SARS-CoV-2, the virus responsible for COVID-19, triggers the synthesis of full-length genomic RNA (gRNA) and subgenomic RNAs (sgRNAs) in host cells upon infection. Traditional PCR aimed at targeting gRNA to detect viral presence is insufficient. sgRNAs serve as novel markers for active viral replication. However, the utility of reverse transcription-quantitative PCR (RT-qPCR) assays targeting sgRNAs as indicators of active viral load and infectivity in rodent models has not been validated. We developed four RT-qPCR assays targeting the SARS-CoV-2 genes-ORF1ab, N, E, and E-sgRNA and two RT-qPCR assays for housekeeping genes Hamster G apdh, and Mouse Actb. We used serial dilutions to establish standard curves for quantification. These assays demonstrated high amplification efficiency (96%-97 %) and a strong correlation between the cycle threshold (Ct) values and logarithmic copy number of the genes (R2 = 0.9933-0.9996). Analyzing 102 residual rodent lung tissue samples, we compared the viral loads quantified using RT-qPCR assays with those determined by viral culture. A strong correlation emerged between the RT-qPCR assays' detection of positivity and the viral culture results. Notably, the quantification of viral loads using the E-sgRNA RT-qPCR assay correlated more closely with viral culture outcomes than with other targets (r = 0.93, p < 0.001). These results underscore the sgRNA RT-qPCR assay's potential for tracking actively replicating viruses in rodent models infected with SARS-CoV-2, offering a reliable alternative to traditional viral culture methods.

Liposomal vaccines have been developed extensively in recent years, and considerable progress has been made in the prevention of infectious diseases. Quaternary chitosan-coated liposomes were used in the present study. Liposomes coated with TMC (namely chitosomes) were synthesized in a single microfluidic step in which liposomes were formed and coated with trimethylchitosan (TMC) via covalent and/or hydrogen bonds in situ. The endocytosis of chitosomes by JAWS II dendritic cells was detected using flow cytometry. Chitosomes covalently coated with TMC were more efficiently internalized than uncoated liposomes, showed lower cytotoxicity than those coated via hydrogen bonds, and could significantly lower the cytotoxicity via protein mixing. In a BALB/c model, covalent modification of chitosomes improved the efficiency of anti-PA IgG induction via anthrax vaccine adsorbed (AnV), compared with those with hydrogen bonds and those with AnV only, where humoral immunity is dominant. These results suggest that covalently TMC-coated chitosomes serve as a promising delivery system for proteins and that they can be reproducibly obtained via rapid, cost-efficient, and automated methods with few scale-up barriers; meanwhile, their versatile practicability favors commercialization. Statement of significance In this study, we proved that liposomes coated with TMC (namely chitosomes) via covalent bonds induced better uptake efficiency, stronger in vivo immune responses, and lower cytotoxicity than those coated via hydrogen bonds. The endocytosis efficiency and cytotoxicity of chitosomes were positively correlated with the concentration of the TMC coating. Additionally, this invention provides a facile single-step microfluidic process for the rapid, low-cost, and high-quality preparation of an innovative high-performance chitosomal delivery system that is convenient and easy to scale up. Based on these benefits, the current disclosure provides an alternative platform for the development of new chitosomal drugs.

