Advancements in understanding the role of ferroptosis in hypoxia-associated brain injury: a narrative review.

Background and Objective: Ferroptosis, a form of programmed cell death driven by lipid peroxidation and dependent on iron ions, unfolds through a sophisticated interplay of multiple biological processes. These include perturbations in iron metabolism, lipid peroxidation, aberrant amino acid metabolism, disruptions in hypoxia-inducible factor-prolyl hydroxylase (HIF-PHD) axis, and endoplasmic reticulum (ER) stress. Recent studies indicate that ferroptosis may serve as a promising therapeutic target for hypoxia-associated brain injury such as hypoxic-ischemic brain damage (HIBD) and cerebral ischemia-reperfusion injury (CIRI). HIBD is a neonatal disease that can be fatal, causing death or mental retardation in newborns. HIBD is a kind of diffuse brain injury, which is characterized by apoptosis of nerve cells and abnormal function and structure of neurons after cerebral hypoxia and ischemia. At present, there are no fundamental prevention and treatment measures for HIBD. The brain is the most sensitive organ of the human body to hypoxia. Cerebral ischemia will lead to the damage of local brain tissue and its function, and CIRI will lead to a series of serious consequences. We hope to clarify the mechanism of ferroptosis in hypoxia-associated brain injury, inhibit the relevant targets of ferroptosis in hypoxia-associated brain injury to guide clinical treatment, and provide guidance for the subsequent treatment of disease-related drugs. Methods: Our research incorporated data on "ferroptosis", "neonatal hypoxic ischemia", "hypoxic ischemic brain injury", "hypoxic ischemic encephalopathy", "brain ischemia-reperfusion injury", and "therapeutics", which were sourced from Web of Science, PubMed, and comprehensive reviews and articles written in English. Key Content and Findings: This review delineates the underlying mechanisms of ferroptosis and the significance of these pathways in hypoxia-associated brain injury, offering an overview of therapeutic strategies for mitigating ferroptosis. Conclusions: Ferroptosis involves dysregulation of iron metabolism, lipid peroxidation, amino acid metabolism, dysregulation of HIF-PHD axis and endoplasmic reticulum stress (ERS). By reviewing the literature, we identified the involvement of the above processes in HIBD and CIRI, and summarized a series of therapeutic measures for HIBD and CIRI by inhibiting ferroptosis. We hope this study would provide guidance for the clinical treatment of HIBD and CIRI in the future.

Progress in the study of mechanisms and pathways related to the survival of random skin flaps.

The clinical application of random flaps in wound repair has been a topic of discussion. Random flaps are prone to necrosis due to the lack of well-defined vascular blood supply during transfer surgery. Their clinical utility is restricted, financial and psychological burdens is imposed on patients due to this limitation. The survival of random skin flaps depends on factors such as ischemia-reperfusion injury, oxidative stress, local inflammatory response, and neovascularization. This review aims to provide an overview of the evidence supporting the use of random flaps in clinical practice. In addition, this review explores the impact of different medications on signaling pathways within the flap's local microcirculation and investigates the interconnections between these pathways.

Corrigendum: Two small-molecule inhibitors of Toxoplasma gondii proliferation in vitro.

[This corrects the article DOI: 10.3389/fcimb.2023.1145824.].

Two small-molecule inhibitors of Toxoplasma gondii proliferation in vitro.

