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Statins are 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors broadly used for the control of hypercholesterolemia. Recently, they are reported to have beneficial effects on certain cancers. In this study, we show that statins inhibited the histone deacetylase (HDAC) activity and increased the accumulation of acetylated histone-H3 and the expression of p21WAF/CIP in human cancer cells. Computational modeling showed the direct interaction of the carboxylic acid moiety of statins with the catalytic site of HDAC2. In the subsequent enzymatic assay, it was shown that lovastatin inhibited HDAC2 activity competitively with a Ki value of 31.6 μmol/L. Sp1 but not p53 sites were found to be the statins-responsive element shown by p21 luciferase-promoter assays. DNA affinity protein binding assay and chromatin immunoprecipitation assay showed the dissociation of HDAC1/2 and association of CBP, leading to the histone-H3 acetylation on the Sp1 sites of p21 promoter. In vitro cell proliferation and in vivo tumor growth were both inhibited by statins. These results suggest a novel mechanism for statins through abrogation of the HDAC activity and promoter histone-H3 acetylation to regulate p21 expression. Therefore, statins might serve as novel HDAC inhibitors for cancer therapy and chemoprevention. [Cancer Res 2008;68(7):2375–83]

Supplementary Figures 1-2 from Statins Increase p21 through Inhibition of Histone Deacetylase Activity and Release of Promoter-Associated HDAC1/2

Type VI CRISPR enzymes cleave target RNAs and are widely used for gene regulation, RNA tracking, and diagnostics. However, a systematic understanding of their RNA binding specificity and cleavage activation is lacking. Here, we describeRNAchip-hybridizedassociation-mappingplatform (RNA-CHAMP), a massively parallel platform that repurposes next-generation DNA sequencing chips to measure the binding affinity for over 10,000 RNA targets containing structural perturbations, mismatches, insertions, and deletions relative to the CRISPR RNA (crRNA). Deep profiling of Cas13d, a compact and widely used RNA nuclease, reveals that it does not require a protospacer flanking sequence (PFS) but is exquisitely sensitive to secondary structure within the target RNA. Cas13d binding is strongly penalized by mismatches, insertions, and deletions in the distal crRNA-target RNA regions, while alterations in the proximal region inhibit nuclease activity without affecting binding. A biophysical model built from these data reveals that target recognition begins at the distal end of unstructured target RNAs and proceeds to the proximal end. Using this model, we designed a series of partially mismatched guide RNAs that modulate nuclease activity to detect single nucleotide polymorphisms (SNPs) in circulating SARS-CoV-2 variants. This work describes the key determinants of RNA targeting by a type VI CRISPR enzyme to improve CRISPR diagnostics andin vivoRNA editing. More broadly, RNA-CHAMP provides a quantitative platform for systematically measuring protein-RNA interactions.

CRISPR-associated transposons (CASTs) have co-opted CRISPR-Cas proteins and Tn7-family transposons for RNA-guided vertical and horizontal transmission. CASTs encode minimal CRISPR arrays but lack all spacer acquisition genes. Here, we define how different CASTs target new invading mobile elements without updating their own CRISPR arrays. A bioinformatic analysis reveals that all CAST sub-families co-exist with defense-associated CRISPR-Cas systems. Using a quantitative transposition assay, we show that type I-F and I-B CASTs use CRISPR RNAs (crRNAs) from these defense systems for horizontal gene transfer. A high-resolution structure of the type I-F CAST-Cascade in complex with a type III-B crRNA reveals a sequence-independent mechanism for direct repeat recognition. Type I CASTs recognize heterologous CRISPR arrays via a short hairpin in the direct repeat of their crRNA. In contrast, type V CASTs require the Cas12k effector protein but not any crRNA for unguided transposition. This transposition causes random genomic insertions via a copy-and-paste mechanism, even with over-expression of the S15 co-factor. Conversely, a single guide RNA, in concert with S15, increases on-target integration for type V CASTs. These discoveries explain how CASTs horizontally transfer to new hosts without updating their own CRISPR arrays. More broadly, this work will guide further efforts to engineer the activity and specificity of CASTs for gene editing applications.

