Molecular-Level Structural Analysis of siRNA-Loaded Lipid Nanoparticles by 1H NMR Relaxometry: Impact of Lipid Composition on Their Structural Properties.

1H NMR relaxometry was applied for molecular-level structural analysis of siRNA-loaded lipid nanoparticles (LNPs) to clarify the impact of the neutral lipids, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol, on the physicochemical properties of LNP. Incorporating DSPC and cholesterol in ionizable lipid-based LNP decreased the molecular mobility of ionizable lipids. DSPC reduced the overall molecular mobility of ionizable lipids, while cholesterol specifically decreased the mobility of the hydrophobic tails of ionizable lipids, suggesting that cholesterol filled the gap between the hydrophobic tails of ionizable lipids. The decrease in molecular mobility and change in orientation of lipid mixtures contributed to the maintenance of the stacked bilayer structure of siRNA and ionizable lipids, thereby increasing the siRNA encapsulation efficiency. Furthermore, NMR relaxometry revealed that incorporating those neutral lipids enhanced PEG chain flexibility at the LNP interface. Notably, a small amount of DSPC effectively increased PEG chain flexibility, possibly contributing to the improved dispersion stability and narrower size distribution of LNPs. However, cryogenic transmission electron microscopy represented that adding excess amounts of DSPC and cholesterol into LNP resulted in the formation of deformed particles and demixing cholesterol within the LNP, respectively. The optimal lipid composition of ionizable lipid-based LNPs in terms of siRNA encapsulation efficiency and PEG chain flexibility was rationalized based on the molecular-level characterization of LNPs. Moreover, the NMR relaxation rate of tertiary amine protons of ionizable lipids, which are the interaction site with siRNA, can be a valuable indicator of the encapsulated amount of siRNA within LNPs. Thus, NMR-based analysis can be a powerful tool for efficiently designing LNP formulations and their quality control based on the molecular-level elucidation of the physicochemical properties of LNPs.

Factors Related to Difficulty in Endoscopic Submucosal Dissection for Superficial Esophageal Cancer.

BACKGROUND: Endoscopic submucosal dissection (ESD) for superficial esophageal cancer is technically challenging, and research on predictive factors related to the difficulty in the procedure is limited. This study aimed to investigate the factors predicting the difficulty in esophageal ESD. METHODS: This retrospective study analyzed 303 lesions treated at our institution between April 2005 and June 2021. The following 13 factors were evaluated: sex, age, tumor location, tumor localization, macroscopic type, tumor size, tumor circumference, preoperative diagnosis of histological type, preoperative diagnosis of invasion depth, previous radiotherapy for esophageal cancer, metachronous lesion located close to post-ESD scar, operator's skill, and use of a clip-and-thread traction method. Difficult esophageal ESD cases were defined as those requiring long procedure time (>120 min). RESULTS: Fifty-one lesions (16.8%) met the defined criterion for difficult cases of esophageal ESD. Logistic regression analysis identified tumor size larger than 30 mm (odds ratio: 9.17, 95% confidence interval: 4.27-19.69, p < 0.001) and tumor circumference more than half that of the esophagus (odds ratio 2.53, 95% confidence interval: 1.15-5.54, p = 0.021) as independent predictive factors related to difficulty in esophageal ESD. CONCLUSION: Tumor size larger than 30 mm and tumor circumference more than half that of the esophagus can predict difficulty in performing esophageal ESD. This knowledge can provide useful information for developing ESD strategies and selecting a suitable operator on a case-by-case basis to achieve favorable clinical outcomes.

Usefulness of magnifying endoscopy with acetic acid and narrow-band imaging for the diagnosis of duodenal neoplasms: proposal of a diagnostic algorithm.

