A Short Fully Covered Self-Expandable Metal Stent for Management of Benign Biliary Stricture Not Caused by Living-Donor Liver Transplantation.

Background: This study evaluated the effectiveness of short fully covered self-expanding metal stents (FCSEMS) with an anti-migration design in treating benign biliary strictures (BBS) not related to living donor liver transplantation (LDLT). Methods: A retrospective analysis was conducted on 75 patients who underwent FCSEMS insertion for BBS management. Stents were initially kept for 3 months and exchanged every 3 months until stricture resolution. Adverse events and stricture recurrence after FCSEMS removal were assessed during follow-up. Results: The study outcomes were technical success, stenosis resolution, and treatment failure. Technical success was 100%, with stricture resolution in 99% of patients. The mean onset time of BBS post-surgery was 4.4 years, with an average stent indwelling period of 5.5 months. Stricture recurrence occurred in 20% of patients, mostly approximately 18.8 months after stent removal. Early cholangitis and stent migration were noted in 3% and 4% of patients, respectively. Conclusions: This study concludes that short FCSEMS demonstrate high efficacy in the treatment of non-LDLT-related BBS, with a low incidence of interventions and complications. Although this is a single-center, retrospective study with a limited sample size, the findings provide preliminary evidence supporting the use of short FCSEMS as a primary treatment modality for BBS. To substantiate these findings, further research involving multicenter studies is recommended to provide additional validation and a broader perspective.

Development of Long Asymmetric siRNA Structure for Target Gene Silencing and Immune Stimulation in Mammalian Cells.

Post-transcriptional regulation of transcript abundances by RNA interference (RNAi) is a widely conserved regulatory mechanism to control cellular processes. We previously introduced an alternative siRNA structure called asymmetric siRNA (asiRNA), and showed that asiRNA exhibits comparable gene-silencing efficiency with reduced off-target effects compared with conventional siRNAs. However, to what extent the length of the guide strand affects the gene-silencing efficiency of asiRNAs is still elusive. In this study, we analyzed in detail the gene-silencing ability of asiRNAs along the guide strand length and immunostimulatory capacity of asiRNAs. We generated asiRNAs containing various guide strand lengths ranging from 25 to 29 nt, called long asiRNA (lasiRNA). We found that the gene-silencing activity of lasiRNAs decreased as the length of the guide strand increased. Nonetheless, the 3'-end overhangs that are complementary to the target gene have higher efficiency for gene silencing compared with mismatched overhangs. In addition, we found that the silencing efficiency of lasiRNAs correlates with their Ago2-binding affinity. Finally, replacing the mismatched overhang with a TLR7- or TLR9-associated immune response motif induced a toll-like receptor (TLR)-specific immune response and retained gene-silencing activity. Our findings demonstrate that lasiRNA structures can be tailored to function as bifunctional siRNA, which trigger a specific immune response combined with target gene silencing. Taken together, we anticipate that our findings provide a road map for the subsequent development of immune-stimulating lasiRNA, which bear the potential to be applied for therapeutic benefits.

Efficacy of Asymmetric siRNA Targeting Androgen Receptors for the Treatment of Androgenetic Alopecia.

Asymmetric small interfering RNAs (asiRNAs) that mediate RNA interference have been investigated for therapeutic use in various tissues, including skin tissue. Androgenetic alopecia (AGA) is caused by a combination of genetic factors, resulting in sensitivity to dihydrotestosterone (DHT), which binds to the androgen receptor (AR) to mediate a series of biomolecular changes leading to hair loss. This study aimed to evaluate the therapeutic potential of a cell-penetrating, AR-targeting asiRNA (cp-asiAR) for AGA treatment, which was designed to silence the AR gene. AGA mouse models were developed by stimulation with DHT, and ex vivo human scalp tissues were also used for analysis. Cp-asiAR-mediated changes in mRNA expression and protein levels of AR were assessed along with the examination of phenotypic improvements in mouse model of AGA. We also assessed downstream signaling associated with AR in primary human dermal papilla (DP) cells. Several cp-asiARs were screened for selecting the optimal sequence of AR using cell lines in vitro. A cholesterol-conjugated, chemically modified cp-asiAR candidate was optimized under passive uptake conditions in vitro. Intradermal cp-asiAR injection efficiently reduced mRNA and protein levels corresponding to AR in mouse models. Moreover, cp-asiAR injection promoted hair growth in mouse models with DHT-induced AGA. In ex vivo human hair follicle culture, the proportion of telogen hair decreased, and the mean hair bulb diameter increased in the cp-asiAR-treated group. In isolated primary human DP cells, AR expression was effectively downregulated by cp-asiAR. Furthermore, cp-asiAR attenuated DHT-mediated increases in interleukin-6, transforming growth factor-ß1, and dickkopf-1 levels. No significant toxicity was observed in DP cells after cp-asiAR treatment. Cp-asiAR treatment showed effective downregulation of AR expression and prevention of DHT-mediated alterations in the hair cycle and hair diameter, indicating its potential as a novel therapeutic option for AGA.