Flexible laser-scribed graphene (LSG) substrates with gold nanoislands have been developed as biochips for in situ electrochemical (EC) and surface-enhanced Raman scattering (SERS) biodetection (biomolecules and viral proteins). A flexible biochip was fabricated using CO2 laser engraving polyimide (PI) films to form a 3D porous graphene-like nanostructure. Gold nanoislands were deposited on the LSG substrates to enhance the intensity of the Raman signals. Moreover, the addition of auxiliary and reference electrodes induced a dual-function EC-SERS biochip with significantly enhanced detection sensitivity. The biochip could selectively and easily capture SARS-CoV-2 S1 protein through the SARS-CoV-2 S1 antibody immobilized on EC-SERS substrates using 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The grafted antibody specifically bound to SARS-CoV-2, resulting in a significant increase in the SERS signal of the target analyte. The limit of detection (LOD) of the SARS-CoV-2 S1 protein was 5 and 100 ng/mL by using EC and SERS detection, respectively. Although the LOD of the SARS-CoV-2 S1 protein detected using SERS is only 100 ng/mL, it can provide fingerprint information for identification. To improve the LOD, EC detection was integrated with SERS detection. The three-electrode detection chip enables the simultaneous detection of SERS and EC signals, which provides complementary information for target identification. The dual-functional detection technology demonstrated in this study has great potential for biomedical applications, such as the rapid and sensitive detection of SARS-CoV-2.

Since its emergence, SARS-CoV-2 has been continuously evolving, hampering the effectiveness of current vaccines against COVID-19. mAbs can be used to treat patients at risk of severe COVID-19. Thus, the development of broadly protective mAbs and an understanding of the underlying protective mechanisms are of great importance. Here, we isolated mAbs from donors with breakthrough infection with Omicron subvariants using a single-B cell screening platform. We identified a mAb, O5C2, which possesses broad-spectrum neutralization and antibody -dependent cell -mediated cytotoxic activities against SARS-CoV-2 variants, including EG.5.1. Single -particle analysis by cryo-electron microscopy revealed that O5C2 targeted an unusually large epitope within the receptor -binding domain of spike protein that overlapped with the angiotensin-converting enzyme 2 binding interface. Furthermore, O5C2 effectively protected against BA.5 Omicron infection in vivo by mediating changes in transcriptomes enriched in genes involved in apoptosis and interferon responses. Our findings provide insights into the development of pan -protective mAbs against SARS-CoV-2.

This study relates to positively surface-charged liposomes ((+)-LPS) coated with cationic polyelectrolytes via covalent bonds and the preparation method and applications thereof. This nanosized (+)-LPS was synthesized via a single-step microfluidic process, in which the formation of LPS and the coating of N-[(2-hydroxy-3-trimethylammonium)propylchitosan (HTCC) on the surface via covalent bonds were completed in situ. Flow cytometric endocytosis analysis of (+)-LPS on JAWS II dendritic cells demonstrated that HTCC coated via covalent bonds showed higher uptake efficiency than those coated via hydrogen bonds and increased uptake by 100-fold compared to LPS without HTCC coating. HTCC-coated LPS, via covalent binding, also reduced the cytotoxicity after incubation with JAWS II cells. In vivo experiments showed that BALB/c mice vaccinated with anthrax vaccine adsorbed (AVA) mixed with HTCC-covalently coated (+)-LPS could induce IgG anti-PA antibodies in a shorter time and at a higher level than mice vaccinated with AVA plus (+)-LPS coated with HTCC via hydrogen bonds and AVA only and that the humoral immune response was more dominant. Moreover, the preparation process allows rapid and high-quality production of (+)-LPS with low cost and simple operation. Thus, the present disclosure is competitive with practicability, providing an advantage in commercialization.

To enhance the cellular uptake of liposomes, we prepared conventional liposomes with targeting molecules and surface-charged liposomes and evaluated their potential as nano-carriers and vaccine adjuvants by comparing their endocytosis efficiencies using immune cells. Surface-charged liposomes were synthesized via a one-step microfluidic method, which provided a novel, simple, fast, and highly reproducible method for preparing liposomes. Flow cytometry revealed that cationic polyelectrolyte-coated liposomes exhibited higher endocytosis efficiencies (of up to a factor of 100) in A774A.1 cells and JAWs II cells compared with uncoated liposomes or those coated with anionic polyelectrolytes. Positively charged liposomes exhibited some cytotoxicity at quaternary-chitosan coating concentrations higher than 6mg/mL; however, significantly lower cytotoxicities (by a factor of almost ten) were obtained by protein mixing. Furthermore, BALB/c mice vaccinated with a mixture of Anthrax vaccine adsorbed (AVA) and quaternary chitosan-coated liposomes showed faster and stronger anti-PA IgG inductions compared to those vaccinated with AVA alone, with titers positively correlating with the amount of cationic liposome used. This finding clearly reveals that quaternary chitosan-coated liposomes act as both nano-carriers and vaccine adjuvants that significantly enhance in-vivo immune responses to vaccines with low immunogenicities.

