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CRISPR-Cas systems and IS200/IS605 transposon-associated TnpBs have been utilized for the development of genome editing technologies. Using bioinformatics analysis and biochemical experiments, here we present a new family of RNA-guided DNA endonucleases. Our bioinformatics analysis initially identifies the stable co-occurrence of conserved RAGATH-18-derived RNAs (reRNAs) and their upstream IS607 TnpBs with an average length of 390 amino acids. IS607 TnpBs form programmable DNases through interaction with reRNAs. We discover the robust dsDNA interference activity of IS607 TnpB systems in bacteria and human cells. Further characterization of the Firmicutes bacteriaIS607 TnpB system (ISFba1 TnpB) reveals that its dsDNA cleavage activity is remarkably sensitive to single mismatches between the guide and target sequences in human cells. Our findings demonstrate that a length of 20 nt in the guide sequence of reRNA achieves the highest DNA cleavage activity for ISFba1 TnpB. A cryo-EM structure of the ISFba1 TnpB effector protein bound by its cognate RAGATH-18 motif-containing reRNA and a dsDNA target reveals the mechanisms underlying reRNA recognition by ISFba1 TnpB, reRNA-guided dsDNA targeting, and the sensitivity of the ISFba1 TnpB system to base mismatches between the guide and target DNA. Collectively, this study identifies the IS607 TnpB family of compact and specific RNA-guided DNases with great potential for application in gene editing.

Low dimensional organic inorganic metal halide materials have shown broadband emission and large Stokes shift, making them widely used in various fields and a promising candidate material. Here, the zero-dimensional lead-free bromide single crystals (C6H14N)3Bi2Br9·H2O (1) and (C6H14N)3Sb3Br12(2) were synthesized. They crystallized in the monoclinic crystal system with the space group of P21and P21/n, respectively. Through ultraviolet–visible–near-infrared (UV–vis–NIR) absorption analysis, the band gaps of (C6H14N)3Bi2Br9·H2O and (C6H14N)3Sb3Br12are found to be 2.75 and 2.83 eV, respectively. Upon photoexcitation, (C6H14N)3Bi2Br9·H2O exhibit broad-band red emission peaking at 640 nm with a large Stokes shift of 180 nm and a lifetime of 2.94 ns, and the emission spectrum of (C6H14N)3Sb3Br12are similar to those of (C6H14N)3Bi2Br9·H2O. This exclusive red emission is ascribed to the self-trapping exciton transition caused by lattice distortion, which is confirmed through both experiments and first-principles calculations. In addition, due to the polar space group structure and the large spin–orbit coupling (SOC) associated with the heavy elements of Bi and Br of crystal 1, an obvious Rashba effect was observed. The discovery of organic inorganic metal bromide material provides a critical foundation for uncovering the connection between 0D metal halide materials’ structures and properties.

Compared to conventional sensing, single-molecule sensing is proven to have extremely high sensitivity and has thus been successfully applied in various fields. However, many single-molecule sensing techniques rely on precise but bulky instruments and complicated operations, thus limiting their scope in on-site applications. To solve the problem, we have reported a series of works on detecting chemicals, proteins, and nucleic acids on-site at a single-molecule level. Though these works deal with diverse targets [avian influenza virus (AIV), hepatitis B virus (HBV), and mercury ion (Hg2+)] and use different sensing devices, they have the same on-site single-molecule sensing tactic, which employs fluorescence-enhanced probes, thus supporting a portable fluorescence microscope equipped with an industrial camera to collect and analyze the single-molecule signal. We have optimized the sensing operations to guarantee that all the procedures can be conveniently implemented on-site using field-portable devices. Therefore, based on the fluorescence-enhanced probes, portable optical platform, and optimized sensing operations, our tactic is proven to have advantages such as high sensitivity, high accuracy, simple procedures, and fast speed. In this article, we describe all the key components of our designed on-site single-molecule sensing tactic, including the sensing system, optical platform, and sensing operations. We compare this tactic with other reported single-molecule sensing approaches and discuss the pros and cons, as well as the prospect of our designed on-site single-molecule sensing. Our tactic will promote the high speed, convenience, and automation of single-molecule sensing and pave the way for the development of on-site single-molecule sensing approaches and instruments.

ObjectiveEarly gastric cardia adenocarcinoma (EGCA) is a highly heterogeneous cancer, and the understanding of its classification and malignant progression is limited. This study explored the cellular and molecular heterogeneity in EGCA using single-cell RNA sequencing (scRNA-seq).DesignscRNA-seq was conducted on 95 551 cells from endoscopic biopsies of low-grade intraepithelial neoplasia, well/moderately/poorly differentiated EGCA and their paired adjacent nonmalignant biopsy samples. Large-scale clinical samples and functional experiments were employed.ResultsIntegrative analysis of epithelial cells revealed that chief cells, parietal cells and enteroendocrine cells were rarely detected in the malignant epithelial subpopulation, whereas gland and pit mucous cells and AQP5+stem cells were predominant during malignant progression. Pseudotime and functional enrichment analyses showed that the WNT and NF-κB signalling pathways were activated during the transition. Cluster analysis of heterogeneous malignant cells revealed that NNMT-mediated nicotinamide metabolism was enriched in gastric mucin phenotype cell population, which was associated with tumour initiation and inflammation-induced angiogenesis. Furthermore, the expression level of NNMT was gradually increased during the malignant progression and associated with poor prognosis in cardia adenocarcinoma. Mechanistically, NNMT catalysed the conversion of nicotinamide to 1-methyl nicotinamide via depleting S-adenosyl methionine, which led to a reduction in H3K27 trimethylation (H3K27me3) and then activated the WNT signalling pathway to maintain the stemness of AQP5+stem cells during EGCA malignant progression.ConclusionOur study extends the understanding of the heterogeneity of EGCA and identifies a functional NNMT+/AQP5+population that may drive malignant progression in EGCA and could be used for early diagnosis and therapy.

