Caffeic acid-grafted chitooligosaccharides downregulate MAPK and NF-kB in RAW264.7 cells.

Chitooligosaccharide (COS) is a derivative of chitosan, which is a natural macromolecular compound. COS has been shown effects in an inflammatory response. Recent reports show that COS derivatives have enhanced anti-inflammatory activity by inhibiting intracellular signals. Evaluation of the anti-inflammatory effect of caffeic acid conjugated COS chain (CA-COS) was performed in this study. The effects of CA-COS on the inflammatory response were demonstrated in lipopolysaccharide-stimulated RAW264.7 macrophages. The results showed that CA-COS inhibited nitric oxide (NO) production and downregulated the gene expression of nitric oxide synthase (iNOS), and cytokines such as tumor necrosis factor-alpha (TNF-α), IL-1ß, and IL-6 without cytotoxic effect. In addition, western blot analysis showed that CA-COS inhibits the protein expression of iNOS and nuclear factor kappa B (NF-kB), including p50 and p65, and mitogen-activated protein kinase (MAPK) signaling pathways. Collectively, these results provide clear evidence for the anti-inflammatory mechanism of CA-COS that show great potential as a novel agent for the prevention and therapy of inflammatory diseases.

Dysphagia and associated factors among patients with acute ischemic stroke in Vietnam.

Background: Dysphagia is considered an important issue in managing and treating acute stroke, but there are currently no studies investigating this issue, especially for patients with acute ischemic stroke in Vietnam. Thus, we conducted this study to determine the prevalence of dysphagia and associated factors of dysphagia in patients with acute ischemic stroke in Vietnam. Materials and methods: From June 2020 to January 2022, a cross-sectional study of patients with acute ischemic stroke was conducted in a tertiary hospital in Vietnam. The dysphagia was evaluated through a bedside screening test using the Gugging Swallowing Screen (GUSS). Factors associated with dysphagia were analysed using univariate and multivariate logistic regression. Results: The prevalence of dysphagia in patients with acute ischemic stroke was 71.6%, in which the mild, moderate and severe dysphagia accounted for 37.5%, 12.4% and 21.7%, respectively. Dysphagia significantly associated with age group 50-59 (OR = 2.2, 95% CI: 1.2-4.2), age group 60-69 (OR = 1.9, 95% CI: 1.04-3.4), age group >70 (OR = 2.2, 95% CI: 1.2-4.2), brainstem (OR = 4.0, 95% CI: 2.1-7.4), having communication disorder (OR = 1.5, 95% CI: 1.1-7.4) and having facial paralysis (OR = 17.9, 95% CI: 12.0-26.8). Conclusion: Our study showed that the prevalence of dysphagia is high among patients with acute ischemic stroke in Vietnam. Intervention solutions should focus more on patient groups of higher age group, brainstem stroke, communication disorder and facial paralysis.

Safety and Efficacy of Rivaroxaban for Extended-Phase Anticoagulation of Patients with Unprovoked or Recurrent Venous Thromboembolism: Real-Life Data from the MAC Project.

Venous thromboembolism (VTE) is a major cause of death in the world. After the acute-phase treatment, the optimal duration of anticoagulation is still debatable. The latest guidelines suggest maintaining long-term anticoagulation in patients with cancer-associated thrombosis (CAT) or with unprovoked VTE and a low bleeding risk. Methods: The MAC Project is an ongoing prospective-cohort, multi-center, observational study in Italy. The project aims to collect real-life clinical information in unselected patients given oral anticoagulants for VTE over a 5-year follow-up period. There were no exclusion criteria, except for life expectancy <6 months and refusal to sign the informed consent form or to attend the planned follow-up visit. All patients were followed-up prospectively with clinical controls scheduled at 3, 6, and 12 months after the index event, and then annually for up to 5 years. The primary efficacy and safety outcomes were symptomatic recurrent VTE and major bleeding. Results: We analyzed 450 consecutive patients treated with rivaroxaban and referred them to the MAC Project database for unprovoked or recurrent VTE. Of these, 267 (55%) were unprovoked VTE, and 377 (87%) were symptomatic. We followed up with the patients for a mean of 22 months (Q1 10.7; Q3 37.4 months). Recurrent VTE occurred in 12 patients on rivaroxaban treatment (IR 1.7 per 100 person-years). Males had more recurrence than women. During the follow-up period, we recorded 13 (2.9%) major bleeding, 12 (2.7%) clinically relevant non-major bleeding, 8 minor bleeding, and no fatal bleeding events. Overall, bleeding events occurred in 33 (7.3%) patients, most occurring within the first 2 years of treatment. In addition, we observed a statistically significant higher incidence of bleeding in patients with a baseline HAS-BLED score of 3 to 4 compared with those with a score of 0 to 2, with most events occurring during the first 3 months of treatment (RR 5.9). Discussion: Rivaroxaban appears to be safe and effective for the long-term treatment of patients with recurrent or unprovoked VTE. Our results match previously published data, and we are confident that the continuation of the follow-up for up to 5 years will confirm these outcomes.