Background: Toxoplasmosis caused by Toxoplasma gondii is a globally distributed zoonosis. Most infections appear asymptomatic in immunocompetent individuals, but toxoplasmosis can be fatal in fetuses and immunocompromised adults. There is an urgent need to research and develop effective and low-toxicity anti-T. gondii drugs because of some defects in current clinical anti-T. gondii drugs, such as limited efficacy, serious side effects and drug resistance. Methods: In this study, 152 autophagy related compounds were evaluated as anti-T. gondii drugs. The activity of ß-galactosidase assay based on luminescence was used to determine the inhibitory effect on parasite growth. At the same time, MTS assay was used to further detect the effects of compounds with over 60% inhibition rate on host cell viability. The invasion, intracellular proliferation, egress and gliding abilities of T. gondii were tested to assess the inhibitory effect of the chosen drugs on the distinct steps of the T. gondii lysis cycle. Results: The results showed that a total of 38 compounds inhibited parasite growth by more than 60%. After excluding the compounds affecting host cell activity, CGI-1746 and JH-II-127 were considered for drug reuse and further characterized. Both CGI-1746 and JH-II-127 inhibited tachyzoite growth by 60%, with IC50 values of 14.58 ± 1.52 and 5.88 ± 0.23 µM, respectively. TD50 values were 154.20 ± 20.15 and 76.39 ± 14.32 µM, respectively. Further research found that these two compounds significantly inhibited the intracellular proliferation of tachyzoites. Summarize the results, we demonstrated that CGI-1746 inhibited the invasion, egress and especially the gliding abilities of parasites, which is essential for the successful invasion of host cells, while JH-II-127 did not affect the invasion and gliding ability, but seriously damaged the morphology of mitochondria which may be related to the damage of mitochondrial electron transport chain. Discussion: Taken together, these findings suggest that both CGI-1746 and JH-II-127 could be potentially repurposed as anti-T. gondii drugs, lays the groundwork for future therapeutic strategies.

Computed tomography-based radiomics nomogram model for predicting adherent perinephric fat.

Aims: We investigated the predictive value of a computed tomography (CT)-based radiomics nomogram model for adherent perinephric fat (APF). Materials and Methods: The data of 220 renal carcinoma patients were collected retrospectively. Patients were divided into training (n = 153) and validation cohorts (n = 67). Radiomics features were extracted from plain CT scans, while radscore was generated by a linear combination of selected radiomics features and their weighting coefficients. Univariate logistic regression was used to screen clinical risk factors. Multivariate logistic regression combined with radscore was used to screen final predictors to construct a radiomics nomogram model. Receiver Operating Characteristic curves were used to evaluate the predictive performance of models. Results: Thirteen radiomics features associated with APF achieved a good predictive effect. The overall area under the curve (AUC) of the radscore model was 0.966, and that of the training and validation cohorts was 0.969 and 0.956, respectively. Gender, age, hypertension, size, perinephric fat thickness, Mayo Adhesive Probability score, neutrophil-to-lymphocyte ratio, monocyte-to-lymphocyte ratio, systemic inflammation response index, and systemic immune-inflammation index were risk factors for APF (P < 0.05). The overall AUC of the radiomics nomogram model based on radiomics features and clinical factors, the training, and validation cohorts was 0.981, 0.997, and 0.949, respectively. Both models had high diagnostic efficiency. However, their differential diagnostic accuracy was higher than that of the clinical model. Additionally, the radiomics nomogram model had higher AUC and specificity. Conclusions: The radiomics nomogram model is a prediction tool based on radiomics features and clinical risk factors and has high prediction ability and clinical application value for APF.

Investigation of Antiparasitic Activity of Two Marine Natural Products, Estradiol Benzoate, and Octyl Gallate, on Toxoplasma gondii In Vitro.

