Hierarchical DNA branch assembly-encoded fluorescent nanoladders for single-cell transcripts imaging.

Spatial visualization of single-cell transcripts is limited by signal specificity and multiplexing. Here, we report hierarchical DNA branch assembly-encoded fluorescent nanoladders, which achieve denoised and highly multiplexed signal amplification for single-molecule transcript imaging. This method first offers independent RNA-primed rolling circle amplification without nonspecific amplification based on circular DNAzyme. It then executes programmable DNA branch assembly on these amplicons to encode virtual signals for visualizing numbers of targets by FISH. In theory, more virtual signals can be encoded via the increase of detection spectral channels and repeats of the same sequences on barcode. Our method almost eliminates nonspecific amplification in fixed cells (reducing nonspecific spots of single cells from 16 to nearly zero), and achieves simultaneous quantitation of nine transcripts by using only two detection spectral channels. We demonstrate accurate RNA profiling in different cancer cells, and reveal diverse localization patterns for spatial regulation of transcripts.

Early discharge hospital at home as alternative to routine hospital care for older people: a systematic review and meta-analysis.

BACKGROUND: The global population of adults aged 60 and above surpassed 1 billion in 2020, constituting 13.5% of the global populace. Projections indicate a rise to 2.1 billion by 2050. While Hospital-at-Home (HaH) programs have emerged as a promising alternative to traditional routine hospital care, showing initial benefits in metrics such as lower mortality rates, reduced readmission rates, shorter treatment durations, and improved mental and functional status among older individuals, the robustness and magnitude of these effects relative to conventional hospital settings call for further validation through a comprehensive meta-analysis. METHODS: A comprehensive literature search was executed during April-June 2023, across PubMed, MEDLINE, Embase, Web of Science, and Cumulative Index of Nursing and Allied Health Literature (CINAHL) to include both RCT and non-RCT HaH studies. Statistical analyses were conducted using Review Manager (version 5.4), with Forest plots and I2 statistics employed to detect inter-study heterogeneity. For I2 > 50%, indicative of substantial heterogeneity among the included studies, we employed the random-effects model to account for the variability. For I2 ≤ 50%, we used the fixed effects model. Subgroup analyses were conducted in patients with different health conditions, including cancer, acute medical conditions, chronic medical conditions, orthopedic issues, and medically complex conditions. RESULTS: Fifteen trials were included in this systematic review, including 7 RCTs and 8 non-RCTs. Outcome measures include mortality, readmission rates, treatment duration, functional status (measured by the Barthel index), and mental status (measured by MMSE). Results suggest that early discharge HaH is linked to decreased mortality, albeit supported by low-certainty evidence across 13 studies. It also shortens the length of treatment, corroborated by seven trials. However, its impact on readmission rates and mental status remains inconclusive, supported by nine and two trials respectively. Functional status, gauged by the Barthel index, indicated potential decline with early discharge HaH, according to four trials. Subgroup analyses reveal similar trends. CONCLUSIONS: While early discharge HaH shows promise in specific metrics like mortality and treatment duration, its utility is ambiguous in the contexts of readmission, mental status, and functional status, necessitating cautious interpretation of findings.

Single femtosecond pulse writing of a bifocal lens.

In this Letter, a method for the fabrication of bifocal lenses is presented by combining surface ablation and bulk modification in a single laser exposure followed by the wet etching processing step. The intensity of a single femtosecond laser pulse was modulated axially into two foci with a designed computer-generated hologram (CGH). Such pulse simultaneously induced an ablation region on the surface and a modified volume inside the fused silica. After etching in hydrofluoric acid (HF), the two exposed regions evolved into a bifocal lens. The area ratio (diameter) of the two lenses can be flexibly adjusted via control of the pulse energy distribution through the CGH. Besides, bifocal lenses with a center offset as well as convex lenses were obtained by a replication technique. This method simplifies the fabrication of micro-optical elements and opens a highly efficient and simple pathway for complex optical surfaces and integrated imaging systems.

Fresnel zone plate sculptured out of diamond by femtosecond laser for harsh environments.