Although humoral immunity is essential for control of SARS-CoV-2, the molecular composition, binding epitopes and effector functions of the immunoglobulin G (IgG) antibodies that circulate in blood plasma following infection are unknown. Proteomic deconvolution of the circulating IgG repertoire (Ig-Seq (1) ) to the spike ectodomain (S-ECD (2) ) in four convalescent study subjects revealed that the plasma response is oligoclonal and directed predominantly (>80%) to S-ECD epitopes that lie outside the receptor binding domain (RBD). When comparing antibodies directed to either the RBD, the N-terminal domain (NTD) or the S2 subunit (S2) in one subject, just four IgG lineages (1 anti-S2, 2 anti-NTD and 1 anti-RBD) accounted for 93.5% of the repertoire. Although the anti-RBD and one of the anti-NTD antibodies were equally potently neutralizing in vitro , we nonetheless found that the anti-NTD antibody was sufficient for protection to lethal viral challenge, either alone or in combination as a cocktail where it dominated the effect of the other plasma antibodies. We identified in vivo protective plasma anti-NTD antibodies in 3/4 subjects analyzed and discovered a shared class of antibodies targeting the NTD that utilize unmutated or near-germline IGHV1-24, the most electronegative IGHV gene in the human genome. Structural analysis revealed that binding to NTD is dominated by interactions with the heavy chain, accounting for 89% of the entire interfacial area, with germline residues uniquely encoded by IGHV1-24 contributing 20% (149 Å (2) ). Together with recent reports of germline IGHV1-24 antibodies isolated by B-cell cloning (3,4) our data reveal a class of shared IgG antibodies that are readily observed in convalescent plasma and underscore the role of NTD-directed antibodies in protection against SARS-CoV-2 infection.

SummaryThe worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to the repeated emergence of variants of concern. The Omicron variant has two dominant sub-lineages, BA.1 and BA.2, each with unprecedented numbers of nonsynonymous and indel spike protein mutations: 33 and 29, respectively. Some of these mutations individually increase transmissibility and enhance immune evasion, but their interactions within the Omicron mutational background is unknown. We characterize the molecular effects of all Omicron spike mutations on expression, human ACE2 receptor affinity, and neutralizing antibody recognition. We show that key mutations enable escape from neutralizing antibodies at a variety of epitopes. Stabilizing mutations in the N-terminal and S2 domains of the spike protein compensate for destabilizing mutations in the receptor binding domain, thereby enabling the record number of mutations in Omicron sub-lineages. Taken together, our results provide a comprehensive account of the mutational effects in the Omicron spike protein and illuminate previously unknown mechanisms of how the N-terminal domain can compensate for destabilizing mutations within the more evolutionarily constrained RBD.

The [10]phenacene and [11]phenacene molecules have been synthesized using a simple repetition of Wittig reactions followed by photocyclization. Sufficient amounts of [10]phenacene and [11]phenacene were obtained, and thin-film FETs using these molecules have been fabricated with SiO2 and ionic liquid gate dielectrics. These FETs operated in p-channel. The averaged measurements of field-effect mobility, <μ>, were 3.1(7) × 10−2 and 1.11(4) × 10−1 cm2 V−1 s−1, respectively, for [10]phenacene and [11]phenacene thin-film FETs with SiO2 gate dielectrics. Furthermore, [10]phenacene and [11]phenacene thin-film electric-double-layer (EDL) FETs with ionic liquid showed low-voltage p-channel FET properties, with <μ> values of 3(1) and 1(1) cm2 V−1 s−1, respectively. This study also discusses the future utility of the extremely extended π-network molecules [10]phenacene and [11]phenacene as the active layer of FET devices, based on the experimental results obtained.