Percutaneous Nephroureteral Tube: A Useful Tool for Management of Intractable Hematuria.

OBJECTIVE: To describe our experience using percutaneous nephroureteral (PCNU) tube placement for the management of intractable gross hematuria. METHODS: We identified patients at our institution who underwent PCNU tube placement from August 2011 to October 2017 for management of gross hematuria who had previously failed management with manual irrigation, continuous bladder irrigation, and cystoscopy with clot evacuation. The primary outcome measured was cessation of bleeding obviating the need for further blood transfusion in a 30 day follow-up period. RESULTS: Six patients were treated with PCNU tube placement for intractable hematuria from either malignant or nonmalignant etiologies. In all patients after PCNU tube placement, hematocrit value remained stable, there were no further transfusions requirements within 30 days, and no immediate or periprocedural complications were encountered. In no instance did a PCNU become obstructed by blood clots and in all cases the bladder and both kidneys were adequately drained. CONCLUSION: In patients with hematuria refractory to conventional management techniques, the placement of a PCNU tube allows for cessation of bleeding, successfully diverts urine with no immediate complications and is not subsequent to clot obstruction of the tube. The use of PCNU tube is a viable treatment in the algorithm of intractable hematuria, specifically before resorting to more morbid and potentially irreversible treatments.

A Critical Comparison of Rejection-Based Algorithms for Simulation of Large Biochemical Reaction Networks.

The rejection-based simulation technique has been applying to improve the computational efficiency of the stochastic simulation algorithm (SSA) in simulating large reaction networks, which are required for a thorough understanding of biological systems. We compare two recently proposed simulation methods, namely the composition-rejection algorithm (SSA-CR) and the rejection-based SSA (RSSA), aiming for this purpose. We discuss the right interpretation of the rejection-based technique used in these algorithms in order to make an informed choice when dealing with different aspects of biochemical networks. We provide the theoretical analysis as well as the detailed runtime comparison of these algorithms on concrete biological models. We highlight important factors that are omitted in previous analysis of these algorithms. The numerical comparison shows that for reaction networks where the search cost is expensive then SSA-CR is more efficient, and for reaction networks where the update cost is dominant, often the case in practice, then RSSA should be the choice.

Reconstruction following abdominoperineal resection (APR): Indications and complications from a single institution experience.

BACKGROUND: Abdominoperineal resection (APR) is the surgical treatment of low-lying rectal cancers and other pelvic malignancies. Plastic surgery offers a means to close these complicated defects through obliterating dead space, providing tension-free closure, and introducing vascularized tissue into a radiated field. The indications for reconstructive surgery and choice of reconstruction are debatable. This study aims to identify when and which reconstruction is preferred. METHODS: A retrospective comparative analysis was performed on all patients undergoing APR at Stanford Hospital between 2007 and 2013. Data points included demographics, disease, operative positioning, and postoperative complications. Univariate analysis and multivariate logistic regression analysis were performed to identify markers of flap reconstruction and complications. RESULTS: A total of 178 APRs were performed, of which 51 underwent flap reconstruction. The odds ratio of all complications between flap and primary closure was not significant at 1.36 (0.69-2.66). Independent predictors for flap reconstruction included prone positioning, anal squamous cell carcinoma (SCC), prior smoking, and neoadjuvant chemoradiation therapy. Univariate predictors of flap reconstruction included female gender and combined vaginectomy. Independent predictors of complications included current and prior smoking. Muscle flap closure had lower recipient site complications than V-to-Y advancement closure (20% vs. 50%, p = 0.039). CONCLUSION: Flap reconstruction following APR is associated with prone positioning, neoadjuvant chemoradiation, female gender, prior smoking, and anal SCC resections. Pedicled muscle flaps had a significantly lower rate of recipient site complications than V-to-Y advancement flaps and therefore should be the flap reconstruction of choice. The vertical rectus abdominis myocutaneous flap was superior to the gracilis flap in terms of the overall reduction of complications.

