Development of PCR primers enabling the design of flexible sticky ends for efficient concatenation of long DNA fragments.

We developed chemically modified PCR primers that allow the design of flexible sticky ends by introducing a photo-cleavable group at the phosphate moiety. Nucleic acid derivatives containing o-nitrobenzyl photo-cleavable groups with a tert-butyl group at the benzyl position were stable during strong base treatment for oligonucleotide synthesis and thermal cycling in PCR reactions. PCR using primers incorporating these nucleic acid derivatives confirmed that chain extension reactions completely stopped at position 1 before and after the site of the photo-cleavable group was introduced. DNA fragments of 2 and 3 kbp, with sticky ends of 50 bases, were successfully concatenated with a high yield of 77%. A plasmid was constructed using this method. Finally, we applied this approach to construct a 48.5 kbp lambda phage DNA, which is difficult to achieve using restriction enzyme-based methods. After 7 days, we were able to confirm the generation of DNA of the desired length. Although the efficiency is yet to be improved, the chemically modified PCR primer offers potential to complement enzymatic methods and serve as a DNA concatenation technique.

Development and Comparison of 4-Thiouridine to Cytidine Base Conversion Reaction.

To study transcriptome dynamics without harming cells, it is essential to convert chemical bases. 4-Thiouridine (4sU) is a biocompatible uridine analogue that can be converted into a cytidine analogue. Although several reactions can convert 4sU into a cytidine analogue, few studies have compared the features of these reactions. In this study, we performed three reported base conversion reactions, including osmium tetroxide, iodoacetamide, and sodium periodate treatment, as well as a new reaction using 2,4-dinitrofluorobenzene. We compared the reaction time, conversion efficacy, and effects on reverse transcription. These reactions successfully converted 4sU into a cytidine analogue quantitatively using trinucleotides. However, the conversion efficacy and effect on reverse transcription vary depending on the reaction with the RNA transcript. OsO4 treatment followed by NH4Cl treatment showed the best base-conversion efficiency. Nevertheless, each reaction has its own advantages and disadvantages as a tool for studying the transcriptome. Therefore, it is crucial to select the appropriate reaction for the target of interest.

Efficacy of ramucirumab and subsequent nivolumab therapy in patients with advanced gastric cancer: A retrospective study.

Nivolumab monotherapy is a standard treatment of metastatic gastric cancer, and this type of cancer involves vascular endothelial growth factor (VEGF) signaling in the tumor immunological environment. The subgroup analysis of the ATTRACTION-2 trial revealed that prior treatment with ramucirumab (RAM), a VEGF inhibitor, affected the therapeutic effect of nivolumab. The present retrospective study aimed to review patients with metastatic gastric cancer who were treated with paclitaxel (PTX) and RAM followed by nivolumab. A total of 29 patients with metastatic gastric cancer were treated with PTX + RAM as second-line treatment, followed by nivolumab monotherapy as third-line treatment. The therapeutic efficacy of nivolumab was compared in 13 patients with progression-free survival (PFS) of <5 months and 16 patients with PFS ≥5 months after PTX + RAM therapy. The present study included 22 male and seven female patients, with a median age of 68 years (range, 45-82 years). Human epidermal growth factor receptor 2 positivity was observed in six patients. The disease control rate was 62.1%. The PFS and overall survival (OS) were 4.4 and 11.9 months, respectively. Patients with PFS ≥5 months after PTX + RAM therapy showed better outcome in both PFS (5.3 months vs. 2.8 months, P=0.039) and OS (6.9 months vs. 15.2 months, P=0.066) after nivolumab treatment than patients with PFS of <5 months after PTX + RAM therapy. However, no significant relationship was observed between the outcome of first-line treatment and nivolumab. The therapeutic effect of nivolumab was associated with prior PTX + RAM treatment in advanced gastric cancer.

Topological capture of mRNA for silencing gene expression.

