Intra- and Inter-cellular Modeling of Dynamic Interaction between Zika Virus and Its Naturally Occurring Defective Viral Genomes.

Here, we examine in silico the infection dynamics and interactions of two Zika virus (ZIKV) genomes: one is the full-length ZIKV genome (wild type [WT]), and the other is one of the naturally occurring defective viral genomes (DVGs), which can replicate in the presence of the WT genome, appears under high-MOI (multiplicity of infection) passaging conditions, and carries a deletion encompassing part of the structural and NS1 protein-coding region. Ordinary differential equations (ODEs) were used to simulate the infection of cells by virus particles and the intracellular replication of the WT and DVG genomes that produce these particles. For each virus passage in Vero and C6/36 cell cultures, the rates of the simulated processes were fitted to two types of observations: virus titer data and the assembled haplotypes of the replicate passage samples. We studied the consistency of the model with the experimental data across all passages of infection in each cell type separately as well as the sensitivity of the model's parameters. We also determined which simulated processes of virus evolution are the most important for the adaptation of the WT and DVG interplay in these two disparate cell culture environments. Our results demonstrate that in the majority of passages, the rates of DVG production are higher inC6/36 cells than in Vero cells, which might result in tolerance and therefore drive the persistence of the mosquito vector in the context of ZIKV infection. Additionally, the model simulations showed a slower accumulation of infected cells under higher activation of the DVG-associated processes, which indicates a potential role of DVGs in virus attenuation. IMPORTANCE One of the ideas for lessening Zika pathogenicity is the addition of its natural or engineered defective virus genomes (DVGs) (have no pathogenicity) to the infection pool: a DVG is redirecting the wild-type (WT)-associated virus development resources toward its own maturation. The mathematical model presented here, attuned to the data from interplays between WT Zika viruses and their natural DVGs in mammalian and mosquito cells, provides evidence that the loss of uninfected cells is attenuated by the DVG development processes. This model enabled us to estimate the rates of virus development processes in the WT/DVG interplay, determine the key processes, and show that the key processes are faster in mosquito cells than in mammalian ones. In general, the presented model and its detailed study suggest in what important virus development processes the therapeutically efficient DVG might compete with the WT; this may help in assembling engineered DVGs for ZIKV and other flaviviruses.

Xenograft-derived mRNA/miR and protein interaction networks of systemic dissemination in human prostate cancer.

BACKGROUND: Distant metastasis formation is the major clinical problem in prostate cancer (PCa) and the underlying mechanisms remain poorly understood. Our aim was to identify novel molecules that functionally contribute to human PCa systemic dissemination based on unbiased approaches. METHODS: We compared mRNA, microRNA (miR) and protein expression levels in established human PCa xenograft tumours with high (PC-3), moderate (VCaP) or weak (DU-145) spontaneous micrometastatic potential. By focussing on those mRNAs, miRs and proteins that were differentially regulated among the xenograft groups and known to interact with each other we constructed dissemination-related mRNA/miR and protein/miR networks. Next, we clinically and functionally validated our findings. RESULTS: Besides known determinants of PCa progression and/or metastasis, our interaction networks include several novel candidates. We observed a clear role of epithelial-to-mesenchymal transition (EMT) pathways for PCa dissemination, which was additionally confirmed by an independent human PCa model (ARCAP-E/-M). Two converging nodes, CD46 (decreasing with metastatic potential) and DDX21 (increasing with metastatic potential), were used to test the clinical relevance of the networks. Intriguingly, both network nodes consistently added prognostic information for patients with PCa whereas CD46 loss predicted poor outcome independent of established parameters. Accordingly, depletion of CD46 in weakly metastatic PCa cells induced EMT-like properties in vitro and spontaneous micrometastasis formation in vivo. CONCLUSIONS: The clinical and functional relevance of the dissemination-related interaction networks shown here could be successfully validated by proof-of-principle experiments. Therefore, we suggest a direct pro-metastatic, clinically relevant role for the multiple novel candidates included in this study; these should be further exploited by future studies.

Cumulative prognostic power of laminin genes in colorectal cancer.

