The structural ensemble of a Holliday junction determined by X-ray scattering interference.

The DNA four-way (Holliday) junction is the central intermediate of genetic recombination, yet key aspects of its conformational and thermodynamic properties remain unclear. While multiple experimental approaches have been used to characterize the canonical X-shape conformers under specific ionic conditions, the complete conformational ensemble of this motif, especially at low ionic conditions, remains largely undetermined. In line with previous studies, our single-molecule Förster resonance energy transfer (smFRET) measurements of junction dynamics revealed transitions between two states under high salt conditions, but smFRET could not determine whether there are fast and unresolvable transitions between distinct conformations or a broad ensemble of related states under low and intermediate salt conditions. We therefore used an emerging technique, X-ray scattering interferometry (XSI), to directly probe the conformational ensemble of the Holliday junction across a wide range of ionic conditions. Our results demonstrated that the four-way junction adopts an out-of-plane geometry under low ionic conditions and revealed a conformational state at intermediate ionic conditions previously undetected by other methods. Our results provide critical information to build toward a full description of the conformational landscape of the Holliday junction and underscore the utility of XSI for probing conformational ensembles under a wide range of solution conditions.

EDSSA: An Encoder-Decoder Semantic Segmentation Networks Accelerator on OpenCL-Based FPGA Platform.

Visual semantic segmentation, which is represented by the semantic segmentation network, has been widely used in many fields, such as intelligent robots, security, and autonomous driving. However, these Convolutional Neural Network (CNN)-based networks have high requirements for computing resources and programmability for hardware platforms. For embedded platforms and terminal devices in particular, Graphics Processing Unit (GPU)-based computing platforms cannot meet these requirements in terms of size and power consumption. In contrast, the Field Programmable Gate Array (FPGA)-based hardware system not only has flexible programmability and high embeddability, but can also meet lower power consumption requirements, which make it an appropriate solution for semantic segmentation on terminal devices. In this paper, we demonstrate EDSSA-an Encoder-Decoder semantic segmentation networks accelerator architecture which can be implemented with flexible parameter configurations and hardware resources on the FPGA platforms that support Open Computing Language (OpenCL) development. We introduce the related technologies, architecture design, algorithm optimization, and hardware implementation of the Encoder-Decoder semantic segmentation network SegNet as an example, and undertake a performance evaluation. Using an Intel Arria-10 GX1150 platform for evaluation, our work achieves a throughput higher than 432.8 GOP/s with power consumption of about 20 W, which is a 1.2× times improvement the energy-efficiency ratio compared to a high-performance GPU.

Determination of the conformational ensemble of the TAR RNA by X-ray scattering interferometry.

The conformational ensembles of structured RNA's are crucial for biological function, but they remain difficult to elucidate experimentally. We demonstrate with HIV-1 TAR RNA that X-ray scattering interferometry (XSI) can be used to determine RNA conformational ensembles. X-ray scattering interferometry (XSI) is based on site-specifically labeling RNA with pairs of heavy atom probes, and precisely measuring the distribution of inter-probe distances that arise from a heterogeneous mixture of RNA solution structures. We show that the XSI-based model of the TAR RNA ensemble closely resembles an independent model derived from NMR-RDC data. Further, we show how the TAR RNA ensemble changes shape at different salt concentrations. Finally, we demonstrate that a single hybrid model of the TAR RNA ensemble simultaneously fits both the XSI and NMR-RDC data set and show that XSI can be combined with NMR-RDC to further improve the quality of the determined ensemble. The results suggest that XSI-RNA will be a powerful approach for characterizing the solution conformational ensembles of RNAs and RNA-protein complexes under diverse solution conditions.

The solution structural ensembles of RNA kink-turn motifs and their protein complexes.

With the growing number of crystal structures of RNA and RNA-protein complexes, a critical next step is understanding the dynamic solution behavior of these entities in terms of conformational ensembles and energy landscapes. To this end, we have used X-ray scattering interferometry (XSI) to probe the ubiquitous RNA kink-turn motif and its complexes with the canonical kink-turn binding protein L7Ae. XSI revealed that the folded kink-turn is best described as a restricted conformational ensemble. The ions present in solution alter the nature of this ensemble, and protein binding can perturb the kink-turn ensemble without collapsing it to a unique state. This study demonstrates how XSI can reveal structural and ensemble properties of RNAs and RNA-protein complexes and uncovers the behavior of an important RNA-protein motif. This type of information will be necessary to understand, predict and engineer the behavior and function of RNAs and their protein complexes.

