The composites were analyzed by various techniques. According to the obtained results, Ag3PO4 QDs with a size of 1–4 nm were uniformly dispersed on rGO nanosheets to form rGO/Ag3PO4 QD composites. The photocatalytic activity of rGO/Ag3PO4 QD composites was evaluated by the decomposition of methylene blue (MB). Meanwhile, effects of the surfactant dosage and the amount of rGO on the photocatalytic activity were also investigated. It was found that rGO/Ag3PO4 QDs (WrGO:Wcomposite = 2.3%) composite exhibited better photocatalytic activity and stability with degrading 97.5% of MB within 5 min. The improved photocatalytic activities and stabilities were majorly related to the synergistic effect between Ag3PO4 QDs and rGO with high specific surface area, which gave rise to efficient interfacial transfer of photogenerated electrons and holes on both materials. Moreover, possible formation and photocatalytic mechanisms of rGO/Ag3PO4 QDs were proposed. The obtained rGO/Ag3PO4 QDs photocatalysts would have great potentials in sewage treatment and water splitting.In this study, a novel graphene/AgThe online version of this article (10.1186/s11671-018-2466-9) contains supplementary material, which is available to authorized users. Al. Al3PO4 05) Fig. . The R-2equation . No diff3PO4 QDs which a relatively narrow size distribution with a diameter of 2.81 ± 1.2 nm were dispersed uniformly on rGO sheet. The lattice spacing was 0.212 and 0.190 nm, which corresponded to the d-spacing of (220) and (310) crystallographic plane of Ag3PO4, respectively. To investigate the effects of ultrasonic, conventional stirring was performed instead of ultrasonic treatment. The results were shown in Additional file 3PO4 particles on rGO which was formed by conventional stirring method did not show uniform structure, and the size of Ag3PO4 became larger than that formed by ultrasonic treatment. The above results indicated that ultrasonic treatment was very effective in dispersing and controlling size of Ag3PO4 particles on rGO layers [Figure O layers .Fig. 2TE+ of Ag3PO4 [3PO4 (529.5 eV) and O1s from rGO (531.3 eV) [3PO4 QDs by C=O bond. The C1s spectrum of GO was divided into three different peaks at 284.8, 286.7, and 287.7 eV, which were assigned to C-C/C=C, C-O, and C=O, respectively [The successful ultrasonic-assisted photo-reduction of GO to rGO can be further confirmed by XPS spectra of GO and R-2.3 composites as shown in Fig. f Ag3PO4   , 39. Theectively , 41  shifted to lower wavenumbers compared to GO, which was consistent with the results of XPS analysis, indicating the existence of charge interaction between rGO and Ag3PO4 in the as-prepared composites.Figure  and C=C , 42, resibration . Ag3PO4 om PO43− . This in3PO4 QDs, and R-2.3. The Raman spectrum of GO showed two characteristic peaks of the D band at 1347 cm− 1 and G band at 1586 cm− 1. The value of ID/IG in R-2.3 and in GO was about 1.039 and 0.9056, respectively. It was obvious that the composite showed relatively high intensity of the D band compared with GO, which confirmed that the GO sheets were partially reduced into rGO [3PO4 QDs and R-2.3 showed three distinct peaks at 410, 566, and 714 cm− 1, and these peaks were accredited to the P-O-P bonds. The strong peak at 907 cm− 1 was raised from the motion of terminal oxygen bond vibration in phosphate chains [Figure into rGO . The Rame chains .3PO4 QD composite was proposed and schematically illustrated in Fig. The synthesis route of rGO/Ag+ and oleate ions interacted electrostatically to form Ag-oleate complexes, hydrolysis of Ag+ ions could be prevented effectively by this process. Ag-oleate complexes interacted with the excess of oleate ions improving its hydrophilic property in order to disperse in water. Oxygen groups on the surface of GO provided hydrophilic property. When GO sheets were added to the Ag- oleate aqueous solution, the Ag-oleate complexes will preferentially adsorb on these oxygen containing functional groups  of Ag3PO4. As a result, rGO/Ag3PO4 QD composite was obtained by photo-ultrasonic-assisted reduction.The total synthesis route could be fallen into four successive stages. Firstly, Agups (Eq. ). Secondace (Eq. ). Thirdl3PO4 QDs and rGO/Ag3PO4 QDs with different mass ratio of rGO were shown in Fig. 3PO4 QDs was shorter than 530 nm; inversely, rGO/Ag3PO4 QD composites structure showed an extended wavelength (> 530 nm) and its intensity increased with increasing rGO contents before which reached 2.3%, and decreased after. This can be attributed to that the presence of carbon in rGO/Ag3PO4 QDs reduces the reflection of light [3PO4 QDs (2.23 eV). The relative narrow band gap energy may be attributed to the synergetic effect that sum of the total effect is superior to the single effect after different types of dispersion to interact between rGO and Ag3PO4 QDs [The UV-vis absorption spectra of Agof light . Accordiof light , we can 3PO4 QDs , which l3PO4 QDs .Fig. 6a 3PO4 QDs size distribution on rGO surface [3PO4 QDs, the concentration of MB decreased rapidly for rGO/Ag3PO4 QD composites  could transfer from the CB of Ag3PO4 QDs to rGO, and rGO in the composites could act as a highway for electron transfer to suppress the e−-h+ recombination, which accounted for the remarkably improved photo-conversion efficiency [To understand the influencing factors on the experimental process to the photocatalytic activity, the effects of different mass of surfactant were investigated as shown in Additional file  surface . This letes Fig. . This reig. 3PO4 . Importamposites , 50. Theficiency . Moreoveficiency , 53. On −-h+ separation. Moreover, the XRD pattern of R-2.3, which was used for five cycles is shown in Fig. 3PO4 QDs and decreased the photocorrosion of Ag3PO4 QDs [To test the stability of the R-2.3 composite, the cycling experiments of the composite for MB were performed Fig. . The res3PO4 QDs .3PO4 was enhanced by combining Ag3PO4 with rGO sheets, which was ascribed to the fast transfer and separation of photo-generated e−-h+ pairs in the composites [−-h+ pairs migration, transfer, and recombination processes in semiconductors [−-h+ pairs compared to Ag3PO4 QDs, indicating that more photogenerated e− and h+ can participate in the reduction and oxidation reaction; this could lead to decline of the recombination of photogenerated e−-h+ pairs in Ag3PO4 in the composites. Therefore, rGO/Ag3PO4 QD composite displayed superior photocatalytic activity than that of Ag3PO4 QDs.The aforementioned experimental results indicated that the photocatalytic performance of Agmposites . The phonductors , 55. Fig3PO4 QDs, MB was used as a pollutant. The results are shown in Fig. 3PO4 QDs was not obviously affected; when EDTA (as hole capture) [2·− scavenger) was added, the deactivation of rGO/Ag3PO4 QDs was unneglectable. The above results illustrated that holes and O2·− radicals were the main active species in the photocatalysis process.To further confirm the main active species in the photocatalysis process over rGO/Agcapture)  was adde3PO4 QDs is shown in Fig. 3PO4 QDs was photoexcited, and electrons were excited from valence band to conduction band; after that the electrons could transfer to rGO due to effect of the electric field, and then electrons retransferred to the surface of rGO to participate in the photocatalytic reaction. rGO could efficiently separate e−-h+ pairs, thus availed the transfer of the electrons [3PO4 QD composites.The mechanism for the photocatalytic degradation of organic dyes by rGO/Aglectrons  and led 3PO4 QD composite was prepared via a facile photo-ultrasonic-assisted reduction method. The obtained rGO/Ag3PO4 QD composites exhibited better photocatalytic activity under visible light and higher than pure Ag3PO4 QDs alone. This was due to the efficient e−-h+ pairs separation and fast electron transfer in these heterojunctions. The rGO sheets effectively promoted the separation of e− and h+ and fast transfer of electrons in the heterostructure photocatalysts. Free radicals trapping experiments indicated that h+ played important roles in the photocatalytic degradation of dyes. It was clear that ultrasonic-assisted method was a facile and economical way to prepare visible-light-responsive and high efficient Ag3PO4 QDs-based composites.A novel rGO/AgAdditional file 1:Figure S1. TEM images of rGO/Ag3PO4 QDs (stirring method). Figure S2. The plots of (αhν)2 versus Eg of Ag3PO4 QDs, R-1.5, R-2, R-2.3, R-2.5, and R-3. Figure S3. (a) Photocatalyticdegradation of MB by R-2.3 prepared by different mass of surfactant and (b) apparent rate constants (k) of samples for photocatalytic degradation of MB. Figure S4. (a) Photocatalytic degradation of MB, MO, and RhB byR-2.3, (b) apparent rate constants (k) of sample for photocatalytic degradation of dyes. (ZIP 12230 kb)


Increasing evidence indicates that PIM1 is a potential prognostic marker and target for cancer treatment but its precise mechanisms of action remain to be determined in salivary adenoid cystic carcinoma (SACC). This study aims to decipher the prognostic and mechanistic role of PIM1 in progression of SACC cells and tumor tissues.A SACC cell line (ACC-M) was transfected with shRNA plasmids targeting the PIM1 gene. The expression levels of PIM1, RUNX3 and p21 were measured by quantitative real-time PCR and western blot. Subcellular translocalization of RUNX3 and p21 proteins was assessed using immunofluorescence, and cell cycle phase was quantified using flow cytometry. A total of 97 SACC patients were retrospectively analyzed by clinicopathologic characteristics and survival outcomes.After down-regulation of PIM1 in ACC-M cells, RUNX3 and p21 proteins were translocated from cytoplasm to nucleus, with a decrease of p21 expression and increase of G0/G1 phase cells. PIM1 and RUNX3 levels show a distinct covariance. PIM1 is associated with T-status, lymph node involvement, nerve invasion, and distant metastasis in SACC tissues. Patients with low PIM1 level had a better outcome than those with higher PIM1 level.PIM1 is multifunctional in ACC-M cells and it serves as a neoteric therapeutic target and potential prognostic marker for SACC patients. Adenoid cystic carcinoma (ACC), a common salivary gland cancer subtype, accounts for 22% of salivary gland malignancies in humans . SalivarPIM1, identified as a preferential integration site for moloney murine leukemia virus to induce T-lymphomas in mice , belongsPIM1 is thought to be constitutively active without the need for posttranslational modification; the level of its kinase activity therefore likely depends on the absolute amount of protein in cells . The preOne potential mechanism for PIM1 in SACC cells is phosphorylation of p21. It is known that the p21 gene encodes a 21kD protein which binds and inhibits a wide range of cyclin/CDK complexes and predominantly blocks G1/S phase cell cycle transition. Also, p21 interacts with proliferating cell nuclear antigen (PCNA) in the nucleus to inhibit DNA replication , 24. MorThe RUNX3 tumor suppressor gene encodes a transcription factor that regulates lineage-specific gene expression in developmental processes and is involved in the formation of multiple cancers. The expression level of RUNX3 can be a prognostic indicator in variety of malignancies . Our prePIM1 is involved in multiple cellular activities and it is likely that PIM1 has more unidentified substrates. As we know, PIM1 regulates the subcellular localization of p21  and RUNXE. coli. We selected 3 clones from each plate for growth in LB liquid medium containing 50 mg/L kanamycin