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Background: Acute liver failure (ALF) is an unpredictable and life-threatening critical illness. The pathological characteristic of ALF is massive necrosis of hepatocytes and lots of inflammatory cells infiltration which may lead to multiple organ failure., Methods: Animals were divided into 3 groups, normal, thioacetamide (TAA, ALF model) and TAA + AGK2. Cultured L02 cells were divided into 5 groups, normal, TAA, TAA + mitofusin 2 (MFN2)-siRNA, TAA + AGK2, and TAA + AGK2 + MFN2-siRNA groups. The liver histology was evaluated with hematoxylin and eosin staining, inositol-requiring enzyme 1 (IRE1), activating transcription factor 6β (ATF6β), protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) and phosphorylated-PERK (p-PERK). C/EBP homologous protein (CHOP), reactive oxygen species (ROS), MFN2 and glutathione peroxidase 4 (GPX4) were measured with Western blotting, and cell viability and liver chemistry were also measured. Mitochondria-associated endoplasmic reticulum membranes (MAMs) were measured by immunofluorescence., Results: The liver tissue in the ALF group had massive inflammatory cell infiltration and hepatocytes necrosis, which were reduced by AGK2 pre-treatment. In comparison to the normal group, apoptosis rate and levels of IRE1, ATF6β, p-PERK, CHOP, ROS and Fe 2+ in the TAA-induced ALF model group were significantly increased, which were decreased by AGK2 pre-treatment. The levels of MFN2 and GPX4 were decreased in TAA-induced mice compared with the normal group, which were enhanced by AGK2 pre-treatment. Compared with the TAA-induced L02 cell, apoptosis rate and levels of IRE1, ATF6β, p-PERK, CHOP, ROS and Fe 2+ were further increased and levels of MFN2 and GPX4 were decreased in the MFN2-siRNA group. AGK2 pre-treatment decreased the apoptosis rate and levels of IRE1, ATF6β, p-PERK, CHOP, ROS and Fe 2+ and enhanced the protein expression of MFN2 and GPX4 in MFN2-siRNA treated L02 cell. Immunofluorescence observation showed that level of MAMs was promoted in the AGK2 pre-treatment group when compared with the TAA-induced group in both mice and L02 cells., Conclusions: The data suggested that AGK2 pre-treatment had hepatoprotective role in TAA-induced ALF via upregulating the expression of MFN2 and then inhibiting PERK and ferroptosis pathway in ALF., (Copyright © 2023 First Affiliated Hospital, Zhejiang University School of Medicine in China. Published by Elsevier B.V. All rights reserved.)