From a structural average to the conformational ensemble of a DNA bulge.

Direct experimental measurements of conformational ensembles are critical for understanding macromolecular function, but traditional biophysical methods do not directly report the solution ensemble of a macromolecule. Small-angle X-ray scattering interferometry has the potential to overcome this limitation by providing the instantaneous distance distribution between pairs of gold-nanocrystal probes conjugated to a macromolecule in solution. Our X-ray interferometry experiments reveal an increasing bend angle of DNA duplexes with bulges of one, three, and five adenosine residues, consistent with previous FRET measurements, and further reveal an increasingly broad conformational ensemble with increasing bulge length. The distance distributions for the AAA bulge duplex (3A-DNA) with six different Au-Au pairs provide strong evidence against a simple elastic model in which fluctuations occur about a single conformational state. Instead, the measured distance distributions suggest a 3A-DNA ensemble with multiple conformational states predominantly across a region of conformational space with bend angles between 24 and 85 degrees and characteristic bend directions and helical twists and displacements. Additional X-ray interferometry experiments revealed perturbations to the ensemble from changes in ionic conditions and the bulge sequence, effects that can be understood in terms of electrostatic and stacking contributions to the ensemble and that demonstrate the sensitivity of X-ray interferometry. Combining X-ray interferometry ensemble data with molecular dynamics simulations gave atomic-level models of representative conformational states and of the molecular interactions that may shape the ensemble, and fluorescence measurements with 2-aminopurine-substituted 3A-DNA provided initial tests of these atomistic models. More generally, X-ray interferometry will provide powerful benchmarks for testing and developing computational methods.

Structural ensemble and microscopic elasticity of freely diffusing DNA by direct measurement of fluctuations.

Precisely measuring the ensemble of conformers that a macromolecule populates in solution is highly challenging. Thus, it has been difficult to confirm or falsify the predictions of nanometer-scale dynamical modeling. Here, we apply an X-ray interferometry technique to probe the solution structure and fluctuations of B-form DNA on a length scale comparable to a protein-binding site. We determine an extensive set of intrahelix distance distributions between pairs of probes placed at distinct points on the surface of the DNA duplex. The distributions of measured distances reveal the nature and extent of the thermally driven mechanical deformations of the helix. We describe these deformations in terms of elastic constants, as is common for DNA and other polymers. The average solution structure and microscopic elasticity measured by X-ray interferometry are in striking agreement with values derived from DNA-protein crystal structures and measured by force spectroscopy, with one exception. The observed microscopic torsional rigidity of DNA is much lower than is measured by single-molecule twisting experiments, suggesting that torsional rigidity increases when DNA is stretched. Looking forward, molecular-level interferometry can provide a general tool for characterizing solution-phase structural ensembles.

Production rate enhancement of size-tunable silicon nanoparticles by temporally shaping femtosecond laser pulses in ethanol.

This paper proposes an efficient approach for production-rate enhancement and size reduction of silicon nanoparticles produced by femtosecond (fs) double-pulse ablation of silicon in ethanol. Compared with a single pulse, the production rate is ~2.6 times higher and the mean size of the NPs is reduced by ~1/5 with a delay of 2 ps. The abnormal enhancement in the production rate is obtained at pulse delays Δt > 200 fs. The production-rate enhancement is mainly attributed to high photon absorption efficiency. It is caused by an increase in localized transient electron density, which results from the first sub-pulse ionization of ethanol molecules before the second sub-pulse arrives. The phase-change mechanism at a critical point might reduce nanoparticle size.

Nanopillar arrays with nanoparticles fabricated by a femtosecond laser pulse train for highly sensitive SERRS.

This work presents a novel method for fabricating repeatable, uniform, large-area, highly sensitive, surface-enhanced resonance Raman scattering (SERRS) substrates combined with silicon nanopillar arrays and silver nanoparticles. The proposed method consists of two steps: (1) induce periodic ripples in deionized water using a linearly polarized femtosecond laser; and (2) generate dense 80-nm-diameter nanopillar arrays with silver nanoparticles in silver nitrate solution with a 90° rotated polarization, femtosecond double-pulse train. As the pulse delay increases from 0 to 1000 fs, the mean size of the silver nanoparticles reduces, and the average number of nanoparticles increases, which, in turn, increases the enhancement factor of SERRS signals up to 1.1×10(9). Furthermore, melamine (down to 125 ppb) was detected by the fabricated SERRS substrates.

