Most severe cases occur among patients secondarily infected with a different dengue virus (DENV) serotype compared with that from the first infection, resulting in antibody-dependent enhancement activity (ADE). Our previous study generated the neutralizing human monoclonal antibody, D23-1B3B9 (B3B9), targeting the first domain II of E protein, which showed strong neutralizing activity (NT) against all four DENV serotypes. However, at sub-neutralizing concentrations, it showed ADE activity In this study, we constructed a new expression plasmid using the existing IgG heavy chain plasmid as a template for Fc modification at position N297Q by site-directed mutagenesis. The resulting plasmid was then co-transfected with a light chain plasmid to produce full recombinant IgG (rIgG) in mammalian cells (N297Q-B3B9). This rIgG was characterized for neutralizing and enhancing activity by using different FcγR bearing cells. To produce sufficient quantities of B3B9 rIgG for further characterization, CHO-K1 cells stably secreting N297Q-B3B9 rIgG were then established.The generated N297Q-B3B9 rIgG which targets the conserved N-terminal fusion loop of DENV envelope protein showed the same cross-neutralizing activity to all four DENV serotypes as those of wild type rIgG. In both FcγRI- and RII-bearing THP-1 cells and FcγRII-bearing K562 cells, N297Q-B3B9 rIgG lacked ADE activity against all DENV serotypes at sub-neutralizing concentrations. Fortunately, the N297Q-B3B9 rIgG secreted from stable cells showed the same patterns of NT and ADE activities as those of the N297Q-B3B9 rIgG obtained from transient expression against DENV2. Thus, the CHO-K1 stably expressing N297Q-B3B9 HuMAb can be developed as high producer stable cells and used to produce sufficient amounts of antibody for further characterization as a promising dengue therapeutic candidate.in vitro. This HuMAb could be further developed as therapeutic candidates.Human monoclonal antibody, targeted to fusion loop of envelope domainII (EDII), was generated and showed cross-neutralizing activity to 4 serotypes of DENV, but did not cause any viral enhancement activity  Flavivirus genus in the Flaviviridae family. There are four antigenically different serotypes of DENV (DENV-1 to DENV-4) (Dengue infection is a leading cause of illness and death in the tropics and subtropics. More than one-third of the world’s population lives in areas at risk . DENV is DENV-4) . Primary DENV-4) . Upon se DENV-4) . Primary DENV-4) .Currently, there is no available antiviral drug specific for all four DENV serotypes. Even though there are many studies of neutralizing antibodies against DENV, fully human monoclonal antibodies (HuMAbs) which are able to neutralize all four DENV serotypes without ADE activity are considered to be the main option for passive immune therapy .in vitro neutralizing activity (NT) against DENV-1 to DENV-4  with strong o DENV-4 . Howevero DENV-4 . To dimio DENV-4 .in vitro and compared with those of wildtype rIgG. We also demonstrate the mimic binding epitope on envelope protein of dengue virus for understanding specificity and functionality of this HuMAb.We constructed plasmids expressing antibody light chain (LC) and modified the Fc region of the heavy chain (HC) constant domain 2 at position N297Q by site-directed mutagenesis. The modified HC plasmid was co-transfected with the LC plasmid into HEK293T mammalian cells to produce full recombinant IgG (rIgG). To evaluate the Fc-modified rIgG as a potential therapeutic candidate for dengue treatment, NT and ADE activity were determined HEK293T cells were maintained in Dulbecco’s modified Eagle medium  with 10% fetal bovine serum (FBS). For the NT test, Vero cells were cultured in minimal essential medium (MEM)  with 10% fetal bovine serum. THP-1 and K562 cells, which were used in ADE assays, were cultured in RPMI 1640 medium (Gibco) with 10% FBS. The DENV strains used in this study were the Mochizuki strain of DENV1, the 16,681 strain of DENV2, the H87 strain of DENV3, and the H241 strain of DENV4. All DENVs were propagated in C6/36 cells, which were maintained in Leibovitz’s L-15 medium (Gibco) supplemented with 10% FBS and 0.3% of BACTO Tryptose Phosphate Broth (TPB) . CHO-K1 cells were maintained in MEM medium supplemented with 10% FBS and 1% non-essential amino acid (Gibco). All cell lines were kindly provided by Research Institute for Microbial Diseases, Osaka University.® HD Cloning Plus; Clontech Laboratories Inc., Shiga, Japan). Primers were designed according to the manufacturer’s instructions to mutate the amino acid at position 297 from asparagine to glutamine. This system combines the action of the In-Fusion HD enzyme with inverse polymerase chain reaction (PCR), which generates linearized DNA from a plasmid template. The PCR reaction was composed of the CloneAmp Hifi PCR premix, 300 nM each of forward and reverse primer, 5 ng of plasmid, and distilled water to final volume of 25 µl. The amplification was performed with 35 cycles of 98 °C for 10 s, 55 °C for 15 s, and 72 °C for 5 s. The inverse PCR products were gel-purified using a PureLink® Quick Gel Extraction Kit  following the manufacturer’s protocol. The linearized plasmids obtained from inverse PCR were then fused by the In-Fusion enzyme, by following the manufacturer’s instructions. The reaction was performed at 50 °C for 15 min. The in-fusion reaction was chemically transformed into Stella chemical competent cells. Individual clones were randomly selected for DNA sequencing.Variable HC and LC sequences of B3B9 HuMAb were previously isolated from hybridoma cells and used for construction of HC and LC expression plasmids producing B3B9 rIgG . This HCE. coli and isolated using a Purelink™ plasmid Midiprep kit (Invitrogen) from 100 ml culture for further transfection in mammalian cells.The DNA sequencing of the mutated HC plasmids were confirmed by Sanger sequencing . Potential N-glycosylation site of wild type and mutated B3B9 HuMAb was identified using NetNGlyc 1.0 software . PlasmidPlasmids expressing N297Q-B3B9 HC and LC were transfected into HEK293T cells to produce whole rIgG with the N297Q mutation as previously described . The culIFAs were used to determine the binding and specificity of N297Q-B3B9 rIgG for all four DENV serotypes. IFA plates were prepared by infecting Vero cells with DENV at a multiplicity of infection of 0.1 as well as mocked infected. The plates were incubated for 3 days before being fixed and permeabilized with 3.7% formaldehyde and 0.1% Triton X-100, respectively. Culture fluid from transfected cells was added, and the plates were incubated at 37 °C for 1 h. AlexaFluor 488-conjugated anti-human IgG  (Invitrogen) was added as secondary antibody. The result was observed under fluorescence microscope . For this assay, non-infected cells were used as a negative control.The IgG isotype of the N297Q-B3B9 rIgG was determined by PCR as previously described  using coH + L) . Foci numbers were counted under a fluorescence microscope. The percent reduction was calculated by comparing the foci number for each antibody concentration with the number of foci obtained from a virus–PBS mixture (negative control).FRNT assay was performed as previously described . DENV at6 cells/ml K562 cells were added. The cell–HuMAb–virus mixture was co-cultured at 37 °C under 5% CO2 for 2 days. The cells were fixed with an acetone/methanol fixing solution at −20 °C. Immunostaining was performed by adding anti-DENV human antibody and incubating at 4 °C overnight. Horseradish peroxidase-conjugated anti-human IgG ( H + L) diluted in 0.05% Tween and 1% FBS in PBS was then added, and the samples were incubated at 37 °C for 1 h. The signal was developed with a DAB substrate solution . Infected cell counts obtained from the test were adjusted with the mean infected cell counts obtained with the four negative controls set in the same experiment. The cut-off value for differentiating enhancing from non-enhancing activities was calculated from the average plus three times the SD of the percentages of infected cells obtained with the four negative controls.The ADE activity of the N297Q-B3B9 rIgG was also assessed on FcγRIIa-bearing K562 cells . Antibod5 cells/ml THP-1 cells, which express both FcγRI and II on their surfaces, were added and the samples were incubated at 37 °C under 5% CO2. After 2 h, RPMI medium with 4% FBS was added. The cell–HuMAb–DENV solution was incubated for 3 days, after which RNA was extracted from the infected cells using TRIzol® reagent (Invitrogen). The viral RNA was quantified by a one-step Realtime PCR using dengue virus specific primer and glyceraldehyde 3-phosphate dehydrogenase (GADPH) as an internal control ) were incubated at room temperature for 30 min with purified B3B9 HuMAb to a final volume of 200 µl TBST. Phage—HuMAb mixture was transferred to the tube containing the blocked magnetic beads and incubated for 20 min at room temperature. After incubation, nonbinding phages that were not captured on magnetic beads were washed with TBST. The bound phages were eluted from magnetic beads with 1 ml glycine elution buffer  and neutralized with 150 µl of 1M Tris–HCl (pH 9.1). Then, the bound phages were directly added to 20 ml early-log Escherichia coli (E. coli) ER2738 culture and incubated at 37 °C with vigorous shaking for 4.5 h. After incubation, the culture was transferred to a 50 ml centrifuge tube and spun at 5,000 rpm for 15 min at 4 °C. The phage-containing supernatant was transferred to a new 50 ml centrifuge tube. These amplified phages were precipitated with 20% PEG 8000/2.5 M NaCl at 4 °C overnight, and centrifuged at 12,000 g for 15 min. Phage pellets were then resuspended with 200 µl TBS and used in the next cycle. Three rounds of selection were performed. Specificity of selected phage clones were confirmed by phage ELISA. Briefly, 96-well microtiter plates were coated in triplicate well of 100 µl B3B9 HuMAb at 4 °C overnight. Then, the wells were washed two times with 0.05% Phosphate Buffered Saline with Tween (PBST). After that, the wells were blocked with blocking buffer (3% BSA in TBST). Then, phage lysate of each selected clones from panning step was added to two wells of coated antibody. At this step, wild type M13 phages were added to another HuMAb-coated well as a no fusion peptide control. Consequently, anti-M13 antibody-HRP conjugated; dilute 1:5,000 in blocking buffer were added. All incubations were performed in humidity chamber at 37 °C, for 1 h. After washing, the reaction was developed using 3, 3′, 5, 5′-Tetramethylbenzidine (TMB) substrate (Sigma-Aldrich), and terminated by adding 100 µl of 2.0 M H2SO4. The absorbance was measured at 450 nm using ELISA reader (TECAN). Plasmids from positive clones of ELISA were isolated and sequenced using -96 gIII sequencing primer 5′-TGA GCG GAT AAC AAT TTC AC-3′. This primer was used to amplify the fusion peptide region. The inserted random 7 and 12 amino acids were identified and analyzed. All inserted sequences were aligned to determine the consensus sequence. The obtained consensus sequences were aligned with dengue viral genome to determine the mimic epitope.Briefly, 50 µl of protein A/G magnetic beads were blocked with BSA by incubating at room temperature for 1 h, and washed with 0.1% Tris Buffered Saline with Tween (TBST). In the meantime, Phages . Absorbance