Novel mRNA adjuvant ImmunER enhances prostate cancer tumor-associated antigen mRNA therapy via augmenting T cell activity.

Prostate cancer (PCa) is characterized as a "cold tumor" with limited immune responses, rendering the tumor resistant to immune checkpoint inhibitors (ICI). Therapeutic messenger RNA (mRNA) vaccines have emerged as a promising strategy to overcome this challenge by enhancing immune reactivity and significantly boosting anti-tumor efficacy. In our study, we synthesized Tetra, an mRNA vaccine mixed with multiple tumor-associated antigens, and ImmunER, an immune-enhancing adjuvant, aiming to induce potent anti-tumor immunity. ImmunER exhibited the capacity to promote dendritic cells (DCs) maturation, enhance DCs migration, and improve antigen presentation at both cellular and animal levels. Moreover, Tetra, in combination with ImmunER, induced a transformation of bone marrow-derived dendritic cells (BMDCs) to cDC1-CCL22 and up-regulated the JAK-STAT1 pathway, promoting the release of IL-12, TNF-α, and other cytokines. This cascade led to enhanced proliferation and activation of T cells, resulting in effective killing of tumor cells. In vivo experiments further revealed that Tetra + ImmunER increased CD8+T cell infiltration and activation in RM-1-PSMA tumor tissues. In summary, our findings underscore the promising potential of the integrated Tetra and ImmunER mRNA-LNP therapy for robust anti-tumor immunity in PCa.

Predictive efficacy of the preoperative neutrophil-lymphocyte ratio in lymph node metastasis of cN0 hormone receptor-positive breast cancer.

Breast cancer, as the most common cancer, has surpassed lung cancer worldwide. The neutrophil-to-lymphocyte ratio (NLR) has been linked to the onset of cancer and its prognosis in recent studies. However, quite a few studies have shown that there is a link between NLR and lymph node metastases in cN0 hormone receptor-positive (HR(+)) breast cancer. The purpose of this study was to evaluate the correlation between NLR and lymph node metastases in cN0 HR(+) breast cancer patients. From January 2012 to January 2022, 220 patients with cN0 HR(+) invasive breast cancers were enrolled in this study. The relationship between NLR and pathological data was statistically examined. The receiver operating characteristic (ROC) curve was used to determine the optimal cutoff of NLR, a chi-squared test was used for the univariate analysis, and logistic analysis was used for the multivariate analysis. The NLR had an optimal cutoff of 2.4 when the Jorden index was at a maximum. Patients with axillary lymph node metastases had a higher NLR (P < 0.05). A Univariate analysis showed that there were significant differences in cN0 HR(+) breast cancer with axillary lymph node metastasis among different clinical stages, histological grades, Ki-67 levels, tumor sizes, and NLR levels (P < 0.05). Clinical stage, tumor size, and NLR were found to be independent risk factors for lymph node metastases in multifactorial analysis. In cN0 HR(+) breast cancer, NLR is an independent risk factor for lymph node metastases. An NLR ≥ 2.4 indicates an increased probability of lymph node metastases. An elevated preoperative NLR has a high predictive value for axillary lymph node metastases.

The impact of coexisting Hashimoto's thyroiditis on the feasibility of endoscopic thyroidectomy in papillary thyroid carcinoma.