We describe herein topological mRNA capture using branched oligodeoxynucleotides (ODNs) with multiple reactive functional groups. These fragmented ODNs efficiently formed topological complexes on template mRNA in vitro. In cell-based experiments targeting AcGFP mRNA, the bifurcated reactive ODNs showed a much larger gene silencing effect than the corresponding natural antisense ODN.

Cap analogs with a hydrophobic photocleavable tag enable facile purification of fully capped mRNA with various cap structures.

Starting with the clinical application of two vaccines in 2020, mRNA therapeutics are currently being investigated for a variety of applications. Removing immunogenic uncapped mRNA from transcribed mRNA is critical in mRNA research and clinical applications. Commonly used capping methods provide maximum capping efficiency of around 80-90% for widely used Cap-0- and Cap-1-type mRNAs. However, uncapped and capped mRNA possesses almost identical physicochemical properties, posing challenges to their physical separation. In this work, we develop hydrophobic photocaged tag-modified cap analogs, which separate capped mRNA from uncapped mRNA by reversed-phase high-performance liquid chromatography. Subsequent photo-irradiation recovers footprint-free native capped mRNA. This approach provides 100% capping efficiency even in Cap-2-type mRNA with versatility applicable to 650 nt and 4,247 nt mRNA. We find that the Cap-2-type mRNA shows up to 3- to 4-fold higher translation activity in cultured cells and animals than the Cap-1-type mRNA prepared by the standard capping method.

The Effect of γ Phosphate Modified Deoxynucleotide Substrates on PCR Activity and Fidelity.

Controlling PCR fidelity is an important issue for molecular biology and high-fidelity PCR is essential for gene cloning. In general, fidelity control is achieved by protein engineering of polymerases. In contrast, only a few studies have reported controlling fidelity using chemically modified nucleotide substrates. In this report, we synthesized nucleotide substrates possessing a modification on Pγ and evaluated the effect of this modification on PCR fidelity. One of the substrates, nucleotide tetraphosphate, caused a modest decrease in Taq DNA polymerase activity and the effect on PCR fidelity was dependent on the type of mutation. The use of deoxyadenosine tetraphosphate enhanced the A : T→G : C mutation dramatically, which is common when using Taq polymerase. Conversely, deoxyguanosine tetraphosphate (dG4P) suppressed this mutation but increased the G : C→A : T mutation during PCR. Using an excess amount of dG4P suppressed both mutations successfully and total fidelity was improved.

Synthesis of nucleoside oligophosphates by electrophilic activation of phosphorothioate.

We herein report a new synthetic method for nucleoside oligophosphates based on electrophilic activation of 5'-phosphorothioate nucleotides. The treatment of phosphorothioate with 2,4-dinitrochlorobenzene (DNCB) efficiently afforded the key activated species, electrophilic thioester nucleotides (EPT-Ns), which were coupled with various phosphate reagents to afford the target nucleoside oligophosphates, including an mRNA cap analog.

Aqueous Povidone-Iodine Versus Normal Saline For Intraoperative Wound Irrigation on The Incidence of Surgical Site Infection in Clean-Contaminated Wounds After Gastroenterological Surgery: A Single-Institute, Prospective, Blinded-Endpoint, Randomized Controlled Trial.

