Inhibition of Immunoproteasome Attenuates NLRP3 Inflammasome Response by Regulating E3 Ubiquitin Ligase TRIM31.

Excessive secretion of pro-inflammatory cytokines leads to the disruption of intestinal barrier in inflammatory bowel disease (IBD). The inflammatory cytokine tumor necrosis factor alpha (TNFα) induces the assembly of the NLRP3 inflammasome, resulting in the augmented secretion of inflammatory cytokines implicated in the pathogenesis of inflammatory bowel disease (IBD). TNFα has also been known to induce the formation of immunoproteasome (IP), which incorporates immunosubunits LMP2, LMP7, and MECL-1. Inhibition of IP activity using the IP subunit LMP2-specific inhibitor YU102, a peptide epoxyketone, decreased the protein levels of NLRP3 and increased the K48-linked polyubiquitination levels of NLRP3 in TNFα-stimulated intestinal epithelial cells. We observed that inhibition of IP activity caused an increase in the protein level of the ubiquitin E3 ligase, tripartite motif-containing protein 31 (TRIM31). TRIM31 facilitated K48-linked polyubiquitination and proteasomal degradation of NLRP3 with an enhanced interaction between NLRP3 and TRIM31 in intestinal epithelial cells. In addition, IP inhibition using YU102 ameliorated the symptoms of colitis in the model mice inflicted with dextran sodium sulfate (DSS). Administration of YU102 in the DSS-treated colitis model mice caused suppression of the NLRP3 protein levels and accompanied inflammatory cytokine release in the intestinal epithelium. Taken together, we demonstrated that inhibiting IP under inflammatory conditions induces E3 ligase TRIM31-mediated NLRP3 degradation, leading to attenuation of the NLRP3 inflammatory response that triggers disruption of intestinal barrier.

Poly(ethylene glycol)-Engrafted Graphene Oxide for Gene Delivery and Nucleic Acid Amplification.

Graphene oxide (GO) is an oxidized form of graphene accommodating various oxygen-containing functional groups such as hydroxyl, epoxy, and carboxyl groups on its surface. GO has been extensively utilized in various biomedical applications including the delivery of biomolecules and the development of biosensors owing to its beneficial properties such as high surface area, nucleic acid adsorption, and fluorescence quenching through fluorescence resonance energy transfer (FRET). However, despite these favorable properties, the direct utilization of GO in these applications is often limited by low dispersibility in a physiological medium, cytotoxicity, low biocompatibility, and a strong binding affinity of nucleic acids to GO surface. The large surface area of GO and the presence of various functional groups on its surface make it highly amenable to facile surface modifications, offering scope for GO surface functionalization to overcome these limitations. When polyethylene glycol (PEG), which is a biocompatible polymer, is conjugated to GO, the PEGylated GO enhances the biocompatibility and dispersibility, reduces cytotoxicity, and allows controlled drug delivery with controllable binding affinity towards nucleic acid. PEG-engrafted GO retains the beneficial properties of GO while effectively addressing its limitations, rendering it suitable for various biomedical applications. In this review, we present the recent advancements of PEGylated GO in gene/drug delivery and the facilitation of nucleic acid amplification techniques, which aid in the development of therapeutic and diagnostic tools, respectively.

Comparative Evaluation of the Efficacy of Combined Intramedullary Pinning with K-Wires Pinning in the Treatment of Fifth Metacarpal Neck Fractures versus Conventional Techniques-K-Wires Pinning and Intramedullary Pinning.