values of each concentration (A) of antibody in solution phase were divided by absorbance values in the absence of antibody (A0), yielding normalized values (A/A0) . Non-rel® 2000) and added to the cells, which were then incubated for 5 h. After that, the medium was replaced with culture medium . The cells were incubated at 37 °C with 5% CO2 for 24 h. After that, the medium was replaced with culture medium containing the two antibiotics at the concentrations specified above. The medium was changed every 3–4 days until 60% of alive cells were observed. The transfected cells were used for cell cloning by limiting dilution on 96 well-cell culture plates and incubated for 10–14 days with selection media. The positive clones that were able to secrete the target anti-DENV rIgG were selected by IFA using the culture supernatant of each well containing a single stable clone. The cells were scaled up for further characterization. The level of IgG contained in the culture fluid of each positive clone was roughly determined by an IgG quantitation enzyme-linked immunosorbent assay . The functionalities of the purified N297Q rIgG secreted from the stable cells were confirmed by IFAs, NT and ADE assays.The HC-expressing constructed plasmid (pQCXIP-CH) contained a puromycin-resistant gene and the LC-expressing constructed plasmid (pQCXIH-CL) contained a hygromycin-resistant gene. After determining the optimal concentrations of the corresponding two antibiotics, stable antibody-secreting CHO-K1 cells were selected using puromycin and hygromycin at 8 µg/ml and 800 µg/ml, respectively. For stable cell generation, briefly, CHO-K1 cells were seeded in a 6-well cell culture plate the day before transfection to obtain a cell density of 90–95% on the next day. The medium was replaced with Opti-MEM I reduced serum medium. Plasmid DNA was mixed with transfection reagent (LipofectamineThe substitution of amino acid at position 297 from Asparagine to glutamine was created and confirmed by DNA sequencing. Five mg of wild type B3B9 and four mg of N297Q-B3B9 HuMAb were obtained. The successful production of wild type and mutant (N297Q) rIgG was verified by the capacity of binding activity observed via IFA. The N297Q-B3B9 rIgG displayed cross-reactivity to all four DENV serotypes similarly to the wild type rIgG –1D.Reverse transcription PCR was performed to determine the B3B9 rIgG isotype. Using B3B9 hybridoma complementary DNA as template, the PCR product with approximately 211 bp of IgG1 was observed .To determine the neutralizing activity of rIgG, we assessed the NT of N297Q-B3B9 rIgG and B3B9 rIgG in Vero cells. N297Q-B3B9 rIgG displayed almost the same level of NT as the B3B9 rIgG against all DENV serotypes. The NT levels of various concentrations of these two antibodies against all four DENV serotypes are shown in B3B9 rIgG was able to neutralize DENV2 and 3 at the minimum concentration, 0.39 and 25 µg/ml respectively –3C, but In THP-1 cells, mutant IgG (N297Q-B3B9) that cannot bind to FcγR showed a complete reduction of ADE activity in all tested antibody concentrations. In contrast, wild type rIgG induced 9541, 43, 3061, and 2020 fold enhancement in the dengue virus RNA copy number compared with the control for DENV1, 2, 3, and 4, respectively –3H.5 to 9.8 × 108 PFU and 6.9 × 105 to 7.7 × 108 PFU respectively, which proposed that specific enrichment had occurred . This clone was continually cultured to collect supernatant for purification. Preliminary study against DENV2 showed that all tested concentrations of the modified rIgG secreted from the stable cell line showed the same result of viral NT  without Due to the complexity of DENV infection and pathogenesis , the ideThe important receptor for ADE mechanism is FcγR (Fc gamma receptor) that, once it binds with immune complex, can stimulate virus infection. The Fc region of antibody contains asparagine residue (N297) which is a single N-linked glycosylation site in its CH2 domain. The nature of this glycan can decisively influence the therapeutic performance of a recombinant antibody, and their absence or modification can leads to changing of conformation and losing Fc effector functions . The Fc Firstly, we evaluated the generated N297Q-B3B9 rIgG for its neutralizing activity in Vero cells and found that the engineering antibody showed proper neutralizing activity comparable to B3B9 rIgG against DENV2, -3, and -4 with some lesser degree of NT activity against DENV1 of N297Q-B3B9 comparing with B3B9 rIgG.in vivo. We then studied the balance of NT and ADE activity using K562 cells, which express FcγRII. In the simplified ADE assay using K562 cells , complement-dependent cytotoxicity (CDC) , and ant10.7717/peerj.4021/supp-1Table S1Click here for additional data file.10.7717/peerj.4021/supp-2Figure S1First lane was 100 bps DNA ladder. Second lane was the sample that amplified from the same forward primer of IgG1 and IgG3; third lane was the sample amplified by forward primer of IgG2 and last lane was the sample amplified by forward primer of IgG4. All of isotypes used the same reverse primer. The expected sizes of the PCR products were 211, 207, 346, and 210 bp for IgG1, IgG2, IgG3, and IgG4, respectively.Click here for additional data file.10.7717/peerj.4021/supp-3Supplemental Information 1The table shows foci number of triplicate obtained from 2 antibodies (B3B9 and N297Q-B3B9 rIgG) against DENV1.Click here for additional data file.10.7717/peerj.4021/supp-4Supplemental Information 2(A) ADE assay on K562 cells of B3B9 and N297Q_B3B9 rIgG against DENV1-4. (B) ADE assays in THP-1.Click here for additional data file.10.7717/peerj.4021/supp-5Supplemental Information 3Click here for additional data file.10.7717/peerj.4021/supp-6Supplemental Information 4The table shows foci number of triplicate of N297Q-B3B9 rIgG derived from a stable and transient CHO-K1 cell line against DENV2.Click here for additional data file.


This work reports the development of Ag-modified BiV1-xMoxO4 composites through a facile hydrothermal synthesis with the subsequent photoinduced reduction of Ag+ at almost neutral pH conditions. Metallic Ag nanoparticles were deposited on the (040) facet of Mo-doped BiVO4 powders. The crystal structure and morphology of the as-prepared samples were studied by XRD and SEM analyses. Moreover, the photocatalytic performance of BiVO4, Ag/BiVO4, and Ag-modified BiV1-xMoxO4 were evaluated by the degradation of rhodamine B (RhB). The Ag/BiV0.9925Mo0.0075O4 composite exhibited the most efficient photocatalytic performance. The present work provides greater insight into the application of BiVO4 in the field of photocatalysis.In recent years, BiVO Phot2WO6 , 4, BiPO2WO6 [4 , 6, Ag3P[4 [3PO4 , 8, and [3PO4 [4 –13, have[3PO4 [4 –16. Addi[3PO4 [4 , 18.4) is a promising n-type semiconductor photocatalyst [4. Various studies have assessed BiVO4 modifications [4 has been shown as the most efficient method to change its carrier transport efficiency. Metal element doping introduces new defects or charges in the crystal lattice [4, leading to the formation of a good crystal structure and considerable photocatalytic activity for water oxidation. Mo has a higher valence than V and therefore strengthens the n-type characteristics of the material [4 is highly dependent on its various crystal facets. Recent studies on the deposition of noble metals, such as Ag, Cu, and Au, on the different facets of BiVO4 have demonstrated good photocatalytic activity [4 composite through the hydrothermal synthesis and photoreduction of Ag deposited on the (040) crystal facets of BiVO4, leading to an enhanced photoelectrochemical performance, as indicated by the fast separation of the electron–hole pairs.Due to its visible photocatalytic activity, wide bandgap of 2.42 eV, high stability, and non-toxicity, bismuth vanadate  facets of the as-produced substrate materials. Ag/BiV0.9925Mo0.0075O4 composite photocatalysts were successfully synthesized and showed enhanced photocatalytic degradation of rhodamine B (RhB) under xenon lamp irradiation (λ > 420 nm) compared to the non-composite Ag-deposited or Mo-doped BiVO4 materials. Herein, we report the preparation, characterization, and photocatalytic activity of BiVO4, Ag/BiVO4, BiV1-xMoxO4, and Ag/BiV1-xMoxO4 composites.In the present study, we build on the facile hydrothermal synthesis approach of Li et al.  to obtai3)3·5H2O, analytical grade), ammonium metavanadate , ammonium carbonate, and ammonium molybdate ((NH4)2MoO4) were obtained from Sigma–Aldrich and used as received, without any further purification. All other chemicals used in the experiments were also of analytical grade, and deionized water was used for the preparation of the solutions. In a typical process, 3.7 mmol of Bi(NO3)3·5H2O, 3.7 mmol of NH4VO3, and 12 mmol of (NH4)2CO3 were dissolved in 75 mL of 1 M HNO3 and stirred for approximately 30 min at room temperature until a clear solution was obtained. The pH of the mixture was adjusted to pH 8 with NaOH (2 M). The mixture was transferred into a 150-mL Teflon-lined stainless autoclave and heated for 12 h at 180 °C under autogenous pressure in an oven. The precipitate was filtered and washed three times with distilled water followed by ethanol and dried for 12 h at 60 °C in a drying oven.Bismuth nitrate pentahydrate  and AgNO3 (0.05 g) were added to a (NH4)2C2O4  aqueous solution in a 250-mL beaker in an ultrasonic bath until an evenly dispersed solution was formed. The resulting yellow mixture was then irradiated with a Xenon lamp for 30 min under magnetic stirring. The color of the system turned from a vivid yellow to grayish-green, indicating the generation of metallic Ag in the reaction system. The resulting samples were then filtered, washed with DI water, and dried at 60 °C for 12 h to obtain the Ag/BiVO4 and Ag/BiV1-xMoxO4 composites.BiVO−1) under ultrasonication in a glass reactor equipped with a cooling water circulator to maintain a reaction system temperature of room temperature. The suspension was stirred for 30 min in the dark to reach the adsorption–desorption equilibrium and was then irradiated for 2 h with a Xenon lamp (300 W) under continuous stirring. A 5-mL aliquot of the suspension was taken every 30 min and centrifuged. The absorption spectrum of the obtained liquid supernatant was measured in reference to the absorption intensity of RhB at 552 nm.Assessment of the photocatalytic activity was performed using the degradation rate of RhB. The experimental system for photodegradation was calibrated at a UV cut-off wavelength below 420 nm, and the irradiation height of the Xenon lamp was close to the height of the 250-mL beaker. In a typical procedure, the as-prepared photocatalyst (0.1 g) was well dispersed in a RhB aqueous solution  and transmission electron microscopy . Elemental analysis was performed by X-ray photoelectron spectroscopy  with a non-monochromatic Mg Kα X-ray source. The crystalline phase of the samples was determined by X-ray diffraction  with Cu Kα radiation. Inductively coupled plasma (ICP) was employed to analyze the elemental composition of the samples. Additionally, UV–vis diffuse reflectance spectrum measurements were performed using a Shimadzu spectrophotometer (UV-2450) to evaluate the bandgap energy of BiVO4, Ag/BiVO4, BiV1-xMoxO4, and Ag/BiV1-xMoxO4 over a wavelength range of 360–800 nm.The