overnight at 37 °C. Clones were analyzed by sequencing . The four target sites for PIM1 shRNA are shown below:PIM1-shRNA-1, 5′-AACATCCTTATCGACCTCAATCGCG-3′;PIM1-shRNA-2, 5′-GTCTCTTCAGAATGTCAGCAT-3′;PIM1-shRNA-3, 5′-GGATCCTGCTGTATGATATGG-3′;PIM1-shRNA-4, 5′-GGGTTTCTCCGGCGTCATTAG-3′.The plasmid pGPU6/GFP/Neo was purchased from GenePharma and the negative control plasmid (no homology to disrupt the sequence) was provided by GenePharma. We designed and synthesized four pairs of inference shRNA oligos . Annealed shRNA oligos were ligated into the pGPU6/GFP/neo vector before transformation into 5 cells/well. After 24 h, transfection complexes were assembled as follows: 4 μg of pGPU6/GFP/Neo-shRNA plasmid or negative control plasmid was resolved in 400 μL RPMI 1640 medium (serum-free and antibiotic-free). To this, 9 μL Lipofectamine™ 2000 transfection reagent was added, thoroughly mixed, and allowed to stand at room temperature for 10 min to form transfection complexes. The transfection mix was added to wells and cells were cultured at 37 °C in a 5% CO2 incubator. The cells were collected 24–48 h after transfection.ACC-M was purchased from the Cell Bank for Type Culture Collection, Chinese Academy of Sciences , and cultured in RPMI-1640 medium (Gibco) supplemented with fetal calf serum (10% Hyclone), penicillin (100 U/mL) and streptomycin (100 μg/mL). ACC-M cells were seeded in a 6-well culture plate at a density of 1 × 10PIM1 forward, 5′-TGTGCTGGGAGAAATACTTGAA-3′ and reverse, 5′-AGGTGGCTCAGCGTTTGG-3′ (size: 160 bp);p21 forward, 5′-GGAGACTCTCAGGGTCGAAAACG-3′ and reverse, 5′-GAGAAGATCAGCCGGCGTTTG-3′ (size: 77 bp);RUNX3 forward, 5′-CCAAGGCACCTCGGAACTGAAC-3′ and reverse, 5′-TCTCCGTGAGGGTTGGCAGC-3′ (size: 83 bp);GAPDH forward, 5′-CATGAGAAGTATGACAACAGCCT-3′ and reverse, 5′-AGTCCTTCCACGATACCAAAGT-3′ (size: 113 bp).RT-PCR and qRT-PCR were performed as described previously . The fol−∆∆Ct method was performed to analyze the relative quantification of transcript expression.After PCR amplification, dissociation of SYBR Green-labeled cDNA  was carried out to affirm that there were no nonspecific PCR products. 2Cells were harvested at 80% confluence and lysed by suspension in RIPA lysis buffer followed by gentle sonication. Protein lysates (20 μg) were separated on 8% SDS polyacrylamide gel by electrophoresis and transferred to PVDF membrane (Millipore). Membranes were then blocked in PBS/Tween (PBS with 5% non-fat powered milk and 0.05% Tween 20) at room temperature for 2 h. The blot was then incubated with affinity-purified rabbit anti-PIM1  overnight. The membranes were washed 3–4 times with PBS/Tween and incubated with HRP-conjugated goat anti-rabbit IgG  for 2 h at room temperature. After repeating the washing steps, the signal was detected by chemiluminescence using the Pierce Super Signal West Pico Systems (Pierce).6/mL) in logarithmic growth phase were plated in 6-well plates and transfected as described above. The cells were fixed with ice-cold 70% ethanol at − 20 °C for 12–24 h. The cells were collected after centrifugation and resuspended in 500 μL of PBS buffer containing RNase A  and incubated at 37 °C for 30 min. Five microliter PI was added and incubated for 30 min at room temperature in dark. Data were acquired by flow cytometry .ACC-M cells , cells were washed three times in PBS and then permeabilized with 0.1% Triton X-100 in PBS for 2 min at room temperature. Samples were blocked with 3% BSA and 0.05% Tween 20 in PBS (blocking solution) for 30 min at room temperature and then incubated overnight at 4 °C with primary antibodies , respectively. For the immunofluorescence staining method, cells were incubated with secondary antibodies (1 µg/mL), DyLight 488 AffiniPure Goat Anti-Mouse IgG (H+L) (EarthOx) at 1:100 for Novus NB200-303 and DyLight 594 AffiniPure Goat Anti-Rabbit IgG (H+L) (EarthOx) at 1:100 for Novus NBP1-40501 and Novus NBP1-46694, There secondary antibodies were conjugated to Alexa-488 green fluorescence . Cells were counterstained with 4′,6-diamidino-2-phenylindole . Samples were visualized by confocal microscopy at 63× magnification.Ninety-seven patients with histopathologically proven SACC in Zhejiang Cancer Hospital between July 2002 and July 2014 were recruited for this study. The study was approved by the Ethics Committee of Zhejiang Cancer Hospital. All patients have signed the informed consent.2O2/methanol, the slides were incubated with antibody for PIM1  and RUNX3  at 4 °C overnight. The immunoreactivity was visualized by using a streptavidin–biotin–peroxidase staining kit  and DAB solution . The results were presented as percentage of nucleus staining positive cells relative to total cells. The scores of staining results were given as negative and positive. In brief, IHC score was determined by combining staining frequency and intensity. In detail, the staining frequency score was defined as no cell stained scored as 0, 1–10% of cells stained as 1, 11–50% of cells stained as 2, 51–80% of cell stained as 3, and 81–100% of cell stained as 4. Staining intensity score was rated on a scale of 0–3, with 0 for negative; 1 for weak; 2 for moderate; and 3 for strong staining. Theoretically, the scores could range from 0 to 12. An IHC score of 9–12 was considered as strong immunoreactivity, 5–8 as moderate, 1–4 as weak and 0 as completely negative. Sections in which the staining could not be well characterized were considered as equivocal. Staining was scored independently by two pathologists who were blinded to each other’s findings. All conflicting calls on scoring were adjudicated by a third individual. We used IHC score 5–12 for normal to high expression (positive) and 0–4 for no to low expression (negative).Paraffin-embedded tissues were cut for 4 μm sections mounted on glass slides  and dried overnight at 37 °C. After deparaffinization, antigen retrieval in 0.01 M citrate buffer, and inactivation of endogenous peroxidase activity in 3% H2 test or the Fisher exact test. Survival analysis was carried out by the Kaplan–Meier method and significant differences were assessed by means of the log-rank test. p values < 0.05 were considered to be statistically significant.SPSS 22.0 was used to perform one-way ANOVA for the discrepancy of three groups or more. The two-tailed Student’s t-test was used to detect any statistically significant difference between two groups. Associations between PIM1, RUNX3 levels, and clinicopathologic parameters were analyzed by using the χIn this study, we used Lipofectamine™ 2000 reagent to optimize the transfection condition. The pGPU6/GFP/Neo-shRNA vector can express green fluorescent protein after being transfected into ACC-M cells. By fluorescence microscopy, we found that the total transfection rate was more than 70% after transfected for 48 h.p < 0.05) in all interference groups. The PIM1-shRNA-3 transfection achieved the most effective knockdown of PIM1. Western blot analysis showed that the protein expression of PIM1 in ACC-M transfected with shRNA for 24 and 48 h was also decreased. PIM1-shRNA-3 and PIM1-shRNA-4 were most effective and thus used in the following experiments.Both PIM1 mRNA and protein levels were significantly decreased in ACC-M cells after shRNA transfection. As shown in Fig. As shown in Fig. p < 0.05), while cells in G2/M stage and S stage decreased from 26.4 to 17.7% and 30.8 to 24.5%, respectively. Transfection with PIM1-shRNA-4 produced similar results; cells in G0/G1 stage increased from 42.3 to 60.5% (p < 0.05), while cells in G2/M stage and S stage decreased from 26.4 to 13.6% and 30.8 to 24.7%, respectively. These data indicated that shRNA transfection in ACC-M cells caused inhibition in the G1 phase, thus resulting in decreased numbers of cells in the S phase  staining in SACC tissues is shown in Fig. p < 0.05).We analyzed the correlation between PIM1 and RUNX3 expression in SACC tissues and clinicopathologic indexes. Table p = 0.01). Patients with low PIM1 levels had much better outcome than those with higher PIM1 levels.Kaplan–Meier survival curves Fig.  indicateThe PIM1 oncogene has been implicated in various human cancers including lymphomas, gastric, colorectal, and prostate carcinomas . Highly In this study, the pGPU6/GFP/Neo-shRNA was used as an optimized expression vector together with green fluorescent protein (GFP) reporter gene to deplete PIM1 levels in SACC cells. Using RNA interference technology, we successfully constructed shRNA recombinant plasmids targeting PIM1 gene expression. After transfection, silencing of PIM1 gene in ACC-M cells was evidenced by decreased PIM1 mRNA and protein expression. Silencing of the PIM1 gene caused ACC-M cell cycle arrest in G1 phase and decreased the proportion of cells in S phase.Similar to other tumor types, interactions exist between oncogenes and tumor suppressor genes involved in the development and maintenance of SACC. Here, we propose a model wherein PIM1 kinase affects expression and subcellular localization of tumor suppressor genes in SACC. Our model is supported by data showing two suppressor genes, p21 and RUNX3, are affected by PIM1 kinase knockdown in ACC-M cells. The p21 protein is one of the most studied inhibitors of cell cycle-dependent kinase (CDK). p21 arrests the cell cycle in G1 phase by inhibiting the CDK activity in G0 phase . RUNX3 gThe present study indicated that the mRNA and protein expression of PIM1 and p21 was decreased in pGPU6/GFP/Neo-shRNA-PIM1 transfected cells, while the mRNA and protein expression of RUNX3 was not affected. The knockdown of PIM1 induced nuclear localization of p21 and RUNX3. The p21 and RUNX3 proteins are involved in multiple cellular functions, especially in cell differentiation and apoptosis. Most of these functions depend on the interaction of p21 and RUNX3 with associated proteins in distinct cellular compartments. The redistribution of p21 and RUNX3 in ACC-M cells may activate cellular apoptosis. Our data suggests that silencing PIM1 regulates p21 and RUNX3 subcellular distribution and influences multiple cellular functions.The IHC results show that a higher PIM1 and lower RUNX3 level is associated with advanced T-stage, lymph node involvement and nerve invasion that translates to aggressive tumor behavior. Importantly, other clinical features including gender, age, tumor location and histological grade type showed no correlation. Higher PIM1 levels are significantly associated with distant metastasis. This evidence suggests the important role of PIM1 in SACC, most likely mediated by RUNX3.The short hairpin RNA expression vector for PIM1 silencing was successfully constructed, and pGPU6/GFP/Neo-shRNA-PIM1 could knockdown the expression of PIM1 gene and regulate the cell cycle. The tumor suppressors p21 and RUNX3 were affected by PIM1 kinase knockdown, which may provide a partial explanation for the pleiotropic effects of this multifunctional kinase. Our study indicates that PIM1 could be a potential prognostic marker for SACC and provides an experimental basis for SACC target therapy although the mechanism needs further elaboration and exploration. Therefore, more studies are needed in order to define mechanisms of PIM1 expression and function in SACC.Our studies demonstrate that down regulation of PIM1 expression effectively decreases p21 expression and disrupts cell cycle. The subcellular re-localization of RUNX3 and p21 caused by PIM1 knockdown may partially explain diverse cellular functions of PIM1. PIM1 serves as a neoteric therapeutic target and potential prognostic marker for SACC patients.


