Proximity proteomics reveals UCH-L1 as an essential regulator of NLRP3-mediated IL-1ß production in human macrophages and microglia.

Activation of the NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome complex is an essential innate immune signaling mechanism. To reveal how human NLRP3 inflammasome assembly and activation are controlled, in particular by components of the ubiquitin system, proximity labeling, affinity purification, and RNAi screening approaches were performed. Our study provides an intricate time-resolved molecular map of different phases of NLRP3 inflammasome activation. Also, we show that ubiquitin C-terminal hydrolase 1 (UCH-L1) interacts with the NACHT domain of NLRP3. Downregulation of UCH-L1 decreases pro-interleukin-1ß (IL-1ß) levels. UCH-L1 chemical inhibition with small molecules interfered with NLRP3 puncta formation and ASC oligomerization, leading to altered IL-1ß cleavage and secretion, particularly in microglia cells, which exhibited elevated UCH-L1 expression as compared to monocytes/macrophages. Altogether, we profiled NLRP3 inflammasome activation dynamics and highlight UCH-L1 as an important modulator of NLRP3-mediated IL-1ß production, suggesting that a pharmacological inhibitor of UCH-L1 may decrease inflammation-associated pathologies.

PCAO2: an ontology for integration of prostate cancer associated genotypic, phenotypic and lifestyle data.

Disease ontologies facilitate the semantic organization and representation of domain-specific knowledge. In the case of prostate cancer (PCa), large volumes of research results and clinical data have been accumulated and needed to be standardized for sharing and translational researches. A formal representation of PCa-associated knowledge will be essential to the diverse data standardization, data sharing and the future knowledge graph extraction, deep phenotyping and explainable artificial intelligence developing. In this study, we constructed an updated PCa ontology (PCAO2) based on the ontology development life cycle. An online information retrieval system was designed to ensure the usability of the ontology. The PCAO2 with a subclass-based taxonomic hierarchy covers the major biomedical concepts for PCa-associated genotypic, phenotypic and lifestyle data. The current version of the PCAO2 contains 633 concepts organized under three biomedical viewpoints, namely, epidemiology, diagnosis and treatment. These concepts are enriched by the addition of definition, synonym, relationship and reference. For the precision diagnosis and treatment, the PCa-associated genes and lifestyles are integrated in the viewpoint of epidemiological aspects of PCa. PCAO2 provides a standardized and systematized semantic framework for studying large amounts of heterogeneous PCa data and knowledge, which can be further, edited and enriched by the scientific community. The PCAO2 is freely available at https://bioportal.bioontology.org/ontologies/PCAO, http://pcaontology.net/ and http://pcaontology.net/mobile/.

Preoperative low plasma creatine kinase levels predict worse survival outcomes in bladder cancer after radical cystectomy.

BACKGROUND AND AIMS: To evaluate the prognostic significance of preoperative creatine kinase (CK) levels in bladder cancer (BCa) patients who underwent radical cystectomy (RC). MATERIALS AND METHODS: 570 BCa patients with RC were identified between 2010 and 2020. 108.5 U/L of CK levels were defined as the cutoff value. Logistic regression analysis and Cox regression models were performed to evaluate the association between CK levels and oncologic outcomes. Subgroup analyses were performed to address cofounding factors. RESULTS: Preoperative low CK levels were associated with worse recurrence-free survival (RFS, log-rank P = 0.001) and overall survival (OS, log-rank P = 0.002). Multivariate analysis revealed that preoperative low CK levels were an independent predictor for worse RFS (hazard ratio [HR]: 1.683; P < 0.001) and OS (HR: 1.567; P = 0.002). CONCLUSIONS: The preoperative low CK level independently predicts worse survival outcomes in BCa after RC. Incorporating it into prediction models might be valuable to assist risk stratification.

Immunization with Live-Attenuated RHΔhad2a Strain Confers Partial Protective Immunity against Acute and Chronic Infection of Toxoplasma gondii in Mice.