We designed a portable Raspberry Pi-based spectrometer, which mainly consists of a white LED acting as the wide-spectrum source, a reflection grating for light dispersion, and a CMOS imaging chip aiming at spectral recording. All the optical elements and Raspberry Pi were integrated using 3-D printing structures with a size of 118 mm × 92 mm × 84 mm, and home-built software was also designed for spectral recording, calibration, analysis, and display implemented with a touch LCD. Additionally, the portable Raspberry Pi-based spectrometer was equipped with an internal battery, thus supporting on-site applications. Tested by a series of verifications and applications, the portable Raspberry Pi-based spectrometer could reach a spectral resolution of 0.065 nm per pixel within the visible band and provide spectral detection with high accuracy. Therefore, it can be used for on-site spectral testing in various fields.

Although the link of white matter to pathophysiology of schizophrenia is documented, loss of myelin is not detected in patients at the early stages of the disease, suggesting that pathological evolution of schizophrenia may occur before significant myelin loss. Disrupted-in-schizophrenia-1 (DISC1) protein is highly expressed in oligodendrocyte precursor cells (OPCs) and regulates their maturation. Recently, DISC1-Δ3, a major DISC1 variant that lacks exon 3, has been identified in schizophrenia patients, although its pathological significance remains unknown. In this study, we detected in schizophrenia patients a previously unidentified pathological phenotype of OPCs exhibiting excessive branching. We replicated this phenotype by generating a mouse strain expressing DISC1-Δ3 gene in OPCs. We further demonstrated that pathological OPCs, rather than myelin defects, drive the onset of schizophrenic phenotype by hyperactivating OPCs’ Wnt/β-catenin pathway, which consequently upregulates Wnt Inhibitory Factor 1 (Wif1), leading to the aberrant synaptic formation and neuronal activity. Suppressing Wif1 in OPCs rescues synaptic loss and behavioral disorders in DISC1-Δ3 mice. Our findings reveal the pathogenetic role of OPC-specific DISC1-Δ3 variant in the onset of schizophrenia and highlight the therapeutic potential of Wif1 as an alternative target for the treatment of this disease.

Objective:Hard-to-heal wounds are a global health challenge, and effective treatments are still lacking. Moist exposed burn ointment (MEBO) and maggots are traditional treatments for promoting wound healing. This study was a preliminary exploration of combined maggot therapy and MEBO in the treatment of hard-to-heal wounds.Method:A coexistence experiment was conducted to determine the survival rates of maggots in MEBO. The maggots were placed in two different existence conditions: one set in MEBO (MEBO group), and another set as the control group (no MEBO) to compare survival rates. Case reports describe the use of the combined application of MEBO and maggots in the treatment of patients with hard-to-heal wounds.Results:The coexistence experiment indicated that maggots in the MEBO group had a higher survival rate. From the therapeutic effect of the clinical cases (n=7), the combined application was safe and effective, with all the reported wounds eventually healing.Conclusion:Based on the findings of this study, we believe the combined application of MEBO and maggots is a promising way of promoting wound healing. Further studies and clinical trials are needed to elucidate the mechanism of the combined application in promoting wound healing and to more persuasively clarify the therapeutic effect.

AbstractSichuan Province is located at the transitional junction regions of the Qinghai-Tibet Plateau and the low-altitude plains. It also serves as the corridor of Sino-Tibetan-speaking population migration and expansion since neolithic expansion of Proto-Tibeto-Burman populations from Middle/Upper Yellow River during Majiayao period (3300–2000 BC). However, the population structure and the corresponding genetic diversity of forensic-related markers in this region remain unclear. Thus, we genotyped 30 insertion-deletion (InDel) markers in 444 samples from four ethnic groups (Han, Tibetan, Hui and Yi) from Sichuan Province using the Investigator®DIPplex kit to explore the characteristics of population genetics and forensic genetic focuses. All the loci were found to be in Hardy-Weinberg Equilibrium (HWE) after applying a Bonferroni correction and no pairwise loci showed prominent linkage disequilibrium. The combined matching probability (CMP) and the combined power of discrimination (CPD) are larger than 1.8089 × 10−11and 0.99999999995, respectively. Principal component analysis, multi-dimensional scaling plots and Neighbour-Joining tree among 65 worldwide populations indicated that Sichuan Hui and Han are genetically close to Hmong-Mien and Tai-Kadai-speaking populations, and Sichuan Tibetan and Yi bear a strong genetic affinity with Tibeto-Burman-speaking populations. The model-based genetic structure further supports the genetic affinity between the studied populations and linguistically close populations.Key PointsForensic parameters of 30 insertion-deletions (InDels) in 444 individuals from four populations are reported, which showed abundant genetic affinity and diversity among populations and high value in personal identification.Genetic similarities existed between the studied populations and ethnically, linguistically close populations.Sichuan Hui and Han are genetically close to Hmong-Mien and Tai-Kadai-speaking populations.Sichuan Tibetan and Yi bear a strong genetic affinity with Tibeto-Burman-speaking populations.