Nonbiodegradable Spiegelmer-Driven Colorimetric Biosensor for Bisphenol A Detection.

Spiegelmers are enantiomers of natural D-oligonucleotides that bind to targets with distinct structures such as aptamers. The high susceptibility of natural D-form aptamers to nucleases greatly hinders their application in biological environments. Here, a nonbiodegradable spiegelmer-based platform for the sensitive detection of bisphenol A (BPA) was developed. Due to the symmetric molecule of BPA, the D-form aptamer can be directly converted into mirror forms via chemical synthesis. Aptamer-target interactions that involve chemically synthesized spiegelmers were characterized by biolayer interferometry, and their stabilities were tested in various biological fluids by exposure to nucleases. We demonstrate for the first time the use of a nuclease-resistant spiegelmer in a simple, label-free gold nanoparticle-based colorimetric assay to detect BPA in a highly sensitive and selective manner. The aptasensor exhibits an LOD of 0.057 ng/mL and dynamic range of 105 (100 pg/mL to 10 mg/mL). With sensing capacity and biological stability, the developed aptasensor shows great potential to utilize in in-field applications such as water quality monitoring.

Novel Dithienopyrrole-Based Conjugated Copolymers: Importance of Backbone Planarity in Achieving High Electrical Conductivity and Thermoelectric Performance.

The development of conjugated polymers with structures that are suitable for efficient molecular doping and charge transport is a key challenge in the construction of high-performance conjugated polymer-based thermoelectric devices. In this study, three novel conjugated polymers based on dithienopyrrole (DTP) are synthesized and their thermoelectric properties are compared. When doped with p-dopant, a donor-acceptor type copolymer, DPP-MeDTP, exhibits higher electrical conductivity and thermoelectric power factor compared to the other donor-donor type copolymers. The high electrical conductivity of DPP-MeDTP compared to the other polymers originates from the high degree of backbone planarity and molecular order, which contributes to its high charge carrier mobility. In addition, the highly crystalline structure of DPP-MeDTP is well maintained upon doping, while the crystalline order of the other polymers decreases significantly upon doping. The findings of this work not only provide insights into the design of DTP-based conjugated polymers for thermoelectric use but also demonstrate the significance of a high degree of molecular order and structural robustness upon doping to achieve high thermoelectric performance.

Validation of IL-7R as an Immunological Biomarker for Human Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer for which no early diagnostic method is available. The immune surveillance hypothesis suggests that the immune system plays crucial roles in tumor development and progression. We validated a PDAC-specific biomarker derived from peripheral blood mononuclear cells (PBMCs) to facilitate early PDAC diagnosis. mRNA levels of interleukin-7R (IL-7R), reportedly a potential immunological marker for PDAC, were measured in PBMCs isolated prospectively from healthy controls (n = 100) and patients with PDAC (n = 135), pancreatic cysts (n = 82), chronic pancreatitis (n = 42), acute pancreatitis (n = 47), and other malignancies (n = 116). The IL-7R level was significantly higher in patients with PDAC than in healthy controls, patients with benign pancreatic disease, and patients with other malignancies. As diagnostic parameters, the sensitivity, specificity, positive predictive value, negative predictive value, and accuracy for IL-7R were 58.5%, 92%, 90.8%, 62.2%, and 72.8%, respectively. The area under the receiver operating characteristic curve (AUROC) was 0.766. IL-7R levels did not differ between resectable and unresectable PDAC cases. The combined measurement of IL-7R and carbohydrate antigen 19-9 (CA19-9) significantly improved the diagnostic parameters and AUROC compared with the use of IL-7R or CA19-9 alone. IL-7R is significantly upregulated in PBMCs in patients with PDAC, and it may be a novel diagnostic marker for PDAC. The combined use of IL-7R and CA19-9 enhanced the diagnostic performance.