Current coronavirus (CoV) vaccines primarily target immunodominant epitopes in the S1 subunit, which are poorly conserved and susceptible to escape mutations, thus threatening vaccine efficacy. Here, we use structure-guided protein engineering to remove the S1 subunit from the Middle East respiratory syndrome (MERS)-CoV spike (S) glycoprotein and develop stabilized stem (SS) antigens. Vaccination with MERS SS elicits cross-reactive β-CoV antibody responses and protects mice against lethal MERS-CoV challenge. High-throughput screening of antibody-secreting cells from MERS SS-immunized mice led to the discovery of a panel of cross-reactive monoclonal antibodies. Among them, antibody IgG22 binds with high affinity to both MERS-CoV and severe acute respiratory syndrome (SARS)-CoV-2 S proteins, and a combination of electron microscopy and crystal structures localizes the epitope to a conserved coiled-coil region in the S2 subunit. Passive transfer of IgG22 protects mice against both MERS-CoV and SARS-CoV-2 challenge. Collectively, these results provide a proof of principle for cross-reactive CoV antibodies and inform the development of pan-CoV vaccines and therapeutic antibodies., Graphical abstract, Hsieh et al. generate MERS-CoV spike stabilized stem (SS) antigens using a structure-guided approach. Mice immunized with MERS SS are protected against MERS-CoV challenge and used for isolation of cross-reactive monoclonal antibodies, including IgG22, which protects mice against MERS-CoV and SARS-CoV-2 challenges. Structures of Fab22-spike complexes reveal a conserved epitope.

The severe acute respiratory syndrome coronavirus 2 spike protein is a critical component of coronavirus disease 2019 vaccines and diagnostics and is also a therapeutic target. However, the spike protein is difficult to produce recombinantly because it is a large trimeric class I fusion membrane protein that is metastable and heavily glycosylated. We recently developed a prefusion-stabilized spike variant, termed HexaPro for six stabilizing proline substitutions, that can be expressed with a yield of >30 mg/L in ExpiCHO cells. This protocol describes an optimized workflow for expressing and biophysically characterizing rationally engineered spike proteins in Freestyle 293 and ExpiCHO cell lines. Although we focus on HexaPro, this protocol has been used to purify over a hundred different spike variants in our laboratories. We also provide guidance on expression quality control, long-term storage, and uses in enzyme-linked immunosorbent assays. The entire protocol, from transfection to biophysical characterization, can be completed in 7 d by researchers with basic tissue cell culture and protein purification expertise.

There is concern about second and subsequent waves of COVID-19 caused by the SARS-CoV-2 coronavirus occurring in communities globally that had an initial disease wave. Metropolitan Houston, TX, with a population of 7 million, is experiencing a massive second disease wave that began in late May 2020. To understand SARS-CoV-2 molecular population genomic architecture and evolution and the relationship between virus genotypes and patient features, we sequenced the genomes of 5,085 SARS-CoV-2 strains from these two waves. Our report provides the first molecular characterization of SARS-CoV-2 strains causing two distinct COVID-19 disease waves., We sequenced the genomes of 5,085 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains causing two coronavirus disease 2019 (COVID-19) disease waves in metropolitan Houston, TX, an ethnically diverse region with 7 million residents. The genomes were from viruses recovered in the earliest recognized phase of the pandemic in Houston and from viruses recovered in an ongoing massive second wave of infections. The virus was originally introduced into Houston many times independently. Virtually all strains in the second wave have a Gly614 amino acid replacement in the spike protein, a polymorphism that has been linked to increased transmission and infectivity. Patients infected with the Gly614 variant strains had significantly higher virus loads in the nasopharynx on initial diagnosis. We found little evidence of a significant relationship between virus genotype and altered virulence, stressing the linkage between disease severity, underlying medical conditions, and host genetics. Some regions of the spike protein—the primary target of global vaccine efforts—are replete with amino acid replacements, perhaps indicating the action of selection. We exploited the genomic data to generate defined single amino acid replacements in the receptor binding domain of spike protein that, importantly, produced decreased recognition by the neutralizing monoclonal antibody CR3022. Our report represents the first analysis of the molecular architecture of SARS-CoV-2 in two infection waves in a major metropolitan region. The findings will help us to understand the origin, composition, and trajectory of future infection waves and the potential effect of the host immune response and therapeutic maneuvers on SARS-CoV-2 evolution.

