A Low-Cost, Sensitive Reporter System Using Membrane-Tethered Horseradish Peroxidase for Efficient Gene Expression Analysis.

Reporter gene assays are essential for high-throughput analysis, such as drug screening or determining downstream signaling activation/inhibition. However, use of this technology has been hampered by the high cost of the substrate (e.g., d-Luciferin (d-Luc)) in the most common firefly luciferase (FLuc) reporter gene assay. Although alternate luciferase is available worldwide, its substrate has remained expensive, and a more affordable option is still in demand. Here, we present a membrane-tethered horseradish peroxidase (mHRP), a new reporter system composed of a cell membrane expressing HRP that can preserve its enzymatic function on the cell surface, facilitates contact with HRP substrates (e.g., ABTS and TMB), and avoids the cell lysis process and the use of the high-priced luciferase substrate. An evaluation of the light signal sensitivity of mHRP compared to FLuc showed that both had comparable signal sensitivity. We also identified an extended substrate half-life of more than 5-fold that of d-Luc. Of note, this strategy provided a more stable detection signal, and the cell lysis process is not mandatory. Furthermore, with this strategy, we decreased the total amount of time taken for analysis and increased the time of detection limit of the reporter assay. Pricing analysis showed a one-third to one twenty-eighth price drop per single test of reporter assay. Given the convenience and stability of the mHRP reporter system, we believe that our strategy is suitable for use as an alternative to the luciferase reporter assay.

Single Virus Detection on Silicon Photonic Crystal Random Cavities.

On-chip silicon microcavity sensors are advantageous for the detection of virus and biomolecules due to their compactness and the enhanced light-matter interaction with the analyte. While their theoretical sensitivity is at the single-molecule level, the fabrication of high quality (Q) factor silicon cavities and their integration with optical couplers remain as major hurdles in applications such as single virus detection. Here, label-free single virus detection using silicon photonic crystal random cavities is proposed and demonstrated. The sensor chips consist of free-standing silicon photonic crystal waveguides and do not require pre-fabricated defect cavities or optical couplers. Residual fabrication disorder results in Anderson-localized cavity modes which are excited by a free space beam. The Q ≈105 is sufficient for observing discrete step-changes in resonance wavelength for the binding of single adenoviruses (≈50 nm radius). The authors' findings point to future applications of CMOS-compatible silicon sensor chips supporting Anderson-localized modes that have detection capabilities at the level of single nanoparticles and molecules.

Genetics behind Cerebral Disease with Ocular Comorbidity: Finding Parallels between the Brain and Eye Molecular Pathology.

Cerebral visual impairments (CVIs) is an umbrella term that categorizes miscellaneous visual defects with parallel genetic brain disorders. While the manifestations of CVIs are diverse and ambiguous, molecular diagnostics stand out as a powerful approach for understanding pathomechanisms in CVIs. Nevertheless, the characterization of CVI disease cohorts has been fragmented and lacks integration. By revisiting the genome-wide and phenome-wide association studies (GWAS and PheWAS), we clustered a handful of renowned CVIs into five ontology groups, namely ciliopathies (Joubert syndrome, Bardet-Biedl syndrome, Alstrom syndrome), demyelination diseases (multiple sclerosis, Alexander disease, Pelizaeus-Merzbacher disease), transcriptional deregulation diseases (Mowat-Wilson disease, Pitt-Hopkins disease, Rett syndrome, Cockayne syndrome, X-linked alpha-thalassaemia mental retardation), compromised peroxisome disorders (Zellweger spectrum disorder, Refsum disease), and channelopathies (neuromyelitis optica spectrum disorder), and reviewed several mutation hotspots currently found to be associated with the CVIs. Moreover, we discussed the common manifestations in the brain and the eye, and collated animal study findings to discuss plausible gene editing strategies for future CVI correction.

Imprint and transfer fabrication of freestanding plasmonic membranes.

