Discovery of new acetamide derivatives of 5-indole-1,3,4-oxadiazol-2-thiol as inhibitors of HIV-1 Tat-mediated viral transcription.

Human immunodeficiency virus-1 (HIV-1) encodes a transcriptional factor called Tat, which is critical for viral transcription. Tat-mediated transcription is highly ordered apart from the cellular manner; therefore, it is considered a target for developing anti-HIV-1 drugs. However, drugs targeting Tat-mediated viral transcription are not yet available. Our high-throughput screen of a compound library employing a dual-reporter assay identified a 1,3,4-oxadiazole scaffold against Tat and HIV-1 infection. Furthermore, a serial structure-activity relation (SAR) study performed with biological assays found 1,3,4-oxadiazole derivatives (9 and 13) containing indole and acetamide that exhibited potent inhibitory effects on HIV-1 infectivity, with half-maximal effective concentrations (EC50) of 0.17 (9) and 0.24 µM (13), respectively. The prominent derivatives specifically interfered with the viral transcriptional step without targeting other infection step(s) and efficiently inhibited the HIV-1 replication cycle in the T cell lines and peripheral blood mononuclear cells infected with HIV-1. Additionally, compared to the wild type, the compounds exhibited similar potency against anti-retroviral drug-resistant HIV-1 strains. In a series of mode-of-action studies, the compounds inhibited the ejection of histone H3 for facilitating viral transcription on the long-terminal repeat (LTR) promoter. Furthermore, SAHA (a histone deacetylase inhibitor) treatment abolished the compound potency, revealing that the compounds can possibly target Tat-regulated epigenetic modulation of LTR to inhibit viral transcription. Overall, our screening identified novel 1,3,4-oxadiazole compounds that inhibited HIV-1 Tat, and subsequent SAR-based optimization led to the derivatives 9 and 13 development that could be a promising scaffold for developing a new class of therapeutic agents for HIV-1 infection.

Autophagosome-lysosome fusion is facilitated by plectin-stabilized actin and keratin 8 during macroautophagic process.

Autophagy is a lysosome-mediated degradative process that removes damaged proteins and organelles, during which autophagosome-lysosome fusion is a key step of the autophagic flux. Based on our observation that intermediate cytofilament keratin 8 (KRT8) enhances autophagic clearance in cells under oxidative stress condition, we investigated whether KRT8 supports the cytoplasmic architectural networks to facilitate the vesicular fusion entailing trafficking onto filamentous tracks. We found that KRT8 interacts with actin filaments via the cytolinker, plectin (PLEC) during trafficking of autophagosome. When PLEC was knocked down or KRT8 structure was collapsed by phosphorylation, autophagosome-lysosome fusion was attenuated. Inhibition of actin polymerization resulted in accumulation of autophagosomes owing to a decrease in autophagosome and lysosome fusion. Furthermore, myosin motor protein was found to be responsible for vesicular trafficking along the actin filaments to entail autolysosome formation. Thus, the autophagosome-lysosome fusion is aided by PLEC-stabilized actin filaments as well as intermediate cytofilament KRT8 that supports the structural integrity of actin filaments during macroautophagic process under oxidative stress condition.

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.

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.

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.

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.

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.

Axial Scanning Metal-Induced Energy Transfer Microscopy for Extended Range Nanometer-Sectioning Cell Imaging.

Nanometer-sectioning optical microscopy has become an indispensable tool in membrane-related biomedical studies. Finally, many nanometer-sectioning imaging schemes, such as variable-angle total internal reflection fluorescence microscopy, metal-induced energy transfer (MIET) imaging, and supercritical-angle fluorescence microscopy have been introduced. However, these methods can measure a single layer of molecules, and the measurement ranges are below 100 nm, which is not large enough to cover the thickness of lamellipodium. This paper proposes an optical imaging scheme that can identify the axial locations of two layers of molecules with an extended measurement range and a nanometer-scale precision by using MIET, axial focal plane scanning, and biexponential analysis in fluorescence lifetime imaging microscopy. The feasibility of the proposed method is demonstrated by measuring an artificial sample of a known structure and the lamellipodium of a human aortic endothelial cell whose thickness ranges from 100 to 450 nm with 18.3 nm precision.

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.

Highly sensitive near-infrared SERS nanoprobes for in vivo imaging using gold-assembled silica nanoparticles with controllable nanogaps.

