The purpose of this study was to determine, for the first time, antioxidant activities of seven peptides (P1–P7) derived from hydrolysis of oat proteins in a cellular model. In the oxygen radical absorbance capacity (ORAC) assay, it was found that P2 had the highest radical scavenging activity /µM peptide) followed by P5, P3, P6, P4, P1, and P7 whose activities were between 0.14–0.61 µM TE/µM). In the hepatic HepG2 cells, none of the peptides was cytotoxic at 20–300 µM. In addition to having the highest ORAC value, P2 was also the most protective (29% increase in cell viability) against 2,2′-azobis(2-methylpropionamidine) dihydrochloride -induced oxidative stress. P1, P6, and P7 protected at a lesser extent, with an 8%–21% increase viability of cells. The protection of cells was attributed to several factors including reduced production of intracellular reactive oxygen species, increased cellular glutathione, and increased activities of three main endogenous antioxidant enzymes. Health benefits associated with the consumption of grains are not only linked to the presence of phytochemicals such as polyphenols but also to their content of fibers and micronutrients . The mecRecent studies have, however, shown that, in addition to fibers and polyphenols, hydrolyzed proteins and peptides also contribute to the health promoting effect of cereals ,8. Lunas3), glutathione reductase (GR), nicotinamide adenine dinucleotide phosphate (NADPH), cumene hydroperoxide, catalase, nitroblue tetrazolium chloride (NBT), xanthine oxidase, 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), 5-sulfosalicylic acid dehydrate, and 96-well and 60-mm tissue culture plates were purchased from Sigma Aldrich . Hydrogen peroxide (H2O2) and fluorescein were from Fisher Scientific Co., . Bathocuproine disulfonic acid (BCS) and xanthine were purchased from MP Biomedical  while 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH) was from Wako Chemicals USA Inc., . Peptides: FNDRLRQGQLL (P1), GLVYIL (P2), GQTV (P3), GQTVFNDRLRQGQLL (P4), YHNAP (P5), YHNAPGLVYIL (P6), and DVNNNANQLEPR (P7) were synthesized by GenScript  at a purity of more than 95%.Reduced L-glutathione (GSH), fatty acid free bovine serum albumin (BSA), potassium phosphate, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), diethylenetriaminepentaacetic acid (DETAPAC), 3--2,5-diphenyltetrazolium bromide (MTT), dimethyl-sulfoxide (DMSO), sodium azide , and ORAC values were expressed as µM Trolox Equivalent (TE)/µM peptide using the standard curve.The ORAC assay was performed as previously described ,14. The 2 and 95% air. Dulbecco’s Minimal Essential Media (DMEM) supplemented with 10% (v/v) Fetal Bovine Serum was used for the maintenance of cells. Initial treatment with 20–500 µM of P2  showed that 50 and 100 µM were optimum concentrations and both were used for all peptides. The MTT -2,5-diphenyltetrazolium bromide) assay, as described in an earlier research, was used for cytotoxicity and cytoprotective tests [4 cells/well in 96-well tissue culture plates were grown for 24 h, washed with phosphate buffer saline solution  followed by 24 h incubation with peptides and another wash, 200 µL of media were then added to cells intended for cytotoxicity and 200 µL of media containing 20 mM of AAPH to those used for cytoprotection evaluation. Following another 24 h incubation, cells were washed twice with 200 µL of PBS. Ten µL of MTT solution (5 mg/mL in PBS) and 100 µL of media were added to each well. After 1 h, MTT solution was removed, and 50 µL DMSO were added. Absorbances were recorded at 570 nm with 630 nm background subtraction using a BioTek Epoch™ microplate reader . Untreated cells were used as negative control (NC) and cells treated with only AAPH were used as positive control (PC). Four peptides  that displayed cytoprotective effect were used for further investigation.Human hepatocellular carcinoma (HepG2) cells were purchased from the American Type Culture Collection . They were cultured at 37 °C in a humidified incubator with 5% COve tests . For bot6 cells/plate in 60 mm tissue culture plates and allowed to grow for 24 h. In each plate, cells were washed twice with 4 mL of PBS (pH 7.2), and treated with 4 mL peptide samples  dissolved in culture media at 50 or 100 µM for 24 h. After removal of peptide solutions, cells were washed twice with 4 mL of PBS/plate and treated with 4 mL of 20 µM AAPH dissolved in culture media and allowed to incubate for 24 h. Cells in each culture plate were harvested by incubation (5 min) with 0.5 mL of 0.25% trypsin, addition of 1 mL of culture media and centrifugation at 1000× g for 5 min. Cell pellets were washed with ice cold PBS (500 µL each) until no pink color was observed, re-suspended in ice cold PBS (300 µL) for the superoxide dismutase (SOD) assay, 500 µL for glutathione peroxidase (GPx) and catalase (CAT) assays. For total glutathione assay, 300 µL of 5% ice cold sulfosalicylic acid bubbled with 100% nitrogen was used for cell lysis. Cells were lysed by sonication on ice for 1 min using a probe-type sonicator  pulsing at 15 s on and 10 s off cycles. Following centrifugation of cell lysate at 13,000× g (4 °C), pellets containing cell debris were discarded, while supernatants were used for determination of glutathione content. Protein contents were determined using a modified Lowry assay.Cells were seeded at 1 × 10The activity of GPx was determined as previously described in a 96-well plate . Briefly2−• generated in situ was followed at 560 nm. SOD activities were calculated and expressed as units per milligram of protein.The SOD activity was measured based on the rate of reduction of nitroblue tetrazolium (2.24 mM) to formazan by xanthine oxidase (13.2 U/mL) at 560 nm . Eight c2O2 (30%) was added, and removal of H2O2 by catalase was followed at 240 nm [To measure the catalase activity, 1790 µL potassium phosphate buffer  was mixed with 200 µL cell lysate in a UV cuvette. Ten µL of Ht 240 nm  using a The assay was based on the enzymatic recycling method adjusted for 96-well plates . All sol4 cells/well in a 96-well tissue culture plate and were treated for 24 h with 100 µM of peptide dissolved in media, with the exception of NC and PC controls. Media was removed, cells washed twice with PBS, incubated (24 h) with 20 mM AAPH and washed again with PBS. Two hundred µL of 40 µM DCFH2-DA dissolved in buffer was added, and fluorescent intensity was immediately recorded at 2 min intervals for a total of 60 min. The percentage increase in fluorescent intensity was calculated as shown in Equation (1).The measurement of intracellular ROS was determined using a method described by Wolfe and Rui, with modifications . HepG2 cn = 4) ± standard error of mean (S.E.M.). Comparison between groups were carried out by one-way analysis of variance using SPSS 11.0 for Windows . Post-hoc Tukey’s honest (HSD) test was used to determine significant differences (p < 0.05).All data were expressed as means (•) scavenging activities of P1–P7 were evaluated using the ORAC assay. Data obtained  compared to that of NC. It was found that P2 greatly promoted the growth of HepG2 cells as evidenced by the nearly 2.7-fold increase in cell viability. The mechanism is unclear but similar findings were reported in hamster ovary cells treated with yeast, soy, and broadbean hydrolysates [The cytotoxicity of each peptide was determined by measuring the viability of HepG2 cells. The assay was initially optimized using P2 (20–500 µM) because of its highest activity in the ORAC assay. It was only cytotoxic above 450 µM. Subsequent tests were then performed using 50 and 100 µM only. As shown in olysates . The groolysates .p < 0.05) as they increased the viability of cells by 15.5% and 8.3%, respectively, compared to PC. Pretreatment of cells with P6 increased the viability of cells from 58.9% ± 2.7% for PC to 88.2% ± 6.4%. The most cytoprotective peptide was P2, which not only completely eliminated AAPH damage, but also increased the viability of HepG2 cells, compared to NC. The protective effect of P2 is partly due to the fact that in the absence of AAPH, it did increased cell viability  above that of normal cells by 30.2% and 38.6%, respectively, while P7 only suppressed the damage caused by AAPH. These data demonstrated that higher cytoprotective effects for P2 and P6 in HepG2 cells might be due to their ability to detoxify hydroperoxides, as they are the main oxidative products of AAPH [As shown in  of AAPH . In the 2O2 were also reported to double CAT activity of rat hepatoma cells while pretreatment with epicatechin tripled the value [Compared to SOD and GPx, CAT is resistant to inactivation by peroxyl radicals, due to the narrowness of its active site which prevents access of large molecules . The inche value . Peptidehe value . In porche value , the deche value ,34. Hencp < 0.05) GSH concentrations above that of normal cells. It has been found that in HepG2 cells, reduced glutathione (GSH) is approximately 30-fold higher than its oxidized form GSSG [The physiological role of GSH has long been of interest, particularly its antioxidant function due to the unique redox chemistry of its cysteinyl thiol residue . The oxiorm GSSG . In the orm GSSG  or HepG2orm GSSG .Results from this study showed that peptides had various scavenging activities in the ORAC assay with P2, P5 and P3 being the most active. Individual peptides alone did not show cytotoxicity in HepG2 cells. However, when followed by AAPH induced oxidative stress, P5 enhanced cells death while P1, P2, P6, and P7 were cytoprotective. The mechanism of protection, particularly for P2 and P6, was related to the increased activities of antioxidant enzymes GPx, SOD and CAT; increase in glutathione synthesis, and reduced production of intracellular reactive oxygen species.


Native myocardial T1 is known to be affected by variables such as age, gender, heart rate and partial voluming from blood pool. Blood T1 itself has a wide (wider) variability. We aimed to investigate causes of blood T1 variability.77 healthy volunteers with no known cardiovascular condition underwent CMR at 1.5T . Mid ventricular short axis native T1 maps by MOLLI (with T1* reconstruction in addition) and ShMOLLI were acquired. Hematocrit (Hct), iron profile and lipid profile were acquired immediately prior to the scan. CVI42  was used for analysis of the maps. A ROI was drawn in the blood pool on the MOLLI T1 map, avoiding papillary muscles and was copied on to the MOLLI T1* and ShMOLLI T1.2 0.530, coeff. -0.728, p < 0.0001). Mean ± SD of blood T1 by MOLLI T1 was 1638 ± 78 ms, MOLLI T1* 1686 ± 111 ms and ShMOLLI T1 1543 ± 77 ms. There was a negative correlation between blood T1 and Hct  of blood T1 variability with anaemia and low iron increasing T1 but with HDL reducing it.