Ornithoctonus huwena (O.huwena) crude venom found that the venom can block neuromuscular transmission of isolated mouse phrenic nerve-diaphragm and sciatic nerve-sartorius preparations. However, little is known about its electrophysiological effects on cardiac myocytes. In this study, electrophysiological activities of ventricular myocytes were detected by 100 μg/mL venom of O.huwena, and whole cell patch-clamp technique was used to study the acute effects of the venom on action potential (AP), sodium current (INa), potassium currents  and L-type calcium current (ICaL). The results indicated that the venom prolongs APD90 in a frequency-dependent manner in isolated neonatal rat ventricular myocytes. 100 μg/mL venom inhibited 72.3 ± 3.6% INa current, 58.3 ± 4.2% summit current and 54 ± 6.1% the end current of IKr, and 65 ± 3.3% ICaL current, yet, didn't have obvious effect on IKs, Ito1 and IK1 currents. In conclusion, the O.huwena venom represented a multifaceted pharmacological profile. It contains abundant of cardiac channel antagonists and might be valuable tools for investigation of both channels and anti- arrhythmic therapy development.Spider venoms are known to contain various toxins that are used as an effective means to capture their prey or to defend themselves against predators. An investigation of the properties of  INa is responsible for the phase 0 depolarization and ICaL is responsible for maintaining plateau (phase 2) of the action potential. The outward currents are carried by four prominent K+ channels, containing the transient outward K+ current Ito1, the rapidly and slowly activating delayed rectifier K+ currents (IKr and IKs) and the inward rectifier K+ current IK1, in cardiac ventricular myocytes. These K+ currents contribute to repolarization of different phases of the action potential }, where Vm is the membrane potential, V1/2 is the half-activation or half-deactivation potential, and K is the inverse slope factor (in mV) reflecting the steepness of the voltage dependence of gating. For G-V curves, Y represents the relative conductance (G/Gmax). Data are given as means ± SE. All experiments were performed at room temperature (23 ± 0.1 °C), except for AP recordings being performed at 30 °C.Patch-clamp data were processed in Clampfit 10.0 and then analyzed in Excel and Origin 9.0. Data for voltage-dependence of activation were fitted to the Boltzmann equation: Y = 1/{1+exp[2(V 90 and APD50 in ventricular myocytes at the frequency of 1 Hz. APD 90 increased from 259.2 ± 12.1 ms to 398.3 ± 14.5 ms (P < 0.01), while APD50 increased from 190.4 ± 11.2 ms to 312.7 ± 10.6 ms (P < 0.01). Notably, action potential amplitude (APA) did not change after the venom addition and washout (P < 0.01) and APD50 increasing from 175.1 ± 8.6 ms to 201.6 ± 10.1 ms. ∆APD 90-50 is the difference between APD90 and APD50 recorded at a constant frequency (∆APD90–50 = APD 90 – APD50). 100 μg/mL venom greatly prolonged ∆APD90–50 to 85.6 ± 10.3 ms compared with the control (∆APD 90–50 = 68.8 ± 12.2 ms) at the frequency of 1 Hz.To test the effect of the venom on action potential duration (APD) in isolated neonatal rat ventricular myocytes, APDs before and after the treatment of 100 μg/mL venom were determined . The dat washout . At the Na was elicited by pulses to -30 mV from a holding potential of -120 mV in in rat ventricular myocytes. As shown in Na currents by 72.3 ± 3.6%, (n>8), indicating that cardiac INa channels antagonists indeed existed in the venom. The current-voltage (I-V) curves before and after the venom treatment showed that the inhibition did not associate with evident changes in the I-V relationships of the cardiac INa currents  = -41 ± 0.4 mV for control and V 1/2 = −43 ± 0.9 mV for 100 μg/mL venom) in Na antagonists with therapeutic potentials from the venom of O.huwena.Nav1.5, as the main voltage-gated sodium channel on ventricular myocytes, generates the fast depolarization of the cardiac action potential and plays a key role in cardiac conduction . INa wascurrents , C and D+ channels contribute to the regulation of ventricular repolarization, including transient outward K+ current (Ito1), the rapid (IKr) and slow (IKs) components of the delayed rectifier current and the inward rectifier current (IK1). Here, we analyzed the effects of O.huwena venom on ventricular myocytes K+ currents.Ventricular myocytes Kto1 is responsible for the initial rapid repolarization (phase 1) and determines the height of the early plateau, thus influencing the activation of other currents that control repolarization, mainly including ICaL and the delayed rectifier K+ currents (IK) [to1 evidently (6.8 ± 4.1%) and not affect the half-maximal activation potential (V1/2), ranged from 26.5 ± 0.3 Mv in control to 27.1 ± 0.4 mV in the presence of the venom. It may indicate that the venom has no significant effect on the activation of Ito1 current in rat ventricular myocytes (Ints (IK) . As showmyocytes  and E.F+ currents (IKs) and rapidly activating delayed rectifier outward K+ currents (IKr). Here, class III antiarrhythmic agent dofetilide (1 μM), a selective blocker of IKr, were used to inhibit IKr and then IKs was recorded independently. Ks assessment in NRVMs by a 3-s-long voltage-clamp pulse protocol. The slowly developing outward current was exhibited in Ks with an inhibition of 8.3 ± 4.4%, without changing the I-V curves.IK comprises two distinct current components: slowly activating delayed rectifier outward K+ current (IK1) diminishes in the adult heart failure, which is the onset of arrhythmias. The strong IK1 is critical for stabilizing the membrane potential in ventricular myocytes. Raw traces before and after 100 μg/mL venom treatment were shown in K1 in NRVMs, leading to approximately about 10.4 ± 5.2% (n >5) reduction of I K1 currents, no changes of the half-maximal activation potential (V1/2) were observed  as described previously [+ currents obtained from a single cardiomyocyte. From a holding potential of -80 mV, depolarizations in a 10-mV increments to voltages between -70 and +80 mV for 1.5 s were applied to evoke currents. Depolarizing steps to voltages above 0 mV induced outward currents, which inactivated in a voltage-dependent manner. The following tail currents at -80 mV displayed an initial rising phase, which is usually described as a “hook,” reflecting the rapid recovery of inactivated channels to the open state before deactivation, and is unique to IKr [+ carried IKr recorded from cardiomyocyte before and after the application of 100 μg/mL venom and the venom inhibited peak currents, currents at the end of 1-s depolarizing steps and the tail currents by 58.3 ± 4.2%, 54 ± 6.1% and 8.3 ± 3.7%, respectively. The I-V relationships of peak currents and currents at the end of 1-s depolarizing step were shown in 1/2) of tail currents (from -38.2 ± 1.7 mV in control to -38.8 ± 1.4mV in the presence of the venom).E-4031-sensitive Ieability . We recoeviously ,22. Figue to IKr . Figure Kr significantly and inhibited other cardiac potassium currents  slightly.In conclusion, our study showed that the venom inhibited I2+ currents (ICaL) are increased during hypertrophy and heart failure [O.huwena on ICaL in NRVMs. The ICaL current was evoked at a test potential of 0 mV for 150 ms from a holding potential of -40 mV. Typical L-type ICa recordings before and after venom treatment are shown in CaL currents by 65 ± 3.3%, without any alteration in the form of the I-V curve , rapid activating delayed rectifier outward K+ current (IKr), slowly activating delayed rectifier outward K+ current (IKs), transient outward K+ current (Ito1), inward rectifier K+ current (IK1) and L-type calcium current (ICaL) . These cThe O.huwena venom prolonged both APD90 and APD50 in ventricular myocytes at both 1 Hz and 2 Hz, but the effect of the venom at 1 Hz frequency is more effectively than 2 Hz. The similar result also observed in Verkerk's study which showed that DIDS  increased APD only at 3.33 Hz, but not at the lower stimulus frequencies . MoreoveCardiac INa contributes to initial rapid repolarization in AP, the inward depolarizing currents (ICaL) plays an important role on the balance of plateau phase in AP. We found that O.huwena venom inhibited the two major inward currents generating the ventricular AP, without affecting the upstroke phase. Although Phase 0 is defined by the activation of voltage-dependent Na+ channels giving rise to inward movement of Na+, the inhibitors of Na+ currents did not affect the AP as various studies described . EfonidiIn conclusion, our work increased the knowledge about the electrophysiological effect of O.huwena venom on the action potential (AP), cardiac Na+, K+ and Ca2+ channels, and indicated that spider venom, containing abundant of cardiac channel antagonists, might be valuable tools for investigation of both channels and anti- arrhythmic therapy development.