morphologies of the pure BiVO4 phase since they were in good agreement with the standard (JCPDS) card no. 14-0688. According to the peak splitting observed at 18.7° and 30.5°, which indicate the (110) and (040) facets, the prepared BiVO4 material possessed a single monoclinic scheelite structure. A diffraction peak at 38.1° was observed in the Ag-related photocatalysts  crystal facet . The crystal lattice fringe at 0.226 nm belonged to the (111) plane of metallic Ag nanoparticles in the Ag/BiVO4 and Ag/BiV0.9925Mo0.0075O4 samples  spectras were taken to investigate the separation efficiency of the photogenerated electron–hole pairs. The PL spectra of pure BiVOλ > 420 nm), confirmed Ag or Mo alone had little effect on the catalytic activity of BiVO4 under light irradiation for 2 h  scavenger 2C2O4)), a superoxide radical (•O2−) scavenger  [0.9925Mo0.0075O4 2C2O4, suggesting that the holes acted as the main active species.To explore the underlying photocatalytic mechanism, RhB degradation was conducted under visible light irradiation , adding one, BQ) , or hydrone, BQ) . Followi5O4 Fig. . The fas2−. In addition, obvious signals  was used. The principle of ESR is to react with free radicals using a spin-trapping agent to generate a relatively stable free radical adduct. A peak intensity was observed under visible light compared with dark conditions Fig. , demonstals Fig.  suggeste0.9925Mo0.0075O4 was illustrated in Fig. 4 photocatalyst. Ag/BiV0.9925Mo0.0075O4 composite photocatalysts were irradiated under visible light, and the photoelectrons in the valence band of BiVO4 could effectively jump to the conduction band to generate electron–hole pairs. The metallic Ag could accept the electrons, which then recombine with the photogenerated holes and enhance the transfer to the surface of the composite photocatalysts, resulting in the improvement of the separation of electrons and holes. The electrons could react to the O2 and transform to •O2−. The holes of BiV0.9925Mo0.0075O4 could react with the adsorbed H2O molecules and transform to •OH. Meanwhile, the h+ could effectively react with the RhB, generating degraded products.According to the discussion above, a possible photocatalytic mechanism of Ag/BiV4 powders. Metallic Ag nanoparticles were then deposited on the (040) crystal facet of BiV0.9925Mo0.0075O4. Thus, a photocatalytic system has been successfully constructed by means of the reduction reaction. These synthesis conditions have been shown to significantly influence the increase in the size of the (040) crystallographic facet, as confirmed by XRD and STEM analyses. The XRD indicated that the peak splitting observed at 30.5° is a result of the (040) facets. Ag nanoparticles deposited on the (040) facets can also be seen from the STEM. Furthermore, Ag/BiV0.9925Mo0.0075O4 showed a highly efficient photocatalytic performance for RhB degradation under visible light irradiation. This work could offer new inspiration for the rational utilization of BiVO4 photocatalysts with high photocatalytic activity and their applications in the fields of energy production and environmental protection.Herein, a simple hydrothermal synthesis procedure at almost neutral pH conditions and using ammonium carbonate as the structure-directing agent is reported for the preparation of Mo-doped BiVO


We previously reported that a clinical isolate of dengue virus (DENV) is capable of causing acute-phase systemic infection in mice harboring knockouts of the genes encoding type-I and -II interferon IFN receptors (IFN-α/β/γR KO mice); in contrast, other virulent DENV isolates exhibited slow disease progression in this mice, yielding lethal infection around 20 days post-infection (p.i.). In the present study, we sought to clarify the dynamics of slow disease progression by examining disease progression of a type-2 DENV clinical isolate (DV2P04/08) in mice.The tissue distributions of DV2P04/08 in several organs of infeted mice were examined at different time points. Whole genome viral sequences from organs were determined.At day 6 p.i., high levels of viral RNA (vRNA) were detected in non-neuronal organs , spleen, kidney, liver, lung, and bone marrow) but not in brain. By day 14 p.i, vRNA levels subsequently decreased in most organs, with the exception of thymus and brain. Sequence analysis of the whole genome of the original P04/08 and those of viruses recovered from mouse brain and thymus demonstrated the presence of both synonymous and non-synonymous mutations. Individual mice showed different virus populations in the brain. The vRNA sequence derived from brain of one mouse was nearly identical to the original DV2P04/08 inoculum, suggesting that there was no need for adaptation of DV2P04/08 for growth in the brain. However, quasispecies  were observed in the thymus of another mouse, and interestingly only mutant population invaded the brain at a late stage of infection.These results suggested that the mouse nearly succeeded in eliminating virus from non-neuronal organs but failed to do so from brain. Although the cause of death by DV2P04/08 infection is likely to be the result of virus invasion to brain, its processes to the death are different in individual mice. This study will provide a new insight into disease progression of DENV in mice.The online version of this article (doi:10.1186/s12985-016-0658-4) contains supplementary material, which is available to authorized users. Aedes aegypti and Aedes albopictus [Recent climate change and urbanization have increased the risk of vector-borne disease –3. Virusbopictus . Clinicabopictus .DENVs possess a single-stranded RNA genome of approximately 10.7 kb . The strBecause the mechanism of severe disease remains obscure –19, deveIFN-α/β receptor KO mice and FcγRIIB receptor KO mice were the kind gifts of (respectively) Prof. Ken J, Ishii, Laboratory of Vaccine Science, WPI Immunology Frontier Research Centre (IFReC)/Laboratory of Adjuvant Innovation, National Institutes of Biomedical Innovation, Health and Nutrition and Prof. Toshiyuki Takai, Department of Experimental Immunology, Institute of Development, Aging and Cancer, Tohoku University. IFN-γ receptor KO mice were purchased from Jackson Laboratory. IFN-α/β/γR KO mice were obtained by breeding IFN-α/β receptor KO mice × IFN-γ receptor KO mice. IFN-α/β/γR/FcγRIIB KO mice were further obtained by breeding IFN-α/β/γR KO × FcγRIIB receptor KO mice. All IFN-receptor and Fcγ receptor KO mice were bred and maintained under specific-pathogen-free conditions in the animal facilities of the Research Institute for Microbial Diseases (RIMD), Osaka University (Osaka), Japan. After breeding, 4- to 5-week-old male and female mice were used for viral quantification and survival experiments, respectively.6 ffu, 3.5 x 105 ffu, and 3.5 x 104 ffu) for the IFN-α/β/γR/FcγRIIB and IFN-α/β/γR KO mouse survival experiments, while a titer of 2.4x106 ffu was used for in vivo quantification of the virus production after infection of IFN-α/β/γR/FcγRIIB KO mice. Trained laboratory personnel performed anesthesia of mice via intraperitoneal injection of a mixture of medetomidine, midazolam, and butorphanol prior to injection of virus and (at a separate time point) euthanasia of mice by cervical dislocation. For the survival experiments, IFN-α/β/γR/FcγRIIB KO mice were injected intraperitoneally with 1 ml of a DV2P04/08 viral suspension at 3.5 x 105 ffu/ml or 3.5 x 104 ffu/ml. IFN-α/β/γR KO mice were injected intraperitoneally with 1 ml of a DV2P04/08 viral suspension at 3.5 x 106 ffu/ml or 3.5 x 104 ffu/ml. Following infection, both the IFN-α/β/γR/FcγRIIB KO and IFN-α/β/γR KO mice were observed daily for any conspicuous clinical manifestations. In a separate experiment, in vivo systemic viral titration was performed by injecting IFN-α/β/γR/FcγRIIB KO mice intraperitoneally with 1 ml of a DV2P04/08 viral suspension at 2.4 x 106 ffu/ml. Following injection, the mice were observed daily; separate subgroups of mice were sacrificed at day 6 or 14 p.i. During mouse necropsies at day 6 and 14, specimens of serum, spleen, kidney, lung, liver, thymus, brain, and BM were collected aseptically and stored at −80 °C pending further processing. At the same time, peritoneal exudate cells (PECs) were collected by injecting 5 ml of sterile PBS-EDTA into the mouse peritoneal cavity at necropsy. Additionally, blood for serum was collected aseptically from the tails of mice on days 2, 5, and 9 p.i. to monitor viremia.DV2P04/08 was a clinical isolate gifted by Dr. Kriengsak Limkittikul, Faculty of Tropical Medicine, Mahidol University, Thailand. Initially, a few generations of the virus were obtained by passaging in the C6/36 mosquito cell line. However, in order to augment the titer, the subsequent 2–3 generations were obtained by passaging in Vero cell lines. The titer of DV2P04/08 was determined by the focus forming assay . We emplViral RNA was isolated from sera (70 μl/specimen) using the QIAmp Viral RNA Mini kit (Qiagen) and from tissue homogenates using TRIzol reagent (Life Technologies) according to the manufacturer’s protocol. Spleen, liver, kidney, thymus, lung, brain, PECs, and BM were homogenized using a Beads Crusher μT-12 (Taitec). Total RNA was extracted using TRIzol and adjusted to 200 μg/ml for use in real-time reverse transcription PCR. RNA was quantified using the One-Step SYBR PrimeScript RT-PCR kit II (Takara) and the following dengue group-specific primers: DN-F, 5’-CAATTGCTGAAACGCGAGAGAAA-3’ and–DN-R, 5’-CCCCATCTATTCAGAATCCCTGCT-3’ . Each 12The viral RNA isolated from the original viral stock and from the virus derived from brain or thymus virus was converted to cDNA using a One-Step SYBR Green RT-PCR Kit II (Takara) following the manufacturer’s instructions. Based on the concentrations of RNA obtained from each of the several organs, RNA samples from the brains of infected mice No. 3 and 6 and from the thymus of infected mouse No. 6 were subsequently chosen for cDNA synthesis. The details of the PCR reaction and the sequence of the primers used were as described previously . The PCRx1 Genetic analyzer.Sequencing reactions were carried out by using different primers spanning the genome . FollowiMultiple sequence alignment (MSA) of whole genomes among DENV reference strain 16681 (accession no.NC_001474), DV2P04/08, and viruses from brains and thymus of infected mice was carried out using GENETYX version18.0.1.The sequences described here have been deposited in the GenBank database under accession numbers LC129169 to LC129171.6 ffu and 3.5 x 104 ffu). Mice infected at the higher viral titer started dying at day 6 p.i., and all of the mice in this group died before day 16 p.i.  . Most vi.i. Fig. . Notably.i. Fig. . As abov4 ffu of virus started dying at day 20 p.i., with all of these animals dying by day 22 p.i. , although the IFN-α/β/γR/FcγRIIB KO mice died within shorter interval (days 20–22 p.i.) than did the IFN-α/β/γR KO mice (days 10–32 p.i.). Interestingly, 60% of IFN-α/β/γR/FcγRIIB KO mice infected with 3.5 x 105 ffu died on days 19 or 20 p.i.  against DENV are thought to play an important role in DENV infection because the interaction between Abs and DENV can result in either immunity or enhanced virus infection by increased uptake of Ab-virus complexes thorough Fcγ receptors (FcγR) . Among F.i. Fig. . Thus, i.i. Fig. . Thus, t6 copies/μg of total RNA) were detected in peritoneal exudate cells (PECs), spleen, kidney, lung, thymus, and bone marrow (BM). Lower levels of vRNA  were detected in liver. Among the organs/tissues examined, the lowest levels of vRNA  were detected in brain , we assumed that adaptation of DV2P04/08 by mutation to a particular genomic sequence was needed to grow in the brain. To characterize this proposed sequence change, we determined the full-length genome sequences of viruses obtained at day 14 p.i. from the brains and thymuses of infected IFN-α/β/γR/FcγRIIB KO mice, and compared those sequences to those of the original (infecting) DV2P04/08 strain. While the DV2P04/08 viruses recovered from the brains and thymuses of infected mice harbored multiple mutations compared to the sequence of the parent virus, the majority of these changes represented synonymous (silent) changes in the structural and non-structural proteins encoded by the virus. However, certain mutations in the segments encoding E, NS1, NS3, NS4, and NS5 proteins represented nonsynonymous changes. Table In the present study, we showed that DENV-2 clinical isolate P04/08 caused lethal infection in IFN-α/β/γR KO mice. Additional KO of the gene encoding inhibitory FcγRIIB appeared not to affect mouse survival. Deficiency of FcγRIIB is expected to increase Ab production by blocking inhibitory effects on plasma cells, amplifying the humoral immune response (as seen for increased autoimmunity in FcγRIIB KO mice) . HoweverIn IFN-α/β/γR/FcγRIIB KO mice, highest production of DV2P04/08 was detected in brain at late stage of disease progression Fig. . SubsequUnfortunately, we were unable to amplify viral sequences from other  organs of the infected animals, precluding sequence analysis of the virus replicating in other organs and tissues. Nevertheless, our result suggests the following hypothesis to explain emergence of mutant viruses in the brain of B6. During the first 5–6 days p.i., DV2 P04/8 replicated in non-neuronal organs. Subsequently (during days 7–13 p.i.), the mouse immune system reacted to the infecting virus and nearly succeeded in clearing virus from non-neuronal organs, with the exception of the thymus. During this interval, the virus started to escape in non-neuronal organs, as evidenced by the diverse viral population in the thymus. Some of the viral population succeeded in escaping from immune pressure, permitting invasion of the brain and maximum viral replication, and resulting in death. We postulate that there must be a release of some unidentified factor from the infected organs to increase the permeability of the BBB at some point. Presumably, only a selected subpopulation of escaping mutant virus produced in other organs succeeds in reaching the brain. However, the true scenario might not be that simple because there was no sequence variation among virus derived from B3 mouse. We cannot exclude other possibilities to explain the sequence variation of virus observed in B6 mouse.Further study will be needed to elucidate the precise molecular mechanisms for DENV neurovirulence. Notably, we will need to clarify the roles of pathogenesis, neurovirulence, and vascular leakage on viral replication, invasion of specific organs/tissues, and evasion of immune regulation. These distinctions will be critical to better understanding of severe DENV manifestations, including DHF and DSS.In summary, DV2P04/08 easily invade into mouse brain and caused lethal infection. IFN-α/β/γR KO mice nearly succeeded in eliminating virus from non-neuronal organs but could not avoid brain invasion. Mutant virus detected in B6 mouse brain was probably the result of escape from mouse immune system and produced in non-neuronal organs such as thymus. Our study provides new insights into the dynamics of viral evolution in individuals.


Prep1 acts at a stage preceding the activation of the differentiation machinery because its DR makes cells more prone to adipogenic differentiation even in the absence of the adipogenic inducers. Prep1 DR expands the DNA binding landscape of C/EBPβ (CCAAT enhancer binding protein β) without affecting its expression or activation. The data indicate that Prep1 normally acts by restricting DNA binding of transcription factors to adipogenic enhancers, in particular C/EBPβ.Transcriptional regulators are crucial in adipocyte differentiation. We now show that the homeodomain-containing transcription factor Prep1 is a repressor of adipogenic differentiation since its down-regulation (DR) in both  Adipogenesis can also be abnormally activated throughout life in obese individuals2, and the mechanisms leading to such dysregulation are not yet totally understood. Mature adipocytes derive from not yet well characterized mesenchymal stem  cells (MSC)3, however several proteins that regulate adipogenesis at different stages of differentiation have been identified mainly via in vitro experiments. In this respect, activation of hormone signaling pathways and chromatin remodeling that occur within hours from induction are critically important6. Adipogenic differentiation can be stimulated in vitro by different soluble inducers7. In the preadipocyte cell line 3T3-L1, frequently used as a model to study the adipogenic process8, the addition of insulin, dexamethasone and 3-isobutyl-1-methylxanthine (IBMX), hence referred to as the complete adipogenic differentiation cocktail, leads to the very early recruitment of transcription factors into well defined chromatin hotspots hosting multiple transcription factor binding sites characterized as adipogenic enhancers9. These hotspots are always occupied by C/EBPβ, which appears to be the most-upstream triggering transcription factor, whereas Pparγ (Peroxisome proliferator-activated receptor gamma), one of the main players in the adipogenic lineage, seems to act downstream10. There is evidence indicating that also the homeoprotein Pbx1 is an important transcription factor in adipogenic differentiation as its down-regulation (DR) activates adipogenesis11. It is known that another homeodomain transcription factor of the TALE class, Prep1, is an essential partner of Pbx112, and we herein investigated its role in adipogenic differentiation.Adipogenesis mostly takes place during embryonic development and childhood , however, it also occurs in adult life, when 10% of fat cells annually renewPrep1−/− embryos die very early at the epiblast stage without reaching gastrulation13 whereas hypomorphic Prep1i/i embryos, that express around 2% of normal Prep1 mRNA, have a milder , not always penetrant, phenotype14. Foetal liver Prep1i/i cells lack active hematopoietic stem cells and are unable to repopulate irradiated hosts15.Prep1 is essential in embryonic development. 17. Indeed, Prep1 cooperates with Pbx1 to regulate Hox gene expression18. We recently described the Prep1 and Pbx1 binding sites to the DNA of mouse embryos, embryonic stem cells and embryonic fibroblasts and showed that Prep1 mostly, but not exclusively, binds DNA as a dimer with Pbx1 and that two thirds of the binding sites are cell-type specific21.Prep1 reaches the nucleus and binds DNA only as a dimer with Pbx1Prep1i/i B and T cells to properly differentiate22, although the mechanisms are still unknown.Prep1 is also involved in cell differentiation as indicated by the inability of ex vivo bone-marrow derived MSCs and in in vitro 3T3-L1 cells, Prep1 DR, similar to the effect of Pbx1 DR11, results in activation of adipogenesis. Interestingly, Prep1 DR triggers adipogenic differentiation even in the absence of complete hormonal induction, since Prep1 down regulated cells are already poised towards adipogenesis before the addition of the inducing hormones. Prep1 DR results in the expansion of the C/EBPβ DNA binding landscape already before induction and its ability to bind DNA is increased after the addition of the adipogenic inducers. Prep1 does not control a specific “differentiating” mechanism, but rather predisposes the cells towards adipogenic differentiation; therefore its possible role appears to be that of restricting DNA binding of transcription factors, in particular C/EBPβ, for example as a chromatin remodeling factor.In this paper we report that in both ex vivo bone marrow-derived MSCs. Initially, we analyzed Prep1 expression, both at the transcriptional and protein levels, in undifferentiated MSCs and at different time points following adipogenic induction, using MesenCult™ Adipogenic Stimulatory Supplement (Stem Cell Technologies). Prep1 protein is present in wild type MSCs, but its expression level rapidly decreases after 3 days of differentiation  . As expected, we detected a significant reduction of Prep1 expression in undifferentiated Prep1i/i MSCs, as compared to wt cells  Fig. . Quantiflls Fig. . Both thlls Fig. . Moreoveion Fig. . InteresPrep1i/i bone marrows (n = 8). This is in perfect agreement with the non-penetrant embryonic and adult phenotype of these mice, as only 75% of the embryos die in utero and only 50% of the adult Prep1i/i mice develop tumors23.We observed these results only in MSC cells derived from 50% of the Prep1i/i mesenchymal stromal cells and the intrinsic high levels of heterogeneity of MSCs, we decided to adopt a more homogeneous cell system to further investigate Prep1 function in the adipogenic process. In particular, we adopted the 3T3-L1 cell line frequently used as a model of in vitro adipogenesis, and infected it with a lentivirus vector carrying a scrambled control virus (henceforth referred to as C cells) or two different Prep1 shRNAs   and 24 hours after induction of differentiation (accessible in GEO at the accession number GSE100057). An expression ratio >2 or <0.5 was used as threshold to identify up- and down-regulated genes, respectively. In C cells, the adipogenic-inducing cocktail affected the expression of 2,800 genes after the first 24 hours of differentiation , dexamethasone (Dex) or IMBXBPα Fig. . Pparγ wSterol Regulatory Element Binding Transcription Factor 1 (Srebf1) in P cells  do not change their expression at the RNA level in P as compared to C cells  were affected by Prep1 DR. However, both total and phosphorylated levels of C/EBPβ  were equally affected in P v. C cells, at all times tested after the induction . The specificity of the C/EBPβ antibody used for ChIP was checked by western blot and proved that C/EBPβ is properly immunoprecipitated  in P versus C cells before differentiation at day −2, and a further 2-fold increase 4 hours after induction of differentiation , or located intragenically (IG), to the corresponding gene and found that the number of C/EBPβ-bound genes was also significantly increased in P cells both before and after differentiation  and “Positive regulation of fat cell differentiation” (p < 10−4). The same GO categories and few others related to adipogenesis  were enriched at 4 hours after induction in P cells; however, these genes were bound by C/EBPβ also in C cells.Regulatory elements regulate gene expression during adipogenic differentiation of 3T3-L1 cells even when they are Mbs away from a corresponding promoterion Fig. . Moreoveion Fig.  was enriThe distribution of the enriched GO categories between P and C cells is shown in Supplementary Table 9, the binding to the additional AP1 consensus sequences was greatly reduced (from 35% to less than 15%) in peaks exclusively bound in P cells 