During invasion, apicomplexan parasites form an intimate circumferential contact with the host cell, the tight junction (TJ), through which they actively glide. The TJ, which links the parasite motor to the host cell cytoskeleton, is thought to be composed of interacting apical membrane antigen 1 (AMA1) and rhoptry neck (RON) proteins. Here we find that, in Plasmodium berghei, while both AMA1 and RON4 are important for merozoite invasion of erythrocytes, only RON4 is required for sporozoite invasion of hepatocytes, indicating that RON4 acts independently of AMA1 in the sporozoite. Further, in the Toxoplasma gondii tachyzoite, AMA1 is dispensable for normal RON4 ring and functional TJ assembly but enhances tachyzoite apposition to the cell and internalization frequency. We propose that while the RON proteins act at the TJ, AMA1 mainly functions on the zoite surface to permit correct attachment to the cell, which may facilitate invasion depending on the zoite-cell combination.

We describe here an efficient method for conditional gene inactivation in malaria parasites that uses the Flp/FRT site-specific recombination system of yeast. The method, developed in Plasmodium berghei, consists of inserting FRT sites in the chromosomal locus of interest in a parasite clone expressing the Flp recombinase via a developmental stage-specific promoter. Using promoters active in mosquito midgut sporozoites or salivary gland sporozoites to drive expression of Flp or its thermolabile variant, FlpL, we show that excision of the DNA flanked by FRT sites occurs efficiently at the stage of interest and at undetectable levels in prior stages. We applied this technique to conditionally silence MSP1, a gene essential for merozoite invasion of erythrocytes. Silencing MSP1 in sporozoites impaired subsequent merozoite formation in the liver. Therefore, MSP1 plays a dual role in the parasite life cycle, acting both in liver and erythrocytic parasite stages.

We describe here a highly efficient procedure for conditional mutagenesis in Plasmodium. The procedure uses the site-specific recombination FLP-FRT system of yeast and targets the pre-erythrocytic stages of the rodent Plasmodium parasite P. berghei, including the sporozoite stage and the subsequent liver stage. The technique consists of replacing the gene under study by an FRTed copy (i.e., flanked by FRT sites) in the erythrocytic stages of a parasite clone that expresses the flip (FLP) recombinase stage-specifically--called the 'deleter' clone. We present the available deleter clones, which express FLP at different times of the parasite life cycle, as well as the schemes and tools for constructing new deleter parasites. We also outline and discuss the various strategies for exchanging a wild-type gene with an FRTed copy and for generating conditional gene knockout or knockdown parasite clones. Finally, we detail the protocol for obtaining sporozoites that lack a protein of interest and for monitoring sporozoite-specific DNA excision and depletion of the target protein. The protocol should allow the functional analysis of any essential protein in the sporozoite, liver stage or hepatic merozoite stages of rodent Plasmodium parasites.

Cancer is a heterogeneous disease with unique genomic and phenotypic features that differ between individual patients and even among individual tumor regions. In recent years, large-scale genomic studies and new next-generation sequencing technologies have uncovered more scientific details about tumor heterogeneity, with significant implications for the choice of specific molecular biomarkers and clinical decision making. Genomic heterogeneity significantly contributes to the generation of a diverse cell population during tumor development and progression, representing a determining factor for variation in tumor treatment response. It has been considered a prominent contributor to therapeutic failure, and increases the likelihood of resistance to future therapies in most common cancers. The understanding of molecular heterogeneity in cancer is a fundamental component of precision oncology, enabling the identification of genomic alteration of key genes and pathways that can be targeted therapeutically. Here, we review the emerging knowledge of tumor genomics and heterogeneity, as well as potential implications for precision medicine in cancer treatment and new therapeutic discoveries. An analysis and interpretation of the TCGA database was included.

Human papilloma viruses (HPV) are linked to an epidemic increase in oropharyngeal head and neck squamous cell carcinomas (HNSCC), which display viral inactivation of tumor suppressors TP53 and RB1 and rapid regional spread. However, the role of genomic alterations in enabling the modulation of pathways that promote the aggressive phenotype of these cancers is unclear. Recently, a subset of HPV HNSCC has been shown to harbor novel genetic defects or decreased expression of (). TRAF3 has been implicated as a negative regulator of alternative NF-κB pathway activation and activator of antiviral type I IFN response to other DNA viruses. How alterations affect pathogenesis of HPV HNSCC has not been extensively investigated. Here, we report that TRAF3-deficient HPV tumors and cell lines exhibit increased expression of alternative NF-κB pathway components and transcription factors NF-κB2/RELB. Overexpression of in HPV cell lines with decreased endogenous TRAF3 inhibited NF-κB2/RELB expression, nuclear localization, and NF-κB reporter activity, while increasing the expression of IFNA1 mRNA and protein and sensitizing cells to its growth inhibition. Overexpression of TRAF3 also enhanced TP53 and RB tumor suppressor proteins and decreased HPV E6 oncoprotein in HPV cells. Correspondingly, TRAF3 inhibited cell growth, colony formation, migration, and resistance to TNFα and cisplatin-induced cell death. Conversely, knockout enhanced colony formation and proliferation of an HPV HNSCC line expressing higher TRAF3 levels. Together, these findings support a functional role of as a tumor suppressor modulating established cancer hallmarks in HPV HNSCC. These findings report the functional role of TRAF3 as a tumor suppressor that modulates the malignant phenotype of HPV head and neck cancers. .

