Promoter-independent synthesis of chemically modified RNA by human DNA polymerase θ variants.

Synthetic RNA oligonucleotides composed of canonical and modified ribonucleotides are highly effective for RNA antisense therapeutics and RNA-based genome engineering applications utilizing CRISPR-Cas9. Yet, synthesis of synthetic RNA using phosphoramidite chemistry is highly inefficient and expensive relative to DNA oligonucleotides, especially for relatively long RNA oligonucleotides. Thus, new biotechnologies are needed to significantly reduce costs, while increasing synthesis rates and yields of synthetic RNA. Here, we engineer human DNA polymerase theta (Polθ) variants and demonstrate their ability to synthesize long (95-200 nt) RNA oligonucleotides with canonical ribonucleotides and ribonucleotide analogs commonly used for stabilizing RNA for therapeutic and genome engineering applications. In contrast to natural promoter-dependent RNA polymerases, Polθ variants synthesize RNA by initiating from DNA or RNA primers, which enables the production of RNA without short abortive byproducts. Remarkably, Polθ variants show the lower capacity to misincorporate ribonucleotides compared to T7 RNA polymerase. Automation of this enzymatic RNA synthesis technology can potentially increase yields while reducing costs of synthetic RNA oligonucleotide production.

Oncological risk of proximal gastrectomy for proximal advanced gastric cancer after neoadjuvant chemotherapy.

PURPOSE: This study assesses the metastasis rate of the key distal lymph nodes (KDLN) that are not routinely dissected in proximal gastrectomy, aiming to explore the oncological safety of proximal gastrectomy for upper gastric cancer who underwent neoadjuvant chemotherapy. METHODS: We analyzed a cohort of 150 patients with proximal locally advanced gastric cancer (cT3/4 before chemotherapy) from two high-volume cancer centers in China who received preoperative neoadjuvant chemotherapy (NAC) and total gastrectomy with lymph node dissection. Metastasis rate of the KDLN (No.5/6/12a) and the risk factors were analyzed. RESULTS: Key distal lymph node metastasis was detected in 10% (15/150) of patients, with a metastasis rate of 6% (9/150) in No. 5 lymph nodes, 6.7% (10/150) in No. 6 lymph nodes, and 2.7% (2/75) in No. 12a lymph nodes. The therapeutic value index of KDLN as one entity is 5.8. Tumor length showed no correlation with KDLN metastasis, while tumor regression grade (TRG) emerged as an independent risk factor (OR: 1.47; p-value: 0.04). Of those with TRG3 (no response to NAC), 80% (12/15) was found with KDLN metastasis. CONCLUSION: For cT3/4 proximal locally advanced gastric cancer patients, the risk of KDLN metastasis remains notably high even after NAC. Therefore, proximal gastrectomy is not recommended; instead, total gastrectomy with thorough distal lymphadenectomy is the preferred surgical approach.

Combined Use of Tumor Markers in Gastric Cancer: A Novel Method with Promising Prognostic Accuracy and Practicality.

BACKGROUND: The effect of a single tumor marker on the prognosis of gastric cancer patients is not ideal. This study explored a novel prognostic assessment method for gastric cancer (GC) patients using a combination of three important tumor markers (CEA, CA72-4, and CA19-9). METHOD: Data from 1966 GC patients who underwent curative gastrectomy at Sun Yat-Sen University Cancer Center (Guangzhou, China) were included. Hazard ratios (HR) for all factors for overall survival (OS) were analyzed by Cox regression. A nomogram and calibration curve were used to establish the survival prediction model. The prediction accuracy was evaluated with the concordance index (C-index). RESULTS: All patients were divided into four groups (C0-C3) according to the number of elevated tumor markers. The 5-year OS rates of the patients in preoperative groups C0-C3 were 83.8% (81.3-86.4%), 72.8% (68.5-77.4%), 58.9% (50.4-68.9%), and 18.5% (4.0-33.0%), respectively, and those in postoperative groups C0-C3 were 82.1% (79.4-84.8%), 76.1% (72.2-80.3%), 57.6% (48.4-68.5%), and 16.8% (5.1-28.5%), respectively, with significant differences between each C0-C3 subgroup in both preoperative and postoperative cohorts. Multivariate analysis showed that preoperative (HR: 6.001, 95% CI: 3.523-10.221) and postoperative (HR: 8.149, 95% CI: 4.962-13.528) elevated tumor markers were independent risk factors for GC patients. The C-index for the combined use of tumor markers was 0.65-0.66, which was higher than that for using a single tumor marker (0.53-0.56). CONCLUSION: The combined use of tumor markers significantly improved the prognostic value compared with using a single tumor marker. The survival prediction model including the combined tumor markers was accurate and effective.