BACKGROUND: Laminins are a major family of extracellular matrix proteins and the main component of basement membranes. Laminins are involved in many if not all stages of cancer progression, and expression of laminin genes has prognostic value in various types of cancer, including colorectal. Only single laminin genes or components of a single laminin trimer with significant differential expression have been regarded as potential biomarkers to date. RESULTS: Here we compared prognostic power of classifiers constructed from sets of laminin genes with that of any single laminin gene. The analysis showed that cumulative prognostic power of sets of laminin genes was higher and was achieved already with pairs and triples of the genes. Interestingly, components of the pairs and the triples did not belong to any known laminin trimer, but, taken together with the gene weights, suggested higher LAMA4/LAMA5 expression ratio in patients with poor prognosis. CONCLUSIONS: Analysis of the laminin expression profile rather than expression of the single genes or components of laminin trimers is useful for colorectal cancer prognosis in patients. High LAMA4/LAMA5 ratio is associated with increased permeability of basement membranes suggesting that basement membranes produced by colorectal tumors might be an important hindrance to their own dissemination in patients.

Knockdown of L1CAM significantly reduces metastasis in a xenograft model of human melanoma: L1CAM is a potential target for anti-melanoma therapy.

Finding additional functional targets for combination therapy could improve the outcome for melanoma patients. In a spontaneous metastasis xenograft model of human melanoma a shRNA mediated knockdown of L1CAM more than sevenfold reduced the number of lung metastases after the induction of subcutaneous tumors for two human melanoma cell lines (MeWo, MV3). Whole genome expression arrays of the initially L1CAM high MeWo subcutaneous tumors revealed unchanged or downregulated genes involved in epithelial to mesenchymal transition (EMT) except an upregulation of Jagged 1, indicating a compensatory change in Notch signaling especially as Jagged 1 expression showed an increase in MeWo L1CAM metastases and Jagged 1 was expressed in metastases of the initially L1CAM low MV3 cells as well. Expression of 17 genes showed concordant regulation for L1CAM knockdown tumors of both cell lines. The changes in gene expression indicated changes in the EMT network of the melanoma cells and an increase in p53/p21 and p38 activity contributing to the reduced metastatic potential of the L1CAM knockdowns. Taken together, these data make L1CAM a highly interesting therapeutic target to prevent further metastatic spread in melanoma patients.

Comprehensive network of miRNA-induced intergenic interactions and a biological role of its core in cancer.

MicroRNAs (miRNAs) are a family of short noncoding RNAs that posttranscriptionally regulate gene expression and play an important role in multiple cellular processes. A significant percentage of miRNAs are intragenic, which is often functionally related to their host genes playing either antagonistic or synergistic roles. In this study, we constructed and analyzed the entire network of intergenic interactions induced by intragenic miRNAs. We further focused on the core of this network, which was defined as a union of nontrivial strongly connected components, i.e., sets of nodes (genes) mutually connected via directed paths. Both the entire network and its core possessed statistically significant non-random properties. Specifically, genes forming the core had high expression levels and low expression variance. Furthermore, the network core did not split into separate components corresponding to individual signalling or metabolic pathways, but integrated genes involved in key cellular processes, including DNA replication, transcription, protein homeostasis and cell metabolism. We suggest that the network core, consisting of genes mutually regulated by their intragenic miRNAs, could coordinate adjacent pathways or homeostatic control circuits, serving as a horizontal inter-circuit link. Notably, expression patterns of these genes had an efficient prognostic potential for breast and colorectal cancer patients.

The Transcriptome of Type I Murine Astrocytes under Interferon-Gamma Exposure and Remyelination Stimulus.

Astrocytes are considered to be an important contributor to central nervous system (CNS) disorders, particularly multiple sclerosis. The transcriptome of these cells is greatly affected by cytokines released by lymphocytes, penetrating the blood-brain barrier-in particular, the classical pro-inflammatory cytokine interferon-gamma (IFNγ). We report here the transcriptomal profiling of astrocytes treated using IFNγ and benztropine, a putative remyelinization agent. Our findings indicate that the expression of genes involved in antigen processing and presentation in astrocytes are significantly upregulated upon IFNγ exposure, emphasizing the critical role of this cytokine in the redirection of immune response towards self-antigens. Data reported herein support previous observations that the IFNγ-induced JAK-STAT signaling pathway may be regarded as a valuable target for pharmaceutical interventions.

Novel biomarkers in cancer: The whole is greater than the sum of its parts.

The major issues hampering progress in the treatment of cancer patients are distant metastases and drug resistance to chemotherapy. Metastasis formation is a very complex process, and looking at gene signatures alone is not enough to get deep insight into it. This paper reviews traditional and novel approaches to identify gene signature biomarkers and intratumoural fluid pressure both as a novel way of creating predictive markers and as an obstacle to cancer therapy. Finally recently developed in vitro systems to predict the response of individual patient derived cancer explants to chemotherapy are discussed.