With the characteristics of low mutation rate, length variation and short amplicon size, insertion/deletion polymorphisms (InDels) have the advantages of both short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs). Herein, people of two ethnicities from Hainan Island were genotyped for the first time using the Investigator DIPplex kit. We investigated the forensic parameters of the 30 InDels and the phylogenetic relationships among different populations. The accumulated powers of discrimination and powers of exclusion were 0.999 999 999 9646 and 0.9897 in the Hainan Han population and 0.999 999 999 9292 and 0.9861 in the Hainan Li population, respectively. Additionally, population comparisons among geographically, ethnically and linguistically diverse populations viacluster heatmap, multidimensional scaling, principal component analysis, phylogenetic tree and STRUCTURE analyses demonstrated that the Hainan Han population had genetic similarities to the other Han, She and Tujia populations, while the Hainan Li population had close genetic relationships to the Zhuang and Miao groups; both populations had a high degree of genetic differentiation from most Turkic-speaking populations. Aforementioned results suggested that the 30 autosomal InDels are highly polymorphic and informative, which are suitable for human identification and population genetics. KEYPOINTSFour hundred and forty-five Chinese individuals from two ethnicities (Hainan Han and Hainan Li) were firstly analyzed by 30 autosomal InDels included in the Investigator DIPplex panel.Forensic parameters of the 30 InDels in the two populations showed high polymorphism and universality for human identification purposes.The Investigator DIPplex panel had a certain capacity of differentiating intercontinental populations and different language populations.

We construct a numerical model for multipulse laser drilling. It is found that the previous laser-pulse-induced temperature accumulation, thermal stress occurrence, and crater morphology change promote subsequent pulse laser drilling. Among them, previous laser-pulse-induced temperature accumulation contributes significantly to the drilled crater depth when the workpiece temperature is higher than its melting point just before the subsequent laser pulse irradiation, especially in a short pulse interval condition. The crater morphology change becomes the main contributor when the workpiece temperature decreases below the melting point, often in a long pulse interval condition. Besides, the previous occurrence of laser-pulse-induced thermal stress always has had little influence on the drilled crater. This work can be a theoretical reference, especially for multipulse laser manufacturing.

Transformer oil used in oil-filled electrical power transformers aims at insulating, stopping arcing and corona discharge, and dissipating transformer heat. Transformer running inevitably induces molecule decomposition, thus leading to gases released into transformer oil. The released gases not only reduce the transformer oil’s performance but also possibly induce transformer fault. To prevent catastrophic failure, approaches using, e.g., chromatography and spectroscopy, precisely measure dissolved gases to monitor transformer oil quality; however, many of these approaches still suffer from complicated operations, expensive costs, or slow speed. To solve these problems, we provide a new transformer oil quality evaluation method based on quantitative phase microscopy. Using our designed phase real-time microscopic camera (PhaseRMiC), under- and over-focus images of gas bubbles in transformer oil can be simultaneously captured during field of view scanning. Further, oil-to-gas-volume ratio can be computed after phase retrieval via solving the transport of intensity equation to evaluate transformer oil quality. Compared with traditionally and widely used approaches, this newly designed method can successfully distinguish transformer oil quality by only relying on rapid operations and low costs, thus delivering a new solution for transformer prognosis and diagnosis.

ABSTRACTIt is now clear that the intercellular transport on microtubules by dynein and kinesin-1 motors has an important role in the replication and spread of many viruses. Porcine epidemic diarrhea virus (PEDV) is an enveloped, single-stranded RNA virus of the Coronavirus family, which can infect swine of all ages and cause severe economic losses in the swine industry. Elucidating the molecular mechanisms of the intercellular transport of PEDV through microtubule, dynein and kinesin-1 will be crucial for understanding its pathogenesis. Here, we demonstrate that microtubule, dynein, and kinesin-1 are involved in PEDV infection and can influence PEDV fusion and accumulation in the perinuclear region but cannot affect PEDV attachment or internalization. Furthermore, we adopted a single-virus tracking technique to dynamically observe PEDV intracellular transport with five different types: unidirectional movement toward microtubule plus ends; unidirectional movement toward microtubule minus ends; bidirectional movement along the same microtubule; bidirectional movement along different microtubules and motionless state. Among these types, the functions of dynein and kinesin-1 in PEDV intercellular transport were further analyzed by single-virus tracking and found that dynein and kinesin-1 mainly transport PEDV to the minus and plus ends of the microtubules, respectively; meanwhile, they also can transport PEDV to the opposite ends of the microtubules different from their conventional transport directions and also coordinate the bidirectional movement of PEDV along the same or different microtubules through their cooperation. These results provided deep insights and references to understand the pathogenesis of PEDV as well as to develop vaccines and treatments.

