Pancreatic juice cytology for diagnosing invasive pancreatic carcinoma/high-grade pancreatic intraepithelial neoplasia without visible tumors on endoscopic ultrasound.

OBJECTIVES: Pancreatic ductal adenocarcinoma (PDAC) with a diameter ≤10 mm and high-grade pancreatic intraepithelial neoplasia (HG-PanIN) require pre-operative diagnosis. Most cases present only indirect imaging findings without visible tumors on endoscopic ultrasound (EUS). Therefore, EUS-guided fine-needle aspiration/biopsy is not applicable. An alternative diagnostic method is pancreatic juice cytology (PJC) via endoscopic naso-pancreatic drainage (ENPD-PJC), which is not the standard practice. This study aimed to investigate ENPD-PJC for diagnosing suspected PDAC/HG-PanIN cases without visible tumors on EUS. METHODS: Data of patients with suspected PDAC/HG-PanIN without visible tumors who underwent PJC were retrospectively evaluated. One PJC sample was collected during endoscopic retrograde pancreatography (ERP-PJC), and 12 samples were collected during ENPD-PJC, 3-hourly for cytological analysis. ERP-PJC, ERP/ENPD-PJC, and ENPD-PJC positivity indicated cytologically positive samples. Patients with positive/negative PJC with follow-up for <4-years were excluded as undiagnosed cases. A non-malignant diagnosis was based on histopathological absence/stable imaging findings for ≥4-years. The primary endpoint was to demonstrate that ERP/ENPD-PJC has a higher diagnostic ability than ERP-PJC. RESULTS: Twenty-two patients with histopathologically diagnosed PDAC/HG-PanIN and 31 with a non-malignant diagnosis were enrolled. ERP-PJC, ERP/ENPD-PJC, and ENPD-PJC showed sensitivities of 36.4 %, 86.4 %, and 77.3 %, specificities of 93.5 %, 87.1 %, and 93.5 %, and accuracies of 69.8 %, 86.7 %, and 86.7 %, respectively. ERP/ENPD-PJC and ENPD-PJC demonstrated superior sensitivity and accuracy compared to ERP-PJC. A greater occurrence of positive outcomes markedly distinguished true positives from false positives. CONCLUSIONS: ERP/ENPD-PJC and ENPD-PJC had higher diagnostic accuracies for PDAC/HG-PanIN without visible tumors on EUS. ENPD-PJC is recommended for the diagnosis of these lesions.

Multivalent dendritic DNA aptamer molecules for the enhancement of therapeutic effects.

Dendritic DNA molecules, referred to as DNA dendrons, consist of multiple covalently linked strands and are expected to improve the cellular uptake and potency of therapeutic oligonucleotides because of their multivalency. In this study, we developed an efficient synthetic method for producing DNA dendrons using strain-promoted azide-alkyne cycloaddition. Integration of the antitumor aptamer AS1411 into DNA dendrons enhanced cellular uptake and antiproliferative activity in cancer cells. These findings demonstrate that the incorporation of multivalent aptamers into DNA dendrons can effectively boost their therapeutic effects.

Development of a Cytosolic DNA Sensor Agonist Using GALA Peptide-Conjugated DNA and Long Single-Stranded DNA.

Cytosolic DNA sensors (CDSs) recognize DNA molecules that are abnormally located in the cytosol, thus leading to the activation of the stimulator of interferon genes (STING) and the induction of type 1 interferon. In turn, type 1 interferon evokes defensive reactions against viral infections and activates the immune system; therefore, the use of agonists of CDSs as cancer therapeutics and vaccine adjuvants is expected. Double-stranded DNA molecules with dozens to thousands of bases derived from bacteria and viruses are agonists of CDSs. However, DNA is a water-soluble molecule with a high molecular weight, resulting in poor cellular uptake and endosomal escape. In contrast, long single-stranded DNA (lssDNA) obtained by rolling circle amplification is efficiently taken up and localized to endosomes. Here we constructed a CDS-targeting lssDNA via the facilitation of its intracellular transport from endosomes to the cytosol. An endosome-disrupting GALA peptide was used to deliver the lssDNA to the cytosol. A peptide-oligonucleotide conjugate (POC) was successfully obtained via the conjugation of the GALA peptide with an oligonucleotide complementary to the lssDNA. By hybridization of the POC to the complementary lssDNA (POC/lssDNA), the CDS-STING pathway in dendritic cells was efficiently stimulated. GALA peptide-conjugated DNA seems to be a helpful tool for the delivery of DNA to the cytosol.