Toxoplasmosis caused by Toxoplasma gondii is an important zoonosis of human and animal health significance. Current chemical therapeutics have side effects, and no commercially available vaccine is licensed for the prevention of toxoplasmosis in humans and most animals. Developing a safe and effective vaccine with long-term protection against T. gondii infection is necessary to control toxoplasmosis. HAD2a is a key member of the haloacid dehalogenase (HAD) phosphatase family, which is essential for T. gondii daughter budding. However, the role of HAD2a in T. gondii virulence remains unknown. In this study, we successfully constructed the had2a gene knockout strain in the T. gondii-type I RH strain (RHΔhad2a) and determined its role in virulence and vaccination. These results demonstrate that HAD2a played an important role in parasite daughter budding and in vitro replication. Knockout of the had2a gene attenuated the virulence of the T. gondii-type I RH strain. Vaccination with RHΔhad2a tachyzoites induced a Th1-biased immune response, provided partial protection against acute T. gondii infection in mice by highly virulent tachyzoites of RH and PYS (ToxoDB#9, Chinese I) strains, and conferred strong protection against challenge infection by cysts and oocysts of the less virulent type II Pru strain. These results demonstrate that T. gondii had2a is important for its in vitro proliferation and virulence in mice and that RHΔhad2a may be used as a candidate strain to generate a multiple gene knockout live-attenuated strain or be collaboratively applied with other live-attenuated strains to confer more effective protection against T. gondii infection.

Cell softness reveals tumorigenic potential via ITGB8/AKT/glycolysis signaling in a mice model of orthotopic bladder cancer.

BACKGROUND: Bladder cancer, characterized by a high potential of tumor recurrence, has high lifelong monitoring and treatment costs. To date, tumor cells with intrinsic softness have been identified to function as cancer stem cells in several cancer types. Nonetheless, the existence of soft tumor cells in bladder tumors remains elusive. Thus, our study aimed to develop a micro-barrier microfluidic chip to efficiently isolate deformable tumor cells from distinct types of bladder cancer cells. METHODS: The stiffness of bladder cancer cells was determined by atomic force microscopy (AFM). The modified microfluidic chip was utilized to separate soft cells, and the 3D Matrigel culture system was to maintain the softness of tumor cells. Expression patterns of integrin ß8 (ITGB8), protein kinase B (AKT), and mammalian target of rapamycin (mTOR) were determined by Western blotting. Double immunostaining was conducted to examine the interaction between F-actin and tripartite motif containing 59 (TRIM59). The stem-cell-like characteristics of soft cells were explored by colony formation assay and in vivo studies upon xenografted tumor models. RESULTS: Using our newly designed microfluidic approach, we identified a small fraction of soft tumor cells in bladder cancer cells. More importantly, the existence of soft tumor cells was confirmed in clinical human bladder cancer specimens, in which the number of soft tumor cells was associated with tumor relapse. Furthermore, we demonstrated that the biomechanical stimuli arising from 3D Matrigel activated the F-actin/ITGB8/TRIM59/AKT/mTOR/glycolysis pathways to enhance the softness and tumorigenic capacity of tumor cells. Simultaneously, we detected a remarkable up-regulation in ITGB8, TRIM59, and phospho-AKT in clinical bladder recurrent tumors compared with their non-recurrent counterparts. CONCLUSIONS: The ITGB8/TRIM59/AKT/mTOR/glycolysis axis plays a crucial role in modulating tumor softness and stemness. Meanwhile, the soft tumor cells become more sensitive to chemotherapy after stiffening, that offers new insights for hampering tumor progression and recurrence.

A newly characterized dense granule protein (GRA76) is important for the growth and virulence of Toxoplasma gondii.