We design a novel phase real-time microscope camera (PhaseRMiC) for live cell phase imaging. PhaseRMiC has a simple and cost-effective configuration only consisting of a beam splitter and a board-level camera with two CMOS imaging chips. Moreover, integrated with 3-D printed structures, PhaseRMiC has a compact size of 136×91×60 mm^3, comparable to many commercial microscope cameras, and can be directly connected to the microscope side port. Additionally, PhaseRMiC can be well adopted in real-time phase imaging proved with satisfied accuracy, good stability and large field of view. Considering its compact and cost-effective device design as well as real-time phase imaging capability, PhaseRMiC is a preferred solution for live cell imaging.

Due to limited depth-of-focus, classical 2D images inevitably lose details of targets out of depth-of-focus, while all-in-focus images break through the limit by fusing multi-focus images, thus being able to focus on targets in extended depth-of-view. However, conventional methods can hardly obtain dynamic all-in-focus imaging in both high spatial and temporal resolutions. To solve this problem, we design REPAID, meaning resolution-enhanced plenoptic all-in-focus imaging using deep neural networks. In REPAID, multi-focus images are first reconstructed from a single-shot plenoptic image, then upsampled using specially designed deep neural networks suitable for real scenes without ground truth to finally generate all-in-focus image in both high temporal and spatial resolutions. Experiments on both static and dynamic scenes have proved that REPAID can obtain high-quality all-in-focus imaging when using simple setups only; therefore, it is a promising tool in applications especially intended for imaging dynamic targets in large depth-of-view.

Studies have indicated that Vascular mimicry (VM) could contribute to the unfavorable prognosis of skin cutaneous melanoma (SKCM). Thus, the objective of this study was to identify therapeutic targets associated with VM in SKCM and develop a novel prognostic model. Gene expression data from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) were utilized to identify differentially expressed genes (DEGs). By intersecting these DEGs with VM genes, we acquired VM-related DEGs specific to SKCM, and then identified prognostic-related VM genes. A VM risk score system was established based on these prognosis-associated VM genes, and patients were then categorized into high- and low-score groups using the median score. Subsequently, differences in clinical characteristics, gene set enrichment analysis (GSEA), and other analyses were further presented between the 2 groups of patients. Finally, a novel prognostic model for SKCM was established using the VM score and clinical characteristics. 26 VM-related DEGs were identified in SKCM, among the identified DEGs associated with VM in SKCM, 5 genes were found to be prognostic-related. The VM risk score system, comprised of these genes, is an independent prognostic risk factor. There were significant differences between the 2 patient groups in terms of age, pathological stage, and T stage. VM risk scores are associated with epithelial biological processes, angiogenesis, regulation of the SKCM immune microenvironment, and sensitivity to targeted drugs. The novel prognostic model demonstrates excellent predictive ability. Our study identified VM-related prognostic markers and therapeutic targets for SKCM, providing novel insights for clinical diagnosis and treatment.

Hybrid improper ferroelectric Ca3Ti2O7and Ca3Ti19Ru0.1O7ceramics were successfully synthesized by conventional solid-state reaction method. Two strongest diffraction peaks located around 2?= 33° shifted towards the lower angle region with Ru substitution, reflecting structure variation. Grain growth and higher oxygen vacancy concentration after doping resulted in a reduction in the coercive field about 20 kV/cm. Optical bandgap estimated by UV-vis diffuse reflectance (DR) spectrum and X-ray photoelectron spectroscopy (XPS) valence band spectra showed a decreasing trend due to the existence of impurity energy level upon Ru doping, which was consistent with the results of first-principles calculations. The origin of the unexpected induced magnetic moments in Ru-dope Ca3Ti2O7is also discussed.

ABSTRACTEmerging evidence has suggested that long noncoding RNAs (lncRNAs) play an essential role in the tumorigenesis of multiple types of cancer including gastric cancer (GC). However, the potential biological roles and regulatory mechanisms of lncRNA in response to cisplatin, which may be involved in cisplatin resistance, have not been fully elucidated. In this study, we identified a novel lncRNA, cisplatin resistance-associated lncRNA (CRAL), that was downregulated in cisplatin-resistant GC cells, impaired cisplatin-induced DNA damage and cell apoptosis and thus contributed to cisplatin resistance in GC cells. Furthermore, the results indicated that CRAL mainly resided in the cytoplasm and could sponge endogenous miR-505 to upregulate cylindromatosis (CYLD) expression, which further suppressed AKT activation and led to an increase in the sensitivity of gastric cancer cells to cisplatin in vitroand in preclinical models. Moreover, a specific small molecule inhibitor of AKT activation, MK2206, effectively reversed the cisplatin resistance in GC caused by CRAL deficiency. In conclusion, we provide the first evidence that a novel lncRNA, CRAL, could function as a competing endogenous RNA (ceRNA) to reverse GC cisplatin resistance via the miR-505/CYLD/AKT axis, which suggests that CRAL could be considered a potential predictive biomarker and therapeutic target for cisplatin resistance in gastric cancer.

A novel optical multi-image encryption method based on focal length multiplexing and multimode phase retrieval is proposed in this study. During the encryption process of the proposed method, multiple secret images were encrypted into a single intensity-only image using focal length multiplexing and a coherent diffractive imaging-based encryption system. A specially designed iterative algorithm based on multimode phase retrieval is proposed for the accurate decryption of the original multiple secret images from the intensity pattern. The advantages of the proposed method include a compact optical setup and high decryption quality. The feasibility, security, and robustness of the proposed method were investigated by numerical simulations.