Roles of long-range tertiary interactions in limiting dynamics of the Tetrahymena group I ribozyme.

We determined the effects of mutating the long-range tertiary contacts of the Tetrahymena group I ribozyme on the dynamics of its substrate helix (referred to as P1) and on catalytic activity. Dynamics were assayed by fluorescence anisotropy of the fluorescent base analogue, 6-methyl isoxanthopterin, incorporated into the P1 helix, and fluorescence anisotropy and catalytic activity were measured for wild type and mutant ribozymes over a range of conditions. Remarkably, catalytic activity correlated with P1 anisotropy over 5 orders of magnitude of activity, with a correlation coefficient of 0.94. The functional and dynamic effects from simultaneous mutation of the two long-range contacts that weaken P1 docking are cumulative and, based on this RNA's topology, suggest distinct underlying origins for the mutant effects. Tests of mechanistic predictions via single molecule FRET measurements of rate constants for P1 docking and undocking suggest that ablation of the P14 tertiary interaction frees P2 and thereby enhances the conformational space explored by the undocked attached P1 helix. In contrast, mutation of the metal core tertiary interaction disrupts the conserved core into which the P1 helix docks. Thus, despite following a single correlation, the two long-range tertiary contacts facilitate P1 helix docking by distinct mechanisms. These results also demonstrate that a fluorescence anisotropy probe incorporated into a specific helix within a larger RNA can report on changes in local helical motions as well as differences in more global dynamics. This ability will help uncover the physical properties and behaviors that underlie the function of RNAs and RNA/protein complexes.

Ablation area quasiperiodic oscillations in semiconductors with femtosecond laser double-pulse delay.

A surprising repeatable phenomenon regarding semiconductor ablation area changes has been discovered. Irradiated by femtosecond double pulses, the ablation area quasiperiodically oscillates as the pulse delay increases from 0 to 1 ps at a material-dependent fluence range. In contrast, the ablation area monotonically decreases as the pulse delay increases beyond 1 ps or if the total fluence increases close to or beyond the single-shot threshold. Similar unexpected patterns of area quasiperiodic oscillations with the double-pulse delay are observed in various semiconductors, including Ge, Si, GaAs, and ZnO. The comparison study shows the same phenomenon in Au-plated ZnO. Yet, its oscillation periods are shorter and more stable than those in bulk ZnO, which implies that the localized carrier density is the key factor in oscillation periods.

A DNA aptamer with high affinity and specificity for molecular recognition and targeting therapy of gastric cancer.

BACKGROUND: Aptamers have emerged as excellent molecular probes for cancer diagnosis and therapy. The aim of the current study was to determine the feasibility of using DNA aptamer cy-apt 20 developed by live cell-SELEX for detecting and targeting gastric cancer. METHODS: The specificity, sensitivity and biostability of cy-apt 20 in detecting gastric cancer were assessed by binding assay, cell fluorescence imaging, and in vivo tumor imaging in animal model in comparison with non-gastric cancers. RESULTS: Flow cytometric analysis showed that cy-apt 20 had higher than 78% of maximal binding rate to gastric cancer cells, much higher than that of non-gastric cancer cells. Cell fluorescence imaging and in vivo tumor imaging showed that the targeting recognition could be visualized by using minimal dose of fluorochrome labeled cy-apt 20. Meanwhile, strong fluorescence signals were detected and lasted for a period of time longer than 50 min in vitro and 240 min in vivo. The fluorescence intensities of gastric cancer were about seven folds in vitro and five folds of that of non-gastric cancers in vivo. CONCLUSION: Our study demonstrated that cy-apt 20 was an excellent molecular probe with high specificity and sensitivity and a certain degree of biostability for molecular recognition and targeting therapy of gastric cancer.

NUP43 promotes PD-L1/nPD-L1/PD-L1 feedback loop via TM4SF1/JAK/STAT3 pathway in colorectal cancer progression and metastatsis.

