CRISPR-Cas13b-mediated suppression of HBV replication and protein expression.

BACKGROUND & AIMS: New antiviral approaches that target multiple aspects of the HBV replication cycle to improve rates of functional cure are urgently required. HBV RNA represents a novel therapeutic target. Here, we programmed CRISPR-Cas13b endonuclease to specifically target the HBV pregenomic RNA and viral mRNAs in a novel approach to reduce HBV replication and protein expression. METHODS: Cas13b CRISPR RNAs (crRNAs) were designed to target multiple regions of HBV pregenomic RNA. Mammalian cells transfected with replication competent wild-type HBV DNA of different genotypes, a HBV-expressing stable cell line, a HBV infection model and a hepatitis B surface antigen (HBsAg)-expressing stable cell line were transfected with PspCas13b-BFP (blue fluorescent protein) and crRNA plasmids, and the impact on HBV replication and protein expression was measured. Wild-type HBV DNA, PspCas13b-BFP and crRNA plasmids were simultaneously hydrodynamically injected into mice, and serum HBsAg was measured. PspCas13b mRNA and crRNA were also delivered to a HBsAg-expressing stable cell line via lipid nanoparticles and the impact on secreted HBsAg determined. RESULTS: Our HBV-targeting crRNAs strongly suppressed HBV replication and protein expression in mammalian cells by up to 96% (p <0.0001). HBV protein expression was also reduced in a HBV-expressing stable cell line and in the HBV infection model. CRISPR-Cas13b crRNAs reduced HBsAg expression by 50% (p <0.0001) in vivo. Lipid nanoparticle-encapsulated PspCas13b mRNA reduced secreted HBsAg by 87% (p = 0.0168) in a HBsAg-expressing stable cell line. CONCLUSIONS: Together, these results show that CRISPR-Cas13b can be programmed to specifically target and degrade HBV RNAs to reduce HBV replication and protein expression, demonstrating its potential as a novel therapeutic option for chronic HBV infection. IMPACT AND IMPLICATIONS: Owing to the limitations of current antiviral therapies for hepatitis B, there is an urgent need for new treatments that target multiple aspects of the HBV replication cycle to improve rates of functional cure. Here, we present CRISPR-Cas13b as a novel strategy to target HBV replication and protein expression, paving the way for its development as a potential new treatment option for patients living with chronic hepatitis B.

Sparse reconstruction of sound field using pattern-coupled Bayesian compressive sensing.

Conventional near-field acoustic holography based on compressive sensing either does not fully exploit the underlying block-sparse structures of the signal or suffers from a mismatch between the actual and predefined block structure due to the lack of prior information about block partitions, resulting in poor accuracy in sound field reconstruction. In this paper, a pattern-coupled Bayesian compressive sensing method is proposed for sparse reconstruction of sound fields. The proposed method establishes a hierarchical Gaussian-Gamma probability model with a pattern-coupled prior based on the equivalent source method, transforming the sound field reconstruction problem into recovering the sparse coefficient vector of the equivalent source strengths within the compressive sensing framework. A set of hyperparameters is introduced to control the sparsity of each element in the sparse coefficient vector of the equivalent source strengths, where the sparsity of each element is determined by both its own hyperparameters and those of its immediate neighbors. This approach enables the promotion of block sparse solutions and achieves better performance in solving for the sparse coefficient vector of the equivalent source strengths without prior information of block partitions. The effectiveness and superiority of the proposed method in reconstructing sound fields are verified by simulations and experiments.

Rapid dynamics of general transcription factor TFIIB binding during preinitiation complex assembly revealed by single-molecule analysis.

Transcription of protein-encoding genes in eukaryotic cells requires the coordinated action of multiple general transcription factors (GTFs) and RNA polymerase II (Pol II). A "step-wise" preinitiation complex (PIC) assembly model has been suggested based on conventional ensemble biochemical measurements, in which protein factors bind stably to the promoter DNA sequentially to build a functional PIC. However, recent dynamic measurements in live cells suggest that transcription factors mostly interact with chromatin DNA rather transiently. To gain a clearer dynamic picture of PIC assembly, we established an integrated in vitro single-molecule transcription platform reconstituted from highly purified human transcription factors and complemented it by live-cell imaging. Here we performed real-time measurements of the hierarchal promoter-specific binding of TFIID, TFIIA, and TFIIB. Surprisingly, we found that while promoter binding of TFIID and TFIIA is stable, promoter binding by TFIIB is highly transient and dynamic (with an average residence time of 1.5 sec). Stable TFIIB-promoter association and progression beyond this apparent PIC assembly checkpoint control occurs only in the presence of Pol II-TFIIF. This transient-to-stable transition of TFIIB-binding dynamics has gone undetected previously and underscores the advantages of single-molecule assays for revealing the dynamic nature of complex biological reactions.

