Polony gels enable amplifiable DNA stamping and spatial transcriptomics of chronic pain.

Methods for acquiring spatially resolved omics data from complex tissues use barcoded DNA arrays of low- to sub-micrometer features to achieve single-cell resolution. However, fabricating such arrays (randomly assembled beads, DNA nanoballs, or clusters) requires sequencing barcodes in each array, limiting cost-effectiveness and throughput. Here, we describe a vastly scalable stamping method to fabricate polony gels, arrays of ∼1-micrometer clonal DNA clusters bearing unique barcodes. By enabling repeatable enzymatic replication of barcode-patterned gels, this method, compared with the sequencing-dependent array fabrication, reduced cost by at least 35-fold and time to approximately 7 h. The gel stamping was implemented with a simple robotic arm and off-the-shelf reagents. We leveraged the resolution and RNA capture efficiency of polony gels to develop Pixel-seq, a single-cell spatial transcriptomic assay, and applied it to map the mouse parabrachial nucleus and analyze changes in neuropathic pain-regulated transcriptomes and cell-cell communication after nerve ligation.

CAZ Composite Photocatalysts for H2 Production and Degradation under Visible Light.

g-C3N4/Ag-ZnO (CAZ) composite photocatalysts were synthesized successfully by the hydrothermal method. The photocatalytic performance of photocatalysts was assessed through experiments measuring both hydrogen (H2) production and the degradation of methylene blue (MB). The H2 production rate of 60% CAZ reached 2.450 mmol·g-1·h-1, which was 8.5 times that of g-C3N4. 25% CAZ degraded 99.14% of MB dye within 40 min, and its degradation rate constant was 12.4 times that of g-C3N4. CAZ composite photocatalysts have good synergistic properties in degradation and hydrogen production and exhibit better photocatalytic performance. A Z-scheme photocatalytic system mechanism of CAZ has been proposed for the enhanced H2 production and photocatalytic degradation rate.

Performance Degradation of a Double-Perovskite PrBaCo2O5+δ Cathode Operating under a CO2/H2O-Containing Atmosphere.

The electrochemical activity and stability of the PBCO electrode are investigated under the annealing processes in an atmosphere containing CO2/H2O for solid oxide fuel cells (SOFCs). The electrochemical impedance spectrum results unequivocally confirm the significant deterioration in PBCO cathode performance upon annealing under ambient air conditions, particularly when exposed to CO2/H2O atmospheres. Microstructure and surface chemical state analyses reveal the segregation of BaO on the PBCO surface, and the formation of insulating BaCO3 degraded the electrochemical performance. CO2 and H2O exhibit a significant induced effect on the segregation of Ba in PBCO to the surfaces, thereby causing a rapid decline in electrode performance. Additionally, the analysis of volume relaxation reveals that the presence of oxygen in the electrode environment can also influence the deposition process occurring on the surface of the electrode. However, this phenomenon is not observed in N2. This study emphasizes the impact of various gases present in the working atmosphere on surface-separated BaO, which consequently plays a pivotal role in the activity and long-term stability of PBCO electrodes.

Dynamic miRNA-mRNA interactions coordinate gene expression in adult Anopheles gambiae.

microRNAs (miRNAs) are increasingly recognized as important regulators of many biological processes in mosquitoes, vectors of numerous devastating infectious diseases. Identification of bona fide targets remains the bottleneck for functional studies of miRNAs. In this study, we used CLEAR-CLIP assays to systematically analyze miRNA-mRNA interactions in adult female Anopheles gambiae mosquitoes. Thousands of miRNA-target pairs were captured after direct ligation of the miRNA and its cognate target transcript in endogenous Argonaute-miRNA-mRNA complexes. Using two interactions detected in this manner, miR-309-SIX4 and let-7-kr-h1, we demonstrated the reliability of this experimental approach in identifying in vivo gene regulation by miRNAs. The miRNA-mRNA interaction dataset provided an invaluable opportunity to decipher targeting rules of mosquito miRNAs. Enriched motifs in the diverse targets of each miRNA indicated that the majority of mosquito miRNAs rely on seed-based canonical target recognition, while noncanonical miRNA binding sites are widespread and often contain motifs complementary to the central or 3' ends of miRNAs. The time-lapse study of miRNA-target interactomes in adult female mosquitoes revealed dynamic miRNA regulation of gene expression in response to varying nutritional sources and physiological demands. Interestingly, some miRNAs exhibited flexibility to use distinct sequences at different stages for target recognition. Furthermore, many miRNA-mRNA interactions displayed stage-specific patterns, especially for those genes involved in metabolism, suggesting that miRNAs play critical roles in precise control of gene expression to cope with enormous physiological demands associated with egg production. The global mapping of miRNA-target interactions contributes to our understanding of miRNA targeting specificity in non-model organisms. It also provides a roadmap for additional studies focused on regulatory functions of miRNAs in Anopheles gambiae.