This paper reports an imprint and transfer approach for the rapid and inexpensive fabrication of the ultra-thin freestanding plasmonic membrane (FPM) that supports surface plasmon resonances. The imprint and transfer fabrication method involves the soft imprint lithography on an ultrathin polymer film, transfer of the perforated polymer film to a supporting frame, subsequent deposition of gold, and final removal of the polymer film. Without using any sophisticated lithography and etching processes, the imprint and transfer method can produce freestanding gold membranes with 2D arrays of submicrometer-sized holes that support plasmonic modes in the mid-wavelength infrared (mid-IR) range. Two FPM devices with an array constant of 4.0 and 2.5 µm have been simulated, fabricated, and measured for their transmittance characteristics. The fabricated FPMs exhibit surface plasmon polariton Bloch mode and extraordinary optical transmission (EOT) with the enhanced local field around the membrane. The effects of membrane thickness and angle dispersion on the FPM were investigated to show the tuning of EOT modes in IR. Furthermore, we demonstrated the refractometric sensing and enhanced IR absorption of the FPM device for its potential in chemical and biomolecule sensing applications.

Exogenous glutamate rapidly induces the expression of genes involved in metabolism and defense responses in rice roots.

BACKGROUND: Glutamate is an active amino acid. In addition to protein synthesis and metabolism, increasing evidence indicates that glutamate may also function as a signaling molecule in plants. Still, little is known about the nutritional role of glutamate and genes that are directly regulated by glutamate in rice. RESULTS: Exogenous glutamate could serve as a nitrogen nutrient to support the growth of rice seedlings, but it was not as effective as ammonium nitrate or glutamine. In nitrogen-starved rice seedlings, glutamate was the most abundant free amino acid and feeding of glutamate rapidly and significantly increased the endogenous levels of glutamine, but not glutamate. These results indicated that glutamate was quickly metabolized and converted to the other nitrogen-containing compounds in rice. Transcriptome analysis revealed that at least 122 genes involved in metabolism, transport, signal transduction, and stress responses in the roots were rapidly induced by 2.5 mM glutamate within 30 min. Many of these genes were also up-regulated by glutamine and ammonium nitrate. Still, we were able to identify some transcription factor, kinase/phosphatase, and elicitor-responsive genes that were specifically or preferentially induced by glutamate. CONCLUSIONS: Glutamate is a functional amino acid that plays important roles in plant nutrition, metabolism, and signal transduction. The rapid and specific induction of transcription factor, kinase/phosphatase and elicitor-responsive genes suggests that glutamate may efficiently amplify its signal and interact with other signaling pathways to regulate metabolism, growth and defense responses in rice.

Decoding and reconstructing disease relations between dry eye and depression: a multimodal investigation comprising meta-analysis, genetic pathways and Mendelian randomization.

INTRODUCTION: The clinical presentations of dry eye disease (DED) and depression (DEP) often comanifest. However, the robustness and the mechanisms underlying this association were undetermined. OBJECTIVES: To this end, we set up a three-segment study that employed multimodality results (meta-analysis, genome-wide association study [GWAS] and Mendelian randomization [MR]) to elucidate the association, common pathways and causality between DED and DEP. METHODS: A meta-analysis comprising 26 case-control studies was first conducted to confirm the DED-DEP association. Next, we performed a linkage disequilibrium (LD)-adjusted GWAS and targeted phenotype association study (PheWAS) in East Asian TW Biobank (TWB) and European UK Biobank (UKB) populations. Single-nucleotide polymorphisms (SNPs) were further screened for molecular interactions and common pathways at the functional gene level. To further elucidate the activated pathways in DED and DEP, a systemic transcriptome review was conducted on RNA sequencing samples from the Gene Expression Omnibus. Finally, 48 MR experiments were implemented to examine the bidirectional causation between DED and DEP. RESULTS: Our meta-analysis showed that DED patients are associated with an increased DEP prevalence (OR = 1.83), while DEP patients have a concurrent higher risk of DED (OR = 2.34). Notably, cross-disease GWAS analysis revealed that similar genetic architecture (rG = 0.19) and pleiotropic functional genes contributed to phenotypes in both diseases. Through protein-protein interaction and ontology convergence, we summarized the pleiotropic functional genes under the ontology of immune activation, which was further validated by a transcriptome systemic review. Importantly, the inverse variance-weighted (IVW)-MR experiments in both TWB and UKB populations (p value <0.001) supported the bidirectional exposure-outcome causation for DED-to-DEP and DEP-to-DED. Despite stringent LD-corrected instrumental variable re-selection, the bidirectional causation between DED and DEP remained. CONCLUSION: With the multi-modal evidence combined, we consolidated the association and causation between DED and DEP.