This study introduced a novel antiviral approach by combining three substances with different antiviral mechanisms: N-(2-hydroxy)propyl-3-trimethylammoniumchitosan (HTC), silver nanoparticles (AgNPs), and oseltamivir. First, positively surface-charged AgNPs were prepared using an environmentally friendly method. The surfaces of these AgNPs were capped with cationic quaternary chitosan HTC. It exhibits a positive zeta potential with extraordinary stability in aqueous solutions and facilitates substantial and rapid cellular uptake including entry into the cell nucleus. HTC-AgNPs display broad-spectrum antiviral activity against three influenza A viruses (H5N1, H3N2, and H1N1) at biocompatible concentrations. When blended with oseltamivir, HTC-AgNPs enhances the antiviral activity from that of oseltamivir alone by at least 20 times. After 24 h of combined treatment, the inhibition efficiency against influenza A virus can attain up to 99.9 %. We anticipate that this combination could reduce the effective dose of Tamiflu by 10-fold when used in clinic, thus shortening recovery period and lowering the medication costs. Moreover, the synergistic effects of the three active substances would reduce the likelihood of the emergence of drug-resistant viral strains. This would, in turn, enhance the effectiveness and safety of this medication.

ABSTRACT Viral RNA-dependent RNA polymerase (RdRp), a highly conserved molecule in RNA viruses, has recently emerged as a promising drug target for broad-acting inhibitors. Through a Vero E6-based anti-cytopathic effect assay, we found that BPR3P0128, which incorporates a quinoline core similar to hydroxychloroquine, outperformed the adenosine analog remdesivir in inhibiting RdRp activity (EC 50 = 0.66 µM and 3 µM, respectively). BPR3P0128 demonstrated broad-spectrum activity against various severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern. When introduced after viral adsorption, BPR3P0128 significantly decreased SARS-CoV-2 replication; however, it did not affect the early entry stage, as evidenced by a time-of-drug-addition assay. This suggests that BPR3P0128’s primary action takes place during viral replication. We also found that BPR3P0128 effectively reduced the expression of proinflammatory cytokines in human lung epithelial Calu-3 cells infected with SARS-CoV-2. Molecular docking analysis showed that BPR3P0128 targets the RdRp channel, inhibiting substrate entry, which implies it operates differently—but complementary—with remdesivir. Utilizing an optimized cell-based minigenome RdRp reporter assay, we confirmed that BPR3P0128 exhibited potent inhibitory activity. However, an enzyme-based RdRp assay employing purified recombinant nsp12/nsp7/nsp8 failed to corroborate this inhibitory activity. This suggests that BPR3P0128 may inhibit activity by targeting host-related RdRp-associated factors. Moreover, we discovered that a combination of BPR3P0128 and remdesivir had a synergistic effect—a result likely due to both drugs interacting with separate domains of the RdRp. This novel synergy between the two drugs reinforces the potential clinical value of the BPR3P0128-remdesivir combination in combating various SARS-CoV-2 variants of concern.

