Klebsiella pneumoniae is a common pathogen associated with nosocomial infections and is characterised serologically by capsular polysaccharide (K) and lipopolysaccharide O antigens. We surveyed a total of 348 non-duplicate K. pneumoniae clinical isolates collected over a 1-year period in a tertiary care hospital, and determined their O and K serotypes by sequencing of the wbb Y and wzi gene loci, respectively. Isolates were also screened for antimicrobial resistance and hypervirulent phenotypes; 94 (27.0%) were identified as carbapenem-resistant (CRKP) and 110 (31.6%) as hypervirulent (hvKP). isolates fell into 58 K, and six O types, with 92.0% and 94.2% typeability, respectively. The predominant K types were K14K64 (16.38%), K1 (14.66%), K2 (8.05%) and K57 (5.46%), while O1 (46%), O2a (27.9%) and O3 (11.8%) were the most common. CRKP and hvKP strains had different serotype distributions with O2a:K14K64 (41.0%) being the most frequent among CRKP, and O1:K1 (26.4%) and O1:K2 (17.3%) among hvKP strains. Serotyping by gene sequencing proved to be a useful tool to inform the clinical epidemiology of K. pneumoniae infections and provides valuable data relevant to vaccine design. Klebsiella pneumoniae (KP) is a common opportunistic pathogen of hospital- and community-acquired infections, resulting in pneumonia, bronchitis, urinary tract and wound infections. The species primarily afflicts patients at the extremes of age who are immunocompromised, have undergone long-term antibiotic therapy and/or with underlying predisposing conditions. The management of these infections is often complicated by the emergence of antimicrobial resistance which can be associated with high morbidity and mortality rates [K. pneumoniae (CRKP) and hypervirulent strains (hvKP) have become widespread in China and also globally, and pose a significant threat to public health [ty rates . In recec health .K. pneumoniae isolates including capsular polysaccharide (K) and lipopolysaccharide (O) antigen characterisation, DNA restriction analysis by pulsed-field gel electrophoresis and multilocus sequence typing, among others [Several methods have been used for the type identification of g others . Both O g others . O-serotg others .Streptococcus pneumoniae. As access to O and K typing antisera is no longer a viable option, and knowledge of serotype distributions among K. pneumoniae isolates in mainland China is limited, we undertook a prevalence study of clinical isolates from our tertiary hospital centre in Shanghai using gene sequencing of O and K serotype-specific chromosomal regions to determine the distribution of strain serotypes.A comprehensive understanding of the seroepidemiology of clinical isolates can be helpful for the design of vaccines to prevent infection through both active and passive immunisation, as has been shown for other encapsulated pathogens, notably K. pneumoniae clinical isolates were collected from patients in different departments between March 2018 and October 2019. Bacterial identification and antimicrobial susceptibility testing were performed by the Vitek2 system  and susceptibility results were reported according to Clinical and Laboratory Standards Institute (CLSI) guidelines. A CRKP strain was defined as an isolate resistant to at least one carbapenem antibiotic. The hvKP phenotype was confirmed by the string test as previously described [wzi gene sequence alignment [wb gene clusters [wzm-wzt loci, and the other determined O1 and O2ac alleles. Isolates unreactive with the latter O type primers but positive for O1/O2 alleles were considered to be of the O2a genotype.Three hundred and forty-eight escribed . Capsulalignment . O genotclusters  using twP < 0.001). Most patients (81.6%) had an indwelling device and likewise for those (81.0%) with CRKP. The mortality rate for all patients was 11.8%, and higher for those harbouring CRKP (12.8%) and hvKP (13.64%) (P < 0.05).Of the 348 isolates, 94 were classed as CRKP and 110 as hvKP phenotypes. Patients ³60 years old accounted for 222 (63.8%) of all isolates; the great majority (299) were from hospitalised patients and the remainder from out-patients. The average length of stay for all inpatients was 38.0 ± 75.6 days, while for patients with CRKP, it was 51.3 ± 90.5 days. In contrast, the average length of stay of patients infected with CSKP (32.5 ± 68.0) or hvKP (27.0 ± 42.8) was significantly lower than for those with CRKP (Most isolates originated from respiratory specimens (58.6%), followed by urinary tract (16.7%) and blood (14.7%). The highest proportion of patients were in ICU (29.0%), followed by the emergency department (10.9%), neurosurgery (10.6%), rheumatology (8.0%) and urology (5.7%). Notably, the highest incidence of CRKP infections occurred in neurosurgery (27.3%) and accounted for 70.3% of all specimens from that department. One hundred and sixty-two (46.5%) patients were classified as nosocomial having acquired the infection after 48 h of admission to hospital. No distinction was made between health care-associated and community-acquired infections.All isolates were resistant to ampicillin, but none was resistant to colistin, tigecycline and polymyxin. Twenty-nine CRKP-infected patients had received carbapenem antibiotics in the 3 months prior to recovery of the isolate. The hvKP isolates were generally susceptible to antibiotics (>80%) compared with CRKP (0–18.1%). CRKP isolates were almost uniformly resistant to antibiotics (>98%) with the exception of amikacin (susceptibility 18.1%) and gentamicin (susceptibility 6.4%).n = 254) fell into 57 K types with six types , accounting for almost half of this phenotype  were assigned to a K type and their distribution according to phenotype is shown in henotype . Likewishenotype . The moshenotype .Fig. 1.Six O genotypes (typeability 94.3%) were identified among all isolates with three types  together accounting for 85.6% of all isolates. PCR products were not obtained for 20 isolates.n = 46), of which 39 and five isolates were CRKP and hvKP, respectively. For the combination O1:K1 ; other frequent combinations were O1:K2 (n = 25) and O3:K57 (n = 16). The latter two serotype combinations accounted for 19 and 14 hvKP isolates, respectively. Eleven hvKP isolates were carbapenem-resistant, including four of O2a:K14K64, two each of O1:K2 and O3:K57, along with single isolates of O1:K1, O1:K14K64 and O3:K5. Regarding associations of capsular and O serotypes, the majority (80.7%) of K14K64 isolates were O2a, while isolates of K1, K2, K19, K5 and K60 were grouped predominantly in serotype O1. Moreover, serotype O3 was strongly associated with K57 isolates but no association of K54 and K63 isolates with an O type was noted. Lastly, five isolates of K47 were O-non-typeable.In total, 80 distinct O:K genotype combinations were identified; 15 isolates were ascribed only to an O or K type. Overall sero-typeability was 98.6% with the most common combinations being O2a:K14K64 . The highest rate of CRKP infections was associated with neurosurgical and ICU departments, the latter having been previously identified as an independent risk factor for acquisition of such strains [In this study, a substantial proportion of hospitalised patients were infected with CRKP. Approximately 80% were nosocomially acquired and strongly associated with the use of indwelling medical devices compared with CSKP isolates , followed by K1 (14.6%) and K2 (8.0%). Differences in the distribution of K types are most likely due to geographical source. Early serotyping surveys reported K2, K8, K9, K21 and K24 to be among the most frequent , but fewOur results showed that the K type distribution among CRKP differed substantially from hvKP isolates with K14K64 (49.5%), K19 (10.5%), K47 (6.3%), K60 (5.3%) and K61 (5.3%) being the most common CRKP serotypes. K64 serotype was also reported to be the major type of CRKP (38%) in Taiwan . In contThe combination of certain O and K serotypes is of interest. Serotype O2a accounted for 80% of K14K64 isolates; O1 was predominantly associated with K1 (88.2%) as well as with K2 (89.3%), and less so with serotypes K19, K5 and K60. Strains of K54 and K63 were evenly distributed in both O types, but notably K47 was linked with strains lacking a demonstrable O type. Despite these associations, O and K combinations may aid the tracking and evolution of strains in outbreak situations if used in conjunction with DNA profiling and sequence-based methods.A significant limitation of our study is that isolates were collected in a single hospital and so the serotype distribution data might not be representative of the wider regional or national situation. Second, hvKP isolates were identified only by their hypermucoviscosity on agar culture and confirmation of the presence of plasmid genes encoding hypervirulence was not undertaken K. pneumoniae provided a useful tool to determine serotype distribution in a large collection of isolates from a tertiary hospital setting in China. The predominant K types were K14K64, K1, K2 and K57. Carbapenem-resistant strains exhibited different serotype distributions from those of the hypervirulent phenotype with serotype combination O2a:K14K64 predominating in the former, and O1:K1 and O1:K2 among the latter. These data are of value to inform vaccine design should they be considered as a potential therapeutic approach.In conclusion, molecular typing of O and K antigens of


In this work, the feasibility of synthesizing the Ca1-xTiO3:Eu3+x phosphor with a good luminescent performance by combining the chemical co-precipitation method and microwave-assisted sintering was studied. The precursor of Ca1-xTiO3:Eu3+x phosphors were prepared by the chemical co-precipitation method. To find an optimized process, we applied both of the traditional (furnace) sintering and the microwave-assisted sintering to synthesize the Ca1-xTiO3:Eu3+x phosphors. We found out that a sintering power of 528 W for 50 min (temperature around 950 °C) by a microwave oven resulted in similar emission intensity results compared to traditional furnace sintering at 900 °C for 2.5 h. The synthesized Ca1-xTiO3:Eu3+x phosphors has an emission peak at 617 nm (5D0→7F2), which corresponds to the red light band. This new synthesized method is an energy efficient, time saving, and environmentally friendly means for the preparation of Ca1-xTiO3:Eu3+x red phosphor with good luminescent performance.The synthesis process has a significant influence on the properties of Ca Particularly, rare earth–activated CaTiO3 phosphors 2−. 