Broad spectrum immunomodulatory effects of Anopheles gambiae microRNAs and their use for transgenic suppression of Plasmodium.

Malaria, caused by the protozoan parasite Plasmodium and transmitted by Anopheles mosquitoes, represents a major threat to human health. Plasmodium's infection cycle in the Anopheles vector is critical for transmission of the parasite between humans. The midgut-stage bottleneck of infection is largely imposed by the mosquito's innate immune system. microRNAs (miRNAs, small noncoding RNAs that bind to target RNAs to regulate gene expression) are also involved in regulating immunity and the anti-Plasmodium defense in mosquitoes. Here, we characterized the mosquito's miRNA responses to Plasmodium infection using an improved crosslinking and immunoprecipitation (CLIP) method, termed covalent ligation of endogenous Argonaute-bound RNAs (CLEAR)-CLIP. Three candidate miRNAs' influence on P. falciparum infection and midgut microbiota was studied through transgenically expressed miRNA sponges (miR-SPs) in midgut and fat body tissues. MiR-SPs mediated conditional depletion of aga-miR-14 or aga-miR-305, but not aga-miR-8, increased mosquito resistance to both P. falciparum and P. berghei infection, and enhanced the mosquitoes' antibacterial defenses. Transcriptome analysis revealed that depletion of aga-miR-14 or aga-miR-305 resulted in an increased expression of multiple immunity-related and anti-Plasmodium genes in mosquito midguts. The overall fitness cost of conditionally expressed miR-SPs was low, with only one of eight fitness parameters being adversely affected. Taken together, our results demonstrate that targeting mosquito miRNA by conditional expression of miR-SPs may have potential for the development of malaria control through genetically engineered mosquitoes.

Regulation of circadian rhythm and sleep by miR-375-timeless interaction in Drosophila.

MicroRNAs are important coordinators of circadian regulation that mediate the fine-tuning of gene expression. Although many studies have shown the effects of individual miRNAs on the circadian clock, the global functional miRNA-mRNA interaction network involved in the circadian system remains poorly understood. Here, we used CLEAR (Covalent Ligation of Endogenous Argonaute-bound RNAs)-CLIP (Cross-Linking and Immuno-Precipitation) to explore the regulatory functions of miRNAs in the circadian system by comparing the miRNA-mRNA interactions between Drosophila wild-type strain W1118 and a mutant of the key circadian transcriptional regulator Clock (Clkjrk ). This experimental approach unambiguously identified tens of thousands of miRNA-mRNA interactions in both the head and body. The miRNA-mRNA interactome showed dramatic changes in the Clkjrk flies. Particularly, among ~300 miRNA-mRNA circadian relevant interactions, multiple interactions involving core clock genes pdp1, tim, and vri displayed distinct changes as a result of the Clk mutation. Based on the CLEAR-CLIP analysis, we found a novel regulation of the circadian rhythm and sleep by the miR-375-timeless interaction. The results indicated that Clk disruption abolished normal rhythmic expression of miR-375 and the functional regulation occurred in the l-LNv neurons, where miR-375 modulated the circadian rhythm and sleep via targeting timeless. This work provides the first global view of miRNA regulation in the circadian rhythm.

