Long-Term Outcomes of Endovascular Aneurysm Repair in Patients Aged ≤70 Years.

Objectives: The efficacy of endovascular aneurysm repair (EVAR) against abdominal aortic aneurysm (AAA) in younger patients remains unknown. Hence, the current study aimed to investigate whether the aneurysm-related mortality rate of EVAR is acceptable among patients aged ≤70 years. Methods: Among 644 patients, 148 underwent EVAR (EVAR group), and 496 received open surgical repair (OSR group). The cumulative incidence rates of aneurysm-related death, any intervention, and serious aneurysm-related events after AAA repair were evaluated using the cumulative incidence function in the presence of competing risks. Results: The EVAR group had higher prevalences of several comorbidities, and overall survival for the EVAR group was significantly inferior to that of the OSR group. The cumulative incidence rates of aneurysm-related death, any intervention, and serious aneurysm-related events at 5 years were 1.5%, 11.7%, and 6.4% in the EVAR group and 1.3%, 5.3%, and 5.9% in the OSR group, respectively. EVAR was not a significant prognostic factor of aneurysm-related mortality and serious aneurysm-related events. However, it was an independent poor prognostic factor of any intervention. Conclusion: EVAR was not a significant prognostic factor of aneurysm-related mortality and serious aneurysm-related events. Therefore, it demonstrated acceptable procedure-related long-term outcomes, at least in high-risk young patients.

Dynamic and static control of the off-target interactions of antisense oligonucleotides using toehold chemistry.

Off-target interactions between antisense oligonucleotides (ASOs) with state-of-the-art modifications and biological components still pose clinical safety liabilities. To mitigate a broad spectrum of off-target interactions and enhance the safety profile of ASO drugs, we here devise a nanoarchitecture named BRace On a THERapeutic aSo (BROTHERS or BRO), which is composed of a standard gapmer ASO paired with a partially complementary peptide nucleic acid (PNA) strand. We show that these non-canonical ASO/PNA hybrids have reduced non-specific protein-binding capacity. The optimization of the structural and thermodynamic characteristics of this duplex system enables the operation of an in vivo toehold-mediated strand displacement (TMSD) reaction, effectively reducing hybridization with RNA off-targets. The optimized BROs dramatically mitigate hepatotoxicity while maintaining the on-target knockdown activity of their parent ASOs in vivo. This technique not only introduces a BRO class of drugs that could have a transformative impact on the extrahepatic delivery of ASOs, but can also help uncover the toxicity mechanism of ASOs.

Exosome-Hijacking Drug Delivery System with Branched Arginine Linker Effectively Deliver Antisense Oligonucleotides into Lung Adenocarcinoma Cells.

Exosomes are a type of extracellular vesicles that contain diverse molecules and are present in our body fluids. They play a crucial role in transporting materials and transmitting signals between cells. Currently, there have been numerous reports on the use of exosomes in drug delivery systems (DDS). However, most existing methods for utilizing exosomes in DDS require the isolation and purification of exosomes, which raises concerns about yield and potential damage to the exosomes. Recently, we have developed a novel DDS called "ExomiR-Tracker" that harnesses exosomes without the need for isolation and purification. This system aims to deliver nucleic acid drugs effectively. ExomiR-Tracker consists of an anti-exosome antibody equipped with nona-D-arginines (9 mer) and nucleic acid drugs which have complementary sequence of target microRNA (anti-miR). In this study, we modified ExomiR-Tracker by incorporating branched nona-D-arginines (9 + 9 mer) molecules (referred to as Branch ExomiR-Tracker) and evaluated its efficacy in lung adenocarcinoma cells (A549 cells). The improved complex formation ability and enhanced cellular uptake of anti-miR, demonstrated by our findings, highlight the advantages of incorporating branched oligoarginine peptides into the ExomiR-Tracker platform. These results represent significant progress in revealing the effectiveness of Branch ExomiR-Tracker against adhesive cancer cells, which has not been shown to be effective with the conventional Linear ExomiR-Tracker.