Objective: ：The safety of endoscopic thyroidectomy in patients with Hashimoto's thyroiditis (HT) is a matter of concern. This study aimed to assess the effect of concomitant HT on the feasibility of endoscopic thyroidectomy in patients with papillary thyroid carcinoma (PTC). Methods: This study is an observational, retrospective study. All patients were histopathologically diagnosed with HT. The study group consisted of 44 patients (40 %) with PTC who also had HT, whereas the remaining 66 patients (60%) without HT were assigned to the control group. The number of dissected lymph nodes, mean operation time, thyroid volume, blood loss, TSH level, and postoperative complications were recorded and statistically analysed. Results: One patient underwent conversion to open thyroidectomy because of recurrent laryngeal nerve (RLN) transection. Another patient required reoperation owing to postoperative haemorrhage. Statistically significant differences were observed in mean operation time (105.4 ± 10.7 vs 98.2 ± 7.4 min, P = 0.0001),mean thyroid lobe volume (12.2 ± 5.8 vs 9.6 ± 3.5 mL [mL], P = 0.0041), TSH level(4.1 ± 1.5 mIU/L vs 3.4 ± 0.9 mIU/L, P ＝ 0.0028), and the number of dissected lymph nodes between groups (4.1 ± 1.5 vs 3.4 ± 0.9,P = 0.0028). The estimated mean blood loss (31.5 ± 6.8 vs 29.5 ± 3.9 mL, P = 0.0529) and rate of complications (15.9% vs 10.6%, P = 0.4136) did not show statistically significant differences between groups. Conclusion: The coexistence of PTC and HT increases the operation time and difficulties in endoscopic thyroidectomy but does not affect postoperative outcomes. Endoscopic thyroidectomy can be safely performed with acceptable complication rates.

Delivery of mRNA Vaccine with 1, 2-Diesters-Derived Lipids Elicits Fast Liver Clearance for Safe and Effective Cancer Immunotherapy.

Messenger RNA (mRNA) vaccine is explored as a promising strategy for cancer immunotherapy, but the side effects, especially the liver-related damage caused by LNP, raise concerns about its safety. In this study, a novel library of 248 ionizable lipids comprising 1,2-diesters is designed via a two-step process involving the epoxide ring-opening reaction with carboxyl group-containing alkyl chains followed by an esterification reaction with the tertiary amines. Owing to the special chemical structure of 1,2-diesters, the top-performing lipids and formulations exhibit a faster clearance rate in the liver, contributing to increased stability and higher safety compared with DLin-MC3-DMA. Moreover, the LNP shows superior intramuscular mRNA delivery and elicits robust antigen-specific immune activation. The vaccinations delivered by the LNP system suppress tumor growth and prolong survival in both model human papillomavirus E7 and ovalbumin antigen-expressing tumor models. Finally, the structure of lipids which enhances the protein expression in the spleen and draining lymph nodes compared with ALC-0315 lipid in Comirnaty is further optimized. In conclusion, the 1, 2-diester-derived lipids exhibit rapid liver clearance and effective anticancer efficiency to different types of antigens-expressing tumor models, which may be a safe and universal platform for mRNA vaccines.

Advantage of log odds of positive lymph nodes in prognostic evaluation of patients with early-onset colon cancer.

BACKGROUND: Colon cancer (CC) is one of the most common cancers of the digestive tract, the third most common cancer worldwide, and the second most common cause of cancer-related deaths. Previous studies have demonstrated a higher risk of lymph node metastasis (LNM) in young patients with CC. It might be reasonable to treat patients with early-onset locally advanced CC with extended lymph node dissection. However, few studies have focused on early-onset CC (ECC) patients with LNM. At present, the methods of predicting and evaluating the prognosis of ECC patients with LNM are controversial. AIM: To compare the prognostic values of four lymph node staging indices and establish the best nomogram for patients with ECC. METHODS: From the data of patients with CC obtained from the Surveillance, Epidemiology, and End Results (SEER) database, data of young patients with ECC (≤ 50 years old) was screened. Patients with unknown data were excluded from the study, while the remaining patients were included. The patients were randomly divided into a training group (train) and a testing group (test) in the ratio of 7:3, while building the model. The model was constructed by the training group and verified by the testing group. Using multiple Cox regression models to compare the prediction efficiency of LNM indicators, nomograms were built based on the best model selected for overall survival (OS) and cause-specific survival (CSS). In the two groups, the performance of the nomogram was evaluated by constructing a calibration plot, time-dependent area under the curve (AUC), and decision curve analysis. Finally, the patients were grouped based on the risk score predicted by the prognosis model, and the survival curve was constructed after comparing the survival status of the high and low-risk groups. RESULTS: Records of 26922 ECC patients were screened from the SEER database. N classification, positive lymph nodes (PLN), lymph node ratio (LNR) and log odds of PLN (LODDS) were considered to be independent predictors of OS and CSS. In addition, independent risk factors for OS included gender, race, marital status, primary site, histology, grade, T, and M classification, while the independent prognostic factors for CSS included race, marital status, primary site, grade, T, and M classification. The prediction model including LODDS is composed of minimal Akaike information criterion, maximal concordance indexes, and AUCs. Factors including gender, race, marital status, primary site, histology, grade, T, M classification, and LODDS were integrated into the OS nomogram, while race, marital status, primary site, grade, T, M classification, and LODDS were included into the CSS nomogram. The nomogram representing both cohorts had been successfully verified in terms of prediction accuracy and clinical practicability. CONCLUSION: LODDS is superior to N-stage, PLN, and LNR of ECC. The nomogram containing LODDS might be helpful in tumor evaluation and clinical decision-making, since it provides an appropriate prediction of ECC.