Pathogenicity of the zoonotic pathogen Toxoplasma gondii largely depends on the secretion of effector proteins into the extracellular milieu and host cell cytosol, including the dense granule proteins (GRAs). The protein-encoding gene TGME49_299780 was previously identified as a contributor to parasite fitness. However, its involvement in parasite growth, virulence and infectivity in vitro and in vivo remains unknown. Here, we comprehensively examined the role of this new protein, termed GRA76, in parasite pathogenicity. Subcellular localization revealed high expression of GRA76 in tachyzoites inside the parasitophorous vacuole (PV). However, its expression was significantly decreased in bradyzoites. A CRISPR-Cas9 approach was used to knock out the gra76 gene in the T. gondii type I RH strain and type II Pru strain. The in vitro plaque assays and intracellular replication showed the involvement of GRA76 in replication of RH and Pru strains. Deletion of the gra76 gene significantly decreased parasite virulence, and reduced the brain cyst burden in mice. Using RNA sequencing, we detected a significant increase in the expression of bradyzoite-associated genes such as BAG1 and LDH2 in the PruΔgra76 strain compared with the wild-type Pru strain. Using an in vitro bradyzoite differentiation assay, we showed that loss of GRA76 significantly increased the propensity for parasites to form bradyzoites. Immunization with PruΔgra76 conferred partial protection against acute and chronic infection in mice. These findings show the important role of GRA76 in the pathogenesis of T. gondii and highlight the potential of PruΔgra76 as a candidate for a live-attenuated vaccine.

The splicing factor SR2 is an important virulence factor of Toxoplasma gondii.

Serine/arginine-rich (SR) proteins are key factors with important roles in constitutive and alternative splicing (AS) of pre-mRNAs. However, the role of SR splicing factors in the pathogenicity of T. gondii remains largely unexplored. Here, we investigated the role of splicing factor SR2, a homolog of Plasmodium falciparum SR1, in the pathogenicity of T. gondii. We functionally characterized the predicted SR2 in T. gondii by gene knockout and studied its subcellular localization by endogenous protein HA tagging using CRISPR-Cas9 gene editing. The results showed that SR2 was localized in the nucleus and expressed in the tachyzoite and bradyzoite stages. In vitro studies including plaque formation, invasion, intracellular replication, egress and bradyzoite differentiation assays showed that deletion of SR2 in type I RH strain and type II Pru strains had no significant effect on the parasite growth and bradyzoite differentiation (p > 0.05). Interestingly, the disruption of SR2 in RH type I (p < 0.0001) and Pru type II (p < 0.05) strains resulted in varying degrees of attenuated virulence. In addition, disruption of SR2 in type II Pru strain significantly reduced brain cyst burden by ~80% (p < 0.0001). Collectively, these results suggest that splicing factor SR2 is important for the pathogenicity of T. gondii, providing a new target for the control and treatment of toxoplasmosis.

ADP-dependent glucokinase controls metabolic fitness in prostate cancer progression.

BACKGROUND: Cell metabolism plays a pivotal role in tumor progression, and targeting cancer metabolism might effectively kill cancer cells. We aimed to investigate the role of hexokinases in prostate cancer (PCa) and identify a crucial target for PCa treatment. METHODS: The Cancer Genome Atlas (TCGA) database, online tools and clinical samples were used to assess the expression and prognostic role of ADP-dependent glucokinase (ADPGK) in PCa. The effect of ADPGK expression on PCa cell malignant phenotypes was validated in vitro and in vivo. Quantitative proteomics, metabolomics, and extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) tests were performed to evaluate the impact of ADPGK on PCa metabolism. The underlying mechanisms were explored through ADPGK overexpression and knockdown, co-immunoprecipitation (Co-IP), ECAR analysis and cell counting kit-8 (CCK-8) assays. RESULTS: ADPGK was the only glucokinase that was both upregulated and predicted worse overall survival (OS) in prostate adenocarcinoma (PRAD). Clinical sample analysis demonstrated that ADPGK was markedly upregulated in PCa tissues vs. non-PCa tissues. High ADPGK expression indicates worse survival outcomes, and ADPGK serves as an independent factor of biochemical recurrence. In vitro and in vivo experiments showed that ADPGK overexpression promoted PCa cell proliferation and migration, and ADPGK inhibition suppressed malignant phenotypes. Metabolomics, proteomics, and ECAR and OCR tests revealed that ADPGK significantly accelerated glycolysis in PCa. Mechanistically, ADPGK binds aldolase C (ALDOC) to promote glycolysis via AMP-activated protein kinase (AMPK) phosphorylation. ALDOC was positively correlated with ADPGK, and high ALDOC expression was associated with worse survival outcomes in PCa. CONCLUSIONS: In summary, ADPGK is a driving factor in PCa progression, and its high expression contributes to a poor prognosis in PCa patients. ADPGK accelerates PCa glycolysis and progression by activating ALDOC-AMPK signaling, suggesting that ADPGK might be an effective target and marker for PCa treatment and prognosis evaluation.