The ongoing COVID-19 (i.e., coronavirus) pandemic continues to adversely affect the human life, economy, and the world's ecosystem. Although significant progress has been made in developing antiviral materials for the coronavirus, much more work is still needed. In this work, N-functionalized graphene quantum dots (GQDs) were designed and synthesized as the antiviral nanomaterial for Feline Coronavirus NTU156 (FCoV NTU156) and Enterovirus 71 (EV71)) with ultra-high inhibition (>99.9%). To prepare the GQD samples, a unique solid-phase microwave-assisted technique was developed and the cell toxicity was established on the H171 and H184 cell lines after 72 h incubation, indicating superior biocompatibility. The surface functionality of GQDs (i.e., the phenolic and amino groups) plays a vital role in interacting with the receptor-binding-domain of the spike protein. It was also found that the addition of polyethylene glycol is advantageous for the dispersion and the adsorption of functionalized GQDs onto the virus surface, leading to an enhanced virus inhibition. The functionality of as-prepared GQD nanomaterials was further confirmed where a functionalized GQD-coated glass was shown to be extremely effective in hindering the virus spread for a relatively long period (>20 h).

The ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), resulting in the emergence of new variants that are resistant to existing vaccines and therapeutic antibodies, has raised the need for novel strategies to combat the persistent global COVID-19 epidemic. In this study, a monoclonal anti-human angiotensin-converting enzyme 2 (hACE2) antibody, ch2H2, was isolated and humanized to block the viral receptor-binding domain (RBD) binding to hACE2, the major entry receptor of SARS-CoV-2. This antibody targets the RBD-binding site on the N terminus of hACE2 and has a high binding affinity to outcompete the RBD. In vitro, ch2H2 antibody showed potent inhibitory activity against multiple SARS-CoV-2 variants, including the most antigenically drifted and immune-evading variant Omicron. In vivo, adeno-associated virus (AAV)-mediated delivery enabled a sustained expression of monoclonal antibody (mAb) ch2H2, generating a high concentration of antibodies in mice. A single administration of AAV-delivered mAb ch2H2 significantly reduced viral RNA load and infectious virions and mitigated pulmonary pathological changes in mice challenged with SARS-CoV-2 Omicron BA.5 subvariant. Collectively, the results suggest that AAV-delivered hACE2-blocking antibody provides a promising approach for developing broad-spectrum antivirals against SARS-CoV-2 and potentially other hACE2-dependent pathogens that may emerge in the future.

In this study, we report that silver nanocubes (AgNCs)can be synthesizedvia the silver mirror reaction (SMR) within 1 min. To extend the reactiontime, the sharp-edge AgNCs quickly denucleated and collapsed intosmall spherical Ag nanoparticles and became aggregates. Based on thisdiscovery, we developed an easy-to-make membrane-based surface-enhancedRaman scattering (SERS) substrate with superior sensitivity and reproducibility.The entire fabrication process, including AgNC synthesis from SMR,membrane pretreatment, and AgNC deposition on the membrane by suctionfiltration, was completed within 30 min. The SERS intensity of R6Gincreased 48-fold after the pretreatment of the polyvinylidene difluoride(PVDF) membrane with methanol and NaCl aqueous solutions. The PVDF-basedSERS substrate successfully verified attomolar (aM) sensitivity toR6G using 3 x 3 mu L of an aqueous sample on a 3 x 3mm(2) substrate. To validate the practicality of the developedSERS substrate as a SERS sensor, it was applied to the detection ofmalachite green, cocaine, 2,6-pyridinedicarboxylic acid, and tetracycline,with limits of detection of 10 ppb, 1 ppm, 1 ppm, and 1 ppm, respectively.However, the SERS substrate maintained up to 80% sensitivity for only3 days. Fortunately, a freshly prepared SERS substrate can be quicklyfabricated on demand; thus, shelf-life or long-term stability is nolonger a concern.