Development and Crosslinking Properties of Psoralen-Conjugated Triplex-Forming Oligonucleotides as Antigene Tools Targeting Genome DNA.

Psoralen-conjugated triplex-forming oligonucleotides (Ps-TFOs) have been utilized for genome editing and anti-gene experiments for over thirty years. However, the research on Ps-TFOs employing artificial nucleotides is still limited, and their photo-crosslinking properties have not been thoroughly investigated in relation to biological activities. In this study, we extensively examined the photo-crosslinking properties of Ps-TFOs to provide fundamental insights for future Ps-TFO design. We developed novel Ps-TFOs containing 2'-O,4'-C-methylene-bridged nucleic acids (Ps-LNA-mixmer) and investigated their photo-crosslinking properties using stable cell lines that express firefly luciferase constitutively to evaluate the anti-gene activities of Ps-LNA-mixmer. As a result, Ps-LNA-mixmer successfully demonstrated suppression activity, and we presented the first-ever correlation between photo-crosslinking properties and their activities. Our findings also indicate that the photo-crosslinking process is insufficient under cell irradiation conditions (365 nm, 2 mW/cm2 , 60 min). Therefore, our results highlight the need to develop new psoralen derivatives that are more reactive under cell irradiation conditions.

Novel strategy of liver cancer treatment with modified antisense oligonucleotides targeting human vasohibin-2.

Vasohihibin-2 (VASH2) is a homolog of vasohibin-1 (VASH1) and is overexpressed in various cancers. Vasohihibin-2 acts on both cancer cells and cancer microenvironmental cells. Previous analyses have shown that VASH2 promotes cancer progression and abrogation of VASH2 results in significant anticancer effects. We therefore propose VASH2 to be a practical molecular target for cancer treatment. Modifications of antisense oligonucleotide (ASO) such as bridged nucleic acids (BNA)-based modification increases the specificity and stability of ASO, and are now applied to the development of a number of oligonucleotide-based drugs. Here we designed human VASH2-ASOs, selected an optimal one, and developed 2',4'-BNA-based VASH2-ASO. When systemically administered, naked 2',4'-BNA-based VASH2-ASO accumulated in the liver and showed its gene-silencing activity. We then examined the effect of 2',4'-BNA-based VASH2-ASO in liver cancers. Intraperitoneal injection of naked 2',4'-BNA-based VASH2-ASO exerted a potent antitumor effect on orthotopically inoculated human hepatocellular carcinoma cells. The same manipulation also showed potent antitumor activity on the splenic inoculation of human colon cancer cells for liver metastasis. These results provide a novel strategy for the treatment of primary as well as metastatic liver cancers by using modified ASOs targeting VASH2.

Unique Crosslinking Properties of Psoralen-Conjugated Oligonucleotides Developed by Novel Psoralen N-Hydroxysuccinimide Esters.

Psoralens and their derivatives, such as trioxsalen, have unique crosslinking features to DNA. However, psoralen monomers do not have sequence-specific crosslinking ability with the target DNA. With the development of psoralen-conjugated oligonucleotides (Ps-Oligos), sequence-specific crosslinking with target DNA has become achievable, thereby expanding the application of psoralen-conjugated molecules in gene transcription inhibition, gene knockout, and targeted recombination by genome editing. In this study, we developed two novel psoralen N-hydroxysuccinimide (NHS) esters that allow the introduction of psoralens into any amino-modified oligonucleotides. Quantitative evaluation of the photo-crosslinking efficiencies of the Ps-Oligos to target single-stranded DNAs revealed that the crosslinking selectivity to 5-mC is the unique feature of trioxsalen. We found that the introduction of an oligonucleotide via a linker at the C-5 position of psoralen can promote favorable crosslinking to target double-stranded DNA. We believe our findings are essential information for the development of Ps-Oligos as novel gene regulation tools.

A Case of Tracheo-innominate Artery Fistula after Tracheostomy Successfully Treated with a Covered Stent.