Synthesis and Biological Evaluation of Novel Piperidine-3-Carboxamide Derivatives as Anti-Osteoporosis Agents Targeting Cathepsin K.

A series of novel piperidamide-3-carboxamide derivatives were synthesized and evaluated for their inhibitory activities against cathepsin K. Among these derivatives, compound H-9 exhibited the most potent inhibition, with an IC50 value of 0.08 µM. Molecular docking studies revealed that H-9 formed several hydrogen bonds and hydrophobic interactions with key active-site residues of cathepsin K. In vitro, H-9 demonstrated anti-bone resorption effects that were comparable to those of MIV-711, a cathepsin K inhibitor currently in phase 2a clinical trials for the treatment of bone metabolic disease. Western blot analysis confirmed that H-9 effectively downregulated cathepsin K expression in RANKL-reduced RAW264.7 cells. Moreover, in vivo experiments showed that H-9 increased the bone mineral density of OVX-induced osteoporosis mice. These results suggest that H-9 is a potent anti-bone resorption agent targeting cathepsin K and warrants further investigation for its potential anti-osteoporosis values.

ApoM suppresses kidney renal clear cell carcinoma growth and metastasis via the Hippo-YAP signaling pathway.

Renal cell carcinoma is one of the most common malignancies worldwide, and kidney renal clear cell carcinoma (KIRC) is the most common histopathological type of renal cell carcinoma. However, the mechanism of KIRC progression remains poorly understood. Apolipoprotein M (ApoM) is a plasma apolipoprotein and a member of the lipid transport protein superfamily. Lipid metabolism is essential for tumor progression, and its related proteins can be used as therapeutic targets for tumors. ApoM influences the development of several cancers, but its relationship with KIRC remains unclear. In this study, we aimed to investigate the biological function of ApoM in KIRC and to reveal its potential molecular mechanisms. We found that ApoM expression was significantly reduced in KIRC and was strongly correlated with patient prognosis. ApoM overexpression significantly inhibited KIRC cell proliferation in vitro, suppressed the epithelial mesenchymal transition (EMT) of KIRC cells, and decreased their metastatic capacity. Additionally, the growth of KIRC cells was inhibited by ApoM overexpression in vivo. In addition, we found that overexpression of ApoM in KIRC attenuated Hippo-YAP protein expression and YAP stability and thus inhibited KIRC growth and progression. Therefore, ApoM may be a potential target for the treatment of KIRC.

Inhibition of ACSS2 attenuates alcoholic liver steatosis via epigenetically regulating de novo lipogenesis.

BACKGROUND AND AIMS: Steatosis is the early pathological change in alcohol-associated liver disease. However, its precise mechanism is still unclear. The present study is aimed to explore the role and mechanism of acetyl-CoA synthetase 2 (ACSS2) in acute alcohol-induced lipogenesis. METHODS: The increase in ACSS2 nuclear import and histone H3 acetylation were observed in mice after intraperitoneally injected with 2 g/kg ethanol or oral administration of 5 g/kg ethanol and also validated in hepatocytes stimulated with ethanol or acetate. The role of ACSS2 was further explored in liver-specific ACSS2 knockdown mice fed with ethanol-containing diet. RESULTS: Alcohol challenge induced hepatic lipid deposition and upregulated lipogenic genes in mice. It also promoted ACSS2 nuclear import and increased histone H3 acetylation. In hepatocytes, ethanol induced similar phenomena whereas ACSS2 knockdown blocked histone acetylation and lipogenic gene induction. P300/CBP associated factor (PCAF), but not general control nonderepressible 5, CREB-binding protein (CBP) and p300, facilitated H3K9 acetylation responding to ethanol challenge. CUT&RUN assay showed the enrichment of acetylated histone H3K9 surrounding Fasn and Acaca promoters. These results indicated that ethanol metabolism promoted ACSS2 nuclear import to support lipogenesis via H3K9 acetylation. In alcohol-feeding mice, liver-specific ACSS2 knockdown blocked the interaction between PCAF and H3K9 and suppressed lipogenic gene induction in the liver, demonstrating the critical role of ACSS2 in lipogenesis. CONCLUSIONS: Our study demonstrated that alcohol metabolism generated acetyl-CoA in the nucleus dependently on nuclear ACSS2, contributing to epigenetic regulation of lipogenesis in hepatic steatosis. Targeting ACSS2 may be a potential therapeutical strategy for acute alcoholic liver steatosis.