Anomalous DNA hybridisation kinetics on gold nanorods revealed via a dual single-molecule imaging and optoplasmonic sensing platform.

Observing the hybridisation kinetics of DNA probes immobilised on plasmonic nanoparticles is key in plasmon-enhanced fluorescence detection of weak emitting species, and refractive index based single-molecule detection on optoplasmonic sensors. The role of the local field in providing plasmonic signal enhancements for single-molecule detection has been studied in great detail. Nevertheless, few studies have compared the experimental results in both techniques for single-molecule studies. Here we developed the first optical setup that integrates optoplasmonic and DNA-PAINT based detection of oligonucleotides to compare these sub-platforms and provide complementary insights into single molecule processes. We record the fluorescence and optoplasmonic sensor signals for individual, transient hybridisation events. The hybridisation events are observed in the same sample cell and over a prolonged time (i.e. towards high binding site occupancies). A decrease in the association rate over the measurement duration is reported. Our dual optoplasmonic sensing and imaging platform offers insight into the observed phenomenon, revealing that irreversible hybridisation events accumulate over detected step signals in optoplasmonic sensing. Our results point to novel physicochemical mechanisms that result in the stabilisation of DNA hybridisation on optically-excited plasmonic nanoparticles.

Differential Effects of Two Tomato Begomoviruses on the Life History and Feeding Preference of Bemisia tabaci.

Tomato yellow leaf curl disease, caused by a group of closely related tomato yellow leaf curl viruses, is a major threat to tomato cultivation worldwide. These viruses are primarily transmitted by the sweet potato whitefly (Bemisia tabaci) in a persistent-circulative manner, wherein the virus circulates in the body of B. tabaci and infects its tissues. The complex relationship between viruses and whiteflies significantly influences virus transmission, with studies showing varying effects of the former on the life history and feeding preference of the latter. Whether these effects are direct or indirect, and whether they are negative, neutral, or positive, appears to depend on the specific interactions between virus and whitefly species. The tomato yellow leaf curl Thailand virus (TYLCTHV) and the tomato leaf curl Taiwan virus (ToLCTV) are two prevalent begomoviruses in fields in Taiwan. This study examined the direct and indirect effects of TYLCTHV and ToLCTV on the life history traits (longevity, fecundity, nymph survival, and nymph developmental time) and feeding preference of B. tabaci Middle East-Asia Minor 1 (MEAM1). The results revealed that TYLCTHV had no effects on these life history traits or the feeding preference of MEAM1 whiteflies. Although ToLCTV did not directly affect the longevity and fecundity of MEAM1 whiteflies, their fecundity and the nymph developmental time were negatively affected by feeding on ToLCTV-infected plants. In addition, ToLCTV infection also altered the feeding preference of MEAM1 whiteflies. The different effects of virus infection may contribute to the lower prevalence of ToLCTV compared to TYLCTHV in fields in Taiwan.

Plasmonic nanogap-enhanced Raman scattering using a resonant nanodome array.