1-xTiO3:Eu3+x phosphors calculated by JADE software (6.5). It can be seen in 3+ content, which is as a reason for the distance decrease of the lattice and the surrounding O2− ion. Indeed, the ion radius (94.7 pm) of Eu3+ is slightly smaller than the ion radius (100 pm) of Ca2+. Moreover, the change of lattice constant also proved that the Eu3+ ions are successfully doped into the crystal structure.To determine the best concentration of Eu1-xTiO3:Eu3+x phosphors doped with various concentrations of Eu3+. The emission spectrum mainly presents four emission peaks, located at 597 nm, 617 nm, 658 nm, and 700 nm, respectively, under excitation wavelength of 398 nm, which correspond to excited state 5D0→7FJ (J = 1~4) transition of the Eu3+ ion. The main emission peak is at 617 nm (5D0→7F2), which corresponds to the red light band. The 5D0→7F2 transition is the electric dipole transition. When the Eu3+ ion occupies the asymmetric position, the electric dipole transition will be the main transition, which also proves the replacement of Eu3+ ions in the crystal structure to Ca2+ ions.3+ ion doping amount, more luminescent centers are formed in the matrix, which increases the luminescence intensity. The emission intensity appears to reduce after it reaches the maximum at x = 0.03 (3 mol%). This is due to the interaction between the luminescent centers by increasing the Eu3+ concentration, the continuous migration of the excitation energy between the centers will not generate effective radiation. Therefore, 3 mol% is the best doping concentration of Eu3+.With the addition of Eu0.97TiO3: Eu3+0.03 phosphor cannot absorb the microwaves; thus, a small crucible with dry Ca0.97TiO3: Eu3+0.03 phosphor powder was placed in a large crucible filled with 3/4 volume of activated carbon. The activated carbon was heated using 2.45 GHz microwave radiation with a maximum power of 800 W. The precursor was heated by heat transfer from the activated carbon to prepare the Ca0.97TiO3: Eu3+0.03 phosphor. We note that the sintering atmosphere in the microwave oven might also slightly change due to the existence of activated carbon underneath the sample crucible. To record the temperature in the microwave oven, we placed a thermocouple into the crucible. The insulation inside the microwave oven is relatively poor, thus, we used an electric potential difference meter to calibrate the instantaneous temperature.Microwave-assisted sintering has many advantages, including rapid heating and energy saving. However, the Ca0.97TiO3:Eu3+0.03 phosphor samples sintered at different power. It can be seen in 0.97TiO3:Eu3+0.03 phosphor can be synthesized with microwave-assisted heating at 800 W for 30 min (at a temperature around 1100 °C) while still in the presence of TiO2 crystals, which indicates 30 min is not long enough for the fully crystallization of the Ca0.97TiO3:Eu3+0.03 phosphor prepared by microwave-assisted sintering at 800 W. The diffraction peaks of the samples are corresponding to the standard cards JCPDS NO.42-0423, which is, the orthorhombic CaTiO3 structure. From 0.97TiO3:Eu3+0.03 phosphor is fully crystallized at the power of 680 W for 50 min (at a temperature around 1025 °C). The results in 0.97TiO3:Eu3+0.03 phosphor were synthesized at 528 W for 50 min (at a temperature around 950 °C). Therefore, a sintering duration of 50 min can synthesize the pure Ca0.97TiO3:Eu3+0.03 phosphor in a microwave oven at different power, where, a power of 528 W for 50 min is the most energy-efficient way.0.97TiO3:Eu3+0.03 prepared at different microwave sintering power . It can be seen from 5D0→7FJ (J = 1~4) transition of Eu3+ ion. Moreover, with the sintering time prolonged, the emission intensity firstly increased to a peak at 50 min and then decreased. A sintering time of 60 min will not significantly increase the emission intensity, which is due to the completion of the crystallization process.0.97TiO3:Eu3+0.03 synthesized by microwave-assisted methods and traditional furnace sintering method. A sintering power of 528 W for 50 min by microwave oven gets the similar emission intensity results as the traditional furnace sintering at 900 °C for 2.5 h. The emission intensity will significantly decrease when we further increase the power. Indeed, the microwave-assisted sintering will greatly save the energy and time for the preparation of Ca0.97TiO3:Eu3+0.03 red phosphor with good luminescent performance.0.97TiO3:Eu3+0.03 phosphors (best doping) synthesized by the different synthesize methods and scanned by SEM. It can be seen from 3:Eu3+0.03 phosphors have a rod shape, which agrees with the orthorhombic perovskite structure from the XRD results. However, the CaTiO3:Eu3+0.03 phosphors synthesized by the microwave-assisted sintering process are slightly bigger while containing numerous defects on the surface compared to the ones prepared by the traditional sintering processes. The imperfection of the shape of the CaTiO3:Eu3+0.03 phosphors is attributed to the rapid and shorter sintering process in a microwave oven.1-xTiO3:Eu3+x phosphor with a good luminescent performance by combining the chemical co-precipitation method and microwave-assisted sintering was studied. First, the precursor was synthesized by the chemical co-precipitation method, which was confirmed by the FT-IR test. Then, we synthesized the Ca1-xTiO3:Eu3+ phosphor by traditional (furnace) sintering based on the TG-DSC analysis of the precursors. We found that the Ca1-xTiO3:Eu3+x phosphor reached a peak emission intensity with a doping amount of 3 mol% Eu3+ and traditional sintering temperature of 900 °C for 2.5 h. Finally, we applied the microwave-assisted sintering with powers from 528 W to 800 W (temperature from 950–1100 °C) to prepare the Ca0.97TiO3:Eu3+0.03 phosphor and found that a sintering power of 528 W for 50 mins by microwave oven resulted in similar emission intensity results compared to traditional furnace sintering at 900 °C for 2.5 h. The new synthesized method will greatly save energy and time and is an environmentally friendly method for the preparation of Ca0.97TiO3:Eu3+0.03 red phosphor with good luminescent performance.In this work, the feasibility of synthesizing Ca


The molecule was also capable of selectively inhibiting UCHL1 in cells and did not demonstrate any discernible off-target toxicity. Finally, the molecule was used for initial probe studies to assess the role of UCHL1 role in proliferation of myeloma cells and migration behavior in small cell lung cancer cells making 34 a new tool to be used in the biological evaluation of UCHL1.The deubiquitinating enzyme (DUB) UCHL1 is implicated in various disease states including neurodegenerative disease and cancer. However, there is a lack of quality probe molecules to gain a better understanding on UCHL1 biology. To this end a study was carried out to fully characterize and optimize the irreversible covalent UCHL1 inhibitor VAEFMK. Structure-activity relationship studies identified modifications to improve activity versus the target and a full cellular characterization was carried out for the first time with this scaffold. The studies produced a new inhibitor,  Ubiquitination of substrate proteins plays a role in a number of cellular pathways including protein trafficking, DNA damage response, and proteasomal degradation ,2,3. TheThe removal of Ub is catalyzed by deubiquitinases (DUBs), of which there are approximately 100 in the human genome . The cysUbiquitin C-terminal Hydrolase L1 (UCHL1) is a 25 kDa DUB whose ectopic expression is linked to a number of cancers . Normall50 value of 670 nM after 30-min pre-incubation while IMP-1710 displayed a value of 38 nM. However, these inhibitors may suffer from apparent non-selective toxicity, particularly in non-UCHL1 expressing cells. Krabill et al. . Fo. FoKI) a5 M−1s−1 . In comp1 bound to UCHL1 confirms that the inhibitor does not bind to the Ub-binding site of UCHL1, but actually approaches and binds to the face opposite of the Ub-site and interacts with the catalytic cysteine . His-UCHL1 was preincubated with an excess of 1 (2 mM) or DMSO overnight at room temperature to ensure full covalent modification of the Cys90 was achieved. The dissociation constant (Kd) of Ub towards UCHL1 pre-treated with DMSO treated control was determined and proved to be similar to previously published affinities  maintain a mono-Ub pool within cells ,66 and , KMS11 and KMS12 (myeloma cell lines). 1 exhibited reduced proliferation compared to DMSO controls after 24, 48, 72 and 96 h timepoints , washed with PBS, then lysed and treated with HA-Ub-VME. The HA-Ub-VME labels any remaining DUBs that have not been inhibited by 1. Blotting for UCHL1 showed a dose-dependent decrease in HA-Ub-UCHL1 complex formation with increasing concentration of 1, confirming on-target engagement of UCHL1 in cells  gel-shift assay was employed . Using tormation . No othe1 and 34 (100 µM) has no effect on the cell viability, whereas chloromethylketones 14 and 37 both kill the cells in a dose-dependent manner with CC50 values of 0.156 µM and 0.176 µM, respectively  with very light bands at other molecular weights that are likely a result of weak non-specific activity at other cysteines  to visualize proteins within the cells that were conjugated to either the fluoromethylketone ysteines C. Converthe cell C. The deUCHL1 has been reported to regulate and contribute to cell invasiveness and metastatic behavior in small cell lung cancer. To assess the role of UCHL1 in cell migration, a wound healing assay was performed using control and UCHL1 depleted SW1721 cells. First, shRNA knockdown of UCHL1 in SW1271 cells was performed and efficient knockdown was observed with shRNA 079 and 274 compared to the vector control pLKO . While t1 and 34 were first assessed for their effect on cell viability at 100 µM to determine if any observed reduction in cell viability may influence the migration assays  in [D6] DMSO or CDCl3 with or without the internal standard of TMS at 0.05 or 0.1% v/v. The purity of all final compounds was > 95% purity as assessed by HPLC. Final compounds were analyzed on a 1200 series chromatograph . The chromatographic methods used were either: (A) Hypersil GOLD C18 column  3 µM particle size, 150 mm length, 4.6 mm ID) or (B) a Thermo Scientific Hypersil GOLD C18 column . UV detection wavelength = 254 nm; flow rate = 1.0 mL min−1; solvent = acetonitrile/water. Both organic and aqueous mobile phase contain 0.1% v/v trifluoroacetic acid. The mass spectrometer used is a CMS-L Compact Mass Spectrometer  with an ESI or an APCI ionization source. Samples are submitted for analysis using either the atmospheric solids analysis probe (ASAP) or flow injection analysis (FIA). Compounds were prepared according to the following protocols and are detailed below. Intermediates 13a  and 13b  were purchased from commercial vendors. These intermediates were left in 21 presented. All other analogs and alternative synthetic procedures reported in Synthesis for analog 2 , benzyl alcohol , p-toluenesulfonic acid monohydrate  in toluene (5.6 mL) were placed in a round bottom flask outfitted with a Dean-Stark apparatus and was heated with stirring to 75 °C in vacuo (27 mm of Hg) until all of the toluene had distilled. Then  for an additional 5 h. The mixture was cooled to 75 °C and isopropanol (15 mL) was added followed by hexanes (30 mL). The mixture was placed in the freezer and allowed to crystalize overnight. The product was filtered, washed with hexanes (2 × 30 mL), and dried overnight in vacuo, yielding 3 as a white solid . 