Trichoderma reesei RUT C30 serves as a well-known industrial workhorse for cellulase production. Therefore, the enhancement of cellulase production by T. reesei RUT C30 is of great importance.Cellulase can convert lignocellulosic feedstocks into fermentable sugars, which can be used for the industrial production of biofuels and chemicals. The high cost of cellulase production remains a challenge for lignocellulose breakdown. ace2-VP16 and DBDcre1-VP16) were designed and expressed in T. reesei RUT C30. Expression of DBDace2-VP16 and DBDcre1-VP16 improved cellulase production under induction (avicel or lactose) and repression (glucose) conditions, respectively. The strain TMTA66 under avicel and TMTA139 under glucose with the highest cellulase activities outperformed other transformants and the parental strain under the corresponding conditions. For TMTA66 strains, the highest FPase activity was approximately 1.3-fold greater than that of the parental strain RUT C30 at 120 h of cultivation in a shake flask using avicel as the sole carbon source. The FPase activity (U/mg biomass) in TMTA139 strains was approximately 26.5-fold higher than that of the parental strain RUT C30 at 72 h of cultivation in a shake flask using glucose as the sole carbon source. Furthermore, the crude enzymes produced in the 7-L fermenter from TMTA66 and TMTA139 supplemented with commercial β-glucosidase hydrolyzed pretreated corn stover effectively.Two sets of novel minimal transcriptional activators  contains supplementary material, which is available to authorized users. Trichoderma reesei, which produces the enzymes necessary for the complete hydrolysis of lignocellulose, has been widely used for the production of commercial cellulase [T. reesei RUT C30 has been proven to be a cellulase hyper-producer [T. reesei RUT C30 as a cellulosic biorefinery is of great importance.Lignocellulosic feedstocks are abundant and renewable resources in nature. They have been used to produce environment-friendly biofuels and chemicals, which have received increased attention for research . The conellulase . T. reesproducer . TherefoT. reesei RUT C30 mainly comprise two cellobiohydrolases (CBHI and CBHII), two endoglucanases (EGI and EGII), and β-glucosidase I (BGLI) that synergistically hydrolyze lignocellulosic materials, together with related xylanases and auxiliary proteins [T. reesei, CBHI and CBHII account for approximately 50–60 and 10–15%, respectively [T. reesei [The cellulase produced in proteins –9. Amongectively . BGLI isectively . XYNI an. reesei . Additio. reesei , 15.T. reesei is regulated by a combination of transcription factors such as XYRI, ACEII, and CREI. Among these factors, XYRI (xylanase regulator 1) is recognized as the primary transcriptional activator with a GAL4-like Zn(II)2Cys6 binuclear cluster domain [2Cys6 DNA-binding domain, which is responsible for the regulation of cellulase-encoding genes in T. reesei under induction conditions [T. reesei RUT C30 is inhibited by CREI when glucose is present [Expression of cellulase and xylanase in r domain . Similarnditions . On the  present .T. reesei [T. reesei strain U3 with enhanced cellulase production by transforming an artificial zinc finger protein library. Similarly, an artificial transcription activator linking the CREI-binding domain with the C-terminus of XYRI was shown to improve cellulase production in the recombinant strain T. reesei zxy-2 with glucose as the sole carbon source [In addition to protein engineering of cellulase chimeras to enhance specific activity , increas. reesei . Zhang e. reesei  screenedn source .ace2-VP16 and DBDcre1-VP16 were transformed into T. reesei RUT C30 to replace the native regulators ACEII and CREI, respectively, in the genome by homologous double exchange. We then investigated the effects of these minimal transcriptional activators (DBDace2-VP16 under induction conditions and DBDcre1-VP16 under repression conditions) on cellulase and xylanase generation. We suggest that the minimal transcriptional activators DBDace2-VP16 and DBDcre1-VP16 can enhance cellulase production.Here, we developed two sets of novel artificial transcriptional activators, which were designed as minimal transcriptional activators containing one DNA binding domain from either ACEII or CREI, and one strong transcriptional activation domain VP16 from the herpes simplex virus . These mace2 DBDs with different lengths were selected to construct the DBDace2-VP16 (MTA58/66/81) minimal transcriptional activators. DBDace2–58 (amino acids 1–58) is the core DBD as previously demonstrated using DNA mobility shift assays [ace2–66 (amino acids 1–66) includes a histidine-rich region (amino acids 53–66) with unknown function that is also found in other Zn(II)2Cys6-type regulatory proteins [ace2–81 (amino acids 1–81) is the longest region responsible for DNA binding and is followed by the region corresponding to the activation domain. Similarly, three sets of cre1 DBDs were selected . However, cre196 does not represent the DNA sequence of amino acids 1–96 of CREI but is a chimera due to the loss of a 2477-bp fragment at position + 262 of cre1 in the T. reesei hyper-cellulolytic mutant RUT C30 [ace2-VP16 (pMTA58/66/81) and DBDcre1-VP16 (pMTA96/109/139)  by homologous double exchange and eliminate the risk of any unpredictable mutagenesis caused by random insertion. We screened nine transformants each for DBDace2-VP16  and for DBDcre1-VP16 . All transformants were identified as correct gene knockout strains harboring single-copy DNA integration  of the native transcription factor and the transcriptional activation domain VP16. The transcription factors ACEII and CREI were used as targets to be replaced. Three sets of t assays . DBDace2proteins . DBDace2ace2-VP16 transformants TMTA58, TMTA66, and TMTA81 were examined in minimal medium (MM) with glucose, lactose, or avicel as the sole carbon source   and CMCase activities (28.6 U/mL), which were about 2.2- and 1.9-fold, respectively, when compared to those of RUT C30 in avicel at 120 h , two major xylanase genes (xyn1 and xyn2), one accessary protein gene (swo1), and two transcription factor genes (xyr1 and cre196) at 12 and 24 h were analyzed using RT-qPCR  and xylanase II activities (5.5 U/mg biomass) in TMTA139 were approximately 11.8- and 11.5-fold, respectively, greater than that of RUT C30 at 72 h. Additionally, the extracellular protein concentration of TMTA96, TMTA109, and TMTA139 in glucose also increased 1.4-, 3.8-, and 7.7-fold compared to that of RUT C30 at 72 h  assay  and FPase (22.4 U/mL) activities after 5 days of cultivation. The TMTA139 strain reached an FPase activity of 4.5 U/mL after 3 days of cultivation (Table The strains T30 Table . The TMTnd FPase 2.4 U/mL MTA66 and TMTA139 was determined via the hydrolysis of pretreated and biodetoxified corn stover [MTA66 and TMTA139 strains showed similar low performances in saccharification of biomass to the glucose yield (66–71%) of the parental strain due to the lack of β-glucosidase produced by T. reesei  and stronger transcriptional activation than that of ACEII [ace2-VP16 increased cellulase expression under induction conditions (avicel and lactose) and DBDcre1-VP16 increased cellulase and xylanase expression under repression conditions (glucose).Cellulase and xylanase genes are majorly regulated at the transcript level in fungi . In T. r domains , 17, 27,of ACEII . Retainiace2-VP16 enhanced cellulase production under induction conditions , a hyper-cellulolytic strain, was used as the parental strain for genetic transformation and chromosomal DNA preparation. Escherichia coli DH5α and Agrobacterium tumefaciens AGL1 were used for plasmid construction and Agrobacterium-mediated transformation, respectively.ace2 and cre196 genes were amplified from T. reesei RUT C30 genomic DNA through PCR using the appropriate primers and used as the 5′ and 3′ homology arms of pMTA58/66/81 and pMTA96/109/139, respectively  and cre96 [T. reesei RUT C30 genomic DNA via PCR, while the DBDcre1–109 and DBDcre1–139 (different truncated versions of the DNA-binding domain sequence of cre1) were cloned from T. reesei Qm9414 genomic DNA using the appropriate primers because the cre1 in RUT C30 was truncated and chimeric. All primers are listed in Additional file PacI/XbaI or SwaI) [The upstream and downstream sequences of nd cre96  were ampor SwaI) , by whicT. reesei RUT C30 via Agrobacterium-mediated transformation [MTA58/66/81 and TMTA96/109/139. The integration of the target gene-deleted constructs was analyzed using diagnostic PCR and sequencing. The single-copy DNA integration in transformed clones was verified by quantitative PCR (qPCR). The details are listed in Additional file The generated plasmids were introduced into ormation . Clone vormation  were per6/mL) of final transformants and the parental strain RUT C30 were inoculated into 100 mL of minimal medium ((NH4)2SO4 5 g/L; Urea 0.3 g/L; KH2PO4 15 g/L; CaCl2 0.6 g/L; MgSO4 0.6 g/L; FeSO4·7H2O 5 mg/L; ZnSO4·7H2O 1.4 mg/L; CoCl2·6H2O 2 mg/L, pH 5.5) supplemented with 20 g/L glucose, lactose, or avicel in 500-mL Erlenmeyer flasks and incubated at 28 °C and 200 rpm for 72 h. Two milliliters of the culture liquid was collected every 12 h for biomass concentration assay. Biomass concentrations from glucose and lactose were measured gravimetrically according to the method of Corder et al. [g at 4 °C for 10 min. Total protein concentration of the suspension was determined by the Modified Lowry Protein Assay Kit . The final protein content was corrected using a set of substrate controls where no inoculum was added to the avicel medium. The biomass dry weight was then calculated assuming an average content of 0.32 g intracellular protein per g of dry cell mass. Each experiment was performed in three biological replicates.Conidia  glucose in a 500-mL shake flask at 28 °C and 200 rpm for 72 h without medium replacement. Each experiment was performed in three biological replicates.To identify the cellulase production of Ti medium  supplemesar1 gene [−ΔΔCt method. The cycling conditions comprised 30 s initial denaturation and polymerase activation at 95 °C, followed by 40 cycles of 5 s at 95 °C and 60 s at 64 °C via an ABI StepOne Plus thermocycler . The primers are described in Additional file Total RNA was extracted using FastRNA Pro Red Kit  according to the manufacturer’s