Toxoplasmosis, caused by Toxoplasma gondii, is a common disease worldwide and could be severe and even fatal in immunocompromised individuals and fetuses. Limitation in current available treatment options drives the need to develop novel therapeutics. This study assessed the anti-T. gondii potential of 103 marine natural products. A luminescence-based ß-galactosidase activity assay was used to screen the marine natural products library. Afterward, those compounds that displayed over 70% parasite inhibition ratio were further chosen to assess their cytotoxicity. Compounds exhibiting low cytotoxicity (≥80% cell viability) were applied to evaluate the inhibition efficacy on discrete steps of the T. gondii lytic cycle, including invasion, intracellular growth, and egress abilities as well as the cell cycle. We found that both estradiol benzoate and octyl gallate caused >70% inhibition of tachyzoite growth with IC50 values of 4.41 ± 0.94 and 5.66 ± 0.35 µM, respectively, and displayed low cytotoxicity with TD50 values of 34.11 ± 2.86 and 26.4 ± 0.98 µM, respectively. Despite their defects in inhibition of invasion and egress of tachyzoite, the two compounds markedly inhibited the tachyzoite intracellular replication. Flow cytometric analyses further suggested that the anti-T. gondii activity of estradiol benzoate, rather than octyl gallate, may be linked to halting cell cycle progression of tachyzoite from G1 to S phase. Taken together, these findings suggest that both estradiol benzoate and octyl gallate are potential inhibitors for anti-T. gondii infection and support the further exploration of marine natural products as a thinkable source of alternative and active agents against T. gondii.

Toxoplasma TgAtg8-TgAtg3 Interaction Primarily Contributes to Apicoplast Inheritance and Parasite Growth in Tachyzoite.

The apicoplast, which harbors key pathways involved in biosynthesis of vital metabolites, is a unique and essential nonphotosynthetic plastid organelle in apicomplexan parasites. Intriguingly, autophagy-related protein 8 (Atg8), a highly conserved eukaryotic protein, can localize to the outermost membrane of the apicoplast and modulate its inheritance in both Toxoplasma and Plasmodium parasites. The Atg8-Atg3 interaction plays a key role in Atg8 lipidation and localization, and our previously work in Toxoplasma has suggested that the core Atg8-family interacting motif (AIM) in TgAtg3, 239FADI242, and the R27 residue of TgAtg8 contribute to TgAtg8-TgAtg3 interaction in vitro. However, little is known about the function of this interaction or its importance in tachyzoite growth in Toxoplasma gondii. Here, we generated two complemented cell lines, TgAtg3F239A/I242A and TgAtg8R27E, based on the TgAtg3 and TgAtg8 conditional knockdown cell lines, respectively. We found that both mutant complemented cell lines were severely affected in terms of tachyzoite growth and displayed delayed death upon conditional knockdown of endogenous TgAtg3 or TgAtg8. Intriguingly, both complemented lines appeared to be defective in TgAtg8 lipidation and apicoplast inheritance. Moreover, we showed that the interaction of TgAtg8 and TgAtg3 is critical for TgAtg8 apicoplast localization. In addition, we found that the TgAtg3F239A/I242A complemented line exhibits an integral mitochondrial network upon ablation of endogenous TgAtg3, which is distinct from TgAtg3-depleted parasites with a fragmented mitochondrial network. Taken together, this work solidifies the contribution of the TgAtg8-TgAtg3 interaction to apicoplast inheritance and the growth of T. gondii tachyzoites. IMPORTANCEToxoplasma gondiiis a widespread intracellular parasite infecting a variety of warm-blooded animals, including humans. Current frontline treatment of toxoplasmosis suffers many drawbacks, including toxicity, drug resistance, and failure to eradicate tissue cysts, underscoring the need to identify novel drug targets for suppression or treatment of toxoplasmosis. TgAtg8 is thought to serve multiple functions in lipidation and is considered essential to the growth and development of both tachyzoites and bradyzoites. Here, we show that Toxoplasma gondii has adapted a conserved Atg8-Atg3 interaction, required for canonical autophagy in other eukaryotes, to function specifically in apicoplast inheritance. Our finding not only highlights the importance of TgAtg8-TgAtg3 interaction in tachyzoite growth but also suggests that this interaction is a promising drug target for the therapy of toxoplasmosis.

A MRI-based radiomics nomogram for evaluation of renal function in ADPKD.