1H NMR  δ 7.64–7.28 , 5.37 , 5.25 ; APCI-MS: m/z 301.1 [M − H]−.A mixture of dimethyl 2-fluoromalonate 3  was suspended in isopropanol (12 mL) and heated to 45 °C. 1.0 M aqueous NaOH  was added dropwise over 1 h. After an additional 10 min, the solution was concentrated to 5 mL and water (2.5 mL) was added. The pH was adjusted to 9 using saturated sodium bicarbonate and washed with DCM (5 × 5 mL) to remove benzyl alcohol. The pH of the solution was adjusted to 2.2 using 5 M HCl, and di-isopropyl ether (5 mL) was used to extract the product. The pH of the aqueous layer was adjusted to 1.9 using 5 M HCl, and further extracted with di-isopropyl ether (5 mL). The combined extracts were washed with brine (5 mL), dried over MgSO4, filtered, and concentrated in vacuo at 35 °C to provide an oily residue. This was triturated with hexanes (7 mL) overnight with stirring to give a solid. The solid was filtered and dried under vacuum to yield 4 as a white solid . 1H NMR  δ 7.38 , 5.40 , 5.32 .Intermediate 4  and THF (2 mL/mmol). This was cooled to 0 °C before adding 2.0 M isopropylmagnesium chloride in THF . The white suspension was stirred for 1 h at 0 °C to generate the magnesium salt 5 to be used for the next reaction.To vial #1 was added S)-2-((tert-butoxycarbonyl)amino)-5-methoxy-5-oxopentanoic acid 6a  and THF (25 mL). The solution was cooled to 0 °C before addition of 1,1′-carbonyldiimidazole . The reaction was stirred for 1 h at 0 °C. After 1 h vial #2 was cooled to −20 °C followed by dropwise addition of magnesium salt 5 from vial #1. The mixture was stirred for 45 min at −20 °C, and then allowed to warm to room temperature and stir for 3.5 h. The reaction contents were then poured onto 1.0 M HCl, extracted with EtOAc, washed in succession with saturated sodium bicarbonate and brine then dried over sodium sulfate. The organic layer was then filtered and concentrated under reduced pressure to provide crude 1-benzyl 7-methyl (4S)-4-((tert-butoxycarbonyl)amino)-2-fluoro-3-oxoheptanedioate 7a. To vial #2 was added  as a white solid. 1H NMR  δ 5.21 , 5.15–4.91 , 4.62 , 3.68 , 2.57–2.31 , 2.27–2.16 , 1.88 , 1.44 . APCI-MS: m/z 278.0 [M + H]+. The residue was dissolved in toluene and Pd/C  was added followed by bubbling with hydrogen gas through the solution for 10 min. The bubbling was stopped and the reaction was allowed to stir under hydrogen atmosphere overnight at room temperature. The mixture was then filtered, concentrated, and purified via flash chromatography (5–60% EtOAc in hexanes) to yield L-phenylalanine  and N-methylmorpholine  in anhydrous THF was added isobutyl chloroformate  at 0 °C. The methyl L-alaninate hydrochloride  was then added and the reaction was allowed to warm slowly to room temperature overnight. The next day the reaction mixture was diluted with EtOAc (25 mL) and washed in succession with 1.0 M HCl (50 mL), 5% sodium carbonate solution (50 mL), brine (50 mL), then dried over sodium sulfate before filtering and concentrating in vacuo. The residue was recrystallized in a DCM:Hexanes solution and filtered, yielding 12g as a white crystalline solid . 1H NMR  δ 8.49 , 7.49 , 7.37–7.02 , 4.88 , 4.39–4.10 , 3.58 , 2.96 , 2.67 , 1.28 ; APCI-MS: m/z 385.1 [M + H]+.To a solution of ((benzyloxy)carbonyl)-12g  was dissolved in THF:H2O  followed by addition of LiOH  at 0 °C. The mixture was stirred at 0 °C for 3 h before acidifying with 1.0 M HCl (2 mL) to < pH 3. The aqueous phase was extracted with EtOAc (3 × 5 mL) and the organic layers were combined and dried over sodium sulfate, filtered, and concentrated to yield 13g as a white solid  which was taken forward crude to the next step.The dipeptide methyl ester 8a  and 4.0 M HCl in 1,4-dioxane . This was allowed to stir for 30 min at room temperature and monitored for completion by TLC, which indicated the formation of a very polar product. Upon completion, the solvent was removed in vacuo, and the oil was triturated with diethyl ether for 10 min and carefully decanted and dried in vacuo. The resulting Boc-removed Glu(OMe)-fluoromethylketone oil was diluted in THF and used directly in the next step.To vial #1 was added 13g  in THF under argon atmosphere. To this solution was added isobutyl chloroformate  and N-methylmorpholine  at 0 °C under argon atmosphere. This was allowed to stir for 45 min at 0 °C after which the deprotected Glu(OMe)-fluoromethylketone in THF from vial #1 was added dropwise at 0 °C and allowed to stir for 15 min before warming to room temperature slowly overnight. The next day the reaction was diluted with EtOAc and washed in succession with 1 N HCl, 5% sodium bicarbonate, and brine. The organic layer was collected and dried over sodium sulfate, filtered, and concentrated in vacuo to provide the crude product. This residue was then recrystallized in DCM:Hexanes overnight, filtered, and dried in vacuo to yield 21 as a white solid . 1H NMR  δ 8.33 , 8.27 , 7.51 , 7.35–7.14 , 5.25–5.07 , 4.93 , 4.40–4.15 , 3.57 , 3.01 , 2.78–2.64 , 2.40–2.26 , 2.12–1.98 , 1.86–1.63 , 1.26 . 13C NMR  δ 203.7 , 173.3, 173.1, 172.0, 156.3, 138.5, 137.4, 129.6, 128.7, 128.4, 128.1, 127.8, 126.6, 84.3 , 65.6, 56.3, 54.7, 51.8, 48.7, 37.7, 29.7, 24.6, 18.0. ESI-MS: m/z 530.0 [M + H]+; HPLC tR = 11.974 min (Column B); HPLC Purity: 98.2%.To vial #2 was added w/v) BSA) to a concentration of 2.5 nM for UCHL1 or 0.025 nM for UCHL3. Stock solutions of Ub-Rhodamine 110 ; 125 nM for UCHL1 assay, and 250 nM for UCHL3 assay) were prepared in the same buffer. A 10 mM stock solution was made for each inhibitor in DMSO, then a dilution of 600 µM in reaction buffer was made followed eight by 1:1 serial dilutions. To each well was added 20 µL of DUB stock solution and 10 µL of inhibitor solutions for nine final inhibitor concentrations of ranging from 0.78 µM–200 µM along with a DMSO only control well. These were allowed to incubate, while sealed, for the 3 h at room temperature. After incubation 20 µL of each Ub-Rho stock solution was added to the respective wells for each DUB to yield final concentration of 50 nM for UCHL1 or 100 nM for UCHL3, respectively. Plates were read immediately and fluorescence of cleaved Rhodamine 110 fluorophore was monitored at λex = 485 nm, λem = 535 nm continuously for 20 min on a Synergy Neo2 instrument  The raw data was loaded into GraphPad Prism 8  and the slope of the linear portion of the fluorescence vs. time curves was calculation for each inhibitor concentration and % activity of the enzyme was determined compared to DMSO-treated controls. The % activity was plotted as a function of inhibitor concentration and the data was fitted with non-linear regression analysis to calculate the IC50 values. Errors are reported as the 95% confidence interval calculated from experiments collected in technical triplicate. The assay was performed in black 384-well plates . DUB stock solutions were diluted in reaction buffer  for inhibition at each concentration. The kobs was plotted as a function of inhibitor concentration and then fit to the equation Y = kinactX/(KI + X) to calculate kinact, KI and kinact/KI as described by Resnick et al. . The concentration of Ub was determined by the BCA assay and diluted to top concentrations into BLI buffer, and 1:1 serial dilutions were completed. The top concentration of Ub was 2 μM. 40 μL of each solution was added to a 384-well tilted-bottom plate . The Ni-NTA biosensor was dipped first into BLI buffer , then into the His-UCH protein wells , then into BLI buffer alone , then into the lowest concentration of UbV , and then into buffer alone . A reference sensor loaded with protein was dipped into wells containing only buffer to adjust for protein-buffer signals. The association-dissociation was repeated with increasing concentrations of UbV. All measurements were taken at 30 °C. This assay was carried out according to our previously published protocol using an Octet RED384 biolayer interferometer  . A solutd.Octet RED384 Data Analysis Software  was used to collect and analyze the raw data for the association and dissociation curves. After subtraction of a reference sensor (loaded sensors dipped into wells containing only buffer), averages of the association responses  were calculated and plotted as a function of UbV concentration in Prism 8. These data were fit to a nonlinear regression one-site specific binding model to determine a KC90A, and UCHL3 (Expressed in pET15b by GenScript) for biochemical assays were grown in LB growth medium at 37 °C to an optical density of 0.6–0.8. After 0.1 mm IPTG induction at 17 °C for 18 h the bacteria were lysed and pelleted at 15,000g, and clarified lysate was purified on HisPur Ni-NTA resin (Thermo Scientific) according to manufacturer’s instructions.UCHL1, UCHL1WT or UCHL1C90A (from pET15b) (49 µL) was incubated with 10 µM inhibitor for 3 h at room temperature in PBS starting block buffer (Thermo Scientific # 37538). After 3 h, 6 µL of a freshly prepared cocktail was added to each sample, consisting of: 3 µL 1.7 mM THPTA in 1:4 DMSO/tBuOH , 1 µL 50 mM CuSO4 , 1 µL TCEP HCl , and 1 uL Cy5-Azide . The samples were allowed to react for 2 h at room temperature protected from light before precipitating the sample by adding 600 µL MeOH, 150 µL chloroform, and 400 µL water, vortexing between each addition. The samples were pelleted by centrifuging at 170,000× g for 5 min. The upper aqueous layer was removed without disturbing the precipitate, and 450 µL MeOH was added again, followed by centrifuging at 17,000× g for 5 min and decanting the solvent (2X). The samples were allowed to air dry for 30 min before resuspending in 1× Laemmli buffer and boiling at 90 °C for 5 min. The samples were then analyzed by SDS-PAGE and imaged on an Odyssey system  and subsequently Coomassie stained.Solutions containing 1 mg/mL recombinant UCHL1WT or UCHL1C90A (from pET15b) (49 µL) was incubated with either 10 µM or 20 µM inhibitor for 3 h at room temperature in PBS starting block buffer (Thermo Scientific # 37538). To quench reaction, chilled acetone was added to the sample to achieve an overall 80% acetone solution. After overnight storage at −20 °C, suspension was centrifuged at 14,000× g at 4 °C for 15 min. Supernatant was discarded, and pellet was dried by vacuum centrifugation for 1 h. Pellet was reconstituted in 50/50 water/acetonitrile with 0.1% formic acid. 25 pmol per sample was analyzed by LC/MS  attached to an Agilent 6129 quadrupole mass spectrometer in positive ion mode.Solutions containing 10 µM recombinant UCHL1m/z.The column was held at 45 °C. Mobile solution A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in acetonitrile. The gradient used was hold at 5% B for 5 min, increase linearly to 100% B for 10, and then hold at 95% B for 5 min. The mass data were collected at a range of 500–1000 m/z range of 670–900 Da, and deconvoluted mass range from 25,000–26,000 Da. Representative data had an abundance threshold of 10–20% for charged state deconvolution calculation. For C90A mutant, this abundance threshold was lowered to 5% to detect the presence of any reacted protein. 