Low-dimensional lead-free organic–inorganic hybrid perovskites have gained increasing attention as having low toxicity, ease of processing, and good optoelectronic properties. Seeking for lead-free and narrow band gap organic–inorganic hybrid perovskites are of great importance for the development and application of photoelectric materials. Here, we reported a Sb-based organic–inorganic hybrid perovskite (MV)[SbI3Cl2], which has one-dimensional inorganic frameworks of the I-sharing double octahedra. (MV)[SbI3Cl2] shows a narrow direct band gap of 1.5 eV, and displays obvious photoresponse for the 532 nm light with rapid response speed of trise= 0.69 s, tdecay= 0.28 s. With an illumination power of 5 mW and a 50 V bias, the responsivities (R) and external quantum efficiency (EQE) for (MV)[SbI3Cl2] photodetector under 532 nm laser are 29.75 mA/W and 6.69% respectively. This Sb-based halide double perovskite material will provide an alternative material for photodetector devices.

With a fundamentally modified structural illumination algorithm, the recently proposed speckle-illuminated Fourier ptychography can be a promising superresolution imaging technique with a large field of view. However, its imaging performance, including image resolution and signal-to-noise ratio, has been discussed less, limiting its further applications. Thus, an in-depth study of this new imaging technique is highly required. In this paper, with theoretical analysis, numerical simulations, and experiments, the influence of both diffuser roughness in the experimental setup and numerical aperture size in iterative reconstruction on the imaging performance of speckle-illuminated Fourier ptychography was studied in detail, and the result explained why a rougher diffuser and larger reconstruction aperture can generate a higher-resolution image with more noise and showed how to get optimized diffuser roughness and reconstruction aperture size by considering the trade-off between imaging resolution and signal-to-noise ratio. This work may be a good reference for high-quality imaging using speckle-illuminated Fourier ptychography.

ObjectiveN6-methyladenosine (m6A) RNA methylation and its associated methyltransferase METTL3 are involved in tumour initiation and progression via the regulation of RNA function. This study explored the biological function and clinical significance of METTL3 in gastric cancer (GC).DesignThe prognostic value of METTL3 expression was evaluated using tissue microarray and immunohistochemical staining analyses in a human GC cohort. The biological role and mechanism of METTL3 in GC tumour growth and liver metastasis were determined in vitro and in vivo.ResultsThe level of m6A RNA was significantly increased in GC, and METTL3 was the main regulator involved in the abundant m6A RNA modification. METTL3 expression was significantly elevated in GC tissues and associated with poor prognosis. Multivariate Cox regression analysis revealed that METTL3 expression was an independent prognostic factor and effective predictor in human patients with GC. Moreover, METTL3 overexpression promoted GC proliferation and liver metastasis in vitro and in vivo. Mechanistically, P300-mediated H3K27 acetylation activation in the promoter of METTL3 induced METTL3 transcription, which stimulated m6A modification of HDGF mRNA, and the m6A reader IGF2BP3 then directly recognised and bound to the m6A site on HDGF mRNA and enhanced HDGF mRNA stability. Secreted HDGF promoted tumour angiogenesis, while nuclear HDGF activated GLUT4 and ENO2 expression, followed by an increase in glycolysis in GC cells, which was correlated with subsequent tumour growth and liver metastasis.ConclusionsElevated METTL3 expression promotes tumour angiogenesis and glycolysis in GC, indicating that METTL3 expression is a potential prognostic biomarker and therapeutic target for human GC.

A single shot large-capacity optical multiple-image encryption method based on wavelength multiplexing and position multiplexing is proposed. In the encryption process of the proposed method, multiple plane waves of different wavelengths are adopted to illuminate secret images that are placed at different positions along the optical axis. All the secret images are encoded into a single grayscale intensity-only image that is recorded by a monochromic camera by applying a diffractive-imaging based double random phase encoding (DRPE) system. In the decryption process, high accuracy images are decrypted without crosstalk from the intensity image through a multimode phase retrieval algorithm and a two-step iterative shrinkage/thresholding (TwIST) algorithm. The feasibility of the proposed method is demonstrated by numerical simulations.

Messenger ribonucleic acid (mRNA) plays an important role in various cellular processes. however, traditional techniques cannot realize mRNA detections in live cells as they rely on mRNA purification or cell fixation. To achieve real-time and quantitative mRNA detections at a single live cell level, a single-strand stem-loop-structured ratiometric molecular beacon (RMB) composed of the phosphorothioate-modified loop domain on the 2′-O-methyl RNA backbone with a reporter dye, quencher, and reference dye is proposed to detect the Hsp27 mRNA as a modeled endogenous mRNA. When the RMB hybridizes with the target, the stem-loop structure opens, causing separation of the reporter dye and the quencher and restores the reporter fluorescent signals; therefore, the Hsp27 mRNA can be quantitatively detected according to the ratio of the reporter fluorescent signal to the reference fluorescent signal. Both the phosphorothioate and 2′-O-methyl RNA modifications obviously reduce the nonspecific opening, and the additional reference dye ensures the detection precision using co-localization analysis. Not only does this remove the false-positive signal caused by the nuclease degradation-generated RMB fragment, but it also corrects variations caused by direct measurement of reporter fluorescence intensities at a single cell level owing to inhomogeneity in probe delivery. The designed RMB could detect the Hsp27 mRNA with high signal-to-noise ratio and sensitivity as well as excellent specificity and antidegradation capability proved in vitro and in live cells. Furthermore, it was successfully adopted in subcellular localization, quantitative copy number measurements, and even real-time monitoring of Hsp27 mRNA in live cells, demonstrating that the proposed RMB can be a potential quantitative endogenous mRNA detection tool, especially at a single live cell level.