Instrumental Mechanoreceptoric Palpation in Gastrointestinal Surgery.

BACKGROUND AND AIMS: Small gastric or colorectal tumours can be visually undetectable during laparoscopic surgeries, and available methods still do not provide a 100% localisation rate. Thus, new methods for further improvements in tumour localisation are highly desirable. In this study, we evaluated the usage of the Medical Tactile Endosurgical Complex (MTEC) in gastrointestinal surgery for localisation of tumours. The MTEC provides the possibility of instrumental mechanoreceptoric palpation, which serves as an analogue of conventional manual palpation. METHODS: Ninety-six elective surgeries were performed, including 48 open surgeries, 43 laparoscopies, and 5 robot-assisted surgeries. The 20 mm version of the MTEC tactile mechanoreceptor was used in open surgeries, and the 10 mm version in laparoscopic and robot-assisted surgeries. RESULTS: The mean time of instrumental mechanoreceptoric palpation was 3 minutes 12 seconds for open surgeries, which constituted the early stage of the learning curve, and 3 minutes 34 seconds for laparoscopic surgeries. No side effects or postoperative complications related to instrumental mechanoreceptoric palpation were observed, and this procedure provided data sufficient for tumour localisation in more than 95% of cases. CONCLUSION: Instrumental mechanoreceptoric palpation performed using MTEC is a simple, safe, and reliable method for tumour localisation in gastrointestinal laparoscopic surgery.

Instrumental tactile diagnostics in robot-assisted surgery.

BACKGROUND: Robotic surgery has gained wide acceptance due to minimizing trauma in patients. However, the lack of tactile feedback is an essential limiting factor for the further expansion. In robotic surgery, feedback related to touch is currently kinesthetic, and it is mainly aimed at the minimization of force applied to tissues and organs. Design and implementation of diagnostic tactile feedback is still an open problem. We hypothesized that a sufficient tactile feedback in robot-assisted surgery can be provided by utilization of Medical Tactile Endosurgical Complex (MTEC), which is a novel specialized tool that is already commercially available in the Russian Federation. MTEC allows registration of tactile images by a mechanoreceptor, real-time visualization of these images, and reproduction of images via a tactile display. MATERIALS AND METHODS: Nine elective surgeries were performed with da Vinci™ robotic system. An assistant performed tactile examination through an additional port under the guidance of a surgeon during revision of tissues. The operating surgeon sensed registered tactile data using a tactile display, and the assistant inspected the visualization of tactile data. First, surgeries where lesion boundaries were visually detectable were performed. The goal was to promote cooperation between the surgeon and the assistant and to train them in perception of the tactile feedback. Then, instrumental tactile diagnostics was utilized in case of visually undetectable boundaries. RESULTS: In robot-assisted surgeries where lesion boundaries were not visually detectable, instrumental tactile diagnostics performed using MTEC provided valid identification and localization of lesions. The results of instrumental tactile diagnostics were concordant with the results of intraoperative ultrasound examination. However, in certain cases, for example, thoracoscopy, ultrasound examination is inapplicable, while MTEC-based tactile diagnostics can be efficiently utilized. CONCLUSION: The study proved that MTEC can be efficiently used in robot-assisted surgery allowing correct localization of visually undetectable lesions and visually undetectable boundaries of pathological changes of tissues.

miRNome of inflammatory breast cancer.

BACKGROUND: Inflammatory breast cancer (IBC) is an extremely malignant form of breast cancer which can be easily misdiagnosed. Conclusive prognostic IBC molecular biomarkers which are also providing the perspectives for targeted therapy are lacking so far. The aim of this study was to reveal the IBC-specific miRNA expression profile and to evaluate its association with clinicopathological parameters. METHODS: miRNA expression profiles of 13 IBC and 17 non-IBC patients were characterized using comprehensive Affymetrix GeneChip miRNA 3.0 microarray platform. Bioinformatic analysis was used to reveal IBC-specific miRNAs, deregulated pathways and potential miRNA targets. RESULTS: 31 differentially expressed miRNAs characterize IBC and mRNAs regulated by them and their associated pathways can functionally be attributed to IBC progression. In addition, a minimal predictive set of 4 miRNAs characteristic for the IBC phenotype and associated with the TP53 mutational status in breast cancer patients was identified. CONCLUSIONS: We have characterized the complete miRNome of inflammatory breast cancer and found differentially expressed miRNAs which reliably classify the patients to IBC and non-IBC groups. We found that the mRNAs and pathways likely regulated by these miRNAs are highly relevant to cancer progression. Furthermore a minimal IBC-related predictive set of 4 miRNAs associated with the TP53 mutational status and survival for breast cancer patients was identified.