Gold (Au) nano-island arrays were deposited on the glass substrate to fabricate surface-enhanced Raman scattering (SERS) substrates by in-situ thermal evaporation (deposited and annealed samples at the same time). The optimal SERS intensity deposited by various thicknesses and in-situ annealing temperatures of Au nano-island arrays would be investigated. The biomolecules (adenine) were dropped on the well-designed SERS substrate for precise and quantitative SERS detection. The characterization of Au nano-island arrays SERS substrate would be evaluated by scanning electron microscope (SEM) and Raman spectroscopy. The results showed that the optimal deposition thickness and annealing temperature of Au nano-island arrays SERS substrate is about 14 nm and 200 & DEG;C respectively, which can construct the smallest interparticle spacing (W)/ particle diameter (D) ratio and the lowest reflection (%) and transmittance (%) to form the strongest SERS intensity. Moreover, finite difference time-domain (FDTD) simulation of the electromagnetic field distributions on Au nano-island arrays displays the similar trend with the experimental results. The 14 nm deposition with 200 & DEG;C in-situ annealing temperature would display the highest density of hot-spots by FDTD simulation. The reproducible Au nano-island arrays SERS substrates with tunable surface roughness, W/D ratio, and lower reflection and transmittance show promising potential for SERS detection of biomolecules, bacteria, and viruses.

The incidence of zoonotic diseases, such as coronavirus disease 2019 and Ebola virus disease, is increasing worldwide. However, drug and vaccine development for zoonotic diseases has been hampered because the experiments involving live viruses are limited to high-containment laboratories. The Ebola virus minigenome system enables researchers to study the Ebola virus under BSL-2 conditions. Here, we found that the addition of the nucleocapsid protein of human coronaviruses, such as severe acute respiratory syndrome coronavirus 2, can increase the ratio of green fluorescent protein-positive cells by 1.5-2 folds in the Ebola virus minigenome system. Further analysis showed that the nucleocapsid protein acts as an activator of the Ebola virus minigenome system. Here, we developed an EBOV MiniG Plus system based on the Ebola virus minigenome system by adding the SARS-CoV-2 nucleocapsid protein. By evaluating the antiviral effect of remdesivir and rupintrivir, we demonstrated that compared to that of the traditional Ebola virus minigenome system, significant concentration-dependent activity was observed in the EBOV MiniG Plus system. Taken together, these results demonstrate the utility of adding nucleocapsid protein to the Ebola virus minigenome system to create a powerful platform for screening antiviral drugs against the Ebola virus.

The flexible, anti-fouling, and bionic surface-enhanced Raman scattering (SERS) biochip, which has a Nepenthes peristome-like structure, was fabricated by photolithography, replicated technology, and thermal evaporation. The pattern of the bionic Nepenthes peristome-like structure was fabricated by two layers of photolithography with SU-8 photoresist. The bionic structure was then replicated by polydimethylsiloxane (PDMS) and grafting the zwitterion polymers (2-methacryloyloxyethyl phosphorylcholine, MPC) by atmospheric plasma polymerization (PDMS-PMPC). The phospholipid monomer of MPC immobilization plays an important role; it can not only improve hydrophilicity, anti-fouling and anti-bacterial properties, and biocompatibility, but it also allows for self-driving and unidirectional water delivery. Ag nanofilms (5 nm) were deposited on a PDMS (PDMS-Ag) substrate by thermal evaporation for SERS detection. Characterizations of the bionic SERS chips were measured by a scanning electron microscope (SEM), optical microscope (OM), X-ray photoelectron spectrometer (XPS), Fourier-transform infrared spectroscopy (FTIR), and contact angle (CA) testing. The results show that the superior anti-fouling capability of proteins and bacteria (E. coli) was found on the PDMS-PMPC substrate. Furthermore, the one-way liquid transfer capability of the bionic SERS chip was successfully demonstrated, which provides for the ability to separate samples during the flow channel, and which was detected by Raman spectroscopy. The SERS intensity (adenine, 10−4 M) of PDMS-Ag with a bionic structure is ~4 times higher than PDMS-Ag without a bionic structure, due to the multi-reflection of the 3D bionic structure. The high-sensitivity bionic SERS substrate, with its self-driving water capability, has potential for biomolecule separation and detection.