BACKGROUND: To take advantages, such as multiplex capacity, non-photobleaching property, and high sensitivity, of surface-enhanced Raman scattering (SERS)-based in vivo imaging, development of highly enhanced SERS nanoprobes in near-infrared (NIR) region is needed. A well-controlled morphology and biocompatibility are essential features of NIR SERS nanoprobes. Gold (Au)-assembled nanostructures with controllable nanogaps with highly enhanced SERS signals within multiple hotspots could be a breakthrough. RESULTS: Au-assembled silica (SiO2) nanoparticles (NPs) (SiO2@Au@Au NPs) as NIR SERS nanoprobes are synthesized using the seed-mediated growth method. SiO2@Au@Au NPs using six different sizes of Au NPs (SiO2@Au@Au50-SiO2@Au@Au500) were prepared by controlling the concentration of Au precursor in the growth step. The nanogaps between Au NPs on the SiO2 surface could be controlled from 4.16 to 0.98 nm by adjusting the concentration of Au precursor (hence increasing Au NP sizes), which resulted in the formation of effective SERS hotspots. SiO2@Au@Au500 NPs with a 0.98-nm gap showed a high SERS enhancement factor of approximately 3.8 × 106 under 785-nm photoexcitation. SiO2@Au@Au500 nanoprobes showed detectable in vivo SERS signals at a concentration of 16 µg/mL in animal tissue specimen at a depth of 7 mm. SiO2@Au@Au500 NPs with 14 different Raman label compounds exhibited distinct SERS signals upon subcutaneous injection into nude mice. CONCLUSIONS: SiO2@Au@Au NPs showed high potential for in vivo applications as multiplex nanoprobes with high SERS sensitivity in the NIR region.

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.

Achieving a high photon count rate in digital time-correlated single photon counting using a hybrid photodetector.

We report an enhanced photon count rate in a digitally implemented time-correlated single-photon counting (TCSPC) system by utilizing a hybrid photodetector (HPD). In our digital TCSPC scheme, the photoelectronic responses from a single photon-sensitive photodetector are digitally analyzed through a high-speed analog-to-digital convertor (ADC). By virtue of the HPD which provides nearly a constant signal gain, the single-photon pulses can be effectively distinguished from pulses of simultaneously detected multiple photons by the pulse heights. Consequently, our digital TCSPC system can selectively collect single-photon signals even in the presence of intense multi-photon detections with its temporal accuracy not to be compromised. In our experiment of fluorescence lifetime measurement, the maximum count rate of single photons nearly reached the theoretical limit given by the Poisson statistics. This demonstrated that the digital TCSPC combined with the HPD provides an ultimate solution for the TCSPC implementation for high photon count rates.

Label-free fluorometric detection of influenza viral RNA by strand displacement coupled with rolling circle amplification.

Since influenza occurs globally every year, it is important to develop a facile and accurate method to detect the influenza virus. This study aimed at developing a sensitive fluorometric assay for detecting influenza viral RNA through tandem gene amplification methods including reverse transcription PCR (RT-PCR), followed by strand displacement amplification (SDA) coupled with rolling circle amplification (RCA). Influenza viral RNA was initially amplified by RT-PCR with a tailed reverse primer containing an additional sequence for SDA. The RT-PCR amplicon was then subjected to SDA, yielding multiple copies of single-stranded DNA (ssDNA) that can be used as a primer for subsequent RCA. Thereafter, a long ssDNA segment harboring tandem repeated G-quadruplexes that were generated through RCA was intercalated by Thioflavin T, yielding a strong fluorescence signal indicating the presence of the target viral RNA. Fluorometric analysis detected influenza viral RNA ranging from 50 pg to 500 pg with a limit of detection of 6.2 pg with a signal-to-background ratio of 10 and identified each influenza virus strain (H1N1, H3N2, and influenza B). Thus, the present method for the label-free fluorometric detection of viral RNA via tandem gene amplifications combining RT-PCR-coupled SDA and G-quadruplex-generating RCA would facilitate the efficient diagnosis of influenza infection.

CT features of malignant and benign oral tumors in 28 dogs.

An improved understanding of the computed tomographic (CT) features for malignant versus benign oral tumors would be helpful for guiding prognosis and treatment planning decisions in dogs. This retrospective, multi-center, observational study compared the CT features of malignant and benign tumors in 28 dogs with 31 oral masses. Malignant tumors were present in 20 dogs, including malignant melanoma (n = 14), squamous cell carcinoma (SCC, n = 4), adenocarcinoma (n = 1), and fibrosarcoma (n = 1). Eight dogs had benign tumors, including giant cell granuloma (n = 2), peripheral odontogenic fibroma (n = 2), acanthomatous ameloblastoma (n = 2), plasmacytoma (n = 1), and oncocytoma (n = 1). Common CT features of malignant tumors included heterogeneous enhancement, tumor invasion into the adjacent bone, tooth loss, and ipsilateral mandibular lymphadenopathy. Malignant tumors were significantly larger than benign tumors. Bone lysis was found in benign tumors (n = 4) such as acanthomatous ameloblastoma, giant cell granuloma, and plasmacytoma. The bone lysis was a well-defined geographic area regardless of malignancy and tumor type. In periosteal reactions, amorphous patterns were seen in both malignant (n = 2) and benign tumors (n = 2); the latter subgroup also showed solid patterns. Bone expansion (n = 2) was identified in malignant melanoma and acanthomatous ameloblastoma. Findings supported a diagnosis of possible malignancy for dogs with oral tumors having the following CT characteristics: large size, heterogeneous contrast enhancement pattern, bone lysis, tooth loss, and ipsilateral lymphadenopathy. However, there was a considerable overlap of CT findings among the different types of oral tumors and between benign and malignant tumors. Histological evaluation therefore remains necessary for definitive diagnosis.