BACKGROUND: This study aimed to clarify the features of superficial non-ampullary duodenal epithelial tumors (SNADETs) on magnifying endoscopy with narrow-band imaging (M-NBI) and magnifying endoscopy with acetic acid and narrow-band imaging (M-AANBI), and evaluate the efficacy of M-NBI/M-AANBI to distinguish high-grade adenomas or adenocarcinomas (HGA/AC) from low-grade adenomas (LGA). METHODS: Clinicopathological data on 62 SNADETs in 58 patients who underwent preoperative M-NBI/M-AANBI and endoscopic resection were retrospectively reviewed. The pathological results were classified into two categories, LGA and HGA/AC. We evaluated microvascular patterns (MVPs) and microsurface patterns (MSPs) observed by M-NBI and MSPs observed by M-AANBI for characterizing LGA and HGA/AC. The kappa value was calculated to assess the interobserver and intraobserver agreements of evaluation of M-AANBI images. RESULTS: Pathologically, 38 lesions (61.3%) were LGA and 24 lesions (38.7%) were HGA/AC. HGA/AC tended to have irregular MVP and/or MSP on M-NBI. M-NBI diagnostic performance to distinguish HGA/AC from LGA showed 62.5% sensitivity, 68.4% specificity, and 66.1% accuracy. SNADETs had irregular MSP on M-AANBI. Three irregularity grades (iG) of MSP were observed by M-AANBI as follows: iG1, mild; iG2, moderate; iG3, significant. HGA/AC lesions had a significantly higher rate of iG3 than LGA lesions (p < 0.001). The iG2 was associated with HGA/AC in elevated lesions and LGA in depressed lesions. The diagnostic performance of M-AANBI was as follows: 95.8% sensitivity, 97.4% specificity, and 96.8% accuracy. The diagnostic accuracy of M-AANBI was significantly higher than that of M-NBI (p < 0.001). The kappa value for interobserver agreement on the diagnosis and irregularity grading of M-AANBI images was 0.742 and 0.719, respectively. These data indicate substantial interobserver agreement. Based on the above-mentioned results, we developed a M-AANBI diagnostic algorithm for SNADETs. CONCLUSION: The diagnostic algorithm for SNADETs using M-AANBI may be useful for differentiating between LGA and HGA/AC.

Clinical utility of endoscopic submucosal dissection using the pocket-creation method with a HookKnife and preoperative evaluation by endoscopic ultrasonography for the treatment of rectal neuroendocrine tumors.

BACKGROUND: Various endoscopic methods have been developed to remove small rectal neuroendocrine tumors (NETs). This study aimed to evaluate the clinical utility of endoscopic submucosal dissection using the pocket-creation method (ESD-PCM) with a HookKnife, following preoperative evaluation by endoscopic ultrasonography (EUS), for the treatment of rectal NETs. METHODS: We analyzed retrospectively consecutive patients who underwent ESD-PCM with a HookKnife for the removal of rectal NETs, with a size less than 10 mm, at Mie University Hospital between June 2015 and December 2019. All the rectal NETs were resected by ESD-PCM with a HookKnife. The R0 resection rate, procedure time, adverse event rate, diagnostic accuracy of tumor size and invasion depth evaluated by preoperative EUS, and follow-up outcome were evaluated retrospectively. RESULTS: The study group comprised 12 patients with 12 resected lesions. The median tumor size of the resected specimens was 5 mm and the size and invasion depth of each tumor was approximately equal to that predicted by preoperative EUS. R0 resection was achieved in all cases, without adverse events. The median procedure time was 50.5 min, which did not differ from previous studies. No recurrence was observed during the median follow-up period of 34.4 months (range, 5.2-60.0 months). CONCLUSIONS: ESD-PCM with a HookKnife provides a favorable clinical utility for removing rectal NETs, with high R0 resection rate and good follow-up outcome. In addition, EUS is useful for evaluating preoperatively the size and invasion depth of rectal NETs.

[Laparoscopic and Endoscopic Cooporative Surgery(LECS)for Superficial Non-Ampullary Duodenal Tumor - A Case Report].

