sulfate ion can boost electron of fucoidan density, allowing it to act as an electron donor [The effect of the extraction method of fucoidan g et al. , sulfate content . Other son donor .S. hystrix on the total antioxidant values is shown in p < 0.05) due to its higher sulfate content (p > 0.05). This is not in line with the higher sulfate content of commercial fucoidans, which increases bioactivity. There is a possibility that other compounds besides the sulfate content in the fucoidan were extracted using methods C and D, which act as antioxidants. According to Alboofetileh et al. [The effect of the extraction method of fucoidan from  content . Accordih et al. , three fh et al. . TherefoSargassum hystrix affect its characteristics and antioxidant activity. Fucoidan from S. hystrix extracted using method A  had the highest yield. Subsequently, the highest sulfate content and antioxidant activities were discovered in method C . The highest antioxidant activity of fucoidan from S. hystrix is from method C which secondary antioxidants were found to be greater in fucoidan S. hystrix extracted with ethanol solvent than primary antioxidants. It has been demonstrated that the DPPH method, which measures primary antioxidant activity, has low activity, whereas the FRAP method, which measures secondary antioxidant activity, has a fairly high antioxidant activity, despite the fact that the FRAP method's antioxidant activity is lower than commercial fucoidans. Similarly, the HRSA method and the total antioxidant approach, which both measure total antioxidant activity, including primary and secondary, both indicate high activity. The antioxidant activity of fucoidan from S. hystrix was influenced by its sulfate content.The extraction methods of fucoidan from


IG‐MYC‐translocation, surface B‐cell receptor (BCR) is expressed, and mutations in the BCR pathway are frequent. Coincidental infections in endemic BL, and specific extra‐nodal sites suggest antigenic triggers. To explore this hypothesis, BCRs of BL cell lines and cases were screened for reactivities against a panel of bacterial lysates, lysates of Plasmodium falciparum, a custom‐made virome array and against self‐antigens, including post‐translationally modified antigens.Burkitt lymphoma (BL) represents the most aggressive B‐cell‐lymphoma. Beside the hallmark of An atypically modified, SUMOylated isoform of Bystin, that is, SUMO1‐BYSL was identified as the antigen of the BCR of cell line CA46. SUMO1‐BYSL was exclusively expressed in CA46 cells with K139 as site of the SUMOylation. Secondly, an atypically acetylated isoform of HSP40 was identified as the antigen of the BCR of cell line BL41. K104 and K179 were the sites of immunogenic acetylation, and the acetylated HSP40 isoform was solely present in BL41 cells. Functionally, addition of SUMO1‐BYSL and acetylated HSP40 induced BCR pathway activation in CA46 and BL41 cells, respectively. Accordingly, SUMO1‐BYSL‐ETA’ immunotoxin, produced by a two‐step intein‐based conjugation, led to the specific killing of CA46 cells. Autoantibodies directed against SUMO1‐BYSL were found in 3 of 14 (21.4%), and autoantibodies against acetylated HSP40 in 1/14(7.1%) patients with sporadic Burkitt‐lymphoma. No reactivities against antigens of the infectious agent spectrum could be observed.These results indicate a pathogenic role of autoreactivity evoked by immunogenic post‐translational modifications in a subgroup of sporadic BL including two EBV‐negative BL cell lines. CRISPR‐F3 genes , 10. SimThe hypothesis that activated BCR signaling by chronic antigen stimulation might contribute to the malignant transformation of B cells is old . For BCRMoraxella catarrhalis was recently reported by us as an antigen in IgD+ nodular lymphocyte predominant Hodgkin lymphoma, with an additive stimulation of LP‐cells by Moraxella superantigen MID/Hag via the Fc domain of sIgD [MYD88 and CD79B [RpoC of the Gram‐negative bacterium  of sIgD . Regardi of sIgD , 25. Fornd CD79B , 27, SAMnd CD79B . In systnd CD79B . Moreovend CD79B . Recentlnd CD79B .This prompted us to screen for, and characterize, possible target antigens of BCRs of BL using expression cloning of primary cryo‐preserved specimens and BL lines, and subsequently screen for infectious antigens and post‐translationally modified autoantigens , 31, 32.2The study had been approved by the local ethics committee (Ärztekammer des Saarlandes 12/13). For expression cloning of BL‐BCRs snap‐frozen specimens of patients were provided by the University Cancer Center Frankfurt (UCT). This study was approved by the institutional review boards of the UCT and the Ethics Committee (SHN‐03‐2018). Sera of a second cohort of patients with BL were obtained from Saarland University Medical Center.BL31, BL41, CA46 and RAMOS derived from EBV‐negative sBL, DAUDI and RAJI derived of EBV‐positive eBL, and BJAB whose cell of origin is debated, were verified by their V genes. In addition, the cell lines CA46 and BL41 were ordered a second time freshly from the DSMZ  and the results were verified. Cell lines of DLBCL and MCL were obtained from ATCC  and DSMZ . All experiments were performed with mycoplasma‐free cells.2.1BCRs from eight BL cell lines . These BL line–derived BCRs and the