OBJECTIVES: This study is aimed to establish a fusion model of radiomics-based nomogram to predict the renal function of autosomal dominant polycystic kidney disease (ADPKD). METHODS: One hundred patients with ADPKD were randomly divided into training group (n = 69) and test group (n = 31). The radiomics features were extracted from T1-weighted fat suppression images (FS-T1WI) and T2-weighted fat suppression images (FS-T2WI). Decision tree algorithm was employed to build radiomics model to get radiomics signature. Then multivariate logistic regression analysis was used to establish the radiomics nomogram based on independent clinical factors, conventional MR imaging variables and radiomics signature. The receiver operating characteristic (ROC) analysis and Delong test were used to compare the performance of radiomics model and radiomics nomogram model, and the decision curve to evaluate the clinical application value of radiomics nomogram model in the evaluation of renal function in patients with ADPKD. RESULTS: Fourteen radiomics features were selected to establish radiomics model. Based on FS-T1WI and FS-T2WI sequences, the radiomics model showed good discrimination ability in training group and test group [training group: (AUC) = 0.7542, test group (AUC) = 0.7417]. The performance of radiomics nomogram model was significantly better than that of radiomics model in all data sets [radiomics model (AUC) = 0.7505, radiomics nomogram model (AUC) = 0.8435, p value = 0.005]. The analysis of calibration curve and decision curve showed that radiomics nomogram model had more clinical application value. CONCLUSION: radiomics analysis of MRI can be used for the preliminary evaluation and prediction of renal function in patients with ADPKD. The radiomics nomogram model shows better prediction effect in renal function evaluation, and can be used as a non-invasive renal function prediction tool to assist clinical decision-making. Trial registration ChiCTR, ChiCTR2100046739. Registered 27 May 2021-retrospectively registered, http://www.ChiCTR.org.cn/showproj.aspx?proj=125955.

Study on the correlation between preoperative inflammatory indexes and adhesional perinephric fat before laparoscopic partial nephrectomy.

OBJECTIVE: This study was aimed to evaluate the effect of preoperative composite inflammatory index on adhesional perinephric fat (APF), providing a help for preoperative risk assessment of laparoscopic partial nephrectomy (LPN) in patients with renal cell carcinoma. MATERIALS AND METHODS: A retrospective study was conducted on 231 patients with renal cell carcinoma, who underwent laparoscopic partial nephrectomy. They were divided into two groups according to whether there was APF during operation. Relevant clinical data, laboratory parameters and imaging examination were obtained before operation to calculate the composite inflammatory index and MAP score. The composite inflammatory index was divided into high value group and low value group by ROC curve method. The related predictive factors of APF were analyzed by logistic regression method. RESULTS: The APF was found in 105 patients (45.5%). In multivariate analysis, systemic immune inflammation index (SII) (high/low), MAP score, tumor size and perirenal fat thickness were independent predictors of APF. The operation time of patients with APF was longer, and the difference of blood loss was not statistically significant. CONCLUSION: SII is an independent predictor of APF before laparoscopic partial nephrectomy. Trial registration ChiCTR, ChiCTR2100045944. Registered 30 April 2021-Retrospectively registered, http://www.chictr.org.cn/showproj.aspx?proj=125703 .

Liver tumor segmentation using 2.5D UV-Net with multi-scale convolution.

Liver tumor segmentation networks are generally based on U-shaped encoder-decoder network with 2D or 3D structure. However, 2D networks lose the inter-layer information of continuous slices and 3D networks might introduce unacceptable parameters for GPU memory. As a result, 2.5D networks were proposed to balance the memory consumption and 3D context. Different from the canonical 2.5D design, which utilizes a 2D network combined with RNN, we propose a new 2.5D design called UV-Net to encode the inter-layer information in the context of 3D convolution, and reconstruct the high-resolution results with 2D deconvolution. At the same time, the multi-scale convolution structure enables multi-scale feature extraction without extra computational cost, which effectively mines structured information, reduces information redundancy, strengthens independent features, and makes feature dimension sparse, to enhance network capacity and efficiency. Combined with the proposed preprocessing method of removing mean energy, UV-Net significantly outperforms the existing methods in liver tumor segmentation and especially improves the segmentation accuracy of small objects on the LiTS2017 dataset.

