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.

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.

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.

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.

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.

Neoadjuvant chemotherapy for patients with locally advanced penile cancer: an updated evidence.

Neoadjuvant chemotherapy (NAC) has shown promising results in patients with locally advanced penile cancer. However, no consensus exists on its applications for locally advanced penile cancer. Thus, it is unclear which kind of chemotherapy regimen is the best choice. Consequently, a systematic search of PubMed, Web of Science, and EMBASE was performed in March 2021 to assess the efficacy and safety of NAC for the treatment of patients with locally advanced penile cancer. The Newcastle-Ottawa Scale was used to assess the risk of bias in each study. This study synthesized 14 published studies. The study revealed that patients who achieved an objective response to NAC obtained a better survival outcome compared with those who did not achieve an objective response. In addition, the objective response rates (ORRs) and pathological complete response (pCR) rates were 0.57 and 0.11, respectively. The incidence of grade ≥3 toxicity was 0.36. Subgroup analysis found that the ORR and pCR of the taxane-platinum (TP) regimen group performed better than those of the nontaxane-platinum (NTP) regimen group (0.57 vs 0.54 and 0.14 vs 0.07, respectively). Moreover, the TP regimen group had more frequent toxicity than the NTP regimen group (0.41 vs 0.26). However, further studies were warranted to confirm the findings.

Holmium laser technologies versus photoselective greenlight vaporization for patients with benign prostatichyperplasia: a meta-analysis.

This study aims to compare the efficacy and safety of holmium laser technologies (HoL-Ts) and photoselective greenlight vaporization (PVP) for the treatment of benign prostatic hyperplasia (BPH), and to perform a meta-analysis according to the Preferred Reporting Items of Systematic Reviews and Meta-Analyses guidelines on PubMed, EMBASE, ClinicalTrial.gov, and the Cochrane Central Register of Controlled Trials up to August 2019. Functional outcomes, perioperative parameters, and complications were included and analyzed. Review Manager 5.3 (Cochrane Collaboration, Oxford, UK) was used to perform all analyses. A total of six articles composed of 2014 patients were included in this review. In comparison with PVP, HoL-Ts had a better performance in 1-, 3-, and 6-month Qmax (P = 0.02, but I2 = 81%), with less postvoid residual urine volume (PVR) (MD = -33.85, 95% CI -52.13 to -15.57, P = 0.0003) and less total energy used (MD = -31.66, 95% CI -58.99 to -4.33, P = 0.02). Moreover, HoL-Ts had a relatively lower risk of conversion rate (OR = 0.08, 95% CI 0.01 to 0.60, P = 0.01) associated with enough enucleation and less intraoperative bleeding. Subgroup analysis of holmium laser enucleation of prostate (HoLEP) versus PVP suggested that HoLEP presented better results in 1-, 3-, 6-month and 1-year Qmax with less PVR, less energy consumption, and lower conversion rate. Compared with PVP, HoL-Ts had higher 1-, 3-, and 6-month Qmax, less PVR, and less total energy consumption with a relatively lower risk of conversion rate. In subgroup analyses, HoLEP had shown better results in accordance with all HoL-Ts. Nevertheless, well-designed RCTs including overall functional indicators are required to confirm our findings.

Transurethral resection of the prostate is an independent risk factor for biochemical recurrence after radical prostatectomy for prostate cancer.

Biochemical recurrence (BCR) is important for measuring the oncological outcomes of patients who undergo radical prostatectomy (RP). Whether transurethral resection of the prostate (TURP) has negative postoperative effects on oncological outcomes remains controversial. The primary aim of our retrospective study was to determine whether a history of TURP could affect the postoperative BCR rate. We retrospectively reviewed patients with prostate cancer (PCa) who had undergone RP between January 2009 and October 2017. Clinical data on age, prostate volume, serum prostate-specific antigen levels (PSA), biopsy Gleason score (GS), metastasis stage (TNM), D'Amico classification, and American Society of Anesthesiologists (ASA) classification were collected. Statistical analyses including Cox proportional hazard models and sensitivity analyses which included propensity score matching, were performed, and the inverse-probability-of-treatment-weighted estimator and standardized mortality ratio-weighted estimator were determined. We included 1083 patients, of which 118 had a history of TURP. Before matching, the non-TURP group differed from the TURP group with respect to GS (P= 0.047), prostate volume (mean: 45.19 vs 36.00 ml, P < 0.001), and PSA level (mean: 29.41 vs 15.11 ng ml-1, P= 0.001). After adjusting for age, PSA level, T stage, N stage, M stage, and GS, the TURP group showed higher risk of BCR (hazard ratio [HR]: 2.27, 95% confidence interval [CI]: 1.13-3.94, P= 0.004). After matching (ratio 1:4), patients who underwent TURP were still more likely to develop BCR according to the adjusted propensity score (HR: 2.00, 95% CI: 1.05-3.79, P= 0.034). Among patients with PCa, those with a history of TURP were more likely to develop BCR after RP.