A 78-year-old man underwent a tracheostomy after embolization for a dural arteriovenous fistula. Seventy days after tracheostomy, arterial bleeding appeared through the tracheal stoma. The bleeding stopped spontaneously. However, two days later, arterial bleeding reappeared, and he was diagnosed with a tracheo-innominate artery fistula (TIF). He then underwent urgent endovascular covered stent placement. After the procedure, there was no bleeding. TIF can be a fatal complication after tracheostomy and it is generally treated with open chest surgery. However, a successful endovascular treatment for TIF has recently been reported and may yield better results.

Chemistry of Therapeutic Oligonucleotides That Drives Interactions with Biomolecules.

Oligonucleotide therapeutics that can modulate gene expression have been gradually developed for clinical applications over several decades. However, rapid advances have been made in recent years. Artificial nucleic acid technology has overcome many challenges, such as (1) poor target affinity and selectivity, (2) low in vivo stability, and (3) classical side effects, such as immune responses; thus, its application in a wide range of disorders has been extensively examined. However, even highly optimized oligonucleotides exhibit side effects, which limits the general use of this class of agents. In this review, we discuss the physicochemical characteristics that aid interactions between drugs and molecules that belong to living organisms. By systematically organizing the related data, we hope to explore avenues for symbiotic engineering of oligonucleotide therapeutics that will result in more effective and safer drugs.

Engineered Cytochrome P450-Catalyzed Oxidative Biaryl Coupling Reaction Provides a Scalable Entry into Arylomycin Antibiotics.

We report herein the first example of a cytochrome P450-catalyzed oxidative carbon-carbon coupling process for a scalable entry into arylomycin antibiotic cores. Starting from wild-type hydroxylating cytochrome P450 enzymes and engineered Escherichia coli, a combination of enzyme engineering, random mutagenesis, and optimization of reaction conditions generated a P450 variant that affords the desired arylomycin core 2d in 84% assay yield. Furthermore, this process was demonstrated as a viable route for the production of the arylomycin antibiotic core on the gram scale. Finally, this new entry affords a viable, scalable, and practical route for the synthesis of novel Gram-negative antibiotics.

Synthesis and Evaluation of Oligonucleotide-Containing 2'-O-{[(4,5',8-Trimethylpsoralen)-4'-ylmethoxy]ethylaminocarb-onyl}adenosine as Photo-crosslinkable Gene Targeting Tools.

Several psoralen-conjugated oligonucleotides (Ps-Oligos) have been developed as photo-crosslinkable oligonucleotides targeting DNA or RNA. To avoid potential off-target effects, it is important to investigate the selective photo-crosslinking reactivity of Ps-Oligos to DNA or RNA. However, the selectivity of these Ps-Oligos has not been reported in detail thus far. In this study, we evaluated the photo-crosslinking properties of two Ps-Oligos, 5'-Ps-Oligo and a novel Ps-Oligo containing 2'-O-{[(4,5',8-trimethylpsoralen)-4'-ylmethoxy]ethylaminocarbonyl}adenosine (APs2-Oligo). Notably, 5'-Ps-Oligo preferentially crosslinked with DNA, whereas APs2-Oligo preferentially crosslinked with RNA. These results demonstrate the interesting crosslinking properties of Ps-Oligos, which will provide useful information for the molecular design of novel Ps-Oligos in future studies.

As(iii) removal through catalytic oxidation and Fe(iii) precipitation.

To remove arsenite (As(iii)) from wastewater effectively, the catalytic oxidation of As(iii) to arsenate (As(v)) and As(v) precipitation with iron ions (Fe(iii)) was investigated. The Pt/SiO2 catalyst functioned as a reaction site for As(iii) with oxygen in the atmosphere. The combination of the Pt/SiO2 catalyst and Fe(iii) precipitant improved the removal of As(iii) in the precipitate; Pt/SiO2 worked as both an As(iii) oxidation site and precipitation site with Fe(iii) precipitant.

Reductive removal of selenate (VI) in aqueous solution using rhodium metal particles supported on TiO2.