23, induction of differentiation of Prep1 DR ES cells has shown dramatic effects19.Since Prep1 belongs to a family of proteins which are essential in embryonic development, it was initially considered as a direct regulator of developmentPrep1i/i mesenchymal adipogenic progenitors may be considered under this angle. The phenotypes observed in both systems appear to be secondary to a fundamental effect, which is already in place before the induction of differentiation, and becomes evident only after the addition of the differentiation cocktail. We hypothesize that Prep1 DR modifies chromatin organization. For example, the increased binding landscape of C/EBPβ might be due to the presence of a more accessible, Prep1-dependent, chromatin architecture. As Prep1 is a regular transcriptional partner of Pbx1 and since Pbx1 DR also shows a similar phenotype to Prep1 DR, one wonders how much of the Pbx1 function is due to a specific differentiation function and how much is due to more basic cellular activities.Therefore, we proposed that also the phenotype of Prep1 DR 3T3-L1 and of ex vivo mesenchymal progenitor cells. It performs its action indirectly, inducing an extensive DNA binding of C/EBPβ at the early stages of the adipogenic process. The mechanism of this induction remains to be elucidated.In conclusion, we have shown for the first time that Prep1 transcription factor is required for adipogenic differentiation of both a preadipocytic cell line and of 2. Medium was changed every 3 days and cells were trypsinized at confluence and reseeded at 2 × 104 cells/cm2 . All experiments were performed at passage 2 (p2). All the experimental protocols on mice were conducted in compliance with DL 26/2014: implementation of European Directive 2010/63 on the protection of animals used for scientific purposes. All animal experiments were approved by the Institutional Animal Care and Use Committee of the CNR Research Campus in Pisa.Mice were sacrificed by cervical dislocation before collecting tibias and femurs. Bone marrow cells were flushed and seeded in culture using MesenCult Basal Medium supplemented with 20% Mesenchymal Mouse Stimulatory Supplement (StemCell Technologies) and 1% Pen-Strept (Life Technologies) (Complete Medium). Cells were grown at 37 °C in humidified atmosphere at 5% CO3T3-L1 cells were cultured in DMEM medium containing 10% BCS. Prep1 down-regulated 3T3-L1 cells were obtained by infection with a pLKO.1-based lentivirus containing Prep1-specific shRNA1 (5′ - AACCAGTTGTTGACTTGAAGC - 3′) or shRNA2 (5′-TTGCTTCCTGACAAAGTTCTC-3′) and control cells were infected with a scrambled shRNA. Cells were selected in puromycin for 3 days. The level of Prep1 expression was verified by immunoblotting using anti-Prep1 monoclonal B2 antibody .4 cells/cm2 and at 80% confluence the Complete Medium was replaced with Adipogenic Induction Medium  and 1% Pen-Strept (Life Technologies) we followed the manufacturer’s recommendations.MCSs were seeded at p2 at 2 × 10For adipogenic differentiation of 3T3-L1 cells, two days after confluence the medium was changed to differentiation medium I containing 1 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, and 1 μg/ml insulin in DMEM containing 10% of FBS. Two days after the induction of differentiation the medium was changed do differentiation medium II containing only 1 μg/ml insulin in the presence of 10% of FBS. After 4 days of differentiation the medium was changed to differentiation medium III containing only 10% of FBS.34. Briefly, cells were fixed in 3.7% of formaldehyde in PBS for 30 min at room temperature and then stained in 0.3% of Oil Red O in 60% of isopropanol for 30 min at room temperature. For quantification assays, Oil Red O was extracted from the stained cells by using 100% isopropanol and absorption was measured at 490 nm.Differentiated 3T3-L1 and MSC cells were stained by Oil-red O using a standard protocolChromatin immunoprecipitations (IP) were performed using standard methods with anti-C/EBPβ C19 antibody . Cells were cross-linked in complete medium (10% FBS) containing 1% formaldehyde for 10 min, and glycine was added to stop the reaction . Fixed cells were washed three times (5 min each) in cold PBS and lysed in LB1 buffer (containing 0.5% NP-40 and 0.25% triton X-100). Nuclei were washed in LB2 buffer (10 mM Tris-HCl pH 8 and 200 mM NaCl) to remove detergents and resuspended in LB3 buffer (containing 0.1% Na-deoxycholate and 0.5% N-lauroylsarcosine). Chromatin was sonicated in Covaris ultrasonicator to obtain fragments ranging 150–250 bps. Sonicated chromatin was incubated with antibody-bound protein G-conjugated magnetic beads . For each IP we used 20 μg of  antibody. IP with rabbit IgG was performed as negative control. After overnight IP at 4 °C the bound complexes were washed twice in WB1 , twice in WB2  and twice in LiCl WB . Immunoprecipitated complexes were eluted from the beads by incubation for 30 min in EB (2% SDS in TE) at 65 °C. The eluted material was reverse cross-linked at 65 °C overnight and incubated for 1 h at 55 °C with proteinase K. DNA was purified with a PCR purification kit . ~10 ng of immunoprecipitated DNA was processed for sequencing. After library preparation DNA was sequenced using an Ion-Torrent system. After eliminating artifacts with FASTX-Toolkit v.0.0.13.2, reads were aligned to the mm9 genome using Bowtie v.0.12.8 (3), allowing up to two mismatches per read and discarding reads with more than one mapping. Duplicate reads were removed using Samtoolrmdup v 0.1.18. Then we down-sampled mapped reads to 8 mln using Samtools v 1.3.1.35, except for p-value threshold (10e-5).The reads were mapped against the mm9 mouse genome. De novo motif discovery was run to identify consensus sequences enriched in the selected regions versus the whole genome using MEME-SUITE as de novo motif finder algorithm36.To identify enriched domains we used MACS version 2.0.10.20131028 with default parameters37, filtering the results using padj <0.1 as a threshold. Gene ontology analysis was performed using Gorilla software38.For RNA-seq, total RNA was purified and the library prepared. For each time point (−2 days and 1 day) and sample (control and Prep1 down-regulated) we used 2 biological replicas. The sequencing was performed using Ion-Torrent system. After eliminating artifacts with FASTX-Toolkit v.0.0.13.2, reads were aligned to the mm9 reference using Tophat v2.0.9. Then GenomicFeatures, GenomicRanges and GenomicAligments R libraries were used to count the number of reads with respect to the annotation reference . Differentially expressed genes between different conditions were retrieved using DESEQ R libraryThe heat maps were drawn plotting the ratios between each of normalized RPKM versus the average of two control replicas at each time point and sample.Srebf1, Klf4, Klf5, Krox20, C/EBPδ, C/EBPβ, Prep1, Glut4 expression analysis, cDNAs were subjected to qRT-PCR on a Roche LightCycler480 (Roche) using predesigned primers . Sequences are available upon request. Results were normalized to Gapdh gene expression.RNA extraction was processed according to the RNeasy (QIAGEN) protocol. After genomic DNA degradation with the RNase-Free DNase kit (QIAGEN), reverse transcription was performed with the Superscript III (Invitrogen). For Antibodies used were anti-Prep1 (Santa Cruz sc-25282), anti-C/EBPα , anti-C/EBPβ , anti-phospho-C/EBPβ (Thr235) , anti-C/EBPδ , anti-Pparγ , anti-pIrs1 (Tyr941) (07-848-I Millipore), anti-pAkt (Ser473) , anti-pAkt (Thr308) , anti-Klf5 (Abcam ab24331), anti-Vinculin (Sigma V9131).The datasets generated during the current study are available in the GEO (accession number GSE100057).Supplementary information


Influenza viruses are divided into three types, A, B, and C. Human influenza A and B viruses can cause seasonal epidemics, but influenza C causes only a mild respiratory illness. Influenza A virus can infect various host species. In 2013, human-infectious avian influenza A (H7N9) was first reported in China. By the second week of 2014, there were 210 laboratory-confirmed human cases in the country, and the mortality rate eventually reached 22 %. Rapid and accurate diagnosis of influenza viruses is important for clinical management and epidemiology.In this assay, a cost-effective chemiluminescence (CL) detection oligonucleotide microarray was developed to genotype and detect avian influenza A (H7N9), avian influenza A (H5N1), 2009 influenza A (H1N1), seasonal influenza A (H1N1), and seasonal influenza A (H3N2). Influenza A viruses and influenza B viruses were also generally detected using this microarray.The results of detection of 40 cultivated influenza virus strains showed that the microarray was able to distinguish the subtypes of these influenza viruses very well. The microarray possessed similar or 10 fold higher limit of detection than the real-time RT-PCR method. Sixty-six clinical swab samples were detected using this microarray and verified with real time RT-PCR to evaluate the efficiency of this microarray for clinical testing.A reliable CL detection oligonucleotide microarray had been developed to genotype and detected these influenza viruses. Influenza viruses are divided into three types, A, B, and C WHO . Human iInfluenza A virus can infect various host species. The greatest diversity of subtypes is found in wildfowl. The ease with which influenza A can recombine causes viral epidemics to spread across different species  were obtained from the Centers for Disease Control (CDC) of Zhejiang Province. The other cultivated influenza virus strains were obtained from the National Institutes for Food and Drug Control. Clinical throat swab samples were collected from patients suspected of having infected influenza in the 307th Hospital of the Chinese People’s Liberation Army for whom it was not possible to determine whether they had taken medication. Non-influenza respiratory viruses were identified and obtained from the National Institute for Viral Disease Control and Prevention and the 302nd Hospital of the Chinese People’s Liberation Army. Lysate had already been added to all these viral samples before they were acquired for the present study. Total RNAs were extracted using the TIANamp Virus RNA Kit as described in the manufacturer’s protocol  and stored at −70 °C until use.Homo sapiens ribonuclease P were designed to monitor specimen extraction, RT-PCR amplification, and microarray hybridization. This marker has been described as a reliable internal positive control marker in several publications , seasonal influenza A (H1N1), influenza A (H3N2), matrix protein (M) gene of influenza A virus, and nonstructural protein (NS) gene of influenza B virus were downloaded from NCBI’s influenza nucleotide database. The sequences were then aligned using AlignX (a component of the Vector NTI Advance 10.3.0) to compare homology. Genotyping primers for these subtypes of influenza A were designed using Primer Premier 5  and they served as the consensus sequences of the HA and NA genes. Universal primers for influenza A virus and influenza B virus were designed and served as the consensus sequences of the relatively well-conserved M and NS genes. Microarray probes ranging from 20 to 40 nt were designed to detect influenza A virus generally, influenza B virus generally, and these subtypes of influenza A virus. Internal standard