Biosensors based on various spectroscopic techniques discriminate the target microRNA (miRNA) from non-target ones with single nucleotide polymorphisms (SNPs) according to the differences in signal intensities which can be caused by other factors besides SNPs. As a result, they are liable to produce false positive results. Herein, we report an attempt to develop a false-positive resistance, sensitive and reliable mass spectrometric platform for miRNA detection. In the proposed platform, the qualitative and quantitative information of the target miRNA was obtained through analyzing mass spectral responses of the multiply charged ions of the residual fragments of the probe DNA produced during exonuclease III assisted signal amplification reaction using an advanced data analysis method. The proposed platform could achieve sensitive and accurate quantitative results for the target miRNA (e.g., miRNA-141) in complex medium with a detection limit of about 1 pM, and unambiguously identify non-target miRNAs with SNPs based on the length distribution patterns of residual fragments of probe DNA. The findings obtained in this study might open an avenue for the design of new miRNA detection methods based on mass spectrometry in combination with various nuclease assisted signal amplification strategies., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Aim: The purpose of the present study was to elucidate the protective effect of histone deacetylase 6 inhibitor ACY1215 on autophagy pathway in acute liver failure (ALF)., Main Methods: Lipopolysaccharide (LPS) and d-galactosamine (D-Gal) were used to induce ALF model in C57BL/6 mice. D-Gal and tumor necrosis factor alpha (TNF-α) were applied in L02 cell. Autophagy inhibitor 3-MA and ACY1215 were conducted to induce 3-MA group, ACY1215 group and ACY1215+3-MA group., Results: ACY1215 improved liver histological and functional changes in ALF mice model, whereas the autophagy inhibitor 3-MA aggravated liver tissue pathological and functional damage in ALF mice model group. The apoptotic levels (including apoptotic index/rate and apoptotic proteins) in ALF mice and L02 cell were ameliorated with treatment ACY1215. 3-MA accentuated the apoptotic levels in ACY1215 group. D-Gal/TNF-α could reduce L02 cell mitochondrial membrane potential (ΔΨm) in control group. ACY1215 increased the ΔΨm in ALF model. 3-MA also further reduced the ΔΨm in ACY1215 group. ACY1215 could induce autophagy in ALF mice and cell model group accompanied with an increase in expression of LC3-II and beclin-1 proteins and down-regulation of p62 protein. Moreover, the expression of LC3-II and beclin1 proteins were greatly reduced and the expression of p62 protein was ascended after intervention with 3-MA in ACY1215 group., Significance: Histone deacetylase 6 inhibitor ACY1215 could protect acute liver failure mice and L02 cell by inhibiting apoptosis pathway through enhancing autophagy way., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Aims: The aim of the present study was to investigate the protective effects of AGK2 as a selective SIRT2 inhibitor on thioacetamide (TAA)-induced acute liver failure (ALF) in mice and its potential mechanism., Main Methods: All male C57BL/6 mice were separated into control, TAA, AGK2 + TAA, and AGK2 groups. The histological changes were observed by hematoxylin and eosin (HE) staining. The apoptosis cells of liver tissues were detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were used to evaluate the damage of liver function. The inflammatory cytokines of iNOS, TNF-α, IL-1β was detected by Western blotting and RT-PCR assay. The expression of mitogen-activated protein kinase (MAPK), NF-κB, and apoptosis pathways was determined by Western blotting., Key Findings: AGK2 improved the damage of TAA-induced liver pathology and function. AGK2 pretreatment also reduced the levels of pro-inflammatory cytokines in ALF liver tissues. AGK2 improved the TAA-induced survival rate. Moreover, AGK2 administration suppressed the increase of phosphorylation NF-κB-p65 and the activation of MAPK pathway. In addition, pretreatment alleviated TAA-induced the liver cells apoptosis., Significance: AGK2 improve TAA-induced survival rate in mice with ALF, suppress the inflammatory responses by inhibition of MAPK and NF-κB signaling pathways, and decrease the hepatocyte necrosis by inhibition of apoptosis. Pharmacologic inhibition of SIRT2 may be a promising approach for the treatment of ALF., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Four experiments were conducted in this study. Experiment 1 was carried out to examine mRNA expressions of nuclear vitamin D receptor (nVDR), membrane vitamin D receptor (mVDR), and type IIb sodium-phosphate cotransporter (NaPi-IIb) in the small intestine of broiler chickens. Experiments 2, 3, and 4 were implemented to evaluate effects of age, non-phytate phosphorus (NPP), and 25-hydroxycholecalciferol (25-OH-D3) on mRNA expressions of nVDR, mVDR, and NaPi-IIb in the duodenum of chickens. Results showed that mRNA expression levels of nVDR and NaPi-IIb were highest in the duodenum of 21-day-old broilers, lower in the jejunum, and lowest in the ileum. By contrast, no differences in mRNA expression levels of mVDR were detected among the duodenum, jejunum, and ileum. Age quadratically affected mRNA expressions of nVDR, mVDR, and NaPi-IIb in the duodenum and 25-hydroxylase in the liver of 7- to 42-day-old broilers, with the highest levels observed at 21 d of age. By contrast, age linearly decreased mRNA expression level of 1α-hydroxylase in kidneys. Dietary NPP levels quadratically affected mRNA expression levels of nVDR and mVDR in the duodenum and 25-hydroxylase in the liver of 21-day-old broilers. The highest mRNA expression levels of nVDR and mVDR and lowest mRNA level of 25-hydroxylase were observed at 0.55% NPP. mRNA expression level of NaPi-IIb linearly declined when dietary NPP levels increased from 0.25 to 0.65%. Addition of 12.5 μg/kg of 25-OH-D3 increased mRNA expression level of 1α-hydroxylase in kidneys and those of nVDR, mVDR, and NaPi-IIb in the duodenum of broilers compared with birds fed the diet without 25-OH-D3. These data indicate that mRNA expressions of nVDR and NaPi-IIb are highest in the duodenum, and the greatest mRNA levels of nVDR, mVDR, and NaPi-IIb are observed at 21 d of age. Dietary NPP levels quadratically increase mRNA expressions of nVDR and mVDR but linearly decrease NaPi-IIb mRNA level. 25-OH-D3 up-regulates the above gene transcription.