Functional Characterization of Eight Zinc Finger Motif-Containing Proteins in Toxoplasma gondii Type I RH Strain Using the CRISPR-Cas9 System.

The Zinc finger protein (ZFP) family is widely distributed in eukaryotes and interacts with DNA, RNA, and various proteins to participate in many molecular processes. In the present study, the biological functions of eight ZFP genes in the lytic cycle and the pathogenicity of Toxoplasma gondii were examined using the CRISPR-Cas9 system. Immunofluorescence showed that four ZFPs (RH248270-HA, RH255310-HA, RH309200-HA, and RH236640-HA) were localized in the cytoplasm, and one ZFP (RH273150-HA) was located in the nucleus, while the expression level of RH285190-HA, RH260870-HA, and RH248450-HA was undetectable. No significant differences were detected between seven RHΔzfp strains (RHΔ285190, RHΔ248270, RHΔ260870, RHΔ255310, RHΔ309200, RHΔ248450, and RHΔ236640) and the wild-type (WT) strain in the T. gondii lytic cycle, including plaque formation, invasion, intracellular replication, and egress, as well as in vitro virulence (p > 0.05). However, the RHΔ273150 strain exhibited significantly lower replication efficiency compared to the other seven RHΔzfp strains and the WT strain, while in vivo virulence in mice was not significantly affected. Comparative expression analysis of the eight zfp genes indicates that certain genes may have essential functions in the sexual reproductive stage of T. gondii. Taken together, these findings expand our current understanding of the roles of ZFPs in T. gondii.

Construction of PANoptosis signature: Novel target discovery for prostate cancer immunotherapy.

PANoptosis pathway gene sets encompassing pyroptosis, apoptosis, and necroptosis were identified from the MSigDB database. We analyzed the perturbations and crosstalk in the PANoptosis pathway in prostate adenocarcinoma (PRAD), including gene mutation, transcription, methylation, and clinical features. By constructing a PANoptosis signature, we accurately predicted the prognosis and immunotherapeutic response of PRAD patients. We further explored the molecular features and immunological roles of the signature, dividing patients into high- and low-score groups. Notably, the high-score group correlated with better survival outcomes and immunotherapeutic responses, as well as a higher mutation frequency and enrichment score in the PANoptosis and HALLMARK pathways. The PANoptosis signature also enhanced overall antitumor immunity, promoted immune cell infiltration, upregulated immune checkpoint regulators, and revealed the cold tumor characteristics of PRAD. We also identified potential drug targets based on the PANoptosis signature. These findings lead the way in identifying novel prognostic markers and therapeutic targets for patients with PRAD.

Clinical benefit and safety associated with mRNA vaccines for advanced solid tumors: A meta-analysis.