Development of potent unmethylated CpG DNA hydrogel by introducing i-motifs into long single-stranded DNA.

Unmethylated cytosine-phosphate-guanine (CpG) DNA is recognized by Toll-like receptor 9, expressed in the endosomes of immune cells, and induces the secretion of proinflammatory cytokines. CpG DNA is, therefore, expected to be used as vaccine adjuvants, but there are many obstacles for its therapeutic application, such as poor cellular uptake and biostability. Long single-stranded DNA (lssDNA) synthesized by rolling circle amplification can be a useful delivery carrier for CpG DNA because of its cellular uptake efficiency, but the immunostimulatory effect is transient because it is easily degraded in endosomes. To improve its stability, we constructed lssDNA which forms hydrogel by i-motifs in an acidic environment mimicking endosome, and incorporated CpG DNA into lssDNA (i-CpG-lssDNA). We synthesized lssDNA containing the optimized i-motif sequence, and confirmed the formation of a DNA hydrogel in an acidic environment. The i-CpG-lssDNA elicited a potent proinflammatory cytokine production in murine macrophages, compared to CpG DNA-containing lssDNA without i-motifs. Consistently, its intradermal administration induced potent inflammatory cytokines at the regional lymph nodes. These results suggested that i-CpG-lssDNA could serve as a novel type of adjuvant for the induction of a potent immune response.

Development of nucleic acid medicines based on chemical technology.

Oligonucleotide-based therapeutics have attracted attention as an emerging modality that includes the modulation of genes and their binding proteins related to diseases, allowing us to take action on previously undruggable targets. Since the late 2010s, the number of oligonucleotide medicines approved for clinical uses has dramatically increased. Various chemistry-based technologies have been developed to improve the therapeutic properties of oligonucleotides, such as chemical modification, conjugation, and nanoparticle formation, which can increase nuclease resistance, enhance affinity and selectivity to target sites, suppress off-target effects, and improve pharmacokinetic properties. Similar strategies employing modified nucleobases and lipid nanoparticles have been used for developing coronavirus disease 2019 mRNA vaccines. In this review, we provide an overview of the development of chemistry-based technologies aimed at using nucleic acids for developing therapeutics over the past several decades, with a specific emphasis on the structural design and functionality of chemical modification strategies.

Pancreatic duct epithelial malignancy suggested by large focal pancreatic parenchymal atrophy in cystic diseases of the pancreas.

BACKGROUND: /Objectives: A cystic lesion is common in the pancreas. Focal pancreatic parenchymal atrophy (FPPA) has been reported as a sign of high-grade pancreatic intraepithelial neoplasia/carcinoma in situ (HGP/CIS). Some cystic lesions accompany FPPA. However, the relationship between a cystic lesion, FPPA, and the histopathological background of the pancreatic duct is unknown. METHODS: We retrospectively evaluated the data of 98 patients with a cystic lesion who underwent serial pancreatic juice aspiration cytologic examination (SPACE) because of accompanying FPPA, increased size of the cystic lesion, and pancreatic duct stricture at the base. RESULTS: The clinical diagnosis of a cystic lesion was intraductal papillary mucinous neoplasia (IPMN) and cysts in 72 (73.5%) and 26 (26.5%) patients, respectively. Ninety of the 98 patients (91.8%) had FPPA. Positive results (adenocarcinoma and suspicion) on SPACE were observed in 56 of all cases (57.1%), 48 of IPMN (66.7%), 8 of cysts (30.8%), and 54 of FPPA (59.3%), and were significantly associated with IPMN (p = 0.002) and the large FPPA (>269.79 mm2,p = 0.0001); moreover, these disorders are considerably related (p = 0.0003). Fifty patients (51.0%) with positive results on SPACE underwent surgery, with the histopathological diagnosis of epithelial malignancy in 42 patients (42.9%, 42/50, 84%). Many cystic lesions clinically diagnosed as IPMN were dilated branches covered by pancreatic intraepithelial neoplasia. CONCLUSIONS: Positive results on SPACE were significantly associated with the clinical diagnosis of IPMN and the large FPPA. Moreover, these disorders are significantly related. Surgery owing to positive results could lead to the histopathological diagnosis of HGP/CIS.