primers and probes based on the sequences of 12 with an amino-labeled 3′-end was connected to the 3′-end of all the probes so that it could be fixed to the aldehyde-chip surface.A repeat sequence of (T)20 with a biotin-labeled 5′-end and an amino-labeled 3′-end served as a quality control probe. The probes were used at 50 μM final concentration. They were spotted and repeated three times in the vertical direction on the surface of the aldehyde-chip using uniform proportional printing buffer as described in previous studies Homo sapiens ribonuclease P, which served as an internal standard. Amplifications were performed on a Veriti 96-Well Thermal Cycler PCR system  under the following conditions: 30 min at 50 °C; 2 min at 94 °C; 45 cycles of 20 s at 94 °C, 20 s at 52 °C, and 20 s at 72 °C; and a final extension of 5 min at 72 °C.The primers were used in three different RT-PCR systems. Each RT-PCR was performed in a 25 μl reaction volume containing 12.5 μl of 2× One Step Buffer, 1.0 μl of PrimeScript One-step Enzyme Mix  Co., Ltd.), 5 μl of total RNA template, and specific primer mix. RT-PCR system A amplified novel avian influenza A (H7N9) and avian influenza A (H5N1). It contained primer H7F1 0.16 μM, H7R2-B 0.8 μM, NA-1Sa 0.12 μM, NA9r-B-4 0.6 μM, NF11 0.1 μM, NR22B 0.5 μM, RP-F4 0.08 μM, and RP-R4 0.4 μM. RT-PCR system B amplified 2009 influenza A (H1N1), seasonal influenza A (H1N1), and influenza A (H3N2). It contained primer F7 0.1 μM, R4aB 0.5 μM, NF5 0.1 μM, NR5B 0.5 μM, NF12 0.1 μM, NR21B 0.5 μM, RP-F1 0.1 μM, and RP-R1B 0.5 μM. RT-PCR system C amplified influenza A and B viruses. It contained primer MF2 0.1 μM, MR2B 0.5 μM, FBF1 0.1 μM, FBR1B 0.5 μM, PR-F 0.05 μM, and RP-RB 0.25 μM. All the three systems contained a pair of primers that could amplify the gene of 2O2  was added to the chip and immediately detected with a micro-light level imaging system .After amplification, the three reaction products derived from the same template were mixed into one tube (10 µl each). After 5 min of denaturation at 95 °C, the mixture was immediately placed on ice 5 min and then mixed with 5 μl of hybridization buffer . A total of 10 μl of hybridization mixture was hybridized on the microarray for 1 h at 45 °C as described   which generally detected influenza A viruses and influenza B viruses, were used to compare results. A panel of non-influenza respiratory viruses, which include parainfluenza virus, adenovirus AD3, AD4, AD30, AD40, measles virus, mumps virus, and respiratory syncytial viruses HK6 and B, were also used to determine the specificity of the microarray.In order to evaluate the sensitivity of this microarray in the detection of various subtypes of influenza virus, the RNA extraction of cultivated virus strains of 5 subtypes of influenza A virus and influenza B virus were serially diluted in tenfold increments and detected using the microarray. Influenza A and B Nucleic Acid Detection Kits (PCR-fluorescent probe)  were also used to compare results. They amplify the conserved M gene of influenza A viruses and the NS gene region of influenza B viruses.To investigate the efficiency of clinical testing, 66 clinical swab samples were collected from the 307th Hospital of the Chinese People’s Liberation Army. Total RNAs were extracted using a TIANamp Virus RNA Kit as described in the manufacturer’s protocol. Then the clinical swab samples were detected using a microarray and verified using four different kinds of real-time RT-PCR kits. These real-time RT-PCR kits included a 2009 Influenza A Virus (H1N1) Nucleic Acid Detection Kit (PCR-fluorescent probe) , 2009 Influenza A Virus (H1N1) Nucleic Acid Detection Kit (PCR-fluorescent probe) , Seasonal Influenza A Virus (H3) Nucleic Acid Detection Kit (PCR-Fluorescent probe) , and Diagnosis Kit for H7N9 Avian Influenza Virus RNA (PCR-fluorescent probe) . These real-time RT-PCR kits identified and determined the concentration of HA gene of 2009 influenza A (H1N1), NA gene of 2009 Influenza A Virus (H1N1), influenza A (H3N2), and avian influenza H7N9 virus. They were all used as described in the manufacturer’s protocol.In this assay, a total of 40 cultivated influenza virus strains were tested to determine the specificity of this microarray. The types or subtypes of these strains included 6 avian influenza A (H7N9), 6 influenza A H3N2), 4 seasonal influenza A H1N1), 2 2009 influenza A (H1N1), 18 avian influenza A (H5N1), and 4 influenza B virus. The results showed that one strain of H3N2 was mixed with H1N1, one strain of H5N1 was mixed with H1N1, another strain of H5N1 was mixed with H3N2, one strain of influenza B was mixed with H1N1, and one strain of H3N2 did not subtype because of its low concentration. The four mixed samples were confirmed by amplifying the M gene (for influenza A viruses) or NS gene (for influenza B viruses) of influenza using universal primers of influenza A and B viruses  are shown in Fig. To assess sensitivity, the microarray detection methods were compared to real-time RT-PCR. Results showed that the microarray have similar (for PH1N1) or tenfold higher limit of detection (LOD) (to the other subtypes of influenza A virus and influenza B virus) than the referenced real-time RT-PCR method. The LOD of general influenza A virus detection was lower than the LOD of genotype detection. The LOD for the two comparator real-time PCR assays was 1 × 10Sixty-six clinical swab samples were detected by microarray. Results indicated that 25 of the samples were 2009 influenza A (H1N1), 3 were influenza A (H3N2), 26 were influenza A, 1 contained both 2009 influenza A (H1N1) and influenza A (H3N2), and 11 were negative. Four different real-time PCR influenza genotyping kits served as reference methods. Results are shown in Table The statistical data of 66 clinical throat swab samples are shown in Table Influenza A virus can infect various host species, and the greatest diversity of subtypes is found in wildfowl. The influenza virus genome comprises 8 independent RNA strands, so it can easily undergo antigenic drift, antigenic shift, and reassortment, which allows it to cause major pandemics  strains have been found to be associated with lower susceptibility to NA inhibitor as indicated by weekly monitoring. Many studies have shown that the severity of the infection is more likely to be reduced if an NA inhibitor is administered within 48 h of the onset of influenza symptoms than when it is administered later . Furthermore, other commercial CL imagers based on CCD imaging technology  could also be used for this CL microarray. The new CL imager had a lower cost and a much faster detection speed than our previous visual microarray system which was based on quantum dot-catalyzed silver deposition. The cost of this CL imaging microarray was also lower than that of our previous fluorescence based microarray system  priority was given to the detection avian influenza A (H7N9) (H7 and N9 were detected simultaneously). (2) Other than avian influenza A (H7N9), only the most common subtypes of influenza virus were detected by genotyping in this microarray. (3) Other than genotyping detection, the microarray also universal detected the more conservative M gene of influenza A viruses and NS gene of influenza B viruses to extend the range of the application. (4) One-step RT-PCR amplification was used to reduce the length of the experimental procedure and so reduce the risk of contamination. (5) Human-derived primers and probes served as internal standards; they were added to all the amplification reactions to monitor extraction of clinical samples, RT-PCR amplification, and microarray hybridization. (6) Low-cost CL imaging technology was used for detection in the microarray. This may allow this system to be used on a large scale.A low-density CL oligonucleotide microarray was developed for genotyping detection of the newest avian influenza A (H7N9), avian influenza A (H5N1), 2009 influenza A (H1N1), seasonal influenza A (H1N1), and influenza A (H3N2). Influenza A viruses and influenza B viruses were also generally detected using this microarray. The microarray kits used here were capable of rapid, high-throughput, highly accurate readings, and the entire detection time from sample extraction to the production of genotyping results was 6–8 h. Six avian influenza A (H7N9) culture viruses and thirty-four other influenza culture viruses served as positive references to confirm the specificity and sensitivity of the microarray. Some non-influenza respiratory viruses were also tested using the microarray to further confirm this specificity. All the results showed that the microarray accurately detected the genotypes of these influenza viruses and of some mixed influenza strains. The LOD of genotyping and universal detection of influenza viruses were similar to or ten-fold higher than reference values for influenza real-time RT-PCR kits. Sixty-six clinical swab samples were assessed using microarray to determine the efficiency of this type of clinical testing and epidemiology. Results showed that the specificity and sensitivity of the microarray met the needs of clinical and epidemiological studies of the influenza virus.This method has some limitations. RT-PCR amplification was here divided into three groups to amplify different subtypes of influenza viruses. This complicated the operation. However, reducing the number of amplification systems (increasing the number of primers in one amplification system) may have decreased sensitivity. Here, 11 pairs of primers were divided into three groups of amplification systems, and the LOD of this microarray was still ten times higher than that of the real-time PCR kits used for reference . The 66 clinical swab samples were detected and microarray failed to show subtypes of samples when the CT value of real-time RT-PCR more than 32. However, general detection of influenza A virus was still positive even when the CT values were as high as 39. Consequently, the sensitivity of this CL detection strategy and microarray could be improved by further optimization. Moreover, avian influenza A (H7N9) and (H5N1) were not actually comparatively tested using the developed microarray and PCR assays due to the deficiency of clinical specimens.Furthermore, the use of this strategy was restricted because of the limitations of detection for only novel avian influenza A (H7N9) and some of the most common human influenza viruses. The other influenza A subtypes such as H5N6 and H9N2, can not be typed. Thus, sequencing or other diagnostic methods are still needed to determine the exact subtypes of those influenza A viruses.A reliable CL detection oligonucleotide microarray was developed to genotype and detected avian influenza A (H7N9), avian influenza A (H5N1), 2009 influenza A (H1N1), seasonal influenza A (H1N1), and seasonal influenza A (H3N2). Influenza A viruses and influenza B viruses were also generally detected using this microarray. The results of detection of 40 cultivated influenza virus strains showed the microarray to be able to distinguish the subtypes of these influenza viruses very well. The microarray possessed similar or tenfold higher LOD than the real-time RT-PCR method. Sixty-six clinical swab samples were detected by microarray and verified using real-time RT-PCR to evaluate the efficiency of this microarray for clinical testing.