Juvenile hormone-regulated alternative splicing of the taiman gene primes the ecdysteroid response in adult mosquitoes.

Juvenile hormone (JH) regulates many aspects of insect development and reproduction. In some processes, JH plays a critical role in defining the action of the steroid hormone 20-hydroxyecdysone (20E). In Aedes aegypti mosquitoes, JH prepares newly emerged female adults to become competent to synthesize vitellogenin in response to 20E after blood ingestion. The molecular basis of this competence is still not well understood. Here, we report that JH regulates pre-mRNA splicing of the taiman gene, which encodes a key transcriptional regulator required for both JH- and 20E-controlled gene expression. JH stimulated the production of the Taiman isoforms A/B, while reducing the levels of the isoforms C/D, in the fat body after adult eclosion. The appearance of the A/B isoforms in maturing mosquitoes was accompanied by acquisition of the competence to respond to 20E. Depletion of the A/B isoforms, by inhibiting the alternative splicing or by isoform-specific RNA interference, considerably diminished the 20E-induced gene expression after a blood meal and substantially impaired oocyte development. In accordance with this observation, further studies indicated that in the presence of 20E, the Taiman A/B isoforms showed much stronger interactions with the 20E receptor complex than the Taiman C/D isoforms. In contrast, all four isoforms displayed similar capabilities of forming active JH receptor complexes with the methoprene-tolerant protein (Met). This study suggested that JH confers the competence to newly emerged female mosquitoes by regulating mRNA splicing to generate the Taiman isoforms that are essential for the vitellogenic 20E response.

COMBINES-CID: An Efficient Method for De Novo Engineering of Highly Specific Chemically Induced Protein Dimerization Systems.

Chemically induced dimerization (CID) systems, in which two proteins dimerize only in the presence of a small molecule ligand, offer versatile tools for small molecule sensing and actuation. However, only a handful of CID systems exist and creating one with the desired sensitivity and specificity for any given ligand is an unsolved problem. Here, we developed a combinatorial binders-enabled selection of CID (COMBINES-CID) method broadly applicable to different ligands. We demonstrated a proof-of-principle by generating nanobody-based heterodimerization systems induced by cannabidiol with high ligand selectivity. We applied the CID system to a sensitive sandwich enzyme-linked immunosorbent assay-like assay of cannabidiol in body fluids with a detection limit of ∼0.25 ng/mL. COMBINES-CID provides an efficient, cost-effective solution for expanding the biosensor toolkit for small molecule detection.

Band gap scaling laws in group IV nanotubes.

By using the first-principles calculations, the band gap properties of nanotubes formed by group IV elements have been investigated systemically. Our results reveal that for armchair nanotubes, the energy gaps at K points in the Brillouin zone decrease as 1/r scaling law with the radii (r) increasing, while they are scaled by -1/r 2 + C at Γ points, here, C is a constant. Further studies show that such scaling law of K points is independent of both the chiral vector and the type of elements. Therefore, the band gaps of nanotubes for a given radius can be determined by these scaling laws easily. Interestingly, we also predict the existence of indirect band gap for both germanium and tin nanotubes. Our new findings provide an efficient way to determine the band gaps of group IV element nanotubes by knowing the radii, as well as to facilitate the design of functional nanodevices.

Electronic and magnetic properties of SnSe monolayers doped by Ga, In, As, and Sb: a first-principles study.

A SnSe monolayer with an orthorhombic Pnma GeS structure is an important two-dimensional (2D) indirect band gap material at room temperature. Based on first-principles density functional theory calculations, we present systematic studies on the electronic and magnetic properties of X (X = Ga, In, As, Sb) atom doped SnSe monolayers. The calculated electronic structures show that the Ga-doped system maintains its semiconducting properties while the In-doped SnSe monolayer is half-metal. The As- and Sb-doped SnSe systems present the characteristics of an n-type semiconductor. Moreover, all considered substitutional doping cases induce magnetic ground states with a magnetic moment of ∼ 1 µB. In addition, the calculated formation energies also show that four types of doped systems are thermodynamically stable. These results provide a new route for the potential applications of doped SnSe monolayers in 2D photoelectronic and magnetic semiconductor devices.