BackgroundOsteoarthritis (OA) is described by an imbalance between anabolic and catabolic processes in the affected joint. This dysregulation of metabolism affects not only chondrocytes within cartilage tissue but also the cells of the synovial membrane across the border of the joint. An important factor in OA is the low viscosity of the synovial fluid. High-molecular-weight hyaluronic acid (HA) can be used to increase the viscosity and also reduce inflammatory processes. The purpose was to establish an in vitro inflammation model and to evaluate the effects of high-molecular-weight HA in a co-cultivation inflammation model of osteoarthritic chondrocytes and M1 macrophages.MethodsFor the establishment of the inflammation model THP-1 cells were, at first, differentiated to M0 macrophages and then activated to the M1 subtype after 5 days of resting period. Surface markers, cytokine release, and gene expression, were analyzed to examine the successful differentiation. In the inflammation model, the defined M1 macrophages were co-cultivated with osteoarthritic chondrocytes for 2 days, with and without the addition of 10 % HA and further analyzed for chondrogenic gene expression markers and the release of cytokines in the supernatant.ResultsThe differentiation and activation process was successful as M1 macrophages expressed higher levels of pro-inflammatory cytokines and specific genes. Similarly, the surface marker CD14 was significantly decreased compared to M0 macrophages. For the co-culture system, the analysis of gene expression showed that HA increased the expression of cartilage-specific genes while catabolic-encoding genes exhibited lower expression levels than the control group. This positive effect of HA was also demonstrated by the measurement of pro-inflammatory cytokines, as their level decreased.ConclusionOur study implies that high-molecular-weight HA has a chondroprotective effect in the present co-cultivation inflammation model, as it decreases pro-inflammatory cytokines and increases anabolic factors.

Colorectal cancer (CRC) is a heterogeneous disease that is associated with a gradual accumulation of genetic and epigenetic alterations. Among all CRC stages, stage II tumors are highly heterogeneous with a high relapse rate in about 20–25 % of stage II CRC patients following surgery. Thus, a comprehensive analysis of gene signatures to identify aggressive and metastatic phenotypes in stage II CRC is desired for a more accurate disease classification and outcome prediction. By utilizing a Cancer Array, containing 440 oncogenes and tumor suppressors to profile mRNA expression, we identified a larger number of differentially expressed genes in poorly differentiated stage II colorectal adenocarcinoma tissues, compared to their matched normal tissues. Ontology and Ingenuity Pathway Analysis (IPA) indicated that these genes are involved in functional mechanisms associated with several transcription factors. Genomic alterations of these genes were also investigated through The Cancer Genome Atlas (TCGA) database, utilizing 195 published CRC specimens. The percentage of genomic alterations in these genes was ranked based on their mRNA expression, copy number variations and mutations. This data was further combined with published microarray studies from a large set of CRC tumors classified based on prognostic features. This led to the identification of eight candidate genes including RPN2, HMGB1, AARS, IGFBP3, STAT1, HYOU1, NQO1 and PEA15 that were associated with the progressive phenotype. In particular, RPN2 and HMGB1 displayed a higher genomic alteration frequency in CRC, compared to eight other major solid cancers. Immunohistochemistry was performed on additional 78 stage I–IV CRC samples, where RPN2 protein immunostaining exhibited a significant association with stage III/IV tumors, distant metastasis, and poor differentiation, indicating that RPN2 expression is associated with poor prognosis. Further, our study revealed significant transcriptional regulatory mechanisms, networks and gene signatures, underlying CRC malignant progression and phenotype warranting future clinical investigations.Electronic supplementary materialThe online version of this article (doi:10.1186/s13578-015-0043-9) contains supplementary material, which is available to authorized users.

During early bone formation, mesenchymal cells condense and then differentiate into collagen type II‐expressing chondrocytes that make up the cartilaginous bone anlagen. This anlage then becomes enclosed by the perichondrium. The mechanisms by which the perichondrium forms are not known. The purpose of this study was to determine whether epiphyseal chondrocytes can differentiate into perichondrial cells. Novel perichondrium markers were identified by expression microarray of microdissected rat perichondrium and growth plate cartilage. A dissection method that allowed for removal of contaminating perichondrium was developed and the absence was confirmed by histological examination and by expression of perichondrium markers. Perichondrium formation surrounding chondrocyte pellets was studied using histology, real‐time PCR, and in situ hybridization for chondrocyte and perichondrium markers. Cultured chondrocyte pellets developed an exterior perichondrium‐like layer. This surrounding tissue did not express chondrocyte markers, collagen‐type II and type X, as assessed by in situ hybridization. Instead, perichondrium markers, periostin, Dickkopf 3 (Dkk3), roundabout 2, cadherin 2, L‐galectin 1 (Lgals1), and thrombospondin 2 (Thbs2) were upregulated following formation of the perichondrium‐like layer as assessed by real‐time PCR. Interestingly, markers specific for the cambium layer, Dkk3, Thbs2, and Lgals1, but not for the fibrous layer, collagen‐type XIV and decorin, were upregulated. The findings suggest that epiphyseal chondrocytes of postnatal animals retain the potential to differentiate into perichondrial cells, supporting the hypothesis that the perichondrium originates from collagen type II‐expressing chondrocytes at the periphery of the cartilaginous bone template. © 2018 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.