Adjuvant Chemotherapy for Patients with Adenocarcinoma of the Esophagogastric Junction: A Retrospective, Multicenter, Observational Study.

BACKGROUND: Although the incidence of adenocarcinoma of the esophagogastric junction (AEG) has been increasing since the past decade, the proportion of AEG cases in two previous clinical trials (ACTS-GC and CLASSIC) that investigated the efficacy of adjuvant chemotherapy was relatively small. Therefore, whether AEG patients can benefit from adjuvant chemotherapy remains unclear. METHODS: Patients who were diagnosed with pathological stage II/III, Siewert II/III AEG, and underwent curative surgery at three high-volume institutions were assessed. Clinical outcomes were analyzed by using Kaplan-Meier curves, log-rank test, and Cox regression model. Propensity score matching (PSM) was used to reduce the selection bias. RESULTS: A total of 927 patients were included (the chemotherapy group: 696 patients; the surgery-only group: 231 patients). The median follow-up was 39.0 months. The 5-year overall survival was 63.1% (95% confidence interval [CI]: 59.0-67.6%) for the chemotherapy group and 50.2% in the surgery-only group (hazard ratio [HR] = 0.69, 95% CI: 0.54-0.88; p = 0.003). The 5-year, disease-free survival was 35.4% for the chemotherapy group and 16.6% for the surgery-only group (HR = 0.66, 95% CI: 0.53-0.83; p < 0.001). After PSM, the survival benefit of adjuvant chemotherapy for AEG was maintained. Multivariate analysis for overall survival and disease-free survival further demonstrated the survival benefit of adjuvant chemotherapy, with HRs of 0.63 (p < 0.001) and 0.52 (p < 0.001), respectively. CONCLUSIONS: Postoperative adjuvant chemotherapy was associated with improved overall survival and disease-free survival in patients with operable stage II or III AEG after D2 gastrectomy.

The role of computed tomography features in assessing response to neoadjuvant chemotherapy in locally advanced gastric cancer.

OBJECTIVE: To compare the computed tomography (CT) images of patients with locally advanced gastric cancer (GC) before and after neoadjuvant chemotherapy (NAC) in order to identify CT features that could predict pathological response to NAC. METHODS: We included patients with locally advanced GC who underwent gastrectomy after NAC from September 2016 to September 2021. We retrieved and collected the patients' clinicopathological characteristics and CT images before and after NAC. We analyzed CT features that could differentiate responders from non-responders and established a logistic regression equation based on these features. RESULTS: We included 97 patients (69 [71.1%] men; median [range] age, 60 [26-75] years) in this study, including 66 (68.0%) responders and 31 (32.0%) non-responders. No clinicopathological variable prior to treatment was significantly associated with pathological response. Out of 16 features, three features (ratio of tumor thickness reduction, ratio of reduction of primary tumor attenuation in arterial phase, and ratio of reduction of largest lymph node attenuation in venous phase) on logistic regression analysis were used to establish a regression equation that demonstrated good discrimination performance in predicting pathological response (area under receiver operating characteristic curve 0.955; 95% CI, 0.911-0.998). CONCLUSION: Logistic regression equation based on three CT features can help predict the pathological response of patients with locally advanced GC to NAC.

Development and verification of a nomogram for predicting the prognosis of resectable gastric cancer with outlet obstruction.