The SARS-CoV-2 spike protein is a critical component of vaccines and a target for neutralizing monoclonal antibodies (nAbs). Spike is also undergoing immunogenic selection with variants that increase infectivity and partially escape convalescent plasma. Here, we describe Spike Display, a high-throughput platform to rapidly characterize glycosylated spike ectodomains across multiple coronavirus-family proteins. We assayed ∼200 variant SARS-CoV-2 spikes for their expression, ACE2 binding, and recognition by 13 nAbs. An alanine scan of all five N-terminal domain (NTD) loops highlights a public epitope in the N1, N3, and N5 loops recognized by most NTD-binding nAbs. NTD mutations in variants of concern B.1.1.7 (alpha), B.1.351 (beta), B.1.1.28 (gamma), B.1.427/B.1.429 (epsilon), and B.1.617.2 (delta) impact spike expression and escape most NTD-targeting nAbs. Finally, B.1.351 and B.1.1.28 completely escape a potent ACE2 mimic. We anticipate that Spike Display will accelerate antigen design, deep scanning mutagenesis, and antibody epitope mapping for SARS-CoV-2 and other emerging viral threats., Graphical abstract, The SARS-CoV-2 spike protein is a critical target of the immune system. Javanmardi et al. report a mammalian cell display platform for rapidly screening the antigenicity and ACE2 affinity of spike variants. This platform, termed Spike Display, accelerates antigen design and antibody epitope mapping for viral glycoproteins.

Stabilizing the prefusion SARS-CoV-2 spike The development of therapeutic antibodies and vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is focused on the spike (S) protein that decorates the viral surface. A version of the spike ectodomain that includes two proline substitutions (S-2P) and stabilizes the prefusion conformation has been used to determine high-resolution structures. However, even S-2P is unstable and difficult to produce in mammalian cells. Hsieh et al. characterized many individual and combined structure-guided substitutions and identified a variant, named HexaPro, that retains the prefusion conformation but shows higher expression than S-2P and can also withstand heating and freezing. This version of the protein is likely to be useful in the development of vaccines and diagnostics. Science , this issue p. 1501

The hyperparameters tuning of machine learning has always been a difficult and time-consuming task in deep learning area. In many practical applications, the hyperparameter tuning directly affects the accuracy. Therefore, the tuning optimization of hyperparameters is an important topic. At present, hyperparameters can only be set manually based on experience, and use Violent Enumeration, Random Search or through Grid Search to try and error, lack of effective automatic search parameters. In this study, we proposed a machine learning hyperparameter fine tuning service on dynamic cloud resource allocation system, which leverages several mainstream hyperparameter tuning methods such as Hyperopt and Optunity. In the meanwhile, various tuning methods are measured and compared by example application in this work. Finally, we dedicated actual case - Heart Sounds, and then tested it. In order to verify that the system service can not only automate the task of tuning, but also break through the limitation of the number of adjustable parameters. Furthermore the proposed hyperparameter fine tune system makes optimization process more efficient.

The physical investigation of surfaces and their properties crucially depends on their quality. Such investigations are commonly performed in an ultra-high vacuum environment. Thereby, the transfer of samples among different setups and under ambient conditions is desirable. The usage of a capping layer for the protection of surfaces against contaminations during long-time storage and transfer, and the subsequent temperature-controlled decapping is an established approach. However, a residual-free removal of the capping layer may present a challenge. Here, we systematically investigate the decapping process of a tellurium protected topological insulator Bi2Te3. We give evidence for the material segregation from the contaminated capping layer surface to the substrates. Therefore, a simple, temperature controlled decapping is not sufficient. We demonstrate that near perfect surfaces can be reliably obtained even after long-time storage through a combination of an initial argon ion sputtering process and a following heating for decapping. This approach is suitable for dedicated analysis systems as well as for industrial applications, large throughput of samples of arbitrary shapes, and is easily implemented in existing setups.