34 adduct is expected to be +486.00 Da after displacement of fluorine.Raw data were processed using MestReNova. For all samples, the deconvoluted mass was calculated with a charged state range from 27 to 36, www.essenbioscience.com, accessed on 31 August 2020). Cells  were seeded in triplicate in 96 well plates the night prior to the addition of compounds. The IncuCyte was set to collect images from five fields per well at 4-h intervals by using the nuclear dye Nuclight Red (Sartorius). At each timepoint, the number of cells per well was deter- mined by taking the mean of the five locations in each well. The relative proliferation at each timepoint was normalized, with 100% set as the number of cells observed for incubated with DMSO (0.1%) controls at 72 h.The proliferation of KMS11 and KMS12 cells was monitored using an IncuCyte devise . HA-Ub-VME was then added to a final concentration of 1 µM and incubated for 1 h at room temperature. An equal volume of 2X Laemmli sample buffer was added, and the samples were heated to 95 °C before separating by gel electrophoresis and analyzed by immunoblot.Twelve hours prior to the addition of compounds to the cells, 1.2 million KMS11 or KMS12 cells mLLuciferase-constructed cancer cells were seeded in 96-well plates at 10,000 cells/well with the compounds at the indicated concentrations. DMSO was used as vehicle control. Luminescence readings were acquired after adding 1% D-luciferin  after 24 h. Cell viability was determined by the ratio of luminescence reading compared to the one at 0 h, and normalized to vehicle control. 2, 0.5 mM EDTA, 5 mM DTT, 2 mM ATP, 0.5% NP-40, 10% v/v glycerol) and incubating on ice for 30 min with vigorous vortexing every 10 min. Cell lysate was clarified by centrifugation  and the supernatant was collected, and the concentration was determined by Bradford (performed according to manufacturer’s protocol). Cells were normalized to 2 mg mL−1. To each reaction mixture was added 36 µL of lysate and 2 µL of HA-Ub-VME or lysis buffer. This was incubated for 30 min at room temperature before quenching with 15 µL of 4X Laemmli buffer and heating to 95 °C for 5 min. Samples were subjected to SDS-PAGE and immunoblot.Cells were treated with 100 µM inhibitor or DMSO for 4 h before being washed, scraped, and collected. Cell pellets were thawed on ice before resuspending in 200 µL lysis buffer  for 30 min on ice with vigorous vortexing every 10 min. Samples were then pelleted at 17,000× g for 10 min before collecting the supernatant and measuring concentration using BCA. Samples were normalized to 2 mg/mL and stored at −80 °C overnight. The following day, samples were thawed. While thawing, a click cocktail was prepared . 50 µL of each sample was added to a new tube, and 6 µL of click cocktail was also added to these tubes. The samples were allowed to react for 2 h at room temperature protected from light before precipitating the sample by adding 600 µL MeOH, 150 µL Chloroform, and 400 µL water, vortexing between each addition. The samples were pelleted by centrifuging at 17,000× g for 5 min. The upper aqueous layer was removed without disturbing the precipitate, and 450 µL MeOH was added again, followed by centrifuging at 17,000× g for 5 min (2X) and decanting the solvent. The samples were allowed to air dry for 30 min before resuspending in 4X Laemmli buffer and boiling at 90 °C for 5 min. The samples were then analyzed by SDS-PAGE and imaged on a Licor Odyssey and subsequently Coomassie stained.Cells were treated with 100 µM inhibitor or DMSO for 4 h before being washed, scraped, and lysed in 200 µL of lysis buffer  was defined as the percentage of area at each timepoint compared to the one at 0 h.1 it was determined that bulky, lipophilic moieties are preferred in the valine position, while alteration of the alanine position was not tolerated. Additionally, it was determined that unnatural D-amino acids abrogate activity. The SAR at these two positions could be rationalized by observations of the VAEFMK-bound UCHL1 crystal structure. Installing an alkyne handle to the benzyloxy capping group of the peptide improved activity when compared to three nearest neighbor analogs suggesting future SAR work on the benzyloxy may improve potency. To round out SAR it was determined that modification of the fluoromethylketone electrophilic site to a more reactive chloromethylketone surprisingly lost activity versus UCHL1 in vitro. Furthermore, when the chloromethylketone derivative was tested in cellular assays it was toxic and shown to non-specifically label numerous proteins. Conversely, the fluoromethylketones were selective for only UCHL1 over the closely related UCHL3. Ultimately, the scaffold was improved from a UCHL1 IC50 value of 24 µM for VAEFMK 1 to 7.7 µM for 34 with no activity against the closest related UCHL3 and only slight inhibition of UCHL5 at 250 µM. Finally, the probes were shown to engage UCHL1 selectively in two different cancer relevant cells lines. Molecule 1 displayed anti-proliferative properties versus KMS11 myeloma cells dependent on UCHL1 while the compound displayed no effect on KMS12 cells that do not express UCHL1. Analog 34 displayed anti-migratory properties in SW1271 small cell lung cancer cell lines consistent with observed behavior from genetic depletion of UCHL1. While the probe displays modest potency toward UCHL1 it is quite selective in cells and allowed for probing UCHL1 without reduction in cell viability as opposed to the shRNA knockdowns, which displayed some reduced viability. A potential way to improve the cellular activity of the scaffold may be to alter the Glu(OMe) moiety. This ester is likely cleaved by cellular esterases leaving the carboxylate side chain, which was shown to be inactive in vitro. The cellular inhibition is likely a competition between inhibitor engagement with UCHL1 and the ester being cleaved. Further optimization of this residue is ongoing. Alternative UCHL1 inhibitors from the cyanopyrrolidine scaffold, although selective in vitro, still exhibit off-target toxicity in the micromolar range while 34 does not exhibit any cellular toxicity up to 100 µM. Analog 34 is yet another option to selectively probe UCHL1 and offers additional capabilities in that an alkyne is already installed to be leveraged for various chemical biology applications.In summary, we have presented an SAR study and biological characterization of an irreversible covalent inhibitor of UCHL1. Starting with the parent compound VAEFMK


Catalysts with high selectivity play key roles in green chemistry. In this work, a granular Raney Ni catalyst using carbon as support (Raney Ni/C) was developed by mixing phenolic resin with Ni-Al alloy, conducting carbonization at high temperature, and leaching with alkaline liquor. The as-prepared Raney Ni/C catalyst is suitable for use in fix-bed reactors. Moreover, it shows high activity and selectivity for catalytic acetone hydrogenation. For instance, at the reaction temperature of 120 °C, the conversion of acetone can reach up to 99.9% and the main byproduct methyl isobutylcarbinol (MIBC) content can be diminished to 0.02 wt%. The Raney Ni/C may represent a new type of shaped Raney metal catalysts, which are important fix-bed catalysts in chemical industry. Catalysts play key roles in the chemical industry, and more than 90% of today’s chemical processes use catalysts ,2. Indus2O3 [2 [2 [3O4 [Isopropanol is a commonly used bulk chemical widely used in organic synthesis, industrial solvent, and fuel cells ,5,6,7. C2O3 , Ni/CeO22O3 [2 , Ni/SiO2O3 [2 [2 , and Pt/ [2 [3O4 . Neverth [2 [3O4 ,17,18,193 temperature-programmed desorption (NH3-TPD), respectively. Its catalytic properties in terms of acetone hydrogenation were studied at different reaction temperatures and compared with a traditional Ni/Al2O3 catalyst.Carbon materials have a variety of advantages  and 1590 cm−1 (G band), which are not separated completely. The D band corresponds to the common feature of all disordered graphitic carbon, while the G band represents the graphitic carbon phase with an sp2 electronic configuration [2-sorption measurements, the specific area and average pore width of Raney Ni/C are determined as 111 m2∙g−1 and 3.3 nm, respectively, by virtue of BET equation; the pore volume is calculated to be 0.092 cm3∙g−1 via Barrett-Joyner-Halenda (BJH) method; and the specific area, average pore width, and pore volume of Ni/Al2O3 are 105 m2∙g−1, 10.8 nm, and 0.29 cm3∙g−1, respectively. Hence, the Raney Ni/C is mesoporous and possesses a more small-aperture pore structure than Ni/Al2O3. SEM image shown in 2O3 were measured by NH3-TPD and are shown in 2O3 show two desorption peaks. Obviously, they have similar weak acidic site desorption peaks at 180 °C; meanwhile, the Raney Ni/C shows a smaller medium-strong acidic sites desorption peak than the Ni/Al2O3 at 350 °C [2O3.The microstructure of Raney Ni/C was characterized by Raman spectrum. As shown in guration . In addiguration . Here, t2O3 are listed in 2O3. Moreover, MIBC is the main byproduct of acetone hydrogenation. When the reaction temperature reached 150 °C, the concentration of MIBC catalyzed by Raney Ni/C was merely 500 ppm (0.05 wt%). As for Ni/Al2O3, the MIBC concentration was 56,000 ppm (5.6 wt%) at the same reaction conditions. Obviously, the Raney Ni/C catalyst can reduce the MIBC byproduct of acetone hydrogenation. This can be attributed to the different acidities of catalyst supports attached to carbon and Al2O3, respectively. Furthermore, in order to testify the effect of acidity on catalytic acetone hydrogenation, a strong acidic cation exchange resin was simply mixed with Raney Ni/C and used as an acetone hydrogenation catalyst. As shown in The numerical results of acetone hydrogenation at 80–150 °C catalyzed by Raney Ni/C and Ni/Al2O3 catalyst was prepared according to the commercial impregnation method. To be specific, 10 g Ni(NO3)2∙6H2O was dissolved in 3 g deionized water, to which 10 g Al2O3 was added and impregnated for 1 h. The above mixture was dried at 120 °C for 12 h, followed with calcined at 360 °C for 4 h. Finally, Ni/Al2O3 was obtained after reduced by hydrogen at 400 °C for 8 h. The Ni-loading of Ni/Al2O3 was 20 wt%.In this study, a typical Raney Ni/C was prepared as follows: 2.5 g Ni-Al alloy powder, 10 g powdery phenol formaldehyde resin, and 1.2 g hexamethylenetetramine were mixed by a high speed mixer . The mixture was filled into a 2 mm mold and compressed tightly at a pressure of 5 Mpa. Then, a 2 mm-thick plate was obtained. Next, the 2 mm-thick plate was dried at 150 °C for 30 min, and mechanically cut into particles with a particle size of approximately 2 mm. Finally, granular Raney Ni/C catalyst was obtained through carbonizing the 2 mm particles at the conditions of 800 °C and nitrogen atmosphere for 2 h, and aluminum of the carbonized particles was leached using alkaline liquid. The Ni-loading of granular Raney Ni/C is 20 wt%. The traditional Ni/AlMore detailed experimental information, including chemical materials, characterization methods, and hydrogenation experiment, is attached in the Raney Ni is shaped as a granular catalyst by using carbon material as support. This Raney Ni/C can be used in fix-bed reactors, which overcomes the disadvantages of commonly used Raney nickel catalyst. Moreover, the Raney Ni/C can dramatically reduce the MIBC byproduct for catalytic acetone hydrogenation, as a result of the weak acidity of the carbon-support. The recycling of the Raney Ni/C would only need calcining in air to get rid of carbon-support, and the remaining metal alloys could be reused directly. We believe that the preparing method of the granular carbon-supported Raney nickel catalyst could be applied to a series of Raney metal catalysts, for instance, Raney cobalt and Raney copper, which are important fix-bed catalysts in chemical industry.