The VWA8 gene was first identified by the Kazusa cDNA project and named KIAA0564. Based on the observation, by similarity, that the protein encoded by KIAA0564 contains a Von Willebrand Factor 8 domain, KIAA0564 was named Von Willebrand Domain-containing Protein 8 (VWA8). The function of VWA8 protein is almost unknown. The purpose of this study was to characterize the tissue distribution, cellular location, and function of VWA8. In mice VWA8 protein was mostly distributed in liver, kidney, heart, pancreas and skeletal muscle, and is present as a long isoform and a shorter splice variant (VWA8a and VWA8b). VWA8 protein and mRNA were elevated in mouse liver in response to high fat feeding. Sequence analysis suggests that VWA8 has a mitochondrial targeting sequence and domains responsible for ATPase activity. VWA8 protein was targeted exclusively to mitochondria in mouse AML12 liver cells, and this was prevented by deletion of the targeting sequence. Moreover, the VWA8 short isoform overexpressed in insect cells using a baculovirus construct had in vitro ATPase activity. Deletion of the Walker A motif or Walker B motif in VWA8 mostly blocked ATPase activity, suggesting Walker A motif or Walker B motif are essential to the ATPase activity of VWA8. Finally, homology modeling suggested that VWA8 may have a structure most confidently similar to dynein motor proteins., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Lenalidomide is an immunomodulatory drug (IMiDs) with clinical efficacy in multiple myeloma (MM) and other late B-cell neoplasms. Although cereblon ( CRBN ) is an essential requirement for IMiD action, the complete molecular and biochemical mechanisms responsible for lenalidomide-mediated sensitivity or resistance remain unknown. Here, we report that IMiDs work primarily via inhibition of peroxidase-mediated intracellular H 2 O 2 decomposition in MM cells. MM cells with lower H 2 O 2 -decomposition capacity were more vulnerable to lenalidomide-induced H 2 O 2 accumulation and associated cytotoxicity. CRBN-dependent degradation of IKZF1 and IKZF3 was a consequence of H 2 O 2 -mediated oxidative stress. Lenalidomide increased intracellular H 2 O 2 levels by inhibiting thioredoxin reductase (TrxR) in cells expressing CRBN, causing accumulation of immunoglobulin light-chain dimers, significantly increasing endoplasmic reticulum stress and inducing cytotoxicity by activation of BH3-only protein Bim in MM. Other direct inhibitors of TrxR and thioredoxin (Trx) caused similar cytotoxicity, but in a CRBN-independent fashion. Our findings could help identify patients most likely to benefit from IMiDs and suggest direct TrxR or Trx inhibitors for MM therapy., (© 2017 by The American Society of Hematology.)

Objective: HIV-infected people who inject drugs (PWID) have a disproportionally low rate of access to antiretroviral therapy (ART). We aimed to assess the impact of ART on 12-month mortality and virological failure of HIV-infected PWID in China, stratified by methadone maintenance treatment (MMT) and active drug use status., Methods: HIV-infected PWID who initiated ART at 29 clinics in 2011 were enrolled and followed in this prospective cohort study. Kaplan-Meier curves and log-rank tests were used to compare the survival probability. Risk factors for mortality and virological failure were evaluated by Cox proportional hazards models and logistic regression analyses., Results: A total of 1,633 participants initiated ART. At the time of initiation, 324 were on MMT, 625 were engaged in active drug use, and 684 had discontinued drug use but were not on MMT. At the 12-month follow-up, 80.3% remained on ART, 13.5% had discontinued ART, and 6.2% had died. Among the MMT group, active drug use group, and drug abstinent group, we observed all-cause mortality of 4.9%, 12.0%, and 1.5% and virological suppression of 51.9%, 41.1%, and 68.7%, respectively. Factors associated with both mortality and virological failure were drug use status, unemployment, and treatment facility type., Conclusion: For HIV-infected PWID receiving ART, engagement in MMT and discontinuation of drug use were more likely to be associated with lower mortality and virological failure compared with active drug use. In order to maximize the clinical impact of ART, HIV treatment programs in China should be further integrated with MMT and social services., (Copyright © 2016. Published by Elsevier Ireland Ltd.)

Objective: We sought to explore the experiences of drug users in China who were recently diagnosed with HIV infection while engaged in methadone maintenance treatment (MMT) and to better understand their perceptions of MMT, HIV risk, and HIV prevention., Methods: We recruited clients of MMT clinics in Chongqing and Kunming who had a baseline HIV-negative test result upon entry to MMT and had been diagnosed with HIV within the past 12 months. We conducted semi-structured qualitative interviews and thematic data analysis to identify situations and factors that increased HIV risk., Results: Among the 27 participants who were interviewed, 15 believed their infection was due to injection drug use, 7 attributed their infection to sexual contact, and 5 were unsure as to how they became infected. High risk behaviors were common; 18 participants continued to use drugs during treatment, and 10 engaged in unprotected sex. Common themes were the difficulty of drug abstinence despite receiving MMT, social pressure to continue using drugs, and low knowledge of effective HIV prevention measures., Conclusion: While MMT is effective in reducing drug usage and needle sharing, many clients remain at risk of HIV infection due to continued injection drug use and unprotected sex. Clients may benefit from additional counseling on HIV prevention methods as well as structural interventions to increase the availability of clean injection equipment., (Copyright © 2016. Published by Elsevier Ireland Ltd.)