With the rapid development of micro-optical applications, there is an increasing demand for micro-optical elements that can be made with minimal processing steps. Current research focuses on practical functionalities of optical performance, lightweight, miniaturization, and easy integration. As an important planar diffractive optical element, the Fresnel zone plate (FZP) provides a compact solution for focusing and imaging. However, the fabrication of FZPs with high quality out of hard and brittle materials remains challenging. Here, we report on the fabrication of diamond FZP by femtosecond laser direct writing. FZPs with the same outer diameter and different focal lengths of 250-1000 µm were made via ablation. The fabricated FZPs possess well-defined geometry and excellent focusing and imaging ability in the visible spectral range. Arrays of FZPs with different focal lengths were made for potential applications in imaging, sensing, and integrated optical systems.

Variable focus convex microlens array on K9 glass substrate based on femtosecond laser processing and hot embossing lithography.

We propose a high-precision method for the fabrication of variable focus convex microlens arrays on K9 glass substrate by combining femtosecond laser direct writing and hot embossing lithography. A sapphire master mold with a blind cylindrical hole array was prepared first by femtosecond laser ablation. The profile control of microlenses dependent on the temperature and the diameter of the blind hole in the sapphire mold was investigated. The curvature radius of the microlens decreased with temperature and increased with diameter. Uniform convex microlens arrays were fabricated with good imaging performance. Further, variable focus convex microlens arrays were fabricated by changing the diameter of the blind hole in sapphire, which produced the image at variable z planes. This method provides a highly precise fabrication of convex microlens arrays and is well suited for batch production of micro-optical elements.

Bioorthogonal Chemical Signature Enabling Amplified Visualization of Cellular Oxidative Thymines.

Cellular oxidative thymines, 5-hydroxymethyluracil (5hmU) and 5-formyluracil (5fU), are found in the genomes of a diverse range of organisms, the distribution of which profoundly influence biological processes and living systems. However, the distribution of cellular oxidative thymines has not been explored because of lacking both specific bioorthogonal labeling and sensitivity methods for single-cell analysis. Herein, we report a bioorthogonal chemical signature enabling amplified visualization of cellular oxidative thymines in single cells. The synthesized ATP-γ-alkyne, an ATP analogue with bioorthogonal tag modified on γ-phosphate can be specifically linked to cellular 5hmU by chemoenzymatic labeling. DNA with 5-alkynephosphomethyluracil were then clicked with azide (N3)-modified 5hmU-primer. Identification of 5fU is based on selective reduction from 5fU to 5hmU, subsequent chemoenzymatic labeling of the newly generated 5hmU, and cross-linking with N3-modified 5fU-primer via click chemistry. Then, all of the 5hmU and 5fU sites are encoded with respective circularized barcodes. These barcodes are simultaneously amplified for multiplexed single-molecule imaging. The above two kinds of barcodes can be simultaneously amplified for differentiated visualization of 5hmU and 5fU in single cells. We find these two kinds of cellular oxidative thymines are spatially organized in a cell-type-dependent style with cell-to-cell heterogeneity. We also investigate their multilevel subcellular information and explore their dynamic changes during cell cycles. Further, using DNA sequencing instead of fluorescence imaging, our proposed bioorthogonal chemical signature holds great potential to offer the sequence information of these oxidative thymines in cells and may provide a reliable chemical biology approach for studying the whole-genome oxidative thymines profiles and insights into their functional role and dynamics in biology.

Click-encoded rolling FISH for visualizing single-cell RNA polyadenylation and structures.

Spatially resolved visualization of RNA processing and structures is important for better studying single-cell RNA function and landscape. However, currently available RNA imaging methods are limited to sequence analysis, and not capable of identifying RNA processing events and structures. Here, we developed click-encoded rolling FISH (ClickerFISH) for visualizing RNA polyadenylation and structures in single cells. In ClickerFISH, RNA 3' polyadenylation tails, single-stranded and duplex regions are chemically labeled with different clickable DNA barcodes. These barcodes then initiate DNA rolling amplification, generating repetitive templates for FISH to image their subcellular distributions. Combined with single-molecule FISH, the proposed strategy can also obtain quantitative information of RNA of interest. Finally, we found that RNA poly(A) tailing and higher-order structures are spatially organized in a cell type-specific style with cell-to-cell heterogeneity. We also explored their spatiotemporal patterns during cell cycle stages, and revealed the highly dynamic organization especially in S phase. This method will help clarify the spatiotemporal architecture of RNA polyadenylation and structures.

Pairwise Proximity-Differentiated Visualization of Single-Cell DNA Epigenetic Marks.