Background and Objectives: Since the neck is the weakest part of the metacarpals, the most common metacarpal fracture is a neck fracture, a type which accounts for 38% of all hand fractures. Such fractures can be fixed using a variety of conventional techniques, including intramedullary pinning and K-wire pinning. However, conventional techniques involve complications, such as angulation, stiffness, and rotational deformity. The purpose of this study was to compare the usefulness of our new technique, combined intramedullary pinning with K-wire pinning (IPKP), with those of intramedullary pinning (IP) and K-wire pinning (KP). Materials and Methods: This was a single-center, randomized controlled trial conducted between January 2005 and April 2023. A total of 158 patients with acute displaced fractures of the fifth-metacarpal neck were randomly assigned to either the IPKP group (n = 48), the KP group (n = 60), or the IP group (n = 50). We radiographically evaluated angulation and shortening in three visits: pre-operatively, post-operatively, and at a 1-year follow-up. We clinically evaluated the ranges of motion and Quick-DASH scores to assess daily living performance and the cosmetic scores, using the SBSES score, to assess patients' satisfaction with their cosmetic outcomes. Results: The IPKP group was superior to the KP group and the IP group regarding radiographical and clinical assessments at the 1-year follow-up visit. The angulation was 15.7° (±7.7) in the KP group, 17.0° (±5.9) in the IP group, and 12.6° (±2.5) in the IPKP group (p < 0.001) at the 1-year follow-up visit. The shortening was 0.9 mm (±0.3) in the KP group, 1.4 mm (±0.2) in the IP group, and 0.4 mm (±0.1) in the IPKP group (m < 0.001) at the 1-year follow-up visit. The TAM was 272.6° (±17.5) in the KP group, 271.1° (±18.0) in the IP group, and 274.1° (±14.9) in the IPKP group (p = 0.42). Four patients (6.6%) in the KP group and two patients (4%) in the IP group were reported as having stiffness, while no patients were found to have stiffness in the IPKP group. The average Quick-DASH score was 2.3 (±0.5) in the KP group, 2.5 (±0.4) in the IP group, and 1.9 (±0.4) in the IPKP group (p > 0.05). The average cosmetic score was 3.7 (±1.2) in the KP group, 3.8 (±0.9) in the IP group, and 4.7 (±0.8) in the IPKP group (p < 0.001). A complication involving nonunion occurred in one case (1.6%) in the KP group, while there were three cases (6%) of rotational deformity in the IP groups. Conclusions: With the IPKP technique, accurate reduction can be achieved to improve hand function and cosmetic outcomes.

Fluorometric Detection of SARS-CoV-2 Single-Nucleotide Variant L452R Using Ligation-Based Isothermal Gene Amplification.

Since the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant was first discovered, several variants showing different infectivity and immune responses have emerged globally. As the conventional method, whole-genome sequencing following polymerase chain reaction (PCR) is currently used for diagnosis of SARS-CoV-2 mutations. However, these conventional PCR-based direct DNA sequencing methods are time-consuming, complicated, and require expensive DNA sequencing modules. Here, we developed a fluorometric method for the accurate detection of a single missense mutation of U to G in the spike (S) gene that changes leucine to arginine (L452R) in SARS-CoV-2 genomic RNA. Our method for the detection of single-nucleotide mutations (SNM) in the viral RNA genome includes RNA sequence-dependent DNA ligation and tandem isothermal gene amplification methods, such as strand displacement amplification (SDA) and rolling circle amplification (RCA) generating G-quadruplex (GQ). In the presence of SNM in the viral RNA, ligation of both ends of the probe DNAs occurs between 5'-phosphorylated hairpin DNA and linear probe DNA that can discriminate a single base mismatch. The ligated DNAs were then extended to generate long-stem hairpin DNAs that are subjected to the first isothermal gene amplification (SDA). SDA produces multitudes of short ssDNA from the long-stem hairpin DNAs, which then serve as primers by annealing to circular padlock DNA for the second isothermal gene amplification (RCA). RCA produces a long stretch of ssDNA containing GQ structures. Thioflavin T (ThT) is then intercalated into GQ and emits green fluorescence, which allows the fluorometric identification of SARS-CoV-2 variants. This fluorometric analysis sensitively distinguished SNM in the L452R variant of SARS-CoV-2 RNA as low as 10 pM within 2 h. Hence, this fluorometric detection method using ligation-assisted tandem isothermal gene amplification can be applied for the diagnosis of SARS-CoV-2 SNM variants with high accuracy and sensitivity, without the need for cumbersome whole-genome DNA sequencing.

Mesoporous Silica Particle as an RNA Adsorbent for Facile Purification of In Vitro-Transcribed RNA.