Tumor mRNA vaccines have been developed for over 20 years. Whether mRNA vaccines could promote a clinical benefit to advanced cancer patients is highly unknown. PubMed and Embase were retrieved from January 1, 2000 to January 4, 2023. Random effects models were employed. Clinical benefit (objective response rate [ORR], disease control rate [DCR], 1-year/2-year progression-free survival [PFS], and overall survival [OS]) and safety (vaccine-related grade 3-5 adverse events [AEs]) were evaluated. Overall, 984 patients (32 trials) were enrolled. The most typical cancer types were melanoma (13 trials), non-small cell lung cancer (5 trials), renal cell carcinoma (4 trials), and prostate adenocarcinoma (4 trials). The pooled ORR and DCR estimates were 10.0% (95%CI, 4.6-17.0%) and 34.6% (95%CI, 24.1-45.9%). The estimates for 1-year and 2-year PFS were 38.4% (95%CI, 24.8-53.0%) and 20.0% (95%CI, 10.4-31.7%), respectively. The estimates for 1-year and 2-year OS were 75.3% (95%CI, 62.4-86.3%) and 45.5% (95%CI, 34.0-57.2%), respectively. The estimate for vaccine-related grade 3-5 AEs was 1.0% (95%CI, 0.2-2.4%). Conclusively, mRNA vaccines seem to demonstrate modest clinical response rates, with acceptable survival rates and rare grade 3-5 AEs.

Vitamin E intake and multiple health outcomes: an umbrella review.

Background: The benefits of vitamin E (VE) for multiple health outcomes have been well evaluated in many recent studies. Objective: The purpose of this umbrella review was to conduct a systematic evaluation of the possible associations between VE intake and various health outcomes. Methods: We systematically searched various databases, such as PubMed, Embase, and the Web of Science, to identify related meta-analyses of observational studies and randomized trials. We estimated the effect size of each association by using the random or fixed effects models and the 95% confidence intervals. We used standard approaches to evaluate the quality of the articles (AMSTAR) and classified the evidence into different levels of quality (GRADE). Results: A total of 1,974 review articles were searched, and 27 articles with 28 health outcomes were yielded according to our exclusion criteria. The intake of VE was inversely associated with the risk of breast cancer, lung cancer, esophageal cancer, gastric cancer, pancreatic cancer, kidney cancer, bladder cancer, cervical neoplasms, cardiovascular disease, Parkinson's disease, depression, age-related cataracts, metabolic syndrome, and fracture. Overall, most of the quality of the evidence was low or very low. Three outcomes (stroke, age-related cataracts, obesity) were identified as having a "moderate" level of quality. The AMSTAR scores for all health outcomes ranged from 5 to 10. Conclusion: Our study revealed that VE intake is beneficially related to multiple health outcomes. However, future studies on recommended doses and recommended populations of VE are also needed. Systematic review registration: http://www.crd.york.ac.uk/PROSPERO/, identifier: CRD42022339571.

Multidimensional controllable fabrication of tumor spheroids based on a microfluidic device.

Multicellular tumor spheroids (MCTSs) are in vitro solid tumor models with physiological relevance. To achieve robust process control, a MCTS fabrication method that combines cell membrane engineering and droplet microfluidic techniques is designed. The fluidic control and the chemical interactions between biotin and streptavidin enable artificial cell aggregation to be accomplished in seconds. Then, spheroids with a uniform size are fabricated within alginate microcapsules. Microfluidic mixing-based cell aggregation regulates the cell aggregate size and the spheroid composition, and the microcapsules regulate the size of spheroids from 120 to 180 µm. The method shows applicability for various cancer cell lines, including HCT116, HepG2, and A549. In addition, composite colon cancer spheroids consisting of HCT116 and NIH3T3 with predetermined cell ratios and uniform distributions are produced. The generated MCTSs are assessed using the ELISA and UPLC-MS/MS techniques. The release of vascular endothelial growth factor (VEGF) and the 5-fluorouracil (5-FU) resistance differ in the monotypic and cocultured colon cancer models. Our method provides a robust way to produce consistent and customized MCTSs in cancer research and drug screening.

Comparison of the survival outcomes between primary and secondary muscle-invasive bladder cancer: a propensity score-matched study.