Saccharomyces cerevisiae, replicative life span (RLS) is primarily affected by the stability of ribosomal DNA (rDNA). The stability of the highly repetitive rDNA array is maintained through transcriptional silencing by the NAD+-dependent histone deacetylase Sir2. Recently, the loss of Smi1, a protein of unknown molecular function that has been proposed to be involved in cell wall synthesis, has been demonstrated to extend RLS in S. cerevisiae, but the mechanism by which Smi1 regulates RLS has not been elucidated. In this study, we determined that the loss of Smi1 extends RLS in a Sir2-dependent manner. We observed that the smi1Δ mutation enhances transcriptional silencing at the rDNA locus and promotes rDNA stability. In the absence of Smi1, the stress-responsive transcription factor Msn2 translocates from the cytoplasm to the nucleus, and nuclear-accumulated Msn2 stimulates the expression of nicotinamidase Pnc1, which serves as an activator of Sir2. In addition, we observed that the MAP kinase Hog1 is activated in smi1Δ cells and that the activation of Hog1 induces the translocation of Msn2 into the nucleus. Taken together, our findings suggest that the loss of Smi1 leads to the nuclear accumulation of Msn2 and stimulates the expression of Pnc1, thereby enhancing Sir2-mediated rDNA stability and extending RLS in S. cerevisiae.In  Saccharomyces cerevisiae  (S. cerevisiae rDNA consists of 100–200 tandemly arrayed copies of a 9.1-kb repeat on chromosome XII (S. cerevisiae.Two distinct life span paradigms have been proposed for revisiae . Chronolrevisiae . Replicarevisiae . A primas (ERCs) . S. ceresome XII , 6. Eachsome XII . Therefo+-dependent histone deacetylase and a key subunit of the RENT complex  complex, which suppresses Pol II-dependent transcription at the rDNA locus and inhibits homologous recombination between rDNA repeats . Sir2 is complex , 9, 10.  complex , 11, 12.+ salvage pathway , which is the Msn2/4 binding site in the PNC1 promoter . To confirm whether this alteration of RLS is truly SMI1-dependent, we introduced SMI1 to Smi1-deficient cells and measured RLS. Re-expression of SMI1 shortened the RLS of smi1Δ cells to a level similar to that of wild-type cells. Additional expression of SMI1 in wild-type cells did not affect the RLS of wild-type cells. These results indicate that the loss of Smi1 extends RLS, even though the overexpression of Smi1 does not affect RLS.To investigate whether Smi1 regulates replicative aging in yeast, we measured the RLS of Smi1-deficient cells. The RLS of pe cells A. To consmi1Δ cells was affected by Sir2. Consistent with the findings of a previous report (B). smi1Δ sir2Δ cells showed a similar RLS to that of sir2Δ cells. These results indicate that the loss of Smi1 increases RLS and that RLS extension in smi1Δ cells depends on the presence of Sir2.Because Sir2 is a well-known factor for extending RLS , we subss report , the RLSs report B. smi1Δ mURA3 silencing reporter gene integrated either inside the NTS1 region of the rDNA locus (RDN1-NTS1::mURA3) or outside the rDNA locus (leu2::mURA3) . Compared with wild-type cells, smi1Δ cells showed enhanced transcriptional silencing of mURA3 at the rDNA locus. In agreement with the RLS data, additional expression of SMI1 in wild-type cells did not alter rDNA silencing, and re-expression of SMI1 in smi1Δ cells restored the rDNA silencing of smi1Δ cells to a level similar to that of wild-type cells (A). As shown previously (sir2Δ cells (A). smi1Δ sir2Δ cells exhibited impaired rDNA silencing similar to that of sir2Δ cells.Given that Sir2 increases rDNA stability by rDNA silencing and thus extends RLS , 11, 12,::mURA3) . Cells wNA locus A and S1Ape cells A. As shoeviously , transcr2Δ cells A. smi1Δ mURA3 gene using real-time reverse transcription-PCR analysis. In wild-type cells, the transcription of the mURA3 gene was effectively silenced at the rDNA locus compared with the outside of the rDNA locus (B). In Smi1-deficient cells, the transcription of the mURA3 gene at the rDNA locus was further silenced. Consistent with the abovementioned spot assay results, transcriptional silencing of mURA3 at the rDNA locus was abolished in sir2Δ and smi1Δ sir2Δ cells. These results suggest that the absence of Smi1 increases Sir2-mediated rDNA silencing.To confirm the spot assay results described above, we measured the transcription levels of the NA locus B. In SmiADE2 marker gene integrated at the rDNA locus . As reported previously . We could not detect a GFP signal of Msn4 (data not shown), probably because of the low expression level of Msn4.Msn2 and Msn4 are transcription factors that regulate the general stress response of titutive . Under vtitutive . A prevititutive . Moreovetitutive , 18. To  nucleus A. We couPNC1 promoter in Smi1-deficient cells, we constructed yeast strains in which the endogenous MSN2 gene was modified to produce a C-terminal TAP fusion protein and conducted a chromatin immunoprecipitation (ChIP) assay. Notably, the loss of Smi1 increased the binding of Msn2 to the PNC1 promoter (B). Next, we examined the expression levels of Pnc1 in Smi1-deficient cells. The transcript levels of PNC1 were increased in smi1Δ cells (C). Consistent with this result, the protein levels of Pnc1 were also increased in smi1Δ cells (D). In the absence of Msn2/4, however, the loss of Smi1 did not increase the expression levels of Pnc1 (C and D). These results suggest that Smi1 deficiency results in an increase in Pnc1 levels by stimulating its transcription and that the effect of Smi1 on PNC1 expression is dependent on the presence of Msn2/4.Given the above observations that the loss of Smi1 enhances rDNA stability and induces the nuclear accumulation of Msn2, we assumed that increased rDNA stability in Smi1-deficient cells might be mediated by Pnc1, which is upregulated by Msn2. To investigate whether nuclear-localized Msn2 binds to the promoter B. Next, 1Δ cells C. Consis1Δ cells D. In the of Pnc1 , C and D+ and produces O-acetyl-ADP ribose and nicotinamide. Nicotinamide is converted to nicotinic acid by the nicotinamidase Pnc1 as part of the NAD+ salvage pathway. Since nicotinamide is a noncompetitive inhibitor of Sir2, nicotinamide clearance by Pnc1 enhances the enzymatic activity of Sir2 . smi1Δ cells showed further enrichment of Sir2 at the NTS1 and NTS2/18S regions. The protein levels of Sir2 were not increased in smi1Δ cells . Taken together, these observations suggest that Smi1 deficiency leads to enhanced association of Sir2 with the rDNA locus by increasing the expression of Pnc1 and extends RLS in an Msn2/4-dependent manner.For the deacetylation reaction, Sir2 consumes NAD of Sir2 . A previilencing . To exam reports , 14, Sir reports E. smi1Δ 1Δ cells , indicatr Msn2/4 F. Taken A). smi1Δ cells did not exhibit a significant change in Cki1 phosphorylation compared with wild-type cells. This result suggests that the cAMP-PKA signaling pathway is not involved in the nuclear accumulation of Msn2 in Smi1-deficient cells.The localization of Msn2/4 is regulated by the cAMP-PKA signaling pathway. While upregulated PKA activity phosphorylates Msn2/4 and inhibits the import of Msn2/4 into the nucleus, downregulated PKA activity dephosphorylates Msn2/4 and mediates the nuclear accumulation of Msn2/4 , 22. To Fig. S3smi1Δ cells. For the determination of TORC1 activity, the phosphorylation of Sch9, a major substrate of TORC1, was analyzed using an SDS-PAGE mobility shift assay (B). smi1Δ cells did not exhibit a significant change in Sch9 phosphorylation compared with wild-type cells. This result suggests that the TOR signaling pathway is not involved in the nuclear accumulation of Msn2 in the absence of Smi1.It has been shown that the intracellular localization of Msn2/4 is also regulated by the TOR signaling pathway. Activated TORC1 induces cytoplasmic retention of Msn2/4 by stimulating the binding of Msn2/4 to the 14-3-3 protein Bmh2, while TORC1 inhibition induces the translocation of Msn2/4 from the cytoplasm to the nucleus , 42. To ft assay . In keepft assay , 43, theft assay B. smi1Δ A). These results suggest that the loss of Smi1 activates Hog1. Next, we investigated the role of Hog1 in the nuclear accumulation of Msn2. Notably, the nuclear accumulation of Msn2 induced by Smi1 deficiency was abolished in the absence of Hog1 , suggesting that the effect of Smi1 on the nuclear accumulation of Msn2 is dependent on the presence of Hog1. To determine whether the loss of Smi1 induces the nuclear localization of Msn2 in a Hog1-dependent manner, we introduced HOG1 into smi1Δ hog1Δ cells and examined the nuclear accumulation of Msn2. Notably, re-expressing Hog1 in smi1Δ hog1Δ cells increased the nuclear accumulation of Msn2 to levels similar to those of smi1Δ cells (B and S4A)Msn2/4 activity is known to be modulated by several protein kinases, including Hog1 . Hog1 isFig. 41Δ cells B and S4Asmi1Δ cells, we examined the protein levels of Pnc1 in the absence of Hog1. The loss of Smi1 did not elevate Pnc1 expression in the absence of Hog1 (C), suggesting that the loss of Smi1 increases Pnc1 expression in a Hog1-dependent manner. In keeping with the nuclear accumulation of Msn2, re-expressing Hog1 in smi1Δ hog1Δ cells enhanced Pnc1 expression to levels similar to those of Smi1-deficient cells (C and D). Taken together, our observations suggest that the loss of Smi1 stimulates the expression of Pnc1 by translocating Msn2 from the cytoplasm to the nucleus, thereby extending RLS, and that this regulation is mediated by Hog1, not by the cAMP-PKA or the TOR signaling pathway.Next, to investigate the role played by Hog1 in Msn2/4-mediated Pnc1 expression in  of Hog1 C, suggesnt cells C and S4Bls C and D. Taken S. cerevisiae; the loss of Smi1 extends RLS by promoting Sir2-mediated rDNA silencing and rDNA stability. It is notable for a protein involved in the regulation of cell wall synthesis and maintenance to play a role in rDNA silencing and RLS regulation. Similar cases have been reported previously. For instance, the loss of Gas1, a β-1,3-glucanosyltransferase that participates in the formation and maintenance of β-1,3-glucan chains and cell wall assembly, increases Sir2-mediated rDNA silencing (smi1Δ cells (A). Second, while a defect in the β-1,3-glucan structure is a common feature of the gas1Δ mutant, smi1Δ mutant, and cells under Congo red treatment (B). In addition, several cell-wall-perturbing agents, such as Calcofluor white, caffeine, SDS, and vanadate, have not been observed to affect transcriptional silencing at the rDNA region , the portion of smi1Δ cells with nuclear-localized Hog1 was very small (data not shown). A similar phenomenon has been reported under prolonged ER stress conditions. Bicknell et al. observed that, under prolonged ER stress conditions, Hog1 phosphorylation was sustained even though Hog1 was retained in the cytosol  and the 2X Real-Time PCR Kit with SFCgreen I (BioFACT). The primers used for the amplification of mURA3, PNC1, and ACT1 are shown in mURA3, which contains the TRP1 promoter instead of the URA3 promoter, has been described previously  to those outside (leu2::mURA3) was calculated. The transcript level of PNC1 was measured and normalized against that of ACT1 and quantified by the 2−ΔΔCT method . cDNA for reverse transcription-PCR was generated using the ReverTra Ace qPCR RT Kit (TOYOBO). The amounts of eviously . For theT method . All relADE2 integrated at the rDNA locus of strain DMY3010 as described previously . The percentage of cells with predominately nuclear fluorescence was determined from three independent experiments. Approximately 200 cells were counted for each experiment and were analyzed statistically by a two-tailed Student's 600 of 1.2∼1.5 and cross-linked with 1% formaldehyde for 15 min. The reaction was quenched with glycine at a final concentration of 125 mM for 5 min. Cells were washed twice with cold phosphate-buffered saline. Cell pellets were resuspended in 400 μl of lysis buffer  and bead-beaten with 0.5-mm glass beads ten times for 1 min at 4 °C. Samples were incubated on ice for 2 min between bead beatings. Cell lysates were sonicated ten times for 15 s with amplitude set at 15% and centrifuged twice at 13,000 rpm for 10 min at 4 °C. The DNA concentration of each cell lysate was quantified and diluted to 2 mg/ml with lysis buffer. To obtain input DNA, 100 μl of lysate was used for each immunoprecipitation reaction. For ChIP experiments, 100 μl of a 50% slurry of prewashed IgG-agarose beads (Amersham Biosciences) was incubated with 1 ml of lysate at 4 °C overnight. Beads were washed twice in lysis buffer, once with wash buffer , and once with TE buffer at room temperature. Beads were eluted by adding 50 μl of elution buffer  and incubating at 65 °C for 10 min. Eluate was transferred to a fresh tube and pooled with a final bead wash of 150 μl of elution buffer. For input DNA, 100 μl of elution buffer was added to 100 μl of lysate. Input and ChIP samples were incubated at 65 °C overnight, combined with 300 μl of TE and 250 μg of proteinase K, and incubated at 37 °C for 2 h. All samples were extracted once with phenol:chloroform:isoamyl alcohol and once with chloroform. NaOAc , and 20 μg of glycogen was added to 400 μl of sample. Precipitated and washed DNA was resuspended in 100 μl of TE with 1 μg of RNase A and incubated at 37 °C for 1 h. ChIP samples were analyzed by quantitative real-time PCR using the QuantStudio 3 Real-Time PCR System (Applied Biosystems) and the 2X Real-Time PCR Kit with SFCgreen I (BioFACT). The primers used for PCR are shown in t-test.ChIP assays were performed as previously described . Yeast cExponentially growing cells were lysed by suspending cells in lysis buffer with protease inhibitors , followed by bead beating. Lysates were clarified by centrifugation at 13,000 rpm for 10 min at 4 °C. Protein concentration was determined by Bradford assay. Protein samples were mixed with 6× SDS sample buffer and boiled for 3 min at 95 °C. SDS-PAGE was performed with 8% separating gel. Western blot analysis was performed by standard methods using an HRP-conjugated mouse anti-GFP antibody  and an HRP-conjugated antimouse IgG antibody . Actin was used as a loading control and was detected by HRP-conjugated mouse antiactin antibody . Images were captured using a luminescent image analyzer, AE-9150 Ez-Capture II (ATTO), and quantified using CS analyzer version 3.0 software (ATTO).600 of ∼1.0 and lysed by suspending cells in lysis buffer with protease inhibitors and phosphatase inhibitors  followed by bead beating. Lysates were clarified by centrifugation at 13,000 rpm for 10 min at 4 °C. Protein concentration was determined by Bradford assay. Protein samples were mixed with 6× SDS sample buffer and boiled for 3 min at 95 °C. SDS-PAGE was performed with 8% separating gel. Cki1 phosphorylation was detected by SDS-PAGE and immunoblotting using an HRP-conjugated mouse anti-Myc antibody . Hog1 phosphorylation was detected by SDS-PAGE and immunoblotting using an anti-phospho-p38 MAPK (Thr180/Tyr182) antibody . Actin was used as a loading control and was detected by an HRP-conjugated mouse antiactin antibody . Analysis of phosphorylated Sch9 was conducted by western blotting, as described previously  followed by heating for 10 min to 65 °C. For 2-nitro-5-thiocyanobenzoic acid cleavage, 30 μl of 0.5 M cyclohexylamino ethane sulfonic acid, pH 10.5, and 20 μl of 7.5 mM 2-nitro-5-thiocyanobenzoic acid were added, and samples were incubated overnight at room temperature. Protein samples were mixed with 6× SDS sample buffer, boiled for 10 min at 65 °C, and clarified by centrifugation at 13,000 rpm for 1 min. SDS-PAGE was performed with an 8% separating gel. Sch9 phosphorylation was detected by SDS-PAGE and immunoblotting using an HRP-conjugated mouse anti-HA antibody . Actin was used as a loading control and was detected by HRP-conjugated mouse antiactin antibody .Analysis of phosphorylated Cki1 and phosphorylated Hog1 was conducted by western blotting, as described previously . Cells weviously . Cells wAll data are contained within the article.The authors declare that they have no conflicts of interest with the contents of this article.


