Analysis of noncanonical calciumdependent protein kinases in Toxoplasma gondii by targeted gene deletion using CRISPR/Cas9

Calcium-dependent protein kinases (CDPKs) are expanded in apicomplexan parasites, especially in Toxoplasma gondii where 14 separate genes encoding these enzymes are found. Although previous studies have shown that several CDPKs play a role in controlling invasion, egress, and cell division in T. gondii, the roles of most of these genes are unexplored. Here we developed a more efficient method for gene disruption using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) that was modified to completely delete large, multiexonic genes from the genome and to allow serial replacement by recycling of the selectable marker using Cre-loxP. Using this system, we generated a total of 24 mutants in type 1 and 2 genetic backgrounds to ascertain the functions of noncanonical CDPKs. Remarkably, although we were able to confirm the essentiality of CDPK1 and CDPK7, the majority of CDPKs had no discernible phenotype for growth in vitro or infection in the mouse model. The exception to this was CDPK6, loss of which leads to reduced plaquing, fitness defect in a competition assay, and reduced tissue cyst formation in chronically infected mice. Our findings highlight the utility of CRISPR/Cas9 for rapid serial gene deletion and also suggest that additional models are needed to reveal the functions of many genes in T. gondii

Dipole: Diagnosis Prediction in Healthcare via Attention-based Bidirectional Recurrent Neural Networks

Predicting the future health information of patients from the historical Electronic Health Records (EHR) is a core research task in the development of personalized healthcare. Patient EHR data consist of sequences of visits over time, where each visit contains multiple medical codes, including diagnosis, medication, and procedure codes. The most important challenges for this task are to model the temporality and high dimensionality of sequential EHR data and to interpret the prediction results. Existing work solves this problem by employing recurrent neural networks (RNNs) to model EHR data and utilizing simple attention mechanism to interpret the results. However, RNN-based approaches suffer from the problem that the performance of RNNs drops when the length of sequences is large, and the relationships between subsequent visits are ignored by current RNN-based approaches. To address these issues, we propose {\sf Dipole}, an end-to-end, simple and robust model for predicting patients' future health information. Dipole employs bidirectional recurrent neural networks to remember all the information of both the past visits and the future visits, and it introduces three attention mechanisms to measure the relationships of different visits for the prediction. With the attention mechanisms, Dipole can interpret the prediction results effectively. Dipole also allows us to interpret the learned medical code representations which are confirmed positively by medical experts. Experimental results on two real world EHR datasets show that the proposed Dipole can significantly improve the prediction accuracy compared with the state-of-the-art diagnosis prediction approaches and provide clinically meaningful interpretation

A stem-cell-derived platform enables complete Cryptosporidium development in vitro and genetic tractability

Despite being a frequent cause of severe diarrheal disease in infants and an opportunistic infection in immunocompromised patients, Cryptosporidium research has lagged due to a lack of facile experimental methods. Here, we describe a platform for complete life cycle development and long-term growth of C. parvum in vitro using air-liquid interface (ALI) cultures derived from intestinal epithelial stem cells. Transcriptomic profiling revealed that differentiating epithelial cells grown under ALI conditions undergo profound changes in metabolism and development that enable completion of the parasite life cycle in vitro. ALI cultures support parasite expansion \u3e 100-fold and generate viable oocysts that are transmissible in vitro and to mice, causing infection and animal death. Transgenic parasite lines created using CRISPR/Cas9 were used to complete a genetic cross in vitro, demonstrating Mendelian segregation of chromosomes during meiosis. ALI culture provides an accessible model that will enable innovative studies into Cryptosporidium biology and host interactions

Rhoptry proteins ROP5 and ROP18 are major murine virulence factors in genetically divergent South American strains of Toxoplasma gondii

Toxoplasma gondii has evolved a number of strategies to evade immune responses in its many hosts. Previous genetic mapping of crosses between clonal type 1, 2, and 3 strains of T. gondii, which are prevalent in Europe and North America, identified two rhoptry proteins, ROP5 and ROP18, that function together to block innate immune mechanisms activated by interferon gamma (IFNg) in murine hosts. However, the contribution of these and other virulence factors in more genetically divergent South American strains is unknown. Here we utilized a cross between the intermediately virulent North American type 2 ME49 strain and the highly virulent South American type 10 VAND strain to map the genetic basis for differences in virulence in the mouse. Quantitative trait locus (QTL) analysis of this new cross identified one peak that spanned the ROP5 locus on chromosome XII. CRISPR-Cas9 mediated deletion of all copies of ROP5 in the VAND strain rendered it avirulent and complementation confirmed that ROP5 is the major virulence factor accounting for differences between type 2 and type 10 strains. To extend these observations to other virulent South American strains representing distinct genetic populations, we knocked out ROP5 in type 8 TgCtBr5 and type 4 TgCtBr18 strains, resulting in complete loss of virulence in both backgrounds. Consistent with this, polymorphisms that show strong signatures of positive selection in ROP5 were shown to correspond to regions known to interface with host immunity factors. Because ROP5 and ROP18 function together to resist innate immune mechanisms, and a significant interaction between them was identified in a two-locus scan, we also assessed the role of ROP18 in the virulence of South American strains. Deletion of ROP18 in South American type 4, 8, and 10 strains resulted in complete attenuation in contrast to a partial loss of virulence seen for ROP18 knockouts in previously described type 1 parasites. These data show that ROP5 and ROP18 are conserved virulence factors in genetically diverse strains from North and South America, suggesting they evolved to resist innate immune defenses in ancestral T. gondii strains, and they have subsequently diversified under positive selection

Toxoplasma gondii infection drives conversion of NK cells into ILC1-like cells

Innate lymphoid cells (ILCs) were originally classified based on their cytokine profiles, placing natural killer (NK) cells and ILC1s together, but recent studies support their separation into different lineages at steady-state. However, tumors may induce NK cell conversion into ILC1-like cells that are limited to the tumor microenvironment and whether this conversion occurs beyond this environment remains unknown. Here, we describ

Genetic diversity of food-medicinal <i>Lycium </i>spp. in China: Insights from chloroplast genome

Objective: Goji (fruits of Lycium spp.) is commonly consumed as food and medicine. The increasing market demand for goji has led to its wide cultivation and broad breeding, which might cause loss of genetic diversity. This study aims to uncover the genetic diversity of the cultivated and wild Lycium. Methods: The chloroplast genome (CPG) of 34 accessions of Chinese food-medicinal Lycium spp., including the popular cultivars and their wild relatives, was re-sequenced and assembled, based on which the genetic diversity was evaluated. Results: Sequence structural comparison shows that CPG is comparatively conserved within species. Phylogenetic analysis indicates that CPG is sufficient for the discrimination of Lycium species; combined with nuclear ribosomal internal transcribed spacer (Nr ITS) sequences, materials with mixed genetic backgrounds can be identified. Nucleotide diversity analysis reveals that the modern cultivars are probably with a common maternal parent, while the wild accessions are with higher level of genetic diversity. Conclusion: For the first time this study reveals the intraspecies genetic diversity of Lycium spp. using CPG, highlighting the urgent conservation demand of wild genetic resources of Lycium. Our study also demonstrates that CPG provides crucial evidence for identification of Lycium species with mixed genetic backgrounds and highlights the importance of the wild relatives in genetic diversity conservation. This CPG-based technology will contribute to the sustainable development of medicinal plants broadly