Electrolyte modification method induced atomic arrangement in FeOx/NF nanosheets for efficient overall water splitting.

To explore transition metal-based electrocatalysts with remarkable energy storage and conversion performance, the rational design and synthesis of electrodes with rich active sites and favorable electrical conductivity are crucial. Herein, using fluoroethylene carbonate (FEC) additive in electrochemical conversion reaction (electrolyte modification method) is proposed as an effective strategy to enhance the catalytic activity of FeOx/NF. The optimal sample FeOx/NF-Li-FEC1 shows optimized HER activity with remarkably low overpotential of 222 mV at a current density of 200 mA cm-2. By employing FeOx/NF-Li-FEC1 as bifunctional electrocatalysts, the overall water-splitting device reaches a current density of 10 mA cm-2 at a low cell voltage of 1.56 V. The outstanding performance is mainly attributed to the atomic arrangement to offer rich active sites as well as the evolved electronic structure and the thin SEI layer to accelerate charge transfer process. This study opens up a novel avenue to rationally design and synthesize low-cost and high-performance electrode materials for electrochemical energy conversion and storage devices.

Spatial mapping of cellular senescence: emerging challenges and opportunities.

Cellular senescence is a well-established driver of aging and age-related diseases. There are many challenges to mapping senescent cells in tissues such as the absence of specific markers and their relatively low abundance and vast heterogeneity. Single-cell technologies have allowed unprecedented characterization of senescence; however, many methodologies fail to provide spatial insights. The spatial component is essential, as senescent cells communicate with neighboring cells, impacting their function and the composition of extracellular space. The Cellular Senescence Network (SenNet), a National Institutes of Health (NIH) Common Fund initiative, aims to map senescent cells across the lifespan of humans and mice. Here, we provide a comprehensive review of the existing and emerging methodologies for spatial imaging and their application toward mapping senescent cells. Moreover, we discuss the limitations and challenges inherent to each technology. We argue that the development of spatially resolved methods is essential toward the goal of attaining an atlas of senescent cells.

Neuroprotective Effect of HIIT against GFAP Hypertrophy through Mitochondrial Dynamics in APP/PS1 Mice.

Alzheimer's disease (AD) is characterized by the accumulation of ß-amyloid (Aß) plaques and tau neurofibrillary tangles in the brain. Although the exact details of the neuronal protective effect of high-intensity interval training (HIIT) on AD remain unclear, the preclinical phase of AD appears to be the important time point for such intervention. The described experiment investigates the neuroprotective effect of HIIT on AD in APP/PS1 mice. In total, 14 C57BL6 healthy control (C) mice and 14 APP/PS1 AD mice were each randomly assigned into two groups, one that did not participate in HIIT (C and AD groups, respectively) and the other subject to HIIT intervention (control HIIT (CE) and AD HIIT (ADE) groups, respectively). Visualization of hippocampal neuronal cells via HE and Congo red staining showed significant improvement in cell status and a significant reduction in amyloidosis in ADE compared with AD. The results of behavioral analysis show that the HIIT intervention significantly improved cognitive decline and reduced spatial exploration in both the C and AD groups. Immunofluorescence showed that the overall brain and the hippocampus of aged rats in the C and AD groups had different degrees of neuroglial responses and astrocyte GFAP proliferation and hypertrophy, with obvious improvement in the CE and ADE groups after 10 weeks of HIIT intervention. These results show that HIIT significantly improves the status of mitochondrial kinetic proteins and related proteins, with the mechanism differing between the normal aging C and the AD groups. 10 weeks of HIIT improved the imbalance in mitochondrial dynamics present in normal control mice and in AD mice. We conclude that preclinical training intervention has a significant positive effect on the exploratory behavior and cognitive functioning of mice.

Prognostic role of microRNA-21 in various carcinomas: a systematic review and meta-analysis.