Two old drugs, NVP-AEW541 and GSK-J4, repurposed against the Toxoplasma gondii RH strain.

BACKGROUND: Toxoplasma gondii is a zoonotic pathogen that causes toxoplasmosis and leads to serious public health problems in developing countries. However, current clinical therapeutic drugs have some disadvantages, such as serious side effects, a long course of treatment and the emergence of drug-resistant strains. The urgent need to identify novel anti-Toxoplasma drugs has initiated the effective strategy of repurposing well-characterized drugs. As a principled screening for the identification of effective compounds against Toxoplasma gondii, in the current study, a collection of 666 compounds were screened for their ability to significantly inhibit Toxoplasma growth. METHODS: The inhibition of parasite growth was determined using a luminescence-based ß-galactosidase activity assay. Meanwhile, the effect of compounds on the viability of host cells was measured using CCK8. To assess the inhibition of the selected compounds on discrete steps of the T. gondii lytic cycle, the invasion, intracellular proliferation and egress abilities were evaluated. Finally, a murine infection model of toxoplasmosis was used to monitor the protective efficacy of drugs against acute infection of a highly virulent RH strain. RESULTS: A total of 68 compounds demonstrated more than 70% parasite growth inhibition. After excluding compounds that impaired host cell viability, we further characterized two compounds, NVP-AEW541 and GSK-J4 HCl, which had IC50 values for parasite growth of 1.17 µM and 2.37 µM, respectively. In addition, both compounds showed low toxicity to the host cell. Furthermore, we demonstrated that NVP-AEW541 inhibits tachyzoite invasion, while GSK-J4 HCl inhibits intracellular tachyzoite proliferation by halting cell cycle progression from G1 to S phase. These findings prompted us to analyse the efficacy of the two compounds in vivo by using established mouse models of acute toxoplasmosis. In addition to prolonging the survival time of mice acutely infected with T. gondii, both compounds had a remarkable ability to reduce the parasite burden of tissues. CONCLUSIONS: Our findings suggest that both NVP-AEW541 and GSK-J4 could be potentially repurposed as candidate drugs against T. gondii infection.

A Bat-Inspired Sparse Recovery Algorithm for Compressed Sensing.

Compressed sensing (CS) is an important research area of signal sampling and compression, and the essence of signal recovery in CS is an optimization problem of solving the underdetermined system of equations. Greedy pursuit algorithms are widely used to solve this problem. They have low computational complexity; however, their recovery performance is limited. In this paper, an intelligence recovery algorithm is proposed by combining the Bat Algorithm (BA) and the pruning technique in subspace pursuit. Experimental results illustrate that the proposed algorithm has better recovery performance than greedy pursuit algorithms. Moreover, applied to the microseismic monitoring system, the BA can recover the signal well.

Protective immune response in mice induced by a suicidal DNA vaccine encoding NTPase-II gene of Toxoplasma gondii.

DNA-based alphaviral RNA replicon vectors, also called suicidal DNA vectors, have been employed to alleviate biosafety concerns attribution to its ability to induce apoptotic cell death of the transfected cells. Toxoplasma gondii nucleoside triphosphate hydrolase-II (TgNTPase-II), which facilitates the parasite to salvage purines from the host cell for survival and replication, have been demonstrated to be a potential vaccine candidate for toxoplasmosis. Herein, we evaluated the immunogenic potential of a suicidal DNA vaccine encoding TgNTPase-II gene, pDREP-TgNTPase-II, delivered intramuscularly in combination with electroporation. Immunization of mice with pDREP-TgNTPase-II elicited specific humoral responses, with high IgG antibody titers and a mixed IgG1/IgG2a response. The cellular immune response was associated with high level production of IFN-γ, IL-2, IL-10 cytokines and low level IL-4 production as well as the increase of the percentage of CD8+ T cells, indicating that a Th1 predominant response was elicited. Furthermore, mice vaccinated with this suicidal DNA vaccine displayed partial protection against acute infection with the virulent RH strain as well as chronic infection with PRU cyst, which shows 77.7% and 71.4% reduction in brain cyst burden in comparison to PBS and pDREP-eGFP control group, respectively. Based on the cellular and antibody responses, the suicidal DNA vaccine elicited a Th1-predominant immune response against T. gondii challenge.