Relationships of Liver X Receptor Antagonists and Atherosclerosis in Drinking Water from Six Chinese Major Cities.

While environmental factors have been considered contributors to atherosclerosis, it remains unclear whether drinking water promotes foam cell formation, the initial event of atherosclerosis. This study revealed that drinking water from six major cities in China, namely, Harbin, Jinan, Shanghai, Wuhan, Chongqing, and Zhuhai, significantly promoted foam cell formation in an in vitro macrophage model at a minimum concentration fold of 2. Moreover, cholesterol efflux was significantly impeded by all samples at 2-16-fold, while cholesterol influx was induced only by samples from Jinan and Chongqing at 16-fold, suggesting the dominant role of efflux in foam cell formation. Interestingly, except for the sample from Jinan, the samples exhibited complete inhibition of liver X receptor α (LXRα) activities at 160-fold, indicating the potential role of chemicals in drinking water in promoting foam cell formation by antagonizing LXRα. Through LXRα protein affinity selection-mass spectrometry, we identified ten LXRα-binding compounds, with efavirenz being revealed for the first time as a significant inducer of foam cell formation through LXRα antagonism. Overall, this study clarifies the atherosclerotic risks posed by drinking water and demonstrates the efavirenz-related atherosclerotic effects.

Urine Excretion, Organ Distribution, and Placental Transfer of 6PPD and 6PPD-Quinone in Mice and Potential Developmental Toxicity through Nuclear Receptor Pathways.

The rubber antioxidant 6PPD has gained significant attention due to its highly toxic transformation product, 6PPD-quinone (6PPDQ). Despite their detection in urines of pregnant women, the placental transfer and developmental toxicity of 6PPD and 6PPDQ are unknown. Here, we treated C57Bl/6 mice with 4 mg/kg 6PPD or 6PPDQ to investigate their urine excretion and placental transfer. Female and male mice exhibited sex difference in excretion profiles of 6PPD and 6PPDQ. Urine concentrations of 6PPDQ were one order of magnitude lower than those of 6PPD, suggesting lower excretion and higher bioaccumulation of 6PPDQ. In pregnant mice treated with 6PPD or 6PPDQ from embryonic day 11.5 to 15.5, 6PPDQ showed ∼1.5-8 times higher concentrations than 6PPD in placenta, embryo body, and embryo brain, suggesting higher placental transfer of 6PPDQ. Using in vitro dual-luciferase reporter assays, we revealed that 6PPDQ activated the human retinoic acid receptor α (RARα) and retinoid X receptor α (RXRα) at concentrations as low as 0.3 µM, which was ∼10-fold higher than the concentrations detected in human urines. 6PPD activated the RXRα at concentrations as low as 1.2 µM. These results demonstrate the exposure risks of 6PPD and 6PPDQ during pregnancy and emphasize the need for further toxicological and epidemiological investigations.

Sparse Reconstruction of Sound Field Using Bayesian Compressive Sensing and Equivalent Source Method.

To solve the problem of sound field reconstruction with fewer measurement points, a sound field reconstruction method based on Bayesian compressive sensing is proposed. In this method, a sound field reconstruction model based on a combination of the equivalent source method and sparse Bayesian compressive sensing is established. The MacKay iteration of the relevant vector machine is used to infer the hyperparameters and estimate the maximum a posteriori probability of both the sound source strength and noise variance. The optimal solution for sparse coefficients with an equivalent sound source is determined to achieve the sparse reconstruction of the sound field. The numerical simulation results demonstrate that the proposed method has higher accuracy over the entire frequency range compared to the equivalent source method, indicating a better reconstruction performance and wider frequency applicability with undersampling. Moreover, in environments with low signal-to-noise ratios, the proposed method exhibits significantly lower reconstruction errors than the equivalent source method, indicating a superior anti-noise performance and greater robustness in sound field reconstruction. The experimental results further verify the superiority and reliability of the proposed method for sound field reconstruction with limited measurement points.