Spatial positioning and proximity of relevant biomolecules such as DNA epigenetic marks are fundamental to a deeper understanding of life. However, it remains poorly explored and technically challenging. Here we report the pairwise proximity-differentiated visualization of single-cell 5-formylcytosine (5fC) and 5-hydroxymethylcytosine (5hmC). These two marks on chromatin in fixed cells are successively labeled and crosslinked with their DNA primer probes via click chemistry. Based on a pairwise proximity-differentiated mechanism, proximal 5fC/5hmC sites and residual 5fC or 5hmC sites are encoded with respective circularized barcodes. These barcodes are simultaneously amplified for multiplexed single-molecule imaging. We thus demonstrate the differentiated visualization of 5fC or 5hmC spatial positioning and their pairwise proximity in single cells. Such multi-level subcellular information may provide insights into regulation functions and mechanisms of chromatin modifications, and the spatial proximity can expose the potential crosstalk or interaction between their reader proteins.

RNA-Primed Amplification for Noise-Suppressed Visualization of Single-Cell Splice Variants.

Splice variants visualization is pivotal for a deeper understanding of cell growth and development. However, it remains technically challenging due to short lengths, similar sequences, and low abundance. The existing single-cell imaging strategies suffer from nonspecific amplification that causes considerable noise during visualization of the splice variants. Herein we develop a new RNA-primed amplification strategy for noise-suppressed visualization of single-cell splice variants. Block probes were designed to specifically identify the conjugated region of exons in mRNA, which was then digested by endonuclease and provided a hydroxyl group at the 3' terminal. The RNA target can act as primer to trigger rolling circle amplification, achieving visualization of splice variants with noise suppressed to nearly zero. We further explored the expression and distribution of BRCA1 splice variants in three breast cell lines, revealing cell-type specific mapping of this cancer suppressor gene.

Visualizing Newly Synthesized RNA by Bioorthogonal Labeling-Primed DNA Amplification.

Monitoring RNA synthesis and spatial distribution can help to understand its role in physiology and diseases. However, visualizing newly synthesized RNA in single cells remains a great challenge. Here, we developed a bioorthogonal labeling-primed DNA amplification strategy to visualize newly synthesized RNA in single cells. The new bioorthogonal N6-allyladenosine nucleoside was prepared to metabolically label cellular newly synthesized RNAs. These allyl-functionalized RNAs then reacted with tetrazine-modified primers. These primers could initiate rolling circle amplification, producing tandem periodic long single DNA strands to capture hundreds of fluorescence probes for signal amplification. Using this method, we explored the subcellular distributions of newly synthesized RNAs. And we found that newly synthesized RNAs are spatially organized in a cell type-specific style with cell-to-cell heterogeneity.

Intracellular Entropy-Driven Multi-Bit DNA Computing for Tumor Progression Discrimination.

Tumor progressions such as metastasis are complicated events that involve abnormal expression of different miRNAs and enzymes. Monitoring these biomolecules in live cells with computational DNA nanotechnology may enable discrimination of tumor progression via digital outputs. Herein, we report intracellular entropy-driven multivalent DNA circuits to implement multi-bit computing for simultaneous analysis of intracellular telomerase and microRNAs including miR-21 and miR-31. These three biomolecules can trigger respective DNA strand displacement recycling reactions for signal amplification. They are visualized by fluorescence imaging, and their signal outputs are encoded as multi-bit binary codes for different cell types. The results can discriminate non-tumorigenic, malignant and metastatic breast cells as well as respective tumors. This DNA computing circuit is further performed in a microfluidic chip to differentiate rare co-cultured cells, which holds a potential for the analysis of clinical samples.

All-in-One Synchronized DNA Nanodevices Facilitating Multiplexed Cell Imaging.

Multifunctional DNA nanodevices perform ever more tasks with applications ranging from in vitro biomarker detection to in situ cell imaging. However, most developed ones consist of a series of split building blocks, which suffer from asynchronous behaviors in complicated cellular microenvironments (endocytosis pathway, diffusion-limited cytoplasm, etc.), causing the loss of stoichiometric information and additional postassembly processes. Herein, we constructed all-in-one DNA nanodevices to achieve synchronous multiplexed imaging. All DNA components, including two sets of probe modules (each containing target-specific walkers, i.e., hairpin tracks with chemically damaged bases), are modified on individual gold nanoparticles. This design not only enables their integrated internalization into cells, circumventing inhomogeneous distribution of different building blocks and increasing the local concentrations of the interacting modules, but also avoids the impact of stochastic diffusion in viscous cytoplasm. A couple of intracellular enzymes in situ actuate the synchronized motion of the modules, all on-particle, after specific recognition of intracellular targets (such as RNAs and proteins), thus facilitating synchronized, multiplexed cell imaging. Finally, the proposed all-in-one nanodevices were successfully applied to monitor intracellular microRNA-21 and telomerase expression levels. The flexible design can be extended to detect other cytoplasmic molecules and monitor related pathways by simply changing the sequences.