BACKGROUND: Studies have classified muscle-invasive bladder cancer (MIBC) into primary (initially muscle-invasive, PMIBC) and secondary subtypes (initially non-muscle-invasive but progresses, SMIBC), for which controversial survival outcomes were demonstrated. This study aimed to compare the survival outcomes between PMIBC and SMIBC patients in China. METHODS: Patients diagnosed with PMIBC or SMIBC at West China Hospital from January 2009 to June 2019 were retrospectively included. Kruskal-Wallis and Fisher tests were employed to compare clinicopathological characteristics. Kaplan-Meier curves and Cox competing proportional risk model were used to compare survival outcomes. Propensity score matching (PSM) was employed to reduce the bias and subgroup analysis was used to confirm the outcomes. RESULTS: A total of 405 MIBC patients were enrolled, including 286 PMIBC and 119 SMIBC, with a mean follow-up of 27.54 and 53.30 months, respectively. The SMIBC group had a higher proportion of older patients (17.65% [21/119] vs. 9.09% [26/286]), chronic disease (32.77% [39/119] vs . 22.38% [64/286]), and neoadjuvant chemotherapy (19.33% [23/119] vs . 8.04% [23/286]). Before matching, SMIBC had a lower risk of overall mortality (OM) (hazard ratios [HR] 0.60, 95% confidence interval [CI] 0.41-0.85, P  = 0.005) and cancer-specific mortality (CSM) (HR 0.64, 95% CI 0.44-0.94, P  = 0.022) after the initial diagnosis. However, higher risks of OM (HR 1.47, 95% CI 1.02-2.10, P  = 0.038) and CSM (HR 1.58, 95% CI 1.09-2.29, P  = 0.016) were observed for SMIBC once it became muscle-invasive. After PSM, the baseline characteristics of 146 patients (73 for each group) were well matched, and SMIBC was confirmed to have an increased CSM risk (HR 1.83, 95% CI 1.09-3.06, P  = 0.021) than PMIBC after muscle invasion. CONCLUSIONS: Compared with PMIBC, SMIBC had worse survival outcomes once it became muscle-invasive. Specific attention should be paid to non-muscle-invasive bladder cancer with a high progression risk.

Utilizing the Lactate Dehydrogenase-to-Albumin Ratio for Survival Prediction in Patients with Bladder Cancer After Radical Cystectomy.

Purpose: Previous studies have suggested that the preoperative lactate dehydrogenase-to-albumin ratio (LAR) is correlated with survival in several cancers except bladder cancer (BCa). This study aimed to determine the prognostic value of the LAR in patients with urothelial carcinoma of the bladder (UCB) after radical cystectomy (RC). Patients and Methods: A total of 595 UCB patients with RC in West China Hospital from December 2010 to May 2020 were enrolled. A receiver operating characteristic (ROC) curve was used to determine the optimal cutoff value of the LAR. Kaplan-Meier curves and Cox regression analyses were applied to evaluate the association of the LAR with overall survival (OS) and recurrence-free survival. Independent factors in multivariate analyses were selected to construct nomograms. Calibration curves, ROC curves, concordance index (C-index) and decision curve analyses were used to evaluate the performance of the nomograms. Results: The optimal cutoff value of the LAR was determined to be 3.8. Preoperative low LAR was associated with decreased OS (P < 0.001) and RFS (P < 0.001), especially in patients with ≥ pT2 disease. LAR was an independent factor for OS (hazard ratio [HR]: 1.719; P <0.001) and RFS (HR: 1.429; P = 0.012). The addition of the LAR into nomograms could result in better prediction performance. The areas under the curves of the nomograms were 0.821 and 0.801 for the prediction of 3-year OS and RFS, respectively. The C-indexes of the nomograms were 0.760 and 0.741 for the prediction of OS and RFS, respectively. Conclusion: The preoperative LAR is a novel and reliable independent prognostic biomarker for survival in UCB after RC.