Selenium and its compounds in high concentration are toxic for humans, especially selenate (VI) is the most toxic due to its high solubility in water. To promote the reductive reaction of Se(vi) to Se(iv) or Se(0), which is relatively easy to remove in water, noble metal particles were added as reaction sites with a reductant. The highest removal performance of selenate in aqueous solution was achieved using rhodium particles supported on TiO2 (Rh/TiO2). Selenate was rapidly reduced with hydrazine on the metal particle, leading to a selenium deposition on the particle inhibiting the stable reductive reaction. On the other hand, when a weaker reductant such as formaldehyde was used for the selenate reduction, the selenium deposition was suppressed due to its low reactivity, resulting in a stable reductive reaction of selenate in water.

Selective Photo-Crosslinking Detection of Methylated Cytosine in DNA Duplex Aided by a Cationic Comb-Type Copolymer.

In the process of cell development and differentiation, C-5-methylation of cytosine (5-methylcytosine: 5-mC) in genome DNA is an important transcriptional regulator that switches between differentiated and undifferentiated states. Further, abnormal DNA methylations are often present in tumor suppressor genes and are associated with many diseases. Therefore, 5-mC detection technology is an important tool in the most exciting fields of molecular biology and diagnosing diseases such as cancers. In this study, we found a novel photo-crosslinking property of psoralen-conjugated oligonucleotide (Ps-Oligo) to the double-stranded DNA (ds-DNA) containing 5-mC in the presence of a cationic comb-type copolymer, poly(allylamine)-graft-dextran (PAA-g-Dex). Photo-crosslinking efficiency of Ps-Oligo to 5-mC in ds-DNA was markedly enhanced in the presence of PAA-g-Dex, permitting 5-mC-targeted crosslinking. We believe that the combination of PAA-g-Dex and Ps-Oligo will be an effective tool for detecting 5-mC in genomic DNA.

Metabolites of soil microorganisms modulate amyloid ß production in Alzheimer's neurons.

Microbial flora is investigated to be related with neuropathological conditions in Alzheimer's disease (AD), and is attracting attention as a drug discovery resource. However, the relevance between the soil microbiota and the pathological condition has not been fully clarified due to the difficulty in isolation culture and the component complexity. In this study, we established a library of secondly metabolites produced in microorganism to investigate the potential effect of microorganisms on the production of amyloid ß (Aß), one of the most representative pathogens of AD. We conducted a library screening to quantify Aß and neuronal toxicity by using cortical neurons from human induced pluripotent stem cells (iPSCs) of AD patients after adding secondary metabolites. Screening results and following assessment of dose-dependency identified Verrucarin A, produced in Myrothecium spp., showed 80% decrease in Aß production. Furthermore, addition of Mer-A2026A, produced in Streptomyces pactum, showed increase in Aß42/40 ratio at the low concentration, and decrease in Aß production at the higher concentration. As a result, established library and iPSC-based phenotyping assay clarified a direct link between Aß production and soil microorganisms. These results suggest that Aß-microorganism interaction may provide insight into the AD pathophysiology with potential therapeutics.

Incubator humidity and temperature control in infants born at 22-23 weeks' gestation.

BACKGROUND: Preterm infants, especially those born at ≤23 gestational weeks (GW), present with extremes in insensible water loss (IWL) and changes in water balance. AIMS: To prevent water loss from the skin and achieve skin maturation without infection, we investigated transepidermal water loss (TEWL), IWL from the skin (IWL-s), and electrolyte balance with differences in high incubator humidity and temperature control from birth to postnatal 1 month in 22-23 GW and 24-25 GW infants. STUDY DESIGN: Prospective cohort study. SUBJECTS: Extremely preterm infants born at 22-23 GW (n = 11) and 24-25 GW (n = 11), admitted to the neonatal intensive care unit between September 2018 and October 2019. OUTCOME MEASURES: Total fluid intake (TFI), fluid output volume, TEWL, IWL-s, and electrolyte balance were compared between the two groups with controlled incubator humidity and temperature, gradually decreasing the humidity and ambient temperature from 95% to 50% and from 37.0 to 34.0 °C, respectively, while maintaining the central body temperature at 36.5-37.5 °C. RESULTS: TEWL and IWL-s between the 22-23 and 24-25 GW was not significantly different for infants at postnatal age. No significant difference in electrolyte imbalance was noted between the two groups, within the first 7 days. Differences in TEWL and IWL-s were eliminated with corresponding humidity and temperature adjustments. CONCLUSIONS: Incubator humidity and temperature control should aid management of 22-23 GW infants to reduce IWL, facilitate skin maturation, and prevent infection.