Mpox virus mRNA-lipid nanoparticle vaccine candidates evoke antibody responses and drive protection against the Vaccinia virus challenge in mice.

In response to the human Mpox (hMPX) epidemic that began in 2022, there is an urgent need for a monkeypox vaccine. Here, we have developed a series of mRNA-lipid nanoparticle (mRNA-LNP)-based vaccine candidates that encode a collection of four highly conserved Mpox virus (MPXV) surface proteins involved in virus attachment, entry, and transmission, namely A29L, A35R, B6R, and M1R, which are homologs to Vaccinia virus (VACV) A27, A33, B5, and L1, respectively. Despite possible differences in immunogenicity among the four antigenic mRNA-LNPs, administering these antigenic mRNA-LNPs individually (5 µg each) or an average mixture of these mRNA-LNPs at a low dose (0.5 µg each) twice elicited MPXV-specific IgG antibodies and potent VACV-specific neutralizing antibodies. Furthermore, two doses of 5 µg of A27, B5, and L1 mRNA-LNPs or a 2 µg average mixture of the four antigenic mRNA-LNPs protected mice against weight loss and death after the VACV challenge. Overall, our data suggest that these antigenic mRNA-LNP vaccine candidates are both safe and efficacious against MPXV, as well as diseases caused by other orthopoxviruses.

Dioxin emission modeling using feature selection and simplified DFR with residual error fitting for the grate-based MSWI process.

Municipal solid waste incineration (MSWI) with grate technology is a widely applied waste-to-energy process in various cities in China. Meanwhile, dioxins (DXN) are emitted at the stack and are the critical environmental indicator for operation optimization control in the MSWI process. However, constructing a high-precision and fast emission model for DXN emission operation optimization control becomes an immediate difficulty. To address the above problem, this research utilizes a novel DXN emission measurement method using simplified deep forest regression (DFR) with residual error fitting (SDFR-ref). First, the high-dimensional process variables are optimally reduced following the mutual information and significance test. Then, a simplified DFR algorithm is established to infer or predict the nonlinearity between the selected process variables and the DXN emission concentration. Moreover, a gradient enhancement strategy in terms of residual error fitting with a step factor is designed to improve the measurement performance in the layer-by-layer learning process. Finally, an actual DXN dataset from 2009 to 2020 of the MSWI plant in Beijing is utilized to verify the SDFR-ref method. Comparison experiments demonstrate the superiority of the proposed method over other methods in terms of measurement accuracy and time consumption.

Nomogram established using risk factors of early gastric cancer for predicting the lymph node metastasis.

BACKGROUND: For the prognosis of patients with early gastric cancer (EGC), lymph node metastasis (LNM) plays a crucial role. A thorough and precise evaluation of the patient for LNM is now required. AIM: To determine the factors influencing LNM and to construct a prediction model of LNM for EGC patients. METHODS: Clinical information and pathology data of 2217 EGC patients downloaded from the Surveillance, Epidemiology, and End Results database were collected and analyzed. Based on a 7:3 ratio, 1550 people were categorized into training sets and 667 people were assigned to testing sets, randomly. Based on the factors influencing LNM determined by the training sets, the nomogram was drawn and verified. RESULTS: Based on multivariate analysis, age at diagnosis, histology type, grade, T-stage, and size were risk factors of LNM for EGC. Besides, nomogram was drawn to predict the risk of LNM for EGC patients. Among the categorical variables, the effect of grade (well, moderate, and poor) was the most significant prognosis factor. For training sets and testing sets, respectively, area under the receiver-operating characteristic curve of nomograms were 0.751 [95% confidence interval (CI): 0.721-0.782] and 0.786 (95%CI: 0.742-0.830). In addition, the calibration curves showed that the prediction model of LNM had good consistency. CONCLUSION: Age at diagnosis, histology type, grade, T-stage, and tumor size were independent variables for LNM in EGC. Based on the above risk factors, prediction model may offer some guiding implications for the choice of subsequent therapeutic approaches for EGC.