recombinantly expressed BCRs (each at a concentration of 10 µg/ml) were pooled and screened on protein macroarrays containing clones of UniPEx 1 and 2 cDNA expression libraries , as previously described , 32. To Plasmodium antigens, all BL BCRs were tested in dot‐blots against inactivated lysates of Plasmodium falciparum obtained from the Institute of Tropical Medicine in Tübingen, Germany.For screening for reactivity of endemic BL BCR against M. catarrhalis, M. osloensis, M. nonliquefaciens, Streptococcus pneumoniae, Streptococcus pyogenes, Haemophilus influenzae, Staphylococcus aureus, coagulase‐negative staphylococci, Klebsiella pneumoniae, Escherichia coli, Acinetobacter radioresistens and Neisseria spp. provided by the Institute of Medical Microbiology and Hygiene in Homburg/Saar, Germany were spotted onto PVDF membranes each with a dose of 10 µg.To screen for potential reactivity of Fabs against bacterial antigens, heat‐inactivated lysates of 13 different bacterial strains or patient isolates including The dot blots of bacterial lysates were blocked in 10% (w/v) non‐fat dry milk powder in TBST  at 4°C overnight, washed with TBST and incubated for 1 h with the individual Fabs (each at 10 µg/ml), followed by further washing steps and subsequent incubation for 1 h at room temperature with biotinylated goat anti‐human heavy and light chain Fab antibody  at a dilution of 1:5,000 (v/v). Subsequently the arrays and blots were incubated for 10 min at room temperature with Strep‐POX  in 2% (w/v) milk/TBST and binding was detected using an ECL system .To screen for potential reactivities against viral antigens a custom‐made array with 2206 viral genes expressed by the NAPPA‐technology of 158 viruses was analyzed for reactivities of the BL Fabs in the Virgina C. Piper Center . Normalized values ≥ 2 were regarded as indicative for potential hits , 35, 36.2.2The expression clones of BYSL and HSP40 fused C‐terminally to a tag consisting of following amino acids DYKDDDDK (FLAG‐tag) in the pSFI vector were recombinantly in HEK293 cells. Additionally, C‐terminally FLAG‐tagged full length and FLAG‐tagged fragments of different length of BYSL and HSP40 were transfected by electroporation into CA46 and BL41 cell lines via pRTS vector .For secondary modification, 500 µl of cell lysates were incubated for 10 min at room temperature with 10 µl anti‐FLAG‐affinity matrix and washed afterwards. Acetylation of HSP40 and SUMOylation of BYSL were performed as previously described , 33. PosFor site‐directed mutagenesis of BYSL and HSP40 the QuickChange II Site‐Directed Mutagenesis Kit  was used. cDNA coding full length BYSL and HSP40 each C‐terminally FLAG‐tagged, and mutants of the amino acids with the highest predicted probability as SUMOylation and acetylation sites were constructed. SUMO1‐sites in BYSL were analyzed by following exchanges of a lysine with an arginine: K36R, K78R, K110R, K139R, K154R, K158R and K194R and acetylation sites in HSP40 by K41R, K104R, K155R, K179R, K254 R, K257R, K259R, K260R, K268R, K306R, K309R, K364R, K368R, K 375R, K376R, K378R, K380R, K384R, K 390R, K403, K422R, K442, K443R, K444R. All FLAG‐tagged mutants were cloned into pRTS vector, and were transfected and expressed in CA46 or BL41 [2.3Pseudomonas exotoxin A (ETA’) according to Nachreiner et al. [N site EFE‐MX1N and the toxin component with MBP, MXC1 as INTC site followed by CEFL and ETA’. Thereafter the BYSL‐antigen component was SUMOylated as described above for 30 min at room temperature by addition of SUMO‐extract and activation buffer. Subsequently, the SUMOylated BYSL‐INTN component was incubated together with the ETA’‐INTC component at room temperature.Recombinantly expressed immunotoxin consisting of BYSL was conjugated to a truncated form of r et al. , and wasr et al.  and expr2.4Acetylated HSP40 and SUMOylated BYSL were confirmed as BCR antigens by ELISA as previously described .2.5Lysates of BL lines or of whole blood from patients were loaded and separated by a 10% SDS‐PAGE and transferred to PVDF membranes using a transblot semidry transfer cell (Bio Rad). After blocking overnight at 4°C in PBS/ 10% nonfat dry milk, a recombinant SUMO1‐BYSL‐ or acetylated HSP40‐reactive His‐tagged Fab were incubated at a concentration of 2 µg/ml for 1 h at room temperature, followed by incubation for 1 h at room temperature with murine anti‐His antibody at 1:2,000 (Qiagen), or with HRP‐labeled anti‐mouse IgG antibody (Bio Rad). Chemiluminescence reagent (New England BioLabs) was used for immunoblot detection. Subsequently DLBCL and MCL lines were screened for the presence of the atypic isoforms of the antigens identified in BL.2.66 cells were incubated with no antigen, BYSL, SUMOylated BYSL, HSP40 or acetylated HSP40, respectively, at 5 µg/ml or anti‐IgM at 1 µg/ml for 3d at 37°C [4/ml CA46 or BL41 cells were seeded in a 200‐µl cell culture medium. Recombinantly expressed BYSL, SUMOylated BYSL, HSP40 and acetylated HSP40 were added at the concentration of 1 µg/ml or the Intein‐based BYSL/ETA’ immunoconjugate [For the analysis of the BCR pathway activation in CA46 cells expressing BCRs with reactivity against SUMO1‐BYSL, and in BL41 cells expressing BCRs with reactivity against acetylated HSP40, 1 × 10 at 37°C . Rabbit