In traditional coded aperture imaging (CAI), the main problem with the deconvolution method is that the noise may dominate the decoding process. Focusing on this problem, a synthetic CAI (s-CAI) method is proposed with a potential to improve the signal-to-noise ratio (SNR) and spatial resolution of CAI. A series of raw coded images are recorded while the pinhole array is varied to different structures, and an iterative decoding algorithm is developed to reconstruct the object image by synthetically using all of these pinhole array structures and corresponding decoded images. Because the proposed iterative decoding algorithm has prominent advantage in noise immunization, and more spatial light components are acquired in comparison with common single-exposure CAI, both the image resolution and SNR are remarkably enhanced.

Dual view transport of intensity phase microscopy is adopted to quantitatively study the regulation of adenosine triphosphate (ATP) on cellular mechanics. It extracts cell phases in real time from simultaneously captured under- and over-focus images. By computing the root-mean-square phase and correlation time, it is found that the cellular fluctuation amplitude and speed increased with ATP compared to those with ATP depletion. Besides, when adenylyl-imidodiphosphate (AMP-PNP) was introduced, it competed with ATP to bind to the ATP binding site, and the cellular fluctuation amplitude and speed decreased. The results prove that ATP is a factor in the regulation of cellular mechanics. To our best knowledge, it is the first time that the dual view transport of intensity phase microscopy was used for live cell phase imaging and analysis. Our work not only provides direct measurements on cellular fluctuations to study ATP regulation on cellular mechanics, but it also proves that our proposed dual view transport of intensity phase microscopy can be well used, especially in quantitative phase imaging of live cells in biological and medical applications.

The wavefront-sensing-based autofocus method can precisely determine the focal plane only with few captured images; however, the required phase retrieval, numerical wavefront propagation, and in-focus determination are often time consuming, inevitably limiting its high-speed applications. To accelerate its processing speed, the pixel-reduced wavefront-sensing-based autofocus (PRWSA) method is proposed: with field of interest selection as pixel reduction in the spatial domain and image compression as pixel reduction in the frequency domain, the wavefront with fewer pixels can be used for autofocusing, significantly decreasing the processing time. With simulations, pixel reduction criteria in both the spatial and frequency domains are first determined and tested; next certificated by experiments, the PRWSA method is proved to be well implemented for different specimens. Considering it can precisely locate the focal plane with simple setup, and accelerate the processing speed, this PRWSA method can be a potential tool for high-speed autofocusing.

Despite the structural conservation of PTEN with dual-specificity phosphatases, there have been no reports regarding the regulatory mechanisms that underlie this potential dual-phosphatase activity. Here, we report that K27-linked polyubiquitination of PTEN at lysines 66 and 80 switches its phosphoinositide/protein tyrosine phosphatase activity to protein serine/threonine phosphatase activity. Mechanistically, high glucose, TGF-β, CTGF, SHH, and IL-6 induce the expression of a long non-coding RNA, GAEA(Glucose Aroused for EMT Activation), which associates with an RNA-binding E3 ligase, MEX3C, and enhances its enzymatic activity, leading to the K27-linked polyubiquitination of PTEN. The MEX3C-catalyzed PTENK27-polyUbactivates its protein serine/threonine phosphatase activity and inhibits its phosphatidylinositol/protein tyrosine phosphatase activity. With this altered enzymatic activity, PTENK27-polyUbdephosphorylates the phosphoserine/threonine residues of TWIST1, SNAI1, and YAP1, leading to accumulation of these master regulators of EMT. Animals with genetic inhibition of PTENK27-polyUb, by a single nucleotide mutation generated using CRISPR/Cas9 (PtenK80R/K80R), exhibit inhibition of EMT markers during mammary gland morphogenesis in pregnancy/lactation and during cutaneous wound healing processes. Our findings illustrate an unexpected paradigm in which the lncRNA-dependent switch in PTEN protein serine/threonine phosphatase activity is important for physiological homeostasis and disease development.

Our previous work found cancer-testis (CT) genes as a new source of epi-driver candidates of cancer. LIN28Bwas a CT gene, but the “driver” ability and the activation mechanism in lung adenocarcinoma (LUAD) remain unclear. We observed that LIN28Bexpression was restricted in testis. It was re-activated in LUAD patients without known genomic alterations in oncogenes and was related to poorer survival. In vitro and In vivo experiments confirmed that the activation of LIN28Bcould promote the proliferation and metastasis of LUAD cells and can influence cell cycle, DNA damage repair, and genome instability. In addition to the known let-7-LIN28B regulation loop, our results further revealed a let-7-independent Cis-regulator of LIN28B: LIN28B-AS1. LIN28B-AS1is a CT long non-coding RNA (CT-lncRNA). It altered the messenger RNA stability of LIN28Bby directly interacting with another CT protein IGF2BP1 but not with LIN28B and constituted a novel regulation network. In sum, we identify that LIN28Bis an “epi-driver” of LUAD and clarify a new lncRNA-activated mechanism of LIN28B, which provide new candidate targets for precise anticancer therapy in the future.