Induction of Autophagy interferes the tachyzoite to bradyzoite transformation of Toxoplasma gondii.

Autophagy process in Toxoplasma gondii plays a vital role in regulating parasite survival or death. Thus, once having an understanding of certain effects of autophagy on the transformation of tachyzoite to bradyzoite this will allow us to elucidate the function of autophagy during parasite development. Herein, we used three TgAtg proteins involved in Atg8 conjugation system, TgAtg3, TgAtg7 and TgAtg8 to evaluate the autophagy level in tachyzoite and bradyzoite of Toxoplasma in vitro based on Pru TgAtg7-HA transgenic strains. We showed that both TgAtg3 and TgAtg8 were expressed at a significantly lower level in bradyzoites than in tachyzoites. Importantly, the number of parasites containing fluorescence-labelled TgAtg8 puncta was significantly reduced in bradyzoites than in tachyzoites, suggesting that autophagy is downregulated in Toxoplasma bradyzoite in vitro. Moreover, after treatment with drugs, bradyzoite-specific gene BAG1 levels decreased significantly in rapamycin-treated bradyzoites and increased significantly in 3-MA-treated bradyzoites in comparison with control bradyzoites, indicating that Toxoplasma autophagy is involved in the transformation of tachyzoite to bradyzoite in vitro. Together, it is suggested that autophagy may serve as a potential strategy to regulate the transformation.

Identification of TgAtg8-TgAtg3 interaction in Toxoplasma gondii.

Autophagy is a catabolic process in eukaryotic cells involved in the targeted degradation of cellular organelles and the cytoplasm. Recent works in Toxoplasma gondii suggest that the autophagy processes may serve as an important pathway in modulating parasite survival or death. As an important modulator of Atg8 lipidation and autophagy, Atg8-Atg3 interaction has been attracting increasing attention. However, there is no direct evidence that TgAtg8-TgAtg3 interaction occurs in the parasite. In this study, we firstly found TgAtg8 partially colocalized with TgAtg3 in GFP-TgAtg8 transgenic strains using IFA. Then, lysates from GFP-TgAtg8 tachyzoites were directly subject to large-scale tandem affinity purification with anti-GFP antibody. Western blot and tandem mass spectrometry (MS/MS) analysis determined the interaction between TgAtg8 and TgAtg3. Additionally, we performed real-time interaction analysis with a surface plasmon resonance biosensor using BIAcore system. As expected, the result demonstrated a concentration-dependent increases in resonance signals and indicated the TgAtg8 could bind directly TgAtg3 in vitro. Noteworthily, A KD of 34.9nM obtained from TgAtg8-TgAtg3 interaction indicate a high-affinity between Atg8-Atg3 in Toxoplasma. Furthermore, homology modeling and sequence alignment showed that TgAtg8 has greatest sequence and structural conservation. Within TgAtg3, this protein possesses the core E2 enzymatic activity structure and a truncated handle region which may contain AIM sequence. Taken together, our findings would help elucidate the formation mechanism of autophagosome in Toxoplasma and provide a possibility for looking into parasitic drug targets.

Correlations between ADC values and molecular markers of Ki-67 and HIF-1α in hepatocellular carcinoma.