Messenger RNA vaccines against SARS-CoV-2 hold great promise for the treatment of a wide range of diseases by using mRNA as a tool for generating vaccination antigens as well as therapeutic proteins in vivo. Increasing interest in mRNA preparation warrants reliable methods for in vitro transcription (IVT) of mRNA, which must entail the elimination of surplus side products such as immunogenic double-stranded RNA (dsRNA). We developed a facile method for the removal of dsRNA from in vitro transcribed RNA with mesoporous silica particles as RNA adsorbents. Various polyamines were tested for the facilitation of RNA adsorption onto mesoporous silica particles in the chromatography. Among the polyamines tested for RNA adsorption, spermidine showed a superior capability of RNA binding to the silica matrix. Mesoporous silica-adsorbed RNA was readily desorbed with elution buffer containing either salt, EDTA, or urea, possibly by disrupting electrostatic interaction and hydrogen bonding between RNA and the silica matrix. Purification of IVT RNA was enabled with the adsorption of RNA to mesoporous silica in a spermidine-containing buffer and subsequent elution with EDTA. By differing EDTA concentration in the eluting buffer, we demonstrated that at least 80% of the dsRNA can be removed from the mesoporous silica-adsorbed RNA. When compared with the cellulose-based removal of dsRNA from IVT RNA, the mesoporous silica-based purification of IVT RNA using spermidine and EDTA in binding and elution, respectively, exhibited more effective removal of dsRNA contaminants from IVT RNA. Thus, mRNA purification with mesoporous silica particles as RNA adsorbents is applicable for the facile preparation of nonimmunogenic RNA suitable for in vivo uses.

How we compare areas: The underlying mechanism of the elongation bias.

Across four experiments, we investigate the mechanism that underlies the elongation bias. We find individuals tasked with assessing the area of two objects do so by comparing the objects' dimensions, and thus subtle changes in the objects' dimensions can impact area assessments. Because a typical elongation bias experiment places two objects side-by-side horizontally and varies the elongation ratio while maintaining the same area, height is generally easier to compare than width. Thus, there will exist a region where the change in height noticeably crosses a perceptual just noticeable difference boundary, but the corresponding change in width does not, and individuals will tend to perceive that the taller object has a greater area or volume. Consistent with this proposed process, we suggest that, although the elongation bias occurs under a comparative judgment, it does not do so under a single judgment situation. This research contributes to our wider understanding of the visual processes underlying area comparisons.

A Novel Time-Resolved Fluorescence Resonance Energy Transfer Assay for the Discovery of Small-Molecule Inhibitors of HIV-1 Tat-Regulated Transcription.

Human immunodeficiency virus-1 (HIV-1) transactivator (Tat)-mediated transcription is essential for HIV-1 replication. It is determined by the interaction between Tat and transactivation response (TAR) RNA, a highly conserved process representing a prominent therapeutic target against HIV-1 replication. However, owing to the limitations of current high-throughput screening (HTS) assays, no drug that disrupts the Tat-TAR RNA interaction has been uncovered yet. We designed a homogenous (mix-and-read) time-resolved fluorescence resonance energy transfer (TR-FRET) assay using europium cryptate as a fluorescence donor. It was optimized by evaluating different probing systems for Tat-derived peptides or TAR RNA. The specificity of the optimal assay was validated by mutants of the Tat-derived peptides and TAR RNA fragment, individually and by competitive inhibition with known TAR RNA-binding peptides. The assay generated a constant Tat-TAR RNA interaction signal, discriminating the compounds that disrupted the interaction. Combined with a functional assay, the TR-FRET assay identified two small molecules (460-G06 and 463-H08) capable of inhibiting Tat activity and HIV-1 infection from a large-scale compound library. The simplicity, ease of operation, and rapidity of our assay render it suitable for HTS to identify Tat-TAR RNA interaction inhibitors. The identified compounds may also act as potent molecular scaffolds for developing a new HIV-1 drug class.

Recent Trends in Lateral Flow Immunoassays with Optical Nanoparticles.

Rapid, accurate, and convenient diagnosis is essential for effective disease management. Various detection methods, such as enzyme-linked immunosorbent assay, have been extensively used, with lateral flow immunoassay (LFIA) recently emerging as a major diagnostic tool. Nanoparticles (NPs) with characteristic optical properties are used as probes for LFIA, and researchers have presented various types of optical NPs with modified optical properties. Herein, we review the literature on LFIA with optical NPs for the detection of specific targets in the context of diagnostics.

Facilitation of Dye-Based Quantitative Real-Time Polymerase Chain Reaction with Poly(ethylene glycol)-Engrafted Graphene Oxide.