Persistent, high-risk human papillomavirus (HPV) infection is the primary cause of cervical cancer. Neutralizing antibodies elicited by L1-only virus-like particles (VLPs) can block HPV infection; however, the lack of high-resolution structures has limited our understanding of the mode of virus infection and the requirement for type specificity at the molecular level. Here, we describe two antibodies, A12A3 and 28F10, that specifically bind to and neutralize HPV58 and HPV59, respectively, through two distinct binding stoichiometries. We show that the epitopes of A12A3 are clustered in the DE loops of two adjacent HPV58 L1 monomers, whereas 28F10 recognizes the HPV59 FG loop of a single monomer. Via structure-based mutagenesis and analysis of antibody binding, we further identified the residues HPV58 D154, S168, and N170 and HPV59 M267, Q270, E273, Y276, K278, and R283, which play critical roles in virus infection. By substituting these strategic epitope residues into other HPV genotypes, we could then redirect the type-specific binding of the antibodies to these genotypes, thus highlighting the importance of these specific residues, HPV58 R161, S168, and N308 and HPV59 Q270, E273, and D281. Overall, our findings provide molecular insights into potential structural determinants of HPV required for infectivity and type specificity. High-risk human papillomaviruses (HPVs) are considered the major causative pathogens of cancers that affect epithelial mucosa, such as cervical cancer. However, because of the lack of high-resolution structural information on the sites of neutralization, we have yet to determine the precise mode of HPV infection and how different types of HPV cause infection. Our crystal structures in this study have uncovered discrete binding stoichiometries for two different antibodies. We show that one A12A3 Fab binds to the center of one HPV58 pentamer, whereas five 28F10 Fabs bind along the top fringe of one HPV59 pentamer. Furthermore, through targeted epitope analysis, we show that 6 to 7 discontinuous residues of the L1 major capsid protein of HPV are determinants, at least in part, for virus infection and type specificity. This knowledge will help us to unravel the process of HPV infection and can potentially be used to drive the development of therapeutics that target neutralization-sensitive sites. High-risk human papillomavirus (HPV) infection is the main cause of cervical and other anogenital cancers , with 154–Cryo-electron microscopy (cryo-EM) structures of the whole virus capsid have provided critical insight into the mechanism of HPV assembly –12, and –15–The HPV capsid is purported to attach to the host cell primarily through heparan sulfate proteoglycans (HSPGs) and the non-HSPG extracellular matrix receptor, laminin-332 (formerly laminin-5), which is secreted by epithelial cells –26. L1 c–34–41–Here, we characterize two type-specific, neutralizing monoclonal antibodies (MAbs), A12A3 and 28F10, against HPV58 and HPV59, respectively, and report the crystal structures of the HPV pentamers in complex with the antigen-binding fragments (Fabs) to resolutions of 3.5 and 3.4 Å, respectively. Through structural and biophysical data, we show that each antibody has a unique binding mode and stoichiometry. Fitting the Fab-bound pentamer crystal structures into corresponding medium-resolution cryo-EM structures of Fab-bound HPV L1 VLPs, we show that these binding modes are compatible with antibody recognition of whole virions. Furthermore, using site-directed mutagenesis, we identified the key epitope residues that are critical for binding type-specific antibodies. Functional studies showed that some residues located on the epitopes for both A12A3 and 28F10 are involved in virus infection and that type-specific antibody binding could be redirected to HPV33 and HPV18 L1 chimeric VLPs, respectively, after swapping key epitope residues. These findings narrow the functional surface area of HPV capsid to the key residues required for virus infection and offer molecular insight into the antibody-mediated HPV neutralization associated with type specificity.4.3; 50% effective concentration [EC50], 5.3 ng/ml) and MAb 28F10 against HPV59 VLPs  (Two murine MAbs—A12A3 (IgG2b) and 28F10 (IgG2a)—were raised against HPV L1-only VLPs derived from HPV58 and HPV59, respectively (see 1 ng/ml) . The typ1 ng/ml) .10.1128/mBio.00787-17.1FIG S1 FIG S1, TIF file, 2.5 MB.Characterization of MAbs A12A3 and 28F10. (A) Type specificity of MAb 12A3 and 28F10. (B and C) Binding of MAb 4B3 to pentamer and VLPs of HPV58 (B) and HPV59 (C). 4B3 is a wide-spectrum linear antibody that was employed to normalize the concentration of pentamer and VLPs used for the binding assays against A12A3 and 28F10, respectively. (D) Representative negative images for both VLP-Fab and VLP-IgG complexes of HPV58:A12A3 and HPV59:28F10. The red arrows indicate the bound Fabs or IgGs. Images are shown to indicate the both A12A3 and 28F10 IgGs are capable of cross-linking HPV58 and HPV59 VLPs, respectively. Download Copyright © 2017 Li et al.2017Li et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 50, 38.5 ng/ml for pentamer and 5.3 ng/ml for VLPs ) is defined as the antibody concentration for achieving 50% binding with the antigen.Wells of 96-well microplates were coated with wild-type or mutant VLPs or pentamers. After plate blocking , 2-fold serial dilutions of the antibody were added to the wells and incubated for 1 h at 37°C. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody  was used to detect the antibody titers, followed by 50 µl of 3,3′,5,5′-tetramethylbenzidine liquid substrate  per well for 30 min at 37°C. The absorbance at 450 nm  was recorded using an automated ELISA reader . Endpoint titers were defined as the highest plasma dilution that resulted in an absorbance value 2 times higher than that of nonimmune plasma with a cutoff value of 0.05. Data are presented as logThe presence of conformational neutralizing epitopes for L1 of different HPV58 and -59 mutant PsVs were determined using a MAb-based sandwich ELISA. The neutralizing MAbs C2D8 and 30A1 were used as capture antibodies, with C2A1-HRP and 24F4-HRP used as detection antibodies, for HPV6 and -11, respectively. These four MAbs are all conformational neutralizing antibodies raised against HPV58 VLPs (MAbs C2D8 and C2A1) and HPV59 VLPs (MAbs 30A1 and 24F4), respectively.280) of 1 in a 1.2-cm path length. The rotor speed was set to 30,000 rpm for all samples. The sedimentation coefficient was obtained using the c(s) method with the Sedfit Software . Samples were diluted to an optical density at 280 nm  . One flow cell of a chip was coated with 13,000 resonance units (RU) of the GAM-Fc, whereas the other flow cell was left uncoated and blocked as a control. The affinity measurements of MAbs A12A3 and 28F10 binding with HPV58 and HPV59 pentamers, respectively, were initiated by passing HBS  over the sensor surface for 100 s at 10 μl/min, followed by injection of 10 μg/ml of MAb A12A3 or 28F10 at 30 μl/min for 3.3 min and then injections of serially diluted antigens at 30 μl/min for 3.3 min. Every measurement on the Biacore 3000 biosensor was performed three times, and the individual values were used to produce the mean affinity constant and standard deviation.An excess of purified A12A3 and 28F10 Fabs were mixed with pentamers of HPV58 and HPV59, respectively, and incubated at 37°C for 2 h for complex formation. The complexes were purified over a Superdex 200  and concentrated to ~5 mg/ml. Crystals were grown by mixing 1 μl complex with 1 μl reservoir solution  using the hanging-drop vapor diffusion method at 20°C. Crystals were cryo-protected in the reservoir solution supplemented with 30% glycerol and flash-cooled at 100 K.http://www.hkl-xray.com). Initial phases were determined by molecular replacement with PHASER (1WEJ); we searched HPV59p:28F10 using two model templates: ensemble 1, the HPV59 L1 pentamer model (PDB no. 5J6R)  beamline BL17U using a Quantum-315r charge-coupled-device (CCD) area detector. All data sets were processed using the HKL-2000 program package . The Consurf server was used to generate the conservation scores (http://tree.bio.ed.ac.uk/software/figtree/).Multiple sequence alignment of the L1 proteins from 36 different HPV serotypes were calculated using Clustal Omega  on the En scores . All alin scores . The phyn scores  on the E2. Particles were manually boxed and extracted with the program Robem . The complexes were then incubated at 37°C for 2 h. An aliquot of a 2-μl sample was deposited onto a glow-discharged Quantifoil holey carbon grid . After 5 s of blotting to remove extra sample, the grid was plunge-frozen into liquid ethane using a FEI Vitrobot. Images were recorded on an FEI Falcon I direct detector camera at a 93,000 nominal magnification in an FEI TF30 FEG microscope at 300 kV, with underfocus settings determined to be between 1.0 and 3.0 µm using CTFFIND3 , and witam Robem . The oriam Robem , and an am Robem . After sam Robem , good paam Robem . The mapam Robem . The cryam Robem .50) of the supernatant was then measured to determine the titers of the PsVs, and the TCID50 values were calculated according to the classical Reed-Muench method (293FT cells for pseudovirus (PsV) production and the subsequent neutralization assay were obtained from American Type Culture Collection (ATCC). Wild-type (WT) or mutant HPV PsVs were produced as described previously , 76, 77.h method . Maturath method .4 cells per well for 6 h. Neutralizing antibodies at a certain concentration were subjected to a 2-fold dilution. PsVs were diluted to 2 × 105 TCID50/µl. Sixty microliters of the PsV diluent and 60 µl of the serially diluted neutralizing antibodies were mixed and incubated at 4°C for 1 h. The negative control was prepared by mixing 60 µl of the PsV diluent with 60 µl of the culture medium. Then, 100 µl of the mixtures described above was added to designated wells and incubated at 37°C for 72 h. Cells were then treated with trypsin and analyzed by flow cytometry. The median inhibitory concentration (IC50 [nanograms per milliliter]) is defined as the antibody concentration for achieving 50% inhibition of PsV.293 FT cells were incubated at 37°C in the wells of a 96-well plate at a density of 1.5 × 10Atom coordinates and structure factors for the HPV58 L1 pentamer, HPV58p:A12A3 Fab, and HPV59p:28F10 Fab have been deposited in the PDB . The electron microscopy (EM) density maps for HPV58v:A12A3 Fab and HPV59v:28F10 Fab have been deposited in the Electron Microscopy DataBank (EMDB) under EMD-6809 and EMD-6814, respectively.