BACKGROUND: The prognosis of patients with gastric cancer (GC) with gastric outlet obstruction (GOO) after gastrectomy is highly variable. In this study, we aimed to develop a nomogram to predict the prognosis of these patients. PATIENTS AND METHODS: Data from 218 GC patients with GOO who underwent gastrectomy at Sun Yat-sen University Cancer Center were retrospectively collected as a training cohort. The data of 59 patients with the same diagnosis who underwent gastrectomy at the First Affiliated Hospital of Guangxi Medical University were collected as an external verification cohort. A nomogram for the overall survival (OS) was developed using the Cox regression model in the training cohort, which was validated in a verification cohort. RESULTS: Multivariate analysis showed that the surgical procedure (P < 0.001), period of chemotherapy (P < 0.001), T stage (P = 0.006), N stage (P = 0.040), systemic immune-inflammatory index (SII) (P < 0.001), and fibrinogen level (P = 0.026) were independent factors affecting OS. The nomogram constructed on the aforementioned factors for predicting the 1- and 3-year OS achieved a Harrell's concordance index (C-index) of 0.756 and 0.763 for the training and verification cohorts, respectively. Compared with the 8th American Joint Committee on Cancer (AJCC) Tumour-Node-Metastasis (TNM) staging system, the nomogram had higher C-index values and areas under the curve (AUCs) and slightly higher net clinical benefit. CONCLUSION: Compared to the 8th AJCC staging system, the newly developed nomogram showed superior performance in predicting the survival of GC patients with GOO after gastrectomy.

Palliative Gastrectomy versus Gastrojejunostomy for advanced Gastric cancer with outlet obstruction: a propensity score matching analysis.

BACKGROUND: Gastric outlet obstruction (GOO) is a late complication of advanced gastric cancer, and it is controversial how to select the therapeutic strategies: gastrojejunostomy and palliative gastrectomy? Therefore, this study was to compare the surgical and survival outcomes of gastrojejunostomy and palliative gastrectomy. METHODS: In total, 199 gastric cancer patients with outlet obstruction treated by surgery between January 2000 and December 2015 at Sun Yat-sen University Cancer Center were retrospectively reviewed. Patients were divided into gastrojejunostomy group and palliative gastrectomy group. Propensity score matching (PSM) was performed to balance the selection bias. RESULTS: After 1:1 PSM, a total of 104 patients were included for final analysis. The median overall survival (OS) times in the gastrojejunostomy group and palliative gastrectomy group were 8.50 and 11.87 months, respectively (P = 0.243). The postoperative complication rates in the gastrojejunostomy group and palliative gastrectomy group were 19.23% (10/52) and 17.31% (9/52), respectively (P = 0.800), and no treatment-related death was observed. Multivariate analysis showed that periton0eal seeding (P = 0.014) and chemotherapy (P < 0.001) were independent prognostic factors. Among them, peritoneal seeding was a risk factor and postoperative chemotherapy was a protective factor. CONCLUSIONS: Our results indicated that although the surgical complications of palliative gastrectomy were manageable, it showed no survival benefit. Therefore, relieving obstruction symptom, improving patients' quality of life and creating better conditions for chemotherapy appear to be the main therapeutic strategies for advanced gastric cancer with GOO.

Disease-free survival as a surrogate endpoint for overall survival in adjuvant trials of pancreatic cancer: a meta-analysis of 20 randomized controlled trials.