The optical properties and surface-enhanced Raman scattering (SERS) of plasmonic nanodome array (PNA) substrates in air and aqueous solution are investigated. PNA substrates are inexpensively and uniformly fabricated with a hot spot density of 6.25 × 10(6) mm(-2) using a large-area nanoreplica moulding technique on a flexible plastic substrate. Both experimental measurement and numerical simulation results show that PNAs exhibit a radiative localized surface plasmon resonance (LSPR) due to dipolar coupling between neighboring nanodomes and a non-radiative surface plasmon resonance (SPR) resulting from the periodic array structure. The high spatial localization of electromagnetic field within the ∼10 nm nanogap together with the spectral alignment between the LSPR and excited and scattered light results in a reliable and reproducible spatially averaged SERS enhancement factor (EF) of 8.51 × 10(7) for Au-coated PNAs. The SERS enhancement is sufficient for a wide variety of biological and chemical sensing applications, including detection of common metabolites at physiologically relevant concentrations.

Improved sensitivity of DNA microarrays using photonic crystal enhanced fluorescence.

DNA microarrays are used to profile changes in gene expression between samples in a high-throughput manner, but measurements of genes with low expression levels can be problematic with standard microarray substrates. In this work, we expand the detection capabilities of a standard microarray experiment using a photonic crystal (PC) surface that enhances fluorescence observed from microarray spots. This PC is inexpensively and uniformly fabricated using a nanoreplica molding technique, with very little variation in its optical properties within- and between-devices. By using standard protocols to process glass microarray substrates in parallel with PCs, we evaluated the impact of this substrate on a one-color microarray experiment comparing gene expression in two developmental stages of Glycine max. The PCs enhanced the signal-to-noise ratio observed from microarray spots by 1 order of magnitude, significantly increasing the number of genes detected above substrate fluorescence noise. PC substrates more than double the number of genes classified as differentially expressed, detecting changes in expression even for low expression genes. This approach increases the dynamic range of a surface-bound fluorescence-based assay to reliably quantify small quantities of DNA that would be impossible with standard substrates.

Surface-enhanced Raman nanodomes.

We demonstrate a surface-enhanced Raman scattering (SERS) substrate consisting of a closely spaced metal nanodome array fabricated on flexible plastic film. We used a low-cost, large-area replica molding process to produce a two-dimensional periodic array of cylinders that is subsequently overcoated with SiO(2) and silver thin films to form dome-shaped structures. Finite element modeling was used to investigate the electromagnetic field distribution of the nanodome array structure and the effect of the nanodome separation distance on the electromagnetic field enhancement. The SERS enhancement from the nanodome array substrates was experimentally verified using rhodamine 6G as the analyte. With a separation distance of 17 nm achieved between adjacent domes using a process that is precisely controlled during thin film deposition, a reproducible SERS enhancement factor of 1.37 × 10(8) was demonstrated. The nanoreplica molding process presented in this work allows for simple, low-cost, high-throughput fabrication of uniform nanoscale SERS substrates over large surface areas without the requirement for high resolution lithography or defect-free deposition of spherical microparticle monolayer templates.

Magnification of photonic crystal fluorescence enhancement via TM resonance excitation and TE resonance extraction on a dielectric nanorod surface.

Using a one-dimensional grating surface photonic crystal (PC), we experimentally demonstrate that the detection of fluorescent molecules on a PC surface can be substantially magnified through the combined effects of resonance-enhanced excitation of the fluorescent dye, resonance-enhanced extraction of the fluorescence emission and a dielectric nanorod surface coating increasing the surface area available for fluorophore-PC interaction. Enhanced excitation is obtained by engineering a high-Q TM resonant mode to efficiently couple with an incident TM-polarized lambda = 633 nm laser for exciting Cyanine-5 (Cy5). Enhanced extraction results from a low-Q TE resonance designed to spectrally overlap the Cy5 emission spectrum for channeling TE-polarized emission towards the detection instrument. The entire PC surface is coated with a porous film of TiO(2) nanorods that allows more fluorophores to penetrate into the region of enhanced near-electric fields. Experimental results reveal a 588-fold enhancement in fluorescence intensity relative to an unpatterned glass surface.