Quantitative real-time polymerase chain reaction (qPCR) is an important and extensively utilized technique in medical and biotechnological applications. qPCR enables the real-time detection of nucleic acid during amplification, thus surpassing the necessity of post-amplification gel electrophoresis for amplicon detection. Despite being widely employed in molecular diagnostics, qPCR exhibits limitations attributed to nonspecific DNA amplification that compromises the efficiency and fidelity of qPCR. Herein, we demonstrate that poly(ethylene glycol)-engrafted nanosized graphene oxide (PEG-nGO) can significantly improve the efficiency and specificity of qPCR by adsorbing single-stranded DNA (ssDNA) without affecting the fluorescence of double-stranded DNA binding dye during DNA amplification. PEG-nGO adsorbs surplus ssDNA primers in the initial phase of PCR, having lower concentrations of DNA amplicons and thus minimizing the nonspecific annealing of ssDNA and false amplification due to primer dimerization and erroneous priming. As compared to conventional qPCR, the addition of PEG-nGO and the DNA binding dye, EvaGreen, in the qPCR setup (dubbed as PENGO-qPCR) significantly enhances the specificity and sensitivity of DNA amplification by preferential adsorption of ssDNA without inhibiting DNA polymerase activity. The PENGO-qPCR system for detection of influenza viral RNA exhibited a 67-fold higher sensitivity than the conventional qPCR setup. Thus, the performance of a qPCR can be greatly enhanced by adding PEG-nGO as a PCR enhancer as well as EvaGreen as a DNA binding dye to the qPCR mixture, which exhibits a significantly improved sensitivity of the qPCR.

Reduced secretion of LCN2 (lipocalin 2) from reactive astrocytes through autophagic and proteasomal regulation alleviates inflammatory stress and neuronal damage.

LCN2/neutrophil gelatinase-associated lipocalin/24p3 (lipocalin 2) is a secretory protein that acts as a mammalian bacteriostatic molecule. Under neuroinflammatory stress conditions, LCN2 is produced and secreted by activated microglia and reactive astrocytes, resulting in neuronal apoptosis. However, it remains largely unknown whether inflammatory stress and neuronal loss can be minimized by modulating LCN2 production and secretion. Here, we first demonstrated that LCN2 was secreted from reactive astrocytes, which were stimulated by treatment with lipopolysaccharide (LPS) as an inflammatory stressor. Notably, we found two effective conditions that led to the reduction of induced LCN2 levels in reactive astrocytes: proteasome inhibition and macroautophagic/autophagic flux activation. Mechanistically, proteasome inhibition suppresses NFKB/NF-κB activation through NFKBIA/IκBα stabilization in primary astrocytes, even under inflammatory stress conditions, resulting in the downregulation of Lcn2 expression. In contrast, autophagic flux activation via MTOR inhibition reduced the intracellular levels of LCN2 through its pre-secretory degradation. In addition, we demonstrated that the N-terminal signal peptide of LCN2 is critical for its secretion and degradation, suggesting that these two pathways may be mechanistically coupled. Finally, we observed that LPS-induced and secreted LCN2 levels were reduced in the astrocyte-cultured medium under the above-mentioned conditions, resulting in increased neuronal viability, even under inflammatory stress.Abbreviations: ACM, astrocyte-conditioned medium; ALP, autophagy-lysosome pathway; BAF, bafilomycin A1; BTZ, bortezomib; CHX, cycloheximide; CNS, central nervous system; ER, endoplasmic reticulum; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; JAK, Janus kinase; KD, knockdown; LCN2, lipocalin 2; LPS, lipopolysaccharide; MACS, magnetic-activated cell sorting; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; MTOR, mechanistic target of rapamycin kinase; NFKB/NF-κB, nuclear factor of kappa light polypeptide gene enhancer in B cells 1, p105; NFKBIA/IκBα, nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha; OVEX, overexpression; SLC22A17, solute carrier family 22 member 17; SP, signal peptide; SQSTM1, sequestosome 1; STAT3, signal transducer and activator of transcription 3; TNF/TNF-α, tumor necrosis factor; TUBA, tubulin, alpha; TUBB3/ß3-TUB, tubulin, beta 3 class III; UB, ubiquitin; UPS, ubiquitin-proteasome system.

AMPK-induced mitochondrial biogenesis decelerates retinal pigment epithelial cell degeneration under nutrient starvation.