onjugate , 24. Aft3Recombinant BCRs in the format of Fabs were successfully synthesized from 3 sBL cases. Moreover, antigen‐binding fragments of ‘natural’ BCRs were obtained by papain digestion from eight sporadic and three endemic BL lines. From eight of these cell lines, recombinant Fabs were generated Table . Sera or3.1P. falciparum and against lysates of bacterial strains did not reveal any specific reactivity, and the screening of the BCRs against 2206 viral proteins of 158 selected viruses did not result in potential hits. In contrast, while the screening of BL Fabs on non‐modified UNIPEX1 and 2 arrays could not identify any antigenic target, screening of the same Fabs on post‐translationally modified protein macro arrays revealed SUMOylated BYSL  and acetylated HSP40  as candidate antigens.The screening against lysate of 3.2ELISA with recombinant BYSL and SUMOylated BYSL or HSP40, and acetylated HSP40 expressed with a C‐terminal FLAG‐tag in HEK293, confirmed SUMOylated BYSL and acetylated HSP40 as target antigens of BL‐derived Fabs Figure  and anti3.3p = 0.0341)In Western‐blots of lysates of BL31, BL41, BJAB, CA46, Daudi, Raji and Ramos Burkitt cell lines, SUMOylated BYSL was exclusively detected in CA46 cells and acetylated HSP40 exclusively in BL41 cells Figure . K139R m3.4Western blot analysis of the stimulation of the BCR pathway upon the addition of recombinant SUMO1‐BYSL and acetylated HSP40 revealed a BCR‐pathway involvement in CA46 and BL41 cells respectively, documented through the upregulation of pTyr525/526 SYK, pTyr96 BLNK, pTyr759 PLCγ2, and pTyr223 BTK. Neither non‐SUMOylated BYSL nor non‐acetylated HSP40 induced this effect Figure . Additio4Here, we report the identification and characterization of two antigens recognized by the BCRs of two cell lines established from sporadic BL. BL41 is a line derived from a tumour of EBV‐negative sporadic BL from an 8‐year‐old boy . CA46 isIG‐MYC rearrangements [TCF3 and ID3 [The genesis of sporadic BL may be explained exclusively intrinsically, mainly by ngements , mutatio and ID3 . Given t and ID3 , 18, 19, and ID3 , 46. WheTherapeutically, cause and prevention of these atypical isoforms might be of interest for secondary prophylaxis. Moreover, our results indicate that artificial BCR‐antigen‐immunotoxins for targeting lymphoma cells specifically through their BCR‐reactivity may become an interesting therapeutic option for the future.P. aeruginosa exotoxin A. However, the application of these identified BCR reactivities to target subgroups of relapsed/refractory lymphoma seems unrealistic at t present because of the rarity of the disease.We have successfully harnessed a two‐step strategy with inteins to fuse SUMOylated Bystin to a truncated recombinant form of Our study is limited by the small number of cases. Unfortunately, it was not possible to obtain blood samples from adult or pediatric patients with BL despite extensive efforts. It would be very interesting to screen not only for the presence of lymphoma‐associated autoantibodies against SUMO1‐Bystin and against acetylated HSP40, but also directly for the presence of the atypical isoforms in PBMCs from patients with sporadic BL. Collecting material, together with clinical data, and making it available to the scientific community for future studies will be an important task of future clinical trials on BL.The frequent extra‐nodal manifestation of BL in the ileocecal junction may point to a possible infectious trigger, and it is obvious that the panel of bacterial lysates that was screened reflects only a small fraction of the microorganisms that are present, for instance, in the microbiome. Additionally, most of the analyzed strains are commonly found as part of the ‘normal’ oral, pharyngeal or intestinal flora in healthy individuals. Finally, with regard to post‐translationally modified self‐proteins and degradation products, our detection tools are still limited, and the two autoantigens reported here likely represent only the tip of the iceberg. Further studies are therefore needed.The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.The study had been approved by the local ethics committee (Ã¿rztekammer des Saarlandes 12/13) and had been approved by the institutional review boards of the UCT and the Ethics Committee (SHN‐03‐2018) of University Cancer Center Frankfurt (UCT).L. Th., N. F., E. R., K. D. P. designed the study. S H and M. L. H. performed microdissection of BL cases. R. M. B., J. H., H. D., S. R., S. L. B., S. L., B. K., S. S., J. R., T. P., R. W., L. Tr. and H. D. were of great help in the acquisition of B. L. samples and clinical data. R. S., E. S., K. R., H. D. and M. H. helped in data interpretation. N. F., T. B. and E. R. performed the protein array experiments and proteomic experiments. E. R. and T. B. performed expression cloning of Fabs and site directed point mutagenesis of HSP40 and Bystin. E. R., N. F. and K. D. P. performed intein‐based two‐step‐approach of expression of SUMOylated‐Bystin‐immunotoxins. L. Th., T. B., M. B. and K. D. P. are responsible for data analysis and interpretation of results. L. Th. wrote the manuscript. K. R., M. B., H. D., S. H., S. L. B., R. M. B., E. C. S., M. H. and R. S. revised the manuscript in detail.