Comparison of the Clinical Features and Outcomes of Gallbladder Neuroendocrine Carcinoma with Those of Adenocarcinoma: A Propensity Score-Matched Analysis.

Neuroendocrine neoplasms (NENs) of the gallbladder (GB) are extremely rare. We aimed to compare the clinical features, disease progression, management, and prognosis of patients with GB-NENs with those of patients with GB-adenocarcinomas (ADCs). A total of 21 patients with GB-NENs and 206 patients with GB-ADCs, treated at three tertiary medical centers between January 2010 and December 2020, were enrolled. Of the 21 patients with GB-NENs, 20 were diagnosed with poorly differentiated small-cell neuroendocrine carcinomas (NECs), and 1 patient had large-cell NEC. All patients presented with advanced stages of cancer with extensive local extension and/or distant metastasis and non-specific symptoms. Tumor-node-metastasis stage IIIB and IV (A/B) tumors were found in 6 and 15 (1/14) patients, respectively. Nine patients with GB-NEC who underwent surgical resection had a significantly better progression-free survival (PFS) than those who did not undergo surgery. After a propensity score matching with a 1:1 ratio using the American Joint Committee on Cancer stage, age, sex, and operation status, 19 pairs of patients were included. Compared with stage-matched patients with GB-ADC, patients with GB-NEC had similar overall survival and PFS. However, as GB-NEC is rarely diagnosed early, further studies investigating methods for the early diagnosis and improvement in the survival of patients with GB-NEC are needed.

L-Type Calcium Channel Blocker Enhances Cellular Delivery and Gene Silencing Potency of Cell-Penetrating Asymmetric siRNAs.

The efficient delivery of small interfering RNAs (siRNAs) to the target cells is critical for the pharmaceutical success of RNA interference (RNAi) drugs. One of the possible strategies to improve siRNA delivery is to identify auxiliary molecules that augment their cellular uptake. Herein, we performed a chemical library screening in an effort to discover small molecules that enhance the potency of cholesterol-conjugated, cell-penetrating asymmetric siRNAs (cp-asiRNAs). Interestingly, three compounds identified from the screen share a common dihydropyridine (DHP) core and function as L-type calcium channel blockers (CCBs). Using confocal microscopy and quantitative analysis of small RNAs, we demonstrated that the L-type CCBs increased the endocytic cellular uptake of cp-asiRNAs. Furthermore, these small molecules substantially improved the potency of cp-asiRNAs, not only in vitro but also in vivo on rat skin. Collectively, our study provides an alternative pharmacological approach for the identification of small molecules that potentiate the effects of therapeutic siRNAs.

Aptamer Affinity-Bead Mediated Capture and Displacement of Gram-Negative Bacteria Using Acoustophoresis.

Here, we report a simple and effective method for capturing and displacement of gram-negative bacteria using aptamer-modified microbeads and acoustophoresis. As acoustophoresis allows for simultaneous washing and size-dependent separation in continuous flow mode, we efficiently obtained gram-negative bacteria that showed high affinity without any additional washing steps. The proposed device has a simple and efficient channel design, utilizing a long, square-shaped microchannel that shows excellent separation performance in terms of the purity, recovery, and concentration factor. Microbeads (10 µm) coated with the GN6 aptamer can specifically bind gram-negative bacteria. After incubation of bacteria culture sample with aptamer affinity bead, gram-negative bacteria-bound microbeads, and other unbound/contaminants can be separated by size with high purity and recovery. The device demonstrated excellent separation performance, with high recovery (up to 98%), high purity (up to 99%), and a high-volume rate (500 µL/min). The acoustophoretic separation performances were conducted using 5 Gram-negative bacteria and 5 Gram-positive bacteria. Thanks to GN6 aptamer's binding affinity, aptamer affinity bead also showed binding affinity to multiple strains of gram-negative bacteria, but not to gram-positive bacteria. GN6 coated bead can capture Gram-negative bacteria but not Gram-positive bacteria. This study may present a different perspective in the field of early diagnosis in bacterial infectious diseases. In addition to detecting living bacteria or bacteria-derived biomarkers, this protocol can be extended to monitoring the contamination of water resources and may aid quick responses to bioterrorism and pathogenic bacterial infections.