Escherichia coli using the autoinduction system, purified, and the proteins were used to raise antisera in rabbits. All antisera detected all three serotypes of PV from infected cell lysates in enzyme-linked immunosorbent assay, immunofluorescence and western blotting.Poliomyelitis, caused by poliovirus, is on the verge of eradication, and the world is preparing to shift from live to inactivated polio vaccine. In view of the requirement of non-infectious reagents, especially protein antigens, for surveillance during the final phase of poliovirus eradication, we have attempted to generate reagents that may be of use for the development of diagnostic tests. Polioviral proteins VP0, VP3, VP1, and 3AB were expressed in  •E. coli using autoinduction.Poliovirus structural and non-structural proteins were expressed in •Purified proteins were used for raising antibodies.•Antisera raised could detect antigens in virus-infected cells by different immunoassays.•Antisera could detect both cleaved and uncleaved as well as native and denatured viral proteins.•These reagents may be useful in vaccine manufacturing and seromonitoring in the future. PV is classified into three serologically distinct types, PV1, PV2, and PV3, each with slight differences in capsid structure, which are differentially recognized in virus neutralization assays. The PV genome is about 7500 nucleotides long, and consists of a single open reading frame flanked by a 5′ non-translated region (NTR), which includes the internal ribosome entry site (IRES), 3′NTR, and a polyA tail. The small viral protein VPg is covalently linked to the 5′ end of the genome. Translation of the viral genome results in the synthesis of a polyprotein, which is cleaved by viral proteases into individual viral proteins, including (a) three structural proteins VP1, VP3 and VP0, the latter of which is further processed into VP4 and VP2 during virion morphogenesis, and (b) seven non-structural proteins . The 2A and 3C proteins are responsible for the proteolytic cleavage of the polyprotein Polio is an acute viral infectious disease of humans, and is caused by poliovirus (PV). PV is a single-stranded positive sense RNA virus belonging to the genus Three major antigenic sites with neutralization epitopes are present on the surface of PV and the location of the antigenic sites differs among serotypes. Antigenic site 1 (residues 89–100 of VP1) is present in PV2 and 3, antigenic site 2a (residues 220–222 of VP1) is present in PV1, antigenic site 2b (residues 164–172 of VP2) is present in PV1 and 3, antigenic site 3a (residues 286–290 of VP1) is present on PV3, whereas antigenic site 3b  is present in PV1 and 3 Seromonitoring of mass vaccination campaigns requires demonstration of high antibody titers against structural proteins, especially VP1. On the other hand, demonstration of anti-NSP antibodies in the sera is an indication of infection. The 3AB protein is absent in purified inactivated vaccines, and antibodies against NSP indicate circulation of PV in a population. Immunological assays developed based on recombinant PV proteins could therefore be useful for demonstration of freedom from infection status after cessation of oral polio vaccine.E. coliVP0, VP3, VP1 and 3AB genes of PV1 in E. coli, and antisera raised using the recombinant proteins was tested for their suitability to detect different serotypes of PV in different immunoassays.The inducible T7 expression system is widely used for expressing foreign genes in 22.1E. coli DH5α strain (Invitrogen) was used for cloning of genes, while E. coli BL21(DE3) strain (Invitrogen) was used for protein expression.The pRSET B  plasmid was used for cloning and expression of genes of interest under the control of the T7 ϕ10 promoter. The The pVS(1)IC-O(T) plasmid , which contains the full length PV1 genome under the control of SV40 promoter 2.2CV1 (ATCC-CCL-70) is a fibroblast cell line derived from male African green monkey kidney. CV1 cells are susceptible for poliovirus infection Sabin strains of PV1, PV2, and PV3 were obtained from Bharat Biotech International Limited, Hyderabad, India. Recombinant PV1 (rPV1) was generated using plasmid pVS(1)IC-O(T) by transfecting CV1 cells (ATCC-CCL-70). For making lysates, all virus infections were carried out at 0.1 multiplicity of infection (MOI), whereas for immunofluorescence, MOI of 0.01 (PV1) or 0.05 (PV2 and PV3) were used. All experiments were carried out in biosafety level II laboratory, following appropriate protocols and procedures.2.3The following bacterial culture media were used in the present study:LB-SP medium: 1% soy peptone, 0.5% yeast extract, 1% sodium chloride.2HPO4, 50 mM KH2PO4, 25 mM (NH4)2SO4, 2 mM MgSO4 and 0.8% glucose.ZYP-0.8G-SP medium: 1% soy peptone, 0.5% yeast extract, 50 mM Na2HPO4, 50 mM KH2PO4, 25 mM (NH4)2SO4, 2 mM MgSO4, 0.5% glycerol, 0.05% glucose and 0.2% α-lactose.ZYP-5052-SP medium for autoinduction: 1% soy peptone, 0.5% yeast extract, 50 mM Na2.4VP0, VP3, VP1 and 3AB genes of Sabin PV1 (AY184219.1) were amplified by RT-PCR using gene-specific primers with flanking restriction sites EcoRI and HindIII ( HindIII  and clon2.5g for 10 min), and the cells were used for analysis.The recombinant plasmids pPV1VP0, pPV1VP3, pPV1VP1, pPV13AB as well as control pRSET B (not containing any insert) were transformed into chemically competent BL21(DE3) cells 2.6One mL of autoinduced culture was pelleted and resuspended in 1X reducing SDS-PAGE loading buffer, boiled at 95 °C for 10 min, subjected to 12% denaturing SDS-PAGE and stained with Coomassie brilliant blue R-250. Gels were blotted to PVDF membranes for western blot analysis. The membrane was blocked with 5% w/v skimmed milk powder (SMP) in phosphate buffered saline, pH 7.2 (PBS), overnight at 4 °C. The membrane was incubated with anti-His monoclonal antibodies  at a dilution of 1:15,000 in 3% SMP in PBS at 37 °C for 1 h, washed three times with PBS containing 0.05% Tween-20 (PBST), followed by incubation with a 1:10,000 dilution of horseradish peroxidase (HRP) conjugated anti-mouse IgG secondary antibody in 3% SMP at 37 °C for 1 h. The membranes were washed three times with PBST, and once with PBS, then incubated with ECL reagent , and His-tagged proteins were detected using X-ray film.2.7g for 10 min. The supernatant and pellet were collected separately and analyzed on reducing SDS-PAGE.Ten mL of autoinduced cultures were pelleted and resuspended in 10 mL of 50 mM Tris (pH 8.0) buffer containing 25% sucrose, 0.5% Triton X-100, and 1 mM phenyl methyl sulfonyl fluoride (PMSF). Cells were lysed by sonication  on ice, and the lysate was pelleted by centrifugation at 16,0002.82.8.1g for 5 min, and used to load a Ni-NTA column pre-equilibrated with 50 mM Tris-HCl, 0.3 M NaCl, 20 mM imidazole, pH 8.0 (soluble protein equilibration buffer). The column was washed with 50 mM Tris–HCl, 0.3 M NaCl, 20 mM imidazole, pH 8.0 (soluble protein wash buffer), until the OD at A280 reached zero, and the column-bound protein was eluted with 50 mM Tris–HCl, 0.3 M NaCl, 250 mM imidazole, pH 8.0 (soluble protein elution buffer). The elution fractions were analyzed on reducing SDS-PAGE, and fractions with the presumed desired protein were pooled and dialyzed against 100 mM Tris, pH 8.0 overnight at 4 °C. Protein concentration was estimated by BCA protein assay kit (Thermo Scientific) according to the manufacturer's instructions.Sonicated lysate was clarified by centrifugation at 16,0002.8.2g for 5 min, and the supernatant was loaded onto a Ni-NTA column pre-equilibrated with denatured protein equilibration buffer. The column was washed with 50 mM Tris-HCl, 8 M urea, 20 mM imidazole, pH 8.0 (denatured protein wash buffer) until OD at A280 reached zero, and column bound proteins were eluted with 50 mMTris–HCl, 8 M urea, 250 mM imidazole, pH 8.0 (denatured protein elution buffer) and collected fractions were analyzed by reducing SDS-PAGE. Fractions with presumed desired protein were pooled, and urea was removed by step dialysis through a graded series of urea  in 100 mM Tris, pH 8.0, for 2 h each, followed by a final dialysis against 100 mM Tris, pH 8.0, overnight, all at 4 °C. Protein concentration was estimated by BCA protein assay kit (Thermo Scientific) according to the manufacturer's instructions.After sonication, the cell pellet was dissolved in 10 mL of 50mMTris-HCl, 8 M urea, pH 8.0 (denatured protein equilibration buffer), clarified by centrifugation at 16,0002.9Animal experiments were conducted with permission from the Institutional Animal Ethics Committee as per the Guidelines of the Committee for the Purpose of Control and Supervision of Experiments in Animals, under the Breeding of and Experiments on Animals (Control and Supervision) Rules, 1998 as amended in 2001 and 2006, Government of India.Five hundred micrograms of the purified recombinant proteins  each were mixed individually with equal volume of Freund's incomplete adjuvant (FIA) to make an emulsion and injected into 2-month-old New Zealand white rabbits subcutaneously. On day 28, the animals were boosted with 250 μg of respective protein emulsions in FIA. On day 35, sera were tested for the ability to detect PV-specific proteins by western blotting. A second booster of 250 μg of respective protein emulsions in FIA was administered on day 56, and sera obtained at day 66 were stored at −20 °C.2.10The sera were tested for their ability to detect three serotypes of PV as well as rPV1. CV1 cells were infected with PV1, PV2, or PV3 viruses, or transfected with pVS(1)IC-O(T) plasmid. At 30% cytopathic effect, cell lysates were prepared by freeze-thawing in PBS, and used for western blotting and ELISA. Similarly, the ability of the antisera to detect the three serotypes of PV by immunofluorescence assay was tested on CV1 cells infected with PV1, PV2, or PV3 viruses.2.10.1o-phenylene diamine (OPD) substrate. The reaction was stopped by addition of 50 μL of 3 M sulphuric acid per well and the OD was read at 490 nm. The ELISA data was analyzed using one-way analysis of variance (GraphPad Prism). The p values were calculated by comparing lysates from control CV1 cells to lysates from CV1 cells infected with PV1, PV2 or PV3, using Bonferroni's multiple comparison test.ELISA plates were coated with PV1, PV2, or PV3 infected or uninfected CV1cell lysates  or recombinant proteins (100 ng/well) overnight at 4 °C. The plates were blocked with 3% SMP in PBS for 1 h at 37 °C. Different polyclonal sera at various dilutions (1:200 to 1:3200) in 1% SMP in PBS were added and incubated at 37 °C for 1 h, washed three times with PBST, followed by addition of HRP-conjugated anti-rabbit IgG (Sigma-Aldrich) at 1:5000 dilution in 1% SMP at 37 °C for 1 h. The plate was washed three times with PBST and once with PBS, and then developed using 2.10.2Lysates of PV1-, PV2-, or PV3-infected CV1 cells, or extracts of pVS(1)IC-O(T) plasmid- or mock transfected CV1 cells were subjected to reducing SDS-PAGE and transferred to a PVDF membrane. Anti-PV1-VP0, -VP3, -VP1or -3AB rabbit sera were