In this study, targeted sequencing to screen 50 multidrug refractory multiple myeloma (rMM) patients was performed by using the Multiple Myeloma Mutation Panel. Patients were pretreated with both immunomodulatory drugs (IMiDs) and proteasome inhibitors (PIs), and 88%, 78%, and 68% were refractory to an IMiD, a PI, or both, respectively. The majority of patients had progressive (82%) or refractory (78%) disease immediately before sampling, with 43% being IMiD refractory and 46% being PI refractory in the most recent line of therapy. Compared with newly diagnosed MM, an increased prevalence of mutations in the Ras pathway genes KRAS, NRAS, and/or BRAF (72%), as well as TP53 (26%), CRBN (12%), and CRBN pathway genes (10%) was observed. Longitudinal analyses performed in 3 patients with CRBN mutations at time of IMiD resistance confirmed that these mutations were undetectable at earlier, IMiD-sensitive time points. Furthermore, the functional introduction of these mutations in MM cells conferred lenalidomide resistance in vitro. These data indicate a differential genetic landscape in rMM associated with drug response., (© 2016 by The American Society of Hematology.)

Background: Previous meta-analyses of pulmonary arterial hypertension (PAH)-specific therapy for PAH pooled PAH-specific combination therapy and monotherapy. This flaw may threaten the authenticity of their findings., Methods: PubMed, Embase, and the Cochrane Library were searched for randomized controlled trials that evaluated any PAH-specific medications in the treatment of PAH. We calculated ORs with 95% CIs for dichotomous data and standardized mean differences for continuous data., Results: In total, 35 randomized controlled trials involving 6,702 patients were included. In monotherapy vs placebo/conventional therapy, significance was obtained in mortality reduction (OR, 0.50 [95% CI, 0.33 to 0.76]; P = .001), 6-min walk test (mean difference, 31.10 m [95% CI, 25.40 to 36.80]; P < .00001), New York Heart Association/World Health Organization functional class (OR, 2.48 [95% CI, 1.51 to 4.07]; P = .0003), and hemodynamic status based on mean pulmonary artery pressure, pulmonary vascular resistance, cardiac index, and incidence of withdrawal due to adverse effects. In combination therapy vs monotherapy, significance was reached for the 6-min walk test (mean difference, 19.96 m [95% CI, 15.35 to 24.57]; P < .00001), functional class (OR, 1.65 [95% CI, 1.20 to 2.28]; P = .002), hemodynamic status, and incidence of withdrawal due to adverse effects (OR, 2.01 [95% CI, 1.54 to 2.61]; P < .00001) but not for mortality reduction (OR, 0.98 [95% CI, 0.57 to 1.68]; P = .94)., Conclusions: Our meta-analysis revealed that PAH-specific monotherapy could improve mortality, exercise capacity, functional class, and hemodynamic status compared with placebo or conventional therapy. However, combination therapy could further improve exercise capacity, functional class, and hemodynamic status compared with monotherapy, but it had no proven effect on mortality. Combination therapy had a much higher incidence of withdrawal due to adverse effects than monotherapy., (Copyright © 2016 American College of Chest Physicians. Published by Elsevier Inc. All rights reserved.)

In this contribution, a multiplicative effects model for generalized multiple-internal-standard method (MEMGMIS) was proposed to solve the signal instability problem of LC-MS over time. MEMGMIS model seamlessly integrates the multiple-internal-standard strategy with multivariate calibration method, and takes full use of all the information carried by multiple internal standards during the quantification of target analytes. Unlike the existing methods based on multiple internal standards, MEMGMIS does not require selecting an optimal internal standard for the quantification of a specific analyte from multiple internal standards used. MEMGMIS was applied to a proof-of-concept model system: the simultaneous quantitative analysis of five edible artificial colorants in two kinds of cocktail drinks. Experimental results demonstrated that MEMGMIS models established on LC-MS data of calibration samples prepared with ultrapure water could provide quite satisfactory concentration predictions for colorants in cocktail samples from their LC-MS data measured 10days after the LC-MS analysis of the calibration samples. The average relative prediction errors of MEMGMIS models did not exceed 6.0%, considerably better than the corresponding values of commonly used univariate calibration models combined with multiple internal standards. The advantages of good performance and simple implementation render MEMGMIS model a promising alternative tool in quantitative LC-MS assays., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The transcription factor c-Maf is extensively involved in the pathophysiology of multiple myeloma (MM), a fatal malignancy of plasma cells. In the present study, affinity chromatography and mass spectrometry were used to identify c-Maf ubiquitination-associated proteins, from which the E3 ligase HERC4 was found to interact with c-Maf and catalyzed its polyubiquitination and subsequent proteasome-mediated degradation. HERC4 mediated polyubiquitination at K85 and K297 in c-Maf, and this polyubiquitination could be prevented by the isopeptidase USP5. Further analysis on the NCI-60 cell line collection revealed that RPMI 8226, a MM-derived cell line, expressed the lowest level of HERC4. Primary bone marrow analysis revealed HERC4 expression was high in normal bone marrow, but was steadily decreased during myelomagenesis. These findings suggested HERC4 played an important role in MM progression. Moreover, ectopic HERC4 expression decreased MM proliferation in vitro, and delayed xenograft tumor growth in vivo. Therefore, modulation of c-Maf ubiquitination by targeting HERC4 may represent a new therapeutic modality for MM., (© 2016 by The American Society of Hematology.)