Carrot and carotene and multiple health outcomes: an umbrella review of the evidence.

In recent years, the benefits of carrots and carotene in different areas of health have been examined. The purpose of this umbrella review was to identify the associations between carrots and carotene and multiple health outcomes. The review considered evidence from meta-analyses of interventional and observational studies of carrots and carotene and any health outcome. We comprehensively searched Web of Science, PubMed, and Embase. For each association, we estimated the summary effect size using random and fixed effects models and the 95% confidence interval. A total of 1329 studies were searched, and 30 meta-analyses with 26 health outcomes were identified that met the eligibility criteria. Carrot intake was associated with a lower risk of multiple cancer outcomes including breast cancer, lung cancer, pancreatic cancer, gastric cancer, urothelial cancer, and prostate cancer. Carotene intake was associated with a lower risk of fracture, age-related cataract, sunburn, Alzheimer's disease, breast cancer, lung cancer, pancreatic cancer, gastric cancer, esophageal cancer, prostate cancer, and head and neck cancer (HNC). Serum carotene was inversely associated with all-cause mortality, breast cancer, and lung cancer. Our study revealed that carrot or carotene intake could reduce the risk of various negative health outcomes. © 2023 Society of Chemical Industry.

Functional Characterization of 15 Novel Dense Granule Proteins in Toxoplasma gondii Using the CRISPR-Cas9 System.

The analysis of the subcellular localization and function of dense granule proteins (GRAs) is of central importance for the understanding of host-parasite interaction and pathogenesis of Toxoplasma gondii infection. Here, we identified 15 novel GRAs and used C-terminal endogenous gene tagging to determine their localization at the intravacuolar network (IVN), parasitophorous vacuole (PV), or PV membrane (PVM) in the tachyzoites and at the periphery of the bradyzoites-containing cysts. The functions of the 15 gra genes were examined in type I RH strain and 5 of these gra genes were also evaluated in the cyst-forming type II Pru strain. The 15 novel gra genes were successfully disrupted by using CRISPR-Cas9 mediated homologous recombination and the results showed that 13 gra genes were not individually essential for T. gondii replication in vitro or virulence in mice during acute and chronic infection. Intriguingly, deletion of TGME49_266410 and TGME49_315910 in both RH and Pru strains decreased the parasite replication in vitro and attenuated its virulence, and also reduced the cyst-forming ability of the Pru strain in mice during chronic infection. Comparison of the transcriptomic profiles of the 15 gra genes suggests that they may play roles in other life cycle stages and genotypes of T. gondii. Taken together, our findings improve the understanding of T. gondii pathogenesis and demonstrate the involvement of two novel GRAs, TGME49_266410 and TGME49_315910, in the parasite replication and virulence. IMPORTANCE Dense granule proteins (GRAs) play important roles in Toxoplasma gondii pathogenicity. However, the functions of many putative GRAs have not been elucidated. Here, we found that 15 novel GRAs are secreted into intravacuolar network (IVN), parasitophorous vacuole (PV), or PV membrane (PVM) in tachyzoites and are located at the periphery of the bradyzoite-containing cysts. TGME49_266410 and TGME49_315910 were crucial to the growth of RH and Pru strains in vitro. Deletion of TGME49_266410 and TGME49_315910 attenuated the parasite virulence in mice. However, disruption of other 13 gra genes did not have a significant impact on the proliferation and pathogenicity of T. gondii in vitro or in vivo. The marked effects of the two novel GRAs (TGME49_266410 and TGME49_315910) on the in vitro growth and virulence of T. gondii are notable and warrant further elucidation of the temporal and spatial dynamics of translocation of these two novel GRAs and how do they interfere with host cell functions.

Hemoglobin, albumin, lymphocyte, and platelet (HALP) score and cancer prognosis: A systematic review and meta-analysis of 13,110 patients.