used as primary antibody (1:1000) and HRP-conjugated anti-rabbit IgG (Sigma-Aldrich) (1:5000) was used as secondary antibody to develop western blot using ECL reagent (Thermo Scientific) to detect respective PV proteins.2.10.3CV1 cells were infected with PV1 (0.01MOI), PV2 (0.05 MOI), or PV3 (0.05MOI), and incubated at 37 °C for 18 h. The cells were fixed with methanol: acetone (1:1) mixture at room temperature for 3 min, and washed twice with PBS. Rabbit anti-VP0, -VP3, -VP1 or -3AB polyclonal sera were added to each well at 1:200 dilution, and incubated at 37 °C for 1 h, and the plates were washed three times with PBS. Then, fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG (Sigma-Aldrich) (1:500 dilution) was added, before incubation at 37 °C for 1 h, and two washes with PBS. The cells were then observed under a fluorescent microscope.33.1VP0, VP3, VP1 and 3AB genes were cloned into pRSET B plasmid, and positive clones  were confirmed by restriction digestion and nucleotide sequencing. All the genes were expressed individually in E. coli BL21(DE3) using autoinduction E. coliThe amplified PCR products for PV1 3.2E. coli expressed recombinant proteins (The anti-PV1-VP0 sera could detect both VP0 (37 kDa) and VP2 (30 kDa) proteins in western blot analysis of lysates from cells infected with all three serotypes of PV, or transfected to obtain rPV1 , panel-Aproteins  as well proteins . ELISA rproteins . SignifiAs expected, all sera also showed signal in cells infected with all three serotypes of PV, and no signal was observed in uninfected cells by immunofluorescence assay . Pre-immDetection of PV proteins in western blot, ELISA and immunofluorescence indicate that the sera could detect both native and denatured proteins. Antibodies developed during this study can be used for vaccine identity test and probably also for potency testing, and we are currently exploring these applications with a vaccine manufacturer. It should be noted that extensive standardization in terms of production, purification, and quality control of the proteins is required to use them as validated reagents. Similarly, the polyclonal sera also need to be extensively characterized. In this context, characterized monoclonal antibodies may provide more standardized antibody reagents, although polyclonal antibodies cover multiple reactive sites on a protein.4Owing to the PV eradication initiative, the incidence of polio due to wild type virus is on the verge of approaching zero. In this transitory period, the oral, live attenuated vaccine is being replaced by the injectable, inactivated vaccine


Alzheimer’s disease (AD) is the most common dementia affecting tens of million people worldwide. The primary neuropathological hallmark in AD is amyloid plaques composed of amyloid-β peptide (Aβ). Several familial mutations found in Aβ sequence result in early onset of AD. Previous studies showed that the mutations located at N-terminus of Aβ, such as the English (H6R) and Tottori (D7N) mutations, promote fibril formation and increase cytotoxicity. However, A2T mutant located at the very N-terminus of Aβ shows low-prevalence incidence of AD, whereas, another mutant A2V causes early onset of AD. To understand the molecular mechanism of the distinct effect and develop new potential therapeutic strategy, here, we examined the effect of full-length and N-terminal A2V/T variants to wild type (WT) Aβ40 by fibrillization assays and NMR studies. We found that full-length and N-terminal A2V accelerated WT fibrillization and induced large chemical shifts on the N-terminus of WT Aβ, whereas, full-length and N-terminal A2T retarded the fibrillization. We further examined the inhibition effect of various N-terminal fragments (NTFs) of A2T to WT Aβ. The A2T NTFs ranging from residue 1 to residue 7 to 10, but not 1 to 6 or shorter, are capable to retard WT Aβ fibrillization and rescue cytotoxicity. The results suggest that in the presence of full-length or specific N-terminal A2T can retard Aβ aggregation and the A2T NTFs can mitigate its toxicity. Our results provide a novel targeting site for future therapeutic development of AD. Alzheimer’s disease (AD) is the most common form of dementia occurred in the elderly that causes huge social and economic impacts in the world. Dementia describes significant loss of certain mental functions such as memory, attention, and abstract thinking. Amyloid-β (Aβ) deposition is the main pathogenic hallmark of AD besides hyperphosphorylated tau in neurofibrillary tangles. CurrentAβ is a proteolyzed peptide product from a type I transmembrane protein, amyloid precursor protein (APP), after sequential cleavages of β- and γ-secretases. The cleavage results in two main isoforms, Aβ40 and Aβ42, comprising 40 and 42 amino acids, respectively. Aβ42 seAβ is an intrinsically disordered peptide containing mostly random coils. Aβ aggrMajority of AD patients are sporadic. There are only less than 10% patients carrying genetic mutations, known as familial AD (FAD) that causes early onset of the symptoms. FAD mutations in APP sequence may occur outside or inside of Aβ region. The mutations can lead to increase of total Aβ production, increase of Aβ42/Aβ40 ratio, alteration in biophysical properties and assembly states of Aβ. The idein vitro and in vivo[Since Aβ aggregation is highly implicated in AD, understanding its misfolding mechanism are essential to develop targeted treatment. Inhibitors preventing Aβ self-assembly are potential therapeutic agents for AD. In factd in vivo. Small md in vivo–41. Dired in vivo, 43. Ford in vivo–44 and td in vivo.In the past, due to the difference of Aβ isoforms resided in the C-terminus, the C-terminus of Aβ have drawn more attention from researchers on amyloid formation. The rol-1 cm-1 and NTFs protein concentration was measured by bicinchoninic acid assay due to lacking of aromatic residues.Aβ40 peptides including WT, A2T, A2V, and A2V NTF(1–10) were synthesized by the peptide synthesis core in Genomics Research Center, Academia Sinica by solid-phase peptide synthesis. A2T NTFs containing amino acids 1-x were purchased from GM biolab. Aβ variants were first dissolved in hexafluoroisopropanol (HFIP) and lyophilized. The lyophilized full-length Aβ peptide powder was re-suspended in DMSO and added to 10 mM phosphate buffer, pH 7.4. The A2T and A2V NTFs were directly dissolved in 10 mM phosphate buffer, pH 7.4. The full-length protein concentration was re-quantified by absorbance at 280 nm with extinction coefficient, 1,280 MAβ at 25 μM were prepared in 10 mM phosphate buffer, pH 7.4, and monitored by addition of 5 μM ThT in quiescence. ThT fluorescence intensity was examined at 485 nm where the excitation was at 442 nm in a microplate reader  at 25°C, The signals were collected every hr automatically with 1 min mixing before the measurement. Finally, the signals were calculated and averaged from three repeats with buffer background subtraction. The signals were normalized to the final plateau of WT Aβ.2O, 100 μl. The washes were performed three times with three separate drops. Then, the grids were stained by 10 μl of 2% filtered uranyl acetate (UA) for 1 min, washed by ddH2O inversely for three times, and air-dried at room temperature overnight. UA is a commonly used negatively stained reagent for TEM studies. UA enhances the contrast by interaction with proteins. The final UA concentration in the samples was 1%. The samples were scanned by Hitachi H-7000 transmission electron microscope. The accelerating voltage at 75 kV was used. Several images were collected in each sample with different magnification to observe amyloid morphology.The 400-mesh Formvar carbon-coated copper grids  were first discharged, then ten μl of end-point Aβ aggregates were placed on the grids for 5 min and washed by inversely placing the grids on top of a drop of distilled deionized HFreshly prepared Aβ variants were dissolved in 3 mM NaOH and sonicated 1 min. After lyophilization, Aβ peptide powder was dissolved in 50 mM phosphate buffer, pH 7.2, and centrifuged at 21,000g at 4°C. Native page was prepared with stacking and running gels containing 4% and 15% acrylamide in 0.375 M Tris-HCl, pH 8.8, respectively. The samples were diluted by 2X sample buffer that contains 62.5 mM Tris-HCl, pH 6.8, 25% glycerol, and 1% bromophenol blue. Thirty μl samples were loaded to the well and the electrophoresis was run at 90 V. The gel was transferred to 0.45 μm PVDF membrane (Millpore) and subjected to western blotting following the standard protocol. The antibodies used for western blotting were mouse anti-Aβ, 1–16 antibody 6E10 (1:5000) (Covance) and anti-mouse IgG (HRP) (GeneTex).Freshly prepared Aβ variants were prepared based on native PAGE protocol. The concentration of Aβ samples was re-quantified and diluted to a final concentration of 50 μM. After preparation, the Aβ variants were immediately examined in a ProteomeLab XL-I centrifuge with an An-60Ti rotor . Sedimentation velocity (SV) experiments was conducted following previous literature[Freshly prepared Aβ variants were dissolved 3 mM NaOH and sonicated 1 min. After lyophilization, Aβ peptide powder was dissolved in 10 mM phosphate buffer, pH 7.2, and centrifuged at 21,000g at 4°C. The supernatant was re-quantified. Then, Aβ variants were diluted to final concentration of 50 μM. All measurements were performed at room temperature, and 10 accumulations were averaged on a JASCO J-815 spectropolarimeter . Spectra were obtained from 190 to 250 nm with buffer background subtraction. The molar ellipticity was calculated and plotted.2O for two times. The pellets were re-suspended by 10 mM phosphate buffer, pH 7.4. Ten μl sample was placed on the sample cell. The spectra were detected by Nicolet™ 6700 FT-IR spectrometers from Thermo Electron Corporation. Spectra were recorded at a resolution of 4 cm-1 and accumulated 30 times at a wave number range from 900 to 4,000 cm-1. The signals were calculated and normalized with the individual peak in max intensity.The end-point products of Aβ were centrifuged at 17,000 x g, 4°C and washed by ddH15N-labeled Aβ40 was expressed in the M9 minimal media containing 15NH4Cl and purified by two affinity columns and one RP-HPLC purification as previously described[15N-labeled Aβ40 in 8 M Tris-buffered guanidine hydrochloride (GdnHCl), pH 7.4, at a concentration of 10 mg/mL, followed by rapid dilution into 10 mM Tris, pH 7.4, containing 10% D2O . Monomeric 15N labeled Aβ was obtained from the supernatant after centrifuging the stock solution at 17,000 × g at 4°C for 10 min to precipitate the aggregates and subjected to NMR studies. Full-length unlabeled WT, A2T, and A2V variants were dissolved in 8 M Tris-buffered GdnHCl, pH 7.4, at 15 mg/mL. Pre-aggregates were removed after centrifuging at 21,000 × g, 4°C for 10 min. Peptide concentration was determined using BCA assay. HSQC spectra were acquired using 50 μM recombinant 15N-labeled Aβ40 in Bruker Avance 600 or 800 MHz NMR spectrometer equipped with 5 mm triple resonance cryoprobe and