On analyzing the results of cell-based assays, we have previously shown that perilla (Perilla frutescens) leaf extract (PLE), a food supplement and orally deliverable traditional Chinese medicine approved by the Taiwan Food and Drug Administration, effectively inhibits SARS-CoV-2 by directly targeting virions. PLE was also found to modulate virus-induced cytokine expression levels. In this study, we explored the anti-SARS-CoV-2 activity of PLE in a hamster model by examining viral loads and virus-induced immunopathology in lung tissues. Experimental animals were intranasally challenged with different SARS-CoV-2 doses. Jugular blood samples and lung tissue specimens were obtained in the acute disease stage (3-4 post-infection days). As expected, SARS-CoV-2 induced lung inflammation and hemorrhagic effusions in the alveoli and perivascular areas; additionally, it increased the expression of several immune markers of lung injury - including lung Ki67-positive cells, Iba-1-positive macrophages, and myeloperoxidase-positive neutrophils. Virus-induced lung alterations were significantly attenuated by orally administered PLE. In addition, pretreatment of hamsters with PLE significantly reduced viral loads and immune marker expression. A purified active fraction of PLE was found to confer higher antiviral protection. Notably, PLE prevented SARS-CoV-2-induced increase in serum markers of liver and kidney function as well as the decrease in serum high-density lipoprotein and total cholesterol levels in a dose-dependent fashion. Differently from lung pathology, monitoring of serum biomarkers in Syrian hamsters may allow a more humane assessment of the novel drugs with potential anti-SARS-CoV-2 activity. Our results expand prior research by confirming that PLE may exert an in vivo therapeutic activity against SARS-CoV-2 by attenuating viral loads and lung tissue inflammation, which may pave the way for future clinical applications.

(Background) The coronavirus disease 2019 (COVID-19) that is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) carries high infectivity and mortality. Efficient intervention strategies are urgently needed. Avian immunoglobulin Y (IgY) showed efficacy against viral infection whereas the in vivo efficacy remains unclear. (Methods) We immunized laying hens with S1, S1 receptor-binding domain (S1-RBD), or S2 subunits of the SARS-CoV-2 spike (S) protein. After immunization, IgYs were collected and extracted from the egg yolks. The neutralization potential of IgYs was examined by the plaque reduction neutralization test (PRNT). The bioutility of IgYs was examined in Syrian hamsters in vivo. (Results) IgYs exhibited typical banding patterns in SDS-PAGE and Western blot and were immunoreactive against S1, S1-RBD, and S2 subunits. The plaque reduction neutralization test (PRNT) showed that all purified IgYs potently neutralized different SARS-CoV-2 strains in vitro. In Syrian hamsters, the combination of IgYs for S1-RBD and S2 subunits administered before or after SARS-CoV-2 infection effectively restored body weight loss and reduced intrapulmonary lesions and the amount of immunoreactive N protein-positive cells, which were caused by SARS-CoV-2 infection. (Conclusions) Collectively, IgYs specific for S protein subunits effectively neutralized SARS-CoV-2 in vitro and in vivo and may serve as prophylactic or therapeutic antibodies in the prevention or treatment of COVID-19.

Infections caused by extensively drug-resistant (XDR) Acinetobacter nosocomialis have become a challenging problem. The frequent use of colistin as the last resort drug for XDR bacteria has led to the emergence of colistin-resistant A. nosocomialis (ColRAN) in hospitals. The mechanism of colistin resistance in A. nosocomialis remains unclear. This study aimed to investigate the mechanisms underlying colistin resistance in clinical ColRAN isolates. We collected 36 A. nosocomialis isolates from clinical blood cultures, including 24 ColRAN and 12 colistin-susceptible A. nosocomialis (ColSAN). The 24 ColRAN isolates clustered with ST1272 (13), ST433 (eight), ST1275 (two), and ST410 (one) by multilocus sequence typing. There was a positive relationship between pmrCAB operon expression and colistin resistance. Further analysis showed that colistin resistance was related to an amino acid substitution, Ser253Leu in PmrB. By introducing a series of recombinant PmrB constructs into a PmrB knockout strain and protein structural model analyses, we demonstrated that the association between Ser253Leu and Leu244 in PmrB was coupled with colistin resistance in ColRAN. To the best of our knowledge, this is the first study demonstrating that the key amino acid Ser253Leu in PmrB is associated with overexpression of the pmrCAB operon and hence colistin resistance. This study provides insight into the mechanism of colistin resistance in A. nosocomialis.