Laparoscopic left hepatectomy in a goat as a training model for laparoscopic anatomic liver resection: results of training courses with a total of 70 goats.

BACKGROUND: To create a suitable animal model for the training of laparoscopic anatomic liver resection, we performed left hepatectomy using a goat and found its suitability. We have since started using goats for wet-lab training and have gradually standardized the relevant procedures. Herein, we report our standardized training procedures using a goat and discuss its feasibility as a novel training model. METHODS: The standardized wet-lab training courses of laparoscopic liver resection conducted on 62 tables with a total of 70 goats were reviewed. The training course began by encircling the hepatoduodenal ligament for the Pringle maneuver, which was repeated during the parenchymal dissection. Following partial liver resection of the left lateral section, left hepatectomy was performed by a standardized procedure for humans in which the liver was split, exposing the entire length of the middle hepatic vein trunk from the dorsal side after extrahepatic transection of the left Glissonean pedicle. If a goat deceased before initiating left hepatectomy, the training was restarted with a new goat. The surgical procedures were performed by surgeons of varying skill levels. RESULTS: A total of 184 surgeons including 10 surgical residents participated in the training. Partial liver resection was initiated in 62 tables, with 8 (13%) dying during or after the procedure of partial liver resection. Subsequently, left hepatectomy was initiated in 61 and completed in 59 tables (98%), regardless of whether the goat survived or deceased, and was not completed in 2 tables (3%) due to time limitation. In 14 tables (23%), the goats deceased during the procedure, however, the procedure was completed. The causes of death were multifactorial, including massive bleeding, reperfusion injury after the Pringle maneuver, and carbon dioxide gas embolism. CONCLUSIONS: Left hepatectomy in a goat is useful as a training model for laparoscopic anatomic liver resection.

Novel Hemostatic Technique During Laparoscopic Liver Parenchymal Transection: Saline-Linked Electrocautery Combined With Wet Oxidized Cellulose (SLiC-WOC) Method.

INTRODUCTION: Although laparoscopic hepatectomy has the potential advantage of reducing intraoperative blood loss, it is more difficult to control bleeding laparoscopically compared to an open approach. We introduced a novel hemostatic technique, the saline-linked electrocautery combined with wet oxidized cellulose (SLiC-WOC) method, during laparoscopic hepatectomy where a combination of saline-linked electrocautery (SLiC) and wet oxidized cellulose (WOC) is used. This study aimed to investigate the feasibility of employing the SLiC-WOC method for laparoscopic hepatectomy. METHODS: Thirteen patients who underwent laparoscopic liver resection with the SLiC-WOC method between 2019 and 2020 were included in this study. The number of bleeding episodes in which the SLiC-WOC method was applied was counted, and the time required to achieve complete hemostasis was measured. RESULTS: Among the bleeding events that were difficult to achieve hemostasis by SLiC alone, 94% were safely and efficiently controlled. Additionally, 69% of hemostasis was achieved within 60 seconds and 91% within 120 seconds. Postoperatively, most patients experienced no complications and no operative mortality was observed. CONCLUSIONS: The SLiC-WOC method can provide safe and time-efficient hemostasis during laparoscopic hepatectomy. This is especially crucial for bleeding, which is difficult to control using electrocautery alone.