High recurrence rates and poor survival of patients with esophageal squamous cell carcinoma (ESCC) after treatment make ongoing research on chemoprevention drugs for ESCC particularly important. In this study, we screened a large number of FDA-approved drugs and found levodopa, a drug used to treat Parkinson’s disease, had an inhibitory effect on the growth of ESCC cells. To elucidate the molecular mechanisms involved, we applied quantitative proteomics to investigate the anti-tumor activity of levodopa on ESCC. The results suggest that levodopa could down-regulate oxidative phosphorylation, non-alcoholic fatty liver disease, and Parkinson’s disease pathways. Major mitochondrial respiratory compounds were involved in the pathways, including succinate dehydrogenase subunit D, NADH-ubiquinone oxidoreductase Fe-S protein 4, and mitochondrial cytochrome c oxidase subunit 3. Down-regulation of these proteins was associated with mitochondrial dysfunction. Western blotting and immunofluorescence results confirmed the proteomics findings. Cell viability assays indicated mitochondrial activity was suppressed after levodopa treatment. Reduced mitochondrial membrane potential was detected using JC-1 staining and TMRE assays. Transmission electron microscopy revealed changes in the morphology of mitochondria. Taken together, these results indicate that levodopa inhibited the growth of ESCC through restraining mitochondria function. Esophageal cancer (EC) ranks seventh and sixth in global incidence and mortality, respectively, for malignant tumors  with esoLevodopa is a natural dopamine biosynthesis precursor and a normal intermediate of human metabolism. It naturally exists in many plants, including as broad beans, cat beans, and mucuna . Levodop2.Human ESCC cell lines KYSE150 and KYSE450 were purchased from the Chinese Academy of Sciences Cell Bank . KYSE150 cells were cultured in RPMI-1640 medium  supplemented with 10% fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin solution. KYSE450 cells were cultured in Dulbecco’s Modified Eagle Medium  supplemented with 10% FBS and 1% (v/v) penicillin/streptomycin solution. All cell cultures were incubated at 37℃ in a humidified incubator with 5% CO3 and 5 × 103 cells/well, respectively, and incubated overnight. The cells were then treated for 24, 48, 72, or 96 h with various concentrations of levodopa . The concentrations of levodopa used were 0, 50, 100, 200, 400, and 600 µM. At each time point, cells were stained with 4ʹ,6-diamidino-2-phenylindole  and the viable cells counted. Cell number curves were plotted for each concentration at the different time points. Each point represented the mean ± standard deviation (SD) of three biological replicates.KYSE150 and KYSE450 cells were seeded into 96-well plates at 3 × 103 cells/well) were suspended in a top layer containing 10% FBS, 45% agar, and the indicated concentrations of levodopa. The cells were cultured for 1 to 2 wk at 37°C in a 5% CO2 incubator. The number of cell clones that formed were then counted and analyzed using a high-content cell imaging analysis system. For each experimental result, three replicates for each concentration were evaluated and the experiment was performed three times.To evaluate anchorage-independent cell growth, Basal Medium Eagle (BME) containing 10% FBS and 40% agar with various concentrations of levodopa  was added to 6-well plates (3 ml/well). After solidification of the bottom BME layer, KYSE150 and KYSE450 cells (8 × 106) were seeded into 15 cm dishes and treated with levodopa (0 or 600 µM) for 24 h. The collected cells and proteins were stored in at −80℃ until use.KYSE150 cells (4.5 × 10g at 4˚C for 10 min and the supernatant then collected. Protein concentrations were determined using a bicinchoninic acid (BCA) kit. Dithiothreitol was added to the protein solutions to a final concentration of 5 mM and then reduced for 30 min at 56˚C. Iodoacetamide (11 mM) was used to alkylate the proteins to for 15 min at room temperature in darkness. Urea concentrations of the samples were diluted to < 2 M by with 100 mM NH4HCO3. Trypsin was added at a trypsin-to-protein mass ratio of 1:50 for a first digestion overnight and then at a ratio of 1:100 for a second 4-h digestion.The samples were sonicated three times on ice in lysis buffer  using a high-intensity ultrasonic processor (Scientz). The lysate was centrifuged at 12,000 × The tryptic peptides were fractionated using high pH reverse-phase HPLC with an Agilent 300 Extend C18 column . The peptides were separated into 60 fractions using a gradient of 8% to 32% acetonitrile (pH 9.0) over 60 min. The peptides were then combined into six fractions and dried by vacuum centrifugation.The peptides were dissolved in solvent A (0.1% formic acid and 2% acetonitrile aqueous solution), and then separated using an ultra-performance liquid chromatography (UPLC) system . The gradient consisted of an increase from 6% to 22% solvent B (0.1% formic acid in 90% acetonitrile) over 40 min, 22% to 35% solvent B over 12 min, 35% to 80% solvent B over 4 min, and rising to 80% solvent B for the final 4 min. A constant flow rate of 700 nl/min was used. The separated peptides were then ionized using a nanospray ionization (NSI) ion source and then analyzed using Orbitrap Fusion™ (Thermo Scientific) mass spectrometry. The electrospray voltage applied was 2.0 kV. Both the peptide parent ions and their secondary fragments were detected and analyzed using high-resolution Orbitrap. The m/z scan range was 350 to 1550 for the full scan and intact peptides were detected in the Orbitrap at a resolution of 60,000. The secondary mass spectrometer scan range was fixed at a starting point of 100 m/z and the resolution was set at 15,000. After the full scan, the first 20 peptides with the highest signal intensities were selected for entry into the higher energy collisional dissociation (HCD) pool and fragmented using 35% of the fragmentation energy. Secondary mass spectrometry was also performed. To improve the effective utilization of mass spectrometry, the automatic gain control (AGC) was set at 5E4, the signal threshold was set to 5000 ions/s, the maximum injection time was set at 200 ms, and the dynamic exclusion time for tandem mass spectrometry scans was set at 30 s to avoid repeated scans of the parent ions.The tandem MS/MS data were indexed using MaxQuant search engine (v.1.5.2.8). Tandem mass spectra were searched against the SwissProt Human database concatenated with a reverse decoy database and common pollution database. Trypsin/P was specified as the cleavage enzyme, allowing for up to 2 missing cleavages. The minimum peptide length was set at 7 amino acid residues and the maximum number of peptide modifications was set at 5. The mass tolerance for precursor ions was set at 20 parts per million (ppm) in the first search and 5 ppm in the main search The mass tolerance for fragment ions was set as 0.02 Da. Alkylation on Cys was specified as a fixed modification while oxidation of Met and N-terminal acetylation were specified as variable modifications. The false discovery rate (FDR) was adjusted to < 1%.Using the GSEA v4.0.3 package, protein group data sets were integrated with gene Set enrichment analysis (GSEA) to analyze differences in protein expression between the treatment group and the control group. Mitochondrial protein complex gene set (GO:0098798) and respiratory electron transport gene set (R-HSA-611105) were selected for enrichment. Through previous functional annotations or experiments, GSEA determined the relevant gene sets, and sequence the genes according to the degree of differential expression of the two types of samples. Gene set enrichment analysis detected gene set expression changes and optimizes the results using P values and false discovery rate (FDR) method. The biological pathway with P value <0.01 and FDR <0.25 was considered statistically significant in the GSEA analysis. Statistical analysis and graphical plotting were conducted utilizing GSEA Software (v4.0.3).6) were seeded into 15-cm dishes and treated with levodopa (0 or 600 μM) for 24 h. Cells were washed twice with cold phosphate-buffered saline (PBS). Cells were scraped from the plates in a modified radioimmunoprecipitation assay (RIPA) buffer containing 50 mM Tris HCl (pH 7.4), 1% NP-40 (v/v), 1.25% SOD (w/v), 150 mM NaCl, 1 mM EDTA•Na2, and 0.1% sodium dodecyl sulfate  supplemented with protease inhibitor (100×), 100 mM Na3VO4, 0.5 μM NaF, and 100 mM phenylmethanesulfonyl fluoride (PMSF). Cells were collected and placed on ice for 30 min followed by centrifugation at 14,000 rpm for 30 min at 4°C. Protein concentrations were determined using a BCA kit (Beyotime). Protein lysates loaded into a 50 μg system and separated using 12% SDS-polyacrylamide gel electrophoresis (PAGE). After separation, the proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). The membranes were blocked with 5% skim milk for 1 h at room temperature and then washed four times with Tris-buffered saline. The membranes were incubated with specific primary antibody at 4°C for 16 to 18 h. Secondary antibody was then added to the membranes and incubated at room temperature for 2 h. The membranes were scanned using an Odyssey System .For Western blot analysis, KYSE150 cells (4.5 × 105 cells/well and incubated overnight. The cells were then treated with levodopa (0 or 600 µM) for 24 h, washed with cold PBS three times, fixed with 4% paraformaldehyde, and placed at −20°C overnight. Cells were permeabilized with 0.5% TritonX-100 solution for 10 min and blocked with 1% bovine serum albumin-PBS-Tween 20 (BSA-PBST) for 1.5 h at room temperature. The cells were then incubated with primary antibodies at 4°C overnight. Cells were subsequently incubated with fluorescent secondary antibody for 1.5 h at room temperature in darkness. Nuclei were stained using DAPI. Images were collected using a confocal microscope and attached camera.KYSE150 cells were seeded into 12-well plates at 1.5 × 103 and 5 × 103 cells/well, respectively, and incubated overnight. Cells were then treated for either 24 or 48 h with various concentrations of levodopa . At each time point, 50 µl XTT detection solution  was added to each well and incubated for another 4 h. Absorbance at 450 nm was measured using a Multiskan Sky spectrophotometer (Thermo Fisher).KYSE150 and KYSE450 cells were seeded into 96-well plates at 3 × 104 and 5 × 104 cells/well, respectively, and incubated overnight. Cells were then treated with levodopa (0 or 600 µM) for 24 h. At each time point, cells in the positive control group were treated with 10 µM carbonyl cyanide 3-chlorophenylhydrazone (CCCP) for 20 min. All cells were then treated with JC-1 dye for 20 min and the nuclei stained with Hoechst 33342 for 30 min. Cells