Detection of Gram-negative bacterial outer membrane vesicles using DNA aptamers.

Infection of various pathogenic bacteria causes severe illness to human beings. Despite the research advances, current identification tools still exhibit limitations in detecting Gram-negative bacteria with high accuracy. In this study, we isolated single-stranded DNA aptamers against multiple Gram-negative bacterial species using Toggle-cell-SELEX (systemic evolution of ligands by exponential enrichment) and constructed an aptamer-based detection tool towards bacterial secretory cargo released from outer membranes of Gram-negative bacteria. Three Gram-negative bacteria, Escherichia coli DH5α, E. coli K12, and Serratia marcescens, were sequentially incubated with the pool of random DNA sequences at each SELEX loop. Two aptamers selected, GN6 and GN12, were 4.2-times and 3.6-times higher binding to 108 cells of Gram-negative bacteria than to Gram-positive bacteria tested, respectively. Using GN6 aptamer, we constructed an Enzyme-linked aptamer assay (ELAA) to detect bacterial outer membrane vesicles (OMVs) of Gram-negative bacteria, which contain several outer membrane proteins with potent immunostimulatory effects. The GN6-ELAA showed high sensitivity to detect as low as 25 ng/mL bacterial OMVs. Aptamers developed in this study show a great potential to facilitate medical diagnosis and early detection of bacterial terrorism, based on the ability to detect bacterial OMVs of multiple Gram-negative bacteria.

Tripodal RNA with 3' Overhangs Prevents RIG-I-Mediated Innate Immune Response.

RNA interference (RNAi) offers great promise in life science research and therapeutic development, as it easily achieves a potent target gene knockdown with high specificity. Since the conventional small interfering RNA (siRNA) structure, known as 19 bp double-stranded RNA (dsRNA) with 2-nucleotide (nt) 3' overhang, has been introduced to successfully elicit the RNAi in mammalian cells, a variety of structural variants of RNAi trigger have been developed. Our group previously reported branched, tripodal interfering RNA (tiRNA) structures as a multigene targeting RNA structure inducing RNAi. However, the immune stimulatory effect of branched tiRNA structure has not been thoroughly evaluated. In this study, we show that tiRNA with blunt ends triggers innate immune response in T98G cell and mouse macrophage cells, which is dependent upon the retinoic acid-inducible gene I (RIG-I), a well-known cytoplasmic dsRNA sensor. Interestingly, immune response triggered by tiRNA can be suppressed by the introduction of 2-nt 3' overhang structure. Our finding expands the structural diversity of RIG-I ligands and provides a guide to develop a safe multitargeting RNA structure for therapeutic application.

Multi-block magnetic nanorods for controlled drug release modulated by Fourier transform surface plasmon resonance.

Stimuli-responsive tunable drug release using nanocarriers is an important subject in smart drug delivery systems. Specifically, magnetic-responsive nanocarriers provide a great opportunity for remote control as well as on-demand command. To effectively utilize magnetic-responsive nanocarriers in vivo and in vitro, drug release should not only be controlled in an efficient way, but also monitored in situ. To satisfy those prerequisites, a template-assisted electrochemical deposition method can be a great option for the synthesis of designer materials that are targeted for specific purposes. Here, we synthesized plasmonic-magnetic nanocarriers by template-assisted electrochemical deposition and covered their surface with a silica shell for drug loading. By appropriately designing the blocks, we synthesized nanocarriers that were plasmonically active and magnetically active with spaces for drug payload. These nanocarriers could be modulated under an external magnetic field and their rotation (or agitation) could be monitored by Fourier transform conversion. Using our nanocarriers, we systematically investigated the tunable release of the anticancer drug doxorubicin as a function of the external magnetic field. Additionally, by applying this modulation system to an in vitro system using HeLa cells we were able to not only monitor the modulation systems but also tailor the drug release in a controlled manner. We expect that our approach will contribute to understanding of nanocarriers in a simulative manner in vitro.