Z-gradient at 278 K. Amide 15N/1H resonance assignment was based on BRMB number 17796[15N-labeled Aβ40 with two molar equivalents of unlabeled WT). NMR chemical shift perturbation is calculated using the equation, CSP = sqrt(δH2+0.2*δN2). The rate of reduction is normalized by assuming intensity of Gly38 peak remains constant and determined by: 1-Int(A2T or A2V variants)/Int(WT control).The described. In brieber 17796 and TOCSMTT assay was employed to examine cell viability. Twenty-five μM of the end-point products of Aβ were diluted by DMEM/F12 media with the final working concentration at 10 μM and treated to human neuroblastoma SH-SY5Y cell line. After 24 hr incubation, MTT solution was added, and the cells were incubated for an additional 3 hr. Cells were lysed by 100% DMSO. The absorbance was measured at a wavelength of 570 nm by an ELISA reader . The signals were subtracted from the buffer control, averaged, and normalized. The statistical analysis was performed by one-way ANOVA and Tukey’s Post Hoc Test in SPSS program . LDH assay was used to examine cytotoxicity. For cytotoxicity of the end-point products, the end-point products of Aβ samples were treated to human neuroblastoma SH-SY5Y in DMEM/F12 media  to the final concentration at 10 μM for one day and subjected to LDH assay. The cells were lysed by 2% Triton X-100 to serve as a positive control for 100% cytotoxicity. The substrate signal was monitored in an ELISA plate reader . The substrate fluorescence was monitored and the kinetics of substrate increase was averaged and normalized to the positive control. The statistical analysis was performed by one-way ANOVA and Tukey’s Post Hoc Test in SPSS program .To gain insight into the underlying mechanism of N-terminal Aβ variants focusing on residue 2, we first compared fibrillization of WT, A2T, and A2V Aβ40 by ThT assay . We moni-1 were measured and normalized. The FTIR spectra showed great similarity among the three fibril samples. The secondary structures of the fibrils were all enriched in β-structures as evidenced by the peak near 1,620 to 1,640 cm-1[-1 for WT and A2V and 1,627 cm-1 for A2T fibrils. We found WT and A2V fibrils have similar spectra around 1,645 to 1,662 cm-1, a region indicates α-helical structures. A2T fibrils have ~13% more spectral intensity for α-helical structures in comparison to that of WT fibrils. In contrast, A2V fibrils do not contain obvious peaks in this region. The CD and FTIR results showed that mutation at residue 2 has the ability to change secondary structure content of freshly prepared and fibrillary Aβ variants.To gain insight into possible structural differences in freshly prepared and fibrillar Aβ variants, we first subjected the freshly prepared Aβ variants, i.e. WT, A2T, and A2V, to native PAGE , SV expe,640 cm-1. The typSince heterozygous APP is appeared in people carrying the familial mutations, we examined the fibrillization of mixtures of WT and A2T or A2V  to mimic1H-15N HSQC of 15N labeled Aβ40 at 50 μM by NMR spectroscopy upon titration with two molar equivalents of unlabeled WT (control), A2T, or A2V variants at 278 K. The total Aβ concentration was 150 μM. The spectra of full-length WT Aβ40 alone (blue cross peaks) and WT Aβ40 with 2 molar equivalent of full-length Aβ40 A2T . These NMR results also demonstrated that the presence of A2V accelerates fibril formation but A2T does not. The results are consistent with our fibrillization assays and the literature[To gain more insight into the effect of A2 mutation to the fibrillization of Aβ, we collected Aβ40 A2T  and A2V Aβ40 A2T   of A2T have the capability to inhibit the fibril formation, we wonder if the effect can be also achieved by shorter NTF peptides. We used A2T NTFs ranging from residue 1 to residues 4 to 10, as listed in Clinically, A2T and A2V that mutate at the same site but different amino acids show either protective or exacerbating effect on AD. It is intriguing why changing amino acids at the same site would cause such an opposite effect even though changing alanine to valine increases hydrophobicity and to threonine increases polarity. Here, in this study we first characterized the properties of full-length A2T and A2V and the effects of full-length A2T and A2V in mixtures with WT. Our data are consistent with previous literature showing It is interesting to discover the effect on N-terminal Aβ even though it is not considered in most of the oligomer and fibril models. However, Some Aβ40 fibril studies also demonstrated N-terminal residues 4–7 are involved in fibril structure–60, in wPeptide inhibitors have been a great interest for AD therapeutic development. The current reported peptides are mostly designed to break β-sheets, 43. SevS1 FigCytotoxicity was examined by LDH assay. The ADDLs of aggregation experiment were treated to neuroblastoma SH-SY5Y cells with final concentration of 25 μM. After 1 day incubation, cytotoxicity was measured by LDH assay. Triton X-100 was used as a positive control for 100% cytotoxicity. The statistical analysis was performed by one-way ANOVA and Tukey’s Post Hoc Test.(TIF)Click here for additional data file.S1 File(PDF)Click here for additional data file.


Scorpiops pococki from Xizang province of China, this research aims to discover a novel functional gene encoding peptidic blocker of Kv1.3, and identify its function.Specific and selective peptidic blockers of Kv1.3 channels can serve as a valuable drug lead for treating T cell-mediated autoimmune diseases, and scorpion venom is an important source of kv1.3 channel inhibitors. Through conducting transcriptomic sequencing for the venom gland of We screened out a new peptide toxin KTX-Sp4 which had 43 amino acids including six cysteine residues. Electrophysiological experiments indicated that recombinant expression products of KTX-Sp4 blocked both endogenous and exogenous Kv1.3 channel concentration-dependently, and exhibited good selectivity on Kv1.3 over Kv1.1, Kv1.2, respectively. Mutation experiments showed that the Kv1 turret region was responsible for the selectivity of KTX-Sp4 peptide on Kv1.3 over Kv1.1.This work not only provided a novel lead compound for the development of anti autoimmune disease drugs, but also enriched the molecular basis for the interaction between scorpion toxins and potassium channels, serving as an important theoretical basis for designing high selective Kv1.3 peptide inhibitors. EM cells) /IC50), where I is the steady-state current and [peptide] is the concentration of toxin. The parameter to be fitted was concentration of half-maximal effect (IC50).Whole-cell patch-clamp recordings were performed using an EPC 9 amplifier  at room temperature (22–24 °C). Pipettes pulled from borosilicate glass  had resistances of 2–4 MΩ when filled with the internal solution. The internal pipette solution for recording Kv currents on DRG neurons contained: 120 mM potassium gluconate, 20 mM KCl, 10 mM EGTA, 10 mM HEPES, 5 mM Naocedures . Kv currScorpiops pococki venom glands, one of the nucleotide sequences obtained displayed an ORF encoding a new putative neurotoxin which was termed KTX-Sp4. The precursor nucleotide sequence of KTX-Sp4 is 312 bp in length, including three parts: 5′UTR, ORF and 3′UTR. The 5′ and 3′ UTRs of KTX-Sp4 are 53 and 67 bp in length  predicted that the precursor of KTX-Sp4 contained a putative signal peptide of 20 residues following a mature toxin of 43 residues with three pairs of disulfide bridges. By sequence alignment with the other toxins .We also investigated the inhibitory effect of KTX-Sp4 on Kv1.3 channels heterologously expressed in HEK293T cells. As expected, KTX-Sp4 reduced the peak amplitude of wild-type mKv1.3-mediated currents in a concentration-dependent manner, which reappeared the phenomenon in the Jurkat T cells. The steady-state current measured at the end of the depolarizing pulse was markedly decreased by KTX-Sp4 with an ICMammalian Kv1.1 and Kv1.2 are highly homologous to the Kv1.3 channel, which affects the selectivity of Kv1.3 channel blockers. Therefore, we also observed the effect of KTX-Sp4 peptides on Kv1.1 and Kv1.2 channels heterologously expressed in HEK293T cells. The addition of 1 μM KTX-Sp4 only reduced the maximum currents of Kv1.1 and Kv1.2 channel by about 20.85 and 7.23%, respectively, which indicated the good selectivity of KTX-Sp4 on Kv1.3 over Kv1.1 and Kv1.2. These electrophysiological results suggested that KTX-Sp4 could serve as a potential drug lead for selectively targeting Kv1.3 channel, thus playing a beneficial role in drug design for treating autoimmune diseases.50 of 12.07 ± 4.08 nM, which is very close to the IC50 of wild Kv1.3 channel. Electrophysiological experiments showed that similar to ADWX-1 [Among all mammalian ion channels, Kv1.1 has the highest homology to Kv1.3. The different residues in Kv1 turret region can well explain the selective blocking effect of some bioactive polypeptid on Kv1.3 over Kv1.1 . To furto ADWX-1 , the selScorpiops pococki has been far less than that from other scorpions such as Buthus martensii Karsch [KTX-Sp4 gene in the cDNA Library of Xizang Scorpiops pococki was screened, and then successfully expressed in prokaryotic expression system. Electrophysiological experiments showed that KTX-Sp4 peptides had a distinctively selective blocking effect on Kv1.3 channel—a new therapeutic target of autoimmune diseases.Venomous animals like scorpions , snakes i Karsch , 26. By 21 of KTX-Sp4, LmKTx8 has the same residue, while J123 has a positively charged residue Arg. In addition, corresponding to the Lys24 of KTX-Sp4, J123 has a negatively charged residue Asp. In selective blocking on Kv1.3 over Kv1.2, the KTX-Sp4 is superior to J123. As shown in Fig. 50 of 26.4 ± 9.3 nM. As shown in Fig. 35 and Thr36 of KTX-Sp4, which suggests that these three PNK residues might be the important structure for J123 to recognize Kv1.2 channel, resulting in low selective blocking of J123 on Kv1.3 over Kv1.2. The spatial structure analysis and amino acid residues mutagenesis would help to thoroughly elucidate the different function between KTX-Sp4 and J123, and then contribute to the molecular design of highly selective and highly active Kv1.3 peptide blockers.KTX-Sp4 was found identical with J123  and LmKTAmong all mammalian ion channels, Kv1.1 is the most homologous channel with Kv1.3. As a result, the lack of selectivity for Kv1.1 and Kv1.3 channels restricts the further development and application of many Kv1.3 channel blockers . SelectiWith selective inhibition on Kv1.3 channels, KTX-Sp4 peptide is a novel lead compound for the development of anti-autoimmune disease drugs. It can be known from this work that different structures between KTX-Sp4 and J123 led to different biological activities and Kv1turret region determined the selective regulation of KTX-Sp4 on Kv1.3 over Kv1.1, which enriches the molecular basis of the interaction between scorpion toxins and potassium channels, and also provides important theoretical basis for designing high selective Kv1.3 channel inhibitors.