were immediately observed under a fluorescence microscope and photographed.KYSE150 cells were seeded into 12-well plates at 3 × 104 and 5 × 104 cells/well, respectively, and incubated overnight. The cells were then treated with levodopa (0 or 600 µM) for 24 h. At each time point, cells in the positive control group were treated with 10 µM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) for 10 min. Subsequently, all cells were treated with TMRE for 30 min and the nuclei stained with Hoechst 33342 for 30 min. Cells were immediately observed under a fluorescence microscope and photographed.KYSE150 cells were seeded into 12-well plates at 3 × 106 cells/dish) were seeded into 15-cm dishes and treated with levodopa (0 or 600 μM) for 24 h. The old media were removed and discarded and 30 ml PBS added to each of the 15 cm dishes. The cells were then digested by the addition of 3 ml Trypsin-EDTA. Cells were transferred to 50-ml tubes and centrifuged at 900 rpm for 5 min at 4°C. Supernatants were discarded and the cells fixed in 2.5% glutaraldehyde within 1 min and moved to 1.5-ml tubes to be centrifuged at 900 rpm for 5 min at 4°C. Again, the supernatants were discarded and the pelleted cells were then placed in 2.5% glutaraldehyde solution on ice and sent for TEM evaluation.KYSE150 and SHEE cells . We first investigated whether levodopa could affect the growth of ESCC cells. KYSE150 and KYSE450 cells were treated with different concentrations of levodopa for various lengths of time and the number of cells then detected using DAPI staining. Results showed that levodopa could significantly inhibit the proliferation of ESCC cells  via the PRIDE partner repository with dataset identifier PXD013824. Project Name: Proteomics reveals the inhibitory mechanism of Levodopa against esophageal squamous cell carcinoma; Project accession: PXD013824.The mass spectrometry proteomics data have been deposited at the ProteomeXchange Consortium  can be found below: Study conception and design: ZGD, ZMD, and KL. Conducted experiments: ZL, XL, XZ, LC, and BL. Analysis and interpretation of data: XH, JZ, and JL. Drafting and critical revision of the manuscript: ZL, XJ, and KL. All authors contributed to the article and approved the submitted version.This work was supported by grants from the National Natural Science Foundations of China (grant numbers 81872335 and 81572812), the Natural Science Foundations of Henan (grant number 161100510300), and National Science & Technology Major Project “Key New Drug Creation and Manufacturing Program,” China (grant number 2018ZX09711002).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.


Epstein–Barr virus (EBV) is a major contributor to nasopharyngeal carcinoma (NPC) tumorigenesis. Mitochondria have been shown to be a target for tumor viral invasion, and to mediate viral tumorigenesis. In this study, we detected that mitochondrial morphological changes in tumor tissues of NPC patients infected with EBV were accompanied by an elevated expression of BHRF1, an EBV encoded protein homologue to Bcl-2. High expression of BHRF1 in human NPC cell lines enhanced tumorigenesis and metastasis features. With BHRF1 localized to mitochondria, its expression induced cyclophlin D dependent mitochondrial membrane permeabilization transition (MMPT). The MMPT further modulated mitochondrial function, increased ROS production and activated mitophagy, leading to enhanced tumorigenesis. Altogether, our results indicated that EBV-encoded BHRF1 plays an important role in NPC tumorigenesis through regulating cyclophlin D dependent MMPT. Nasopharyngeal carcinoma (NPC) is a squamous cell carcinoma that develops in the nasopharynx . EpidemiMitochondria are ubiquitous organelles in eukaryotic cells whose primary role is to generate energy in the form of ATP through oxidative phosphorylation . The mitApoptosis is a highly conserved cell death pathway that regulates cell homeostasis. Apoptosis can be activated intrinsically through mitochondrial pathways . IntrinsIn this study, we set out to understand the role of mitochondria in EBV-mediated NPC tumorigenesis. We initiated our investigation by comparing nasopharyngeal cancer tissues and nasopharyngitis tissues obtained from 60 patients with nasopharyngeal disease. We then further studied the mechanism with molecular and biochemical experiments in two EBV-negative human NPC cell lines, namely CNE1 and 5-8F. Some of these molecular results were then verified in NPC patient samples.Nasopharyngeal cancer tissues and nasopharyngitis tissues were obtained from 60 patients with nasopharyngeal disease who underwent endoscopy at The First Affiliated Hospital of Wenzhou Medical University from 10 April 2015 to 21 September 2016. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Wenzhou Medical University (2014006).2 and 37 °C.CNE1 and 5-8F are EBV negative, human NPC cell lines. C666-1 is an EBV positive, human NPC cell line. CNE1 and 5-8F cell lines were obtained from Dr. Meng , and the C666-1 cell line was provided by Anburui Biological Technology Co., Ltd. . CNE1-BHRF1 and 5-8F-BHRF1 are NPC cell lines that stably express BHRF1. All cell lines were grown in DMEM (Dulbecco’s Modification of Eagle’s Medium) with high glucose  supplemented with 10% fetal bovine serum plus antibiotics. All cells were cultured in a humidified incubator maintaining 5% COAccording to the sequence information in Gene Bank (NCBI: NC_007605), we designed the primers 5′-CACTAGTCCAGTGTGGTGGCCACCATGGCCTATTCAACAAGGGAGATA-3′ (forward) and 5′-TGCTGGATATCTGCAGTGTCTTCCTCTGGAGATAAA-3′ (reverse) to clone the BHRF1 gene from the B95-8 cell line by PCR. The recombinant vector PCDH-BHRF1-flag was created and verified transformed by the Shanghai Bio-Engineering Company.2 at 37 °C. Finally, the cells were screened for the BHRF1 gene with 0.9 μg/mL puromycin for about 10 days and the clonal cell clusters were verified using a western blotting assay.To construct a stable-transfected cell line that expresses BHRF1 protein, the 293T as the prepared cells, was transfected with packaging plasmid  and PCDH-BHRF1-flag plasmid using lipofectamine-3000 reagent, establishing the BHRF1 virions. 24 h later, the filtered virus was harvested with a 0.45 μm filter. When the CNE1 and 5-8F cells achieved 40–50% confluence in 6-well plates, the cell culture medium was changed to 1 mL DMEM high glucose medium and 1 mL BHRF1 virus. After 10 h, the medium was replaced with medium supplemented with 10% FBS and incubated for a further 48 h in 5% COg at 4 °C for 3 min, and then the supernatant was transferred to a new Eppendorf tube and subjected to a centrifugation again under the same conditions. Then, the supernatant was collected and centrifuged at 15,000× g for 2 min at 4 °C. The freshly extracted mitochondria (M) were suspended with 60 μL TD buffer and centrifuged. After removing the supernatant, the sediment underwent lysis with 30 μL 0.1 mM Na2CO3 for 30 min on ice and centrifugation at 75,000× g at 4 °C for 40 min. The reaction mixtures were centrifuged at 170,000× g at 4 °C for 30 min to separate the precipitate  and supernatant  fractions. The fractions were subjected to SDS-PAGE followed by Western blots.Cells were suspended in TD buffer (W), and were homogenized on ice until 80% of the cells were stained blue by trypan blue under a microscope. The lysates were centrifuged at 12,000× For separating the inner and outer membrane of mitochondria, Proteinase K (Pro K) was used to digest the mitochondrial outer membrane, Pro K with Triton × 100 (20%) was used to digest the inner and outer mitochondrial membrane, and only Triton × 100 (20%) was used to digest neither. Then, 3.33 μL 1 mg/mL Pro K was added to the 30 μL freshly extracted mitochondria for 1 h and then mixed with 1 μL PMSF to obtain the mitochondrial inner membrane. A mixture of 3.33 μL 1 mg/mL Pro K and 3.33 μL Triton × 100 (20%) was added to the 30 μL freshly extracted mitochondria for 1 h and then mixed with 1 μL PMSF, which yielded nothing. Subsequently, 3.33 μL Triton × 100 (20%) alone was added into the 30 μL freshly extracted mitochondria for 1 h and then mixed with 1 μL PMSF in order to harvest everything. The reaction mixtures were separated by SDS-PAGE and analyzed by Western blots.2 cells/mL in six-well plates, seeding 2 mL cell suspensions per well and incubating with 5% CO2 at 37 °C for 7–10 days. The cells were washed with PBS and fixed in 4% paraformaldehyde for 20 min, then were washed twice with PBS and stained with 1% crystallized purple dye for 30 min. After washing three times with PBS, the number and size of cell colonies were analyzed by a microscope.The cells were diluted to 4 × 104 cells were placed on 96-well plates in 100 μL of DMEM and were cultured at 37 °C for 0, 24, 48, or 72 h. The medium was replaced the following day with 10% FBS DMEM medium with 10 μL CCK-8 for 1 h at 37 °C. Cell counting and viability analysis were performed using a microplate reader by a Varioskan™ Flash Multimode Reader .Multiple identical samples of 1 × 105 cells/mL in 24-well plates, seeding 0.5 mL cell suspensions per well with 10% FBS DMEM medium, and then incubated in 5% CO2 at 37 °C for 1 day to prepare cells achieving 80% confluence. Then, a pin tool or needle was used to scratch and remove cells from a discrete area of the confluent monolayer to form a cell-free zone into which cells at the edges of the wound can migrate. After washing three times with PBS, cells were grown in DMEM medium with high glucose. Images of cell movement were captured at 0, 24, 48, and 72 h and the rate of wound closure was then calculated.The cells were diluted to 2 × 102 at 37 °C for 12 h. Then, the transwells were put in 24-well plates with 600 μL 10% FBS DMEM medium. After growing in serum-free DMEM medium for 6 h, 1 × 103 cells were resuspended in 200 μL of serum-free DMEM medium, then transferred to the prepared transwells and cultured in 5% CO2 at 37 °C for 48 h. The transwells were washed with PBS and fixed in 4% paraformaldehyde for 20 min, then were washed twice with PBS and stained with 0.1% crystallized purple dye for 20 min. The numbers of purple cells were examined by a microscope after eliminating covered cells under the transwell with PBS. The transwell migration assay was conducted in a similar way as the invasion assay, but without the matrigel solution.The 45 μL matrigel solution per transwell was prepared by combining 5 μL matrigel (melted at 4 °C) and 45 μL high glucose DMEM with serum-free medium and incubated in 5% COThe prepared cells were fixed in 2.5% glutaraldehyde fixative  for 2 h at 4 °C. Cells were fixed with 1% osmic acid for 1 h and stained with 1% uranium acetate for the same amount of time. Then, the cells were dehydrated in increasing concentrations of acetone  for 15 min each