OBJECTIVE: This trial evaluated the superiority of intraoperative wound irrigation (IOWI) with aqueous povidone-iodine (PVP-I) compared with that with saline for reducing the incidence of surgical site infection (SSI). BACKGROUND: IOWI with aqueous PVP-I is recommended for the prevention of SSI by the World Health Organization and the Centers for Disease Control and Prevention, although the evidence level is low. METHODS: This single institute in Japan, prospective, randomized, blinded-endpoint trial was conducted to assess the superiority of IOWI with aqueous PVP-I in comparison with IOWI with saline for reducing the incidence of SSI in clean-contaminated wounds after gastroenterological surgery. Patients 20 years or older were assessed for eligibility, and the eligible participants were randomized at a 1:1 ratio using a computer-generated block randomization. In the study group, IOWI was performed for 1 minute with 40 mL of aqueous 10% PVP-I before skin closure. In the control group, the procedure was performed with 100 mL of saline. Participants, assessors, and analysts were masked to the treatment allocation. The primary outcome was the incidence of incisional SSI in the intention-to-treat set. RESULTS: Between June 2019 and March 2022, 941 patients were randomized to the study group (473 patients) or the control group (468 patients). The incidence of incisional SSI was 7.6% in the study group and 5.1% in the control group (risk difference 0.025, 95% CI -0.006 to 0.056; risk ratio 1.484, 95% CI 0.9 to 2.448; P =0.154). CONCLUSION: The current recommendation of IOWI with aqueous PVP-I should be reconsidered.

Enhancement of DNA hypomethylation alterations by gastric and bile acids promotes chromosomal instability in Barrett's epithelial cell line.

Gastric and bile acid reflux leads to chronic inflammation, resulting in methylation alterations in Barrett's esophagus (BE) together with chromosomal instability (CIN). We investigated DNA hypomethylation following acid exposure and confirmed its significance in BE-related carcinogenesis by inducing CIN in vitro. OACP4C, an esophageal cancer cell line, and CP-A, a non-dysplastic cell line originating from BE, were exposed to acidic conditions using deoxycholic acid. CP-A exhibited substantially increased DNA hypomethylation of alpha satellite sequences in the centromere region, as well as increased levels of alpha satellite transcripts, but no changes were observed in the long interspersed nucleotide element-1 sequences distributed throughout the entire genome. These changes were not clearly found in OACP4C. Copy number changes at specific chromosomes were identified in CP-A, along with an increased number of cells exhibiting abnormal segregations, whereas these changes were rarely observed in OACP4C. The changes were maintained after several cell divisions. These findings suggest that alpha satellites are likely targets of DNA hypomethylation induced by acid exposure. CP-A was more sensitive to acid exposure than OACP4C, indicating that acid-induced DNA hypomethylation is involved in cancer development rather than progression, which could be involved in the underlying mechanism of esophagogastric junction carcinoma development.

A 2'-modified uridine analog, 2'-O-(methylthiomethoxy)methyl uridine, for siRNA applications.

The medicinal applications of siRNAs have been intensively examined but are still hindered by their low molecular stability under biological conditions and off-target effects, etc. The introduction of chemical modifications to the nucleoside is a promising strategy for solving these limitations. Herein, we describe the development of a new uridine analog, U*, that has a (methylthiomethoxy)methoxy group at the 2' position. The phosphoramidite reagent corresponding to U* was easily synthesized and the RNA oligonucleotides containing U* were stably prepared using a standard protocol for oligonucleotide synthesis. The introduction of U* into the siRNA resulted in positive or negative effects on the targeted gene silencing in a position-dependent manner, and the positive effects were attributed to the improved stability under biological conditions. The thermodynamic analysis of the U*-modified RNAs revealed a slight destabilization of the dsRNA, based depending on which U was strategically utilized to restrain the off-target effects of the siRNA. This study describes a rare example of nucleoside analogs with a large substitution at the 2'-position in the context of an siRNA application and is informative for the development of other analogs to further improve the molecular properties of siRNAs for medicinal applications.

Modified keyhole plus technique with partial release of posterior rectus sheath for parastomal hernia repair after ileal conduit.

Parastomal hernia (PH) is a common complication of ileal conduit diversions. The Sugarbaker technique has a lower recurrence rate than the keyhole (KH) technique and is typically preferred. However, it may not be feasible in some cases because of anatomical features including the length of the conduit and torsion of the ureter. An 80-year-old woman with complaints of abdominal distention was diagnosed with PH 5 years after radical cystectomy. Computed tomography revealed a 90 × 20-mm muscular layer defect on the cranial side of the ileal conduit. Therefore, we performed the KH technique with intracorporeal closure of the defect using a relief incision of the posterior rectus sheath, avoiding the possibility of torsion of the ureteral ileal anastomosis. No hernia recurrence was observed at postoperative 10 months. The proposed KH plus technique may be an effective method for PH after ileal conduit diversion, thus preventing urinary complications.