Control of diameter and numerical aperture of microlens by a single ultra-short laser pulse.

We demonstrate a versatile method for fast and flexible fabrication of either one or an array of microlenses. Multi-foci axial intensity distribution generated by a phase pattern displayed on a spatial light modulator irradiates silica, causing ablation and its internal modification. The following wet etching step defines the diameter r, while the radius of curvature R (hence, the focal length f) is maintained the same. As a result, the numerical aperture NA=r/f changes from 0.2 to 0.4 for the same pulse energy (but different number of multi-foci) during ablation. An isotropic wet etching of silica becomes highly anisotropic for the initial stages of etching following the irradiated pattern. Subsequent evolution of the shape is governed by an isotropic silica etch and forms a spherical lens. This method can be extended to other materials and geometries of micro-optical elements.

MnO2-Nanosheet-Powered Protective Janus DNA Nanomachines Supporting Robust RNA Imaging.

Both biomarker and probe degradations cause serious false assay results. However, protecting a target or a target and a probe simultaneously has rarely been explored. Herein, MnO2-nanosheet-powered target- and probe-protective Janus DNA nanomachines are reported. It is formed in living cells by an RNA-responsive assembly of two chemically modified DNA partzymes and one substrate probe. MnO2 nanosheets are used to facilitate the cellular uptake of DNA reagents and generate Mn2+, which are indispensable DNAzyme cofactors for efficient catalytic cleavage. We find that DNA partzymes with modified sugar moieties (e.g., LNA or ones with 2'-O-methylation) protect the RNA of RNA-DNA hybrids from RNase H degradation. LNA blocks RNase H recruitment on the hybrid best because of its 2'-O, 4'-C methylene bridge structure. In contrast, modifications at DNA phosphate moieties fail to protect the RNA. RNA protection can exclude target-degradation-induced false negative results. In addition, the phosphorothioate-modified substrate probe is known to resist nuclease degradation, which minimizes false positive interference. Compared to canonical DNA systems without chemical modifications, the protective Janus nanomachine avoids false results and supports robust RNA imaging.

Single-pulse writing of a concave microlens array.

This work developed a method of femtosecond laser (fs-laser) parallel processing assisted by wet etching to fabricate 3D micro-optical components. A 2D fs-laser spot array with designed spatial distribution was generated by a spatial light modulator. A single-pulse exposure of the entire array was used for parallel processing. By subsequent wet etching, a close-packed hexagonal arrangement, 3D concave microlens array on a curved surface with a radius of approximately 120 µm was fabricated, each unit lens of which has designable spatial distribution. Characterization of imaging was carried out by a microscope and showed a unique imaging property in multi-planes. This method provides a parallel and efficient technique to fabricate 3D micro-optical devices for applications in optofluidics, optical communication, and integrated optics.

Wet-etching-assisted femtosecond laser holographic processing of a sapphire concave microlens array.

We report rapid and mask-free fabrication of a sapphire concave microlens array by a combined method of femtosecond laser holographic processing and wet etching. The method features high fabrication efficiency, as crater arrays can be created on sapphire through a parallel processing manner, and the subsequent wet etching facilitates the formation of microlens arrays with a smooth surface. More importantly, the size and spacing of the concave microlenses can be well tuned by varying the distance of craters and etching time. Two types of microlens arrays with a spacing of 25 and 40 µm have been successfully fabricated, both of which showed good imaging performance. This method holds great promise for developing sapphire-based micro-optical components.

Accurate Electrochemistry Analysis of Circulating Methylated DNA from Clinical Plasma Based on Paired-End Tagging and Amplifications.