BACKGROUND: Recent studies have shown that microRNAs (miRNA) could play a potential role as diagnostic and prognostic biomarkers of cancers. The aim of this meta-analysis is to summarize the global predicting role of miR-21 for survival in patients with a variety of carcinomas. DESIGN: Eligible studies were identified and assessed for quality through multiple search strategies. Data were collected from studies comparing overall, relapse-free or cancer-specific survival (CSS) in patients with cancer having higher miR-21 expression with those having lower levels. Pooled hazard ratios (HRs) of miR-21 for survival were calculated. RESULTS: A total of 17 studies dealing with various carcinomas were included for this global meta-analysis. For overall survival (OS), the pooled hazard ratio (HR) of higher miR-21 expression in cancerous tissue was 1·69 (95% CI: 1·33-2·16, P < 0·001), which could significantly predict poorer survival in general carcinomas. For relapse-free or CSS, elevated miR-21 was also a significant predictor, with a pooled HR of 1·48 (95% CI: 1·03-2·11, P = 0·033). Importantly, subgroup analysis suggested that higher expression of miR-21 correlated with worse OS in head and neck squamous cell carcinoma (HNSCC) (HR 1·46, 95% CI: 1·13-1·87, P = 0·004) and carcinomas in digestion system (HR 1·56, 95% CI: 1·08-2·26, P = 0·018). CONCLUSIONS: Our findings suggest that miR-21 detection has a prognostic value in patients with cancer, especially in HNSCC and digestion system cancers.

Creating Red Light-Switchable Protein Dimerization Systems as Genetically Encoded Actuators with High Specificity.

Protein dimerization systems controlled by red light with increased tissue penetration depth are a highly needed tool for clinical applications such as cell and gene therapies. However, mammalian applications of existing red light-induced dimerization systems are hampered by limitations of their two components: a photosensory protein (or photoreceptor) which often requires a mammalian exogenous chromophore and a naturally occurring photoreceptor binding protein typically having a complex structure and nonideal binding properties. Here, we introduce an efficient, generalizable method (COMBINES-LID) for creating highly specific, reversible light-induced heterodimerization systems independent of any existing binders to a photoreceptor. It involves a two-step binder screen (phage display and yeast two-hybrid) of a combinatorial nanobody library to obtain binders that selectively engage a light-activated form of a photoswitchable protein or domain not the dark form. Proof-of-principle was provided by engineering nanobody-based, red light-induced dimerization (nanoReD) systems comprising a truncated bacterial phytochrome sensory module using a mammalian endogenous chromophore, biliverdin, and light-form specific nanobodies. Selected nanoReD systems were biochemically characterized, exhibiting low dark activity and high induction specificity, and further demonstrated for the reversible control of protein translocation and activation of gene expression in mice. Overall, COMBINES-LID opens new opportunities for creating genetically encoded actuators for the optical manipulation of biological processes.

Strong Valence Electrons Dependent and Logical Relations of Elemental Impurities in 2D Binary Semiconductor: a Case of GeP3 Monolayer from Ab Initio Studies.

Using first-principle calculations within density functional theory, we investigate the electronic property and stability of substitutionally doped 2D GeP3 monolayer with dopants from group III to VI. The conducting properties are found to be dramatically modified by both the doping sites and the number of valence electrons of dopants. Specifically, substitution on Ge site exhibits metal-semiconductor oscillations as a function of the number of valence electrons of dopants, while such oscillations are totally reversed when substitution on P site. Moreover, we also study the case of co-doping in GeP3, showing that co-doping can produce a logical "AND" phenomenon, that is, the conducting properties of co-doped GeP3 can be deduced via a simple logical relation according to the results of single doping. Finally, we investigate the formation energy of dopants and find that the electron-hole and hole-hole co-doped systems are much more energetically favorable due to the Coulomb attraction. Our findings not only present a comprehensive understanding of 2D doping phenomenon, but also propose an intriguing route to tune the electronic properties of 2D binary semiconductors.

Bioinformatic Analysis of MicroRNA Sequencing Data.