Engineering Dielectric Screening for Potential-well Arrays of Excitons in 2D Materials.

Tailoring of the band gap in semiconductors is essential for the development of novel devices. In standard semiconductors, this modulation is generally achieved through highly energetic ion implantation. In two-dimensional (2D) materials, the photophysical properties are strongly sensitive to the surrounding dielectric environment presenting novel opportunities through van der Waals heterostructures encompassing atomically thin high-κ dielectrics. Here, we demonstrate a giant tuning of the exciton binding energy of the monolayer WSe2 as a function of the dielectric environment. Upon increasing the average dielectric constant from 2.4 to 15, the exciton binding energy is reduced by as much as 300 meV in ambient conditions. The experimentally determined exciton binding energies are in excellent agreement with the theoretical values predicted from a Mott-Wannier exciton model with parameters derived from first-principles calculations. Finally, we show how texturing of the dielectric environment can be used to realize potential-well arrays for excitons in 2D materials, which is a first step toward exciton metamaterials.

Garlic essential oil mediates acute and chronic mild stress-induced depression in rats via modulation of monoaminergic neurotransmission and brain-derived neurotrophic factor levels.

Garlic essential oil (GEO) and its major organosulfur component (diallyl disulfide, DADS) possess diverse biological properties; however, limited information on their antidepressant-like effects is available. This study is the first to investigate these effects of GEO using the forced swimming test (FST) and unpredictable chronic mild stress (UCMS) induced depression in rats. After oral administration for 28 consecutive days, GEO (25 and 50 mg per kg bw) significantly reduced the immobility time in the FST. Additionally, GEO and DADS significantly reversed the sucrose preference index decrease induced by 5 weeks of UCMS. GEO (25 mg per kg bw) effectively decreased the frontal cortex turnover ratio of serotonin (5-HT) and dopamine (DA), thus increasing the 5-HT and DA levels, with no hippocampal effects. Chronic GEO treatment increased hippocampal brain-derived neurotrophic factor (BDNF), c-AMP response element binding protein (CREB), and protein kinase B (AKT) expression, exhibiting its effects via monoamine neurotransmitter modulation and the BDNF-related signaling pathway.

Deposition of Silicon-Based Stacked Layers for Flexible Encapsulation of Organic Light Emitting Diodes.

In this study, inorganic silicon oxide (SiOx)/organic silicon (SiCxHy) stacked layers were deposited by a radio frequency inductively coupled plasma chemical vapor deposition system as a gas diffusion barrier for organic light-emitting diodes (OLEDs). The effects of thicknesses of SiOx and SiCxHy layers on the water vapor transmission rate (WVTR) and residual stress were investigated to evaluate the encapsulation capability. The experimental results showed that the lowest WVTR and residual stress were obtained when the thicknesses of SiOx and SiCxHy were 300 and 30 nm, respectively. Finally, different numbers of stacked pairs of SiOx/SiCxHy were applied to OLED encapsulation. The OLED encapsulated with the six-pair SiOx/SiCxHy exhibited a low turn-on voltage and low series resistance, and device lifetime increased from 7 h to more than 2000 h.

Label-Free Optical Single-Molecule Micro- and Nanosensors.

Label-free optical sensor systems have emerged that exhibit extraordinary sensitivity for detecting physical, chemical, and biological entities at the micro/nanoscale. Particularly exciting is the detection and analysis of molecules, on miniature optical devices that have many possible applications in health, environment, and security. These micro- and nanosensors have now reached a sensitivity level that allows for the detection and analysis of even single molecules. Their small size enables an exceedingly high sensitivity, and the application of quantum optical measurement techniques can allow the classical limits of detection to be approached or surpassed. The new class of label-free micro- and nanosensors allows dynamic processes at the single-molecule level to be observed directly with light. By virtue of their small interaction length, these micro- and nanosensors probe light-matter interactions over a dynamic range often inaccessible by other optical techniques. For researchers entering this rapidly advancing field of single-molecule micro- and nanosensors, there is an urgent need for a timely review that covers the most recent developments and that identifies the most exciting opportunities. The focus here is to provide a summary of the recent techniques that have either demonstrated label-free single-molecule detection or claim single-molecule sensitivity.