Therapeutic application of sequence-specific binding molecules for novel genome editing tools.

Genome editing has been expected to widely increase the available treatment options for various diseases and permit pharmaceutical interventions in previously untreatable conditions. The availability of genome editing tools was dramatically increased by the development of the CRISPR-Cas9 system. However, a number of issues limit the use of the CRISPR-Cas9 system and other gene-editing tools in the clinical treatment of diseases. This review summarized the history and types of genome editing tools and limitations of their use. In addition, the study addressed several next-generation technologies aiming to overcome the limitations of current gene therapy protocols in an effort to accelerate the clinical development of potential treatment options. This review has provided an extensive foundation of the current state of genome editing technology and its clinical development. This review also indicate that the study additionally highlighted the need for multidisciplinary approaches to overcome current bottlenecks in the development of genome editing.

Drug discovery and development scheme for liver-targeting bridged nucleic acid antisense oligonucleotides.

Antisense oligonucleotides (ASOs) containing bridged nucleic acids (BNAs) have been proven to be very powerful. However, ensuring a reliable discovery and translational development scheme for this class of ASOs with wider therapeutic windows remains a fundamental challenge. We here demonstrate the robustness of our scheme in the context of the selection of ASOs having two different BNA chemistries (2,'4'-BNA/locked nucleic acid [LNA] and amido-bridged nucleic acid [AmNA]) targeting human proprotein convertase subtilisin/kexin type 9 (PCSK9). The scheme features a two-step process, including (1) a unique and sensitive in vitro screening approach, called Ca2+ enrichment of medium (CEM) transfection, and (2) a ligand-targeted drug delivery approach to better reach target tissues, averting unintended accumulation of ASOs. Using CEM screening, we identified a candidate ASO that shows >70% cholesterol-lowering action in monkeys. An N-acetylgalactosamine (GalNAc) ligand then was appended to the candidate ASO to further broaden the therapeutic margin by altering the molecule's pharmacokinetics. The GalNAc conjugate, HsPCSK9-1811-LNA, was found to be at least ten times more potent in non-human primates (compared with the unconjugated counterpart), with reduced nephrotoxicity in rats. Overall, we successfully showed that our drug development scheme is better suited for selecting clinically relevant BNA-based ASOs, especially for the treatment of liver-associated diseases.

Teachers' visual processing of children's off-task behaviors in class: A comparison between teachers and student teachers.

As teachers are responsible for responding instantaneously to students' statements and actions, the progress of the class, and their teaching purpose, they need to be able to engage in responsive teaching. Teachers obtain information about students' learning by observing them in the classroom, and subsequently make instructional decisions based on this information. Teachers need to be sensitive to student behaviors and respond accordingly, because there are students who follow the teacher's instructions and those who do not in every classroom. Skilled teachers may distribute their gaze over the entire class and discover off-task behaviors. So how does a teacher's visual processing and noticing ability develop? It is important to clarify this process for both experienced teachers and student teachers. Therefore, the purpose of this study was to investigate whether there is a difference in visual processing and the ability to notice off-task behaviors in class between teachers and student teachers through gaze analysis. Using an eye tracking device, 76 teachers and 147 student teachers were asked to watch a video, and gaze measurements were collected. In the video, students exhibiting off-task behaviors in class were prompted by their classroom teacher to participate in the lesson. After the video, the participants were asked if they could identify the students who had displayed off-task behaviors and whom the teachers had warned. The results showed that teachers gazed at students engaging in off-task behaviors in class more often and noticed them at a higher rate than student teachers did. These results may be attributed to differences in the experiences of visual processing of relevant information in the classroom between teachers and student teachers. Thus, the findings on teachers' visual processing by direct measurement of gaze will be able to contribute to teachers' development.