Temperature, as the main driving factor of carbon fixation during phytoplankton photosynthesis, is closely related to TEP production. However, little is known about the effects of temperature on TEP the production, DOC secretion, and carbon pool allocation of phytoplankton. This study analyzed the effect of temperature on the carbon pool allocation of phytoplankton and the significance of TEP in the marine carbon pool. Our results suggest that increased temperature affects carbon pool allocation in phytoplankton cells by promoting DOC exudation and extracellular TEP formation. This study provides an important basis for understanding the contribution of TEP to the allocation of POC and DOC and will benefit the prediction of phytoplankton TEP production and the marine carbon cycle under the background of global warming.TEP is the bridge mediating DOC and POC conversion in the ocean, which is a key component of the marine carbon cycle. TEP accelerates the accumulation or deposition rate of organic carbon at the sea surface and promotes the short- and long-term sequestration of carbon, counteracting atmospheric CONitzschia closterium and Chaetoceros affinis) and two model dinoflagellate species (Prorocentrum micans and Scrippisella trichoidea). The results showed that temperature had a significant impact on TEP production in all species. First, increased temperature promoted the TEP production of all four species. Second, elevated temperature affected the carbon pool allocation, with enhanced dissolved organic carbon (DOC) exudation in the form of TEP in all species. The TEP-C/DOC percentages of N. closterium and P. micans were 93.42 ± 5.88% and 82.03 ± 21.36% at the highest temperature (24 °C), respectively, which was approximately two to five times higher than those percentages at 16 °C. In contrast, TEP’s contribution to the POC pool is lower than that to the DOC pool, ranging from 6.74 ± 0.79% to 28.31 ± 1.79% for all species. Moreover, phytoplankton TEP production may be related to cellular size and physiology. The TEP content produced by the smallest N. closterium (218.96 ± 15.04 fg Xeq./μm3) was ~5 times higher compared to P. micans, S. trichoidea, or C. affinis. In conclusion, TEP production is temperature sensitive and species specific, which should be taken into consideration the regarding TEP-mediated oceanic carbon cycle, particularly in the context of global warming.Transparent exopolymer particles (TEP) are sticky polymeric substances that are commonly found in the periphery of microbial cells or colonies. They can naturally flocculate smaller suspended particles into larger aggregates and thus play a crucial role in the biological pump and the global carbon cycle. Phytoplankton are the major contributors to marine TEP production, whereas the way TEP production interacts with abiotic factors at the species level is generally unknown but critical for estimating carbon fluxes. In this study, the effects of temperature on TEP production and carbon allocation were studied in two representative diatom species ( Phytoplankton can exude organic matter  such as Nitzschia closterium and Chaetoceros affinis) or dinoflagellate (Prorocentrum micans and Scrippisella trichoidea) species in the South China Sea [Phytoplankton blooms are the main source of marine TEP production ,13,14. Ihina Sea ,16. Sedihina Sea . Therefohina Sea  discoverhina Sea . Temperahina Sea ,21,22. Phina Sea ,16. Thishina Sea ,24,25 anhina Sea ,27,28. HNitzschia closterium and Chaetoceros affinis) and the two dinoflagellate species (Scrippsiella trichoidea and Prorocentrum micans) used in this study are the dominant species of coastal phytoplankton blooms in the northern regions of the South China Sea (2·s2) in a 12 h:12 h illumination–darkness cycle. To ensure axenic cultivation, penicillin and streptomycin sulfate were added to final concentrations of 0.04 μg/mL and 0.01 μg/mL, respectively.The two diatom species  below [Aliquots of 5 mL cultures were taken every day and preserved with 0.5 mL of acidic Lugol’s iodine solution. The cells were counted, and cell dimensions were determined using an inverted microscope (OLYMPUS-CKX41). When calculating the cell volumes, we assumed that 1) below .(1)μ=lnμ is the specific growth rate; N1 and N0 are the numbers of cells at the end and beginning of the exponential phase, respectively; and t0 and t1 represent the beginning and end time of the exponential phase, respectively.Here, a were determined by filtering 15 mL of culture through a 0.7 µm glass fiber membrane. Chl a samples were extracted in the darkness in 90% acetone solution for 24 h, and fluorescence of extracted Chl a was measured with a UV–vis spectrophotometer.Chl During the stationary phase, 3 mL of culture was taken from each species and filtered through a 0.4 μm pore-sized polycarbonate membrane with a negative pressure lower than 0.02 MPa. The filter membrane was stained in 1 mL of 0.02% Alcian blue  for five seconds and then washed twice with 1 mL ultrapure water. The filter membrane was transferred to a 25 mL beaker with 6 mL of 80% sulfuric acid solution and incubated for 2 h. Finally, the absorbance value of the solution was measured by a UV–vis spectrophotometer at the wavelength of 787 nm, and the content of TEP was calculated by constructing a xanthan gum (Xeq.) standard curve .TEP carbon content (TEP-C) was calculated by the following formula:color refers to the TEP concentration measured by the colorimetric method, and 0.51–0.88 is the range of the coefficient for different phytoplankton species [TEP species . The ran species .To prepare POC samples, 30 mL of algal culture were filtered through a combusted  0.7μm glass fiber membrane. The organic matter caught on the membrane was used to measure POC, and the filtrate was used to measure DOC. Then the membrane was wrapped with tinfoil, dried at 40 °C for 48 h and stored at −20 °C. The filtrate was transferred to a 30 mL scintillation flask and stored at −20 °C. To measure the POC content, 5 mol/L hydrochloric acid was used to wash the membrane and then incubated at 40 °C to dry for 48 h. The membrane was loaded into a tin boat, using the membrane filtered with artificial seawater as a blank control. The POC content was determined by an element stable isotope ratio mass spectrometer. For DOC content measurement, samples were first thawed at room temperature, and then measured by a TOC measuring instrument using artificial seawater as the blank and potassium hydrogen phthalate as standards.GraphPad Prism 9.0 was used to process and plot the data, and SPSS 25.0 was used for statistical analysis. One-way ANOVA was conducted to analyze the TEP concentration and DOC concentration. Canoco 5 was used to analyze the effects of temperature on different physiological parameters of phytoplankton by principal component analysis (PCA). The significance level cutoff was set to 0.05 in this study.C. affinis and S. trichoidea were not significantly affected by varying temperatures (p > 0.05) (S. trichoidea and C. affinis at 16 °C were significantly lower than that at 20/24 °C (p < 0.05) (N. closterium and P. micans increased significantly with the increase of temperature (p < 0.05) (The four species grew faster at higher temperature (20 °C and 24 °C) than at low temperature (16 °C) a–d, but  > 0.05) . The cel < 0.05) c,d. Howe < 0.05) . As a re < 0.05) a,b, respa ranged from 2.15 to 13.47 μg Xeq./Chl a. P. micans, C. affinis, N. Closterium, and S. trichoidea had significantly promoted TEP production with the increase of temperature (p < 0.05) , comparing to S. trichoidea with unchanged productions (p > 0.05) (All four species could produce TEP at the three temperatures . TEP con < 0.05) . However > 0.05) .P. micans and N. closterium are the largest, (15.19 ± 5.24) × 103 μm3, and the smallest, (0.10 ± 0.02) × 103 μm3, respectively   , while Pp < 0.05) in most species, rather than POC or TOC (p > 0.05) (P. micans and S. trichoidea decreased significantly with the increase of temperature (p < 0.05)   (Increased temperature mainly affected the DOC content per cell significantly ( > 0.05) a–d. The  < 0.05) a,b, whil > 0.05) d, and C.at 20 °C c. This w affinis g but dec < 0.05) g.p < 0.001) except S. trichoidea (p < 0.05)   e and theichoidea f. The TEat 24 °C e, respec < 0.05) e. The TE < 0.05) f. The DOP. micans and S. trichoidea) were correlated with POC, TOC, and cell volume, and POC had a strong correlation with TOC, while the two diatoms (C. affinis and N. closterium) had good correlations with the growth rate and Chl a.  was used to explore the influences of temperature on different physiological parameters of phytoplankton. A total of 63.4% and 25.0% of the variance of physiological variables were explained by the first and second axes, respectively . Temperad Chl a. .S. trichoidea was 17 °C–25 °C, and the optimal growth temperature was 20 °C–22 °C [S. trichoidea grew better at 20 °C–24 °C. It was also reported that the temperature around the growth of P. micans ranged from 18 °C to 28 °C [Temperature is one of the important environmental factors that affect the growth and reproduction of phytoplankton . Previou°C–22 °C . This wato 28 °C . Here, tto 28 °C  reportedto 28 °C ,28.N. closterium strain was higher than that used by Engel et al. [C. affinis strain versus the CCMP 159 used by Passow [a of S. trichoidea was not affected by temperature variation, whereas it increased under higher temperatures with respect to P. micans, C. affinis, and N. closterium. Claquin et al. [Lepidodinium chlorophorum in consistency with our findings. However, Claquin et al. [Thalassiosira pseudonana [The TEP productions of sister strains could be considerably disparate. In this study, the TEP concentration of the l et al. , while iy Passow  who usedn et al.  found inn et al.  found thn et al.  found ineudonana . Other seudonana ,35. Thereudonana ,37,38. TN. closterium had the highest TEP content, about 5 times higher than the other three species. Yet, the largest-sized P. micans, which was ~150 times larger than the smallest N. closterium, only had a TEP content 1/5 of N. closterium. Fukao et al. [Coscinodiscus granii (103.00 ± 6.24) × 103 μm3 was 400 times larger than that of Skeletonema sp. (0.26 ± 0.05) × 103 μm3, while the concentration of TEP per cell volume of the former (341.6 ± 56.33 fg Xeq/μm3) was 5 times higher than that of the latter (68.30 ± 3.28 fg Xeq/μm3). This was opposite to our findings and likely indicates that TEP content does not change linearly with phytoplankton cell volume. According to Wu et al. [N. closterium belongs to the class of pennatae diatoms and usually lives an adherent life to solid matrices. The dinoflagellates P. micans, S. trichoidea, and centrales diatom C. affinis are generally planktonic. Most diatoms have longitudinal shell fissure connecting cytoplasm and extracellular environment which can determine the attaching and gliding ability of diatom cells on a solid surface [N. closterium was several orders of magnitude higher than that of C. affinis. The gliding and attaching functions of benthic diatoms depend on the exopolysaccharides on the cell surface [N. closterium produces