Efficacy and safety of phosphodiesterase type 5 inhibitors for the treatment of distal ureteral calculi of 5 to 10 mm in size: A systematic review and network meta-analysis.

To evaluate the Efficacy and safety of phosphodiesterase type 5 inhibitors as a medical therapy for distal ureteral calculi by means of a systematic review and network meta-analysis (NMA). We searched the Embase, Medline, and the Cochrane Central Register of Controlled Trials for randomised controlled trials (RCTs) published before May, 2017. Stone passage rate as the primary outcome. We used random effects model for pairwise meta-analyses and Bayesian random effects model for NMA. We evaluated the quality of evidence by the GRADE framework for each network estimate. Five RCTs (861 patients) comparing four different interventions. The results of NMA showed that compared with tamsulosin alone, tamsulosin combined with tadalafil group was associated with significantly higher stone passage rate (odds radio [OR] 2.55, 95% credible intervals [Crl] 1.11 to 5.89). When considering stone expulsion rate, compared with tamsulosin, silodosin was ranked best (OR 3.58, 95% Crl 1.13 to 11.91), followed by tamsulosin combined with tadalafil (OR 2.55, 95% Crl 1.11 to 5.89) and tadalafil alone (OR 1.86, 95% Crl 0.95 to 4.25). No significant difference was found considering safety profiles between any interventions. This meta-analysis indicates that tamsulosin combined with tadalafil is an effective treatment option for ureteral stones with a low occurrence of side effects. Clinicians should take all known safety and compliance of patients into account when choosing an optimal strategy. Since sample size of included studies, further RCTs are strongly encouraged to address the clinical question.

Time-effect relationship of acupuncture on histopathology, ultrastructure, and neuroethology in the acute phase of cerebral hemorrhage.

Many clinical studies have addressed the treatment of acute cerebral hemorrhage using acupuncture. However, few studies have examined the relationship between time of acupuncture and curative effect on cerebral hemorrhage. By observing the effect of acupuncture on changes in histopathology, ultrastructure, and neuroethology in a cerebral hemorrhage model of rats, we have directly examined the time-effect relationship of acupuncture. The rat model of cerebral hemorrhage was produced by slowly injecting autologous blood to the right caudate nucleus. The experimental groups were: 3-, 9-, 24-, and 48-hour model groups; and 3-, 9-, 24-, and 48-hour acupuncture groups. The sham-operation group was used for comparison. Acupuncture was performed at the Neiguan (PC6) and Renzhong (DU26) acupoints, twice a day, 6 hours apart, for 5 consecutive days. Brain tissue changes were observed by light microscopy and transmission electron microscopy. Neuroethology was assessed using Bederson and Longa scores. Our results show that compared with the sham-operation and model groups, Bederson and Longa scores were lower in each acupuncture group, with visibly improved histopathology and brain tissue ultrastructure. Further, the results were better in the 3- and 9-hour acupuncture groups than the 24- and 48-hour acupuncture groups. Our findings show that acupuncture treatment can relieve pathological and ultrastructural deterioration and neurological impairment caused by the acute phase of cerebral hemorrhage, and may protect brain tissue during this period. In addition, earlier acupuncture intervention following cerebral hemorrhage (by 3 or 9 hours) is associated with a better treatment outcome.

Pu-erh tea powder preventive effects on cisplatin-induced liver oxidative damage in Wistar rats.

BACKGROUND: Chemotherapy is one of the major means for control of malignancies, with cisplatin (CDDP) as one of the main agents, widely used for the treatment of various malignant solid tumors. However, prevention of hepatotoxicity from cisplatin is one of the urgent issues in cancer chemotherapy. In this study, we aimed to investigate the effects of pu-erh tea on hepatotoxicity through body weight and tissue antioxidant parameters like, liver coefficient, serum alanine aminotransferase (ALT), serum aspartate aminotransferase (AST), superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), malondialdehyde(MDA) and glutathione (GSH) levels, and light microscopic evaluation by histological findings. MATERIALS AND METHODS: The rats were randomly divided into five groups: Control (n=10), cisplatin (3 mg/kg p.i., n=10), cisplatin+pu-erh (0.32 g/kg/day i.g., n=10), cisplatin+pu-erh (0.8 g/kg/day i.g., n=10) and cisplatin+pu-erh (1.6 g/kg/day i.g., n=10). Pu-erh tea powder was administrated for 31 consecutive days. The rats were sacrificed at the end on the second day after a single dose of cisplatin treatment for measuring indices. RESULTS: Pu-erh tea powder exhibited a protective effect by decreasing MDA and GSH and increasing the SOD and GSH-PX levels and GSH-PX/MDA ratio in comparison with the control group. Besides, pu-erh tea was also able to alleviate the pathological damage to some extent. CONCLUSION: Pu-erh tea powder is protective against cisplatin-induced liver oxidative damages, especially at the medium dosage (0.8 g/kg/d).