Enhanced Immunostimulatory Activity of Covalent DNA Dendrons.

The present study focused on the design and synthesis of covalent DNA dendrons bearing multivalent cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODNs) that can stimulate the immune system through the activation of TLR9. These dendrons were synthesized using branching trebler phosphoramidite containing three identical protecting groups that enabled the simultaneous synthesis of multiple strands on a single molecule. Compared with linear ODNs, covalent DNA dendrons were found to be more resistant to nuclease degradation and were more efficiently taken up by macrophage-like RAW264.7 cells. Cellular uptake was suggested to be mediated by macrophage scavenger receptors. The covalent DNA dendrons composed of multivalent immunostimulatory branches enhanced the secretion of proinflammatory cytokines TNF-α and IL-6 from RAW264.7 cells, and 9-branched DNA dendrons showed the highest enhancement. Given their enhanced efficacy, we expect covalent DNA dendrons to be useful structures of oligonucleotide medicines.

[Retrospective Analysis of Treatment Strategies for Local Recurrence following Radical Resection of Rectal Cancer].

We report the findings from a retrospective study to determine the optimum treatment strategy for local recurrence following radical resection of rectal cancer. In our department, among all 430 patients that underwent radical resection of rectal cancer from 2012 to 2018, there were 28 patients that developed local recurrence. Of those patients, 12 underwent surgical treatment(Op group)and 16 did not(N-Op group). In the Op group, 8 patients underwent radical resection, of which 2 patients remained recurrence-free, and the other 6 patients developed recurrence. In the N-Op group, 6 patients were treated with systemic chemotherapy alone, a further 6 patients had palliative irradiation in addition to systemic chemotherapy, and the other 4 selected best supportive care(2 patients were treated with palliative irradiation). In the 8 patients who had palliative irradiation, 7 showed a decrease in numerical rating scale(NRS)after irradiation. The adverse events of palliative irradiation were scrotal dermatitis in 1 patient and perianal inflammation in another 3 patients. Our surgical results for local recurrence of rectal cancer in our department were worse in terms of recurrence rate, so these findings suggest that the preoperative surgical strategy could be reviewed, as well as the actual surgical methods such as the optimal circumferential resection margin. Palliative irradiation was found to be useful for pain control. However, the occurrence of adverse events remains a concern.

Telomere-specific chromatin capture using a pyrrole-imidazole polyamide probe for the identification of proteins and non-coding RNAs.

BACKGROUND: Knowing chromatin components at a DNA regulatory element at any given time is essential for understanding how the element works during cellular proliferation, differentiation and development. A region-specific chromatin purification is an invaluable approach to dissecting the comprehensive chromatin composition at a particular region. Several methods (e.g., PICh, enChIP, CAPTURE and CLASP) have been developed for isolating and analyzing chromatin components. However, all of them have some shortcomings in identifying non-coding RNA associated with DNA regulatory elements. RESULTS: We have developed a new approach for affinity purification of specific chromatin segments employing an N-methyl pyrrole (P)-N-methylimidazole (I) (PI) polyamide probe, which binds to a specific sequence in double-stranded DNA via Watson-Crick base pairing as a minor groove binder. This new technique is called proteomics and RNA-omics of isolated chromatin segments (PI-PRICh). Using PI-PRICh to isolate mouse and human telomeric components, we found enrichments of shelterin proteins, the well-known telomerase RNA component (TERC) and telomeric repeat-containing RNA (TERRA). When PI-PRICh was performed for alternative lengthening of telomere (ALT) cells with highly recombinogenic telomeres, in addition to the conventional telomeric chromatin, we obtained chromatin regions containing telomeric repeat insertions scattered in the genome and their associated RNAs. CONCLUSION: PI-PRICh reproducibly identified both the protein and RNA components of telomeric chromatin when targeting telomere repeats. PI polyamide is a promising alternative to simultaneously isolate associated proteins and RNAs of sequence-specific chromatin regions under native conditions, allowing better understanding of chromatin organization and functions within the cell.