OBJECTIVE: Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths. Cell proliferativity and hypoxia have important impact on the response to radiotherapy or chemotherapy. The purpose of this study was to investigate the association of apparent diffusion coefficient (ADC) values and the molecular markers Ki-67 and hypoxia inducible factor-α (HIF-α) in hepatocellular carcinoma (HCC). MATERIALS AND METHODS: Forty-seven patients diagnosed with HCC were included in this study. All patients performed diffusion-weighted magnetic resonance imaging (DW-MRI) before any anticancer treatment. The ADC maps were automatically calculated on a Syngo workstation. The Ki-67 and HIF-1α expression were assessed by immunohistochemistry. The Pearson correlation test was used to assess the correlation between ADC values and Ki-67 and HIF-1α expression. RESULTS: Ki-67 staining was clearly identified based on the brown nuclear staining in tumor cells. High Ki-67 expression was correlated with low differentiation (p=0.028). A significant correlation was observed between HIF-1α expression and maximum diameter (p=0.014). The mean ADC value was (0.983±0.21)×10(-3) mm(2)/s. The level of Ki-67 expression was correlated inversely with the ADC values (r=-0.371, p=0.01). There was a significant positive correlation between the ADC values and HIF-1α expression (r=0.389, p=0.007). CONCLUSION: The ADC values were observed to correlate significantly with the molecular markers Ki-67 and HIF-1α. Our results suggest that the ADC values on DW-MRI may be used as a measure of cell proliferativity and hypoxia in hepatocellular carcinoma.

[Preparation and identification of polyclonal antibody against small peptides of beta-tubulin of Toxoplasma gondii].

The amino acid sequences of beta-tubulin from Toxoplasma gondii stains (GT1 and ME49) and human were aligned by ClustalW2 software. Based on the alignment result, the C-terminal peptides of beta-tubulin of T. gondii were artificially synthesized. Rabbits were immunized with 0.5 mg synthesized peptides for five times at 2-week intervals. Serum samples were collected at the second week after the final immunization, and were analyzed for specific antibodies by ELISA. Finally, the specific-beta-tubulin polyclonal antibody was evaluated by Western blotting with the total protein of RH strain, ME49 strain, and PRU strain of T. gondii, respectively. The results showed that beta-tubulin of T. gondii stains (GT1 and ME49) shared 100% amino-acid sequence identity, and there was 98% amino acid homology between T. gondii and human. The main variable region was the C-terminus. After the fifth immunization, the titers of polyclonal antibody reached 1 : 52,800. Western blotting result indicated that the specific-beta-tubulin polyclonal antibody reacted with beta-tubulin in all the three strains (RH, ME49, and PRU), respectively.

[Preparation and antigenicity analysis of recombinant aegyptin-like protein of Aedes albopictus].

OBJECTIVE: To clone and express the aegyptin-like protein (alALP) encoding gene from Aedes albopictus salivary gland, and analyze its antigenicity. METHODS: The homology, secondary structure and antigen peptides of alALP and aegyptin protein (GenBank No. ABF18122.1) was analyzed by bioinformatics software tools. Total RNA was extracted from Ae. albopictus salivary gland. The coding region of alALP (GenBank No. AY826121) was amplified by PCR. RT-PCR product was digested with restriction enzyme and ligated into a pGEX-6P-1 vector. The recombinant pGEX-6P-1-alALP plasmid was transformed into E. coli BL21 and induced by IPTG. The recombinant soluble GST-alALP fusion protein was purified with Glutathione Sepharose 4B. The expression product was analyzed by SDS-PAGE and Western blotting. Mice were immunized each with 60 microg purified GST-alALP at every 2 weeks for 3 times, and mouse anti-GST-alALP serum was prepared. Western blotting assay with mice anti-GST-alALP serum and serum of mice exposed to Ae. albopictus bites was used to analyze its antigenicity. RESULTS: Bioinformatics prediction results showed that alALP and aegyptin had 65.58% homology with a similar secondary structure, and a conservative polypeptide. The product of RT-PCR was 762 bp. The recombinant plasmid pGEX-6P-1-alALP was confirmed by double restriction enzyme digestion, PCR and sequencing. SDS-PAGE and Western blotting analysis showed that the bacteria containing recombinant plasmid pGEX-6p-1-alALP expressed a soluble recombinant fusion protein (M(r) 56 000) after being induced with IPTG. Western blotting analysis revealed that GST-alALP protein was recognized by mouse anti-GST-alALP serum and serum of mice ex- posed to Ae. albopictus bites. CONCLUSION: Mature peptide gene of alALP can be expressed in prokaryotic expression system, and the recombinant protein shows antigenicity.