TEP consistently to become resistant to the frequent environmental changes. In conclusion, the amount of TEP produced by phytoplankton may be associated with the species’ physiologies and growth patterns, so the relationship between TEP and physiologies should be further studied.The TEP content per unit cell volume was variable for the four phytoplankton species in this study. The smallest-sized o et al.  reportedu et al. , the abs surface ,43,44. T surface ,46. Thes surface . TherefoS. trichoidea (34.09 ± 2.33%) and P. micans (16.72 ± 2.24%) ranked at the top and bottom, respectively, of which the latter lay within the reported range (~5–30%) by Baine et al. [This study showed that the POC and TOC contents per cell of the four species did not change with temperature, in consistence with the study by Montagnes et al.  who conce et al. . Consumpe et al. . In genee et al.  argued tP. micans and S. trichoidea decreased, and yet the TEP-C/DOC ratios increased as temperature increased. The DOC content per cell of N. closterium was not affected by temperature, and the TEP-C/DOC ratio also increased with the increase of temperature. For C. affinis, the DOC content was the lowest at 24 °C, but the TEP-C/DOC ratio reached the highest value. Furthermore, the DOC/TOC fraction decreased with increasing temperature. These results indicate that higher temperature can expedite the conversion of DOC to TEP-C, causing enhanced DOC secretion in the form of TEP . These results indicate that higher temperatures can promote the conversion from DOC to TEP-C, and as a result, more DOC is secreted by cells in the form of TEP. Moreover, the production of TEP in phytoplankton may be related to the cell size and physiological status. The content of TEP produced by the smallest-sized N. closterium was about 5 times higher than that of P. micans, S. trichoidea, and C. affinis. These observations overturn our hypothesis that the contribution of DOC and POC to TEP production is independent of temperature and improved our understanding of other factors affecting TEP production, which is required to make realistic projections of carbon fluxes in a warmer ocean.The four phytoplankton species grew better at high temperatures in this study, and the specific growth rate was affected by temperature change with interspecies differences. The increase of temperature promoted the production of TEP in


Agaricomycetes) employ enzymatic and nonenzymatic cellulose degradation mechanisms, the latter presumably relying on Fenton-generated radicals. The effects of the two mechanisms on the cellulose microfibrils structure remain poorly understood. We examined cellulose degradation caused by litter decomposers and wood decomposers, including brown-rot and white-rot fungi and one fungus with uncertain wood decay type, by combining small- and wide-angle X-ray scattering. We also examined the effects of commercial enzymes and Fenton-generated radicals on cellulose using the same method. We detected two main degradation or modification mechanisms. The first characterized the mechanism used by most fungi and resembled enzymatic cellulose degradation, causing simultaneous microfibril thinning and decreased crystalline cellulose. The second mechanism was detected in one brown-rot fungus and one litter decomposer and was characterized by patchy amorphogenesis of crystalline cellulose without substantial thinning of the fibers. This pattern did not resemble the effect of Fenton-generated radicals, suggesting a more complex mechanism is involved in the destruction of cellulose crystallinity by fungi. Furthermore, our results showed a mismatch between decay classifications and cellulose degradation patterns and that even within litter decomposers two degradation mechanisms were found, suggesting higher functional diversity under current ecological classifications of fungi.Mushroom-forming fungi (IMPORTANCE Cellulose degradation by fungi plays a fundamental role in terrestrial carbon cycling, but the mechanisms by which fungi cope with the crystallinity of cellulose are not fully understood. We used X-ray scattering to analyze how fungi, a commercial enzyme mix, and a Fenton reaction-generated radical alter the crystalline structure of cellulose. Our data revealed two mechanisms involved in crystalline cellulose degradation by fungi: one that results in the thinning of the cellulose fibers, resembling the enzymatic degradation of cellulose, and one that involves amorphogenesis of crystalline cellulose by yet-unknown pathways, resulting in a patchy-like degradation pattern. These results pave the way to a deeper understanding of cellulose degradation and the development of novel ways to utilize crystalline cellulose. Cellulose is the most abundant carbohydrate  and a vaAgaricomycetes). Brown-rot fungi secrete endoglucanases but no cellobiohydrolases , suggesting that they could harbor both enzymatic and nonenzymatic cellulose degradation mechanisms  and another that is connected to cellulose amorphogenesis (patchy-like) but did not match the effect of Fenton-based radicals. Moreover, the two mechanisms did not match the current ecological or wood decay classifications that separate mushroom-forming fungi into white-rot and brown-rot wood decomposers and litter decomposers, suggesting that functional diversity among the three groups is more complex than we currently think.We used small- and wide-angle X-ray scattering (SAXS and WAXS), which have been used previously to study the structure of intact cellulose , to examod decay , 28. We chanisms , 29, 30.drolases . We