BRCA1-associated protein 1 (BAP1) is a nuclear localizing UCH, having tumor suppressor activity and is widely involved in many crucial cellular processes. BAP1 has garnered attention for its links with cancer, however, the molecular mechanism in the regulation of cancer by BAP1 has not been established. Amongst the four UCHs, only BAP1 and UCHL5 are able to hydrolyze small and large ubiquitin adducts but UCHL5 hydrolyzes only when it is present in the PA700 complex of the proteasome. The ability of BAP1 to cleave large ubiquitin derivatives is because of its relatively longer active-site crossover loop than other UCHs. The mechanism of ubiquitin recognition has not been studied for BAP1. The comparative enzymatic analysis of ubiquitin C-terminal hydrolase L1 (UCHL1), ubiquitin C-terminal hydrolase L3 (UCHL3), ubiquitin C-terminal hydrolase L5 (UCHL5N), and BAP1N has confirmed that enzymatically BAP1 is similar to UCHL5, which corroborates with the bioinformatics analysis done earlier. We have undertaken extensive mutational approaches to gain mechanistic insight into BAP1–ubiquitin interaction. Based on the homology-modeled BAP1 structure, we have identified a few BAP1 residues which possibly play a crucial role in ubiquitin interaction of which a few mutations have been identified in many cancers. Our comparative thermodynamic analysis reveals that BAP1–ubiquitin interaction is majorly driven by entropy factor which is unique amongst UCHs. Our study sheds light on BAP1 interaction with ubiquitin, which will be useful in understanding its enzymatic function. BRCA1-associated protein 1 (BAP1) is a nuclear localizing deubiquitinating enzyme and a tumor suppressor protein, which was initially identified as an interactor of BRCA1 –5. BAP1 BAP1 is a cysteine protease with a conserved catalytic UCH domain sharing 44, 26, and 23% of sequence identity with ubiquitin C-terminal hydrolase L5 (UCHL5), ubiquitin C-terminal hydrolase L3 (UCHL3), and ubiquitin C-terminal hydrolase L1 (UCHL1), respectively. On the basis of sequence similarity, it is predicted that BAP1 has a UCHL5-like domain (ULD) at C-terminal that is potentially important in regulating its enzymatic activity by interacting with its binding partners. It is likely that ULD of BAP1 is structurally and functionally similar to that of UCHL5. Recent studies reveal that mechanism of H2A deubiquitination by BAP1–ASXL1 in PR–DUB complex is similar to UCHL5–RPN13 complex ,30 whereAmongst four UCHs, physiological substrates for UCHL1 and UCHL3 are not known but they can hydrolyze C-terminal peptide tail of small ubiquitin derivatives . They faCatalogue Of Somatic Mutations In Cancer (COSMIC) database. To gain a further mechanistic insight into UCHs function, the comparative thermodynamic analysis of ubiquitin binding to BAP1N and other UCH members has been performed. The thermodynamic studies of UCH members and ubiquitin disclose a differential nature of substrate interactions in UCH members.In the present work, we have shown how BAP1 interacts with the ubiquitin by studying the enzyme kinetics and thermodynamics of BAP1N–ubiquitin interaction. We have predicted a few residues in BAP1N that might play an important role in BAP1–ubiquitin binding based on the ubiquitin-binding interface of analogous UCHs. It is interesting to note that some of the putative ubiquitin interacting residues are found to be mutated in many cancers as observed in the Escherichia coli Rosetta 2 cells  and proteins were purified according to the standard GST purification protocol. Briefly, Rosetta 2 cells containing pGEX-6P-1-BAP1N plasmid DNA were grown to 0.6 OD and then induced with 1 mM/l of IPTG at 18°C for 16 h. Cells were harvested and the pellet was suspended in 1× PBS with 400 mM KCl buffer containing 10 μg/ml lysozyme. Homogenized solution was loaded onto a GST column  that was previously equilibrated with 1× PBS with 400 mM KCl. Protein was eluted in 50 mM Tris/HCl, pH 8.0, 500 mM NaCl, and 20 mM GSH. GST tag was removed by Pre-Scission protease . Eluted protein was dialyzed extensively with 1× PBS with 400 mM KCl. Protein was concentrated and loaded onto HiLoad 16/600 Superdex 75 column  for gel filtration in 50 mM Tris/HCl, pH 7.4, 150 mM NaCl, and 5 mM DTT buffer. Eluted fractions were concentrated and purity was verified by SDS/PAGE.Flag-HA tagged full-length BAP1, UCHL3, and UCHL5 were obtained from Addgene, U.S.A. UCHL3, UCHL5, and the catalytic domain of BAP1N (1–240) were subcloned into pGEX-6P-1 using standard cloning procedure. The pGEX-6P-1 UCHL1 plasmid was a gift from Dr. Chittaranjan Das, Purdue University, Department of Chemistry, U.S.A. Mutations in the catalytic domain of BAP1 were generated by site-directed mutagenesis using Quick-change Site Directed Mutagenesis Kit . Catalytic domain BAP1N and all cancer-associated mutants were expressed in kcat and Km values were determined from double-reciprocal plots assuming Michaelis–Menten model. The Km value reported is the average of at least three independent measurements.Multiple concentrations of Ub-aminomethylcoumarin (AMC) (100 nM–7.5 μM)  were mixed in reaction buffer  and constant concentration of BAP1N  was added to initiate the enzymatic reaction . Rate of AMC released was monitored at 25°C with a SpectramaxM5 plate reader  with excitation at 355 nm and emission at 455 nm. Initial velocity was calculated from the initial slope of each progress curve and The homology-modeled BAP1N structure was generated in SWISS-MODEL  using UCStock solution of BAP1N and catalytic domain mutants E7Q, S10A, E31Q, Y33A, E148Q, I214A, T218A, R227A, F228H, and L230A were diluted with reaction buffer containing 50 mM Tris/HCl, pH 7.6, 5 mM DTT, 0.5 mM EDTA, 0.1% of BSA in the individual wells of 96-well plate to a final concentration of 250 pM. Ub-AMC  was added to each well at a final concentration of 600 nM. Final reaction volume in each well was 100 μl. The rate of Ub-AMC hydrolysis was monitored at 25°C for 90 min using SpectramaxM5 plate reader . The excitation and emission wavelength used in this measurement were 355 and 455 nm, respectively. The amount of AMC released due to hydrolysis by BAP1 was quantitated using 7-amido-4-methylcoumarin as a standard (Sigma–Aldrich). Effect of salt concentration on catalysis was monitored by using NaCl salt concentrations ranging from 0 to 1.5 M in the reaction buffer along with Ub-AMC in a final concentration of 450 nM.KD) was calculated by steady state affinity binding model using Biacore T200 evaluation software (v. 1.0).Measurements of interactions using surface plasmon resonance (SPR) were carried out on a Biacore T200 . Ubiquitin in 10 mM sodium acetate with pH 4.5 was immobilized on to a dextran coated CM5 sensor chip  using a standard program, aiming for a density of 1250 response units (RU). Sensograms were collected at 25°C in 50 mM Tris at pH 7.4, 150 mM NaCl, and 0.5 mM DTT  at a flow rate of 30 μl/min, contact time of 120 s, and dissociation time of 600 s. Experiments were run in triplicate in the range of 0–50 μM for each BAP1 mutants dissolved in the same buffer. The binding affinity  experiments were performed using a Nano-ITC  at 25°C. Ubiquitin from bovine erythrocytes was purchased from Sigma–Aldrich. Amino acid sequences between human and bovine ubiquitin are identical. Samples of UCHL1 C90S, UCHL3 C95S, UCHL5 C88S, and BAP1N C91S, and ubiquitin were extensively dialyzed against 50 mM Tris/HCl, pH 7.6, and 50 mM NaCl buffer which was then degassed to remove dissolved air. Enzyme concentration of 10 μM was used for UCHL1 C90S and UCHL5 C95S and ubiquitin concentration used for both was 100 μM. For UCHL5 C88S and BAP1N C91S concentrations used were 250 and 120 μM, respectively, ubiquitin used in the present study was ten-fold higher concentration than the respective enzyme concentrations. Titrations consisted of 2.5 μl of 20 injections of ubiquitin into the sample cell containing proteins with 5-min time intervals to ensure that each peak was returned to baseline. The heat of dilution was subtracted from each data by performing ubiquitin buffer titration experiment. All data were analyzed by the program included with the system (TA Instruments). The data were fitted to one-site binding model (one molecule of ubiquitin binding to one molecule of UCHs). Estimates of −2·dmol−1). For determination of thermal melting point, wild-type and mutant BAP1N proteins were dissolved in 50 mM Tris/HCl, pH 7.4 to a final concentration of 7 μM. Tm for each protein was estimated from the unfolded protein fraction at 222 nm for varying temperature from 25 to 75°C maintained by the Peltier-type temperature controller.CD spectra for wild-type and different mutants were recorded in the wavelength region from 260 to 190 nm. Briefly, wild-type and mutant BAP1N proteins were diluted in 50 mM phosphate buffer with pH 7.4 to a final concentration of 7 μM, and the spectra were recorded using JASCO J815 CD spectrometer at 25°C in wavelength scan mode. Typically, data were obtained as an average of ten scans by using a quartz cuvette of 0.2-cm path. The protein CD spectra were reported as the mean residue ellipticity (deg·cmKm) for BAP1N was ~2.9 μM which was 3.6-fold higher than UCHL5N (1–240) [kcat) for BAP1N was calculated to be 2.1 s−1, which was ~4-fold and 2.8-fold lower than hydrolysis rate of UCHL5N and UCHL3, respectively. BAP1N exhibited ~72-fold higher turnover rate than UCHL1. The kcat/Km value defines the catalytic efficiency of an enzyme and the estimated efficiency of the BAP1N enzyme was 7.10 × 10−5 M−1s−1 that is comparable with UCHL5 and UCHL1. Interestingly, BAP1N exhibited 390-fold lower enzymatic efficiency than UCHL3, which suggests that BAP1 is not an efficient enzyme like UCHL3   Table 1Km for BA (1–240) . The turTill date, there is no structural information available for BAP1 full length or domain wise. Thus the 3D model structure of BAP1N 5–240) was generated based on the coordinates of UCHL5N structure with the help of bioinformatics tool SWISS-MODEL . Amongst was geneTsUCH37, a lower organism homolog of human UCHL5 is characterized from an infectious Trichinella spiralis, which shares 45% of its identity to that of human UCHL5 [TsUCH37 and tried to reciprocate on to UCHL5-based homology model of BAP1N (8–Thr9) of N-terminal β-hairpin of ubiquitin  have been reported ,44 FiguA,B. Crysan