A Near-Infrared Fluorogenic Pyrrole-Imidazole Polyamide Probe for Live-Cell Imaging of Telomeres.

Telomeres are closely associated with cellular senescence and cancer. Although some techniques have been developed to label telomeres in living cells for study of telomere dynamics, few biocompatible near-infrared probes based on synthetic molecules have been reported. In this study, we developed a near-infrared fluorogenic pyrrole-imidazole polyamide probe (SiR-TTet59B) to visualize telomeres by conjugating a silicon-rhodamine (SiR) fluorophore with a tandem tetramer pyrrole-imidazole polyamide targeting 24 bp in the telomeric double-stranded (ds) DNA. SiR-TTet59B was almost nonfluorescent in water but increased its fluorescence dramatically on binding to telomeric dsDNA. Using a peptide-based delivery reagent, we demonstrated the specific and effective visualization of telomeres in living U2OS cells. Moreover, SiR-TTet59B could be used to observe the dynamic movements of telomeres during interphase and mitosis. This simple imaging method using a synthetic near-infrared probe could be a powerful tool for studies of telomeres and for diagnosis.

How to dissect the liver parenchyma: Excavation with cavitron ultrasonic surgical aspirator.

BACKGROUND: A large amount of blood flows in and out of the liver through the inflow system consisting of the portal vein and hepatic artery within the Glissonean cord and the outflow system constituted by the hepatic veins. METHODS: During liver parenchymal dissection, useful methods to maintain a dry operative field are to control the inflow system with the Pringle maneuver and the outflow system by managing the central venous pressure. Additionally, mature techniques of dissecting the liver parenchyma, which can prevent injury to the blood vessels and appropriately and promptly stop bleeding, are fundamental. Similar to archaeological excavation, in which buried remains are unearthed and exposed in intact form, the Glissonean cords and hepatic veins buried in the liver parenchyma should be exposed or isolated without causing injury to these structures during liver parenchymal dissection. RESULTS: The cavitron ultrasonic surgical aspirator (CUSA) is useful as a surgical device for excavation because it has multiple functions in one device. However, there have been no systematic guidelines on how to use it effectively during hepatectomy. CONCLUSION: We herein describe how to use the CUSA, based on our knowledge and experiences.

Basic knowledge of and a small trick for atraumatic needle driving in laparoscopic suturing.

Highlight Although recent advances in robotic surgery have enabled more precise movements of the needle driver, a sophisticated laparoscopic suturing technique is still desired. Honda and colleagues describe the basic knowledge of and a small but useful trick for atraumatic needle driving in laparoscopic suturing, based on mechanistic considerations.

Laparoscopic Liver Resection of Segments 7 and 8: from the Initial Restrictions to the Current Indications.

Since the beginning of laparoscopic liver surgery, resection of the posterosuperior segments has been considered one of the most challenging procedure due to its difficult access. The main drawbacks of the laparoscopic approach to dome lesions are poor visualization, the difficulty of instrumentation and the greater complexity in the control of bleeding. In the evolution of minimally invasive techniques from hybrid techniques to the current purely laparoscopic approaches, the different authors have established gradually the currents indications and surgical techniques to operate these segments with a similar feasibility and safety than open approach. The standardization in the patient position, the use of intercostal trocars, the learning curve in laparoscopic liver surgery, the management of the hepatic blood flow and the refinement of the technique in the extrahepatic and intrahepatic Glissonean pedicle approaches, has allowed to leave behind the initial contraindications about the laparoscopic approach in these segments. In the present review of the literature, the accumulated experience of the different groups in minimally invasive liver surgery together with the technological advances in the different laparoscopic devices have facilitated the resection of tumors in segments 7 and 8 with similar and even better results than open surgery.

Submolecular dissection reveals strong and specific binding of polyamide-pyridostatin conjugates to human telomere interface.