instructions. Synthesis of cDNA with 1000 ng total RNA was performed using TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR  following the manufacturer’s instructions. The transcript levels of target genes were assessed using real-time quantitative PCR (RT-qPCR) and normalized to that of the ar1 gene  using thg for 10 min at 4 °C) were used for enzyme and secreted protein concentration assays. The FPase and CMCase activities were measured via the DNS method using glucose as a standard. One unit represents the amount of enzyme that formed 1 µmol of reducing sugar per minute during the hydrolysis reaction. The pNPCase and pNPGase activities were measured against p-nitrophenol-d-cellbioside (pNPC) and p-nitrophenyl β-d-glucopyranoside (pNPG) , respectively. One unit of pNPCase and pNPGase activity was defined as 1 μmol of p-nitrophenol released per minute during the hydrolysis reaction. Xylanase I and II activities were determined by xylan degradation at pH values of 3.7 and 5.0 [The supernatants collected via centrifugation  with a final working volume of 3 L. The cultivations TMTA66 and parental strain RUT C30 were performed as follows. Conidia (106/mL) of strains were inoculated into 200 mL of Mandels-Andreotti medium supplemented with 1% (w/v) glucose and 1% (w/v) avicel in a 1-L shake flask and subsequently incubated with shaking (200 rpm) at 28 °C for 2 days. The culture was added to 2.8-L of fresh Mandels-Andreotti medium supplemented with 6% (w/v) avicel in a jar fermenter. Cultivation was carried out at 28 °C with 25% dissolved oxygen and 2 vvm (volumes of air per volume of liquid per minute) of aeration for 5 days. The pH was controlled within the range of 4.0–4.3 for the first 2 days and 5.0–5.2 thereafter.Selected dominant strains TMTA139 and the parental strain RUT C30 were performed as follows. Conidia (106/mL) of strains were inoculated into 200 mL of Mandels-Andreotti medium supplemented with 2% (w/v) glucose in a 1-L shake flask and incubated with shaking (200 rpm) at 28 °C for 2 days. The culture was poured into 2.8 L of Mandels-Andreotti medium fresh supplemented with 2% (w/v) glucose in the jar fermenter. Cultivation was carried out at 28 °C with 25% dissolved oxygen and 2 vvm of aeration for 3 days. The pH was controlled within the range of 5.0–5.2.The cultures of TT. reesei strains were supplemented with/without β-glucosidase (SUNSON®) with a CBU/FPA ratio of two to hydrolyze the corn stover. Hydrolysis experiments were performed in a flask containing 10% (w/v) pretreated corn stover as the substrate and FPase loading (15 U/g dry biomass) at 50 °C and pH 5.0 for 72 h. The methods for glucose analysis and calculation of glucose yield were based on the study by Li et al. [Pretreated and biodetoxified corn stover was donated by Professor Jie Bao . The prei et al. .Additional file 1: Table S1. All primers used for this study. Figure S1. The verification of the single-copy DNA integration in transformed clones by diagnostic PCR and qPCR. Figure S2. SDS-PAGE analysis of extracellular proteins secreted by T. reesei RUT C30 and TMTA96, TMTA109, TMTA139. Figure S3. Hydrolysis of pretreated corn stover by CTec2 and the crude enzyme from TMTA66, TMTA139 and RUT C30 using the same FPase loading.


Little is known about the roles of Notch signaling in cholangiocarcinoma (CC). The expression of hairy and enhancer of split 1 (Hes-1) has not been investigated yet in resected specimens of CC. Notch signaling has been reported to be related to cancer stem cell (CSC) like properties in some malignancies. Our aim is to investigate the participation of Notch signaling in resected specimens of extrahepatic CC (EHCC) and to evaluate the efficacy of CC cells with CSC-like properties by Notch signaling blockade.+CD44+ cells, which are surface markers of CSCs in EHCC, after exposure with GSI IX, gemcitabine (GEM), and the combination of GSI IX plus GEM were assessed by flow cytometry using the human CC cell lines, RBE, HuCCT1 and TFK-1. Also, anchorage-independent growth and mice tumorigenicity in the cells recovered by regular culture media after GSI IX exposure were assessed.First, the expression of Notch1, 2, 3, 4 and Hes-1 was examined by immunohistochemistry in 132 resected EHCC specimens. The clinicopathological characteristics in the expression of Notch receptors and Hes-1 were investigated. Second, GSI IX, which is a γ-secretase-inhibitor, was used for Notch signaling blockade in the following experiment. Alterations of the subpopulation of CD24P = 0.008 and 0.053). The patients with the expression of at least any one of Notch1-3 receptors, who were in 80.3 % of the total, exhibited poorer survival (P = 0.050). Similarly, the expression of Hes-1 tended to show poor survival (P = 0.093). In all of the examined CC cell lines, GSI IX treatment significantly diminished the subpopulation of CD24+CD44+ cells. Although GEM monotherapy relatively increased the subpopulation of CD24+CD44+ cells in all lines, GSI IX plus GEM attenuated it. Anchorage-independent growth and mice tumorigenicity were inhibited in GSI IX-pretreated cells in RBE and TFK-1 (P < 0.05).Notch1, 2, 3, 4 and Hes-1 in the resected EHCC specimens were expressed in 50.0, 56.1, 42.4, 6.1, and 81.8 % of the total cohort, respectively. Notch1 and 3 expressions were associated with poorer histological differentiation (Aberrant Notch signaling is involved with EHCC. Inhibition of Notch signaling is a novel therapeutic strategy for targeting cells with CSC-like properties. Cholangiocarcinoma (CC) arises from epithelial cells lining the bile duct. The incidence of CC is the highest in East and South Asia and has been increasing worldwide , 2. ChroNotch signaling is an evolutionarily conserved pathway that plays an important role in various cellular and developmental processes . AberranHes-1. γ-secretase inhibitor (GSI) has been reported to have antitumor effects as Notch antagonism by suppression of the Notch receptor cleavage against cancers linked with aberrant activation of Notch signaling in vitro and in vivo /2.Nonobese diabetic/severe combined immunodeficiency (NOD/SCID) female mice were purchased from CLEA Japan, Inc. . CC cell lines were pretreated ex vivo with media containing 0.5 % FBS with DMSO or GSI IX (40 μM) for 96 h, followed by recovery in full serum conditions for an additional 24 h before subcutaneous implantation. Viable 3 × 10 formula : Tumor Sχ2 test was used to compare categorical variables and the Kaplan-Meier method was used to generate survival curves. The association between clinicopathological factors and Notch receptors/Hes-1 was assessed with the Pearson correlation coefficient. Analyzed data were described as the mean ± S.E. A Wilcoxon test was used for statistical analysis with JMP Pro 11.2.0 . Significant difference between experimental groups was determined as a P-value < 0.05.The n = 100: 75.8 %) was the highest. Lymph node metastases were observed in 91 patients (68.9 %). The number of patients with Stage I, II, III and IV were 20 (15.2 %), 57 (43.2 %), 18 (13.6 %) and 37 (28.0 %), respectively. R0 resection was achieved in 98 patients (74.2 %).One hundred thirty-two patients with EHCC, comprising 92 men and 40 women (median age: 68 y.o.), were diagnosed with 83 perihilar and 49 distal CC. Among the 132 patients, pathological arterial and portal invasion was observed in 6 and 22 patients, respectively , 74 (56.1 %), 56 (42.4 %), 8 (6.1 %) and 108 (81.8 %) specimens, respectively, and cases with positive immunostaining in at least any one of Notch1, 2 and 3 were shown in 106 specimens (80.3 %) Fig. . On the P = 0.008) (Table P = 0.053). In terms of the Tumor category, T1/2 was higher than T3/4 in the expression of Noch3 (P = 0.049). According to the stage classification, there was no significant difference in the expression of any Notch receptors and Hes-1. 92 specimens of cases with at least one expression of Notch1, 2 and 3 (86.8 %) also showed positive staining of Hes1 and there was significant correlation between them (P = 0.005). By Pearson’s correlation analysis, there was no significant correlation between the clinicopathological factors and expression of Notch receptors/Hes-1.The number of patients with Notch1 expression was significantly greater in those with Grade 2/3 than Grade 1 (P = 0.050) .In the 98 patients with R0 resection, there was no significant survival difference between patients with and without the expression of each Notch receptor (data not shown). However, those with at least one expression of Notch1, 2 and 3 exhibited a poorer prognosis than those with no expression of Notch1, 2 or 3 : 57.6 % vs 70.2 %, 50) Fig. . SimilarP < 0.05)  Fig. . These r05) Fig. .Fig. 2Al+CD44+ cells by treatment with GSI IX  (P < 0.05). In the other CC cell lines, GSI treatment also decreased the subpopulation of CD24+CD44+ cells   Fig. . The res05) Fig. .To confirm the effectiveness of treatment with GSI on cells with CSC-like properties, we investigated the alteration of anchorage-independent growth and mice tumorigenicity after pretreatment with GSI IX Fig. –d. The aRecently, several reports have discussed the participation of Notch signaling in CC –20. Howe+CD44+ cells showed CSC-like properties in EHCC [+CD44+ in all CC cell lines and induced a significant reduction of anchorage-independent growth and delayed tumor engraftment in mice. The present study first elucidated the therapeutic effect of GSI on CC cells with CSC-like properties, similar to the effect found in other cancers.GSI inhibits the γ-secretase-dependent cleavage of all Notch receptors as a pan-Notch inhibitor. GSI induces apoptosis through the regulation of nuclear factor-κB  and inhi in EHCC . In this+CD44+ cells compared with GEM alone. Combination therapy of GEM plus GSI was reported to show a synergy effect in pancreatic cancer xenograft [The CSCs hypothesis is based on the idea that cancer tissue has a minute proportion of cells with stem cell-like properties, which possess a great ability for self-renewal and produce heterogeneous progeny. CSCs, which drive tumorigenesis and maintain tumor proliferation, are located at the top of a hierarchy of tumor cells –27. Cellenograft , 22. Morenograft . Notch sAberrant Notch signaling is involved in EHCC. In CC, inhibition of Notch signaling could be a novel therapeutic strategy for targeting cells with CSC-like properties.