E. coli and yeast Hsp104 are homologous molecular chaperones and members of the AAA+ (ATPases Associated with various cellular Activities) superfamily of ATPases. They are required for thermotolerance and function in disaggregation and reactivation of aggregated proteins that form during severe stress conditions. ClpB and Hsp104 collaborate with the DnaK or Hsp70 chaperone system, respectively, to dissolve protein aggregates both in vivo and in vitro. In yeast, the propagation of prions depends upon Hsp104. Since protein aggregation and amyloid formation are associated with many diseases, including neurodegenerative diseases and cancer, understanding how disaggregases function is important. In this study, we have explored the innate substrate preferences of ClpB and Hsp104 in the absence of the DnaK and Hsp70 chaperone system. The results suggest that substrate specificity is determined by nucleotide binding domain-1.ClpB of  All cells have a protein network involved in maintaining the proteome following periods of stress. Maintenance of the proteome utilizes energy-dependent molecular machines that facilitate protein remodeling, reactivation, disaggregation and degradation of misfolded, aggregated or inactive proteins. Members of the Clp/Hsp100 family of ATP-dependent AAA+ proteins are molecular chaperones found in bacteria, archea, and the organelles of metazoans. Hsp104 and ClpB are two members of the Clp/Hsp100 family and are found in yeast and bacteria, respectively, where they are essential for growth following extreme stress, such as high temperature  containing pClpBwt (pET24b vector) or pClpBE279A, E678A (pET24b vector) were grown at 30°C to OD600 of ~0.6 and then induced overnight with 0.1 mM IPTG. All purification steps were carried out at 4°C. Clarified cellular extracts were purified over a Q-Sepharose column  in 20 mM Tris-HCl, pH 7.5, 80 mM NaCl, 5 mM MgCl2, 0.5 mM EDTA, 20% glycerol (vol/vol) and 1 mM DTT. Proteins were eluted from the column with a linear gradient of 80–1,000 mM NaCl in the same buffer. Fractions containing ClpB were further purified using Sephacryl S-200 chromatography in 50 mM Tris-HCl, pH 7.5, 200 mM KCl, 10% glycerol, 20 mM MgCl2, 1 mM EDTA, and 1 mM DTT.ClpB wild-type and ClpBE285A, E687A  by electroporation. Transformed cells were plated on LB plates supplemented with 50 μg/mL ampicillin and 10 μg/mL chloramphenicol and grown overnight at 32°C. Transformations were optimized to yield several hundred colony-forming units on each plate. The fresh transformants were used to inoculate Hsp104 expression cultures as follows: 5 mL of LB broth was added to each plate and the cells were resuspended using a sterile glass or plastic rod; cells from a single plate were used to inoculate 1 L of LB broth supplemented with 100 μg/mL carbenicillin (chloramphenicol was not added) in a 2 L baffled flask. Typically, 2–4 L of culture were grown at the same time for one preparation. The cultures were incubated with shaking at 25°C and 250 rpm to an OD600 = 0.25; the incubator temperature was reduced to 18°C and Hsp104 expression induced with the addition of IPTG to a final concentration of 0.1 mM; growth was continued overnight (14–16 h) at 18°C with shaking at 250 rpm. Cells were harvested by centrifugation in a pre-chilled rotor at 5,000 × g  for 10 min at 4°C. The cell pellet from each 1 L culture was resuspended in 25 mL ice cold Q104 buffer  containing EDTA-free complete protease inhibitor cocktail (Roche), which was prepared by mixing 1 tablet/50 mL of Q104 buffer. The resuspended cells were lysed by two or three passages through an ice-cold French Pressure cell . The cell lysate was collected at the sample outlet tube with a vessel submerged in an ice bath. Cell debris was removed by centrifugation at 12,000 × g  for 15 min at 4°C. The resulting supernatant was then centrifuged at 130,000 × g  for 30 min at 4°C. As an alternative, a single centrifugation step at 34,500 × g  for 90 min at 4°C will produce similar results. The supernatant was used for subsequent purification. The supernatant must be subjected to the first column purification step without interruption or overnight storage or Hsp104 activity will be significantly reduced or lost. All purification steps were performed at 4°C using pre-chilled buffers. The clarified lysate was applied to a 20 mL Q-sepharose Fast Flow  column equilibrated with Q104 buffer at 1 mL/min using a peristaltic pump. The column was washed with two column volumes of Q104 buffer and protein was eluted with a 100 mL, 80–500 mM NaCl linear gradient in Q104 buffer. Column fractions of 3 mL each were collected. At this point, fractions containing Hsp104 can be stored at −80°C. Next, a 3 mL Q-sepharose Hsp104 peak fraction was applied onto a 40 mL Sephacryl S-200 High Resolution  column (1.5 cm I.D. × 30 cm length) equilibrated with SE104 buffer , 5 mM ATP) at 0.5 mL/min using a peristaltic pump. Fractions (1 mL) were collected and those containing purified Hsp104 were stored at −80°C. When thawed for use, individual fractions are divided into 100–200 μL aliquots and stored at -80°C to minimize the number of freeze-thaw cycles. Using this procedure, the Hsp104 activity is stable for at least 1 year.Hsp104 wild-type and Hsp104clpP- transformed with pNM-His were grown in LB (30 μg/mL Kan and 10 μg/mL Cam) at 37°C to an OD600 of ~0.6–0.8 and induced with 1 mM IPTG for 2 h. Cells were harvested by centrifugation and resuspended in 40 mM Hepes, pH 7.4, and lysed using a French Press. Urea was added to a final concentration of 8 M and the lysate kept at room temperature (~23°C) for the remaining preparation. Insoluble material was removed by centrifugation. NM-His was precipitated with the addition of MeOH to 70% (vol/vol) and the precipitate collected by centrifugation. The protein pellet was resuspended in NM Buffer  and then incubated with TALON resin for 30 min. The slurry was poured into an empty chromatography column and washed with 10 bed volumes of NM Buffer. NM-His was eluted with NM Buffer containing 50 mM Imidazole. NM-His containing fractions were precipitated with MeOH as above, the pellet was resuspended in 70% MeOH and the sample was stored at -80°C in small aliquots. NM-His was stable for ~6 months.NM-His was purified as previously described , an ATP regenerating system (20 mM creatine phosphate and 6 μg creatine kinase), 0.4 μM GFP or GFP fusion protein, 3.0 μM GroELTrap and 1 μM ClpB or Hsp104. GroELTrap is a mutant form of GroEL that binds but does not release unfolded proteins and was included in the reactions to prevent the GFP fusion proteins from refolding .Reaction mixtures (100 μL) contained buffer A , 0.005% Triton X-100 (vol/vol), 0.2 mg/mL BSA, 10 mM MgCl30-H6, or GFP-X7-H6 (0.4 μM) with or without ClpBE279A, E678A or Hsp104E285A, E687A (2 μM) were incubated in buffer A, 0.005% Triton-X100, 5 mM ATP, and 10 mM MgCl2 for 45 min at room temperature. Reaction mixtures were fractionated on a Sephacryl S200 column  equilibrated with 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 20 mM KCl, 0.1 mM EDTA, 10% glycerol, 5 mM ATP, and 10 mM MgCl2 at room temperature. Fractions (100 μL) were collected and GFP fluorescence was measured in a Tecan Infinite M200Pro plate reader at 25°C as described above. The percentage of the GFP fusion protein signal that was shifted upon chaperone binding was determined by calculating the area under the shifted peak compared to the total area under all peaks. The elution profile of ClpBE279A, E678A or Hsp104E285A, E687A (2 μM) was determined in the absence of GFP fusion protein by measuring protein in each fraction using the Bradford assay.Reaction mixtures (100 μL) containing GFP-15, GFP-X2, 2 mM DTT) with 5 mM ATPγS in the presence or absence of 0.5 μM ClpB or Hsp104 as previously described  was denatured at 42°C in Buffer B  were initiated by diluting denatured NM-His in 8 M urea  100-fold to a final concentration of 0.2 μM with assembly buffer  in the presence or absence of 0.5 μM ClpB or Hsp104 , 0.5 μM ClpB, 1.3 μM DnaK, 0.2 μM DnaJ and 0.1 μM GrpE or 0.5 μM Hsp104, 1.3 μM human Hsp70 (HSPA1A) and 0.2 μM Ydj1. GFP fluorescence was monitored over time at 23°C using a Varian Cary Eclipse fluorescence spectrophotometer with a plate reader. Excitation and emission wavelengths were 395 and 510 nm, respectively. Reactivation was determined compared to a non-denatured GFP-38 control.GFP-38 disaggregation was performed as previously described  were used for these experiments because they bind but do not hydrolyze ATP and therefore limit the protein remodeling pathway to substrate interaction  was ~4-fold higher for ClpB than for Hsp104 with this substrate  at a faster rate than Hsp104 while Hsp104 unfolded two (GFP-X30-H6 and GFP-X42-H5) faster than ClpB (Figure In our unfolding studies, we observed that ClpB and Hsp104 discriminate between GFP fusion proteins with different polypeptide tags fused at an end. Three of the substrates tested share almost the same 13 C-terminal residues, however, ClpB unfolded one (GFP-XThe studies presented here using chimeras of Hsp104 and ClpB indicate that the N-domain of Hsp104 and ClpB does not affect the substrate discrimination observed with the two substrates tested. Previous studies addressed the role of the ClpB N-domain in substrate binding and unfolding and showed that the N-domain of ClpB is important for stabilizing ClpB and interaction with substrate (Nagy et al., Understanding the mechanism of the intrinsic chaperone activity of ClpB/Hsp104 is providing the groundwork for understanding the more complex and biologically important reaction carried out by ClpB/Hsp104 in physical and functional collaboration with DnaK/Hsp70.DJ, MM, JH, SW, and SD designed the experiments. DJ, MM, JH, and SD performed the experiments. All authors were involved in data interpretation and discussion. SW and SD wrote the manuscript with contributions from JH.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.