Efficacy and outcome of extensive intraoperative peritoneal lavage plus surgery vs surgery alone with advanced gastric cancer patients.

BACKGROUND: Gastric cancer (GC) is one of the most common malignant tumors. After resection, one of the major problems is its peritoneal dissemination and recurrence. Some free cancer cells may still exist after resection. In addition, the surgery itself may lead to the dissemination of tumor cells. Therefore, it is necessary to remove residual tumor cells. Recently, some researchers found that extensive intraoperative peritoneal lavage (EIPL) plus intraperitoneal chemotherapy can improve the prognosis of patients and eradicate peritoneal free cancer for GC patients. However, few studies explored the safety and long-term outcome of EIPL after curative gastrectomy. AIM: To evaluate the efficacy and long-term outcome of advanced GC patients treated with EIPL. METHODS: According to the inclusion and exclusion criteria, a total of 150 patients with advanced GC were enrolled in this study. The patients were randomly allocated to two groups. All patients received laparotomy. For the non-EIPL group, peritoneal lavage was washed using no more than 3 L of warm saline. In the EIPL group, patients received 10 L or more of saline (1 L at a time) before the closure of the abdomen. The surviving rate analysis was compared by the Kaplan-Meier method. The prognostic factors were carried out using the Cox appropriate hazard pattern. RESULTS: The basic information in the EIPL group and the non-EIPL group had no significant difference. The median follow-up time was 30 mo (range: 0-45 mo). The 1- and 3-year overall survival (OS) rates were 71.0% and 26.5%, respectively. The symptoms of ileus and abdominal abscess appeared more frequently in the non-EIPL group (P < 0.05). For the OS of patients, the EIPL, Borrmann classification, tumor size, N stage, T stage and vascular invasion were significant indicators. Then multivariate analysis revealed that EIPL, tumor size, vascular invasion, N stage and T stage were independent prognostic factors. The prognosis of the EIPL group was better than the non-EIPL group (P < 0.001). The 3-year survival rate of the EIPL group (38.4%) was higher than the non-EIPL group (21.7%). For the recurrence-free survival (RFS) of patients, the risk factor of RFS included EIPL, N stage, vascular invasion, type of surgery, tumor location, Borrmann classification, and tumor size. EIPL and tumor size were independent risk factors. The RFS curve of the EIPL group was better than the non-EIPL group (P = 0.004), and the recurrence rate of the EIPL group (24.7%) was lower than the non-EIPL group (46.4%). The overall recurrence rate and peritoneum recurrence rate in the EIPL group was lower than the non-EIPL group (P < 0.05). CONCLUSION: EIPL can reduce the possibility of perioperative complications including ileus and abdominal abscess. In addition, the overall survival curve and RFS curve were better in the EIPL group.

Assessment of PCDD/Fs formation and emission characteristics at a municipal solid waste incinerator for one year.