BACKGROUND: We aimed to assess whether disease-free survival (DFS) could serve as a reliable surrogate endpoint for overall survival (OS) in adjuvant trials of pancreatic cancer. METHODS: We systematically reviewed adjuvant randomized trials for non-metastatic pancreatic cancer after curative resection that reported a hazard ratio (HR) for DFS and OS. We assessed the correlation between treatment effect (HR) on DFS and OS, weighted by sample size or precision of hazard ratio estimate, assuming fixed and random effects, and calculated the surrogate threshold effect (STE). We also performed sensitivity analyses and a leave-one-out cross validation approach to evaluate the robustness of our findings. RESULTS: After screening 450 relevant articles, we identified a total of 20 qualifying trails comprising 5170 patients for quantitative analysis. We noted a strong correlation between the treatment effects for DFS and OS, with coefficient of determination of 0.82 in the random effect model, 0.82 in the fixed effect model, and 0.80 in the sample size weighting; the robustness of this finding was further verified by the leave-one-out cross-validation approach. Sensitivity analyses with restriction to phase 3 trials, large trials, trials with mature follow-up periods, and trials with adjuvant therapy versus adjuvant therapy strengthened the correlation (0.75 to 0.88) between DFS and OS. The STE was 0.96 for DFS. CONCLUSIONS: Therefore, DFS could be regarded as a surrogate endpoint for OS in adjuvant trials of pancreatic cancer. In future similar adjuvant trials, a hazard ratio for DFS of 0.96 or less would predict a treatment impact on OS.

Structural determinants of APOBEC3B non-catalytic domain for molecular assembly and catalytic regulation.

The catalytic activity of human cytidine deaminase APOBEC3B (A3B) has been correlated with kataegic mutational patterns within multiple cancer types. The molecular basis of how the N-terminal non-catalytic CD1 regulates the catalytic activity and consequently, biological function of A3B remains relatively unknown. Here, we report the crystal structure of a soluble human A3B-CD1 variant and delineate several structural elements of CD1 involved in molecular assembly, nucleic acid interactions and catalytic regulation of A3B. We show that (i) A3B expressed in human cells exists in hypoactive high-molecular-weight (HMW) complexes, which can be activated without apparent dissociation into low-molecular-weight (LMW) species after RNase A treatment. (ii) Multiple surface hydrophobic residues of CD1 mediate the HMW complex assembly and affect the catalytic activity, including one tryptophan residue W127 that likely acts through regulating nucleic acid binding. (iii) One of the highly positively charged surfaces on CD1 is involved in RNA-dependent attenuation of A3B catalysis. (iv) Surface hydrophobic residues of CD1 are involved in heterogeneous nuclear ribonucleoproteins (hnRNPs) binding to A3B. The structural and biochemical insights described here suggest that unique structural features on CD1 regulate the molecular assembly and catalytic activity of A3B through distinct mechanisms.

Bursectomy for advanced gastric cancer: an update meta-analysis.

BACKGROUND: The present meta-analysis was to explore the surgical and oncological outcomes of bursectomy for advanced gastric cancer (AGC). METHODS: Relevant studies that evaluated the role of bursectomy for AGC were comprehensively examined to perform a meta-analysis. The primary outcomes were overall survival (OS) and disease-free survival (DFS). The secondary outcomes were the number of harvested lymph nodes (LNs), operation time, operative bleeding, hospital stay, postoperative complication and mortality. RESULTS: A total of seven studies comprising 2633 cases (1176 cases in the bursectomy group and 1457 cases in the non-bursectomy group) were finally included. There was no significant difference in OS (HR 0.95, P = 0.647) and DFS (HR 0.99, P = 0.936) between the two groups. Even for patients with serosa-penetrating tumours, OS was comparable between the two groups (HR 0.87, P = 0.356). The operation time of the bursectomy group was longer (weighted mean difference, WMD 32.76 min, P = 0.002). No significant difference was found between the two groups in terms of the number of dissected LNs (WMD 5.86, P = 0.157), operative bleeding (WMD 66.99 ml, P = 0.192) and hospital stay (WMD - 0.15 days, P = 0.766). The overall postoperative complication (relative risk, RR 1.08, P = 0.421) and mortality (RR 0.44, P = 0.195) were similar between two groups. CONCLUSIONS: This meta-analysis indicated that bursectomy is time-consuming without increasing the number of harvested LNs. Although bursectomy can be safely performed without increasing complications and mortality, it does not prolong the OS and DFS of AGC patients, including patients with serosa-penetrating tumours. Therefore, bursectomy should not be recommended as a standard procedure for AGC.

Enhancing Received Signal Strength-Based Localization through Coverage Hole Detection and Recovery.