Complete Chemical Synthesis of Minimal Messenger RNA by Efficient Chemical Capping Reaction.

Site-specific chemical modification of mRNA can improve its translational efficiency and stability. For this purpose, it is desirable to develop a complete chemical synthesis method for chemically modified mRNA. The key is a chemical reaction that introduces a cap structure into the chemically synthesized RNA. In this study, we developed a fast and quantitative chemical capping reaction between 5'-phosphorylated RNA and N7-methylated GDP imidazolide in the presence of 1-methylimidazole in the organic solvent dimethyl sulfoxide. It enabled quantitative preparation of capping RNA within 3 h. We prepared chemically modified 107-nucleotide mRNAs, including N6-methyladenosine, insertion of non-nucleotide linkers, and 2'-O-methylated nucleotides at the 5' end and evaluated their effects on translational activity in cultured HeLa cells. The results showed that mRNAs with non-nucleotide linkers in the untranslated regions were sufficiently tolerant to translation and that mRNAs with the Cap_2 structure had higher translational activity than those with the Cap_0 structure.

Development of Fluorophosphoramidate as a Biocompatibly Transformable Functional Group and its Application as a Phosphate Prodrug for Nucleoside Analogs.

Synthetic phosphate-derived functional groups are important for controlling the function of bioactive molecules in vivo. Herein we describe the development of a new type of biocompatible phosphate analog, a fluorophosphoramidate (FPA) functional group that has characteristic P-F and P-N bonds. We found that FPA with a primary amino group was relatively unstable in aqueous solution and was converted to a monophosphate, while FPA with a secondary amino group was stable. Furthermore, by improving the molecular design of FPA, we developed a reaction in which a secondary amino group is converted to a primary amino group in the intracellular environment and clarified that the FPA group functions as a phosphate prodrug of nucleoside. Various FPA-gemcitabine derivatives were synthesized and their toxicity to cancer cells were evaluated. One of the FPA-gemcitabine derivatives showed superior toxicity compared with gemcitabine and its ProTide prodrug, which methodology is widely used in various nucleoside analogs, including anti-cancer and anti-virus drugs.

Prophylactic use of pegfilgrastim enables the management of severe neutropenia without dose delays in patients with metastatic colorectal cancer treated with TAS-102 plus bevacizumab.

Combined treatment with bevacizumab and trifluridine/tipiracil (TAS-102) leads to an increased chance of survival in patients with refractory metastatic colorectal cancer (mCRC); however, this treatment is associated with an increased frequency of severe neutropenia (number of neutrophils <1,000), which should ideally be managed without dose delays. The present study provided a retrospective review of 35 patients with mCRC, and aimed to elucidate the benefits of prophylactic pegfilgrastim for the treatment of severe neutropenia. Patients received TAS-102 (35 mg/m2) orally twice daily on days 1-5 and 8-12 of each 28-day treatment cycle, along with intravenous bevacizumab (5 mg/kg) on days 1 and 15. Moreover, the patients received 3.6 mg pegfilgrastim on day 15 of each cycle. The incidence of adverse events (AEs), disease control rate (DCR), progression-free survival (PFS) and overall survival (OS) were assessed. In the first and subsequent cycles, 23 and 12 patients, respectively, received pegfilgrastim. The most common AE experienced was grade 3/4 neutropenia (8 patients; 22.9%). Among these 8 patients, 6 (17.1%) and 3 (8.6%) exhibited neutropenia prior to receiving pegfilgrastim or following discontinuation of pegfilgrastim administration, respectively. Moreover, 1 individual among these 8 patients (2.9%) demonstrated grade 3 neutropenia both prior to receiving pegfilgrastim and following discontinuation of pegfilgrastim. A total of 2 patients (5.7%) exhibited grade 3 bone pain, which prevented sustainable administration of pegfilgrastim and resulted in grade 3 neutropenia. Dose delays and dose reduction of TAS-102 due to neutropenia were required in 5 (14.3%) and 2 (5.7%) patients, respectively, during the treatment period. None of the patients exhibited severe neutropenia during chemotherapy after pegfilgrastim administration, thereby preventing dose delays and dose reduction of TAS-102. The relative dose intensity was 96.8% (65.0-100.0%), and the DCR was 54.3%. The median PFS and median OS were 4.4 and 14.9 months, respectively. In conclusion, prophylactic pegfilgrastim may facilitate the management of severe neutropenia without dose delays in patients with mCRC treated with TAS-102 plus bevacizumab.