INTRODUCTION: We report a case ofsuperf icial non-ampullary duodenal tumor(SNADT)resected by laparoscopic and endoscopic cooperative surgery(LECS)technique. CASE PRESENTATION: A 55-year-old man underwent screening esophagogastroduodenoscopy. Endoscopy revealed 0- II a+ II c mucosal lesion measuring 15mm in size located the portion ofduodenum contralateral to the ampulla ofVater. During observation, irregularity in depressed mucosa was observed and malignant alteration was suspected. So, we performed local resection with LECS as diagnostic therapy. During operation, endoscopic mucosal resection(ESD)was performed first. Next, duodenum was mobilized laparoscopically and the floor of the ulcer was closed with endoscopy guided laparoscopic suturing technique. Histopathology revealed tubular adenoma and the resection margin was negative. DISCUSSION: SNADT is rare condition and therapeutic strategy for SNADT has not established. Further study are needed.

Crohn's disease complicated by hepatitis B virus successfully treated with the use of adsorptive depletion of myeloid lineage leucocytes to suppress inflammatory cytokine profile.

BACKGROUND AIMS: In patients with inflammatory bowel disease infected with hepatitis B virus (HBV), immunosuppressive therapy required to suppress active inflammatory bowel disease may promote HBV reactivation. METHODS: A 27-year-old corticosteroid-naive woman with Crohn's disease (CD) activity index of 249.8 complicated by HBV infection was offered Entecavir to control HBV reactivation during immunosuppressive therapy for CD. The patient refused Entecavir, fearing that it might adversely affect her pregnancy outcome. Instead, we applied intensive granulocyte/monocyte adsorptive apheresis (GMA) at two sessions per week to deplete inflammatory cytokine-producing leucocytes as an immunosuppressive therapy in this case. RESULTS: GMA induced stable remission (CD activity index, I 105) and endoscopic improvement without HBV reactivation or safety concern. Furthermore, CD remission was paralleled by suppression of tumor necrosis factor and interleukin as measured in serum samples. CONCLUSIONS: Immunosuppressive therapy required to treat an active CD potentially can promote HBV reactivation and worsen liver function. In this study involving a CD case complicated by chronic HBV infection, intensive GMA as a non-pharmacologic treatment intervention was associated with clinical remission and endoscopic improvement without HBV reactivation. Furthermore, GMA was well-tolerated and was without any safety concern. However, suppression of tumor necrosis and interleukin-6by GMA in this clinical setting is potentially very interesting.

Endoscopic submucosal dissection for superficial esophageal cancer in a diverticulum: A case report with literature review.

Superficial esophageal cancer (SEC) in a diverticulum is rare and has a high risk of perforation during endoscopic resection. Although endoscopic submucosal dissection (ESD) is a standard treatment option, it is challenging to perform. Here, we describe the case of a 79-year-old male patient with a history of ESD for SEC. Surveillance esophagogastroduodenoscopy identified a 20-mm-sized reddish depressed lesion in a diverticulum in the middle esophagus. The lesion was confirmed to be squamous cell carcinoma by biopsy. Magnification endoscopy with narrow-band imaging showed intraepithelial papillary capillary loops of type B1 according to the magnified endoscopic classification of the Japan Esophageal Society. Endoscopic ultrasonography revealed the presence of the muscular layer of the esophagus wall in the diverticulum. Therefore, the lesion was diagnosed as SEC, confined to the epithelium or lamina propria mucosae, in a Rokitansky diverticulum. Based on these findings, ESD was considered technically feasible. Traction-assisted ESD using clip with line was performed, and en bloc resection was achieved without adverse events. The resected specimen pathologically revealed a squamous cell carcinoma confined to the lamina propria mucosae without lymphovascular invasion, suggesting a curative resection. The patient recovered well, and no recurrence has been observed for 5 years after the ESD. Whether ESD is appropriate for the treatment of SEC in a diverticulum remains unclear. However, our case shows that it can be a treatment option in such cases due to its minimal invasiveness and good effectiveness.

Tibial nerve dynamics during ankle dorsiflexion: The relationship between stiffness and excursion of the tibial nerve.