and twice with 100% acetone for 20 min every time. Next, the samples were embedded in epoxy resin  at 45 °C for 6 h and 65 °C for 48 h. After resin embedding, ultra-thin sections were cut with an ultramicrotome using a diamond knife and collected onto 150 mesh copper grids . These sections were observed using a transmission electron microscope .4 cells/mL in six-well plates, seeding 2 mL cell suspensions per well and were incubated in 5% CO2 at 37 °C for 2–3 days to prepare cells achieving 60–80% confluence. Cells were washed once with PBS and incubated with TMRE , which was diluted with 10% FBS medium to 30 μM. We applied 1 mL dilute TMRE per well in 5% CO2 at 37 °C for 15 min and then cells were observed and photographed under a fluorescence microscope. To determine the membrane potential, we used fluorescence ratio (F-F0)/F0 to represent the potential, where F0 is the baseline fluorescence signal.The cells were diluted to 4 × 102 at 37 °C for 5 min. Then, cells were washed twice with HBSS and fluorescence was recorded by a Varioskan™ Flash Multimode Reader .Mitochondrial ROS were measured according to our team’s published article . In shorg for 1 min. ATP content was determined by measuring the luminescence of supernatants mixed with luciferase assay buffer using a Varioskan™ Flash Multimode Reader . ATP luminescence was normalized via protein concentration.According to the manufacturer’s instructions, an ATP measurement kit  was used as the main reagent to measure ATP. In short, the prepared cells achieving 60–80% confluence in 6-well plates were washed with PBS and then boiled in 100 μL boiling buffer  at 100 °C for 90 seconds. Supernatants were retrieved via centrifugation at 10,000× 6 cells each, seeding 200 μL cell suspensions per well, and then treated or untreated with the concentrations as described in the text of CsA for 48 h with 5% CO2 at 37 °C. Cells were washed once with PBS and resuspended in 500 μL PBS. Then, 200 μL of cell suspensions were transferred into a tube and then incubated with 1.5 μL Annexin V and 1 μL 7-AAD  for 30 min. After supernatants were discarded via centrifugation at 1500 rpm/min for 5 min, the cells were resuspended with 200 μL binding buffer and stained with 1 μL APC-Annexin V (BD Biosciences) for 15 min. The apoptosis analysis was performed using flow cytometry .Fluorochrome-labeled Annexin V (Annexin V-FITC) was used to specifically target and identify early apoptotic cells. Late apoptotic cells, which have lost membrane integrity, are recognized by 7 -aminoactinomycin D (7-AAD). In contrast, necrotic cells lose membrane integrity early and become 7-AAD positive. The prepared cells were plated in 96-well plates at 1 × 10Proteins were resolved by 12% SDS-polyacrylamide gel electrophoresis under reducing conditions. The resolved proteins were transferred electrophoretically to a polyvinylidene fluoride membrane. After incubating with 5% non-fat milk in tris-buffered saline with tween (TBS-T) , 0.05% Tween 20) for at least 1 h at room temperature, the membrane was incubated with primary antibodies  and Flag ) for the appropriate time, washed extensively with TBS-T and then incubated with HRP-conjugated secondary antibody (1:2000 dilution). Protein bands were detected using a Super Signal West Pico Chemi-Luminescent Substrate Kit . Densitometric analyses were done using Image J software.5 cells/mL in six-well plates in 5% CO2 at 37 °C for 2–3 days to prepare cells achieving 60% confluence. Cells were washed once with PBS, then mixed with 1 mL RNAiso Plus  for 5 min and 200 µL chloroform for 10 min. After centrifuging at 14,000 r/min for 20 min at 4 °C, the supernatant was collected and added 400 µL isopropanol for 10 min and repeat centrifuging in the same condition. Then, 1 mL 75% precooled ethanol was put into the precipitate and centrifugation at 7500 r/min for 1 min at 4 °C. After removing the supernatant, we applied 20µL ddH2O and then RNA were saved. The reaction mixtures 1  stay in 42 °C for 2 min. The reaction mixtures 2 , 10 µL reaction mixtures 1 and 4 µL RNase Free dH2O) stay in 37 °C for 15 min, 85 °C for 5 s and 4 °C forever. The reaction mixtures 3 were performed in a final volume of 25 µL containing 2 µL reaction mixtures 3 (cDNA), 0.4 µL upstream primer, 0.4 µL downstream primer, 10 µL 2 × plus SYBR real-time mixture , 0.4 µL ROX I and 6.8 µL dH2O. The quality of the specimens was assessed by a real-time RT-PCR targeting the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. Amplification was performed on a Bio-Rad IQ real-time PCR system . Sterile diethyl pyrocarbonatetreated water was used as an RT- and PCR-negative control.The cells were diluted to 2 × 10t-test, or a one-way analysis of variance followed by a Dunnett’s multiple comparison test when appropriate.Data in graphs are reported as ± SE and depict the average of at least three independent experiments. Morphological images are representative of at least three independent experiments with similar results. Statistical analysis was performed using GraphPad software  with Student’s paired The nasopharyngeal carcinoma (NPC) and nasopharyngitis are two common pathological changes in nasopharynx. The initial clinical symptoms of nasopharyngeal carcinoma such as epistaxis, nasal congestion and stuffy nose are generally consistent with nasopharyngitis. Patients with such symptoms were consulted by doctors in otolaryngology department of the hospital, and were followed with examinations by electronic fiber laryngoscope. The tissues (2 × 2 mm) were obtained from the posterior of parapharyngeal. To directly address the mitochondrial implications in EBV mediated NPC, we collected 41 NPC tissue specimens, and compared them to the tissue of 19 patients with nasopharyngitis with similar age and sex distribution from the same location . We usedp < 0.0001) , which have previously been implicated in tumorigenesis and mitochondrial regulation . Among B 0.0001) C. For co 0.0001) . In addi 0.0001) .To further explore the potential role of BHRF1 in EBV mediated NPC tumorigenesis, we selected two EBV-negative NPC cell lines with different metastatic properties, CNE1  and 5-8Fp < 0.05)  C.In order to investigate the contribution of BHRF1 to NPC carcinogenesis, we analyzed tumorigenesis features in BHRF1 transfectants and control lines. In a colony formation assay, we found BHRF1-expressing cells formed larger and more clones than controls A. As a cBHRF1 has been previously suggested to localize to mitochondria ,22. To cp < 0.005) and 5-8F cells (p < 0.05) (p < 0.05)  with TMRE. As shown in  < 0.05) C. To det < 0.05) D. To inv < 0.05) E.To further investigate the underlying mechanisms of putative BHFR1 mediated NPC tumorigenesis with compromised oxidative phosphorylation, increased ROS production and increased cell survival, we decided to look into the role of the mitochondrial membrane permeability transition (MMPT). This could be a promising new link in the mechanism, as mitochondrial swelling and compromised MMP have been associated with the MMPT, which is also considered as the critical stage for cell death/survival. Cyclosporine A (CsA) is a potent inhibitor for the MMPT, binding to mitochondrial cyclophilin to prevent the opening of mitochondrial permeability transition pores . As showTo further explore how the MMPT induced by BHRF1 may mediate tumorigenesis, we repeated the tumorigenesis assays performed in the previous sections in presence of the MMPT inhibitor CsA. In a colony formation assay, the number and volume of colonies all decreased after CsA treatment in NPC cells with BHRF1 expression A. The CCTo determine the molecular mechanism by which the BHRF1-reguated MMPT modulates NPC tumorigenesis, we analyzed the protein levels by Western blot of critical factors that have the potential to mediate the retrograde signaling from mitochondrial alteration to tumorigenesis. Among them, we found multiple proteins related to mitochondrial dynamics and mitophagy exhibited significant changes. As shown in EBV was the first human virus to be directly implicated in tumorigenesis, and has been linked to the development of cancers originating from both lymphoid and epithelial cells . InteresEBV encodes nearly a hundred genes, and among them the EBV latent genes expressed in NPC are clearly essential in the carcinogenic process. While the EBNA proteins have been shown to be important in replication and segregation of the EBV genome and viral gene expression, LMP proteins have been shown to play various roles contributing to tumorigenic phenotypes ,27. EBV In an effort to investigate the role of mitochondria in EBV dependent NPC tumorigenesis, we explored the abnormal mitochondrial morphology A associaThe expression of BHFR1 in two NPC cell lines reproduced our findings in the NPC tumor tissues that showed changes in the mitochondrial morphology . FurtherApoptosis, mainly regulated by mitochondria, is one of the important regulatory pathways in tumorigenesis. Mechanistically, MMPT associated with an increase in the permeability of the mitochondrial membrane leads mitochondrial swelling and rupture of the outer mitochondrial membrane. This has been demonstrated to mediate the depolarization of the transmembrane potential as illustrated by the reduction of the MMP, release of apoptotic factors, and decrease of oxidative phosphorylation ,34. ThusOne interesting finding of this study is that the CypD dependent MMPT is regulated by Bcl-2 homolog BHRF1. The coupling of transitions of out mitochondrial membrane (OMM) and inner mitochondrial membrane (IMM) has previously been shown that the activation of the permeability transition pore (PTP) at least in vitro, resulted in passive swelling of the IMM and rupture of the OMM, with ensuing release of IMS components. In particular, over expression of Bcl-2 increased the resistance of IMM transition . In addiThe MMPT has been considered to be a ‘point of no return’ during apoptosis, which once was suggested to inevitably lead to cell death. One key event after the MMPT is the triggering of autophagy . Quite aAlterations in the autophagy pathway have been associated with the formation of various cancers. Autophagy is also an adaptive catabolic process that happens in response to metabolic stress, including hypoxia and ROS. One possible mechanism here is that NPC exploits the enhancement of autophagy for survival to adapt to the hypoxia of a tumor microenvironment ,43. Our It is also important to note that our studies don’t necessarily exclude the contributions of other EBV encoded protein in NPC tumorigenesis. Rather, our results describe a scenario in which EBV encoded BHRF1 induces MMPT, and such MMPT further regulates NPC tumorigenesis through ROS production and mitophagy activation (as shown in the graphic abstract).