Municipal solid waste incineration (MSWI) has become a predominant emission source of polychlorinated dibenzo-p-dioxins and furans (PCDD/Fs). Research focusing on the impact of operating conditions, environmental changes, and operating time on the generation and emissions of PCDD/Fs has not been resolved. To this end, this study tracked and investigated the PCDD/Fs and 17 congener emissions of a typical grate incinerator (800 t/d) continuously for one year. Results showed that the PCDD/Fs concentration at the boiler outlet, stack inlet, and bag filter, including normal and abnormal operation conditions, ranges from 2.11E-02-41.86 ng I-TEQ/Nm3, 7.00E-04-6.76 ng I-TEQ/Nm3, and 1.12-2.90E+03 ng I-TEQ/Nm3, respectively. The 2,3,4,7,8-P5CDF has the highest contribution in all samples, in which a proportion of TEQ ranged from 30 % to 77.73 %. Moreover, by applying the correlation analysis between PCDD/Fs and operating parameters, the emission characteristic is mainly affected by incinerators and boilers during the normal period, and it is affected by the whole MSWI process under abnormal conditions. In addition, the PCDD/Fs emission from the MSWI plant gradually increases from spring to winter. This study is beneficial for supporting the control of PCDD/Fs emission reduction and assisting the operators to optimize the relevant operating parameters of the MSWI plant to achieve a stable and up-to-substandard emissions during the operation period.

Investigation on dioxins emission characteristic during complete maintenance operating period of municipal solid waste incineration.

The dioxins (DXN) are a set of pollutants encompass polychlorinated dibenzo-p-dioxin/dibenzofuran (PCDD/F), their emissions from municipal waste incineration processes (MSWI) are normally detected under steady operating conditions. However, limited studies have focused on the PCDD/F emission characteristics under a complete maintenance operating period (CMOP), which includes shut-down, cooling, maintenance, heating, startup, and normal operations. In this article, the shutdown process (SDP) starts from the normal operation, followed by shutdown, and then cooling; while the startup process (SUP) commences from heating, followed by startup, and then normal operation. The detection and analysis were conducted at the SDP and SUP stages. The PCDD/F mass and total toxic equivalent quantity (TEQ) concentrations were measured in the flue gas and bag filter fly ash (BF-FA) during a CMOP of Beijing MSWI plant. The highest PCDD/F concentrations in the flue gas were found in the "cooling" and "startup" phases; in the FA, this condition occurred in the "startup" phase. Further, the results show that the most heightened concentrations were observed for 5-6 chlorinated PCDF and 4-5 chlorinated PCDD among the 17 PCDD/F congeners in most cases. More importantly, the air pollution control devices (APCDs) which include activated carbon, lime, and BF, have high removal efficiency for PCDD/F (especially PCDD) during the "startup" phase. APCDs also easily release a considerable amount of PCDD/F because of the memory effect, which emits more PCDD/F at the "shutdown" phase than at the "startup" one. Besides, the annual PCDD/F emission in the flue gas of the MSWI plant was estimated to be 67.72 mg I-TEQ, of which the emission accounts for approx. 20% during the CMOP. Moreover, the experiment shows that the PCDD/F emissions of the MSWI plant in Beijing under unsteady conditions are more miniature than those reported earlier in other areas.

Tree Broad Learning System for Small Data Modeling.

Broad learning system based on neural network (BLS-NN) has poor efficiency for small data modeling with various dimensions. Tree-based BLS (TBLS) is designed for small data modeling by introducing nondifferentiable modules and an ensemble strategy to the traditional broad learning system (BLS). TBLS replaces the neurons of BLS with the tree modules to map the input data. Moreover, we present three new TBLS variant methods and their incremental learning implementations, which are motivated by deep, broad, and ensemble learning. Their major distinction is reflected in the incremental learning strategies based on: 1) mean square error (mse); 2) pseudo-inverse; and 3) pseudo-inverse theory and stack representation. Therefore, this study further explores the domain of BLS based on the nondifferentiable modules. The simulations are compared with some state-of-the-art (SOTA) BLS-NN and tree methods under high-, medium-, and low-dimensional benchmark datasets. Results show that the proposed method outperforms the BLS-NN, and the modeling accuracy is remarkably improved with the small training data of the proposed TBLS.

Psychrophilic phage VSW-3 RNA polymerase reduces both terminal and full-length dsRNA byproducts in in vitro transcription.