UCHL5 ,48 .On the basis of ubiquitin-bound UCHs structures, residues involved in interactions were mapped onto the BAP1 sequence, by sequence alignment. Approximately ten residues E7, S10, E31, Y33, E148, I214, T218, R117, R228, and L230 were predicted to play an important role in BAP1N–ubiquitin interaction  of BAP1N C91S for ubiquitin was 4 ± 1.6 µM (Supplementary Table S4). The ubiquitin affinity for E7Q and E148Q did not change significantly while catalytically inactive mutants S10A, E31Q, and Y33A showed 21-, 27-, and 13-fold decrease in ubiquitin binding. Rest of the mutants I214, R227A, F228H, and L230A showed 11-, 13-, 9-, and 6-fold lower binding affinity toward ubiquitin, respectively. Interestingly, T218A, which was catalytically as efficient as wild-type BAP1N, showed ~four-fold decreased affinity toward ubiquitin (Supplementary Table S4).Catalytic inactive BAP1N double mutants were made by site-directed mutagenesis to check if mutations contributed significantly to ubiquitin binding. Catalytic cysteine at position 91 was replaced by serine (C91S) in all the mutants to make it catalytically inactive. The secondary structure analysis of all double mutants was checked by CD spectra analysis. The double mutation did not perturb the overall structure of BAP1N mutants  making it an ideal system for binding analysis. Binding experiments were executed by SPR to measure the affinity of ubiquitin for BAP1N double mutants. The ubiquitin was immobilized on a dextran-coated CM5 chip and probed for BAP1N double mutants binding ability at pH 7.4. Time-dependent binding and dissociation of BAP1N mutants to ubiquitin at varied protein concentrations were shown in Kd) for BAP1N (C91S) and ubiquitin interaction was 3.2 µM at pH 7.4, which was consistent with Km value obtained from the enzymatic kinetics analysis and SPR binding experiment . Accordingly, higher concentrations of UCHL5 C88S (250 µM) and ubiquitin (2.5 mM) were used in the present ITC study. The binding parameters for UCHL5 and ubiquitin interaction were Kd ~10.1 µM, ΔH ~ –18.6 kJ/mol, ΔS ~33.2 J/mol·K, and ΔG ~ –28.3 kJ M−1. Thermodynamic parameters of UCHL3–ubiquitin interactions were found to be Kd value of ~0.01 µM, ΔH ~ –49.6 kJ/mol, ΔS ~ –14.9 J/mol·K, and ΔG ~ –45.3 kJ M−1 , UCHL5 (C88S), UCHL3 (C95S), and UCHL1 (C90S) were titrated with ubiquitin in ITC at 25°C . The thekcal/mol . Indeed,3 kJ M−1  which waKm and high kcat values amongst all the UCH members. However, UCHL1 is considered to be a poor enzyme despite its comparable Km with UCHL3 due to a misaligned active site and compromised kcat. The catalytic efficiency of UCHL5N is comparable with the UCHL1 mainly because of its high kcat contribution. BAP1N shows a very much similar catalytic efficiency (kcat/Km) to UCHL5. Similar catalytic efficiencies of both enzymes correlate well with a support of common ancestry for BAP1 and UCHL5 regulators in PR–DUB and INO80 chromatin remodeling complexes.In the present study, we have investigated the enzymatic properties of BAP1N and compared it with other UCH members. We have used the catalytic domain of BAP1 for our study as full-length BAP1 protein expression is very less compared with the catalytic domain and our earlier report has demonstrated that the truncation of a middle and C-terminal domain of BAP1 does not lower the catalytic activity rather BAP1N shows higher activity. It is hypothesized that the C-terminal ULD domain of BAP1 blocks catalytic cleft of BAP1 making it a less efficient enzyme . AccordiAn important finding of our work is the identification of residues of BAP1N playing a crucial role in ubiquitin interaction. Some of the mutants have shown a varying degree of catalytic activity impairment and a few mutants have shown complete loss of activity. The catalytic activity of all mutants has suggested that either these residues are crucial in ubiquitin interaction or they are essential for structural stability. We have reported previously that cancer-associated BAP1N mutants show reduced structure stability and increased aggregation propensity . The comTsUCH37 shows similar bilobal architecture where a major change is observed from E51 to D75 with the presence of loop instead of a helix (TsUCH37. A notable difference in side chain orientation was found for S10, Y33, F228, and R227 residues whereas I214 and T218 displayed spatial difference. No alteration was observed in confirmation of E31 and L230 residues compared with E32 and L218 of TsUCH37, respectively (The superposition of BAP1N homology model with the crystal structure of UCHL1, UCHL3, and  a helix A. Amongsectively B,C.The thermodynamic analysis reveals that BAP1–ubiquitin interaction is entropically driven. Entropy-driven interactions are majorly associated with hydrophobic interactions and a large amount of static water displacement upon ubiquitin binding. Large structural rearrangement upon ubiquitin binding may contribute to increased entropy. An earlier report has suggested that ASXL1 binding to BAP1 decreases the entropy significantly  and stabOur comprehensive biochemical analysis of ubiquitin binding to UCHs provides a framework for future work on the possible role of divergence in ubiquitin interaction despite having conserved sequences in these UCHs. Clearly, the present study offers an opportunity to test this model and proposed sites for BAP1–ubiquitin interaction analysis. The molecular insight into BAP1–ubiquitin complex will assist researchers to design a potent inhibitor of this enzyme.


The aims of this study were to detect HPV E6/E7 mRNA expression in women with high-risk genotypes  analysing its relationship with tissue pathology and 2) 2-year follow-up of E6/E7 mRNA tested group.Our samples were genotyped and classified by pathologists according to Bethesda system. After RNA extraction, E6/E7 oncogene mRNA detection was performed by NucliSens® EasyQ® HPV v1 Test (bioMérieux).The results of the present study showed that E6/E7 mRNA positivity rate was 68.29 % in women tested once and 69.56 % in women tested twice. According to tissue pathology, all samples with high-grade lesions were positive for mRNA. Among women with low-grade lesions varied over the years from 89.28 to 84 % in women tested once and from 77.77 to 70 % in tested twice. Among women without lesion, positivity rate maintained in women tested once (from 50 to 41.38 %) and decreased in tested twice, from 63.63 to 44.44 %. Regarding lesion evolution, mRNA positivity was higher in women with lesion progression (53.13 %) and in women with positive results in two tested samples (83.33 %).HPV E6/E7 mRNA detection may be an effective screening test and biomarker for cervical cancer in women infected with these five genotypes. Nonetheless, further studies are needed to standardize as routine triage test. Human papillomavirus (HPV) infection is responsible of cervical cancer, which is the second most frequent cancer affecting woman worldwide –3.More than 100 genotypes have been described; 12 of them are classified as high-risk genotypes (HR-HPV) due to their high oncogenic potential . Among tHPV viral oncoprotein expression differs among infection state  , 11. HPVTo date, cervical cancer screening is based on cytology and this has been a powerful tool to reduce the incidence and mortality of this type of cancer , 15, butThe present study was designed to 1) study HPV E6/E7 mRNA expression and analyse its relationship with tissue pathology in order to test mRNA as an effective biomarker for cervical cancer and 2) 2-year follow-up of a group of women and test E6/E7 mRNA expression.Samples from women which were referred from Consultation of Sexually Transmitted Diseases and Gynaecological consultation at Basurto University Hospital  were analyzed due to possible HPV infection during the last 7 years (2007–2014). All patients gave written and informed consent prior to their inclusion in the study.The samples were categorized by pathologists according to Bethesda system: 1) negative (no lesion was found) 2) Atypical squamous cells of undetermined significance (ASCUS) 3) Low-grade squamous intraepithelial lesion (LSIL) and 4) High-grade squamous intraepithelial lesion (HSIL).Samples were analyzed with Cobas® HPV Test  that detects 14 high-risk HPV DNA genotypes, 16 and 18 separately and other 12 high-risk HPV genotypes altogether. Samples that were positive for other HR were genotyped with Linear Array HPV Genotyping Test kit (Roche Molecular Diagnostics), a line-blot assay that detects 37 genotypes .RNA was extracted from 200 μl of samples by NucliSENS Lysis Buffer and NucliSENS® miniMAG®  according to manufacturer’s instructions.E6/E7 oncogene mRNA was detected by NucliSens® EasyQ® HPV v1 Test (bioMérieux) following manufacturer’s instructions. This test detects the E6/E7 oncogene expression of five HR-HPV genotypes . The quality of extracted RNA was monitored by an internal control: human U1 small nuclear ribonucleoprotein specific protein A (U1A). If neither U1A nor mRNA were detected, the test result was analyzed as invalid. Invalid runs were repeated once again.In the present study, E6/E7 mRNA expression was analyzed in a total of 128 samples (corresponding to 105 women). Eighty-two women’s samples were analyzed once and in 23 women mRNA expression was studied twice . All these women were positive for HPV DNA (DNA positivity rate was 100 %).HPV-16 was the most prevalent genotype , followed by 18 , 45 , 31  and 33 . Nevertheless, regarding oncogene expression, HPV-33 was the most expressed , followed by 18 , 16 , 31  and 45 .Eighty-two women samples were tested for E6/E7 mRNA oncogene expression only once. The mean age was 33.62 ± 10.57 years (range 17–63). Most of them were infected with multiple HPV infection genotypes (54/82) whereas 34.15 % of them had single HPV genotype infection.More frequently detected genotype was 16 (67.01 %) followed by 18, 45, 31 and 33 .The E6/E7 mRNA positivity rate was 68.29 % (56/82). The mean age of E6/E7 positive and negative women was similar (33.53 ± 11.12 vs 33.8 ± 9.62 years). HPV 16 E6/E7 mRNA was detected in 71.42 % of positive samples (40/56), 18 in 10.71 % (6/56), 33 in 3.57 % (2/56), 16 plus 33 multiple infection in 3.57 % (2/56), and the following genotypes were only detected in 1.78 % of positive samples (1/56) .The E6/E7 mRNA results were analyzed regarding their cytology