In wireless sensor networks (WSNs), Radio Signal Strength Indicator (RSSI)-based localization techniques have been widely used in various applications, such as intrusion detection, battlefield surveillance, and animal monitoring. One fundamental performance measure in those applications is the sensing coverage of WSNs. Insufficient coverage will significantly reduce the effectiveness of the applications. However, most existing studies on coverage assume that the sensing range of a sensor node is a disk, and the disk coverage model is too simplistic for many localization techniques. Moreover, there are some localization techniques of WSNs whose coverage model is non-disk, such as RSSI-based localization techniques. In this paper, we focus on detecting and recovering coverage holes of WSNs to enhance RSSI-based localization techniques whose coverage model is an ellipse. We propose an algorithm inspired by Voronoi tessellation and Delaunay triangulation to detect and recover coverage holes. Simulation results show that our algorithm can recover all holes and can reach any set coverage rate, up to 100% coverage.

Naturally Occurring Single Amino Acid Substitution in the L1 Major Capsid Protein of Human Papillomavirus Type 16: Alteration of Susceptibility to Antibody-Mediated Neutralization.

Background: Each vaccine for human papillomavirus type 16 (HPV16) has been developed on the basis of a single variant, and whether these vaccines can prevent infection due to naturally occurring variants was not clear. Methods: To examine this question, constructs of 39 naturally occurring single amino acid substitutions in L1 were generated for pseudovirion production, based on the analysis of 1204 HPV16 L1 protein sequences from the National Center for Biotechnology Information and Papilloma Virus Episteme. Results: Thirty-one of 39 HPV16 L1 mutants produced infectious pseudovirions that exhibited similar particle-to-infectivity ratios, compared with reference pseudovirions. Twenty-one of 31 pseudovirion-producing mutants showed different susceptibilities to monoclonal antibodies, with 6 resulting in complete loss of reactivity to some of the tested monoclonal antibodies. The vaccinated sera neutralized all 31 variants. Mean neutralization titers of most variants changed by approximately 4-fold, compared with the reference pseudovirions, with the C428W and K430Q mutations displaying 9-fold and 11-fold lower susceptibilities, respectively, to neutralization by the sera than the reference pseudovirions. Conclusions: These results suggest that the current HPV vaccines may not offer equal protection against all of the naturally occurring HPV16 variants discovered so far.

Unsupervised 2D Dimensionality Reduction with Adaptive Structure Learning.

In recent years, unsupervised two-dimensional (2D) dimensionality reduction methods for unlabeled large-scale data have made progress. However, performance of these degrades when the learning of similarity matrix is at the beginning of the dimensionality reduction process. A similarity matrix is used to reveal the underlying geometry structure of data in unsupervised dimensionality reduction methods. Because of noise data, it is difficult to learn the optimal similarity matrix. In this letter, we propose a new dimensionality reduction model for 2D image matrices: unsupervised 2D dimensionality reduction with adaptive structure learning (DRASL). Instead of using a predetermined similarity matrix to characterize the underlying geometry structure of the original 2D image space, our proposed approach involves the learning of a similarity matrix in the procedure of dimensionality reduction. To realize a desirable neighbors assignment after dimensionality reduction, we add a constraint to our model such that there are exact [Formula: see text] connected components in the final subspace. To accomplish these goals, we propose a unified objective function to integrate dimensionality reduction, the learning of the similarity matrix, and the adaptive learning of neighbors assignment into it. An iterative optimization algorithm is proposed to solve the objective function. We compare the proposed method with several 2D unsupervised dimensionality methods. K-means is used to evaluate the clustering performance. We conduct extensive experiments on Coil20, AT&T, FERET, USPS, and Yale data sets to verify the effectiveness of our proposed method.

Study of SV40 large T antigen nucleotide specificity for DNA unwinding.