[Preparation and application of the polyclonal antibody of Toxoplasma gondii autophagy protein 8 (TgAtg8)].

OBJECTIVE: To prepare and evaluate specific-TgAtg8 polyclonal antibody. METHODS: The known Saccharomyces cerevisiae Atg protein sequences were used to identify Toxoplasma gondii homologous protein through bioinformatics analysis. TgAtg8 cDNA was amplified and cloned into prokaryotic expression vector pGEX-6p-1. The constructed pGEX-6p-1-TgAtg8 was transformed into E. coli BL21 cells and induced with IPTG for expression. The expression product was analyzed through SDS-PAGE and Western blotting. The recombinant TgAtg8 protein with an N-terminal glutathione-S transferase tag was used to immunize rabbits and raise specific polyclonal antibody against TgAtg8. Subsequently, the antibody was applied for Western blotting and IFA assay. RESULTS: Recombinant expression plasmid of pGEX-6p-1-TgAtg8 was confirmed correct by restriction enzyme digestion and sequencing. SDS-PAGE and Western blotting analysis showed that the recombinant TgAtg8 protein with the predicted molecular weight (M(r)40000) was expressed highly in E. coli BL21. After immunization, the specific antibodies against TgAtg8 protein were produced. The anti-TgAtg8 polyclonal antibody reacted specifically with TgAtg8 fusion protein or endogenous TgAtg8. Importantly, IFA assay determined that the TgAtg8 signal was generally distributed throughout the cytoplasm of the tachyzoites. However, the green fluorescence signal gathered into one or more green spots after induction of autophagy. CONCLUSION: The specific polyclonal antibody against TgAtg8 could be used to observe the dynamics of autophagosome formation in T. gondii, which is useful tool to investigate the autophagic machinery in this parasite.

[Cloning and expression of Toxoplasma gondii autophagy-related protein 3 gene and preparation of its polyclonal antibody].

OBJECTIVE: To clone and express autophagy-related protein 3 (TgAtg3) gene of Toxoplasma gondii, and obtain the specific polyclonal antibody against TgAtg3. METHODS: TgAtg3 cDNA was inserted into prokaryotic expression vector pET28a. After identification, the constructed plasmid pET28a-TgAtg3 was transformed into E. coli Rosetta cells, and induced by special induction medium for expression of the protein. The recombinant protein was purified via Ni-NTA affinity chromatography. Western blotting assay was performed with anti-His tag mouse monoclonal antibody as the primary antibody. Rabbits were immunized with 125 µg purified TgAtg recombinant protein. Each rabbit received 4 immunizations at 2-week intervals with the same dose of antigen. The specific anti-TgAtg3 polyclonal antibody was obtained, and analyzed by Western blotting and indirect immunofluorescence assay (IFA). RESULTS: pET28a-TgAtg3 plasmid was identified by restriction enzyme digestion, PCR amplification and sequencing. SDS-PAGE and Western blotting analysis showed that the recombinant TgAtg3 protein (about Mr 44,000) was expressed in E. coli Rosetta cells. TgAtg3 protein from tachyzoite lysates was recognized by the specific anti-TgAtg3 polyclonal antibody. IFA assay determined that the specific polyclonal antibody bound to TgAtg3 protein from the cytoplasm of tachyzoites. CONCLUSION: The obtained soluble polyclonal antibody against TgAtg3 can specifically react to the endogenous TgAtg3 protein.