Genome­wide DNA hypomethylation drives a more invasive pancreatic cancer phenotype and has predictive occult distant metastasis and prognosis potential.

Genome­wide DNA hypomethylation is the most common molecular feature in human cancers associated with chromosomal instability (CIN), which is involved in the mechanisms that regulate pancreatic cancer (PC) metastasis. It was investigated whether genome­wide DNA hypomethylation affects the phenotype in PC via CIN in vitro, and its significance on the biological behavior of PC was verified. The relative demethylation level (RDL) of long interspersed nucleotide element­1 (LINE­1) in human PC cell lines was used to characterize DNA hypomethylation using methylation­specific quantitative (q)PCR. CIN was estimated by changes in chromosomal copy number using comparative genomic hybridization analysis. Abnormal segregation of chromosomes was assessed by immunocytochemistry, and the DNA damage response was evaluated using the number of anti­Î³H2AX positive cells. Invasion ability was assessed using a Matrigel invasion assay. Clinical specimens from 49 patients with PC who underwent curative surgery were evaluated for a correlation of DNA hypomethylation with clinical outcome. Successful induction of genome­wide DNA hypomethylation in PC cells led to copy number changes in specific chromosomal regions. The number of cells with abnormal segregation of chromosomes significantly increased with the number of anti­Î³H2AX positive cells. The invasive potential of these cells also significantly increased. The occurrence of occult distant metastasis in the clinical specimens and receiver operating characteristic analysis clearly identified those who were and were not likely to have occult distant metastasis, with high LINE­1 RDL significantly correlated with the presence of occult distant metastasis (P=0.035) and poor prognosis (P=0.048). The significance of genome­wide DNA hypomethylation on the biological behavior of PC, which promotes a more invasive phenotype via CIN in vitro and predicts the susceptibility to occult distant metastasis and poor prognosis in patients with PC was revealed.

Impact of sarcopenia on surgical and oncologic outcomes of laparoscopic surgery for colorectal cancer.

BACKGROUND: The study aimed to evaluate the impact of sarcopenia on short- and long-term outcomes for laparoscopic colorectal cancer surgery. METHODS: Study participants were 209 patients who underwent laparoscopic surgery for any stage of colorectal cancer between 2016 and 2017. Skeletal muscle indices were calculated with preoperative computed tomography. Patients were divided into sarcopenic and non-sarcopenic groups based on index cut-off values and variables were compared. RESULTS: The prevalence of sarcopenia was 41.1%. Sarcopenic patients experienced shorter operative times and a lower incidence of surgical site infections; however, the incidence of severe postoperative complications and readmission were increased for this group. Although the 3-year disease-free survival rate was not statistically different between groups, sarcopenic patients had a significantly worse 3-year overall survival rate compared with than the non-sarcopenic group. CONCLUSION: Sarcopenia has both favorable and unfavorable effects on patients who underwent laparoscopic colorectal cancer surgery.

Antisense Oligonucleotide Modified with Disulfide Units Induces Efficient Exon Skipping in mdx Myotubes through Enhanced Membrane Permeability and Nucleus Internalization.