A three-phase interleaved boost converter stage of an ac-dc PFC converter that uses GaN switches is developed that utilizes a feed-forward band-stop filter control method to suppress low-frequency output voltage ripple. To increase power density, the converter switches at 500 kHz, operates in triangular current mode (TCM) to achieve zero voltage switching (ZVS), and uses GaN switching devices. A 1-kW laboratory prototype was designed and tested to verify feasibility. With the proposed controller, output ripple was reduced by 70%. Overall efficiency is greater than 96.5% and power density is 65.028 W/in3.

Here we found loss of c-Cbl, an E3 ligase, expression in non-small cell lung cancer (NSCLC) compared with its adjacent normal tissue in patient specimens. HDAC inhibition by WJ or knockdown of HDAC 1, HDAC2, HDAC3 or HDAC6 all induced c-Cbl. Ectopic expression of c-Cbl induced decreased EGFR, inhibited growth in NSCLC cells. Knockdown of EGFR inhibited NSCLC growth. Mutation of EGFR at Y1045 decreased WJ-induced growth inhibition as well as in vivo anti-cancer effect and EGFR degradation mediated by WJ. Time-lapse confocal analysis showed co-localization of c-Cbl and EGFR after WJ treatment. Furthermore, WJ inhibited lung tumor growth through c-Cbl induction in orthotopic and tail vein injected models. C-Cbl up-regulation induced by HDACi is a potential strategy for NSCLC treatment.

This paper proposes a new solar power generation system, which is composed of a dc/dc power converter and a new seven-level inverter. The dc/dc power converter integrates a dc-dc boost converter and a transformer to convert the output voltage of the solar cell array into two independent voltage sources with multiple relationships. This new seven-level inverter is configured using a capacitor selection circuit and a full-bridge power converter, connected in cascade. The capacitor selection circuit converts the two output voltage sources of dc-dc power converter into a three-level dc voltage, and the full-bridge power converter further converts this three-level dc voltage into a seven-level ac voltage. In this way, the proposed solar power generation system generates a sinusoidal output current that is in phase with the utility voltage and is fed into the utility. The salient features of the proposed seven-level inverter are that only six power electronic switches are used, and only one power electronic switch is switched at high frequency at any time. A prototype is developed and tested to verify the performance of this proposed solar power generation system.

An 8-channel closed-loop neural-prosthetic SoC is presented for real-time intracranial EEG (iEEG) acquisition, seizure detection, and electrical stimulation in order to suppress epileptic seizures. The SoC is composed of eight energy-efficient analog front-end amplifiers (AFEAs), a 10-b delta-modulated SAR ADC (DMSAR ADC), a configurable bio-signal processor (BSP), and an adaptive high-voltage-tolerant stimulator. A wireless power-and-data transmission system is also embedded. By leveraging T-connected pseudo-resistors, the high-pass (low-pass) cutoff frequency of the AFEAs can be adjusted from 0.1 to 10 Hz (0.8 to 7 kHz). The noise-efficiency factor (NEF) of the AFEA is 1.77, and the DMSAR ADC achieves an ENOB of 9.57 bits. The BSP extracts the epileptic features from time-domain entropy and frequency spectrum for seizure detection. A constant 30- μA stimulus current is delivered by closed-loop control. The acquired signals are transmitted with on-off keying (OOK) modulation at 4 Mbps over the MedRadio band for monitoring. A multi-LDO topology is adopted to mitigate the interferences across different power domains. The proposed SoC is fabricated in 0.18- μm CMOS and occupies 13.47 mm2. Verified on Long Evans rats, the proposed SoC dissipates 2.8 mW and achieves high detection accuracy (> 92%) within 0.8 s.