The spike (S) protein is a leading vaccine candidate against SARS-CoV-2 infection. The S1 domain of S protein, which contains a critical receptor-binding domain (RBD) antigen, potentially induces protective immunoreactivities against SARS-CoV-2. In this study, we presented preclinical evaluations of a novel insect cell-derived SARS-CoV-2 recombinant S1 (rS1) protein as a potent COVID-19 vaccine candidate. The native antigenicity of rS1 was characterized by enzyme-linked immunosorbent assay with a neutralizing monoclonal antibody targeting the RBD antigen. To improve its immunogenicity, rS1-adjuvanted with fucoidan/trimethylchitosan nanoparticles (FUC-TMC NPs) and cytosine-phosphate-guanosine-oligodeoxynucleotides (CpG-ODNs) were investigated using a mouse model. The S1-specific immunoglobulin G (IgG) titers, FluoroSpot assay, pseudovirus- and prototype SARS-CoV-2-based neutralization assays were assessed. The results showed that the rS1/CpG/ FUC-TMC NPs (rS1/CpG/NPs) formulation induced a broad-spectrum IgG response with potent, long-lasting, and cross-protective neutralizing activity against the emerging SARS-CoV-2 variant of concern, along with a Th1-biased cellular response. Thus, the rS1/CpG/NPs formulation presents a promising vaccination approach against COVID-19.

We report an extremely sensitive and ultra-low-cost PVDF-based SERS substrate using an easy fabrication process that can be conducted in a laboratory and scaled up for industrial applications. Hydrophilic PVDF membranes were sequentially dipped in ethanol and aqueous sodium chloride solution, following which multi-shaped Ag nanoparticles were deposited on the surface of the membranes by suction filtration. The simple solvating pretreatment led to an approximately 188-fold enhancement in the SERS signal intensity of R6G. Using the hydrophilic PVDF-based SERS substrate, subnanomolar sensitivity for R6G was achieved using 3 mu L of sample volume on a 3x3 mm(2) SERS substrate within 1 min detection time and without the need for concentration or drying processes. To demonstrate the feasibility of the developed SERS substrate as a sensor for bacterial spore detection, it was applied for the detection of the representative biomarker dipicolinic acid (DPA). The limits of detection of DPA and Bacillus spores were measured as 1 ppm and 5x10(3) spores/mL, respectively. The optimized configuration of low substrate area and wet micro-analyte volume makes the PVDF-based SERS substrate a simple and low-cost tool for the trace-level detection of small molecules in a rapid, sensitive, and high-throughput manner.

In obese adults, nonalcoholic fatty liver disease (NAFLD) is accompanied by multiple metabolic dysfunctions. Although upregulated hepatic fatty acid synthesis has been identified as a crucial mediator of NAFLD development, the underlying mechanisms are yet to be elucidated. In this study, we reported upregulated expression of gene related to anergy in lymphocytes (GRAIL) in the livers of humans and mice with hepatic steatosis. Grail ablation markedly alleviated the high-fat diet-induced hepatic fat accumulation and expression of genes related to the lipid metabolism, in vitro and in vivo. Conversely, overexpression of GRAIL exacerbated lipid accumulation and enhanced the expression of lipid metabolic genes in mice and liver cells. Our results demonstrated that Grail regulated the lipid accumulation in hepatic steatosis via interaction with sirtuin 1. Thus, Grail poses as a significant molecular regulator in the development of NAFLD.