The implications of nutrient starvation due to aging on the degeneration of the retinal pigment epithelium (RPE) is yet to be fully explored. We examined the involvement of AMPK activation in mitochondrial homeostasis and its relationship with the maintenance of a healthy mitochondrial population and epithelial characteristics of RPE cells under nutrient starvation. Nutrient starvation induced mitochondrial senescence, which led to the accumulation of reactive oxygen species (ROS) in RPE cells. As nutrient starvation persisted, RPE cells underwent pathological epithelial-mesenchymal transition (EMT) via the upregulation of TWIST1, a transcription regulator which is activated by ROS-induced NF-κB signaling. Enhanced activation of AMPK with metformin decelerated mitochondrial senescence and EMT progression through mitochondrial biogenesis, primed by activation of PGC1-α. Thus, by facilitating mitochondrial biogenesis, AMPK protects RPE cells from the loss of epithelial integrity due to the accumulation of ROS in senescent mitochondria under nutrient starvation. [BMB Reports 2023; 56(2): 84-89].

Highly Bright Silica-Coated InP/ZnS Quantum Dot-Embedded Silica Nanoparticles as Biocompatible Nanoprobes.

Quantum dots (QDs) have outstanding optical properties such as strong fluorescence, excellent photostability, broad absorption spectra, and narrow emission bands, which make them useful for bioimaging. However, cadmium (Cd)-based QDs, which have been widely studied, have potential toxicity problems. Cd-free QDs have also been studied, but their weak photoluminescence (PL) intensity makes their practical use in bioimaging challenging. In this study, Cd-free QD nanoprobes for bioimaging were fabricated by densely embedding multiple indium phosphide/zinc sulfide (InP/ZnS) QDs onto silica templates and coating them with a silica shell. The fabricated silica-coated InP/ZnS QD-embedded silica nanoparticles (SiO2@InP QDs@SiO2 NPs) exhibited hydrophilic properties because of the surface silica shell. The quantum yield (QY), maximum emission peak wavelength, and full-width half-maximum (FWHM) of the final fabricated SiO2@InP QDs@SiO2 NPs were 6.61%, 527.01 nm, and 44.62 nm, respectively. Moreover, the brightness of the particles could be easily controlled by adjusting the amount of InP/ZnS QDs in the SiO2@InP QDs@SiO2 NPs. When SiO2@InP QDs@SiO2 NPs were administered to tumor syngeneic mice, the fluorescence signal was prominently detected in the tumor because of the preferential distribution of the SiO2@InP QDs@SiO2 NPs, demonstrating their applicability in bioimaging with NPs. Thus, SiO2@InP QDs@SiO2 NPs have the potential to successfully replace Cd-based QDs as highly bright and biocompatible fluorescent nanoprobes.

Identification of 3-Oxindole Derivatives as Small Molecule HIV-1 Inhibitors Targeting Tat-Mediated Viral Transcription.

The heterocyclic indole structure has been shown to be one of the most promising scaffolds, offering various medicinal advantages from its wide range of biological activity. Nonetheless, the significance of 3-oxindole has been less known. In this study, a series of novel 3-oxindole-2-carboxylates were synthesized and their antiviral activity against human immunodeficiency virus-1 (HIV-1) infection was evaluated. Among these, methyl (E)-2-(3-chloroallyl)-4,6-dimethyl-one (6f) exhibited the most potent inhibitory effect on HIV-1 infection, with a half-maximal inhibitory concentration (IC50) of 0.4578 µM but without severe cytotoxicity (selectivity index (SI) = 111.37). The inhibitory effect of these compounds on HIV-1 infection was concordant with their inhibitory effect on the viral replication cycle. Mode-of-action studies have shown that these prominent derivatives specifically inhibited the Tat-mediated viral transcription on the HIV-1 LTR promoter instead of reverse transcription or integration. Overall, our findings indicate that 3-oxindole derivatives could be useful as a potent scaffold for the development of a new class of anti-HIV-1 agents.

Enhanced Specificity in Loop-Mediated Isothermal Amplification with Poly(ethylene glycol)-Engrafted Graphene Oxide for Detection of Viral Genes.

Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification method that allows the simple, quick, and low-cost detection of various viral genes. LAMP assays are susceptible to generating non-specific amplicons, as high concentrations of DNA primers can give rise to primer dimerization and mismatched hybridizations, resulting in false-positive signals. Herein, we reported that poly(ethylene glycol)-engrafted nanosized graphene oxide (PEG-nGO) can significantly enhance the specificity of LAMP, owing to its ability to adsorb single-stranded DNA (ssDNA). By adsorbing surplus ssDNA primers, PEG-nGO minimizes the non-specific annealing of ssDNAs, including erroneous priming and primer dimerization, leading to the enhanced specificity of LAMP. The detection of complementary DNAs transcribed from the hepatitis C virus (HCV) RNA was performed by the PEG-nGO-based LAMP. We observed that the inclusion of PEG-nGO significantly enhances the specificity and sensitivity of the LAMP assay through the augmented difference in fluorescence signals between the target and non-target samples. The PEG-nGO-based LAMP assay greatly facilitates the detection of HCV-positive clinical samples, with superior precision to the conventional quantitative real-time PCR (RT-qPCR). Among the 20 clinical samples tested, all 10 HCV-positive samples are detected as positive in the PEG-nGO-based LAMP, while only 7 samples are detected as HCV-positive in the RT-qPCR. In addition, the PEG-nGO-based LAMP method significantly improves the detection precision for the false-positive decision by 1.75-fold as compared to the LAMP without PEG-nGO. Thus, PEG-nGO can significantly improve the performance of LAMP assays by facilitating the specific amplification of target DNA with a decrease in background signal.

Inhibition of immunoproteasome attenuates NLRP3 inflammasome formation in tumor necrosis factor α-stimulated intestinal epithelial cell.

Excessive release of inflammatory cytokines has been considered as a major cause of chronic inflammation, resulting in intestinal barrier disruption that leads to inflammatory bowel disease (IBD). Tumor necrosis factor α (TNFα) is one of the well-known inflammatory cytokines that activates formation of NLRP3 inflammasome, thus resulting in excessive secretion of inflammatory cytokines causing IBD. Although immunoproteasome inhibitors have been reported to inhibit inflammatory cytokine release, immunoproteasome inhibition has not yet been addressed for attenuation of NLRP3 inflammasome activity in intestinal epithelial cell. Here, we observed that NLRP3 inflammasome assembly was attenuated by peptide epoxyketone YU102, a LMP2 subunit immunoproteasome inhibitor, in intestinal epithelial cell. YU102 also inhibited maturation of active caspase-1 and secretion of IL-1ß, which are subsequent inflammatory cascade after the formation of NLRP3 inflammasome. Progression of epithelial-mesenchymal transition and increase of cellular permeability, which were induced by TNFα, were also suppressed through inhibition of immunoproteasome. Furthermore, we found that YU102 does not inhibit degradation of IкBα and its following NF-кB activation that leads to transcription of NLRP3. These findings suggest that inhibition of immunoproteasome with YU102 offers a potential therapeutic premise for prevention of TNFα-induced chronic inflammation through attenuation of NLRP3 inflammasome assembly.

Aptamer-conjugated nano-liposome for immunogenic chemotherapy with reversal of immunosuppression.

Cancer cells have various immune evasion mechanisms that resist the immune cells by reprogramming the tumor microenvironment (TME), such as programmed death-ligand 1 (PD-L1) and indoleamine 2,3-dioxygenase-1 (IDO1) overexpression. One of the approaches to restore antitumor immune response by T-cells is through induction of immunogenic cell death (ICD). Thus, drug carrier containing IDO1 siRNA and ICD inducer would be effective anticancer regimen to modulate the immunosuppressive TME by reversing the IDO1-mediated immunosuppression in a synergistic combination with ICD induction. However, numerous nanocarrier platforms for co-delivery of multiple drugs mostly depend on the enhanced permeation and retention (EPR), which is insufficient to achieve selectivity in tumor sites harboring various types of cells. We designed a targeted drug delivery system using nano-sized liposomes functionalized with anti-CD44 and anti-PD-L1 DNA aptamers, which target breast cancer cells and inhibit PD-1/PD-L1 interaction between cancer cells and T-cells. To reverse immunosuppressive TME and reactivate immune response, cancer-targeting nano-liposomes were prepared to contain immunogenic cell death inducer (Doxorubicin, DOX) and IDO1 siRNA, namely Aptm[DOX/IDO1]. The Aptm[DOX/IDO1] specifically delivered the loaded DOX and IDO1 siRNA into target breast cancer cells through aptamer-mediated endocytosis. Cancer-targeted DOX/IDO1 siRNA delivery enhanced ICD and suppressed IDO1 expression with significantly high toxicity in cancer cells. We demonstrated that Aptm[DOX/IDO1] could achieve synergistic antitumor effects by facilitating ICD response and simultaneous reversal of the immunosuppressive TME with IDO1 knockdown in the subcutaneous breast cancer model mice, thus reducing tumor size. These antitumor effects were exerted with intratumoral infiltration of CD8+ cytotoxic T lymphocyte as well as attenuation of regulatory T-cell recruitment in the tumor sites. We further proved that our Aptm[DOX/IDO1] strategy significantly reduced tumor metastasis in tumor-xenograft mice through a synergistic combination of cancer cell-targeted ICD induction and reversal of the IDO1-mediated immunosuppressive TME. Our nanocarrier platform based on cationic liposomes containing DOX and IDO1 siRNA, which are conjugated with two DNA aptamers targeting the cancer cell surface, accomplished synergistic chemoimmunotherapy through tumor-specific immune modulation into immune-favorable TME in vivo.