BACKGROUND AND AIMS: We sought to explore the prognostic role of the hemoglobin, albumin, lymphocyte, and platelet (HALP) score on survival outcomes in patients with solid tumors by performing a systematic review and meta-analysis. MATERIALS AND METHODS: PubMed, EMBASE and Web of Science were searched for relevant articles through 02 Nov 2022. Hazard ratios (HRs) with 95% confidence intervals (95% CIs) were extracted and pooled to assess the association of the HALP score with survival outcomes in solid tumors using Stata 15.0. The primary outcome was overall survival (OS), and the secondary outcomes were cancer-specific survival (CSS) and progression/disease/recurrence-free survival (PFS/DFS/RFS). RESULTS: A total of 28 studies (31 cohorts) comprising 13,038 patients were enrolled. A low HALP score was associated with decreased OS (HR, 1.61; 95 % CI, 1.44-1.81; P < 0.001), CSS (HR, 1.80; 95 % CI: 1.54-2.09; P < 0.001) and PFS/DFS/RFS (HR, 1.61; 95 % CI: 1.45-1.80; P < 0.001). The prognostic value of HALP on OS was observed across various tumor types and tumor stages. meta-regression found that tumor stage was the major source of the heterogeneity for OS. CONCLUSION: A low pretreatment HALP score is a reliable and negative prognostic biomarker for survival outcomes in patients with cancer.

Effect of deleting four Toxoplasma gondii calcium-binding EGF domain-containing proteins on parasite replication and virulence.

Several calcium-binding proteins including calcium-dependent protein kinases play important roles in several facets of the intracellular infection cycle of the apicomplexan protozoan parasite Toxoplasma gondii. However, the role of the calcium-binding epidermal growth factor (EGF) domain-containing proteins (CBDPs) remains poorly understood. In this study, we examined the functions of four CBDP genes in T. gondii RH strain of type I by generating knock-out strains using CRISPR-Cas9 system. We investigated the ability of mutant strains deficient in CBDP1, CBDP2, CBDP3, or CBDP4 to form plaques, replicate intracellularly, and egress from the host cells. The results showed that no definite differences between any of these four CBDP mutant strains and the wild-type strain in terms of their ability to form plaques, intracellular replication, and egress. Additionally, CBDP mutants did not exhibit any significant attenuated virulence compared to the wild-type strain in mice. The expression profiles of CBDP2-4 genes were conserved among T. gondii strains of different genotypes, life cycle stages, and developmental forms. Whether other CBDP genes play any roles in the pathogenicity of T. gondii strains of different genotypes remains to be elucidated.

Theoretical insights into dissociative-associative mechanism for enhanced electrochemical nitrate reduction to ammonia.

The development of electrochemical nitrate reduction reaction (NO3RR) is a "two birds-one stone" method, which can not only remove NO3- pollutant, but also produce valuable ammonia (NH3). However, a mechanistic understanding of the nitrate reduction process remains very limited. Herein, we highlighted a dissociative-associative mechanism for the NO3RR, in which the N-O bond of nitrate is initially broken to form *O and *NO2 intermediate adsorbed on two active sites (dissociation process) and then subsequently hydrogenated and reduced to ammonia (association process). By taking a series of diatomic site catalysts (CuTM/g-CN and CuTM/N6C, TM= Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn) as models, we systematically investigate the dissociative-associative mechanism for the NO3RR and compared with the Cu-based single-atom catalysts which follows the traditional directly associative mechanism. Density functional theory (DFT) calculations show that dissociative-associative mechanism is energetically favorable on seven catalysts (CuTi/g-CN, CuV/g-CN, CuMn/g-CN, CuCo/g-CN, CuV/N6C, CuCr/N6C and CuFe/N6C) with the significantly reduced limiting potential of - 0.14 V to - 0.47 V. Specifically, an efficiently screening strategy was proposed to determine the dissociative-associative or directly associative mechanism for NO3RR. This work can provide useful guideline for the rational design and development of NO3RR electrocatalysts.