Construction of histidine-containing hydrocarbon stapled cell penetrating peptides for in vitro and in vivo delivery of siRNAs.

A hydrocarbon stapled peptide based strategy was used to develop an optimized cell penetrating peptide for siRNA delivery. Various stapled peptides, having amphipathic Leu- and Lys-rich regions, were prepared and their cell penetrating potentials were evaluated. One peptide, stEK, was found to have high cell penetration and siRNA delivery abilities at low nanomolar concentrations. In order to improve its ability to promote gene silencing, stEK was modified by replacing several Lys residues with His moieties. The modified peptide, LKH-stEK, was found to facilitate endosomal escape and to display >90% knock-down with 50 nM of a siRNA targeting cyclophilin B in HeLa cells. The results of an in vivo animal wound healing model study demonstrate that LKH-stEK promotes delivery of an siRNA, which targets the connective tissue growth factor, and that this process leads to efficient gene silencing by the siRNA at a nanomolar level in mouse skin.

Cell-SELEX-Based Identification of a Human and Mouse Cross-Reactive Endothelial Cell-Internalizing Aptamer.

Increased interest and insights gained by researchers on the roles of endothelial cells in the pathophysiology of cancer, inflammatory, and cardiovascular diseases have led to the design of pharmacological interventions aimed at the endothelium lining in the diseased sites. Toward this end, we used established brain microvascular endothelial cell lines mouse (bEND3), human (hCMEC/D3), and Toggle Cell-SELEX to identify a species cross-reactive, endothelial cell-internalizing aptamer R11-3. This 2'F-modified RNA aptamer is specific for endothelial cells as no internalization was seen with cells of nonendothelial origin. R11-3 was truncated in size, and its potential in endothelial targeted therapeutics was established using VEGFR2 targeting long interfering RNA (liRNA) aptamer chimera. Due to its specificity for both mouse and human endothelial cells, we believe that this aptamer not only fits for development of endothelial targeted drug development for human diseases but is also suitable for preclinical evaluation in mice.

RNA Interference-Mediated Gene Silencing by Branched Tripodal RNAs Does Not Require Dicer Processing.

Specific gene silencing through RNA interference (RNAi) holds great promise as the next-generation therapeutic development platform. Previously, we have shown that branched, tripodal interfering RNA (tiRNA) structures could simultaneously trigger RNAi-mediated gene silencing of three target genes with 38 nt-long guide strands associated with Argonaute 2. Herein, we show that the branched RNA structure can trigger effective gene silencing in Dicer knockout cell line, demonstrating that the Dicer-mediated processing is not required for tiRNA activity. The finding of this study confirms the flexibility of the structure of RNAi triggers as well as the length of the guide strand in RNAi-mediated gene silencing.

Cell-SELEX Based Identification of an RNA Aptamer for Escherichia coli and Its Use in Various Detection Formats.

Escherichia coli are important indicator organisms, used routinely for the monitoring of water and food safety. For quick, sensitive and real-time detection of E. coli we developed a 2'F modified RNA aptamer Ec3, by Cell-SELEX. The 31 nucleotide truncated Ec3 demonstrated improved binding and low nano-molar affinity to E. coli. The aptamer developed by us out-performs the commercial antibody and aptamer used for E. coli detection. Ec3(31) aptamer based E. coli detection was done using three different detection formats and the assay sensitivities were determined. Conventional Ec3(31)-biotin-streptavidin magnetic separation could detect E. coli with a limit of detection of 1.3 × 106 CFU/ml. Although, optical analytic technique, biolayer interferometry, did not improve the sensitivity of detection for whole cells, a very significant improvement in the detection was seen with the E. coli cell lysate (5 × 104 CFU/ml). Finally we developed Electrochemical Impedance Spectroscopy (EIS) gap capacitance biosensor that has detection limits of 2 × 104 CFU/mL of E. coli cells, without any labeling and signal amplification techniques. We believe that our developed method can step towards more complex and real sample application.

Development of Cell-Penetrating Asymmetric Interfering RNA Targeting Connective Tissue Growth Factor.

