Malaria parasites have a complex life cycle with multiple developmental stages in mosquito and vertebrate hosts, and different developmental stages express unique sets of genes. Unexpectedly, many transcription factors (TFs) commonly found in eukaryotic organisms are absent in malaria parasites; instead, a family of genes encoding proteins similar to the plant Apetala2 (ApiAP2) transcription factors is expanded in the parasites. Several malaria ApiAP2 genes have been shown to play a critical role in parasite development; however, the functions of the majority of the ApiAP2 genes remain to be elucidated. In particular, no study on the Plasmodium yoelii ApiAP2 (PyApiAP2) gene family has been reported so far. This study systematically investigated the functional roles of PyApiAP2 genes in parasite development. Twenty-four of the 26 PyApiAP2 genes were selected for disruption, and 12 were successfully knocked out using the clustered regularly interspaced short palindromic repeat-CRISPR-associated protein 9 (CRISPR-Cas9) method. The effects of gene knockout (KO) on parasite development in mouse and mosquito stages were evaluated. Ten of 12 successfully disrupted genes, including two genes that have not been functionally characterized in any Plasmodium species previously, were shown to be critical for P. yoelii development of sexual and mosquito stages. Additionally, seven of the genes were labeled for protein expression analysis, revealing important information supporting their functions. This study represents the first systematic functional characterization of the P. yoelii ApiAP2 gene family and discovers important insights on the roles of the ApiAP2 genes in parasite development.IMPORTANCE Malaria is a parasitic disease that infects hundreds of millions of people, leading to an estimated 0.35 million deaths in 2015. A better understanding of the mechanism of gene expression regulation during parasite development may provide important clues for disease control and prevention. In this study, systematic gene disruption experiments were performed to study the functional roles of members of the Plasmodium yoelii ApiAP2 (PyApiAP2) gene family in parasite development. Genes that are critical for the development of male and female gametocytes, oocysts, and sporozoites were characterized. The protein expression profiles for seven of the PyApiAP2 gene products were also analyzed, revealing important information on their functions. This study provides expression and functional information for many PyApiAP2 genes, which can be explored for disease management.
Malaria parasites are unicellular organisms residing inside the red blood cells, and current methods for editing the parasite genes have been inefficient. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats and Cas9 endonuclease-mediated genome editing) system is a new powerful technique for genome editing and has been widely employed to study gene function in various organisms. However, whether this technique can be applied to modify the genomes of malaria parasites has not been determined. In this paper, we demonstrated that Cas9 is able to introduce site-specific DNA double-strand breaks in the Plasmodium yoelii genome that can be repaired through homologous recombination. By supplying engineered homologous repair templates, we generated targeted deletion, reporter knock-in, and nucleotide replacement in multiple parasite genes, achieving up to 100% efficiency in gene deletion and 22 to 45% efficiencies in knock-in and allelic replacement. Our results establish methodologies for introducing desired modifications in the P. yoelii genome with high efficiency and accuracy, which will greatly improve our ability to study gene function of malaria parasites. Importance: Malaria, caused by infection of Plasmodium parasites, remains a world-wide public health burden. Although the genomes of many malaria parasites have been sequenced, we still do not know the functions of approximately half of the genes in the genomes. Studying gene function has become the focus of many studies; however, editing genes in malaria parasite genomes is still inefficient. Here we designed several efficient approaches, based on the CRISPR/Cas9 system, to introduce site-specific DNA double-strand breaks in the Plasmodium yoelii genome that can be repaired through homologous recombination. Using this system, we achieved high efficiencies in gene deletion, reporter tagging, and allelic replacement in multiple parasite genes. This technique for editing the malaria parasite genome will greatly facilitate our ability to elucidate gene function.
To combat future severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants and spillovers of SARS-like betacoronaviruses (sarbecoviruses) threatening global health, we designed mosaic nanoparticles that present randomly arranged sarbecovirus spike receptor-binding domains (RBDs) to elicit antibodies against epitopes that are conserved and relatively occluded rather than variable, immunodominant, and exposed. We compared immune responses elicited by mosaic-8 (SARS-CoV-2 and seven animal sarbecoviruses) and homotypic (only SARS-CoV-2) RBD nanoparticles in mice and macaques and observed stronger responses elicited by mosaic-8 to mismatched (not on nanoparticles) strains, including SARS-CoV and animal sarbecoviruses. Mosaic-8 immunization showed equivalent neutralization of SARS-CoV-2 variants, including Omicrons, and protected from SARS-CoV-2 and SARS-CoV challenges, whereas homotypic SARS-CoV-2 immunization protected only from SARS-CoV-2 challenge. Epitope mapping demonstrated increased targeting of conserved epitopes after mosaic-8 immunization. Together, these results suggest that mosaic-8 RBD nanoparticles could protect against SARS-CoV-2 variants and future sarbecovirus spillovers.