Peripheral nerves extend with a gradual increase in stiffness and also with excursion, namely reduction of fiber bundle waviness, to adapt to joint movements. Although the close relationships between the tibial nerve (TN) excursion and stiffness during ankle dorsiflexion in cadaver studies, the precise in vivo their relationships remain unclear. We hypothesized that the excursion of the TN can be estimated from its stiffness in vivo using shear-wave elastography. This study aimed to analyze the relationships between the TN stiffness at the plantarflexion and dorsiflexion and TN excursion during dorsiflexion using ultrasonography. Twenty-one healthy adults participated in constant-velocity movements of the ankle joint with a 20° range from the maximum dorsiflexion, and the TN was imaged using an ultrasound imaging system. The maximum flow velocity value and the TN excursion distance per dorsiflexion were then calculated as indexes of excursion using the application software Flow PIV. The shear wave velocities of the TN at plantarflexion and dorsiflexion were also measured. Based on our single linear regression, the shear wave velocities of the TN at the plantarflexion had the strongest effect on the excursion indexes, followed by the those at dorsiflexion. Ultrasonographic shear wave velocity could predict the TN excursion if measured under mild plantarflexion of the ankle joint, and might have a close biomechanical relation to the total waviness of the TN.

Risk Factors Associated with Painful Colonoscopy and Prolonged Cecal Intubation Time in Female Patients.

Objectives: Few studies have examined risk factors leading to painful colonoscopy and prolonged cecal intubation time in female patients. We aimed to determine the factors associated with painful colonoscopy and prolonged cecal intubation time in female patients. Methods: This retrospective study analyzed prospectively collected data from a randomized controlled trial with female patients who underwent colonoscopy. Multivariate logistic and linear regression analyses were performed using the following factors that might be associated with painful colonoscopy and prolonged cecal intubation time, respectively: age, body mass index, history of colonoscopy, previous abdominal surgery, routine use of laxatives, inadequate bowel preparation, sigmoid colon diverticulosis, use of a small-caliber colonoscope, and an inexperienced operator. Results: The study enrolled 219 female patients aged >20 years. Using the receiver operating characteristic curve, painful colonoscopy was defined in cases where the visual analogue scale of overall pain was ≥50 mm. Logistic regression analysis for risk factors associated with painful colonoscopy revealed that sigmoid colon diverticulosis [odds ratio (OR), 2.496; 95% confidence interval (CI), 1.013-5.646; p=0.028] was a risk factor for painful colonoscopy; conversely, the use of a small-caliber colonoscope was a negative factor for painful colonoscopy (OR, 0.436; 95% CI, 0.214-0.889, p=0.022). In linear regression analysis, inadequate bowel preparation was significantly associated with prolonged cecal intubation time (ß-coefficient, 3.583; 95% confidence interval, 0.578-6.588; p=0.020). Conclusions: Female patients with sigmoid colon diverticulosis are more likely to experience severe pain during colonoscopy, and those with inadequate bowel preparation may require more time for cecal intubation.

Obscure Gastrointestinal Bleeding Caused by a Small Intestinal Lymphatic-venous Malformation: A Case Report with a Literature Review.

A 44-year-old woman presented with severe anemia. We strongly suspected gastrointestinal bleeding; however, esophagogastroduodenoscopy, colonoscopy, and computed tomography showed no bleeding sources. Video capsule endoscopy revealed an actively bleeding submucosal lesion within the jejunum. Double-balloon enteroscopy revealed a 20-mm continuously bleeding submucosal lesion in the distal jejunum. We suspected small intestinal vascular malformation and performed surgical resection. The resected specimen pathologically comprised dilated, thin-walled lymphatic channels and blood vessels involving the small intestinal submucosa. Therefore, the patient was diagnosed with small intestinal lymphatic-venous malformation. Postoperatively, the patient recovered well, and recurrence was not observed.

Negative effect of prolonged cecal intubation time on adenoma detection in female patients.