LDV-induced stroboscopic digital image correlation for high spatial resolution vibration measurement.

Vibration measurement, particularly mode shape measurement, is an important aspect of structural dynamic analysis since it can validate finite element or analytical vibration models. Scanning laser Doppler vibrometry (LDV) and high-speed digital image correlation have become dominant methods for experimental mode shape measurement. However, these methods have high equipment costs and several disadvantages regarding spatial or temporal performance. This paper proposes a laser Doppler vibrometer induced stroboscopic digital image correction for non-contact mode shape and operational deflection shape measurement. Our results verify that single-point LDV and normal rate cameras can be used obtain high spatial resolution mode shape and operational deflection shape. Measurement frequency range is much higher than the camera capturing rate. We also show that the proposed approach coincides well with time-averaged electronic speckle pattern interferometry.

Affordable Ab Initio Path Integral for Thermodynamic Properties via Molecular Dynamics Simulations Using Semiempirical Reference Potential.

Path integral molecular dynamics (PIMD) is becoming a routinely applied method for incorporating the nuclear quantum effect in computer simulations. However, direct PIMD simulations at an ab initio level of theory are formidably expensive. Using the protonated 1,8-bis(dimethylamino)naphthalene molecule as an example, we show in this work that the computational expense for the intramolecular proton transfer between the two nitrogen atoms can be remarkably reduced by implementing the idea of reference-potential methods. The simulation time can be easily extended to a scale of nanoseconds while maintaining the accuracy on an ab initio level of theory for thermodynamic properties. In addition, postprocessing can be carried out in parallel on massive computer nodes. A 545-fold reduction in the total CPU time can be achieved in this way as compared to a direct PIMD simulation at the same ab initio level of theory.

A Fringe Phase Extraction Method Based on Neural Network.

In optical metrology, the output is usually in the form of a fringe pattern, from which a phase map can be generated and phase information can be converted into the desired parameters. This paper proposes an end-to-end method of fringe phase extraction based on the neural network. This method uses the U-net neural network to directly learn the correspondence between the gray level of a fringe pattern and the wrapped phase map, which is simpler than the exist deep learning methods. The results of simulation and experimental fringe patterns verify the accuracy and the robustness of this method. While it yields the same accuracy, the proposed method features easier operation and a simpler principle than the traditional phase-shifting method and has a faster speed than wavelet transform method.

A New Pattern Quality Assessment Criterion and Defocusing Degree Determination of Laser Speckle Correlation Method.

The laser speckle correlation method has found widespread application for obtaining information from vibrating objects. However, the resolution and accuracy of the laser speckle correlation method as they relate to the defocusing degree have not been analyzed sufficiently. Furthermore, the possible methods for speckle pattern quality assessment and enhancement have not been studied. In this study, the resolution and accuracy of the laser speckle correlation method are analyzed, and it is found that they are affected by the defocusing degree and speckle pattern quality, respectively. A new speckle pattern quality criterion combining the mean intensity gradient and frequency spectrum was proposed, called CMZ. The quality of the speckle pattern is higher when the CMZ is closer to zero. The proposed criterion was verified by simulated speckle patterns and real speckle patterns with different speckle sizes, densities, and gray contrasts. In the experimental setup stage, a suitable defocusing degree can be selected based on the resolution requirement and optimal speckle size, and other experimental parameters can be determined according to the CMZ criterion. Rotation and vibration experiments verified the effectiveness of the laser speckle correlation method and confirmed the reliability of the experiment preparation based on proposed CMZ criterion.

Discovery of a widespread metabolic pathway within and among phenolic xenobiotics.