Localized malignant pleural sarcomatoid mesothelioma misdiagnosed as benign localized fibrous tumor.

Localized malignant pleural mesothelioma (LMPM) is a rare tumor with good prognosis by surgical resection. We report an atypical case of malignant pleural sarcomatoid mesothelioma (SM) in an asymptomatic 65-year-old woman, who had no history of exposure to asbestos. She presented with a small pleural mass without pleural effusion and was misdiagnosed as a benign localized fibrous tumor (BLFT) on pathologic examination through a surgical tumor specimen. However, seven months later, the patient returned with serious cancerous symptoms. A large recurrent tumor mass was found within the chest wall invading at the old surgical resection site. SM, a subtype of LMPM, was confirmed with histopathogy and immunohistochemisty. In conclusion, malignant pleural mesothelioma (MPM) can present with typical radiologic finding similar to a BLFT, and has a wide histopathologic presentation in biopsy specimen. A thorough pathologic investigation should be attempted even when a pleural mass resembles benign, localized, and small on radiologic studies.

Equivocal effect of intraoperative fluorescence angiography on colorectal anastomotic leaks.

BACKGROUND: Intraoperative fluorescence angiography is beneficial in several surgical settings to assess tissue perfusion. It is also used to assess bowel perfusion, but its role in improving outcomes in colorectal surgery has not been studied. OBJECTIVE: The purpose of this work was to determine whether intraoperative angiography decreases colorectal anastomotic leaks. DESIGN: This was a case-matched retrospective study in which patients were matched 1:1 with respect to sex, age, level of anastomosis, presence of a diverting loop ileostomy, and preoperative pelvic radiation therapy. SETTINGS: The study was conducted at an academic medical center. PATIENTS: Patients who underwent colectomy or proctectomy with primary anastomoses were included. INTERVENTIONS: The intraoperative use of fluorescence angiography to assess perfusion of the colon for anastomosis was studied. MAIN OUTCOME MEASURES: Anastomotic leak within 60 days and whether angiography changed surgical management were the main outcomes measured. RESULTS: Case matching produced 173 pairs. The groups were also comparable with respect to BMI, smoking status, diabetes mellitus, surgical indications, and type of resection. In patients who had intraoperative angiography, 7.5% developed anastomotic leak, whereas 6.4% of those without angiography did (p value not significant). Univariate analysis revealed that preoperative pelvic radiation, more distal anastomosis, surgeon, and diverting loop ileostomy were positively associated with anastomotic leak. Multivariate analysis demonstrated that level of anastomosis and surgeon were associated with leaks. Poor perfusion of the proximal colon seen on angiography led to additional colon resection before anastomosis in 5% of patients who underwent intraoperative angiography. LIMITATIONS: The retrospective study design with the use of historical control subjects, selection bias, and small sample size were limitations to this study. CONCLUSIONS: Intraoperative fluorescence angiography to assess the perfusion of the colon conduit for anastomosis was not associated with colorectal anastomotic leak. Perfusion is but one of multiple factors contributing to anastomotic leaks. Additional studies are necessary to determine whether this technology is beneficial for colorectal surgery.

Efficient rejection-based simulation of biochemical reactions with stochastic noise and delays.

We propose a new exact stochastic rejection-based simulation algorithm for biochemical reactions and extend it to systems with delays. Our algorithm accelerates the simulation by pre-computing reaction propensity bounds to select the next reaction to perform. Exploiting such bounds, we are able to avoid recomputing propensities every time a (delayed) reaction is initiated or finished, as is typically necessary in standard approaches. Propensity updates in our approach are still performed, but only infrequently and limited for a small number of reactions, saving computation time and without sacrificing exactness. We evaluate the performance improvement of our algorithm by experimenting with concrete biological models.

Dual-color ultrasensitive bright-field RNA in situ hybridization with RNAscope.

In situ hybridization (ISH) techniques have been important to the study of gene expression signatures in cells and tissues. The ability to detect multiple targets simultaneously is especially valuable, since it allows dissecting gene expression of distinct cell types with precise cellular and subcellular resolution within morphological context. Recently, we have reported using a novel dual-color ultrasensitive bright-field RNA in situ hybridization for detection of clonally restricted immunoglobulin light chain mRNA expression in B cell lymphomas. Here, we present detailed protocols of RNAscope 2-Plex assays for FFPE tissue sections. The protocols describe the tissue preparation, pretreatment, probe hybridization, signal amplification, visualization, and analysis, as well as emphasize the critical steps for ensuring successful staining.