Programmed cell death-ligand 1 (PD-L1) has a significant role in tumor progression and metastasis, facilitating tumor cell evasion from immune surveillance. PD-L1 can be detected in the tumor cell nucleus and exert an oncogenic effect by nuclear translocation. Colorectal cancer (CRC) progression and liver metastasis (CCLM) are among the most lethal diseases worldwide, but the mechanism of PD-L1 nuclear translocation in CRC and CCLM remains to be fully understood. In this study, using CRISPR-Cas9-based genome-wide screening combined with RNA-seq, we found that the oncogenic factor NUP43 impacted the process of PD-L1 nuclear translocation by regulating the expression level of the PD-L1 chaperone protein IPO5. Subsequent investigation revealed that this process could stimulate the expression of tumor-promoting factor TM4SF1 and further activate the JAK/STAT3 signaling pathway, which ultimately enhanced the transcription of PD-L1, thus establishing a PD-L1-nPD-L1-PD-L1 feedback loop that ultimately promoted CRC progression and CCLM. In conclusion, our study reveals a novel role for nPD-L1 in CRC, identifies the PD-L1-nPD-L1-PD-L1 feedback loop in CRC, and provides a therapeutic strategy for CRC patients.

A single-stranded junction modulates nanosecond motional ordering of the substrate recognition duplex of a group I ribozyme.

Rigid spinning: Site-directed spin-labeling studies using a rigid nitroxide spin label (Ç) reveal that both length and sequence of a single-stranded junction (J1/2) modulate nanosecond motional ordering of the substrate-recognition duplex (P1) of the 120 kD group I ribozyme. The studies demonstrate an approach for experimental measurements of nanosecond dynamics in high-molecular-weight RNA complexes.

Femtosecond double-pulse fabrication of hierarchical nanostructures based on electron dynamics control for high surface-enhanced Raman scattering.

This Letter presents a simple, efficient approach for high surface-enhanced Raman scattering by one-step controllable fabrication of hierarchical structures (nanoparticles+subwavelength ripples) on silicon substrates in silver nitrate solutions using femtosecond double pulses based on nanoscale electron dynamics control. As the delays of the double pulses increase from 0 fs to 1 ps, the hierarchical structures can be controlled with (1) nanoparticles--the number of nanoparticles in the range of 40-100 nm reaches the maximum at 800 fs and (2) ripples--the subwavelength ripples become intermittent with decreased ablation depths. The redistributed nanoparticles and the modified ripple structures contribute to the maximum enhancement factor of 2.2×10(8) (measured by 10(-6) M rhodamine 6G solution) at the pulse delay of 800 fs.

Femtosecond laser-induced periodic structure adjustments based on electron dynamics control: from subwavelength ripples to double-grating structures.

This study proposes a method for adjusting subwavelength ripple periods and the corresponding double-grating structures formed on fused silica by designing femtosecond laser pulse trains based on localized transient electron density control. Four near-constant period ranges of 190-490 nm of ripples perpendicular to the polarization are obtained by designing pulse trains to excite and modulate the surface plasmon waves. In the period range of 350-490 nm, the double-grating structure is fabricated in one step, which is probably attributable to the grating-assisted enhanced energy deposition and subsequent thermal effects.

Rational design of wettability-patterned microchips for high-performance attomolar surface-enhanced Raman detection.

Recent progress in wettability-patterned microchips has facilitated the development of ultra-trace detection in multiple biomedical and food safety fields. The existence of a superhydrophilic trap can realize targeted deposition of the analyte. However, the wetting transition from the Cassie-Baxter state to the Wenzel state usually occurs during evaporation and leads to a larger deposition footprint, which has a strong impact on the detection sensitivity and uniformity. In this paper, we report an integrated design, fabrication, and evaporation strategy to avoid the transition for high-performance attomolar surface-enhanced Raman scattering (SERS) detection. An improved force balance model was proposed to design the microstructures of wettability-patterned microchips, which were fabricated by nanosecond laser direct writing and surface fluorination. The microchips were composed of superhydrophobic micro-grooves and superhydrophilic traps, by which the targeted deposition of Au nanoparticles and rhodamine 6G (R6G) onto a minimal area of ∼70 × 70 µm2 was realized after a two-step heated evaporation. Accordingly, the detection limit was down to the attomolar level (5 × 10-18 M) with SERS enhancement factors (EFs) exceeding 1010. More importantly, the Raman signals showed good uniformity (RSD of 11.5%) for the concentration of 2 × 10-17 M. A good linear relationship was obtained in the quantitative concentration range of 10-12 M to 5 × 10-18 M with a high correlation coefficient (R2) of 0.996. These wettability-patterned microchips exhibit high performance (that is, both good sensitivity and good uniformity) in the detection of ultra-trace molecules in aqueous solutions, avoiding the need for expensive equipment and considerable skill in operations. The proposed strategy could also be applied to other microfluidic devices for rapid and simple analyte pre-concentration.

A role for a single-stranded junction in RNA binding and specificity by the Tetrahymena group I ribozyme.