RNA research and applications are underpinned by in vitro transcription (IVT), but RNA impurities resulting from the enzymatic reagents severely impede downstream applications. To improve the stability and purity of synthesized RNA, we have characterized a novel single-subunit RNA polymerase (RNAP) encoded by the psychrophilic phage VSW-3 from a plateau lake. The VSW-3 RNAP is capable of carrying out in vitro RNA synthesis at low temperatures (4-25°C). Compared to routinely used T7 RNAP, VSW-3 RNAP provides a similar yield of transcripts but is insensitive to class II transcription terminators and synthesizes RNA without redundant 3'-cis extensions. More importantly, through dot-blot detection with the J2 monoclonal antibody, we found that the RNA products synthesized by VSW-3 RNAP contained a much lower amount of double-stranded RNA byproducts (dsRNA), which are produced by transcription from both directions and are significant in T7 RNAP IVT products. Taken together, the VSW-3 RNAP almost eliminates both terminal loop-back dsRNA and full-length dsRNA in IVT and thus is especially advantageous for producing RNA for in vivo use.

Macromolecules in polysorbate 80 for injection: an important cause of anaphylactoid reactions.

Polysorbate 80 for injection (TW80) is a common excipient used for injection whose macromolecular impurities, including those that cause anaphylactoid reactions, are frequently ignored. The main aim of this study was to prove that the macromolecular impurities in the excipient are an important cause of anaphylactoid reactions. Component A (containing macromolecules > 100 kDa), Component B (containing macromolecules from 10 to 100 kDa), and Component C (containing substances < 10 kDa) were prepaired from the original TW80 using ultrafilters. The original TW80 contained numerous substances with molecular weights > 10kD. The original TW80 and Components A and B caused strong anaphylactoid reactions in both guinea pigs and rabbits by intravenous administration. Moreover, the original TW80 and Components A and B even caused strong passive cutaneous anaphylactoid (PCA) reactions and pulmonary capillary permeability. The PCA reaction and increased permeability were partly prevented by cromolyn sodium. Additionally, the original TW80 and Components A and B caused vasodilation and severe hemolysis in vitro. The anaphylactoid reactions were associated with histamine release but not with mast cell degranulation. Nevertheless, Component C almost caused no anaphylactoid reactions or hemolysis and was weaker in the few reactions that ocurred. Taken together, these results suggest that macromolecular substances are one of the main risk factors responsible for anaphylactoid reactions and hemolysis caused by TW80.

The Learning Curve and Importance of Collaboration in Endoscopic Thyroidectomy Via Breast Areola Approach: A Single Surgical Team's Experience of 100 Patients.

BACKGROUND: Endoscopic thyroidectomy is popular among young patients because of its excellent cosmetic outcomes. But it takes a long time to become proficient and competent for surgeons. In addition, collaboration plays a critical role in endoscopic thyroidectomy. Our research aims to evaluate the learning curve of endoscopic thyroidectomy via breast areola approach, provide details of this approach, and demonstrate the importance of collaboration. METHODS: The authors retrospectively analyzed 100 cases of benign and malignant thyroid disease who underwent endoscopic thyroidectomy via breast areola approach between January 2015 and December 2020, which were performed by the same group of surgeons with little experience of endoscopic thyroidectomy. The learning curve was analyzed by moving average method. The mean operation time, blood loss, tumor size, postoperative complications were used to determine learning curve progression. RESULTS: The learning curve in the first 30 cases were uplifted, stable at 30 to 60 cases and declined in the following cases. The mean operation time and blood loss decreased significant after the first 30 cases and again after the first 60 cases. And there was no difference in postoperative complications. CONCLUSIONS: A well-trained surgeon with experience in conventional open thyroidectomy can significantly reduce the total operation time by studying the learning curve. The key steps including establishment of working space and reaching for recurrent laryngeal nerve. A stable level can be achieved after 30 cases. More than 60 cases are required to become proficient. A successful endoscopic thyroid surgery requires a stable team.

Dioxin emission prediction based on improved deep forest regression for municipal solid waste incineration process.