Circulating methylated DNA has been a new kind of cancer biomarker, yet its small fraction of trace total DNA from clinical samples impairs the accurate analysis. Though fluorescence methods based on quantitative methylation specific PCR (qMSP) have been adopted routinely, yet alternative electrochemistry assay of such DNA from clinical samples remains a great challenge. Herein, we report accurate electrochemistry analysis of circulating methylated DNA from clinical plasma samples based on a paired-end tagging and amplifications strategy. Two DNA primers each labeled with digoxigenin (Dig) and biotin are designed for the recognition and amplification of methylated DNA. Paired-end tagging amplicons and avidin-HRP molecules are successively captured on the electrode modified with Anti-Dig. Then HRP executes catalytic reaction to generate amplified signal. The design of paired-end tagging can readily integrate downstream electrochemical amplified reaction, and two heterogeneous amplifications enable high assay sensitivity. As little as 40 pg of methylated genomic DNA (∼10 genomic equivalents) is well identified, and our strategy can even distinguish as low as 1% methylation level. Tumor-specific methylated DNA is clearly detected in the plasma of 10 of 11 NSCLC patients. The high clinical sensitivity of 91% (10/11) indicates the good consistency with clinical diagnosis. Excellent spatial control of electrochemistry allows simpler detection of more methylation patterns compared to fluorescence methods. The developed electrochemical assay is a promising liquid biopsy tool for the analysis of tumor-specific circulating DNA.

Nano-ablation of silica by plasmonic surface wave at low fluence.

Formation of ripples by ablation of surfaces of laser-irradiated materials is an example of ultrafast energy delivery. Herein, we report on fs-laser optical imprinting of periodic nano-grooves on silica substrate at only 25% of the laser ablation threshold via an interface plasmonic light localization at the ZnS film (top) interface with silica (bottom) by plasmonic surface wave. The nano-grooves were formed throughout ZnS with the same period and orientation imprinted onto the underlying silica. Based on a detailed account of the multi-photon and avalanche ionization using the Drude model, laser-induced plasmonic ablation describes quantitatively the energy deposition from the top ZnS to the substrate of silica.

A potent bioreducible ionizable lipid nanoparticle enables siRNA delivery for retinal neovascularization inhibition.

Small interfering RNA (siRNA) is emerging as a promising treatment for retinal neovascularization due to its specific inhibition of the expression of target genes. However, the clinical translation of siRNA drugs is hindered by the efficiency and safety of delivery vectors. Here, we describe the properties of a new bioreducible ionizable lipid nanoparticle (LNP) 2N12H, which is based on a rationally designed novel ionizable lipid called 2N12B. 2N12H exhibited degradation in response to the mimic cytoplasmic glutathione condition and ionization with a pKa value of 6.5, which remaining neutral at pH 7.4. At a nitrogen to phosphorus ratio of 5, 2N12H efficiently encapsulated and protected siRNA from degradation. Compared to the commercial vehicle Lipofectamine 2000, 2N12H demonstrated similar silencing efficiency and improved safety in the in vitro cell experiments. 2N12H/siVEGFA reduced the expression of VEGFA in retinal pigment epithelium cells and mouse retina, consequently suppressing cell migration and retinal neovascularization. In the mouse model, the therapeutic effect of 2N12H/siVEGFA was comparable to that of the clinical drug ranibizumab. Together, these results suggest the potential of this novel ionizable LNP to facilitate the development of nonviral ocular gene delivery systems.

pH-Responsive polymer boosts cytosolic siRNA release for retinal neovascularization therapy.

Small interfering RNA (siRNA) has a promising future in the treatment of ocular diseases due to its high efficiency, specificity, and low toxicity in inhibiting the expression of target genes and proteins. However, due to the unique anatomical structure of the eye and various barriers, delivering nucleic acids to the retina remains a significant challenge. In this study, we rationally design PACD, an A-B-C type non-viral vector copolymer composed of a hydrophilic PEG block (A), a siRNA binding block (B) and a pH-responsive block (C). PACDs can self-assemble into nanosized polymeric micelles that compact siRNAs into polyplexes through simple mixing. By evaluating its pH-responsive activity, gene silencing efficiency in retinal cells, intraocular distribution, and anti-angiogenesis therapy in a mouse model of hypoxia-induced angiogenesis, we demonstrate the efficiency and safety of PACD in delivering siRNA in the retina. We are surprised to discover that, the PACD/siRNA polyplexes exhibit remarkable intracellular endosomal escape efficiency, excellent gene silencing, and inhibit retinal angiogenesis. Our study provides design guidance for developing efficient nonviral ocular nucleic acid delivery systems.