Exercise immunology meets MiRNAs.

A large body of evidence indicates modified expression of protein-coding genes in response to different kinds of physical activity. Recent years have exposed another level of regulation of cellular processes mediated by non-coding RNAs. MicroRNAs (miRNAs) are one of the largest families of non-coding RNAs. MiRNAs mediate post-transcriptional regulation of gene expression. The amount of data supporting the key role of miRNAs in the adaptation of the immune and other body systems to exercise steadily grows. MiRNAs change their expression profiles after exercise and seem to be involved in regulation of exercise-responsive genes in immune and other cell types. Here we discuss existing data and future directions in the field.

Dynamically regulated miRNA-mRNA networks revealed by exercise.

BACKGROUND: MiRNAs are essential mediators of many biological processes. The aim of this study was to investigate the dynamics of miRNA-mRNA regulatory networks during exercise and the subsequent recovery period. RESULTS: Here we monitored the transcriptome changes using microarray analysis of the whole blood of eight highly trained athletes before and after 30 min of moderate exercise followed by 30 min and 60 min of recovery period. We combined expression profiling and bioinformatics and analysed metabolic pathways enriched with differentially expressed mRNAs and mRNAs which are known to be validated targets of differentially expressed miRNAs. Finally we revealed four dynamically regulated networks comprising differentially expressed miRNAs and their known target mRNAs with anti-correlated expression profiles over time. The data suggest that hsa-miR-21-5p regulated TGFBR3, PDGFD and PPM1L mRNAs. Hsa-miR-24-2-5p was likely to be responsible for MYC and KCNJ2 genes and hsa-miR-27a-5p for ST3GAL6. The targets of hsa-miR-181a-5p included ROPN1L and SLC37A3. All these mRNAs are involved in processes highly relevant to exercise response, including immune function, apoptosis, membrane traffic of proteins and transcription regulation. CONCLUSIONS: We have identified metabolic pathways involved in response to exercise and revealed four miRNA-mRNA networks dynamically regulated following exercise. This work is the first study to monitor miRNAs and mRNAs in parallel into the recovery period. The results provide a novel insight into the regulatory role of miRNAs in stress adaptation.

High-throughput identification of reference genes for research and clinical RT-qPCR analysis of breast cancer samples.

BACKGROUND: Quantification and normalization of RT-qPCR data critically depends on the expression of so called reference genes. Our goal was to develop a strategy for the selection of reference genes that utilizes microarray data analysis and combines known approaches for gene stability evaluation and to select a set of appropriate reference genes for research and clinical analysis of breast samples with different receptor and cancer status using this strategy. METHODS: A preliminary search of reference genes was based on high-throughput analysis of microarray datasets. The final selection and validation of the candidate genes were based on the RT-qPCR data analysis using several known methods for expression stability evaluation: comparative ∆Ct method, geNorm, NormFinder and Haller equivalence test. RESULTS: A set of five reference genes was identified: ACTB, RPS23, HUWE1, EEF1A1 and SF3A1. The initial selection was based on the analysis of publically available well-annotated microarray datasets containing different breast cancers and normal breast epithelium from breast cancer patients and epithelium from cancer-free patients. The final selection and validation were performed using RT-qPCR data from 39 breast cancer biopsy samples. Three genes from the final set were identified by the means of microarray analysis and were novel in the context of breast cancer assay. We showed that the selected set of reference genes is more stable in comparison not only with individual genes, but also with a system of reference genes used in commercial OncotypeDX test. CONCLUSION: A selection of reference genes for RT-qPCR can be efficiently performed by combining a preliminary search based on the high-throughput analysis of microarray datasets and final selection and validation based on the analysis of RT-qPCR data with a simultaneous examination of different expression stability measures. The identified set of reference genes proved to be less variable and thus potentially more efficient for research and clinical analysis of breast samples comparing to individual genes and the set of reference genes used in OncotypeDX assay.