Dioxin (DXN) emission concentration is an important environmental indicator in the municipal solid waste incineration (MSWI) process. The prediction model of DXN emission can be used for pollution control to realize actual requirements of operation optimization. Therefore, a DXN emission concentration prediction model based on improved deep forest regression (ImDFR) is proposed in this study. A feature reduction layer based on out-of-bagging error is first introduced into the ImDFR to eliminate redundant variables and feed all confidence information on DXN emission into the feature enhancement layer of the MSWI process. A deep ensemble stacking model is subsequently built to depict deep features and increase diversity and accuracy using random forests, completely random forests, GBDT, and XGBoost as subforests. Finally, the predicted value of the DXN prediction model is determined in the decision layer. The DXN emission prediction model is verified using actual historical data of two incinerators operated with a daily processing capacity of 800 tons. The experimental results showed that the proposed prediction model presents higher accuracy and better generalization ability than state-of-the-art models.

mRNA produced by VSW-3 RNAP has high-level translation efficiency with low inflammatory stimulation.

In vitro preparation of mRNA is a key step for mRNA therapeutics. The widely used T7 RNA polymerase (RNAP) was shown to have many by-products during in vitro transcription (IVT) process, among which double-stranded RNA (dsRNA) is the major by-product to activate the intracellular immune response. Here, we describe the use of a new VSW-3 RNAP that reduced dsRNA production during IVT and the resulting mRNA exhibited low inflammatory stimulation in cells. Compared to T7 RNAP transcripts, these mRNA exhibited superior protein expression levels, with an average of 14-fold increase in Hela cells and 5-fold increase in mice. In addition, we found that VSW-3 RNAP did not require modified nucleotides to improve protein production of IVT products. Our data suggest that VSW-3 RNAP could be a useful tool for mRNA therapeutics.

Uricase deficiency causes mild and multiple organ injuries in rats.

Uricase-deficient rats could be one of the optimal model animals to study hyperuricemia. The present study aimed to find the biological differences between uricase-deficient (Kunming-DY rats) and wild-type male rats. Uricase-deficient rats and wild-type rats were commonly bred. Their body weight, water and food consumption, 24-h urine and feces, uric acid in serum and organs, and serum indexes were recorded or assayed. Organs, including the heart, liver, spleen, lung, kidney, thymus, stomach, duodenum, and ileum, were examined using a routine hematoxylin-eosin staining assay. We found that the growth of male uricase-deficient rats was retarded. These rats excreted more urine than the wild-type rats. Their organ indexes (organ weight body weight ratio), of the heart, liver, kidney, and thymus significantly increased, while those of the stomach and small intestine significantly decreased. The uricase-deficient rats had a significantly higher level of serum uric acid and excreted more uric acid via urine at a higher concentration. Except for the liver, uric acid increased in organs and intestinal juice of uricase-deficient rats. Histological examination of the uricase-deficient rats showed mild injuries to the heart, liver, spleen, lung, kidney, thymus, stomach, duodenum, and ileum. Our results suggest that uricase-deficient rats have a different biological pattern from the wild-type rats. Uricase deficiency causes growth retardation of young male rats and the subsequent increase in serum uric acid results in mild organs injuries, especially in the kidney and liver.

Aboriginal Bacterial Flora in the Uricase-Deficient Rat Gut is Not the Main Factor Affecting Serum Uric Acid.

The relationship between intestinal bacteria and hyperuricemia is a hot research topic. To better understand this relationship, uricase-deficient Sprague-Dawley rats (Kunming-DY rats) were used. The wild-type rats and Kunming-DY rats were used as controls. Kunming-DY rats were treated with ampicillin (90 mg/kg) and ciprofloxacin (150 mg/kg) for 5 days. Bacterial 16S rDNA in the fresh stool was sequenced, and the abundance was calculated. The rats' serum uric acid (SUA) level was assayed, and the rats' intake and output in 24 h were recorded. The bacterial diversity in three groups' fresh stool was analyzed. The gut bacterial diversity and abundance changed in the Kunming-DY rats. More than 99% of bacteria were inhibited or killed by the combination of antibiotics. In contrast to each of the antibiotics alone, the combination of antibiotics lowered the Kunming-DY rats' SUA level; it also caused mild diarrhea, which increased uric acid excretion through stool. These results suggested that the aboriginal gut bacteria in uricase-deficient rats play a minor role in determining the SUA levels. It is too early to conclude that aboriginal gut bacteria are a tempting target for lowering SUA levels.