Manipulation of TRAIL receptor 2 (DR5) pathway is a promising therapeutic strategy to overcome TRAIL-resistant lung cancer cells. Preclinical studies have shown that proteasome inhibitors enhance TRAIL-induced apoptosis in lung cancer cells, but the underlying mechanism has not been fully elucidated. In this study, we demonstrated the enhancement of TRAIL-mediated apoptosis in human alveolar epithelial cells by proteasome inhibitors that up-regulate DR5 expression. This effect was blocked by DR5-neutralizing Ab. Using reporter assay, we demonstrated that the p53 and NF-κB elements on the DR5 first intron region were involved in proteasome inhibitor-induced DR5 expression. Both p53 small interfering RNA and NF-κB inhibitor suppressed DR5 expression, strengthening the significance of p53 and NF-κB in DR5 transcription. The protein stability, Ser392 phosphorylation and Lys373/Lys382 acetylation of p53 were enhanced by MG132. In addition to p53, IκBα degradation and NF-κB translocation was also observed. Moreover, the binding of p53 and p65 to the first intron of DR5 was demonstrated by DNA affinity protein-binding and chromatin immunoprecipitation assays. Intracellular reactive oxygen species (ROS) generation after MG132 treatment contributed to p53, but not p65 nuclear translocation and DNA-binding activity. ROS scavenger dramatically inhibited the apoptosis induced by proteasome inhibitors plus TRAIL. The p53-null H1299 cells were resistant to proteasome inhibitor-induced DR5 up-regulation and enhancement of TRAIL-induced apoptosis. These findings reveal that proteasome inhibitor-mediated NF-κB and ROS-dependent p53 activation are contributed to intronic regulation of DR5 transcription, and resulted in the subsequent enhancement of TRAIL-induced apoptosis in human lung cancer cells.

The high unemployment rate, decreasing salary, and housing pressure, etc. in Taiwan have led to unhappiness and frustration among people. To address this problem, the question our team proposes for exploring a possible technological solution is: “Is every one able to possess eudemonic well-being?” This study expects to identify a web/mobile-based Orange Technology application that enables the sense of eudemonic well-being of its users. Because happiness (i.e., a subjective measure of eudemonic well-being) is shown to be resulted from being socially connected, such application could be a “social communication and interactive tool” that handles interactions between a pair or group of users. The targeted users are those whose ages fall between the generations X and Y (twenties to forties). The adopted technologies include those for developing web platforms (e.g., web-based programming languages and protocols) and analyzing real time data (e.g., Ensemble Empirical Mode Decomposition, Cross-Correlation, and Relaxed Variable Kernel Density Estimation).

We experimentally investigate fluctuations of entropy production in a coupled driven-RC circuit. In particular, we focus on the hidden-variable problem, where part of the circuit is neglected intentionally. In the two versions of the reduced descriptions we provide for the system, the fluctuation theorem (FT) is valid in all timescales for weak coupling. However, FT fails in the strong-coupling regime, in the short-time limit for one version, and in the long-time limit for the other. In these timescales where FT fails, both descriptions still give FT-like behavior. The failure of FT implies non-Markovian dynamics, meaning there exists a hidden variable that cannot be incorporated into the heat bath. We argue that FT can be restored with the introduction of a timescale-dependent effective noise.

This paper presents a radial force control scheme for self-bearing operation of a surface permanent magnet synchronous motor. The control scheme is based on a self-bearing motor which consists of separate torque and suspension windings. Torque winding produces rotational torque, and the suspension winding produces radial force for rotor levitation. Calculated winding currents are combined internally in the controller. Thus, only a set of winding is required to produce controllable radial force and rotational torque. Analysis, implementation, and experimental verifications of the proposed scheme are presented.