Following the observation of candidate chiral doublet bands in 78,80Br, triaxial deformations with corresponding configurations in odd-odd bromine isotopes have been investigated using adiabatic and configuration-fixed constrained triaxial relativistic-mean-field calculations with an aim of finding the boundary of chirality in the chain of Br isotopes. Several minima with triaxial deformation and the proper particle-hole configuration were obtained in 74,76,78,80,82Br, where the chiral doublet bands have the possibility of occurrence. Furthermore, the possible existence of multiple chiral doublet (MχD) bands is demonstrated in 74,76,78,80,82Br. Experiments to explore the chirality and MχD properties of Br isotopes are conducted to verify the predictions.

Classical differential interference contrast (DIC) microscopy is an excellent tool for rendering high-contrast images to observe transparent specimen. Though the monochromatic shadow-cast image qualitatively reflects the specimen configuration, its phase can hardly be extracted, inevitably limiting its application in quantitative measurements. In order to extend its application, we propose a quantitative DIC (qDIC) microscopy technique, which can extract the specimen phase from phase-shifting beam-shearing interference images combining principal component analysis (PCA) and phase integration methods. Proved by both simulations and experiments, the PCA-based qDIC microscopy can rapidly and accurately retrieve the quantitative specimen phase. The new proposed technique shares with conventional DIC the advantage of high-contrast imaging, especially for transparent label-free specimens, but transforms DIC microscopy into a quantitative phase microscopy technique. We envision PCA-based qDIC microscopy as a future quantitative biological imaging modality.

RNA helicase A (RHA) as a member of the DExH/D-box subgroup of helicase superfamily II is involved in virtually all aspects of RNA metabolism. It exhibits robust RNA helicase activity in vitro. However, little is known about the molecular and physical determinants for RHA substrate recognition and RHA translocation along the nucleic acids. Here, our nondenaturing polyacrylamide gel electrophoresis (PAGE)-based unwinding assays of chemical and structural modified substrates indicate that RHA translocates efficiently along the 3′ overhang of RNA, but not DNA, with a requirement of covalent continuity. Ribose-phosphate backbone lesions on both strands of the nucleic acids, especially on the 3′ overhang of the loading strand, affect RHA unwinding significantly. Furthermore, RHA requires RNA on the 3′ overhang which directly or indirectly connects with the duplex region to mediate productive unwinding. Collectively, these findings propose a basic mechanism of the substrate determinants for RHA backbone tracking during duplex unwinding.

Via the transport of intensity phase microscopy, quantitative phase can be retrieved directly from captured multi-focal intensities. The accuracy of the retrieved phases depends highly on the quality of the recorded images; therefore, the exposure time should be carefully chosen for high-quality intensity captures. However, it is difficult to record well-exposure intensities to maintain rather a high signal to noise ratio and to avoid over-exposure due to the complex samples. In order to simplify the exposure determination, here the adaptive dual-exposure fusion-based transport of intensity phase microscopy is proposed: with captured short- and long-exposure images, the well-exposure multi-focal images can be numerically reconstructed, and then high-accurate phase can be computed from these reconstructed intensities. With both simulations and experiments provided in this paper, it is proved that the adaptive dual-exposure fusion-based transport of intensity phase microscopy not only provides numerically reconstructed well-exposure image with simple operation and fast speed but also extracts highly accurate retrieved phase. Moreover, the exposure time selection scope of the proposed method is much wider than that based on single exposure, and even though there is an over-exposure region in the long-exposure image, a well-exposure image can still be reconstructed with high precision. Considering its advantages of high accuracy, fast speed, simple operation, and wide application scope, the proposed technique can be adopted as quantitative phase microscopy for high-quality observations and measurements.

Because of the limited dynamic range of common detectors, high-frequency components weaker than the threshold of the detector used are lost in digital holography (DH) even with the aid of coherent diffraction imaging (CDI)-based iterative computation, and this makes DH less preferable for many interesting applications. To overcome this problem, the partially saturated diffraction pattern was adopted to enhance the resolution of DH iteratively. Since more high-frequency components were retrieved in comparison with our former CDI-based digital holographic method, the resolution of DH was improved further. The feasibility of the proposed method is demonstrated both numerically and experimentally.

Although EZH2 enzymatic inhibitors have shown antitumor effects in EZH2-mutated lymphoma and ARID1A-mutated ovarian cancer, many cancers do not respond because EZH2 can promote cancer independently of its histone methyltransferase activity. Here we identify ZRANB1 as the EZH2 deubiquitinase. ZRANB1 binds, deubiquitinates, and stabilizes EZH2. Depletion of ZRANB1 in breast cancer cells results in EZH2 destabilization and growth inhibition. Systemic delivery of ZRANB1 small interfering RNA (siRNA) leads to marked antitumor and antimetastatic effects in preclinical models of triple-negative breast cancer (TNBC). Intriguingly, a small-molecule inhibitor of ZRANB1 destabilizes EZH2 and inhibits the viability of TNBC cells. In patients with breast cancer, ZRANB1 levels correlate with EZH2 levels and poor survival. These findings suggest the therapeutic potential for targeting the EZH2 deubiquitinase ZRANB1.