We have investigated the role of a single-stranded RNA junction, J1/2, that connects the substrate-containing P1 duplex to the remainder of the Tetrahymena group I ribozyme. Single-turnover kinetics, fluorescence anisotropy, and single-molecule fluorescence resonance energy transfer studies of a series of J1/2 mutants were used to probe the sequence dependence of the catalytic activity, the P1 dynamics, and the thermodynamics of docking of the P1 duplex into the ribozyme's catalytic core. We found that A29, the center A of three adenosine residues in J1/2, contributes 2 orders of magnitude to the overall ribozyme activity, and double-mutant cycles suggested that J1/2 stabilizes the docked state of P1 over the undocked state via a tertiary interaction involving A29 and the first base pair in helix P2 of the ribozyme, A31·U56. Comparative sequence analysis of this group I intron subclass suggests that the A29 interaction sets one end of a molecular ruler whose other end specifies the 5'-splice site and that this molecular ruler is conserved among a subclass of group I introns related to the Tetrahymena intron. Our results reveal substantial functional effects from a seemingly simple single-stranded RNA junction and suggest that junction sequences may evolve rapidly to provide important interactions in functional RNAs.

Subwavelength ripples adjustment based on electron dynamics control by using shaped ultrafast laser pulse trains.

This study reveals that the periods, ablation areas and orientations of periodic surface structures (ripples) in fused silica can be adjusted by using designed femtosecond (fs) laser pulse trains to control transient localized electron dynamics and corresponding material properties. By increasing the pulse delays from 0 to 100 fs, the ripple periods are changed from ~550 nm to ~255 nm and the orientation is rotated by 90°. The nearwavelength/subwavelength ripple periods are close to the fundamental/second-harmonic wavelengths in fused silica respectively. The subsequent subpulse of the train significantly impacts free electron distributions generated by the previous subpulse(s), which might influence the formation mechanism of ripples and the surface morphology.

METTL3-Mediated ADAMTS9 Suppression Facilitates Angiogenesis and Carcinogenesis in Gastric Cancer.

The role of methyltransferase-like 3 (METTL3), which participates in catalyzing N-methyladenosine (m6A) RNA modification, in gastric cancer (GC) is unclear. Here, we found that METTL3 was overexpressed in human GC. Functionally, we verified that METTL3 promoted tumor cell proliferation and angiogenesis through a series of phenotypic experiments. Subsequently, ADAMTS9 was identified as the downstream effector of METTL3 in GC, which could be degraded by the YTHDF2-dependent pathway. Finally, the data suggested that METTL3 might facilitate GC progression through the ADAMTS9-mediated PI3K/AKT pathway. Our study unveiled the fundamental mechanisms of METTL3 in GC progression. The clinical value of METTL3 in GC deserves further exploration.

Comprehensive Analysis of VCAN Expression Profiles and Prognostic Values in HCC.

Background: Hepatocellular carcinoma (HCC) is the world's most common cause of cancer death. Therefore, more molecular mechanisms need to be clarified to meet the urgent need to develop new detection and treatment strategies. Methods: We used TCGAportal, Kaplan-Meier Plotter, the Cistrome DB Toolkit Database, MExpress, GEPIA2, and other databases to discuss the expression profiles, possible biological function, and potential prognostic value of versican (VCAN) in HCC. We conducted cell experiments such as Transwell migration and invasion assays, wound healing assay, and CCK8 experiment to explore the function of VCAN in HCC. Result: We selected three HCC transcriptome databases GSE124535, GSE136247, and GSE144269 and analyzed the overexpressed genes contained in them. The overlapping genes were found by the Venn map, and two interacting network modules were found by Mcode. Module 1 was mainly related to mitosis and cell cycle, and module 2 was mainly related to EMT, angiogenesis, glycolysis, and so on. We found that the seed gene in module 2 is VCAN. Data from TCGAportal showed that compared with normal tissues, the expression of VCAN was up-regulated in HCC tissues. The patients with high expression of VCAN had shorter distant recurrence-free survival and overall survival. Multiple possible VCAN interactions had also been identified. These results revealed that the level of VCAN was higher in the subtypes of HCC with higher malignant degree and was connected to the poor prognosis. In addition, the treatment of VCAN with DNA methyltransferase inhibitors and transcription factor inhibitors may improve the prognosis of patients with HCC. Conclusion: Our findings systematically elucidated the expression profile and different prognostic values of VCAN in HCC, which may provide new therapeutic targets and potential prognostic biomarkers for HCC patients.