Epidermal growth factor receptor (EGFR), a receptor tyrosine kinase which promotes cell proliferation and survival, is abnormally overexpressed in numerous tumors of epithelial origin, including colorectal cancer (CRC). EGFR monoclonal antibodies have been shown to increase the median survival and are approved for the treatment of colorectal cancer. Histone deacetylases (HDACs), frequently overexpressed in colorectal cancer and several malignancies, are another attractive targets for cancer therapy. Several inhibitors of HDACs (HDACi) are developed and exhibit powerful antitumor abilities. In this study, human colorectal cancer cells treated with HDACi exhibited reduced EGFR expression, thereby disturbed EGF-induced ERK and Akt phosphorylation. HDACi also decreased the expression of SGLT1, an active glucose transporter found to be stabilized by EGFR, and suppressed the glucose uptake of cancer cells. HDACi suppressed the transcription of EGFR and class I HDACs were proved to be involved in this event. Chromatin immunoprecipitation analysis showed that HDACi caused the dissociation of SP1, HDAC3 and CBP from EGFR promoter. Our data suggested that HDACi could serve as a single agent to block both EGFR and HDAC, and may bring more benefits to the development of CRC therapy.

TGF-beta regulates diverse biologic effects including cell growth, cell death or apoptosis, cell differentiation, and extracellular matrix (ECM) synthesis. Connective tissue growth factor (CTGF), induced by TGF-beta has been reported to mediate stimulatory action of TGF-beta-induced ECM. Although TNF-alpha was reported to suppress theTGF-beta-induced CTGF gene expression, the molecular mechanism is not well clarified. In this study, we found the inhibitory effect of TNF-alpha on TGF-beta-induced CTGF expression in WT but not p65-/-MEF cells. TNF-alpha neither induced Smad7 expression nor affected TGF-beta-induced Smad2 phosphorylation and nuclear translocation. We demonstrated that p300 physically associated with p65 rather than Smad4 in the presence of both TNF-alpha and TGF-beta. Moreover, the TGF-beta-induced binding of p300 and acetylated H4, but not Smad4 to the CTGF promoter was disturbed by TNF-alpha treatment. Overall, our data showed that suppression of TNF-alpha on TGF-beta-induced CTGF expression is due to the competition of p300 by p65 and Smad4.

Elevated levels of NF-kappaB are frequently detected in many inflammatory diseases and cancers. Blocking the IKK-NF-kappaB pathway has been seen as a promising approach for new therapies. By employing the dominant-negative mutant of IKKbeta, our data revealed that loss of IKKbeta activity reduces not only the proliferation and invasion of lung adenocarcinoma A549 cells in vitro but also the tumour formation, metastasis and angiogenesis in mouse xenograft model. Treatment of IKKbeta inhibitors (CYL-19s and CYL-26z) leads to the arrest of cell cycle progression at G1 and G2/M, followed by apoptosis. IKKbeta inhibitors can increase the protein stability, nuclear accumulation and promoter-binding activity of p53, leading to the p21 gene transcription. Furthermore, knockdown of IKKbeta by siRNA increased the stability and expression of p53 and p21 promoter activity. In addition, IKKbeta inhibitor-induced p53 and p21 expressions were augmented in the presence of IKKbeta siRNA. Correlation between p53 acetylation and its protein stabilization was also seen after treatment with IKKbeta inhibitors. These results suggest that loss of IKKbeta activation is important for the enhancement of p53 stability, leading to p21 expression and cell cycle arrest and apoptosis of tumour cells.

HDAC inhibitors are promising anticancer agents that induce cell cycle arrest and apoptosis. However, the role of HDACs in cancer progression, such as angiogenesis and metastasis, remains largely unexplored. Among various HDAC inhibitors, we demonstrate that TSA and SAHA upregulated the expression of angiostatic ADAMTS1 in A549 cells. HDAC6 inhibitor tubacin, and knockdown of HDAC6, also lead to ADAMTS1 upregulation. By reporter, DAPA, and ChIP assays, the proximal GC boxes were demonstrated to be essential for ADAMTS1 induction. Decreased binding of SP1 and HDAC6 to the ADAMTS1 promoter after TSA treatment was also seen. These data suggest the involvement of HDAC6 and SP1 in the HDACi-induced expression of angiostatic ADAMTS1.