Quantitative ultrasensitive bright-field RNA in situ hybridization with RNAscope.

RNA in situ hybridization (ISH) can provide valuable morphological context for molecular markers on one hand and enable morphological analysis in molecular context on the other hand. It has become increasingly important, thanks to increasing interest in new biomarkers and noncoding RNAs in both research and clinical applications. We have developed an ultrasensitive RNA ISH technology, RNAscope, employing a unique probe design strategy that allows target-specific signal amplification while suppressing background noise. This approach enables single RNA molecule detection in formalin-fixed paraffin-embedded (FFPE) specimens under standard bright-field microscopy and is capable of multiplex detection at the single cell level. After staining, target-specific signals appear as punctate dots present in individual cells in well-preserved tissue morphological context, which facilitates both semiquantitative manual scoring and software-assisted quantitative analysis. Here, we present detailed protocols of RNAscope for FFPE tissue sections. The step-by-step protocols describe tissue preparation, pretreatment, probe hybridization, signal amplification, visualization, and analysis. We also highlight the critical steps for ensuring successful staining.

RNAscope for in situ detection of transcriptionally active human papillomavirus in head and neck squamous cell carcinoma.

The 'gold standard' for oncogenic HPV detection is the demonstration of transcriptionally active high-risk HPV in tumor tissue. However, detection of E6/E7 mRNA by quantitative reverse transcription polymerase chain reaction (qRT-PCR) requires RNA extraction which destroys the tumor tissue context critical for morphological correlation and has been difficult to be adopted in routine clinical practice. Our recently developed RNA in situ hybridization technology, RNAscope, permits direct visualization of RNA in formalin-fixed, paraffin-embedded (FFPE) tissue with single molecule sensitivity and single cell resolution, which enables highly sensitive and specific in situ analysis of any RNA biomarker in routine clinical specimens. The RNAscope HPV assay was designed to detect the E6/E7 mRNA of seven high-risk HPV genotypes (HPV16, 18, 31, 33, 35, 52, and 58) using a pool of genotype-specific probes. It has demonstrated excellent sensitivity and specificity against the current 'gold standard' method of detecting E6/E7 mRNA by qRT-PCR. HPV status determined by RNAscope is strongly prognostic of clinical outcome in oropharyngeal cancer patients.

Automated quantitative RNA in situ hybridization for resolution of equivocal and heterogeneous ERBB2 (HER2) status in invasive breast carcinoma.

Patient management based on HER2 status in breast carcinoma is an archetypical example of personalized medicine but remains hampered by equivocal testing and intratumoral heterogeneity. We developed a fully automated, quantitative, bright-field in situ hybridization technique (RNAscope), applied it to quantify single-cell HER2 mRNA levels in 132 invasive breast carcinomas, and compared the results with those by real-time quantitative PCR (qPCR) and Food and Drug Administration-approved methods, including fluorescence in situ hybridization (FISH), IHC, chromogenic in situ hybridization, and dual in situ hybridization. Both RNAscope and qPCR were 97.3% concordant with FISH in cases in which FISH results were unequivocal. RNAscope was superior to qPCR in cases with intratumoral heterogeneity or equivocal FISH results. This novel assay may enable ultimate HER2 status resolution as a reflex test for current testing algorithms. Quantitative in situ RNA measurement at the single-cell level may be broadly applicable in companion diagnostic applications.

RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues.

In situ analysis of biomarkers is highly desirable in molecular pathology because it allows the examination of biomarker status within the histopathological context of clinical specimens. Immunohistochemistry and DNA in situ hybridization (ISH) are widely used in clinical settings to assess protein and DNA biomarkers, respectively, but clinical use of in situ RNA analysis is rare. This disparity is especially notable when considering the abundance of RNA biomarkers discovered through whole-genome expression profiling. This is largely due to the high degree of technical complexity and insufficient sensitivity and specificity of current RNA ISH techniques. Here, we describe RNAscope, a novel RNA ISH technology with a unique probe design strategy that allows simultaneous signal amplification and background suppression to achieve single-molecule visualization while preserving tissue morphology. RNAscope is compatible with routine formalin-fixed, paraffin-embedded tissue specimens and can use either conventional chromogenic dyes for bright-field microscopy or fluorescent dyes for multiplex analysis. Unlike grind-and-bind RNA analysis methods such as real-time RT-PCR, RNAscope brings the benefits of in situ analysis to RNA biomarkers and may enable rapid development of RNA ISH-based molecular diagnostic assays.