Immunomodulation is an established treatment strategy in multiple myeloma with thalidomide and its derivatives lenalidomide and pomalidomide as its FDA approved representatives. Just recently the method of action of these cereblon binding molecules was deciphered and results from large phase 3 trials confirmed the backbone function of this drug family in various combination therapies. This review details the to-date knowledge concerning mechanism of IMiD action, clinical applications and plausible escape mechanisms in which cells may become resistant/refractory to cereblon binding molecule based treatment., (Published by Elsevier Ltd.)

In this contribution, surface-enhanced Raman spectroscopy (SERS) based on conical holed glass substrates deposited with silver colloids was reported for the first time. It combines the advantages of both dry SERS assays based on plane films deposited with silver colloids and wet SERS assays utilizing cuvettes or capillary tubes. Compared with plane glass substrates deposited with silver colloids, the conical holed glass substrates deposited with silver colloids exhibited five-to ten-folds of increase in the rate of signal enhancement, due to the internal multiple reflections of both the excitation laser beam and the Raman scattering photons within conical holes. The application of conical holed glass substrates could also yield significantly stronger and more reproducible SERS signals than SERS assays utilizing capillary tubes to sample the mixture of silver colloids and the solution of the analyte of interest. The conical holed glass substrates in combination with the multiplicative effects model for surface-enhanced Raman spectroscopy (MEMSERS) achieved quite sensitive and precise quantification of 6-mercaptopurine in complex plasma samples with an average relative prediction error of about 4% and a limit of detection of about 0.02 μM using a portable i-Raman 785H spectrometer. It is reasonable to expect that SERS technique based on conical holed enhancing substrates in combination with MEMSERS model can be developed and extended to other application areas such as drug detection, environmental monitoring, and clinic analysis, etc., (Copyright © 2015 Elsevier B.V. All rights reserved.)

To identify molecular targets that modify sensitivity to lenalidomide, we measured proliferation in multiple myeloma (MM) cells transfected with 27 968 small interfering RNAs in the presence of increasing concentrations of drug and identified 63 genes that enhance activity of lenalidomide upon silencing. Ribosomal protein S6 kinase (RPS6KA3 or RSK2) was the most potent sensitizer. Other notable gene targets included 5 RAB family members, 3 potassium channel proteins, and 2 peroxisome family members. Single genes of interest included I-κ-B kinase-α (CHUK), and a phosphorylation dependent transcription factor (CREB1), which associate with RSK2 to regulate several signaling pathways. RSK2 knockdown induced cytotoxicity across a panel of MM cell lines and consistently increased sensitivity to lenalidomide. Accordingly, 3 small molecular inhibitors of RSK2 demonstrated synergy with lenalidomide cytotoxicity in MM cells even in the presence of stromal contact. Both RSK2 knockdown and small molecule inhibition downregulate interferon regulatory factor 4 and MYC, and provides an explanation for the synergy between lenalidomide and RSK2 inhibition. Interestingly, RSK2 inhibition also sensitized MM cells to bortezomib, melphalan, and dexamethasone, but did not downregulate Ikaros or influence lenalidomide-mediated downregulation of tumor necrosis factor-α or increase lenalidomide-induced IL-2 upregulation. In summary, inhibition of RSK2 may prove a broadly useful adjunct to MM therapy., (© 2015 by The American Society of Hematology.)

Cereblon (CRBN) mediates immunomodulatory drug (IMiD) action in multiple myeloma (MM). Using 2 different methodologies, we identified 244 CRBN binding proteins and established relevance to MM biology by changes in their abundance after exposure to lenalidomide. Proteins most reproducibly binding CRBN (>fourfold vs controls) included DDB1, CUL4A, IKZF1, KPNA2, LTF, PFKL, PRKAR2A, RANGAP1, and SHMT2. After lenalidomide treatment, the abundance of 46 CRBN binding proteins decreased. We focused attention on 2 of these-IKZF1 and IKZF3. IZKF expression is similar across all MM stages or subtypes; however, IKZF1 is substantially lower in 3 of 5 IMiD-resistant MM cell lines. The cell line (FR4) with the lowest IKZF1 levels also harbors a damaging mutation and a translocation that upregulates IRF4, an IKZF target. Clinical relevance of CRBN-binding proteins was demonstrated in 44 refractory MM patients treated with pomalidomide and dexamethasone therapy in whom low IKZF1 gene expression predicted lack of response (0/11 responses in the lowest expression quartile). CRBN, IKZF1, and KPNA2 levels also correlate with significant differences in overall survival. Our study identifies CRBN-binding proteins and demonstrates that in addition to CRBN, IKZF1, and KPNA2, expression can predict survival outcomes., (© 2014 by The American Society of Hematology.)