Background and aims: Currently there is considerable interest in ketone metabolism owing to recently reported benefits of ketosis for human health. Traditionally, ketosis has been achieved by following a high-fat, low-carbohydrate “ketogenic” diet, but adherence to such diets can be difficult. An alternative way to increase blood D-β-hydroxybutyrate (D-βHB) concentrations is ketone drinks, but the metabolic effects of exogenous ketones are relatively unknown. Here, healthy human volunteers took part in three randomized metabolic studies of drinks containing a ketone ester (KE); (R)-3-hydroxybutyl (R)-3-hydroxybutyrate, or ketone salts (KS); sodium plus potassium βHB.Methods and Results: In the first study, 15 participants consumed KE or KS drinks that delivered ~12 or ~24 g of βHB. Both drinks elevated blood D-βHB concentrations , which returned to baseline within 3–4 h. KS drinks were found to contain 50% of the L-βHB isoform, which remained elevated in blood for over 8 h, but was not detectable after 24 h. Urinary excretion of both D-βHB and L-βHB was <1.5% of the total βHB ingested and was in proportion to the blood AUC. D-βHB, but not L-βHB, was slowly converted to breath acetone. The KE drink decreased blood pH by 0.10 and the KS drink increased urinary pH from 5.7 to 8.5. In the second study, the effect of a meal before a KE drink on blood D-βHB concentrations was determined in 16 participants. Food lowered blood D-βHB Cmax by 33% , but did not alter acetoacetate or breath acetone concentrations. All ketone drinks lowered blood glucose, free fatty acid and triglyceride concentrations, and had similar effects on blood electrolytes, which remained normal. In the final study, participants were given KE over 9 h as three drinks (n = 12) or a continuous nasogastric infusion (n = 4) to maintain blood D-βHB concentrations greater than 1 mM. Both drinks and infusions gave identical D-βHB AUC of 1.3–1.4 moles.min.Conclusion: We conclude that exogenous ketone drinks are a practical, efficacious way to achieve ketosis. Produced continuously under physiological conditions, blood ketone concentrations increase during starvation  and ketone salts (KS). The metabolic effects of KS ingestion have been reported in rats . Secondly, in a randomized five-arm cross-over study, inter- and intra-participant repeatability of ketosis was examined following ingestion of identical KE drinks, twice whilst fed and twice whilst fasted. As a control, participants also consumed one isocaloric  dextrose drink . Finally, blood d-βHB was measured after equal amounts of KE were given as three drinks (n = 12) or a constant nasogastric (NG) infusion (n = 4)  over 9 h.Firstly, in a randomized four-arm cross-over study, blood βHB concentrations were compared following ingestion of equal amounts of βHB as a KE or a KS at two doses by healthy volunteers at rest  and were conducted in accordance with the Declaration of Helsinki . StudiesParticipants refrained from alcohol and caffeine for 24 h prior to each visit AND were asked to consume a similar meal the night before each visit. All studies were carried out at the University of Oxford Human Physiology Laboratories and started at 0800 h following an overnight (>8 h) fast, with a minimum of 72 h between visits. Visit order was randomized prior to commencement by an administrative investigator using a pseudo-random number generator to produce a list of combinations of visit order, which were then allocated based on order of enrolment by a different investigator.Fasting blood samples were collected prior to all interventions. Following consumption of study drinks (details below), blood, expired gas and urine samples were collected at regular intervals for 4 h. Water was freely permitted and participants remained sedentary at the test facility throughout the visit. A subset of participants returned for samples 8 and 24 h after the ketone drinks (Study 1).n = 7) and immediately analyzed for pH and electrolytes using a clinical blood gas analyser .Venous blood samples (2 ml) were obtained during all visits using a 22 G catheter inserted percutaneously into an antecubital vein. The catheter was kept patent using a saline flush following each sample collection. Additionally, during Study 1, arterialized blood from a catheter inserted into a heated hand . Samples were stored on ice, centrifuged and duplicate plasma aliquots stored at −80°C. All urine passed during the visit was collected, the total volume recorded, and 1 ml aliquots taken, frozen and retained for analysis.d-βHB were assayed using a commercial semi-automated bench-top analyzer , and insulin was measured using a commercially available ELISA assay . Both the pure liquid KS and KE, and a subset of plasma (n = 5) and urine (n = 10) samples from a subset of participants in Study 1 underwent analysis using GC-MS and a chiral column, and the concentrations of l-βHB was calculated using the enzymatically determined concentration of d-βHB and the ratio of the d/l-βHB peaks obtained through GC-MS. Acetoacetate was assayed using an enzymatic method , triglycerides (TG) and urinary n = 15) consumed 1.6 and 3.2 mmol.kg−1 of βHB as KE  or as KS  sodium and potassium βHB, containing 1.6–3.2 g of each cation), plus 6 g of sweetener containing 19 kCal (4 g of carbohydrate) , diluted to 300 ml using water. Drink blinding was not possible due to unmaskable differences in taste .Over four visits, participants (n = 16) consumed either 4.4 mmol.kg−1 of βHB : twice whilst fasted, and twice following a standardized meal, or an isocaloric dextrose drink without a meal. To improve palatability, drinks were diluted to 500 ml with a commercially available, citrus flavored drink containing 65 kCal (5 g of carbohydrate) . The dextrose drink was taste-matched using a bitterness additive . The standard meal consisted of porridge oats (54 g), semi-skimmed milk (360 ml) and banana (120 g), giving 600 kCal per person, with a macronutrient ratio of Carbohydrate: Protein: Fat of 2:1:1.Over five visits, participants  over 9 h, either as 3 drinks of 4.4 mmol.kg−1 of βHB at 3 h intervals (n = 12), or as an initial bolus of 4.4 mmol.kg−1 of βHB given through a nasogastric tube, followed by an infusion of 1.1 mmol.kg.h−1, beginning 60 min after the initial bolus, for 8 h (n = 4). Two participants completed both conditions . In both conditions, the KE was diluted to 1.5 L using the same citrus water as used in Study 2.Participants consumed 13.2 mmol.kgd-βHB appearance was estimated using d-βHB concentrations at baseline and 30 min post-drink, and d-βHB elimination was estimated using the AUC between the post-drink peak (60 min) and trough (180 min) d-βHB concentrations, with a baseline correction to the value at 180 min.For all studies, the area under the curve (AUC) of blood [βHB] was calculated using the trapezium rule. In Study 3, for each of the three drinks, the initial rate of p < 0.05. Initial tests were undertaken to ensure that normality and sphericity assumptions were not violated. Subsequently, either one or two way repeated measures ANOVA, or Freidman's test with post-hoc Tukey or Dunnet's correction were performed, to compare changing concentrations of substrates, electrolytes, pH, insulin, breath and urinary βHB: both over time and between study interventions. In Study 2, data from each of the two study visits in each condition (fed and fasted) completed by an individual were included in the analysis.Unless otherwise stated, statistical analysis was conducted using Prism 6™ software. Values, expressed as means ± SEM, were considered significantly different at t-test with equal SD was used to compare urine βHB concentrations. Additionally, a linear mixed effects model was constructed to estimate partitions of variance in R, using the lme4 and blme packages  were analyzed to reveal higher l-βHB than d-βHB, the total βHB Cmax being 3.4 ± 0.2 mM  consumed 3.2 mmol.kg−1 of βHB as KE and KS with hourly blood and breath sample collection up to 4 h, plus additional samples at 8 h and 24 h post-drink. l-βHB was found to be 1.1 ± 0.1 mM at 4 h, and 0.7 ± 0.2 mM after 8 h, but undetectable after 24 h  were present at 24 h, presumably due to endogenous production. Both ketone drinks significantly increased breath acetone concentration, but at a slower rate than blood d-βHB, reaching a peak after 3 h that was twice as high following the KE (87 ± 9 ppm) than the KS (44 ± 10 ppm), suggesting that d-βHB was readily converted to acetone, but l-βHB was not , the amount of d-βHB excreted in the urine increased with d-βHB intake, but was <1.5% of the total βHB ingested and was not different between matched doses of KE vs. KS  but was significantly alkalinized by KS consumption (pH 8.5 ± 0.1. p < 0.001 vs. baseline)  Figure .p < 0.05). Concentrations were the same as at baseline by 4 h, with FFA at 0.6 mM, TG at 0.9 mM and glucose 5.1 mM  the effect of 3.2 mmol.kg−1 of βHB as KE and KS on blood pH and electrolytes after ketone drinks was investigated. Blood d-βHB kinetics were similar to those in the initial experiment , but remained within the normal range . Despite the differences in d-βHB kinetics after the meal, there were no effects of food on urinary ketone excretion , <1% to visit order, 16% to inter-participant variability, and the residual 24% variability due to non-specific random effects.The concentrations of blood  Figures . The d-βd-βHB concentrations were elevated for longer after KE drinks than glucose after dextrose drinks. Insulin increased to ~ 35 mU.ml−1 after both the meal and the dextrose drink, but also increased to 13 ± 2 mU.ml−1 when KE was consumed whilst fasted owing to the 15 g of glucose in the flavored drink used as a diluent . The effect was not altered by a meal Figures . Dextrost Figure .d-βHB rose and then fell, but had not returned to baseline (~ 0.1 mM) when the next drink was consumed. There was no significant difference in d-βHB Cmax between drinks 2 and 3 (3.4 ± 0.2 mM vs. 3.8 ± 0.2 mM p = 0.3), as the rate of d-βHB appearance fell slightly with successive drinks (0.07 ± 0.01 mmol.min−1 and 0.06 ± 0.01 mmol.min−1p = 0.6). d-βHB elimination was the same after each bolus . When KE was given via a nasogastric tube, the initial bolus raised blood d-βHB to 2.9 ± 0.5 mM after 1 h, thereafter continuous infusion maintained blood d-βHB between 2–3 mM. Total d-βHB appearance in the blood was identical for both methods of administration .Serial drinks or a continuous NG infusion of KE effectively kept blood ketone concentrations >1 mM for 9 h Figure . With drd-βHB > 50% higher than KS drinks, the latter significantly increasing blood l-βHB, which was metabolized more slowly by the body. Both drinks had similar effects on FFA, TG, glucose and electrolyte concentrations, although