Background and Aim: Withdrawal time of the colonoscope is associated with adenoma detection. However, the association between cecal intubation time and adenoma detection remains unclear. This study aimed to evaluate the association between cecal intubation time and adenoma detection. Methods: This retrospective study analyzed prospectively collected data from a randomized controlled trial on female patients who underwent colonoscopy in an academic hospital. The primary outcome was the mean number of all adenomas detected per patient. Secondary outcomes included the mean number of advanced, diminutive, small/large, right-sided colonic, and left-sided colonic adenomas detected per patient. Furthermore, the detection rates of all categories of adenoma were evaluated. Results: The analysis included 216 female patients aged ≥20 years. The correlation analysis did not reveal a significant relationship (P = 0.473) between cecal intubation and withdrawal times. The mean number of all adenomas detected per patient declined by approximately 30% (1.05-0.70) from the fastest to the slowest insertion time quartile. Adjusted regression analysis showed a significant decrease in the mean number of all adenomas detected per patient with increased intubation time (relative risk, RR = 0.87; 95% confidence interval, 0.76-0.99, P = 0.045), whereas the mean number of other categories of adenomas detected per patient and the detection rates of all categories of adenoma were not associated with the cecal intubation time. Conclusions: This study showed a significant association between prolonged cecal intubation time and decreased adenoma detection. The cecal intubation time may be a significant quality indicator for colonoscopy.

Long-term outcomes after endoscopic submucosal dissection for colorectal epithelial neoplasms in patients with severe comorbidities.

Background and Aim: Long-term outcomes after endoscopic submucosal dissection (ESD) for colorectal epithelial neoplasms (CENs) in patients with severe comorbidities have not been clarified; the current study aimed to examine these long-term outcomes and compared them with those in patients with non-severe comorbidities. Methods: We included 231 patients with CENs who underwent ESD between April 2005 and March 2023. Patients with comorbidities were categorized according to the American Society of Anesthesiologists Physical Status (ASA-PS). We conducted a propensity score-matched analysis and compared long-term outcomes of the two groups after ESD for CENs. Results: Of the 156 patients enrolled in the study, 43 and 113 had severe (ASA-PS III) and non-severe (ASA-PS I/II) comorbidities, respectively. The 1:1 propensity score analysis matched 36 patients with severe comorbidities to 36 patients with non-severe comorbidities. After matching, there was no difference in the procedural outcomes of ESD between both groups. Regarding long-term outcomes, the 5-year overall survival rates after matching in the ASA-PS I/II and III groups were 100% and 73.5%, respectively, and patients in the ASA-PS III group exhibited significantly shorter overall survival than those in the ASA-PS I/II group (hazard ratio 7.209; 95% confidence interval 1.592-32.646; P = 0.010). No colorectal cancer-related deaths were noted in either group. Conclusion: Overall survival after ESD for CENs was shorter in patients with severe comorbidities than in those with non-severe comorbidities. Clinicians should carefully determine whether the benefits of CEN resection with ESD outweigh the procedural risks in patients with severe comorbidities.

Incomplete activation of Alyref and Gabpb1 leads to preimplantation arrest in cloned mouse embryos.

Differentiated cell nuclei can be reprogrammed after nuclear transfer (NT) to oocytes and the produced NT embryos can give rise to cloned animals. However, development of NT embryos is often hampered by recurrent reprogramming failures, including the incomplete activation of developmental genes, yet specific genes responsible for the arrest of NT embryos are not well understood. Here, we searched for developmentally important genes among the reprogramming-resistant H3K9me3-repressed genes and identified Alyref and Gabpb1 by siRNA screening. Gene knockout of Alyref and Gabpb1 by the CRISPR/Cas9 system resulted in early developmental arrest in mice. Alyref was needed for the proper formation of inner cell mass by regulating Nanog, whereas Gabpb1 deficiency led to apoptosis. The supplement of Alyref and Gabpb1 mRNA supported efficient preimplantation development of cloned embryos. Alyref and Gabpb1 were silenced in NT embryos partially because of the repressed expression of Klf16 by H3K9me3. Thus, our study shows that the H3K9me3-repressed genes contain developmentally required genes, and the incomplete activation of such genes results in preimplantation arrest of cloned embryos.