Connective tissue growth factor (CTGF) is a multifunctional matricellular protein, playing a role as a central mediator in tissue remodeling and fibrosis. A number of reports have shown the pivotal roles of CTGF in the progression of fibrosis, suggesting CTGF as a promising therapeutic target for the treatment of fibrotic disorders including hypertrophic scars and keloids. In this study, we present the development of an interfering RNA molecule that efficiently inhibits the expression of CTGF via RNA interference mechanism both in vitro and in vivo. Chemical modifications were introduced to the asymmetric interfering RNA (asiRNA) backbone structure. The resulting RNA molecule, termed cell-penetrating asiRNA (cp-asiRNA), entered into cells and triggered RNA interference-mediated gene silencing without delivery vehicles. The gene-silencing activity of cp-asiRNA targeting CTGF (cp-asiCTGF) was examined both in vitro and in vivo. Furthermore, the administration of cp-asiCTGF in the rat skin excision wound model efficiently reduced the induction of CTGF and collagens during the wound-healing process. These results suggest that the cp-asiCTGF molecule could be developed into antifibrotic therapeutics such as antiscar drugs.

Diagnostic performance of contrast-enhanced multidetector computed tomography and gadoxetic acid disodium-enhanced magnetic resonance imaging in detecting hepatocellular carcinoma: direct comparison and a meta-analysis.

The purpose of this study was to directly (head-to-head) compare the per-lesion diagnostic performance of contrast-enhanced computed tomography (CT) (also referred to as CT hereafter) and gadoxetic acid disodium (Gd-EOB-DTPA)-enhanced magnetic resonance (MR) imaging (also referred to as MRI hereafter) for the detection of hepatocellular carcinoma (HCC). Studies reporting direct per-lesion comparison data of contrast-enhanced multidetector CT and Gd-EOB-DTPA-enhanced MR imaging that were published between January 2000 and January 2015 were analyzed. The data of each study were extracted. Systematic review, paired meta-analysis, and subgroup analysis were performed. Twelve studies including 627 patients and 793 HCC lesions were analyzed. The sensitivity estimates of MRI and CT were, respectively, 0.86 (95% CI 0.76-0.93) and 0.70 (95% CI 0.58-0.80), with significant difference (P < 0.05). The sensitivity estimates were both 0.94 (95% CI 0.92-0.96) (Chi-square 4.84, degrees of freedom = 1, P > 0.05). In all subgroups, Gd-EOB-DTPA-enhanced MR imaging was more sensitive than multidetector CT for the detection of HCC, and specificity estimates of both tests maintained at a similarly high level in all conditions: sensitivity estimates of both tests were reduced in studies where patients were diagnosed with HCC solely by liver explant or in those where HCC lesions were small (≤2 cm, especially when ≤1 cm). But in all situations, sensitivities of MRI were higher than those of CT with or without significance. Gd-EOB-DTPA-enhanced MR imaging showed better per-lesion diagnostic performance than multidetector CT for the diagnosis of HCC in patients with cirrhosis and in small hepatic lesions.

Formation and decay of charge carriers in aggregate nanofibers consisting of poly(3-hexylthiophene)-coated gold nanoparticles.

Thin nanofibers (NFs) of J-dominant aggregates with a thickness of 15 nm and thick NFs of H-dominant aggregates with a thickness of 25 nm were fabricated by the self-assembly of poly(3-hexylthiophene)-coated gold nanoparticles. The formation and decay dynamics of the charge carriers, which are dependent on the aggregate types of NFs, was investigated by time-resolved emission and transient-absorption spectroscopy. With increasing excitation energy, the fraction of the fast emission decay component decreased, suggesting that the fast formation of polaron pairs (PP), localized (LP), and delocalized polarons (DP) results from higher singlet exciton states produced by the singlet fusion. The faster decay dynamics of DP and LP in the thick NFs than in thin NFs is due to the increased delocalization of DP and LP. As the interchain aggregation is weaker than intrachain aggregation, PP decays faster in thin NFs than in thick NFs. In both thin and thick NFs, although triplet (T1) excitons were barely observed with excitation at 532 nm on a nanosecond time scale, they were observed with excitation at 355 nm, showing that T1 excitons within NFs are generated mainly through the singlet fission from a higher singlet exciton state rather than through intersystem crossing.