BACKGROUND: Simian Virus 40 (SV40) Large Tumor Antigen (LT) is an essential enzyme that plays a vital role in viral DNA replication in mammalian cells. As a replicative helicase and initiator, LT assembles as a double-hexamer at the SV40 origin to initiate genomic replication. In this process, LT converts the chemical energy from ATP binding and hydrolysis into the mechanical work required for unwinding replication forks. It has been demonstrated that even though LT primarily utilizes ATP to unwind DNA, other NTPs can also support low DNA helicase activity. Despite previous studies on specific LT residues involved in ATP hydrolysis, no systematic study has been done to elucidate the residues participating in the selective usage of different nucleotides by LT. In this study, we performed a systematic mutational analysis around the nucleotide pocket and identified residues regulating the specificity for ATP, TTP and UTP in LT DNA unwinding. METHODS: We performed site-directed mutagenesis to generate 16 LT nucleotide pocket mutants and characterized each mutant's ability to unwind double-stranded DNA, oligomerize, and bind different nucleotides using helicase assays, size-exclusion chromatography, and isothermal titration calorimetry, respectively. RESULTS: We identified four residues in the nucleotide pocket of LT, cS430, tK419, cW393 and cL557 that selectively displayed more profound impact on using certain nucleotides for LT DNA helicase activity. CONCLUSION: Little is known regarding the mechanisms of nucleotide specificity in SV40 LT DNA unwinding despite the abundance of information available for understanding LT nucleotide hydrolysis. The systematic residue analysis performed in this report provides significant insight into the selective usage of different nucleotides in LT helicase activity, increasing our understanding of how LT may structurally prefer different energy sources for its various targeted cellular activities.

Endoscopic ultrasonography compared with multidetector computed tomography for the preoperative staging of gastric cancer: a meta-analysis.

BACKGROUND: The current study sought to perform a meta-analysis to compare the preoperative staging of endoscopic ultrasonography (EUS) and multidetector computed tomography (MDCT) in gastric carcinoma. METHODS: Articles published between January 1, 2000, and April 1, 2016, that compared EUS with MDCT were included, and data were presented as 2 × 2 tables. The sensitivities, specificities and summary receiver operating characteristic (ROC) curves for T and N staging were calculated using a bivariate mixed effects model. Data were weighted by generic variance and then pooled by random-effects modeling. RESULTS: Eight studies comprising 1736 patients were included in this meta-analysis. For T1 staging, the sensitivity value for EUS (82%) was significantly higher than that for MDCT (41%) (relative risk (RR): 2.06, 95% confidence interval (CI) 1.07-3.94; P = 0.030). For lymph node involvement, the sensitivity value for EUS (91%) was also significantly higher than that for MDCT (77%) (RR 1.14, 95% CI 1.05-1.23; P = 0.001). However, the specificity values of both EUS and MDCT were quite low, at 49 and 63%, respectively. No significant differences in T2-4 staging between EUS and MDCT were noted. CONCLUSION: This meta-analysis indicates that EUS may be superior to MDCT in preoperative T1 and N staging. Additionally, the low specificity values of EUS and MDCT for N staging merits attention.

A Study of Pattern Prediction in the Monitoring Data of Earthen Ruins with the Internet of Things.

An understanding of the changes of the rammed earth temperature of earthen ruins is important for protection of such ruins. To predict the rammed earth temperature pattern using the air temperature pattern of the monitoring data of earthen ruins, a pattern prediction method based on interesting pattern mining and correlation, called PPER, is proposed in this paper. PPER first finds the interesting patterns in the air temperature sequence and the rammed earth temperature sequence. To reduce the processing time, two pruning rules and a new data structure based on an R-tree are also proposed. Correlation rules between the air temperature patterns and the rammed earth temperature patterns are then mined. The correlation rules are merged into predictive rules for the rammed earth temperature pattern. Experiments were conducted to show the accuracy of the presented method and the power of the pruning rules. Moreover, the Ming Dynasty Great Wall dataset was used to examine the algorithm, and six predictive rules from the air temperature to rammed earth temperature based on the interesting patterns were obtained, with the average hit rate reaching 89.8%. The PPER and predictive rules will be useful for rammed earth temperature prediction in protection of earthen ruins.