Metabolism is an organism's primary defense against xenobiotics, yet it also increases the production of toxic metabolites. It is generally recognized that phenolic xenobiotics, a group of ubiquitous endocrine disruptors, undergo rapid phase II metabolism to generate more water-soluble glucuronide and sulfate conjugates as a detoxification pathway. However, the toxicological effects of the compounds invariably point to the phase I metabolic cytochrome P450 enzymes. Here we show that phenolic xenobiotics undergo an unknown metabolic pathway to form more lipophilic and bioactive products. In a nontargeted screening of the metabolites of a widely used antibacterial ingredient: triclosan (TCS), we identified a metabolic pathway via in vitro incubation with weever, quail, and human microsomes and in vivo exposure in mice, which generated a group of products: TCS-O-TCS. The lipophilic metabolite of TCS was frequently detected in urine samples from the general population, and TCS-O-TCS activated the constitutive androstane receptor with the binding activity about 7.2 times higher than that of the parent compound. The metabolic pathway was mediated mainly by phase I enzymes localized on the microsomes and widely observed in chlorinated phenols, phenols, and hydroxylated aromatics. The pathway was also present in different phenolic xenobiotics and formed groups of unknown pollutants in organisms (e.g., TCS-O-bisphenol A and TCS-O-benzo(a)pyrene), thus providing a cross-talk reaction between different phenolic pollutants during metabolic processes in organisms.

Phase Transition of Ice at High Pressures and Low Temperatures.

The behavior of ice under extreme conditions undergoes the change of intermolecular binding patterns and leads to the structural phase transitions, which are needed for modeling the convection and internal structure of the giant planets and moons of the solar system as well as H2O-rich exoplanets. Such extreme conditions limit the structural explorations in laboratory but open a door for the theoretical study. The ice phases IX and XIII are located in the high pressure and low temperature region of the phase diagram. However, to the best of our knowledge, the phase transition boundary between these two phases is still not clear. In this work, based on the second-order Møller-Plesset perturbation (MP2) theory, we theoretically investigate the ice phases IX and XIII and predict their structures, vibrational spectra and Gibbs free energies at various extreme conditions, and for the first time confirm that the phase transition from ice IX to XIII can occur around 0.30 GPa and 154 K. The proposed work, taking into account the many-body electrostatic effect and the dispersion interactions from the first principles, opens up the possibility of completing the ice phase diagram and provides an efficient method to explore new phases of molecular crystals.

Mitochondrial accumulation of amyloid ß (Aß) peptides requires TOMM22 as a main Aß receptor in yeast.

Mitochondrial accumulation of intracellular ß-amyloid (Aß) peptides is present in the brains of individuals with Alzheimer's disease (AD) as well as in related mouse models of AD. This accumulation is extremely toxic because Aß disrupts the normal functions of many mitochondrial proteins, resulting in significant mitochondrial dysfunction. Therefore, understanding the mitochondrial accumulation of Aß is useful for future pharmaceutical design of drugs to address mitochondrial dysfunction in AD. However, the detailed molecular mechanism of this accumulation process remains elusive. Here, using yeast mitochondria, we present direct experimental evidence suggesting that Aß is specifically recognized by translocase of outer mitochondrial membrane subunit 22 (Tom22 in yeast; TOMM22 in human), a noncanonical receptor within the mitochondrial protein import machinery, and that this recognition is critical for Aß accumulation in mitochondria. Furthermore, we found that residues 25-42 in the Aß peptide mediate the specific interaction with TOMM22. On the basis of our findings, we propose that cytosolic Aß is recognized by TOMM22; transferred to another translocase subunit, TOMM40; and transported through the TOMM channel into the mitochondria. Our results not only confirm that yeast mitochondria can be used as a model to study mitochondrial dysfunction caused by Aß peptides in AD but also pave the way for future studies of the molecular mechanism of mitochondrial Aß accumulation.

A de novo protein binding pair by computational design and directed evolution.

The de novo design of protein-protein interfaces is a stringent test of our understanding of the principles underlying protein-protein interactions and would enable unique approaches to biological and medical challenges. Here we describe a motif-based method to computationally design protein-protein complexes with native-like interface composition and interaction density. Using this method we designed a pair of proteins, Prb and Pdar, that heterodimerize with a Kd of 130 nM, 1000-fold tighter than any previously designed de novo protein-protein complex. Directed evolution identified two point mutations that improve affinity to 180 pM. Crystal structures of an affinity-matured complex reveal binding is entirely through the designed interface residues. Surprisingly, in the in vitro evolved complex one of the partners is rotated 180° relative to the original design model, yet still maintains the central computationally designed hotspot interaction and preserves the character of many peripheral interactions. This work demonstrates that high-affinity protein interfaces can be created by designing complementary interaction surfaces on two noninteracting partners and underscores remaining challenges.