We have found that antisense oligonucleotides and siRNA molecules modified with repeat structures of disulfide units can be directly introduced into the cytoplasm and exhibit a suppressive effect on gene expression. In this study, we analyzed the mechanism of cellular uptake of these membrane-permeable oligonucleotides (MPONs). Time-course analysis by confocal microscopy showed that the uptake of MPONs from the plasma membrane to the cytoplasm reached 50 % of the total uptake in about 5 min. In addition, analysis of the plasma membrane proteins to which MPONs bind, identified several proteins, including voltage-dependent anion channel. Next, we analyzed the behavior of MPONs in the cell and found them to be abundant in the nucleus as early as 24 h after addition with the amount increasing further after 48 and 72 h. The amount of MPONs was 2.5-fold higher than that of unmodified oligonucleotides in the nucleus after 72 h. We also designed antisense oligonucleotides and evaluated the effect of MPONs on mRNA exon skipping using DMD model cells; MPONs caused exon skipping with 69 % efficiency after 72 h, which was three times higher than the rate of the control. In summary, the high capacity for intracytoplasmic and nuclear translocation of MPONs is expected to be useful for therapeutic strategies targeting exon skipping.

Laparoscopic surgery for strangulated diaphragmatic hernia after radiofrequency ablation for hepatocellular carcinoma: a case report.

BACKGROUND: Percutaneous radiofrequency ablation (RFA) is an effective treatment for hepatocellular carcinoma (HCC), but delayed thermal damage can cause diaphragmatic hernia (DH). Surgery is recommended for DH, and open surgery is widely accepted. This report presents a case of laparoscopic surgery for strangulated DH that occurred after RFA. CASE PRESENTATION: An 80-year-old woman with a history of hepatitis C-induced liver cirrhosis and HCC was admitted to our institution owing to sudden-onset intense epigastric pain. Twenty-two months earlier, she received RFA treatment for HCC located in segment 6/7. Contrast-enhanced computed tomography revealed herniation of the small intestine into the thoracic cavity, with mesenteric fat haziness. Emergency laparoscopic surgery was performed, and the patient was diagnosed with strangulated DH associated with the prior RFA. The defect was closed using absorbable sutures, and the ischaemic small intestine was resected via mini-laparotomy. The patient was discharged on the 10th postoperative day without complications, and no evidence of DH recurrence 15 months after surgery was noted. CONCLUSIONS: Laparoscopic surgery seems useful and feasible for strangulated DH.

Completely Chemically Synthesized Long DNA Can be Transcribed in Human Cells.

Chemical ligation reaction of DNA is useful for the construction of long functional DNA using oligonucleotide fragments that are prepared by solid phase chemical synthesis. However, the unnatural linkage structure formed by the ligation reaction generally impairs the biological function of the resulting ligated DNA. We achieved the complete chemical synthesis of 78 and 258 bp synthetic DNAs via multiple chemical ligation reactions with phosphorothioate and haloacyl-modified DNA fragments. The latter synthetic DNA, coding shRNA for luciferase genes with a designed truncated SV promoter sequence, successfully induced the expected gene silencing effect in HeLa cells.

Structure, Synthesis and Inhibition Mechanism of Nucleoside Analogues as HIV-1 Reverse Transcriptase Inhibitors (NRTIs).

Acquired immunodeficiency syndrome (AIDS) is caused by infection with the human immunodeficiency virus (HIV). Although treatments against HIV infection are available, AIDS remains a serious disease that causes many deaths annually. Although a variety of anti-HIV drugs have been synthesized and marketed to treat HIV-infected patients, nucleoside analogue reverse transcriptase inhibitors (NRTIs), which mimic nucleosides, are used extensively and remain a subject of interest to medicinal chemists. However, HIV has acquired drug resistance against NRTIs, and thus the struggle to find novel therapies continues. In this review, we trace the trajectory of NRTIs, focusing on the synthesis, mechanisms of action and applications of NRTIs that have been developed.