Sublingual (s.l.) immunization has been described as a convenient and safe way to induce mucosal immune responses in the respiratory and genital tracts. We constructed a helper-dependent adenoviral (HDAd) vector expressing a condon-optimized soluble fusion glycoprotein (sFsyn) of respiratory syncytial virus (HDAd-sFsyn) and explored the potential of s.l. immunization with HDAd-sFsyn to stimulate immune responses in the respiratory mucosa. The RSV specific systemic and mucosal immune responses were generated in BALB/c mice, and the serum IgG with neutralizing activity was significantly elevated after homologous boost with s.l. application of HDAd-sFsyn. Humoral immune responses could be measured even 14weeks after a single immunization. Upon challenge, s.l. immunization with HDAd-sFsyn displayed an effective protection against RSV infection. These findings suggest that s.l. administration of HDAd-sFsyn acts as an effective and safe mucosal vaccine against RSV infection, and may be a useful tool in the prevention of RSV infection., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The longitudinal evolution of a myeloma genome from diagnosis to plasma cell leukemia has not previously been reported. We used whole-genome sequencing (WGS) on 4 purified tumor samples and patient germline DNA drawn over a 5-year period in a t(4;14) multiple myeloma patient. Tumor samples were acquired at diagnosis, first relapse, second relapse, and end-stage secondary plasma cell leukemia (sPCL). In addition to the t(4;14), all tumor time points also shared 10 common single-nucleotide variants (SNVs) on WGS comprising shared initiating events. Interestingly, we observed genomic sequence variants that waxed and waned with time in progressive tumors, suggesting the presence of multiple independent, yet related, clones at diagnosis that rose and fell in dominance. Five newly acquired SNVs, including truncating mutations of RB1 and ZKSCAN3, were observed only in the final sPCL sample suggesting leukemic transformation events. This longitudinal WGS characterization of the natural history of a high-risk myeloma patient demonstrated tumor heterogeneity at diagnosis with shifting dominance of tumor clones over time and has also identified potential mutations contributing to myelomagenesis as well as transformation from myeloma to overt extramedullary disease such as sPCL.

The precise molecular mechanism of action and targets through which thalidomide and related immunomodulatory drugs (IMiDs) exert their antitumor effects remains unclear. We investigated the role of cereblon (CRBN), a primary teratogenic target of thalidomide, in the antimyeloma activity of IMiDs. CRBN depletion is initially cytotoxic to human myeloma cells, but surviving cells with stable CRBN depletion become highly resistant to both lenalidomide and pomalidomide, but not to the unrelated drugs bortezomib, dexamethasone, and melphalan. Acquired deletion of CRBN was found to be the primary genetic event differentiating isogenic MM1.S cell lines cultured to be sensitive or resistant to lenalidomide and pomalidomide. Gene expression changes induced by lenalidomide were dramatically suppressed in the presence of CRBN depletion, further demonstrating that CRBN is required for lenalidomide activity. Downstream targets of CRBN include interferon regulatory factor 4 (IRF4) previously reported to also be a target of lenalidomide. Patients exposed to, and putatively resistant to, lenalidomide had lower CRBN levels in paired samples before and after therapy. In summary, CRBN is an essential requirement for IMiD activity and a possible biomarker for the clinical assessment of antimyeloma efficacy.

The molecular target(s) cooperating with proteasome inhibition in multiple myeloma (MM) remain unknown. We therefore measured proliferation in MM cells transfected with 13 984 small interfering RNAs in the absence or presence of increasing concentrations of bortezomib. We identified 37 genes, which when silenced, are not directly cytotoxic but do synergistically potentiate the growth inhibitory effects of bortezomib. To focus on bortezomib sensitizers, genes that also sensitized MM to melphalan were excluded. When suppressed, the strongest bortezomib sensitizers were the proteasome subunits PSMA5, PSMB2, PSMB3, and PSMB7 providing internal validation, but others included BAZ1B, CDK5, CDC42SE2, MDM4, NME7, RAB8B, TFE3, TNFAIP3, TNK1, TOP1, VAMP2, and YY1. The strongest hit CDK5 also featured prominently in pathway analysis of primary screen data. Cyclin-dependent kinase 5 (CDK5) is expressed at high levels in MM and neural tissues with relatively low expression in other organs. Viral shRNA knockdown of CDK5 consistently sensitized 5 genetically variable MM cell lines to proteasome inhibitors (bortezomib and carfilzomib). Small-molecule CDK5 inhibitors were demonstrated to synergize with bortezomib to induce cytotoxicity of primary myeloma cells and myeloma cell lines. CDK5 regulation of proteasome subunit PSMB5 was identified as a probable route to sensitization.