The bacterial endoribonuclease RNase E can cleave RNA in the absence of the RNA chaperone Hfq.

RNase E is a component of the RNA degradosome complex and plays a key role in RNA degradation and maturation in Escherichia coli RNase E-mediated target RNA degradation typically involves the RNA chaperone Hfq and requires small guide RNAs (sRNAs) acting as a seed by binding to short (7-12-bp) complementary regions in target RNA sequences. Here, using recombinantly expressed and purified proteins, site-directed mutagenesis, and RNA cleavage and protein cross-linking assays, we investigated Hfq-independent RNA decay by RNase E. Exploring its RNA substrate preferences in the absence of Hfq, we observed that RNase E preferentially cleaves AU-rich sites of single-stranded regions of RNA substrates that are annealed to an sRNA that contains a monophosphate at its 5'-end. We further found that the quaternary structure of RNase E is also important for complete, Hfq-independent cleavage at sites both proximal and distal to the sRNA-binding site within target RNAs containing monophosphorylated 5'-ends. Of note, genetic RNase E variants with unstable quaternary structure exhibited decreased catalytic activity. In summary, our results show that RNase E can degrade its target RNAs in the absence of the RNA chaperone Hfq. We conclude that RNase E-mediated, Hfq-independent RNA decay in E. coli requires a cognate sRNA sequence for annealing to the target RNA, a 5'-monophosphate at the RNA 5'-end, and a stable RNase E quaternary structure.

Identification of novel compounds against Tat-mediated human immunodeficiency virus-1 transcription by high-throughput functional screening assay.

Trans-activator (Tat)-mediated human immunodeficiency virus type 1 (HIV-1) transcription is essential for the replication of HIV-1 and is considered a potent therapeutic target for HIV-1 inhibition. In this study, the Library of Pharmacologically Active Compounds (LOPAC1280) was screened using our dual-reporter screening system for repositioning as Tat-inhibitory compounds. Consequently, two compounds were found to be potent, with low cytotoxicity. Of these two compounds, Roscovitine (CYC202) is already known to be a Tat inhibitor, while gemcitabine has been newly identified as an inhibitor of Tat-mediated transcription linked to viral production and replication. In an additional screening using the ribonucleoside analogues of gemcitabine, two analogues (2'-C-methylcytidine and 3-deazauridine) showed a specific Tat-inhibitory effect linked to their anti-HIV-1 activity. Interestingly, these compounds did not affect Tat protein directly, while the mechanism underlying their inhibition of Tat-mediated transcription was linked to pyrimidine biosynthesis, rather than to alteration of the dNTP pool, influenced by the inhibition of ribonucleotide reductase. Taken together, the proposed functional screening system is a useful tool for the identification of inhibitors of Tat-mediated HIV-1 transcription from among a large number of compounds, and the inhibitory effect of HIV-1 transcription by gemcitabine and its analogues may suggest a strategy for developing a new class of therapeutic anti-HIV drugs.

Deoxygenative Amination of Azine-N-oxides with Acyl Azides via [3 + 2] Cycloaddition.

A transition-metal-free deoxygenative C-H amination reaction of azine-N-oxides with acyl azides is described. The initial formation of an isocyanate from the starting acyl azide via a Curtius rearrangement can trigger a [3 + 2] dipolar cycloaddition of polar N-oxide fragments to generate the aminated azine derivative. The applicability of this method is highlighted by the late-stage and sequential amination reactions of complex bioactive compounds, including quinidine and fasudil. Moreover, the direct transformation of aminated azines into various bioactive N-heterocycles illustrates the significance of this newly developed protocol.

Label-free fluorometric detection of microRNA using isothermal rolling circle amplification generating tandem G-quadruplex.

MicroRNAs (miRNAs) play an important role in various biological processes and have been regarded as promising diagnostic biomarkers for solid tumors in the field of clinical diagnostics. In this study, we developed a simple label/quencher-free fluorometric system for sensitive and selective miRNA detection using isothermal gene amplification such as rolling circle amplification generating tandem G-quadruplex DNA structures (GQ-RCA). The closed-circular dumbbell-shaped padlock DNA was designed to be complementary to its corresponding target miRNA. In the presence of the target miRNA, a long stretch of ssDNA with tandem G-quadruplex sequence repeats was readily generated by RCA, initiated by phi29 DNA polymerase through DNA synthesis priming at the 3'-OH of the target miRNA annealed to the padlock DNA. The RCA product harboring tandem G-quadruplex was monitored with fluorophore Thioflavin T (ThT) that emits strong fluorescence only when it intercalates into the G-quadruplex. The GQ-RCA assay enabled us to detect miRNA as low as 4.9 fM with a linear range from 25.6 fM to 80 pM within 0.5 h. In addition, our system was applied to the miRNA samples present in human plasma, showing its potential use in the clinical diagnosis of cancer.