Synthesis of Clean Cabbagelike (111) Faceted Silver Crystals for Efficient Surface-Enhanced Raman Scattering Sensing of Papaverine.

Clean cabbagelike (111) faceted silver crystals were synthesized via a facile galvanic replacement reaction of [Ag(NH3)2]OH and a commercial aluminum foil, a surfactant-free formation process. The cabbagelike silver crystals consisted of interconnected nanoplates and exhibited a single-crystal structure along with preferential (111) facet oriented growth. These silver crystals showed high and reliable surface-enhanced Raman scattering (SERS) activity due to electromagnetic mechanism, and they could be easily transferred onto other rigid or flexible surfaces, making their SERS applications more versatile. Since Ag (111) with low surface energy could preferentially adsorb papaverine molecules, which was verified by molecular dynamics simulation, the cabbagelike silver crystals were further employed as a promising SERS assay for efficient sensing of papaverine, a nonnarcotic alkaloid. A linear range of 0.1-1000 µM was achieved, along with a detection limit of 10 nM and good selectivity relative to other excitability drugs. This SERS assay has successfully been used to determine the concentration of papaverine in hot pot seasonings and drugs with satisfactory recoveries and relative standard deviations.

Efficient Computation of Free Energy Surfaces of Diels⁻Alder Reactions in Explicit Solvent at Ab Initio QM/MM Level.

For Diels⁻Alder (DA) reactions in solution, an accurate and converged free energy (FE) surface at ab initio (ai) quantum mechanical/molecular mechanical (QM/MM) level is imperative for the understanding of reaction mechanism. However, this computation is still far too expensive. In a previous work, we proposed a new method termed MBAR+wTP, with which the computation of the ai FE profile can be accelerated by several orders of magnitude via a three-step procedure: (I) an umbrella sampling (US) using a semi-empirical (SE) QM/MM Hamiltonian is performed; (II) the FE profile is generated using the Multistate Bennett Acceptance Ratio (MBAR) analysis; and (III) a weighted Thermodynamic Perturbation (wTP) from the SE Hamiltonian to the ai Hamiltonian is performed to obtain the ai QM/MM FE profile using weight factors from the MBAR analysis. In this work, this method is extended to the calculations of two-dimensional FE surfaces of two Diels⁻Alder reactions of cyclopentadiene with either acrylonitrile or 1-4-naphthoquinone at ai QM/MM level. The accurate activation free energies at the ai QM/MM level, which are much closer to the experimental measurements than those calculated by other methods, indicate that this MBAR+wTP method can be applied in the studies of complex reactions in condensed phase with much-enhanced efficiency.

Mono-2-ethylhexyl phthalate inhibits human extravillous trophoblast invasion via the PPARγ pathway.

Concerns over the adverse reproductive outcomes in human have been raised, more evidence including the underlying mechanism are required. Since extravillous trophoblast (EVT) invasion is an important physiological step during early development, the effects of mono-2-ethylhexyl phthalate (MEHP), the bioactive metabolite of DEHP, on EVT invasion were investigated using Matrigel-coated transwell chambers and cell line HTR-8/SVneo. In the transwell-based invasive assay, MEHP exposure inhibited EVT invasion as judged by decreased invasion index. Further analysis showed that MEHP exposure significantly inhibited the activity of matrix metalloproteinase-9 (MMP-9), which is an important positive regulator of EVT invasion. Meanwhile, the protein levels of tissue inhibitor matrix metalloproteinase-1 (TIMP-1), one key negative regulator of EVT invasion, were upregulated by MEHP treatment. Finally, inactivation of PPARγ pathway by either PPARγ inhibitors or PPARγ shRNA knockdown rescued the MEHP-induced inhibited invasion of HTR-8/SVneo cells, which is accompanied by the recovery of inhibited MMP-9 expression. The present study provides the evidence that MEHP exposure inhibits trophoblast invasion via PPARγ at concentrations comparable to those found in humans, which provides an insight in understanding the mechanisms of DEHP-associated early pregnancy loss.