To modulate biological functions, G-quadruplexes in genome are often non-specifically targeted by small molecules. Here, specificity is increased by targeting both G-quadruplex and its flanking duplex DNA in a naturally occurring dsDNA-ssDNA telomere interface using polyamide (PA) and pyridostatin (PDS) conjugates (PA-PDS). We innovated a single-molecule assay in which dissociation constant (Kd) of the conjugate can be separately evaluated from the binding of either PA or PDS. We found Kd of 0.8 nM for PA-PDS, which is much lower than PDS (Kd ∼ 450 nM) or PA (Kd ∼ 35 nM). Functional assays further indicated that the PA-PDS conjugate stopped the replication of a DNA polymerase more efficiently than PA or PDS. Our results not only established a new method to dissect multivalent binding into actions of individual monovalent components, they also demonstrated a strong and specific G-quadruplex targeting strategy by conjugating highly specific duplex-binding molecules with potent quadruplex ligands.

Effect of antithrombic therapy on bleeding complications in patients receiving emergency cholecystectomy for acute cholecystitis.

BACKGROUND: The risk of developing hemorrhagic complications during or after emergency cholecystectomy in patients with antithrombic therapy (ATT) remains uncertain. In this study, we evaluate outcomes in patients with ATT undergoing emergency cholecystectomy and assess the relevance between ATT and perioperative complications including bleeding complications. METHODS: We retrospectively evaluated 296 patients who were diagnosed as acute cholecystitis and underwent emergency cholecystectomy between 2005 and 2017. One hundred and thirty-three of them (45%) were on ATT. The primary outcome measures were intraoperative blood loss over 500 ml and postoperative complications including bleeding complications. This study was approved by our institutional review board (#13072904). RESULTS: There were 23 patients (8%) who experienced intraoperative blood loss over 500 ml and nine postoperative bleeding complications (3%). Multivariable analyses showed that male sex (P = 0.027), Performance Status 2-4 (P = 0.031) and grade II or III acute cholecystitis (P = 0.033) were independent risk factors for intraoperative bleeding over 500 ml, whereas not single antiplatelet therapy (APT) use but multiple APT (P = 0.034) and anticoagulation therapy (ACT) (P = 0.032) were independently associated with postoperative bleeding complications. Additionally, laparoscopic surgery, but not ATT, was a significant prognostic factor for severe postoperative complications. CONCLUSIONS: Single APT was not remained as an independently associated factor of intraoperative excessive bleeding or severe postoperative complications including bleeding complications. However, patients treated with multiple APT or ACT still represent a challenging group and must be carefully managed to avoid postoperative bleeding complications.

TAMRA-polypyrrole for A/T sequence visualization on DNA molecules.

Fluorophore-linked, sequence-specific DNA binding reagents can visualize sequence information on a large DNA molecule. In this paper, we synthesized newly designed TAMRA-linked polypyrrole to visualize adenine and thymine base pairs. A fluorescent image of the stained DNA molecule generates an intensity profile based on A/T frequency, revealing a characteristic sequence composition pattern. Computer-aided comparison of this intensity pattern with the genome sequence allowed us to determine the DNA sequence on a visualized DNA molecule from possible intensity profile pattern candidates for a given genome. Moreover, TAMRA-polypyrrole offers robust advantages for single DNA molecule detection: no fluorophore-mediated photocleavage and no structural deformation, since it exhibits a sequence-specific pattern alone without the use of intercalating dyes such as YOYO-1. Accordingly, we were able to identify genomic DNA fragments from Escherichia coli cells by aligning them to the genomic A/T frequency map based on TAMRA-polypyrrole-generated intensity profiles. Furthermore, we showed band and interband patterns of polytene chromosomal DNA stained with TAMRA-polypyrrole because it prefers to bind AT base pairs.

Sequence-specific DNA binding Pyrrole-imidazole polyamides and their applications.

Pyrrole-imidazole polyamides (Py-Im polyamides) are cell-permeable compounds that bind to the minor groove of double-stranded DNA in a sequence-specific manner without causing denaturation of the DNA. These compounds can be used to control gene expression and to stain specific sequences in cells. Here, we review the history, structural variations, and functional investigations of Py-Im polyamides.