ABSTRACTSudden cardiac death (SCD) is defined as an unexpected natural death without any obvious non-cardiac causes that occurs within 1 h with witnessed symptom onset or within 24 h without witnessed symptom onset. Genetic studies conducted during the past decade have markedly illuminated the genetic basis of the cardiac disorders associated with SCD. Macrophage migration inhibitory factor (MIF) is an upstream immunoregulatory cytokine associated with the pathogenesis of many inflammatory diseases including atherosclerosis and myocardial infarction. Previous studies have reported that the functional −794(CATT)5–8polymorphism in MIF is unrelated to sudden infant death syndrome susceptibility. However, there are no reports concerning the association between the polymorphism and adult SCD susceptibility. In the current study, we investigated the association between the −794(CATT)5–8polymorphism and adult SCD susceptibility using 79 adult SCD cases and 313 healthy controls. All samples were analysed using a conventional polymerase chain reaction (PCR) technique. We found that CATT6and 5–6 were the most common allele and genotype in both groups, respectively, while no significant association was found between the −794(CATT)5–8polymorphism and SCD susceptibility. We also summarized the allele frequencies of −794(CATT)5–8in cohorts of healthy people from different countries and found that the allele frequency distributions of the polymorphism in Chinese populations were quite different from that of American and European populations (P= 0.005, P= 0.0001, respectively), but similar to Japanese populations (P= 0.827). In conclusion, this study indicates that the −794(CATT)5–8polymorphism may not be associated with adult SCD susceptibility in Chinese populations. Different allele frequency distributions of the polymorphism in multiple populations may provide a useful reference for further genetic association studies.

To reduce the long data acquisition time of the common mechanical scanning based Ptychographic Iterative Engine (PIE) technique, the digital micro-mirror device (DMD) is used to form the fast scanning illumination on the sample. Since the transverse mechanical scanning in the common PIE is replaced by the on/off switching of the micro-mirrors, the data acquisition time can be reduced from more than 15 minutes to less than 20 seconds for recording 12 × 10 diffraction patterns to cover the same field of 147.08 mm^2. Furthermore, since the precision of DMD fabricated with the optical lithography is always higher than 10 nm (1 μm for the mechanical translation stage), the time consuming position-error-correction procedure is not required in the iterative reconstruction. These two improvements fundamentally speed up both the data acquisition and the reconstruction procedures in PIE, and relax its requirements on the stability of the imaging system, therefore remarkably improve its applicability for many practices. It is demonstrated experimentally with both USAF resolution target and biological sample that, the spatial resolution of 5.52 μm and the field of view of 147.08 mm^2 can be reached with the DMD based PIE method. In a word, by using the DMD to replace the translation stage, we can effectively overcome the main shortcomings of common PIE related to the mechanical scanning, while keeping its advantages on both the high resolution and large field of view.

Bone metastases remain a serious health concern because of limited therapeutic options. Here, we report that crosstalk between ROR1–HER3 and the Hippo–YAP pathway promotes breast cancer bone metastasis in a long noncoding RNA-dependent fashion. Mechanistically, the orphan receptor tyrosine kinase ROR1 phosphorylates HER3 at a previously unidentified site Tyr1307, following neuregulin stimulation, independently of other ErbB family members. p-HER3 Tyr1307 recruits the LLGL2–MAYA–NSUN6 RNA–protein complex to methylate Hippo/MST1 at Lys59. This methylation leads to MST1 inactivation and activation of YAP target genes in tumour cells, which elicits osteoclast differentiation and bone metastasis. Furthermore, increased ROR1, p-HER3 Tyr1307 and MAYA levels correlate with tumour metastasis and unfavourable outcomes. Our data provide insights into the mechanistic regulation and linkage of the ROR1–HER3 and Hippo–YAP pathway in a cancer-specific context, and also imply valuable therapeutic targets for bone metastasis and possible therapy-resistant tumours.

Phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3or PIP3) mediates signalling pathways as a second messenger in response to extracellular signals. Although primordial functions of phospholipids and RNAs have been hypothesized in the ‘RNA world’, physiological RNA–phospholipid interactions and their involvement in essential cellular processes have remained a mystery. We explicate the contribution of lipid-binding long non-coding RNAs (lncRNAs) in cancer cells. Among them, long intergenic non-coding RNA for kinase activation (LINK-A) directly interacts with the AKT pleckstrin homology domain and PIP3at the single-nucleotide level, facilitating AKT–PIP3interaction and consequent enzymatic activation. LINK-A-dependent AKT hyperactivation leads to tumorigenesis and resistance to AKT inhibitors. Genomic deletions of the LINK-A PIP3-binding motif dramatically sensitized breast cancer cells to AKT inhibitors. Furthermore, meta-analysis showed the correlation between LINK-A expression and incidence of a single nucleotide polymorphism (rs12095274: A > G), AKT phosphorylation status, and poor outcomes for breast and lung cancer patients. PIP3-binding lncRNA modulates AKT activation with broad clinical implications.