compq, from 0.004 to 2.9 Å−1. The various cellulose treatments examined are listed in I(q)] of the original  filter paper (OP), together with control filter papers (autoclaved), incubated for 40 days in the liquid medium without fungi. The six different patterns showing the scattering intensity over the q range [I(q)] were identical, and the scattered intensity and the I(q) profile were reproducible. This suggests that the sterilization process and incubation of the autoclaved paper in the absence of fungi did not alter the fiber structure of cellulose. Therefore, we used the pattern from the OP as a reference for investigating the impact of the treatments.Changes in cellulose structure were investigated by using SAXS and WAXS covering almost 3 orders of magnitude in the scattering vector, I(q) had the following features: (i) At lower q values (0.004 to 0.04 Å−1), the intensity decayed as a power law, I(q) = Aq−4, with A being a constant. (ii) At an intermediate q of ≈0.1 Å−1, there was a small bump in the scattering curve. (iii) In the high-q WAXS regimen (q > 0.8 Å−1), the diffraction pattern demonstrated the presence of crystalline cellulose phases Iα and Iβ  = Aq−4. As the fiber diameter was much greater than a qmin−1 of ≈250 Å , the q−4 decay of the scattered intensity can be described in terms of Porod’s law int = B[exp(−kq2) + bg], where k is a constant. Assuming that this structural feature is homogeneous throughout the fibers, bg is a q-independent (background) contribution associated with the nonperiodic part of the local molecular packing. The WAXS part of the overall pattern, for q of >0.8 Å−1, with a series of Bragg peaks, indicates mainly the presence of crystalline domains in the fiber. Cellulose degradation results in a decrease in the intensity, while the diffraction pattern remains constant. We can thus describe the intensity as Icr(q) = CScr(q), where the structure factor Scr(q) describes the diffraction pattern and C is a measure of the diffracted intensity, which is proportional to the amount of crystalline cellulose. Assuming that the crystalline domains are homogeneously distributed in the fibers, then C, just like B, is proportional to the total fiber volume in the X-ray beam.We interpreted the low od’s law , where tray beam . We attre fibers . Previoue fibers , 36, 37.q range of 0.004 to 0.4 Å, using A and B. As a measure of the factor C, in the WAXS region, we used the peak intensity of the most intense Bragg peak (Miller index of 200) in the diffraction pattern, at q = 1.63 ± 0.04 Å−1. The full scattering profiles were normalized considering the sample transmission, T. Thus, T is equal to:iI and tI are the intensities of incident beam and transmitted beam, respectively, δ is the thickness, and d is the sample density, while the ratio μ/ρ is the mass attenuation. For small dδ:T can also be used as measure of material loss, as nontransmitted X-rays (1 − T) are either adsorbed or scattered. For OP, T is 0.86, and in T as obtained from the different experiments. The transmission here was relatively high, so that (1 − T) was approximately proportional to the total volume of scatterers. Gloeophyllum sp. OMC-1627, Leucoagaricus leucothites (LEL), Coprinellus angulatus (COP), Agrocybe pediades (AGP), and enzymatic and Fenton treatments revealed lower values of (1 − T), consistent with a higher material loss.The SAXS region can be modeled as follows:A = A0, B = B0, and C = C0, we characterized the cellulose degradation and modification patterns caused by fungi 2, 0B/B, and 0C/C values or all the investigated samples. As mentioned above, if the fibers were homogeneous in crystallinity, then C would be expected to be proportional to the fiber volume. In contrast, A is proportional to the interfacial area, and in the case of cylindrical fibers, to the fiber radius R. Thus, for cylindrical fibers we expect A2 to be proportional to the fiber volume. This means that if the effect of cellulose degradation were a homogeneous thinning of the fibers, then we would expect to have (0A/A)2 = 0C/C. And, if the average fiber radius decreased from the initial value, 0R, to R, then R/0R = 0A/A = (0C/C)1/2. The crystallinity of cellulose filter papers of this kind is typically high: a value of 76% was reported for filter paper similar to the one used in this study (q = 1.38 Å−1 is considered to have a significant contribution from amorphous cellulose (38α) between amorphous and crystalline cellulose varied across treatments, we calculated the ratio between the intensity at q of 1.38 Å−1 and the sum of peak intensities in the q range of 1 to 1.6 Å−1  over the entire q range (0A/A)2 ≈ 0C/C, consistent with a homogeneous thinning of the fibers  over the entire q range (α (0A/A)2 and (0C/C) (0A/A), the amount of crystalline cellulose did not decrease proportionally to the radius, which further suggests fundamental differences between the mode of action of enzymes and Fenton reaction radicals during cellulose degradation. It should also be mentioned that in the case of both enzymatic  and Fenton (Fe-12h and Fe-24h) degradation, the loss of material was so large that the number of fibers was most likely decreased. In the case that the fibers were degraded one by one, this would result in a gradual decrease of the filter paper thickness, which would then correspond to a decrease in scattering (fiber) volume at constant fiber radius, R. Therefore, we expect A/A0~C/C0  . This re/A0~C/C0 . For theS. commune (uncertain wood decay type), all representing four orders  were examined for their ability to degrade or modify cellulose