Statins are 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors broadly used for the control of hypercholesterolemia. Recently, they are reported to have beneficial effects on certain cancers. In this study, we show that statins inhibited the histone deacetylase (HDAC) activity and increased the accumulation of acetylated histone-H3 and the expression of p21WAF/CIP in human cancer cells. Computational modeling showed the direct interaction of the carboxylic acid moiety of statins with the catalytic site of HDAC2. In the subsequent enzymatic assay, it was shown that lovastatin inhibited HDAC2 activity competitively with a Ki value of 31.6 μmol/L. Sp1 but not p53 sites were found to be the statins-responsive element shown by p21 luciferase-promoter assays. DNA affinity protein binding assay and chromatin immunoprecipitation assay showed the dissociation of HDAC1/2 and association of CBP, leading to the histone-H3 acetylation on the Sp1 sites of p21 promoter. In vitro cell proliferation and in vivo tumor growth were both inhibited by statins. These results suggest a novel mechanism for statins through abrogation of the HDAC activity and promoter histone-H3 acetylation to regulate p21 expression. Therefore, statins might serve as novel HDAC inhibitors for cancer therapy and chemoprevention. [Cancer Res 2008;68(7):2375–83]

Here we found loss of c-Cbl, an E3 ligase, expression in Non-small cell lung cancer (NSCLC) compared with its adjacent normal tissue in patient specimens. HDAC inhibition by WJ or knockdown of HDAC 1, HDAC2, HDAC3 or HDAC6 all induced c-Cbl. Ectopic expression of c-Cbl induced decreased EGFR, inhibited growth in NSCLC cells. Knockdown of EGFR inhibited NSCLC growth. Mutation of EGFR at Y1045 decreased WJ-induced growth inhibition as well as in vivo anti-cancer effect and EGFR degradation mediated by WJ. Time-lapse confocal analysis showed co-localization of c-Cbl and EGFR after WJ treatment. Furthermore, WJ inhibited lung tumor growth through c-Cbl induction in orthotopic and tail vein injected models. C-Cbl up-regulation induced by HDACi is a potential strategy for NSCLC treatment. Citation Format: Chen Ching-Chow, Tzu-Tang Wei, Yu-Chin Lin, Pei-Hua Lin, Jin-Yuan Shih, Chia-Wei Chou. Induction of c-Cbl is a novel mechanism for the anti-cancer effect of HDAC inhibitor in lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-250. doi:10.1158/1538-7445.AM2015-LB-250

CRISPR-associated transposons (CASTs) co-opt CRISPR-Cas proteins and Tn7-family transposons for RNA-guided vertical and horizontal transmission. CASTs encode minimal CRISPR arrays but can't acquire new spacers. Here, we show that CASTs instead co-opt defense-associated CRISPR arrays for horizontal transmission. A bioinformatic analysis shows that all CAST sub-types co-occur with defense-associated CRISPR-Cas systems. Using an E. coli quantitative transposition assay, we show that CASTs use CRISPR RNAs (crRNAs) from these defense systems for horizontal gene transfer. A high-resolution structure of the type I-F CAST-Cascade in complex with a type III-B crRNA reveals that Cas6 recognizes direct repeats via sequence-independent π - π interactions. In addition to using heterologous CRISPR arrays, type V CASTs can also transpose via a crRNA-independent unguided mechanism, even when the S15 co-factor is over-expressed. Over-expressing S15 and the trans-activating CRISPR RNA (tracrRNA) or a single guide RNA (sgRNA) reduces, but does not abrogate, off-target integration for type V CASTs. Exploiting new spacers in defense-associated CRISPR arrays explains how CASTs horizontally transfer to new hosts. More broadly, this work will guide further efforts to engineer the activity and specificity of CASTs for gene editing applications., Competing Interests: Declaration of Interests K.H., C.O.W. and I.J.F. have filed a patent application relating to CRISPR-associated transposons.