A paucity of validated kinase targets in human multiple myeloma has delayed clinical deployment of kinase inhibitors in treatment strategies. We therefore conducted a kinome-wide small interfering RNA (siRNA) lethality study in myeloma tumor lines bearing common t(4;14), t(14;16), and t(11;14) translocations to identify critically vulnerable kinases in myeloma tumor cells without regard to preconceived mechanistic notions. Fifteen kinases were repeatedly vulnerable in myeloma cells, including AKT1, AK3L1, AURKA, AURKB, CDC2L1, CDK5R2, FES, FLT4, GAK, GRK6, HK1, PKN1, PLK1, SMG1, and TNK2. Whereas several kinases (PLK1, HK1) were equally vulnerable in epithelial cells, others and particularly G protein-coupled receptor kinase, GRK6, appeared selectively vulnerable in myeloma. GRK6 inhibition was lethal to 6 of 7 myeloma tumor lines but was tolerated in 7 of 7 human cell lines. GRK6 exhibits lymphoid-restricted expression, and from coimmunoprecipitation studies we demonstrate that expression in myeloma cells is regulated via direct association with the heat shock protein 90 (HSP90) chaperone. GRK6 silencing causes suppression of signal transducer and activator of transcription 3 (STAT3) phosphorylation associated with reduction in MCL1 levels and phosphorylation, illustrating a potent mechanism for the cytotoxicity of GRK6 inhibition in multiple myeloma (MM) tumor cells. As mice that lack GRK6 are healthy, inhibition of GRK6 represents a uniquely targeted novel therapeutic strategy in human multiple myeloma.

Helper-dependent adenoviral (HDAd) vectors were developed primarily for genetic disease therapy by deleting all coding regions for attenuating the host cellular immune response to adenovirus (Ad) and long-lasting gene expression. Recently Harui et al. reported that HDAd vaccine could stimulate superior transgene-specific cytotoxic T lymphocyte (CTL) and antibody responses via the intraperitoneal route, compared to first-generation adenoviral (FGAd) vaccine. This prompted us to explore the potential of HDAd as a vaccine vector administrated intranasally. In this study, we prepared HDAd and FGAd vectors expressing enhanced green fluorescent protein (EGFP), respectively, and compared their efficacy in mice. Mice were immunized intranasally with 5x10(9) vp HDAd or FGAd vector particles. Despite stimulating similar anti-Ad antibody responses with FGAd vaccine in the prime/boost strategy, HDAd vector expressing EGFP displayed superior transgene-specific serum IgG, mucosal IgA and cellular immune response, with the characterization of balanced or mixed Th1/Th2 CD4+ T-cell responses. Meanwhile, a single dose of intranasal (i.n.) vaccine of HDAd-EGFP induced a serum IgG response with more efficacy than FGAd-EGFP. In addition, i.n. boost immunization enhanced transgene-specific humoral and cellular responses, compared to single i.n. HDAd-EGFP immunization. Our results suggest that HDAd has potential for a mucosal vaccine vector via i.n. route, which will be useful for the development of vaccines against respiratory viruses, such as respiratory syncytial virus and influenza virus., (Copyright 2009 Elsevier Inc. All rights reserved.)

As multiple myeloma tumors universally dysregulate cyclin D genes we conducted high-throughput chemical library screens for compounds that induce suppression of cyclin D2 promoter transcription. The top-ranked compound was a natural triterpenoid, pristimerin. Strikingly, the early transcriptional response of cells treated with pristimerin closely resembles cellular responses elicited by proteosome inhibitors, with rapid induction of heat shock proteins, activating transcription factor 3 (ATF3), and CHOP. Enzymatic assays and immunoblotting confirm that pristimerin rapidly (< 90 minutes) and specifically inhibits chymotrypsin-like proteosome activity at low concentrations (< 100 nM) and causes accumulation of cellular ubiquitinated proteins. Notably, cytotoxic triterpenoids including pristimerin inhibit NF-kappaB activation via inhibition of IKK alpha or IKK beta, whereas proteosome inhibitors instead suppress NF-kappaB function by impairing degradation of ubiquitinated I kappaB. By inhibiting both IKK and the proteosome, pristimerin causes overt suppression of constitutive NF-kappaB activity in myeloma cells that may mediate its suppression of cyclin D. Multiple myeloma is exquisitely sensitive to proteosome or NF-kappaB pathway inhibition. Consistent with this, pristimerin is potently and selectively lethal to primary myeloma cells (IC(50) < 100 nM), inhibits xenografted plasmacytoma tumors in mice, and is synergistically cytotoxic with bortezomib--providing the rationale for pharmaceutical development of triterpenoid dual-function proteosome/NF-kappaB inhibitors as therapeutics for human multiple myeloma and related malignancies.