status. There were 77 women with cytology and E6/E7 mRNA test. Pathogenicity was divided into three groups: 1) normal or negative (no lesion was found), 2) ASCUS and LSIL and 3) HSIL. Among E6/E7 mRNA positive samples, 47.89 % belonged to women with ASCUS or LSIL . HPV single infection was detected in 52.17 % (12/23) and multiple HPV infection in 47.83 % (11/23). The most detected genotype was 16 (73.47 %) followed by 18 (14.29 %) and 31, 33 and 45 genotypes (4.08 % of incidence each one).The E6/E7 mRNA positivity rate was 69.56 % (32/46) and the oncogene expression was mostly detected in HPV-16 in 68.75 % of samples (22/32), followed by 18 in 15.63 % (5/32), 16 plus 31 multiple infection in 6.26 % (2/32), and the followings genotypes in 3.12 % (1/32): 33; 16 plus 18 and 16, 31 plus 33.According to E6/E7 mRNA positivity rate, 55.56 % had some type of lesion (38.89 % ASCUS or LSIL and 16.67 % HSIL) in samples taken first   or had a previous negative result in mRNA but then mRNA was detected (16.67 %) Fig. . OtherwiSingle HPV infection was detected in 40 of 105 analyzed women and 65 women were infected with multiple HPV infection. Among women with single HPV infection E6/E7 mRNA positivity rate was 67.50 % whereas that rate was 70.77 % in women with multiple HPV infection. The 60 % of women with single HPV infection had normal cytology results whereas the 40 % showed some lesion (high grade or low grade lesion). On the other hand, the 58.33 % of women with multiple HPV infection had some lesion and only the 41.66 % has normal cytology results. Among women with multiple HPV infection and positive for E6/E7 mRNA the 19.56 % were expressing more than one genotype oncogenes.Human papillomavirus is closely related with cervical cancer. HPV infections usually show a regression in the next 2 years, but few patients infected with HR-HPV genotypes are at risk of developing cervical cancer , 22. DurDNA positivity rate was less valuable in clinical use than mRNA as it has been observed in previous studies , 23, 24.Among mRNA positive women HPV 16 single infection transcript was the most recurrent, though, the main majority of women tested had multiple HPV infection. HPV 16 tended to express more than other genotypes, which may be connected with the fact that it is considered the most carcinogenic genotype . NeverthIt was proved that E6/E7 mRNA positivity rate was higher in women with high-grade lesion than in samples with low-grade lesion or without lesion , 19, 26.According to E6/E7 mRNA as cervical disease biomarker, mRNA positivity increased over time in specimens with lesions in women tested once and in tested twice, namely, E6/E7 mRNA positive samples showed a malignancy progress during the next years. These results were expected since E6 and E7 oncogene are over expressed in precancerous lesions and it has been proved how these oncogenes and cervical lesion severity increases at the same time . TherefoNevertheless, in women who were tested twice, there was no difference in mRNA negativity between samples tested first and specimens tested one or two years later, so it seemed that there were a significant number of specimens that had minor cervical lesions but were negative for E6/E7 mRNA. This event could be related with the fact that low-grade cervical lesions normally disappears in a few months without treatment . Even soFinally, E6/E7 mRNA could be a good prognosis marker since it was observed that women who were positive for E6/E7 mRNA had more probabilities to develop lesion progression. Besides, women in which samples mRNA was negative had more possibilities to remain the same pathological grade or have infection clearance. These findings are in concordance with previous studies where E6/E7 mRNA was considered a short-term prognostic factor for high-grade lesions . NeverthE6 and E7 oncogene mRNA detection seems to be a useful instrument as a triage test in women infected with HPV-16, -18, -31, -33 or -45 genotypes. Moreover, it could be a promising biomarker since it has been proved its relationship with lesion grade, making possible the reduction of HPV positive women referred to colposcopy. Even so, further studies are needed to use NucliSens® EasyQ® HPV v1 Test as standard screening method in HPV positive women.


The high cost of enzymes is one of the key technical barriers that must be overcome to realize the economical production of biofuels and biomaterials from biomass. Supplementation of enzyme cocktails with lytic polysaccharide monooxygenase (LPMO) can increase the efficiency of these cellulase mixtures for biomass conversion. The previous studies have revealed that LPMOs cleave polysaccharide chains by oxidization of the C1 and/or C4 carbons of the monomeric units. However, how LPMOs enhance enzymatic degradation of lignocellulose is still poorly understood.TrAA9A, formerly known as TrCel61A) from Trichoderma reesei] and a cellobiohydrolase I (TlCel7A from T. longibrachiatum) with bacterial microcrystalline cellulose (BMCC) as a substrate. Cellulose conversion by TlCel7A alone was enhanced from 46 to 54% by the addition of TrAA9A. Conversion by a mixture of TlCel7A, endoglucanase, and β-glucosidase was increased from 79 to 87% using pretreated BMCC with TrAA9A for 72 h. AFM imaging demonstrated that individual TrAA9A molecules exhibited intermittent random movement along, across, and penetrating into the ribbon-like microfibril structure of BMCC, which was concomitant with the release of a small amount of oxidized sugars and the splitting of large cellulose ribbons into fibrils with smaller diameters. The dividing effect of the cellulose microfibril occurred more rapidly when TrAA9A and TlCel7A were added together compared to TrAA9A alone; TlCel7A alone caused no separation.In this study, we combined enzymatic assays and real-time imaging using atomic force microscopy (AFM) to study the molecular interactions of an LPMO [TrAA9A increases the accessible surface area of BMCC by separating large cellulose ribbons, and thereby enhances cellulose hydrolysis yield. By providing the first direct observation of LPMO action on a cellulosic substrate, this study sheds new light on the mechanisms by which LPMO enhances biomass conversion.The online version of this article (10.1186/s13068-018-1023-1) contains supplementary material, which is available to authorized users. Non-food plant biomass is a sustainable source of fermentable sugars for the production of biofuels and chemicals –3. CelluThe most widespread method of cellulose conversion requires three types of cellulases to act synergistically to convert cellulose to glucose: endo-β-1,4-glucanases (EGs) cleave the internal bonds in the cellulose chain, cellobiohydrolases (CBHs) processively hydrolyze cellulose from the chain ends to produce cellobiose, and β-glucosidases (BGs) hydrolyze cellobiose to glucose . Lytic pin situ interactions of an LPMO  and a CBH I (TlCel7A), alone and together, with bacterial microcrystalline cellulose (BMCC). Our goal was to visualize in real time the molecular motion of LPMO and its effects on the structure of the cellulose surface, to deepen our understanding of LPMO's role in cellulose hydrolysis.Atomic force microscopy (AFM), especially high-speed AFM, can image with sub-nanometer resolution under aqueous conditions at video rate. AFM has been used to visualize the interactions between enzymes and substrates in real time . AFM hasTrAA9A and TlCel7A converted 8% more cellulose  than TlCel7A alone  to analyze the soluble products after 72 h incubation of TrAA9A with BMCC, and detected small amounts of oxidized forms of cellobiose and cellotriose  , 22. To TrAA9A interaction with BMCC under AFM, a small piece of BMCC was transferred onto a glass slide pre-coated with poly-lysine. As the negative control, BMCC was imaged under the same buffer used for the enzyme reactions. We observed primarily large ribbon-like cellulose structures of 30–150-nm width, with a relatively smooth surface. About 10 min after adding TrAA9A, we observed particles of 6–10 nm in diameter accumulating on the surface of the BMCC ribbon  in three patterns: in both directions along one cellulose ribbon, across one ribbon, or moving from one ribbon to another  [TrAA9A [TrAA9A molecules seemed to form into groups that moved together towards a particular cellulose ribbon where eventually many molecules accumulated  to 89.8 ± 16.7 nm (n = 16), but the surface roughness increased greatly, with obvious grooves and edges observed on the surface  in width, which was approximately 52% of their original ribbon width. Similar structural change was also observed recently [TrAA9A to penetrate inside the cellulose ribbon leads to the dividing of a large cellulose ribbon into multiple smaller microfibrils. Considering that a BMCC ribbon is composed of multiple cellulose elementary fibrils (CEF) that can be as small as 2–4 nm [TrAA9A oxidation disrupts the glucan chains between these CEFs. We further studied the overall BMCC structure changes by incubating TlCel7A with and without TrAA9A. After 72-h incubation with both enzymes, BMCC was broken into small pieces. In contrast, treatment with TlCel7A alone resulted in softening of BMCC floating on the top of the reaction buffer. There was no significant structural change of BMCC viewed by eye after TrAA9A treatment alone .Gluconacetobacter xylinus  strain ATCC 53524 from the American-Type Culture Collection  was maintained on Hestrin and Schramm (HS) medium containing 1.5% agar. HS medium contains 20-g/L glucose, 5-g/L peptone, 5-g/L yeast extract, 2.7-g/L Na2HPO4, and 1.15-g/L citric acid monohydrate. The pH was adjusted to 6.0 with NaOH or HCl.TlCel7A from Trichoderma longibrachiatum  was purchased from Megazyme, Ltd., Bray, Ireland. TrAA9A (GenBank CAA71999), endo-β-1,4-glucanase  and β-glucosidase  from T. reesei were expressed in Pichia pastoris as previously described [escribed . PronaseG. xylinus colonies and incubated at 30°C for 1–3 days in static until cellulose pellicle was visible. The culture was vigorously shaken for 30 min to release active cells embedded in the pellicle. Then, 450-mL fresh HS medium in a plastic tray (18 × 6 × 2.5 in.) was inoculated with the culture broth and incubated in stationary for another 3–5 days until the cellulose pellicle was grown on the entire medium surface.The bacterial microcrystalline