Early molecular events associated with nitrogen deficiency in rice seedling roots.

Nitrogen (N) deficiency is one of the most common problems in rice. The symptoms of N deficiency are well documented, but the underlying molecular mechanisms are largely unknown in rice. Here, we studied the early molecular events associated with N starvation (-N, 1 h), focusing on amino acid analysis and identification of -N-regulated genes in rice roots. Interestingly, levels of glutamine rapidly decreased within 15 min of -N treatment, indicating that part of the N-deficient signals could be mediated by glutamine. Transcriptome analysis revealed that genes involved in metabolism, plant hormone signal transduction (e.g. abscisic acid, auxin, and jasmonate), transporter activity, and oxidative stress responses were rapidly regulated by -N. Some of the -N-regulated genes encode transcription factors, protein kinases and protein phosphatases, which may be involved in the regulation of early -N responses in rice roots. Previously, we used similar approaches to identify glutamine-, glutamate-, and ammonium nitrate-responsive genes. Comparisons of the genes induced by different forms of N with the -N-regulated genes identified here have provided a catalog of potential N regulatory genes for further dissection of the N signaling pathwys in rice.

Thin-Film Coated Plastic Wrap for Food Packaging.

In this study, the antimicrobial property and food package capability of polymethylpentene (PMP) substrate with silicon oxdie (SiOx) and organic silicon (SiCxHy) stacked layers deposited by an inductively coupled plasma chemical vapor deposition system were investigated. The experimental results show that the stacked pair number of SiOx/SiCxHy on PMP is limited to three pairs, beyond which the films will crack and cause package failure. The three-pair SiOx/SiCxHy on PMP shows a low water vapor transmission rate of 0.57 g/m²/day and a high water contact angle of 102°. Three-pair thin-film coated PMP demonstrates no microbe adhesion and exhibits antibacterial properties within 24 h. Food shelf life testing performed at 28 °C and 80% humidity reports that the three-pair thin-film coated PMP can enhance the food shelf-life to 120 h. The results indicate that the silicon-based thin film may be a promising material for antibacterial food packaging applications to extend the shelf-life of food products.

Metal-Enhanced Fluorescence of Silver Island Associated with Silver Nanoparticle.

The coupling plasmon of a hybrid nanostructure, silver island (SI) associated with silver nanoparticle (SNP), on metal-enhanced fluorescence (MEF) was studied theoretically. We used the multiple multipole method to analyze the plasmon-mediated enhancement factor on the fluorescence of a molecule immobilized on SNP and located in the gap zone between SI and SNP; herein, the SI was modeled as an oblate spheroid. Numerical results show that the enhancement factor of the hybrid nanostructure is higher than that of a SNP or a SI alone due to the coupled gap mode. This finding is in agreement with the previous experimental results. In addition, the plasmon band of the structure is broadband and tunable, which can be red-shifted and broadened by flattening or enlarging SI. Based on this property, the hybrid nanostructure can be tailored to obtain the optimal enhancement factor on a specific molecule according to its excitation spectrum. Moreover, we found that there is an induced optical force allowing SNP be attracted by SI. Consequently, the gap is reduced gradually to perform a stronger MEF effect.

Resonantly Increased Optical Frequency Conversion in Atomically Thin Black Phosphorus.

Optical frequency conversion via the nonlinear effect of third harmonic generation is shown to be resonantly enhanced in few-layer black phosphorus. This feature is believed to be a consequence of exciton-related resonance, as the enhancement is strongly correlated with the observation of exciton-recombination photoluminescence. Few-layer thicknesses are obtained both via mechanical exfoliation and laser thinning.