The vital role of microRNAs (miRNAs) involved in gene expression regulation has been confirmed in many biological processes. With the growing power and reducing cost of next-generation sequencing, more and more researchers turn to apply this high-throughput method to solve their biological problems. For miRNAs with known sequences, their expression profiles can be generated from the sequencing data. It also allows us to identify some novel miRNAs and explore the sequence variations under different conditions. Currently, there are a handful of tools available to analyze the miRNA sequencing data with separated or combined features, such as reads preprocessing, mapping and differential expression analysis. However, to our knowledge, a hands-on guideline for miRNA sequencing data analysis covering all steps is not available. Here we will utilize a set of published tools to perform the miRNA analysis with detailed explanation. Particularly, the miRNA target prediction and annotation may provide useful information for further experimental verification.

Association of microRNAs with Argonaute proteins in the malaria mosquito Anopheles gambiae after blood ingestion.

Drastic changes in gene expression occur after adult female mosquitoes take a blood meal and use the nutrients for egg maturation. A growing body of evidence indicates that microRNAs (miRNAs) contribute to this tightly controlled tissue- and stage-specific gene expression. To investigate the role of miRNAs, we monitored miRNA expression in the mosquito Anopheles gambiae during the 72-h period immediately after blood feeding. We also measured the association of miRNAs with Argonaute 1 (Ago1) and Argonaute 2 (Ago2) to assess the functional status of individual miRNA species. Overall, 173 mature miRNAs were precipitated with Ago1 and Ago2, including 12 new miRNAs, the orthologs of which are found thus far only in other Anopheles species. Ago1 is the predominant carrier of miRNAs in Anopheles gambiae. The abundance and Ago loading of most of the mature miRNAs were relatively stable after blood ingestion. However, miRNAs of the miR-309/286/2944 cluster were considerably upregulated after blood feeding. Injection of the specific antagomir for miR-309 resulted in smaller developing oocytes and ultimately fewer eggs. In addition, the Ago association of some miRNAs was not proportional to their cellular abundance, suggesting that integration of miRNAs into the Ago complexes is regulated by additional mechanisms.

Let-7c-5p inhibits cell proliferation and induces cell apoptosis by targeting ERCC6 in breast cancer.

In this study, we found that let-7c-5p expression was clearly downregulated in breast cancer tissues compared with that of corresponding adjacent tissues. Furthermore, overexpression of let-7c-5p in MCF-7 breast cancer cells could significantly inhibit cell proliferation and induce cell apoptosis. The target genes of let-7c-5p were predicted by the way of bioinformatics, and validated by dual luciferase reporter assay and western blotting demonstrating that excision repair cross complementing 6 (ERCC6) gene was a direct target. Collectively, the present study suggested that let-7c-5p acted as a tumor suppressor in breast cancer possibly by negatively regulating ERCC6, which took an important part in nucleotide excision repair and it may provide a new potential strategy for breast cancer therapy.

Protein kinase C modulates transcriptional activation by the juvenile hormone receptor methoprene-tolerant.

Juvenile hormone (JH) controls many biological events in insects by triggering dramatic changes in gene expression in target cells. The Methoprene-tolerant (MET) protein, an intracellular JH receptor, acts as a transcriptional regulator and binds to the promoters of tissue- and stage-specific JH target genes when JH is present. Our recent study has demonstrated that the transcriptional activation by MET is modulated by a membrane-initiated JH signaling pathway, involving phospholipase C (PLC) and calcium/calmodulin-dependent protein kinase II (CaMKII). Here we report that protein kinase C (PKC) is another essential intermediate of this pathway. PKC was activated by JH and this action was PLC-dependent. Inhibition of the PKC activity substantially weakened the JH-induced gene expression in mosquito cells. RNAi experiments indicated that several PKC isoforms were involved in the JH action during the post-emergence development of adult female mosquitoes. JH treatment considerably increased the binding of MET to the promoters of JH response genes in cultured mosquito abdomens that were collected from newly emerged female adults. The JH-induced DNA binding of MET was hindered when the abdomens were treated with a PKC inhibitor and JH. Therefore, the results suggest that PKC modulates the transactivation activity of MET by enhancing the binding of MET to JH response elements in the JH target genes. This mechanism may allow for variable and stage- and tissue-specific genomic responses to JH.