Detection of SARS-CoV-2 RNA through tandem isothermal gene amplification without reverse transcription.

Diagnosis of SARS-CoV-2 infection through rapid, accurate, and sensitive testing is the most important and fundamental step in coping with the COVID-19 epidemic. We have developed a sensitive fluorometric assay to detect SARS-CoV-2 viral RNA without thermal cycling. This assay system, based on tandem isothermal gene amplification (TIGA), is composed of ternary rolling circle amplification (t-RCA) and subsequent strand displacement amplification (SDA) coupled with G-quadruplex-generating RCA (SDA/GQ-RCA). Without the need to convert viral RNA into cDNA, viral RNA forms a ternary complex composed of hairpin primer (HP) and dumbbell padlock DNA during the t-RCA process. t-RCA generates a long chain of single-stranded DNA (ssDNA) with tandemly repeated hairpin structures that are subjected to SDA. SDA produces multiple short ssDNAs from t-RCA products, which then serve as primers for the second RCA reaction. A long ssDNA harboring repeated copies of the G-quadruplex is produced in the second round of RCA. Emission of enhanced fluorescence by thioflavin T, which intercalates into the G-quadruplex, allows fluorometric detection of amplified viral genes. This fluorometric analysis sensitively detected SARS-CoV-2 RNA as low as 5.9 aM, with a linear range between 0.2 fM and 200 fM within 1 h. Hence, this isothermal gene amplification method without reverse transcription of viral RNA can be applied to diagnose COVID-19 with high sensitivity and accuracy as an alternative to current PCR-based diagnosis.

Use of nanosecond excitation pulses in fluorescence lifetime measurement via phasor analysis.

We investigated the possibility of using long excitation pulses in fluorescence lifetime imaging microscopy (FLIM) using phasor analysis. It has long been believed that the pulse width of an excitation laser must be shorter than the lifetime of a fluorophore in a time-domain FLIM system. Even though phasor analysis can effectively minimize the pulse effect by using deconvolution, the precision of a measured lifetime can be degraded seriously. Here, we provide a fundamental theory on pulse-width-dependent measurement precisions in lifetime measurement in the phasor plane. Our theory predicts that high-precision lifetimes can be obtained even with a laser whose pulse width is four times larger than the lifetime of a fluorophore. We have experimentally demonstrated this by measuring the lifetimes of fluorescence probes with 2.57 ns and 3.75 ns lifetimes by using various pulse widths (0.52-38 ns) and modulation frequencies (10-200 MHz). We believe our results open a new possibility of using long pulse-width lasers for high-precision FLIM.

Bipolar Resistive Switching in Lanthanum Titanium Oxide and an Increased On/Off Ratio Using an Oxygen-Deficient ZnO Interlayer.

The present study pioneered an oxygen migration-driven metal to insulator transition Mott memory, a new type of nonvolatile memory using lanthanum titanium oxide (LTO). We first show the reset first bipolar property without an initial electroforming process in LTO. We used oxygen-deficient ZnO as an interlayer between LTO and a W electrode to clarify whether oxygen migration activates LTO as the Mott transition. ZnO oxygen deficiency provides oxygen ion migration paths as well as a reservoir, facilitating oxygen migration from LTO to the W electrode. Thus, including the ZnO interlayer improved oxygen migration between LTO and the W electrode, achieving a 10-fold increased on/off current ratio. The current research contributes to a better understanding of valence change Mott memory by exploring the LTO resistive switching mechanism and ZnO interlayer influences on the oxygen migration process.