cellulose (BMCC) was produced based on the previous study with modifications , 35. HS 2O) to rinse off the medium as much as possible. Washed cellulose was boiled in 1% NaOH with stirring for 30 min to remove remaining bacteria. Finally, alkali-treated cellulose was washed several times with ddH2O until the water pH reached 7.0, freeze-dried, and cut to 1-cm strip for enzymatic degradation. Native BMCC without alkali treatment was stored in 0.02% sodium azide at 4 °C for AFM imaging.The cellulose pellicle was collected by filtering through 2-layer Miracloth. Then, the pellicle was washed repeatedly with distilled deionized water  with deionized water. The samples were kept in the water for all of the imaging and measurement process.TrAA9A penetration experiment, we lowered the scan rate to 2 Hz to obtain images with better resolution. For static observations, the scan rate was 1 Hz with the resolution of 1024 × 1024 pixels.All the AFM experiments were conducted at room temperature on a Dimension AFM with Nanoscope controller V  with an acoustic and vibration isolation system. Probes we used were SCANASYST-FLUID+  for imaging under fluid. The AFM operation software (Nanoscope V9.1) was used to control the scan size, setpoint, and gain. Before AFM imaging, the scanner has been carefully calibrated using calibration kit  to make sure that all the measurements are very close to their actual value. To dynamically capture the movement of enzyme molecules, the scan rate was normally set as 10–20 Hz for the continuous observation with low resolution (256 × 128 pixels or 128 × 64 pixels). However, when we conducted the imaging on single All off-line data analysis was based on the Nanoscope Analysis v1.8 software . The height and peak force error images were analyzed using plane fit filter at one order for images presented in all figures. The color bar was manually modified according to the best presence of each image. The images used for height and width measurement were raw data without any processing and all width and height measurements were conducted with the “section” function in the Nanoscope Analysis software.TrAA9A, 1-mM L-ascorbic acid, and 0.02% sodium azide for 72 h in 50-mM pH 4.8 sodium acetate buffer. All reactions in triplicate were conducted at 150 rpm, 50 °C in shaking incubator. After 72-h incubation, BMCC residue was recovered by filtering through two layers of Miracloth . Filtrates from all reactions were saved for TrAA9A product analysis. Then, TrAA9A remained on cellulose residue was removed according to the previous study with modifications [2O. Then, it was incubated with 10-mg/mL Pronase E in 50-mM pH 7.5 Tris buffer overnight at 37 °C, 100 rpm for complete proteolysis of remaining TrAA9A. Next, BMCC residue was collected by filtering through two layers of Miracloth, washed with ddH2O, 1 M NaCl, and ddH2O again to remove Pronase E. Finally, the BMCC residue was freeze-dried and stored at 4 °C.2-mg/mL BMCC was incubated with 100-μg/mL ications . The resTrAA9A was centrifuged in 70% ethanol for 20 min at 4 °C to precipitate TrAA9A and supernatant was collected. Filtrate of BMCC incubated under the same condition without TrAA9A was also prepared in the same way for mass spectrometry analysis. Samples were analyzed on a Waters Xevo G2-XS Q-TOF system coupled to a Waters I-Class UPLC system. Carbohydrates were separated by an ACQUITY UPLC BEH Amide column  maintained at 40 °C, with the injection volume at 10 μL. Solvent A was 10-mM ammonium formate, and solvent B was 100% acetonitrile. The solutes were eluted at 0.2 mL/min starting at 95% B, followed by a linear gradient to 35% B over 14 min. The proportion of solvent B was maintained at 35% for 2 min and then increased back to 95% and kept for 4 min for re-equilibration.Filtrate obtained after 72-h reaction with The operation condition for mass spectrometer was capillary voltage of 3.0 kV, sample cone voltage of 80 V, source temperature of 100 °C, desolvation temperature of 350 °C, and desolvation gas flow of 600 L/h. Mass spectra were acquired in positive ion mode across the 50–2000-m/z range. Data processing was performed using the MassLynx software .TrAA9A and TlCel7A on cellulose degradation, 2-mg/mL BMCC was incubated with 34-μg/mL TlCel7A and 6-μg/mL TrAA9A, alone or together, in 10-mL reactions containing 1-mM L-ascorbic acid and 0.02% sodium azide. Reactions were conducted in triplicate in 50-mM sodium acetate, pH 4.8, for 72 h at 150 rpm, 50 °C. To detect cellulose conversion rate, 100-μL supernatant was incubated with 40-μg/mL β-glucosidase in 50-mM pH 4.8 sodium acetate buffer for 30 min at 50 °C. Free glucose was measured by enzyme-linked colorimetry as described [To test the synergism between escribed .TrAA9A-treated cellulose, TrAA9A-treated BMCC was compared against untreated BMCC for enzymatic hydrolysis at pH 4.8. For all reactions, cellulose loading was 2 mg/mL and cellulase mixture loading 100 μg/mL, which contained TlCel7A, EG, and BG with 6:3:1 ratio. Each reaction in triplicate was carried at 150 rpm, 50 °C in 0.02% sodium azide. Glucose yield was measured during 72-h incubation.To test the digestibility of Additional file 1: Figure S1. ESI-MS analysis of oxidized cellobiose and cellotriose released from BMCC by TrAA9A. BMCC (2 mg/mL) was incubated with 100 μg/mL TrAA9A and 1 mM L-ascorbic acid in 50 mM sodium acetate, pH 4.8, for 72 h at 50 °C and 100 rpm. The reactions were conducted in triplicate. As the negative control, BMCC alone was incubated for 72 h under the same condition. (a) Small amount of cellobiose was detected in the negative control. (b), (c), and (d) Cellobiose (DP2), cellotriose (DP3), and two oxidation products derived from DP2 and DP3 were detected in all replicates of TrAA9A-containing samples. Lytic polysaccharide monooxygenase (LPMO) degrades cellulose to cello-oligosaccharides and oxidized cello-oligomers. Three types of LPMOs were described based on their oxidation products. Type 1 enzymes oxidize the C1 carbon of the glucose unit and produce aldonolactone and its hydrated product aldonic acid [TrAA9A were analyzed by mass spectrometry. After 72 h incubation, BMCC alone produced a small amount of cellobiose (DP2) but no other cellodextrin or oxidized sugar . In contrast, TrAA9A generated cellobiose (DP2), cellotriose (DP3), and two types of oxidation products derived from DP2 and DP3 after 72 h reaction . Cellodextrin and oxidized sugar products were all associated with Na+ in mass spectrum probably because the reactions were conducted in sodium acetate buffer [TrAA9A-containing reactions. Therefore, cellobiose is probably one of the products of TrAA9A. In contrast to many studies, cello-oligomer and oxidized cello-oligomer of higher degree of polymerization (DP) were not detected after 72 h TrAA9A reaction. One possible explanation is that TrAA9A might also be active on cello-oligosaccharide [TrAA9A activity [nic acid , 37, 38.nic acid , 39. Typnic acid –43. C1 oe buffer . Althougccharide  and thatactivity .Additional file 2: Video S1. AFM (Height) of TrAA9A molecules moved and diffused across on BMCC cellulose ribbons for approximately 10 min. The images were recorded at 128 x 64 pixels, and 17-Hz scan rate. The height scale was fixed at 100 nm. The movie is played back at ~650 times of actual speed.Additional file 3: Video S2. AFM (Height) of TrAA9A molecules moved and diffused along on BMCC cellulose ribbons for approximately 10 min. The images were recorded at 128 x 42 pixels, and 17-Hz scan rate. The height scale was fixed at 35.5 nm. The movie is played back at ~650 times of actual speed.Additional file 4: Video S3. AFM (Peak force error) of TrAA9A molecules moved and jumped between BMCC cellulose ribbons for approximately 6 hours. The images were recorded at 256 x 256 pixels, and 1.5-Hz scan rate. The height scale was fixed at 1.3 nN. The movie is played back at ~1800 times of actual speed.Additional file 5: Figure S2.TrAA9A penetrating and moving inside BMCC ribbon. Time-lapse images showing TrAA9A (indicated by the cyan arrows) moving in and out of the surface of a cellulose ribbon during 210 min incubation. Scale bar is 100 nm and color bar 75 nm.Additional file 6: Video S4. AFM (Peak force error) of TrAA9A molecules penetrated and moved inside BMCC cellulose ribbons for approximately 3 hours. The images were recorded at 256 x 256 pixels, and 1.5-Hz scan rate. The height scale was fixed at 0.8 nN. The movie is played back at ~1800 times of actual speed.Additional file 7: Figure S3. Changes in morphology of BMCC ribbon when incubated with TrAA9A (indicated by cyan arrows) during 2 h continuous AFM observation. Pictures were taken from Video S5. Scale bar is 50 nm. Color bar is 50 nm and 830 pN in height (left) and peak force error (right) channels, respectively.Additional file 8: Video S5. AFM (Height) of TrAA9A molecules moved and caused morphology change of BMCC cellulose ribbon for approximately 2 h. The images were recorded at 128 x 64 pixels, and 17-Hz scan rate. The height scale was fixed at 50 nm. The movie is played back at ~650 times of actual speed.Additional file 9: Figure S4. Morphology change of BMCC after 72 h incubation with or without enzymes. Reaction conditions were the same as Fig. 1. BMCC was cut to 1-cm strip for enzymatic digestion. Cellulose loading was 2 mg/mL, TlCel7A 34 μg/mL, and TrAA9A 6 μg/mL. Reactions were conducted in triplicates in 50 mM, pH 4.8, sodium acetate buffer at 150 rpm and 50°C. No obvious morphological change of cellulose strip after TrAA9A treatment was observed. In contrast, after 72 h incubation with TlCel7A, the length of the strip shortened to approximately half of its original length with almost 50% cellulose degradation .Additional file 10: Video S6. AFM (Height) of morphological changes of cellulose ribbon during incubation with TrAA9A and TlCel7A for approximately 6 hours. The images were recorded at 256 x 256 pixels, and 10-Hz scan rate. The height scale was fixed at 100 nm. The movie is played back at ~1000 times of actual speed.Additional file 11: Figure S5. Comparison of cellulase hydrolysis of untreated and TrAA9A-treated BMCC. Cellulose loading was 2 mg/mL and total cellulase loading was 100 μg/mL with TlCel7A: EG: BG ratio of 6:3:1. The reactions in triplicate were conducted in 50 mM, pH 4.8, sodium acetate buffer at 150 rpm, 50°C for 72 h.