fibers without nitrogen limitation (I(q) profiles (Gloeophyllum sp. OMC-1627), showed similar efficiency in degrading cellulose. This was obvious from all calculated values of 0A/A, 0B/B, and 0C/C (0A/A and 0C/C); however, we observed a more pronounced decrease of 0B/B under high-nitrogen conditions .Three species of white-rot fungi, six species of brown-rot fungi, eight species of litter decomposers, and mitation . In addimitation . The degprofiles , right aprofiles  and 5. Tprofiles . Overalland C/C0  and 5, wnditions , particuBoletales, which implies that nitrogen might not have a regulatory effect under these experimental conditions. For most samples, the relationship of (0A/A)2 ≈ 0C/C was maintained, i.e., there was a decrease of the overall volume V of the fibers (thinning)  or wood decay strategy (white versus brown rot), most fungi had a similar effect on cellulose structure. Similarly, the two different nitrogen concentrations did not alter the patterns of cellulose degradation by brown-rot hinning)  and a prhinning) –45 and winning) –, 18, 19.inning) –. The strellulose . The thiellulose . Our finGloeophyllum sp. and the strain of the litter decomposer Leucoagaricus leucothites were the only two strains that did not agree with the findings described above. Both strains showed an unusual pattern of cellulose degradation that did not match the enzymatic or Fenton treatments. Cellulose degradation by both species manifested itself in a strong decrease in the diffraction peak intensities (0 =C/C 0.5) and the intermediate peak intensity (0B/B = 0.3)  . In controminent . As discrization . This aprization . AlthougGloeophyllum sp.) and one litter decomposer (L. leucothites), led to a decrease of crystallinity without proportional thinning of the fibers  production of hydroxyl radicals in close proximity to cellulose. Second, the cellulolytic gene composition of L. leucothites showed similarity to white-rot fungi , which cause brown rot, but they also code for enzymes involved in crystalline cellulose degradation . In addition to the nitrogen source, the Highley medium contained (per liter): 2 g KH2PO4, 0.5 g MgSO4 · 7H2O, 0.1 g CaCl2 · 2H2O, 0.57 mg HBO3, 0.036 mg MnCl2 · 4H2O, 0.31 mg ZnSO4 · 7H2O, 0.039 mg CuSO4 · 5H2O, 0.018 mg (NH4)6Mo7O24 · 4H2O, 0.015 mg FeSO4 · 7H2O, and 10 mL of a vitamin stock solution. The vitamin stock solution contained (per liter): 1 g inositol, 0.01 g thiamine-HCl, 0.0025 g biotin, 0.01 g pyrodoxine, 0.01 g riboflavine, 0.01 g nicotinamide, 0.01 g p-aminobenzoic acid, 0.01 g Ca-panthotenate. A Nylon 66 membrane  was placed on top of the paper, and an inoculum (7 mm) from actively growing cultures of the strains was placed at the center of each dish. The cultures were incubated at 20 or 25°C . Nine or 18 μL of the enzymatic mix was added in a final volume of 10 mL sodium acetate buffer  to form concentrations C1 and C2, respectively. Filter paper (100 mg) was incubated for 24 and 48 h at 50°C under rotation (280 rpm). At the end of the incubation, the liquid was removed and the paper was washed twice with MilliQ water and dried at room temperature for 24 h. Earlier experiments had shown that these condition are suitable for the advanced degradation of filter paper cellulose . Cellic 2+ and hydrogen peroxide were 0.005 M and 0.8 M, respectively. The surplus of hydrogen peroxide was used to ensure a continuous Fenton reaction for 48 h. The pH was measured every hour for the first 6 h and at 8, 10, 12, 14, 16, 19, and 24 h. It remained close to 3.65 throughout the experiment. After 24 h, the liquid was removed and the paper was washed twice with MilliQ water and dried at room temperature for 24h.The Fenton reaction was performed in 5 mL sodium acetate buffer  at room temperature. The final concentrations of freshly prepared FeI(q) was recorded with the detector placed at three sample-to-detector distances, yielding scattering vectors (q) from 0.004 to 2.9 Å−1. Samples were located in a sandwich cell holder . The temperature was controlled by an external recirculating water bath fixed to 25°C (accuracy of ca. 0.2°C). The two-dimensional (2D) scattering pattern was recorded using a 2D 300 k Pilatus detector  and radially averaged using SAXSGui software to obtain I(q). The SAXS and WAXS data were normalized by the intensity of the transmitted beam through the sample, whereas thickness was fixed to 0.430 mm, i.e., the thickness of the original paper (OP). The overall thickness of the decomposed filter paper changed very little with respect to the OP, making any measurement of the thickness unsuitable. The deconvolution of the WAXS patterns is described in Fig. S1 in the supplemental material. The SAXS-WAXS method was chosen over other methods because it allowed us not only to estimate the crystallinity, but also to evaluate fiber thinning.Paper samples, of approximately 0.5 cm by 0.5 cm, before and after fungal degradation were placed in a “sandwich” sample holder without windows. All measurements were performed under vacuum. The sample holder was scanned by means of transmission experiments to locate accurately the sample positions. The sample was taken about 1 cm away from where the fungal inoculum had been placed to ensure prolonged exposure of the cellulose to the fungus. SAXS and WAXS measurements were performed using a SAXSLab Ganesha 300XL instrument , which includes a pinhole-collimated system equipped with a Genix 3D X-ray source . The scattering intensity All data are available in the main text or the supplemental material.The raw scattering data are available upon request.