D-cyclins are regulators of cell division that act in a complex with cyclin-dependent kinases to commit cells to a program of DNA replication. D-cyclins are overexpressed in many tumors, including multiple myeloma and leukemia, and contribute to disease progression and chemoresistance. To better understand the role and impact of D-cyclins in hematologic malignancies, we conducted a high throughput screen for inhibitors of the cyclin D2 promoter and identified the drug cyproheptadine. In myeloma and leukemia cells, cyproheptadine decreased expression of cyclins D1, D2, and D3 and arrested these cells in the G(0)/G(1) phase. After D-cyclin suppression, cyproheptadine induced apoptosis in myeloma and leukemia cell lines and primary patient samples preferentially over normal hematopoietic cells. In mouse models of myeloma and leukemia, cyproheptadine inhibited tumor growth without significant toxicity. Cyproheptadine-induced apoptosis was preceded by activation of the mitochondrial pathway of caspase activation and was independent of the drug's known activity as an H1 histamine and serotonin receptor antagonist. Thus, cyproheptadine represents a lead for a novel therapeutic agent for the treatment of malignancy. Because the drug is well tolerated and already approved in multiple countries for clinical use as an antihistamine and appetite stimulant, it could be moved directly into clinical trials for cancer.

Objective: The purpose of this study was to evaluate the clinical result of 546 tetracycline-stained teeth restored with a porcelain laminate veneer system (Cerinate, Den-Mat, USA) for aesthetic reasons., Methods: Tetracycline-stained teeth (546) were restored with a porcelain veneer system, and bonded with Ultra Bond resin cement. The restorations were recalled after 0.5, 1.5 and 2.5 years, respectively. Modified Ryge criteria were used to evaluate the veneers marginal adaptation, interfacial staining, secondary caries, postoperative sensitivity and the patients' satisfaction of the colour of the restorations., Results: This study found that 99% veneers had excellent marginal adaptations; and less than 1% veneers required rebonding in the first 6 months; the colour of the veneers was stable and no evident staining was found. Almost all patients were satisfied with the colour match of their restorations 1 year after placement., Conclusions: The research indicated that the porcelain veneer restoration system under investigation provided a reliable and highly satisfactory choice for the aesthetic restoration of tetracycline-stained teeth.

Two helper-dependent (HD) adenoviral vectors encoding a canine factor VIII B-domain-deleted transgene (cFVIII) were constructed and evaluated in 4 hemophilia A dogs. One vector was regulated by the cytomegalovirus (CMV) promoter (HD-CMV-cFVIII), while the other vector contained a tissue-restricted promoter comprised of the human FVIII proximal promoter with an upstream concatemer of 5 hepatocyte nuclear factor 1 binding sites (HD-HNF-cFVIII). We detected no toxicity at low dose (5 x 10(11) vp/kg), but at higher vector doses (> 1 x 10(12) vp/kg) transient hepatotoxicity and thrombocytopenia were observed. Low-level increases in FVIII activity were detected in all 3 HD-HNF-cFVIII-treated dogs, which corresponded with decreased whole blood clotting times. None of the animals receiving the HD-HNF-cFVIII vector developed FVIII inhibitors, and in 1 of the 3 animals, FVIII activity was sustained for over 6 months after treatment. One animal, which received the HD-CMV-cFVIII vector, achieved peak levels of FVIII above 19 000 mU/mL, but FVIII activity disappeared within 1 week, coincident with the development of a potent anti-canine FVIII antibody response. This study supports previous demonstrations of improved safety using HD gene transfer and suggests that these vectors can provide transient FVIII expression with minimal, acute toxicity in the absence of inhibitor formation.

Background: Adenoviral-based methods of gene therapy have been ineffective at providing sustained factor (F)VIII expression in outbred populations of large animal hemophilic models primarily due to the immunogenicity of these vectors. Improvements have been made in vector design leading to the development of the helper-dependent adenoviral (HD) system. Unfortunately, it remains unclear whether these modifications are sufficient to circumvent the induction of inhibitor formation associated with adenoviral gene transfer., Objective: To develop an HD vector capable of mediating sustained FVIII expression and to determine the variables that influence inhibitor development., Methods: HD vectors were constructed encoding the canine FVIII B-domain deleted transgene under the control of either the cytomegalovirus (CMV) promoter or a tissue-restricted hybrid element consisting of five HNF-1 binding sites, located upstream of the human FVIII proximal promoter. Inbred and outbred populations of hemophilic mice were treated, and monitored for vector-induced toxicity, therapeutic efficacy, and inhibitor formation., Results: When HD vectors utilizing the CMV promoter were administered, all hemophilic mice developed high levels of FVIII inhibitors. In contrast, vectors under the control of the HNF/FVIII element were capable of achieving sustained elevations of FVIII for over 6 months. Strain-specific differences were also observed, with outbred animals showing a greater propensity towards inhibitor development in response to treatment., Conclusions: HD vectors can be used to provide long-term FVIII expression in hemophilic animals, but treatment outcome and the induction of inhibitors is dependent on a number of variables including the transgene promoter, the vector dose, and the genetic background of the host.