Objective:To examine serum sIgE of allergic rhinitis patients for Candida albicans.Methods:The serum sIgE of sixty allergic rhinitis patients with positive skin test of Candida albicans were detected by RAST EIA method and CAP system.Results:The positive rates of sIgE for RAST EIA method and CAP system were 56.7% and 61 7% respectively,which were highly correlated.Conclusion:Forty percent of patients with positive skin test for Candida albicans were not genuinely supersensitive to Candida albicans.
Summary The CO 2 concentrating mechanism (CCM) is a key component of the carbon assimilation strategy of aquatic microalgae. Induced by limiting CO 2 and tightly regulated, the CCM enables these microalgae to respond rapidly to varying environmental CO 2 supplies and to perform photosynthetic CO 2 assimilation in a cost‐effective way. A functional CCM in eukaryotic algae requires Rubisco sequestration, rapid interconversion between CO 2 and catalyzed by carbonic anhydrases (CAs), and active inorganic carbon (Ci) uptake. In the model microalga Chlamydomonas reinhardtii , a membrane protein HLA3 is proposed to be involved in active Ci uptake across the plasma membrane. In this study, we use an artificially designed transcription activator‐like effector ( dTALE ) to activate the expression of HLA3. The successful activation of HLA3 expression demonstrates dTALE as a promising tool for gene‐specific activation and investigation of gene function in Chlamydomonas . Activation of HLA3 expression in high CO 2 acclimated cells, where HLA3 is not expressed, resulted in increased Ci accumulation and Ci‐dependent photosynthetic O 2 evolution specifically in very low CO 2 concentrations, which confirms that HLA3 is indeed involved in Ci uptake, and suggests it is mainly associated with transport in very low CO 2 concentrations, conditions in which active CO 2 uptake is highly limited.
Since neutralizing antibodies (NAb) targeting the human cytomegalovirus (HCMV) pentamer complex (PC) potently block HCMV host cell entry, anti-PC NAb induction is thought to be important for a vaccine formulation to prevent HCMV infection. By developing a vaccine strategy based on soluble PC protein and using a previously generated Modified Vaccinia Ankara vector co-expressing all five PC subunits (MVA-PC), we compared HCMV NAb induction by homologous immunization using prime-boost vaccine regimen employing only PC protein or MVA-PC and heterologous immunization using prime-boost combinations of PC protein and MVA-PC. Utilizing a recently isolated anti-PC NAb, we produced highly pure soluble PC protein that displayed conformational and linear neutralizing epitopes, interfered with HCMV entry, and was recognized by antibodies induced by HCMV during natural infection. Mice vaccinated by different immunization routes with the purified PC protein in combination with a clinically approved adjuvant formulation elicited high-titer and durable HCMV NAb. While MVA-PC and soluble PC protein either alone or in combination elicited robust HCMV NAb, significantly different potencies of these vaccine approaches were observed in dependence on immunization schedule. Using only two immunizations, vaccination with MVA-PC alone or prime-boost combinations of MVA-PC and PC protein was significantly more effective in stimulating HCMV NAb than immunization with PC protein alone. In contrast, with three immunizations, NAb induced by soluble PC protein either alone or combined with two boosts of MVA-PC increased to levels that exceeded NAb titer stimulated by MVA-PC alone. These results provide insights into the potency of soluble protein and MVA to elicit NAb by the HCMV PC via homologous and heterologous prime-boost immunization, which may contribute to develop clinically deployable vaccine strategies to prevent HCMV infection.
Two experiments were conducted to study the effect of Banzyladenin and Gibberellin on increasing runner production and some vegetative traits of three strawberry cultivars in greenhouse. The experiments were carried out in a Completely Randomized Design (CRD), with two factors (hormonal treatments and three strawberry cultivars). In the first year (2006), hormonal treatments had two levels (a combination of BA at 1200 ppm and GA in 300 ppm, and control). Three strawberry cultivars (Pajaro, Queen Eliza and Paros) were sprayed in May and July. In the second experiment (2007), hormonal treatments had five levels (1- Control (0 ppm), 2- BA 1200 ppm + GA 300 ppm sprayed in May, 3- BA 1200 ppm + GA 300 ppm sprayed in July, 4- BA 1200 ppm sprayed in May, 5- GA 300 ppm sprayed in May). Effects of hormonal treatments were not significant on any of the vegetative traits in the first year, but not the second year. Highest number of plantlets with a standard diameter of 6.15 mm was produced by applying the second treatment (mixture of BA 1200 ppm and GA 300 ppm sprayed in May) and it was chosen as the best treatment. However, highest number of runners in each mother plant and highest diameter of plantlets were observed in the control (0 ppm). Cultivars responded differently to hormones. Number of runners in Paros cultivar decreased dramatically with application of hormones, but it did not change in Queen Eliza cultivar.
Key words: Hormone, daughter plant, plantlet, crown, strawberry.
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