with disparate effects on pH. A prior meal decreased total blood d-βHB appearance after a KE drink. Finally, either three KE drinks or nasogastric feeding effectively maintained nutritional ketosis over 1 mM for 9 h.Exogenous ketones drinks are growing in popularity as a method to elevate blood ketone concentrations and mimic a ketogenic diet without the need for dietary changes , allowing βHB Palmer, .d-βHB concentrations than KS, we investigated factors that may be important in the use of ketone drinks to achieve nutritional ketosis. Initially we determined the repeatability of blood ketosis following KE drinks and found little variation in kinetic parameters between individuals. Variability between participants was less than within the population, and accurate individual prediction of the d-βHB Cmax following a body-weight adjusted KE drink was achieved. Variability within individuals was likely due to normal daily changes in GI function, including gastric emptying, portal blood flow or intestinal transit time, which may alter KE hydrolysis and absorption.As KE drinks achieved a significantly higher +:NADH may have altered the conversion of βHB to AcAc  reportedly inhibited ketone clearance from the blood, however the underlying mechanism is unknown , as there was no significant dose-response relationship between increasing blood βHB and the small changes in plasma FFA, TG or glucose across all of the study drinks  represented ~1.5% of the total consumed (≈23.7 g), it appears that the major fate of exogenous d-βHB was oxidation in peripheral tissues. These results suggest that neither breath acetone nor urinary ketone measurements accurately reflect the rapid changes in blood ketone concentrations after ketone drinks, and that blood measurement should be the preferred method to quantitatively describe ketosis. That said, it should be noted that although commercial handheld monitors are the most practical and widely available tool for measuring blood ketones, they can overestimate blood D-βHB compared to laboratory measures  (Study 1) and London Queen's Square REC (14/LO/0288) (Study 2 and 3). The studies were carried out in accordance with the recommendations of the Declaration of Helsinki, apart from pre-registration in a database. All subjects gave written informed consent in accordance with the Declaration of Helsinki.BS, KC, and PC designed the research studies. BS, PC, RE, SM, and PS carried out the studies. SH provided the gas analyser used in the study on behalf of NTT DOCOMO Inc. BS, MS, and SM analyzed the data and performed statistical analysis in collaboration with JM. BS wrote the paper with help from KC, PC, and OF. KC had primary responsibility for final content. All authors read and approved the final manuscript.Intellectual property covering uses of dietary ketone and ketone ester supplementation is owned by BTG Ltd., the University of Oxford, the National Institute of Health and TΔS Ltd. Should royalties ever accrue from these patents, KC and PC, as inventors, will receive a share of the royalties under the terms prescribed by the University of Oxford. KC is a director of TΔS Ltd., a company spun out of the University of Oxford to develop and commercialize products based on the science of ketone bodies in human nutrition. At the time of data collection and manuscript preparation, BS was an employee of TΔS Ltd., funded by the Royal Commission for the Exhibition of 1851. SH is an employee of NTT DOCOMO, Inc. (Japan). The other 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.


High-resolution oligo-SNP array allowed the identification of extremely small pathogenic deletions at numerous clinically relevant regions. In our clinical practice, we found that small pathogenic deletions were frequently encountered at chromosome 9p and 9q terminal regions.DOCK8, KANK1, EHMT1 genes. The remaining 47 cases were due to interstitial or terminal deletions larger than 1 Mb or unbalanced translocations. The small pathogenic deletions of DOCK8, KANK1 and EHMT1 genes were more prevalent than small pathogenic deletions of NRXN1, DMD, SHANK3 genes and were only second to the 16p11.2 deletion syndrome, 593-kb (OMIM #611913).A review of 531 cases with reportable copy number changes on chromosome 9 revealed142 pathogenic copy number variants (CNVs): 104 losses, 31 gains, 7 complex chromosomal rearrangements. Of 104 pathogenic losses, 57 were less than 1 Mb in size, enriched at 9p24.3 and 9q34.3 regions, involving the DOCK8, KANK1 and EHMT1 genes.This study corroborated comprehensive genotype-phenotype large scale studies at 9p24.3 and 9q24.3 regions for a better understanding of the pathogenicity caused by haploinsufficiency of the None; it is not a clinical trial, and the cases were retrospectively collected and analyzed.The online version of this article (doi:10.1186/s13039-016-0291-3) contains supplementary material, which is available to authorized users. Chromosomal microarray analysis (CMA) has been widely utilized for the genome-wide screening of microdeletion and microduplication syndromes . The sizNRXN1 and SHANK3 in the past [DOCK8 and/or KANK1 gene [EHMT1 gene at 9q34.3 [High-resolution oligo-SNP array is able to reveal a variety of chromosomal disorders including uniparental disomy or extremely small pathogenic deletions which would be missed by low-resolution oligonucleotide CMA. Our and other previous studies showed the cases with uniparental disomy were relatively limited in number on chromosome 9 as compared to chromosome 15, 11 and 7 , 3. In cthe past , 5. To tNK1 gene –8, and aThe purpose of this study is to evaluate 1): the incidence of small (< 1 Mb) pathogenic deletions in postnatal specimens, 2): whether the small pathogenic deletions at 9p24.3 and 9q34.3 constituted a significant proportion of small deletions, 3): what proportion of deletions on chromosome 9 was caused by small pathogenic deletions at 9p24.3 and 9q34.3, 4): the efficacy of identifying extremely small homozygous pathogenic deletions using high-resolution oligo-SNP array.NRXN1 (35 cases), DMD (31 cases), DOCK8 and/or KANK1 (30 cases), and other chromosomal regions such as 16p11.2 , 16p12.2 , 22q13.33 (OMIM #606232).Approximately 38,000 postnatal specimens were studied by high-resolution oligo-SNP array in our laboratory from 2011 through 2015. Of these, we reported approximately 13,000 (34 %) pathogenic variants or variants of uncertain clinical significance (VOUS). The detection rate was consistent with our previous study [DOCK8 and/or KANK1 genes at 9p24.3  to 790 kb (case 35), with an average of 384 kb (Table PTCH1 gene (OMIM 601309) and diagnosis of Gorlin syndrome (OMIM #109400), a 1,349-kb deletion (case 15) involving the STXBP1 gene and diagnosis of early infantile epileptic encephalopathy (OMIM #612164), and two 9q34.3 deletions of 1,218 kb and 1,268 kb (case 31 and 32). The small (57 cases) and relative small (4 cases) deletions established 59 % (61/104) of pathogenic copy number losses on chromosome 9.Of 13,000 cases with reported copy number variants (CNVs), 531 cases were from chromosome 9, and 142 were pathogenic, including 104 losses, 31 gains, 7 complex chromosomal rearrangements with both losses and gains . Besides that, a 50-kb heterozygous deletion was also found in the 5′ region of the GLDC gene. Parental study showed the mother was a carrier of a 25-kb heterozygous deletion and the father was a carrier of a 75-kb heterozygous deletion of the GLDC gene. The 25-kb maternally inherited deletion was located within the 75-kb paternally inherited deletion, and therefore inheritance of abnormal allele from both parents led to a 25-kb homozygous and a 50-kb heterozygous deletion in the proband : a new born baby girl who presented with a metabolic disorder  was revealed to contain a 25-kb homozygous deletion in the and Fig. ; 2): a 1The subtelomeric region such as 1p36 is known to be gene-rich and prone to have deletions, supported by a study with a large cohort of over 5,000 cases . The casEHMT1 gene was only reported in one case: a 40-kb intragenic deletion of the EHMT1 gene with uncertain phenotypic consequence [EHMT1 gene , the sensitivity to haploinsufficiency for DOCK8 and KANK1 genes was not proven  [DOCK8 and KANK1 displayed two distinct phenotypes [KANK1 gene implied that an imprinting mechanism may play a role in the phenotypic variation in this family. The authors suggested KANK1 is a maternally imprinted gene and only expressed in the paternal allele [KANK1 were found in 6 cases  . A recenenotypes . The stul allele . In our GLDC and CDK5RAP2 genes were discovered in this cohort. In our previous study, we demonstrated the autosomal recessive disorders could be linked to regions of homozygosity (ROH) containing gene with point mutation which was inherited from related parents [GLDC gene : high-resolution oligo-SNP array was capable of identifying homozygous deletions as small as 25 kb in size.This study demonstrated 1): the incidence of recurrent small pathogenic deletions (< 1 Mb) was approximately 3 %  of all reported CNVs; 2): the small pathogenic deletions at 9p24.3 and 9q34.3 constituted 12 % of small pathogenic deletions from all the chromosomes, 3): 59 % of pathogenic deletions on chromosome 9 were due to small (<1 Mb) or relatively small (1–1.5 Mb) pathogenic deletions; 4): 81 % (46/57) of small pathogenic deletions were enriched at 9p24.3 and 9q34.3 regions involving the Patients with a broad range of clinical indications including intellectual disability, developmental delay, multiple congenital anomalies, dysmorphic features and pervasive developmental disorders were referred to our laboratory for oligo-SNP array studies. The data for this study were compiled from de-identified results of 38,000 consecutive patient specimens referred to our laboratory for constitutional oligo-SNP array study from 2011 to 2015. The patients were majorly from general population in the United States, with < 5 % from Mexico and other countries.http://dgv.tcag.ca/dgv/app/home?ref=GRCh37/hg19) and present in the general population were excluded from reporting.Oligo-SNP array analysis was performed on either Human SNP Array 6.0 (in 2011) or CytoScan® HD (2012–2015), using genomic DNA extracted from whole blood. The Human SNP Array 6.0 has 1.8 million genetic markers, including about 906,600 SNPs and 946,000 probes for the detection of CNVs. The CytoScan® HD has more than 2.67 million probes, including 1.9 million non-polymorphic copy number probes and 750,000 SNP probes. The overall resolutions are approximately 1.7 kb for Human SNP Array 6.0 and 1.15 kb for CytoScan® HD. For chromosome 9, the probes for Human SNP Array 6.0 covered: 9p and 9q; for CytoScan HD®: 9p  and 9q . Genomic coordinates were based upon genome build 37/hg19 (2009). Hybridization, data extraction, and analysis were performed as per manufacturers’ protocols. The Affymetrix® Chromosome Analysis Suite (ChAS) Software version 2.0 was used for data analysis, review, and reporting. For genome-wide screening, thresholds were set at > 200 kb for gains and > 50 kb for losses. For cytogenetically relevant regions, thresholds were set at > 100 kb for gains and > 20 kb for losses. Benign CNVs that are documented in the database of genomic variations (


Human papillomavirus genotypes 52 and 58 (HPV52/58) are frequently detected in patients with cervical intraepithelial neoplasia (CIN) and invasive cervical cancer (ICC) in East Asian countries including Japan. As with other HPV genotypes, HPV52/58 consist of multiple lineages of genetic variants harboring less than 10% differences between complete genome sequences of the same HPV genotype. However, site variations of nucleotide and amino acid sequences across the viral whole-genome have not been fully examined for HPV52/58. The aim of this study was to investigate genetic variations of HPV52/58 prevalent among Japanese women by analyzing the viral whole-genome sequences.The entire genomic region of HPV52/58 was amplified by long-range PCR with total cellular DNA extracted from cervical exfoliated cells isolated from Japanese patients with CIN or ICC. The amplified DNA was subjected to next generation sequencing to determine the complete viral genome sequences. Phylogenetic analyses were performed with the whole-genome sequences to assign variant lineages/sublineages to the HPV52/58 isolates. The variability in amino acid sequences of viral proteins was assessed by calculating the Shannon entropy scores at individual amino acid positions of HPV proteins.n = 20; CIN2/3, n = 21; ICC, n = 11), 50 isolates belonged to lineage B (sublineage B2) and two isolates belonged to lineage A (sublineage A1). Among 48 isolates of HPV58 , 47 isolates belonged to lineage A (sublineages A1/A2/A3) and one isolate belonged to lineage C. Single nucleotide polymorphisms specific for individual variant lineages were determined throughout the viral genome based on multiple sequence alignments of the Japanese HPV52/58 isolates and reference HPV52/58 genomes. Entropy analyses revealed that the E1 protein was relatively variable among the HPV52 isolates, whereas the E7, E4, and L2 proteins showed some variations among the HPV58 isolates.Among 52 isolates of HPV52  contains supplementary material, which is available to authorized users. Human papillomaviruses (HPVs) constitute a large family of small DNA viruses, having a circular double-stranded DNA genome of approximately 8000 base pairs . Their gAmong the high-risk HPVs, HPV16 and HPV18 are the most and second most prevalent genotypes in ICC, respectively, in total accounting for about 70% of ICC cases worldwide . In JapaHPV genomes with less than 10% differences in their L1 sequences, recognized as intra-type variants, constitutes an additional level of HPV genetic complexity. Based on complete viral genome sequences, intra-type variants are phylogenetically classified into different lineages and sublineages, which are defined as containing 1.0–10.0% and 0.5–1.0% nucleotide variations, respectively . As suchMany lines of evidence attribute a higher risk of progression to ICC to some distinct variant lineages of high-risk HPVs. Intriguingly, recent large-scale studies revealed that different HPV16 variant sublineages were associated with different risks for cervical squamous cell carcinoma or adenocarcinoma –15. A stWhile HPV52/58 infections are common in East Asia, comprehensive surveys have not been conducted regarding their variant distributions in Japan. Determining the complete viral genome sequence is the most reliable and accurate procedure to assign HPV variant lineages/sublineages compared with utilizing only limited sequences in the HPV genome, which was generally done in previous studies on HPV52/58 variant classification , 20–22. n = 52) or HPV58 (n = 47) were selected for subsequent analyses of the whole-genome sequences of HPV52/58. The study protocol was approved by the Ethics Committees at each hospital and the National Institute of Infectious Diseases, and written informed consent for study participation was obtained from each patient.Cervical exfoliated cells were collected in ThinPrep media  using a cytoblush from Japanese patients diagnosed with CIN1, CIN2, CIN3 or ICC at Keio University Hospital and Tsukuba University Hospital from 2012 to 2016. The total cellular DNA was extracted from the recovered cells on a MagNA Pure LC 2.0 , and subjected to PCR with PGMY09/11 primers to amplify HPV L1 DNA, followed by reverse blot hybridization for HPV genotyping, as described previously . Based ohttps://gph.niid.go.jp/cgi-bin/virustap/index.cgi). The accuracy of the reconstructed whole-genome sequences was verified by read mapping with Burrows-Wheeler Aligner v0.7.12 [Full-circle PCR or overlapping PCR was performed with PrimeSTAR® GXL DNA polymerase  to amplify the whole-genome sequences of HPV52/58 as described previously . The seq v0.7.12  and subs v0.7.12 .n = 52) or HPV58 (n = 48) isolates were aligned against each other using MAFFT v7.309 [The complete genome sequences of HPV52  specific for variant lineages and sublineages. The multiple sequence alignments of the whole-genome sequences of HPV52/58 were sorted according to variant lineage/sublineage and the number of mismatched bases in order to visually differentiate lineage/sublineage-specific SNPs.Amino acid variations at individual positions of viral proteins were calculated on the basis of Shannon’s equation :\documenH(i), p(xi), and i indicate the amino acid entropy score of a given position, the probability of occurrence of a given amino acid at the position, and the number of positions, respectively. An H(i) score of zero indicates absolute conservation, whereas a score of 4.4 indicates complete randomness. The deduced amino acid sequences of eight HPV proteins (E6/E7/E1/E2/E4/E5/L2/L1) of the HPV52/58 isolates were concatenated and aligned with each other using MAFFT. The entropy calculation was performed on the multiple sequence alignments using R v2.11.1 (https://cran.r-project.org) with bio3d package v1.1–6 [where e v1.1–6 .P value <0.05 was regarded as statistically significant. The relative risk for progression from CIN1 to CIN2/3/ICC among HPV58 A sublineages was estimated by calculating adjusted odds ratio with its 95% confidence interval.All statistical analyses were performed using R v3.3.2. Fisher’s exact test was performed to evaluate a difference in HPV58 A sublineages distribution across histological categories. n = 20; CIN2/3, n = 21; ICC, n = 11), and 47 HPV58-positive cases . The mean age ± standard deviation of the cases in each histological grade was as follows: for HPV52: CIN1, 38.6 ± 12.0 years; CIN2/3, 38.6 ± 8.3 years; ICC, 59.2 ± 15.7 years; for HPV58: CIN1, 35.6 ± 7.4 years; CIN2/3, 37.1 ± 7.5 years; ICC, 55.8 ± 16.2 years.The study subjects consisted of 52 HPV52-positive cases , 50 isolates belonged to lineage B (sublineage B2) and two isolates belonged to lineage A (sublineage A1). As shown in Fig. n = 21; CIN2/3, n = 19; ICC, n = 8), 47 isolates belonged to lineage A  and one isolate belonged to lineage C. Interestingly, one HPV58-positive CIN1 specimen yielded two distinct genome sequences of HPV58 (#065 and #066), and these were classified into two different sublineages (A1 and A2), which indicates co-infections with two closely related sublineages of HPV58 in a single patient. The presence of these two sublineages was further confirmed by cloning and Sanger sequencing of HPV58 PCR products obtained from the original DNA sample (data not shown).Phylogenetic analyses were conducted with the whole-genome sequences of the HPV52/58 isolates, together with those of reference HPV52/58 genomes that represent individual variant lineages/sublineages. As shown in Fig. n = 33; HPV58, n = 36) are shown in Table P = 0.97). Furthermore, no significant difference in the relative risk for progression from CIN1 to CIN2/3/ICC was observed among HPV58 sublineages A1/A2/A3  for HPV52 and lineage A for HPV58. The distributions of HPV52/58 variant lineage/sublineage according to cervical histology status when restricted to single infection , and thus supposed to generate internally deleted E2/E4 proteins, which may have altered biological activities for viral transcription, replication and segregation . Of partBy comparing the whole-genome sequences of our Japanese HPV52/58 isolates with those of reference HPV52/58 genomes representing all variant lineages/sublineages, we have presented for the first time a comprehensive list of specific SNPs for discriminating their variant lineages/sublineages. In general, these diagnostic SNPs are dispersed throughout the viral genome, as was reported for HPV16  and HPV6Previously, a nucleotide substitution in the L1 region of HPV52  was reported to generate a mismatched base to the GP6+ primer, one of the consensus primers of GP5+/6+ PCR to amplify L1 DNA of multiple HPV types . Among tAlphapapillomaviruse-9) [The variability of amino acid residues in viral proteins reflects the degree of non-synonymous substitution in the nucleotide sequences of ORFs, and the higher variation at certain amino acid positions suggests that these positions are either neutral or under diversifying selection pressure during viral evolution. From such an evolutionary point of view, the different patterns of amino acid variations in the viral proteins observed in HPV52 and HPV58 are unexpected because these two viruses are genetically closely related to each other and positioned on the same branch of an HPV phylogenetic tree (iruse-9) . We specAn important unanswered question is why HPV52/58 infections are so common in Asia compared to other parts of the world. During the long history of HPV evolution and spread across the globe, these genotypes might have matched a characteristic property of Asian people through as-yet-unknown mechanisms of viral adaptation. Since HPV52/58 infections pose a significant disease burden of cervical cancer on Asian women, further work based on the viral whole-genome sequences, together with elucidation of the genetic background of Asian people including human leukocyte antigen polymorphism, will be required for a better understanding of cervical carcinogenesis driven by these Asia-prevalent HPVs.Among the HPV52/58-positive specimens from Japanese women with CIN/ICC, the variant distributions were strongly biased toward lineage B for HPV52 and lineage A for HPV58 across histological categories. Different patterns of amino acid variations were observed in HPV52 and HPV58 across the viral whole-genome.